JP2005151810A - Method for determining onset risk of alzheimer's disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a molecule associated with normal formation of a respiratory complex of cells and to provide a method for detecting Alzheimer's disease and the risk thereof by a new method. <P>SOLUTION: An MIRTD [mitochondrial respiration generator of truncated DLST (dihydrolipoamide succinyltransferase)] which is a transcription product subjected to transcription initiation from any base in intron 7 of a DLST gene and a nucleic acid encoding the MIRTD are provided. An onset risk to the Alzheimer's disease is determined by using the expression level of the MIRTD as an index. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to Alzheimer's disease and a method for detecting the risk thereof.

  Alzheimer's disease (AD) is a progressive neurological disorder characterized by loss of cognitive function and dementia. Although the biological mechanism of this disease is not well understood, for familial early-onset AD, three genes are involved: the amyloid precursor protein gene on chromosome 21, the presenilin-1 gene on chromosome 14, and Mutations in the presenilin-2 gene on chromosome 1 have been identified by linkage analysis and positional cloning. For 40-50% of sporadic AD, a specific allele of the apolipoprotein E gene on chromosome 19 is associated with accelerated AD development.

  On the other hand, mammalian α-ketoglutarate dehydrogenase complex (KGDHC) exists in mitochondria, and succinyl-CoA is synthesized in the Krebs cycle by catalyzing the rate-limiting step of oxidative decarboxylation of α-ketoglutarate. To do. This complex consists of three different enzymes: α-ketoglutarate decarboxylase, dihydrolipoamide dehydrogenase, and the core enzyme dihydrolipoamide succinyltransferase (DLST). It has been reported that the enzyme activity of this complex is often decreased in the brain and cultured fibroblasts of AD patients (see Mastrogiacomo F et al., J Neurochem 1993; 61: 2007-2014). In addition, according to the study of the present inventors, in sporadic AD patients, the frequency at which the 19117th and 19183th base substitutions of the DLST gene are homozygous is compared to that of healthy individuals. It has been found to be significantly higher (see Patent Document 1).

Japanese Patent Laid-Open No. 11-308996 THE LANCET Volume 350 Page 9088 Eur. J. Biochem., 224, 179-189 (1994)

  The present invention relates to a mitochondrial respiration-produced truncated DLST protein (Mitochondrial Respiration Generator of), a transcription product initiated from any base in intron 7 of the DLST gene, which is a molecule involved in the respiratory function of cells. Truncated DLST; MIRTD), and nucleic acids encoding it. A further object of the present invention is to provide a method for detecting Alzheimer's disease and its risk.

  The present inventors express a DLST truncated protein expressed in a neuron, and the protein is a DLST exon derived from a transcription product initiated from any base within intron 7 of the DLST gene. It was found to consist of 8 and 9, and was named Mitochondrial Respiration Generator of Truncated DLST (hereinafter referred to as MIRTD). Furthermore, it has been found that MIRTD mRNA and MIRTD protein are decreased in the brains of AD patients compared to healthy individuals.

  Furthermore, it was found that a reduction in the amount of MIRTD mRNA was observed in a haplotype in which the gene at position 10579 in the DLST gene was T.

As described above, the present invention provides the following 1. The following protein (a) or (b):
(a) a protein having the amino acid sequence shown in SEQ ID NO: 1
(b) having the amino acid sequence in which one to several amino acid residues are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and having the same activity as the protein of (a), ie hydrogen peroxide A protein having an activity that maintains oxidative stress tolerance due to extracellular stimulation such as and / or promotes the formation of a mitochondrial respiratory chain enzyme complex (particularly complex I and / or IV);
2. A nucleic acid encoding the protein according to 1 above;
3. The following nucleic acids (a) or (b):
(a) a nucleic acid having the base sequence shown in SEQ ID NO: 2
(b) a nucleic acid capable of hybridizing under stringent conditions with a nucleic acid complementary to the nucleic acid having the base sequence shown in SEQ ID NO: 2;
4). Quantifying mitochondrial respiratory-producing truncated DLST protein (MIRTD) mRNA, a transcription product initiated from a base in intron 7 of a dihydrolipoamide succinyltransferase (DLST) gene in cells collected from a subject A method for determining Alzheimer's disease or the risk of developing it;
5). A method for determining Alzheimer's disease or the risk of developing it, comprising quantifying MIRTD protein in cells collected from a subject;
6). 6. The method according to 4 or 5 above, wherein the cell is derived from any one selected from the group consisting of nerve tissue, heart, liver and kidney, or any other tissue;
7). A method for determining the risk of developing Alzheimer's disease, comprising examining whether or not the 10579th base of a dihydrolipoamide succinyltransferase (DLST) gene of a subject is T;
8). 8. The method according to 7 above, wherein the gene is extracted from peripheral blood;
9. In order to determine Alzheimer's disease or the risk of developing it, including a primer pair for detecting MIRTD mRNA which is a transcription product initiated from any base in intron 7 in the dihydrolipoamide succinyltransferase (DLST) gene Diagnostic kit;
10. 10. The diagnostic kit according to 9 above, wherein the primer pair for MIRTD mRNA detection comprises 5′-ctcatttcagacagtgccagtg-3 ′ (SEQ ID NO: 5) and 5′-tggctcagctagtggtggg-3 ′ (SEQ ID NO: 6);
11. A TaqMan probe for quantifying MIRTD mRNA, comprising a probe having a luminescent dye as a reporter at the 5 ′ end of 5′-cctccttctggcaaacctgtgtctct-3 ′ (SEQ ID NO: 7) and a quencher for the luminescent dye added at the 3 ′ end The diagnostic kit according to 9 or 10, further comprising:
12 Any of the above 9 to 11, further comprising 5'-acctacagcagcggcagt-3 '(SEQ ID NO: 3) and 5'-ctcctggctcagctagtggt-3' (SEQ ID NO: 4) as a primer pair for measuring the amount of full-length DLST mRNA for standardization A diagnostic kit according to claim 1;
13. A diagnostic kit for determining Alzheimer's disease or its risk of developing, comprising an antibody specifically recognizing MIRTD for detecting MIRTD protein;
14 An antibody specifically recognizing MIRTD, which is produced using a C-terminal fragment of DLST containing a region downstream from methionine of exon 8 of DLST, comprising the sequence shown in SEQ ID NO: 1;
15. 14. The antibody according to 14 above, which is a monoclonal antibody;
16. 14. The kit according to 13 above, comprising the antibody according to 14 or 15 above;
About.

  The present invention provides MIRTD proteins that promote the formation of respiratory chain complexes (particularly complex I and complex IV) in cells (particularly nerve cells). Further, the present invention makes it possible to determine the risk of developing AD by quantifying the expression level or mRNA level of MIRTD, and to determine the risk of developing AD by a method based on the identification of the haplotype of the DLST gene. According to the method based on the identification of the haplotype, it is possible to use it as an index of the risk of developing AD only by identifying one base in a chromosome, and provide useful information for a subject.

The present invention is described in detail below.
DLST is a core enzyme of α-KGDHC that synthesizes succinyl-CoA by catalyzing the rate-limiting step of oxidative decarboxylation of α-ketoglutarate in the Krebs cycle, and its human gene is q24 on chromosome 14. Although located at 2-q24.3 (Nakano et al., 1993, Biochim. Biophys. Acta, 1216, 360-368), it consists of a total of 15 exons, ie there are 14 introns in the gene (Nakano et al., 1994, Eur. J. Biochem. 224 179-189).

