Use of cryab in medical diagnosis and therapy

Abstract

The invention discloses application of an alpha B crystal protein (CRYAB) as a marker in diagnosis or prediction of narcolepsy encephalopathy, especially epilepsy. The invention also discloses application of the alpha B crystal protein in preparation of drugs or kits for preventing or treating narcolepsy encephalopathy, especially epilepsy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of αB-crystallin (CRYAB) as a marker in diagnosing paroxysmal encephalopathy, especially epilepsy, and also to the use of CRYAB in preparing a drug or kit for preventing or treating paroxysmal encephalopathy, especially epilepsy. Background Art

[0002] Epilepsy is a clinical syndrome characterized by recurrent epileptic seizures caused by abnormal synchronized discharges of brain neurons. There are approximately 65 million epilepsy patients worldwide, and the World Health Organization has listed it as one of the five major neuropsychiatric diseases that are the focus of global prevention and treatment (Asadi-Pooya, 2023). Most epilepsy patients can control epileptic seizures with anti-epileptic drugs, but 30% of patients still do not respond to drug treatment. There is an urgent need to have a deeper understanding of its etiology and pathogenesis in order to develop new anti-epileptic drugs and treatments. According to traditional views, epilepsy is a gray matter disease or a neuronal disease. A breakthrough in recent years is the recognition that epilepsy is also a white matter disease or a myelin disease (Hogan, 2020). Driven by neuronal activity, the myelin structure of the myelin sheath changes according to the activity of the neurons, which is called adaptive myelination. In the epileptic state, repeated abnormal activity of neurons leads to increased myelin content and myelin formation in nerve fibers, which is called maladaptive myelination. The relationship between myelin plasticity and disease (epilepsy) is considered to be the forefront of neuroscience and clinical neurology research in the next decade (Bonetto, 2021; Xin, 2020). αB-crystallin (CRYAB) is a small molecule chaperone protein of the heat shock protein family (Hayashi, 2020) and is highly expressed in oligodendrocytes (OL) that form myelin.

[0003] The prior art mainly involves the diagnosis and treatment of CRYAB in autoimmune diseases, especially multiple sclerosis, and the biggest difference from the present application is that it does not involve diseases such as paroxysmal encephalopathy, especially epilepsy. At present, there is no application of CRYAB in the diagnosis and treatment of paroxysmal encephalopathy, especially epilepsy. Summary of the invention

[0004] A first aspect of the present invention provides a use of CRYAB or a detection reagent of CRYAB.

[0005] Specifically, provided is the use of CRYAB or a detection reagent of CRYAB in preparing a reagent or a kit for diagnosing paroxysmal encephalopathy, wherein the amino acid sequence of CRYAB is shown in SEQ ID NO: 1.

[0006] Also provided is the use of CRYAB or a detection reagent of CRYAB in preparing a reagent or a kit for predicting the onset of paroxysmal encephalopathy, wherein the amino acid sequence of CRYAB is shown in SEQ ID NO: 1.

[0007] In a preferred embodiment, the above use further comprises analyzing CRYAB or the expression and / or protein level of CRYAB in a biological sample, wherein the biological sample is selected from the group consisting of blood, serum, plasma, saliva, cerebrospinal fluid, urine, feces and tissue samples.

[0008] In a preferred embodiment, the above use further comprises detecting CRYAB or the expression of CRYAB in oligodendrocytes or exosomes derived from oligodendrocytes.

[0009] A second aspect of the present invention provides a use of CRYAB, a CRYAB derivative or a CRYAB exosome in the preparation of a drug or a kit for preventing, alleviating or treating paroxysmal encephalopathy, wherein the amino acid sequence of the CRYAB is shown in SEQ ID NO: 1.

[0010] The CRYAB derivative comprises a conjugate of CRYAB and / or a pharmaceutically acceptable salt of CRYAB.

[0011] A third aspect of the invention provides a method for treating episodic encephalopathy.

[0012] In a preferred embodiment, a therapeutically effective dose of CRYAB is administered to a patient by parenteral administration, wherein the dose is effective in inhibiting the progression of an established disease.

[0013] In a preferred embodiment, the pharmaceutical composition is administered to a subject intra-arterially, intranasally, intraperitoneally, intravenously, intramuscularly, subcutaneously, transdermally, or orally.

[0014] In a preferred embodiment, the administration comprises the step of administering to the patient an effective amount of the conjugate and / or a mixture comprising a pharmaceutically acceptable carrier.

[0015] The paroxysmal encephalopathy described in the present invention refers to a brain disease with paroxysmal characteristics, which is often characterized by suddenness, recurrence, self-limitation, etc., and manifests as epileptic seizures and / or non-epileptic seizures.

[0016] The paroxysmal encephalopathy in the present invention is preferably epilepsy.

[0017] The paroxysmal encephalopathy / paroxysmal nervous system disease mentioned in the present invention refers to epilepsy, paroxysmal disorientation, lethargy and the like.

[0018] The term "episodic encephalopathy" is used interchangeably with "seizure disorder" and refers to a clinical brain disease characterized by sudden onset, recurrence, and self-limitation, including epileptic encephalopathy (DEE) and recurrent encephalopathy.

[0019] The CRYAB or CRYAB detection reagents described in the present invention include CRYAB-specific antibodies and detection methods such as high-resolution mass spectrometry.

[0020] Technical Effects

[0021] The inventors have found that CRYAB can be used as a pathological marker of epileptogenic foci, and it is mainly expressed in oligodendrocytes. In the cortical tissue of epileptic patients, the increase of CRYAB is accompanied by the increased expression of inflammatory factors. In the plasma of epileptic patients, the expression of CRYAB in oligodendrocyte-derived exosomes is increased. Therefore, CRYAB can be used as a diagnostic marker for paroxysmal diseases, especially epilepsy.

