JP2005130791A - GENE INHIBITING CELL DEATH CAUSED BY AMYLOID beta PROTEIN, AND PROTEIN - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a gene utilizable for prevention and treatment of amyloid protein-storage disease such as Alzheimer's disease, and to provide a protein. <P>SOLUTION: The new gene is BF5-2 or BF5-1 which is a splice valiant thereof. The protein BF5-1 is encoded thereby. The gene and the protein inhibit the storage of the amyloid protein to enable them to be used for a medicinal composition or the like for preventing or treating the amyloid protein-storage disease such as the Alzheimer's disease. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel gene BF5-2 and its splice variant BF5-1 and the protein BF5-1 encoded thereby. These novel genes and protein BF5-1 suppress cell death observed in human cells, preferably human nerve cells, by amyloid β protein (hereinafter sometimes abbreviated as “Aβ” or “Aβ protein”). New genes and new proteins. These genes and proteins suppress the toxicity of amyloid β protein in diseases related to amyloid β toxicity, such as Alzheimer's disease.

  The present invention also relates to a pharmaceutical composition that suppresses amyloid β toxicity and gene therapy using the above gene sequence. Furthermore, the present invention relates to a protein and a low molecular weight compound that suppress or promote cell death through an interaction with Aβ that causes cell death directly or with protein BF5-1, or diagnosis and prevention using them.

In recent years, the prevalence of Alzheimer's disease due to aging has increased. Alzheimer's disease imposes a great burden not only on patients but also on the side of caregiving, and it is expected to establish an effective treatment or prevention method. Currently, there is Donepezil hydrochloride (Aricept, Eisai Co., Ltd.) as a therapeutic drug for Alzheimer's disease on the market. This drug improves dementia symptoms by inhibiting cholinesterase in the brain. However, since it is not a fundamental therapy that improves the etiology of Alzheimer's disease, the actual condition is that the progression of the disease cannot be stopped (Drug 61, 41 (2001)). Therefore, development of a drug that realizes this fundamental treatment is expected.
Senile plaques are frequently observed in brains with Alzheimer's disease. This senile plaque is composed of a protein called Aβ (Proc. Natl. Acad. Sci. USA 82, 4245 (1985)). This Aβ is a component of amyloid precursor protein (APP), which is cleaved by the action of two secretases and produced as one of the fragments (Science 286, 735 (1999); Mol. Cell Neurosci. 14, 419 ( 1999); Nature, 402,533 (1999); Nature 405,689 (2000); Nature Cell Biol. 2, 428 (2000)). This protein has the property of self-aggregating and forms a polymer based on a β-sheet structure. In experiments using cultured neurons, it has been reported that this protein polymer is toxic (Cell Mol. Life Sci. 57, 705 (2000)). In familial Alzheimer's disease, it has been reported that the production of Aβ1-42 (amyloid β protein 1-42), which is more likely to aggregate, is increased by mutation of the amino acid sequence of APP or secretase (Science 235,880 (1987). Science 235, 885 (1987)). Against this background, Aβ is considered as a pathogen of Alzheimer's disease (see Non-Patent Documents 1 and 2, etc.), and a method for reducing the toxicity of this protein to neurons is being sought.
Trend Pharmacol. Sci. 12, 383 (1991) Ann. Neurol. 37, 287 (1995)

  The present invention solves the problem of finding a novel gene / new protein that suppresses the toxicity of amyloid β protein in diseases related to Aβ toxicity, such as Alzheimer's disease, and applying these to the treatment of diseases related to Aβ toxicity, such as Alzheimer's disease. It is a challenge to try.

  Therefore, we used a human neuroblast SH-SY5Y cell, which was induced to differentiate into a neuron by retinoic acid and BDNF (Brain derived neurotrophic factor), and developed a cDNA library for detecting only the expression of mRNA derived from the cDNA library. Targeted differential display was performed, and novel genes BF5-2 and BF5-1 encoding proteins that suppress Aβ toxicity were found from a human fetal gene library.

