WO2020209469A1 - Biomarker for diagnosing each subtype of peripheral neuropathy - Google Patents

Abstract

The present invention provides a method for providing information for predicting or diagnosing a risk of developing each subtype of peripheral neuropathy by using a combination of biomarkers; a composition for diagnosing each subtype; and a drug screening method for treating peripheral neuropathy using same. The method of the present invention measures an expression level of a biomarker to effectively provide information for predicting or diagnosing the risk of developing each subtype of peripheral neuropathy, and the diagnostic composition and kit of the present invention can diagnose specific subtypes of peripheral neuropathy with high accuracy.

Description

Biomarkers for diagnosis by subtype of peripheral neuropathy

The present invention relates to a biomarker capable of effectively diagnosing various peripheral neuropathies by subtype.

The peripheral nervous system includes the motor and sensory roots, the dorsal root ganglion, the nerve plexus, and the peripheral nerves, and the cranial nerves and ganglions excluding the optic and olfactory nerves, as well as the autonomic and autonomic nerves. Include.

In peripheral neuropathy, there are various pathological findings that invade the peripheral nerve, and when physical or chemical damage to axons occurs, Waller degeneration occurs at the distal part of the damaged area, and metabolic disorders of neurons. Axonal degeneration, which proceeds from the distal to the proximal part of the axon (dying-back), neuropathy that causes the degeneration of the cell body and axon at the same time, and the function of the axon is relatively maintained while myelinated ( Myelin sheath) is primarily destroyed, such as demyelinopathy, which can be mixed in certain peripheral neuropathies.

Symptoms of peripheral neuropathy include sensory symptoms, motor symptoms, and autonomic symptoms, and sensory symptoms and motor symptoms are divided into positive and negative symptoms. Voice motor symptoms are due to the loss of a conduction block or axon of the motor nerve and appear as muscle weakness. On the other hand, the symptoms of benign movements appear as fasciculation, myokymia, tremor, and muscle cramp due to abnormal activity in the peripheral nerve. Benign sensory symptoms include hypersensitivity, including pain, and dysethesia, and negative sensory symptoms may include decreased sensitivity and numbness. Autonomic nervous system disorders can be accompanied by digestive disorders or general autonomic disorders in the body.

The causes of peripheral neuropathy are secondary neurodegeneration following physical nerve damage such as compression or entrapment, acquired immunodeficiency syndrome, infectious diseases such as leprosy, Lyme disease, medical diseases such as diabetes, ischemic disease, There are a wide variety of cases, such as paraneoplastic syndrome, nutritional deficiencies, toxic diseases, inflammatory demyelinating diseases, and genetic diseases. In addition, the causes are very diverse, and even a hospital specializing in peripheral neuropathy cannot determine the cause of about 25%. Therefore, in order to accurately diagnose peripheral neuropathy, a step-by-step and systematic approach is required through various laboratory tests including detailed medical history, physical and neurological examination, and electrophysiological examination. The most common criterion for discriminating those subtypes is electrophysiology, and if you use this, you can get an index to distinguish between axon type and demyelinating type. In the case of hereditary nature, the patient's family history and gene mutation test are used to help diagnose.

Demyelinating peripheral neuropathy is partially differentiated from axonopathy by electrophysiological examination, but there is still no method for discriminating and diagnosing inflammatory or hereditary causes within demyelination using specific proteins in the blood, and the introduction of such a simple diagnostic marker is a demyelination disease. It is also very important in selecting treatment strategies through rapid diagnosis of subtypes.

The scientific background for the invention of a subtype-specific biomarker for peripheral neuropathy is due to the study of transformation of Schwann cells directly involved in demyelination. Mature Schwann cells that produce myelin are transformed into immature Schwann cells during inflammatory demyelination, contributing to demyelination, and express a specific phenotypic factor. On the other hand, in the case of hereditary demyelination, it shows a phenotype that maintains the immature state during development or causes abnormal differentiation. In addition, Schwann cells when axonal damage occurs are also dedifferentiated and transformation into immature Schwann cells occurs. When these Schwann cells are transformed, it is expected that various factors that are specifically expressed and secreted by demyelination can be discovered and systematically studied whether they are found in the patient's serum, thereby suggesting the possibility of use as a diagnostic tool for peripheral neuropathy. .

The present invention provides a method of providing information for predicting or diagnosing the risk of developing peripheral neuropathy by using a combination of biomarkers, providing a diagnostic composition for each subtype, and providing a drug screening method for treating peripheral neuropathy using this Has its purpose.

1. Measuring the expression level of p75 and NCAM protein in the sample isolated from the individual; And comparing the measured expression level with a control subject. And predicting or determining whether the individual has a risk of developing hereditary peripheral neuropathy or non-hereditary peripheral neuropathy according to the comparison result;

The hereditary peripheral neuropathy is Charcomaritus disease 1a (CMT1a), and the non-hereditary peripheral neuropathy is acute motor axonal neuropathy (AMAN), chronic inflammatory demyelinating polymyelopathy (CIDP, Chronic inflammatory neuropathy). Demyelinating polyradiculoneuropathy) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP), a method of providing information for predicting or diagnosing the risk of developing peripheral neuropathy subtypes.

2. In the above 1, if there is no significant difference in the expression level of p75 protein compared to the control individual, and the expression level of NCAM is significantly higher than that of the control individual, the method of predicting that the risk of developing hereditary peripheral neuropathy is higher.

3, in the above 1, a method for predicting that the risk of developing non-hereditary peripheral neuropathy is higher if the expression level of p75 and NCAM protein is significantly higher than that of the control individual.

4. The method of 1 above, further comprising the step of measuring the expression level of the CXCL13 protein.

5. In the above 4, if the expression level of p75, NCAM and CXCL13 protein is significantly higher than that of the control individual, the method of predicting that the risk of developing inflammatory demyelinating polyradiculoneuropathy is higher.

6. The method according to any one of the above 1 to 5, wherein the sample is at least one selected from the group consisting of serum, plasma, nerve cells, immune cells, cerebrospinal fluid, and exosomes.

7. Comparing the expression levels of p75 and NCAM proteins before and after the treatment by treating the test material on the sample isolated from the peripheral neuropathy subject; And determining whether the test substance is a candidate substance for prevention or treatment for each subtype of peripheral neuropathy according to the comparison result,

The peripheral neuropathy is an inherited peripheral neuropathy or a non-hereditary peripheral neuropathy,

The hereditary peripheral neuropathy is Charcomaritus disease 1a (CMT1a), and the non-hereditary peripheral neuropathy is acute motor axonal neuropathy (AMAN), chronic inflammatory demyelinating polymyelopathy (CIDP, Chronic inflammatory neuropathy). Demyelinating polyradiculoneuropathy) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP), a method for screening candidates for prevention or treatment of peripheral neuropathy subtypes.

8. In the above 7, when the expression level of p75 and NCAM protein is significantly reduced compared to before treatment of the test material, it is known that Charcomaritus disease 1a (CMT1a), acute motor axonal neuropathy (AMAN), Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP) as a candidate for the prevention or treatment of chronic inflammatory demyelinating polyradiculoneuropathy.

9. In the above 7, if there is no significant difference in the expression level of p75 protein compared to before treatment of the test substance, and the expression level of NCAM protein is significantly reduced, it is a candidate substance for preventing or treating Charcomaritus disease 1a (CMT1a). How to sort by.

10. The method according to the above 7, further comprising the step of comparing the expression level of the CXCL13 protein before and after treatment of the test substance.

11. In the above 10, if the expression level of p75, NCAM, and CXCL13 protein is significantly reduced compared to before treatment of the test substance, it is selected as a candidate for preventing or treating inflammatory demyelinating polyradiculoneuropathy. .

12. The method of 7 above, wherein the sample is at least one selected from the group consisting of serum, plasma, nerve cells, immune cells, cerebrospinal fluid, and exosomes.

13. A nucleotide sequence of a gene encoding p75 and NCAM protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance that specifically binds to a protein encoded by the nucleotide sequence,

For diagnosis of the onset of inherited peripheral neuropathy or non-hereditary peripheral neuropathy,

The hereditary peripheral neuropathy is Charcomaritus disease 1a (CMT1a), and the non-hereditary peripheral neuropathy is acute motor axonal neuropathy (AMAN), chronic inflammatory demyelinating polymyelopathy (CIDP, Chronic inflammatory neuropathy). Demyelinating polyradiculoneuropathy) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP), a composition for diagnosing peripheral neuropathy subtypes.

