WO2016172955A1 - 一种筛选用于治疗阿尔茨海默病的药物和治疗靶点的方法 - Google Patents
一种筛选用于治疗阿尔茨海默病的药物和治疗靶点的方法 Download PDFInfo
- Publication number
- WO2016172955A1 WO2016172955A1 PCT/CN2015/078070 CN2015078070W WO2016172955A1 WO 2016172955 A1 WO2016172955 A1 WO 2016172955A1 CN 2015078070 W CN2015078070 W CN 2015078070W WO 2016172955 A1 WO2016172955 A1 WO 2016172955A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- rbo
- protein
- pi4kiiiα
- drosophila
- gene
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims description 64
- 208000024827 Alzheimer disease Diseases 0.000 title claims description 62
- 239000003814 drug Substances 0.000 title claims description 41
- 229940079593 drug Drugs 0.000 title claims description 37
- 230000001225 therapeutic effect Effects 0.000 title claims description 35
- 238000012216 screening Methods 0.000 title claims description 23
- 230000007466 Aβ secretion Effects 0.000 claims abstract description 35
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 claims description 105
- 230000028327 secretion Effects 0.000 claims description 21
- 238000002965 ELISA Methods 0.000 claims description 20
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 16
- 230000001965 increasing effect Effects 0.000 claims description 14
- 101000823051 Homo sapiens Amyloid-beta precursor protein Proteins 0.000 claims description 11
- 102000046783 human APP Human genes 0.000 claims description 11
- 230000002018 overexpression Effects 0.000 claims description 7
- 229940000406 drug candidate Drugs 0.000 claims description 5
- 238000003018 immunoassay Methods 0.000 claims description 5
- 230000003248 secreting effect Effects 0.000 claims description 5
- 238000011984 electrochemiluminescence immunoassay Methods 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 142
- 102000004169 proteins and genes Human genes 0.000 abstract description 92
- 230000000694 effects Effects 0.000 abstract description 66
- 241000255588 Tephritidae Species 0.000 abstract description 52
- 210000002569 neuron Anatomy 0.000 abstract description 51
- 101150030426 efr3 gene Proteins 0.000 abstract description 40
- 101000845180 Homo sapiens Tetratricopeptide repeat protein 7A Proteins 0.000 abstract description 35
- 102100031282 Tetratricopeptide repeat protein 7A Human genes 0.000 abstract description 35
- 101000925095 Homo sapiens Protein EFR3 homolog A Proteins 0.000 abstract description 30
- 101000925087 Homo sapiens Protein EFR3 homolog B Proteins 0.000 abstract description 30
- 102100033963 Protein EFR3 homolog A Human genes 0.000 abstract description 30
- 102100033970 Protein EFR3 homolog B Human genes 0.000 abstract description 30
- 239000003112 inhibitor Substances 0.000 abstract description 26
- 102000004190 Enzymes Human genes 0.000 abstract description 15
- 108090000790 Enzymes Proteins 0.000 abstract description 15
- 108010052285 Membrane Proteins Proteins 0.000 abstract description 14
- 102000018697 Membrane Proteins Human genes 0.000 abstract description 11
- 230000007082 Aβ accumulation Effects 0.000 abstract description 9
- 230000002068 genetic effect Effects 0.000 abstract description 6
- 230000004064 dysfunction Effects 0.000 abstract description 3
- 210000005036 nerve Anatomy 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 73
- BQVCCPGCDUSGOE-UHFFFAOYSA-N phenylarsine oxide Chemical compound O=[As]C1=CC=CC=C1 BQVCCPGCDUSGOE-UHFFFAOYSA-N 0.000 description 61
- 229920013639 polyalphaolefin Polymers 0.000 description 58
- 230000014509 gene expression Effects 0.000 description 51
- 210000000170 cell membrane Anatomy 0.000 description 48
- 238000010175 APPswe/PSEN1dE9 Methods 0.000 description 35
- 241000699670 Mus sp. Species 0.000 description 32
- 230000002401 inhibitory effect Effects 0.000 description 32
- 239000012528 membrane Substances 0.000 description 29
- 230000035772 mutation Effects 0.000 description 28
- 238000011282 treatment Methods 0.000 description 28
- 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 27
- 102000002659 Amyloid Precursor Protein Secretases Human genes 0.000 description 26
- 108010043324 Amyloid Precursor Protein Secretases Proteins 0.000 description 26
- 101710137189 Amyloid-beta A4 protein Proteins 0.000 description 26
- 101710151993 Amyloid-beta precursor protein Proteins 0.000 description 26
- 102100022704 Amyloid-beta precursor protein Human genes 0.000 description 26
- 230000035508 accumulation Effects 0.000 description 25
- 238000009825 accumulation Methods 0.000 description 25
- 239000000126 substance Substances 0.000 description 24
- 238000012360 testing method Methods 0.000 description 21
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 18
- 241000255925 Diptera Species 0.000 description 18
- 108010001515 Galectin 4 Proteins 0.000 description 18
- 102100039556 Galectin-4 Human genes 0.000 description 18
- 206010064571 Gene mutation Diseases 0.000 description 18
- 101150023392 Pi4ka gene Proteins 0.000 description 18
- 210000004556 brain Anatomy 0.000 description 18
- 230000006870 function Effects 0.000 description 18
- 230000013016 learning Effects 0.000 description 18
- 230000005062 synaptic transmission Effects 0.000 description 18
- 230000003828 downregulation Effects 0.000 description 17
- 238000003197 gene knockdown Methods 0.000 description 17
- 241000699666 Mus <mouse, genus> Species 0.000 description 16
- 230000015572 biosynthetic process Effects 0.000 description 15
- 238000002474 experimental method Methods 0.000 description 15
- -1 small molecule compound Chemical class 0.000 description 15
- 108700019146 Transgenes Proteins 0.000 description 14
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 14
- 238000001514 detection method Methods 0.000 description 14
- 230000003834 intracellular effect Effects 0.000 description 14
- 239000002773 nucleotide Substances 0.000 description 14
- 125000003729 nucleotide group Chemical group 0.000 description 14
- 230000002829 reductive effect Effects 0.000 description 14
- 238000013518 transcription Methods 0.000 description 14
- 230000035897 transcription Effects 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 13
- 239000002609 medium Substances 0.000 description 13
- 101150115776 Efr3a gene Proteins 0.000 description 12
- 101100171882 Mus musculus Efr3a gene Proteins 0.000 description 12
- 150000001413 amino acids Chemical class 0.000 description 12
- 238000003119 immunoblot Methods 0.000 description 12
- 239000000872 buffer Substances 0.000 description 11
- 230000007423 decrease Effects 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 230000005764 inhibitory process Effects 0.000 description 11
- 239000002502 liposome Substances 0.000 description 11
- 230000037361 pathway Effects 0.000 description 11
- 229920001184 polypeptide Polymers 0.000 description 11
- 108090000765 processed proteins & peptides Proteins 0.000 description 11
- 102000004196 processed proteins & peptides Human genes 0.000 description 11
- 230000001737 promoting effect Effects 0.000 description 11
- 230000001419 dependent effect Effects 0.000 description 10
- 230000003993 interaction Effects 0.000 description 10
- 229910052751 metal Inorganic materials 0.000 description 10
- 239000002184 metal Substances 0.000 description 10
- 229920000642 polymer Polymers 0.000 description 10
- 230000001105 regulatory effect Effects 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 101150095590 Efr3b gene Proteins 0.000 description 9
- 101100171886 Mus musculus Efr3b gene Proteins 0.000 description 9
- 230000027455 binding Effects 0.000 description 9
- 239000012634 fragment Substances 0.000 description 9
- 230000004770 neurodegeneration Effects 0.000 description 9
- 230000009467 reduction Effects 0.000 description 9
- 230000000638 stimulation Effects 0.000 description 9
- 241000124008 Mammalia Species 0.000 description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 8
- 210000004958 brain cell Anatomy 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 210000004295 hippocampal neuron Anatomy 0.000 description 8
- 102100024013 Golgi SNAP receptor complex member 2 Human genes 0.000 description 7
- 101000904234 Homo sapiens Golgi SNAP receptor complex member 2 Proteins 0.000 description 7
- 230000037396 body weight Effects 0.000 description 7
- 230000000875 corresponding effect Effects 0.000 description 7
- 108020001507 fusion proteins Proteins 0.000 description 7
- 102000037865 fusion proteins Human genes 0.000 description 7
- 230000001976 improved effect Effects 0.000 description 7
- 150000002632 lipids Chemical class 0.000 description 7
- 230000015654 memory Effects 0.000 description 7
- 239000002679 microRNA Substances 0.000 description 7
- 238000001543 one-way ANOVA Methods 0.000 description 7
- 238000004445 quantitative analysis Methods 0.000 description 7
- 238000013519 translation Methods 0.000 description 7
- 230000014616 translation Effects 0.000 description 7
- 208000012902 Nervous system disease Diseases 0.000 description 6
- 102000038030 PI3Ks Human genes 0.000 description 6
- 108091007960 PI3Ks Proteins 0.000 description 6
- 108091030071 RNAI Proteins 0.000 description 6
- 230000033228 biological regulation Effects 0.000 description 6
- 210000005013 brain tissue Anatomy 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 230000009368 gene silencing by RNA Effects 0.000 description 6
- 210000003128 head Anatomy 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000000178 monomer Substances 0.000 description 6
- 150000002894 organic compounds Chemical class 0.000 description 6
- 150000003905 phosphatidylinositols Chemical class 0.000 description 6
- 238000006116 polymerization reaction Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 231100000419 toxicity Toxicity 0.000 description 6
- 230000001988 toxicity Effects 0.000 description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 6
- 241000713666 Lentivirus Species 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 108091000080 Phosphotransferase Proteins 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 230000034994 death Effects 0.000 description 5
- 231100000517 death Toxicity 0.000 description 5
- 210000003520 dendritic spine Anatomy 0.000 description 5
- 230000012202 endocytosis Effects 0.000 description 5
- 108091070501 miRNA Proteins 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000008194 pharmaceutical composition Substances 0.000 description 5
- 150000003904 phospholipids Chemical class 0.000 description 5
- 102000020233 phosphotransferase Human genes 0.000 description 5
- 230000002028 premature Effects 0.000 description 5
- 238000003757 reverse transcription PCR Methods 0.000 description 5
- 230000009261 transgenic effect Effects 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 208000037259 Amyloid Plaque Diseases 0.000 description 4
- 206010003694 Atrophy Diseases 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 208000020358 Learning disease Diseases 0.000 description 4
- 102000004317 Lyases Human genes 0.000 description 4
- 108090000856 Lyases Proteins 0.000 description 4
- 208000026139 Memory disease Diseases 0.000 description 4
- 208000028389 Nerve injury Diseases 0.000 description 4
- 208000025966 Neurological disease Diseases 0.000 description 4
- 102000017343 Phosphatidylinositol kinases Human genes 0.000 description 4
- 108050005377 Phosphatidylinositol kinases Proteins 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 241000700159 Rattus Species 0.000 description 4
- 230000037444 atrophy Effects 0.000 description 4
- 210000003169 central nervous system Anatomy 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000010226 confocal imaging Methods 0.000 description 4
- 230000009193 crawling Effects 0.000 description 4
- 210000001787 dendrite Anatomy 0.000 description 4
- 230000002222 downregulating effect Effects 0.000 description 4
- 239000000835 fiber Substances 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 238000003304 gavage Methods 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 201000003723 learning disability Diseases 0.000 description 4
- 238000001325 log-rank test Methods 0.000 description 4
- 230000007087 memory ability Effects 0.000 description 4
- 230000008764 nerve damage Effects 0.000 description 4
- 208000015122 neurodegenerative disease Diseases 0.000 description 4
- 229920003023 plastic Polymers 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- 231100000331 toxic Toxicity 0.000 description 4
- 230000002588 toxic effect Effects 0.000 description 4
- 238000012549 training Methods 0.000 description 4
- 230000007730 Akt signaling Effects 0.000 description 3
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 3
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 3
- 101100126626 Drosophila melanogaster Itpr gene Proteins 0.000 description 3
- 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 3
- 241000282412 Homo Species 0.000 description 3
- 241000699660 Mus musculus Species 0.000 description 3
- 108020004485 Nonsense Codon Proteins 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 3
- 101710184528 Scaffolding protein Proteins 0.000 description 3
- 108091027967 Small hairpin RNA Proteins 0.000 description 3
- 108020004459 Small interfering RNA Proteins 0.000 description 3
- 210000001015 abdomen Anatomy 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 101150049912 bin3 gene Proteins 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000009194 climbing Effects 0.000 description 3
- 238000000749 co-immunoprecipitation Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 108091006047 fluorescent proteins Proteins 0.000 description 3
- 102000034287 fluorescent proteins Human genes 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 230000000971 hippocampal effect Effects 0.000 description 3
- 238000001114 immunoprecipitation Methods 0.000 description 3
- 238000012744 immunostaining Methods 0.000 description 3
- 230000001418 larval effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 230000037434 nonsense mutation Effects 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 3
- IEKVHGDUBCZHRB-UHFFFAOYSA-N phenylarsenic Chemical compound [As]C1=CC=CC=C1 IEKVHGDUBCZHRB-UHFFFAOYSA-N 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000002924 silencing RNA Substances 0.000 description 3
