JP2018117638A - Methods for screening compounds for treating and/or preventing diseases associated with tdp-43 agglomerate accumulation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide TDP-43 variants having agglomerate-forming ability and cytotoxicity in the full length sequence, and methods for screening compounds for treating and/or preventing diseases associated with TDP-43 agglomerate accumulation using the TDP-43 variants.SOLUTION: The invention provides a TDP-43 variant comprising an amino acid sequence where cysteine at position 173 and/or position 175 in the amino acid sequence of TDP-43 is/are substituted. The invention also provides a method for screening a compound for treating and/or preventing a disease associated with TDP-43 agglomerate accumulation, which method comprises the steps of: (1) contacting the TDP-43 variant with a candidate compound; (2) detecting an activity of the compound to inhibit agglomerate formation; and (3) selecting a compound that inhibits the agglomerate formation.SELECTED DRAWING: Figure 6

Description

The present invention relates to a TDP-43 mutant having the ability to form aggregates, and an RRM1 domain in the TDP-43 mutant,
The present invention also relates to a method for screening a compound for treating and / or preventing a disease in which an aggregate of TDP-43 using the TDP-43 mutant or RRM1 domain accumulates. The present invention also provides TDP-
The present invention relates to a transgenic non-human animal that is a model of a disease in which 43 aggregates accumulate. Furthermore, the present invention relates to an antibody or antibody fragment that binds to an aggregate of TDP-43, a diagnostic agent containing the antibody or antibody fragment, a diagnostic kit, and a pharmaceutical composition.

TDP-43, a nuclear protein, is the causative protein of frontotemporal lobar degeneration (FTLD), a major dementia type, and amyotrophic lateral sclerosis (ALS), the most refractory fatal neurological disease (TAR DNA-b
inding protein of 43 kDa) has been identified. TDP-43 has decreased nuclear localization in FTLD and ALS, and forms pathological aggregates in the cytoplasm, but the mechanism is unknown.

TDP-43 has been found to be found at a very high rate in ubiquitinated inclusion bodies in FTLD and ALS. Diseases presenting abnormal pathological findings of TDP-43 are currently positioned as a group of diseases called TDP-43 proteinopathy, and various reports indicate that TDP-43 dysfunction is likely to be the essence of ALS pathology.

Thus, elucidating the physiological and pathological functions of TDP-43 may lead to overcoming ALS, and energetic research is underway all over the world. The most clear and important pathological findings of the TDP-43 proteinopathy are reduced nuclear staining of TDP-43 and inclusion body formation in the cytoplasm. Elucidation of the function of this ectopic localization is indispensable for understanding the pathology of ALS.

The molecular structure of TDP-43 has two RNA binding regions (RRM), a glycine-rich region on the carboxyl (C) terminal side, a nuclear translocation signal (NLS), and a nuclear export signal (NES).

TDP-43 is originally a nuclear protein, but FTLD and ALS are characterized by extranuclear escape and aggregate formation, which undergo ubiquitination and phosphorylation. At that time, TDP-43
Is known to be cleaved into 35 kDa and 25 kDa highly cohesive C-terminal fragments (C-terminal fragments), a common finding in lesions. However, from the accumulation of research so far, this phenomenon is in the advanced stage of the disease, and the structural change at the early stage of the disease causing protein structural abnormality is still unclear.

The C-terminal fragment of TDP-43 actually exists in patient tissues, and is widely known to form inclusion bodies in the cytoplasm in cultured cells and genetically modified mice (for example, Non-Patent Documents 1 and 2), Drug screening using this system exists. However, there is no evidence that these fragmentations appear early in the onset of disease, and overexpression of these fragments is atypical and incomplete as an ALS model in animal experiments.

In addition to FTLD and ALS, diseases that exhibit ectopic localization of TDP-43 include low-grade glioma, Alzheimer's disease, Huntington's disease, Pick's disease, Parkinson's disease, Lewy body disease,
Cortical basal ganglia degeneration, inclusion body myositis, B cell lymphoma (M stage), etc. have been reported.

TDP-43 has been reported to be transiently ectopically localized in the cytoplasm due to head trauma, axon pull-out damage, and crushing damage. Does not show FTLD or ALS disease type.

T. Nonaka et al .; Human Molecular Genetics, 2009, Vol.18, No.18, 3353-3364 Y-J Zhang et al .; Journal of Neuroscience, 2007, Vol.27, No.39, 10530-10534 T Sato et al .; Neuroscience, 2009, Vol.164, 1565-1578

However, the appearance of the C-terminal fragment in TDP-43 transgenic (Tg) mice is a late phenomenon of the appearance of the phenotype, and cell death does not occur only by overexpression of the C-terminal fragment. The C-terminal fragment TDP-43 Tg mouse does not show an ALS expression system. Thus, TDP-43 C in vivo
There is no evidence that end fragments can trigger disease. In addition, TDP-43 using full-length TDP-43
There is currently no model of proteinopathy, and structural changes leading to misfolding of full-length TDP-43 are also unknown.

Therefore, the present invention treats and / or prevents a TDP-43 mutant having aggregate-forming ability and cytotoxicity in the full-length sequence, and a disease in which aggregates of TDP-43 using the TDP-43 mutant accumulate. An object of the present invention is to provide a screening method for compounds. The present invention also provides:
An object of the present invention is to provide a transgenic non-human animal that is a model of a disease in which aggregates of TDP-43 accumulate.

The present inventors have identified the structural change of RRM1 in TDP-43 aggregate formation, and further identified an intramolecular sequence important for the structural change of RRM1, and the mutant was cultured with FTLD and ALS patients. The knowledge that it becomes the disease model which shows the same pathological change was acquired.

The present invention has been completed based on these findings, and provides the following TDP-43 mutant, RRM1 domain, compound screening method, transgenic non-human animal, and the like.

(I) TDP-43 mutant, RRM1 domain
(I-1) A TDP-43 variant comprising an amino acid sequence in which cysteine at position 173 and / or 175 is substituted in the amino acid sequence of TDP-43.
(I-2) The RRM1 domain in the TDP-43 mutant according to (I-1).

(II) Compound screening method (A)
(II-1) A method for screening a compound for treating and / or preventing a disease in which an aggregate of TDP-43 accumulates, comprising the following steps:
(1) contacting the candidate compound with the TDP-43 mutant according to (I-1) or the RRM1 domain according to (I-2);
(2) a step of detecting the aggregate formation inhibitory activity of the compound, and (3) a step of selecting a compound that inhibits aggregate formation.

(III) Transgenic non-human animals
(III-1) A transgenic non-human animal into which a DNA encoding the TDP-43 mutant according to (I-1) is introduced.
(III-2) the non-human animal is a mouse, a rat, a nematode or a fruit fly, (III-1)
A transgenic non-human animal according to 1.
(III-3) To treat and / or prevent a disease in which an aggregate of TDP-43 accumulates, characterized by using the transgenic non-human animal described in (III-1) or (III-2) Screening method.

