JP6281831B2 - Regulators of neurodegenerative diseases - Google Patents

Description

  The present invention relates to laboratory diagnosis for detecting the presence of Amyotrophic Lateral Sclerosis (ALS) and the use of factors useful as therapeutic targets for the disease.

  Since neurodegenerative diseases including dementia are generally progressive and involve irreversible degeneration of brain neurons, it is important to diagnose early and start treatment. However, an effective biomarker has not been established at present, a diagnostic method using a serological test method has not been established, and a reliable treatment method has not been established. As a result, it is refractory and the situation in which daily life is destroyed, not only the physical burden of the patient himself, but also a great burden on the family. Neurodegenerative diseases are not only for patients themselves but also for caregivers, and there is a real situation that leads to a great loss of vitality for the whole society. In Japan, where a super-aging society is advancing, countermeasures against neurodegenerative diseases have become an important national issue. That is, (1) It is a fundamental issue of medical welfare administration in an aging society, (2) To reduce huge economic loss due to dementia (equivalent to 5 trillion yen per year), (3) Japan through medical innovation Promote the development of innovative treatments from Japan, eliminate the import deficit in Japan (over 2 trillion yen), and (4) focus diseases (cancer, It is designated as intractable disease / rare disease, hepatitis, infection, diabetes, cerebrocardiovascular disease, neuropsychiatric disease, pediatric disease). Thus, the development of new biomarkers, the development of diagnostic reagents, and the development of therapeutic drugs and treatment methods are urgent issues.

  With regard to laboratory diagnostic methods, if disease-related biomarkers are identified and serological diagnostic methods enable simple early diagnosis and monitoring of pathological conditions, early treatment becomes possible, and many patients are irreversible. Damage can be minimized. If early diagnosis becomes possible, there is no doubt that it will greatly contribute to improving the QOL of not only patients but also caregivers, and thus maintaining the vitality of the entire Japanese society that has become super-aged. Regarding the treatment method, not only the pathologic-improving drug but also the progression inhibitor is sufficiently medically significant, and it is important to analyze the involvement of the biomarker in the nerve function.

  In vivo factors related to the onset and pathogenesis of neurodegenerative diseases have been extensively studied from genes to cells, but there are so many unexplained parts, and no useful biomarkers have yet been found to lead to diagnosis or treatment . Among intractable neurodegenerative diseases, Amyotrophic Lateral Sclerosis (ALS) is a disease that leads to severe neurological dysfunction and death, and the development of useful biomarkers has become an urgent issue Yes.

  In recent years, research on proteolytic systems for life support has advanced greatly. In addition to the mechanism for creating a new protein, the cell also has a mechanism that degrades the protein when it is no longer needed. There are two main mechanisms: (1) the ubiquitin-proteasome system and (2) autophagy. Exists. The ubiquitin-proteasome system is a selective proteolytic system in which each protein to be degraded is recognized and degraded by the proteasome by binding multiple ubiquitin molecules, and each protein is degraded. In contrast, autophagy degrades many proteins at once. For this reason, proteolysis by autophagy is also called a bulk degradation system. The mechanism of autophagy is well preserved from yeast to higher animals and plants.

  Autophagy is thought to be involved in the maintenance of homeostasis and various diseases. In neurodegenerative diseases, involvement in Parkinson's disease and Alzheimer's disease has been suggested (Non-patent Document 1). In neurodegenerative diseases, intracellular inclusions are observed pathologically, and aggregates are observed in specific proteins. This intracellular inclusion body is thought to be the result of a failure of the intracellular proteolytic system, which may cause a disorder of neuronal function. It may lead to treatment. Actually, regarding the use for diagnosis and treatment of neurodegenerative diseases, use of acidic β-glucocerebrosidase (GBA) polypeptides and cathepsin D has been proposed for the treatment of synucleinopathies (Patent Document 1). ).

  In ALS, aggregates such as SOD1, TARDBP (TDP-43), FUS, and Optinurin are known. Among them, activation of autophagy aimed at degradation of TARDBP aggregates is FTLD, which is an ALS disease state model. -There is a report that the disease state of U mice has been improved (Non-patent Document 2). On the other hand, inhibition of mTOR (mammalian target of rapamycin), which is a regulator of tissue regeneration and repair in cells, promotes autophagy. Therefore, rapamycin, a typical mTOR inhibitor, is administered to activate autophagy. However, there is also a report that motor neurodegeneration of ALS model mice (SOD1-G93A) did not improve, and conversely there were many early deaths (Non-patent Document 3). That is, in the ALS model mouse, there are both reports that the disease state has been improved and reports that have been deteriorated by the activation of autophagy, and these are all model mice that have enhanced the characteristic lesions of ALS, Since there is no actual human ALS effect, there is a limit to the unified interpretation.