- 239000004055 small Interfering RNA Substances 0.000 description 3
- 230000003976 synaptic dysfunction Effects 0.000 description 3
- 230000000946 synaptic effect Effects 0.000 description 3
- 238000011830 transgenic mouse model Methods 0.000 description 3
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 3
- 102000001556 1-Phosphatidylinositol 4-Kinase Human genes 0.000 description 2
- 108010029190 1-Phosphatidylinositol 4-Kinase Proteins 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- GUQQBLRVXOUDTN-XOHPMCGNSA-N 3-[dimethyl-[3-[[(4r)-4-[(3r,5s,7r,8r,9s,10s,12s,13r,14s,17r)-3,7,12-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]propyl]azaniumyl]-2-hydroxypropane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CC(O)CS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 GUQQBLRVXOUDTN-XOHPMCGNSA-N 0.000 description 2
- 108020005544 Antisense RNA Proteins 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 108700011906 Drosophila gt Proteins 0.000 description 2
- 108700007479 Drosophila tau Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- 101001001516 Homo sapiens Phosphatidylinositol 4-kinase alpha Proteins 0.000 description 2
- 101000730433 Homo sapiens Phosphatidylinositol 4-kinase beta Proteins 0.000 description 2
- 108091008585 IP3 receptors Proteins 0.000 description 2
- 102000007640 Inositol 1,4,5-Trisphosphate Receptors Human genes 0.000 description 2
- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 102100032619 Phosphatidylinositol 4-kinase beta Human genes 0.000 description 2
- 102100038931 Proenkephalin-A Human genes 0.000 description 2
- 101100221606 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) COS7 gene Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 101150052440 YPP1 gene Proteins 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 108010064539 amyloid beta-protein (1-42) Proteins 0.000 description 2
- 238000000540 analysis of variance Methods 0.000 description 2
- 229910000413 arsenic oxide Inorganic materials 0.000 description 2
- 229960002594 arsenic trioxide Drugs 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 210000004720 cerebrum Anatomy 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 210000003703 cisterna magna Anatomy 0.000 description 2
- 239000003184 complementary RNA Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 210000000172 cytosol Anatomy 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 150000001982 diacylglycerols Chemical class 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000000104 effect on dyskinesia Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 230000000763 evoking effect Effects 0.000 description 2
- 230000002964 excitative effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 210000000609 ganglia Anatomy 0.000 description 2
- 230000030279 gene silencing Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000001320 hippocampus Anatomy 0.000 description 2
- 239000005457 ice water Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 239000003547 immunosorbent Substances 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 229960003299 ketamine Drugs 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 206010027175 memory impairment Diseases 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000007659 motor function Effects 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 230000009251 neurologic dysfunction Effects 0.000 description 2
- 208000015015 neurological dysfunction Diseases 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 231100000915 pathological change Toxicity 0.000 description 2
- 230000036285 pathological change Effects 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000000750 progressive effect Effects 0.000 description 2
- 229940121649 protein inhibitor Drugs 0.000 description 2
- 239000012268 protein inhibitor Substances 0.000 description 2
- 238000012764 semi-quantitative analysis Methods 0.000 description 2
- 238000000528 statistical test Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000007470 synaptic degeneration Effects 0.000 description 2
- 230000003977 synaptic function Effects 0.000 description 2
- 108010026424 tau Proteins Proteins 0.000 description 2
- 102000013498 tau Proteins Human genes 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 108700026220 vif Genes Proteins 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 230000031836 visual learning Effects 0.000 description 2
- 230000003313 weakening effect Effects 0.000 description 2
- BPICBUSOMSTKRF-UHFFFAOYSA-N xylazine Chemical compound CC1=CC=CC(C)=C1NC1=NCCCS1 BPICBUSOMSTKRF-UHFFFAOYSA-N 0.000 description 2
- 229960001600 xylazine Drugs 0.000 description 2
- CNWINRVXAYPOMW-FCNJXWMTSA-N 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-1D-myo-inositol 4,5-biphosphate Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(=O)O[C@H](COC(=O)CCCCCCCCCCCCCCCCC)COP(O)(=O)O[C@@H]1[C@H](O)[C@H](O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H]1O CNWINRVXAYPOMW-FCNJXWMTSA-N 0.000 description 1
- SNKZJIOFVMKAOJ-UHFFFAOYSA-N 3-Aminopropanesulfonate Chemical compound NCCCS(O)(=O)=O SNKZJIOFVMKAOJ-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 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
- AJOGHKUZDLYXKS-UHFFFAOYSA-N 5-[2-amino-3-(4-morpholin-4-ylphenyl)benzimidazol-5-yl]-n-(2-fluorophenyl)-2-methoxypyridine-3-sulfonamide Chemical compound COC1=NC=C(C=2C=C3N(C=4C=CC(=CC=4)N4CCOCC4)C(N)=NC3=CC=2)C=C1S(=O)(=O)NC1=CC=CC=C1F AJOGHKUZDLYXKS-UHFFFAOYSA-N 0.000 description 1
- 206010001497 Agitation Diseases 0.000 description 1
- 208000000044 Amnesia Diseases 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- DWJXYEABWRJFSP-XOBRGWDASA-N DAPT Chemical compound N([C@@H](C)C(=O)N[C@H](C(=O)OC(C)(C)C)C=1C=CC=CC=1)C(=O)CC1=CC(F)=CC(F)=C1 DWJXYEABWRJFSP-XOBRGWDASA-N 0.000 description 1
- 206010011906 Death Diseases 0.000 description 1
- 206010012289 Dementia Diseases 0.000 description 1
- 108010035533 Drosophila Proteins Proteins 0.000 description 1
- 108700009567 Drosophila stmA Proteins 0.000 description 1
- 241001269524 Dura Species 0.000 description 1
- 102000043859 Dynamin Human genes 0.000 description 1
- 108700021058 Dynamin Proteins 0.000 description 1
- 208000012661 Dyskinesia Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 229940125373 Gamma-Secretase Inhibitor Drugs 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000845201 Homo sapiens Tetratricopeptide repeat protein 7B Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 231100000111 LD50 Toxicity 0.000 description 1
- 231100000002 MTT assay Toxicity 0.000 description 1
- 238000000134 MTT assay Methods 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 102000007474 Multiprotein Complexes Human genes 0.000 description 1
- 108010085220 Multiprotein Complexes Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 101150037263 PIP2 gene Proteins 0.000 description 1
- WSLBJQQQZZTFBA-MLUQOLBVSA-N PIP[4'](17:0/20:4(5Z,8Z,11Z,14Z)) Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(=O)O[C@H](COC(=O)CCCCCCCCCCCCCCCC)COP(O)(=O)OC1C(O)C(O)C(OP(O)(O)=O)[C@@H](O)C1O WSLBJQQQZZTFBA-MLUQOLBVSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102100036161 Phosphatidylinositol 4-kinase alpha Human genes 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 101100262439 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) UBA2 gene Proteins 0.000 description 1
- 102100031270 Tetratricopeptide repeat protein 7B Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 102000014384 Type C Phospholipases Human genes 0.000 description 1
- 108010079194 Type C Phospholipases Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- UJVUMTUBMCYKBK-BNOPZSDTSA-N [(2r)-2-hexadecanoyloxy-3-[hydroxy-[(2r,3r,5s,6r)-2,3,5,6-tetrahydroxy-4-phosphonooxycyclohexyl]oxyphosphoryl]oxypropyl] hexadecanoate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@@H](OC(=O)CCCCCCCCCCCCCCC)COP(O)(=O)OC1[C@H](O)[C@H](O)C(OP(O)(O)=O)[C@H](O)[C@H]1O UJVUMTUBMCYKBK-BNOPZSDTSA-N 0.000 description 1
- IKWTVSLWAPBBKU-UHFFFAOYSA-N a1010_sial Chemical compound O=[As]O[As]=O IKWTVSLWAPBBKU-UHFFFAOYSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 230000000386 athletic effect Effects 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000002886 autophagic effect Effects 0.000 description 1
- 230000003376 axonal effect Effects 0.000 description 1
- 229950001863 bapineuzumab Drugs 0.000 description 1
- 238000009227 behaviour therapy Methods 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 230000013629 beta-amyloid clearance Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000003990 capacitor Substances 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 230000007248 cellular mechanism Effects 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 230000003920 cognitive function Effects 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000011977 dual antiplatelet therapy Methods 0.000 description 1
- 210000001951 dura mater Anatomy 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- QELUYTUMUWHWMC-UHFFFAOYSA-N edaravone Chemical compound O=C1CC(C)=NN1C1=CC=CC=C1 QELUYTUMUWHWMC-UHFFFAOYSA-N 0.000 description 1
- 229950009041 edaravone Drugs 0.000 description 1
- 231100001129 embryonic lethality Toxicity 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000036749 excitatory postsynaptic potential Effects 0.000 description 1
- 230000028023 exocytosis Effects 0.000 description 1
- 230000000494 facilitatory effect Effects 0.000 description 1
- 150000004665 fatty acids Chemical group 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000003540 gamma secretase inhibitor Substances 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Natural products O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000007154 intracellular accumulation Effects 0.000 description 1
- 210000004020 intracellular membrane Anatomy 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- YWXYYJSYQOXTPL-SLPGGIOYSA-N isosorbide mononitrate Chemical compound [O-][N+](=O)O[C@@H]1CO[C@@H]2[C@@H](O)CO[C@@H]21 YWXYYJSYQOXTPL-SLPGGIOYSA-N 0.000 description 1
- 230000008386 key pathological change Effects 0.000 description 1
- 229940043355 kinase inhibitor Drugs 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000006386 memory function Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 210000001087 myotubule Anatomy 0.000 description 1
- UPSFMJHZUCSEHU-JYGUBCOQSA-N n-[(2s,3r,4r,5s,6r)-2-[(2r,3s,4r,5r,6s)-5-acetamido-4-hydroxy-2-(hydroxymethyl)-6-(4-methyl-2-oxochromen-7-yl)oxyoxan-3-yl]oxy-4,5-dihydroxy-6-(hydroxymethyl)oxan-3-yl]acetamide Chemical compound CC(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@H](O)[C@@H](NC(C)=O)[C@H](OC=2C=C3OC(=O)C=C(C)C3=CC=2)O[C@@H]1CO UPSFMJHZUCSEHU-JYGUBCOQSA-N 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 230000018352 negative regulation of endocytosis Effects 0.000 description 1
- 210000000118 neural pathway Anatomy 0.000 description 1
- 230000010004 neural pathway Effects 0.000 description 1
- 210000002682 neurofibrillary tangle Anatomy 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 231100000878 neurological injury Toxicity 0.000 description 1
- 210000000715 neuromuscular junction Anatomy 0.000 description 1
- 230000016273 neuron death Effects 0.000 description 1
- 230000006764 neuronal dysfunction Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- ZPIRTVJRHUMMOI-UHFFFAOYSA-N octoxybenzene Chemical compound CCCCCCCCOC1=CC=CC=C1 ZPIRTVJRHUMMOI-UHFFFAOYSA-N 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 206010033675 panniculitis Diseases 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- RLLPVAHGXHCWKJ-UHFFFAOYSA-N permethrin Chemical group CC1(C)C(C=C(Cl)Cl)C1C(=O)OCC1=CC=CC(OC=2C=CC=CC=2)=C1 RLLPVAHGXHCWKJ-UHFFFAOYSA-N 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000008105 phosphatidylcholines Chemical class 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000003757 phosphotransferase inhibitor Substances 0.000 description 1
- 230000007505 plaque formation Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 108010074732 preproenkephalin Proteins 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 108010041071 proenkephalin Proteins 0.000 description 1
- 210000001176 projection neuron Anatomy 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 230000007026 protein scission Effects 0.000 description 1
- 210000002763 pyramidal cell Anatomy 0.000 description 1
- 238000012207 quantitative assay Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000033764 rhythmic process Effects 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 230000018448 secretion by cell Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 210000003625 skull Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000006886 spatial memory Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 210000004304 subcutaneous tissue Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
- 230000007617 synaptic impairment Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 229960003570 tramiprosate Drugs 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 229960000281 trometamol Drugs 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Images
Classifications
-
- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- 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
-
- 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; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/70—Invertebrates
- A01K2227/706—Insects, e.g. Drosophila melanogaster, medfly
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; 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
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
- A01K67/0337—Genetically modified Arthropods
- A01K67/0339—Genetically modified insects, e.g. Drosophila melanogaster, medfly
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/136—Screening for pharmacological compounds
-
- 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
Definitions
- the present invention was completed under the auspices of the Ministry of Science and Technology 973 project (2013CB530900) and the National Natural Science Foundation of China (81371400 and 81071026).