[0022] The inventors found that the administration of kainate (KA) to the corpus callosum and cortex of epileptic mice led to increased expression of CRYAB positive fibers, and CRYAB exerted anti-epileptic effects by inhibiting inflammatory factors. The inventors' research showed that CRYAB can be used to prepare therapeutic drugs for paroxysmal diseases, especially epilepsy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 a Paraffin section of human brain tissue stained with Laufer fast blue, white matter part of the temporal lobe cortex of an epileptic patient; Figure 1 b Comparison of LFB optical density values ​​between epilepsy group and control group; Figure 1 c Paraffin section of human brain tissue stained with immunohistochemistry, white matter part of temporal lobe cortex of epileptic patients; Figure 1 d Comparison of CRYAB-positive cells between epilepsy group and control group; Figure 1 e Western Blot gel image of crystallin; Figure 1 fComparison of crystallin expression levels between epilepsy group and control group;

[0024] Figure 2 To identify oligodendrocyte lineage cells expressing CRYAB using different markers. Figure 2 a: Olig 2 marker was used; Figure 2 b: SOX10 marker was used; Figure 2 c is the use of NG2 markers; Figure 2 d: GFAP marker was used; Figure 2 e: Olig 1 marker was used; Figure 2 f: Olig 3 marker was used; Figure 2 g: Olig SP marker was used; Figure 2 h used Iba1 marker; Figure 2 i means using CRYAB markers; Figure 2 j: NeuN marker was used; Figure 2 k is a common marker of CRYAB and NeuN; Figure 2 m means using CRYAB markers; Figure 2 n is the use of PV markers; Figure 2 o is a common marker of CRYAB and PV; Figure 2 l and Figure 2 p Figure 2 k and Figure 2 The enlarged image in the white box in o. Figure 2 The scale bars in a, b, c, and d are 10 μm; Figure 2 The scale bars in e, f, g, and h are 50 μm; Figure 2 The scale bars in i, j, and k are 100 μm; Figure 2 The scale bars for m, n, and o are 100 μm; Figure 2 The scale bars of l and p are 50 μm.

[0025] Figure 3 a is Western Blot detection of the expression of CRYAB, an inflammatory factor in the temporal cortex tissue; Figure 3 b is a statistical chart of CRYAB expression of inflammatory factors, * indicates P < 0.05, ** indicates p < 0.01, **** indicates p < 0.001.

[0026] Figure 4 a The expression of related proteins in human brain tissue, human plasma and plasma exosomes; Figure 4 b shows the expression of marker proteins from different cell sources, and proteins were purified using antibodies from different cell sources; Figure 4 c is the morphological and structural characteristics of purified exosomes shown by transmission electron microscopy; Figure 4 d is the exosome nanotracer technique (NTA) showing that the particle size of purified exosomes is distributed between 50-200 nm; Figure 4 e shows the expression of CRYAB and CD81 in exosomes from different sources; Figure 4 f shows the expression levels of CRYAB and CD81 in plasma exosomes of the control group and epilepsy patients; Figure 4 g Comparison of CRYAB expression levels in plasma exosomes between the control group and epilepsy patients, ** indicates p < 0.01.

[0027] Figure 5 a shows the mouse behaviors with different scoring levels recorded by the three-dimensional behavior video monitoring system; Figure 5b is the original recording of the mouse EEG at the time corresponding to figure a; Figure 5 c is a screenshot of the behavior video recording;

[0028] Figure 5 d is CRYAB immunofluorescence staining of the corpus callosum and cortex of mice after KA modeling; Figure 5 e: The number of cells co-expressing CRYAB, olig2 and Iba1 in the hippocampus.

[0029] Figure 6 a is the changes in seizures and related protein expression levels in epilepsy model mice after CRYAB administration. 6a is a statistical chart of the number of grand mal seizures in mice in different co-treatment groups; 6b is a band chart of immunoblotting of inflammatory factors in different treatment groups; 6c is a statistical chart of the relative expression of inflammatory factors in different treatment groups.

[0030] Figure 7 To demonstrate the effect of chemical genetics specifically upregulating Cryab expression, the displayed image shows the staining of Cryab-cre mouse brain sections injected with hM3Dq-mCherry virus in both hippocampi. Figure 7 a: The control group was labeled with Cryab using fluorescein 488; Figure 7 b is the spontaneous mCherry signal of the control group; Figure 7 c is Figure 7 a Partial enlarged image; Figure 7 d is Figure 7 b Partial enlarged image; Figure 7 e: CNO activated group with Cryab labeled with fluorescein 488; Figure 7 f is the spontaneous signal of mCherry in the CNO activated group; Figure 7 g is Figure 7 e Partial enlarged image; Figure 7 h is Figure 7 f Local enlarged image; Figure 7 i is the control group, in which Cryab was labeled with fluorescein 488, and c-fos and DAPI signals were labeled with fluorescein 647; Figure 7 j is Figure 7 i Partial magnified image of the hippocampal CA1 region; Figure 7 k is Figure 7 i Partial magnified image of the hippocampal CA3 region; Figure 7 l is Figure 7 i Partial magnified image of the hippocampal DG region; Figure 7 m: CNO activation group, Cryab was labeled with fluorescein 488, and c-fos and DAPI signals were labeled with fluorescein 647; Figure 7 n is Figure 7 m Partial magnification of the hippocampal CA1 region; Figure 7 o is Figure 7 m Partial magnification of the hippocampal CA3 area; Figure 7 p is Figure 7 n Partial magnification of the hippocampal DG region; Figure 7 The scale bars of a, b, e, f, i, and m are 800 μm. Figure 7 The scale bars in c, d, g, h, j, k, l, n, o, and p are 80 μm.

[0031] Figure 8 A is the ROC and DCA analysis diagram of CRYAB concentration. A is the ROC curve of CRYAB concentration in diagnosing gliomas with preoperative epilepsy and gliomas without preoperative epilepsy; Figure 8 B is the ROC curve of CRYAB concentration in diagnosing epilepsy; Figure 8 C is the ROC curve of CRYAB concentration in diagnosing refractory epilepsy and epilepsy; Figure 8 D is the decision curve of plasma CRYAB circulating levels for gliomas with preoperative epilepsy and gliomas without preoperative epilepsy; Figure 8 E is the decision curve of plasma CRYAB circulating levels in epilepsy patients and healthy controls; Figure 8 F is the decision curve of plasma CRYAB circulating levels for refractory epilepsy and epilepsy. DETAILED DESCRIPTION

[0032] The experimental methods described in the following examples, unless otherwise specified, are conventional methods and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0033] Unless otherwise specified, the materials, reagents, etc. described in the following examples can be obtained from commercial sources.

