JP2020065482A - Parkinson's disease diagnostic agent and model animal - Google Patents

Abstract

To provide novel causative genes for Parkinson's disease and to provide novel model animals.SOLUTION: Provided is a genetic testing method for Parkinson's disease, which detects a prosaposin gene mutation in a subject-derived sample.SELECTED DRAWING: None

Description

  The present invention relates to a diagnostic agent for Parkinson's disease and a non-human animal model for Parkinson's disease.

  Parkinson's disease (PD) is the second most common neurodegenerative disease in Japan. PD is a disease whose main condition is mesencephalic dopaminergic neuron loss, and movement disorders with tremor, akinesia, muscular rigidity, and postural reflex disorder as the main symptoms develop in middle-aged and older people as the prevalent age Proceed to. The treatment is limited to symptomatic treatment, mainly dopamine replacement therapy by oral administration, and the fundamental treatment has not been developed yet. The effect of symptomatic treatment is often impaired as the disease progresses, and in severely advanced cases, patients often become bedridden due to difficulty walking or falling.

  Most of the PDs are sporadic onsets with no family history, although 5-10% have hereditary PD with a clear family history. Until now, functional analyzes of the causative gene product of hereditary PD have shown the involvement of various pathological conditions such as mitochondrial disorder, oxidative stress, ubiquitin / protease system disorder, and autophagy / lysosomal system disorder in nigral dopamine neuronal cell death. (Patent Document 1).

Annu Rev Pathol 2011.6; 193-222. J Biol Chem 2010.285; 20423-20427 J Biol Chem 2003.24; 25009-25013

  An object of the present invention is to provide a new causative gene of hereditary PD and a new model animal.

  Therefore, the present inventor has focused on the lysosomal function, focused on the prosaposin (PSAP) gene involved in the lysosomal function, and examined the PSAP gene of PD patients. As a result, the PSAP gene mutation is present in autosomal dominant PD patients. It was also found that the clinical symptoms of PD patients having the PSAP gene mutation are very similar to those of sporadic PD. Furthermore, it was found that when the PSAP gene mutation was introduced into mice, the same motor disorders and dopaminergic nerve loss as those in PD patients were caused.

  That is, the present invention provides the following [1] to [8].

[1] A genetic test method for Parkinson's disease, which comprises detecting a prosaposin gene mutation in a sample derived from a subject.
[2] The detection method according to [1], wherein the prosaposin gene mutation is a mutation in the region encoding the saposin D domain.
[3] A diagnostic agent for Parkinson's disease containing a prosaposin gene mutation detection reagent.
[4] The diagnostic agent according to [3], wherein the prosaposin gene mutation is a mutation in the region encoding the saposin D domain.
[5] A Parkinson's disease model non-human animal having a prosaposin gene mutation.
[6] The model non-human animal according to [5], wherein the prosaposin gene mutation is a mutation in the region encoding the saposin D domain.
[7] A method for screening a preventive / therapeutic drug for Parkinson's disease using a non-human animal model of Parkinson's disease having a prosaposin gene mutation.
[8] The screening method according to [7], wherein the prosaposin gene mutation is a mutation in the region encoding the saposin D domain.

  According to the present invention, a prosaposin gene mutation-positive PD can be diagnosed by detecting a prosaposin gene mutation in a subject. Further, by using the model animal of the present invention, it can be used for searching for a new drug for preventing or treating PD.

3 shows prosaposin gene mutations in autosomal dominant PD disease in 3 families. All three gene mutations in the prosaposin gene are heterozygous mutations in the site encoding the saposin D region. The sequence of II-2 is shown in SEQ ID NO: 1, the sequence of II-1 is shown in SEQ ID NO: 2, and the sequence of II-3 is shown in SEQ ID NO: 3. The electron microscopic image of the skin fibroblast of a PD disease patient who has a prosaposin gene mutation is shown. Weakly magnified image (upper scale bar 5.0 μm) and strong magnification (lower scale bar 1.0 μm) are shown. N indicates a nucleus. Lysosomal-like structures contain bilayer vesicles. The autophagic flux assay result of iPS cell-derived midbrain dopamine neurons is shown. It is shown that PSAP-PD increases α-synuclein in the Triton X-100 insoluble fraction of midbrain dopamine neurons derived from iPS cells. It is shown that mice introduced with a gene mutation (C509S) in the saposin D domain exhibit movement disorders associated with aging. Fig. 7 shows the loss of substantia nigra tyrosine hydroxylase-positive neurons in mice introduced with a gene mutation for the saposin D domain (C509S).