- the present invention belongs to the field of medicine, and relates to a method for treating Alzheimer's disease by using a related inhibitor to down-regulate PI4KIII ⁇ kinase and a membrane protein complex formed thereof with RBO/EFR3/EFR3A/EFR3B protein and TTC7 protein;
- the invention relates to a method for screening for drugs and therapeutic targets for the treatment of Alzheimer's disease depending on whether or not the A ⁇ secretion of the cells is promoted.
- AD Alzheimer's disease
- a ⁇ Alzheimer's disease
- a ⁇ accumulates not only outside the cell but also in neurons.
- a large amount of evidence suggests that A ⁇ accumulates in various intracellular organs of neurons and participates in pathological changes of AD such as synaptic disorder, amyloid plaque formation, and neuronal death.
- oligo-A ⁇ which is thought to be the most destructive to synaptic and cognitive functions, is formed intracellularly and accumulates in the brain neurons of AD patients and APP transgenic mice or accumulates in the cell membrane. These membrane-coupled A ⁇ may be pinched on the cell membrane of the neuron or on the cell membrane of the cell. There may be many pathways leading to accumulation of A ⁇ in the accumulated neurons, such as extracellular A ⁇ endocytosis, decreased intracellular A ⁇ secretion and retention, autophagic vacuoles production and accumulation of A ⁇ , and decreased intracellular A ⁇ clearance.
- a ⁇ accumulates inside and outside the cell
- the concentration of A ⁇ 42 in the cerebrospinal fluid of patients with AD and those with early AD is reduced, about half that of the control population.
- the concentration of A ⁇ 42 in cerebrospinal fluid and brain tissue fluid was also reduced in age, and A ⁇ dimer was not detected in brain tissue fluid.
- the concentration of A [beta] 42 in CSF and brain tissue has been speculated to reduce fluid may be due to uptake of extracellular amyloid plaques, reducing A [beta] 42 secretion, induced by A [beta] 42 accumulation in the brain or neuronal cell membrane.
- a ⁇ 42 can be inserted into the lipid membrane to bind to acidic phospholipids, and the combined acidic phospholipids promote A ⁇ 42 transition from the disordered configuration to the folded beta], resulting in the polymerization of A ⁇ 42 to lipid membranes or membrane; 2) culturing phosphatidylinositol 4,5 bisphosphate (phosphatidylinositol 4,5-phosphate in the cell membranes of cells, PIP2) level and the amount of cellular secretion of A ⁇ 42 was negatively correlated; 3) Drosophila neuronal dysfunction caused by A ⁇ 42 expression and learning and memory disorders can be repaired by the inhibition of PI3K.
- the rolling blackout (rbo) gene in Drosophila encodes a membrane protein RBO with some homology to the diacylglycerol lyase.
- RBO proteins play a role in phospholipid metabolism in Drosophila, light transmission in visual cells, synaptic transmission, and bulk endocytosis in bulk. RBO proteins are conserved from yeast to humans.
- RBO homologous protein in yeast EFR3 and two homologous proteins of mouse (EFR3A and EFR3B) on the cell membrane with III ⁇ -type phosphatidylinositol 4 kinase (PI4KIII ⁇ ) and a scaffolding protein (called in mammals) Forming a complex for Tetratricopeptide repeat domain 7, TTC7, see Baird D, Stefan C, et al., 2008, J Cell Biol; Nakatsu F, Baskin JF, et al., 2012, J Cell Biol), thereby anchoring PI4KIII ⁇ On the cell membrane, and further regulating the levels of phosphatidylinositol 4-phosphate (PI 4 P) and PIP 2 on the cell membrane.
- PI 4 P III ⁇ -type phosphatidylinositol 4 kinase
- pancreatic neuronal expression of A ⁇ 42 fused to a secretion signal peptide results in accumulation of A ⁇ 42 and neurodegenerative changes in neurons.
- the inventors expressed A ⁇ 42 containing a secretory signal peptide in a simple neural pathway of adult Drosophila, the giant fiber pathway (GF), and found that A ⁇ 42 accumulated in neurons and led to age-dependent (age-dependent) obstacles such as outstanding delivery failure and decreased athletic ability.
- This Drosophila expressing A ⁇ 42 provides a good platform for studying the role of some genes that may be associated with AD in the accumulation of A ⁇ 42 in neurons and the synaptic dysfunction caused by it.
- the inventors used this model to detect mutations or overexpression of genes rbo, PI4KIII ⁇ and ttc7, and the effects of commonly used PI4KIII ⁇ protein inhibitors on neurodegenerative diseases in this model.
- the inventors also studied the atrophy of hippocampal neurons by a mouse homolog Efr3a, which knocks down the rbo gene, in APP/PS1 transgenic mice, and the use of the common PI4KIII ⁇ kinase small molecule inhibitor PAO to administer learning and memory.
- the present invention discloses the use of genetic means or related inhibitors to inhibit down-regulation of PI4KIII ⁇ protein, RBO/EFR3/EFR3A/EFR3B membrane protein, TTC7 protein, and the amount of membrane protein complexes they form or their related enzyme activities can promote neuronal cells A ⁇ (especially A ⁇ 42 ) secretes and correspondingly reduces A ⁇ accumulation in neurons, thereby reducing neurological dysfunction in AD model Drosophila and mice.
- the present invention thus discloses an important role of neuronal A ⁇ 42 secretion in the treatment of AD, and provides a novel strategy for the treatment of AD; at the same time, the present invention also provides a new drug for treating AD, and further screening for drugs for treating AD and The therapeutic target points to a new direction.
- the invention provides a method of screening for a medicament for treating Alzheimer's disease and a therapeutic target, the method comprising screening for a drug candidate or therapeutic target that increases the level of A ⁇ secretion by the cell.
- the method for screening for a drug for treating Alzheimer's disease and a therapeutic target comprises the steps of: 1) applying the drug to an overexpressing APP or a secretion signal peptide A ⁇ cell line or Drosophila tissue or the therapeutic target in a cell line or Drosophila tissue that overexpresses APP or A ⁇ secreting a signal peptide; and 2) detection of the overexpressed APP or by immunoassay a cell line secreting a signal peptide of A ⁇ or a cell in a Drosophila tissue, wherein the drug or therapeutic target is increased if the application of the drug or the modulation of a therapeutic target results in an increase in the amount of A ⁇ secreted by the cell.
- the detected A ⁇ is A ⁇ 42 ; and the immunoassay method for detecting the secretion of A ⁇ is an enzyme-linked immunosorbent Determination or electrochemiluminescence immunoassay;
- the cell line overexpressing APP or A ⁇ containing a secretion signal peptide is a HEK293T, N2a or SH-SY5Y cell line stably transfecting human APP or A ⁇ containing a secretion signal peptide, or
- the Drosophila tissue overexpressing APP or A ⁇ containing a secretion signal peptide is a third instar larva tissue of Drosophila.
- Figure 1 shows that mutation of the rbo gene alleviates neurological damage of Drosophila expressing A ⁇ arc , reduces the expression of PI4KIII ⁇ protein or attenuates the interaction of PI4KIII ⁇ protein with RBO protein.
- Figure 2 shows down-regulation of PI4KIII ⁇ protein expression or drug inhibition of PI4KIII ⁇ enzyme activity to alleviate neurological impairment of Drosophila expressing A ⁇ arc .
- Figure 3 shows that down-regulation of RBO/PI4KIII ⁇ protein expression or function reduces the accumulation of A ⁇ in neurons.
- Figure 4 shows that down-regulation of RBO/PI4KIII ⁇ protein expression or function promotes secretion of A ⁇ 42 .
- Figure 5 shows the effect of knockdown of the Efr3a gene on the dendritic diameter and dendritic spine density of hippocampal CA3 and DG neurons in APP/PS1 and control mice.
- Figure 6 shows the effect of PAO treatment on learning and memory ability of APP/PS1 mice and control mice, as well as CSF and brain cell membrane-coupled A ⁇ 42 levels.
- Figure 7 shows expression rbo mutation alleviate neurological injury Drosophila A ⁇ 42.
- Figure 8 shows the effect of rbo gene and shibire gene mutations on the motility and longevity of Drosophila overexpressing A ⁇ arc or Drosophila Tau protein, respectively.
- Figure 9 shows the effect of mutations in the rbo S358A gene and the itpr SV35 gene on expression of A ⁇ arc fruit flies.
- Figure 10 shows the effect of the outer cells 42 and A [beta] rbo PI4KA genes and gene mutation gene knockdown Efr3a N2a cells on the endocytosis of A ⁇ arc Drosophila transcription expression of A ⁇ arc.
- Figure 11 shows the efficiency of knockdown of the Efr3a gene or PI4KA gene in HEK293 cells or mouse primary cultured hippocampal neurons.
- Figure 12 shows that insertion of a transposon into one copy of the PI4KA gene of APP/PS1 mice significantly improved learning and memory in mice.
- Figure 13 shows that PI 4 P promotes the formation of oligomers of A ⁇ 42 in liposomes.
- Figure 14 shows the effect of PAO on APP expression and ⁇ , ⁇ , ⁇ secretase activity.
- Figure 15 shows the effect of ttc7 gene mutation and overexpression on neurological impairment of A ⁇ arc Drosophila.
- rbo/Efr3/Efr3a/Efr3b gene refers to a rbo gene derived from Drosophila, a yeast-derived Efr3, or a mammalian Efr3a gene and an Efr3b gene; the term “RBO/EFR3” as used in the present invention.
- the /EFR3A/EFR3B protein refers to a protein derived from a fruit fly-derived rbo gene, a yeast-derived Efr3 gene, or a mammal-derived Efr3a/Efr3b gene.
- PI4KIII ⁇ /PI4KA gene refers to a PI4KIII ⁇ gene or a PI4KA gene derived from Drosophila or a mammal;
- PI4KIII ⁇ protein as used in the present invention means a gene encoded by the PI4KIII ⁇ /PI4KA gene in Drosophila or mammal. Protein.
- ttc7 gene refers to a ttc7 gene derived from a mammal
- TTC7 protein refers to a protein encoded by the ttc7 gene in the aforementioned mammal.
- inhibitor refers to a substance capable of reducing, reducing, or eliminating the amount of a target, a specific function, and a specific property.
- the target may be a protein, a polypeptide, a nucleic acid, or the like, and the inhibitor may directly or indirectly act on the number, specific function, and specific property of the target, and the number and specific function of the target may be made. And the corresponding properties are correspondingly reduced, reduced or eliminated.
- the inhibitor may be a protein, a polypeptide, a nucleic acid, a small molecule compound or the like.
- PI4KIII ⁇ inhibitor means the stability of the PI4KIII ⁇ protein capable of reducing, reducing, and eliminating the expression, transcription, translation, and/or formation thereof of the PI4KIII ⁇ /PI4KA gene, and the membrane protein RBO ⁇ EFR3 ⁇ EFR3A ⁇ EFR3B and the binding ability of TTC7 protein, and various factors such as phosphokinase activity, including but not limited to inhibitory nucleotides specific for PI4KIII ⁇ /PI4KA gene, anti-PI4KIII ⁇ protein antibody, and ability to inhibit PI4KIII ⁇ kinase An active small molecule compound, and/or various substances capable of inhibiting the interaction of the PI4KIII ⁇ protein with other membrane proteins.
- RBO/EFR3/EFR3A/EFR3B inhibitor refers to an RBO/ capable of inhibiting, reducing, or eliminating the expression, transcription, translation, and/or formation of the rbo/Efr3/Efr3a/Efr3b gene.
- EFR3/EFR3A/EFR3B protein The stability of EFR3/EFR3A/EFR3B protein, its ability to bind to PI4KIII ⁇ protein, and other substances, including but not limited to inhibitory nucleotides specific for rbo/Efr3/Efr3a/Efr3b gene, anti-RBO/EFR3
- the antibody of the /EFR3A/EFR3B protein and various substances capable of inhibiting the formation of a complex of the RBO/EFR3/EFR3A/EFR3B protein and the PI4KIII ⁇ protein The antibody of the /EFR3A/EFR3B protein and various substances capable of inhibiting the formation of a complex of the RBO/EFR3/EFR3A/EFR3B protein and the PI4KIII ⁇ protein.
- TTC7 inhibitor refers to a TTC7 protein which is capable of reducing, reducing, and eliminating the expression, transcription, translation, and/or formation of the ttc7 gene, and the membrane protein RBO ⁇ EFR3 ⁇ EFR3A ⁇ EFR3B.