  In the research leading to the present invention, the inventors of the present invention used a terminal in the human neural model cell line whose transcription initiation point is any base in the seventh intron of the gene (for example, the base at position 10556 and its vicinity). It was found that truncated DLST (referred to herein as MIRTD) mRNA is present. The present inventors have obtained the knowledge that the MIRTD will be present in all tissues including the brain, heart, kidney and liver.

  The amino acid sequence of human MIRTD is shown in SEQ ID NO: 1. The protein is present between the outer and inner mitochondrial membranes as described in the examples herein and is involved in the formation of mitochondrial respiratory chain enzyme complexes (particularly complex I and complex IV). The formation of these complexes is remarkably suppressed by the lack of the protein. Therefore, it is considered that by increasing the amount of the protein in the cell, respiratory failure is improved and cell damage or cell death can be suppressed. In particular, in neurodegenerative diseases including AD, cell damage or cell death can be suppressed by increasing the amount of MIRTD in cells accompanied with degeneration, thereby avoiding or suppressing its onset or its progression. It is considered possible. In fact, when the amount of MIRTD in AD brain was confirmed, it was found that the mRNA level of MIRTD was significantly reduced in AD brain compared to that of healthy individuals (non-AD humans). The inventors have found.

  Accordingly, the present invention relates to the human MIRTD protein shown in SEQ ID NO: 1. In the amino acid sequence shown in SEQ ID NO: 1, 1 to 50, preferably 1 to 10, more preferably 1 to 5, particularly preferably 1 to 3 amino acid residues have been deleted, substituted or added. A protein having an amino acid sequence and having the same activity as the MIRTD protein, that is, a mitochondrial respiratory chain enzyme complex-forming activity, or a protein functionally equivalent to the MIRTD protein can also be included in the present invention. Furthermore, when the homology with the amino acid sequence shown in SEQ ID NO: 1 is calculated using BLAST or the like (for example, using the BLAST default or initial condition parameters), 75% or more, 80% or more, A protein having an amino acid sequence of 85% or more or 90% or more, preferably 95% or more, more preferably 98% or more, and most preferably 99% or more, and has the same activity as the MIRTD protein, ie, mitochondrial respiratory chain enzyme complex Proteins having somatic activity or proteins functionally equivalent to MIRTD proteins can also be encompassed by the present invention.

  Whether the target protein has the same activity as the MIRTD protein is determined by forcibly expressing the target protein in a cell that does not express MIRTD, and this cell is the respiratory chain complex described in the Examples below. It can be determined by subjecting it to an activity assay or the like. In addition, it can be achieved by exposing the forcedly expressed cells to hydrogen peroxide and examining the increase in resistance to hydrogen peroxide. The MIRTD protein is essential for the formation of a respiratory chain complex, and it has been revealed by the present inventors that its expression improves resistance to oxidative stress caused by extracellular hydrogen peroxide stimulation. For details, refer to the examples of this specification.

  The present invention also includes nucleic acids (including DNA and RNA) encoding the above-described proteins. A person skilled in the art can design and synthesize or produce a nucleic acid encoding the protein based on the amino acid sequence of the protein. SEQ ID NO: 2 shows an example of a DNA sequence encoding MIRTD consisting of the amino acid sequence shown in SEQ ID NO: 1. However, the present invention is not limited to the sequence shown in SEQ ID NO: 2, but also includes DNA having a degenerate relationship therewith. Furthermore, a nucleic acid that can hybridize under stringent conditions with a nucleic acid that is complementary to a DNA having the sequence shown in SEQ ID NO: 2 or a sequence that is degenerately related thereto or an RNA corresponding to the sequence of the DNA, Included in the present invention. Here, stringent conditions refer to, for example, conditions in which the sodium concentration is 10 mM to 300 mM, preferably 42 to 55 ° C., more preferably 42 ° C. Furthermore, a nucleic acid comprising a nucleotide sequence in which one or more bases have been deleted, substituted or added in the nucleotide comprising the nucleotide sequence shown in SEQ ID NO: 2 is also included in the present invention. Here, the number of bases to be deleted, substituted or added is not particularly limited, but is preferably 1 to 50, more preferably 1 to several, and most preferably 1 to 3. As the nucleotide sequence of such a nucleotide, when the homology with the nucleotide sequence shown in SEQ ID NO: 2 is calculated using BLAST or the like (for example, when BLAST default, that is, initial condition parameters are used), 70 % Or more, preferably 90% or more, more preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Such a nucleotide is substantially identical to the nucleotide having the base sequence shown in SEQ ID NO: 2. In addition, the “nucleic acid” referred to in the present specification includes DNAs and RNAs having various modifications and analogs thereof.

  Furthermore, a cell containing the nucleic acid as a foreign gene is also included in the present invention. The nucleic acid can be introduced into cells by a gene gun or various known methods. For the introduction, the nucleic acid can be inserted into various plasmids or viral vectors, and the vector can be incorporated into a host cell. Therefore, vectors (including expression vectors) containing the nucleic acid are also included in the scope of the present invention. The present invention also includes a cell in which the nucleic acid is introduced into a cell as a foreign gene and the nucleic acid is integrated into the genome by homologous recombination or the like, or a cell including a vector containing the nucleic acid. These techniques can be performed using conventional techniques in the art, see Sambrook et al., Molecular Cloning: A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, etc. Good.

  In addition, since the amount of MIRTD mRNA in AD brain is significantly reduced in AD brain compared to that of healthy subjects (non-AD humans), the amount of MIRTD mRNA in cells derived from the tissues of subjects Can be used to determine whether or not the patient from which the tissue is derived has AD disease or whether the risk of developing AD is high or low. MIRTD is highly expressed in the heart and nerve tissues (especially the brain) and has been confirmed to be present in the liver and kidney, but it is thought to be universally expressed like DLST. Cells derived from any of (including blood) may be used, and cells derived from a tissue selected from the group consisting of neural tissue, heart, liver and kidney are preferably used. Particularly preferably, cells derived from central nervous tissue are used.

  That is, a specific tissue is collected from a subject, the tissue sample is processed according to a conventional method, and MIRTD mRNA contained in the tissue is quantified. Various methods are known as this quantification method. For example, mRNA can be purified and quantified by quantitative RT-PCR. More preferably, quantification is performed by a real-time RT-PCR method such as TaqMan method.

  For quantification of MIRTD mRNA, a primer pair consisting of a sequence that hybridizes to the 3 ′ end side of intron 7 of the DLST gene may be used. In order to distinguish it from full-length DLST mRNA, a sequence that hybridizes within intron 7 More preferably, a forward primer consisting of For example, from forward primer 5'-ctcatttcagacagtgccagtg-3 '(SEQ ID NO: 5) that hybridizes to the sequence in intron 7 and reverse primer 5'-tggctcagctagtggtggg-3' (SEQ ID NO: 6) that hybridizes to the sequence in exon 9 Quantitative RT-PCR can be performed using the following primer pair.

  Furthermore, when quantifying by the real-type quantitative PCR method using TaqMan method, a luminescent dye is used as a reporter at the 5 ′ end of 5′-cctccttctggcaaacctgtgtct-3 ′ (SEQ ID NO: 7), and a quencher for the luminescent dye is used at the 3 ′ end. MIRTD mRNA can be quantified by performing TaqMan method according to a conventional method using a probe to which is added and an appropriate MIRTD primer pair such as the above primer pair. Examples of the combination of the luminescent dye and the quencher corresponding thereto include FAM and TAMRA, but are not limited thereto, and any known one may be used in combination.