[0034] In the following examples, the protein immunoblotting detection, flow cytometry and other experimental methods are all conventional experimental methods well known to those skilled in the art. If the experimental method does not specify specific conditions, it is usually performed under conventional conditions.

[0035] The human brain tissue sections described in the following examples were obtained from volunteer donation samples of the Chinese Brain Tissue Resource Bank. Plasma from epilepsy patients was obtained from clinical research samples of Huashan Hospital Affiliated to Fudan University. Plasma from glioma patients was obtained from clinical research samples of Huashan Hospital Affiliated to Fudan University. Temporal cortex tissue was obtained from clinical research samples of Huashan Hospital Affiliated to Fudan University.

[0036] Example 1: Staining of paraffin sections of human brain tissue using the Laufer fast blue staining method

[0037] The paraffin sections of human brain tissue were stained with the Laufer fast blue staining method as follows:

[0038] 1. Dewax the sections to water: (xylene → gradient alcohol → distilled water), soak in xylene I and II for 10 min each, 100% alcohol I and II → 95% alcohol → 90% alcohol → 80% alcohol → 70% alcohol for 5 min each, and distilled water for 5 min;

[0039] 2. Place tissue sections in Luxol Fast Blue (LFB) stain at room temperature overnight;

[0040] 3. Wash with 95% ethanol until no blue particles remain on the tissue surface;

[0041] 4. Wash with deionized water for 5 minutes;

[0042] 5. Differentiate with 0.05% lithium carbonate for 10 seconds (differentiation needs to be observed under a light microscope) until the gray and white matter are clearly distinguished;

[0043] 6. Wash with deionized water for 5 minutes;

[0044] 7. Re-dye with tar violet dye and place the dye vat in a constant temperature oven at 37°C for 10 minutes;

[0045] 8. Wash with deionized water for 5 minutes;

[0046] 9. Differentiate in tar violet differentiation solution for 5 seconds (differentiation needs to be observed under a light microscope);

[0047] 10. Wash with deionized water for 5 minutes;

[0048] 11. Dehydration and transparency: (gradient alcohol → xylene) immerse in 70% alcohol → 80% alcohol → 90% alcohol for 5 seconds each, immerse in 100% alcohol I and II for 3 minutes each, and immerse in xylene I and II for 10 minutes each;

[0049] Note: After each soaking step, lift the slice rack up and down several times before transferring it to the next liquid for better results.

[0050] 12. Neutral gum sealing: wet sealing, (lower the fume hood glass to arm's height for protection) drop a small amount of gum on the tissue, cover with a coverslip, place in the fume hood and wait for the gum to dry, then observe under a light microscope and store at room temperature.

[0051] Human brain tissue paraffin sections stained with Laufer fast blue Figure 1 As shown in a, myelin staining in the temporal cortex white matter of epilepsy patients increased significantly. The LFB optical density (OD) value was significantly increased in the epilepsy group (n=9) compared with the healthy control group (n=10), ****p<0.001( Figure 1 b).

[0052] Example 2: Immunohistochemical staining of crystallins in paraffin sections of human brain tissue

[0053] The crystallin immunohistochemistry method was used to stain paraffin sections of human brain tissue. The steps are as follows:

[0054] 1. Dewax the sections to water: (xylene → gradient alcohol → distilled water), soak in xylene I and II for 10 min each, 100% alcohol I and II → 95% alcohol → 90% alcohol → 80% alcohol → 70% alcohol for 5 min each, and distilled water for 5 min;

[0055] Note: At the end of each soaking step, lift the slice rack up and down several times before transferring it to the next liquid for better results.

[0056] 2. Transfer the slices to a glass staining jar and add PBS, place on a shaker and shake slowly, wash with PBS buffer 3 times, 10 minutes each time;

[0057] 3. Antigen repair: Use TrisHCl pH 9.0 buffer to repair the slices in an antigen repair pressure cooker. After completion, cool naturally to room temperature before taking out the slices;

[0058] 4. Place the removed slices in a glass staining jar and add PBS, place on a shaker and shake slowly, and wash with PBS buffer 3 times, 10 minutes each time;

[0059] 5. Permeabilization: (This solution should be prepared and used immediately, and should be protected from light during preparation) First, prepare 0.3% Triton with PBS, put it in a 37°C water bath and stir it thoroughly to dissolve it. Add 0.3% hydrogen peroxide and mix it thoroughly before use. Pour the permeabilization solution into the staining tank with the glass slide, and treat it at 37°C in the dark for 30 minutes to increase the permeability of the slices;

[0060] 6. Wash with PBS buffer 3 times, 10 minutes each time (shake slowly on a shaker);

[0061] 7. Blocking: Prepare reagents: 5% donkey serum in PBST;

[0062] Operation method: Take out the slices, absorb the moisture around the tissue with filter paper, draw a circle around the tissue with an immunohistochemical pen, put it in a wet box, and drop about 150ul of donkey serum in the circle (to completely cover the tissue), then put the wet box in a constant temperature water bath (37℃) for 30 minutes;

[0063] 8. Incubate with antibody I: Prepare reagents: dilute the antibody (Anti-Alpha B Crystallin antibody [1B6.1-3G4], Mouse, ab13496, Abcam) with PBST at a dilution of 1:500 to prepare the antibody I working solution;

[0064] Operation method: Take out the wet box, gently remove the donkey serum on the slice with a pipette, add about 150ul of Ⅰ antibody working solution (to completely cover the tissue), put the wet box into a constant temperature water bath (37℃) for 60 minutes.

