US20060167227A1 - Truncated tau proteins - Google Patents
Truncated tau proteins Download PDFInfo
- Publication number
- US20060167227A1 US20060167227A1 US10/521,140 US52114005A US2006167227A1 US 20060167227 A1 US20060167227 A1 US 20060167227A1 US 52114005 A US52114005 A US 52114005A US 2006167227 A1 US2006167227 A1 US 2006167227A1
- Authority
- US
- United States
- Prior art keywords
- tau
- lys
- type
- ser
- molecules
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108010026424 tau Proteins Proteins 0.000 title claims description 445
- 102000013498 tau Proteins Human genes 0.000 title claims description 444
- 238000000034 method Methods 0.000 claims abstract description 66
- 238000012216 screening Methods 0.000 claims abstract description 32
- 102000029749 Microtubule Human genes 0.000 claims description 148
- 108091022875 Microtubule Proteins 0.000 claims description 148
- 210000004688 microtubule Anatomy 0.000 claims description 148
- 230000001575 pathological effect Effects 0.000 claims description 64
- 241001465754 Metazoa Species 0.000 claims description 54
- 208000024827 Alzheimer disease Diseases 0.000 claims description 46
- 238000003556 assay Methods 0.000 claims description 45
- 230000002401 inhibitory effect Effects 0.000 claims description 41
- 230000003472 neutralizing effect Effects 0.000 claims description 40
- 238000000338 in vitro Methods 0.000 claims description 34
- 230000009261 transgenic effect Effects 0.000 claims description 31
- 230000001737 promoting effect Effects 0.000 claims description 29
- 210000005013 brain tissue Anatomy 0.000 claims description 26
- 238000012360 testing method Methods 0.000 claims description 23
- 230000027455 binding Effects 0.000 claims description 18
- 125000001433 C-terminal amino-acid group Chemical group 0.000 claims description 14
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 claims description 14
- 150000001875 compounds Chemical class 0.000 claims description 8
- 238000011282 treatment Methods 0.000 claims description 5
- 230000002265 prevention Effects 0.000 claims description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims 4
- 230000029115 microtubule polymerization Effects 0.000 claims 1
- 238000003745 diagnosis Methods 0.000 abstract description 5
- 238000002560 therapeutic procedure Methods 0.000 abstract description 2
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 115
- 108090000623 proteins and genes Proteins 0.000 description 83
- 210000004027 cell Anatomy 0.000 description 79
- 102000004169 proteins and genes Human genes 0.000 description 68
- 230000000694 effects Effects 0.000 description 54
- 108010003885 valyl-prolyl-glycyl-glycine Proteins 0.000 description 49
- 102000004243 Tubulin Human genes 0.000 description 48
- 108090000704 Tubulin Proteins 0.000 description 48
- 108010060857 isoleucyl-valyl-tyrosine Proteins 0.000 description 37
- 210000004556 brain Anatomy 0.000 description 35
- 108010026333 seryl-proline Proteins 0.000 description 34
- 230000015572 biosynthetic process Effects 0.000 description 31
- 108090000765 processed proteins & peptides Proteins 0.000 description 30
- 239000000203 mixture Substances 0.000 description 29
- 108010065920 Insulin Lispro Proteins 0.000 description 28
- 150000001413 amino acids Chemical class 0.000 description 28
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 28
- 229940000406 drug candidate Drugs 0.000 description 27
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 27
- LBRCLQMZAHRTLV-ZKWXMUAHSA-N Ile-Gly-Ser Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O LBRCLQMZAHRTLV-ZKWXMUAHSA-N 0.000 description 26
- CPTQYHDSVGVGDZ-UKJIMTQDSA-N Val-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](C(C)C)N CPTQYHDSVGVGDZ-UKJIMTQDSA-N 0.000 description 26
- RLVTVHSDKHBFQP-ULQDDVLXSA-N Val-Tyr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)CC1=CC=C(O)C=C1 RLVTVHSDKHBFQP-ULQDDVLXSA-N 0.000 description 26
- 108700019146 Transgenes Proteins 0.000 description 25
- 102000004196 processed proteins & peptides Human genes 0.000 description 25
- 108010072986 threonyl-seryl-lysine Proteins 0.000 description 25
- OINVDEKBKBCPLX-JXUBOQSCSA-N Ala-Lys-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OINVDEKBKBCPLX-JXUBOQSCSA-N 0.000 description 24
- RLZDUFRBMQNYIJ-YUMQZZPRSA-N Lys-Cys-Gly Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CS)C(=O)NCC(=O)O)N RLZDUFRBMQNYIJ-YUMQZZPRSA-N 0.000 description 24
- 239000003814 drug Substances 0.000 description 24
- 229940079593 drug Drugs 0.000 description 22
- 210000002569 neuron Anatomy 0.000 description 22
- 239000003112 inhibitor Substances 0.000 description 21
- DXTOOBDIIAJZBJ-BQBZGAKWSA-N Pro-Gly-Ser Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CO)C(O)=O DXTOOBDIIAJZBJ-BQBZGAKWSA-N 0.000 description 20
- OOZJHTXCLJUODH-QXEWZRGKSA-N Pro-Val-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 OOZJHTXCLJUODH-QXEWZRGKSA-N 0.000 description 20
- 238000012217 deletion Methods 0.000 description 20
- 230000037430 deletion Effects 0.000 description 20
- 230000006870 function Effects 0.000 description 20
- AMSSKPUHBUQBOQ-SRVKXCTJSA-N Leu-Ser-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N AMSSKPUHBUQBOQ-SRVKXCTJSA-N 0.000 description 19
- OFTXTCGQJXTNQS-XGEHTFHBSA-N Val-Thr-Ser Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](C(C)C)N)O OFTXTCGQJXTNQS-XGEHTFHBSA-N 0.000 description 19
- 108010017391 lysylvaline Proteins 0.000 description 19
- 239000000126 substance Substances 0.000 description 19
- 108020004414 DNA Proteins 0.000 description 18
- 101000891579 Homo sapiens Microtubule-associated protein tau Proteins 0.000 description 17
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 17
- 102000057063 human MAPT Human genes 0.000 description 17
- 229920001184 polypeptide Polymers 0.000 description 17
- KMCRKVOLRCOMBG-DJFWLOJKSA-N Asn-Ile-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC(=O)N)N KMCRKVOLRCOMBG-DJFWLOJKSA-N 0.000 description 16
- XYBJLTKSGFBLCS-QXEWZRGKSA-N Asp-Arg-Val Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC(O)=O XYBJLTKSGFBLCS-QXEWZRGKSA-N 0.000 description 16
- JUWISGAGWSDGDH-KKUMJFAQSA-N Asp-Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=CC=C1 JUWISGAGWSDGDH-KKUMJFAQSA-N 0.000 description 16
- KPNWAJMEMRCLAL-GUBZILKMSA-N Gln-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)N)N KPNWAJMEMRCLAL-GUBZILKMSA-N 0.000 description 16
- BBFCMGBMYIAGRS-AUTRQRHGSA-N Gln-Val-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O BBFCMGBMYIAGRS-AUTRQRHGSA-N 0.000 description 16
- ILWHFUZZCFYSKT-AVGNSLFASA-N Glu-Lys-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O ILWHFUZZCFYSKT-AVGNSLFASA-N 0.000 description 16
- DDXZHOHEABQXSE-NKIYYHGXSA-N Glu-Thr-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O DDXZHOHEABQXSE-NKIYYHGXSA-N 0.000 description 16
- KMSGYZQRXPUKGI-BYPYZUCNSA-N Gly-Gly-Asn Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC(N)=O KMSGYZQRXPUKGI-BYPYZUCNSA-N 0.000 description 16
- TTYKEFZRLKQTHH-MELADBBJSA-N His-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CC2=CN=CN2)N)C(=O)O TTYKEFZRLKQTHH-MELADBBJSA-N 0.000 description 16
- XDUVMJCBYUKNFJ-MXAVVETBSA-N Ile-Lys-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N XDUVMJCBYUKNFJ-MXAVVETBSA-N 0.000 description 16
- GMUYXHHJAGQHGB-TUBUOCAGSA-N Ile-Thr-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N GMUYXHHJAGQHGB-TUBUOCAGSA-N 0.000 description 16
- ZURHXHNAEJJRNU-CIUDSAMLSA-N Leu-Asp-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O ZURHXHNAEJJRNU-CIUDSAMLSA-N 0.000 description 16
- GAHJXEMYXKLZRQ-AJNGGQMLSA-N Lys-Lys-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O GAHJXEMYXKLZRQ-AJNGGQMLSA-N 0.000 description 16
- ZUGVARDEGWMMLK-SRVKXCTJSA-N Lys-Ser-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN ZUGVARDEGWMMLK-SRVKXCTJSA-N 0.000 description 16
- XNCUYZKGQOCOQH-YUMQZZPRSA-N Ser-Leu-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O XNCUYZKGQOCOQH-YUMQZZPRSA-N 0.000 description 16
- JAKHAONCJJZVHT-DCAQKATOSA-N Val-Lys-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)O)N JAKHAONCJJZVHT-DCAQKATOSA-N 0.000 description 16
- 108010028295 histidylhistidine Proteins 0.000 description 16
- 108010003700 lysyl aspartic acid Proteins 0.000 description 16
- SLKLLQWZQHXYSV-CIUDSAMLSA-N Asn-Ala-Lys Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O SLKLLQWZQHXYSV-CIUDSAMLSA-N 0.000 description 15
- YWAQATDNEKZFFK-BYPYZUCNSA-N Gly-Gly-Ser Chemical compound NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O YWAQATDNEKZFFK-BYPYZUCNSA-N 0.000 description 15
- OIQSIMFSVLLWBX-VOAKCMCISA-N Lys-Leu-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OIQSIMFSVLLWBX-VOAKCMCISA-N 0.000 description 15
- MPGJIHFJCXTVEX-KKUMJFAQSA-N Phe-Arg-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O MPGJIHFJCXTVEX-KKUMJFAQSA-N 0.000 description 15
- 108010029485 Protein Isoforms Proteins 0.000 description 15
- 102000001708 Protein Isoforms Human genes 0.000 description 15
- 239000012634 fragment Substances 0.000 description 15
- 230000014509 gene expression Effects 0.000 description 15
- 241000880493 Leptailurus serval Species 0.000 description 14
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 14
- 230000000875 corresponding effect Effects 0.000 description 14
- 238000011534 incubation Methods 0.000 description 14
- 238000006386 neutralization reaction Methods 0.000 description 14
- 108010073969 valyllysine Proteins 0.000 description 14
- ILQCHXURSRRIRY-YUMQZZPRSA-N Asp-His-Gly Chemical compound C1=C(NC=N1)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CC(=O)O)N ILQCHXURSRRIRY-YUMQZZPRSA-N 0.000 description 13
- 241000894006 Bacteria Species 0.000 description 13
- 239000000523 sample Substances 0.000 description 13
- 238000002965 ELISA Methods 0.000 description 12
- QITBQGJOXQYMOA-ZETCQYMHSA-N Gly-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)CN QITBQGJOXQYMOA-ZETCQYMHSA-N 0.000 description 12
- FBOMZVOKCZMDIG-XQQFMLRXSA-N His-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N FBOMZVOKCZMDIG-XQQFMLRXSA-N 0.000 description 12
- PWRITNSESKQTPW-NRPADANISA-N Val-Gln-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CO)C(=O)O)N PWRITNSESKQTPW-NRPADANISA-N 0.000 description 12
- 230000001413 cellular effect Effects 0.000 description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 12
- 230000003993 interaction Effects 0.000 description 12
- 102100040543 FUN14 domain-containing protein 2 Human genes 0.000 description 11
- FQCILXROGNOZON-YUMQZZPRSA-N Gln-Pro-Gly Chemical compound NC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O FQCILXROGNOZON-YUMQZZPRSA-N 0.000 description 11
- 101000893764 Homo sapiens FUN14 domain-containing protein 2 Proteins 0.000 description 11
- REPBGZHJKYWFMJ-KKUMJFAQSA-N Leu-Lys-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N REPBGZHJKYWFMJ-KKUMJFAQSA-N 0.000 description 11
- 230000005764 inhibitory process Effects 0.000 description 11
- 230000000986 microtubule polymerisation Effects 0.000 description 11
- 238000002156 mixing Methods 0.000 description 11
- 108010020755 prolyl-glycyl-glycine Proteins 0.000 description 11
- 108010069117 seryl-lysyl-aspartic acid Proteins 0.000 description 11
- ZFXQNADNEBRERM-BJDJZHNGSA-N Ala-Ala-Pro-Pro Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 ZFXQNADNEBRERM-BJDJZHNGSA-N 0.000 description 10
- NINQYGGNRIBFSC-CIUDSAMLSA-N Ala-Lys-Ser Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CO)C(O)=O NINQYGGNRIBFSC-CIUDSAMLSA-N 0.000 description 10
- BJFKXBOBGVWFCT-GUBZILKMSA-N Ala-Val-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@H](C)N)C(C)C)C(O)=O BJFKXBOBGVWFCT-GUBZILKMSA-N 0.000 description 10
- HPSVTWMFWCHKFN-GARJFASQSA-N Arg-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O HPSVTWMFWCHKFN-GARJFASQSA-N 0.000 description 10
- GNYUVVJYGJFKHN-RVMXOQNASA-N Arg-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N GNYUVVJYGJFKHN-RVMXOQNASA-N 0.000 description 10
- GMFAGHNRXPSSJS-SRVKXCTJSA-N Arg-Leu-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O GMFAGHNRXPSSJS-SRVKXCTJSA-N 0.000 description 10
- VENMDXUVHSKEIN-GUBZILKMSA-N Arg-Ser-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O VENMDXUVHSKEIN-GUBZILKMSA-N 0.000 description 10
- DNLQVHBBMPZUGJ-BQBZGAKWSA-N Arg-Ser-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O DNLQVHBBMPZUGJ-BQBZGAKWSA-N 0.000 description 10
- ZJBUILVYSXQNSW-YTWAJWBKSA-N Arg-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)O ZJBUILVYSXQNSW-YTWAJWBKSA-N 0.000 description 10
- IARGXWMWRFOQPG-GCJQMDKQSA-N Asn-Ala-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IARGXWMWRFOQPG-GCJQMDKQSA-N 0.000 description 10
- CQZDZKRHFWJXDF-WDSKDSINSA-N Gly-Gln-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CN CQZDZKRHFWJXDF-WDSKDSINSA-N 0.000 description 10
- AQLHORCVPGXDJW-IUCAKERBSA-N Gly-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)CN AQLHORCVPGXDJW-IUCAKERBSA-N 0.000 description 10
- WNGHUXFWEWTKAO-YUMQZZPRSA-N Gly-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN WNGHUXFWEWTKAO-YUMQZZPRSA-N 0.000 description 10
- CYHYBSGMHMHKOA-CIQUZCHMSA-N Ile-Ala-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N CYHYBSGMHMHKOA-CIQUZCHMSA-N 0.000 description 10
- QSXSHZIRKTUXNG-STECZYCISA-N Ile-Val-Tyr Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QSXSHZIRKTUXNG-STECZYCISA-N 0.000 description 10
- PWPBLZXWFXJFHE-RHYQMDGZSA-N Leu-Pro-Thr Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O PWPBLZXWFXJFHE-RHYQMDGZSA-N 0.000 description 10
- LZWNAOIMTLNMDW-NHCYSSNCSA-N Lys-Asn-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCCN)N LZWNAOIMTLNMDW-NHCYSSNCSA-N 0.000 description 10
- QQPSCXKFDSORFT-IHRRRGAJSA-N Lys-Lys-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCCN QQPSCXKFDSORFT-IHRRRGAJSA-N 0.000 description 10
- 108010079364 N-glycylalanine Proteins 0.000 description 10
- BNBBNGZZKQUWCD-IUCAKERBSA-N Pro-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H]1CCCN1 BNBBNGZZKQUWCD-IUCAKERBSA-N 0.000 description 10
- KPDRZQUWJKTMBP-DCAQKATOSA-N Pro-Asp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@@H]1CCCN1 KPDRZQUWJKTMBP-DCAQKATOSA-N 0.000 description 10
- AFXCXDQNRXTSBD-FJXKBIBVSA-N Pro-Gly-Thr Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O AFXCXDQNRXTSBD-FJXKBIBVSA-N 0.000 description 10
- MHHQQZIFLWFZGR-DCAQKATOSA-N Pro-Lys-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O MHHQQZIFLWFZGR-DCAQKATOSA-N 0.000 description 10
- WFIVLLFYUZZWOD-RHYQMDGZSA-N Pro-Lys-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WFIVLLFYUZZWOD-RHYQMDGZSA-N 0.000 description 10
- KDBHVPXBQADZKY-GUBZILKMSA-N Pro-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 KDBHVPXBQADZKY-GUBZILKMSA-N 0.000 description 10
- PCWLNNZTBJTZRN-AVGNSLFASA-N Pro-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 PCWLNNZTBJTZRN-AVGNSLFASA-N 0.000 description 10
- FDMKYQQYJKYCLV-GUBZILKMSA-N Pro-Pro-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 FDMKYQQYJKYCLV-GUBZILKMSA-N 0.000 description 10
- RCYUBVHMVUHEBM-RCWTZXSCSA-N Pro-Pro-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O RCYUBVHMVUHEBM-RCWTZXSCSA-N 0.000 description 10
- MKGIILKDUGDRRO-FXQIFTODSA-N Pro-Ser-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H]1CCCN1 MKGIILKDUGDRRO-FXQIFTODSA-N 0.000 description 10
- MUARUIBTKQJKFY-WHFBIAKZSA-N Ser-Gly-Asp Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O MUARUIBTKQJKFY-WHFBIAKZSA-N 0.000 description 10
- MIJWOJAXARLEHA-WDSKDSINSA-N Ser-Gly-Glu Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O MIJWOJAXARLEHA-WDSKDSINSA-N 0.000 description 10
- LVHHEVGYAZGXDE-KDXUFGMBSA-N Thr-Ala-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)N1CCC[C@@H]1C(=O)O)N)O LVHHEVGYAZGXDE-KDXUFGMBSA-N 0.000 description 10
- CQNFRKAKGDSJFR-NUMRIWBASA-N Thr-Glu-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O CQNFRKAKGDSJFR-NUMRIWBASA-N 0.000 description 10
- KERCOYANYUPLHJ-XGEHTFHBSA-N Thr-Pro-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O KERCOYANYUPLHJ-XGEHTFHBSA-N 0.000 description 10
- UMSZZGTXGKHTFJ-SRVKXCTJSA-N Tyr-Ser-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 UMSZZGTXGKHTFJ-SRVKXCTJSA-N 0.000 description 10
- WANVRBAZGSICCP-SRVKXCTJSA-N Val-Pro-Met Chemical compound CSCC[C@H](NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)C(C)C)C(O)=O WANVRBAZGSICCP-SRVKXCTJSA-N 0.000 description 10
- 108010005233 alanylglutamic acid Proteins 0.000 description 10
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 10
- 108010087823 glycyltyrosine Proteins 0.000 description 10
- 108010034529 leucyl-lysine Proteins 0.000 description 10
- 108010064235 lysylglycine Proteins 0.000 description 10
- 230000004048 modification Effects 0.000 description 10
- 238000012986 modification Methods 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 10
- 108010077112 prolyl-proline Proteins 0.000 description 10
- 108010093296 prolyl-prolyl-alanine Proteins 0.000 description 10
- 108010029020 prolylglycine Proteins 0.000 description 10
- 239000010453 quartz Substances 0.000 description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 10
- IBIDRSSEHFLGSD-UHFFFAOYSA-N valinyl-arginine Natural products CC(C)C(N)C(=O)NC(C(O)=O)CCCN=C(N)N IBIDRSSEHFLGSD-UHFFFAOYSA-N 0.000 description 10
- 101100402621 Homo sapiens MSANTD4 gene Proteins 0.000 description 9
- MUXNCRWTWBMNHX-SRVKXCTJSA-N Lys-Leu-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O MUXNCRWTWBMNHX-SRVKXCTJSA-N 0.000 description 9
- 102100031642 Myb/SANT-like DNA-binding domain-containing protein 4 Human genes 0.000 description 9
- 206010029260 Neuroblastoma Diseases 0.000 description 9
- 230000004071 biological effect Effects 0.000 description 9
- 201000010099 disease Diseases 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 238000002955 isolation Methods 0.000 description 9
- 108010054155 lysyllysine Proteins 0.000 description 9
- 230000026731 phosphorylation Effects 0.000 description 9
- 238000006366 phosphorylation reaction Methods 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- UMHUHHJMEXNSIV-CIUDSAMLSA-N Asp-Leu-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(O)=O UMHUHHJMEXNSIV-CIUDSAMLSA-N 0.000 description 8
- KZZCOWMDDXDKSS-CIUDSAMLSA-N Leu-Ser-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O KZZCOWMDDXDKSS-CIUDSAMLSA-N 0.000 description 8
- RIPJMCFGQHGHNP-RHYQMDGZSA-N Lys-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCCN)N)O RIPJMCFGQHGHNP-RHYQMDGZSA-N 0.000 description 8
- 239000007990 PIPES buffer Substances 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- NCGICGYLBXGBGN-UHFFFAOYSA-N 3-morpholin-4-yl-1-oxa-3-azonia-2-azanidacyclopent-3-en-5-imine;hydrochloride Chemical compound Cl.[N-]1OC(=N)C=[N+]1N1CCOCC1 NCGICGYLBXGBGN-UHFFFAOYSA-N 0.000 description 7
- IBLAOXSULLECQZ-IUKAMOBKSA-N Asn-Ile-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CC(N)=O IBLAOXSULLECQZ-IUKAMOBKSA-N 0.000 description 7
- ZYPWIUFLYMQZBS-SRVKXCTJSA-N Asn-Lys-Lys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N ZYPWIUFLYMQZBS-SRVKXCTJSA-N 0.000 description 7
- AKJRHDMTEJXTPV-ACZMJKKPSA-N Glu-Asn-Ala Chemical compound C[C@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCC(O)=O)C(O)=O AKJRHDMTEJXTPV-ACZMJKKPSA-N 0.000 description 7
- ZYRXTRTUCAVNBQ-GVXVVHGQSA-N Glu-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZYRXTRTUCAVNBQ-GVXVVHGQSA-N 0.000 description 7
- MFVQGXGQRIXBPK-WDSKDSINSA-N Gly-Ala-Glu Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O MFVQGXGQRIXBPK-WDSKDSINSA-N 0.000 description 7
- BGVYNAQWHSTTSP-BYULHYEWSA-N Gly-Asn-Ile Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BGVYNAQWHSTTSP-BYULHYEWSA-N 0.000 description 7
- QPDUVFSVVAOUHE-XVKPBYJWSA-N Gly-Gln-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCC(N)=O)NC(=O)CN)C(O)=O QPDUVFSVVAOUHE-XVKPBYJWSA-N 0.000 description 7
- AKAPKBNIVNPIPO-KKUMJFAQSA-N His-His-Lys Chemical compound C([C@@H](C(=O)N[C@@H](CCCCN)C(O)=O)NC(=O)[C@@H](N)CC=1NC=NC=1)C1=CN=CN1 AKAPKBNIVNPIPO-KKUMJFAQSA-N 0.000 description 7
- UXSATKFPUVZVDK-KKUMJFAQSA-N His-Lys-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC1=CN=CN1)N UXSATKFPUVZVDK-KKUMJFAQSA-N 0.000 description 7
- PNDMHTTXXPUQJH-RWRJDSDZSA-N Ile-Glu-Thr Chemical compound N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H]([C@H](O)C)C(=O)O PNDMHTTXXPUQJH-RWRJDSDZSA-N 0.000 description 7
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 7
- JQSXWJXBASFONF-KKUMJFAQSA-N Leu-Asp-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JQSXWJXBASFONF-KKUMJFAQSA-N 0.000 description 7
- WSXTWLJHTLRFLW-SRVKXCTJSA-N Lys-Ala-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O WSXTWLJHTLRFLW-SRVKXCTJSA-N 0.000 description 7
- FLCMXEFCTLXBTL-DCAQKATOSA-N Lys-Asp-Arg Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N FLCMXEFCTLXBTL-DCAQKATOSA-N 0.000 description 7
- IVFUVMSKSFSFBT-NHCYSSNCSA-N Lys-Ile-Gly Chemical compound OC(=O)CNC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN IVFUVMSKSFSFBT-NHCYSSNCSA-N 0.000 description 7
- YSPZCHGIWAQVKQ-AVGNSLFASA-N Lys-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCCN YSPZCHGIWAQVKQ-AVGNSLFASA-N 0.000 description 7
- 108010085220 Multiprotein Complexes Proteins 0.000 description 7
- 102000007474 Multiprotein Complexes Human genes 0.000 description 7
- HAAQQNHQZBOWFO-LURJTMIESA-N Pro-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H]1CCCN1 HAAQQNHQZBOWFO-LURJTMIESA-N 0.000 description 7
- QKQDTEYDEIJPNK-GUBZILKMSA-N Ser-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CO QKQDTEYDEIJPNK-GUBZILKMSA-N 0.000 description 7
- NLOAIFSWUUFQFR-CIUDSAMLSA-N Ser-Leu-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O NLOAIFSWUUFQFR-CIUDSAMLSA-N 0.000 description 7
- NOWXWJLVGTVJKM-PBCZWWQYSA-N Thr-Asp-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N)O NOWXWJLVGTVJKM-PBCZWWQYSA-N 0.000 description 7
- WRQLCVIALDUQEQ-UNQGMJICSA-N Thr-Phe-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O WRQLCVIALDUQEQ-UNQGMJICSA-N 0.000 description 7
- 108010036533 arginylvaline Proteins 0.000 description 7
- 230000003833 cell viability Effects 0.000 description 7
- 239000002299 complementary DNA Substances 0.000 description 7
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 7
- 108010036413 histidylglycine Proteins 0.000 description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 7
- 230000036542 oxidative stress Effects 0.000 description 7
- 230000001717 pathogenic effect Effects 0.000 description 7
- 239000008194 pharmaceutical composition Substances 0.000 description 7
- 238000007423 screening assay Methods 0.000 description 7
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 6
- KIUYPHAMDKDICO-WHFBIAKZSA-N Ala-Asp-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O KIUYPHAMDKDICO-WHFBIAKZSA-N 0.000 description 6
- GGNHBHYDMUDXQB-KBIXCLLPSA-N Ala-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)N GGNHBHYDMUDXQB-KBIXCLLPSA-N 0.000 description 6
- SUHLZMHFRALVSY-YUMQZZPRSA-N Ala-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)NCC(O)=O SUHLZMHFRALVSY-YUMQZZPRSA-N 0.000 description 6
- SVABRQFIHCSNCI-FOHZUACHSA-N Asp-Gly-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O SVABRQFIHCSNCI-FOHZUACHSA-N 0.000 description 6
- NVFSJIXJZCDICF-SRVKXCTJSA-N Asp-Lys-Lys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N NVFSJIXJZCDICF-SRVKXCTJSA-N 0.000 description 6
- OHUKZZYSJBKFRR-WHFBIAKZSA-N Gly-Ser-Asp Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O OHUKZZYSJBKFRR-WHFBIAKZSA-N 0.000 description 6
- YKBSXQFZWFXFIB-VOAKCMCISA-N Lys-Thr-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CCCCN)C(O)=O YKBSXQFZWFXFIB-VOAKCMCISA-N 0.000 description 6
- LBSWWNKMVPAXOI-GUBZILKMSA-N Met-Val-Ser Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O LBSWWNKMVPAXOI-GUBZILKMSA-N 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- JLPMFVAIQHCBDC-CIUDSAMLSA-N Ser-Lys-Cys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CO)N JLPMFVAIQHCBDC-CIUDSAMLSA-N 0.000 description 6
- LLSLRQOEAFCZLW-NRPADANISA-N Ser-Val-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O LLSLRQOEAFCZLW-NRPADANISA-N 0.000 description 6
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 6
- 108010016616 cysteinylglycine Proteins 0.000 description 6
- 238000003205 genotyping method Methods 0.000 description 6
- 108010015792 glycyllysine Proteins 0.000 description 6
- 230000028993 immune response Effects 0.000 description 6
- 230000002163 immunogen Effects 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 230000010189 intracellular transport Effects 0.000 description 6
- 108020004999 messenger RNA Proteins 0.000 description 6
- 230000009257 reactivity Effects 0.000 description 6
- 210000003705 ribosome Anatomy 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- ACKNRKFVYUVWAC-ZPFDUUQYSA-N Asn-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N ACKNRKFVYUVWAC-ZPFDUUQYSA-N 0.000 description 5
- BUVNWKQBMZLCDW-UGYAYLCHSA-N Asp-Asn-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BUVNWKQBMZLCDW-UGYAYLCHSA-N 0.000 description 5
- PMNHJLASAAWELO-FOHZUACHSA-N Gly-Asp-Thr Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PMNHJLASAAWELO-FOHZUACHSA-N 0.000 description 5
- FJWYJQRCVNGEAQ-ZPFDUUQYSA-N Ile-Asn-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N FJWYJQRCVNGEAQ-ZPFDUUQYSA-N 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 5
- 239000004365 Protease Substances 0.000 description 5
- PPNPDKGQRFSCAC-CIUDSAMLSA-N Ser-Lys-Asp Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)CO)C(=O)N[C@@H](CC(O)=O)C(O)=O PPNPDKGQRFSCAC-CIUDSAMLSA-N 0.000 description 5
- CKDXFSPMIDSMGV-GUBZILKMSA-N Ser-Pro-Val Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O CKDXFSPMIDSMGV-GUBZILKMSA-N 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 238000010171 animal model Methods 0.000 description 5
- 210000004899 c-terminal region Anatomy 0.000 description 5
- 238000012512 characterization method Methods 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000005194 fractionation Methods 0.000 description 5
- 108010050848 glycylleucine Proteins 0.000 description 5
- 230000001939 inductive effect Effects 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 230000002018 overexpression Effects 0.000 description 5
- 230000004962 physiological condition Effects 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 229960005486 vaccine Drugs 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 4
- QUIGLPSHIFPEOV-CIUDSAMLSA-N Ala-Lys-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O QUIGLPSHIFPEOV-CIUDSAMLSA-N 0.000 description 4
- RKRSYHCNPFGMTA-CIUDSAMLSA-N Arg-Glu-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O RKRSYHCNPFGMTA-CIUDSAMLSA-N 0.000 description 4
- LLQIAIUAKGNOSE-NHCYSSNCSA-N Arg-Val-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N LLQIAIUAKGNOSE-NHCYSSNCSA-N 0.000 description 4
- XTHUKRLJRUVVBF-WHFBIAKZSA-N Cys-Gly-Ser Chemical compound SC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O XTHUKRLJRUVVBF-WHFBIAKZSA-N 0.000 description 4
- OCDLPQDYTJPWNG-YUMQZZPRSA-N Gly-Asn-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)CN OCDLPQDYTJPWNG-YUMQZZPRSA-N 0.000 description 4
- QPTNELDXWKRIFX-YFKPBYRVSA-N Gly-Gly-Gln Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O QPTNELDXWKRIFX-YFKPBYRVSA-N 0.000 description 4
- YADRBUZBKHHDAO-XPUUQOCRSA-N His-Gly-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](C)C(O)=O YADRBUZBKHHDAO-XPUUQOCRSA-N 0.000 description 4
- AMSYMDIIIRJRKZ-HJPIBITLSA-N Ile-His-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N AMSYMDIIIRJRKZ-HJPIBITLSA-N 0.000 description 4
- FMEICTQWUKNAGC-YUMQZZPRSA-N Leu-Gly-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O FMEICTQWUKNAGC-YUMQZZPRSA-N 0.000 description 4
- ODRREERHVHMIPT-OEAJRASXSA-N Leu-Thr-Phe Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 ODRREERHVHMIPT-OEAJRASXSA-N 0.000 description 4
- QBEPTBMRQALPEV-MNXVOIDGSA-N Lys-Ile-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN QBEPTBMRQALPEV-MNXVOIDGSA-N 0.000 description 4
- PDIDTSZKKFEDMB-UWVGGRQHSA-N Lys-Pro-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O PDIDTSZKKFEDMB-UWVGGRQHSA-N 0.000 description 4
- JMNRXRPBHFGXQX-GUBZILKMSA-N Lys-Ser-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O JMNRXRPBHFGXQX-GUBZILKMSA-N 0.000 description 4
- GIKFNMZSGYAPEJ-HJGDQZAQSA-N Lys-Thr-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O GIKFNMZSGYAPEJ-HJGDQZAQSA-N 0.000 description 4
- 102000009664 Microtubule-Associated Proteins Human genes 0.000 description 4
- 108010020004 Microtubule-Associated Proteins Proteins 0.000 description 4
- OQTDZEJJWWAGJT-KKUMJFAQSA-N Phe-Lys-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O OQTDZEJJWWAGJT-KKUMJFAQSA-N 0.000 description 4
- LRWBCWGEUCKDTN-BJDJZHNGSA-N Ser-Lys-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LRWBCWGEUCKDTN-BJDJZHNGSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- FKIGTIXHSRNKJU-IXOXFDKPSA-N Thr-His-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)[C@H](O)C)CC1=CN=CN1 FKIGTIXHSRNKJU-IXOXFDKPSA-N 0.000 description 4
- YUPVPKZBKCLFLT-QTKMDUPCSA-N Thr-His-Val Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](C(C)C)C(=O)O)N)O YUPVPKZBKCLFLT-QTKMDUPCSA-N 0.000 description 4
- UEHRGZCNLSWGHK-DLOVCJGASA-N Val-Glu-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O UEHRGZCNLSWGHK-DLOVCJGASA-N 0.000 description 4
- UGFMVXRXULGLNO-XPUUQOCRSA-N Val-Ser-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O UGFMVXRXULGLNO-XPUUQOCRSA-N 0.000 description 4