14. The composition of the above 13, further comprising a nucleotide sequence of a gene encoding CXCL13 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance that specifically binds to a protein encoded by the nucleotide sequence .

15. The composition of 13 above, wherein the composition is for diagnosis of inflammatory demyelinating polymuscular neuropathy.

16. Including the composition of any one of the above 13 to 15,

For diagnosis of the onset of inherited peripheral neuropathy or non-hereditary peripheral neuropathy,

The hereditary peripheral neuropathy is Charcomaritus disease 1a (CMT1a), and the non-hereditary peripheral neuropathy is acute motor axonal neuropathy (AMAN), chronic inflammatory demyelinating polymyelopathy (CIDP, Chronic inflammatory neuropathy). Demyelinating polyradiculoneuropathy) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP), a diagnostic kit for peripheral neuropathy subtypes.

17. The kit according to the above 16, wherein the kit is a protein array or a protein chip including a substance specifically binding to a protein encoded by the nucleotide sequence.

The information providing method of the present invention can effectively provide information for predicting or diagnosing the risk of developing a peripheral neuropathy subtype by measuring the expression level of a biomarker.

The screening method of the present invention can effectively screen a candidate therapeutic drug for peripheral neuropathy by measuring the expression level of a biomarker.

The diagnostic composition and kit of the present invention can effectively diagnose peripheral neuropathy.

1A to 1C: (A) Workflow for biomarker development in demyelinating neuropathy, (B) Venn diagram depicting the overlap of the proteome in the mouse Schwann cell exosomes of this study and the ExoCarta mouse exosomes, (C ) Characteristics of Schwann cell exosomes by Western blot analysis (SCL: Schwann cell lysate, Rab5b: cytoplasmic protein marker), (D, E) detection results of p75 and NCAM in the serum of peripheral neuropathy patients by ELISA (ANOVA Significant differences between the patient group and the normal control group by (represented by **(p<0.01) and ***(p<0.005)), (F, G) ROC curves of p75 and NCAM detection by ELISA.

Figures 2a to 2b: (A, B) is the expression profile of (A) p75 and (B) NCAM in the neuromuscular of human CIDP and CMT1a patients, where arrows are demyelinating Schwann cells, arrowheads are NCAMs in non-myelinated Schwann cells. , Asterisks indicate excess onion progenitor Schwann cells, double arrows indicate demyelinating Schwann cells, respectively (scale bar, 20 μm). (C) is the expression profile of NCAM in the nerves of human CIDP and CMT1a patients, and NCAM staining of the longitudinal section (left two panels) showed differences in NCAM expression in the splenic nerves of CIDP and CMT1a (MBP: myelin basic protein, Asterisk: Schmidt-Lanterman incisures, scale bar, 20 μm). The right panel is an enlarged image of onion progenitor cells of CMT1a (arrow: nucleus). All concentric cytoplasmic layers of onion progenitor Schwann cells expressed NCAM. (D) is a graph showing the percentage of demyelinating Schwann cells expressing p75 or NCAM among 600 to 1800 MBP positive Schwann cells in the nerve sections of CIDP and CMT1a patients (Unpaired Student's t-test, *: p <0.05, **: p <0.01). (E) is the fluorescence intensity values of p75 and NCAM staining in an in vivo nerve biopsy (Unpaired Student's t-test, **: p <0.01).

3A to 3B: (A) shows that the osteotomy induced the expression of p75, not NCAM, while demyelination of the SC (DSC, arrow), and only immature Schwann cells (arrowheads) showed NCAM expression before and after axonal dissection. (Scale bar, 20 μm). (B) is a Western blot analysis result showing that the expression of p75, not NCAM, was induced in the sciatic nerve after axonal incision. (C) shows the quantitative values of p75 and NCAM expression levels (mean±SEM, unpaired Student's t-test, **: p<0.01 in three independent experiments). (D) shows that demyelinating Schwann cells at the beginning and peak stages of EAN showed p75 induction (arrow), but NCAM staining increased in immature Schwann cells (arrowheads). (E) is a Western blot analysis result showing that p75 and NCAM are induced in the sciatic nerve of EAN. (F) is a graph showing the quantitative values of p75 and NCAM expression levels (mean unpaired Student's t-test from 3 independent experiments, *: p <0.05, **: p <0.01).

Figure 4: Western blot analysis results of analyzing the expression patterns of p75 and NCAM in primary cultured Schwann cells by cytokines and growth factors. Pro-inflammatory cytokines such as INF-γ and TNF-α increased NCAM expression in cultured primary SCs, but ER stress inducers, including proteasome inhibitors (Bortezomib, BTZ) and thapsigargin (TG) 45, did not. The expression of p75 was not significantly changed by pro-inflammatory cytokine treatment. The graph shows the quantitative value of the NCAM expression level in three independent experiments (Unpaired student t-test; p: * <0.05, ** <0.01, *** <0.001).

5A to 5B: Regarding the proinflammatory environment associated with M1-macrophages having CXCL13 expression in B7-2KO neurons, (A) cytokine arrays in B7-2KO neurons and injured neurons. (B) Schematic of the cytokine expression profile in B7-2KO neurons and injured C57BL/6 neurons, where the levels of CXCL13, CCL5, MIP-1 and CXCL10 were specifically increased in B7-2KO neurons. (C) Expression of CXCL13 was induced in CD68+ macrophages (arrowheads), but was not expressed in S100-positive SCs in B7-2KO neurons. CXCL13 positive cells were not present in the NOD nerve or the damaged C57BL/6 nerve (6dPI, 6 days after injury) (WD: Wallerian degeneration, scale bar, 20 μm). (D) Using antibodies against CD206 and CD197, M2 and M1 macrophages were detected and analyzed in the nerve section, respectively. In B7-2KO neurons, most of the CD68+ macrophages were CD206, and some CD197+ cells were present. During WD, most of the CD68+ macrophages were CD206+ (arrowheads) and a few were CD197+ (scale bar, 20 μm).

6A to 6C: (A) The number of B cells and T cells in the B7-2KO sciatic nerve and non-obesity diabetic (NOD) nerve, and the dot indicates the number of the sciatic nerve part (Unpaired student t-test; ** *, p <0.001). (B) Profiles of CXCR5 expression in B7-2KO neurons, with double immunostaining for specific markers of CXCR5 and T cells (CD4), macrophages (CD68), and dedifferentiated Schwann cells (p75). It was shown that CXCR5 levels were increased in B7-2KO neurons (arrowheads indicate double positive cells, scale bar, 20 μm). (C) Western blot analysis showed that the cultured primary SC expressed CXCL5 when exposed to neuregulin (NRG) (spleen was used as a positive control).

Figure 7: (A) The serum CXCL13 concentration of patients with peripheral neuropathy was investigated using ELISA. Serum concentrations of CXCL13 were significantly increased in patients with acute (AIDP; p <0.05) and chronic (CIDP; p <0.01) inflammatory demyelinating polyneuromyopathy compared to healthy controls, acute motor axon neuropathy (AMAN), and CMT1a group. I did. (Significant differences between the patient group and the healthy control group by repeated measurement ANOVA are expressed as * (p <0.05) and ** (p <0.01)) (B, C) CXCL13 expression in neuromuscular muscles of human CIDP and CMT1a patients With arrows, CXCL13+ cells in the cranial nerves are indicated. The gastrocnemius nerve of CMT1a patient showed myelin dysplasia without significant expression of CXCL13 (scale bar, 50 μm).

8A to 8D: (A) Myelination profile of the sciatic nerve of wild type (WT) and C22 mice, Semitin plastic sections showed significant hypomyelination in C22 mice (5W; postnatal 5 weeks, scale bar, 20μm), (B) NCAM expression profile of the sciatic nerve of C22 and WT mice at 8 weeks postnatal (8W), NCAM expression in adult normal mice (WT) was non-myelinated SC (arrowhead) And a number of small NCAM positive staining (arrows) were observed in C22 mice (scale bar, 20 μm). (C) The number of NCAM-positive DAPI staining in the sciatic nerve of WT and C22 adult mice, and the dot represents the number in the unit region of the sciatic nerve part (Unpaired student t-test; ***, p <0.001). Increased expression of NCAM in C22 neurons was confirmed by Western blot analysis, and expression of c-jun was used as a marker for immature SC. (D) Invasion of CD68+ macrophages in C22 neurons was minimized (arrowhead, scale bar, 20μm). (E) As a result of NCAM expression of human CMT1a nerve, all concentric SC layers (arrowheads) of non-myelinated SC (arrow) and onion-bulb showed NCAM expression (MBP; myelin basic protein, scale bar, 50 μm (inserted at the top) ) Or 20 μm (bottom insert)). (F) As a result of differential macrophage infiltration in the gastrointestinal nerve of CMT1a and CIDP patients, Iba-1 staining showed minimal infiltration of macrophages in human CMT1a (scale bar, 20 μm).