(IV) Antibody or antibody fragment
(IV-1) An antibody or antibody fragment that binds to a peptide consisting of an amino acid sequence corresponding to positions 108 to 116 of TDP-43.
(IV-2) A diagnostic agent for a disease in which an aggregate of TDP-43 containing the antibody or antibody fragment described in (IV-1) accumulates.
(IV-3) A diagnostic kit for a disease in which aggregates of TDP-43 containing the antibody or antibody fragment described in (IV-1) accumulate.
(IV-4) A pharmaceutical composition comprising the antibody or antibody fragment described in (IV-1).
(IV-5) The composition according to (IV-4), which is used for treating and / or preventing a disease in which an aggregate of TDP-43 accumulates.

(V) Compound screening method (B)
(V-1) A method for screening a compound for treating and / or preventing a disease in which an aggregate of TDP-43 accumulates, comprising the following steps:
(1) A step of bringing the antibody or antibody fragment described in (IV-1) and the candidate compound into contact with the TDP-43 mutant described in (I-1) or the RRM1 domain described in (I-2) ,
(2) detecting the binding inhibitory activity of the compound, and (3) selecting a compound that inhibits binding of the antibody or antibody fragment.
(V-2) A method for screening a compound for diagnosis of a disease in which an aggregate of TDP-43 accumulates, comprising the following steps:
(1) A step of bringing the antibody or antibody fragment described in (IV-1) and the candidate compound into contact with the TDP-43 mutant described in (I-1) or the RRM1 domain described in (I-2) ,
(2) detecting the binding inhibitory activity of the compound, and (3) selecting a compound that inhibits binding of the antibody or antibody fragment.

(VI) Peptides, nucleic acids
(VI-1) A peptide comprising an amino acid sequence corresponding to positions 108 to 116 of TDP-43.
(VI-2) A nucleic acid encoding the peptide according to (VI-1).
(VI-3) A pharmaceutical composition for stimulating or enhancing the production of an antibody against an aggregate of TDP-43, comprising the peptide according to (VI-1) or the nucleic acid according to (VI-2).
(VI-4) The composition according to (VI-3), which is a vaccine.

According to the present invention, functional domains and key sequences involved in aggregate formation of TDP-43 were identified for the first time. Moreover, the cultured cells expressing the TDP-43 mutant of the present invention are the first effective cultured cell model that reproduces the pathological findings of patients.

The TDP-43 mutant and RRM1 domain of the present invention are compounds for treating and / or preventing a disease in which aggregates of TDP-43 accumulate by using aggregate formation inhibition in vitro or in cells as an index. It can be used for screening.

In addition, by introducing a DNA encoding the TDP-43 mutant of the present invention into an animal, it may be possible to create a model animal for a disease in which aggregates of TDP-43 accumulate (for example, ALS).

Since the antibodies and antibody fragments of the present invention can specifically recognize TDP-43 cytoplasmic inclusions, TDP-43
It is expected to be applied to early diagnosis, treatment, and prevention of diseases in which aggregates of these are accumulated. Further, the antibody or antibody fragment of the present invention is expected to be applied to a method for screening a compound for diagnosis and treatment of a disease in which an aggregate of TDP-43 accumulates.

In addition, since the peptide or nucleic acid of the present invention can stimulate or enhance the production of antibodies against a pathological condition before TDP-43 aggregate formation, it can be used for treatment and prevention of diseases in which TDP-43 aggregates accumulate. Is expected to be applied.