That is, the present invention provides DNAs, proteins, vectors, transformants, pharmaceutical compositions and kits described in the following (1) to (20).
(1) DNA having the DNA sequence set forth in SEQ ID NO: 1 in the Sequence Listing.
(2) DNA that hybridizes with the DNA of (1) above under stringent hybridization conditions.
(3) DNA having 70% or more homology with the DNA of (1) or (2) above.
(4) DNA having the DNA sequence set forth in SEQ ID NO: 2 in the Sequence Listing.
(5) A DNA that hybridizes with the DNA of (4) above under stringent hybridization conditions.
(6) DNA having 70% or more homology to the DNA of (4) or (5) above.
(7) DNA having a nucleotide sequence of nucleotides 184 to 282 set forth in SEQ ID NO: 3 in the sequence listing.
(8) DNA that hybridizes with the DNA of (7) above under stringent hybridization conditions.
(9) DNA having 70% or more homology to the DNA of (7) or (8) above.
(10) RNA complementary to the DNA according to any one of (1) to (9) above.
(11) A BF5-1 protein having the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing.
(12) A DNA encoding a protein having one or several amino acids inserted, deleted or substituted in the protein of (11) and having an Aβ toxicity-inhibiting effect equivalent to that of the BF5-1 protein.
(13) A BF5-1 protein encoded by the DNA or RNA of (1) to (3), (7) to (10) or (12) above, or a protein having an Aβ toxicity inhibitory effect equivalent thereto.
(14) A vector comprising the DNA sequence or RNA sequence of (1) to (3), (7) to (10) or (12) above.
(15) A transformant transformed with the vector according to (14) above.
(16) DNA or RNA containing the DNA sequence or RNA sequence of (1) to (3), (7) to (10) or (12) above, or an Aβ accumulating disease containing the vector of (14) above A therapeutic pharmaceutical composition.
(17) Inhibition of Aβ toxicity by the DNA or RNA of (1) to (3), (7) to (10) or (12), or the BF5-1 protein encoded by the vector of (14) or equivalent thereto A pharmaceutical composition for the treatment of an Aβ accumulating disease comprising a protein having an effect.
(18) DNA or RNA comprising the DNA sequence or RNA sequence of (1) to (3), (7) to (10) or (12) above, or the vector of (14) above, to a cell obtained from a patient A method for treating an Aβ-accumulating disease, comprising introducing, culturing the cell, and then introducing the cell into the patient.
(19) A protein or low molecular weight compound that suppresses or promotes cell death through interaction with amyloid β protein directly or with protein BF5-1.
(20) A kit or pharmaceutical composition comprising the protein or low molecular weight compound according to (19) above for diagnosis or prevention of an amyloid β protein accumulation disease.

  According to the present invention, there are provided novel genes BF5-2 and BF5-1 encoding a protein that suppresses Aβ toxicity, and a novel protein BF5-1 encoded by them, such as diseases related to amyloid β toxicity, such as Alzheimer It can be applied to the treatment of diseases.

The present invention is described in further detail below.
In one embodiment, the present invention relates to a DNA having the DNA sequence set forth in SEQ ID NO: 1 in the Sequence Listing, that is, a BF5-1 full-length gene. The present invention also relates to DNA that hybridizes under stringent conditions to the BF5-1 full-length gene. Stringent hybridization conditions are described in many textbooks and literature, and are well known to those skilled in the art. For example, at least 70% or more, preferably 80% or more, more preferably 90%, with respect to a certain DNA. %, More preferably 95% or more of the DNA sequence having a nucleotide sequence homology only. As an example, conditions for hybridizing at 42 ° C. in a solution containing 6 × SSC, 5 × Denhardt's reagent and 0.5% SDS, 100 μg / ml salmon sperm DNA can be mentioned. Further, the present invention provides at least 70%, preferably 80% or more, more preferably 90% or more, most preferably 95% or more of the BF5-1 full-length gene or DNA hybridized thereto under stringent conditions. It also relates to DNA having nucleotide sequence homology. In the present specification, at least 70% of the DNA that hybridizes under stringent conditions to the BF5-1 full-length gene and the DNA that hybridizes thereto under stringent conditions under the BF5-1 full-length gene. In some cases, DNAs having nucleotide sequence homology of 80% or more, more preferably 90% or more, and most preferably 95% or more are collectively referred to as BF5-1 gene variants.

  In one aspect, the present invention relates to a DNA having the DNA sequence set forth in SEQ ID NO: 2 in the Sequence Listing, that is, the BF5-2 full-length gene. The present invention also relates to DNA that hybridizes under stringent conditions to the BF5-2 full-length gene. Stringent hybridization conditions are as described above. Further, the present invention provides at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more of the BF5-2 full-length gene or DNA hybridized thereto under stringent conditions. It also relates to DNA having nucleotide sequence homology. As described in the Examples section, the BF5-1 gene was found to be a splice variant of the BF5-2 gene. In the present specification, at least 70% or more of DNA that hybridizes under stringent conditions to the BF5-2 full-length gene, and DNA that hybridizes to it under stringent conditions or BF5-2. DNAs having nucleotide sequence homology of preferably 80% or more, more preferably 90% or more, and most preferably 95% or more may be collectively referred to as BF5-2 gene variants.