  The difference in results in ALS model mice seems to be related to that mTOR acts not only on autophagy but also on the immune system, and that excessive autophagy can lead to cell death. Therefore, it is suggested that proper regulation of autophagy is important for disease treatment. At the same time, it is suggested that verification analysis using ALS patient-derived tissue as much as possible is important regardless of model mice.

  As described above, in ALS, the development of diagnostics and treatment methods focusing on factors related to proteolytic systems has attracted attention, and the use of disease biomarkers useful for laboratory diagnosis or drug discovery is eagerly desired. A method for directly using autophagy-related factors for treatment has not yet been established. There is a strong need to elucidate the etiology and research and develop disease biomarkers using human samples as much as possible.

Special table 2010-535153

MIzushima N and Komatsu M, Cell. 2011; 147 (4): 728-41. Wang IF et al., Autophagy. 2012; 9 (2): 1-2. Zhang X et al., Autophagy. 2011 Apr; 7 (4): 412-25.

As described above, testing methods and treatment methods for refractory ALS have not been established, which is a major medical problem. If a factor that can be a biomarker is found from autophagy-related factors that are assumed to be involved in a disease, and a method for using the factor is established, it can be applied to a new test diagnostic method, disease state monitoring method, and therapeutic drug.
An object of the present invention is to find a factor that becomes a biomarker of ALS and to apply it to clinical examinations, pathological monitoring, and treatment methods.

The present invention relates to the following inventions:
[1] A muscle atrophic lateral cord comprising a step of measuring miRNA in a sample collected from a subject, or a polypeptide or mRNA of a target gene AMBRA1 thereof, wherein the miRNA is at least one of the miRNAs described in Table 1. How to detect sclerosis.
[2] A biomarker for detecting amyotrophic lateral sclerosis, which is a miRNA selected from the group consisting of miRNAs described in Table 1, or a polypeptide or mRNA of its target gene AMBRA1.
[3] including a step of collecting a sample from a subject treated for amyotrophic lateral sclerosis, and a step of measuring a miRNA in the sample, or a polypeptide or mRNA of a target gene AMBRA1 thereof, The method for determining the therapeutic effect, which is at least one of the miRNAs listed in Table 1.
[4] A pharmaceutical composition comprising an AMBRA1 polypeptide or mRNA as an active ingredient.
[5] A pharmaceutical composition comprising as an active ingredient an oligonucleotide that suppresses at least one of the miRNAs described in Table 1.
[6] The pharmaceutical composition according to [4] or [5], which is used for treatment of amyotrophic lateral sclerosis.
[7] A method for screening a therapeutic agent for amyotrophic lateral sclerosis using at least one expression of the miRNA described in Table 1 as an index.
[8] A method for screening a therapeutic agent for amyotrophic lateral sclerosis using the AMBRA1 polypeptide or mRNA as an index.

  In the present specification, a description with a gene name such as “AMBRA1” means the gene or a molecule (polypeptide or the like) produced from the gene unless otherwise specified. A person skilled in the art can easily understand which state is meant.

  According to the present invention, a therapeutic agent for amyotrophic lateral sclerosis (ALS) can be provided. In addition, the AMBRA1 regulatory miRNA or AMBRA1 gene product in the present invention can be used for examination diagnosis and treatment of the same disease. Furthermore, it can utilize for the screening of the compound used as the therapeutic agent of the disease.

Results of immunostaining of FFPE (formalin-fixed paraffin-embedded) specimens of brain motor areas obtained from 3 ALS patients (right side of FIG. 1) and 3 normal controls (left side of FIG. 1) are shown. It is an optical micrograph.

Except as specifically described below, the detection method, therapeutic effect determination method, therapeutic method, pharmaceutical composition, and therapeutic drug screening method of the present invention can be referred to each other as appropriate.
In the present invention, among factors involved in autophagy, it was found that AMBRA1 protein was decreased at the lesion site of ALS patients, and it was shown that autophagy failure occurred. Therefore, by inducing or enhancing the expression of the AMBRA1 gene product (polypeptide or messenger RNA (hereinafter referred to as mRNA)), it can be expected that the autophagy function is improved and that it leads to treatment of ALS.
AMBRA1 (activating molecule in BECN1-regulated autophagy protein 1; NM_017749, NM_001267782, NM_001267783) is one of the factors involved in autophagy and is present in mitochondria and activates BECN1 (Beclin1), the main factor of autophagy To promote autophagy (Fimia GM et al., Oncogene, 2012 Oct15, doi: 10.1038).