- 230000002159 abnormal effect Effects 0.000 description 4
- 230000009471 action Effects 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000006907 apoptotic process Effects 0.000 description 4
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 4
- 230000008335 axon cargo transport Effects 0.000 description 4
- 230000030833 cell death Effects 0.000 description 4
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 210000002257 embryonic structure Anatomy 0.000 description 4
- 108010049041 glutamylalanine Proteins 0.000 description 4
- 230000000977 initiatory effect Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 210000003470 mitochondria Anatomy 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 108010018625 phenylalanylarginine Proteins 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 150000003384 small molecules Chemical class 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000035882 stress Effects 0.000 description 4
- 229940126585 therapeutic drug Drugs 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 108010021889 valylvaline Proteins 0.000 description 4
- MQLACMBJVPINKE-UHFFFAOYSA-N 10-[(3-hydroxy-4-methoxyphenyl)methylidene]anthracen-9-one Chemical compound C1=C(O)C(OC)=CC=C1C=C1C2=CC=CC=C2C(=O)C2=CC=CC=C21 MQLACMBJVPINKE-UHFFFAOYSA-N 0.000 description 3
- 101710137189 Amyloid-beta A4 protein Proteins 0.000 description 3
- 101710151993 Amyloid-beta precursor protein Proteins 0.000 description 3
- 102100022704 Amyloid-beta precursor protein Human genes 0.000 description 3
- PQKSVQSMTHPRIB-ZKWXMUAHSA-N Asn-Val-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O PQKSVQSMTHPRIB-ZKWXMUAHSA-N 0.000 description 3
- GCACQYDBDHRVGE-LKXGYXEUSA-N Asp-Thr-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H]([C@H](O)C)NC(=O)[C@@H](N)CC(O)=O GCACQYDBDHRVGE-LKXGYXEUSA-N 0.000 description 3
- 206010012289 Dementia Diseases 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- LVXFNTIIGOQBMD-SRVKXCTJSA-N His-Leu-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O LVXFNTIIGOQBMD-SRVKXCTJSA-N 0.000 description 3
- CCHSQWLCOOZREA-GMOBBJLQSA-N Ile-Asp-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCSC)C(=O)O)N CCHSQWLCOOZREA-GMOBBJLQSA-N 0.000 description 3
- WZVSHTFTCYOFPL-GARJFASQSA-N Lys-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCCCN)N)C(=O)O WZVSHTFTCYOFPL-GARJFASQSA-N 0.000 description 3
- OVTOTTGZBWXLFU-QXEWZRGKSA-N Met-Val-Asp Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC(O)=O OVTOTTGZBWXLFU-QXEWZRGKSA-N 0.000 description 3
- 101710115937 Microtubule-associated protein tau Proteins 0.000 description 3
- 102100040243 Microtubule-associated protein tau Human genes 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 108010038807 Oligopeptides Proteins 0.000 description 3
- 102000015636 Oligopeptides Human genes 0.000 description 3
- 108010067902 Peptide Library Proteins 0.000 description 3
- QBFONMUYNSNKIX-AVGNSLFASA-N Pro-Arg-His Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O QBFONMUYNSNKIX-AVGNSLFASA-N 0.000 description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
- BKZYBLLIBOBOOW-GHCJXIJMSA-N Ser-Ile-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O BKZYBLLIBOBOOW-GHCJXIJMSA-N 0.000 description 3
- NUEHQDHDLDXCRU-GUBZILKMSA-N Ser-Pro-Arg Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O NUEHQDHDLDXCRU-GUBZILKMSA-N 0.000 description 3
- PURRNJBBXDDWLX-ZDLURKLDSA-N Ser-Thr-Gly Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CO)N)O PURRNJBBXDDWLX-ZDLURKLDSA-N 0.000 description 3
- DJDSEDOKJTZBAR-ZDLURKLDSA-N Thr-Gly-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O DJDSEDOKJTZBAR-ZDLURKLDSA-N 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 description 3
- LLJLBRRXKZTTRD-GUBZILKMSA-N Val-Val-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)O)N LLJLBRRXKZTTRD-GUBZILKMSA-N 0.000 description 3
- DZHSAHHDTRWUTF-SIQRNXPUSA-N amyloid-beta polypeptide 42 Chemical compound C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C(C)C)C1=CC=CC=C1 DZHSAHHDTRWUTF-SIQRNXPUSA-N 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 230000008827 biological function Effects 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 238000000423 cell based assay Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 238000007877 drug screening Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 230000005714 functional activity Effects 0.000 description 3
- 238000002523 gelfiltration Methods 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 108010025306 histidylleucine Proteins 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000002438 mitochondrial effect Effects 0.000 description 3
- 230000004770 neurodegeneration Effects 0.000 description 3
- 208000015122 neurodegenerative disease Diseases 0.000 description 3
- 231100000255 pathogenic effect Toxicity 0.000 description 3
- 230000035790 physiological processes and functions Effects 0.000 description 3
- 229940124606 potential therapeutic agent Drugs 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- RLMISHABBKUNFO-WHFBIAKZSA-N Ala-Ala-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O RLMISHABBKUNFO-WHFBIAKZSA-N 0.000 description 2
- WKOBSJOZRJJVRZ-FXQIFTODSA-N Ala-Glu-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WKOBSJOZRJJVRZ-FXQIFTODSA-N 0.000 description 2
- LMFXXZPPZDCPTA-ZKWXMUAHSA-N Ala-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N LMFXXZPPZDCPTA-ZKWXMUAHSA-N 0.000 description 2
- OBVSBEYOMDWLRJ-BFHQHQDPSA-N Ala-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N OBVSBEYOMDWLRJ-BFHQHQDPSA-N 0.000 description 2
- 238000009010 Bradford assay Methods 0.000 description 2
- 208000028698 Cognitive impairment Diseases 0.000 description 2
- 108700024394 Exon Proteins 0.000 description 2
- 201000011240 Frontotemporal dementia Diseases 0.000 description 2
- INKFLNZBTSNFON-CIUDSAMLSA-N Gln-Ala-Arg Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O INKFLNZBTSNFON-CIUDSAMLSA-N 0.000 description 2
- RKAQZCDMSUQTSS-FXQIFTODSA-N Gln-Asp-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N RKAQZCDMSUQTSS-FXQIFTODSA-N 0.000 description 2
- LFIVHGMKWFGUGK-IHRRRGAJSA-N Gln-Glu-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)N)N LFIVHGMKWFGUGK-IHRRRGAJSA-N 0.000 description 2
- XXCDTYBVGMPIOA-FXQIFTODSA-N Glu-Asp-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O XXCDTYBVGMPIOA-FXQIFTODSA-N 0.000 description 2
- RTOOAKXIJADOLL-GUBZILKMSA-N Glu-Asp-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)N RTOOAKXIJADOLL-GUBZILKMSA-N 0.000 description 2
- MTAOBYXRYJZRGQ-WDSKDSINSA-N Glu-Gly-Asp Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O MTAOBYXRYJZRGQ-WDSKDSINSA-N 0.000 description 2
- NTNUEBVGKMVANB-NHCYSSNCSA-N Glu-Val-Met Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCSC)C(O)=O NTNUEBVGKMVANB-NHCYSSNCSA-N 0.000 description 2
- IWAXHBCACVWNHT-BQBZGAKWSA-N Gly-Asp-Arg Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IWAXHBCACVWNHT-BQBZGAKWSA-N 0.000 description 2
- INLIXXRWNUKVCF-JTQLQIEISA-N Gly-Gly-Tyr Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 INLIXXRWNUKVCF-JTQLQIEISA-N 0.000 description 2
- LLZXNUUIBOALNY-QWRGUYRKSA-N Gly-Leu-Lys Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCCN LLZXNUUIBOALNY-QWRGUYRKSA-N 0.000 description 2
- GBMSSORHVHAYLU-QTKMDUPCSA-N His-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC1=CN=CN1)N)O GBMSSORHVHAYLU-QTKMDUPCSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 206010020772 Hypertension Diseases 0.000 description 2
- HIIZIQUUHIXUJY-GUBZILKMSA-N Lys-Asp-Gln Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HIIZIQUUHIXUJY-GUBZILKMSA-N 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- LNICFEXCAHIJOR-DCAQKATOSA-N Pro-Ser-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O LNICFEXCAHIJOR-DCAQKATOSA-N 0.000 description 2
- 108010059712 Pronase Proteins 0.000 description 2
- 108010003201 RGH 0205 Proteins 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- PMCMLDNPAZUYGI-DCAQKATOSA-N Ser-Lys-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O PMCMLDNPAZUYGI-DCAQKATOSA-N 0.000 description 2
- 238000000692 Student's t-test Methods 0.000 description 2
- 239000012505 Superdex™ Substances 0.000 description 2
- LMMDEZPNUTZJAY-GCJQMDKQSA-N Thr-Asp-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O LMMDEZPNUTZJAY-GCJQMDKQSA-N 0.000 description 2
- LHNNQVXITHUCAB-QTKMDUPCSA-N Thr-Met-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N)O LHNNQVXITHUCAB-QTKMDUPCSA-N 0.000 description 2
- IQPWNQRRAJHOKV-KATARQTJSA-N Thr-Ser-Lys Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN IQPWNQRRAJHOKV-KATARQTJSA-N 0.000 description 2
- KCPFDGNYAMKZQP-KBPBESRZSA-N Tyr-Gly-Leu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O KCPFDGNYAMKZQP-KBPBESRZSA-N 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 108010047495 alanylglycine Proteins 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 210000005056 cell body Anatomy 0.000 description 2
- 210000003793 centrosome Anatomy 0.000 description 2
- 208000010877 cognitive disease Diseases 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000006735 deficit Effects 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 230000004064 dysfunction Effects 0.000 description 2
- 230000002275 effect on microtubule Effects 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 108010078144 glutaminyl-glycine Proteins 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 108010040030 histidinoalanine Proteins 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 108010071185 leucyl-alanine Proteins 0.000 description 2
- FKDHHVKWGRFRTG-UHFFFAOYSA-N linsidomine Chemical group [N-]1OC(=N)C=[N+]1N1CCOCC1 FKDHHVKWGRFRTG-UHFFFAOYSA-N 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 230000007257 malfunction Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 2
- 108010056582 methionylglutamic acid Proteins 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 230000030544 mitochondrion distribution Effects 0.000 description 2
- 239000003068 molecular probe Substances 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 210000002682 neurofibrillary tangle Anatomy 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 231100000915 pathological change Toxicity 0.000 description 2
- 230000036285 pathological change Effects 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 230000001991 pathophysiological effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 108010012581 phenylalanylglutamate Proteins 0.000 description 2
- 208000028591 pheochromocytoma Diseases 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 230000004481 post-translational protein modification Effects 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000001350 scanning transmission electron microscopy Methods 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000007470 synaptic degeneration Effects 0.000 description 2
- 238000012353 t test Methods 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000011820 transgenic animal model Methods 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 230000034512 ubiquitination Effects 0.000 description 2
- 238000010798 ubiquitination Methods 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- WDIYWDJLXOCGRW-ACZMJKKPSA-N Ala-Asp-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WDIYWDJLXOCGRW-ACZMJKKPSA-N 0.000 description 1
- HOVPGJUNRLMIOZ-CIUDSAMLSA-N Ala-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)N HOVPGJUNRLMIOZ-CIUDSAMLSA-N 0.000 description 1
- IOFVWPYSRSCWHI-JXUBOQSCSA-N Ala-Thr-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C)N IOFVWPYSRSCWHI-JXUBOQSCSA-N 0.000 description 1
- 229940124717 Alzheimer's therapeutics Drugs 0.000 description 1
- 208000037259 Amyloid Plaque Diseases 0.000 description 1
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 1
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 1
- 108010039627 Aprotinin Proteins 0.000 description 1
- MHJKFLZILGFTFC-UHFFFAOYSA-N C1CCC1.C1CCC1 Chemical compound C1CCC1.C1CCC1 MHJKFLZILGFTFC-UHFFFAOYSA-N 0.000 description 1
- UZFQSUHEMAOGKP-UHFFFAOYSA-N C1CCC1.C1CCC1.C1CCC1.C1CCC1 Chemical compound C1CCC1.C1CCC1.C1CCC1.C1CCC1 UZFQSUHEMAOGKP-UHFFFAOYSA-N 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 208000032928 Dyslipidaemia Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- FFVXLVGUJBCKRX-UKJIMTQDSA-N Gln-Ile-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CCC(=O)N)N FFVXLVGUJBCKRX-UKJIMTQDSA-N 0.000 description 1
- QXUPRMQJDWJDFR-NRPADANISA-N Glu-Val-Ser Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O QXUPRMQJDWJDFR-NRPADANISA-N 0.000 description 1
- ZLCLYFGMKFCDCN-XPUUQOCRSA-N Gly-Ser-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CO)NC(=O)CN)C(O)=O ZLCLYFGMKFCDCN-XPUUQOCRSA-N 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- ZRLUISBDKUWAIZ-CIUDSAMLSA-N Leu-Ala-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(O)=O ZRLUISBDKUWAIZ-CIUDSAMLSA-N 0.000 description 1
- XBBKIIGCUMBKCO-JXUBOQSCSA-N Leu-Ala-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XBBKIIGCUMBKCO-JXUBOQSCSA-N 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- SPCHLZUWJTYZFC-IHRRRGAJSA-N Lys-His-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C(C)C)C(O)=O SPCHLZUWJTYZFC-IHRRRGAJSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 102100022033 Presenilin-1 Human genes 0.000 description 1
- 108010036933 Presenilin-1 Proteins 0.000 description 1
- 108010036908 Presenilin-2 Proteins 0.000 description 1
- HJSCRFZVGXAGNG-SRVKXCTJSA-N Pro-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H]1CCCN1 HJSCRFZVGXAGNG-SRVKXCTJSA-N 0.000 description 1
- IIRBTQHFVNGPMQ-AVGNSLFASA-N Pro-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@@H]1CCCN1 IIRBTQHFVNGPMQ-AVGNSLFASA-N 0.000 description 1
- 108010001267 Protein Subunits Proteins 0.000 description 1
- 102000002067 Protein Subunits Human genes 0.000 description 1
- 238000010240 RT-PCR analysis Methods 0.000 description 1
- 206010038997 Retroviral infections Diseases 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- WNDUPCKKKGSKIQ-CIUDSAMLSA-N Ser-Pro-Gln Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O WNDUPCKKKGSKIQ-CIUDSAMLSA-N 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 208000030886 Traumatic Brain injury Diseases 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- KGSDLCMCDFETHU-YESZJQIVSA-N Tyr-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CC2=CC=C(C=C2)O)N)C(=O)O KGSDLCMCDFETHU-YESZJQIVSA-N 0.000 description 1
- TZVUSFMQWPWHON-NHCYSSNCSA-N Val-Asp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)N TZVUSFMQWPWHON-NHCYSSNCSA-N 0.000 description 1
- YODDULVCGFQRFZ-ZKWXMUAHSA-N Val-Asp-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O YODDULVCGFQRFZ-ZKWXMUAHSA-N 0.000 description 1
- DEGUERSKQBRZMZ-FXQIFTODSA-N Val-Ser-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O DEGUERSKQBRZMZ-FXQIFTODSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 229960004405 aprotinin Drugs 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000003376 axonal effect Effects 0.000 description 1
- 238000003705 background correction Methods 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- PXXJHWLDUBFPOL-UHFFFAOYSA-N benzamidine Chemical compound NC(=N)C1=CC=CC=C1 PXXJHWLDUBFPOL-UHFFFAOYSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000009875 biological transport Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000012832 cell culture technique Methods 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 238000007878 drug screening assay Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 230000009760 functional impairment Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- CEAZRRDELHUEMR-UHFFFAOYSA-N gentamicin Chemical compound O1C(C(C)NC)CCC(N)C1OC1C(O)C(OC2C(C(NC)C(C)(O)CO2)O)C(N)CC1N CEAZRRDELHUEMR-UHFFFAOYSA-N 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036732 histological change Effects 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 230000006951 hyperphosphorylation Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 210000004201 immune sera Anatomy 0.000 description 1
- 229940042743 immune sera Drugs 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000007852 inverse PCR Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 102000021160 microtubule binding proteins Human genes 0.000 description 1
- 108091011150 microtubule binding proteins Proteins 0.000 description 1
- 230000025090 microtubule depolymerization Effects 0.000 description 1
- 230000026326 mitochondrial transport Effects 0.000 description 1
- 230000025608 mitochondrion localization Effects 0.000 description 1
- 238000007479 molecular analysis Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 210000000944 nerve tissue Anatomy 0.000 description 1
- 210000002241 neurite Anatomy 0.000 description 1
- 230000016273 neuron death Effects 0.000 description 1
- 230000006764 neuronal dysfunction Effects 0.000 description 1
- 230000005015 neuronal process Effects 0.000 description 1
- 210000002511 neuropil thread Anatomy 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000006864 oxidative decomposition reaction Methods 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 108010091212 pepstatin Proteins 0.000 description 1
- 229950000964 pepstatin Drugs 0.000 description 1
- FAXGPCHRFPCXOO-LXTPJMTPSA-N pepstatin A Chemical compound OC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)CC(C)C FAXGPCHRFPCXOO-LXTPJMTPSA-N 0.000 description 1
- CMFNMSMUKZHDEY-UHFFFAOYSA-M peroxynitrite Chemical compound [O-]ON=O CMFNMSMUKZHDEY-UHFFFAOYSA-M 0.000 description 1
- 229940080469 phosphocellulose Drugs 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 238000011533 pre-incubation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000011775 sodium fluoride Substances 0.000 description 1
- 235000013024 sodium fluoride Nutrition 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000010972 statistical evaluation Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- -1 superoxide anions Chemical class 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 230000009529 traumatic brain injury Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000002268 wool Anatomy 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0278—Humanized animals, e.g. knockin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
- G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0306—Animal model for genetic diseases
- A01K2267/0312—Animal model for Alzheimer's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2821—Alzheimer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the invention relates to N- and C-terminally truncated diseased forms of tau proteins discovered specifically in Alzheimer's disease and related disorders.
- the invention further relates to methods for screening and testing potential drugs effective in inhibiting, neutralising and eliminating N- and C-terminally double truncated tau proteins or preventing the formation of thereof and to procedures for screening and testing potential drugs of which the mode of action is based on neutralising the modification of microtubule assembly and/or dynamics caused by said double truncated diseased forms of tau proteins.
- Alzheimer's disease is the most common cause of dementia. In less than 5% of the cases Alzheimer's disease cosegregates almost completely with one or more specific mutations in the amyloid precursor protein, presenilin-1 or presenilin-2 genes (1) and in over 95% of the cases, the exact disease cause is not clear.
- Alzheimer's disease is characterized histopathologically by the presence of numerous neurons with neurofibrillary tangles of paired helical filaments (PHF) and extracellular deposits of amyloid ⁇ as the major component of senile plaques in the brain.
- PHF neurofibrillary tangles of paired helical filaments
- amyloid ⁇ the major component of senile plaques in the brain.
- neurofibrillary degeneration appears to be required for the clinical expression of the disease, i.e. dementia (2,3,4).
- Neurofibrillary degeneration is represented by neurofibrillary tangles, dystrophic neurites and neuropil threads.
- the major protein subunit of these structures is microtubule associated protein tau (5,6).
- tau proteins differ whether they contain three (t3L, t3S, or t3) or four (t4L, t4S, or t4) tubulin binding domains (repeats, R) of 31 or 32 amino acids near the C-terminal and two (t3L, t4L), one (t3s, t4S), or no (t3, t 4) inserts of 29 amino acids each in the N-terminal part of the molecule (7,8). Under physiological conditions tau protein is involved in assembly, spatial organisation, stabilisation and behaviour of microtubules.
- tau protein Under physiological conditions the protein appears in six isoforms in healthy human brains.
- tau protein is known to undergo a number of different post-translational modifications (hyperphosphorylation, ubiquitination, glycosylation).
- FTDP-17 disease frontotemporal dementia with Parkinsonism linked to chromosome 17
- certain abnormalities in the tau protein can be a primary cause of neurodegeneration and dementia in affected individuals (9,10).
- the molecular events leading to tau modification and paired helical filament (PHF) formation in Alzheimer's disease are unknown.
- PHF assembled tau protein reacts with certain antibodies in a phosphorylation dependent manner, suggesting a special phosphorylation status (15,16). Furthermore it has been observed that PHF derived tau protein shows a reduced electrophoretic mobility in SDS gels which may be related to its phosphorylation pattern (Steiner et al., EMBO J. 9 (1990), 3539-3544).
- tau is one of the most soluble proteins known (19,20,21) and therefor its aggregation in Alzheimer's disease is particularly enigmatic.
- Phosphorylation of tau affects the potential of tau to form aggregates, producing either stimulatory or inhibitory effects on microtubule polymerisation, presumably depending on the site of phosphorylation (22-27).
- Kontsekova et al. J. Immunol. Meth. 185 (1995), 245-248 disclose a quick purification method of recombinant human truncated tau proteins for immunoanalysis in which heat resistancy of human tau protein is used. Neither structural nor biological properties or functions of recombinant tau analoga used therein have been described.
- Fasulo et al. (Alzheimer's Research 2(5) (1996), 195-200) report that over-expression of recombinant analogue of PHF core tau is not sufficient to induce tau aggregation and assembly thereof in paired helical filaments. These data are in contrast to a publication of Abraha et al. (J. Cell. Science (113) (21) (2000), 3737-3745) obviously due to the unusual non-physiological assay system described in this publication (cell lines from monkey kidneys).
- Esposito et al. J. Peptide Science 6 (2000), 550-559 describe the C terminal 19 amino acids of tau protein and normal healthy tau protein.
- the articles of Novak et al. (Chem. Papers 52 (1998), 429-430) and Ugolini et al. (NeuroReport 8 (1997) 3709-3712) also relates to the C terminal truncated tau protein also with respect to apoptosis. More recent publications show that Alzheimer's disease is not related to apoptosis processes.
- WO 94/18560 Al discloses an immuno assay for detecting human tau protein in a cerebrospinal fluid for detecting patients with cell central nervous cytopathies. This assay does not discriminate between normal tau and tau of patients with central nervous cytopathis but detects the total amount of tau protein in a sample.
- the present invention therefore provides N- and C-terminally double truncated tau molecules, which are characterized by the following features (“type IA tau molecules”):
- N- and C-terminally double truncated tau proteins is used to describe two groups of truncated tau derivatives which appear in Alzheimer's disease brains and which are closely correlated with pathological dysfunction of Alzheimer's disease neurons.
- these proteins represent a group of molecules which exert their pathological function by modifying microtubule associated biological functions such as microtubule assembly or intracellular transport.
- protein complexes is used for N- and C-terminally double truncated tau proteins in the form of homodimeric, heterodimeric or multimeric complexes that are composed of molecules that are physically associated with tau and/or double truncated tau proteins.
- tau refers to the group of shortest naturally occurring isoforms present in healthy human brain containing three repeats (tau44) and four repeats (tau43) in their microtubule binding domain as previously described (39, 40): tau43 (383 amino acids, missing exons 2 and 3 [pos 45-102]) tau44 (352 amino acids, missing exons 2,3 and 10 [pos 45-102 and 275-307, resp.]).
- tau43 383 amino acids, missing exons 2 and 3 [pos 45-102]
- tau44 352 amino acids, missing exons 2,3 and 10 [pos 45-102 and 275-307, resp.]
- wild type tau is used synonymously for “normal tau protein” and refers to tau protein derived from healthy brains.
- microtubule assembly assays are e.g. described in (19) and (20).
- microtubule polymerisation assays are e.g. described in (19) and (20).
- the term “preventing” includes any significant inhibition of 20% or more, preferably 50% or more of normal tau promoting activity.
- Specifically preferred type IA tau molecules according to the present invention comprise an amino acid sequence selected from the group of SEQ ID NOs 1 to 3.
- N- and C-terminally double truncated tau molecules which are characterized by the following features (“type IB tau molecules”):
- Preferred type IB tau molecules are characterized in that the comprise an amino acid sequence selected from the group of SEQ ID NOs 4 to 10.
- the present invention also provides N- and C-terminally double truncated tau molecules, which are characterized by the following features (“type IIA tau molecules”):
- the enhanced microtubule assembly proting activity is at least 20% higher, especially at least 50% higher than wild type tau when measured spectrophotometrically.
- Preferred type IIA tau molecules are characterized in that the comprise an amino acid sequence selected from the group of SEQ ID NOs 11 to 18.
- N- and C-terminally double truncated tau molecules which are characterized by the following features (“type IIB tau molecules”):
- Preferred type IIB tau molecules according to the present invention are characterized in that they comprise an amino acid sequence selected from the group of SEQ ID NOs 19 and 20.
- novel tau polypeptides according to the present invention have typical and unique localisation characteristics since they exclusively localize in Alzheimer's diseased brain tissue. Moreover, also the interaction of these polypeptides with non-polymerized tubulin (alpha/beta dimers) and polymerized form (as microtubule) is unique.
- the present invention provides a method for the preparation of molecules according to the present invention (type IA, IB, IIA, IIB), characterized in by the following steps:
- the truncations are as defined above for type IA, IB, IIA and IIB molecules.
- the microtubule assembly assay activity is preferably as defined above, especially as for IA.
- the present invention provides a method for the preparation of molecules according to the present invention, characterized in by the following steps:
- microtubule assembly assay activity is preferably as defined above, especially as for IA.
- the present invention further provides a method for testing substances effective in disassembling a complex of type IA molecules and tubulin, comprising the following steps:
- the present invention also provides a method for testing substances effective in inhibiting type IA molecules from initiating the formation of complexes with tubulin in a cellular system expressing wild type tau comprising the following steps:
- the present invention also provides a method for in vitro conversion of microtubules into a pathological state characterized by incubating tubulin protein with type IIA under physiological conditions which allow the interaction of said type IIA molecules with microtubules generating pathological microtubules.
- the present invention provides a method for screening substances capable of neutralising the pathological effects of a type IIA molecules for their property to eliminate and/or neutralize type IIA molecules and to restore physiological microtubule parameters and functions caused by type II molecules comprising the following steps:
- a method for testing substances effective in inhibiting the in vivo activity of type IIA molecules in promoting abnormal microtubule formation and function in a cellular system expressing type IIA molecules comprises the following steps:
- the present invention also provides transgenic animals expressing a molecule according to the present invention (type IA, IB, IIA or IIB), especially IA an/or IIA.
- the present invention also relates to the use of a transgenic animal according to the present invention as animal model for Alzheimer's disease, especially for screening and testing drugs for the treatment of Alzheimer's disease.
- a vaccine which comprises a molecule according to the present invention (IA, IB, IIA or IIB), especially IA and IIA, and a pharmaceutically acceptable carrier, especially an adjuvant.