Schematic representation of RRM1 residue. Aggregated RRM1 pronase resistant fragments were analyzed by LC-MS / MS; regions with monoisotopic m / z> 800 are shown. Amino acid residues indicated with asterisks indicate irreversible changes in NMR chemical shifts after 2 kbar pressure treatment and are indicated as cores -a, -b and -c associated with misfolding. (A) shows the results of Western blot analysis of WT or one RRM1 C / S mutant (C173S or C175S) under reducing conditions (DTT (+)) or non-reducing conditions (DTT (−)). The protein was incubated at 4 ° C. for 7 days after 0 h (−) or 16 h (+) shaking. (B) It is a graph which shows the result of a density | concentration analysis of a dimer / monomer ratio. This indicates that one C / S mutation promotes intermolecular disulfide bond formation. The results of thioflavin T (ThT) fluorescence analysis showing that RRM1 aggregates form amyloid fibrils are shown. RRM1 protein (0.03 mM) was incubated at 4 or 37 ° C. for 24 hours and then reacted with ThT derivative (BTA-1). Data show ThT ratio compared to control. * p <0.05 vs. control by one way ANOVA Each value represents the mean of three times ± standard error of the mean (SE). The result of the confocal analysis of HEK293A cell which overexpressed RRM1-C / S mutant transiently is shown. Nuclei were stained with DAPI. B is a confocal microscopic observation of cytoplasmic translocation type TDP-43 with NLS modified in all constructs. Scale bar 10μm (A) shows the result of confocal microscopic analysis of SHSY-5Y into which TDP-43-EGFP full-length WT or RRM1 C / S mutant was introduced. Cells were stained with antibodies targeting phospho-TDP-43 (a-i) and K48 conjugated ubiquitin (j-o) at S409 / S410. Nuclei were stained with DAPI. Phosphorylated or ubiquitinated inclusion bodies in the cytoplasm are indicated by arrows. Cytoplasmic inclusion bodies are strongly stained by both antibodies. Scale bar 10 μm Upper: TDP-43-EGFP, Middle: antibody, Lower: TDP-43-EGFP + antibody + DAPI (B) TDP-43 with RRM1-C / S substitution is suppressed by degradation of proteasome by lactacystin, It is the result of the Western blot which shows that it oligomerizes. TDP-43-FLAG was overexpressed in HEK293A cells; SDS-treated whole cell lysates were analyzed by SDS-PAGE and reacted with antibodies targeting FLAG and GAPDH. (C) Western blot results of TDP-43 with RRM1-C / S substitution using an antibody targeting TDP-43 phosphorylated at S409 / S410. TDP-43 with RRM1-C / S substitution was more strongly phosphorylated and lactacystin treatment increased phosphorylated TDP-43 (lanes 5-10). (A) shows the result of confocal microscopic analysis of SHSY-5Y that transiently overexpressed TDP-43-EGFP with full-length WT or RRM1 C / S mutation of TDP-43-EGFP and modified NLS (mNLS) . Cells were stained with antibodies targeting phospho-TDP-43 (a-i) and K48-conjugated ubiquitin (j-o) at S409 / S410. Nuclei were stained with DAPI. Phosphorylated or ubiquitinated inclusion bodies in the cytoplasm are indicated by arrows. Cytoplasmic inclusion bodies are strongly stained by both antibodies. Scale bar 10μm Upper: TDP-43-EGFP, middle: antibody, lower: TDP-43-EGFP + antibody + DAPI (B) TDP-43 with RRM1-C / S substitution is an oligomer due to inhibition of proteasome degradation by lactacystin It is the result of the Western blot which shows becoming. TDP-43-FLAG was overexpressed in HEK293A cells; SDS-treated whole cell lysates were analyzed by SDS-PAGE and reacted with antibodies targeting FLAG and GAPDH. (C) shows the results of Western blotting of TDP-43 with RRM1-C / S substitution using an antibody targeting TDP-43 phosphorylated at S409 / S410. TDP-43 having mutant NLS and RRM1-C / S substitution was more strongly phosphorylated than the mutant having only RRM1-C / S substitution, and the enhancement effect by lactacystin treatment was also strong (lanes 5-10). (A) WT or mutant TDP-43-EGFP was transiently overexpressed in HEK cells and analyzed using a rabbit polyclonal anti-TDP-43 antibody. Scale bar 50 μm Upper: TDP-43-EGFP + antibody, Lower: TDP-43-EGFP + antibody + DAPI (B) Results of quantification of TDP-43-EGFP-expressing cells with TDP-43 ectopically localized in the cytoplasm Show. DCS, double C / S mutant data are expressed as mean ± SE (n = 4). In each experiment, 21-70 cells were counted. * P <0.05 vs WT by one way ANOVA using Newman-Keuls test FIG. 6 shows the results of immunofluorescence analysis showing that TDP-43 nuclear or cytoplasmic aggregates with RRM1-C / S substitution adsorb nuclear WT TDP-43. HEK293A cells were co-expressed with WT TDP-43-FLAG with WT or C / S mutant TDP-43-EGFP (with normal NLS (af) or with modified NLS (gl)) . Co-localized TDP-43-EGFP and TDP-43-FLAG are indicated by arrows. Scale bar 10 μm Upper: TDP-43-EGFP, Middle: antibody, Lower: TDP-43-EGFP + antibody + DAPI Introduced C175S mutation into EGFP-fused TDP-43 protein with TDP-43 mutant (A315T and Q331K) identified in familial ALS (df) (control without C175S mutation was used) (ac)), and transiently overexpressed in HEK293A cells. This was observed with a confocal microscope, a digital image was obtained blindly, and the number of aggregate-expressing cells in the total cells expressing TDP-43-EGFP was counted. Data represent mean ± SE (n = 8 images). 20-120 cells were counted in each image. * P <0.05 with one way ANOVA using Newman-Keuls test (A) Confocal micrograph showing intracellular distribution and inclusion body formation of TDP-43-EGFP in NSC34. Left column: TDP-43-EGFP, middle column: antibody, right column: TDP-43-EGFP + antibody + DAPI (B) Mouse motor neuron NSC34 cells, WT or NLS (mNLS) and / or C173 / C175 (C173S, TDP-34-FLAG of various mutants including C175S, C173S / C175S (DCS)) was transiently overexpressed. After 48 hours incubation in differentiation medium, NSC34 cells were given a mixture of fluorescent indicator containing cell-permeable GF-AFC for live cell count and cell-impermeable bis-AAF-R110 for dead cell count And reacted at 37 ° C. for 2 hours at the same time. Data represent mean ± SE (n = 6 wells from 2 independent experiments). Each value indicates the ratio to the vector control. * P <0.05 vs vector control with one-way ANOVA for Newman-Keuls test p <0.05 vs WT TDP-43 with one-way ANOVA for Newman-Keuls test Top: Polyclonal antibodies against core-a (aa 108-116), -b (aa 134-142), -c (aa 163-173) (shown as pAb-RRM1-a, -b, -c, respectively) ) Is a schematic diagram showing the antigen sequence in RRM1 used to generate. Asterisks indicate residues that showed irreversible chemical shifts in NMR. Bottom: Confocal micrograph showing SHSY-5Y cells that transiently express EGFP-fused WT or C / S mutant TDP-43. Cells were immunostained with an antibody targeting core-a (pAb-RRM1-a). Nuclei were stained with DAPI. Scale bar 10 μm Upper: TDP-43-EGFP, Middle: antibody, Lower: TDP-43-EGFP + antibody + DAPI Spinal cord sections from sporadic ALS patients (a-b) and non-ALS subjects (c) were stained with antibodies targeting RRM1-a. The arrow indicates a Lewy body-like hyaline inclusion body. Scale bar 50μm

  Hereinafter, the present invention will be described in detail.

The present inventors focused on the RRM1 domain, which is the main RNA binding site in TDP-43. RRM1
By high-pressure NMR using stable isotope protein and mass analysis of RRM1 aggregate, RRM1 has easy aggregation property, and three specific amino acid sequences (aa113-122, 133-147, 166-173) are included in the aggregation. Determined to be involved. In particular, the β-sheet structure containing aa166-173 is an association surface when RRM1 abnormally associates under abnormal stress such as pressure, shaking stimulation, or oxidative stress, and two cysteine residues (C173 and C175) present at the same site It was found that the formation of aggregates is promoted if is not free.

Mutants in which C173 and C175 are replaced with serine form marked inclusions in the nucleus and in the cell, and ubiquitination and phosphorylation are observed in the cytoplasm as seen in ALS and FTLD patients. It was revealed that the cells are neurotoxic. Interestingly, C173 / C175
Mutational changes in RRM1 markedly exacerbated the agglutinating properties of TDP-43 mutations reported in hereditary ALS, with wild-type TDP-43 and mutant TDP-43 previously unknown. The difference was clearly suggested to be involved in the abnormal structural change of TDP-43 in ALS.

A rabbit polyclonal antibody against these three amino acid sequences was prepared and cultured in aa113
It was confirmed that antibodies against -122 and 166-173 did not recognize normal nuclear TDP-43, and recognized intracellular aggregates due to C173 and C175 mutations. Tissue staining using the spinal cord of ALS patients revealed that disease-specific TDP-43 inclusions were stained with an antibody against aa113-122.
It was confirmed that the inclusion body model of C175 reflects the pathology of the disease.

Diseases in which aggregates of TDP-43 in the present invention accumulate are not limited to these, for example, amyotrophic lateral sclerosis, frontotemporal dementia, low-grade glioma, Alzheimer's disease Huntington's disease, Pick's disease, Parkinson's disease, Lewy body disease, basal ganglia degeneration, inclusion body myositis, B-cell lymphoma (stage M), etc., especially amyotrophic lateral sclerosis and frontal side Head dementia.

TDP-43 variant, RRM1 domain The TDP-43 variant of the present invention is characterized by comprising an amino acid sequence in which cysteine at positions 173 and / or 175 is substituted in the amino acid sequence of TDP-43.