  Furthermore, since the present invention shows that cells transfected with the BF5-1 gene become resistant to Aβ toxicity, as shown in the Examples section, the present invention particularly relates to the BF5-1 gene. The present invention relates to a novel protein BF5-1 encoded by BF5-1 ORF. As explained in the Examples section, BF5-1 protein has the effect of suppressing Aβ toxicity. As further described in the Examples section, protein BF5-1 was found to be encoded by the DNA sequence from nucleotides 184 to 282 of the BF5-1 full-length gene shown in SEQ ID NO: 1. Therefore, the present invention relates to a DNA having the base sequence from nucleotide 184 to 282 of SEQ ID NO: 3. In this specification, this DNA is referred to as BF5-1ORF (ORF is an abbreviation for open reading frame) and is described in SEQ ID NO: 3. The present invention also relates to DNA that hybridizes with BF5-1 ORF under stringent hybridization conditions. Stringent hybridization conditions are as described above. The present invention also relates to a nucleotide sequence of at least 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more with respect to BF5-1ORF or DNA that hybridizes under stringent conditions. It also relates to DNA having homology.

  The present invention also relates to DNA having the DNA sequence set forth in SEQ ID NO: 1 in the sequence listing, that is, DNA that hybridizes under stringent conditions to the BF5-1 full-length gene, BF5-1 full-length gene, or BF5 -1 having at least 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more nucleotide sequence homology to a full-length gene or DNA that hybridizes to it under stringent conditions It also relates to proteins encoded by DNA (sometimes referred to as polypeptides). In particular, the present invention relates to BF5-1ORF shown in SEQ ID NO: 3, DNA that hybridizes to BF5-1ORF under stringent hybridization conditions, or DNA that hybridizes to BF5-1ORF or stringent conditions under this condition. And at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more. In the present specification, at least 70% or more of DNA that hybridizes under stringent conditions to the BF5-1 full-length gene, or DNA that hybridizes thereto under stringent conditions under the BF5-1 full-length gene, DNA having nucleotide sequence homology of preferably 80% or more, more preferably 90% or more, and most preferably 95% or more may be collectively referred to as a BF5-1ORF variant. In the present specification, the protein (also referred to as polypeptide) encoded by the BF5-1 ORF shown in SEQ ID NO: 3 is referred to as BF5-1 protein (also referred to as polypeptide). Further, in the present specification, at least 70% or more, preferably at least 70% or more of DNA that hybridizes with BF5-1ORF under stringent hybridization conditions, and DNA that hybridizes with BF5-1ORF or stringent conditions under it, preferably Proteins encoded by DNA having a nucleotide sequence homology of 80% or more, more preferably 90% or more, and most preferably 95% or more may be collectively referred to as BF5-1 protein variants. As described above, the BF5-1 protein and the BF5-1 protein variant have the same effect of suppressing Aβ toxicity. For example, a protein having one or several amino acids inserted, deleted or substituted in the amino acid sequence of the BF5-1 protein and having an Aβ toxicity inhibitory effect equivalent to that of the BF5-1 protein is also included in the BF5-1 protein variant. Is done. Again, stringent conditions include the examples described above and are well known to those skilled in the art. In general, such BF5-1 protein variants have at least 70%, preferably 80% or more, more preferably 90% or more, most preferably 95% or more amino acid sequence homology to the BF5-1 protein. .

  Therefore, the protein or polypeptide encoded by the above-mentioned BF5-1 gene and BF5-1 gene variants, particularly BF5-1 ORF and BF5-1 ORF variants, has an effect of suppressing Aβ toxicity.

  The present invention further relates to RNA complementary to the above DNA. Accordingly, the BF5-1 protein or BF5-1 variant of the present invention may be encoded by RNA complementary to the BF5-1 ORF or BF5-1 ORF variant.

  The method for obtaining DNA or RNA may be an appropriate combination of methods known in the art. For example, the DNA of the present invention can be obtained by transforming a cDNA library (preferably a human cDNA library) into a mammalian cell (preferably a human cDNA library). It can be obtained by appropriately combining a differential hybridization method and a PCR method. One example is described in detail in the Examples section. General methods for determining and estimating DNA and amino acid sequences can also be used.

  The Aβ toxicity-inhibiting effect of the DNA, RNA or protein of the present invention is the cell death of cells given Aβ (preferably human cells) and cells given Aβ given the DNA, RNA or protein to be tested. It can be examined from the ratio. For example, the cell death rate when Aβ (for example, Aβ1-40) is given to cells into which the vector of the present invention has been introduced and cells that have not been introduced may be examined. Such tests are also detailed in the Examples section.

  Furthermore, the present invention, in another aspect, includes DNA containing the nucleotide sequence of the BF5-1 full-length gene, BF5-1 gene variant, BF5-1 ORF, or BF5-1 ORF variant, or RNA complementary to these DNAs. Provide vector. Furthermore, the present invention also provides a transformant transformed with such a vector.