- the present invention also provides inhibitor of the initiation of the formation of complexes of a type IA molecule with wild type tau.
- inhibitors are substances comprising a binding moiety as the monoclonal antibody DC44 deposited under the deposition number 02060767 at the European Collection of Cell Cultures (ECACC), Porton Down, Salisbury, UK, especially DC44 or binding fragments thereof, such as the Fab.
- the present invention provides:
- the present invention relates to the characterisation of N- and C-terminally double truncated forms of pathological tau protein and their epitopes which are specifically occurring in Alzheimer's disease.
- wt wild type tau
- 'wt covers all 6 naturally occurring isoforms of tau protein normally found in the brain of healthy individuals.
- Various short truncation forms of at found in Alzheimer diseased brain were produced in bacteria, purified to various extent with aim to probe physiological function of at proteins, to map their domains and phosphorylation epitopes or in experiments trying to understand the mechanisms of paired helical assembly in Alzheimer's disease and other neurodegenerative disorders, with equivocal results (23-27,34,41,42).
- N- and C-terminally double truncated forms of tau proteins refers to any tau protein in Alzheimer's disease with loss at least one of its amino acids at both ends of molecule.
- double truncated tau in extracts from Alzheimer diseased brains it was found in the course of the present invention that some of these molecules displayed structurally and functionally distinct characteristics which allowed to discriminate them from other tau fragments found in Alzheimer's diseased brain tissue.
- Type I and Type II tau molecules Two major classes of pathogenic molecules of N- and C-terminally double truncated tau molecules distinct from healthy tau.
- These groups can further be subdivided into two subclasses each based on the molecular structure and are designated type IA and B, and type IIA and B, respectively.
- Type IA and type IIA represent structurally and functionally distinct types of diseased molecules derived from microtubule associated protein tau generated by pathological processing.
- N- and C-terminally truncated tau molecules represent diseased molecules, derived from microtubule associated protein tau and emerging during specific pathological processes characteristic of Alzheimer's disease. This is a common feature of all four groups of tau derived proteins. Further common features of all groups are an N- and C-terminal truncations, their intra- and extraneuronal localisation and functional distinction from normal, healthy tau.
- type IA The group of molecules designated ‘type IA’ is described by the examples SEQ ID 1-3. These truncated tau molecules differ from normal tau in acting as key (central), active units, and driving force for interaction of pathological tau and tubulin. Type IA as well as type IB molecules do not have any promoting activity in microtubule assembly. Surprisingly type IA is able to prevent normal tau from promoting microtubule assembly (Example 1). Despite of similar primary sequence features and molecular masses, type IB, does not show this functional activity in vitro (Example 2). This is suggestive for a strong binding activity of type IA to tubulin and thereby providing a dominant negative effect on tau physiology.
- Type IA molecules are therefore most likely responsible for continuous, chronic depletion of neurons from functional microtubular network and for taking part in neurofibrillary structures which directly correlate with the clinical severeness of Alzheimer's disease.
- type IB e.g. SEQ.ID.NO: 4-10
- type IIA double truncated tau derivatives bind microtubules and promote their pathological assembly (Example 3).
- pathological microtubules Surprisingly molecules with similar sequences and ranges of molecular weights (Type IIB) are lacking these high microtubule polymerisation capabilities. In microtubule assembly assays they perform to the levels seen with full length tau protein (see Example 3).
- N- and C-terminally truncated tau derivatives of both groups interfere at the cellular level with axonal transport leading to synaptic loss which ultimately results in neuronal dysfunction and cognitive impairment in Alzheimer's disease patients.
- afflicted neurons are vulnerable to various forms of stress such as oxidative stress (Example 4).
- Type IIB despite of having similar molecular sizes than type IIA additionally promote microtubule assembly to levels seen for full length healthy tau (wild type tau) when measured spectrophotometrically.
- said type IA group of N- and C-terminally double truncated members comprises the following amino acid sequences:
- R4 (239-333,R4)
- SEQ ID NO: 1 ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala lys ala lys thr asp his gly ala glu 2
- said type IB group of N- and C-terminally double truncated members comprises the following amino acid sequences: (239-326,R4) SEQ ID NO: 4 ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala (239-328), SEQ ID NO: 4
- R3 (208-302,R3) SEQ ID NO: 10 leu lys his gln pro gly gly gly lys val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala lys ala lys thr asp his gly ala ly
- tau protein There may be one or more epitopes of tau protein which specifically occur in type IA or type IIA members in N- and C-terminally double truncated diseased forms of tau proteins.
- said epitopes are specifically located within the primary structure of type IA (SEQ ID 1-3) and type IIA (SEQ ID 11-18) group members and their number, heterogeneity and specificity depends on and is added by specific structural conformation of each individual group member. Therefore the singularity of each molecule is not solely based on its primary structure together with its effects on microtubule assembly, but also on its secondary and ternary structure which makes up its epitopes. Some of them can form particularly important “conformational regions” contributing significantly to the activity of said molecules.
- formational region refers to epitopes clustered to one region of molecule contributing to its activity.
- the conformational region encompassed in type I and type II molecules comprising amino acids “ile lys his val pro gly gly gly ser val gin ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu” is corresponding to residues 239-267 (SEQ ID NO: 1-9 and 11-14,19 R4) and comprising amino acids “val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu” corresponding to residues 217-236 (SEQ ID NO: 10.15-18,20 R3) was designated sequence A.
- said epitopes in said conformational region were identified and their relative contribution determined by deletion mutagenesis. The significance of all these epitopes and their relationship to function on microtubules are demonstrated by the mutant forms which showed that they are contributing at various extent to the activity of type IA molecules (Example 5).
- These individual epitopes comprise the following amino acid sequences: A: ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu
- the epitope deletion mutant has SEQ ID NO: 21 (268-333,R4;del 239-267) gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu ala glu A1: ile lys his val pro gly gly gly ser
- the deletion mutant has SEQ ID NO: 22 (248-333,R4;del 239-247) val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala lys thr asp his gly ala lys thr asp his gly ala lys thr
- the deletion mutant has SEQ ID NO: 23 (258-333,R4;del 239-257) ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu ala lys thr asp his gly ala glu ala glu A3: ile lys his val pro gly gly gly ser val gln ile val t
- the deletion mutant has SEQ ID NO: 24 (263-333,R4;del 239-262) lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu ala lys thr asp his gly ala glu ala glu A4: ser val gln ile val tyr lys pro val asp leu ser lys val thr ser (corresponding to residues
- the epitope deletion mutant has SEQ ID NO: 25 (239-333, R4;del 248-262) ile lys his val pro gly gly gly lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu ala lys thr asp his gly ala glu ala glu A5: asp leu ser lys val thr ser corresponding to residues
- the epitope deletion mutant has SEQ ID NO: 26 (239-333,R4;del 256-262) ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala lys ala lys thr asp his gly ala lys ala ly
- A6 lys cys gly ser leu corresponding to residues 263-267 in SEQ ID NO: 1-9 and 11-14,19 and to residues 232-236,R3 in SEQ ID NO: 10, 15-18,20
- the epitope deletion mutant has SEQ ID NO: 27 (239-333,R4;del 263-267) ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala lys ala lys thr asp his gly ala lys ala
- said type IA diseased tau proteins have the following properties:
- the present invention relates to type IA group of N- and C-terminally truncated diseased tau forms SEQ ID NO 1-3 and their “conformational region” (sequence “A”) and epitopes A1-A6.
- Type IB tau proteins differ with respect to the following properties:
- Another embodiment of the present invention is the combination of the presented approach comprising various extraction methods, many of them per se known in the art, combined with functional assays with the mentioned double truncated forms of tau leading to the identification of further molecules affecting tau and microtubule functions.
- the yield of tau protein from brain extract may vary in functionality of extracted N- and C-terminally double truncated tau molecules depending on the staging of the particular brain tissue sample (Example 6). The person skilled in the art knows how to employ the method of the present invention for a variety of different purposes which all fall under the scope of protection of the present invention.
- the present invention relates especially to SEQ ID NO: 1 as a prototype type IA molecule group member.
- Still another object of the invention is to provide a method for the in vitro conversion of normal tau protein into Alzheimer protein wherein tubulin is incubated with a type IA molecule of the present invention under conditions which allow the interaction of said tubulin with said type IA molecule.
- allowing the interaction of type IA molecules or peptide derivatives thereof with tubulin refers to conditions which allow the activity, preferably the optimal activity, of type IA molecules. This activity results in binding to tubulin and inhibiting its physiological function in microtubule assembly.
- type IA molecules could be inhibited or neutralized by derivatives thereof.
- type IA peptides and derivatives thereof such as peptides containing deletions or mutations can be tested or screened for their effects on microtubule polymerisation.
- Normal tau protein may be derived from natural or recombinant sources. However for the purpose of carrying out the method of the present invention, it is expedient to use recombinant material.
- An inhibitor useful in the composition of the present invention is therefore any inhibitor capable of modulating the pathological interaction of type IA molecules with tubulin.
- the mode of action of such an inhibitory molecule consists of an interaction with either type IA or normal tau.
- inhibitors may be specific for the epitope or epitopes encompassed in type IA molecules, by e.g. blocking the epitope or may be directed to various domains on type IA molecules, as long as they prevent or disturb its pathological or biological activity.
- the inhibitory effect can be defined quantitatively by measuring residual microtubule assembly promoting activity by normal tau.
- As a source of inhibitors can be used libraries of small molecules of defined chemical structure and composition, peptide libraries, antibody libraries free in the solution or displayed on the surface of synthetic surfaces of phages or bacteria or ribosomes (ribosomal display) and similar technologies known in the art.
- a further object of the invention is to provide a method for testing molecules and compounds effective in disassembling type IA complexes (type I in vitro assay) comprising the following steps:
- allowing the formation of complexes between type IA molecules or peptides derived thereof and tubulin refers to condition which allows interaction of type IA molecule with said tubulin resulting in inhibition of microtubule formation.
- the present invention relates to a method for testing drugs effective in inhibiting type IA molecules from initiating the formation of complexes in a cellular system expressing tau or tau derived proteins (type I cellular assay) comprising the following steps:
- cell expressing tau protein refers to cells which have the capacity to express N- and C-terminally double truncated tau forms from a gene construct encoding a type IA molecule or a derivative thereof.
- the person skilled in the art is aware of the fact that the sequence of experimental steps of the introduction of the genes encoding the type IA molecules is irrelevant for the purpose of the method of the invention.
- Said method is particularly advantageous since the screening system is based on the continuously growing cell lines which provide a close image of the in vivo situation. Moreover, ample supply of type IA molecules located intracellularly allows screening for drugs effective in inhibiting the biological effects of type IA molecules.
- said cell expressing type IA molecules is a neuroblastoma, or pheochromocytoma cell or a primary culture of nerve cells derived from transgenic animal expressing type IA molecules.
- the group of molecules designated ‘type II’ consists of N- an C-terminally double truncated tau protein molecules (e.g. sequences described in SEQ ID 11-20). Representatives of this group localize intra- and extraneuronally and are functionally different from normal, healthy tau.
- type IIA molecules promote pathological microtubule assembly significantly higher than microtubule assembly promoted by normal healthy tau isoforms when measured spectrophotometrically (see Examples 1 and 3, resp.).
- type IIB N- and C-terminally double truncated tau molecules with similar sequences and ranges of molecular weights
- this subgroup of molecules performs to the levels seen with full length tau protein (Example 3).
- the present invention relates to a new type of modified tau protein found in Alzheimer's disease, called type IIA group of tau proteins.
- the group consist of N- and C-terminally double truncated tau molecules (SEQ ID 11-18).
- type II molecules refers to members of the group significantly different in structure and function not only from normal healthy tau but from type IA and -B tau group as well. Molecules of this subgroup bind microtubules and promote their pathological assembly that is significantly more pronounced than normal microtubule assembly by healthy tau isoforms (Example 3).
- Type IIA N- and C-terminally double truncated tau molecules interfere at the cellular level with axonal transport of constituents leading to synaptic loss and neuronal malfunction ultimately leading to cognitive impairment of the whole organism in Alzheimer's disease neurons and under experimental conditions (Examples 15 and 16, resp.). Simultaneously, afflicted neurons are vulnerable to various forms of stress such as for example oxidative stress (Example 4).
- said type IIA group of N- and C-terminally double truncated members comprises the amino acid sequences:
- R4 69-333,R4
- SEQ ID NO: 11 met val ser lys ser lys asp gly thr gly ser asp asp lys lys ala lys gly ala asp gly lys thr lys ile ala thr pro arg gly ala ala pro pro gly gln lys gly gln ala asn ala thr arg ile pro ala lys thr pro pro pro ala pro lys thr pro pro ser ser gly glu pro pro pro lys ser gly asp arg ser gly tyr ser ser pro gly ser pro gly thr pro gly ser arg ser thr pro ser leu pro thr pro pro thr arg glu pro lys lys val ala val val arg thr pro pro pro
- R3 Derived from three repeat tau (tau 44) are labeled R3 (93-302,R3) SEQ ID NO: 15 ile ala thr pro arg gly ala ala pro pro gly gln lys gly gln ala asn ala thr arg ile pro ala lys thr pro pro pro ala pro lys thr pro pro ser ser gly glu pro pro pro lys ser gly asp arg ser gly tyr ser ser pro gly ser pro gly thr pro gly ser arg ser arg thr pro ser leu pro thr pro pro thr arg glu pro lys lys val ala val val arg thr pro pro pro lys ser pro ser ser ala lys ser arg leu gln thr ala pro val pro met pro asp leu lys asn val lys ser ile gly ser
- said type II B group of N- and C-terminally double truncated members comprises the amino acid sequences: (6-378,R4) SEQ ID NO: 19 gln glu phe glu val met glu asp his ala gly thr tyr gly leu gly asp arg lys asp gln gly gly tyr thr met his gln asp gln glu gly asp thr asp ala gly leu lys ala glu glu ala gly ile gly asp thr pro ser leu glu asp glu ala ala gly his val thr gln ala arg met val ser lys ser lys asp gly thr gly ser asp asp lys lys ala lys gly ala asp gly lys thr ly
- said type IIA diseased tau proteins have the following properties:
- epitopes of type IIA and B molecules were identified in a similar way as described for type I molecules.
- the significance for type II molecules of all these epitopes and their relationship to function on microtubules are demonstrated by the mutant forms which showed that they are contributing at various extent to the activity of N- and C-terminally double truncated tau molecules such as shown in the example of type IA.
- An inhibitor useful in the composition of the present invention is therefore any inhibitor capable of modulating the pathological interaction of type IIA molecules with microtubules resulting in, pathological microtubules’.
- the term ,pathological microtubules’ as used herein refers to microtubules modified by type II molecules.
- the mode of action of such an inhibitory molecule consists of an interaction with either microtubules, microtubule associated molecules including tau and pathological derivatives thereof.
- As a source of inhibitors can be used libraries of small molecules of defined chemical structure and composition, peptide libraries, antibody libraries free in the solution or displayed on synthetic surfaces, or on phages or bacteria or ribosomes (ribosomal display) and similar technologies known in the art.
- these ‘inhibitors’ may be specific for the epitope or epitopes encompassed in type IIA molecules, by e.g. blocking the epitope or may be directed to various domains on type IIA molecules, as long as they prevent or disturb its pathological or biological activity in vitro or in vivo.
- the inhibitory effect can be defined quantitatively e.g. by measuring residual microtubule assembly promoting activity by normal tau or by measuring intracellular microtubule parameters such as outgrowth, stability or intracellular transport.
- type IIA molecules can be inhibited or neutralized by derivatives thereof for example as dominant negative proteins expressed in the respective cell.
- type IIA peptides and derivatives thereof such as peptides containing deletions or mutations can be tested or screened for their effects on inhibiting the pathological effects of N- and C-terminally double truncated tau molecules.
- the therapeutic effect is achieved by inhibiting impairment of microtubule structure and functions.
- Another object of the invention is to provide pharmaceutical compositions containing a specific inhibitor for the type IIA tau molecules of the invention, optionally in combination with a pharmaceutically acceptable carrier and/or diluent.
- the present invention relates especially to SEQ ID NO: 11 as a prototype of type IIA group molecules.
- Still another object of the invention is to provide a method for the in vitro conversion of normal microtubules into a pathological state wherein normal tau protein is incubated with type IIA or -B of the present invention under physiological conditions which allow the interaction of said type IIA or -B with microtubules generating pathological microtubules.
- the invention further relates to a screening assay allowing screening any molecule libraries for compounds capable of neutralising the pathological effects of type IIA molecules.
- test molecules are screened for their property to eliminate and/or neutralize type IIA molecules and to restore physiological microtubule parameters and functions caused by type II molecules.
- the drug screening assay consists of the following steps:
- inhibitors may be specific for the epitope or epitopes encompassed in type IIA molecules, by e.g. blocking the epitope or may be directed to various domains on type IIA molecules, as long as they prevent or disturb its activity.
- the inhibitory effect can be quantified by measuring microtubule assembly dynamics.
- inhibitors can be used libraries of small molecules of defined chemical structure and composition, peptide libraries, antibody libraries free in the solution or displayed on the surface of synthetic surfaces of phages or bacteria or ribosomes (ribosomal display) and similar technologies known in the art.
- the drug to be tested is effective in reducing the amount of type IIA molecules and/or their activity, thus fulfilling a supplementary therapeutic effect, although a total removal of the type IIA activity is preferred.
- a further object of the invention is to provide a method for the validation of drugs in living cells i.e. neurons or neurone like cells expressing type II molecules (type II Cellular assay).
- living cells i.e. neurons or neurone like cells expressing type II molecules (type II Cellular assay).
- type II Cellular assay Alternatively primary neuronal culture derived from transgenic animals or other primary neuronal cells derived from various sources expressing type IIA molecules can be used.
- neurotrophic expressing type II molecules refers to cells which stably express the molecules or which have the capacity to express type IIA molecules and into which a functional type IIA gene has been introduced either by cell culture techniques or via transgenesis as exemplified below.
- said cell expressing type IIA molecules is a neuroblastoma, or pheochromocytoma cell or a primary culture of nerve cells derived from transgenic animal expressing type IIA molecules.
- neurons expressing these molecules under appropriate conditions causes the perturbance of intracellular transport processes. Furthermore neurons expressing type IIA molecules undergo cell death under appropriate stress conditions (Example 4).
- Said method is particularly advantageous, since the system involved which is based on the use of continuously growing cell lines which provide a close image of the in vivo situation provide an ample supply of type IIA molecules located intracellularly is generated allowing drug screening for compounds effective in alleviation of intracellular type IIA effects.
- the readout of this cellular assay is adapted for low- or high throughput quantification systems.
- appropriate conditions in connection with mentioned phenotypes leading to disruption or impairment of microtubular transport and/or to neuronal death refers to any condition which allows appearance of said phenotypes as shown in the example.
- the potential drug either screened by this system, or validated in the system or drug of the third origin, is effective in the reduction of the scale of the phenotypes, thus fulfilling a supplementary function in therapy, although a total elimination or reduction of the diseased phenotypes by the drug is preferred.
- the respective invention can also be extended to an analogous readout system using cells derived from whole animals which express type IIA or -B molecules in their neurons (The transgenic animal model will be exemplified below).
- said cells and transgenic animals stably expressing N- and C-terminally double truncated type IIA tau forms allow mapping of disease pathways yielding precious information leading to new molecules relevant to pathogenesis of Alzheimer's disease, its diagnosis and treatment.
- screening and identification procedures include mRNA expression based screening technologies as well as protein based technologies.
- said type I and type IIA and -B molecules or derivatives thereof provide also a recombinant DNA construct which can be introduced into the genome of non-human animals for the purpose of providing a transgenic animal model carrying and expressing the pathogenic N- and C-terminally double truncated forms of type IA, type IIA and -B described above.
- Transgenic animals according to the invention include animals into which the construct has been introduced directly as well as progeny of such animals which retain the ability to express the construct.
- the transgene sequence is a polynucleotide sequence functionally linked to a ubiquitously expressed or otherwise to a tissue specific promoter.
- the transgene DNA encoding type IA and type IIA and -B molecules is preferentially cDNA and/or genomic DNA derived from either animal or human sources.
- Transgenic animals expressing said type I and type IIA and -B molecules are expected to develop functional changes at the cellular and/or the organ level which are phenotypically related to Alzheimer's disease. These include histological changes, RNA expression changes, changes of cellular physiological parameters and preferably behavioural changes characteristic of AD. In mature neurons of transgenic animals the expression of Type I type and IIA and -B molecules has not previously been tested. It is to expect that the level at which type I, type IIA and -B transgenes are expressed in the transgenic animal (i.e.
- transgene mRNA is an important parameter for obtaining consistent pathophysiological defects in the transgenic animal.
- the pathological features can be enhanced, attenuated or otherwise modulated such as e.g. by introducing the transgene into animal strains currently serving as disease models, animals expressing other transgenes or animals lacking functional expression of genes (see Example 14).
- the present invention provides a transgenic non-human animal cell, wherein DNA encoding a human type I and type IIA and -B molecule is expressed under the transcriptional control of suitable ubiquitous or otherwise tissue specific promoters including regulable modifications thereof.
- Cells manipulated according to the invention may be prepared by any known transfection technique.
- the DNA sequence may be introduced by direct genetic manipulation or into an earlier generation of the cell.
- the cells may be obtained from transgenic animals and cultured in vitro.
- the transgenic animals may be generated according to well established methods, such as manipulation of embryos, e.g. by gene transfer into embryonic stem cells, retroviral infection of early embryos or pronuclear microinjection.
- the results obtained in the offspring can be analysed using various techniques well known in the art.
- Models based on cells and animals of the invention may be used for example to identify and assess the efficacy of potential therapeutic agents in neurodegenerative diseases where tau and N- and C-terminally double truncated tau derived molecules but also other molecules related to Alzheimer's disease such as APP and derivatives thereof can be analysed.
- models may be used in screening or characterisation assays for detecting agents likely to prevent the pathogenic effects of N- and C-terminally double truncated tau derived molecules described here.
- the invention comprises a method for testing a potential therapeutic agent for a specified condition, in particular a neurodegenerative disease, preferably AD, wherein a cell derived from a transgenic animal expressing the said double truncated forms of tau is used as target cell. More particularly it comprises such a method, wherein the therapeutic agent such as e.g. antibodies or their derivatives is administered to a transgenic animal of the invention or introduced by crossbreeding or genetic manipulation and further tested by assay systems presented above. Moreover the invention comprises a screening or characterisation assay consisting in or including such a method, as well as a screening assay kit comprising cells of the invention. Methods for screening potential therapeutic agents using cell lines expressing type I and type IIA and -B molecules of the present invention are given in the present invention (see Example 15). The cells and animals of the present invention may be used in analogous manner.
- a neurodegenerative disease preferably AD
- Another object of the invention is to provide pharmaceutical compositions containing a specific inhibitor for N- and C-terminally double truncated forms of tau proteins optionally in combination with pharmaceutically acceptable carrier and/or diluent.
- specific inhibitor for the N- and C-terminally double truncated tau refers to substances which specifically inhibit the actions of said double truncated tau proteins.
- the nature of an inhibitor can be an antibody, an engineered, derived molecule thereof, any peptide or defined chemical composition exhibiting the desired inhibitory activity in the test systems of the present invention.
- Another object of the invention is an antibody or derivative thereof which specifically recognises an epitope of the invention and is able to partially or completely inhibit the pathological activities of N- and C-terminally double truncated tau molecules.
- oligo- or polypeptide comprising an epitope, or epitopes of the invention refers to peptides which in their two- or three-dimensional structure reconstitute the epitope of the invention which is specifically recognized by an antibody directed thereto. Moreover, said oligo- or polypeptides may solely consist of the amino acids representing said epitope(s) or they may comprise additional amino acids. The construction of such oligo- or polypeptides is well known in the art.
- the present invention relates to monoclonal antibodies and derivatives thereof either native or recombinant, immobilised, free in solution or displayed on the surface of various molecules or bacteria, viruses, or other surfaces.
- the antibodies and their derivatives are able to partially or completely inhibit the biologic activities of N- and C-terminally double truncated tau molecules.
- Such a specific antibody activity has been shown using the monoclonal antibody DC44 raised against said double truncated tau molecules isolated from Alzheimer diseased brain tissue (Examples 10 and 11, resp.).
- Said antibody(-ies) has many other variants (DC82, DC136, etc.) and may be a serum derived or a monoclonal antibody or any derivative thereof.
- the production of both monoclonal and polyclonal antibodies to a desired epitope is well known in the art (43).
- said antibody may be a natural or an antibody derived by genetic engineering, such as a chimeric antibody derived by techniques which are well understood in the art.
- said antibody also refers to a fragment of an antibody which has retained its capacity to bind the specific epitope, such as a Fab fragment or single chain Fv minibody, or intracellularly expressed single chain antibodies called intrabodies.
- the present invention relates to a pharmaceutical composition for use in the treatment of Alzheimer's disease.
- said pharmaceutical composition may be administered to a patient in need thereof by route and in dosage which is deemed appropriate by the physician handling the case.
- said pharmaceutical composition contains as the specific inhibitor at least one monoclonal antibody or small molecule or derivative thereof binding any part or group of epitopes listed above leading to their alteration and/or neutralisation, partial or complete thereof (see Examples 10, 11 and 12, resp.).
- Another object of the invention is to provide diagnostic compositions for the detection and/or monitoring of Alzheimer's disease comprising a) an epitope(s) of the invention; b) an antibody of the invention or a derived molecule thereof.
- the diagnostic composition of the invention may comprise for example an antibody of the invention which specifically recognizes one member of type IA or type II group molecule or its epitope(s) or an enhanced level of type IA or type IIA molecules in a sample to be tested.
- said diagnostic composition may comprise an antibody of the invention directed to one of the epitopes of the invention.
- an Alzheimer disease state correlating sample may be detected by treating said sample with an antibody recognising the epitope of the invention.
- the antibody- epitope (hapten) complex may be visualized using a second antibody directed to the antibody of the invention and being labelled according to methods known in the art (43).
- said diagnostic composition may consist of an epitope of the invention and an antibody of the invention. Treatment of a sample with said antibody may give rise to conclusions with regard to the disease state of the corresponding patent, if the binding of said antibody to said sample is brought in relation to binding of said antibody to said epitope of the invention used as a reference sample.
- the diagnostic composition may comprise type IA or type IIA molecules and an antibody of the invention. Activity of both types of molecules may be monitored with respect to normal tau neutralising capacity of the sample, compared to the recombinant type IA molecule (e.g. SEQ ID NO:1) and IIA molecules (SEQ. ID NO: 11-18) of the invention. From the quantified aberrant activity of type I molecule, the level of the molecules contained in said sample and therefore the disease state of the patient may be deduced. The type IA activity may e.g. be deduced by measuring the residual activity of normal tau left unreacted with type I molecules. Type II activity may be deduced by measuring further activity of type II molecules in a microtubule-assembly assay.
- type IA molecule e.g. SEQ ID NO:1
- IIA molecules SEQ. ID NO: 11-18
- Another object of the invention is to provide a method for the in vitro diagnosis and/or monitoring of Alzheimer's disease comprising assaying cerebrospinal fluid isolates of a patient, carrying out a biopsy of nerve tissue for the presence of N- and C-terminally double truncated tau molecules of type IA and type IIA molecule or its epitope(s) and for the level of their normal tau inhibitory activity.
- the ‘cerebrospinal fluid isolate of a patient’ is obtained by standard medical procedures.
- the invention relates to type I and type II molecules that are identical or homologous to the said amino acid sequence of type IA and type IIA, respectively molecules and immunogenic fragments derived thereof capable of inducing an immune response in animals.
- type I and type II molecules can be used (a) as immunogens for production of inhibitory antibodies and as central part of vaccines used for immunisation against the disease.
- type I and II molecules or derivatives thereof are capable of inducing an immune response directed against the primary, secondary and/or the ternary structure of said molecules.
- the resulting immune response is therefore capable of distinguishing between healthy and diseased forms of tau and its derivatives.
- This characteristic of the invention can be used as vaccine emphasising on the unique quality of these N- and C-terminally double truncated tau forms in inducing a disease-specific immune response.
- a type IA and IIA N- and C-terminally double truncated tau polypeptide is used for e.g. vaccination purposes or for raising antibodies, it is however not necessary to use the whole polypeptide described in the present invention. It is also possible to use a fragment of these polypeptides that are capable of inducing an immune response against that entire polypeptide, a so-called immunogenic fragment.
- this embodiment of the invention not only relates to polypeptides according to the invention, but also to derived fragments of those polypeptides that are still capable of inducing an immune response against the polypeptides (so-called immunogenic fragments).
- FIG. 1 Microtubule assembly with N- and C-terminally double truncated tau type IA and type IB molecules.
- FIG. 2 Inhibition of microtubule assembly by N- and C-terminally double truncated tau type IA and type IB molecules.
- FIG. 3 Activity of N- and C-terminally double truncated tau type IIA and IIB molecules in microtubule assembly.
- FIG. 4 Type IIA N- and C-terminally double truncated tau expressed in neuronal cells significantly increases their sensitivity to oxidative stress.
- FIG. 5 Affinity of monoclonal antibody to diseased tau type IA protein and its deletion mutants. Apparent affinity of monoclonal antibody to diseased tau type IA protein and its deletion mutants.
- the middle column are indicated epitopes of the present invention. Apparent affinities stated in the last column were measured by competitive ELISA, and shown as the concentrations of corresponding antigen needed for 50% competition with the prototype tau type IA protein.
- FIG. 6 Fractionation of tau proteins from AD-brain on Superdex 200-column
- FIG. 7 Type IA inhibitory activity in fraction No.19 from three separate isolations from AD brains.
- FIG. 8 Demonstration of N- and C-terminally double truncated tau type I molecules in AD brain.
- FIG. 9 Presence of tau type I in AD brain but not in healthy brain.
- FIG. 10 Immunoreactivity of N- and C-terminally double truncated tau type II molecules.
- FIG. 11 Construction of recombinant tau type I-II (SEQ ID 1-24).
- FIG. 12 Inhibitory effect of AD-brain derived and recombinant tau type IA on normal healthy tau.
- FIG. 13 First round screening for drug candidates neutralizing tau type IA molecules (step 1).