As a technique for inducing or enhancing the expression of the AMBRA1 gene product, it is possible to bind to AMBRA1 mRNA and to suppress miRNA targeting AMBRA1 that regulates the expression (hereinafter sometimes referred to as AMBRA1 regulatory miRNA). Is mentioned. Examples of the AMBRA1 control miRNA include the AMBRA1 control miRNA shown in Table 1 according to miRNA and target prediction databases (Target Scan Human, relase 6.2 and microRNA.org-Targets and Expression). In particular, hsa-miR-7 is a molecule that is conserved on the AMBRA1 gene and is preferred. The miRNA shown in Table 1 can be used alone or in combination. In order to increase accuracy, it is preferable to use a combination of plural. For example, it is desirable to use 1-20 miRNA, more preferably 2-10 miRNA in combination.
The pharmaceutical composition (therapeutic agent) of the present invention may be of a single type, but it is desirable to target a plurality of AMBRA1 regulatory miRNAs.

  As shown in the specific experimental data in the examples described later, in ALS patients, the miRNA in Table 1 that controls the expression of the AMBRA1 gene product was increased at the brain lesion site compared to healthy controls. Therefore, AMBRA1 can be regulated and treated by administering an antisense oligonucleotide against these to a subject.

Diagnosis (detection) of ALS can be performed by detecting the miRNA in Table 1 or the polypeptide or mRNA of AMBRA1 that is a target gene thereof from a subject sample. In particular, these miRNAs in samples of organ tissues, cells, blood, cerebrospinal fluid, saliva, tears, etc. collected from patients suspected of neurodegenerative diseases or patients with neurodegenerative diseases (hereinafter referred to as miRNA for ALS detection) It is possible to determine whether it is ALS by measuring at least one of AMBRA1 polypeptide or mRNA, or a combination thereof according to a method known per se. In particular, it is preferable to use a brain disease site (brain motor field lesion site or the like) or a peripheral site (cerebrospinal fluid or the like) as a sample.
Moreover, it can also utilize combining well-known miRNA and / or polypeptide or mRNA of the target gene.

  In the present invention, ALSRA1 regulatory miRNA (particularly preferably hsa-miR-7), or its target gene, AMBRA1 polypeptide or mRNA (preferably polypeptide), is used as a biomarker to diagnose or diagnose ALS. Treatment or screening for therapeutic agents for the disease can be performed. Furthermore, gene products (polypeptides or mRNA) of various genes shown in Table 2 known as autophagy-related factors, or miRNAs that control the expression thereof, together with the biomarkers or alone By using it, ALS can be diagnosed or treated, or a therapeutic drug for the disease can be screened.

When miRNA or AMBRA1 mRNA is used as a biomarker in the detection method of the present invention, it can be measured by a known genetic test method such as a hybridization method using a nucleic acid probe or a PCR method using a PCR primer. it can.
In addition, when an AMBRA1 polypeptide is used as a biomarker in the detection method of the present invention, a protein analysis method known per se, for example, an immunological analysis method using an antibody, a biochemical analysis method such as electrophoresis, or a mass It can be carried out by an analysis method, and an automatic analyzer for clinical examination can also be used. Depending on the analysis method used (for example, mass spectrometry method), the AMBRA1 polypeptide can be analyzed by analyzing the peptide derived from AMBRA1.

  Samples used in the method of the present invention include, for example, brain disease sites, cerebrospinal fluid, blood samples (eg, peripheral blood, plasma, serum), saliva, tears, urine, lymph, and other body fluids, preferably brain diseases A part or its peripheral part (cerebrospinal fluid etc.) can be used. Furthermore, organ tissues, pathological tissues, cells collected by biopsy, or components extracted therefrom can also be used.

In the detection method of the present invention, the effects of various treatments in which the miRNAs shown in Table 1 (ALS detection miRNA) or the target gene AMBRA1 polypeptide or mRNA are used for ALS patients are used as biomarkers in the detection method of the present invention. It can also be used to determine.
The therapeutic effect determination method of the present invention includes a step of collecting a sample from a subject treated for ALS, and a step of measuring miRNA or AMBRA1 polypeptide or mRNA in the sample.