  Various vectors described in the literature or various commercially available vectors can be used as vectors used for transformation of nerve cells by inserting any of the above DNAs. For example, pVgRXR, pIND, pIND / V5 Viruses such as His, pIND / GFP, pcDNA3.1 or pcDNA3.1 / myc or pcDNA3.1 / His or pcDNA3.1 / V5-His or pcDNA3.1-CT-GFP-TOPO®, or LNCX2 There are vectors. Known transformation methods include calcium phosphate method, Super Fector reagent method, lipid-mediated method using LipofectAMINE reagent and the like. Any cell may be used as a cell for screening, but a nerve cell is preferable, and examples of preferable nerve cell include PC12 cell, NB2a, Neuro-2A, B104, SH-SY5Y, primary cultured nerve cell and the like. . In particular, SH-SY5Y cells are preferred. The conditions for constructing the vector, cell transformation, and cell culture are determined according to individual cells, and are within the knowledge of those skilled in the art or can be easily determined. Such a transformant is also included in the present invention. It is desirable to attach a detectable tag to the protein to be expressed so that the expression of the BF5-1 protein or BF5-1 protein variant can be easily checked. Tags can be added at the DNA level, for example, expressed by fusing green fluorescent protein DNA, red fluorescent protein DNA, myc gene, etc. to a DNA sequence encoding BF5-1 protein or BF5-1 protein variant Can be made. In particular, a fusion product with green fluorescent protein is recommended as a simple and highly sensitive one. After selecting the expression cells, it is necessary to check the cell death inhibitory effect by the screening assay described herein.

  Cells expressing BF5-1 protein or BF5-1 protein variants are extracted by appropriate means known to those skilled in the art (for example, use of immunoprecipitation with BF5-1 protein or BF5-1 protein variant-specific antibodies). Then, BF5-1 protein or BF5-1 protein variant can be isolated by purification (use of a Sepharose column or the like using the above antibody as a carrier).

  Furthermore, the present invention provides a pharmaceutical composition for the treatment of Aβ-accumulating disease containing the BF5-1 full-length gene, the BF5-1 gene variant, RNA complementary thereto, or the above vector. These polynucleotides may be administered directly into the patient's body, introduced into cells obtained from the patient, and returned to the patient after culturing.

  The present invention also provides a pharmaceutical composition for the treatment of Aβ-accumulating diseases, which contains a protein encoded by the above DNA or RNA or vector, specifically, a BF5-1 protein or a BF5-1 protein variant. .

  Here, the Aβ accumulating disease refers to a disease caused by accumulation of Aβ in a tissue, for example, dementia such as Alzheimer's disease or Down syndrome when Aβ accumulates in the brain, or other dementia. In addition, there are Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar degenerative disease, Parkinson's disease and the like.

  The amyloid β protein in the present specification may be of any type, and examples thereof include Aβ1-39, Aβ1-40, Aβ1-42, Aβ1-43 and the like. The definition of amyloid β protein and the notation of such amyloid β protein are also well known in the art.

  The production method and dosage form of the pharmaceutical composition and the pharmaceutically acceptable carrier or excipient are selected according to the condition of the subject, the treatment site, the administration route, etc., and those skilled in the art can easily select the pharmaceutical composition. Can be obtained.

  Furthermore, the present invention also provides a substance that regulates (promotes or suppresses, respectively an agonist or antagonist) the cell death inhibitory action of the BF5-1 protein or BF5-1 protein variant. Examples of the substance that regulates the cell death inhibitory action of such BF5-1 protein or BF5-1 protein variant include natural or synthetic proteins, peptides, nucleic acids, etc., but are not limited to these, and natural or synthetic low molecular weight compounds. It may be. Screening for agonists and antagonists of such BF5-1 protein or BF5-1 protein variant is performed by, for example, comparing cell death using cells fed with Aβ alone and cells fed with Aβ and a test substance. be able to.

  Further, for example, BF5-1 protein or an agonist of BF5-1 protein variant is added to a BF5-1 protein or BF5-1 protein variant-containing pharmaceutical composition to further enhance the cell death inhibitory effect, thereby causing cell death such as Alzheimer's disease. It is also possible to treat or prevent related diseases more effectively.

  On the other hand, the substance that promotes cell death of the present invention is useful for, for example, establishment of a cell death system, various studies related to cell death, for example, diseases related to cell death of nerve cells, such as the mechanism of Alzheimer's disease. Useful for elucidation. Furthermore, the substance that promotes cell death of the present invention is useful for the treatment and / or prevention of diseases caused by excessive proliferation of cells, such as cancer and autoimmune diseases.

  Therefore, in another aspect, the present invention relates to a pharmaceutical composition for treating and / or preventing a disease caused by cell hyperproliferation, comprising the cell death promoting substance described above. The production method and dosage form of the pharmaceutical composition and the pharmaceutically acceptable carrier or excipient are selected according to the condition of the subject, the treatment site, the administration route, etc., and can be easily selected by those skilled in the art. The present invention also relates to a method for treating and / or preventing a disease caused by cell hyperproliferation in a subject, which comprises administering the cell death promoting substance to the subject. The present invention also relates to the use of the aforementioned cell death promoting substance in the manufacture of a medicament for the treatment and / or prevention of diseases caused by cell overgrowth. The gene therapy as described above may also be applied in the treatment and / or prevention of diseases caused by cell hyperproliferation.

