Use of cryab in medical diagnosis and treatment

Abstract

The use of αB crystallin (CRYAB) as a marker in the diagnosis or prediction of episodic encephalopathy, especially epilepsy, and the use of αB crystallin in the preparation of a drug or kit for preventing or treating episodic encephalopathy, especially epilepsy.

Description

Applications of CRYAB in Medical Diagnosis and Treatment Technical Field

[0001] This invention relates to the biomedical field, specifically to the use of αB-crystallin (CRYAB) as a biomarker in the diagnosis of paroxysmal encephalopathy, especially epilepsy, and also to the use of CRYAB in the preparation of drugs or kits for the prevention or treatment of paroxysmal encephalopathy, especially epilepsy. Background Technology

[0002] Epilepsy is a clinical syndrome characterized by recurrent epileptic seizures caused by abnormal, synchronized discharges of neurons in the brain. Globally, approximately 65 million people suffer from epilepsy, and it has been listed by the World Health Organization as one of the five most important neuropsychiatric disorders requiring global prevention and control (Asadi-Pooya, 2023). Most epilepsy patients can control their seizures with antiepileptic drugs, but 30% of patients do not respond to medication. A deeper understanding of its etiology and pathogenesis is urgently needed to develop new antiepileptic drugs and treatments. Traditionally, epilepsy was considered a gray matter disorder or neuronal disorder. A recent breakthrough is the recognition that epilepsy is also a white matter disorder or myelin disorder (Hogan, 2020). Driven by neuronal activity, the myelin structure of the myelin sheath changes according to neuronal activity, a process called adaptive myelination. In epileptic states, repeated abnormal neuronal activity leads to increased myelin content and myelin formation in nerve fibers, a phenomenon known as maladaptive myelination. The relationship between myelin plasticity and disease (epilepsy) is considered a frontier in neuroscience and clinical neurology research for the next decade (Bonetto, 2021; Xin, 2020). αB-crystallin (CRYAB) is a small chaperone protein of the heat shock protein family (Hayashi, 2020) and is highly expressed in oligodendrocytes (OLs) that form myelin sheaths.

[0003] Existing technologies mainly involve the diagnostic and therapeutic effects of CRYAB in autoimmune diseases, especially multiple sclerosis. The biggest difference between these technologies and this application is that they do not involve paroxysmal encephalopathy, especially epilepsy. Currently, there is no application of CRYAB in the diagnosis and treatment of paroxysmal encephalopathy, especially epilepsy. Summary of the Invention

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

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

[0006] The use of CRYAB or a CRYAB detection reagent in the preparation of reagents or kits for predicting the onset of paroxysmal encephalopathy is also provided, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] In a preferred embodiment, the above-described uses further include analyzing the expression and / or protein levels of CRYAB or CRYAB in biological samples selected from blood, serum, plasma, saliva, cerebrospinal fluid, urine, feces, and tissue samples.

[0008] In a preferred embodiment, the above-described uses further include detecting the expression of CRYAB or CRYAB in oligodendrocytes or exosomes derived from oligodendrocytes.

[0009] A second aspect of the present invention provides the use of CRYAB, CRYAB derivatives or CRYAB exosomes in the preparation of medicaments or kits for the prevention, relief or treatment of paroxysmal encephalopathy, wherein the amino acid sequence of CRYAB is shown in SEQ ID NO: 1.

[0010] The CRYAB derivatives therein comprise conjugates of CRYAB and / or pharmaceutically acceptable salts of CRYAB.

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

[0012] In a preferred embodiment, a therapeutically effective dose of CRYAB is administered to a patient via 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 an individual via intra-arterial, intranasal, intraperitoneal, intravenous, intramuscular, subcutaneous, transdermal, or oral administration.

[0014] In a preferred embodiment, the administration includes the following steps: administering to the patient an effective amount of the conjugate and / or a mixture containing a pharmaceutically acceptable carrier.