Figure 9: Schematic explaining the selective expression of NCAM in inflammatory demyelinating neuropathy. Axonal injury induced co-activation of innate immunity with SC dedifferentiation and M2 macrophage polarization. In inflammatory demyelinating neuropathy such as acute (AIDP) and chronic (CIDP) inflammatory demyelinating neuropathy, the inflammatory environment of M1-type macrophages expressing CXCL13 induces SC dedifferentiation and NCAM expression. SCs dedifferentiated in both conditions generally express c-Jun, p75 and CCL2 and contribute to myelination.

Figure 10: As a pathological immature Schwann cell model in peripheral neuropathy, a schematic showing the difference between demyelinating Schwann cells and pathologically differentiated Schwann cells in peripheral neuropathy based on p75 and NCAM expression. Demyelinating SC (p75+/NCAM-) and extra SC (p75-/NCAM+) are pathological immature Schwann cells that contribute to the onset of peripheral neuropathy, and their presence in neuropathic nerves was detected by human serum ELISA. Can be (ID: inflammatory demyelination).

Hereinafter, the present invention will be described in detail.

First, the peripheral nervous system refers to the rest of the nervous system of our body 　 central nervous system 　 that is, the brain and spinal cord, and is distributed in almost all organs in the body and is involved in the regulation of its functions. The peripheral nervous system is largely motor nervous system, sensory nervous system, autonomic It is classified as the nervous system.

Peripheral neuropathy is classified into congenital or hereditary peripheral neuropathy, acquired peripheral neuropathy depending on the age of occurrence and family history, and it is classified into sensory, motility, autonomic neuropathy, and complex depending on the area where the disorder mainly appears.In addition, pathology There are classifications according to phenomena and classification according to the distribution of invading nerves.

For example, as congenital or hereditary peripheral neuropathy, Charcot-Marie-Tooth disease (CMT), hereditary compression neuropathy (HNPP, Hereditary 　 neuropathy 　with 　liability 　to 　 pressure 　palsies), HMN , Hereditary　motor　neuropathy), hereditary sensory autonomic neuropathy (HSAN, Hereditary　sensory　and　autonomic　neuropathy), etc., as a subtype of the CMT disease, including all of CMT1a, CMT1b, CMT1c, CMT1d, CMT2, and CMT3. can do.

For example, as acquired or non-hereditary peripheral neuropathy, all peripheral neuropathies except congenital or hereditary peripheral neuropathy may be included. Metabolic neuropathy, addictive neuropathy, allergic neuropathy, cancerous neuropathy, other neuropathy It may include.

Specific examples of acquired peripheral neuropathy include Inflammatory demyelinating polyradiculoneuropathy, Acute motor axonal neuropathy (AMAN), Guillain-Barre syndrome (GBS), Miller Fisher syndrome (MFS, Miller-Fisher syndrome), diabetic peripheral neuropathy, vasculitis neuropathy, etc. can be included, and the inflammatory demyelinating polyradiculoneuropathy is chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). , Chronic inflammatory demyelinating polyradiculoneuropathy), Acute inflammatory demyelinating polyradiculoneuropathy (AIDP), Acute sensory ataxic neuropathy (ASAN), Multifocal motor neuropathy (MMN) And Lewis-sumner syndrome (LSS).

The p75 of the present invention is a neurotrophin receptor, which contains four TNFR cysteine-rich motifs, a transmembrane region, and an intracellular region containing a death domain, and thus, LINGO-1/Nogo-66 receptor signals It has been known that it is a component of the ring pathway and can mediate the survival and death of neurons, but its role in relation to specific peripheral neuropathy is unknown.

NCAM (Neural cell adhesion molecule 1) of the present invention is known as a binding glycoprotein expressed on the surface of neurons, glial cells or skeletal muscle, also called CD56, but no known about its role in relation to specific peripheral neuropathy.

CXCL13 (C-X-C motif chemokine ligand 13) of the present invention belongs to the CXC chemokine family, and plays an important role in lymphoid organ formation and development, B cell follicle formation, and B cell supplementation. It is ectopically produced in inflammatory tissues of multiple chronic inflammatory diseases, and is considered to play an important role in maintaining local B and T cell activity and inflammation, but no known role is known in relation to peripheral neuropathy.

The present invention provides a method of providing information for predicting and diagnosing the risk of development of each subtype of peripheral neuropathy, including measuring the expression level of p75 and NCAM proteins in a sample isolated from an individual.

The present invention finds that the expression patterns of p75 and NCAM in samples obtained from patients with peripheral neuropathy are different for each peripheral neuropathy subtype, and based on this, the combination of p75 and NCAM is a peripheral neuropathy subtype-specific biomarker or indicator It is based on discovering its applicability as

The individual refers to an animal including humans, and specifically, may mean at least one selected from human, rat, rabbit, mouse, etc., but is not particularly limited if it is a possible target of peripheral neuropathy.

The method may measure the expression level of p75 and NCAM proteins, and according to the result, the risk of developing hereditary peripheral neuropathy or non-hereditary peripheral neuropathy may be predicted or determined whether to develop.

The method may further include predicting that the risk of developing hereditary peripheral neuropathy is higher if there is no significant difference in the expression level of the p75 protein compared to the control individual, and the expression level of NCAM is significantly higher than that of the control individual. For a more specific example, the hereditary peripheral neuropathy may be Charcomaritus disease 1a (CMT1a).

The method may further include predicting that the risk of developing non-hereditary peripheral neuropathy is higher when the expression level of p75 and NCAM protein is significantly higher than that of the control individual. For a more specific example, the non-hereditary peripheral neuropathy is acute motor axonal neuropathy (AMAN), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), or acute inflammatory demyelinating polymuscular neuropathy. It may be a disease (AIDP, Acute inflammatory demyelinating polyradiculoneuropathy).

The method is CMT1a hereditary peripheral neuropathy; Or, it is possible to predict and determine the risk of onset or onset of non-hereditary peripheral neuropathy, which is AMAN, CIDP, or AIDP.

The method may further include the step of measuring the expression level of the CXCL13 protein.In this case, if the expression level of the p75, NCAM and CXCL13 protein is significantly higher than that of the control individual, inflammatory demyelinating polyradiculoneuropathy (Inflammatory demyelinating polyradiculoneuropathy) Predicting that the risk of developing is higher may be further included. The inflammatory demyelinating polymuscular neuropathy may include chronic or acute inflammatory demyelinating polymuscular neuropathy.

In the interpretation of the'significance', it is preferable to interpret the expression level measurement data by a well-known statistical processing method in the art, and for a specific example, the criterion is a confidence level of 95% (p<0.05). Alternatively, it may be a confidence level of 99% (p<0.01), for example, by the Unpaired Student t-test, but is not limited to a specific test method and level of statistical significance.

The sample of the individual and the sample of the control group are biological samples, meaning all samples obtained from individuals whose p75, NCAM or CXCL13 protein of the present invention can be detected, and the biological samples include biopsy, blood, immune cells, It may be any one selected from the group consisting of nerve cells and skin tissues, preferably any one selected from the group consisting of serum, plasma, nerve cells, immune cells, cerebrospinal fluid, and exosomes, but is not particularly limited thereto, It can be prepared by processing in a method commonly used in the technical field of the present invention.

As a method of measuring the expression level, a method of measuring the concentration in a sample of mRNA, which is a transcription material of a gene encoding p75, NCAM, or CXCL13 protein, or a concentration of the protein in a sample, may be selected, but is not limited thereto. It can be carried out by selecting a method commonly used in the technical field of the invention.

As a method of measuring the concentration of the mRNA in a sample, reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (Competitive RT-PCR), real-time reverse transcriptase polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blotting, DNA chip, and the like, but are not limited thereto.