  In the diagnosis of prosaposin gene mutation-positive PD in the present invention, prosaposin gene mutation in a sample derived from a subject is detected.

In recent years, the involvement of lysosomal dysfunction in the elucidation of PD pathology has been reported many times. In particular, the involvement of lysosomal disease, which is a group of diseases that cause lysosomal dysfunction, has attracted particular attention. In some lysosomal diseases, it is known that deficiency of enzymes involved in intracellular sphingolipid metabolism causes sphingolipidosis in which sphingolipid, which is a substrate thereof, accumulates, and is accompanied by severe central nervous system disorder (non- Patent Document 2). The importance of the pathophysiology of lysosomal disease in PD is that the ATP13A2 gene, which is the causative gene of neuronal ceroid lipofuscinosis, which is a type of lysosomal disease, is also the causative gene of hereditary PD, and the most frequent occurrence of lysosomal disease. It is also suggested that the GBA gene mutation, which is the causative gene of Gaucher disease, is the greatest genetic risk of PD. Furthermore, in lysosomes, α-synuclein, which is a major constituent protein of Lewy bodies, which is characteristic of brain pathology of PD patients, is decomposed (Non-patent Document 3).
Although the prosaposin (PSAP) gene is extremely rare, its pathological mutation is a gene capable of causing lysosomal disease. However, it was completely unknown whether or not the PSAP gene mutation occurred in PD patients. As a result of the study by the present inventor, it was found for the first time that a PSAP gene mutation occurred in an autosomal dominant PD patient of three kindreds (FIG. 1).
Since all PSAP gene mutations were found in the saposin D domain-encoding region, mutations in the saposin D domain-encoding region are particularly useful for the diagnosis of hereditary PD.

  Furthermore, as a mutation in the saposin D domain, a mutation that replaces at least one of the six cysteine residues constituting the three pairs of disulfide bonds in the saposin D domain, or a mutation in the vicinity of the region encoding the six cysteine residues Mutations are preferred. Here, the six cysteine residues are C409, C412, C440, C451, C476, C482 in the case of human and C442, C445, C473, C484, C509, C515 in the case of mouse. Among these, a mutation in which a cysteine residue selected from C409, C412, C440 and C482 is replaced with another amino acid residue is preferable.

  Subjects include humans with PD who wish to undergo genetic testing. It is particularly suitable as a subject if there is an autosomal dominant PD family history, but it can also be performed in sporadic cases or autosomal recessive PD cases. The sample used for the test may be a human-derived sample containing a gene, and examples thereof include blood, urine, sweat, saliva, and a part of tissue.

  As a means for detecting the PSAP gene mutation, conventionally known means, for example, DNA sequence, DNA chip, DNA microarray, RFLP, PCR-SSOP-Luminex method can be used. Therefore, examples of the reagent for detecting a PSAP gene mutation of the present invention include reagents used for these detection means.

  When a PSAP gene mutation is detected in a sample derived from the subject, the subject can be diagnosed as PSAP gene mutation positive PD. According to the study by the present inventor, since the clinical symptoms of PSAP gene mutation positive PD patients closely resemble the clinical characteristics of sporadic PD, the PSAP gene mutation is not only a causative gene of hereditary PD but also sporadic It is considered to be deeply involved in the formation of the pathological mechanism of PD.

