WO2022075243A1 - Biomarqueur de la schizophrénie - Google Patents

Biomarqueur de la schizophrénie Download PDF

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WO2022075243A1
WO2022075243A1 PCT/JP2021/036560 JP2021036560W WO2022075243A1 WO 2022075243 A1 WO2022075243 A1 WO 2022075243A1 JP 2021036560 W JP2021036560 W JP 2021036560W WO 2022075243 A1 WO2022075243 A1 WO 2022075243A1
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crmp2
phosphorylated
subject
schizophrenia
sample derived
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良郎 五嶋
宗孝 野本
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公立大学法人横浜市立大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • the present invention relates to a schizophrenia biomarker.
  • Schizophrenia is a psychiatric disorder characterized by impaired cognition, perception, emotions, and behavior. Diagnosis of schizophrenia is made by symptom-based scoring, and the development of quantitative biomarkers is awaited. Under such circumstances, Bader et al. Reported that a change was observed in one of the CRMP family molecules called CRMP1 (Non-Patent Document 1: Hum Mol Genet, 21, 4406-4418, 2012). However, the method of Bader et al. Had to adjust the lymphocytes, which was complicated.
  • An object of the present invention is to provide a biomarker for schizophrenia that can be detected by a simple method.
  • the present inventors may find that an increase in the expression level of non-phosphate Collapsin Response Mediator Protein 2 (CRMP2) in a blood sample and / or a decrease in the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio may be useful for diagnosing schizophrenia. It was found that there is, and the present invention was completed.
  • CRMP2 Collapsin Response Mediator Protein 2
  • the gist of the present invention is as follows.
  • a method for testing schizophrenia which comprises measuring the expression level of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or phosphorylated CRMP2 in a sample derived from a subject.
  • CRMP2 Collapsin Response Mediator Protein 2
  • phosphorylated CRMP2 ratio indicates that the possibility or risk of developing schizophrenia is high.
  • the possibility or risk of developing schizophrenia is that the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio in the subject's peripheral blood mononuclear cell fraction is 0.25 or less.
  • a method for diagnosing schizophrenia in a subject Obtaining a sample from the subject, b.
  • the method comprising determining the likelihood or risk of developing schizophrenia in a subject based on the expression level of non-phosphorylated CRMP2 and / or the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio.
  • a method for diagnosing and treating schizophrenia in a subject Obtaining a sample from the subject, b. Measuring the expression levels of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or phosphorylated CRMP2 in a sample derived from the subject. c.
  • the method described above comprising initiating treatment of the subject with schizophrenia if the likelihood or risk of developing schizophrenia is determined to be high.
  • the expression level of non-phosphorylated CRMP2 in the sample derived from the subject is higher than the expression level of non-phosphorylated CRMP2 in the sample derived from a healthy person who has not developed schizophrenia, and / or the sample derived from the subject.
  • Phosphorylated CRMP2 Non-phosphorylated CRMP2 ratio in a sample derived from a healthy person who has not developed schizophrenia is lower than the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio in the possibility or risk of developing schizophrenia.
  • Integration including a reagent capable of measuring the expression level of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or a reagent capable of measuring the expression level of phosphorylated CRMP2 in a sample derived from a subject.
  • CRMP2 Collapsin Response Mediator Protein 2
  • the present invention enables quantitative diagnosis of schizophrenia.
  • CRMP2 ratio may be useful in diagnosing schizophrenia.
  • This specification includes the contents described in the Japanese patent application, Japanese Patent Application No. 2020-169396 and / or the drawings which are the basis of the priority of the present application.
  • CRMP2 pCRMP2 phosphorylated with serine 522 was examined by quantitative Western blot using certain well-certified antibodies.
  • a single band of 64 kDa was detected using 9F in brain lysates from wt, crmp1 -/- and crmp2 ki / ki mice, but not in crmp2 -/- mice.
  • a single band was detected with anti-pCRMP1 / 2 antibodies in the brain lysates from wt and crmp1 -/- , but in crmp1 -/- ; crmp2 -/- and crmp2 ki / ki mice. There wasn't.