[0065] 9. Remove the wet box from the water bath and place it in a 4°C refrigerator overnight (for more than 16 hours);

[0066] 10. Remove the wet box from the refrigerator and leave it at room temperature for 60 minutes to return to room temperature;

[0067] 11. Pour away the antibody, put the slide into the staining tank and wash it three times with PBS, 10 minutes each time;

[0068] 12. Incubate with antibody II: Prepare reagents: Prepare antibody II (Horse Anti-Mouse IgG Antibody (H+L), Biotinylated, BA-2000, VECTOR) at 1:200 PBST to prepare antibody II working solution; Operation method: Use filter paper to absorb the water around the circle of the washed slide, add about 150ul of antibody II working solution (to completely cover the tissue) and put it into a wet box in a constant temperature water bath (37°C) for 120 minutes;

[0069] 13. Take out the wet box, pour out the II-antibody working solution, put the slide into the staining tank and wash it three times with PBS, 10 minutes each time;

[0070] 14. Incubate with ABC solution: Use filter paper to absorb the water around the glass circle, add about 150ul ABC solution (ABC-HRP Kit, PK-4000, VECTOR) (1:200, prepared 30 minutes in advance) with a pipette to completely cover the tissue, and place the wet box in a constant temperature water bath (37°C) for 60 minutes;

[0071] 15. Remove the wet box, place the slide in the staining tank and wash it three times with PBS, 10 minutes each time;

[0072] 16. Color development: Quickly drop DAB working solution (1% DAB 50ul; 30% hydrogen peroxide 1ul plus PBST to 1ml, DAB (D5637, SIGMA)), observe the staining, and quickly pour off the staining solution;

[0073] Notes: ①DAB is contact toxic, so be sure to wear latex gloves and operate carefully in this step. ②Observe each slice with the naked eye first. If there is yellowing, quickly put it under a microscope to check for positivity (the background just turns yellow, and the positive result appears sesame-like brown). Use PBS to terminate the reaction. The longest color development time is 15 minutes.

[0074] 17. Place the slide in a staining jar and wash with PBS for 10 minutes, then wash with double distilled water for 10 minutes;

[0075] 18. Dehydration and transparency: (gradient alcohol → xylene) 70% alcohol → 80% alcohol → 90% alcohol → 100% alcohol. Soak in alcohol Ⅰ and Ⅱ for 5 minutes respectively, and soak in xylene Ⅰ and Ⅱ for 10 minutes respectively.

[0076] Note: After each soaking step, lift the slice rack up and down several times before transferring it to the next liquid for better results.

[0077] 19. Neutral gum sealing: For wet sealing, (lower the fume hood glass to arm’s height for protection) drop a small amount of gum on the tissue, cover with a coverslip, place in the fume hood and wait for the gum to dry. Then observe under a light microscope and store at room temperature.

[0078] Immunohistochemical staining of paraffin sections of human brain tissue Figure 1 As shown in c, CRYAB is selectively expressed in the white matter. Myelin staining in the temporal cortex white matter of epileptic patients is significantly increased. The number of CRYAB positive cells in the epilepsy group (n=18) is significantly increased compared with the healthy control group (n=27), *p<0.01( Figure 1 d).

[0079] Example 3: Human brain tissue crystallin-Western Blot experiment

[0080] The SDS-PAGE gel was prepared as shown in Table 1:

[0081] Table 1

[0082]

[0083]

[0084] Sample loading: Add 5ul prestained protein marker (WJ101, Yazyme) to the second well of each plate, and add 10ul tissue protein to the remaining wells in sequence. (Note: It is best not to use the first and last wells of each plate to obtain better protein bands)

[0085] Perform SDS-PAGE electrophoresis: 120v constant voltage electrophoresis for 70 minutes. (Note: The electrophoresis time depends on the position of the pre-stained marker band and the size of the target protein. The molecular weight of the crystal protein in this experiment is about 25KD.)

[0086] Cut a piece of PVDF membrane (0.45um, IPVH00010, Millipore) that is the same size as the gel, mark one end with a pen, soak it in methanol for about 5 minutes, and then soak it in transfer buffer. At the same time, the cut gel, filter paper and sponge used in the transfer are also soaked in transfer buffer. Then, stack the membrane and gel in the order of black porous plate - sponge - filter paper - gel - membrane - sponge - white porous plate, close the clamp, and put it into the transfer tank in the direction of "black to black, white to red". Add an ice box, pour in the transfer buffer, and place the transfer tank in ice to keep it low.

[0087] Transfer: 280mA constant current transfer for 120 minutes.

[0088] After the transfer is completed, take out the membrane, stain it with 1x Ponceau Red for about 1 minute, then rinse it with ddH2O until the bands are visible to observe the transfer status.

[0089] The membrane with the target protein was blocked in blocking solution and shaken at room temperature for 60 minutes.

[0090] Incubation with antibody I: Prepare the antibody (Anti-Alpha B Crystallin antibody [1B6.1-3G4], Mouse, ab13496, Abcam) at a dilution of 1:1000 in 0.5% TBST buffer, and prepare the internal reference (GAPDH) at a dilution of 1:2000 in 0.5% TBST buffer to prepare the antibody I working solution. Place the membrane in a wet box and use a pipette to slowly and evenly cover the membrane with the working solution. Then place the wet box in a 4°C refrigerator overnight.

[0091] The next day, the wet box was removed and placed at room temperature for 60 minutes.

[0092] Wash the membrane with TBS-T three times for 15 minutes.

[0093] Incubate with secondary antibody: prepare the antibody (Anti-Mouse IgG (H+L), HRP Conjugate, W4021, Promega) at a dilution of 1:2000 with TBS-T at room temperature, place the membrane in a wet box, use a pipette to slowly and evenly cover the membrane with the working solution, and then place the wet box at room temperature for 120 minutes.

[0094] Wash the membrane with TBS-T three times for 15 minutes.

[0095] ECL development: ECL is composed of two reagents (enhanced luminescent liquid A, BL520B-1; stabilizer liquid B. BL520B-2, Biosharp) in a 1:1 ratio. Prepare them for immediate use. Note that you need to change the pipette tip when pipetting the two reagents to prevent them from mixing. Avoid direct light during incubation. Use a WB developer to develop and save the image.