  For quantification, the amount of mRNA serving as an endogenous control, such as GAPDH mRNA or β-actin mRNA, was quantified in the same manner in a sample containing mRNA purified from the tissue, and normalized to the relative amount of MIRTD mRNA. A quantitative value should be obtained. For example, when quantifying GAPDH mRNA, a primer pair having a sequence consisting of 5′-gggaaggtgaaggtcgga-3 ′ (SEQ ID NO: 8) and 5′-gcagccctggtgaccag-3 ′ (SEQ ID NO: 9) can be used. When GAPDH mRNA is quantified by the TaqMan method, a luminescent dye is added as a reporter to the 5 'end of 5'-caacggatttggtcgtattgggcg-3' (SEQ ID NO: 10), and a probe with a quencher for the luminescent dye added to the 3 'end. When used together with appropriate primer pairs, GAPDH mRNA can be quantified.

  Alternatively, the amount of full-length DLST mRNA can be quantified, and the amount of MIRTD mRNA can be quantified as a relative quantitative value for this amount. An example of a primer pair for measuring the amount of full-length DLST mRNA for standardization is a primer pair consisting of 5'-acctacagcagcggcagt-3 '(SEQ ID NO: 3) and 5'-ctcctggctcagctagtggt-3' (SEQ ID NO: 4). Furthermore, as a TaqMan probe for quantifying full-length DLST mRNA, the above TaqMan probe for MIRTD can be used.

  Furthermore, those skilled in the art can easily design primers for mRNA quantification and TaqMan probes, and are not limited to those having the sequences exemplified above.

  Regardless of which quantification method is used, comparison of control MIRTD mRNA levels measured with the same treatment using the same tissue collected from humans with no DL type suspicion and / or having a DLST gene other than ac type as a control If the subject is about 60% or less, preferably 50% or less, it can be determined that the subject is suffering from AD or has a high risk of developing AD. Such a control tissue is preferably collected from a human who has no suspicion of AD disease as a clinical finding and has a DLST gene other than the ac type in order to exclude humans at risk of onset. This is because, as described later, humans having an ac-type DLST gene are considered to have a relatively high expression level of MIRTD even if there is no suspicion of AD disease in clinical findings.

Therefore, a primer pair for MIRTD quantification can be provided as a diagnostic kit for determining AD or its risk of developing, and such a diagnostic kit is also included in the scope of the present invention. Further, the kit may contain a primer for mRNA quantification for standardization as described above, optionally further a TaqMan probe, and further, the total RNA or polyA obtained from the control tissue or the tissue. ⁺ May contain RNA.

  In addition, since the amount of MIRTD protein in the brain of AD patients is reduced in response to the amount of mRNA, it is possible to detect AD disease or its risk by quantifying the amount of MIRTD protein in the tissue of the subject. It is.

  When the amount of MIRTD protein is quantified, various immunochemical methods can be used, but it is preferable to use a method that specifically detects the truncated MIRTD protein without detecting the full-length DLST protein. Therefore, for full-length DLST, detect the MIRTD protein in the sample after pull-down with an antibody that recognizes its N-terminal side, or use the difference in molecular weight between full-length DLST and MIRTD. It can be quantified by an immunochemical method by Western blotting. For example, an antibody that specifically recognizes both DLST and MIRTD, which is produced using the C-terminal region of DLST including a region downstream from methionine of exon 8 shown in SEQ ID NO: 1 as an immunogen, can be used. Preferably, a monoclonal antibody is used. The molecular weight of DLST is about 55 kDa and the molecular weight of MIRTD is about 30 kDa. Can be distinguished, and MIRTD can be quantified semi-quantitatively.

  Therefore, a diagnostic kit for determining Alzheimer's disease or its risk of developing comprising an antibody that specifically recognizes MIRTD as described above is also included in the scope of the present invention. In the present specification, “an antibody capable of specifically recognizing MIRTD” is not limited to an antibody specifically recognizing only MIRTD, but also recognizes DLST, but human cell-derived proteins other than MIRTD and DLST Refers to an antibody that can detect MIRTD (and DLST) so that MRTD and DLST can be clearly discriminated from proteins other than MIRTD and DLST because they are not recognized or only slightly recognized.

  The antibody may be a polyclonal antibody or a monoclonal antibody. Furthermore, any molecule may be used as long as it includes a variable region of an antibody that can specifically recognize MIRTD, such as a chimeric antibody or an antibody fragment.

  Furthermore, in the present invention, a genotype associated with a decrease in the amount of MIRTD has been found. The present inventors have already found that there are 3 types of haplotypes of human DLST gene: ac type, at type, and gc type (see Patent Document 1). ), 11044th position a, 11104th position t, 13845th position t, 15456th position t, 17107th position g, 19117th position a and 19183th position c). The base at position 10579, which is in the immediate vicinity of position 10556, which is the transcription start point, is T in contrast to A in the other types. It was found that the transcription amount of MIRTD mRNA was decreased in cells with an ac type having a point mutation from A to T at position 10579, compared to cells having a gc type with position 10579 being A. doing.

  Therefore, humans with a DLST gene at position 10579 of T or humans with DLST gene haplotype ac type are compared to humans with base A or DLST genes other than ac type. It can be said that the risk of AD is high.

  Therefore, the risk of AD can be detected by examining the base at position 10579 of the DLST gene on the chromosome of the subject. In addition, whether or not it is ac type may be determined by detecting a mutation specific to the ac type described above, particularly by examining whether or not position 19117 is a and position 19183 is c. .

  The method for investigating such bases is a synthetic oligonucleotide of about 20 bases that is effective for the purpose of detecting only the base substitution at a specific position, such as the base sequence determination method (sequence method) of the target region, which is the most direct method. Allele-specific oligonucleotide hybridization method (MASA method) using presence / absence of hybridization between target RNA and target nucleotide sequence, treatment of hybrid of standard RNA probe and standard DNA fragment with ribonuclease A and cleavage of probe RNA at mismatch position When the ribonuclease A mismatch cleavage method (RNase protection method), which knows the existence and approximate position of the nucleotide sequence in DNA, or when a DNA sequence is amplified by PCR, a GC-rich base sequence (GC clamp) is attached to one end of the DNA sequence. To detect single base substitution by performing denaturing gradient gel electrophoresis (DGGE) (PCR- DGGE method) can be used.

  A method of detecting single base substitution as a difference in mobility by dissociating a double-stranded DNA fragment into single strands and performing polyacrylamide gel electrophoresis on a unique higher-order structure depending on the base sequence (PCR) -SSCP method), hybridization method using nucleotide sequence specific probe (SSOP) (PCR-SSOP method), modified methods (PCR-RD method and PCR-MPH method), and PCR method for certain regions PCR-RFLP method in which the site where the point mutation has occurred is treated with a restriction enzyme that recognizes it as a cleavage site and detected as an allele having a different size from the DNA that did not cause the point mutation. -308999 gazette), annealing a detection primer to a nucleic acid sequence adjacent to the nucleotide site at the 3 'end of the target DNA, and a single label that is complementary to the nucleotide to be detected using DNA polymerase Nucleotide triphosphorus A method of extending such a primer with an acid (mini-sequencing method) or the like can also be used.

  It can also be detected by various known SNP typing methods such as TaqMan PCR method and invader method. For example, in the case of TaqMan PCR method, a probe designed to hybridize to a sequence of about 20 bases surrounding T at the position corresponding to position 10579 is prepared. A fluorescent dye is attached to the 5 'end of the probe, a quencher is attached to the 3' end, and the 3 'end is phosphorylated. The probe is then hybridized to a template DNA extracted from a tissue sample (which can be any tissue) collected from a subject, and PCR primers and Taq DNA polymerase designed to further amplify the probe region are used. Perform PCR reaction. Since Taq DNA polymerase has 5 'nuclease activity, when the extension reaction from the primer reaches the 5' end of the probe, the 5 'end of the probe is cleaved. This causes the fluorescent dye to fluoresce away from the quencher. By measuring the fluorescence, it can be determined whether or not the probe has hybridized to the template, and thereby whether or not the base at position 10579 is T is determined. Specific methods of the TaqMan PCR method are disclosed in various documents.