Human-derived TDP-43 is a protein consisting of 414 amino acids, and its primary structure has high homology with the heteroribonucleoprotein (hnRNA) family. TDP-43 has two highly conserved RNA recognition motifs (RRM1, RRM2) and contains a glycine-rich region on the C-terminal side,
This glycine-rich region binds to members of the hnRNP family.

The amino acid sequence of human-derived TDP-43 protein is registered as GenBank Accession No. NP_031401, and is shown in SEQ ID NO: 1. In addition, a gene encoding TDP-43 protein derived from human is registered as GenBank Accession No. NM_007375.3, and cDNA is shown in SEQ ID NO: 2.

TDP-43 in the present invention is a mutation of a protein comprising the amino acid sequence represented by SEQ ID NO: 1 as long as it has a biological activity equivalent to that of the protein comprising the amino acid sequence represented by SEQ ID NO: 1. It may be a body.

The TDP-43 of the present invention is usually derived from an animal, preferably from a mammal, particularly preferably from a human.

The “cysteine at positions 173 and / or 175” of the present invention is basically a cysteine at positions 173 and / or 175 of the amino acid sequence represented by SEQ ID NO: 1, but has a different amino acid sequence. The same position applies to -43.

The type of amino acid that substitutes cysteine at positions 173 and / or 175 is not particularly limited as long as the effect of the present invention can be obtained, but is preferably serine or alanine.

The TDP-43 mutant of the present invention is preferably further modified in such a way that the nuclear localization signal (NLS) does not function.

The TDP-43 mutant of the present invention forms inclusion bodies in the nucleus and cells, and exhibits neurotoxicity to motor neuron cultured cells. Moreover, even if only the RRM1 domain in the TDP-43 mutant is used, an aggregate is easily formed. Therefore, the TDP-43 mutant and RRM1 domain of the present invention are TDP-
It can be used in a method for screening a compound for treating and / or preventing a disease in which 43 aggregates accumulate.

  The RRM1 domain is a portion at positions 103 to 183 in the amino acid sequence represented by SEQ ID NO: 1.

Compound screening method (A)
The compound screening method of the present invention comprises the following steps:
(1) contacting a candidate compound with the TDP-43 mutant or the RRM1 domain;
(2) a step of detecting the aggregate formation inhibitory activity of the compound, and (3) a step of selecting a compound that inhibits aggregate formation.

The TDP-43 mutant or the compound that inhibits the RRM1 domain aggregate formation selected by the above method may be effective for the treatment and / or prevention of diseases in which the aggregate of TDP-43 accumulates.

Candidate compounds can be used without particular limitation, for example, low molecular compounds,
Examples thereof include polymer compounds, biopolymers (proteins, nucleic acids, polysaccharides, etc.), and various compound libraries containing such compounds can be used.

“Contacting” in the above step (1) means that a candidate compound is brought into contact with the TDP-43 mutant or the RRM1 domain in vitro, and a cultured cell expressing the TDP-43 mutant is regarded as a candidate. Both contacting with the compound.

Due to the presence of a candidate compound, in vitro formation of the TDP-43 mutant or the RRM1 domain aggregate is 10% or more, preferably 25% or more, more preferably 50% or more, and even more preferably 75%. If this is reduced, the compound can be treated for diseases in which aggregates of TDP-43 accumulate and / or
Or it can select as an effective thing in prevention.

As a method for measuring the aggregate formation inhibitory activity in vitro, it can be used without particular limitation.
Examples thereof include a method for confirming the presence of aggregates by using an absorptiometer, ELISA method, SDS-PAGE or Western blotting method.

Further, due to the presence of a candidate compound, the formation of inclusion bodies (preferably in the cytoplasm) in the cultured cells expressing the TDP-43 mutant is 10% or more, preferably 25% or more, more preferably 50 %
As mentioned above, when it falls more preferably 75% or more, the said compound can be selected as a thing effective in the treatment and / or prevention of the disease which the aggregate of TDP-43 accumulates.

The method for measuring the aggregate formation inhibitory activity using cultured cells can be used without particular limitation.For example, observing cultured cells expressing the above TDP-43 mutant bound with EGFP using a fluorescence microscope Can be used to confirm the presence of inclusion bodies.

Transgenic non-human animal The transgenic non-human animal of the present invention is characterized in that a DNA encoding the TDP-43 mutant is introduced.

The non-human animal in the present invention means a vertebrate or invertebrate that does not include humans, and is not particularly limited as long as it is a non-human animal capable of producing a transgenic animal. For example, a mouse, a hamster, a guinea pig, Examples include rats, rabbits, chickens, dogs, cats, goats, sheep, cows, pigs, monkeys, nematodes, and fruit flies.
Nematodes and Drosophila are preferred, and mice are particularly preferred.

Methods for producing transgenic non-human animals are known. Specifically, DNA encoding the above TDP-43 mutant is introduced into an totipotent cell of an animal, and this cell is generated into an individual. The target transgenic non-human animal can be produced by selecting individuals from which the transgene has been incorporated into somatic cells and germ cells from the individuals obtained.
Examples of totipotent cells into which genes are introduced include cultured cells such as ES cells in addition to fertilized eggs and early embryos.

The transgenic non-human animal of the present invention includes a DNA encoding the TDP-43 mutant.
Any generation of transgenic non-human animals may be used. In addition, the transgenic non-human animal of the present invention may be one that retains the DNA encoding the TDP-43 mutant in either a heterozygous or homozygous manner.

In the transgenic non-human animal of the present invention, the introduced TDP-43 mutant forms inclusion bodies in the nucleus and in the cells, and exhibits neurotoxicity in cultured motor neuron cells. Therefore, aggregates of TDP-43 accumulate. It can be used as a model animal of the disease to be treated, and it is possible to evaluate the therapeutic effect and the preventive effect of the disease in which aggregates of TDP-43 accumulate. Therefore, by using the transgenic non-human animal of the present invention, a compound for treating and / or preventing a disease in which aggregates of TDP-43 accumulate can be screened.

For example, by administering a candidate compound to the transgenic non-human animal of the present invention, and evaluating the ability of the transgenic non-human animal to exercise, etc., a disease in which aggregates of TDP-43 of the compound accumulate The therapeutic effect and the preventive effect can be evaluated. Further, by administering a candidate compound to the transgenic non-human animal of the present invention and analyzing TDP-43 inclusion bodies in cells in the brain after administration, aggregates of TDP-43 of the compound accumulate. It is also possible to evaluate the therapeutic effect and preventive effect on the disease.

Antibody or Antibody Fragment The antibody or antibody fragment of the present invention is characterized by binding to a peptide consisting of an amino acid sequence corresponding to positions 108 to 116 of TDP-43.

Here, the peptides to be bound include both those in a partial state of TDP-43 and those consisting only of the peptide.