  • FIG. 1 A typical immunoblotting method using an anti-CRMP2 antibody against a human peripheral blood mononuclear cell (PBMC) fraction of a normal healthy control subject [left panel] was compared with a positive control brain lysate. In the right panel, it can be seen that the band is successfully blocked by the antigenic peptide of CRMP2. (See also Figure 3 for further validation of the antibodies used in this study). Further validation of the antibodies used in the immunoblotting in this study and evidence that the CRMP1 isoform is not associated with the phenomenon described in this study. The difference seen in SCZ and non-affected patients was also demonstrated by the use of antibodies against CRMP1 to be more important in the difference in the abundance of CRMP2 isoforms than in CRMP1 isoforms.
  • PBMC peripheral blood mononuclear cell
  • the present invention provides a method for testing schizophrenia, which comprises measuring the expression levels of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or phosphorylated CRMP2 in a sample derived from a subject.
  • CRMP2 Collapsin Response Mediator Protein 2
  • Schizophrenia is a mental illness of unknown cause that often occurs in adolescence. Although genetic predisposition to develop the disease is recognized, the onset is considered to be due to the interaction between environmental factors and genetic factors as well as genetic factors. Symptoms are characterized by peculiar thought disorders, inappropriate or dull feelings and motivation, autistic or playful lifestyles, and social and occupational dysfunction. Anomalous experiences such as hallucinations and delusions, ego disorders such as acts and affected experiences, and lack of insight are shown in the form of psychotic episodes. Although there are cognitive impairments such as proper selection, judgment, and execution of information, consciousness is clear and general intelligence is maintained. There are no clear physical findings. The course indicates either acute or chronic course, but most have a chronic course.
  • neuregulin 1, DISC 1, and dysbindin which are potential risk genes for schizophrenia, are more highly expressed in the fetal brain than in adults and are involved in the formation of synapses and neural circuits (Harrison). PJ, Weinberger DR. Mol Psychiatry. 2005 Jan; 10 (1): 40-68; image 5.). Glantz and Lewis (2000) also reported that Golgi's staining reduced the density of dendrite spines in pyramidal neurons in the third layer of the dorsolateral prefrontal cortex (Glantz LA, Lewis DA. Arch). Gen Psychiatry. 2000 Jan; 57 (1): 65-73.).
  • CRMP2 Collapsin Response Mediator Protein 2
  • CRMPs are cytoplasmic proteins, and five subtypes (CRMP1-5) have been identified so far. All of these show high levels of expression during the developmental stage of the brain, but each shows a specific expression distribution and time of expression. Of these, CRMP2 shows high expression levels in the sensory system, hippocampus, cerebral cortex, cerebellum, etc. even in the adult brain.
  • CRMPs are homologous molecules of C. elegans Unc-33, and mutations in C. unc-33 cause axonal elongation and abnormal guidance in C. elegans neurons.
  • CRMPs are phosphorylated downstream of Sema3A, where cyclin dependent kinase 5 (Cdk5) phosphorylates the 522nd Serin residue, thereby continuing to glycogen synthase kinase (GSK3b) Ser517, Thr514, Thr509 residues. It is a protein that undergoes two-step phosphorylation modification, and its regulation of phosphorylation plays an important role in the development and maturation of nerves.
  • Cdk5 cyclin dependent kinase 5
  • GSK3b glycogen synthase kinase
  • CRMP2 undergoes various post-translational modifications such as Rho / ROCK kinase (ROCK1), Fyn (FYN), Fes (FES) tyrosine kinase, and SUMOylation.
  • ROCK1 Rho / ROCK kinase
  • FYN Fyn
  • FES Fes
  • SUMOylation phosphorylation modification
  • CRMP2 changes its interaction with molecules such as tuberin and calcium channels, and through this, it plays a role as a mediator of neuronutrient factors and other external signals, including Sema3A, as well as polarity and axon formation. It has been reported to be involved in various nerve functions and pathological conditions such as nerve cell migration, synaptogenesis, synaptic plasticity, and nerve disease (Nakamura et al, Front Cell Neurosci.
  • CRMP2 is phosphorylated by Cdk5 (cyclin-dependent kinase 5, CDK5) and GSK3b (GSK3b, glycogensynthase3b), which reduces the interaction with tuberin and suppresses protrusion elongation.