  Therefore, the present invention provides a protein or low molecular weight compound that suppresses or promotes cell death through interaction with Aβ directly or with protein BF5-1. The present invention also provides a kit or a pharmaceutical composition containing the above protein or low molecular weight compound for the prevention or treatment of Aβ accumulation disease.

The present invention will be described more specifically and in detail below with reference to examples. However, the examples are only for illustrative purposes of the present invention, and should not be construed as limiting the present invention.
A. Experimental method 1. LDH activity measurement method for evaluating cell death The LDH activity in the culture medium was measured using Cytotoxicity Detection Kit (Roche). 100 μl of the culture solution was recovered, centrifuged at 5,000 rpm × 3 minutes, and 50 μl of the supernatant was used as a sample. 50 μl of reaction mixture was added and incubated for 30 minutes at room temperature. After stopping the reaction by adding 25 μl of 1N hydrochloric acid, the absorbance at 490 nm was measured with a plate reader (Spectra Max 250, Molecular Devices).

2. Cell culture method SH-SY5Y cells were purchased from ECACC (European Collection of Animal Cell Cultures). D-MEM medium (catalog number: 12430-054, Invitrogen) containing penicillin / streptomycin (100 units / ml, catalog number: 15140-148, Invitrogen) and serum (15% v / v, catalog number: 2916754, Lot: 3846E) ) (Hereinafter, culture medium) and cultured in a CO ₂ incubator (catalog number: MCO-345, SANYO) at 37 ° C. and 5% CO ₂ . Plates and dishes coated with poly-L-lysine were used. According to the method of Encinas et al. (J Neurochem 75, 991, (2000)), cells differentiated like neurons were used. That is, SH-SY5Y into which a cDNA library or a control vector was introduced was suspended in a culture medium, and then seeded in a 6-well plate (catalog number: 356515, Becton Dickinson) at a rate of 200,000 cells / 3 ml / well.
After 24 hours, the culture medium was replaced with a culture medium containing all-trans retinoic acid (10 μM, catalog number: R2625, Sigma). After culturing for 5 days, the culture medium is removed, and D-MEM containing penicillin / streptomycin (100 units / ml, catalog number: 15140-148, Invitrogen) and 0.1% BSA (catalog number: A-4503, Sigma) Washing was performed twice with 3 ml per well using a medium (catalog number: 12430-054, Invitrogen) (hereinafter, serum-free medium). A serum-free medium containing BDNF (50 ng / ml, catalog number: 10908-010, Invitrogen) was added, and the cells were further cultured for 5 days. Through the above process, SH-SY5Y cells differentiated like neurons.

3. Gene Transfer Method The human fetal gene library was purchased from Takara (Code: 9549) and Invitrogen (Catalog Number: 10661-013). Each of these cDNA libraries is prepared with a pAP3neo vector or a pCMVsport2 vector and can be expressed in mammalian cells.
SH-SY5Y cells were seeded at 200,000 cells / 3 ml / dish in a 60 mm dish (catalog number: 356517, Becton Dickinson) coated with poly-L-lysine. After culturing for 24 hours, gene transfer was performed using Effectene (registered trademark) transfection reagent (Cat. 301425, Qiagen). Cells were treated with 3 μg of cDNA library, 150 μl of EC buffer, 8 μl of Enhancer, and 25 μl of Effectene. After 24 hours, the medium was changed and further cultured for 48 hours. Geneticin (catalog number: G9516, Sigma) was added at 600 μg / ml to obtain stable transgenic cells. Cells were obtained by culturing until colonies appeared. The obtained cells were stored frozen at −80 ° C. in a freezing medium (a culture medium containing 10% DMSO) until a cell death experiment was performed.

4). Cell death induction and cell death evaluation method by Aβ1-40 By adding Aβ1-40 (20 μM: adjusted by incubation at 37 ° C. for about 24 hours) to SH-SY5Y cells differentiated into these neurons, 8 Cell death seen after the day was measured. Cell death was performed using the MTT assay. This method utilizes the fact that when MTT (methylthiazoyl diphenyl-tetrazolium bromide, catalog number: M2128, Sigma) is taken up by living cells, insoluble formazan (blue) is produced by reduction reaction in mitochondria. Yes (T. Mosmann. J Immunol. Methods 65, 55 (1983)). MTT was dissolved in D-PBS (catalog number: 14190-144, Invitrogen) to a concentration of 3 mg / ml. After filter sterilization, cells were added to a final concentration of 0.3 mg / ml. After incubation for 4 hours in a CO ₂ incubator (37 ° C., 5% CO ₂ ), the same amount of detergent (10% SDS containing 1 mM NH ₄ OH) was added, and 24 hours in the CO ₂ incubator. Incubated for hours. Thereafter, the absorbance at 550 nm was measured with a plate reader (Spectra Max 250, Molecular Devices).