The “amino acid sequence corresponding to positions 108 to 116” is basically VLGLPWKTT in the amino acid sequence represented by SEQ ID NO: 1, but the same amino acid sequence applies to TDP-43 having other amino acid sequences. .

The amino acid residues at positions 108 to 116 of TDP-43 are easily exposed to antibodies because they are externally exposed due to structural changes in the aggregates of TDP-43, but the wild type has a structure that is difficult to bind to antibodies. It is thought that it is taking.

The antibody or antibody fragment can be obtained according to a known method, for example, by the method described in the Examples. The antibody may be either a monoclonal antibody or a polyclonal antibody, or may be a humanized chimeric antibody.
Examples of antibody fragments include Fab, Fab ′, F (ab ′) ₂ , Fv, scFv, and the like, and the antibody or antibody fragment may be modified with a fluorescent dye or the like. The peptide can be detected by using the antibody or antibody fragment in an ELISA method, Western blotting method, protein chip analysis method or the like.

The diagnostic agent and diagnostic kit of the present invention comprise the above antibody or antibody fragment,
Aggregates of P-43 can be detected. Therefore, the diagnostic agent and diagnostic kit of the present invention may enable diagnosis of diseases in which aggregates of TDP-43 accumulate.

The pharmaceutical composition of the present invention is administered to mammals including humans, and comprises the above antibody or antibody fragment, and is used for the treatment and / or prevention of diseases in which aggregates of TDP-43 accumulate. It can have the effect. This is presumably because the amino acid residues at positions 108 to 116 of TDP-43 undergo structural changes due to diseases and mutations, and become structures that easily bind to antibodies.

The pharmaceutical composition of the present invention can optionally contain biologically acceptable carriers, excipients and the like depending on the usage form. The pharmaceutical composition of the present invention can be produced according to conventional methods.
For example, orally as tablets, capsules, elixirs, microcapsules, etc. with sugar coating or enteric coating as needed, or as an external agent such as an ointment or plaster, transdermally as a spray, inhalant, etc. It can be used nasally or tracheally, parenterally in the form of injections such as sterile solutions and suspensions with water or other pharmaceutically acceptable fluids.

The dosage of the pharmaceutical composition of the present invention can be appropriately determined finally based on the judgment of a doctor in consideration of the type of dosage form, administration method, patient age and weight, patient symptom, and the like.

Compound screening method (B)
The compound screening method of the present invention is characterized by comprising the following steps:
(1) contacting the TDP-43 mutant or the RRM1 domain with the antibody or antibody fragment and a candidate compound;
(2) detecting the binding inhibitory activity of the compound, and (3) selecting a compound that inhibits binding of the antibody or antibody fragment.

The compound that inhibits the binding of the antibody or antibody fragment to the TDP-43 variant or the RRM1 domain selected by the above method is a disease and / or prevention in which an aggregate of TDP-43 accumulates, and TDP- It may be useful for diagnosis of diseases in which 43 aggregates accumulate.

Candidate compounds can be used without particular limitation, for example, low molecular compounds,
Examples thereof include polymer compounds, biopolymers (proteins, nucleic acids, polysaccharides, etc.), and various compound libraries containing such compounds can be used.

When the binding of the antibody or antibody fragment described above is reduced by 10% or more, preferably 25% or more, more preferably 50% or more, still more preferably 75% or more due to the presence of a candidate compound, the compound is reduced to TDP- It can be selected as effective for the treatment and / or prevention of diseases in which 43 aggregates accumulate, and in the diagnosis of diseases in which aggregates of TDP-43 accumulate. The method for measuring the binding inhibitory activity can be used without particular limitation, and examples thereof include an ELISA method.

Peptide, Nucleic Acid The peptide of the present invention is characterized by including an amino acid sequence corresponding to positions 108 to 116 of TDP-43, and the nucleic acid of the present invention is characterized by encoding the peptide.

The “amino acid sequence corresponding to positions 108 to 116” is basically VLGLPWKTT in the amino acid sequence represented by SEQ ID NO: 1, but the same amino acid sequence applies to TDP-43 having other amino acid sequences. .

The peptide of the present invention preferably consists of an amino acid sequence corresponding to positions 108 to 116 of TDP-43.

The pharmaceutical composition of the present invention is administered to mammals including humans, contains the peptide or the DNA, and can stimulate or enhance the production of an antibody against an aggregate of TDP-43. . The amino acid residues corresponding to positions 108 to 116 of TDP-43 undergo structural changes due to diseases and mutations, resulting in a structure that easily binds to antibodies, but wild-type TDP-43 has a structure that is difficult to bind to antibodies. It is thought that it has taken. Therefore, the pharmaceutical composition of the present invention can function as a vaccine having an effect of preventing diseases in which aggregates of TDP-43 accumulate. In addition, the pharmaceutical composition of the present invention has TD
It may have an effect of treating and / or preventing a disease in which an aggregate of P-43 accumulates.

The pharmaceutical composition of the present invention can optionally contain biologically acceptable carriers, excipients and the like depending on the usage form. The pharmaceutical composition of the present invention can be produced according to conventional methods.
For example, orally as tablets, capsules, elixirs, microcapsules, etc. with sugar coating or enteric coating as needed, or as an external agent such as an ointment or plaster, transdermally as a spray, inhalant, etc. It can be used nasally or tracheally, parenterally in the form of injections such as sterile solutions and suspensions with water or other pharmaceutically acceptable fluids.

In addition, the pharmaceutical composition of the present invention may include a non-viral vector or a viral vector using DNA capable of expressing the peptide in cells or tissues.
As an administration method using a non-viral vector, a method of introducing DNA using a liposome,
Examples include a microinjection method and a method of transferring DNA into cells together with a carrier using a gene gun. Examples of the method of administration using a viral vector include a method using a viral vector such as a recombinant adenovirus and a retrovirus. The vector expressing the peptide is introduced into a detoxified adenovirus, a retrovirus, and the like. A gene can be introduced into a cell or tissue by infecting the cell or tissue with this virus.

The dosage of the pharmaceutical composition of the present invention can be appropriately determined finally based on the judgment of a doctor in consideration of the type of dosage form, administration method, patient age and weight, patient symptom, and the like.

Examples are given below to illustrate the present invention in more detail. However, the present invention is not limited to these examples.