As a method of measuring the concentration of the protein in a sample, the amount of protein may be determined using an antibody that specifically binds to the protein. Analysis methods for this include immunotaxonomy, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion, and rocket immunoelectricity. Electrophoresis, tissue immunostaining (immunohistochemistry), immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), FACS (fluorescence-activated cell sorting) and protein chip (protein chip), and the like, but are not limited thereto. .

The NCAM, p75, and CXCL13 may be those of the judgment object, for example, in the case of human, the mRNA sequence may be a sequence of SEQ ID NO: 1, 2, and 3, respectively, and the protein is, for example, SEQ ID NO: 4, 5, It may be the sequence of 6, but is not limited thereto.

The present invention provides a method for screening a candidate material for prevention or treatment for each subtype of peripheral neuropathy comprising the step of treating a test substance in a sample isolated from a peripheral neuropathy individual to compare the expression levels of p75 and NCAM proteins before and after the treatment. .

The method may further include the step of selecting a candidate material for preventing or treating peripheral neuropathy when the level of expression of p75 and NCAM protein is significantly reduced compared to before treatment of the test material. The peripheral neuropathy may be hereditary peripheral neuropathy or non-hereditary peripheral neuropathy, and specific examples are Charcomaritus disease 1a (CMT1a), acute motor axonal neuropathy (AMAN), chronic inflammatory demyelination. It may be chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP).

The method further comprises the step of selecting a candidate material for preventing or treating hereditary peripheral neuropathy when there is no significant difference in the expression level of the p75 protein compared to before treatment of the test material, and the level of expression of the NCAM protein is significantly reduced. can do. For a specific example, the hereditary peripheral neuropathy may be Charcomaritus disease 1a (CMT1a).

The method may further include comparing the expression level of the CXCL13 protein before and after treatment with the test substance. In this case, when the expression levels of p75, NCAM and CXCL13 proteins are significantly reduced compared to before treatment with the test substance, it is inflammatory. It may further include the step of selecting as a candidate material for preventing or treating inflammatory demyelinating polyradiculoneuropathy. The inflammatory demyelinating polymuscular neuropathy may include chronic or acute inflammatory demyelinating polymuscular neuropathy.

The test substance is a newly synthesized or known compound, and may include, without limitation, substances that are expected to exhibit an effect on the prevention or treatment of peripheral neuropathy subtypes. For example, nucleic acids, nucleotides, proteins, peptides, amino acids, It may be at least one selected from the group consisting of sugars, lipids, and compounds, but is not particularly limited thereto.

In addition, in the screening method of the present invention, matters related to the individual, the sample, the peripheral neuropathy, and the measurement of the degree of expression are as described above.

The present invention is a peripheral neuropathy subtype comprising a nucleotide sequence of a gene encoding p75 and NCAM protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide or a substance that specifically binds to a protein encoded by the nucleotide sequence It provides a composition for star diagnosis.

The composition may be used for diagnosis of the onset of hereditary peripheral neuropathy or non-hereditary peripheral neuropathy, and specifically, the hereditary peripheral neuropathy is Charcomaritus disease 1a (CMT1a), and the non-hereditary peripheral neuropathy is acute motor axon neuropathy. (AMAN, Acute motor axonal neuropathy), Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP).

The composition may further comprise a nucleotide sequence of a gene encoding CXCL13 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance that specifically binds to a protein encoded by the nucleotide sequence. The composition of the case may be a composition for diagnosing inflammatory demyelinating polyradiculoneuropathy.

The substance specifically binding to the protein may specifically be an antibody, and the antibody refers to a specific immunoglobulin directed against an antigenic site, and the 　 antibody is specifically directed to p75, NCAM or CXCL13 protein. It refers to an antibody that binds, and a gene encoding p75, NCAM or CXCL13 is cloned into an expression vector to obtain a p75, NCAM or CXCL13 protein, and an antibody can be prepared from the obtained protein according to a conventional method in the art. The form of the 　 antibody includes polyclonal 　 antibody 　 or monoclonal 　 antibody, and all immunoglobulin 　 antibodies are included. The 　 antibody is not only in complete form with two full-length light chains and two full-length heavy chains, but also has two light chains and two heavy chains, and does not have the structure of an antibody, but is directed against the antigenic site. It also includes functional fragments of 　antibodies 　molecules having a specific antigen-binding site (binding domain) and possessing an antigen-binding function. In the diagnosis of peripheral neuropathy using the composition of the present invention, by performing hybridization using p75, NCAM or CXCL13 protein and the antibody, and measuring the expression level of p75, NCAM or CXCL13 through the degree of hybridization, peripheral neuropathy Can be diagnosed. The selection and hybridization conditions of an appropriate antibody can be appropriately selected according to techniques known in the art.

The nucleotide sequence, a sequence complementary to the nucleotide sequence, and a substance that specifically binds to the fragment of the nucleotide may be specifically a probe or a primer.

The probe refers to a nucleotide fragment such as RNA or DNA corresponding to a few bases or hundreds of bases that can specifically bind to a nucleotide such as mRNA, and is labeled with a radioactive element, so that a specific mRNA exists. You can check the presence or absence and content (amount of expression). The probe may be prepared in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe probe, etc., and encodes p75, NCAM or CXCL13 protein. Peripheral neuropathy can be diagnosed by performing hybridization using a probe complementary to the mRNA of the gene, and measuring the expression level of the mRNA through the degree of hybridization. Selection of an appropriate probe and conditions for hybridization can be appropriately selected according to techniques known in the art.

The primer is a nucleotide sequence having a short free 3-terminal hydroxyl group, which can form a base pair with a complementary template, and refers to a short nucleotide sequence serving as a starting point for template strand copying. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase/polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature, and p75, Peripheral neuropathy can be diagnosed through PCR amplification using a primer of an mRNA of a gene encoding NCAM or CXCL13 protein and measuring the expression level of a desired p75, NCAM or CXCL13 protein. The PCR conditions and the length of the primer set may be appropriately selected according to techniques known in the art.

The nucleotide sequence of a gene encoding the p75, NCAM or CXCL13 protein, a sequence complementary to the nucleotide sequence, or a 　 probe or primer that specifically binds to a fragment of the nucleotide is the nucleotide sequence of a gene encoding p75, NCAM or CXCL13 protein As is known, a person skilled in the art can design the primer 　 or 　 probe according to a conventional method in the art based on the sequence.

The 　probe or 　primer can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known method, and the length is 10 to 100 nucleotides (hereinafter referred to as'nt'), 10 to 90 nt, 10 to 80 nt, 10 to 70 nt, 10 to 60 nt, 10 to 50 nt, 10 to 40 nt, 10 to 30 nt, 10 to 25 nt, 20 to 100 nt, 30 to 90 nt, 40 to 80 nt, 50 to 70 nt, 20 to 60 nt, 20 to 50 nt, 30 to 40 nt, 20 to 30 nt, or 20 to 25 nt.

In addition, in the diagnostic composition of the present invention, matters related to peripheral neuropathy and the like are as described above.

The present invention provides a diagnostic kit for each subtype of peripheral neuropathy comprising the composition.

The kit can diagnose peripheral neuropathy by measuring the expression level of p75, NCAM or CXCL13, the mRNA of the gene encoding the p75, NCAM or CXCL13 protein, or the expression level of the p75, NCAM or CXCL13 protein.

The peripheral neuropathy may be hereditary peripheral neuropathy or non-hereditary peripheral neuropathy, and specific examples are Charcomaritus disease 1a (CMT1a), acute motor axonal neuropathy (AMAN), chronic inflammatory demyelination. It may be chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP).

The kit may include a nucleotide sequence of a gene encoding a p75, NCAM or CXCL13 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance that specifically binds to a protein encoded by the nucleotide sequence. In addition, the kit may include one or more other constituents/compositions, solutions, or devices suitable for an analysis method for measuring the expression level of p75, NCAM or CXCL13 protein used.

When the kit is a kit for measuring the expression level of the mRNA of the gene encoding the p75, NCAM or CXCL13 protein, it may be a kit including essential elements necessary for performing RT-PCR. RT-PCR kits include test tubes or other suitable containers, reaction buffers, deoxyribonucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase, in addition to each pair of primers specific for the mRNA of the marker gene. Inhibitors, DEPC-water, sterile water, and the like. In addition, it may include a 　 primer 　 pair specific to the gene used as a quantitative control.