When the ALS detection miRNA shown in Table 1 (miRNA that controls the expression of the AMBRA1 gene) is used as a biomarker in the therapeutic effect determination method of the present invention, the miRNA value is normal in the ALS patient before the treatment. There is a tendency to show a higher value than the average person. When the miRNA value decreases by treatment, it can be determined that the treatment was effective. On the other hand, if the treatment did not decrease, it can be determined that the treatment was ineffective.
When an AMBRA1 polypeptide or the same mRNA is used as a biomarker, the expression level of ALS patients before the treatment tends to be lower than that of healthy subjects. When the expression level increases by performing treatment, it can be determined that the treatment was effective. On the other hand, when the treatment does not increase, it can be determined that the treatment has no effect.
Since excessive autophagy leads to cell death and the possibility of exacerbating neurodegenerative lesions cannot be denied, autophagy is not only promoted but is most preferably adjusted appropriately. Therefore, when the ALS detection miRNA and AMBRA1 polypeptide or mRNA are used as a therapeutic index, it is most preferable to evaluate the change before and after treatment rather than the increase or decrease relative to the healthy control value.

  In the therapeutic method of the present invention, an inhibitory oligonucleotide against the AMBRA1-regulated miRNA described in Table 1 whose abundance is increased in ALS, for example, an oligo comprising a complementary sequence capable of hybridizing with the full length or a part of the miRNA ALS can be treated with nucleotides (antisense oligonucleotides) or by supplementing AMBRA1 polypeptide or mRNA that is reduced with ALS. In this specification, the term “treatment” includes “treatment” in a narrow sense for treating a patient after the onset of a disease and “prevention” for treating a patient before the onset of the disease.

  The method of administering to the patient the inhibitory oligonucleotide for miRNA shown in Table 1 can be performed according to a method known per se, a method of encapsulating the oligonucleotide in a liposome imitating an exosome, Method of slow release as a complex, utilization of chemically modified substance by 4'-thioRNA conversion in which O (oxygen) of RNA sugar moiety is replaced with S (sulfur), 2'-F of sugar moiety of oligonucleotide It can be replenished stably in vivo by using a chemically modified product modified with 2′-O-methyl or 2′-O-methoxyethyl. At the same time, since miRNA has a high possibility of controlling a target gene with a plurality of molecules, replenishment with a plurality of nucleic acid cocktails can be preferably used for the above-described nucleic acid supplementation method.

  The pharmaceutical composition of the present invention can be used for the treatment of ALS, and as an active ingredient, an inhibitory oligonucleotide against at least one of miRNAs listed in Table 1 (full length or a part thereof), or polymorphic AMBRA1 The peptide can be included, and can optionally further include a pharmaceutically acceptable carrier.

In addition, the miRNA listed in Table 1 used as a biomarker in the detection method of the present invention, or the polypeptide or mRNA of its target gene AMBRA1 can be used for screening ALS therapeutics using AMBRA1 as an index. it can. In this case, methods for screening candidate substances (for example, compounds) that target the AMBRA1 protein include in vitro methods for examining candidate substances that directly bind to the target by spectroscopic changes and exposing the candidate substances to cultured cells. And a method for examining the expression of a target (miRNA or gene product of the target gene) in cultured cells is applied, and in vivo, a method using an improvement in neurodegenerative symptoms or a neurodegenerative pathological image as an index in a model animal Etc. apply.
In this aspect, for example, the step of bringing the polypeptide or a cell, tissue, or animal individual (particularly a non-human animal) expressing the polypeptide into contact with the candidate substance, and the binding of the candidate substance to the polypeptide, It can be carried out by a method comprising a step of analyzing a change in the expression level or activity of a polypeptide, or a symptom change in an animal individual.

Alternatively, screening of therapeutic agents can be performed in the same manner by examining the expression of mRNA of AMBRA1 or the miRNA described in Table 1.
In this embodiment, for example, a step of administering a candidate substance to a cultured cell or an animal individual (particularly a non-human animal), a step of collecting a sample from the individual, and measurement of miRNA or AMBRA1 mRNA in the sample It can implement by the method including the process to do.

  Candidate substances to be evaluated by the screening method of the present invention are not particularly limited, but in addition to various compounds, various extracts such as culture supernatants of microorganisms, natural organisms or extracts derived from various organisms or tissues thereof Can be mentioned. Furthermore, the cells used are cultured cells isolated from tissues, established cell lines, ES cells, iPS cells, Muse cells, etc. In non-human animals, specific genes were knocked out or knocked in. Model mice, rats, etc. can be mentioned.