<Plasmid construction and protein purification>
Human RRM1 (aa 103-183) cDNA was cloned by PCR using a previously reported construct (pcDNA3-TDP-43-FLAG) as a template (1). Cysteine to serine (C
/ S) substitution mutations, familial ALS-related mutants (A315T and Q331K), and NLS mutants (mNLS) were generated using site-directed mutagenesis as previously reported (1). RRM1 of human TDP-43
A deletion mutant (ΔRRM1) was generated by PCR by designing primers to the deleted site to be removed. These cDNAs are pcDNA3, pEGFP-N2 (Clontech, Palo
Alto. CA) and pGEX-6p-1 to produce recombinant protein, respectively, BamHI / XhoI
Subcloned at the XhoI / BamHI and BamHI / XhoI sites. Recombinant protein was produced and purified in E. coli as previously reported (1).
(1) J. Neurosci. Res. 88, 784-797 (2010)

<Antibody>
Rabbit polyclonal anti-TDP-43 antibody (Sigma, St. Louis, MO) was used for Western blot analysis of RRM1 domain (1: 1000 dilution). Another rabbit polyclonal anti-TFP-43 antibody (Proteintech, Chicago, IL) was used for immunofluorescence (1: 1000). Mouse monoclonal anti-FLAG (M2) (Sigma) was used at 1: 500 dilution for both immunofluorescence staining and Western blotting. Rabbit polyclonal anti-phospho-TDP-43 antibody was used at 1: 500 dilution for both immunofluorescence and Western blotting. Rabbit monoclonal anti-K48-specific ubiquiti
n (Millipore, Temecula, CA) was used at a 1: 500 dilution for immunofluorescence. Methods for generating antisera against misfold-related cores of the RRM1 domain are described below.

<NMR measurement>
Protein samples were prepared at 1.1 mM, pH 7.0 in 20 mM d-Tris-HCl buffer. ¹ H-NMR and ¹⁵ N / ¹ H HSQC measurements were performed at 25 ° C. from 30 bar to 2000 bar every 500 bar. The analysis was performed as previously reported with a DRX600 spectrometer (Br
uker biospin Co., Fallanden, Switzerland) (2). The three-dimensional structure of RRM1 in solution was determined by conventional triple resonance NMR techniques. ^{The 1} H chemical shift directly referred to the DSS methyl resonance, while the ¹⁵ N chemical shift indirectly referred to the DSS ¹ H chemical shift.
(2) Chemical Reviews 106, 1814-1835 (2006)

<Biochemical analysis of recombinant protein and cultured cell lysate>
The recombinant protein was shaken with a multi-vortex mixer at 800 rpm for 16 hours at 22 ° C. and then incubated at 4 ° C. The protein is then added to the LDS sampling buffer (I
nvitrogen, Carlsbad, CA) and denaturation by reaction at 70 ° C. for 20 minutes, followed by SDS-PAGE. Perfluorooctanoic acid (PFO, Fluorochem Ltd, Derbyshire, UK) is a non-dissociating surfactant that preserves protein-protein interactions of both cytoplasmic and membrane proteins. The protein sample should contain the same amount of PFO sampling buffer (100 mM Tris base, 2%
NaPFO, 20% glycerol, 0.005% Bromophenol Blue, pH 8.0) and incubated at 22 ° C. for 1 hour. Protein samples are pre-cooled running buffer (0.5% NaPF
O, 25 mM Tris-HCl, 192 mM Glycine, pH 8.5) were used to separate on a 4-12% gradient polyacrylamide gel (Novex, Tris-Bis gel, Invitrogen) at 140V. For SDS-PAGE or PFO-PAGE, samples are prepared with protease inhibitor cocktail (Roche) and / or phosphatase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) with or without dithiothreitol (DTT).
) With 2% SDS-sample buffer. Samples are separated by SDS-PAGE,
Transferred to PVDF membrane for Western blotting. Antibody detection was performed using enhanced chemiluminescence (Nacalai Tesque). The gel after PFO-PAGE was stained with Coomassie Brilliant Blue (CBB, Nacalai Tesque).

<Thioflavin T assay>
Amyloid formation by heat-denatured RRM1 was assessed using the thioflavin T assay. Following post-incubation at the indicated time of 4 ° C., the protein solution was reacted with an equimolar amount of the thioflavin derivative BTA-1 (Sigma). Fluorescence is obtained from a multiplate reader (Tecan, Man
nedorf, Switzerland) at 440 nm (excitation) and 480 nm (emission).

<LC-MS / MS analysis>
LC-MS / MS analysis of pronase-degraded RRM1 protein aggregates was generally performed according to previously reported methods (3). RRM1 protein aggregate (25 μg) was added to 100 μL of digestion buffer (50
resuspended in 5 mM Tris, 100 mM NaCl, 5 mM CaCl ₂ , pH 8.0) and 5 μg pronase (Millipore)
And incubated at 37 ° C. for 1 hour. The protein in the reaction mixture is 100,000 g at 4 ° C.
Sedimented by ultracentrifugation for 30 minutes. The pellet is 20 μL of buffer (50 mM Tris, 500 mM NaC
l, 6 M GdnHCl, 5 mM EDTA, 5 mM DTT, pH 8.0) and collected in NuTip C-18 (Glygen Co, Co
lumbia, MD) for desalting. Buffer is 2% acetonitrile and 0.1% in distilled water
Substituted with trifluoroacetic acid (Nacalai, Kyoto, Japan). The degraded fragment is LC-
MS / MS (Paradigm MS4, AMR, Kyoto, Japan; Finigan LCQ Advantage MAX, Thermo Electr
on, Waltham, MA).
(3) J. Biol. Chem. 286, 18664-18672 (2011)

<Cell culture, transduction>
All cultured cells were in a state of 5% CO ₂ , 100% humidity and 37 ° C. HEK293A cells (Invitrogen)
Is maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and penicillin / streptomycin (PS), and the human neuroblastoma cell line SHSY-5Y contains 15% horse serum, DMEM / Mouse motor neuron cell line NSC3 cultured in F12-Ham medium
Four cells (Cellutions biosystems, Vancouver, BC) were maintained in culture with DMEM containing 10% FBS.
For differentiation, the medium was changed to a differentiation medium containing 3% FBS in DMEM / F12-Ham containing 1% non-essential amino acids (NEAA). FuGene HD transfection kit (Roche, Basel, Switzerland) was used for plasmid introduction. Cells were subjected to various analyzes 48 hours after transfection.

<Immunocytochemistry and confocal microscopy>
After fixation with 4% PFA, the cells were incubated for 1 hour at room temperature in PBS containing 5% goat serum and 0.2% Triton X100. Cells were reacted with various primary antibodies in PBS containing 0.1% Triton X100 for 1 hour at room temperature, and then fluorescent secondary antibodies (Alexa 488, Invitrogen; CF 568, Bi in the same buffer).
otium, Hayward, CA). After three washes with PBS, nuclei were stained by incubating cells in PBS containing 2 μg / mL. Stained cells are confocal laser microscope
(Nikon, C1 SI, Tokyo, Japan). For cell counting experiments, 7-8 pictures were taken randomly (20-50 GFP positive cells per image). Cell counts of total fluorescent cells and inclusion body positive cells were performed in a blinded fashion using ImageJ software.