- Various substances in combination including but not limited to inhibitory nucleotides specific for the ttc7 gene, anti-TTC7 protein antibodies, and/or inhibiting the interaction of the TTC7 protein with the membrane protein RBO ⁇ EFR3 ⁇ EFR3A ⁇ EFR3B Various substances, etc.
- PI 4 P inhibitor refers to a substance capable of inhibiting, reducing, and eliminating the level of PI 4 P on a cell membrane, including but not limited to an anti-PI 4 P antibody, and capable of being specific to PI 4 P. Binding OSH2-PH2X fusion protein or OSH2-2x-PH fusion protein, and the like.
- antibody refers to any immunoglobulin or intact molecule that binds to a particular epitope, as well as fragments thereof. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, and fragments and/or portions of intact antibodies, as long as these fragments or portions retain the antigen binding ability of the parent antibody.
- anti-PI4KIII ⁇ antibody refers to a monoclonal antibody, a polyclonal antibody, a single-chain antibody, and an immunologically active fragment or portion thereof capable of specifically binding to a PI4KIII ⁇ protein, or a functional variant or a functional fragment thereof.
- terms such as “PI4KIII ⁇ antibody”, “anti-PI4KIII ⁇ antibody”, and “antibody against PI4KIII ⁇ ” are used interchangeably.
- “functional variant” refers to a protein or polypeptide of the invention having one or more amino acids altered in its amino acid sequence.
- the alteration may be a "conservative” change (wherein the substituted amino acid has similar structural or chemical properties) or a “non-conservative” change; similar changes may also include amino acid deletions or insertions or both.
- alterations in these amino acid residues or deletions or insertions of amino acids should not substantially alter or disrupt the biological or immunological activity and function of the original amino acid sequence.
- “functional fragment” refers to any part of a protein or polypeptide of the present invention which retains its function as A portion of a protein or polypeptide (ie, a "parent” protein or polypeptide) has substantially similar or identical biological or immunological activities and functions.
- inhibitory nucleotide refers to a nucleotide compound capable of binding to and inhibiting expression of a specific gene.
- Typical inhibitory nucleotides include, but are not limited to, antisense oligonucleotides, triple helix DNA, aptamers, ribozymes, short-suppressed ribonucleosides Glycosylate (siRNA), short hairpin RNA (shRNA) and microRNA. These nucleotide compounds are capable of binding to the specific gene with greater affinity than other nucleotide sequences, thereby inhibiting the expression of a particular gene.
- small molecule compound refers to an organic compound having a molecular weight of less than 3 kilodaltons, which may be natural or chemically synthesized.
- derivative refers to a compound produced by modifying a parent organic compound by one or more chemical reactions, which has a similar structure to the parent organic compound and has a similar effect in function.
- analog refers to an organic compound which is not necessarily obtained by chemical modification of a parent organic compound, but which is structurally similar to a parent organic compound and functionally It also has a similar effect.
- AD Alzheimer's disease
- senile neurodegenerative disease in which progressive learning and memory disorders are prominent clinical symptoms.
- Most AD patients have extracellular beta-amyloid plaques in the middle and advanced stages, and there are neurofibrillary tangles composed of Tau protein or synaptic and nerve cell loss.
- the disease can exist in both humans and animals, such as dogs.
- a ⁇ refers to a series of polypeptides which are between 38-48 amino acids in length and which are cleaved by secretases from Amyloid Precursor Protein (APP), mainly A ⁇ 38 .
- a ⁇ can also be cleaved by other protein cleavage enzymes expressed by a method of infecting cells with a transgene or a viral vector, for example, the N-terminus of A ⁇ can be derived from Drosophila.
- Secretion signal peptide of the protein encoded by the necrotic gene (amino acid sequence: MASKVSILLLLTVHLLAAQTFAQDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) (SEQ ID NO: 1) or secretion signal peptide derived from rat Proenkephalin precursor (pre-proenkephalin) (amino acid sequence: MAQFLRLCIWLLALGSCLLATVQA (SEQ ID NO: 2) ))
- a ⁇ secretion refers to a process in which A ⁇ is discharged extracellularly via a cell membrane after intracellular or cell membrane production, which may result in a decrease in intracellular A ⁇ accumulation.
- the secretion of A ⁇ 42 specifically refers to A ⁇ 42 in the cell or after discharged to the cell membrane via extracellular membrane process, the process may lead to reduced intracellular accumulation of A ⁇ 42.
- the term "therapeutic target” as used in the present invention refers to various substances which can be used for the treatment of a certain disease and their action targets in an animal or a human body.
- the action of the substance on the target of the action can have an effect of treating the disease.
- the substance may be a protein, a polypeptide, a nucleic acid, a small molecule compound or the like, and the target may be a certain gene (including a specific sequence of the gene), a certain protein (including a specific site of the protein),
- a substance entity such as a certain protein complex (including a specific binding site thereof) may also be a certain characteristic of the aforementioned gene and/or protein, a certain function, a relationship with a surrounding substance, an environment, etc., as long as The substance can affect the gene, protein, protein complex, or its specific characteristics, functions, relationships, etc., thereby functioning to treat the disease.
- treating refers to reversing, alleviating or inhibiting the progression of a disease to which the term is applied, or one or more symptoms of a disease.
- the term also includes preventing the disease, including preventing the onset of the disease or any symptoms associated therewith, as well as reducing the severity of the condition or any condition prior to its onset.
- the terms “inhibiting,” “weakening,” “down-regulating,” “eliminating,” and the like, are meant to mean a reduction or reduction in quantity or degree. This reduction or reduction is not limited to any magnitude, Just show this trend. For example, the reduction or reduction may be 100%, 50%, or 1% or less relative to the original amount or degree.
- the present invention discloses down-regulating the expression of RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein or PI4KIII ⁇ protein, weakening the interaction between RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein, and PI4KIII ⁇ protein, and inhibiting the enzyme activity of PI4KIII ⁇ .
- a series of behaviors can alleviate defects such as synaptic dysfunction and loss of nerve cells expressing A ⁇ 42 with increasing age, and disclose that these effects are promoted by promoting the secretion of A ⁇ (especially A ⁇ 42 ) in nerve cells. Reduce the accumulation of A ⁇ (especially A ⁇ 42 ) in nerve cell membranes or nerve cells.
- the inventors found that knockdown of the Efr3a gene can reduce dendritic and spine atrophy of hippocampal neurons in APP/PS1 double transgenic mice, and a commonly used PI4KIII ⁇ protein inhibitor, phenylarsenic oxide (Phenylarsine Oxide). , PAO) can significantly improve the learning and memory ability of APP/PS1 mice, and reduce the content of A ⁇ 42 , especially polymerized A ⁇ 42 , which is coupled to the cell membrane of brain tissue, although the process is accompanied by A ⁇ 42 in cerebrospinal fluid. The content is increased.
- PAO phenylarsenic oxide
- the inventors have found that genetically downregulating the expression of RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein, and PI4KIII ⁇ protein in cells or neurons, or preventing them from forming protein complexes, reduces the membrane distribution of PI4KIII ⁇ protein, or inhibits
- the phosphokinase activity of PI4KIII ⁇ protein can promote the secretion of A ⁇ 42 in cells or neurons, reduce the accumulation of A ⁇ 42 in neurons, alleviate AD-related neurodegenerative changes and dysfunction, but at the same time, whether it is A ⁇ 42
- the expression level of APP or the activity of ⁇ , ⁇ and ⁇ secretase of APP was not significantly affected.
- PI 4 P a product of the PI4KIII ⁇ protein, promotes polymerization of A ⁇ 42 monomers in liposomes to form multimers, and the promotion is better than the precursor of PI 4 P (PI) and its derivative PI 4 , The promotion of 5 P is much stronger.
- a ⁇ (including A ⁇ 42 ) is produced on the plasma membrane or in intracellular organs, and the resulting A ⁇ may be released by passive release, exocytosis, lysosomal-mediated release, or other undiscovered pathways.
- Secreted cells The inventors believe that A ⁇ (including A ⁇ 42 ) is produced on the plasma membrane or in intracellular organs, and the resulting A ⁇ may be released by passive release, exocytosis, lysosomal-mediated release, or other undiscovered pathways. Secreted cells. However, no matter where A ⁇ is produced, how it is secreted, the cytoplasmic membrane should be the last pathway that A ⁇ must pass after leaving the cell. Due to the hydrophobicity of A ⁇ , on the cytoplasmic membrane, A ⁇ is inserted into the hydrophobic fatty acid chain on the one hand, and on the other hand with phosphatidylinositol (especially PI 4 P in phosphorylated phosphatidylinositol) and other acidic phospholipids
- PI 4 P is one of the main components of phosphorylated phosphatidylinositol on the cell membrane, and its promoting effect on the formation of A ⁇ polymer is stronger than that of PI and PI 4,5 P.
- the inventors have found that the promotion effect of PI 4 P on the formation of a polymer of A ⁇ 42 in a lipid is in a dose-dependent manner. Down-regulation of RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein or PI4KIII ⁇ protein expression, inhibition of their formation of a complex, or inhibition of the kinase function of the PI4KIII ⁇ protein will reduce the amount of PI 4 P produced on the cell membrane.
- a ⁇ 42 in Drosophila cells activates PI3K and its associated PI3K/Akt signaling pathway, thereby inducing AD-related synaptic impairment and long-term memory loss; accordingly, related studies suggest inhibition of PI3K The activity can also be used as a means of treating AD.
- the inventors believe that the accumulation of A ⁇ 42 in cells is not caused by the activation of the PI3K/Akt signaling pathway associated with phosphatidylinositol kinase (including PI3K), but more directly due to the plasma membrane.
- the phosphatidylinositol kinase involved in the present invention is mainly PI4K, and is not PI3K involved in the PI3K/Akt signaling pathway.
- PI4K secreted A ⁇ 42 having high specificity but not sensitive PI3K phosphatidylinositol kinase inhibitors, for example PAO, i.e. at lower concentrations may be effective in promoting cell.
- the present invention discloses that in order to achieve the purpose of treating AD, a method of promoting A ⁇ secretion, particularly A ⁇ 42 secretion, may be employed to reduce accumulation of A ⁇ (including A ⁇ 42 ) in or on nerve cells.
- a ⁇ including A ⁇ 42
- the increased secretion of A ⁇ cannot be attributed to an increase in the expression level of APP or an increase in the production of A ⁇ caused by the activities of ⁇ , ⁇ and ⁇ secretase.
- promoting A ⁇ secretion by nerve cells can take many forms, including impairing the binding or interaction of A ⁇ with sugar, lipids, and proteins on the cell membrane.
- the present invention also discloses that the regulation of RBO/EFR3/EFR3A/EFR3B protein, the amount of TTC7 protein and PI4KIII ⁇ protein and their ability to form complexes, and the regulation of the phosphokinase activity of PI4KIII ⁇ protein can reduce the formation of A ⁇ on the cell membrane.
- the polymer which promotes the secretion of A ⁇ (especially A ⁇ 42 ) in cells, so these proteins and the interrelationship of these proteins can constitute potential therapeutic targets for the treatment of AD.
- the RBO/EFR3/EFR3A/EFR3B inhibitors include, but are not limited to, inhibitory nucleotides of the rbo/Efr3/Efr3a/Efr3b gene (including antisense RNA, siRNA or miRNA, etc.), and anti-RBO/EFR3/EFR3A/EFR3B Protein antibodies, etc.
- inhibitory nucleotides of the rbo/Efr3/Efr3a/Efr3b gene and methods for their preparation have been disclosed (for example, see www.genecards.org, and existing products: ORIGENE, Cat. #SR308056 and Cat.#TR303768).
- methods for preparing anti-RBO/EFR3/EFR3A/EFR3B antibodies have also been disclosed (for example, see www.genecards.org, and existing products: Novus, Cat. #NBP1-81539; Thermo Fisher Scientific Cat.# PA5-24904).
- the expression, transcription, and translation of the PI4KIII ⁇ /PI4KA gene are regulated, and the stability of the PI4KIII ⁇ protein encoded by the PI4KIII ⁇ /PI4KA gene is regulated, and the protein and membrane proteins RBO/EFR3/EFR3A/EFR3B and TTC7 are regulated.
- the ability of a protein to form a protein complex, as well as the enzyme activity that regulates the phosphokinase, can also be used as a means of treatment for AD.
- the anti-PI4KIII ⁇ protein antibody can inhibit the formation of a membrane protein complex between the PI4KIII ⁇ protein and the membrane protein, and a small molecule compound inhibitor capable of inhibiting the kinase activity, etc., can be used for the treatment of AD. .
- the inhibitor is a small molecule compound, for example, PAO (phenylarsenic oxide or Phenilarsine Oxide), a derivative of PAO, A1, G1, or A1 and an analog of G1. More preferably, the inhibitor may be PAO or a derivative thereof.