  Any method capable of detecting a single-base mutation can be used in the present invention without being limited to the above method.

  The numbering of the base positions of the DLST gene in this specification was as described in Nakano K et al., Eur J Biochem. 1994 Aug 15; 224 (1): 179-189. Although there is a deviation of one base from the description of EMBO J. 22 2913-2923 (2003), base number 19183 in the description of this paper is an error that should originally be 19182. Changed.

Materials and methods
Cell culture human neuroblastoma cell line SH-SY5Y is in Dulbecco's modified Eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 2 mM glutamine, 20 IU / ml penicillin and 20 μg / ml streptomycin. The cells were cultured at 37 ° C. under 5% CO ₂ . Rat pheochromocytoma PC12 was cultured in DMEM supplemented with 5% FBS, 5% inactivated horse serum, 100 IU / ml penicillin and 100 μg / ml streptomycin.

Brain sample
Brain samples from AD patients and control samples were taken from the cortex from 40 to 660 minutes (255.0 174.9) after death. The average age of AD patients was 77.7 ± 6.5 years (60-86 years), while the average age of controls was 65.9 ± 9.0 years (48-79 years). The frequency of ac-type carriers was 32% and 24% in the AD and control groups, respectively.

Quantitative RT-PCR
Total RNA was isolated from human brain and cultured cell samples using the RNeasy Mini Kit (Qiagen) or ISOGEN (Wako) according to the manufacturer's recommended protocol. Poly (A) ⁺ RNA was purified from total RNA using the QuickPrep mRNA Purification Kit (Amersham Bioscience). First strand cDNA was synthesized by SUPERSCRIPT II RT (Invitrogen) using oligo (dT) primers (Invitrogen).

  The amount of mRNA was quantified by real-time quantitative RT-PCR using ABI PRISM 7700 Sequence Detector System (Applied Biosystems) (Holland et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 7276-7280 .; Livak et al., 1995, PCR Methods Appl., 4, 357-362.). The measurement was performed at least 3 times to obtain an average value. Primers and probes designed by Primer Express software program (Applied Biosystems) are as follows: DLST forward primer; 5'-acctacagcagcggcagt-3 '(exon 8: SEQ ID NO: 3), DLST reverse primer; 5'-ctcctggctcagctagtggt- 3 '(exon 9: SEQ ID NO: 4), MIRTD forward primer; 5'-ctcatttcagacagtgccagtg-3' (intron 7: SEQ ID NO: 5), MIRTD reverse primer; 5'-tggctcagctagtggtggg-3 '(exon 9: SEQ ID NO: 6) TaqMan probes for both DLST and MIRTD; 5'-cctccttctggcaaacctgtgtctct-3 '(strand exons 8 to 9: SEQ ID NO: 7), GAPDH forward primer; 5'-gggaaggtgaaggtcgga-3' (SEQ ID NO: 8), GAPDH Reverse primer; 5'-gcagccctggtgaccag-3 '(SEQ ID NO: 9), TaqMan probe for GAPDH; 5'-caacggatttggtcgtattgggcg-3' (SEQ ID NO: 10). For measurement of the amount of mRNA of cytochrome c oxidase subunit IV, forward primer 5'-tgtgtgtgtacgagctcatgaaag-3 '(SEQ ID NO: 11), reverse primer 5'-gtggtcacgccgatccat-3' (SEQ ID NO: 12), and TaqMan probe 5 '-ttgtgaagagcgaagacttttcgctccc-3' (SEQ ID NO: 13). Values were normalized to GAPDH cDNA. The TaqMan probe contains a luminescent dye FAM as a reporter at the 5 ′ end and TAMRA as a quencher at the 3 ′ end.

Determination of transcription start site of MIRTD mRNA
The transcription start site of MIRTD mRNA is determined by oligonucleotide capping method (Yamabe et al., 1998, Mol. Cell Biol., 18, 18) using human brain Cap Site cDNA ^™ (Nippon Gene, Toyama, Japan) according to the manufacturer's recommended protocol. 6191-6200.). The first PCR was performed using 5'-caaggtacgccacagcgtatg-3 '(1RC, Nippon Gene: SEQ ID NO: 14) and 5'-aagtaacgaatgtctactgggtatcagca-3' (SEQ ID NO: 15). Samples were amplified for 40 cycles at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 45 seconds. A part of the first reaction product was used as a template for the second PCR. The second PCR (nested PCR) was performed using 5′-gtacgccacagcgtatgatgc-3 ′ (2RC2; Nippon Gene: SEQ ID NO: 16) and 5′-actgggtatcagcaatatgcaaag-3 ′ (SEQ ID NO: 17), and the DNA sequence was determined.

For western blotting whole cell lysate, the cultured cells were washed with PBS and then collected with PBS. Cells were harvested by centrifugation and lysed with 2% SDS, 20 mM Tris-HCl pH 6.8, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The protein concentration was measured by BCA assay (Pierce). The sample was subjected to SDS-PAGE after adding 2-mercaptoethanol, and then subjected to Western blotting using an antibody.

Preparation of anti-DLST monoclonal antibody and other antibodies The C-terminal region of DLST, including the region downstream from the methionine of exon 8, was expressed in E. coli along with glutathione S-transferase. Inclusion bodies containing the fusion protein were isolated and subjected to continuous preparative SDS-PAGE. The fusion protein was eluted from the excised gel, concentrated and dialyzed against phosphate buffered saline (PBS). Spleen cells of BALB / c mice immunized with this were fused with PAI myeloma cells (Ohta and Schatz, 1984 EMBO J., 3, 651-657), and 6 hybridoma cells producing antibodies were purified. Screening was performed by solid-phase enzyme immunoassay using a DLST C-terminal region recombinant having histidine. Positive cells were cloned by limiting dilution.

  Antibodies against mitochondria Tom40 and Tom20 were a gift from Prof. K. Mihara, Kyushu University, Fukuoka, Japan. All antibodies against the subunits of the respiratory chain complex were purchased from Molecular Probes.

Preparation of PC12 cells harboring human DLST gene Genomic DNA was isolated from peripheral blood and partially digested with Sau3A I. The digested DNA was inserted into the BamHI site of the cosmid vector SuperCos 1 (Stratagene). The ligated DNA was packaged using Gigapack III Gold packaging extract (Stratagene) according to the manufacturer's instructions. Positive clones were selected by colony hybridization using a PCR fragment corresponding to the promoter region of the DLST gene (Nakano et al., 1994, Eur. J. Biochem., 224, 179-189.). Was confirmed to include the full length by amplifying the upstream region, exons 1, 8, 14 and downstream region. Lipofectin (Invitrogen) was used to co-transfect PC12 cells with a cosmid and geneticin resistance gene including an ac-type or gc-type DLST gene. By selecting geneticin resistant cells for 14 days, the ratio of transfected cells was increased.