In the screening method of the present invention, when at least one of the miRNAs listed in Table 1 is measured, a substance capable of reducing the amount of the miRNA can be selected as a candidate for a therapeutic agent for ALS.
Further, when measuring AMBRA1 polypeptide or mRNA, a substance capable of increasing the expression level or activity of the gene product can be selected as a candidate for an ALS therapeutic.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Example 1: Analysis of AMBRA1 in brain lesion tissue
A brain motor area lesion site of a patient with definite diagnosis of amyotrophic lateral sclerosis (ALS) and a normal site of a healthy human brain as a normal control group were collected after autopsy, fixed with 10% formalin, and paraffin. Embedded in FFPE (formalin fixed paraffin embedded) specimen.
Among many autophagy-related factors, AMBRA1 that has not been fully elucidated was selected and analyzed by immunostaining of FFPE samples. For this purpose, spinal FFPE was prepared from 5 sporadic ALS and 5 normal controls, and anti-AMBRA1 antibody (SDIX, Newark, DE, USA; 1: 500) was used. The FFPE was stained with an immune antibody and observed with an optical microscope.

FIG. 1 shows micrographs of 3 ALS cases (right side of FIG. 1) and 3 normal controls (left side of FIG. 1).
In the normal control, the cell body of the motor neuron in the anterior horn of the spinal cord was diffusely stained in fine granules (brown part). In ALS, the staining of the anterior horn neurons was highly attenuated. Human AMBRA1 binds to Beclin1, which is involved in autophagosome formation. Moreover, when the expression of AMBRA1 is suppressed in a human fibrosarcoma cell line, autophagy is suppressed. Considering these results together, it is considered that the autophagy function is suppressed in ALS motoneurons, which causes a failure in the degradation system of abnormal proteins (such as TDP-43) and causes cell death.
The present results indicate that AMBRA1 polypeptide or mRNA can serve as a detection marker for ALS, and that control molecules targeting AMBRA1 and AMBRA1 can serve as therapeutic agents for ALS. Thus, it is the first in the world to find a decrease in AMBRA1 in ALS patient brain pathological tissue.

<< Example 2: Analysis of miRNA in brain lesion tissue >>
Prediction of AMBRA1-regulated miRNA is performed using Target Scan Human, release 6.2 (http://www.targetscan.org/vert_61/) and microRNA.org-Targets and Expression (http: / /www.microrna.org/microrna/home.do). As a result, AMBRA1 control miRNA shown in Table 1 was extracted. Among these, hsa-miR-7 was the most conserved molecule on the AMBRA1 gene (indicated by “◎” in Table 1).
Specific methods and results are shown below. As a reference, the behavior of miRNA described in Table 1 at the brain lesion site was examined. Specifically, FFPE specimens (5 × 5 mm, 5 μm, 2 to 4 pieces) were used as materials, and RNA was extracted from specimens of 3 ALS patients and 3 ALS normal controls, respectively, and miRNA analysis was performed using a microarray.

  The microarray uses a 3D-gene chip (manufactured by Toray Industries, Inc.), a miRNA extract designated by the manufacturer, a hybridization reagent set, and a human miRNA Oligo chip (about 1,800 human miRNA probes selected from the database miRBase Release 17.0). The change in the signal intensity of the patient group relative to the target group was determined. From this, the rate of change in miRNA increase / decrease was determined, and the increased miRNA relative to the healthy control group was examined. As a result, hsa-miRNA-7 selected from the above Target Scan Human database and assumed to have the highest AMBRA1 control ability increased to a healthy subject relative value of 0.16 (Log2 display) at the ALS brain lesion. It was verified that AMBRA1 tends to be suppressed. Furthermore, this result is consistent with the pathological specimen result shown in FIG.

  The present invention can be used for examination diagnosis and treatment of ALS. Furthermore, it can utilize for the screening of the compound used as the therapeutic agent of the disease.

Claims (3)

A method for assisting detection of amyotrophic lateral sclerosis, comprising a step of measuring AMBRA1 polypeptide or mRNA in a sample collected from a subject. A method for measuring AMBRA1 polypeptide or mRNA in a sample collected from a subject in order to detect amyotrophic lateral sclerosis. Comprising the step of measuring the AMBRA1 polypeptide or mRNA in a sample from a subject were treated for amyotrophic lateral sclerosis, the method for acquiring data for determining the therapeutic effect.