- PAO phenylarsenic oxide or Phenilarsine Oxide
- the inhibitor may be PAO or a derivative thereof.
- PAO is an arsenic oxide-based and benzene ring-based structure.
- a small molecule compound which has a strong inhibitory effect on the phosphokinase activity of the PI4KIII ⁇ protein.
- the chemical structure of PAO is:
- Phenyl arsenic oxide (oxo(phenyl)arsane)
- A1 and G1 are small molecule compound inhibitors of the PI4KIII ⁇ protein, and have a similar structure.
- the chemical structure of A1 is:
- A1 and G1 Methods for the preparation of A1 and G1 are disclosed (for example, see Bojjireddy, N., et al. (2014), JBC 289: 6120-6132 and Leivers, AL, et al. (2014), JMC 57: 2091-2106 ).
- structural analogs of A1 and G1 can also be used for the treatment of AD as long as they have a function of inhibiting the phosphokinase activity of the PI4KIII ⁇ protein.
- methods for preparing such structural analogs are also disclosed.
- TTC7 inhibitor includes, but is not limited to, an inhibitory nucleotide of the ttc7 gene (including antisense RNA, siRNA or miRNA, etc.), an antibody against the TTC7 protein, and the like.
- downregulating the expression of the RBO/EFR3/EFR3A/EFR3B protein and the PI4KIII ⁇ protein, or preventing them from forming a complex to reduce the membrane distribution of the PI4KIII ⁇ protein, or inhibiting the phosphokinase activity of the PI4KIII ⁇ protein substantially results in The decrease in PI 4 P on the cell membrane promotes A ⁇ secretion from the cells. Accordingly, those of ordinary skill in the art understand, any time or inhibitor reduce the number of method PI 4 P level on cell membrane, in turn, can reduce the role of A ⁇ multimers formed in the cell membrane, it can also play the treatment of AD.
- the PI 4 P inhibitors may be capable of binding to PI 4 P specific antibodies or other molecules.
- Current methods for the preparation of anti-PI 4 P antibodies have been disclosed (see Brown BK and Karasavass N, et al., 2007, J virol; Wassef NM and Roerdink F, et al. 1984, Mol Immuol).
- it can be a broad-spectrum neutralizing antibody 4E10 and other anti-PI4P antibodies of human origin.
- the above various substances provided by the present invention may be used for the treatment of AD or a substance having therapeutic AD (collectively referred to as "the substance of the present invention"), including but not limited to an anti-RBO/EFR3/EFR3A/EFR3B antibody, an anti-PI4KIII ⁇ antibody, and an anti-PI4KIII ⁇ antibody.
- TTC7 antibody anti-PI 4 P antibody
- inhibitory nucleotide specific for rbo/Efr3/Efr3a/Efr3b gene inhibitory nucleotide specific for PI4KIII ⁇ /PI4KA gene
- inhibitory effect on phosphokinase of PI4KIII ⁇ protein The small molecule compounds and the like may all be isolated, purified, synthesized, and/or recombinant.
- compositions such as pharmaceutical compositions.
- the invention provides pharmaceutical compositions comprising any of the foregoing antibodies, inhibitory nucleotides, or/and small molecule compounds, and the corresponding pharmaceutically acceptable carriers.
- the pharmaceutical composition comprising any of the substances of the invention may comprise more than one substance of the invention, for example: antibodies and small molecule compounds, inhibitory nucleotides and antibodies, or two or more different antibodies or small molecule compounds and the like.
- the pharmaceutical composition may also include a combination with another or more pharmaceutically active agents or drugs.
- an anti-A ⁇ antibody drug such as Bapineuzumab, or a compound that binds to A ⁇ or ⁇ amyloid plaques outside the brain's nerve cells, blocks A ⁇ polymerization or promotes polymerization of A ⁇ , such as marine sulfuric acid, may be included.
- Oligosaccharides HSH971 and its analogs, tramiprosate and its analogs, Edaravone and its analogs, and the like may be included.
- the pharmaceutical composition promotes the secretion of A ⁇ from the neurons on the one hand, and promotes the clearance of A ⁇ outside the neurons on the one hand, thereby achieving a better therapeutic effect on AD.
- the invention also discloses a novel method for screening for a drug or therapeutic target for the treatment of AD.
- the design of this method is based on the novel discovery of the present invention described above, that is, the manner of promoting secretion of A? 42 can reduce the accumulation of intracellular A? 42 , thereby alleviating and preventing AD-related neurodegeneration and dysfunction. Therefore, the criteria for screening candidate drugs or therapeutic targets is to achieve the effect of promoting A ⁇ secretion of cells after the drug is administered or modulating the therapeutic target, especially the secretion of A ⁇ 42 , and the increased A ⁇ secretion cannot be attributed to the up-regulation.
- the expression level of APP, or the change in the activity of ⁇ , ⁇ or ⁇ secretase leads to an increase in the production of A ⁇ .
- the regulation of the therapeutic target refers to direct or indirect action of the therapeutic target with the related substance, thereby causing a change in the function, properties or correlation with the surrounding environment of the therapeutic target, thereby enabling Produces or triggers the corresponding effect of promoting A ⁇ secretion from cells, especially the secretion of A ⁇ 42 .
- the cell line that can be used for the screening test can be a eukaryotic cell line of mammals, insects, etc., such as: HEK293, COS7, N2a, SH. -SY5Y, S2 and sf9, etc.
- the method comprises detecting whether the candidate drug can reduce A ⁇ accumulation in the cell membrane or cells, especially the accumulation of A ⁇ 42 , thereby selecting an effective drug for treating Alzheimer's disease.
- the detection assay for whether the A ⁇ 42 secretion is increased can be carried out using a cell line overexpressing APP (such as HEK293, COS7, N2a, SH-SY5Y cell lines stably transfected with human APP), or using a fruit fly model. Preferably, it is a third instar larva tissue of Drosophila. Whether the A ⁇ 42 secretion is increased or not can be detected by an immunoassay method, including an enzyme-linked immunosorbent assay (ELISA) or an electrochemiluminescence immunosorbent assay (ECLIA) method.
- ELISA enzyme-linked immunosorbent assay
- ELIA electrochemiluminescence immunosorbent assay
- the method for screening for a drug for treating AD or for modulating a target may comprise first observing the effect of the candidate drug or the application of the regulatory target on the activity of the PI4KIII ⁇ enzyme, if the candidate drug or the application of the regulatory target enables the detection system
- a negative change in the effect of PI4KIII ⁇ kinase, ie, attenuation of PI4KIII ⁇ enzyme activity or a decrease in PI4P levels on the cell membrane indicates that the drug, agent or target is a potential drug or regulatory target for the treatment of AD.
- it is further tested whether it causes a decrease in the accumulation of intracellular A ⁇ (especially A ⁇ 42 ) and whether it promotes the secretion of A ⁇ into the extracellular. With such a method, the screening efficiency of candidate objects can be greatly improved.
- the method for screening a drug or a therapeutic target for treating AD can be performed by directly detecting the application of the drug candidate or the regulation of the therapeutic target to enable the PI4KIII ⁇ protein to be localized in the cell membrane.
- the distribution of the pulp, thereby reducing the amount of PI4KIII ⁇ protein on the cell membrane, results in a reduction in the conversion of A ⁇ monomer to the polymer on the cell membrane and an increase in extracellular secretion.
- the fluorescently labeled PI4KIII ⁇ such as fluorescent protein-labeled PI4KIII ⁇ (GFP-PI4KIII ⁇ )
- GFP-PI4KIII ⁇ fluorescent protein-labeled PI4KIII ⁇
- the fluorescently labeled PI4KIII ⁇ can be selectively observed on the cell membrane to observe whether the fluorescently labeled PI4KIII ⁇ is transferred from the cell membrane to the cytosol.
- the method of screening for a drug or therapeutic target for the treatment of AD can also be carried out by a method comprising detecting the interaction of a protein with a protein, such as co-immunoprecipitation.
- a protein such as co-immunoprecipitation.
- the application of a drug candidate or the regulation of a therapeutic target can attenuate the interaction of the RBO/EFR3/EFR3A/EFR3B protein with the TTC7 protein and the PI4KIII ⁇ protein, ie, the ability of the drug or target to impair the ability of the PI4KIII ⁇ protein to form a membrane egg complex white.
- the transformation of the A ⁇ monomer on the cell membrane to the polymer is reduced, and the secretion to the extracellular is increased.
- the method for screening a drug or a therapeutic target for treating AD can also be performed by directly detecting the application of the drug candidate or whether the regulation of the therapeutic target can lower the PI 4 P level of the cell membrane.
- fluorescence-microscopic, confocal or two-photon microscopy can be used to observe fluorescently labeled molecules that specifically bind to PI 4 P on the cell membrane, such as fluorescent protein-labeled OSH2-PH2X or OSH2-2x-PH fusion protein, on the cell membrane. Whether it is reduced, or whether it is transferred from the cell membrane to the cytosol.
- Example 1 Drosophila strain and genetic method
- the standard medium was circulated alternately under the conditions of 12 hours of light and 12 hours of darkness, and cultured at a constant temperature of 25 °C.
- the present invention employs the following transgenic Drosophila strains: rbo S358A , [UAS] A ⁇ arc , [UAS] A ⁇ 42 , [UAS] dtau, [UAS] mCD8-gfp (provided by Dr. Z. Wang), [UAS] shibire tsl (by Dr.A Guo), [Gal4] A307 ( supplied by Dr.O'Kane).
- the rbo S358A gene is a transgene constructed by site-directed mutagenesis of wild-type genomic DNA containing the rbo gene, and its expression is driven by the pre-driver of the rbo gene itself.
- [Gal4]A307 expresses the transcription factor Gal4, which is used to drive the [UAS]A ⁇ arc , [UAS]A ⁇ 42 , [UAS]dtau, [UAS]mCD8-gfp, [UAS]shibire ts1 transgene in the giant fiber pathway of Drosophila ( Giant Fiber system) Dynamin expressing neurons of A ⁇ arc , A ⁇ 42 , dTau, mCD8-GFP or temperature-sensitive mutations in neurons and a small number of other neurons.
- the Drosophila mutants used were: rbo ts1 (temperature-sensitive missense mutation), rbo 2 (knockout mutation), itpr sv35 (nonsense mutation, Bloomington Stock # 30740), PI4KIII ⁇ def (deleted PI4KIII ⁇ gene and its vicinity) Mutations in DNA, Bloomington Stock # 9518), PI4KIII ⁇ GS27 and PI4KIII ⁇ GJ86 (both nonsense mutations).
- all transgenic and mutant flies were backcrossed with a wild isogenic line (isogenic w 1118 , Bloomington stock #5 905) before use 5 Generations above.
- Drosophila (A ⁇ 42 or A ⁇ arc Drosophila) expressing wild-type or arctic mutant A ⁇ 42 in the GF pathway exhibits accumulation of A ⁇ 42 in neurons and age-dependent synaptic transmission. Failure and shortened life. These fruit flies also showed a decline in age-dependent crawling ability.
- two mutations of the rbo gene, the missense mutation (rbo ts1 ) and the knockout mutation (rbo 2 ) were introduced into the A ⁇ arc flies. They were tested for their effects on synaptic transmission, crawling ability, and longevity.
- Each group of fruit flies contains 1-2 lines, of which "ctrl” refers to the wild type control fruit fly with the [Gal4]A307 transgene; “rbo ts1/+ “ and “rbo 2/+ “ means One copy of [Gal4]A307 transgenic rbo ts1/+ and rbo 2/+ heterozygous Drosophila; "A ⁇ arc “ refers to a fruit fly with ([Gal4]A307/[UAS]A ⁇ arc double transgene; "A ⁇ arc -rbo ts1/+ ” and “A ⁇ arc -rbo 2/+ ” are respectively referred to as [Gal4]A307-rbo ts1 /[UAS]A ⁇ arc and [Gal4]A307-rbo 2 /[UAS]A ⁇ arc . Drosophila does not express A ⁇ arc and is classified as “non-A ⁇ flies
- Example 2 Detection of rbo gene mutation by synaptic transmission specifically inhibits A ⁇ 42 expression in Drosophila neurological disorders
- the excitatory junction potential (EJP) method of intracellular excitability was recorded in the escape pathway.
- Adult female flies of a specific age were fixed on a glass slide with a low melting wax, tackiwax (Boekel Scientific), with the abdomen down.
- the recording system includes a reference electrode on the abdomen, two stimulating electrodes inserted into the eye, and a recording electrode inserted into the longitudinal muscle cells of the back. Electrical stimulation (100 Hz, 50 pulses) was given to both eyes.
- the stimulation intensity is 5-20 Volts with a duration of 0.2 ms, which is about 150% of the threshold stimulation intensity.