The kit provides immunological detection of a nucleotide sequence of a gene encoding a p75, NCAM or CXCL13 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance that specifically binds to the protein encoded by the nucleotide sequence. For this purpose, a substrate, a suitable buffer solution, a secondary antibody labeled with a color developing enzyme or a fluorescent substance, and a color developing substrate may be included. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a glass slide glass, and the color developing enzyme is peroxidase, alkaline phosphatase ( alkaline phosphatase) can be used, fluorescent materials can be used FITC, RITC, etc., and the color developing substrate is 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or o- Phenylenediamine (OPD), tetramethyl benzidine (TMB), and the like may be used.

As a specific example, the kit may be a microarray for peripheral neuropathy 　 diagnosis capable of measuring the mRNA expression level of a p75, NCAM or CXCL13 protein or a gene encoding a protein thereof. The microarray can be easily manufactured by a person skilled in the art according to a method known in the art, and according to one embodiment, the cDNA of the sequence corresponding to the mRNA of the gene encoding the p75, NCAM or CXCL13 protein or a fragment thereof is It may be a microarray attached to a substrate as a probe.

As a specific example, the kit may be a protein array or a protein chip for peripheral neuropathy diagnosis capable of measuring the expression level of p75, NCAM or CXCL13 protein. The protein array or protein chip can be easily manufactured by a person skilled in the art according to a method known in the art, and according to one embodiment, p75, NCAM, or CXCL13 protein that is immobilized in the kit by extracting a sample from a patient Peripheral neuropathy can be diagnosed by subtype by confirming the reaction between a sample of a patient and a substance capable of measuring the expression level of p75, NCAM, or CXCL13 protein such as antibodies, receptors, nucleic acids, carbohydrates, etc. that can specifically bind to .

In addition, in the kit of the present invention, matters related to the diagnostic composition, sample, or peripheral neuropathy included in the kit are as described above.

Hereinafter, examples will be described in detail to illustrate the present invention in detail.

Experiment method

1. Antibodies and reagents

Antibodies against β-actin, p75 neurotrophin receptor (p75), CD68, CD4, CD63, Hsp70, and myelin basic protein (MBP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). NCAM and human CXCL13 antibodies were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against CD206, Rab5b and CXCR5 were obtained from Abcam (Cambridge, UK), and the Alexa-Fluor 488 conjugated CD197 antibody was purchased from Biolegend (San Diego, CA, USA). Antibodies against CXCL13 and myelin basic protein were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Horseradish peroxidase (HRP) binding anti-rabbit IgG and anti-mouse IgG were obtained from Cell Signaling technology (Danvers, MA, USA). Alexa Fluor 488 or Cy3 secondary antibody was purchased from Molecular probes (Carlsbad, CA, USA). All recombinant cytokines used were obtained from Peprotech (Rocky Hill, NJ, USA) and R&D Systems, and unless otherwise specified, all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2. Animal model

Non-obesity diabetes (NOD) and NOD-B7-2 knockout (B7-2KO) mice were purchased from Jackson Lab (Bar Harbor, Stock No. 004762, USA). The genotype was determined, and neuropathy was evaluated weekly from 20 weeks after birth. Tail-drop and hind-limb were investigated. The clinical progression of motor deficit was divided into 5 grades: Grade(G)0, no symptoms; G1, floppy tail; G2, mild paraparesis or unilateral hind limb paralysis; G3, severe catastrophic paralysis; G4, tetraparesis; G5, dying condition or death. PMP22 transgenic mice (C22) 19 were obtained from Samsung Medical Center (Seoul, Korea). The mouse model contains seven human peripheral myelinated protein 22 (PMP22) genes that cause demyelinating neuropathy. All surgical operations were performed according to the animal testing guidelines established by Dong-A University Animal Testing Society (No. DIACUC-16-21) and the protocol approved by Dong-A University Animal Testing Committee.

In case of sciatic nerve injury, after anesthesia with a mixture of 10% ketamine hydrochloride (Sanofi-Ceva, Dusseldorf, Germany; 0.1 ml/100 g body weight) and Rompun (Bayer, Leverkusen, Germany; 0.05 ml/100 g body weight) , Using fine scissors (FST Inc, Foster City, CA), the sciatic nerve of mature C57BL/6 mice was incised toward the center of the body 5 mm from the tibioperoneal bifurcation.

For morphological analysis of the degenerative nerve, distal stumps of 1 mm length from the lesion site were discarded, and distal stumps of 5 mm length were collected at the indicated time.

3. Human serum sampling and ELISA

Serum samples included 36 CIDPs (10 females, 26 males), 14 AIDPs (3 females, 11 males), 20 AMANs (7 females, 13 males) and 39 CMT1a (17 females, 21 males) patients and 20 healthy controls (14 females, 6 males). The blood was centrifuged at 3000 rpm for 10 minutes to separate the serum (plain tube, no anticoagulant), and the collected serum was stored at -80°C until use. The diagnosis of CIDP and GBS (AIDP, AMAN) was performed according to clinical and laboratory criteria, respectively. AMAN was classified according to the positive anti-ganglioside GM1 antibody using ELISA. All serum samples of CIDP and GBS were collected by Dong-A University Neuroimmunology Team (DAUNIT) for ganglioside antibody testing as a presumptive diagnosis of immune-mediated neuropathy in cooperation with the Korean Inflammatory Neuropathy Consortium (KINC). Serum of CMT1a patient was obtained from Samsung Medical Center. The research protocol was approved by the Institutional Review Board of Dong-A University (HR-004-02), Dong-A University Hospital (13-042), and Samsung Medical Center (2017-11-152). Measurements of serum NCAM and CXCL13 were performed using commercially available ELISA kits (#DCX130 and DY2408, R&D Systems).

4. Primary Schwann cell culture

Sciatic nerve sections were taken at 37° C. for 80 minutes, collagenase NB4 (0.26 U/ml, Serva, Heidelberg, Germany) and dispase ± (neutral protease, grade ±, 0.94 U/ml, Roche, CA, USA). ) Was digested with an enzyme solution containing. Then, the mixture was centrifuged at 1000 rpm for 10 minutes, and the supernatant was removed, and then the cell pellet was neuregulin-1 (neuregulin-1, 30 ng/ml, R&D Systems), N2 supplement (Invitrogen, Carlsbad, CA), 5 μM It was cultured in a medium containing forskolin, 1% fetal bovine serum (FBS, Hyclone, Melbourne, Australia), and penicillin-streptomycin (Gibco, NY, USA). After 48 hours, cells were treated with 0.2% dispase ± diluted in DMEM for 20 minutes, and then kept by shaking horizontally for 1-3 minutes to enrich the SC in the culture flask. Subsequently, the suspended cells were collected by centrifugation at 1000 rpm for 5 minutes, and after removing the supernatant, the pellet was resuspended and plated on a flask at a density of 2 to 2.5 x 10 ⁴ cells/cm 2. For biochemistry and collection of conditioned media, cells were used in 2-4 generations.

5. Purification of exosomes derived from Schwann cells

SCs were incubated with DMEM containing 5 μM forskolin, 30 ng/mL neuregulin-1, and 1% exosome-free FBS (obtained by serum ultracentrifugation at 100,000 g for 12 hours). After incubation for 3 days, the culture solution was collected and continuously centrifuged at 4° C. for 30 minutes at 300 g and 60 minutes at 10,000 g, and the supernatant was filtered through a membrane filter (0.2 μm, Sartorius Biotech, Goettingen, Germany), and 4° C. Ultracentrifugation was performed at 100,000 g for 90 minutes at (70 Ti rotor, Beckman). The pellet was washed with phosphate buffered saline (PBS, pH 7.4) and centrifuged again for 60 minutes at 100,000 g at 4°C. Purified exosomes were modified with 1% phenylmethylsulfonylfluoride and 1% protease inhibitor cocktail (Sigma-Aldrich) in RIPA buffer (1% Triton X-100, 50 mM Tris-HCl, pH 6.8, 2 mM EDTA) Dissolved in, and quantified by Bradford assay or microBCA assay (Thermo Fisher Scientific).

6. SDS-PAGE and In-gel trptic digestion

Proteins in exosomes were separated by electrophoresis in precast Bolt ^TM 4-12% Bis-Tris Plus SDS-PAGE Gel (Thermo Fisher Scientific) and stained with Coomassie Brilliant Blue R-350 (GE Healthcare, Uppsala, Sweden). The gel lane is excised into 14 sections, and then cut into 1-2 mm cubes with a clean scalpel. The gel pieces were washed twice with 30% methanol and removed in 50% acetonitrile in 100 mM ammonium hydrogen carbonate. The sample was then reduced with 10 mM DTT for 1 hour at 56° C. and alkylated with 20 mM iodoacetamide for 1 hour at room temperature in the dark. After removing the solution, acetonitrile was added to dehydrate the gel pieces, and then treated with 50 mM ABC, trypsin (Sequencing grade modified, Promega, Madison, WI, USA) w) at 37°C at a ratio of 1:50 enzyme to protein. I did.