- FIG. 14 Second round screening for drug candidates neutralizing type IA molecules with selectivity against normal tau (step 2).
- FIG. 15 First round screening for drug candidates neutralizing tau type IIA.
- FIG. 16 Second round screening for drug candidates capable to neutralize tau type IIA molecules and discriminate them from normal tau (step 2).
- FIG. 17 Specific antibody levels in prefused mice sera determined by ELISA.
- FIG. 18 ELISA reactivity of monoclonal antibodies with AD-brain derived tau (fraction #19) and control healthy brain-derived tau (DC 20: monoclonal antibody with irrelevant specificity. Shown data represent mean values from three parallel experiments).
- FIG. 19 ELISA reactivity of monoclonal antibodies with recombinant tau molecules (DC 20: monoclonal antibody with irrelevant specificity. Shown data represent mean values from three parallels).
- FIG. 20 Screening for neutralizing antibodies directed against AD-brain derived tau type IA (fraction #19).
- FIG. 21 Screening for neutralizing antibodies directed against recombinant tau type IA (SEQ ID NO:1).
- FIG. 22 Screening for drug candidates capable of neutralizing tau type IA molecules and of discriminating them from healthy tau.
- FIG. 23 Neutralisation of pathological activity of recombinant tau type IIA (SEQ ID NO.12) by monoclonal antibodies.
- FIG. 24 Levels of antibodies against recombinant tau type IIA (SEQ ID NO.:12) detected by ELISA.
- FIG. 25 Genotyping of transgenic animals.
- Panel B Genotyping of animals from F1 generation. Genomic DNA was extracted from tail tips and double truncated tau specific DNA sequence was identified and are shown in lanes 1. Lane 2 and 3 show negative controls. Identification of a tau specific DNA fragment in the F1 generation confirms the inheritability of these transgenes.
- FIG. 26 Gene expression of double truncated human tau transcripts in the F1 generation of transgenic animals.
- RNA was extracted from flash frozen tissue of transgenic animals and subjected to reverse transcription followed by specific amplification of the cDNA.
- An example shows transgene expressing animals in lanes number 1 and 2. Lanes 3-5 represent non-expressing controls while lane 5 shows a non-specific signal typically emerging in non-transgenic animal when using this method. This example indicates the presence of double truncated tau specific mRNA expressed from the transgene in experimental animals.
- FIG. 27 Cell death caused by type IIA molecule overexpression after 6 day in vitro differentiation. Comparison of the cell viability of SY5Y cells transfected with double truncated tau type IIA (type IIA) and non-transfected control neuron-like cells (mock), respectively.
- FIG. 28 A: Increased binding affinity of type IIA molecules to microtubules is showed by using cellular fractionation of tau from stably transfected cells expressing type IIA double truncated molecules and full-length tau. Isolation of free tau (FT), microtubule bound tau (MT) and nucleus associated tau (NAT) was performed as described. B: Inhibition of tau type IA and IIA, microbutule polymerisation assay, respectively, by organic compound F123; C: Direct comparison between absorption measurement and microtubule polymerisation shown by electromicroscopic analysis.
- FT free tau
- MT microtubule bound tau
- NAT nucleus associated tau
- FIG. 29 Logarithmically growing SH—SY5Y cells stained with MitoFluor. Regular distribution of mitochondria in cell bodies and processes.
- FIG. 30 Logarithmically growing tau tpe IIA molecule expressing SH—SY5Y cells stained with MitoFluor. Perinuclear clustering of green-labelled mitochondria around the centrosome area of the cell.
- the physiological function of healthy tau consists in stabilizing microtubules (MTs). This function can be measured by a microtubule assembly assay (MAA).
- MAA microtubule assembly assay
- the MAA reactions were carried out using three types of tau molecules: normal healthy human tau, recombinant forms of tau type IA (SEQ ID NO: 1) and tau type IB (SEQ ID. NO: 4). Normal human tau, tau type IA and type IB were assayed individually in separate reactions.
- tau type IA and IB molecules lack functional activity when applied in a the MT assembly assay (MAA).
- tau type IA molecules show an inhibitory effect on tubulin in microtubule assembly.
- type IB proteins (despite similar primary structure) do not inhibit functional activity of tubulin in MAA.
- recombinant forms of tau type IA SEQ ID NO:1
- type IB SEQ ID NO:4
- the assembly-inhibition reactions were carried out separately using type IA and type IB proteins. Human tubulin (2 mg/ml) was mixed with either type IA molecules (0.2 mg/ml) or type IB molecules (0.2 mg/ml).
- type-IIA double truncated tau derivatives were surprisingly found to promote pathological microtubule assembly (see FIG. 3 and FIG. 28C ).
- the microtubule assembly reactions were carried out using three types of molecules: natural healthy human tau isoforms, Alz. tau type IIA (SEQ ID NO: 12) and tau type IIB (SEQ ID NO: 19). Three separate reactions were performed, each with single preparation of respective tau (healthy tau, recombinant tau type IIA or type IIB).
- recombinant tau type IIA exhibited extremely high (threefold) promotion of pathological microtubule assembly ( FIG. 3 , top curve 1) as compared to physiological microtubule assembly by healthy tau ( FIG. 3 , curve 2).
- type IIB molecules despite being N- and C-terminally double truncated are not able to perform in MAA as type IIA and promote microtubule assembly only to the level seen with healthy tau ( FIG. 3 , curve 3).
- the cells were grown in MEM/F12 with 10% FCS, 2 mM L-Glutamine, 1% NEAA, 50 U/L gentamicine.
- 3-morpholinosydnonimine (SIN-1) was diluted from 1 M stock solution in serum-free medium (e.g., 47.5 mg into 230 ml).
- MTT stock solution (2.6 mg/ml) was prepared in MEM/F12 w/o serum and sterilized by filtration.
- the cells were cultivated by the methods that are well known in the art. 96 well plates were seeded with 2 ⁇ 10 4 cells/well. One half of the plate was seeded with cells expressing tau type II molecules and the other half of the plate was seeded with non-expressing cells. The medium was changed every 36-48 hours. After five day, SIN-1 was added in concentrations ranging from 0 to 3.3 mM and the plates were incubated for 24 hours. Each concentration was assayed in hexaplicate. After SIN-1 incubation, MTT stock solution was added to final concentration 200 mg/ml and the plates were incubated for another 1 hour. The medium was discarded; the surface of the plate was dried up by paper wool.
- FIG. 4 The results of stated example ( FIG. 4 ) contributes to an explanation of the pathogenic effect of diseased form of tau protein.
- the chart according to FIG. 4 represents the decrease in relative resistance to oxidative stress of neuronal cells in the presence of tau type IIA. Resistance of cells non-harboring the said protein (control) is expressed as 100% (left bar) and resistance of neuronal cells expressing the diseased tau protein are shown as % of the control value (middle and right bar). Resistance is defined as the concentration of free radicals generated by SIN-1 in culture medium, where 50% of the cells die.
- the results represent measurement of double truncated tau proteins type IIA SEQ ID NO:12 (93-333, R4) and SEQ ID NO:18 (69-332, R3), respectively.
- tau type IA (segment A) or its parts was determined by sequential deletion either of whole conformation region (segment A) or its individual parts called epitopes and designated A1-A6. Since the conformation of type IA molecules strongly correlates with their function, the contribution of each epitope (A1-A6) to the overall conformation of the ‘segment A’ was measured on the basis of its reactivity when using a tau monoclonal antibody ( FIG. 5 ).
- the prototype tau type IA (SEQ ID NO:1) has an affinity of 10 nM.
- the affinity is dramatically decreased by three orders of magnitude of the affinity of prototype tau type IA.
- Preparation of Alzheimer's brain derived tau type I and type II molecules Diseased human brain tissue from neuropathologically confirmed cases of Alzheimer's disease were used as a source for isolation of double truncated tau IA,-B and IIA proteins. Preparation of tau from Alzheimer brain is based on the combination of homogenization of tissue in TRIS buffer and fractionation of lysates by saturated ammonium sulfate precipitation.
- the tissue was homogenized in cold 20 mM TRIS pH 8, 0.32 mM sucrose, 10 mM b-merkaptoethanol, 5 mM EGTA, 10 mM EDTA, 5 mM MgSO 4 , 1 mM phenylmethylsulfonyl fluoride, 50 mM sodium fluoride, 5 mM benzamidine, 5 ⁇ g/ml leupeptin, 1.5 ⁇ g/ml pepstatin, 2 ⁇ g/ml aprotinin with Heidolph DIAX 900 homogenizer for 10 min at 4° C. The homogenate was spun at 27 000 g for 30 min at 4° C. to remove cellular debris.
- Tau proteins were precipitated from brain tissue supernatant by adding 44.12% (v/v) of saturated ammonium sulfate. After incubation for 20 min at 25° C. and gently mixing, the sample was centrifuged at 20 000 g for 10 min at 25° C. Pellet was resuspended in 500 ⁇ l of 100 mM PIPES pH 6.9, 2 mM EGTA, 1 mM MgSO 4 and dialysed against the same buffer.
- This preparation was fractionated by gel filtration on a Superdex 200-column (Amersham-Pharmacia-Biotech) and the fractions were resolved by SDS-PAGE (gradient 5-20% polyacrylamide) and tau proteins were detected by immunoblotting according to standard procedure using anti tau antibodies DC25 ( FIG. 6 ). Te effect of individual fractions on microtubule assembly was tested.
- Fraction #15 ( FIG. 6 ) containing the tau molecules corresponding to the molecular mass of 30 kDa is representative of double truncated type IIA molecules. Fraction #15 showed the abnormally high microtubule assembly promoting activity. This fraction was characterized by Western blot analysis using three anti tau antibodies: DC25 recognizes both truncated and full length proteins, DC39 (specific for intact C-terminus) and Alz50 (specific for intact N-terminus) ( FIG. 10 ). The immunoreactivity of these antibodies demonstrated the presence of N- and C-terminally double truncated type II proteins only in fractions derived from from AD-brain. The concentration of tau proteins was determined by sandwich RIA. Total protein concentration was determined using the Bradford assay.
- N- and C-terminally double truncated tau molecules were prepared by PCR amplification of the relevant regions from cDNA. Specific primers introducing translation initiation start (ATG), stop (TGA) codons and NdeI , EcoRI restriction sites were used.
- Plasmids carrying deletion of A4-A6 epitopes (SEQ ID 25-27) in the tau cDNA were generated by inverse PCR as shown in FIG. 11 (bottom panel).
- AD-brain extracts as well as recombinant molecules of tau type IA are capable to inhibit microtubule assembly promotion when using natural healthy tau isoforms.
- healthy human tau was isolated from brains of age matched controls and tau type IA was isolated from brains of AD patients (see Example 6, FIG. 6 , fraction #19).
- Recombinant tau type IA SEQ ID NO: 1
- type IB SEQ ID NO:4, negative control
- brain-derived healthy tau isoforms 0.1 mg/ml
- AD-brain derived or recombinant type IA tau or type IB were mixed with tubulin. Each combination was assayed separately.
- test mixtures were incubated 1 hr at 37° C. in a water bath with gently shaking.
- GTP and/or normal tau final concentration of tubulin is 1 mg/ml and GTP 1 mM
- polymerisation buffer 100 mM PIPES, pH 6.9, 1 MM MgSO 4 , 2 mM EGTA.
- the samples were pipetted into quartz microcuvettes and equilibrated at 37° C. in a thermostatically controlled spectrophotometer (Beckman Coulter DU640). The turbidity changes were measured at 340 nm in 10 s intervals for a period of 5 min.
- a screening assay was designed for selection of compounds capable of neutralizing the inhibitory activity of type IA molecules.
- Diseased tau type IA can be derived from AD-brains or recombinant sources, however it is expedient to use recombinant material.
- the neutralizing effect of drug candidate can be defined quantitatively by measuring residual capacity of normal healthy tau to promote microtubule assembly. The assay is performed in two steps:
- tau type IIA molecules have unexpectedly high potency to promote tubulin polymerization forms a basis for a screening assay for selection of compounds neutralizing said activity of type IIA proteins.
- the neutralization of type IIA can be quantified by measuring residual microtubule assembly activity of type IIA molecules. The assay is performed in two steps:
- 17 represents levels of specific antibodies in one of the immunized mice.
- serum from the mouse immunized with irrelevant protein.
- Mouse spleen cells were fused with NS/0 myeloma cells, using a modified procedure well known in the art (M. Kohler and C. Milstein, 1975).
- monoclonal antibodies DC44, DC82 and DC136 recognize N- and C-terminally double truncated type IA and type IIA molecules from Alzheimer brain. For these antibodies no reactivity was observed with tau isolates from normal human brain prepared by the same method ( FIG. 18 ) By contrast, monoclonal antibody DC25 reacts in ELISA with the said proteins from pathological as well as from normal healthy brain ( FIG. 18 ). This antibody does not discriminate between pathological form (AD-tau) of tau and normal human tau. After this primary screening, hybridomas were subcloned in soft agarose, a technique well-know to those skilled in the art, finally resulting in homogenous hybridoma populations secreting antibodies with an identical idiotype.
- Selected monoclonal antibodies DC44, DC82 , DC136 and DC25 were further characterized for their ability to neutralize the activity of native tau type IA isolated from Alzheimer brain (see Example 6).
- Said tau isolate (final concentration of 100 mg/ml) and tested antibodies (final concentration 50 mg/ml) were preincubated for 1 hr/37° C. After incubation tubulin, normal human tau and GTP were added to the mixture (the final concentration: tubulin 1 mg/ml, healthy human tau ⁇ 100 mg/ml, GTP-1 mM) at +4° C. After rapid mixing the samples were pipetted into quartz microcuvettes and equilibrated at 37° C. in a thermostatically controlled spectrophotometer.
- the next selection step was aimed at antibodies capable to discriminate between healthy and type IA molecules.
- Mixtures of normal healthy tau (final concentration 100 mg/ml) and tested antibody (final concentration 50 mg/ml) were preincubated 1 hr/37° C. After incubation tubulin and GTP were added to the mixture (the final concentration: tubulin 1 mg/ml, GTP-1 mM) at +4° C.
- the samples were pipetted into quartz microcuvettes and equilibrated at 37° C. in a thermostatically controlled spectrophotometer. The turbidity changes were measured at 340 nm. None of antibodies DC136, DC44, DC82 and DC25 was able to inhibit normal healthy tau in microtubule assembly ( FIG.
- Antibodies previously isolated for their tau type IA neutralizing activity were tested for their neutralizing activity against recombinant tau type IIA (SEQ ID NO:12) using the method described in Example 8B. All three neutralizing monoclonal antibodies DC44, DC82 and DC136 were able to reduce the pathological activity of N- and C-terminally double truncated tau type IIA molecules ( FIG. 23 ; antibodies were preincubated with recombinant tau type IIA and then mixed with tubulin and GTP. The formation of microtubules was determined spectrophotometrically after 5 min at 37° C. The bars represent the mean value of three independent experiments.
- said recombinant tau type IA and IIA proteins are used for vaccination purposes or for raising antibodies which specifically neutralize the pathogenic activity of diseased tau type IA and IIA molecules.
- recombinant N- and C-terminally double truncated tau type type IIA (SEQ ID NO: 12) was used as an immunogen.
- Balb/c mice were primed subcutaneously with said proteins (50 mg/ mouse) in complete Freund's adjuvant and boosted intraperitoneally 3 times thereafter at 4-week intervals with the 50 mg/mouse of the same proteins in incomplete Freund's adjuvant. Immune sera were collected and the level of specific antibodies against respective recombinant antigens tau were determined by ELISA ( FIG. 24 ).
- Genoptyping ( FIG. 25 ): Specific amplification of transgenes encoding double truncated tau forms was performed on genomic DNA derived from the parental generation of transgenic animals and is shown in FIG. 25A . Further analysis of genomic DNA of the F1 generation revealed that transgenes are heritable since they were also identified in the offspring of parental generation. Transgenes encoding double truncated tau are therefore fixed in chromosomal DNA of the animals ( FIG. 25B —Genotyping of F1 generation).
- the animals used in this example are of a specific genetic background characterized by spontaneous hypertension and other Alzheimer's disease associated risk factors, such as dyslipidaemia or diabetes. This animal strain therefore represents a unique experimental Alzheimer model by combining the most frequently occurring Alzheimer's disease risk factors such as hypertension and diabetes.
- cDNA encoding double truncated tau was introduced into an expression vector linked to a promoter directing an expression in ubiquitous or tissue specific manner.
- the gene fragment was introduced into one day embryos via pronuclear injection (non limited).
- Resulting offspring was genotyped using genomic DNA from the tail tip.
- FIG. 26 Analysis of transgene expression ( FIG. 26 ): Expression of mRNA derived from the transgenes were assessed by RT-PCR analysis, applying generally known methods such as RT-PCR and agarose gel electrophoresis.
- Panel B of FIG. 25 Genotyping of animals from F1 generation. Genomic DNA was extracted from tail tips and double truncated tau specific DNA sequence was identified and are shown in lanes 1. Lane 2 and 3 show negative controls. Identification of a tau specific DNA fragment in the F1 generation confirms the inheritability of these transgenes.
- FIG. 26 RNA was extracted from flash frozen tissue of transgenic animals and subjected to reverse transcription followed by specific amplification of the cDNA.
- An example shows transgene expressing animals in lanes number 1 and 2. Lanes 3-5 represent non-expressing controls while lane 5 shows a non-specific signal typically emerging in non-transgenic animal when using this method. This example indicates the presence of double truncated tau specific mRNA expressed from the transgene in experimental animals.
- neuroblastoma cell line SH-SY5Y cell death caused by type IIA molecule was demonstrated using standardized in vitro differentiation conditions known to the person skilled in the art. The effect was tested in stably transfected cells expressing type IIA double truncated tau and compared with non-transfected cells. Cell viability was quantified manually using a trypan blue exclusion assay in triplicates and statistical evaluation was performed using the One-way ANOVA test. Significant differences in cell viability between cells overexpressing type IIA double truncated tau and wild type cells were found after 6 day of in vitro differentiation (P ⁇ 0.001).
- Type II double truncated tau molecules show increased binding affinity to the microtubular system.
- FT free tau
- MT microtubule bound tau
- NAT nucleus associated tau
- Normal healthy tau and Alzheimer tau type II are analysed with respect to their microtubule assembly promotion capacity.
- Normal tau in this example represented by tau 43 forms typical microtubules shown in electron microscopy (see FIG. 28C ).
- Alzheimer tau type II produces pathological microtubules with typical pattern (see FIG. 28C ).
- the pathological phenotype showing altered transport of mitochondria caused by overexpression of a type IIA molecules was performed in the neuroblastoma cell line SH-SY5Y.
- the influence of the N- and C-terminally double truncated tau type II molecules was examined by comparing mitochondrial redistribution in living wild type SH-SY5Y cells with transfected cells.
- Cell biological transport assays known to the person skilled in the art were used. In brief, cells were cultivated on LabTekII chambers (Nunc) with equal density (70% confluent) according to standard laboratory techniques and transfection was performed using Fugene 6 (Roche) according to the instructions of the manufacturer.
- mitochondrial localization was compared in induced and non-induced SH-SY5Y cells.
- the staining confirmed the negative effect of type IIA double truncated tau molecules on mitochondrial transport in SH-SY5Y cells resulting in perinuclear mitochondrial clustering near the centrosome indicative of a functional dominance of the minus end directed intracellular forces ( FIG. 30 ).
- FIG. 29 logarithmically growing cells reveal a regular distribution of mitochondria in the cell body as well as in the cell periphery.
- the N- and C-terminally double truncated type IIA proteins are therefore able to influence intracellular transport mechanism which affect mitochondrial redistribution.
- the present-experimental setting shows a suitable method for testing inhibitory activities directed against type IIA molecules.
Abstract
Described are novel N- and C-terminally double truncated tau molecules, (“type IA, IB, IIA and IIB tau molecules”) as well as methods for providing these molecules, both from recombinant and biological sources. Moreover, screening methods using these molecules in connection with Alzheimer's diagnosis and therapy are provided.
Description
- The invention relates to N- and C-terminally truncated diseased forms of tau proteins discovered specifically in Alzheimer's disease and related disorders.
- The invention further relates to methods for screening and testing potential drugs effective in inhibiting, neutralising and eliminating N- and C-terminally double truncated tau proteins or preventing the formation of thereof and to procedures for screening and testing potential drugs of which the mode of action is based on neutralising the modification of microtubule assembly and/or dynamics caused by said double truncated diseased forms of tau proteins.
- Alzheimer's disease is the most common cause of dementia. In less than 5% of the cases Alzheimer's disease cosegregates almost completely with one or more specific mutations in the amyloid precursor protein, presenilin-1 or presenilin-2 genes (1) and in over 95% of the cases, the exact disease cause is not clear.
- Independent of etiology, Alzheimer's disease is characterized histopathologically by the presence of numerous neurons with neurofibrillary tangles of paired helical filaments (PHF) and extracellular deposits of amyloid β as the major component of senile plaques in the brain. Although the exact nature of a direct relationship, if any, between these two hallmark lesions of Alzheimer's disease is presently not understood, the presence of neurofibrillary degeneration appears to be required for the clinical expression of the disease, i.e. dementia (2,3,4). Neurofibrillary degeneration is represented by neurofibrillary tangles, dystrophic neurites and neuropil threads. The major protein subunit of these structures is microtubule associated protein tau (5,6).
- In healthy human brain tau appears in six protein isoforms generated by alternative mRNA splicing of a transcript derived from a single gene locus. tau proteins differ whether they contain three (t3L, t3S, or t3) or four (t4L, t4S, or t4) tubulin binding domains (repeats, R) of 31 or 32 amino acids near the C-terminal and two (t3L, t4L), one (t3s, t4S), or no (t3, t 4) inserts of 29 amino acids each in the N-terminal part of the molecule (7,8). Under physiological conditions tau protein is involved in assembly, spatial organisation, stabilisation and behaviour of microtubules. Under physiological conditions the protein appears in six isoforms in healthy human brains. However in AD, tau protein is known to undergo a number of different post-translational modifications (hyperphosphorylation, ubiquitination, glycosylation). The recent discovery of cosegregation of specific mutations in the tau gene with the disease frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) has confirmed that certain abnormalities in the tau protein can be a primary cause of neurodegeneration and dementia in affected individuals (9,10). The molecular events leading to tau modification and paired helical filament (PHF) formation in Alzheimer's disease are unknown. This explains the observation of a broad spectrum of pathophysiological events such as pathological redistribution of tau protein, failure of axonal transport or a failure to maintain axonal microtubule function (11,12,13). To date the significance of PHF fibril formation in Alzheimer's disease is questioned in the light of the recent discovery that any protein can form fibrils in vitro (14).
- Many authors believe that formation of paired helical fibrils in Alzheimer's disease represents a primary event in neurofibrillry pathology which is based on abnormal phosphorylation. PHF assembled tau protein reacts with certain antibodies in a phosphorylation dependent manner, suggesting a special phosphorylation status (15,16). Furthermore it has been observed that PHF derived tau protein shows a reduced electrophoretic mobility in SDS gels which may be related to its phosphorylation pattern (Steiner et al., EMBO J. 9 (1990), 3539-3544). Similarly it has been suggested that due to phosphorylation, PHF derived tau has lower affinity for microtubules compared to normal tau protein, since a similar effect was found when normal tau was phosphorylated in vitro by certain kinases (17,18). tau is one of the most soluble proteins known (19,20,21) and therefor its aggregation in Alzheimer's disease is particularly enigmatic. On the other side the mechanisms by which tau protein is modified in a manner which leads to filament formation in Alzheimer's disease are unknown. Phosphorylation of tau affects the potential of tau to form aggregates, producing either stimulatory or inhibitory effects on microtubule polymerisation, presumably depending on the site of phosphorylation (22-27). Many in vitro studies demonstrate that in the presence of the reducing agent dithiothreitol (DTT), unsaturated free fatty acids, RNA or glycosaminoglycans, normal tau can be transformed into filaments (28-31,38). Furthermore, the process of filament formation can also be accelerated by the presence of cross-linked tau generated through oxidation at Cys322 (32). The parameters that varied in different filament assembly studies including tau protein concentration, pH and ionic strength were manifold higher than in the cytoplasm under physiological conditions. Examination of in vitro formed tau filaments by scanning transmission electron microscopy (STEM) showed, that these filaments differ from native paired helical filaments (33). In the absence of glycans or RNA, no PHF-like filaments are detectable in samples containing unphosphorylated or phosphorylated wild type tau. Moreover it has been suggested that phosphorylation could play a protective role in Alzheimer's disease (34). Similar suggestions for modification of tau leading to assembly of PHF with resulting microtubule disassembly and interference with vital neuronal processes, such as axonal transport, were made for ubiquitination and glycosylation (30,35,36,37). However none of above mentioned post-translational modifications alone could provide molecular explanation for the initiation of tau changes leading to its malfunction that correlate with clinical expression in Alzheimer's disease.
- Therefore it remains unclear which of above mentioned modifications of tau are involved in the pathogenesis of Alzheimer's disease.
- To date no reliable data on the mode or regulation of post-translational events leading to the formation of early tau protein complexes are available. For the prevention of the formation of such complexes and for neutralisation of any associated pathogenic effects thereof, the precise molecular nature of diseased tau and the regulatory mechanism transforming normal tau to its N- and C-terminally double truncated forms need to be clarified. This detailed knowledge would allow to construct tools for Alzheimer therapeutics and diagnostics.
- Zelman et al. (J. P. Neurochem. 72(2) (1999), 741-750) suggest that the cleavage product of microtubule binding protein tau occurs in cerebrospinal fluid of persons with traumatic brain injury and reflects damage of neurons. A connection with Alzheimer's disease, however, is not made in this report.
- Novak (Acta Virologica 38 (1994), 173-189) reports in this review article concerning “Tauons” the minimal protease resistant unit of PHFs (“paired helical filaments”) produced artificially with broad spectrum protease “Pronase”.
- Kontsekova et al. (J. Immunol. Meth. 185 (1995), 245-248) disclose a quick purification method of recombinant human truncated tau proteins for immunoanalysis in which heat resistancy of human tau protein is used. Neither structural nor biological properties or functions of recombinant tau analoga used therein have been described.
- In Novak et al. (EMBO J. 12(1) (1993), 365-370) paired helical filaments (PHF core) have been prepared in vitro artificially, wherein a minimal protease resistant tau unit was recovered by in vitro digestion with the protease pronase. Monoclonal antibody MN423 was used to detect the minimal protease resistant tau unit. The tau polypeptides described in this article, however, do not have biological structural pathological properties common with “real world” tau proteins, especially tau proteins being connected with Alzheimer's disease.
- Fasulo et al. (Alzheimer's Research 2(5) (1996), 195-200) report that over-expression of recombinant analogue of PHF core tau is not sufficient to induce tau aggregation and assembly thereof in paired helical filaments. These data are in contrast to a publication of Abraha et al. (J. Cell. Science (113) (21) (2000), 3737-3745) obviously due to the unusual non-physiological assay system described in this publication (cell lines from monkey kidneys).
- Fasulo et al (J. Neurochem. 75 (2000), 624-633) describe tau fragments which induce apoptosis. However, none of the Alzheimer's disease related tau proteins described in the present invention can induce apoptosis.
- Esposito et al. (J. Peptide Science 6 (2000), 550-559) describe the
C terminal 19 amino acids of tau protein and normal healthy tau protein. The articles of Novak et al. (Chem. Papers 52 (1998), 429-430) and Ugolini et al. (NeuroReport 8 (1997) 3709-3712) also relates to the C terminal truncated tau protein also with respect to apoptosis. More recent publications show that Alzheimer's disease is not related to apoptosis processes. - In Abraha et al. (J. Cell Science 113 (21) (2000), 3737-3745) in vitro experiments are described in order to show the contribution of single domains of tau protein for formation of filaments. Therefore a set of recombinant tau molecules have been assembled which have been produced in vitro. Neither biological nor pathological activities of these proteins in bacteria have been generated or determined. Moreover, no data with respect to tau proteins derived from brains from Alzheimer's disease patients have been described in this article.
- In Jicha et al. (J. Neuroscience Research 55 (1999), 713-723) a molecular analysis of the epitope of the monoclonal antibodies Alz50 and MC-1 is described. Both antibodies depend on a functional N-terminus of tau molecule, especially amino acid positions 7-9. Tau truncations are not mentioned in this document.
- Brandt et al. (J. Biol. Chem. 268 (1993), 3414-3419) have analysed different domains of normal healthy human tau proteins. For this, recombinant tau fragments have been produced in bacteria. Alzheimer related truncated tau fragments, however, are not described in this document.
- Philippe et al. (J. Neuroscience Research 46 (1996), 709-719) disclose monoclonal anti amyloid precursor protein antibodies. The authors describe the generation of a tau reactive antibody, although this antibody originally was raised against amyloid precursor protein. Pathology related Alzheimer tau fragments are not disclosed in this document.
- WO 94/18560 Al discloses an immuno assay for detecting human tau protein in a cerebrospinal fluid for detecting patients with cell central nervous cytopathies. This assay does not discriminate between normal tau and tau of patients with central nervous cytopathis but detects the total amount of tau protein in a sample.
- It is therefore an object of the present invention to provide such reliable markers correlated with pathological dysfunction of Alzheimer's disease neurons. Moreover, suitable tools for verifying the presence and assaying the activity of such tau derived polypeptides would be valuable means for Alzheimer diagnostics and therapeutics.
- The present invention therefore provides N- and C-terminally double truncated tau molecules, which are characterized by the following features (“type IA tau molecules”):
-
- the molecules have at least the first 236 N-terminal amino acids and at least the last 45 C-terminal amino acids of the 4 repeat containing tau43 truncated,
- the molecules are detectable in Alzheimer's diseased brain tissue whereas the molecules are not detectable in normal healthy brain tissue and
- the molecules prevent normal tau protein from promoting microtubule assembly in an in vitro microtubule assembly assay,
- said prevention of the promotion of microtubule assembly can be eliminated by specific inhibitory, neutralising monoclonal antibodies against said molecules in a microtubule assembly assay.