[0015] The paroxysmal encephalopathy described in this invention refers to a brain disease characterized by paroxysmal episodes, often exhibiting features such as sudden onset, recurrence, and self-limitation, manifesting as epileptic seizures and / or non-epileptic seizures.

[0016] The paroxysmal encephalopathy described in this invention is preferably epilepsy.

[0017] In this invention, paroxysmal encephalopathy / paroxysmal neurological disorders refer to epilepsy, paroxysmal disorientation, stupor, etc.

[0018] The terms "episodic encephalopathy" and "episodic brain disease" are used interchangeably. They refer to clinical brain diseases characterized by sudden onset, recurrence, and self-limitation, including epileptic encephalopathy (DEE) and recurrent encephalopathy.

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

[0020] Technical effect

[0021] The inventors discovered that CRYAB can serve as a pathological marker for epileptogenic foci, primarily expressed in oligodendrocytes. In the cortical tissue of epilepsy patients, increased CRYAB levels are accompanied by elevated expression of inflammatory factors. In the plasma of epilepsy patients, oligodendrocyte-derived exosomal CRYAB expression is elevated. Therefore, CRYAB can serve as a diagnostic marker for paroxysmal disorders, especially epilepsy.

[0022] The inventors' research found that kaempferol (KA) administration to the corpus callosum and cortex of epileptic mice led to increased expression of CRYAB-positive fibers, and that CRYAB exerts its anti-epileptic effect by inhibiting inflammatory factors. The inventors' research suggests that CRYAB can be used to prepare therapeutic drugs for paroxysmal disorders, especially epilepsy. Attached Figure Description

[0023] Figure 1a: Paraffin section of human brain tissue stained with Laufer Quick Blue, temporal lobe white matter of an epileptic patient; Figure 1b: Comparison of LFB optical density values ​​between the epilepsy group and the control group; Figure 1c: Paraffin section of human brain tissue stained with immunohistochemistry, temporal lobe white matter of an epileptic patient; Figure 1d: Comparison of CRYAB positive cells between the epilepsy group and the control group; Figure 1e: Western Blot gel image of crystal proteins; Figure 1f: Comparison of crystal protein expression levels between the epilepsy group and the control group.

[0024] Figure 2 shows the oligodendrocyte lineages expressing CRYAB identified using different markers. Figure 2a shows the lineage using Olig 2 marker; Figure 2b shows the lineage using SOX10 marker; Figure 2c shows the lineage using NG2 marker; Figure 2d shows the lineage using GFAP marker; Figure 2e shows the lineage using Olig 1 marker; Figure 2f shows the lineage using Olig 3 marker; Figure 2g shows the lineage using Olig SP marker; Figure 2h shows the lineage using Iba1 marker; Figure 2i shows the lineage using CRYAB marker; Figure 2j shows the lineage using NeuN marker; Figure 2k shows the lineage using a combination of CRYAB and NeuN markers; Figure 2m shows the lineage using CRYAB marker; Figure 2n shows the lineage using PV marker; Figure 2o shows the lineage using a combination of CRYAB and PV markers; Figures 2l and 2p are magnified views of the white boxes in Figures 2k and 2o, respectively. In Figure 2a, b, c, and d, the scale bar is 10 μm; in Figure 2e, f, g, and h, the scale bar is 50 μm; in Figure 2i, j, and k, the scale bar is 100 μm; in Figure 2m, n, and o, the scale bar is 100 μm; and in Figure 2l and p, the scale bar is 50 μm.

[0025] Figure 3a shows the expression of CRYAB, an inflammatory factor, in the temporal lobe cortex tissue detected by Western blotting; Figure 3b is a statistical graph of CRYAB expression of inflammatory factors. * indicates P<0.05, ** indicates p<0.01, and **** indicates p<0.001.