  • Cdk5 cyclin-dependent kinase 5, CDK5
  • GSK3b glycogensynthase3b
  • CRMP2 is highly expressed mainly in the central nervous system, especially in the cerebral spinal cord tissue during development. Among the CRMP family molecules, the expression level of CRMP2 is maintained even in the relatively mature stage, and its role in adults such as learning, memory, and cognitive function is presumed. The expression of CRMP2 is also expressed in cardiovascular system, immune system, and digestive system tissues, but its role is often unknown. CRMP2 undergoes various post-translational modifications, including phosphorylation, as described above. An important modification is phosphorylation modification by Cdk5, GSK3b, Rho / ROCK kinase at the C-terminus of CRMP2.
  • Cdk5 phosphorylates CRMP2 with Ser522, which promotes phosphorylation modification of CRMP2 by GSK3 ⁇ in Thr514 and Ser518.
  • This two-step phosphorylation modification mode is called prime phosphorylation (Nakamura et al, Front Cell Neurosci. 2020 Jun 23; 14: 188.).
  • prime phosphorylation Nakamura et al, Front Cell Neurosci. 2020 Jun 23; 14: 188.
  • phosphorylated CRMP2 has reduced interaction with tubulin and reduced cytoskeletal system function.
  • CRMP2 interacts with tubulin better and the formation of a cytoskeletal system occurs.
  • the phosphorylated CRMP2 is sometimes referred to as an inactive form, and the dephosphorylated form is sometimes referred to as an active form.
  • Other known physiological significance of CRMP2 post-translational modification is the interaction of CRMP2 with sodium and calcium channels and their activity regulation (Chew and Khana, Neuronal Signal. 2018; 2 (1): NS20170220.).
  • the phosphorylated CRMP2 is preferably CRMP2 phosphorylated by Ser522.
  • the subject is a mammal suspected of developing schizophrenia, but all mammals at risk of developing schizophrenia may be targeted. It is typically human. Humans are preferably under the age of 40, more preferably under the age of 30. Samples derived from the subject include cells, tissues, body fluids, etc. obtained from the subject, specifically, the subject's blood (for example, whole blood, serum, plasma, plasma exchange external fluid, etc.), brain tissue, saliva, tears, etc. Etc. can be exemplified. A blood sample such as whole blood, serum or plasma obtained by a normal blood test (clinical test) may be used as a blood sample. The blood sample may be a peripheral blood mononuclear cell fraction.
  • the expression of non-phosphorylated CRMP2 and / or phosphorylated CRMP2 in a sample derived from a subject may be measured by measuring the abundance of the above protein or a fragment thereof in the sample.
  • the measuring means is not particularly limited, and a known method may be used. It is preferably measured at the protein level, but may be measured at the nucleic acid level.
  • an antibody that specifically recognizes the protein may be either a monoclonal antibody or a polyclonal antibody. These antibodies can be produced by known methods, or commercially available ones may be used. In the examples described below, an anti-phosphorylated CRMP1 / 2 (S522) (p-S522-CRMP1 / 2) polyclonal antibody that recognizes CRMP2 phosphorylated by Ser522 was used. Typical methods include immunoassays such as an ELISA method and an immunochromatography method.
  • the immunoassay method does not require a special device or technique and can easily and quickly detect and quantify the target protein, it can be preferably used for the measurement of the above protein in the present invention as well.
  • Antibodies to the above proteins are known, and commercially available products also exist. Further, as described above, since the amino acid sequence of the above protein and the base sequence encoding these are also known, a specific antibody against each protein may be prepared by preparing a general hybridoma.
  • the immunoassay itself is well known in this field.
  • a sandwich method a competitive method, an agglutination method, a Western blotting method and the like, and based on the label, there are enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, luminescence immunoassay and the like.
  • any immunoassay method capable of quantitative detection may be used.
  • a sandwich method such as sandwich ELISA can be preferably used.
  • the sandwich method the antibody that binds to the target protein is immobilized and reacted with the sample.
  • the target protein bound to the immobilized antibody is measured using a detection antibody labeled with an enzyme or the like.
  • the detection antibody it is preferable to use an antibody that binds to the target protein at a site different from that of the immobilized antibody.