- In the following the designation ‘N- and C-terminally double truncated tau proteins’ is used to describe two groups of truncated tau derivatives which appear in Alzheimer's disease brains and which are closely correlated with pathological dysfunction of Alzheimer's disease neurons. In particular, these proteins represent a group of molecules which exert their pathological function by modifying microtubule associated biological functions such as microtubule assembly or intracellular transport. In the following the term ‘protein complexes’ is used for N- and C-terminally double truncated tau proteins in the form of homodimeric, heterodimeric or multimeric complexes that are composed of molecules that are physically associated with tau and/or double truncated tau proteins.
- As used herein, the term ‘tau’ refers to the group of shortest naturally occurring isoforms present in healthy human brain containing three repeats (tau44) and four repeats (tau43) in their microtubule binding domain as previously described (39, 40): tau43 (383 amino acids, missing
exons 2 and 3 [pos 45-102]) tau44 (352 amino acids, missingexons - Suitable microtubule assembly assays (alternatively often also termed “microtubule polymerisation assays”) are e.g. described in (19) and (20). The term “preventing” includes any significant inhibition of 20% or more, preferably 50% or more of normal tau promoting activity.
- Specifically preferred type IA tau molecules according to the present invention comprise an amino acid sequence selected from the group of
SEQ ID NOs 1 to 3. - Further, the present invention provides N- and C-terminally double truncated tau molecules, which are characterized by the following features (“type IB tau molecules”):
-
- the molecules have at least the first 238 N-terminal amino acids and at least the last 40 C-terminal amino acids of the 4 repeat containing tau43 or the first 207 N-terminal amino acids and at least the last 50 C-terminal amino acids of the 3 repeat containing tau44 truncated
- the molecules are detectable in Alzheimer's diseased brain tissue whereas the molecules are not detectable in normal healthy brain tissue and
- the molecules do not prevent wild type tau from promoting microtubule assembly in an in vitro microtubule assembly assay.
- Preferred type IB tau molecules are characterized in that the comprise an amino acid sequence selected from the group of
SEQ ID NOs 4 to 10. - The present invention also provides N- and C-terminally double truncated tau molecules, which are characterized by the following features (“type IIA tau molecules”):
-
- the molecules have at least the first 68 N-terminal amino acids and at least the last 40 C-terminal amino acids of the 4 repeat containing tau43 or the first 68 N-terminal amino acids and at least the last 20 C-terminal amino acids of the 3 repeat containing tau44 truncated,
- the molecules are detectable in Alzheimer's diseased brain tissue, whereas the molecules are not detectable in normal healthy brain tissue,
- the molecules have a higher microtubule assembly promoting activity than wild type tau in an in vitro microtubule assembly assay,
- said microtubule assembly promoting activity can be eliminated by specific inhibitory, neutralising monoclonal antibodies against said molecules in a microtubule assembly assay and
- the pathologic activity of said molecules relies their binding to the microtubular network defined by the microtubule polymerisation promoting activity.
- Preferably, the enhanced microtubule assembly proting activity is at least 20% higher, especially at least 50% higher than wild type tau when measured spectrophotometrically.
- Preferred type IIA tau molecules are characterized in that the comprise an amino acid sequence selected from the group of
SEQ ID NOs 11 to 18. - Moreover, the present invention provides N- and C-terminally double truncated tau molecules, which are characterized by the following features (“type IIB tau molecules”):
-
- the molecules have at least the first 68 N-terminal amino acids and at least the last 40 C-terminal amino acids of the 4 repeat containing tau43 or the first 68 N-terminal amino acids and at least the last 20 C-terminal amino acids of the 3 repeat containing tau44 truncated,
- the molecules are detectable in Alzheimer's diseased brain tissue, whereas the molecules are not detectable in normal healthy brain tissue,
- the molecules have a pathological microtubule assembly promoting activity different from wild type tau in an in vitro microtubule assembly assay.
- Preferred type IIB tau molecules according to the present invention are characterized in that they comprise an amino acid sequence selected from the group of
SEQ ID NOs - The novel tau polypeptides according to the present invention (IA, IB, IIA and IIB) have typical and unique localisation characteristics since they exclusively localize in Alzheimer's diseased brain tissue. Moreover, also the interaction of these polypeptides with non-polymerized tubulin (alpha/beta dimers) and polymerized form (as microtubule) is unique.
- According to another aspect, the present invention provides a method for the preparation of molecules according to the present invention (type IA, IB, IIA, IIB), characterized in by the following steps:
-
- a) construction of a recombinant prokaryotic expression plasmids carrying coding sequences for a double truncated tau molecule with deletions covering at least the first 236 and the last 40 amino acids or the first 68 and the last 20 amino acids or combinations thereof,
- b) growing said bacteria under conditions allowing expression of said N- and C-terminally double truncated tau molecule,
- c) collecting of bacteria, preferably by centrifugation,
- d) resuspending the bacterial pellet,
- e) sonicating said bacteria,
- f) fractionating said sonicated bacteria by gel filtration and
- g) monitoring the activity of the obtained fractions by a microtubule assembly assay thereby identifying the different activities of type I and type II tau molecules.
- Preferably, the truncations are as defined above for type IA, IB, IIA and IIB molecules. The microtubule assembly assay activity is preferably as defined above, especially as for IA.
- Moreover the present invention provides a method for the preparation of molecules according to the present invention, characterized in by the following steps:
-
- a) providing Alzheimer's diseased brain tissue,
- b) homogenising said diseased brain tissue in a buffer, especially in Tris buffer,
- c) ammonium sulfate precipitation of said homogenized brain tissue,
- d) redissolving in PIPES buffer,
- e) fractionating said redissolved material by gel filtration and
- f) monitoring the activity of the obtained fractions by a microtubule assembly assay thereby identifying the different activities of type I and type II tau molecules.
- The microtubule assembly assay activity is preferably as defined above, especially as for IA.
- The present invention further provides a method for testing substances effective in disassembling a complex of type IA molecules and tubulin, comprising the following steps:
-
- a) allowing the formation of protein complexes between type IA molecules and tubulin and
- b) incubating the protein complexes with a substance to be tested and identifying those substances which allow the restoration of the microtubule assembly promoting capacity of wild type tau.
- Further, the present invention also provides a method for testing substances effective in inhibiting type IA molecules from initiating the formation of complexes with tubulin in a cellular system expressing wild type tau comprising the following steps:
-
- a) introducing a functional gene encoding a type IA molecule under the control of suitable regulatory regions into a cell expressing normal tau protein,
- b) allowing the formation of protein complexes between type IA molecules and tubulin molecules,
- c) applying the substance to be tested to the cells harboring said complexes an
- d) examining the effect of said substance on type IA biological activity as defined above.
- The present invention also provides a method for in vitro conversion of microtubules into a pathological state characterized by incubating tubulin protein with type IIA under physiological conditions which allow the interaction of said type IIA molecules with microtubules generating pathological microtubules.
- According to another aspect, the present invention provides a method for screening substances capable of neutralising the pathological effects of a type IIA molecules for their property to eliminate and/or neutralize type IIA molecules and to restore physiological microtubule parameters and functions caused by type II molecules comprising the following steps:
-
- a) formation of pathological microtubules in the presence of type IIA molecules and tubulin,
- b) incubation of a mixture of the substance, type IIA and tubulin with the substance to be screened and
- c) examination of the result with respect to diminishing the formation of pathological microtubules caused by type IIA molecules.
- According to the present invention, also a method for testing substances effective in inhibiting the in vivo activity of type IIA molecules in promoting abnormal microtubule formation and function in a cellular system expressing type IIA molecules is provided, which comprises the following steps:
-
- a) introducing a functional gene encoding type IIA molecules under the control of suitable regulatory regions into a cell expressing wild type tau,
- b) allowing the formation of complexes between type IIA tau molecules and microtubules, whereby said complexes are involved in the formation of pathological microtubules,
- c) applying the substance to be tested to the cells harboring said complexes and
- d) examining the effect of said substance on type IIA biological activity, especially on the modifications of the microtubule network and its associated functions.
- According to another aspect, the present invention also provides transgenic animals expressing a molecule according to the present invention (type IA, IB, IIA or IIB), especially IA an/or IIA.
- The present invention also relates to the use of a transgenic animal according to the present invention as animal model for Alzheimer's disease, especially for screening and testing drugs for the treatment of Alzheimer's disease.
- With the present invention a vaccine is provided which comprises a molecule according to the present invention (IA, IB, IIA or IIB), especially IA and IIA, and a pharmaceutically acceptable carrier, especially an adjuvant.
- The present invention also provides inhibitor of the initiation of the formation of complexes of a type IA molecule with wild type tau. A specific example for such inhibitors are substances comprising a binding moiety as the monoclonal antibody DC44 deposited under the
deposition number 02060767 at the European Collection of Cell Cultures (ECACC), Porton Down, Salisbury, UK, especially DC44 or binding fragments thereof, such as the Fab. - Thus, the present invention provides:
-
- (1) molecular and functional identification and characterisation of N- and C-terminally truncated diseased forms of tau proteins. These molecules exert their pathological function in Alzheimer's disease by modifying microtubule associated biological functions such as microtubule assembly or intracellular transport.
- (2) antibodies specific for the protein epitopes
- (3) antibodies neutralising pathological activities of said proteins
- (4) methods for screening and testing therapeutic drug candidates (including antibodies) effective in inhibiting, neutralising and eliminating N- and C-terminally double truncated tau proteins or preventing formation thereof
- (5) the development of animal models bearing gene constructs encoding for the respective double truncated tau proteins as transgene or transgene-combinations which can be used for drug screening
- (6)pharmaceutical compositions comprising inhibitors to said double truncated tau proteins and to proteases involved in their origin
- (7) methods for screening molecules which generate N- and C-terminally double truncated tau molecules
- (8) diagnostic and therapeutic compositions recognising and/or interacting with said molecules
- (9) the development of vaccines based on the antigenicity of said double truncated proteins
- (10) methods involving said proteins and their epitopes and/or antibodies or other specific probes for in vitro and in vivo diagnosis of Alzheimer's disease and other disorders related to pathological changes of tau.
- Accordingly, the present invention relates to the characterisation of N- and C-terminally double truncated forms of pathological tau protein and their epitopes which are specifically occurring in Alzheimer's disease.
- Degradation of proteins is a general phenomenon occurring during physiological elimination of proteins encompassing production of intermediate truncation products of various size, usually of short half life at protein is no exception and undergoes this process in healthy brains containing wt (=wild type) tau. In the following the term 'wt at covers all 6 naturally occurring isoforms of tau protein normally found in the brain of healthy individuals. Various short truncation forms of at found in Alzheimer diseased brain were produced in bacteria, purified to various extent with aim to probe physiological function of at proteins, to map their domains and phosphorylation epitopes or in experiments trying to understand the mechanisms of paired helical assembly in Alzheimer's disease and other neurodegenerative disorders, with equivocal results (23-27,34,41,42). The general term “N- and C-terminally double truncated forms of tau proteins” refers to any tau protein in Alzheimer's disease with loss at least one of its amino acids at both ends of molecule. Throughout the analysis of double truncated tau in extracts from Alzheimer diseased brains it was found in the course of the present invention that some of these molecules displayed structurally and functionally distinct characteristics which allowed to discriminate them from other tau fragments found in Alzheimer's diseased brain tissue. On the basis of this discrimination a novel scheme was provided which defines two major classes of pathogenic molecules of N- and C-terminally double truncated tau molecules distinct from healthy tau: Type I and Type II tau molecules. These groups can further be subdivided into two subclasses each based on the molecular structure and are designated type IA and B, and type IIA and B, respectively.
- Type IA and type IIA represent structurally and functionally distinct types of diseased molecules derived from microtubule associated protein tau generated by pathological processing. N- and C-terminally truncated tau molecules, represent diseased molecules, derived from microtubule associated protein tau and emerging during specific pathological processes characteristic of Alzheimer's disease. This is a common feature of all four groups of tau derived proteins. Further common features of all groups are an N- and C-terminal truncations, their intra- and extraneuronal localisation and functional distinction from normal, healthy tau.
- The group of molecules designated ‘type IA’ is described by the examples SEQ ID 1-3. These truncated tau molecules differ from normal tau in acting as key (central), active units, and driving force for interaction of pathological tau and tubulin. Type IA as well as type IB molecules do not have any promoting activity in microtubule assembly. Surprisingly type IA is able to prevent normal tau from promoting microtubule assembly (Example 1). Despite of similar primary sequence features and molecular masses, type IB, does not show this functional activity in vitro (Example 2). This is suggestive for a strong binding activity of type IA to tubulin and thereby providing a dominant negative effect on tau physiology. Type IA molecules are therefore most likely responsible for continuous, chronic depletion of neurons from functional microtubular network and for taking part in neurofibrillary structures which directly correlate with the clinical severeness of Alzheimer's disease. Unexpectedly, type IB (e.g. SEQ.ID.NO: 4-10), despite having similar molecular mass and sequences as the type IA group of molecules, display none of pathological activities of group IA members (see Example 2). As opposed to these groups, type IIA double truncated tau derivatives bind microtubules and promote their pathological assembly (Example 3). In the following Type IIA promoted microtubules are referred to as ‘pathological microtubules’. Surprisingly molecules with similar sequences and ranges of molecular weights (Type IIB) are lacking these high microtubule polymerisation capabilities. In microtubule assembly assays they perform to the levels seen with full length tau protein (see Example 3).
- N- and C-terminally truncated tau derivatives of both groups (type IIA and B) interfere at the cellular level with axonal transport leading to synaptic loss which ultimately results in neuronal dysfunction and cognitive impairment in Alzheimer's disease patients. Simultaneously, afflicted neurons are vulnerable to various forms of stress such as oxidative stress (Example 4). Type IIB despite of having similar molecular sizes than type IIA additionally promote microtubule assembly to levels seen for full length healthy tau (wild type tau) when measured spectrophotometrically.
- In a further preferred embodiment of the type IA and -B and type IIA and B molecules of the invention the recombinant versions of said molecules can be obtained by carrying out the following steps:
-
- (a) Construction of a recombinant prokaryotic expression plasmids carrying coding sequences for said double truncated tau molecules (type I and II)
- (b) growth of bacteria under conditions allowing expression an N- and C-terminally double truncated tau molecules (type I and II)
- (c) collecting of bacteria by centrifugation
- (d) resuspending the bacterial pellet from 500 ml cultivation in buffer A: (20 mM PIPES pH 6.9, 50 mM NaCl, 1 mM EGTA, 1 mM MgSO4, 2 mM DTT, 0.1 mM PMSF)
- (e) sonication on ice for 1 min (3 times) centrifugation at 45 000 rpm, 15 min at +2° C. (rotor TLA-120, 2, Beckmann Optima TLX)
- (f) chromatography on Phosphocellulose, or
MONO S HR 5/5 or 5 ml HiTrap SP Sepharose HP column in linear gradient 0-1M NaCl in buffer “A” identifying the obtained proteins by SDS-PAGE and Western blot analysis.
- In a preferred embodiment of the invention, said type IA group of N- and C-terminally double truncated members comprises the following amino acid sequences:
- Derivatives from four repeat tau (tau 43) will be labeled R4
(239-333,R4) SEQ ID NO: 1 ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu (237-333,R4) SEQ ID NO: 2 asp asn ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu (239-318,R4) SEQ ID NO: 3 ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu - In a preferred embodiment of the invention, said type IB group of N- and C-terminally double truncated members comprises the following amino acid sequences:
(239-326,R4) SEQ ID NO: 4 ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala (239-328,R4) SEQ ID NO: 5 ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr (239-331,R4) SEQ ID NO: 6 ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly (239-334,R4) SEQ ID NO: 7 ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu ile (239-340,R4) SEQ ID NO: 8 ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu ile val tyr lys ser pro val (239-343,R4) SEQ ID NO: 9 ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu ile val tyr lys ser pro val val ser gly - Derivatives from three repeat tau (tau 44) will be labeled R3
(208-302,R3) SEQ ID NO: 10 leu lys his gln pro gly gly gly lys val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu - There may be one or more epitopes of tau protein which specifically occur in type IA or type IIA members in N- and C-terminally double truncated diseased forms of tau proteins.
- In the present embodiment of the invention, said epitopes are specifically located within the primary structure of type IA (SEQ ID 1-3) and type IIA (SEQ ID 11-18) group members and their number, heterogeneity and specificity depends on and is added by specific structural conformation of each individual group member. Therefore the singularity of each molecule is not solely based on its primary structure together with its effects on microtubule assembly, but also on its secondary and ternary structure which makes up its epitopes. Some of them can form particularly important “conformational regions” contributing significantly to the activity of said molecules.
- The term “conformational region” as used herein refers to epitopes clustered to one region of molecule contributing to its activity.
- In a particularly preferred embodiment the conformational region encompassed in type I and type II molecules comprising amino acids “ile lys his val pro gly gly gly ser val gin ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu” is corresponding to residues 239-267 (SEQ ID NO: 1-9 and 11-14,19 R4) and comprising amino acids “val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu” corresponding to residues 217-236 (SEQ ID NO: 10.15-18,20 R3) was designated sequence A.
- In still another preferred embodiment of the invention said epitopes in said conformational region were identified and their relative contribution determined by deletion mutagenesis. The significance of all these epitopes and their relationship to function on microtubules are demonstrated by the mutant forms which showed that they are contributing at various extent to the activity of type IA molecules (Example 5). These individual epitopes comprise the following amino acid sequences:
A: ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu - (corresponding to residues 239-267 in SEQ ID NO: 1-9 and 11-14,19). The epitope deletion mutant has SEQ ID NO: 21 (268-333,R4;del 239-267)
gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu A1: ile lys his val pro gly gly gly ser - (corresponding to residues 239-247 in SEQ ID NO: 1-9 and 11-14,19). The deletion mutant has SEQ ID NO: 22 (248-333,R4;del 239-247)
val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu A2: ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu - (corresponding to residues 239-257 in SEQ ID NO: 1-9 and 11-14,19). The deletion mutant has SEQ ID NO: 23 (258-333,R4;del 239-257)
ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu A3: ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser - (corresponding to residues 239-262 in SEQ ID NO: 1-9 and 11-14,19). The deletion mutant has SEQ ID NO: 24 (263-333,R4;del 239-262)
lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu A4: ser val gln ile val tyr lys pro val asp leu ser lys val thr ser
(corresponding to residues 246-262 in SEQ ID NO: 1-9 and 11-14,19). - The epitope deletion mutant has SEQ ID NO: 25 (239-333, R4;del 248-262)
ile lys his val pro gly gly gly lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu A5: asp leu ser lys val thr ser
corresponding to residues 256-262 in SEQ ID NO: 1-9 and 11-14,19, and to residues 225-231,R3 SEQ ID NO:10, 15-18,20 - The epitope deletion mutant has SEQ ID NO: 26 (239-333,R4;del 256-262)
ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu - A6: lys cys gly ser leu corresponding to residues 263-267 in SEQ ID NO: 1-9 and 11-14,19 and to residues 232-236,R3 in SEQ ID NO: 10, 15-18,20
- The epitope deletion mutant has SEQ ID NO: 27 (239-333,R4;del 263-267)
ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu - Again it is to be understood that not all of the amino acids of the peptide necessarily contribute to the specific site actually recognised by specific antibodies.
- In a preferred embodiment of the invention, said type IA diseased tau proteins have the following properties:
-
- a) the proteins are N- and C-terminally truncated (Example 6)
- b) the proteins are present in Alzheimer's diseased tissue whereas the proteins are absent in normal healthy brain (Example 6)
- c) in an in vitro microtubule assembly assay they prevent normal tau protein from promoting microtubule assembly (Examples 7)
- d) in a microtubule assembly assay using normal tau, their inhibitory activity can be eliminated by specific inhibitory, neutralising monoclonal antibodies (Example 11)
- e) their pathologic activity relies on combination of the amino acid sequence and structural conformations not present in normal healthy tau (Example 6)
- f) the proteins appear to be are conformationally different from normal tau proteins (Example 6).
- In a most preferred embodiment the present invention relates to type IA group of N- and C-terminally truncated diseased tau forms SEQ ID NO 1-3 and their “conformational region” (sequence “A”) and epitopes A1-A6.
- Type IB tau proteins differ with respect to the following properties:
-
- a) Type IB proteins are N- and C-terminally truncated (Example 6)
- b) the proteins may be present in normal healthy human brain
- c) in an in vitro microtubule assembly assay they do not prevent normal tau protein from promoting microtubule assembly (Examples 2 and 7, resp.)
- d) they do not show pathologic activity in modification of microtubule assembly (Examples 2 and 7, resp.).
- e) type IB molecules appear to be are conformationally different from normal tau (Example 6).
- Another embodiment of the present invention is the combination of the presented approach comprising various extraction methods, many of them per se known in the art, combined with functional assays with the mentioned double truncated forms of tau leading to the identification of further molecules affecting tau and microtubule functions. The yield of tau protein from brain extract may vary in functionality of extracted N- and C-terminally double truncated tau molecules depending on the staging of the particular brain tissue sample (Example 6). The person skilled in the art knows how to employ the method of the present invention for a variety of different purposes which all fall under the scope of protection of the present invention.
- In another preferred embodiment the present invention relates especially to SEQ ID NO: 1 as a prototype type IA molecule group member.
- Still another object of the invention is to provide a method for the in vitro conversion of normal tau protein into Alzheimer protein wherein tubulin is incubated with a type IA molecule of the present invention under conditions which allow the interaction of said tubulin with said type IA molecule.
- The term “allowing the interaction of type IA molecules or peptide derivatives thereof with tubulin” refers to conditions which allow the activity, preferably the optimal activity, of type IA molecules. This activity results in binding to tubulin and inhibiting its physiological function in microtubule assembly.
- In another embodiment type IA molecules could be inhibited or neutralized by derivatives thereof. As described in the present invention for screening inhibitory molecules, type IA peptides and derivatives thereof such as peptides containing deletions or mutations can be tested or screened for their effects on microtubule polymerisation.
- Normal tau protein may be derived from natural or recombinant sources. However for the purpose of carrying out the method of the present invention, it is expedient to use recombinant material.
- The methods described above specifically provide sufficient amounts of type IA N- and C-terminally double truncated tau proteins for a variety of purposes: An in vitro screening system for new inhibitors may be established which prevents the inhibition of microtubule assembly caused by pathological, double truncated tau type IA.
- An inhibitor useful in the composition of the present invention is therefore any inhibitor capable of modulating the pathological interaction of type IA molecules with tubulin. The mode of action of such an inhibitory molecule consists of an interaction with either type IA or normal tau.
- These ‘inhibitors’ may be specific for the epitope or epitopes encompassed in type IA molecules, by e.g. blocking the epitope or may be directed to various domains on type IA molecules, as long as they prevent or disturb its pathological or biological activity. The inhibitory effect can be defined quantitatively by measuring residual microtubule assembly promoting activity by normal tau. As a source of inhibitors can be used libraries of small molecules of defined chemical structure and composition, peptide libraries, antibody libraries free in the solution or displayed on the surface of synthetic surfaces of phages or bacteria or ribosomes (ribosomal display) and similar technologies known in the art.
- A further object of the invention is to provide a method for testing molecules and compounds effective in disassembling type IA complexes (type I in vitro assay) comprising the following steps:
-
- a) allowing the formation of protein complexes between type IA molecules or peptides derived thereof and tubulin or other molecules interacting with type IA molecules
- b) incubating the protein complexes with drug to be tested
- c) examining the result of the incubation of step (b) with respect to the restoration of the microtubule assembly promoting capacity of the healthy tau isoforms.
- Still another object of the invention is to provide a method for testing drugs effective in the prevention or reduction of the inhibition of normal in vitro activity of healthy tau isoforms comprising the following steps:
-
- a) A given drug to be tested in combination with type IA molecules or peptides derived thereof is expected not to interfere with normal tau and its in vitro functions.
- b) Incubation of a type IA molecule with a drug to be tested in the presence of normal tau and tubulin
- c) Examining the result of the incubation of step a) and b) with respect to the presence or absence of inhibiting activity of type IA molecules on microtubule polymerisation (Example 8).
- The term “allowing the formation of complexes between type IA molecules or peptides derived thereof and tubulin” in the absence of said drug refers to condition which allows interaction of type IA molecule with said tubulin resulting in inhibition of microtubule formation.
- The person skilled in the art knows how to employ the method of the present invention for a variety of different purposes which all fall under the scope of protection of the present invention.
- In a further aspect, the present invention relates to a method for testing drugs effective in inhibiting type IA molecules from initiating the formation of complexes in a cellular system expressing tau or tau derived proteins (type I cellular assay) comprising the following steps:
-
- a) introducing a functional gene encoding type IA molecules under the control of suitable regulatory regions into a cell expressing normal tau protein
- b) allowing the formation of protein complexes between type IA tau and tubulin molecules
- c) applying the drug to be tested to the cells harboring said complexes
- d) examining the effect of said drug on type IA biological activity such as structural and functional modifications of microtubules.
- The term ‘cell expressing tau protein’ as used in step (a), refers to cells which have the capacity to express N- and C-terminally double truncated tau forms from a gene construct encoding a type IA molecule or a derivative thereof. The person skilled in the art is aware of the fact that the sequence of experimental steps of the introduction of the genes encoding the type IA molecules is irrelevant for the purpose of the method of the invention.
- Said method is particularly advantageous since the screening system is based on the continuously growing cell lines which provide a close image of the in vivo situation. Moreover, ample supply of type IA molecules located intracellularly allows screening for drugs effective in inhibiting the biological effects of type IA molecules.
- In a preferred embodiment said cell expressing type IA molecules is a neuroblastoma, or pheochromocytoma cell or a primary culture of nerve cells derived from transgenic animal expressing type IA molecules.
- The group of molecules designated ‘type II’ consists of N- an C-terminally double truncated tau protein molecules (e.g. sequences described in SEQ ID 11-20). Representatives of this group localize intra- and extraneuronally and are functionally different from normal, healthy tau.
- The discovery and isolation of this group of proteins underlying the present invention provides (1) a molecular description and characterisation of tau modifications leading to specific microtubule binding and abnormal promotion of microtubule assembly (Example 3) with pathological consequences to its carrier (Example 4), (2) antibodies specific for the protein epitopes and (3) antibodies neutralising pathological activities of said type II molecules (Example 12), (4) methods for screening and testing therapeutic drug candidates effective in inhibiting, neutralising and eliminating said type II proteins or (5) methods screening and testing therapeutic drug candidates effective in inhibiting formation of tau derived proteins such as type II molecules, (6) the development of animal models bearing gene constructs encoding for the respective N- and C-terminally double truncated tau proteins as a transgene or transgene-combinations which can be used for drug screening (7) pharmaceutical compositions comprising inhibitors to said double truncated tau proteins and their proteases, (8) diagnostic and therapeutic compositions recognising/interacting with said molecules, (9) the development of vaccines based on said double truncated proteins (10) methods involving said proteins and their epitopes and/or antibodies or other specific probes for in vitro and in vivo diagnosis of Alzheimer's disease and other disorders related to pathological changes of tau.
- As opposed to the groups type IA and B, type IIA molecules promote pathological microtubule assembly significantly higher than microtubule assembly promoted by normal healthy tau isoforms when measured spectrophotometrically (see Examples 1 and 3, resp.). Surprisingly a subgroup of N- and C-terminally double truncated tau molecules with similar sequences and ranges of molecular weights (type IIB) are lacking these “high” microtubule polymerisation capabilities. In microtubule assembly assays, this subgroup of molecules performs to the levels seen with full length tau protein (Example 3).
- Accordingly, the present invention relates to a new type of modified tau protein found in Alzheimer's disease, called type IIA group of tau proteins. The group consist of N- and C-terminally double truncated tau molecules (SEQ ID 11-18).
- The term type II molecules refers to members of the group significantly different in structure and function not only from normal healthy tau but from type IA and -B tau group as well. Molecules of this subgroup bind microtubules and promote their pathological assembly that is significantly more pronounced than normal microtubule assembly by healthy tau isoforms (Example 3). Type IIA N- and C-terminally double truncated tau molecules interfere at the cellular level with axonal transport of constituents leading to synaptic loss and neuronal malfunction ultimately leading to cognitive impairment of the whole organism in Alzheimer's disease neurons and under experimental conditions (Examples 15 and 16, resp.). Simultaneously, afflicted neurons are vulnerable to various forms of stress such as for example oxidative stress (Example 4).