[0026] Figure 4a shows the expression of relevant proteins in human brain tissue, human plasma, and plasma exosomes; Figure 4b shows the expression of marker proteins from different cell sources, purified using antibodies from different cell sources; Figure 4c shows the morphological and structural characteristics of purified exosomes as displayed by transmission electron microscopy; Figure 4d shows the particle size distribution of purified exosomes between 50-200 nm as displayed by exosome nanotracing (NTA); Figure 4e shows the expression of CRYAB and CD81 in exosomes from different sources; Figure 4f shows the expression levels of CRYAB and CD81 in plasma exosomes from the control group and epilepsy patients; Figure 4g compares the CRYAB expression levels in plasma exosomes from the control group and epilepsy patients, ** indicates p<0.01.

[0027] Figure 5a shows the mouse behavior at different rating levels recorded by the three-dimensional behavioral video monitoring system; Figure 5b shows the original EEG recording of the mouse at the time corresponding to Figure a; Figure 5c shows a screenshot of the behavioral video recording; Figure 5d shows the CRYAB immunofluorescence staining of the corpus callosum and cortex of mice after KA modeling; Figure 5e shows the number of cells in the hippocampus co-expressing CRYAB, olig2, and Iba1.

[0028] Figure 6a shows the changes in seizures and related protein expression levels in a mouse epilepsy model after CRYAB administration. 6a is a statistical graph of the number of grand mal seizures in mice in different co-treatment groups; 6b is an immunoblot plot of each inflammatory factor in different treatment groups; 6c is a statistical graph of the relative expression levels of each inflammatory factor in different treatment groups.

[0029] Figure 7 illustrates the effect of chemogenetic-specific upregulation of Cryab expression. The figure shows a stained brain section of a Cryab-cre mouse injected with hM3Dq-mCherry virus into the hippocampus of mice with bilateral infection. Figure 7a shows Cryab labeled with fluorescein 488 in the control group; Figure 7b shows spontaneous mCherry signals in the control group; Figure 7c is a magnified view of Figure 7a; Figure 7d is a magnified view of Figure 7b; Figure 7e shows Cryab labeled with fluorescein 488 in the CNO-activated group; Figure 7f shows spontaneous mCherry signals in the CNO-activated group; Figure 7g is a magnified view of Figure 7e; Figure 7h is a magnified view of Figure 7f; Figure 7i shows Cryab labeled with fluorescein 488, c-fos labeled with fluorescein 647, and DAPI signals in the control group; Figure 7j shows the CA1 region of the hippocampus in Figure 7i. Magnified partial views; Figure 7k is a magnified partial view of the CA3 region of the hippocampus in Figure 7i; Figure 7l is a magnified partial view of the DG region of the hippocampus in Figure 7i; Figure 7m shows the CNO-activated group using fluorescein 488-labeled Cryab, fluorescein 647-labeled c-fos, and DAPI signals; Figure 7n is a magnified partial view of the CA1 region of the hippocampus in Figure 7m; Figure 7o is a magnified partial view of the CA3 region of the hippocampus in Figure 7m; Figure 7p is a magnified partial view of the DG region of the hippocampus in Figure 7n; The scale bars for Figures 7a, b, e, f, i, and m are 800 μm, and the scale bars for Figures 7c, d, g, h, j, k, l, n, o, and p are 80 μm.

[0030] Figure 8 shows the ROC and DCA analysis of CRYAB concentration. Figure 8A shows the ROC curve of CRYAB concentration in diagnosing gliomas with and without preoperative epilepsy; Figure 8B shows the ROC curve of CRYAB concentration in diagnosing epilepsy; Figure 8C shows the ROC curve of CRYAB concentration in diagnosing refractory epilepsy and epilepsy; Figure 8D shows the decision curve of plasma circulating CRYAB level in gliomas with and without preoperative epilepsy; Figure 8E shows the decision curve of plasma circulating CRYAB level in epilepsy patients and healthy controls; Figure 8F shows the decision curve of plasma circulating CRYAB level in refractory epilepsy and epilepsy. Detailed Implementation

[0031] Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0032] Unless otherwise specified, all materials and reagents described in the following examples are commercially available.

[0033] In the following embodiments, the experimental methods such as Western blot detection and flow cytometry are conventional experimental methods well known to those skilled in the art. Where no specific conditions are specified in the experimental methods, they are usually operated according to conventional conditions.