7. Nano-LC-MS/MS and protein identification

Trypsin-degrading peptides derived from Schwann cell exosomes are easy nLC 1000 coupled online with reversed-phase nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) (LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific)). system (Thermo Fisher Scientific)). For each nLC-MS/MS experiment, a trapping column in which peptides were desalted with 0.1% formic acid in 5% ACN (Acclaim® PepMap® 100 C18, 2 cm x 75 μm, 3 μm particle size, 100Å pores, Thermo Fisher Scientific ) (FA) was stirred for 10 minutes, and then separated by an in-house microcapillary column (C18, 20 cm x 100 µm, particle size 3 µm) at a flow rate of 300 nL/min using a 3 hour gradient. The chromatographic gradient was 5% B (0.1% FA in ACN) over 15 minutes, 5-10% B over 5 minutes, 10-40% B over 135 minutes, 95% over 1 minute. A profile of increase to B, 95% B over 13 minutes, increase to 5% B over 1 minute, and 10 minutes equilibration at 5% B.

The eluent was sprayed and ionized at 1.9 kV in a nano-electron spray source of a mass spectrometer operated in a data dependent acquisition mode. MS survey scans were performed in an orbitlab with a resolution of 60,000 FWHM (200 m/z) over a mass range of 400-2,000 m/z, followed by a collision energy of 35%, an activation time of 10 ms, and a linear trap quadrupole. trap quadrupole, LTQ) collision-induced dissociation MS/MS fragmentation was performed on the fifteen most intense ions with an isolation window of 2 Da. For dynamic exclusion, the number of repetitions was set to 2 and the exclusion period was set to 60 seconds. For the identification of proteins, mass spectrometry was performed using Proteome Discover software (ver.2.2) for mouse proteins in the UniProt/Swiss-Prot database (April 2018 version, www.uniprot.org). Identification of the peptide was performed by setting mass tolerances of 10 ppm and 0.6 Da for precursor ions and fragment ions, respectively, allowing up to two missed cleavages. Cysteine carbamidomethylation and methionine oxidation were set as fixed and variable modifications, respectively, and the false discovery rate (FDR) was set to 1% at both the peptide and protein levels. The list of the proteins identified as a result was compared with the exosome database ExoCarta (www.exocarta.org), and the gene ontology was analyzed using a database for annotation, visualization and integration discovery (DAVID, v6.8).

8. Western Blot Analysis

Schwann cells cultured for western immunoblotting were homogenized in modified RIPA buffer. The lysate was fractionated on an SDS-PAGE gel and transferred to a nitrocellulose membrane (Millipore). Primary antibody diluted in TBST containing 3% fat-free milk for 1 hour at room temperature with 5% skim milk in Tris buffered saline containing 0.05% Tween-20 (TBST) and the membrane at 4°C overnight Was incubated with. After washing three times in TBST, it was reacted with HRP-conjugated secondary antibody for 1 hour at room temperature, and then washed again with TBST. For detection, an enhanced chemiluminescence western blot system (Amersham, Piscataway, USA) was used, and images were analyzed with LuminoGraph 2 (ATTO, Tokyo, Japan). A software CS analyzer (ATTO, Tokyo, Japan) was used to calculate the relative gray value (gray value of target band / gray value of β-actin band) of each target protein. For quantitative analysis, at least 3 independent experiments were performed.

9. Histological staining

Mouse sciatic nerves fixed with 4% paraformaldehyde were cryopreserved with 20% sucrose solution. Sections having a thickness of 14 μm were made using a cryostat (Frigocut, Leica, Bensheim, Germany), and stored in a deep freezer until use. Slides were blocked with PBS containing 0.2% Triton X-100 and 2% bovine serum albumin for 1 hour. Sections were incubated with the primary antibody at 4° C. for 16 hours and washed 3 times with PBS. Then, the slides were incubated with Cy3- or Alexa 488-conjugated secondary antibody for 3 hours at room temperature, followed by DAPI staining for 30 minutes. Four formalin-fixed, paraffin-inserted human gastrointestinal nerves were provided at Yonsei University Severance Hospital, and a continuous 4 μm section was fabricated using a microtome. The sections were then deparaffinized and rehydrated with graded ethanol. After antigen recovery, the sections were blocked with 5% fetal bovine serum for 1 hour at room temperature, and the same immunostaining protocol was applied as above. For optical microscopic analysis, stained sections were examined with a Zeiss AxioImager 2 microscope equipped with ApoTome (Carl Zeiss, Gottingen, Germany). To count the number of DAPI-labeled nuclei or immune-reactive cells in the sciatic nerve section, 8-10 images (500 µm X 650 µm) of 3 animals in each group were taken with a 403/1.2NA water immersion lens using Zen 2.3 Pro imaging software. Captured.

10. Mouse Cytokine Array

Each sciatic nerve of NOD, B7-2KO, intact C57BL/6 mice and injured C57BL/6 mice was homogenized in modified RIPA buffer, and the lysate was subjected to 40 cytokines (Mouse Cytokine Array Panel A, #ARY006, R&D Systems) was used for a cytokine antibody array designed to monitor the expression level. Briefly, lysates (300 μg) were incubated overnight with an antibody-coated membrane on an incubated platform, washed three times with PBS to remove unbound material, and then aligned capture antibody and biotinylated ( biotinylated) detection antibodies were used to continuously detect bound cytokines via a sandwich ELISA format. After washing three times with PBS, chemiluminescence detection reagent (Amersham) was added, and then proceeded according to the manufacturer's instructions.

11. Statistical method

Results were expressed as mean ± standard error, and ELISA data were analyzed by one-way analysis of variance by Kruskal-Wallis test and Sidak's multiple comparisons test using GraphPad Prism version 6.01 (GraphPad Software Inc., La Jolla, CA). . An unpaired student t-test was performed for the Western blot and statistical analysis of the number of immune response cells, and a value of p <0.05 was considered significant.

Experiment result

1. Discovery of p75 and NCAM, biomarkers for peripheral neuropathy diagnosis

In order to discover potential serum biomarkers for pathological immature Schwann cells (SCs) in human neuropathic neurons, proteins in exosomes were confirmed in immature SCs cultured by proteomic analysis (Fig. 1A A ). Peptide-containing fractions in exosomes were analyzed by nLC-MS/MS, and raw MS data were processed using Proteome Discover ^™ software. 13,116 peptides corresponding to a total of 1,356 protein groups were found in mouse Schwann cell exosomes (false discovery rate <1%). Of the 1,356 proteins, 696 were classified as exosome proteins extracted from mice in the ExoCarta database (FIG. 1B). Notably, the protein in the exosomes derived from mouse Schwann cells included p75, NCAM and well-known immature Schwann cell proteins, and significant enrichment of p75 and NCAM proteins in mouse Schwann cell-derived exosomes was confirmed through western blot. (Fig. 1a C).

Next, the levels of p75 and NCAM in the serum of various peripheral neuropathy patients were examined using ELISA (Fig. 1B D, E). At least 3 times higher p75 in serum from CIDP (256 ± 31.26 pg/mL, p <0.001) and AIDP (207.3 ± 37.85 pg/mL, p <0.001) mL) patients compared to healthy controls (73.69 ± 40.58 pg/mL) Indicated the concentration. The serum NCAM concentrations of patients with CIDP (4,960 ± 476 pg/mL, p <0.001) and AIDP (4,729 ± 661 pg/mL, p <0.05) were also higher than that of the healthy control group (2,298 ± 303 pg/mL). However, in the AMAN group, the serum concentration of NCAM and p75 was higher than that of the control group, but it was not significant. Interestingly, in the serum of CMT1a patients, the highest level of NCAM concentration (6,663 ± 277 pg/mL) was observed, while the p75 concentration (12.74 ± 5.315 pg/mL) was similar to that of the control group.

Next, from the ROC curve, we determined the optimal cut-off values for p75 and NCAM according to each group (Fig. 1C).