  • the immobilized antibody and the detection antibody may be a polyclonal antibody or a monoclonal antibody, and an antigen-binding fragment of the antibody may be used.
  • the target protein bound to the immobilized antibody is reacted with the detection antibody, washed, and then the amount of the bound detection antibody is measured by a signal from the substance labeled on the antibody.
  • the substrate of the enzyme may be added into the reaction system, and the amount of color development, fluorescence, and luminescence generated by the enzyme reaction may be measured by a corresponding device.
  • Perform immunomeasurement on a standard sample containing a target protein with a known concentration prepare a calibration curve plotting the relationship between the signal of the labeling substance and the concentration, and perform the same operation on a sample with an unknown target protein concentration. By applying the obtained signal measurement value to the calibration curve, the target protein in the sample can be quantified.
  • nucleic acid probe that can specifically hybridize with the mRNA of the above protein (when measuring by Northern blotting).
  • at least one pair of nucleic acid primers capable of specifically amplifying the cDNA synthesized using the mRNA of the above protein as a template may be used (when measured by the RT-PCR method).
  • Nucleic acid probes and nucleic acid primers can be designed based on the genetic information (described above) of the above proteins.
  • a nucleic acid probe of about 15 to 1500 bases is usually suitable.
  • the nucleic acid probe may be labeled with a radioactive element, a fluorescent dye, an enzyme or the like.
  • nucleic acid primer one having about 15 to 30 bases is usually suitable.
  • Nucleic acid primers may be labeled with radioactive elements, fluorescent dyes, enzymes and the like.
  • non-phosphorylated CRMP2 If an increase in the expression level of non-phosphorylated CRMP2 and / or a decrease in the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio is confirmed, it is highly possible that the patient has schizophrenia or develops schizophrenia. It can be determined that the possibility (risk of onset) is high. Therefore, the present invention can assist in the diagnosis of schizophrenia. INDUSTRIAL APPLICABILITY The present invention can be used for evaluation of the possibility or risk of developing schizophrenia, differentiation of schizophrenia (distinguishing from other diseases), and the like.
  • schizophrenia can be diagnosed according to the following criteria.
  • a blood sample for example, plasma, serum
  • a higher value is obtained in a blood sample collected from an unaffected person (for example, a healthy person).
  • Subjects are evaluated to have developed schizophrenia or have a high probability of developing schizophrenia (risk of developing schizophrenia).
  • the expression levels of non-phosphorylated CRMP2 and phosphorylated CRMP2 in blood samples (eg, plasma, serum) collected from the subject were measured, and the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio was calculated, and this ratio was determined from the set value.
  • This preset value can be appropriately set by those skilled in the art. For example, a 95% confidence interval of the quantitative value of a healthy person who has not developed schizophrenia can be used as a reference value, or a cutoff value can be set from the ROC curve. As one embodiment, when the age is less than 30 years old in FIG. 1A and the reference value for the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio is set to 0.25, the ratio in the measured value is equal to or less than the reference value. Can be determined to be more likely to have schizophrenia.
  • non-phosphorylated CRMP2 expression in samples derived from subjects determined to be highly likely to have schizophrenia was measured once or multiple times at different times, and the expression level was close to the cutoff value or the reference value. If the level drops to a level, it is determined that the patient has recovered from schizophrenia by treatment, and if the level is high or does not decrease, it is determined that the patient has not recovered from schizophrenia by treatment or the recovery is insufficient. can do.
  • the expression of non-phosphorylated CRMP2 and phosphorylated CRMP2 in samples derived from subjects judged to have a high possibility of schizophrenia was measured once or multiple times at different times, and phosphorylated CRMP2: non-phosphorylated.
  • the method of the present invention can be used not only for diagnosing schizophrenia, but also for prognostic examination and confirmation of therapeutic effect.
  • drug treatment will be the basis, and lifestyle guidance, psychotherapy, and community care will be added to promote social recovery.
  • the purpose of treatment is to reduce or suppress psychiatric symptoms and dysfunction, prevent recurrence, and reduce social and occupational activity restrictions and participation restrictions, that is, rehabilitation.
  • Drug therapy is based on drug selection according to the symptoms.