[0096] Western Blot further demonstrated that compared with the healthy control group, the expression of CRYAB in the temporal cortex white matter of the epilepsy group was significantly increased. Figure 1 As shown in e and f.

[0097] Example 4: Increased expression of CRYAB is accompanied by increased expression of inflammatory factors

[0098] The Western Blot method described in Example 3 was used to detect the expression of inflammatory factor proteins and CRYAB in temporal cortical tissues, where the control group was derived from healthy subjects (n=14) and the epilepsy group (n=16) was derived from epilepsy patients. The results showed that compared with the control group, the expression of CARYAB in the temporal cortical tissues of the epilepsy group was significantly increased, accompanied by an increase in the expression of inflammatory cytokines IL-1b, TNFα, NFκB, and pNFκB, as shown in Figure 3. Figure 3 As shown in a and b.

[0099] Example 5: Markers of the Oligodendrocyte Lineage

[0100] To further identify oligodendrocytes expressing CRYAB ( Figure 2 The markers of oligodendrocyte lineage were systematically observed. Olig 1, Olig 2, Olig 3 and OligSP ( Figure 2 a, e, f, g (red) is an essential transcription factor for oligodendrocyte development; SOX10 ( Figure 2 b) is a transcription factor that guides neural stem cells to develop into glial cells; NG2 ( Figure 2 c red) is a membrane chondroitin sulfate proteoglycan expressed by oligodendrocyte precursor cells and is now used as one of the markers of oligodendrocyte precursor cells. CRYAB coexists with multiple markers derived from the oligodendrocyte lineage and can be used in combination with various markers to identify oligodendrocytes ( Figure 2 ah).

[0101] GFAP, a marker of astrocytes Figure 2 d red) and microglial marker Iba1 ( Figure 2 h red) and CRYAB cell bodies were not co-expressed. CRAYB (green) and the neuronal marker NeuN (red) ( Figure 2 i, j, k, l) and PV (red), a marker of interneurons ( Figure 2 m, n, o, p) are not co-expressed.

[0102] Example 6: Preparation of CRYAB exosomes and nano-tracing and Western Blot detection of CRYAB expression in exosomes

[0103] (1) Preparation of exosomes

[0104] 1. Take 200 μL of plasma sample from epilepsy patients and thaw it at room temperature until it is completely liquid, then place it on ice for later use.

[0105] 2. Centrifuge the plasma sample at 2000 x g for 20 min at 4°C to remove cells and debris, and place the clarified supernatant into a new tube.

[0106] 3. Centrifuge the sample at 10000 x g for 20 min at 4 °C and place the clear supernatant into a new tube and place it on ice for later use.

[0107] 4. Add 100 μL PBS to 200 μL clear plasma and pipette to mix. Add 60 μL coprecipitation reagent to the mixed solution. Pipet to mix and incubate at room temperature for 10 minutes. Centrifuge the sample at 10,000 x g for 5 minutes at room temperature. Discard the supernatant and precipitate the total exosomes.

[0108] 5. Add 200 μL PBS to a new tube. Add 0.4 μL CD81 antibody (Santa, sc-7637) to the test tube to prepare CD81 antibody working solution. Add 1 μL biotin antibody (VECTOR, BA-2000) to the CD81 antibody working solution and incubate at room temperature for 2 hours to prepare biotinylated antibody working solution. Add 10 μL resin to the incubated biotinylated antibody working solution and incubate on a rotating mixer at room temperature for 1 hour to prepare resin antibody working solution.

[0109] 6. To enrich neuron-derived exosomes (NDE), astrocyte-derived exosomes (ADE), microglia-derived exosomes (MDE) and oligodendrocyte-derived exosomes (OED), different antibodies and methods were used to prepare biotinylated antibody working solutions. To enrich ADE, EAAT2 antibody (Abcam, ab41621) was used for binding. To enrich MDE, TMEM119 antibody (Biolegend, 853302) was used for binding. To enrich OED, MOG antibody (Abcam, ab243034) was used for binding. To enrich NDE, 0.4 μL CD171 antibody (Invitrogen, 13-1719-82) (a commercial antibody labeled with biotin) was used. The dosage was based on 200 μL of antibody working solution, and the antibody culture conditions were as described in Example (1) 5 above.

[0110] 7. Mix the resin antibody working solution evenly, take 200 μL into the test tube containing the exosome precipitate and pipette to mix. Incubate overnight at 4°C on a rotating mixer. Centrifuge the sample at 2500 x g for 3 min at 4°C, discard the supernatant, and then add 200 μL PBS to resuspend, repeat 2 times. Centrifuge the sample at 2500 x g for 3 min at 4°C, discard the supernatant.

[0111] After that, different resuspension methods were used based on subsequent experiments: ① Collect exosomes for nano-tracing and electron microscopy observation, add 45μL 1M Tris-HCl (pH=8) to resuspend, and incubate at room temperature for 5min. Add 5μL 50mM Gly-HCl (pH=3) and stop elution. Centrifuge at 2500g for 3min at 4℃, and place the supernatant into a new centrifuge tube; ② Prepare protein solution for Western Blot, add 50μL RIPA lysis buffer, lyse at 4℃ for 1h, and use for protein concentration determination.

[0112] (2) Electron microscopy observation of exosomes

[0113] 1. Place the carbon nano support membrane in a hydrophilic instrument and ionize for 10 seconds.

[0114] 2. Pick up the carbon nano support membrane with self-locking pointed tweezers, with the carbon membrane facing up, add 2.5 μL of exosome suspension, and adsorb for 90 seconds.

[0115] 3. Use filter paper to absorb excess liquid, immediately add an appropriate amount of 2% uranyl acetate, and then absorb it with filter paper. Repeat twice.

[0116] 4. Add 2% uranyl acetate onto the carbon film and adsorb for 90 seconds.

[0117] 5. Use filter paper to absorb the liquid and dry it under a heat lamp to complete the transmission electron microscope sample preparation.

[0118] 6. TECNAI T12 120KV transmission electron microscope was used to observe the exosomes, showing that the morphological structure of the purified exosomes was a 50-200nm circular single-layer membrane structure. Figure 4 As shown in c.