<Cell death assay>
A commercially available double-fluorescence assay (Multitox-Fluor Multiplex) that can measure live and dead cells simultaneously by measuring protease activity derived from living cells and extracellular space released from dead cells. The cytotoxicity assay (Promega) was used to assess the viability and toxicity of overexpressed cells. This method can minimize different transfection efficiencies between wells. NSC34 cells, one day before transfection at a concentration per well 1 × 10 ⁴ 96-well fluorescence observation black plastic culture vessels (
CN137101, Nunc). WT or various mutant Flag tag TDP-43 plasmid
It was introduced at 0.2 μg / well using HD transfection kit (Roche) and replaced with differentiation medium the next day. 48 hours after transfection, cell-permeable GF-AFC and cell-impermeable bis-AAF-R110, fluorescent indicators for live and dead cells, respectively, were added. After 2 hours incubation, use multiplate reader (Infinite 200, TECAN, Mannedorf, Switzerland) using 400/505 nm for live cells and 485/520 nm for dead cells.
I got RFU. Bis-AAF-R110 fluorescence was used as an internal standard for GF-AFC to assess viability. The cytotoxicity ratio was expressed as a ratio to the vector control.

<Generation of antibodies that target aggregation-related RRM1>
Keyhole limpet hemocyanin-binding peptides (CVLGLPWKTT, CQVKKDLKTG, SQRHMIDGRWC; RRM1-a, RRM1-a, respectively) identified as aggregation-related sequences by LC-MS / MS and high-pressure NMR analysis
RRM1-b and RRM1-c are called). Antisera obtained after immunization with RRM1-a and RRM1-c showed specific and high reactivity against recombinant RRM1 and full-length TDP-43 protein. Antibody specificity was confirmed by Western blotting and ELISA. IgG purified using protein A was used for immunofluorescence and immunohistochemistry (1: 500).

<Immunohistochemistry of specimens from ALS patients>
The experimental procedure was approved by the guidelines of the Ethics Committee of Kyoto University and conducted under the guidelines. 3 patients with a definitive diagnosis of sporadic ALS and 3 age-matched non-A
Lumbar spinal cord from LS specimen was used. After excision, the lumbar spinal block was immediately incubated in 10% buffered formalin and embedded in paraffin. Sections (6 μm) were processed by autoclave for antigen activation (121 ° C. 10 min in 10 mM sodium citrate buffer) and P containing 3% BSA
Incubated overnight at 4 ° C. with primary antibodies against RRM1-a or RRM1-c in BS. The antibody reaction was performed with the chromogen 3,3'-diaminobenzidine tetrahydrochloride along with the Vectastain Elite ABC kit (Vector
Visualized using Laboratories, Burlingame, CA). Staining specificity is 3% BS for primary antibody
It was evaluated by replacing with an appropriate amount of PBS containing A and not observing staining.

<Statistics>
Multiple comparisons were analyzed by one way ANOVA in Newman-Keuls multiple comparison test using Prism software (Graphpad, La Jolla, CA).

Test example 1
Variations in the site-specific conformation of the RRM1 domain were investigated using high pressure NMR spectroscopy. Pressure was used to increase partially disturbed conformation, which is a potential risk of molecular association. Before and after pressure treatment at 2000 bar, which usually induces reversible structural changes in many proteins,
Overlapping the ¹⁵ N / ¹ H HSQC spectrum of the domain at 30 bar reveals irreversible changes in chemical shifts (especially for many residues at aa 113-122, 133-147, and 166-173) became.

To further investigate the role of the three RRM1 core regions identified by NMR, mass spectral analysis of shake-induced RRM1 aggregates degraded with pronase, a non-specific protease, was performed. A number of undegraded peptides have been identified, indicating that during physical stress these core regions have undergone structural changes that render the protease inaccessible. Interestingly, the three clusters of protease resistant fragments matched those identified by NMR (core-a, core-b, core-c, FIG. 1). In particular, core-c located in the 4th and 5th β-strands was rich in non-degradation products most resistant to proteases (FIG. 1).

Test example 2
RRM1 aggregates contained a fraction of oligomers by disulfide bonds mediated by C173 and C175 of the fifth β-strand. Substitution of C173 and C175 cysteines with serine has been previously reported that C173 and C175 are oxidized to form oligomers of TDP-43.
It was investigated whether (C / S mutant) could suppress oligomer formation. However, one C /
S mutants (C173S and C175S) promoted the formation of DTT-sensitive oligomers even when left standing (FIG. 2A, B). In addition, the double C / S mutant (C173S / C175 / S) was more unstable and formed irregular oligomers and aggregates even in the absence of shaking. Also in the thioflavin T assay, cysteine substitution increased β-sheet formation and the double C / S mutant showed higher thioflavin T fluorescence than one C / S mutant (FIG. 3).

Test example 3
The role of C173 and C175 in the higher-order structure of full-length TDP-43 was investigated. WT or C / S mutant (C173S,
The C-terminal EGFP fusion TDP-43 construct containing C175S, C173S / C175S) RRM1 was overexpressed in HEK293A cells for confocal microscopy analysis. Consistent with the data obtained from RRM1 domain analysis, one or double C / S mutations resulted in the formation of significant full-length TDP-43 aggregates or inclusion bodies. They are mainly in the nucleus, but some were also present in the cytoplasm (FIG. 4A). It has been reported that when RRM1 loses its ability to bind RNA, it aggregates by localizing to chromosomal chromatin. Cytoplasmic T with C174S and / or C175S by modifying NLS to exclude the possibility that TDP-43 nuclear inclusions with RRM1 C / S mutation simply cannot bind to DNA / RNA
A DP-43 mutant was generated. As a result, TDP-43 containing RRM1 C / S mutation and mutant NLS (mNLS) frequently formed round inclusion bodies even in the cytoplasm where chromatin was not present (FIG. 4B).

Test example 4
In order to assess the degree of pathogenic features of proteinopathy shared by RRM1-C / S mutants, immunocytochemistry against ubiquitination or phosphorylation was performed. In human neuronal SHSY-5Y cells, most of the nuclear inclusions of RRM1-C / S mutant TDP-43 are not phosphorylated, while the cytoplasmic inclusions are phospho-TDP-43 targeted antibodies (FIG. 5A, di) or K48 binding It showed strong reactivity with the ubiquitin antibody (FIG. 5A, mo). Furthermore, cytoplasmic TDP-43 inclusion bodies caused by RRM1-C / S mutations with modified NLS signals were frequently phosphorylated (FIG. 6A, di) and ubiquitinated (FIG. 6A, mo) at S409 and S410. . It is noteworthy that all cytoplasmic inclusion bodies are not modified by ubiquitin or phosphorylation, indicating that aggregation occurs prior to such modification in the cytoplasm. Without RRM1 C / S substitution, cytosolic TDP-43 is anti-phospho-TDP-43 antibody (
FIG. 6A, ac) or anti-ubiquitin antibody (FIG. 6A, jl) did not show clear reactivity. WT (
FIG. 5B) or Western blot analysis of FLAG-tagged TDP-43 containing RRM1-C / S substitution with mutant NLS (FIG. 6B) shows that RRM1-C / S TDP-43 is easily oligomerized in the presence of proteasome inhibitors. It was made clear that Furthermore, TDP-43 protein containing RRM1-C / S substitution was more phosphorylated at S409 / S410 than WT TDP-43 protein, especially in the presence of the proteasome inhibitor lactacystin (FIG. 5C). Furthermore, the ectopic localized form of the RRM1-C / S mutant was more sensitive to phosphorylation of S409 and S410, even in the absence of lactacystin (FIG. 6C).