- In a preferred embodiment of the invention, said type IIA group of N- and C-terminally double truncated members comprises the amino acid sequences:
- Derivatives from four repeat tau (tau 43) are labeled R4
(69-333,R4) SEQ ID NO: 11 met val ser lys ser lys asp gly thr gly ser asp asp lys lys ala lys gly ala asp gly lys thr lys ile ala thr pro arg gly ala ala pro pro gly gln lys gly gln ala asn ala thr arg ile pro ala lys thr pro pro ala pro lys thr pro pro ser ser gly glu pro pro lys ser gly asp arg ser gly tyr ser ser pro gly ser pro gly thr pro gly ser arg ser arg thr pro ser leu pro thr pro pro thr arg glu pro lys lys val ala val val arg thr pro pro lys ser pro ser ser ala lys ser arg leu gln thr ala pro val pro met pro asp leu lys asn val lys ser lys ile gly ser thr glu asn leu lys his gln pro gly gly gly lys val gln ile ile asn lys lys leu asp leu ser asn val gln ser lys cys gly ser lys asp asn ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu (93-333,R4) SEQ ID NO: 12 ile ala thr pro arg gly ala ala pro pro gly gln lys gly gln ala asn ala thr arg ile pro ala lys thr pro pro ala pro lys thr pro pro ser ser gly glu pro pro lys ser gly asp arg ser gly tyr ser ser pro gly ser pro gly thr pro gly ser arg ser arg thr pro ser leu pro thr pro pro thr arg glu pro lys lys val ala val val arg thr pro pro lys ser pro ser ser ala lys ser arg leu gln thr ala pro val pro met pro asp leu lys asn val lys ser lys ile gly ser thr glu asn leu lys his gln pro gly gly gly lys val gln ile ile asn lys lys leu asp leu ser asn val gln ser lys cys gly ser lys asp asn ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu (69-363,R4) SEQ ID NO: 13 met val ser lys ser lys asp gly thr gly ser asp asp lys lys ala lys gly ala asp gly lys thr lys ile ala thr pro arg gly ala ala pro pro gly gln lys gly gln ala asn ala thr arg ile pro ala lys thr pro pro ala pro lys thr pro pro ser ser gly glu pro pro lys ser gly asp arg ser gly tyr ser ser pro gly ser pro gly thr pro gly ser arg ser arg thr pro ser leu pro thr pro pro thr arg glu pro lys lys val ala val val arg thr pro pro lys ser pro ser ser ala lys ser arg leu gln thr ala pro val pro met pro asp leu lys asn val lys ser lys ile gly ser thr glu asn leu lys his gln pro gly gly gly lys val gln ile ile asn lys lys leu asp leu ser asn val gln ser lys cys gly ser lys asp asn ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu ile val tyr lys ser pro val val ser gly asp thr ser pro arg his leu ser asn val ser ser thr gly ser ile asp met val asp (93-363,R4) SEQ ID NO: 14 ile ala thr pro arg gly ala ala pro pro gly gln lys gly gln ala asn ala thr arg ile pro ala lys thr pro pro ala pro lys thr pro pro ser ser gly glu pro pro lys ser gly asp arg ser gly tyr ser ser pro gly ser pro gly thr pro gly ser arg ser arg thr pro ser leu pro thr pro pro thr arg glu pro lys lys val ala val val arg thr pro pro lys ser pro ser ser ala lys ser arg leu gln thr ala pro val pro met pro asp leu lys asn val lys ser lys ile gly ser thr glu asn leu lys his gln pro gly gly gly lys val gln ile ile asn lys lys leu asp leu ser asn val gln ser lys cys gly ser lys asp asn ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu ile val tyr lys ser pro val val ser gly asp thr ser pro arg his leu ser asn val ser ser thr gly ser ile asp met val asp - Derived from three repeat tau (tau 44) are labeled R3
(93-302,R3) SEQ ID NO: 15 ile ala thr pro arg gly ala ala pro pro gly gln lys gly gln ala asn ala thr arg ile pro ala lys thr pro pro ala pro lys thr pro pro ser ser gly glu pro pro lys ser gly asp arg ser gly tyr ser ser pro gly ser pro gly thr pro gly ser arg ser arg thr pro ser leu pro thr pro pro thr arg glu pro lys lys val ala val val arg thr pro pro lys ser pro ser ser ala lys ser arg leu gln thr ala pro val pro met pro asp leu lys asn val lys ser lys ile gly ser thr glu asn leu lys his gln pro gly gly gly lys val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu (69-302,R3) SEQ ID NO: 16 met val ser lys ser lys asp gly thr gly ser asp asp lys lys ala lys gly ala asp gly lys thr lys ile ala thr pro arg gly ala ala pro pro gly gln lys gly gln ala asn ala thr arg ile pro ala lys thr pro pro ala pro lys thr pro pro ser ser gly glu pro pro lys ser gly asp arg ser gly tyr ser ser pro gly ser pro gly thr pro gly ser arg ser arg thr pro ser leu pro thr pro pro thr arg glu pro lys lys val ala val val arg thr pro pro lys ser pro ser ser ala lys ser arg leu gln thr ala pro val pro met pro asp leu lys asn val lys ser lys ile gly ser thr glu asn leu lys his gln pro gly gly gly lys val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu (93-332,R3) SEQ ID NO: 17 ile ala thr pro arg gly ala ala pro pro gly gln lys gly gln ala asn ala thr arg ile pro ala lys thr pro pro ala pro lys thr pro pro ser ser gly glu pro pro lys ser gly asp arg ser gly tyr ser ser pro gly ser pro gly thr pro gly ser arg ser arg thr pro ser leu pro thr pro pro thr arg glu pro lys lys val ala val val arg thr pro pro lys ser pro ser ser ala lys ser arg leu gln thr ala pro val pro met pro asp leu lys asn val lys ser lys ile gly ser thr glu asn leu lys his gln pro gly gly gly lys val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu ile val tyr lys ser pro val val ser gly asp thr ser pro arg his leu ser asn val ser ser thr gly ser ile asp met val asp (69-332,R3) SEQ ID NO: 18 met val ser lys ser lys asp gly thr gly ser asp asp lys lys ala lys gly ala asp gly lys thr lys ile ala thr pro arg gly ala ala pro pro gly gln lys gly gln ala asn ala thr arg ile pro ala lys thr pro pro ala pro lys thr pro pro ser ser gly glu pro pro lys ser gly asp arg ser gly tyr ser ser pro gly ser pro gly thr pro gly ser arg ser arg thr pro ser leu pro thr pro pro thr arg glu pro lys lys val ala val val arg thr pro pro lys ser pro ser ser ala lys ser arg leu gln thr ala pro val pro met pro asp leu lys asn val lys ser lys ile gly ser thr glu asn leu lys his gln pro gly gly gly lys val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu ile val tyr lys ser pro val val ser gly asp thr ser pro arg his leu ser asn val ser ser thr gly ser ile asp met val asp - In a preferred embodiment of the invention, said type II B group of N- and C-terminally double truncated members comprises the amino acid sequences:
(6-378,R4) SEQ ID NO: 19 gln glu phe glu val met glu asp his ala gly thr tyr gly leu gly asp arg lys asp gln gly gly tyr thr met his gln asp gln glu gly asp thr asp ala gly leu lys ala glu glu ala gly ile gly asp thr pro ser leu glu asp glu ala ala gly his val thr gln ala arg met val ser lys ser lys asp gly thr gly ser asp asp lys lys ala lys gly ala asp gly lys thr lys ile ala thr pro arg gly ala ala pro pro gly gln lys gly gln ala asn ala thr arg ile pro ala lys thr pro pro ala pro lys thr pro pro ser ser gly glu pro pro lys ser gly asp arg ser gly tyr ser ser pro gly ser pro gly thr pro gly ser arg ser arg thr pro ser leu pro thr pro pro thr arg glu pro lys lys val ala val val arg thr pro pro lys ser pro ser ser ala lys ser arg leu gln thr ala pro val pro met pro asp leu lys asn val lys ser lys ile gly ser thr glu asn leu lys his gln pro gly gly gly lys val gln ile ile asn lys lys leu asp leu ser asn val gln ser lys cys gly ser lys asp asn ile lys his val pro gly gly gly ser val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu ile val tyr lys ser pro val val ser gly asp thr ser pro arg his leu ser asn val ser ser thr gly ser ile asp met val asp ser pro gln leu ala thr leu ala asp glu val ser ala ser leu (6-347,R3) SEQ ID NO: 20 gln glu phe glu val met glu asp his ala gly thr tyr gly leu gly asp arg lys asp gln gly gly tyr thr met his gln asp gln glu gly asp thr asp ala gly leu lys ala glu glu ala gly ile gly asp thr pro ser leu glu asp glu ala ala gly his val thr gln ala arg met val ser lys ser lys asp gly thr gly ser asp asp lys lys ala lys gly ala asp gly lys thr lys ile ala thr pro arg gly ala ala pro pro gly gln lys gly gln ala asn ala thr arg ile pro ala lys thr pro pro ala pro lys thr pro pro ser ser gly glu pro pro lys ser gly asp arg ser gly tyr ser ser pro gly ser pro gly thr pro gly ser arg ser arg thr pro ser leu pro thr pro pro thr arg glu pro lys lys val ala val val arg thr pro pro lys ser pro ser ser ala lys ser arg leu gln thr ala pro val pro met pro asp leu lys asn val lys ser lys ile gly ser thr glu asn leu lys his gln pro gly gly gly lys val gln ile val tyr lys pro val asp leu ser lys val thr ser lys cys gly ser leu gly asn ile his his lys pro gly gly gly gln val glu val lys ser glu lys leu asp phe lys asp arg val gln ser lys ile gly ser leu asp asn ile thr his val pro gly gly gly asn lys lys ile glu thr his lys leu thr phe arg glu asn ala lys ala lys thr asp his gly ala glu ile val tyr lys ser pro val val ser gly asp thr ser pro arg his leu ser asn val ser ser thr gly ser ile asp met val asp ser pro gln leu ala thr leu ala asp glu val ser ala ser leu - In a preferred embodiment of the invention, said type IIA diseased tau proteins have the following properties:
-
- a) the proteins are N- and C-terminally truncated (Example 6)
- b) are efficient pathological promoters of microtubule assembly (Example 3;
FIG. 28C ) - c) their pathological microtubule assembly promoting activity can be removed by specific compounds such as for example inhibitory monoclonal antibodies or derivatives thereof (Example 12)
- d) the proteins are not present in normal healthy brain (Example 6)
- e) significantly impair intracellular transport functions (Example 16)
- f) their pathologic activity relies on high affinity binding to microtubular network and its functional impairment (Example 3)
- g) they appear to be are conformationally different from normal tau (Example 6).
- In another preferred embodiment of the invention type IIB molecules have the following properties:
-
- a) the proteins are N- and C-terminally truncated
- b) are less effective in promoting microtubule assembly than type IIA
- c) the proteins are not present in normal healthy brain
- d) are likely to impair microtubule function by binding to it however to a lesser extent than observed for type IIA
- e) They appear to be conformationally different from normal tau.
- In still another preferred embodiment of the invention the epitopes of type IIA and B molecules were identified in a similar way as described for type I molecules. The significance for type II molecules of all these epitopes and their relationship to function on microtubules are demonstrated by the mutant forms which showed that they are contributing at various extent to the activity of N- and C-terminally double truncated tau molecules such as shown in the example of type IA.
- An inhibitor useful in the composition of the present invention is therefore any inhibitor capable of modulating the pathological interaction of type IIA molecules with microtubules resulting in, pathological microtubules’. The term ,pathological microtubules’ as used herein refers to microtubules modified by type II molecules. The mode of action of such an inhibitory molecule consists of an interaction with either microtubules, microtubule associated molecules including tau and pathological derivatives thereof. As a source of inhibitors can be used libraries of small molecules of defined chemical structure and composition, peptide libraries, antibody libraries free in the solution or displayed on synthetic surfaces, or on phages or bacteria or ribosomes (ribosomal display) and similar technologies known in the art.
- In a preferable embodiment these ‘inhibitors’ may be specific for the epitope or epitopes encompassed in type IIA molecules, by e.g. blocking the epitope or may be directed to various domains on type IIA molecules, as long as they prevent or disturb its pathological or biological activity in vitro or in vivo. The inhibitory effect can be defined quantitatively e.g. by measuring residual microtubule assembly promoting activity by normal tau or by measuring intracellular microtubule parameters such as outgrowth, stability or intracellular transport.
- In another embodiment type IIA molecules can be inhibited or neutralized by derivatives thereof for example as dominant negative proteins expressed in the respective cell. As described in the present invention for screening inhibitory molecules, type IIA peptides and derivatives thereof such as peptides containing deletions or mutations can be tested or screened for their effects on inhibiting the pathological effects of N- and C-terminally double truncated tau molecules.
- The therapeutic effect is achieved by inhibiting impairment of microtubule structure and functions.
- Accordingly, another object of the invention is to provide pharmaceutical compositions containing a specific inhibitor for the type IIA tau molecules of the invention, optionally in combination with a pharmaceutically acceptable carrier and/or diluent.
- In another preferred embodiment the present invention relates especially to SEQ ID NO: 11 as a prototype of type IIA group molecules.
- Still another object of the invention is to provide a method for the in vitro conversion of normal microtubules into a pathological state wherein normal tau protein is incubated with type IIA or -B of the present invention under physiological conditions which allow the interaction of said type IIA or -B with microtubules generating pathological microtubules.
- The invention further relates to a screening assay allowing screening any molecule libraries for compounds capable of neutralising the pathological effects of type IIA molecules. In the present test molecules are screened for their property to eliminate and/or neutralize type IIA molecules and to restore physiological microtubule parameters and functions caused by type II molecules. The drug screening assay consists of the following steps:
-
- (1) formation of pathological microtubules in the presence of type IIA molecules and tubulin under appropriate conditions (Examples 3 and 4, resp.).
- (2) incubation of these pathological microtubules with the candidate drug to be tested
- (3) examination of the result with respect to neutralising the effect of type IIA molecules on microtubules. (Examples 9 and 12, resp.).
- An in vitro screening system for inhibitors may be established which alleviates its effect on microtubules caused by pathological, N- and C-terminally double truncated tau type IIA. These ‘inhibitors’ may be specific for the epitope or epitopes encompassed in type IIA molecules, by e.g. blocking the epitope or may be directed to various domains on type IIA molecules, as long as they prevent or disturb its activity. The inhibitory effect can be quantified by measuring microtubule assembly dynamics. As a source of inhibitors can be used libraries of small molecules of defined chemical structure and composition, peptide libraries, antibody libraries free in the solution or displayed on the surface of synthetic surfaces of phages or bacteria or ribosomes (ribosomal display) and similar technologies known in the art.
- For the object of the present invention it is sufficient that the drug to be tested is effective in reducing the amount of type IIA molecules and/or their activity, thus fulfilling a supplementary therapeutic effect, although a total removal of the type IIA activity is preferred.
- The person skilled in the art knows how to employ the method of the present invention for a variety of different purposes which all fall under the scope of protection of the present invention.
- A further object of the invention is to provide a method for the validation of drugs in living cells i.e. neurons or neurone like cells expressing type II molecules (type II Cellular assay). Alternatively primary neuronal culture derived from transgenic animals or other primary neuronal cells derived from various sources expressing type IIA molecules can be used.
- The term “neurons expressing type II molecules” as used above, refers to cells which stably express the molecules or which have the capacity to express type IIA molecules and into which a functional type IIA gene has been introduced either by cell culture techniques or via transgenesis as exemplified below.
- In a preferred embodiment said cell expressing type IIA molecules is a neuroblastoma, or pheochromocytoma cell or a primary culture of nerve cells derived from transgenic animal expressing type IIA molecules.
- The person skilled in the art is aware of the fact that the sequence of the introduction of the genes encoding the type IIA molecules is irrelevant for the purpose of the method of the invention.
- The present invention relates to a method for testing drugs effective in inhibiting type IIA in promoting abnormal microtubule formation and function in a cellular system expressing type IIA molecules comprising the following steps:
-
- a) introducing a functional gene encoding type II molecules under the control of suitable regulatory regions into a cell expressing normal tau protein
- b) allowing the formation of complexes between type IIA tau and microtubules (pathological microtubules)
- c) applying the drug to be tested to the cells harboring the resulting complexes
- d) examining the effect of said drug on type IIA biological activity such as modifications of the microtubule network and its associated functions.
- In still another most preferable embodiment of the present invention is the phenotype of neurons expressing type IIA molecules. Neurons expressing these molecules under appropriate conditions causes the perturbance of intracellular transport processes. Furthermore neurons expressing type IIA molecules undergo cell death under appropriate stress conditions (Example 4).
- Said method is particularly advantageous, since the system involved which is based on the use of continuously growing cell lines which provide a close image of the in vivo situation provide an ample supply of type IIA molecules located intracellularly is generated allowing drug screening for compounds effective in alleviation of intracellular type IIA effects.
- In a preferred embodiment the readout of this cellular assay is adapted for low- or high throughput quantification systems. The term “appropriate conditions” in connection with mentioned phenotypes leading to disruption or impairment of microtubular transport and/or to neuronal death refers to any condition which allows appearance of said phenotypes as shown in the example.
- For the object of the present invention it is sufficient that the potential drug either screened by this system, or validated in the system or drug of the third origin, is effective in the reduction of the scale of the phenotypes, thus fulfilling a supplementary function in therapy, although a total elimination or reduction of the diseased phenotypes by the drug is preferred.
- In addition to stably growing cell lines or primary cells, the respective invention can also be extended to an analogous readout system using cells derived from whole animals which express type IIA or -B molecules in their neurons (The transgenic animal model will be exemplified below).
- The person skilled in the art knows how to employ the method of the present invention for a variety of different purposes which all fall under the scope of protection of the present invention.
- In a preferred embodiment said cells and transgenic animals stably expressing N- and C-terminally double truncated type IIA tau forms allow mapping of disease pathways yielding precious information leading to new molecules relevant to pathogenesis of Alzheimer's disease, its diagnosis and treatment. These screening and identification procedures include mRNA expression based screening technologies as well as protein based technologies.
- In a preferred embodiment said type I and type IIA and -B molecules or derivatives thereof provide also a recombinant DNA construct which can be introduced into the genome of non-human animals for the purpose of providing a transgenic animal model carrying and expressing the pathogenic N- and C-terminally double truncated forms of type IA, type IIA and -B described above. Transgenic animals according to the invention include animals into which the construct has been introduced directly as well as progeny of such animals which retain the ability to express the construct. The transgene sequence is a polynucleotide sequence functionally linked to a ubiquitously expressed or otherwise to a tissue specific promoter. The transgene DNA encoding type IA and type IIA and -B molecules is preferentially cDNA and/or genomic DNA derived from either animal or human sources. Transgenic animals expressing said type I and type IIA and -B molecules are expected to develop functional changes at the cellular and/or the organ level which are phenotypically related to Alzheimer's disease. These include histological changes, RNA expression changes, changes of cellular physiological parameters and preferably behavioural changes characteristic of AD. In mature neurons of transgenic animals the expression of Type I type and IIA and -B molecules has not previously been tested. It is to expect that the level at which type I, type IIA and -B transgenes are expressed in the transgenic animal (i.e. the level of transgene mRNA), is an important parameter for obtaining consistent pathophysiological defects in the transgenic animal. By breeding and intercrossing animals carrying the transgenes, the pathological features can be enhanced, attenuated or otherwise modulated such as e.g. by introducing the transgene into animal strains currently serving as disease models, animals expressing other transgenes or animals lacking functional expression of genes (see Example 14).
- More particularly the present invention provides a transgenic non-human animal cell, wherein DNA encoding a human type I and type IIA and -B molecule is expressed under the transcriptional control of suitable ubiquitous or otherwise tissue specific promoters including regulable modifications thereof.
- Cells manipulated according to the invention may be prepared by any known transfection technique. The DNA sequence may be introduced by direct genetic manipulation or into an earlier generation of the cell. Thus, the cells may be obtained from transgenic animals and cultured in vitro. Also the transgenic animals may be generated according to well established methods, such as manipulation of embryos, e.g. by gene transfer into embryonic stem cells, retroviral infection of early embryos or pronuclear microinjection. The pronuclear microinjection technique is preferred. Transcription units obtained from a recombinant DNA construct of the invention are injected into pronuclei of animal embryos and the obtained founder transgenics are bred. The results obtained in the offspring can be analysed using various techniques well known in the art. Models based on cells and animals of the invention may be used for example to identify and assess the efficacy of potential therapeutic agents in neurodegenerative diseases where tau and N- and C-terminally double truncated tau derived molecules but also other molecules related to Alzheimer's disease such as APP and derivatives thereof can be analysed. In particular such models may be used in screening or characterisation assays for detecting agents likely to prevent the pathogenic effects of N- and C-terminally double truncated tau derived molecules described here.
- Accordingly in a further aspect the invention comprises a method for testing a potential therapeutic agent for a specified condition, in particular a neurodegenerative disease, preferably AD, wherein a cell derived from a transgenic animal expressing the said double truncated forms of tau is used as target cell. More particularly it comprises such a method, wherein the therapeutic agent such as e.g. antibodies or their derivatives is administered to a transgenic animal of the invention or introduced by crossbreeding or genetic manipulation and further tested by assay systems presented above. Moreover the invention comprises a screening or characterisation assay consisting in or including such a method, as well as a screening assay kit comprising cells of the invention. Methods for screening potential therapeutic agents using cell lines expressing type I and type IIA and -B molecules of the present invention are given in the present invention (see Example 15). The cells and animals of the present invention may be used in analogous manner.
- Another object of the invention is to provide pharmaceutical compositions containing a specific inhibitor for N- and C-terminally double truncated forms of tau proteins optionally in combination with pharmaceutically acceptable carrier and/or diluent.
- The term ‘specific inhibitor for the N- and C-terminally double truncated tau’ refers to substances which specifically inhibit the actions of said double truncated tau proteins. The nature of an inhibitor can be an antibody, an engineered, derived molecule thereof, any peptide or defined chemical composition exhibiting the desired inhibitory activity in the test systems of the present invention.
- Another object of the invention is an antibody or derivative thereof which specifically recognises an epitope of the invention and is able to partially or completely inhibit the pathological activities of N- and C-terminally double truncated tau molecules.
- The term ‘oligo- or polypeptide comprising an epitope, or epitopes of the invention’ refers to peptides which in their two- or three-dimensional structure reconstitute the epitope of the invention which is specifically recognized by an antibody directed thereto. Moreover, said oligo- or polypeptides may solely consist of the amino acids representing said epitope(s) or they may comprise additional amino acids. The construction of such oligo- or polypeptides is well known in the art.
- In a preferred embodiment the present invention relates to monoclonal antibodies and derivatives thereof either native or recombinant, immobilised, free in solution or displayed on the surface of various molecules or bacteria, viruses, or other surfaces. The antibodies and their derivatives are able to partially or completely inhibit the biologic activities of N- and C-terminally double truncated tau molecules. Such a specific antibody activity has been shown using the monoclonal antibody DC44 raised against said double truncated tau molecules isolated from Alzheimer diseased brain tissue (Examples 10 and 11, resp.).
- Said antibody(-ies) has many other variants (DC82, DC136, etc.) and may be a serum derived or a monoclonal antibody or any derivative thereof. The production of both monoclonal and polyclonal antibodies to a desired epitope is well known in the art (43). Furthermore, said antibody may be a natural or an antibody derived by genetic engineering, such as a chimeric antibody derived by techniques which are well understood in the art. Moreover, said antibody also refers to a fragment of an antibody which has retained its capacity to bind the specific epitope, such as a Fab fragment or single chain Fv minibody, or intracellularly expressed single chain antibodies called intrabodies.
- In a most preferred embodiment the present invention relates to a pharmaceutical composition for use in the treatment of Alzheimer's disease.
- Again, said pharmaceutical composition may be administered to a patient in need thereof by route and in dosage which is deemed appropriate by the physician handling the case.
- In another preferred embodiment of the present invention, said pharmaceutical composition contains as the specific inhibitor at least one monoclonal antibody or small molecule or derivative thereof binding any part or group of epitopes listed above leading to their alteration and/or neutralisation, partial or complete thereof (see Examples 10, 11 and 12, resp.).
- Another object of the invention is to provide diagnostic compositions for the detection and/or monitoring of Alzheimer's disease comprising a) an epitope(s) of the invention; b) an antibody of the invention or a derived molecule thereof.
- The diagnostic composition of the invention may comprise for example an antibody of the invention which specifically recognizes one member of type IA or type II group molecule or its epitope(s) or an enhanced level of type IA or type IIA molecules in a sample to be tested. In another embodiment, said diagnostic composition may comprise an antibody of the invention directed to one of the epitopes of the invention. Thus an Alzheimer disease state correlating sample may be detected by treating said sample with an antibody recognising the epitope of the invention. The antibody- epitope (hapten) complex may be visualized using a second antibody directed to the antibody of the invention and being labelled according to methods known in the art (43).
- In still another embodiment of the present invention, said diagnostic composition may consist of an epitope of the invention and an antibody of the invention. Treatment of a sample with said antibody may give rise to conclusions with regard to the disease state of the corresponding patent, if the binding of said antibody to said sample is brought in relation to binding of said antibody to said epitope of the invention used as a reference sample.
- In still another embodiment, the diagnostic composition may comprise type IA or type IIA molecules and an antibody of the invention. Activity of both types of molecules may be monitored with respect to normal tau neutralising capacity of the sample, compared to the recombinant type IA molecule (e.g. SEQ ID NO:1) and IIA molecules (SEQ. ID NO: 11-18) of the invention. From the quantified aberrant activity of type I molecule, the level of the molecules contained in said sample and therefore the disease state of the patient may be deduced. The type IA activity may e.g. be deduced by measuring the residual activity of normal tau left unreacted with type I molecules. Type II activity may be deduced by measuring further activity of type II molecules in a microtubule-assembly assay.
- The person skilled in the art is in the position to design other test systems which combine any of the above objects of the invention. It is to be understood that all conceivable combinations fall within the scope of protection of the present invention.
- Another object of the invention is to provide a method for the in vitro diagnosis and/or monitoring of Alzheimer's disease comprising assaying cerebrospinal fluid isolates of a patient, carrying out a biopsy of nerve tissue for the presence of N- and C-terminally double truncated tau molecules of type IA and type IIA molecule or its epitope(s) and for the level of their normal tau inhibitory activity.
- The ‘cerebrospinal fluid isolate of a patient’ is obtained by standard medical procedures.
- In a further embodiment the invention relates to type I and type II molecules that are identical or homologous to the said amino acid sequence of type IA and type IIA, respectively molecules and immunogenic fragments derived thereof capable of inducing an immune response in animals. In accordance with the present invention, it was found that both type I and type II molecules can be used (a) as immunogens for production of inhibitory antibodies and as central part of vaccines used for immunisation against the disease.
- Upon parenteral application, all sequences and epitopes listed above and type I and II isolated from diseased brain tissue are immunogenic and lead to the production of antibodies specifically directed against said type I and II proteins and derivatives thereof (Examples 10 and 13, resp.).
- In a most preferred embodiment type I and II molecules or derivatives thereof are capable of inducing an immune response directed against the primary, secondary and/or the ternary structure of said molecules. In the host, the resulting immune response is therefore capable of distinguishing between healthy and diseased forms of tau and its derivatives. This characteristic of the invention can be used as vaccine emphasising on the unique quality of these N- and C-terminally double truncated tau forms in inducing a disease-specific immune response.
- It is understood that, for the pathogenic N- and C-terminally double truncated tau polypeptides embraced herein, natural variations are existing amongst individual cases of Alzheimer's diseases. These variations may exist in (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. Such amino acid substitutions of the exemplary embodiments of this invention are within the scope of the invention. Thus, natural variations not essentially influencing the immunogenicity of the polypeptide, are considered immunologically equivalent variants of the said double truncated forms of tau polypeptides according to the invention.
- When a type IA and IIA N- and C-terminally double truncated tau polypeptide is used for e.g. vaccination purposes or for raising antibodies, it is however not necessary to use the whole polypeptide described in the present invention. It is also possible to use a fragment of these polypeptides that are capable of inducing an immune response against that entire polypeptide, a so-called immunogenic fragment.
- Therefore, this embodiment of the invention not only relates to polypeptides according to the invention, but also to derived fragments of those polypeptides that are still capable of inducing an immune response against the polypeptides (so-called immunogenic fragments).
- For the purpose of giving an example, the immunogenicity in animals of either a recombinant type IA and IIA peptide or a fraction of type IA and IIA N- and C-terminally double truncated diseased tau derived from a diseased human Alzheimer brain is given (Example 3)
- The invention is further described by the following examples and the drawing figures, yet without being restricted thereto.