[0034] The human brain tissue sections described in the following examples were obtained from volunteer donations from the Chinese Brain Tissue Resource Bank. Plasma from epilepsy patients was obtained from clinical research samples from Huashan Hospital affiliated with Fudan University. Plasma from glioma patients was obtained from clinical research samples from Huashan Hospital affiliated with Fudan University. Temporal lobe cortex tissue was obtained from clinical research samples from Huashan Hospital affiliated with Fudan University.

[0035] Example 1: Staining of paraffin sections of human brain tissue using the Laufer Quick Blue staining method

[0036] The paraffin sections of human brain tissue were stained using the Laufer Quick Blue staining method, and the steps are as follows:

[0037] 1. Dewaxing the sections to water: (xylene → graded alcohol → distilled water), soak in xylene I and II for 10 min each, soak in 100% alcohol I and II → 95% alcohol → 90% alcohol → 80% alcohol → 70% alcohol for 5 min each, and soak in distilled water for 5 min;

[0038] 2. Place the tissue sections in Luxol Fast Blue (LFB) staining solution and incubate overnight at room temperature;

[0039] 3. Wash with 95% ethanol until no blue particles remain on the tissue surface;

[0040] 4. Rinse with deionized water for 5 minutes;

[0041] 5. Differentiate with 0.05% lithium carbonate for 10 seconds (observation under a light microscope is required during differentiation) until the gray and white matter are clearly distinguishable;

[0042] 6. Rinse with deionized water for 5 minutes;

[0043] 7. For re-dyeing with tar purple dye, place the dye vat in a 37℃ constant temperature oven for 10 minutes;

[0044] 8. Rinse with deionized water for 5 minutes;

[0045] 9. Differentiate in tar purple solution for 5 seconds (observation under a light microscope is required during differentiation);

[0046] 10. Rinse with deionized water for 5 minutes;

[0047] 11. Dehydration and transparency: (gradient alcohol → xylene) Soak in 70% alcohol → 80% alcohol → 90% alcohol for 5 seconds each, soak in 100% alcohol I and II for 3 minutes each, and soak in xylene I and II for 10 minutes each;

[0048] Precautions: After each soaking step, lift the slicer up and down several times before transferring it to the next liquid for better results;

[0049] 12. Neutral resin mounting: wet mounting (lower the fume hood glass to arm height for protection) drop a small amount of resin onto the tissue, cover with a coverslip, place in the fume hood and wait for the resin to dry. It can then be observed under a light microscope and stored at room temperature.

[0050] Figure 1a shows paraffin sections of human brain tissue stained with Laufer Quick Blue. The myelin sheath staining in the white matter of the temporal lobe cortex of epilepsy patients was significantly increased. Compared with the healthy control group (n=10), the LFB optical density value (OD) of the epilepsy group (n=9) was significantly increased, p<0.001 (Figure 1b).

[0051] Example 2: Immunohistochemical staining of crystal proteins in paraffin sections of human brain tissue

[0052] The paraffin sections of human brain tissue were stained using crystalloid protein immunohistochemistry, and the steps are as follows:

[0053] 1. Dewaxing the sections to water: (xylene → graded alcohol → distilled water), soak in xylene I and II for 10 min each, soak in 100% alcohol I and II → 95% alcohol → 90% alcohol → 80% alcohol → 70% alcohol for 5 min each, and soak in distilled water for 5 min;

[0054] Precautions: After each soaking step, lift the slicer up and down several times before transferring it to the next liquid for better results;

[0055] 2. Transfer the slides to a glass staining jar and add PBS. Place the jar on a shaker and shake slowly. Wash the slides three times with PBS buffer, 10 minutes each time.

[0056] 3. Antigen retrieval: The sections were retrievald in an antigen retrieval pressure cooker using TrisHCl pH9.0 buffer. After retrieval, the sections were allowed to cool to room temperature before being removed.