  Further, as shown in Examples below, morphological analysis by electron microscopy of PD (PSAP-PD) -derived skin fibroblasts having a PSAP mutation revealed abnormal accumulation of lysosomal structures (Fig. 2). This structure was considered to be an autolysosome because it contained bilayer vesicles. The accumulation was consistent with the findings suggesting lysosomal dysfunction, and it was shown that the saposin D domain mutation causes lysosomal dysfunction. Moreover, in order to prove the lysosomal dysfunction in PSAP-PD midbrain dopamine neurons, autophagy flux assay was performed using lysosomal inhibitor E64d and pepstatin A. As a result, as shown in FIG. 3, PSAP-PD-derived midbrain dopamine neurons showed a decrease in autophagy flux, indicating lysosomal dysfunction. Further, when α-synuclein in PSAP-PD-derived dopamine cells was evaluated, Triton X-100 insoluble α-synuclein was increased, suggesting that α-synuclein, which tends to aggregate, accumulates.

  The present invention also provides a PD model non-human animal having a PSAP gene mutation and a method for screening a PD preventive / therapeutic agent using this model non-human animal.

  The PSAP gene mutation is preferably a mutation in the region encoding the saposin D domain, and in particular, at least one amino acid of 6 cysteine residues forming 3 pairs of disulfide bonds in the saposin D molecule is substituted. Mutations or mutations near the region encoding the six cysteine residues are preferred. Here, the six cysteine residues are C409, C412, C440, C451, C476, C482 in the case of human and C442, C445, C473, C484, C509, C515 in the case of mouse. Among these, mutations in which the cysteine residue selected from C442, C445, C473, C484 and C515 is replaced with another amino acid residue are preferred.

  The non-human animal is preferably a non-human mammal. Non-human mammals include, for example, cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, etc. Rodents with relatively short cycles and easy breeding, especially mice or rats, are preferred.

In order to introduce a PSAP gene mutation into a non-human animal, a method usually used for modifying a gene on a chromosome can be applied. Preferably, a targeting vector having a homologous recombination region and a Cre-loxP system region is used. The case of a mouse will be described below.
First, a homologous recombination region for producing a mouse PSAP gene mutation is prepared. The homologous recombination region determines the region of the PSAP gene to be mutated based on the nucleotide sequence information obtained from a database or the like. Particularly preferred is a mutation in which at least one amino acid of 6 cysteine residues forming 3 pairs of disulfide bonds in the saposin D molecule is substituted, or a mutation near the region encoding 6 cysteine residues. This genetic mutation (C509S) is expected to disrupt one pair of three disulfide bonds within the saposin D molecule. Diseases due to gene mutations in the saposin D domain are extremely rare, but mutations that destroy a pair of disulfide bonds in saposin B and saposin C cause human deficiencies in saposin B and C are known. . Since each of the four saposin molecules is a molecule with extremely high homology, it is expected that the mutation that disrupts the disulfide bond in saposin D is also likely to have a pathological significance. In addition, the mutation found in familial PD also includes a mutation at a site encoding a cysteine residue formed in three pairs of disulfide bonds in saposin D.

  Further, as the targeting vector, the Cre-loxP system containing a marker gene for selecting cells into which the vector has been incorporated or cells in which the desired homologous recombination is likely to occur is used. Examples of the marker gene inserted between the two loxPs include neomycin resistance gene (neo), hygromycin resistance gene (hyg), herpesvirus thymidine kinase gene (HSV-tk), diphtheria toxin A fragment gene (DT-A). , A thymidine kinase gene (tk) and the like which are usually used for drug selection are used. For example, the neomycin resistance gene is a marker gene for positive selection that enables selection of cells in which homologous recombination has occurred by using the neomycin analog G418. In addition, a marker gene used for negative selection for selecting a target cell, for example, a thymidine kinase gene (using ganciclovir as a selection agent and selectively removing non-homologous recombinants depending on its sensitivity), diphtheria toxin A fragment gene ( (A non-homologous recombinant is selectively removed by the diphtheria toxin expressed by DT-A) and the like can also be used together with a marker gene for positive selection. In the targeting vector, the marker gene for positive selection is preferably located in a region flanked by upstream and downstream homologous regions, and is located in a region flanked by recombinase target sequences since the marker gene is excised in the presence of recombinase. More preferably. The marker gene for negative selection is preferably located in a region outside the homologous region upstream or downstream.