  • antipsychotic drugs chlorpromazine and haloperidol, which have a strong effect of suppressing acute symptoms such as hallucinations and delusions, are used. Since these are potent dopamine D2 receptor antagonists, extrapyramidal symptoms such as Parkinson's disease-like symptoms can be problematic as side effects.
  • these drugs generally do not improve, but even worsen, for negative symptoms such as cognitive impairment, withdrawal, and decreased motivation.
  • negative symptoms such as cognitive impairment, withdrawal, and decreased motivation.
  • Lybalvi which is a combination of olanzapine and the opioid receptor antagonist Samiddlefan, and aripiprazole, which has the effect of promoting the release of dopamine and noradrenaline by presynaptic D2 receptor antagonist, are used.
  • the anxiolytics lorazepam, paroxetine, the hypnotic solpitem, flunitrazepam, etc. are prescribed according to the psychological symptoms.
  • the present invention provides a method for diagnosing schizophrenia. That is, the present invention is a method for diagnosing schizophrenia in a subject. Obtaining a sample from the subject, b. Measuring the expression levels of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or phosphorylated CRMP2 in a sample derived from the subject, and c. Provided above are the methods comprising determining the likelihood or risk of developing schizophrenia in a subject based on the expression level of non-phosphorylated CRMP2 and / or the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio.
  • CRMP2 Collapsin Response Mediator Protein 2
  • the present invention also provides a method of treating schizophrenia in combination with the diagnosis of schizophrenia. That is, the present invention is a method for diagnosing and treating schizophrenia in a subject. Obtaining a sample from the subject, b. Measuring the expression levels of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or phosphorylated CRMP2 in a sample derived from the subject. c. Determining the likelihood or risk of developing schizophrenia in a subject based on the expression level of non-phosphorylated CRMP2 and / or the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio, and d. Provided above are methods that include initiating treatment for schizophrenia for a subject if the likelihood or risk of developing schizophrenia is determined to be high.
  • CRMP2 Collapsin Response Mediator Protein 2
  • CRMP2 non-phosphorylated CRMP2 ratio
  • the expression level of non-phosphorylated CRMP2 in the sample derived from the subject is higher than the expression level of non-phosphorylated CRMP2 in the sample derived from a healthy person who has not developed schizophrenia.
  • the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio in the sample derived from the subject is lower than the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio in the sample derived from a healthy person who has not developed schizophrenia, schizophrenia. It can be determined that the possibility of developing or the risk of developing the disease is high.
  • the expression level of non-phosphorylated CRMP2 in the sample derived from the subject is higher than the set value, and / or the phosphorylated CRMP2: non-phosphorylated CRMP2 ratio in the sample derived from the subject is high.
  • it is lower than the set value it can be determined that the possibility of developing schizophrenia or the risk of developing schizophrenia is high.
  • the set values are described above.
  • the present invention includes a reagent capable of measuring the expression level of non-phosphorylated Collapsin Response Mediator Protein 2 (CRMP2) and / or a reagent capable of measuring the expression level of phosphorylated CRMP2 in a sample derived from a subject. Kits for testing for schizophrenia are also provided.
  • CRMP2 Collapsin Response Mediator Protein 2
  • the kit of the present invention contains as a reagent an antibody capable of specifically recognizing non-phosphorylated CRMP2 and / or phosphorylated CRPM2.
  • the antibody should be capable of recognizing CRMP2 phosphorylated by Ser522.
  • the antibody may be either a monoclonal antibody or a polyclonal antibody.
  • the antibody may be immobilized on a microtiter plate, magnetic beads, a cellulose membrane or a substrate.
  • the kit may further include an instrument for collecting a sample derived from a subject, an anticoagulant, a set of reagents for detecting the protein, an instruction manual, and the like.
  • the instruction manual should also describe the evaluation and / or discrimination criteria for schizophrenia.
  • the kit of the present invention contains a nucleic acid probe that can specifically hybridize with CRMP2 mRNA as a reagent.
  • the nucleic acid probe may be immobilized on the substrate.
  • the kit also includes equipment for collecting biological samples, anticoagulants, reagents for extracting RNA from subject-derived samples, reagents for detecting RNA, instruction manuals, etc. May be good.
  • the instruction manual should also describe the evaluation and / or discrimination criteria for schizophrenia.