ＰＣＲ後、アガロース電気泳動を行い、コントロール培地処置群とＡβ１−４０処置群の間でバンドの比較を行い、同様の強度で発現しているバンドを候補遺伝子としてゲルより切り出した。 5). Differential Display Method Targeting cDNA Library Aβ1-40 (Catalog Number: 4307-v, Peptide Institute) 0.55 mg was added 0.1% NH ₄ OH 150 μl and D-PBS 150 μl, and about 24 at 37 ° C. Incubated for hours. 6 ml of serum-free medium was added to adjust the concentration of Aβ1-40 to 20 μM. As a control, 6 ml of serum-free medium was prepared by adding 150 μl of 0.1% NH ₄ OH and 150 μl of D-PBS. Aβ1-40 or control medium was added to SH-SY5Y seeded in two wells and differentiated like neuronal cells. After culturing for 8 days, total RNA was recovered using TRIZOl (registered trademark) reagent (catalog number: 15596-026, Invitrogen). The obtained RNA was dissolved in 30 μl of DEPC water, and 3 μl of 3M sodium acetate solution (pH 5.2) and 80 μl of ethanol were added. After ethanol precipitation at room temperature for 10 minutes, it was stored at -80 ° C. 10 μl of this was used as a sample for the differential display.
About 10 μl of the above RNA (corresponding to about 1 μg of RNA in the control group), reverse transcription reaction was performed using an oligo dT primer having an adapter sequence and Superscript ^™ II RNase H reverse transcriptase (catalog number: 18064-014, Invitrogen). CDNA was prepared. The oligo dT primer having the following sequence was used (Sigma Genosys).

5'- AACTGGAAGAATTCGCGGCCGCAGGAATTTTTTTTTTTTTTTTTT 3 '
(SEQ ID NO: 5)

The prepared cDNA was subjected to PCR according to the following protocol. For PCR, Taq PCR CORE Kit (catalog number: 201223, Qiagen) was used.

PCR reaction solution (per bottle)
1) 5 μl of 10 X PCR buffer
2) 10 μl of 5 x Q solution
3) d-NTP mix 1 μl
4) Taq polymerase 0.25 μl
5) Distilled water 30.75 μl
6) Primer 1 (20 μM) 1 μl
7) Primer 2 (20 μM) 1 μl
8) 2 μl template (cDNA corresponding to 1 μl RNA)

Primer set 1 (for Takara Library)
Primer 1: 5'- ACC GGG CCC TTA GGA CGC GTA ATA 3 '
(SEQ ID NO: 6)
Primer 2: 5'- AAG AAT TCG CGG CCG CAG GAA TTT TTT 3 '
(SEQ ID NO: 7)

Primer set 2 (for Invitrogen Library)
Primer 1: 5'- TGA CAC TAT AGA AGG TAC GCC TGC AGG TAC 3 '
(SEQ ID NO: 8)
Primer 2: 5'- AAG AAT TCG CGG CCG CAG GAA TTT TTT 3 '
(SEQ ID NO: 9)

After PCR, agarose electrophoresis was performed, the band was compared between the control medium treatment group and the Aβ1-40 treatment group, and the band expressed at the same intensity was cut out from the gel as a candidate gene.

増幅したＤＮＡをアガロース電気泳動後精製し、ＥｃｏＲＶ処理ｐＡＰ３ｎｅｏベクターまたはＳｍａＩ処理ｐＣＭＶｓｐｏｒｔ２ベクター（両ベクターとも脱リン酸化処理済み）にライゲーションした。ライゲーションにはDNA Ligation Kit Ver II（カタログ番号：６０２２、タカラ）を用いた。作成したプラスミドを用いてシークエンスを行った。 6). cDNA Cloning Method A gene was extracted from the gel by adding 100 μl of Tris EDTA buffer to the cut out gel and heating at 95 ° C. for 10 minutes. The next PCR was performed using 5 μl of the gene-extracted solution as a sample. Pyrobest (registered trademark) DNA Polymerase (catalog number: R005A, TAKARA) was used for PCR.

PCR reaction solution (per bottle)
1) Distilled water 73 μl
2) 10 x buffer 10 μl
3) 8 μl of dNTP mixture
4) Template 5 μl
5) Pyrobest 0.5 μl
6) Primer 1 (100 μM) 2 μl
7) Primer 2 (100 μM) 2 μl

The primer used was the same sequence as that used in the differential display targeting the cDNA library and the 5 ′ end was phosphorylated.

The amplified DNA was purified after agarose electrophoresis, and ligated to EcoRV-treated pAP3neo vector or SmaI-treated pCMVsport2 vector (both vectors were dephosphorylated). For the ligation, DNA Ligation Kit Ver II (catalog number: 6022, Takara) was used. Sequencing was performed using the prepared plasmid.