-
FIG. 1 : Microtubule assembly with N- and C-terminally double truncated tau type IA and type IB molecules. -
FIG. 2 : Inhibition of microtubule assembly by N- and C-terminally double truncated tau type IA and type IB molecules. -
FIG. 3 : Activity of N- and C-terminally double truncated tau type IIA and IIB molecules in microtubule assembly. -
FIG. 4 : Type IIA N- and C-terminally double truncated tau expressed in neuronal cells significantly increases their sensitivity to oxidative stress. -
FIG. 5 : Affinity of monoclonal antibody to diseased tau type IA protein and its deletion mutants. Apparent affinity of monoclonal antibody to diseased tau type IA protein and its deletion mutants. In the first column are listed: the ‘prototype’ tau type IA protein (SEQ ID NO 1) and respective deletion mutants. In the middle column are indicated epitopes of the present invention. Apparent affinities stated in the last column were measured by competitive ELISA, and shown as the concentrations of corresponding antigen needed for 50% competition with the prototype tau type IA protein. -
FIG. 6 : Fractionation of tau proteins from AD-brain on Superdex 200-column -
FIG. 7 : Type IA inhibitory activity in fraction No.19 from three separate isolations from AD brains. -
FIG. 8 : Demonstration of N- and C-terminally double truncated tau type I molecules in AD brain. -
FIG. 9 : Presence of tau type I in AD brain but not in healthy brain. -
FIG. 10 : Immunoreactivity of N- and C-terminally double truncated tau type II molecules. -
FIG. 11 : Construction of recombinant tau type I-II (SEQ ID 1-24). -
FIG. 12 : Inhibitory effect of AD-brain derived and recombinant tau type IA on normal healthy tau. -
FIG. 13 : First round screening for drug candidates neutralizing tau type IA molecules (step 1). -
FIG. 14 : Second round screening for drug candidates neutralizing type IA molecules with selectivity against normal tau (step 2). -
FIG. 15 : First round screening for drug candidates neutralizing tau type IIA. -
FIG. 16 : Second round screening for drug candidates capable to neutralize tau type IIA molecules and discriminate them from normal tau (step 2). -
FIG. 17 : Specific antibody levels in prefused mice sera determined by ELISA. -
FIG. 18 : ELISA reactivity of monoclonal antibodies with AD-brain derived tau (fraction #19) and control healthy brain-derived tau (DC 20: monoclonal antibody with irrelevant specificity. Shown data represent mean values from three parallel experiments). -
FIG. 19 : ELISA reactivity of monoclonal antibodies with recombinant tau molecules (DC 20: monoclonal antibody with irrelevant specificity. Shown data represent mean values from three parallels). -
FIG. 20 : Screening for neutralizing antibodies directed against AD-brain derived tau type IA (fraction #19). -
FIG. 21 : Screening for neutralizing antibodies directed against recombinant tau type IA (SEQ ID NO:1). -
FIG. 22 : Screening for drug candidates capable of neutralizing tau type IA molecules and of discriminating them from healthy tau. -
FIG. 23 : Neutralisation of pathological activity of recombinant tau type IIA (SEQ ID NO.12) by monoclonal antibodies. -
FIG. 24 : Levels of antibodies against recombinant tau type IIA (SEQ ID NO.:12) detected by ELISA. -
FIG. 25 : Genotyping of transgenic animals. Panel A shows genotyping of the parental generation of transgenic animals. Specific amplification of double truncated sequence of DNA from genomic DNA inlanes lanes 1.Lane -
FIG. 26 : Gene expression of double truncated human tau transcripts in the F1 generation of transgenic animals. RNA was extracted from flash frozen tissue of transgenic animals and subjected to reverse transcription followed by specific amplification of the cDNA. An example shows transgene expressing animals inlanes number lane 5 shows a non-specific signal typically emerging in non-transgenic animal when using this method. This example indicates the presence of double truncated tau specific mRNA expressed from the transgene in experimental animals. -
FIG. 27 : Cell death caused by type IIA molecule overexpression after 6 day in vitro differentiation. Comparison of the cell viability of SY5Y cells transfected with double truncated tau type IIA (type IIA) and non-transfected control neuron-like cells (mock), respectively. -
FIG. 28 : A: Increased binding affinity of type IIA molecules to microtubules is showed by using cellular fractionation of tau from stably transfected cells expressing type IIA double truncated molecules and full-length tau. Isolation of free tau (FT), microtubule bound tau (MT) and nucleus associated tau (NAT) was performed as described. B: Inhibition of tau type IA and IIA, microbutule polymerisation assay, respectively, by organic compound F123; C: Direct comparison between absorption measurement and microtubule polymerisation shown by electromicroscopic analysis. -
FIG. 29 : Logarithmically growing SH—SY5Y cells stained with MitoFluor. Regular distribution of mitochondria in cell bodies and processes. -
FIG. 30 : Logarithmically growing tau tpe IIA molecule expressing SH—SY5Y cells stained with MitoFluor. Perinuclear clustering of green-labelled mitochondria around the centrosome area of the cell. - The physiological function of healthy tau consists in stabilizing microtubules (MTs). This function can be measured by a microtubule assembly assay (MAA). In this examples, the MAA reactions were carried out using three types of tau molecules: normal healthy human tau, recombinant forms of tau type IA (SEQ ID NO: 1) and tau type IB (SEQ ID. NO: 4). Normal human tau, tau type IA and type IB were assayed individually in separate reactions. Single preparations of tau at 0.1 mg/ml were mixed with purified porcine brain tubulin at a final concentration of 1 mg/ml and 1 mM GTP, all materials in polymerisation buffer (100 mM PIPES, pH 6.9, 1 mM MgSO4, 2 mM EGTA). Tau was added last to initiate the promotion of MT assembly. After gentle and rapid mixing, the samples were pipetted into quartz microcuvettes and equilibrated at 37° C. in a thermostatically controlled spectrophotometer (Beckman Coulter DU640). The turbidity was continuously monitored at 340 nm in 10 s intervals for a period of 20 min. The top curve 1 (
FIG. 1 ) shows microtubule assembly promotion capacity of normal healthy tau. In contrast, neither type IA (curve 2) nor type IB (curve 3) exhibited this activity of normal tau and lacked any MT assembly promotion in MAA. - Both tau type IA and IB molecules lack functional activity when applied in a the MT assembly assay (MAA). Surprisingly, tau type IA molecules show an inhibitory effect on tubulin in microtubule assembly. In contrast, type IB proteins (despite similar primary structure) do not inhibit functional activity of tubulin in MAA. For inhibition of microtubule assembly, recombinant forms of tau type IA (SEQ ID NO:1) and type IB (SEQ ID NO:4) were used. The assembly-inhibition reactions were carried out separately using type IA and type IB proteins. Human tubulin (2 mg/ml) was mixed with either type IA molecules (0.2 mg/ml) or type IB molecules (0.2 mg/ml). The mixtures were incubated 1 hr at 37° C. with gently shaking. To the mixtures kept on ice normal human tau (0.1 mg/ml) and GTP (final concentration of tubulin in the mixture is 1 mg/ml and
GTP 1 mM) were added in polymerization buffer (100 mM PIPES, pH 6.9, 1 mM MgSO4, 2 mM EGTA). After gentle and rapid mixing, the samples were pipetted into quartz microcuvettes and equilibrated at 37° C. in a thermostatically controlled spectrophotometer (Beckman Coulter DU640). The turbidity changes were measured at 340 nm in 10 sec intervals over a period of 5 min. The top curve 1 (FIG. 2 ) demonstrates that normal human tau alone was fully capable to induce tubulin polymerisation. Preincubation of tubulin with type IA abolished microtubule assembly (FIG. 2 , bottom curve 2). On the contrary, incubation of tubulin with type IB does not inhibit the microtubule assembly capacity of normal tau (FIG. 2 , curve 3), despite having molecular mass in the same range than type IA.TABLE Influence of N- and C-terminal of double truncated tau molecules on microtubule polymerisation
numbering SEQ ID NO: 11-14; 19 according to the shortest R4 isoform
numbering SEQ ID NO: 15-18; 20 according to the shortest R3 isoform
- As opposed to the group IA molecules, type-IIA double truncated tau derivatives were surprisingly found to promote pathological microtubule assembly (see
FIG. 3 andFIG. 28C ). The microtubule assembly reactions were carried out using three types of molecules: natural healthy human tau isoforms, Alz. tau type IIA (SEQ ID NO: 12) and tau type IIB (SEQ ID NO: 19). Three separate reactions were performed, each with single preparation of respective tau (healthy tau, recombinant tau type IIA or type IIB). Individual tau preparations at 0.1 mg/ml were mixed with tubulin and GTP (final concentration of tubulin is 1 mg/ml andGTP 1 mM), all reagencies in polymerisation buffer (100 mM PIPES, pH 6.9, 1 mM MgSO4, 2 mM EGTA). After gentle and rapid mixing, the samples were pipetted into quartz microcuvettes and equilibrated at 37° C. in a thermostatically controlled spectrophotometer (Beckman Coulter DU640). The turbidity changes were measured at 340 nm in 10 s intervals for a period of 5 min. In this experiment, recombinant tau type IIA exhibited extremely high (threefold) promotion of pathological microtubule assembly (FIG. 3 , top curve 1) as compared to physiological microtubule assembly by healthy tau (FIG. 3 , curve 2). In contrast, type IIB molecules despite being N- and C-terminally double truncated are not able to perform in MAA as type IIA and promote microtubule assembly only to the level seen with healthy tau (FIG. 3 , curve 3). - In the present example, the oxidative decomposition of 3-morpholinosydnonimine (SIN-1) was used which generates superoxide anions and nitric oxide, which react and thereby form peroxynitrite. This very reactive radical can further oxidize mainly cellular membrane systems. Persons skilled in art will be able to also apply another sources of oxidative stress to neuroblastoma cells in culture and will be able to obtain the same effect here described by the use on SIN-1.
- The effect of vulnerability was tested as follows:
-
- 1. SIN-1 was applied at various concentrations (0-3.32 mm) to human neuronal cell lines expressing tau type IIA protein
SEQ ID NO 15 andSEQ ID NO 11, respectively. - 2. The cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay to determine the effective concentration of hydrogen peroxide for 50% cell viability (EC50). Persons skilled in the art are aware of different ways for evaluating the cell viability for measuring the effect that is described in the present invention.
- 3. The EC50 values were compared for neuroblastoma cell lines in the presence or absence of diseased tau type IIA protein expression and the statistical significance of EC50 value differences was assessed by t-test.
- 1. SIN-1 was applied at various concentrations (0-3.32 mm) to human neuronal cell lines expressing tau type IIA protein
- The cells were grown in MEM/F12 with 10% FCS, 2 mM L-Glutamine, 1% NEAA, 50 U/L gentamicine. 3-morpholinosydnonimine (SIN-1) was diluted from 1 M stock solution in serum-free medium (e.g., 47.5 mg into 230 ml). MTT stock solution (2.6 mg/ml) was prepared in MEM/F12 w/o serum and sterilized by filtration.
- The cells were cultivated by the methods that are well known in the art. 96 well plates were seeded with 2×104 cells/well. One half of the plate was seeded with cells expressing tau type II molecules and the other half of the plate was seeded with non-expressing cells. The medium was changed every 36-48 hours. After five day, SIN-1 was added in concentrations ranging from 0 to 3.3 mM and the plates were incubated for 24 hours. Each concentration was assayed in hexaplicate. After SIN-1 incubation, MTT stock solution was added to
final concentration 200 mg/ml and the plates were incubated for another 1 hour. The medium was discarded; the surface of the plate was dried up by paper wool. 50 ml of DMSO per well were added and the plates were incubated overnight at room temperature. The absorbance at 540 nm with background correction at 690 nm was measured on ELISA reader and the background-subtracted values were used for EC50 calculation, as it is well known in the art. - The significance of differences in log EC50 concentration between neuroblastoma cells expressing type IIA protein and non-expressing said protein was tested using the t-test, the P value was for both type IIA diseased tau protein P<0.001. Expression of tau protein SEQ ID NO: 12 and SEQ ID NO:18 decreased the resistance of neuroblastoma cells to oxidative stress by 50%.
- The results of stated example (
FIG. 4 ) contributes to an explanation of the pathogenic effect of diseased form of tau protein. - The person skilled in the art is in the position to design other test systems that combine any of the above objects of the invention. It is to be understood that all conceivable combinations fall within the scope of protection of the present invention.
- The chart according to
FIG. 4 represents the decrease in relative resistance to oxidative stress of neuronal cells in the presence of tau type IIA. Resistance of cells non-harboring the said protein (control) is expressed as 100% (left bar) and resistance of neuronal cells expressing the diseased tau protein are shown as % of the control value (middle and right bar). Resistance is defined as the concentration of free radicals generated by SIN-1 in culture medium, where 50% of the cells die. The results represent measurement of double truncated tau proteins type IIA SEQ ID NO:12 (93-333, R4) and SEQ ID NO:18 (69-332, R3), respectively. - The significance of the ‘conformational region’ in tau type IA (segment A) or its parts was determined by sequential deletion either of whole conformation region (segment A) or its individual parts called epitopes and designated A1-A6. Since the conformation of type IA molecules strongly correlates with their function, the contribution of each epitope (A1-A6) to the overall conformation of the ‘segment A’ was measured on the basis of its reactivity when using a tau monoclonal antibody (
FIG. 5 ). - The prototype tau type IA (SEQ ID NO:1) has an affinity of 10 nM. Individual deletion mutants SEQ ID NO: 22, 23, 26, with deleted epitopes A1, A2 and A5, respectively, showed that the contribution of these regions is reflected in 2-4-fold decrease in affinity (20-40 nM) whereas the deletion of epitopes A3, A4, A6 in SEQ ID NO. 24, 25 and 27, respectively, contributed to greater, 10-30-fold loss of affinity (100-300 nM). Only after deletion of the entire segment A (mutant SEQ ID NO: 21), the affinity is dramatically decreased by three orders of magnitude of the affinity of prototype tau type IA.
- Preparation of Alzheimer's brain derived tau type I and type II molecules: Diseased human brain tissue from neuropathologically confirmed cases of Alzheimer's disease were used as a source for isolation of double truncated tau IA,-B and IIA proteins. Preparation of tau from Alzheimer brain is based on the combination of homogenization of tissue in TRIS buffer and fractionation of lysates by saturated ammonium sulfate precipitation. The tissue was homogenized in cold 20
mM TRIS pH 8, 0.32 mM sucrose, 10 mM b-merkaptoethanol, 5 mM EGTA, 10 mM EDTA, 5 mM MgSO4, 1 mM phenylmethylsulfonyl fluoride, 50 mM sodium fluoride, 5 mM benzamidine, 5 μg/ml leupeptin, 1.5 μg/ml pepstatin, 2 μg/ml aprotinin with Heidolph DIAX 900 homogenizer for 10 min at 4° C. The homogenate was spun at 27 000 g for 30 min at 4° C. to remove cellular debris. Tau proteins were precipitated from brain tissue supernatant by adding 44.12% (v/v) of saturated ammonium sulfate. After incubation for 20 min at 25° C. and gently mixing, the sample was centrifuged at 20 000 g for 10 min at 25° C. Pellet was resuspended in 500 μl of 100 mM PIPES pH 6.9, 2 mM EGTA, 1 mM MgSO4 and dialysed against the same buffer. This preparation was fractionated by gel filtration on a Superdex 200-column (Amersham-Pharmacia-Biotech) and the fractions were resolved by SDS-PAGE (gradient 5-20% polyacrylamide) and tau proteins were detected by immunoblotting according to standard procedure using anti tau antibodies DC25 (FIG. 6 ). Te effect of individual fractions on microtubule assembly was tested. - Isolation of tau type IA and IB: Fraction #19 (
FIG. 7 ) contains the tau molecules corresponding to the molecular mass of (12 kDa) representative of double truncated type IA and IB molecules—this fraction showed the highest inhibitory capacity. This fraction was characterized by Western blot analysis using three anti tau antibodies: DC25 recognizes both, truncated and full length proteins, DC39 (specific for intact C-terminus) and Alz50 (specific for intact N-terminus) (FIG. 8 ). The immunoreactivity of these antibodies demonstrated the lack of N- and C-terminally double truncated type I proteins only in fractions from AD-brain. Corresponding fractions prepared by the same method from normal healthy brain showed neither inhibitory activity nor specific immunoreactivity (FIG. 9 ). The concentration of tau proteins was determined by sandwich RIA. The total protein concentration was determined using the Bradford assay. Preparation of tau were stored at −20° C. until use. - Isolation of tau type IIA: Fraction #15 (
FIG. 6 ) containing the tau molecules corresponding to the molecular mass of 30 kDa is representative of double truncated type IIA molecules.Fraction # 15 showed the abnormally high microtubule assembly promoting activity. This fraction was characterized by Western blot analysis using three anti tau antibodies: DC25 recognizes both truncated and full length proteins, DC39 (specific for intact C-terminus) and Alz50 (specific for intact N-terminus) (FIG. 10 ). The immunoreactivity of these antibodies demonstrated the presence of N- and C-terminally double truncated type II proteins only in fractions derived from from AD-brain. The concentration of tau proteins was determined by sandwich RIA. Total protein concentration was determined using the Bradford assay. - Cloning, expression and purification of recombinant tau type I and type II proteins: Genes for recombinant truncated tau proteins were derived from human cDNAs for isoforms tau43 and tau44. cDNA inserts were cloned in pET17b (Novagen) vector using NdeI-EcoRI restriction sites. (
FIG. 11 ) (Studier et al., Meth. Enzym. 185 (1990), 60-89). - Recombinant N- and C-terminally double truncated tau molecules (SEQ ID 1-24) were prepared by PCR amplification of the relevant regions from cDNA. Specific primers introducing translation initiation start (ATG), stop (TGA) codons and NdeI , EcoRI restriction sites were used.
- Plasmids carrying deletion of A4-A6 epitopes (SEQ ID 25-27) in the tau cDNA were generated by inverse PCR as shown in
FIG. 11 (bottom panel). - AD-brain extracts as well as recombinant molecules of tau type IA are capable to inhibit microtubule assembly promotion when using natural healthy tau isoforms. For these experiments healthy human tau was isolated from brains of age matched controls and tau type IA was isolated from brains of AD patients (see Example 6,
FIG. 6 , fraction #19). Recombinant tau type IA (SEQ ID NO: 1) and type IB (SEQ ID NO:4, negative control) were produced and purified as shown in Example 6. In these experiments, brain-derived healthy tau isoforms (0.1 mg/ml), AD-brain derived or recombinant type IA tau or type IB (0.2 mg/ml) were mixed with tubulin. Each combination was assayed separately. The test mixtures were incubated 1 hr at 37° C. in a water bath with gently shaking. To the mixture kept on ice was added GTP and/or normal tau (final concentration of tubulin is 1 mg/ml andGTP 1 mM) all reagencies in polymerisation buffer (100 mM PIPES, pH 6.9, 1 MM MgSO4, 2 mM EGTA). After gentle and rapid mixing, the samples were pipetted into quartz microcuvettes and equilibrated at 37° C. in a thermostatically controlled spectrophotometer (Beckman Coulter DU640). The turbidity changes were measured at 340 nm in 10 s intervals for a period of 5 min. Data show that both AD brain derived as well as recombinant double truncated type IA molecules inhibit the capacity of normal tau to promote microtubule assembly (FIG. 12 ,curve 2,3). In contrast, recombinant type IB is not able to inhibit the tubulin polymerization promoting capacity of induced by normal human tau (FIG. 12 , curve 4). Curve 1 (FIG. 12 ) represents microtubule assembly promoted by normal tau. - Using the capacity of double truncated tau type IA molecules to inhibit activity of healthy normal tau to promote tubulin polymerization, a screening assay was designed for selection of compounds capable of neutralizing the inhibitory activity of type IA molecules. Diseased tau type IA can be derived from AD-brains or recombinant sources, however it is expedient to use recombinant material. The neutralizing effect of drug candidate can be defined quantitatively by measuring residual capacity of normal healthy tau to promote microtubule assembly. The assay is performed in two steps:
-
- 1. Screening for drug candidates neutralizing tau type I A. Prototype recombinant type IA molecules (SEQ. ID NO: 1) (
final concentration 100 mg/ml) mixed separately with individual drug candidates (final concentration 50 mg/ml) were preincubated for 1 hr/37° C. Following incubation, tubulin, GTP and healthy tau were added to the mixture (the final concentration: tubulin-1 mg/ml, GTP-1 mM, healthy tau −100 mg/ml) at +4° C. After rapid mixing, the samples were loaded into quartz microcuvettes and equilibrated at 37° C. in a thermostatically controlled spectrophotmeter. The turbidity changes were measured at 340 nm. Drug candidates with capacity to neutralize type IA-activity were selected by measuring residual microtubule assembly promoting potential of normal healthy tau (FIG. 13 ; a drug candidate was pre-incubated with type IA molecule and efficiency of type IA neutralization was assayed in microtubule assembly.Bottom curve 1 andtop curve 2 represent negative (no neutralization) and positive (100%) neutralizing activity of tested drug candidate against diseased type IA molecules. Middle curves indicate various efficiencies of type IA-neutralization by three different drug candidates). It is obvious that the threshold for selection of positive drugs is arbitrary and may vary from total neutralization of type IA to partial neutralization of thereof. - 2. Selection of drug candidates neutralizing type IA molecules and discriminating them from normal healthy tau. Selected candidates with neutralizing activity against tau type IA molecules were screened for reactivity with normal healthy tau to select molecules specific only for type IA. Separate mixtures of normal healthy tau (
final concentration 100 mg/ml) with individual drug candidates (final concentration 50 mg/ml) were pre-incubated 1 hr/37° C. After incubation tubulin and GTP were added to the mixtures (the final concentration:tubulin 1 mg/ml, GTP-1 mM) at +40C. Following rapid mixing, the samples were loaded into quartz microcuvettes. Turbidity changes were measured at 340 nm. Those drug candidates were selected which showed no interference with the MT polymerization promoting activity of healthy tau (FIG. 14 ).
- 1. Screening for drug candidates neutralizing tau type I A. Prototype recombinant type IA molecules (SEQ. ID NO: 1) (
- The present invention shows that tau type IIA molecules have unexpectedly high potency to promote tubulin polymerization forms a basis for a screening assay for selection of compounds neutralizing said activity of type IIA proteins. The neutralization of type IIA can be quantified by measuring residual microtubule assembly activity of type IIA molecules. The assay is performed in two steps:
-
- 1. Screening for therapeutic drug candidates neutralizing tau type IIA. The separate mixtures of tau type IIA (SEQ ID NO:12) (
final concentration 100 mg/ml) with single drug candidates (final concentration 50 mg/ml) were preincubated for 1 hr/37° C. Following incubation tubulin and GTP were added to the mixtures (the final concentration:tubulin 1 mg/ml, GTP-1 mM) at +4° C. After rapid mixing, the samples were pipetted into quartz microcuvettes and equilibrated at 37° C. in a thermostatically controlled spectrophotometer. The turbidity changes were measured at 340 nm. Drug candidates which significantly decreased microtubule assembly rate were selected for second step of the assay (FIG. 15 ; drug candidate was preincubated with type IIA molecule and efficiency of type IIA neutralization was assayed in microtubule assembly.Bottom curve 1 represents positive (100%) neutralizing activity of respective drug candidate andtop curve 2 indicates no neutralization of diseased type IIA molecules. Middle curves indicate different efficiency of various drug candidates in type IIA-neutralization). - 2. Selection of drug candidates neutralizing type IIA molecules and discriminating them from normal healthy tau. Separate mixtures of drug candidates (
final concentration 50 mg/ml) with normal healthy tau (final concentration 100 mg/ml) were preincubated for 1 hr/37° C. Then tubulin and GTP were added to the mixtures (the final concentration:tubulin 1 mg/ml, GTP-1 mM) at +4° C. After rapid mixing, the samples were pipetted into quartz microcuvettes and equilibrated at 37° C. in a thermostatically controlled spectrophotometer. The turbidity changes were measured at 340 nm. Drug candidates with no interference with healthy tau were selected (FIG. 16 ; drug candidates selected instep 1 were preincubated with healthy tau and the effect on microtubule assembly was assayed. The bottom curve (1) represents maximal inhibition of healthy tau and the top curve (2) indicates no inhibition of healthy tau. Middle curves show drug candidates with different inhibitory activity against healthy tau).
- 1. Screening for therapeutic drug candidates neutralizing tau type IIA. The separate mixtures of tau type IIA (SEQ ID NO:12) (
- Immunization protocol and fusion procedure: N- and C-terminally double truncated tau type I proteins isolated from human Alzheimer brains (
Fraction # 19, Example 6) were used as a immunogen. Balb/c mice were primed subcutaneously with said proteins (50 mg/ mouse) in complete Freund's adjuvant and boosted intraperitoneally 3 times thereafter at 4-week intervals with the 50 mg/mouse of the same proteins. Prefusion sera were collected and the level of specific antibodies against tau were tested by ELISA (FIG. 17 ; the levels of specific antibodies in sera of mice immunized with AD derived tau were tested in ELISA on the same antigen. All five sera showed high anti-tau binding activity to said tau protein.FIG. 17 represents levels of specific antibodies in one of the immunized mice. As a control was used serum from the mouse immunized with irrelevant protein). Mouse spleen cells were fused with NS/0 myeloma cells, using a modified procedure well known in the art (M. Kohler and C. Milstein, 1975). - According to the results shown in
FIG. 18 , monoclonal antibodies DC44, DC82 and DC136 recognize N- and C-terminally double truncated type IA and type IIA molecules from Alzheimer brain. For these antibodies no reactivity was observed with tau isolates from normal human brain prepared by the same method (FIG. 18 ) By contrast, monoclonal antibody DC25 reacts in ELISA with the said proteins from pathological as well as from normal healthy brain (FIG. 18 ). This antibody does not discriminate between pathological form (AD-tau) of tau and normal human tau. After this primary screening, hybridomas were subcloned in soft agarose, a technique well-know to those skilled in the art, finally resulting in homogenous hybridoma populations secreting antibodies with an identical idiotype. - These cloned hybridomas clones were further checked for reactivity to recombinant full length tau isoforms and double truncated tau type IA (SEQ ID NO: 1) and type IIA (SEQ ID NO: 12) molecules, in ELISA identical to the screening assay.
- Selected monoclonal antibodies DC44, DC82 , DC136 and DC25 were further characterized for their ability to neutralize the activity of native tau type IA isolated from Alzheimer brain (see Example 6). Said tau isolate (final concentration of 100 mg/ml) and tested antibodies (
final concentration 50 mg/ml) were preincubated for 1 hr/37° C. After incubation tubulin, normal human tau and GTP were added to the mixture (the final concentration:tubulin 1 mg/ml, healthy human tau −100 mg/ml, GTP-1 mM) at +4° C. After rapid mixing the samples were pipetted into quartz microcuvettes and equilibrated at 37° C. in a thermostatically controlled spectrophotometer. The turbidity changes were measured at 340 nm. Monoclonal antibodies DC136, DC44 and DC82 were able to inhibit the pathological activity of said protein (FIG. 20 ; antibodies were preincubated with native tau type IA (fraction #19) and subsequently mixed with healthy human tau, tubulin and GTP. The formation of microtubules was determined spectrophotometrically after 5 min at 370C. The bars represent a mean value of three independent experiments. MAA-microtubule assembly assay with healthy human tau. MAIA-microtubule assembly inhibition assay with healthy human tau preincubated with tau type IA (without antibody)). In an analogous experiment, antibodies were tested with the recombinant prototype of tau type IA (SEQ ID NO: 1), showing a similar pattern of neutralizing activity (FIG. 21 ). Control antibody DC25 recognizes all forms of truncated and normal tau tested by ELISA and Western blotting however does not interfere with AD-brain derived type IA activity in microtubule assembly assays. These results suggest that antibody DC25 reacts with the distinct region of tau comparing to the antibodies DC136, DC44 and DC82. In contrast, antibodies DC136, DC44 and DC82 bind epitope(s) involved in pathological of type IA molecules. - The next selection step was aimed at antibodies capable to discriminate between healthy and type IA molecules. Mixtures of normal healthy tau (
final concentration 100 mg/ml) and tested antibody (final concentration 50 mg/ml) were preincubated 1 hr/37° C. After incubation tubulin and GTP were added to the mixture (the final concentration:tubulin 1 mg/ml, GTP-1 mM) at +4° C. Following rapid mixing, the samples were pipetted into quartz microcuvettes and equilibrated at 37° C. in a thermostatically controlled spectrophotometer. The turbidity changes were measured at 340 nm. None of antibodies DC136, DC44, DC82 and DC25 was able to inhibit normal healthy tau in microtubule assembly (FIG. 22 ; antibodies neutralizing tau type IA were preincubated with healthy tau and subsequently mixed with tubulin and GTP. The formation of microtubules was determined spectrophotometrically after 5 min at 370C. The bars show the mean value of three independent experiments. MAA-microtubule assembly assay with healthy tau. As a negative control an antibody neutralizing healthy tau was used). The present data demonstrated that antibodies DC136, DC44 , DC82 recognize specific epitope(s) involved in interaction of truncated diseased forms of tau with healthy tau proteins. - Antibodies previously isolated for their tau type IA neutralizing activity were tested for their neutralizing activity against recombinant tau type IIA (SEQ ID NO:12) using the method described in Example 8B. All three neutralizing monoclonal antibodies DC44, DC82 and DC136 were able to reduce the pathological activity of N- and C-terminally double truncated tau type IIA molecules (
FIG. 23 ; antibodies were preincubated with recombinant tau type IIA and then mixed with tubulin and GTP. The formation of microtubules was determined spectrophotometrically after 5 min at 37° C. The bars represent the mean value of three independent experiments. MAA-microtubule assembly assay with tau type IIA (without antibody)), This suggests that the epitope(s) of said antibodies is shared at least by type I A SEQ ID NO:1 and type II A SEQ ID NO:12. For antibody DC25 no type IIA-inhibitory activity was observed. - Immunization protocol: In a preferred embodiment of the invention, said recombinant tau type IA and IIA proteins are used for vaccination purposes or for raising antibodies which specifically neutralize the pathogenic activity of diseased tau type IA and IIA molecules. In the given example recombinant N- and C-terminally double truncated tau type type IIA (SEQ ID NO: 12) was used as an immunogen. Balb/c mice were primed subcutaneously with said proteins (50 mg/ mouse) in complete Freund's adjuvant and boosted intraperitoneally 3 times thereafter at 4-week intervals with the 50 mg/mouse of the same proteins in incomplete Freund's adjuvant. Immune sera were collected and the level of specific antibodies against respective recombinant antigens tau were determined by ELISA (
FIG. 24 ). - DNA extracted from tail tips: Genomic DNA was extracted by DNeasy tissue kit, Qiagen.
- Genoptyping (
FIG. 25 ): Specific amplification of transgenes encoding double truncated tau forms was performed on genomic DNA derived from the parental generation of transgenic animals and is shown inFIG. 25A . Further analysis of genomic DNA of the F1 generation revealed that transgenes are heritable since they were also identified in the offspring of parental generation. Transgenes encoding double truncated tau are therefore fixed in chromosomal DNA of the animals (FIG. 25B —Genotyping of F1 generation). The animals used in this example are of a specific genetic background characterized by spontaneous hypertension and other Alzheimer's disease associated risk factors, such as dyslipidaemia or diabetes. This animal strain therefore represents a unique experimental Alzheimer model by combining the most frequently occurring Alzheimer's disease risk factors such as hypertension and diabetes. - For transgene generation, standard techniques of molecular biology were used as described in Sambrook et al., Molecular Cloning A Laboratory Manual, CSH Laboratory, New York (2001). cDNA encoding double truncated tau was introduced into an expression vector linked to a promoter directing an expression in ubiquitous or tissue specific manner. The gene fragment was introduced into one day embryos via pronuclear injection (non limited).
- Resulting offspring was genotyped using genomic DNA from the tail tip.
- Analysis of transgene expression (
FIG. 26 ): Expression of mRNA derived from the transgenes were assessed by RT-PCR analysis, applying generally known methods such as RT-PCR and agarose gel electrophoresis. - Panel A of
FIG. 25 shows genotyping of the parental generation of transgenic animals. Specific amplification of double truncated sequence of DNA from genomic DNA inlanes FIG. 25 : Genotyping of animals from F1 generation. Genomic DNA was extracted from tail tips and double truncated tau specific DNA sequence was identified and are shown inlanes 1.Lane -
FIG. 26 : RNA was extracted from flash frozen tissue of transgenic animals and subjected to reverse transcription followed by specific amplification of the cDNA. An example shows transgene expressing animals inlanes number lane 5 shows a non-specific signal typically emerging in non-transgenic animal when using this method. This example indicates the presence of double truncated tau specific mRNA expressed from the transgene in experimental animals. - In neuroblastoma cell line SH-SY5Y, cell death caused by type IIA molecule was demonstrated using standardized in vitro differentiation conditions known to the person skilled in the art. The effect was tested in stably transfected cells expressing type IIA double truncated tau and compared with non-transfected cells. Cell viability was quantified manually using a trypan blue exclusion assay in triplicates and statistical evaluation was performed using the One-way ANOVA test. Significant differences in cell viability between cells overexpressing type IIA double truncated tau and wild type cells were found after 6 day of in vitro differentiation (P<0.001). The over-production of type IIA double truncated tau (0.5% of the total protein amount) caused a 3×-decreased viability rate of the cells (
FIG. 27 ; comparison of the cell viability of SY5Y cells transfected with double truncated tau type IIA (type IIA) and non-transfected control neuron-like cells (mock), respectively). - In analogy to the previously shown constructions a similar system has been established using constructs encoding for double truncated type I molecules.
- Type II double truncated tau molecules show increased binding affinity to the microtubular system.