  Such a targeting vector can be prepared by an ordinary DNA recombination technique. For example, it can be obtained by using a PSAP mutant gene or BAC clone cloned according to a conventional method and cleaving it with an appropriate restriction enzyme. If a fragment or a DNA fragment or the like prepared by amplification by the PCR method or the like is combined with the synthesized linker DNA, a reporter gene, a fragment containing a drug resistance marker gene or the like in an appropriate order according to the above design Good.

  Next, the prepared targeting vector for homologous recombination is introduced into an appropriate cell which is usually used for producing a chimeric animal. The cells used here include, for example, egg cells and embryonic stem cells (ES cells), and ES cells are preferable because they have pluripotency capable of differentiating into all kinds of cells in the living body.

  ES cells are cell lines that are established from the inner cell mass (ICM) of blastocysts and that can be proliferated and cultured while maintaining an undifferentiated state. A gene transfer method using ES cells has been established for mice. (Mansour, SL et al., Nature 336: 348 (1988)). As the ES cells, already established cell lines may be used. For example, in the case of mice, ES cells derived from mouse strains such as TT2 and C57BL / 6 can be mentioned. Alternatively, a newly established one may be used. ES cells may be subcultured according to a conventional method.

  The targeting vector can be introduced into cells by a conventional method, for example, an electroporation method, a microinjection method, a calcium phosphate method, a lipofection method, a DEAE-dextran method, or the like. Of these, the electroporation method is preferably used because it is simple and can treat a large number of cells. The conditions for gene transfer by the electroporation method may be those for normal gene transfer into animal cells.

  ES cells into which the targeting vector has been introduced can be cultured on feeder cells according to a conventional method to obtain single cell-derived colonies. At this time, in the ES cell in which homologous recombination has occurred, the marker gene is also integrated into the chromosome together with the PSAP gene mutation in the vector. Thus, the target ES cell can be selected. In addition, ES cells in which homologous recombination has occurred can also be selected by the PCR method, the Southern hybridization method or the like using the genomic DNA extracted from the colony. In the case of the PCR method, for example, a primer designed outside the homologous region and a primer designed in the marker gene are used to perform PCR using genomic DNA as a template to determine whether a band of a desired length can be obtained. can do. In the case of the Southern hybridization method, for example, a DNA fragment obtained by digesting genomic DNA with a restriction enzyme that facilitates observation of changes in the cleavage pattern due to homologous recombination, a probe designed outside the homologous region and a probe designed for a marker gene are used. Southern hybridization can be carried out using this to determine whether a band can be obtained at the desired position.

  Next, chimeric animals are produced using the ES cells that have undergone homologous recombination, according to the methods generally used for producing chimeric animals, such as the injection method and the aggregation method. Specifically, the ES cells that have undergone homologous recombination are injected into an embryo or blastocyst at an appropriate time in the early stage of embryogenesis, for example, at the 8-cell stage, and the obtained embryo is transferred to a pseudo-pregnant female foster mother. By transplanting into the uterus, a chimeric animal composed of cells having a normal PSAP locus and cells having an ES cell-derived PSAP locus mutation can be obtained. Selection of an animal of which strain to obtain the host embryo may be performed according to a conventional method so that the ES cell-derived cells and the host embryo-derived cells can be distinguished by a phenotype such as coat color.

When a part of the germ cells of the chimeric animal has a PSAP gene locus mutation derived from the ES cell, all tissues derived from the population obtained by mating the chimeric individual with a normal individual have the PSAP gene derived from the ES cell. Individuals (PSAP F / +) composed of cells having a locus mutation can be selected by a discrimination method such as hair color.
For example, when a chimeric mouse obtained by using a mouse ES cell of the J1 strain and a host embryo of the C57BL / 6J strain is crossed with the C57BL / 6J strain, the delivered offspring are homologous to the germ cell of the chimeric mouse. If it is derived from a modified ES cell, it exhibits the same wild color (agouti) as the mouse from which the ES cell is derived, and if it is derived from the host embryo, it exhibits the same black color as the mouse from which the host embryo is derived.