- Figure 1 a representative record of brain stimulation-induced EJP in four groups of Drosophila at different ages (see Figure 1(a)), and a quantitative analysis of the success rate of induced EJPs (see Figure 1(b)) .
- the rbo mutation significantly inhibits age-dependent neurosynaptic transmission failure caused by A ⁇ arc .
- high-frequency electrical stimulation 100 Hz, 50 pulses
- EJP endplate potential
- Example 3 Detection of rbo gene mutation by creep test specifically inhibits A ⁇ 42 expression in Drosophila neurological disorders
- the ability to climb the tube was measured by measuring the height of 10 fruit flies crawling up from the bottom of an upright transparent plastic tube for 7 seconds.
- a highly reproducible fruit fly creep test capability test device was developed.
- the device comprises: 1) a rectangular metal frame (32 cm wide, Height 21cm), 10 transparent plastic tubes (inner diameter 2.1cm, height 19.0cm) are fixed vertically in the frame; 2) an electric motor is used to drive the metal frame to move in the vertical direction; 3) a stepper drive is used To control the working rhythm of the electric motor to make the metal frame move up and down continuously 4 times every 1 minute, each time moving up and down by a predetermined height; 4) a video recorder for recording the creeping process; 5) a set of analysis Software used to analyze the crawling position of fruit flies at specific times in the video.
- Example 4 Analysis of rbo gene mutations by lifespan specifically inhibits A ⁇ 42 expression in Drosophila neurological disorders
- Each genotype of 100 or 200 fruit flies was equally distributed to 5 or 10 tubes containing standard Drosophila food and dry yeast, and cultured at 25 °C. Drosophila was exchanged into tubes containing new food and dry yeast every 3 days and the number of dead fruit flies was recorded. Final use SPSS 11 Kaplan-Meier Software statistics survival rate.
- the rbo gene mutation has an effect on dyskinesia (left) and premature death caused by overexpression of Drosophila Tau protein in the Drosophila giant fiber pathway.
- the shibire gene mutation has an effect on dyskinesia (left) and premature death caused by overexpression of A ⁇ arc in the Drosophila giant fiber pathway.
- the toxic effects of rbo gene mutations against A ⁇ 42 cannot be attributed to a general effect on the accumulation of toxic proteins in neurons, as mutations in the rbo gene did not improve the lifespan of Drosophila overexpressing Tau protein (Fig. 8a).
- the toxic effect of the rbo gene mutation against A ⁇ 42 cannot be attributed to a general effect that may be based on synaptic function or down-regulation of endocytosis, since the introduction of a shibire gene mutation (Shibire ts1 ) into A ⁇ arc Drosophila does not alleviate A ⁇ arc
- the phenomenon of premature death of fruit flies Fig. 8b.
- mutations in the shibire ts1 gene also cause temperature-dependent synaptic transmission, bulk endocytosis, and motor function changes.
- Examples 1-4 the results show that mutation or deficiency of the rbo gene can specifically improve neurodegenerative diseases expressing wild-type and mutant A ⁇ 42 Drosophila.
- Example 5 Detection of PI4KIII ⁇ enzyme lacking or inhibiting interaction with RBO protein by immunoprecipitation to alleviate nerve damage in A ⁇ arc Drosophila
- Tris buffer formulation was as follows: 50 mM Tris, 50 mM KCl, 1 mM EDTA, 1% cocktail protease inhibitor (Calbiochem), adjusted to pH 7.4.
- the tissue homogenate was centrifuged at 10000 g for 10 minutes, and the supernatant was taken, and about 1 ⁇ g of a mouse-derived Drosophila RBO monoclonal antibody or a rabbit polyclonal antibody against Drosophila PI4KIII ⁇ was used for co-immunoprecipitation and immunoblotting.
- the above two antibodies were constructed in cooperation with Shanghai Abmart and China Abgent.
- RBO antibody polypeptide construct used is 251 th -500 th amino acid subtype C RBO Drosophila protein, polypeptide construct is against the Drosophila PI4KIII ⁇ NH2-KRSNRSKRLQYQKDSYC-CONH2 (SEQ ID NO: 3).
- the dilution ratio of the anti-Drosophila RBO and PI4KIII ⁇ antibodies was 1:2000.
- Anti-Drosophila RBO and PI4KIII ⁇ antibodies were verified by head tissue homogenate of wild-type and corresponding homozygous mutants, respectively.
- RBO is a putative diacylglycerol (DAG) lyase
- DAG diacylglycerol
- the homologous protein of the RBO protein in yeast cells and mice binds to the PI4KIII ⁇ protein and forms a complex on the cell membrane. Consistent with this, the RBO protein specifically immunoprecipitated with the Drosophila PI4KIII ⁇ protein (Fig. 1e).
- removal of one copy of the rbo gene (rbo 2/+ ) in A ⁇ arc -rbo Drosophila significantly reduced the expression levels of RBO and PI4KIII ⁇ proteins (Fig. 1f), while the rbo ts1/+ gene mutation did not decrease.
- the expression levels of RBO protein and PI4KIII ⁇ protein but significantly impaired the interaction between RBO protein and PI4KIII ⁇ protein (Fig. 1g). It is worth noting that the two rbo gene mutations did not alter the transcription of the PI4KIII ⁇ gene (Fig. 1h).
- a ⁇ arc Drosophila deletions are introduced into chromosomes (chromosome DNA fragment containing the deleted gene PI4KIII ⁇ , Pi4k def/+ ) and a nonsense mutation of PI4KIII ⁇ (PI4KIII ⁇ GS27/+ ).
- the A ⁇ staining method in the Drosophila central system is as follows.
- the antigenic determinants were exposed by washing with PBS for 30 minutes, followed by treatment with formic acid (70% in water) for 45 minutes, and then washed repeatedly with PBS-0.25% Triton-5% BSA.
- the primary antibody (6E10, 1:100 dilution) was incubated for 10-12 hours at 4 °C.
- FIG. 3(a) full-spectrum A ⁇ staining of the ventral ganglia of the control flies (upper row) and A ⁇ arc expressing Drosophila (middle row) expressing 21-day-old days of mCD8-GFP Confocal imaging; the expression of both mCD8-GFP and A ⁇ arc is driven by [Gal4]A307. Each set of stains was repeated twice; the lower row is an enlargement of the squared area in the middle row.
- the nerve damage caused by the expression of A ⁇ arc in the GF pathway was attributed to the accumulation of intracellular A ⁇ amyloid.
- the introduction of the uas-mCD8-gfp transgene into A ⁇ arc Drosophila further confirmed the accumulation of A ⁇ amyloid in neurons.
- the uas-mCD8-gfp transgene expresses the mCD8-GFP fluorescent protein localized to the cell membrane, which is driven by the same driver as A ⁇ arc , so neurons expressing A ⁇ arc are labeled by GFP, confocal microscopy It was revealed that most of the A ⁇ immunostaining signals co-localized with the GFP signal (Fig. 3(a)), thus confirming the phenomenon of A ⁇ accumulation in neurons in this Drosophila model.
- a ⁇ immunostaining was performed in A ⁇ arc , A ⁇ arc -rbo, and A ⁇ arc -PI4KIII ⁇ Drosophila.
- the A ⁇ immune response signal of A ⁇ arc -rbo and A ⁇ arc -PI4KIII ⁇ Drosophila was found to be significantly weaker than that of A ⁇ arc Drosophila (Fig. 3(b)).
- the A ⁇ 42 Human ELISA Kit (invitrogen) was used for the ELISA and experiments were performed according to the instructions for use.
- RT-PCR method detects the representative figure of the efficiency of knockdown of EFR3a gene in N2 cells (left side) and normalized quantitative analysis (middle), and the right side shows that knockdown of EFR3a gene does not affect N2a cells. Endocytosis of extracellular A ⁇ 42 .
- the sequence used to construct the knockdown Efra RNAi was: 5'-AGGTATCATTCAGGTTCTGTT-3' (SEQ ID NO: 4).
- Example 8 Preparation of PI4KIII ⁇ inhibitor PAO, A1 and G1 solutions and their toxicity test
- PAO PAO-containing fruit flies
- 50, 100, 200, 300, Foods of 400 and 600 ⁇ M PAO were cultured in wild-type Drosophila, starting from the embryonic stage. It was found that PAO of 200 ⁇ M and below had no significant effect on the emergence rate and creeping energy after emergence. Thus, A ⁇ arc fruit flies and control fruit flies were cultured at concentrations of 25, 50, 100 and 150 ⁇ M PAO.
- HEK239 cells When testing the toxicity of PAO and A1 on HEK239 cells, HEK239 cells were cultured in DMEM medium containing 50, 100, 150, 200, 250, 300, 400 and 600 nM PAO or A1 for 12 hours, and found to be 250 nM and above by MTT assay. Concentrations of both PAO and A1 kill most cells, and concentrations of 150 nM and below do not. Therefore, 25, 50, 100, and 150 nM PAO and A1 concentrations were selected for culture.
- PAO powder was dissolved in DMSO to prepare a mother liquor of 30 mg/ml. It was then diluted with distilled water to the desired concentration and the final concentration of DMSO was adjusted to avoid differences in DMSO concentration affecting the experimental results.
- Three-month-old C57BL/6 mice were first gavaged at doses of 18, 10, and 6 mg/kg body weight, and two mice were intragastrically administered per dose. On day 2, all mice were found dead. Then, the rats were intragastrically administered at a dose of 4.5 and 2.0 mg/kg body weight, and 5 mice were intragastrically administered per dose. For the 4.5 mg/kg body weight dose, one per day was administered, and after 5 consecutive days, 4 out of 5 mice survived.
- the patient was intragastrically administered once a week from week 1 to week 5 and stopped at the weekend. After 2 consecutive weeks, 5 mice were found to be alive.
- the median lethal dose of PAO is 2-6 mg/kg body weight, about 4 mg/kg body weight. Therefore, we selected 0.1, 0.3, and 1.0 mg/kg body weight to administer APP/PS1 and control mice, and administrated once a week from week 1 to week 5, and discontinued on weekends for 6 weeks.
- Example 9 Down-regulation of RBO/PI4KIII ⁇ promotes secretion of A ⁇ 42 by larval tissue culture expressing A ⁇
- the third instar larvae of Drosophila were sterilized with water and disinfected with 70% alcohol for 2 minutes, cut along the midline of the larvae in Schneider's (Sigma) medium, and the trachea, viscera and adipose tissue of the larvae were carefully removed.
- the dissected larvae were washed in Schneider's medium and transferred to a 2 ml centrifuge tube containing 150 ul of Schneider's medium and gentamicin (20 mg/ml). There were 5 dissected larvae in each tube and the tubes were placed in a humid, dark, constant temperature environment at 25 °C for 8 hours. Then, 100 ul of each tube was used to quantitatively test A ⁇ 42 by ELISA.
- the A ⁇ 42 Human ELISA Kit (invitrogen) was used for the ELISA.
- Fig. 4(a)-(c) show normalized quantitative analysis of different PAO concentration treatments, rbo gene and PI4KIII ⁇ gene mutations in cultured anatomically expressed A ⁇ 42 in medium expressing A ⁇ arc Drosophila third instar larvae. The impact of the level.
- Example 10 Detection of down-regulation of RBO/PI4KIII ⁇ promotes secretion of A ⁇ 42 by HEK293T cell culture expressing human APP
- HEK293T cell culture expressing human APP
- HEK293T cells stably transfected with human APP were cultured in DMEM medium (Hyclone), and 10% FBS (Gibco), penicillin and streptomycin, and G418 (100 ⁇ g/ml) were added.
- the HEK293T cells stably transfected with APP were cultured in a 12-well plate, and the concentration of PAO in the culture solution was 0, 25, 50, 100 or 150 nM, and after 6-8 hours of culture, an equal amount of cells were collected.
- cells were separately lysed with 500 ⁇ l of TBS buffer, centrifuged at low temperature (13,000 g) for 15 minutes, the supernatant was retained, and the pellet was resuspended in 500 ⁇ l of TBS buffer.
- a 2-fold reaction solution 50 mM Tris-HCl, pH 6.8, 4 mM EDTA, 0.5% CHAPSO (w/v)
- a specific fluorogenic substrate Calbiochem Cat. No. 565764
- the collected cells were lysed with a protease inhibitor (1% cocktail, invitrogen) in TBS buffer, and then subjected to immunoblotting using an anti-APP/A ⁇ antibody (6E10).
- a protease inhibitor 1% cocktail, invitrogen
- Figure 14 (b) shows immunoblotting A representative graph showing the amount of APP expression of HEK293T cells treated with different PAO concentrations, which was repeated 3 or more.
- a ⁇ ⁇ -amyloid precursor protein (APP) 42 detects a stably overexpressing secretion of A ⁇ HEK293T cells 42 of the human APP.