[0057] 4. Place the extracted slides into a glass staining jar and add PBS. Place the jar on a shaker and shake slowly. Wash with PBS buffer three times, 10 minutes each time.

[0058] 5. Permeability Adjustment: (This solution should be prepared fresh and protected from light during preparation.) First, prepare 0.3% Triton solution with PBS, then dissolve it thoroughly in a 37°C water bath by stirring. Before use, add 0.3% hydrogen peroxide and mix thoroughly. Pour the permeability adjustment solution into a staining jar containing glass slides and incubate at 37°C in the dark for 30 minutes to increase the permeability of the slides.

[0059] 6. Wash three times with PBS buffer, 10 minutes each time (shake gently on a shaker);

[0060] 7. Blocking: Reagent preparation: 5% donkey serum was prepared with PBST;

[0061] Procedure: Remove the slide, blot the water around the tissue with filter paper, draw a circle around the tissue with an immunohistochemical pen, place it in a humidified chamber, and drop about 150 μL of donkey serum into the circle (to completely cover the tissue). Then place the humidified chamber in a constant temperature water bath (37°C) for 30 minutes.

[0062] 8. Incubation of Antibody I: Preparation of reagents: Prepare the antibody (Anti-Alpha B Crystallin antibody [1B6.1-3G4], Mouse, ab13496, Abcam) with PBST at a ratio of 1:500 to prepare the working solution of Antibody I;

[0063] Procedure: Remove the humidified chamber, gently remove the donkey serum from the slides with a pipette, add about 150 μL of I-antibody working solution (enough to completely cover the tissue), and place the slides back into the humidified chamber in a constant temperature water bath (37°C) for 60 min.

[0064] 9. Remove the wet box from the water bath and place it in a 4°C refrigerator overnight (more than 16 hours);

[0065] 10. Remove the humidifier box from the refrigerator and let it sit at room temperature for 60 minutes to allow it to return to room temperature;

[0066] 11. Discard the antibody, place the slide in a staining jar, and wash it three times with PBS, 10 minutes each time;

[0067] 12. Incubation of Antibody II: Preparation of reagents: Prepare Antibody II (Horse Anti-Mouse IgG Antibody (H+L), Biotinylated, BA-2000, VECTOR) at a ratio of 1:200 PBST to make Antibody II working solution; Procedure: After washing the slide, use filter paper to absorb the water around the circle, add about 150 μL of Antibody II working solution (to completely cover the tissue), place it in a humidified box and put it in a constant temperature water bath (37°C) for 120 minutes;

[0068] 13. Remove the humidified chamber, discard the working solution of antibody II, place the slide in the staining jar and wash it three times with PBS, 10 minutes each time;

[0069] 14. Incubation with ABC solution: Use filter paper to absorb the water around the circle of the slide, add about 150ul of 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 humidified box in a constant temperature water bath (37℃) for 60 minutes.

[0070] 15. Remove the humidified chamber, place the slides in the staining jar, and wash three times with PBS, 10 minutes each time;

[0071] 16. Color development: Quickly add DAB working solution (50ul of 1% DAB; 1ul of 30% hydrogen peroxide plus PBST to 1ml, DAB (D5637, SIGMA)), observe the staining, and quickly discard the staining solution;

[0072] Precautions: ① DAB is toxic by contact. Be sure to wear latex gloves and handle with care during this step. ② First, observe each slide with the naked eye. If any slides turn yellow, quickly place them under a microscope to confirm a positive result (the background just turns yellow and the positive result appears as a sesame-like brown). Terminate the reaction with PBS. The maximum color development time is 15 minutes.

[0073] 17. Place the slide in a staining jar and wash with PBS for 10 minutes, then wash with double-distilled water for 10 minutes;

[0074] 18. Dehydration and transparency: (gradient alcohol → xylene) Soak in 70% alcohol → 80% alcohol → 90% alcohol → 100% alcohol I and II for 5 minutes each, and soak in xylene I and II for 10 minutes each.