  • the kit of the present invention contains at least one pair of nucleic acid primers as a reagent capable of specifically amplifying cDNA synthesized using mRNA of CRMP2 as a template.
  • the kit also includes equipment for collecting subject-derived samples, anticoagulants, reagents for extracting RNA from subject-derived samples, reagents for detecting RNA, instruction manuals, etc. It is good to be.
  • the instruction manual should also describe the evaluation and / or discrimination criteria for schizophrenia.
  • the kit of the present invention may include a standard protein, a buffer, a substrate (when the antibody is enzyme-labeled), a reaction stop solution, a washing solution, a reaction vessel, and the like.
  • the kit of the present invention can be used as a pharmaceutical product for diagnosing a disease.
  • SCZ Schizophrenia
  • PBMC peripheral blood mononuclear cells
  • CRMP2 and pCRMP2 in peripheral blood are the basis for relatively rapid, minimally invasive, sensitive and specific auxiliary diagnostic tests for early SCZ (together with testing and imaging). It suggests that it may form.
  • Increased CRMP2 and / or decreased pCRMP2: CRMP2 ratio is useful for diagnosis in newly developed young patients with suspected SCZ, especially when trying to rule out other mimicry disorders (eg, manic episodes in BPD). there is a possibility.
  • SCZ Treatment Introduction Schizophrenia
  • SCZ is an idiopathic psychiatric disorder with substantial morbidity and mortality, and enormous personal and social costs. It is characterized by impaired cognition, perception, emotion and behavior. The pathogenic mechanism by the underlying pathogenic molecule that causes and / or promotes the disease is unknown. However, it is known that there are important genetic components (1). Nonetheless, the concordance rate for disease between identical twins remains at 50%, and SCZ is likely to be multigenic, epigenetic, and multifactorial in its pathogenicity and expression. It suggests that.
  • SCZ The underlying pathophysiology of SCZ is not understood, but it is believed to be related to changes in the structure and function of the neural network of the brain (2-5).
  • neural circuits are formed through neurogenesis, differentiation, axonal guidance, dendrite development, synaptogenesis, and activity-dependent refinement of immature synapses (6).
  • synapses are sites of neurotransmitter signaling, dendritic spine dysfunction may play an important pathogenic role in SCZ.
  • numerous genetic linkages and related studies suggest that abnormalities in genes involved in synaptogenesis and / or maturation may be risk factors for SCZ (4, 6-8).
  • CRMP2 Cold Response Mediator Protein 2
  • DPYSL2 Dihydropyrimidinase-like 2
  • CRMP2 Collapsin Response Mediator Protein 2
  • DPYSL2 Dihydropyrimidinase-like 2
  • the CRMP family of proteins is currently known to be composed of five homologous cytoplasmic proteins from CRMP1 to CRMP5 (10, 11).
  • CRMP2 actively binds to non-phosphorylated active cytoskeletal elements. Phosphorylation of CRMP2, a two-step process, inactivates CRMP2 and induces the release of cytoskeletal elements. Cdk5 first phosphorylates CRMP2 with Ser522 and primes glycogen synthase kinase 3 ⁇ (GSK3 ⁇ ) to phosphorylate CRMP2 with Thr514 and Ser518 (12). We now know that "toggling" between inactive (phosphorylated) and active (non-phosphorylated) CRMP2 is a continuous physiological adaptive mechanism to prevent the germination of abnormal neurons. ing.
  • Crmp2- / -Mice show hyperactivity, emotional behavioral disorders, decreased social interaction, mild contextual learning disabilities, and high susceptibility to methamphetamine (16, 17).
  • Bipolar disorder BPD
  • the "set value" of the ratio of phosphorylated (inactive) CRMP2 to non-phosphorylated (active) is abnormally high in LiR human brains and patient-specific human-induced stem cell (hiPSC) -derived neurons.
  • CRMP2 is also expressed in lymphocytes, can these brain abnormalities be reflected in readily accessible peripheral blood mononuclear cells (PBMCs) and serve as the basis for practical clinical diagnostic markers for SCZ? I decided. The difference in blood was found to be remarkable. CRMP2 levels were elevated compared to a healthy age-matched control group. The ratio of pCRMP2: CRMP2 is abnormally low (based on an increase in the denominator).