[0119] (3) Exosome Nanotracing

[0120] 1. Collect 1 mL of the exosome suspension obtained in (1).

[0121] 2. Nanosight 300, a nanoparticle tracking analyzer, was used to perform nanotracing detection, which showed that the particle size of the purified exosomes was distributed between 50 and 200 nm, which is consistent with the exosome particle size range, such as Figure 4 As shown in d.

[0122] (4) Western Blot detection of CRYAB expression in exosomes

[0123] 1. Add BCA reagent (Pierce TM BCA Protein Assay Kits, 23225, Thermo Scientific TM ) Prepare BCA working solution by mixing solution A, solution B and solution C in a ratio of 25:24:1.

[0124] 2. Add 20 μL protein solution or standard, 80 μL PBS, and 100 μL BCA working solution to a 96-well plate, incubate in a 37°C incubator for 30 min, and use an ELISA reader to measure the absorbance at 595 nm.

[0125] 3. Calculate the sample concentration, using the lowest concentration sample as the standard, and dilute each sample to the same concentration using RIPA lysis buffer (RIPA lysis buffer (medium), P0013C, Bio-Tech). Add an equal volume of 2X loading buffer and boil in a boiling water bath for 6 minutes.

[0126] 4. Prepare 10% separation gel + 5% concentration gel SDS-PAGE plate, load 10 μL of sample into each well, and perform electrophoresis at 120V constant voltage for 80 min.

[0127] 5. Using a 0.44 μm PVDF membrane, perform electrophoresis at a constant current of 280 mA for 2 h in a buffer solution containing 20% ​​methanol.

[0128] Block with 6.5% skim milk powder at room temperature for 1 h.

[0129] 7. Incubate different antibodies in different strips at 4°C overnight. Wash the strips three times with TBST, 10 min each time.

[0130] 8. Incubate with HRP-labeled secondary antibody (Anti-Mouse IgG (H+L), HRP Conjugate, W402B, PROMEGA) / (Anti-Rabbit IgG (H+L), HRP Conjugate, W401B, PROMEGA), 1:2000, at room temperature for 2 hours. Wash the strips 3 times with TBST, 10 minutes each time.

[0131] 9. Expose and color, and calculate the gray value of each band and the relative expression of protein.

[0132] 10. The band exposure results showed that the concentrations of other neural cell markers in single-source exosomes were low. This scheme can separate single-source exosomes.

[0133] By comparing the grayscale values ​​and calculating the relative protein expression, the results showed that sufficient amount and purity of exosomes were obtained in this experiment ( Figure 4 a); Exosomes derived from oligodendrocytes in plasma of epilepsy patients have higher CRYAB expression levels ( Figure 4 b); CRYAB is most abundantly expressed in exosomes derived from oligodendrocytes and microglia ( Figure 4 e); Compared with the control group (n=6), the CRYAB level in plasma exosomes of epilepsy patients (n=6) was significantly increased (Figure f, g).

[0134] Example 7: Preparation of CRYAB mouse model for epilepsy treatment

[0135] It is known that kainate (KA) is widely used to induce acute brain epileptic seizures and is often used to construct mouse epilepsy models.

[0136] 1. Grouping of CRYAB treatment of epilepsy experiment. The experiment was divided into three groups: CRYAB treatment group, KA treatment group and control group. In the experiment, epilepsy model was established by intraperitoneal injection of KA in both CRYAB treatment group and KA treatment group. CRYAB was pre-injected before KA injection in CRYAB treatment group, and saline was pre-injected before KA injection in KA treatment group. The control group received saline twice.

[0137] 2. The experiment used 8-week-old male C57BL / 6 mice (Spefox Biotechnology Co., Ltd.). One week before the modeling, the mice were installed with electrode mother sockets, 4 screws were installed to weld the electrode mother sockets, and dental cement was used to fix them.

[0138] 3. Place the mice in an EEG observation cage or a 3D behavior recorder box, connect a camera to ensure clear and complete recording of the mouse behavior, and use a three-dimensional behavior video monitoring system (Animal Behavior Acquisition and Analysis System (3D)), BA-DC01, Shenzhen Yiwan Life Science Technology Co., Ltd.) to record the mouse seizures and score them according to the Racine grading system.

[0139] 4. The mice in the CRYAB treatment group were intraperitoneally injected with 1 mg / kg CRYAB, and the mice in the KA treatment group and the control group were injected with an equal volume of normal saline. The behavior of the mice was observed and recorded for 30 minutes.

[0140] 5. The mice in the CRYAB treatment group and the KA treatment group were intraperitoneally injected with 25 mg / kg KA, and the mice in the control group were injected with an equal volume of saline. The epileptic behavior of the mice was observed and recorded for 120 minutes.

[0141] 6. After recording, the mice were housed individually. After 72 hours, 80 mg / kg sodium pentobarbital was injected to anesthetize the mice. PBS and PFA were perfused and the whole brain was removed, or fresh brain tissue was obtained after anesthesia.

[0142] 7. Record the epileptic status of each mouse, which is divided into 1 to 5 levels: 1. Rigid behavior; 2. Continuous nodding; 3. Convulsion of the upper body and forelimbs; 4. Whole body convulsion, standing; 5. Whole body convulsion, standing imbalance, jumping up, lying on the back. Epilepsy behaviors of level 4 and above are recorded as grand mal seizures. Record the number and duration of grand mal seizures of each mouse, record the duration and number of epileptic waves on the computer, and calculate the average value of each group as the criterion for judging the degree of epileptic seizures.

[0143] 8. Compared with the KA-treated group, the number of grand mal seizures above grade 4 in the CRYAB-treated group was significantly reduced, and pre-injection of CRYAB had an anti-epileptic effect in this acute epilepsy model.

[0144] The video monitoring system records the behavior of mice, such as Figure 5 The original EEG recordings of mice with epileptic status scores of 2, 3, 4, and 5 are shown in a and c. Figure 5 As shown in b; Behavioral video recording shows that the mouse has facial clonus, nodding, and unilateral limb clonus, which are grade 2 / 3 epileptic seizures. Note that the head is implanted with EEG recording electrodes, as shown in Figure 5 As shown in c.