[0075] Precautions: After each soaking step, lift the slicer up and down several times before transferring it into the next liquid for better results;

[0076] 19. Neutral resin mounting: wet mounting (lower the fume hood glass to arm height for protection) drop a small amount of resin onto the tissue, cover with a coverslip, place in the fume hood and wait for the resin to dry. After drying, it can be observed under a light microscope and stored at room temperature.

[0077] The paraffin sections of human brain tissue stained by immunohistochemistry are shown in Figure 1c. CRYAB is selectively expressed in white matter. Myelin staining is significantly increased in the temporal lobe white matter of epileptic patients. Compared with the healthy control group (n=27), the epileptic group (n=18) showed a significant increase in CRYAB positive cells, *p<0.01 (Figure 1d).

[0078] Example 3: Human brain tissue crystal protein-Western Blot experiment

[0079] SDS-PAGE adhesive is prepared according to the following table 1:

[0080] Table 1

[0081] Sample loading: Add 5 µL of pre-stained protein marker (WJ101, Yaxin) to the second well of each gel, and add 10 µL of tissue protein to each of the remaining wells in sequence. (Note: It is best not to use the first and last wells of each gel to obtain better protein bands.)

[0082] SDS-PAGE electrophoresis was performed at a constant voltage of 120V for 70 minutes. (Note: The electrophoresis time depends on the position of the pre-stained marker bands and the size of the target protein. The molecular weight of the crystalline protein in this experiment was approximately 25 kDa.)

[0083] Cut a PVDF membrane (0.45µm, IPVH00010, Millipore) 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, soak the cut gel, filter paper and sponge used in the transfer in the transfer buffer. Then stack the membrane and gel in the following order: black multiwell plate - sponge - filter paper - gel - membrane - sponge - white multiwell plate, close and clamp them, and place them in 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 at a low temperature.

[0084] Transfer: 280mA constant current transfer for 120 minutes.

[0085] After the transfer is complete, remove the membrane, stain it with 1x Ponceau S for about 1 minute, then rinse it with ddH2O until bands are visible, and observe the transfer process.

[0086] The membrane containing the target protein was placed in a blocking solution and sealed, and then shaken at room temperature for 60 minutes.

[0087] Incubation of Antibody I: Antibody (Anti-Alpha B Crystallin antibody [1B6.1-3G4], Mouse, ab13496, Abcam) was prepared at a ratio of 1:1000 with 0.5% TBST buffer, and internal control (GAPDH) was prepared at a ratio of 1:2000 with 0.5% TBST buffer to prepare Antibody I working solution. The membrane was placed in a humidified chamber, and the working solution was slowly and evenly spread on the membrane with a pipette. The humidified chamber was then placed in a 4°C refrigerator overnight.

[0088] The next day, remove the humidifier and leave it at room temperature for 60 minutes.

[0089] TBS-T wash mask, 3 times for 15 minutes each time.

[0090] Secondary antibody incubation: Prepare the working solution of secondary antibody (Anti-Mouse IgG (H+L), HRP Conjugate, W4021, Promega) at a ratio of 1:2000 with TBS-T at room temperature. Place the membrane in a humidified chamber and slowly and evenly cover the membrane with the working solution using a pipette. Then place the humidified chamber at room temperature for 120 minutes.

[0091] TBS-T wash mask, 3 times for 15 minutes each time.

[0092] ECL Development: ECL is prepared by mixing two reagents (enhanced luminescence solution A, BL520B-1; stabilizer solution B, BL520B-2, Biosharp) in a 1:1 ratio. Prepare the solution immediately before use. When drawing the two reagents, use separate pipette tips to avoid mixing them. Avoid direct sunlight during incubation. Develop the solution using a Western blotting (WB) developer and save the image.

[0093] Western blotting further demonstrated that, compared with the healthy control group, CRYAB expression was significantly increased in the temporal lobe white matter of the epilepsy group, as shown in Figures 1e and 1f.

[0094] Example 4: Increased CRYAB expression accompanied by increased expression of inflammatory factors

[0095] Using the Western blotting method described in Example 3, the expression of inflammatory factor proteins and CRYAB in temporal lobe cortex tissue was detected. The control group was derived from healthy individuals (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 CRYAB in the temporal lobe cortex tissue 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 Figures 3a and 3b.