  The thus obtained individual is a heterozygous type having a homologous recombinant ES cell-derived PSAP locus mutation on one homologous chromosome (PSAP F / +). By mating these F1 heterozygous individuals, F2 heterozygous and F2 homozygous (PSAP F / F) individuals can be obtained. To make the function of the PSAP gene mutation defective in a tissue-specific manner, it is preferable to use a homozygous type.

  The PSAP F / F mouse can be produced by the above-mentioned method or the like, but an existing PSAP F / F mouse may be used, for example, as described in Matsusuda et al. , Human Molecular Genetics 2004; 13 (21): 2709-2723.

  When the present inventor reared mice having this PSAP gene mutation to an old age, it was found that aging causes the same movement disorder and dopaminergic nerve loss as PD patients. Therefore, a non-human animal having a PSAP gene mutation is useful as a Parkinson's disease model non-human animal. Here, the movement disorder can be confirmed by the deterioration of the movement function by the rotarod method. The loss of dopamine cells can be measured by staining with an anti-tyrosine kinase (TH) antibody.

  If a non-human animal model of Parkinson's disease having a PSAP gene mutation is used, it can be applied to screening for a preventive / therapeutic drug for Parkinson's disease. For example, by administering a test drug to a model non-human animal having this PSAP gene mutation and observing the progress thereof, it can be easily determined whether or not the test drug is useful as a prophylactic / therapeutic drug for Parkinson's disease. it can.

  Next, the present invention will be described in more detail with reference to examples.

Example 1
As a result of gene analysis of autosomal dominant PD patients of 3 families, it was found that there was a pathological mutation in the PSAP gene (Fig. 1). All PSAP gene mutations found in the three families were found in the region encoding the saposin D domain. The fact that the clinical features of PD patients with PSAP mutation closely resemble the clinical features of sporadic PD suggests that PSAP mutation may be involved in the pathological mechanism of sporadic PD development. it was thought. PSAP is a protein involved in lysosomal function, and attention was paid to lysosomal dysfunction in Parkinson's disease patients (PSAP-PD) having a PSAP gene mutation. First, morphological analysis of PSAP-PD-derived skin fibroblasts by electron microscopy revealed abnormal accumulation of lysosome-like structures (Fig. 2). This structure was considered to be an autolysosome because it contained bilayer vesicles. The accumulation was consistent with the findings suggesting lysosomal dysfunction, and it was shown that the saposin D domain mutation causes lysosomal dysfunction.

Example 2
Analysis of dopamine neurons differentiated from induced pluripotent stem cells (iPSC) was performed. Specifically, iPSCs were established using Sendai virus vectors expressing Yamanaka factor (Klf4, Oct3 / 4, Sox2, c-Myc) in monocytes obtained by blood collection. Differentiation into midbrain dopamine neurons was performed according to Proc Natl Acad Sci USA 2018; 115: E5815-5823. In order to prove the lysosomal dysfunction in PSAP-PD midbrain dopamine neurons, autophagy flux assay using lysosomal inhibitor E64d and pepstatin A was performed. Specifically, the above two agents were added to the medium of healthy control and midbrain dopamine neurons derived from PSAP-PD. Twenty-four hours after the drug treatment, cells were collected with RIPA buffer, and the cell lysate was evaluated for the protein amount of LC3, which is an autophagosome marker, by Western blotting.
That is, the lysosomal inhibitor E64d (15 μM) / pepstatin A (Pepstatin A 30 μM) was added to the medium of iPS cell-derived midbrain dopamine neurons, and cells were collected 24 hours later to give an antibody against LC3, which is an autophagosome marker. Was used for Western blotting. Drug-non-added cells are steady-state cells, and the LC3-II / GAPDH ratio is increased in midbrain dopamine neurons derived from PSAP-PD patients (Fig. 3). The ratio of LC3-II / GAPDH after drug addition and LC3-II / GAPDH of non-added cells was calculated to evaluate the autophagic flux. The amount of protein was evaluated by densitometry using Image J software. An anti-GAPDH antibody was used as a loading control. Statistical analysis was performed by Student's t-test (n = 4, * p <0.05, ** p <0.01).
As shown in FIG. 3, PSAP-PD-derived midbrain dopamine neurons showed a decrease in autophagy flux, indicating lysosomal dysfunction.