  • CRMP2 findings are in stark contrast to the LiRBPD situation, where the pCRMP2: CRMP2 ratio was always abnormally high and CRMP2 was always normal, distinguishing between the two states of undiagnosed young patients. May provide molecular and biochemical methods for, and may not be clear in the first presentation (manic episodes and schizophrenia "breaks" may resemble each other). These changes in the production of CRMP2 in SCZ not only provide rapid, sensitive, specific non-invasively accessible biomarkers and diagnostic support, but also underlie the neuropathological changes seen in SCZ. Begins to provide insights into certain molecular mechanisms.
  • CRMP2 was not significantly different from age-appropriate non-affected patients, but the pCRMP2: CRMP2 ratio was significantly higher in LiR BPD patients compared to non-affected controls (similarly, other psychology, including LiNR BPD). Compared to patients with neurological disorders).
  • the denominator (CRMP2) remained unchanged, with pCRMP2 (numerator) being abnormally high in LiR BPD patients. Differences in CRMP2 between SCZ patients and unaffected controls were significant, as detected through this more accessible peripheral blood-derived cell type assay (pCRMP2: CRMP2 ratio, we In addition to making it a suspicious and informative metric).
  • CRMP2 is a master cytoskeletal regulator and, therefore, a neural network modulator. Phosphorylation of CRMP2 alters the binding affinity for specific subsets of cytoskeletons and related proteins such as tubulin, RhoA, and filamine-A (11, 20, 21). These changes in phosphorylation state affect the regulation of cytoskeletal dynamics.
  • the “togling" between the inactive (phosphorylated) CRMP2 and the active (non-phosphorylated) CRMP2 is physiological (14, 18). However, imbalances between active and inactive CRMP2 (one too much) can cause abnormalities in synaptogenesis, synaptic maturation, and synaptic transmission (11, 22).
  • CRMP2 is also present in lymphocytes.
  • studies have shown similarities between receptor expression and transduction processes in cells and lymphocytes from the nervous system (23).
  • PBMC assay we found that blood levels of CRMP2 were higher compared to age-matched non-affected controls.
  • the net effect of increasing the denominator while maintaining a constant numerator was that the ratio of pCRMP2: CRMP2 in the SCZ group was lower than that in the control group. ..
  • These differences were most pronounced in the youngest group of SCZ patients. In SCZ patients younger than 30 years, the pCRMP2: CRMP2 ratio was lower than in control patients, up to the p ⁇ 0.01 significance level.
  • CRMP2 levels and ratios are SCZ-related behaviors because pCRMP2 begins to increase with age, especially with the development of Alzheimer's disease, when followed over time and over time in the same patient. May help distinguish between aging and age-related behaviors. In fact, in one patient, if the patient is already elderly and has many other comorbidities (24, 25), we nail the use of this biomarker for the first time. The specificity of the pCRMP2: CRMP2 ratio is likely to be worse in the older group.
  • CRMP2 the levels of master cytoskeletal modulator and neural network determinant, CRMP2 are higher than in non-affected age-matched controls.
  • CRMP2 eg, LiRBPD
  • higher active CRMP2 levels with constant inactive pCRMP2 levels are non-affected controls with a pCRMP2: CRMP2 ratio. It results in a unique reduction that is lower than that of the subject. This disruption in the normal equilibrium between the active and inactive CRMP2 can lead to an imbalance in the neural network as well.
  • CRMP2 appears to be involved in the pathology of SCZ in some way (in fact, some have been considered risk genes (26))
  • the dynamic regulation of the cytoskeleton and neural network Further research may lead to greater insight into the molecular mechanisms underlying the neuropathological changes seen in SCZ, as well as new therapeutic interventions.
  • the table summarizes the demographics of SCZ patients and healthy control patients (range 18-40 years) who collected PBMCs. Such data include age and gender, family history, smoking history, drinking history, education, background and medication history. The subjects were inpatients or outpatients at Yokohama City University Hospital, Yokohama Medical Center, and Yokohama Maioka Hospital (belonging to Yokohama City University). This study was approved by the ethics review board of each hospital. Each SCZ patient was diagnosed by at least two psychiatrists based on the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Illness (DSM-IV). The severity of symptoms was assessed using PANSS (Positive and Negative Symptom Scale). Volunteers were enrolled by general open call for conforming non-affected controls. Psychiatrists interviewed each volunteer to rule out mental illness. Informed consent was obtained from all participants.