Sub-organelle fractionation of rat liver mitochondria To isolate complete mitochondria, fresh rat liver was pre-chilled in disruption buffer (0.22 M mannitol, 0.07 M sucrose, 20 mM Hepes-KOH pH 7.4, and It was washed with 1 mM EDTA) and weighed. It was cut with a scissors and suspended in the disruption buffer. The suspension was homogenized with a Potter-Elvehjem glass homogenizer using a Teflon pestle. The homogenate was overlaid with 0.34 M sucrose and centrifuged at 1,000 xg for 10 minutes. The upper layer (post-nuclear supernatant) was centrifuged at 10,000 xg for 15 minutes to remove the precipitate (P10,000 fraction). The obtained supernatant was centrifuged again at 100,000 × g for 2 hours to separate into a precipitate (P100,000 fraction) and a supernatant (S100,000). To prepare mitoplasts, mitochondria (10 mg / ml) were diluted 10-fold with 20 mM Hepes-KOH pH 7.4 and 1 mM EDTA. The mitochondrial and mitoplast samples were then aliquoted and treated with 0, 10, or 50 μg / ml proteinase K for 30 minutes on ice. Mitochondria or mitoplasts were re-separated by centrifugation and analyzed by SDS-PAGE and Western blotting.

Establishment of cells that stably express maxizyme
Chemically synthesized oligonucleotides encoding the maxizyme and pol III termination sequences for MIRTD mRNA were converted to double stranded sequences by PCR. After digestion with Csp45 I and Sal I, each appropriate fragment was chemically synthesized between the EcoR I and Sal I sites of pUC19, and the pUC-dt tRNA ^Val promoter (including the promoter for the human tRNA ^Val gene) (Kuwabara et al. 1999, Proc. Natl. Acad. Sci. USA, 96, 1886-1891.). The base sequence of the construct was confirmed by direct sequencing. SH-SY5Y cells were co-transfected with maxizyme and geneticin resistance gene using Effectene reagent (Qiagen) according to the manufacturer's recommended protocol. Selection with Geneticin (Invitrogen) isolated several independent clones stably expressing the maxizyme or monomer control.

Respiratory Chain Complex Activity Assay The activity of each mitochondrial respiratory chain complex was measured spectrophotometrically at 30 ° C. according to previous reports (Trounce et al., 1996; Birch-Machin and Turnbull, 2001). For the assay of NADH-cytochrome c oxidoreductase (complex I + III) activity, frozen thawed mitochondria (15 μg protein / ml) were added at 30 ° C. for 3 minutes, 25 mM KPi pH 7.2, 5 mM MgCl ₂ and Preincubation in 2.5 mg / ml bovine serum albumin (BSA) containing 2 mM KCN. Thereafter, cytochrome c (III) (50 μM) and NADH (130 μM) were added, and the change in absorbance between 550 nm and 580 nm (attenuation coefficient 19 mM ⁻¹ cm ⁻¹ ) of cytochrome c was measured. Two minutes later, rotenone (2 μg / ml) was added and the ratio of rotenone insensitivity was measured. For assay of succinate-cytochrome c oxidoreductase (complex II + III) activity, frozen thawed mitochondria (15 μg / ml) were prepared at 25 ° C. for 10 minutes at 25 mM KPi pH 7.2, 5 mM MgCl ₂ and 20 mM sodium succinate And preincubation in 2.5 mg / ml BSA containing 2 mM KCN, 2 μg / ml rotenone. Thereafter, cytochrome c (III) (30 μM) was added, and the change in absorbance was measured. For assay of ubiquinol-cytochrome c oxidoreductase (complex III) activity, 35 μM ubiquinol and cytochrome c (III) were added to 25 mM KPi pH 7.2, 5 mM MgCl ₂ and 2 mM KCN, 2 μg / ml rotenone, It was added to 2.5 mg / ml BSA containing 0.6 mM n-dodecyl β-D-maltoside and the non-enzymatic ratio was measured. Thereafter, mitochondria (10 μg protein / ml) was added, and the change in absorbance of cytochrome c was measured. Cytochrome c oxidase (complex IV) activity was measured by measuring the change in the absorbance of cytochrome c from 550 nm to 580 nm (attenuation coefficient 19 mM ^-1 cm ^-1 ). That is, reduced cytochrome c (15 μM) was added to 20 mM KPi pH 7.2 and 0.45 mM n-dodecyl β-D-maltoside, and the non-enzymatic ratio was measured. Thereafter, mitochondria (10 μg protein / ml) was added, and the change in absorbance of cytochrome c was measured.

result
Identification of truncated transcripts of DLST We have found that truncated forms of DLST are present in myocytes (Matsuda et al., 1997). Based on this fact, we thought that a DLST gene truncated protein might induce cell death, and first we searched for a truncated DLST mRNA whose transcription was initiated from an intron of the DLST gene.

Reverse transcription was performed from poly A ⁺ RNA derived from human neuroblastoma SH-SY5Y cells using reverse transcriptase (RT), and this was subjected to PCR amplification using the primers shown in FIG.

  When a primer pair consisting of a sequence in intron 7 and a sequence in exon 9 was used, amplification of a fragment consisting of about 240 bp was detected (FIG. 2B lane 1). It coincided with the base length of the amplification product estimated from the position of the primer, and was considered to be a transcription product having a sequence following intron 7 to exon 8 and exon 9. Furthermore, since the length does not include intron 8, it was clear that the amplification product was derived from mRNA.

Therefore, when the transcription start site of mRNA from which this amplification product was derived was identified by a previously reported oligonucleotide capping method (Yamabe et al., 1998, Mol. Cell Biol., 18, 6191-6200), position 10556 of the DLST gene was determined. It was found to be an mRNA having a base of a as a transcription initiation site. The truncated protein derived from this novel mRNA was named MIRTD (MItochondrial Respiration Generator of Truncated DLST).
Subsequently, the analysis of the function of the protein that is the translation product of the mRNA was advanced.

MIRTD mRNA expression is significantly decreased in the brain of AD patients
MIRTD mRNA levels were compared by real-time quantitative RT-PCR using poly A ⁺ RNA derived from cerebral cortex of AD patients and healthy individuals. For mRNA derived from the cerebral cortex of healthy controls, the ratio of MIRTD mRNA to DLST mRNA and the ratio of MIRTD mRNA to GAPDH mRNA (GAPDH was measured for standardization control) were (0.0621 ± 0.0505), respectively. In contrast, the above ratios were (0.0248 ± 0.0368) × 10 ⁴ , (0.731 ± 1.15) × 10 for mRNA derived from the cerebral cortex of AD patients, compared to × 10 ⁴ and (1.62 ± 1.47) × 10 ^{4. 4. A} significant difference was observed between healthy AD patients at any ratio (p = 0.004 for the ratio of MIRTD mRNA to DLST mRNA, and p = 0.018 for the ratio of MIRTD mRNA to GAPDH mRNA). Therefore, it was shown that MITRD mRNA is significantly reduced in the cerebral cortex of AD patients. Therefore, this suggests that MITRD is related to AD.

Control of MIRTD expression by DLST haplotype The DLST haplotype affects the expression of MIRTD mRNA because the polymorphism of DLST gene exists near position 10556 of the MIRTD mRNA transcription start site specified above. Whether to give or not.

  First, the ac-type and gc-type haplotype full-length human DLST genes shown in FIG. 1 were each cloned into a cosmid vector and transfected into a rat PC12 cell line. The amount of human MIRTD mRNA and the amount of human DLST mRNA in the transfectant were measured by real-time quantitative RT-PCR to determine the amount of human MIRTD mRNA relative to the amount of human DLST mRNA (FIG. 4). Primers that specifically amplify only human DLST and MIRTD were selected and used without amplifying the endogenous rat mRNA of PC12 cells. Actually, only exogenous human DLST and MIRTD mRNA were detected, and PC12 endogenous mRNA was not detected (FIGS. 4A and B). In 10579T (ac haplotype) cells, the ratio of MIRTD mRNA to DLST mRNA was 3.6 ± 1.9% and 4.6 ± 2.3%, but in 10579A (gc haplotype) cells, the ratio of MIRTD mRNA to DLST mRNA was They were 8.7 ± 2.4% and 9.7 ± 3.2% (respectively measured in two independent clones: FIG. 4C). There was a statistically significant difference between the two (see the column of FIG. 4 in the brief description of the drawing), suggesting that the expression of human MIRTD mRNA depends on the haplotype of the DLST gene.