[0145] 9. After KA administration to establish epilepsy mouse model, immunofluorescence staining was performed and image analysis was performed. CRYAB immunofluorescence staining (green) of the corpus callosum and cortex of mice after KA modeling showed that the number of CRYAB immune-positive nerve fiber markers increased compared with the control group, such as Figure 5 d; A1-A3, B1-B3, C1-C3 are high-power micrographs of the boxed area in the left figure; Statistical analysis showed that the number of cells co-expressing CRYAB, olig2, and Iba1 in the hippocampus of epileptic mice increased, as shown in Figure 5 As shown in e.

[0146] Example 8: Western Blot Detection of Inflammatory Factor Levels in Mouse Brain Tissue Samples

[0147] 1. Take -80℃ frozen mouse hippocampal tissue, add RIPA lysis buffer at a ratio of 250μL per 10mg tissue, grind and crush with a tissue grinder at 65Hz, 30s, 2 times, and let stand at 4℃ for 1h.

[0148] 2. Centrifuge the sample at 10,000 x g for 5 min at 4°C and place the clarified supernatant into a new tube for protein concentration determination.

[0149] 3. Prepare BCA working solution by mixing BCA reagent A solution, B solution and C solution in a ratio of 25:24:1.

[0150] 4. Add 20 μL protein solution or standard, 80 μL PBS, and 100 μL BCA working solution to a 96-well plate, incubate in a 37°C incubator for 30 min, and use an ELISA reader to measure the absorbance at 595 nm.

[0151] 5. Calculate the sample concentration, using the lowest concentration sample as the standard, and dilute each sample to the same concentration using RIPA lysis buffer. Add an equal volume of 2X loading buffer and boil in a water bath for 6 minutes.

[0152] 6. Prepare 10% separation gel + 5% concentration gel SDS-PAGE plate, load 10 μL of sample into each well, and perform electrophoresis at 120V constant voltage for 80 min.

[0153] 7. Using a 0.44 μm PVDF membrane, perform constant current electrophoresis at 280 mA for 2 h in a buffer solution containing 20% ​​methanol.

[0154] Block with 8.5% skim milk powder at room temperature for 1 h.

[0155] 9. Incubate different inflammatory factor antibodies in different strips at 4°C overnight. Wash the strips three times with TBST, 10 min each time.

[0156] 10. Incubate with HRP-labeled secondary antibody at 1:2000 for 2 h at room temperature. Wash the strips 3 times with TBST for 10 min each time.

[0157] 11. Expose and color development, calculate the gray value of each band and the relative expression of protein.

[0158] 12. The strip exposure results showed that the levels of some inflammatory factors in the mouse tissue samples treated with CRYAB were reduced. Pre-injection of CRYAB may play a role in reducing the level of inflammation during KA-induced epilepsy.

[0159] The results showed that CRYAB significantly inhibited KA-induced epileptic seizure behavior in epileptic mice after administration of CRYAB. Figure 6 a); Immunoblotting shows the expression of representative inflammatory factor proteins ( Figure 6 b); Statistical analysis showed that CRYAB administration significantly reduced the expression of inflammatory factors (NFkB, TNF-α, TLR4) induced by KA ( Figure 6 c). Therefore, it can be proved that CRAYB exerts anti-epileptic effect by intervening in the inflammatory factor signaling pathway.

[0160] Example 9: Chemical genetic stimulation increases cellular expression of CRYAB

[0161] hM3Dq, modified from the human muscarinic acetylcholine receptor M3 (hM3D), is an artificial receptor (DREADD) that only responds to clozapine-N-oxide (CNO) and is no longer activated by acetylcholine. CNO acts on cells expressing hM3Dq, causing them to depolarize and enhance cell excitability. In some neurons, after CNO binds to hM3D, it activates the Gq protein-coupled phosphatase PLCβ, causing phosphatidylinositol 4,5-bisphosphate (PIP2) to be degraded, thereby opening the KCNQ outward potassium ion channel closed by PIP2, so that the cell membrane is depolarized and an action potential is formed. Cryab-cre mice were constructed, and the constructed pAAV-hSyn-DIO-hM3Dq-mCherry virus was injected bilaterally into the hippocampus of the mice, where hSyn is a neuron-specific promoter; the DIO sequence allows cre-positive cells to express the target gene, and mCherry is a commonly used red fluorescent protein. In this system, CRYAB cells specifically express hM3Dq, which can be specifically activated after injection of CNO. Chemical genetic excitation increases CRYAB cell expression. Figure 7 ah showed that after injection of CNO to activate hM3Dq, the expression of CRYAB cells in the entire hippocampus was significantly increased. Figure 7 After ip injection of CNO to activate hM3Dq, 40μm frozen sections were made on the brain tissue of the experimental individual mice, and fluorescent staining was used to mark CRYAB and c-fos (a protein expressed by an early gene, which characterizes changes in neuronal activity). Saline was used as a control. The results showed that the expression of CRYAB and c-fos in the CA1, CA3 and DG regions of the hippocampus increased significantly ( Figure 7 ).

[0162] Example 10: ROC and DCA analysis of CRYAB as a diagnostic marker

[0163] 1. CRYAB testing in patients with glioma and epilepsy

[0164] The plasma was collected from patients with glioma (including those with and without preoperative epilepsy), epilepsy patients, healthy controls, and patients with refractory epilepsy, and the circulating levels of CRYAB in the plasma were determined using the method described in Example 6.

[0165] 2. ROC curve analysis and decision curve analysis

[0166] Figure 8 The receiver operating characteristic (ROC) curve analysis of A showed that the AUC value was 0.9135>0.9, indicating that the diagnostic accuracy was high, so the CRYAB concentration had a strong diagnostic ability in distinguishing gliomas with preoperative epilepsy from gliomas without preoperative epilepsy. Figure 8The ROC curve analysis of B showed that the AUC value was 0.9474>0.9, indicating that the diagnostic accuracy was high. Therefore, CRYAB concentration has a strong diagnostic ability in distinguishing epilepsy from healthy controls. Figure 8 The ROC curve analysis of C showed that the AUC value was 0.5, indicating that this diagnosis had no diagnostic value, and therefore CRYAB concentration had no diagnostic ability in distinguishing refractory epilepsy from epilepsy controls.