ＰＣＲ終了後、使用説明書に従いエタノール沈澱によりサンプルを精製し、Hi-Di ホルムアミド２５μｌに溶解した。その後、ABI PRISM（登録商標） 3100ジェネティックアナライザ（アプライドバイオシステムズ）によりシークエンスの解析を行った。ＰＣＲによる遺伝子変異補正の為、各遺伝子につき最低５クローンのシークエンスを行った。
7). 4. BF5-1 and BF5-2 cDNA sequencing method and amino acid analysis method Sequencing was performed using the plasmid prepared in (1). About 300 ng of the plasmid was mixed with the reaction solution of BigDye (registered trademark) Terminator v3.0 Ready Reaction Cycle Sequencing Kit (catalog number: 4390242, Applied Biosystems), and the following PCR reaction was performed.

After completion of PCR, the sample was purified by ethanol precipitation according to the instruction manual and dissolved in 25 μl of Hi-Diformamide. Thereafter, the sequence was analyzed by ABI PRISM (registered trademark) 3100 Genetic Analyzer (Applied Biosystems). For gene mutation correction by PCR, at least 5 clones were sequenced for each gene.

B. Experimental results Confirmation of cytotoxicity of Aβ1-40 against SH-SY5Y after induction of differentiation 1) Cell death by Aβ1-40 FIG. 1 shows a microscopic image of cells 4 days after addition of Aβ1-40 (20 μM) to differentiated SH-SY5Y cells. It is shown. Compared with the control group (left side), in the Aβ1-40 addition group (right side), the cells that caused cell death did not remain in their original form and were morphologically observed to be atrophy in a circle.
2) Time course of cytotoxicity by Aβ1-40 Aβ1-40 (20 μM) was added to differentiated SH-SY5Y cells, and Aβ1 was measured using MTT activity and LDH activity of cultured cells after 1, 2, 4 and 8 days as indicators. Cell death due to -40 cytotoxicity was assessed. As a result, it was revealed that cell death was almost completed in 4 to 8 days compared to the control group, and 40 to 60% of cells were killed in 8 days (FIG. 2).
From these results, it was found that the cytotoxicity of Aβ1-40 can be examined using cell shape change and MTT activity and LDH activity of cultured cells as indicators.

2. Using stably expressing cells in which the cDNA libraries 3 to 7 were introduced into SH-SY5Y cells, the cells were differentiated like neurons with all-trans retinoic acid (10 μM) and BDNF (50 ng / ml). Aβ1-40 incubated at 37 ° C. for about 24 hours was added to the cells, and after 8 days, differential display was carried out targeting mRNA derived from the cDNA library from the cells in which cell death was induced, and only expression of mRNA derived from the expression vector was performed. Detected. Only the cell group transfected with the cDNA library 5 containing BF5-1 and BF5-2 showed cell death resistance against Aβ1-40 (FIG. 3). The full-length genes of BF5-1 and BF5-2 were ligated again to the expression vector pAP3neo, and after each was transfected into cells, the effect on Aβ toxicity was examined. As a result, it was shown that cells transfected with the BF5-1 gene became resistant to Aβ toxicity (FIG. 4).

3. The BF5-1 gene is a sequence lacking about 120 bases in the center of the BF5-2 gene, and is a splicing variant of the BF5-2 gene (FIG. 5). These genes had a sequence called ATGTAG upstream, and the downstream reading frame was a non-translatable gene sequence, but splice site search (Splice Site Prediction by Neural Network, http://www.fruitfly.org/seq_tools /splice.html ), it was predicted that the region containing this ATGTAG would be excluded by splicing. In addition, one of the splice products predicted from the BF5-2 gene sequence completely matched the BF5-1 gene. (The full-length gene sequence of BF5-1 is shown in SEQ ID NO: 1, and the full-length gene sequence of BF5-2 is shown in SEQ ID NO: 2.) From the analysis of the gene sequences of BF5-1 and BF5-2, a reading frame encoding the protein was predicted. Further, in consideration of the splicing site on the BF5-1 gene, an open reading frame (BF5-1ORF) encoding 32 amino acids with 99 bases was determined (nucleotides 184 to 282 of SEQ ID NO: 1, and SEQ ID NO: 3). ).

4). In order to analyze whether the predicted ORF (open reading frame) is actually expressed as a protein, a plasmid in which the GFP base sequence is linked to the 3 ′ side of the predicted ORF using the full-length gene was prepared, and the plasmid was introduced into SH-SY5Y cells. After transfection, GFP expression was observed. As a result, GFP expression was observed in both BF5-1 and BF5-2, suggesting that the predicted ORF was translated as a protein (FIG. 6).

5). Total protein was collected from the cells expressing the above GFP fusion protein, and after SDS-PAGE, Western blotting was performed using an anti-GFP antibody (catalog number: 8362-1, Clontech). In cells expressing the GFP-fused BF5-1 gene, a band was observed at the predicted position (GFP-fused BF5-1 protein, MW: 27 kD), confirming that the protein encoded by BF5-1 was stably expressed. It was. On the other hand, the protein encoded by the BF5-2 gene is a very unstable protein with no band in the corresponding size (GFP-fused BF5-2 protein, MW: 33 kD) (FIG. 7).