In the case of p75 of F, as a graph analyzing the difference with other groups based on the CIDP result of FIG. 1BD, the center dotted line is the result of CIDP, and for the NCAM of G, the result of CMT1a of FIG. 1BE As a graph analyzing the difference from other groups based on, the center dotted line is the result value of CMT1A.

In the case of p75, the CIDP group showing the highest level showed a sensitivity of 92.1% and a specificity of 95.0% compared to the CMT1a group at 56.45 pg/mL (AUC = 0.986, p <0.001) (FIG. 1C F). CIDP was 103.40 pg / mL (AUC = 0.782, 63.2% sensitivity and 85.0% specificity, p <0.001) AIDP (AUC = 0.596, 84.6% sensitivity and 40.0% specificity, with AMAN showing high levels of p75 expression, p = 0.365) also showed specificity and was clearly distinguished. In the case of NCAM, the CMT1a group with the highest level was CIDP and 7038.69 pg/mL (AUC = 0.672, 39.5% sensitivity and 100% specificity, p = 0.017), and AIDP was 6896.14 pg/mL (AUC = 0.679, 42.1). % Sensitivity and 100% specificity, p=0.033), AMAN showed a clear difference at 3937.07 pg/mL (AUC = 0.852, 97.4% sensitivity and 72.2% specificity, p <0.001). These results suggest that measuring the level of NCAM and p75 expression in the serum of neuropathy patients can be a useful tool for predicting and diagnosing the risk of developing peripheral neuropathy by subtype.

In order to confirm whether the differential induction of p75 and NCAM in the patient's sera, in the pathologically immature Schwann cells of the neuropathy patients with peripheral neuropathy, the expression of these proteins was specifically changed, in the neurological tissues of patients with peripheral neuropathy. The expression profiles of p75 and NCAM were investigated. About 25% of Schwann cells with myelin in the neural tissues of CIDP patients expressed p75 in the cytoplasm (FIGS. 2AA, 2BD). In contrast, the expression of p75 was hardly observed in the nerves of CMT1a patients (Figs. 2AA, 2BD). In addition, when comparing the immunofluorescence intensity of p75, there was a significant difference between the two groups (FIG. 2BE), which indicates a correlation with p75 in the serum of the two patient groups. In the spinal nerves of CIDP patients, NCAM staining was observed in both immature Schwann cells and myelin-bearing Schwann cells (FIG. 2). Consistent with the ELISA data that showed the highest serum NCAM expression in the CMT1a group among peripheral neuropathy patients, the expression of NCAM in the spinal nerves of CMT1a patients was much higher than in CIDP patients, and non-myeloid immature, including onion progenitor cells. It was observed not only in Schwann cells but also in demyelinating Schwann cells (Fig. 2a B, 2b C).

In order to determine whether the induction of p75 and NCAM in the serum indicates immature demyelinating SC (DSC), first, in an animal model of experimental allergic neuritis (EAN) and Wallerian degeneration (WD), which are mouse AIDP models, the expression profile of p75 and NCAM Investigated. On the 6th day after axonal cutting, a high level of p75 expression was induced in almost all DSCs (FIG. 3AA), and p75 was induced after axonal injury in Western blot analysis (FIG. 3AB, 3AC). In contrast, axonal cleavage did not induce the expression of NCAM in DSC, and did not significantly increase the expression of NCAM in non-myelinating SC (FIGS. 3A A, B, 3B C). In the sciatic nerve of EAN, the expression of p75, not NCAM, was dramatically induced in DSC, and the expression of NCAM was increased in non-myelinated SC (Fig. 3D). In addition, in the gastrocnemius biopsy of CIDP patients, a large number of DSCs in the abaxonal cytoplasm showed p75 expression (Figs. 3bE, 3bF). In contrast, high levels of NCAM were mainly found in the non-myelinated SC of the gastrocnemius (Figs. 3BE, 3BF). ELISA data showed normal levels of p75 and highest NCAM levels in CMT1a patients among neuropathic patients. Consistent with this, by NCAM staining in all layers of Onion-bulb SC with very rare p75 positive cells, NCAM expression in CMT1a gastrocnemius was much higher than in CIDP patients.

2. NCAM Expression in Serum and Gastric Nerve of Patients with Inflammatory Demyelinating Peripheral Neuropathy

To determine whether expression of NCAM is induced by inflammatory cytokines in cultured primary SCs, inflammatory cytokines known to be associated with demyelination were tested. According to western blot analysis, it can be seen that IFN-γ, TNF-α and nitric oxide donor sodium nitroprusside significantly induce NCAM expression (FIG. 4). However, neuregulin, TGF-β, and endoplasmic reticulum stress inducers did not upregulate NCAM expression. In contrast to the expression of NCAM, p75 expression in SC was not induced by the inflammatory cytokines tested (FIG. 5A ). These results suggest that pro-inflammatory cytokines can induce SC NCAM expression in demyelinating nerves.

3. Pre-inflammatory environment in inflammatory demyelinating nerves containing CXCL13+ macrophages

The pro-inflammatory state-related NCAM expression in dedifferentiated SC was induced to compare the cytokine expression profile in the neuropathic sciatic nerve and the damaged C57BL/6 nerve in B7-2KO mice (in this case, natural immunity was observed during WD. Activated). Expression levels of ICAM-1, interleukin-1 receptor antagonist, metalloproteinase 1 tissue inhibitor, CCL2 and IL-16 were increased in both the sciatic nerve of B7-2KO mice and injured C57BL/6 mice, and CCL5, macrophages It was found that inflammatory protein-1 and CXCL10 were specifically upregulated in B7-2KO neurons compared to NOD and damaged C57BL/6 mouse neurons (Figs. 5A A, 5A B). CCL5 and CXCL10 are cytokines associated with INF-γ-induced type-1 macrophage (M1), and both are known to be involved in inflammatory neuropathy. Interestingly, CXCL13, a B cell recruitment factor, is constitutively expressed in follicular cells and macrophages in secondary lymph node tissues, and is selectively upregulated in B7-2KO neurons, but not in WD (Figs. 5A A, 5A B).

Consistent with the cytokine array data, IF staining revealed a number of CXCL13 positive cells in B7-2KO neurons, but not in NOD neurons (Fig. 5BC). Double immunostaining for CXCL13 and macrophage marker CD68 or SC marker S100 showed that expression of CXCL13 was restricted to CD68+ macrophages in B7-2KO neurons. Conversely, even if a large number of invasive CD68+ macrophages were present in the C57BL/6 nerve, no CXCL13 positive cells were observed, suggesting that the pre-inflammatory state of the B7-2KO nervous system is related to the expression of CXCL13 (Fig. 5b C, 5b. D).

Assessing the differentiation of macrophages into M1 and/or M2 is related to the expression of CXCL13 in B7-2KO neurons, and IF staining with antibodies against M1 and M2 macrophage markers was performed on sciatic nerve sections (Fig. 5BD). . In this experiment, while CD68+ macrophages of the B7-2KO nervous system did not express the M2 marker CD206, a number of macrophages CD197 positive for the M1 marker were present. In contrast, the sciatic nerve undergoing WD showed a large number of CD206+/CD97- macrophages (Fig. 5BD).

Taken together, from these results, it can be seen that the demyelinating B7-2KO neuron exhibits a pro-inflammatory environment associated with M1-macrophages, which is associated with selective CXCL13 and NCAM expression in inflammatory demyelination.

4. Localized CXCL13/CXCR5-mediated immune response in inflammatory demyelinating nerves

Expression of CXCL13 in B7-2KO neurons may indicate local B cell-related immune activation through CXCR5, a receptor for CXCL13. Invasion of CD19+ B cells in the B7-2KO and NOD nerves was investigated. It was found that B cells penetrated the B7-2KO nerve a lot, but this was not the case in the NOD nerve (Fig. 6A). The cellular localization of CXCR5 in the B7-2KO nerve was examined using IF staining. The expression level of CXCR5 was low in the axon and SC of the NOX nerve, but the expression of CXCR5 was rapidly increased in the B7-2KO nerve. Double immunostaining showed that many of the CXCR5 positive mononuclear cells in B7-2KO neurons were CD4+ T cells and CD68+ macrophages (Figs. 6A A, 6B B). In addition, some p75 positive dedifferentiated SCs also express CXCR5 in B7-2KO neurons (Fig. 6BB). Expression of CXCR5 in the SC was confirmed by Western blot analysis in the cultured primary SC (FIG. 6CC), which is the pathology of CXCL13 and CXCR5/CD4+ T cells, B cells and SC in autoimmune inflammatory demyelination in mice. This suggests that it could be involved as an enemy.