- FIG. 4 (d) - (g) the normalized quantitative analysis of different concentrations A1 and PAO, EFR3a PI4KA knockdown and culture medium human APP stably transfected HEK293 cells the level of A ⁇ 42 influences.
- PAO treatment produced similar effects in increasing the concentration of A ⁇ 42 in the medium (Fig. 4(d)), and PAO improved culture even in the presence of the ⁇ -secretase inhibitor DAPT (1uM).
- the concentration of A ⁇ 42 in the base Fig.
- PAO promoted the secretion of A ⁇ 42 by HEK293 cells stably transfected with APP, but did not change the activity of ⁇ , ⁇ , and ⁇ secretase of splicing APP.
- Figure 14 (a) did not cause an increase in APP levels.
- Figure 4 (b) did not cause an increase in APP levels.
- Example 11 Viral construction packaging and microinjection in mice
- This most potent miRNA vector is recombined with pDONRTM221 and pLenti6/V5 DEST
- the recombinant reaction yielded the pLENT6/V5-GW/ ⁇ EmGFP-miRNA vector.
- Lentiviruses were obtained by co-transfection of pLENT6/V5-GW/ ⁇ EmGFP-miRNA vector and Packaging Mix. Viral concentrations were obtained by serial dilution in HEK293T cells. EGFP positive cells were then counted every three days. Silencing efficiency was further obtained by lentiviral transfection of primary cultured hippocampal neurons.
- APP/PS1 transgenic male rats B6.Cg-Tg(APPswe, PSEN1dE9) 85Dbo/Mmjax (MMRRC ID 034832-JAX) were cross-bred by mouse hybridization with F1 band (C57BL/6 and C3H).
- mice were anesthetized with 100 mg/kg Ketamine plus 20 mg/kg Xylazine, fixed on a stereotactic device, and placed on the electric blanket on the abdomen. The head hair was removed, the skin was cut, and a small hole was drilled through the skull.
- the pump (Harvard Apparatus) was injected through a cannula system (external diameter, 0.29 mm, internal diameter, 0.1 mm, RWD Life Science Co., Ltd.) into 2 ⁇ l of lentivirus solution (virus concentration: 6x10 -7 ) to 2.1 in 20 minutes. Mm posterior to bregma, 2.3mm lateral and 1.9mm ventral. 5 minutes after injection, the needle was removed, the skin was sutured, and the mice were moved to an environment containing sufficient food and water at 25 ° C. At 12 months of age, the anesthesia was small again. Rats were perfused with 4% paraformaldehyde (PFA) in PBS. Experiments were performed on mice following the American Society of Neuroscience policy on animal use.
- PFA paraformaldehyde
- Example 12 Detection of knockdown of EFR3a gene by GFP staining of mouse brain slices can repair dendritic atrophy of APP/PS1 mouse neurons
- Brain slices were blocked with PBS-0.3% triton-5% BSA for 1 hour, and incubated overnight at 4 ° C using rabbit-derived anti-GFP antibody (A11122, invitrogen, 1:100 dilution). It was then washed with PBS and incubated overnight at 4 °C with biotinylated sheep-derived anti-rabbit IgG antibody (H+L) (AbboMax, Inc, 1:100 dilution). The cells were washed again with PBS, and incubated with Cy3-Streptavidin (Jackson ImmunoResearch Laboratories Inc, 1:1000 dilution) for 2 hours at room temperature.
- RT-PCR method detects knockdown of the intrinsic EFR3a gene (a) and PI4KA gene (b) in HEK293 cells, knockdown of overexpressed mice in HEK293 cells
- the EFR3a-gfp recombinant gene (c) The EFR3a-gfp recombinant gene (c), and the efficiency of knockdown of the mouse internal EFR3a gene (d) in mouse primary cultured hippocampal neurons.
- the representative figure is on the top, and the normalized method is quantitatively analyzed.
- RNAi that knock down the human Efr3a and PI4KA genes are: 5'-GGTTATTGAAATTCGAACT-3' (SEQ ID NO: 5) and 5'-TGCTCATTAGCAGTAAAGA-3' (SEQ ID NO: 6), respectively.
- n 3-5, the T test yields a P value.
- confocal imaging showed a full-screen (upper) hippocampal slice of anti-GFP immunostaining and a pyramidal cell (lower) of the CA3 region transfected with lentivirus.
- a fragment of the dendritic segment (between the two arrows) of about 30 ⁇ m in length was selected to quantify the diameter of the dendrite and the density of the dendritic spines.
- the scale bars are 500 microns (top) and 50 microns (bottom).
- EFR3a gene Mouse and humans have two rbo homologous genes, EFR3a gene and EFR3b gene, which are enriched in the AD susceptible region such as hippocampus (Allen brain atlas).
- AD susceptible region such as hippocampus (Allen brain atlas).
- Example 13 Analysis by detecting PAO CSF and brain membrane fractions isolated from mouse improvement APP / PS1 mice memory and learning, the increase in CSF A ⁇ 42, A ⁇ 42 but reduced in brain membranes
- mice were anesthetized with ketamine and xylazine, and the adapter was used to protect the head of the mouse.
- the hair of the mouse neck was shaved and the skin was cut, and the underlying subcutaneous tissue and muscle were separated to the sides with forceps to expose the portion of the dura mater covering the cisterna magna.
- the capillary is successfully inserted into the cisterna magna, and the cerebrospinal fluid is drawn into the capillary, collecting approximately 10-20 ⁇ l.
- the collected cerebrospinal fluid was transferred to a microcentrifuge tube and stored at -80 ° C until use.
- the detergent-soluble A ⁇ 42 was obtained by a series of extractions, and 5 times the volume of the cerebral hemisphere corresponding to the mouse cerebral hemisphere was ground with tromethamine buffer (TBS), ground to homogenate, and then placed in a centrifuge. The supernatant was centrifuged at 100,000 g for 60 min at 4 ° C, and the supernatant was a TBS extract. The lower precipitate was collected, and 5 volumes of TBS buffer containing 1% polyethylene glycol octylphenyl ether was added, ground again and centrifuged, and the upper layer was TBS-Triton extract.
- TBS tromethamine buffer
- the lower layer of the precipitate was collected, and 5 volumes of TBS buffer containing 1% SDS was added for a third grinding and centrifugation, and the supernatant was a TBS-SDS extract. Collect the last three clear liquids separately, put them in the refrigerator at -80 °C, and store them for ELISA.
- Fig. 6(a) the water maze experimental training curves of APP/PS1 mice (left panel) treated with different concentrations of PAO and wild-type mice born in littermates (right panel). For the convenience of comparison, the learning curve of APP/PS1 mice with a PAO concentration of 0 was shown in both the left and right images.
- Figure 6(b) the search time of the post-training control and APP/PS1 mice in the target quadrant as a percentage of the total search time.
- the ELISA method was used to quantitatively analyze the cerebrospinal fluid of APP/PS1 mice treated with different concentrations (see Figure (c)), and the brain cell membrane fraction extracted from TBS buffer of 1% Triton and 1% SDS (see Figure 6(d)). A ⁇ 42 levels.
- the ELISA method was used to quantify the A ⁇ 42 assay in the cerebral cell membrane fraction extracted from the TBS buffer of APP/PS1 mice (left panel) and 1% SDS at 100 °C for different time. influences.
- the ELISA method quantitatively analyzes the A ⁇ 42 level in the brain cell membrane fraction extracted from TBS buffer of 1% Triton and 1% SDS after 60 minutes of treatment at 100 °C.
- the amount of A? 42 released from each brain cell membrane fraction extracted from 1% Triton and 1% SDS TBS buffer after 100 minutes of treatment at 100 °C accounted for a percentage of the total amount.
- P values were analyzed by one-way ANOVA.
- Example 14 Detection of mutant mouse PI4KIII ⁇ by water maze test improves learning and memory ability of APP/PS1 mice
- Example 15 Effect of PI, PI 4 P, PI 4,5 P on the polymerization of A ⁇ 42 in liposomes by liposome assay
- a ⁇ 42 and various lipids were mixed together at a ratio of Table 1, and then the organic solvent in the mixture was drained with a freeze dryer.
- the effects and comparisons of PI, PI 4 P, PI 4 , 5 P on the formation of A ⁇ 42 oligomers in liposomes were analyzed.
- the left column of Figure 13 shows the PI 4 P versus A ⁇ 42 oligos in liposomes.
- the promotion of polymer formation is concentration-dependent, and the upper and lower parts are the result of relatively short and long-term post-exposure development of the same immunoblotting membrane; please note the promotion of A ⁇ 42 oligomer formation at a concentration of 80 ⁇ M PI 4 P The effect is weaker than 40 ⁇ M.
- the right column of Figure 13 shows the effect of PI, PI 4 P and PI 4,5 P on the polymerization of A ⁇ 42 in liposomes, and the upper and lower parts are the result of relatively short and long-term post-exposure development of the same immunoblotting film. It is noted that the effect of PI 4 P is significantly better than that of PI and PI 4,5 P, and that PI and PI 4 P promote the formation of A ⁇ 42 trimer and above oligomers more strongly than PI 4,5 P.
- Example 16 RBO/EFR3/EFR3A/EFR3B, PI4KIIIa and TTC7 form a complex on the cell membrane
- yeast EFR3 protein and PI4KIIIa and a scaffolding protein YPP1 form a complex on the cell membrane, and clustered (PIK patchs), The PI 4 P levels of the membrane, even the PI 4, 5 P levels, are controlled together.
- YPP1 interacts directly with the N-terminal and intermediate regions of the yeast PI4KIIIa protein and plays a key role in the construction and stabilization of PIK patchs (Baird D, Stefan C, et al., 2008, J Cell Biol).
- composition and function of PIK patchs are also conserved in mammalian cells (Nakatsu F, Baskin JF, et al., 2012, J Cell Biol).
- the Drosophila homologous protein of TTC7 in Drosophila is encoded by the lethal(2)k14710[(l(2)k14710) gene.
- transpon-mediated transgenes were introduced into A ⁇ arc Drosophila, one for P ⁇ lacW ⁇ l(2). ) k14710k 15603 , (Bloomington Cat. #11134)
- the transposon is inserted in the first exon of the (l) k14710 gene, preventing transcription of the (l) k14710 gene; the other is P ⁇ EPgy2 ⁇ bin3 EY09582 , (Bloomington Cat. #20043).
- This experiment constructed a total of 4 groups of fruit flies: control fruit fly (ctrl), A ⁇ arc fruit fly, A ⁇ containing one copy of P ⁇ lacW ⁇ l(2)k14710k 15603 Arc flies (A ⁇ arc -dttc7 +/- , TTC7 down-regulated) and A ⁇ arc fruit flies (A ⁇ arc -dttc7-OE, TTC7 overexpressing) containing a copy of P ⁇ EPgy2 ⁇ bin3 EY09582 .
- control fruit fly ctrl
- a ⁇ arc fruit fly A ⁇ containing one copy of P ⁇ lacW ⁇ l(2)k14710k 15603
- Arc flies A ⁇ arc -dttc7 +/- , TTC7 down-regulated
- a ⁇ arc fruit flies A ⁇ arc -dttc7-OE, TTC7 overexpressing
- FIG 15 shows the effect of down-regulation and overexpression of TTC7 on neurotransmission in A ⁇ arc Drosophila, respectively.