  • PBMCs were isolated from all blood samples taken using mononuclear cell preparation tubes (BD Vacutainer CPT: Becton-Dickinson, NJ, USA). Whole blood specimens were centrifuged for 30 minutes (1500 g, 25 ° C) and the PBMC layer was collected in a 15 ml tube. These samples were then washed with phosphate buffered saline minus (PBS) and centrifuged at 427 g for 10 minutes at 4 ° C. three times. After discarding the supernatant, the pellet was resuspended in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and ampicillin.
  • FBS fetal bovine serum
  • the resuspended sample was sown on an uncoated Petri dish and cultured at 37 ° C for 2 days in a CO2 incubator. A supernatant fraction containing non-adhesive lymphocytes was collected and transferred to a 15 ml test tube. The sample was centrifuged at 200 g for 5 minutes at 4 ° C. After discarding the supernatant, the pellet was resuspended in 1 ml PBS and transferred to a 1.5 ml tube. After centrifugation at 200 g for 5 minutes at 4 ° C, the supernatant was discarded, the pellet was frozen in liquid nitrogen and stored at -80 ° C until use. CRMP2 and pCRMP2 levels were then evaluated in these samples as described below.
  • Anti-human CRMP2 monoclonal antibody (9F) was generated by injecting Balb / c mice into the C-terminal region of human CRMP2 (amino acids 486-528) (27).
  • An anti-phosphorylated CRMP1 / 2 (S522) (p-S522-CRMP1 / 2) polyclonal antibody that recognizes phosphorylated CRMP2 with Ser522 was produced in rabbits as previously reported (12).
  • the specificity of the two antibodies was verified by peptide blocking experiments in immunoblot analysis of human PMBC samples as previously reported (12, 27, 28). (See Figures 2 and 3)
  • CRMPs critical molecules for neurite morphogenesis and neuropsychiatric diseases. Mol Psychiatry. 2015; 20 (9): 1037-45. 11. Yamashita N, and Goshima Y. Collapsin response mediator proteins regulate neuronal development and plasticity by switching their phosphorylation status. Molecular neurobiology. 2012; 45 (2): 234-46. 12. Uchida Y, Ohshima T, Sasaki Y, Suzuki H, Yanai S, Yamashita N, et al. Semaphorin3A signalling is mediated via sequential Cdk5 and GSK3beta phosphorylation of CRMP2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer's disease.
  • the present invention can assist in the diagnosis of schizophrenia.

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Abstract

L'invention concerne un biomarqueur de la schizophrénie. L'invention concerne une méthode d'examen de la schizophrénie consistant à mesurer le niveau d'expression de la protéine 2 médiatrice de la réponse à la collapsine (CRMP2) non phosphorylée et/ou de la CRMP2 phosphorylée dans un échantillon issu d'un sujet.
PCT/JP2021/036560 2020-10-06 2021-10-04 Biomarqueur de la schizophrénie WO2022075243A1 (fr)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2012108548A1 (fr) * 2011-02-10 2012-08-16 住友化学株式会社 TRANSFORMÉ DANS LEQUEL ONT ÉTÉ INTRODUITS LE GÈNE Gm1, LE GÈNE GPCR, ET LE GÈNE CRMP2
WO2021100685A1 (fr) * 2019-11-19 2021-05-27 国立大学法人東京大学 Prévention ou traitement d'une maladie psychiatrique, reposant sur le moteur kif3, et criblage de médicament

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WO2012108548A1 (fr) * 2011-02-10 2012-08-16 住友化学株式会社 TRANSFORMÉ DANS LEQUEL ONT ÉTÉ INTRODUITS LE GÈNE Gm1, LE GÈNE GPCR, ET LE GÈNE CRMP2
WO2021100685A1 (fr) * 2019-11-19 2021-05-27 国立大学法人東京大学 Prévention ou traitement d'une maladie psychiatrique, reposant sur le moteur kif3, et criblage de médicament

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