Intracellular distribution of MIRTD protein
The existence of MIRTD at the protein level was proved. Experiments were performed using rat liver. The reason for using the liver is that it is easy to fractionate intracellular organelles. In the rat liver, a short mRNA molecule, that is, MIRTD mRNA, was detected by Northern blotting (FIG. 5A), so it was considered that the MIRTD protein was expressed in the rat liver.

  In order to clarify the intracellular distribution of MIRTD protein, rat liver was subjected to cell fractionation according to a conventional method, and Western blotting was performed for each fraction (FIG. 5B). 55 kDa and 30 kDa proteins were detected in the mitochondrial fraction (P10000 fraction). The 55 kDa protein corresponds to the full-length DLST, and the 30 kDa protein is a translation product from the first AUG codon present in exon 8 of the DLST gene (the estimated molecular weight of the product is 29.7 kDa, and the reading frame is the same as the full-length DLST) It is considered to be a translation product from Therefore, MIRTD, which was detected in the mitochondrial fraction, suggests that the protein is mainly distributed in mitochondria.

  In order to examine the distribution of MIRTD in more detail, mitochondria isolated from rat liver were treated with proteinase K and subjected to Western blotting. Tom40, Tom20 and full-length DLST (matrix protein) were used as endogenous controls. Tom40 and Tom20 are components of the translocation complex used to translocate the mitochondrial precursor protein encoded by nuclear DNA to mitochondria. Tom40 is exposed on the inner membrane side, and Tom20 is exposed outside the mitochondria. Protein (Pfanner and Geisller, 2001, Nat. Rev. Mol. Cell Biol., 2, 339-349). Tom20 was readily digested with 10 μg / ml proteinase K, but Tom40 and DLST and MIRTD proteins were not digested (FIG. 5C, lanes 1-3). On the other hand, when mitochondria were used as mitoplast, the band of MIRTD disappeared (FIG. 5C, lane 4), but the bands of Tom40 and Tom20 were detected. From this, it is presumed that the MIRTD protein is localized in the space between the inner and outer mitochondrial membranes.

Establishing a cell line for digesting MIRTD mRNA Next, we identify the role of the protein in the cell by specifically digesting MIRTD mRNA and reducing the expression level of MIRTD protein using maxizyme technology. Tried. A maxizyme is a new ribozyme that recognizes a target sequence and specifically cleaves the target RNA. In order to express the maxizyme under the control of a strong promoter, genes encoding each maxizyme monomer (MzL and MzR) were inserted downstream of the human tRNA ^Val promoter sequence. The design of the human tRNA ^Val construct was performed according to the method described in Koseki et al., 1999. A sequence of a maxizyme that takes an active three-dimensional structure only in the presence of mRNA corresponding to the ligation site of intron 7-exon 8 of the DLST gene (FIG. 6A) was designed to cleave only at a specific location. The maxizyme has been found to specifically cleave MIRTD mRNA and not DLST mRNA (Tanabe et al., 2000, Biomacromolecules, 1, 108-117.).

Poly A ⁺ RNA was isolated from human neuroblastoma SH-SY5Y cells that transfected and expressed DNA fragments encoding MzL and MzR, and MIRTD mRNA was quantified by real-time quantitative PCR. In the maxizyme-expressing cells, the MIRTD mRNA level was also significantly decreased (FIG. 6B). The amount of DLST and MIRTD proteins was detected semi-quantitatively by Western blot (Figure 6C). The amount of DLST protein was not affected by the introduction of maxizyme, but MIRTD protein was significantly reduced (FIG. 6C, lanes Z5 and Z21). Therefore, maxizyme specifically digested MIRTD mRNA, and the 30 kDa protein was proved to be a translation product of MIRTD mRNA.

Characteristics of MIRTD-deficient cell lines
We tried to elucidate the action of MIRTD by examining the traits that appear by specifically digesting MIRTD mRNA. First, cells expressing maxizyme or its monomer (non-active form) were exposed to 0 to 0.6 mM hydrogen peroxide-containing medium, and the numbers of live and dead cells were determined as iodine propidium (PI) and Hoechst 33342, respectively. Investigated by dye staining. In the maxizyme-expressing cells, depending on the exposure amount and time of hydrogen peroxide, the cell activity against hydrogen peroxide was significantly decreased compared to the control (monomer-expressing cells) (FIGS. 7A and B). When the cells were differentiated into neurons by retinoic acid treatment, the sensitivity of maxizyme-expressing cells to hydrogen peroxide was further increased (FIG. 7C). Even when differentiated into nerve cells, the sensitivity to hydrogen peroxide did not change greatly, so it was estimated that this phenomenon was independent of the state of the cells.

  When MRTTD expression in the above-mentioned transfectant cells was examined by real-time RT-PCR and Western blotting, both mRNA and protein levels were slightly increased in the control cells at an early stage, but gradually thereafter. It was maintained at a low level in the maxizyme-expressing cells (FIGS. 7D and E). This result suggests that the decrease in MIRTD is involved in the sensitivity of cells to external stimulation by hydrogen peroxide.

The cells were also tested for sensitivity to drugs that induce endogenous oxidative stress. Menadione is a quinone derivative that produces a superoxide anion from the mitochondrial respiratory chain, which is immediately converted to hydrogen peroxide. When maxizyme-expressing cells were exposed to menadion, resistance to the drug increased compared to control cells (FIG. 7G). Hydrogen peroxide production from menadion is a hydrogen peroxide-specific fluorescent dye [5- (and-6-) chloromethyl 2 ', 7'-dichlorodihydrofluorescin diacetate acetyl ester (CM-H ₂ DCFDA)] Was examined by flow cytometry.

  In the control cell clone L1, the amount of hydrogen peroxide produced was increased by the menadione treatment, whereas in the maxizyme-expressing cell clone Z21, no increase in the amount of hydrogen peroxide was observed (FIG. 7H). Therefore, it was found that MIRTD-deficient cells resulted in decreased superoxide anion production and increased resistance to menadione.

Respiratory insufficiency in maxizyme-expressing cells In order to investigate the reason why the MIRTD-deficient cells found above are insensitive to menadione, the mitochondrial respiratory chain in maxizyme-expressing cells was examined. First, when the oxygen consumption rate of whole cells was examined using an oxygen electrode, respiration was hardly observed in the maxizyme-expressing cell clones (Z5, Z21), but respiration was observed in one control cell clone (L1 and R2). Was observed (FIG. 8A). Therefore, when mitochondria were isolated from each clone and the enzyme activity of each complex of the respiratory chain was measured (FIG. 8B), the activity of complex IV (cytochrome c oxidase) was not detected in the MIRTD-deficient clone (Z5, Z21). It was. Furthermore, in these clones, the activity of NADH-cytochrome c oxidoreductase (complex I + III) was remarkably reduced, but the activity of succinate cytochrome c oxyreductase (complex II + III) was not reduced. As a result, the activity of cytochrome c oxidase was completely lost, and the activity of complex I (NADH-ubiquinone oxidoreductase) was significantly affected but not completely lost. Was found to bring about the disappearance of