Test Example 5
Focusing on the characteristics of TDP-43 proteinopathy, functional evaluation of misfolded TDP-43 caused by RRM1-C / S mutation was performed.

In particular, the formation of intranuclear aggregates of TDP-43 with RRM1-C / S induces ectopic localization in the cytoplasm of nuclear TDP-43. Immune cells using anti-TDP-43 antibodies Revealed by chemistry (Figure 7). In addition, both nuclear and cytoplasmic inclusions of TDP-43 with RRM1-C / S were co-expressed WT TD
Co-localized with P-43-FLAG protein (FIGS. 8, f and i). This indicates that misfolded TDP-43 can adsorb wild-type nuclear TDP-43 to aggregates in both nucleus and cytoplasm.

Test Example 6
The mutant protein of TDP-43 identified so far in patients with familial ALS does not show a marked difference in aggregation tendency of mutant TDP-43 compared to WT, despite having a dominant trait (Fig. 9A ac
, B). However, introduction of RRM1-C / S in the familial ALS (FALS) -related mutation TDP-43 of A315T and Q331K clearly strengthened the aggregation tendency of the FALS mutant as compared to WT (FIG. 9A df, B).

The pathogenic structure of TDP-43 that is directly linked to neurotoxicity in the TDP-43 proteinopathy is unknown.
In particular, there is no certain opinion about the presence or absence of inclusion body toxicity. Therefore, simultaneous measurement of markers for live and dead cells was performed, and the toxicity of TDP-43 nuclei or cytoplasmic aggregates was investigated using the motor neuron cell line NSC34 cells. As a result, RRM1-C / with all modified NLS
While the S mutant significantly increased toxicity compared to the vector control, ectopic localization in the nucleus did not show significant toxicity (FIGS. 10A, B). In addition, nuclear aggregation forms (C173S, C175
S) and the FALS-related mutation TDP-43 did not cause obvious toxicity (FIG. 10B).

Test Example 7
To investigate whether TDP-43 inclusion bodies caused by RRM1-C / S mutants are related to the pathology of ALS, a rabbit polyclonal antibody (pAbs) against three RRM1 core regions (FIG. 11)
Was generated. Two antisera that showed high antibody titers against both RRM1 and full-length TDP-43 are core-
Obtained for a and core-c (pAb-RRM1-a and pAb-RRM1-c). In cultured cells, pHb-RRM1-a and pAb
-RRM1-c hardly reacted with endogenous or external WT TDP-43 except for TDP-43 expressed at high concentration in the nucleus (FIG. 11). On the other hand, aggregates of TDP-43 carrying the RRM1-C / S mutation were clearly recognized by both pAbs (FIG. 11). These results indicate that the core associated with aggregation is TDP-43 RRM1-
This shows that the C / S mutant aggregates are externally exposed.

We tested whether the pAbs recognize TDP-43 inclusions in ALS patients. Immunohistochemical analysis revealed that pAb-RRM1-a recognized Lewy body-like hyaline inclusions in spinal motor neurons of sporadic ALS patients (FIG. 12). Nuclear TDP-43 was not stained by pAb-RRM1-a in motor neurons despite the presence of cytoplasmic TDP-43 aggregates. This is consistent with the results for cultured cells. These results suggest that RRM1-C / S substitution-induced inclusion bodies of TDP-43 share common conformational changes with those of ALS.

Claims (17)

A TDP-43 variant comprising an amino acid sequence in which cysteine at position 173 and / or 175 is substituted in the amino acid sequence of TDP-43.   The RRM1 domain in the TDP-43 variant of claim 1. A method for screening a compound for treating and / or preventing a disease in which an aggregate of TDP-43 accumulates, comprising the following steps:
(1) contacting the candidate compound with the TDP-43 mutant according to claim 1 or the RRM1 domain according to claim 2;
(2) a step of detecting the aggregate formation inhibitory activity of the compound, and (3) a step of selecting a compound that inhibits aggregate formation. A transgenic non-human animal into which a DNA encoding the TDP-43 mutant according to claim 1 has been introduced. The transgenic non-human animal according to claim 4, wherein the non-human animal is a mouse, a rat, a nematode or a fruit fly. TD, characterized by using the transgenic non-human animal according to claim 4 or 5.
A method for screening a compound for treating and / or preventing a disease in which an aggregate of P-43 accumulates. An antibody or antibody fragment that binds to a peptide consisting of an amino acid sequence corresponding to positions 108 to 116 of TDP-43.   A diagnostic agent for a disease in which an aggregate of TDP-43 containing the antibody or antibody fragment according to claim 7 accumulates. A diagnostic kit for a disease in which aggregates of TDP-43 containing the antibody or antibody fragment according to claim 7 accumulate.   A pharmaceutical composition comprising the antibody or antibody fragment according to claim 7. The composition according to claim 10, which is used for treating and / or preventing a disease in which aggregates of TDP-43 accumulate. A method for screening a compound for treating and / or preventing a disease in which an aggregate of TDP-43 accumulates, comprising the following steps:
(1) contacting the TDP-43 mutant according to claim 1 or the RRM1 domain according to claim 2 with the antibody or antibody fragment according to claim 7 and a candidate compound;
(2) detecting the binding inhibitory activity of the compound, and (3) selecting a compound that inhibits binding of the antibody or antibody fragment. A method for screening a compound for diagnosis of a disease in which an aggregate of TDP-43 accumulates, comprising the following steps:
(1) contacting the TDP-43 mutant according to claim 1 or the RRM1 domain according to claim 2 with the antibody or antibody fragment according to claim 7 and a candidate compound;
(2) detecting the binding inhibitory activity of the compound, and (3) selecting a compound that inhibits binding of the antibody or antibody fragment.   A peptide comprising an amino acid sequence corresponding to positions 108 to 116 of TDP-43.   A nucleic acid encoding the peptide according to claim 14. A pharmaceutical composition for stimulating or enhancing the production of an antibody against an aggregate of TDP-43, comprising the peptide according to claim 14 or the nucleic acid according to claim 15.   The composition according to claim 16, which is a vaccine.

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