  Furthermore, Triton X-100 (TX) insoluble α-synuclein was evaluated. Specifically, healthy control cells and PSAP-PD-derived dopamine neurons were dissolved in a 1% TX-containing Tris buffer, allowed to stand on ice for 20 minutes, and then ultracentrifuged at 100,000 g for 20 minutes. The resulting precipitate was used as a TX insoluble fraction (insoluble) and the supernatant was used as a TX soluble (soluble) fraction, and the amount of α-synuclein was evaluated using Western blotting. As shown in FIG. 4, α-synuclein in TX insoluble was increased, which is a finding suggesting that α-synuclein having an aggregation tendency is accumulated.

Example 3
An attempt was made to evaluate the presence or absence of dyskinesia and the presence or absence of midbrain substantia nigra dopamine neurons in mice into which the saposin D domain gene mutation of the PSAP gene was introduced. The mouse (C509S) described in Human Molecular Genetics 2004; 13 (21): 2709-2723 was used as the saposin D domain gene mutation-introduced mouse.
The motility disorders of saposin D mutation-introduced mice and the number of midbrain dopamine neurons were evaluated using wild-type (WT) mice using heterozygous (PSAP ^{wt / mt} ) mice and homozygous (PSAP ^{mt / mt} ) mice. Was analyzed by comparing
First, movement disorders were analyzed by the rotarod method. Specifically, the 0-70 week old mice were subjected to an accelerated rotation test using a rotary machine equipped with an automatic timer and a drop sensor. Five PSAP ^{wt / mt} mice, 5 PSAP ^{mt / mt} mice and WT were placed on a rubber-covered rotating rod having a diameter of 3 cm (5 males each), and the rotation speed was accelerated from 3 rpm to 35 rpm over 5 minutes. The time (in seconds) from the start of rotation to the mouse falling was recorded. As shown in FIG. 5, the time from the 35th week of age to the fall of the PSAP ^{mt / mt} mouse began to show a shortening tendency, and especially at the 70th week of age, both the PSAP ^{wt / mt} and the PSAP ^{mt / mt} had significant time to WT. Shortening was observed (n = 5, * p <0.05, ** p <0.01, *** p <0.001).
Next, the number of substantia nigra dopamine neurons was evaluated. The left side of FIG. 6 shows that the number of cells stained with anti-tyrosine hydroxylase (TH) antibody in PSAP ^{wt / mt} mouse and PSAP ^{mt / mt} mouse is significantly reduced as compared with WT. There is. TH is a rate-limiting enzyme for dopamine biosynthesis in the first step of the catecholamine biosynthesis system, and biosynthesizes L-DOPA from L-tyrosine. L-DOPA is then converted to dopamine through a decarboxylation reaction. Since there is always TH in the mesencephalic substantia nigra dopamine neurons, it is considered that the decrease in the number of cells stained with anti-TH antibody indicates the loss of dopamine neurons. As shown in the right figure of FIG. 6, the number of dopamine neurons at 18 months of age was measured, and a significant decrease in the number of dopamine neurons in PSAP ^{wt / mt} mice and PSAP ^{mt / mt} mice was observed.

Claims (8)

  A genetic test method for Parkinson's disease, which detects a prosaposin gene mutation in a sample derived from a subject.   The detection method according to claim 1, wherein the prosaposin gene mutation is a mutation in the region encoding the saposin D domain.   A diagnostic agent for Parkinson's disease containing a prosaposin gene mutation detection reagent.   The diagnostic agent according to claim 3, wherein the prosaposin gene mutation is a mutation in the region encoding the saposin D domain.   Parkinson's disease model non-human animal having a prosaposin gene mutation.   The model non-human animal according to claim 5, wherein the prosaposin gene mutation is a mutation in the region encoding the saposin D domain.   A method for screening a preventive / therapeutic drug for Parkinson's disease using a non-human animal model of Parkinson's disease having a prosaposin gene mutation.   The screening method according to claim 7, wherein the prosaposin gene mutation is a mutation in the region encoding the saposin D domain.