6). In order to confirm whether the sequence encoded by the predicted ORF actually shows an effect, a fragment designed to express this portion was prepared, and after the cells were transfected, the effect on Aβ1-40 toxicity was examined. As a result, it was confirmed that the predicted ORF functions to suppress Aβ1-40 toxicity. In addition, not only the ORF but also the stability of expression differs depending on the presence or absence of a downstream base sequence, and it was suggested that the downstream sequence is particularly necessary for the stability of BF5-1 mRNA (FIG. 8).

  The present invention provides novel genes and novel proteins that suppress amyloid protein accumulation, and can be used for the prevention and treatment of amyloid protein accumulation diseases such as Alzheimer's disease. Therefore, the gene, protein and the like of the present invention can be used for the manufacture of a medicament for the prevention and treatment of amyloid protein accumulation diseases.

It is a figure which shows the cytotoxicity of A (beta) 1-40 with respect to SH-SY5Y after differentiation induction. The scale bar is 100 microns. It is a figure which shows the time course of the cytotoxicity after A (beta) 1-40 addition. It is a figure which shows the result of the differential display which made the cDNA library a target. A is Aβ1-40 (20 μM), B is a control (vehicle) lane. It is a figure which shows the influence of BF5-1 and BF5-2 with respect to A (beta) toxicity in the differentiated SH-SY5Y cell. MTT activity was measured 8 days after Aβ1-40 treatment. Statistical processing was performed using ANOVA. A represents Aβ1-40 (20 μM), and B represents a control (vehicle). It is a figure which shows the expression of mRNA in the cell which gene-transferred BF5-1 or BF5-2. It is a figure which shows the expression in SH-SY5Y cell of GFP fusion BF5-1 and BF5-2 protein. The GFP fusion gene to be expressed is shown on the left, and the fluorescence image of the GFP fusion protein is shown on the right. It is a figure which shows the result of the western blotting of each fusion protein of BF5-1 and BF5-2 and GFP. It is a figure which shows the expression of each fragment of BF5-1, and a cell death suppression effect.

Claims (20)

  DNA having the DNA sequence set forth in SEQ ID NO: 1 in the Sequence Listing.   A DNA that hybridizes with the DNA of claim 1 under stringent hybridization conditions.   DNA having 70% or more homology with the DNA according to claim 1 or 2.   DNA having the DNA sequence set forth in SEQ ID NO: 2 in the Sequence Listing.   A DNA that hybridizes with the DNA according to claim 4 under stringent hybridization conditions.   DNA having 70% or more homology to the DNA according to claim 4 or 5.   DNA having a nucleotide sequence of nucleotides 184 to 282 set forth in SEQ ID NO: 3 in the Sequence Listing.   A DNA that hybridizes with the DNA of claim 7 under stringent hybridization conditions.   DNA having 70% or more homology to the DNA according to claim 7 or 8.   RNA complementary to the DNA of any one of claims 1-9.   BF5-1 protein which has an amino acid sequence of sequence number: 4 of a sequence table. A DNA encoding a protein having one or more amino acids inserted, deleted or substituted in the protein according to claim 11 and having an Aβ toxicity-inhibiting effect equivalent to that of the BF5-1 protein.   A BF5-1 protein encoded by the DNA or RNA according to any one of claims 1 to 3, 7 to 10, or 12, or a protein having an Aβ toxicity inhibitory effect equivalent thereto.   A vector comprising the DNA sequence or the RNA sequence according to any one of claims 1 to 3, 7 to 10 or 12.   A transformant transformed with the vector according to claim 14.   A pharmaceutical composition for the treatment of Aβ accumulating diseases, comprising DNA or RNA comprising the DNA sequence or RNA sequence according to any one of claims 1 to 3, 7 to 10 or 12, or the vector according to claim 14.   A DNA or RNA according to any one of claims 1 to 3, 7 to 10 or 12, or a BF5-1 protein encoded by the vector according to claim 14 or a protein having an Aβ toxicity inhibitory effect equivalent thereto. A pharmaceutical composition for treating an Aβ accumulating disease.   A DNA or RNA comprising the DNA sequence or RNA sequence according to any one of claims 1-3, 7-10 or 12 or the vector according to claim 14 is introduced into a cell obtained from a patient, A method for treating an Aβ-accumulating disease, comprising culturing and then introducing the cell into the patient.   A protein or low molecular weight compound that inhibits or promotes cell death through interaction with amyloid β protein directly or with protein BF5-1. A kit or a pharmaceutical composition containing the protein or low molecular weight compound according to claim 19 for the prevention or treatment of amyloid β protein accumulation disease.

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