To explore the mechanism behind the respiratory defect, constitutive levels of subunits involved in the respiratory chain were examined by Western blot (FIG. 8C). The amount of complex II, III and V subunits encoded on the nuclear chromosome was independent of MIRTD deficiency. In contrast, the amount of complex I 15K subunit encoded on the nuclear chromosome was slightly reduced in MIRTD-deficient cells. Furthermore, the amount of complex IV subunits (CO I and CO II) encoded on the mitochondrial chromosome was significantly reduced. In addition, the amount of complex IV subunit (CO IV) encoded on the nuclear chromosome was also significantly affected. These trends were consistent with a decrease in enzyme activity. It is a drug that inhibits the translation in the mitochondria and the cytoplasmic translation mechanism in order to investigate whether or not the mitochondrial translation mechanism is involved in the decrease of subunits (CO I and CO II) encoded on the mitochondrial chromosome The gene product was examined by pulse labeling with [ ³⁵ S] methionine in the presence of emetine. Labeled translation products were separated by SDS-PAGE and detected by autoradiography (FIG. 8D). The signal intensity did not change much for each clone, and the change could not explain the decrease in constitutive levels of CO I and CO II. Thus, a decrease in constitutive levels of complex IV subunits appears to have occurred during the post-translational process. Furthermore, the amount of subunit CO IV mRNA encoded on the complex IV nuclear chromosome was independent of MIRTD (FIG. 8E). From these facts, MIRTD deficiency affects the complex formation of complex IV and also has some effect on the complex formation of complex I, which loses activity and does not participate in complex formation. The theory is that it may be digested by mitochondrial proteinases.

FIG. 1 is a schematic diagram of the DLST gene structure. Nucleotide residues are counted based on a revised sequence starting at 1 from the transcription start site of DLST mRNA (Nakano et al., 1994). Haplotypes are defined by two nucleotide variations at positions 19117 (A or G) and 19183 (C or T). Nucleotides specific for the ac haplotype are listed. The transcription start site of MIRTD mRNA is around 10556. FIG. 2 shows the presence of truncated mRNA transcribed from intron 7. (A) Schematic diagram of the primer (ad) and TaqMan probe (e) used. The arrow indicates the position of the primer. In order to detect the truncated mRNA (MIRTD mRNA), PCR was performed by 35 cycles of 95 ° C. for 15 seconds and 57 ° C. for 1 minute at 95 ° C. for 10 minutes. Primers a, b, c and d are respectively MIRTD forward primer; 5'-ctcatttcagacagtgccagtg-3 '(Intron 7: SEQ ID NO: 5), MIRTD reverse primer; 5'-tggctcagctagtggtggg-3' (Exon 9: SEQ ID NO: 6) DLST forward primer; 5′-acctacagcagcggcagt-3 ′ (exon 8: SEQ ID NO: 3) and DLST reverse primer; 5′-ctcctggctcagctagtggt-3 ′ (exon 9: SEQ ID NO: 4). (B) Poly (A) ⁺ RNA was isolated from SH-SY5Y cells. Complementary DNA was synthesized using oligo (dT) primers, and PCR was performed using primers a and b (lane 1) or primers c and d (lane 2). The Poly (A) ⁺ RNA fraction was not treated with reverse transcriptase but subjected to PCR (lane 3). Lane 4 shows a marker of φX174 DNA digested with Hinc II. Arrows indicate the respective amplified fragments. (C) The start site of MIRTD mRNA was determined using the oligonucleotide capping method (Yamabe et al., 1998, Mol.) Using the Cap Site cDNA ^™ of human brain (Nippon Gene, Toyama, Japan) as described in Materials and Methods. Cell Biol., 18, 6191-6200.). FIG. 3 is a diagram showing a decrease in the amount of MIRTD mRNA in the brain of Alzheimer's disease patients. MIRTD mRNA levels in AD patients or control brains were measured by real-time quantitative RT-PCR as described above and normalized to DLST (A) or GAPDH mRNA (B). Sample numbers, mean values with standard deviation (± SD), and p-values tested by Student's t-test are shown in the figure. FIG. 4 shows a decrease in human MIRTD expression in rat PC12 cells containing the ac-type human DLST gene. Lipofectin (Invitrogen) was used to co-transfect PC12 cells with a cosmid containing an ac- or gc-type DLST gene and a geneticin resistance gene. Recombinants were enriched by selecting 14 days of geneticin resistant cells and Poly (A) ⁺ RNA was prepared from the recombinant pool. Human MIRTD and DLST mRNA levels were measured by real-time quantitative RT-PCR. (A) Real-time quantitative RT-PCR profile showing the increase in PCR products by human DLST and MIRTD mRNA. (B) Real-time quantitative RT-PCR profile using Poly (A) ⁺ RNA from untransfected PC12 cells in which neither DLST nor MIRTD mRNA is detected. (C) The relative value of MIRTD with respect to DLST mRNA is shown as an average value ± SD obtained from four independent experiments. The differences were significant in 1 and 3 (p = 0.039), 1 and 4 (p = 0.049), 2 and 3 (p = 0.016) and 2 and 4 (p = 0.016). FIG. 5 is a diagram showing identification and localization of MIRTD protein. (A) Total RNA (50 μg) isolated from rat liver was analyzed by Northern blotting. DLST and MIRTD mRNA were detected by ³² P-labeled rat DLST cDNA. 28S and 18S ribosomal RNAs are shown on the right. Arrows indicate mRNA corresponding to DLST and MIRTD. (B) Fractionation of rat liver was performed as described above. The precipitate fraction (P10,000) obtained by centrifuging the supernatant excluding nuclei at 10,000 xg contained most mitochondria. The supernatant was centrifuged at 100,000 xg and the precipitate (P100,000) and supernatant (S100,000) contained microsomes and cytosol, respectively. Samples were analyzed by Western blotting using anti-DLST monoclonal antibody. (C) Isolated rat liver mitochondria were diluted with isotonic buffer (lanes 1-3) or hypotonic buffer (lanes 4-6) to prepare mitoplasts. Samples were aliquoted and treated with 0 (lanes 1, 4), 10 (lanes 2, 5), or 50 μg / ml (lanes 3, 6) proteinase K. Samples were analyzed by Western blotting using anti-DLST monoclonal antibody, anti-Tom40 polyclonal antibody, or anti-Tom20 polyclonal antibody. The asterisk represents the degradation product of DLST. PK; proteinase K, M; mitochondria, MP; mitoplast. FIG. 6 is a view showing a decrease in the amount of MIRTD mRNA due to the introduction of maxizyme. (A) Sequences of MIRTD mRNA and maxizyme heterodimer, MzR and MzL. The maxizyme heterodimer can form a three-dimensional structure having activity only when a junction between intron 7 and exon 8 of the DLST gene is present. A triangular mark indicates a cleavage site by maxizyme. (B) Independent clones (Z5, Z7 and Z21) or monomeric controls (L1 and Z21) that co-transfect maxizyme and geneticin resistance gene DNA into neuroblastoma SH-SY5Y cells and constitutively express maxizyme After isolation of R2), relative values of MIRTD mRNA relative to DLST mRNA were obtained as described in Materials and Methods. (C) Upper panel; Protein (50 μg) of each sample was subjected to SDS-PAGE, followed by Western blotting using an antibody against the C-terminal region of DLST protein. Lower panel; MIRTD protein levels were quantified and normalized to DLST protein levels. The results are shown as the mean ± SD of three experiments. FIG. 7 is a graph showing sensitivity to oxidative stress in MIRTD-deficient cells. (A) Maxizyme expressing cells (Z5, Z7 and Z21) and control cells (L1 and R2) were exposed to media containing 0.2, 0.4 or 0.6 mM H ₂ O ₂ . After 24 hours, cells were stained with Hoechst 33342 (blue; dead and live cells) and propidium iodide (pink; dead cells). Cell viability was calculated by counting 200 cells for each clone exposed to various concentrations of H ₂ O ₂ . (B) Z5, Z21, L1 and R2 cells were exposed to 0.4 mM H ₂ O ₂ for the indicated times. Cell viability was calculated as described above. (C) Recombinants (L1, R2, Z7 and Z21) were induced to differentiate into neuron-like cells with 10 μM retinoic acid for 5 days in the presence of 1% fetal calf serum, and then in the presence of 1% fetal calf serum. Exposed to various concentrations of H ₂ O ₂ for 12 hours. Cell viability was calculated as described above. (D) Prepare total protein from L1 and Z21 cells exposed to 0.2 mM H ₂ O _{2 for 0} , 6, 12, and 24 hours, SDS-PAGE the protein of each sample (50 μg), then C-terminus of DLST protein The sample was subjected to Western blotting using an antibody against the region. Arrows indicate MIRTD and full-length DLST protein. (E) Poly (A) ⁺ RNA was isolated from L1 and Z21 cells exposed to 0.2 mM H ₂ O _{2 for 0} , ₃ , 6, 12 or 24 hours. MIRTD mRNA levels were measured by real-time quantitative RT-PCR and normalized to DLST mRNA levels. (F) MIRTD protein amount was quantified by Western blotting in (D) and normalized to DLST protein amount. (G) Z5, Z21, L1, and R2 cells were exposed to media containing the indicated concentrations of menadione. After 24 hours, cell viability was calculated as described in (A). (H) Z21 and L1 cells were cultured for 3 hours in the presence or absence of 10 M menadione, then 5-(-6) -chloromethyl-2 ', 7'-dichlorodihydrofluorescein diacetate, acetyl ester ( CM-H ₂ DCFDA, Molecular Probes) (5 μM) was treated for 1 hour. After the cells were trypsinized, the cells were subjected to flow cytometry using an EPICS ELITE flow cytometer (Beckman Coulter) in order to measure the fluorescence signal of DCF. In each experiment, 20,000 cells were measured and the average value was determined. (AC) and (EH) results are shown as the mean ± SD obtained from 3 or 4 experiments. FIG. 8 shows the activity of the mitochondrial respiratory chain complex and a decrease in its amount. (A) Semi-confluent cells were collected by trypsin treatment. The collected cells were suspended in a culture medium at 0.7-1.5 × 10 ⁷ cells / ml and placed in an oxygen electrode chamber (50 l). The oxygen consumption rate by the cells was measured at 37 ° C. using a Clark electrode (Strathkelvin Instruments, Glasgow). Left; follow-up results of oxygen consumption by cells at 1 x 10 ⁷ cells / ml. Right; mean and standard deviation of 3 independent experiments normalized to cell number. (B) NADH-cytochrome c oxidoreductase (I + III), succinate-cytochrome c oxidoreductase (II + III), ubiquinol-cytochrome c oxidoreductase (III) and cytochrome c oxidase (IV) as described above The activity of was measured. Results are shown as relative activity. The level of activity in mitochondria isolated from SH-SY5Y cells was taken as 100. Note that complex IV activity was not detected in mitochondria isolated from Z5 and Z21 clones. Mean values and standard deviations were obtained from three independent experiments. (C) The protein (10 μg) of the mitochondrial fraction prepared from each clone was subjected to Western blotting using the respective antibodies against the respiratory complex and the ATP synthase subunit. COI and II are mitochondrial encoded proteins, while others are encoded by nuclear genes. F ₁ β; F ₁ -ATPase: β subunit. (D) Mitochondrial translation products labeled with [ ³⁵ S] methionine in vivo were analyzed as described (Hayashi et al., 1993). Briefly, semi-confluent cells were labeled with [ ³⁵ S] methionine in the presence of emetine (0.2 mg / ml) for 60 minutes at 37 ° C. The protein of the mitochondrial fraction was separated by SDS-PAGE (15-25% gradient gel). The translation product was analyzed using bioimaging analyzer FLA2000 (Fuji Film, Tokyo). ND 1-5; complex I (NADH-ubiquinone oxidoreductase) subunit, COI-III; cytochrome c oxidase (complex IV) subunit, cyt b; ubiquinol-cytochrome c oxidoreductase (complex III) ) Cytochrome b subunit, ATP6 and 8; ATP synthase (complex V) subunit. (E) Poly (A) ⁺ RNA from each clone was converted to cDNA, and CO IV mRNA was quantified by real-time RT-PCR as described in Materials and Methods. Average values and standard deviations from triplicate experiments are shown after normalization to GAPDH cDNA.