[0167] Figure 8 D's decision curve analysis (DCA) showed the clinical benefit of predicting glioma patients with preoperative epilepsy or glioma patients without preoperative epilepsy by reducing the circulating plasma CRYAB levels, so reducing the circulating plasma CRYAB levels provides a greater net benefit in clinical decision-making in predicting glioma patients with preoperative epilepsy. Figure 8 The decision curve analysis of E showed the clinical benefit of reducing the circulating levels of plasma CRYAB in predicting epilepsy, so the reduction of circulating CRYAB levels in the blood provides a large net benefit in clinical decision-making for predicting epilepsy. Figure 8 The decision curve for F showed that circulating levels of plasma CRYAB had no added net benefit in predicting refractory epilepsy.

[0168] References

[0169] 1. Asadi-Pooya AA, Brigo F, Lattanzi S, Blumcke I (2023) Adultepilepsy. Lancet 402:412–424.

[0171] 2. Hogan RE(2020) Epilepsy as a Disease of White Matter. Epilepsy Curr21:27–29.

[0172] 3. Bonetto G, Belin D, Káradóttir RT (2021) Myelin: A gatekeeper of activity-dependent circuit plasticity? Science 374:eaba6905.

[0174] 4. Xin W, Chan JR (2020) Myelin plasticity: sculpting circuits in learning and memory. NatRev Neurosci 21:682–694.

[0175] 5.Hayashi J,Carver JA(2020)The multifaceted nature of αB-crystallin.Cell Stress

[0176] Chaperones 25:639–654.

[0177] Entertainment

[0178] SEQ ID NO: 1CRYAB promoter

[0179] RRPFFPFHSP SRLFDQFFGE HLLEDLFPT STSLSPFYLR

[0180] PPSFLRAPSW FDTGLSEMRL EKDRFSVNLD VKHFSPEELK VKVLGDVIEV

[0181] HGKHEERQDE HGFISREFHR KYRIPADVDP LTITSSLSSD GVLTVNGPRK

[0182] QVSGPERTIP ITREEKPAVT APPLICATION ID NO:2CRYAB CLASSIFICATION

[0183] aggaggagga ggcaccaaac ggcctgggtg gatagaaggg ggacaaggag gcacacccag

[0184] gccggcaaag agcagctcag tgagtactgg gtatgtgtca cattgccaaa tcccggatca

[0185] caagtctcca tgaactgctg gtgagctagg fatheraaac ccctgacatc accattccag

[0186] aagcttcaca agactgcata fatherggggc tggctgtagc tgcagctgaa ggagctgacc

[0187] agccagctga cccctcacac tcacctagcc acctggaca tcgccatcca ccacccctgg

[0188] atccgccgcc ccttctttcc tttccactcc cccagccgcc tctttgacca gttcttcgga

[0189] gagcacctgt tggagtctga tcttttcccg acgtctactt ccctgagtcc cttctacctt

[0190] cggccaccct ccttcctgcg ggcacccagc tggtttgaca ctggactctc agagatgcgc

[0191] ctggagaagg acaggttctc tgtcaacctg gatgtgaagc acttctcccc agaggaactc

[0192] aaagttaagg tgttgggaga tgtgattgag gtgcatggaa aacatgaaga gcgccaggat

[0193] gaacatggtt tcatctccag ggagttccac aggaaatacc ggatcccagc tgatgtagac

[0194] cctctcacca ttacttcatc cctgtcatct gatggggtcc tcactgtgaa tggaccaagg

[0195] aaacaggtct ctggccctga gcgcaccatt cccatcaccc gtgaagagaa gcctgctgtc

[0196] accgcagccc ccaagaaata gatgcccttt cttgaattgc attttttaaa acaagaaagt

[0197] ttccccacca gtgaatgaaa gtcttgtgac tagtgctgaa gcttattaat gctaagggca

[0198] ggcccaaatt atcaagctaa taaaatatca ttcagcaaca gataaaaaaa aaaaaaaaaa

[0199] aaaa

[0200] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Use of CRYAB or a detection reagent of CRYAB in preparing a reagent or a kit for diagnosing paroxysmal encephalopathy, wherein the amino acid sequence of CRYAB is shown in SEQ ID NO: 1, and the nucleotide sequence of CRYAB is shown in SEQ ID NO:

2.

2. Use of CRYAB or a detection reagent of CRYAB in the preparation of a reagent or a kit for predicting the onset of paroxysmal encephalopathy, wherein the amino acid sequence of CRYAB is shown in SEQ ID NO: 1, and the nucleotide sequence of CRYAB is shown in SEQ ID NO:

2.

3. The use according to claim 1 or 2, further comprising detecting CRYAB or the expression of CRYAB in a biological sample, wherein the biological sample is selected from blood, serum, plasma, saliva, cerebrospinal fluid, urine, feces or a tissue sample. 4 . The use according to claim 1 or 2 , further comprising detecting CRYAB or the expression of CRYAB in oligodendrocytes or exosomes derived from oligodendrocytes.

5. Use of CRYAB, CRYAB derivatives or CRYAB exosomes in the preparation of a drug or kit for preventing, alleviating or treating paroxysmal encephalopathy, wherein the amino acid sequence of CRYAB is shown in SEQ ID NO: 1, and the nucleotide sequence of CRYAB is shown in SEQ ID NO:

2. The use according to claim 5, wherein the CRYAB derivative comprises a conjugate of CRYAB and / or a pharmaceutically acceptable salt of CRYAB.

7. The use according to any one of claims 1, 2 or 5, wherein the paroxysmal encephalopathy refers to a brain disease with paroxysmal characteristics, which manifests as epileptic seizures and / or non-epileptic seizures.

8. The use according to any one of claims 1, 2 or 5, wherein the paroxysmal encephalopathy is epilepsy.

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