[0096] Example 5: Markers for oligodendrocyte lineages

[0097] To further identify CRYAB-expressing oligodendrocytes (green in Figure 2), markers of oligodendrocyte lineages were systematically observed. Olig 1, Olig 2, Olig 3, and OligSP (red in Figures 2a, e, f, and g) are essential transcription factors for oligodendrocyte development; SOX10 (red in Figure 2b) is a transcription factor that guides neural stem cells towards glial cell development; NG2 (red in Figure 2c) is a membrane chondroitin sulfate proteoglycan expressed by oligodendrocyte precursor cells and is now used as one of the markers for oligodendrocyte precursor cells. CRYAB coexists with multiple markers derived from oligodendrocyte lineages, and various markers can be used in combination to identify oligodendrocytes (Figures 2a-h).

[0098] The astrocyte marker GFAP (Fig. 2d, red) and the microglia marker Iba1 (Fig. 2h, red) were not co-expressed with the cell body of CRYAB. CRAYB (green) was also not co-expressed with the neuronal marker NeuN (red) (Fig. 2i, j, k, l) and the interneuron marker PV (red) (Fig. 2m, n, o, p).

[0099] Example 6: Preparation of CRYAB exosomes and their nano-tracing and Western Blot detection of CRYAB expression in exosomes

[0100] (1) Preparation of exosomes

[0101] 1. Take 200 μL of plasma sample from an epilepsy patient and thaw it at room temperature until it is completely liquid, then place it on ice for later use.

[0102] 2. Centrifuge the plasma sample at 2000x g for 20 min at 4°C to remove cells and debris, and transfer the clear supernatant into a new tube.

[0103] 3. Centrifuge the sample at 10000x g for 20 min at 4°C, and transfer the clear supernatant into a new tube and place it on ice for later use.

[0104] 4. Add 100 μL of PBS to 200 μL of clear plasma and mix by blowing and aspiration. Add 60 μL of coprecipitation reagent to the mixture. Mix by blowing and aspiration, and incubate at room temperature for 10 minutes. Centrifuge the sample at 10000 x g for 5 minutes at room temperature. Discard the supernatant; the precipitate is total exosomes.

[0105] 5. Add 200 μL of PBS to a new tube. Add 0.4 μL of CD81 antibody (Santa, sc-7637) to the tube to prepare the CD81 antibody working solution. Add 1 μL of biotinylated antibody (VECTOR, BA-2000) to the CD81 antibody working solution and incubate at room temperature for 2 hours to prepare the biotinylated antibody working solution. Add 10 μL of resin to the incubated biotinylated antibody working solution and incubate at room temperature on a rotary mixer for 1 hour to prepare the resin antibody working solution.

[0106] 6. To enrich neuronal exosomes (NDE), astrocyte-derived exosomes (ADE), microglia-derived exosomes (MDE), and oligodendrocyte-derived exosomes (OED), biotinylated antibody working solutions were prepared using different antibodies and methods. EAAT2 antibody (Abcam, ab41621) was used to enrich ADE. TMEM119 antibody (Biolegend, 853302) was used to enrich MDE. MOG antibody (Abcam, ab243034) was used to enrich OED. For NDE enrichment, 0.4 μL of CD171 antibody (Invitrogen, 13-1719-82) (a commercially available biotinylated antibody) was used. Doses were based on 200 μL of antibody working solution, and antibody culture conditions were as described in Example (1) 5 above.

[0107] 7. Mix the resin antibody working solution thoroughly, and transfer 200 μL to a test tube containing the exosome precipitate; mix well by pipetting. Incubate overnight at 4°C on a rotary mixer. Centrifuge the sample at 2500 x g for 3 min at 4°C, discard the supernatant, and then resuspend in 200 μL of PBS. Repeat twice. Centrifuge the sample at 2500 x g for 3 min at 4°C, and discard the supernatant.