Claims (16)

Protein of either of the following (a) or (b).
(a) a protein having the amino acid sequence shown in SEQ ID NO: 1
(b) a protein having an amino acid sequence in which one to several amino acid residues are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and having the same activity as the protein of (a)   A nucleic acid encoding the protein according to claim 1. The following nucleic acid (a) or (b):
(a) a nucleic acid having the base sequence shown in SEQ ID NO: 2
(b) a nucleic acid capable of hybridizing under stringent conditions with a nucleic acid complementary to the nucleic acid having the base sequence shown in SEQ ID NO: 2   Quantifying mitochondrial respiratory-producing truncated DLST protein (MIRTD) mRNA, a transcription product initiated from a base in intron 7 of a dihydrolipoamide succinyltransferase (DLST) gene in cells collected from a subject A method for determining Alzheimer's disease or the risk of developing it.   A method for determining Alzheimer's disease or the risk of developing it, comprising quantifying MIRTD protein in cells collected from a subject.   The method according to claim 4 or 5, wherein the cells are derived from any one selected from the group consisting of neural tissue, heart, liver and kidney.   A method for determining the risk of developing Alzheimer's disease, comprising examining whether or not the 10579th base of a dihydrolipoamide succinyltransferase (DLST) gene of a subject is T.   8. The method according to claim 7, wherein the gene is extracted from peripheral blood.   In order to determine Alzheimer's disease or the risk of developing it, including a primer pair for detecting MIRTD mRNA which is a transcription product initiated from any base in intron 7 in the dihydrolipoamide succinyltransferase (DLST) gene Diagnostic kit.   The diagnostic kit according to claim 9, wherein the primer pair for MIRTD mRNA detection consists of 5'-ctcatttcagacagtgccagtg-3 '(SEQ ID NO: 5) and 5'-tggctcagctagtggtggg-3' (SEQ ID NO: 6).   A TaqMan probe for quantifying MIRTD mRNA, comprising a probe having a luminescent dye as a reporter at the 5 ′ end of 5′-cctccttctggcaaacctgtgtctct-3 ′ (SEQ ID NO: 7) and a quencher for the luminescent dye added at the 3 ′ end The diagnostic kit according to claim 9 or 10, further comprising:   The primer pair according to any one of claims 9 to 11, further comprising 5'-acctacagcagcggcagt-3 '(SEQ ID NO: 3) and 5'-ctcctggctcagctagtggt-3' (SEQ ID NO: 4) as a primer pair for measuring the amount of full-length DLST mRNA for normalization The diagnostic kit according to claim 1.   A diagnostic kit for determining Alzheimer's disease or its risk of developing comprising an antibody that specifically recognizes MIRTD for detecting MIRTD protein.   An antibody that specifically recognizes MIRTD, which is produced by using a C-terminal fragment of DLST containing a region downstream from methionine of exon 8 of DLST, comprising the sequence shown in SEQ ID NO: 1.   The antibody according to claim 14, which is a monoclonal antibody.   The kit according to claim 13, comprising the antibody according to claim 14 or 15.

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