5. Effect of high concentration of CXCL13 in serum on inflammatory peripheral neuropathy

Serum CXCL13 levels of patients with peripheral neuropathy were examined. Serum of patients with CIDP (71.41 ± 5.83 pg/mL, p <0.01) and AIDP (71.34 ± 13.73 pg/mL, p <0.05) patients with control (37.35 ± 6.75 pg/mL) mL) of serum and found a higher level of CXCL13. In contrast, CXCL13 levels in serum of AMAN (17.99 ± 7.02 pg/mL) and CMT1a (47.39 ± 3.98 pg/mL) were not significantly different from those of control (p>0.05, Fig. 7). Consistent with the ELISA data, CXCL13+ cell infiltration was found in the gastrocnemius in CIDP patients, but not in CMT1a patients. These results indicate that CXCL13 may be a specific marker of inflammatory peripheral demyelination.

6. Relationship between the increase of immature Schwann cells and the onion-bulb and the increase of the NCAM concentration in CMT1a neuropathy

ELISA data showed a high level of NCAM in the serum of CMT1a patients (Fig. 1).Since immature SC generally expresses NCAM at the beginning of birth, the expression of Pmp22 was wrong, resulting in NCAM expression of many abnormally differentiated immature SCs. This may be associated with high NCAM levels in the serum of CMT1a patients. In order to test this possibility, the expression of NCAM in the sciatic nerve of C22 mice, which is a PMP22 overexpressing CMT1a mouse model, was investigated. Mice showed abnormal gait at 2 weeks of age and were maintained until the mice were sacrificed. The sciatic nerve was not well myelinated at 5 and 8 weeks after birth in C22 mice (Fig. 8A A). At 8 weeks postnatal, NCAM immune responsive cells were found more frequently in the sciatic nerve of C22 mice compared to the control nerve (Figs. 8AB, 8BC). High levels of NCAM expression in C22 neurons were also confirmed by Western blot analysis (Fig. 8BC). Most of the NCAM staining in the C22 nerve was patch-shaped, but it was found to be different from the abaxonal perimyelin staining of demyelinating SC in B7-2KO mice (Fig. 8A B), and the large number of NCAM-positive cells in the C22 nerve was extra immature SC This suggests that may be the cause (Figs. 8AB, 8BC). In fact, the number of DAPI-positive cells was about 2 times higher in the C22 sciatic nerve (786 ± 73, p <0.001) compared to the WT control (322 ± 26), but macrophage infiltration was mild (45.7 ± 5.6 [C22]). 26.3 ± 11.7 [WT], p <0.001, Fig. 8BD), mostly CD206+/CXCL13-. IF staining of the human CMT1a gastrocnemius showed that NCAM expression was found not only in a number of dot-shaped non-myelinated SCs, but also in all the SC layers of onion-bulb (FIG. 8C E ). The level of NCAM expression in human CMT1a nerve segments was much higher than that in human CIDP neurons (FIG. 1 ), which is in good agreement with differentiated serum NCAM levels. In addition, compared with a large number of macrophages in the gastrointestinal nerve of CIDP patients, only a few IBA-1 positive macrophages were found in the gastrointestinal nerve CMT1a (FIG. 8DF).

Taken together, high levels of NCAM expression in the serum of CMT1a patients may reflect a large number of immature SCs and/or abnormally differentiated SCs that are not associated with inflammatory responses.

Claims (17)

Measuring the expression level of p75 and NCAM protein in the sample isolated from the individual; And comparing the measured expression level with a control subject. And predicting or determining whether the individual has a risk of developing hereditary peripheral neuropathy or non-hereditary peripheral neuropathy according to the comparison result; The hereditary peripheral neuropathy is Charcomaritus disease 1a (CMT1a), and the non-hereditary peripheral neuropathy is acute motor axonal neuropathy (AMAN), chronic inflammatory demyelinating polymyelopathy (CIDP, Chronic inflammatory neuropathy). Demyelinating polyradiculoneuropathy) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP), a method of providing information for predicting or diagnosing the risk of developing peripheral neuropathy subtypes. The method of claim 1, wherein if there is no significant difference in the expression level of p75 protein compared to the control individual, and the expression level of NCAM is significantly higher than that of the control individual, the risk of developing hereditary peripheral neuropathy is higher. The method of claim 1, wherein when the expression level of p75 and NCAM protein is significantly higher than that of the control individual, the risk of developing non-hereditary peripheral neuropathy is higher. The method of claim 1, further comprising measuring the expression level of the CXCL13 protein. The method of claim 4, wherein when the expression level of p75, NCAM, and CXCL13 protein is significantly higher than that of the control individual, the risk of developing inflammatory demyelinating polyradiculoneuropathy is higher. The method according to any one of claims 1 to 5, wherein the sample is at least one selected from the group consisting of serum, plasma, nerve cells, immune cells, cerebrospinal fluid, and exosomes. Treating a test substance on a sample isolated from a peripheral neuropathy individual to compare the expression levels of p75 and NCAM proteins before and after the treatment; And determining whether the test substance is a candidate substance for prevention or treatment for each subtype of peripheral neuropathy according to the comparison result, The peripheral neuropathy is an inherited peripheral neuropathy or a non-hereditary peripheral neuropathy, The hereditary peripheral neuropathy is Charcomaritus disease 1a (CMT1a), and the non-hereditary peripheral neuropathy is acute motor axonal neuropathy (AMAN), chronic inflammatory demyelinating polymyelopathy (CIDP, Chronic inflammatory neuropathy). Demyelinating polyradiculoneuropathy) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP), a method for screening candidates for prevention or treatment of peripheral neuropathy subtypes. The method according to claim 7, wherein when the expression level of p75 and NCAM protein is significantly reduced compared to before treatment of the test substance, it is Charcomaritus disease 1a (CMT1a), acute motor axonal neuropathy (AMAN), chronic inflammatory disease. A method of screening as a candidate for the prevention or treatment of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP). The method according to claim 7, if there is no significant difference in the expression level of the p75 protein compared to before treatment of the test substance, and the expression level of the NCAM protein is significantly reduced, it is selected as a candidate for preventing or treating Charcomaritus disease 1a (CMT1a). How to. The method of claim 7, further comprising comparing the expression level of the CXCL13 protein before and after treatment with the test substance. The method of claim 10, wherein when the expression levels of p75, NCAM, and CXCL13 proteins are significantly reduced compared to before treatment of the test substance, it is selected as a candidate substance for preventing or treating inflammatory demyelinating polyradiculoneuropathy. The method of claim 7, wherein the sample is at least one selected from the group consisting of serum, plasma, nerve cells, immune cells, cerebrospinal fluid, and exosomes. a nucleotide sequence of a gene encoding p75 and NCAM protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance that specifically binds to a protein encoded by the nucleotide sequence, For diagnosis of the onset of inherited peripheral neuropathy or non-hereditary peripheral neuropathy, The hereditary peripheral neuropathy is Charcomaritus disease 1a (CMT1a), and the non-hereditary peripheral neuropathy is acute motor axonal neuropathy (AMAN), chronic inflammatory demyelinating polymyelopathy (CIDP, Chronic inflammatory neuropathy). Demyelinating polyradiculoneuropathy) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP), a composition for diagnosing peripheral neuropathy subtypes. The composition of claim 13, further comprising a nucleotide sequence of a gene encoding a CXCL13 protein, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a substance that specifically binds to a protein encoded by the nucleotide sequence. The composition according to claim 13, wherein the composition is for diagnosis of inflammatory demyelinating polymuscular neuropathy. Comprising the composition of any one of claims 13 to 15, For diagnosis of the onset of inherited peripheral neuropathy or non-hereditary peripheral neuropathy, The hereditary peripheral neuropathy is Charcomaritus disease 1a (CMT1a), and the non-hereditary peripheral neuropathy is acute motor axonal neuropathy (AMAN), chronic inflammatory demyelinating polymyelopathy (CIDP, Chronic inflammatory neuropathy). Demyelinating polyradiculoneuropathy) or acute inflammatory demyelinating polyradiculoneuropathy (AIDP), a diagnostic kit for peripheral neuropathy subtypes. The kit according to claim 16, wherein the kit is a protein array or a protein chip including a substance specifically binding to a protein encoded by the nucleotide sequence.

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