- Isolation of free tau fractions (FT), microtubule associated fractions (MT) and nuclear fractions (NAT) from stably transfected SH-SY5Y cells expressing type IIA double truncated tau and full length tau was performed. Quantification of tau association with microtubules showed an increased affinity of double truncated type IIA tau to microtubules (more than 50%) in comparison with the full-length form (
FIG. 28A ; increased binding affinity of type IIA molecules to microtubules is demonstrated by using cellular fractionation of stably transfected cells expressing type IIA double truncated molecules and full-length tau. Isolation of free tau (FT), microtubule bound tau (MT) and nucleus associated tau (NAT) was performed as described). The amount of tau was quantified according to standard cell biological fractionation methods used in the art followed by Western blot analysis. Calibration curves were calculated using recombinant tau protein with defined amounts. - B: Organic substance F123 [C34-59O14-23H32-44N6-8]n is analysed with respect to its inhibitory effect in a microtubule polymerisation assay:
-
- (1) Incubation of F123 with tau (normal tau and tau type IA′; conc=100 μg/ml; tau type IIA conc=60 μg/ml) for 1 h at 37° C.
- (2) addition of tubulin (conc=1 mg/ml)
- (3) measurement of 340 nm/5 min (remark: all dilution with PIPES buffer
- C: Normal healthy tau and Alzheimer tau type II are analysed with respect to their microtubule assembly promotion capacity. Normal tau in this example represented by
tau 43 forms typical microtubules shown in electron microscopy (seeFIG. 28C ). However, Alzheimer tau type II produces pathological microtubules with typical pattern (seeFIG. 28C ). - The pathological phenotype showing altered transport of mitochondria caused by overexpression of a type IIA molecules was performed in the neuroblastoma cell line SH-SY5Y. The influence of the N- and C-terminally double truncated tau type II molecules was examined by comparing mitochondrial redistribution in living wild type SH-SY5Y cells with transfected cells. Cell biological transport assays known to the person skilled in the art were used. In brief, cells were cultivated on LabTekII chambers (Nunc) with equal density (70% confluent) according to standard laboratory techniques and transfection was performed using Fugene 6 (Roche) according to the instructions of the manufacturer. Staining of mitochondria (MitoFluor Red 594, Molecular Probes) was performed following the instructions of the manufacturer. Living cells were examined with an Axiovert 200M fluorescence microscope (ZEISS) equipped with an 63× oil-immersion objective and fluorescence filters. Pictures were taken with a CCD camera (Photometrics, Cool snap HQ; Hamamatsu) in combination with the software program MetaMorph (Universal Imaging).
- Using the mitochondria-specific dye MitoFluor (Molecular Probes), mitochondrial localization was compared in induced and non-induced SH-SY5Y cells. The staining confirmed the negative effect of type IIA double truncated tau molecules on mitochondrial transport in SH-SY5Y cells resulting in perinuclear mitochondrial clustering near the centrosome indicative of a functional dominance of the minus end directed intracellular forces (
FIG. 30 ). - As a control, logarithmically growing cells (
FIG. 29 ) reveal a regular distribution of mitochondria in the cell body as well as in the cell periphery. In conclusion, the N- and C-terminally double truncated type IIA proteins are therefore able to influence intracellular transport mechanism which affect mitochondrial redistribution. The present-experimental setting shows a suitable method for testing inhibitory activities directed against type IIA molecules. -
- 1. Finch C, Tanzi R E, (1997) Science 278, 407-411
- 2. Blessed G, Tomlinson B E, Roth M (1968 Br J Psychiatry 114: 797-811
- 3. Tomlinson B E, Blessed G, Roth M J, (1970)
Neurol Sci 11, 205-242 - 4. Arigada P A, Growdon J H, Hedley-White E T, Hyman B T (1992) Neurology 42, 631-639
- 5. Wischik C M, Novak M, Edwards P C, Klug A, Tichelaar W, Crowther R A (1988a) Proc Natl Acad Sci USA 85: 4884-4888
- 6. Wischik C M, Novak M, Trogersen H C, Edwards P C, Runswick M J, Jake R, Walker J E, Milstein C, Roth M, Klug A (1988b) Proc Natl Acad USA 85: 4506-4510
- 7. Himmler A, Drechsel D, Kirschner M W, Martin D W jr, (1989)
Mol Cell Biol 9, 1381-1388 - 8. Goedert M, Spillantini M G, Jakes R, Rutherford D., Crowther R A (1989) Neuron #, 519-526
- 9. Hutton M, Lendon C L, Rizzu P, Baker M, Froelich S., Houlden H, Pickering-Brown S, Chackraverty S, Isaacs A, Grover A (1998) Nature 393, 702-705
- 10. Spillantini N G, Murrell J R, Goedert M, Farlow M R, Klug A, Ghetti B (1998) Proc Natl Acad Sci USA 95, 7737-7741.
- 11. Selkoe D J, (1999) Nature 399, A23-31
- 12. Isacson O, Seo H, Lin L, Albeck D, Granholm A C H, (2002)
Trends Neurosci 25, 79-84 - 13. Mudher A, Lovestone S, (2002)
Trends Neurosci 25, 22-26 - 14. Couzin J, (2002) Science 296, 28-29
- 15. Grundke-Iqbal I, Iqbal K, Tung Y-C, Quinlan M, Wisniewski H M, Binder L I, (1986) Proc Natl Acad Sci USA, 83, 4913-4917
- 16. Gustke N, Steiner B, Mandelkow E M, Biernat J, Meyer H E, Goedert M, Mandelkow E (1992) FEBS Lett 307, 199-205
- 17. Lindwall G, Cole R D (1984) J Biol Chem 259, 12241-12245
- 18. Kopke E, Tung Y-Ch, Shaikh S, Alonso A C, Iqbal K, Grundke-Iqbal I (1993)
J Biol Chem 268, 24374-24384 - 19. Cleveland D W, Hwo S Y, Kirschner M W (1977a) J Mol Biol 116: 227-247
- 20. Cleveland D W, Hwo S Y, Kirschner M W (1977b) J Mol Biol 116: 207-225
- 21. Lee M Y, Balin B J, Otvos L, Trojanowski J Q (1991) Science 251, 675-678
- 22. Alonso A C, Zaidi T, Grundke-Iqbal I, Iqbal K (1994) Proc Natl Acad Sci U S A 91:5562-5566
- 23. Wille H, Mandelkow E M, Mandelkow E (1992) J Biol Chem 267:10737-10742
- 24. Alonso A C, Grundke-Iqbal I, Iqbal K (1996) Nat Med 2:783-787
- 25. von Bergen M, Barghorn S, Li L, Marx A, Biernat J, Mandelkow E M, Mandelkow E (2001) J Biol Chem 276:48165-48174
- 26. Friedhoff P, von Bergen M, Mandelkow E M, Mandelkow E (2000) Biochim Biophys Acta 1502:122-132 Review
- 27. Illenberger S, Zheng-Fischhofer Q, Preuss U, Stamer K, Baumann K, Trinczek B, Biernat J, Godemann R, Mandelkow E M, Mandelkow E (1998) Mol Biol Cell 9:1495-1512
- 28. Kampers T, Friedhoff P, Biernat J, Mandelkow E M, Mandelkow E (1996) FEBS Lett 399: 344-349
- 29. Perez M, Valpuesta J M, Medina M, Montejo de Garcini E, Avila J (1996) J Neurochem 67: 1183-1190
- 30. Wang J-Z; Grundke-Iqbal I; Iqbal K(1996) Nature Med 2: 871-875
- 31. Wilson D M, Binder L I (1997) Am J Pathol 150: 2181-2195
- 32. Schweers O, Mandelkow E M, Biernat J, Mandelkow E (1995) Proc Natl Acad Sci USA 92: 8463-8467
- 33. Ksiezak-Reding H, Yang G, Simon M, Wall J S (1998) Brain Res. 814: 86-98
- 34. Schneider A, Biernat J, Von Bergen M, Mandelkow E, Mandelkow EM (1999) Biochemistry 38: 3549-3558
- 35 Yan S-D, Chen X, Schmid A-M, Brett J, Godman G, Zou Y-S, Scott C W, Caputo C, Frappier T, Smith M A, Perry G, Yen S H, Stern D (1994) Proc Natl Acad Sci USA 91: 7787-7791
- 36. Smith M A, Taneda S, Richey P L, Miyata S, Yan S D, Stern D, Sayre L M, Monnier V M, Perry G (1994) Proc Natl Acad Sci U S A 91:5710-5714
- 37. Mori H, Kondo J, Ihara Y (1987) Science 235: 1641-1644
- 38. Paudel H, Li W (1999). J Biol Chem 274: 8029-8038.
- 39.Goedert M, Spillantini M G, Potier M C, Ulrich J, Crowther R A, (1989) EMBO J. 8, 393
- 40. Goedert M, Spillantini M G, Jakes R, Rutherford D, Crowther R A (1989)
Neuron 4, 519 - 41. Crowther R A, Olesen O F, Jakes R, Goedert M (1992) FEBS Lett 309:199-202
- 42. Crowther R A, Olesen O F, Smith M J, Jakes R, Goedert M (1994) FEBS Letter 337: 135-138
- 43. (see e.g. M. Kohler and C. Milstein, in “Continuous Cultures of Fused Cells Secreting Antibody of Pre-Defined Specificity”, Nature, 256, pp. 495-497, 1975; and Harlow and Lane, in “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988)
Claims (12)
1-23. (canceled)
24. An N- and C-terminally double truncated tau molecule further defined as a type IA tau molecule, type IB tau molecule, type IIA tau molecule, or type IItau molecule.
25. The N- and C-terminally double truncated tau molecule of claim 24 , further defined as a type IA tau molecule:
having at least the first 236 N-terminal amino acids and at least the last 45 C-terminal amino acids of the 4 repeat containing tau43 truncated;
detectable in Alzheimer's diseased brain tissue but not detectable in normal healthy brain tissue; and
preventing normal tau protein from promoting microtubule assembly in an in vitro microtubule assembly assay, wherein prevention of the promotion of microtubule assembly can be eliminated by specific inhibitory, neutralizing monoclonal antibodies against the molecules in a microtubule assembly assay.
26. The type IA tau molecule of claim 25 , further defined as comprising an amino acid sequence of any of SEQ ID NO: 1 to 3.
27. The N- and C-terminally double truncated tau molecule of claim 24 , further defined as a type IB tau molecule:
having at least the first 238 N-terminal amino acids and at least the last 40 C-terminal amino acids of the 4 repeat containing tau43 or the first 207 N-terminal amino acids and at least the last 50 C-terminal amino acids of the 3 repeat containing tau44 truncated;
detectable in Alzheimer's diseased brain tissue whereas the molecules are not detectable in normal healthy brain tissue; and
not capable of preventing wild type tau from promoting microtubule assembly in an in vitro microtubule assembly assay.
28. The type IB tau molecule of claim 27 , further defined as comprising an amino acid sequence of any of SEQ ID NO: 4 to 10.
29. The N- and C-terminally double truncated tau molecule of claim 24 , further defined as a type IIA tau molecule:
having at least the first 68 N-terminal amino acids and at least the last 40 C-terminal amino acids of the 4 repeat containing tau43 or the first 68 N-terminal amino acids and at least the last 20 C-terminal amino acids of the 3 repeat containing tau44 truncated;
detectable in Alzheimer's diseased brain tissue, whereas the molecules are not detectable in normal healthy brain tissue;
having higher microtubule assembly promoting activity than wild type tau in an in vitro microtubule assembly assay, wherein the microtubule assembly promoting activity can be eliminated by specific inhibitory, neutralizing monoclonal antibodies against the molecules in a microtubule assembly assay; and
wherein pathologic activity of the molecule relies on binding to the microtubular network defined by the microtubule polymerization promoting activity.
30. The type IIA tau molecule of claim 29 , further defined as comprising the amino acid sequence of any of SEQ ID NO: 11 to 18.
31. The N- and C-terminally double truncated tau molecule of claim 24 , further defined as a type IIB tau molecule:
having at least the first 68 N-terminal amino acids and at least the last 40 C-terminal amino acids of the 4 repeat containing tau43 or the first 68 N-terminal amino acids and at least the last 20 C-terminal amino acids of the 3 repeat containing tau44 truncated;
detectable in Alzheimer's diseased brain tissue, whereas the molecules are not detectable in normal healthy brain tissue; and
having a pathological microtubule assembly promoting activity different from wild type tau in an in vitro microtubule assembly assay.
32. The type IIB tau molecule of claim 31 , further defined as comprising the amino acid sequence of any of SEQ ID NO: 19 to 20.
33. A transgenic animal expressing a molecule of claim 24 .
34. A method of screening or testing a candidate compound for utility in the treatment of Alzheimer's disease comprising obtaining a transgenic animal according to claim 33 and using the animal to screen or test the candidate compound.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/574,414 US9485972B2 (en) | 2002-07-12 | 2009-10-06 | Truncated tau proteins |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AT10532002 | 2002-07-12 | ||
PCT/EP2003/007389 WO2004007547A2 (en) | 2002-07-12 | 2003-07-09 | Truncated tau proteins |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/574,414 Division US9485972B2 (en) | 2002-07-12 | 2009-10-06 | Truncated tau proteins |
Publications (1)
Publication Number | Publication Date |
---|---|
US20060167227A1 true US20060167227A1 (en) | 2006-07-27 |
Family
ID=30120907
Family Applications (5)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/521,140 Abandoned US20060167227A1 (en) | 2002-07-12 | 2003-07-09 | Truncated tau proteins |
US10/521,049 Expired - Lifetime US8288608B2 (en) | 2002-07-12 | 2003-07-09 | Transgenic animal expressing alzheimer's tau protein |
US12/574,414 Active 2025-05-08 US9485972B2 (en) | 2002-07-12 | 2009-10-06 | Truncated tau proteins |
US13/618,946 Expired - Fee Related US9161520B2 (en) | 2002-07-12 | 2012-09-14 | Transgenic animal expressing Alzheimer's tau protein |
US14/850,683 Abandoned US20160106077A1 (en) | 2002-07-12 | 2015-09-10 | Transgenic Animal Expressing Alzheimer's Tau Protein |
Family Applications After (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/521,049 Expired - Lifetime US8288608B2 (en) | 2002-07-12 | 2003-07-09 | Transgenic animal expressing alzheimer's tau protein |
US12/574,414 Active 2025-05-08 US9485972B2 (en) | 2002-07-12 | 2009-10-06 | Truncated tau proteins |
US13/618,946 Expired - Fee Related US9161520B2 (en) | 2002-07-12 | 2012-09-14 | Transgenic animal expressing Alzheimer's tau protein |
US14/850,683 Abandoned US20160106077A1 (en) | 2002-07-12 | 2015-09-10 | Transgenic Animal Expressing Alzheimer's Tau Protein |
Country Status (10)
Country | Link |
---|---|
US (5) | US20060167227A1 (en) |
EP (3) | EP1521774B1 (en) |
JP (3) | JP4414332B2 (en) |
CN (2) | CN100572392C (en) |
AT (2) | ATE391781T1 (en) |
AU (2) | AU2003246664B2 (en) |
DE (2) | DE60320258T2 (en) |
DK (2) | DK1521774T3 (en) |
ES (2) | ES2304146T3 (en) |
WO (2) | WO2004007547A2 (en) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8703137B2 (en) | 2011-01-31 | 2014-04-22 | Intellect Neurosciences Inc. | Treatment of tauopathies |
US8926974B2 (en) | 2012-08-16 | 2015-01-06 | Ipierian, Inc. | Methods of treating a tauopathy |
US20150050215A1 (en) * | 2011-09-19 | 2015-02-19 | Axon Neuroscience Se | Protein-based therapy and diagnosis of tau-mediated pathology in alzheimer's disease |
US8980270B2 (en) | 2013-01-18 | 2015-03-17 | Ipierian, Inc. | Methods of treating a tauopathy |
US9834596B2 (en) | 2012-07-03 | 2017-12-05 | Washington University | Antibodies to tau |
US9957317B2 (en) | 2014-06-27 | 2018-05-01 | C2N Diagnostics, Llc | Humanized anti-tau antibodies |
US10400018B2 (en) | 2014-02-14 | 2019-09-03 | Ipierian, Inc. | Tau peptides, anti-tau antibodies, and methods of use thereof |
US11155609B2 (en) | 2019-04-05 | 2021-10-26 | TauC3 Biologies Limited | Anti-TAUC3 antibodies and uses thereof |
Families Citing this family (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100572392C (en) * | 2002-07-12 | 2009-12-23 | 阿克松神经科学研究和发展股份有限公司 | The brachymemma Protein tau |
DE10303974A1 (en) | 2003-01-31 | 2004-08-05 | Abbott Gmbh & Co. Kg | Amyloid β (1-42) oligomers, process for their preparation and their use |
US7609947B2 (en) | 2004-09-10 | 2009-10-27 | Panasonic Corporation | Method and apparatus for coordinating playback from multiple video sources |
KR20180058863A (en) | 2005-11-30 | 2018-06-01 | 애브비 인코포레이티드 | Monoclonal antibodies against amyloid beta protein and uses thereof |
US8691224B2 (en) | 2005-11-30 | 2014-04-08 | Abbvie Inc. | Anti-Aβ globulomer 5F7 antibodies |
KR100734815B1 (en) | 2006-02-15 | 2007-07-09 | 대한민국 | 24 Transgenic dementia mice expressing htau24 and the method for preparing thereof |
US8455626B2 (en) | 2006-11-30 | 2013-06-04 | Abbott Laboratories | Aβ conformer selective anti-aβ globulomer monoclonal antibodies |
US20100311767A1 (en) | 2007-02-27 | 2010-12-09 | Abbott Gmbh & Co. Kg | Method for the treatment of amyloidoses |
US8987419B2 (en) | 2010-04-15 | 2015-03-24 | AbbVie Deutschland GmbH & Co. KG | Amyloid-beta binding proteins |
CN103298833B (en) | 2010-08-14 | 2015-12-16 | Abbvie公司 | Amyloid beta associated proteins |
CN102058619A (en) * | 2010-12-08 | 2011-05-18 | 山西医科大学 | Preparation method of composite animal model with Alzheimer disease |
CN103476945A (en) * | 2010-12-31 | 2013-12-25 | 宙斯科技公司 | Improved methods for determining cell viability using molecular nucleic acid-based techniques |
US9506051B2 (en) | 2011-05-20 | 2016-11-29 | Oligomerix, Inc. | Tau protease compositions and methods of use |
US9200068B2 (en) | 2012-12-18 | 2015-12-01 | Regents Of The University Of Minnesota | Compositions and methods related to tauopathy |
WO2015165961A1 (en) | 2014-04-29 | 2015-11-05 | Affiris Ag | Treatment and prevention of alzheimer's disease (ad) |
EP3221349B1 (en) | 2014-11-19 | 2020-11-04 | Axon Neuroscience SE | Humanized tau antibodies in alzheimer's disease |
MA41586A (en) * | 2015-02-04 | 2017-12-13 | Hoffmann La Roche | ANTISENSE OLIGOMERS OF TAU PROTEIN AND THEIR USES |
WO2017172764A1 (en) * | 2016-04-01 | 2017-10-05 | The Regents Of The University Of California | Modified cell line and method of determining tauopathies |
KR101997319B1 (en) | 2016-06-21 | 2019-07-08 | 전남대학교산학협력단 | Manufacturing and Applications of flagellin-Adjuvanted Vaccine which induces Conformer recognizing Antibodies |
CA3047132A1 (en) * | 2016-12-30 | 2018-07-05 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Methods for the detection of tau protein aggregates |
CA3057289A1 (en) * | 2017-03-21 | 2018-09-27 | The Jackson Laboratory | A genetically modified mouse expressing human apoe4 and mouse trem2 p.r47h and methods of use thereof |
CA3095443A1 (en) | 2018-03-28 | 2019-10-03 | Axon Neuroscience Se | Antibody-based methods of detecting and treating alzheimer's disease |
WO2021024209A1 (en) | 2019-08-06 | 2021-02-11 | Aprinoia Therapeutics Inc. | Antibodies that bind to pathological tau species and uses thereof |
CN110679549B (en) * | 2019-11-05 | 2021-08-20 | 南通大学 | Construction method of Alzheimer disease mouse model |
TW202328449A (en) * | 2021-09-24 | 2023-07-16 | 美商艾拉倫製藥公司 | Microtubule associated protein tau (mapt) irna agent compositions and methods of use thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020010947A1 (en) * | 2000-01-21 | 2002-01-24 | Gurney Mark E. | Transgenic mouse model of human neurodegenerative disease |
US20060112437A1 (en) * | 2002-07-12 | 2006-05-25 | Eva Kontsekova | Transgenic animal expressing alzheimer's tau protein |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06239899A (en) | 1993-02-12 | 1994-08-30 | Teijin Ltd | Antibody for human tau protein and determination of human tau protein in body fluid utilizing the same |
GB9506197D0 (en) * | 1995-03-27 | 1995-05-17 | Hoffmann La Roche | Inhibition of tau-tau association. |
WO1999062548A1 (en) | 1998-06-01 | 1999-12-09 | Advanced Research And Technology Institute | Methods and compositions for diagnosing tauopathies |
NZ515956A (en) * | 1999-07-02 | 2004-04-30 | Janssen Pharmaceutica Nv | Transgenic animals as models for neurodegenerative disease |
JP2003508788A (en) * | 1999-09-09 | 2003-03-04 | マックス−プランク−ゲゼルシャフト ツール フォルデルング デル ヴィッセンシャフテン エー.ファウ. | Minimal tau peptide for nucleation of paired helical filaments |
AU2001241849A1 (en) * | 2000-02-29 | 2001-09-12 | Karen Duff | Transgenic mice comprising a genomic human tau transgene |
GB0100119D0 (en) * | 2001-01-03 | 2001-02-14 | Univ Aberdeen | Materials and methods relating to protein aggregation in neurodegenerative disease |
GB0101049D0 (en) * | 2001-01-15 | 2001-02-28 | Univ Aberdeen | Materials and methods relating to protein aggregation in neurodegenerative disease |
AT500379B8 (en) * | 2001-02-02 | 2009-08-15 | Axon Neuroscience | TAU PROTEINS |
US20020164657A1 (en) * | 2001-02-23 | 2002-11-07 | Sharma Satish K. | Assays for assessing A beta-Tau aggregation |
-
2003
- 2003-07-09 CN CNB03816647XA patent/CN100572392C/en not_active Expired - Lifetime
- 2003-07-09 EP EP03763763A patent/EP1521774B1/en not_active Expired - Lifetime
- 2003-07-09 ES ES03763764T patent/ES2304146T3/en not_active Expired - Lifetime
- 2003-07-09 AU AU2003246664A patent/AU2003246664B2/en not_active Expired
- 2003-07-09 CN CN03816645A patent/CN100577803C/en not_active Expired - Lifetime
- 2003-07-09 JP JP2004520542A patent/JP4414332B2/en not_active Expired - Lifetime
- 2003-07-09 EP EP03763764A patent/EP1521831B1/en not_active Expired - Lifetime
- 2003-07-09 DE DE60320258T patent/DE60320258T2/en not_active Expired - Lifetime
- 2003-07-09 DK DK03763763T patent/DK1521774T3/en active
- 2003-07-09 AT AT03763764T patent/ATE391781T1/en active
- 2003-07-09 JP JP2004520541A patent/JP4308760B2/en not_active Expired - Fee Related
- 2003-07-09 EP EP08014706A patent/EP1995255A1/en not_active Withdrawn
- 2003-07-09 WO PCT/EP2003/007389 patent/WO2004007547A2/en active IP Right Grant
- 2003-07-09 DK DK03763764T patent/DK1521831T3/en active
- 2003-07-09 AT AT03763763T patent/ATE406383T1/en active
- 2003-07-09 US US10/521,140 patent/US20060167227A1/en not_active Abandoned
- 2003-07-09 AU AU2003253044A patent/AU2003253044B2/en not_active Expired
- 2003-07-09 DE DE60323231T patent/DE60323231D1/en not_active Expired - Lifetime
- 2003-07-09 WO PCT/EP2003/007390 patent/WO2004007722A2/en active IP Right Grant
- 2003-07-09 US US10/521,049 patent/US8288608B2/en not_active Expired - Lifetime
- 2003-07-09 ES ES03763763T patent/ES2311734T3/en not_active Expired - Lifetime
-
2008
- 2008-12-02 JP JP2008307564A patent/JP2009143906A/en active Pending
-
2009
- 2009-10-06 US US12/574,414 patent/US9485972B2/en active Active
-
2012
- 2012-09-14 US US13/618,946 patent/US9161520B2/en not_active Expired - Fee Related
-
2015
- 2015-09-10 US US14/850,683 patent/US20160106077A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020010947A1 (en) * | 2000-01-21 | 2002-01-24 | Gurney Mark E. | Transgenic mouse model of human neurodegenerative disease |
US20060112437A1 (en) * | 2002-07-12 | 2006-05-25 | Eva Kontsekova | Transgenic animal expressing alzheimer's tau protein |
Cited By (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8703137B2 (en) | 2011-01-31 | 2014-04-22 | Intellect Neurosciences Inc. | Treatment of tauopathies |
US11673944B2 (en) | 2011-01-31 | 2023-06-13 | Tauc3 Biologics Limited | Treatment of tauopathies |
US10894822B2 (en) | 2011-01-31 | 2021-01-19 | Tauc3 Biologics Limited | Treatment of tauopathies |
US20180282401A1 (en) * | 2011-09-19 | 2018-10-04 | Axon Neuroscience Se | Protein-based therapy and diagnosis of tau-mediated pathology in alzheimer's disease |
US9828421B2 (en) | 2011-09-19 | 2017-11-28 | Axon Neuroscience Se | Protein-based therapy and diagnosis of tau-mediated pathology in Alzheimer's disease |
US11098106B2 (en) | 2011-09-19 | 2021-08-24 | Axon Neuroscience Se | Protein-based therapy and diagnosis of tau-mediated pathology in alzheimer's disease |
US20150050215A1 (en) * | 2011-09-19 | 2015-02-19 | Axon Neuroscience Se | Protein-based therapy and diagnosis of tau-mediated pathology in alzheimer's disease |
US9518101B2 (en) * | 2011-09-19 | 2016-12-13 | Axon Neuroscience Se | Protein-based therapy and diagnosis of tau-mediated pathology in alzheimer's disease |
US9845352B2 (en) | 2011-09-19 | 2017-12-19 | Axon Neuroscience Se | Protein-based therapy and diagnosis of tau-mediated pathology in Alzheimer's disease |
US9834596B2 (en) | 2012-07-03 | 2017-12-05 | Washington University | Antibodies to tau |
US9567395B2 (en) | 2012-08-16 | 2017-02-14 | Ipierian, Inc. | Methods of treating a tauopathy |
US10040847B2 (en) | 2012-08-16 | 2018-08-07 | Ipierian, Inc. | Methods of treating a tauopathy |
US8926974B2 (en) | 2012-08-16 | 2015-01-06 | Ipierian, Inc. | Methods of treating a tauopathy |
US9777058B2 (en) | 2013-01-18 | 2017-10-03 | Ipierian, Inc. | Methods of treating a tauopathy |
US8980271B2 (en) | 2013-01-18 | 2015-03-17 | Ipierian, Inc. | Methods of treating a tauopathy |
US8980270B2 (en) | 2013-01-18 | 2015-03-17 | Ipierian, Inc. | Methods of treating a tauopathy |
US9447180B2 (en) | 2013-01-18 | 2016-09-20 | Ipierian, Inc. | Methods of treating a tauopathy |
US9051367B2 (en) | 2013-01-18 | 2015-06-09 | Ipierian, Inc. | Methods of treating a tauopathy |
US10400018B2 (en) | 2014-02-14 | 2019-09-03 | Ipierian, Inc. | Tau peptides, anti-tau antibodies, and methods of use thereof |
US9957317B2 (en) | 2014-06-27 | 2018-05-01 | C2N Diagnostics, Llc | Humanized anti-tau antibodies |
US11155609B2 (en) | 2019-04-05 | 2021-10-26 | TauC3 Biologies Limited | Anti-TAUC3 antibodies and uses thereof |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9485972B2 (en) | Truncated tau proteins | |
AU732508B2 (en) | Nucleic acids and proteins related to Alzheimer's disease, and uses therefor | |
Peng et al. | Decreased brain-derived neurotrophic factor depends on amyloid aggregation state in transgenic mouse models of Alzheimer's disease | |
CZ339097A3 (en) | Genetic sequences and proteins concerning alzheimer's disease and their use | |
Agca et al. | Development of transgenic rats producing human β-amyloid precursor protein as a model for Alzheimer's disease: Transgene and endogenous APP genes are regulated tissue-specifically | |
US6670195B1 (en) | Mutant genes in Familial British Dementia and Familial Danish Dementia | |
US20020010947A1 (en) | Transgenic mouse model of human neurodegenerative disease | |
Horie et al. | Characterization of novel dystonia musculorum mutant mice: implications for central nervous system abnormality | |
EP1516930A1 (en) | Cellular model of tauopathies for lead identification and drug discovery | |
JP2000516087A (en) | Gene sequences and proteins associated with Alzheimer's disease and uses thereof | |
JP4824902B2 (en) | Double transgenic animals with Alzheimer's disease | |
US7135610B2 (en) | Mouse showing neurofibril change due to senile dementia | |
Kim et al. | Calsyntenin-3 directly interacts with neurexins to orchestrate excitatory synapse development in the hippocampus | |
JP6323876B2 (en) | Knock-in mouse | |
JP2002543767A (en) | Non-human transgenic animals whose germ cells and somatic cells contain a knockout mutation in the DNA encoding 4E-BP1 | |
Armbrust | Murine models of spinocerebellar ataxia type 5 | |
CA2244412A1 (en) | Nucleic acids and proteins related to alzheimer's disease, and uses therefor |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: AXON NEUROSCIENCE FORSCHUNGS-UND ENTWICKLUNGS GMBH Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:KONTSEKOVA, EVA;REEL/FRAME:017167/0418 Effective date: 20041221 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |
|
AS | Assignment |
Owner name: AXON NEUROSCIENCE SE, SLOVAKIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:AXON NEUROSCIENCE FORSCHUNGS- UND ENTWICKLUNGS GMBH;REEL/FRAME:028848/0956 Effective date: 20120101 |