- Drosophila ctrl
- overexpression of TTC7 A ⁇ arc-dttc7-OE
- down-regulation of TTC7 expression A ⁇ arc-dttc7+/-
Abstract
Description
Claims (6)
- 一种筛选用于治疗阿尔茨海默病的药物以及治疗靶点的方法,所述方法包含筛选能提高细胞分泌Aβ水平的候选药物或治疗靶点。
- 如权利要求1所述的筛选用于治疗阿尔茨海默病的药物以及治疗靶点的方法,所述方法包括以下步骤:(1)将所述药物应用于过量表达APP或含分泌信号肽的Aβ的细胞系或果蝇组织或调节过量表达APP或含分泌信号肽的Aβ的细胞系或果蝇组织中的所述治疗靶点;以及(2)利用免疫分析方法检测所述过量表达APP或含分泌信号肽的Aβ的细胞系或果蝇组织中细胞分泌Aβ的情况,其中,如果所述药物的应用或治疗靶点的调节能导致细胞分泌Aβ的量增加,则所述药物或治疗靶点可用于治疗阿尔茨海默病。
- 如权利要求1或2中任一所述的筛选用于治疗阿尔茨海默病的药物以及治疗靶点的方法,所述Aβ为Aβ42。
- 如权利要求2中所述的筛选用于治疗阿尔茨海默病的药物以及治疗靶点的方法,所述检测Aβ的分泌情况的免疫分析方法为酶联免疫吸附剂测定或电化学发光免疫分析方法。
- 如权利要求2中所述的筛选用于治疗阿尔茨海默病的药物以及治疗靶点的方法,所述过量表达APP或含分泌信号肽的Aβ的细胞系为稳转人源APP或含分泌信号肽的Aβ的HEK293T、N2a或SH-SY5Y细胞系。
- 如权利要求2中所述的筛选用于治疗阿尔茨海默病的药物以及治疗靶点的方法,所述过量表达APP或含分泌信号肽的Aβ的果蝇组 织为果蝇三龄幼虫组织。
Priority Applications (11)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2015392754A AU2015392754B2 (en) | 2015-04-30 | 2015-04-30 | Method for screening drug and therapeutic target used for treating Alzheimer's disease |
KR1020217008474A KR102488987B1 (ko) | 2015-04-30 | 2015-04-30 | 알츠하이머 질환을 치료하는데 사용되는 약물 및 치료 표적제를 스크리닝하는 방법 |
CN201580079376.9A CN107849608A (zh) | 2015-04-30 | 2015-04-30 | 一种筛选用于治疗阿尔茨海默病的药物和治疗靶点的方法 |
KR1020207002081A KR20200011567A (ko) | 2015-04-30 | 2015-04-30 | 알츠하이머 질환을 치료하는데 사용되는 약물 및 치료 표적제를 스크리닝하는 방법 |
KR1020177034631A KR20180012274A (ko) | 2015-04-30 | 2015-04-30 | 알츠하이머 질환을 치료하는데 사용되는 약물 및 치료 표적제를 스크리닝하는 방법 |
ES15890335T ES2913526T3 (es) | 2015-04-30 | 2015-04-30 | Método de cribado de fármacos y dianas terapéuticas para el tratamiento de la enfermedad de Alzheimer |
DK15890335.1T DK3290525T3 (da) | 2015-04-30 | 2015-04-30 | Fremgangsmåde til screening af lægemiddel og terapeutisk mål der anvendes til behandling af alzheimers sygdom |
PCT/CN2015/078070 WO2016172955A1 (zh) | 2015-04-30 | 2015-04-30 | 一种筛选用于治疗阿尔茨海默病的药物和治疗靶点的方法 |
US15/570,672 US11236378B2 (en) | 2015-04-30 | 2015-04-30 | Method for screening drug and therapeutic targets used for treating Alzheimer's disease |
EP15890335.1A EP3290525B1 (en) | 2015-04-30 | 2015-04-30 | Method for screening drug and therapeutic target used for treating alzheimer's disease |
JP2018507760A JP6636614B2 (ja) | 2015-04-30 | 2015-04-30 | アルツハイマー病の治療に用いられる薬物および治療標的のスクリーニング方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/CN2015/078070 WO2016172955A1 (zh) | 2015-04-30 | 2015-04-30 | 一种筛选用于治疗阿尔茨海默病的药物和治疗靶点的方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2016172955A1 true WO2016172955A1 (zh) | 2016-11-03 |
Family
ID=57198995
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2015/078070 WO2016172955A1 (zh) | 2015-04-30 | 2015-04-30 | 一种筛选用于治疗阿尔茨海默病的药物和治疗靶点的方法 |
Country Status (9)
Country | Link |
---|---|
US (1) | US11236378B2 (zh) |
EP (1) | EP3290525B1 (zh) |
JP (1) | JP6636614B2 (zh) |
KR (3) | KR102488987B1 (zh) |
CN (1) | CN107849608A (zh) |
AU (1) | AU2015392754B2 (zh) |
DK (1) | DK3290525T3 (zh) |
ES (1) | ES2913526T3 (zh) |
WO (1) | WO2016172955A1 (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021057955A1 (zh) * | 2019-09-27 | 2021-04-01 | 江苏挪贝肽医药科技有限公司 | 一种治疗心境障碍的方法 |
WO2021197396A1 (zh) * | 2020-03-31 | 2021-10-07 | 挪贝肽医药科技(上海)有限公司 | 氘代氧化苯砷化合物及其应用 |
WO2023051805A1 (zh) * | 2021-09-30 | 2023-04-06 | 挪贝肽医药科技(上海)有限公司 | 卤代氧化苯砷化合物及其应用 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114807114A (zh) * | 2022-04-14 | 2022-07-29 | 广州医科大学附属第二医院 | 一种记录果蝇大脑单细胞长时程增强电生理信号的方法 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1329674A (zh) * | 1998-12-07 | 2002-01-02 | 阿文蒂斯药物德国有限公司 | Aβ-肽的筛选方法 |
CN1339066A (zh) * | 1998-09-24 | 2002-03-06 | 法玛西雅厄普约翰美国公司 | 阿尔茨海默氏疾病分泌酶 |
CN102265806A (zh) * | 2010-05-13 | 2011-12-07 | 中国科学院上海生命科学研究院 | 抗阿尔茨海默症转基因果蝇模型及其在药物筛选中的应用 |
CN103529182A (zh) * | 2012-07-06 | 2014-01-22 | 中国科学院上海生命科学研究院 | rbo/Efr3a/Efr3b基因或其蛋白在诊断和治疗阿尔茨海默病中的应用 |
CN104046676A (zh) * | 2013-03-13 | 2014-09-17 | 中国科学院上海生命科学研究院 | 清除淀粉样多肽的细胞及其应用 |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994001772A1 (en) * | 1992-07-13 | 1994-01-20 | The Children's Medical Center Corporation | SCREEN FOR ALZHEIMER'S DISEASE THERAPEUTICS BASED ON β-AMYLOID PRODUCTION |
WO2003072037A2 (en) * | 2002-02-27 | 2003-09-04 | Pharmacia & Upjohn Company | High-level production of amyloid-beta peptides from imr-32 cells |
EP1680120A2 (en) * | 2003-11-03 | 2006-07-19 | Probiodrug AG | Combinations useful for the treatment of neuronal disorders |
WO2006096529A2 (en) * | 2005-03-07 | 2006-09-14 | Novartis Ag | Genes involved in neurodegenerative conditions |
CA2617294A1 (en) * | 2005-08-03 | 2007-02-08 | Boehringer Ingelheim International Gmbh | Substituted ethane-1,2-diamines for the treatment of alzheimer's disease ii |
CN1907287A (zh) * | 2006-08-23 | 2007-02-07 | 华东师范大学 | β-淀粉样肽抑制剂及其筛选方法 |
ITMI20071975A1 (it) * | 2007-10-12 | 2009-04-13 | Fond I R C C S Istituto Neur O | Prodotti e loro uso per la diagnosi prevenzione e-o cura di patologie umane e-o animali caraterizzate dalla anomala deposizione di sostanza b-amiloide e-o similamiloide in organi e tesstui umani e-o animali e metodo di screening per la determinazione |
-
2015
- 2015-04-30 JP JP2018507760A patent/JP6636614B2/ja active Active
- 2015-04-30 US US15/570,672 patent/US11236378B2/en active Active
- 2015-04-30 KR KR1020217008474A patent/KR102488987B1/ko active IP Right Grant
- 2015-04-30 KR KR1020207002081A patent/KR20200011567A/ko active Application Filing
- 2015-04-30 DK DK15890335.1T patent/DK3290525T3/da active
- 2015-04-30 KR KR1020177034631A patent/KR20180012274A/ko not_active Application Discontinuation
- 2015-04-30 ES ES15890335T patent/ES2913526T3/es active Active
- 2015-04-30 WO PCT/CN2015/078070 patent/WO2016172955A1/zh active Application Filing
- 2015-04-30 CN CN201580079376.9A patent/CN107849608A/zh active Pending
- 2015-04-30 EP EP15890335.1A patent/EP3290525B1/en active Active
- 2015-04-30 AU AU2015392754A patent/AU2015392754B2/en active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1339066A (zh) * | 1998-09-24 | 2002-03-06 | 法玛西雅厄普约翰美国公司 | 阿尔茨海默氏疾病分泌酶 |
CN1329674A (zh) * | 1998-12-07 | 2002-01-02 | 阿文蒂斯药物德国有限公司 | Aβ-肽的筛选方法 |
CN102265806A (zh) * | 2010-05-13 | 2011-12-07 | 中国科学院上海生命科学研究院 | 抗阿尔茨海默症转基因果蝇模型及其在药物筛选中的应用 |
CN103529182A (zh) * | 2012-07-06 | 2014-01-22 | 中国科学院上海生命科学研究院 | rbo/Efr3a/Efr3b基因或其蛋白在诊断和治疗阿尔茨海默病中的应用 |
CN104046676A (zh) * | 2013-03-13 | 2014-09-17 | 中国科学院上海生命科学研究院 | 清除淀粉样多肽的细胞及其应用 |
Non-Patent Citations (1)
Title |
---|
See also references of EP3290525A4 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021057955A1 (zh) * | 2019-09-27 | 2021-04-01 | 江苏挪贝肽医药科技有限公司 | 一种治疗心境障碍的方法 |
WO2021197396A1 (zh) * | 2020-03-31 | 2021-10-07 | 挪贝肽医药科技(上海)有限公司 | 氘代氧化苯砷化合物及其应用 |
EP4130014A4 (en) * | 2020-03-31 | 2024-05-15 | Nuo Beta Pharmaceutical Tech Shanghai Co Ltd | DEUTERATED OXOPHENYLARSINE COMPOUND AND ITS USE |
WO2023051805A1 (zh) * | 2021-09-30 | 2023-04-06 | 挪贝肽医药科技(上海)有限公司 | 卤代氧化苯砷化合物及其应用 |
Also Published As
Publication number | Publication date |
---|---|
ES2913526T3 (es) | 2022-06-02 |
AU2015392754B2 (en) | 2019-03-14 |
DK3290525T3 (da) | 2022-03-14 |
US20190211371A1 (en) | 2019-07-11 |
US11236378B2 (en) | 2022-02-01 |
KR20210035917A (ko) | 2021-04-01 |
KR20200011567A (ko) | 2020-02-03 |
CN107849608A (zh) | 2018-03-27 |
JP6636614B2 (ja) | 2020-01-29 |
EP3290525A4 (en) | 2018-12-19 |
KR20180012274A (ko) | 2018-02-05 |
EP3290525B1 (en) | 2022-02-23 |
JP2018520367A (ja) | 2018-07-26 |
EP3290525A1 (en) | 2018-03-07 |
AU2015392754A1 (en) | 2017-12-21 |
KR102488987B1 (ko) | 2023-01-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2016172952A1 (zh) | PI4KIIIα蛋白及相关的膜蛋白复合体在治疗阿尔茨海默病中的应用 | |
Zhang et al. | The X-linked intellectual disability protein PHF6 associates with the PAF1 complex and regulates neuronal migration in the mammalian brain | |
WO2016172955A1 (zh) | 一种筛选用于治疗阿尔茨海默病的药物和治疗靶点的方法 | |
Odfalk et al. | Microglia: Friend and foe in tauopathy | |
Zhang et al. | Downregulation of RBO-PI4KIIIα facilitates Aβ42 secretion and ameliorates neural deficits in Aβ42-expressing Drosophila | |
ES2923015T3 (es) | Fijación como objetivo de sinaptogirina-3 en el tratamiento de tauopatías | |
Meng et al. | TMEM59 haploinsufficiency ameliorates the pathology and cognitive impairment in the 5xFAD mouse model of alzheimer’s disease | |
US20240173287A1 (en) | Application Of PI4KIIIA Protein And Related Membrane Protein Complex In Treating Alzheimer's Disease | |
CN114601928B (zh) | 一种钙超载介导神经元死亡的标志物及应用 | |
Yi | Multi-facet Roles of MG29, a Synaptophysin Family Protein, in Skeletal Muscle Development, Regeneration, and Metabolic Function | |
Morozova | Mechanism of Tau Propagation: Putative Therapeutic Approaches | |
Sarah | Targeting the M1 muscarinic acetylcholine receptor in neurodegeneration | |
王萱 et al. | Cellular and Genetic Studies of α-Synuclein Propagation in the C. elegans Neuronal Circuit. | |
CA3099946A1 (en) | Use of nod2 agonist for the treatment, prophylaxis and/or delay of the onset of multiple sclerosis and alzheimer's disease | |
Hoang | Role of the 39-kDa receptor-associated protein (RAP) in Alzheimer's disease | |
Tournoy | Physiological Study of Presenilins and Baces, Two Proteases Involved in the Pathogenesis of Alzheimer's Disease | |
JP2009102231A (ja) | 肥満細胞の脱顆粒抑制剤 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 15890335 Country of ref document: EP Kind code of ref document: A1 |
|
DPE1 | Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101) | ||
ENP | Entry into the national phase |
Ref document number: 2018507760 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 20177034631 Country of ref document: KR Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2015890335 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2015392754 Country of ref document: AU Date of ref document: 20150430 Kind code of ref document: A |