[0108] Subsequently, different resuspension methods were used for subsequent experiments: ① Exosomes were collected for nano-tracing and electron microscopy observation, resuspended in 45 μL of 1M Tris-HCl (pH=8), and incubated at room temperature for 5 min. 5 μL of 50 mM Gly-HCl (pH=3) was added and elution was stopped. The mixture was centrifuged at 2500 g for 3 min at 4 °C, and the supernatant was transferred to a new centrifuge tube; ② Protein solutions were prepared for Western blotting, with 50 μL of RIPA lysis buffer added, and lysed at 4 °C for 1 h for protein concentration determination.

[0109] (2) Electron microscopic observation of exosomes

[0110] 1. Place the carbon nanotube support membrane in a hydrophilometer and ionize it for 10 seconds.

[0111] 2. Use self-locking pointed tweezers to pick up the carbon nanotube support membrane with the carbon membrane side facing up, add 2.5 μL of exosome suspension, and allow it to adsorb for 90 seconds.

[0112] 3. Use filter paper to absorb excess liquid, immediately add an appropriate amount of 2% uranium acetate, then absorb it again with filter paper. Repeat twice.

[0113] 4. Add 2% uranium acetate to the carbon film and allow it to adsorb for 90 seconds.

[0114] 5. Use filter paper to absorb the liquid, place the sample under a heat lamp to dry, and complete the preparation of the transmission electron microscope sample.

[0115] 6. Exosomes were observed using a TECNAI T12 120KV transmission electron microscope. The purified exosomes were shown to be circular monolayer membrane structures with a diameter of 50-200 nm, as shown in Figure 4c.

[0116] (3) Exosome nano-tracing

[0117] 1. Collect 1 mL of the exosome suspension obtained in (1).

[0118] 2. Nanosight 300 nanoparticle tracking analyzer was used for nanoparticle tracking detection, which showed that the purified exosomes were distributed in size between 50-200 nm, which is consistent with the exosome size range, as shown in Figure 4d.

[0119] (4) Western Blot detection of CRYAB expression in exosomes

[0120] 1. Add BCA reagent (Pierce) TM BCA Protein Assay Kits, 23225, Thermo Scientific TM Mix solutions A, B, and C in a ratio of 25:24:1 to prepare BCA working solution.

[0121] 2. Add 20 μL of protein solution or standard, 80 μL of PBS, and 100 μL of BCA working solution to a 96-well plate, incubate at 37°C for 30 min, and measure the absorption at 595 nm using a microplate reader.

[0122] 3. Convert the sample concentration. Using the lowest concentration sample as the standard, dilute each sample to the same concentration using RIPA lysis buffer (RIPA lysis buffer (medium), P0013C, Beyotime). Add an equal volume of 2X loading buffer and incubate in a boiling water bath for 6 minutes.

[0123] 4. Prepare an SDS-PAGE plate with 10% separating gel + 5% stacking gel, load 10 μL of sample into each well, and perform electrophoresis at 120V for 80 min.

[0124] 5. Using a 0.44μm PVDF membrane, perform electrophoresis at a constant current of 280mA for 2 hours in a buffer solution containing 20% ​​methanol.

[0125] 6.5% skim milk powder sealed at room temperature for 1 hour.

[0126] 7. Incubate different antibodies in different bands overnight at 4°C. Wash the bands three times with TBST for 10 minutes each time.

[0127] 8. Incubate with HRP-labeled secondary antibodies (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 bands three times with TBST for 10 minutes each time.

[0128] 9. Expose and develop colors, and calculate the gray values ​​of each band and the relative expression of the protein.

[0129] 10. The band exposure results showed that the concentrations of other neuronal markers in exosomes from a single source were low, and this method can isolate exosomes from a single source.

[0130] By comparing gray values ​​and calculating relative protein expression, the results showed that sufficient quantity and purity of exosomes were obtained in this experiment (Figure 4a); exosomes derived from oligodendrocytes in the plasma of epilepsy patients had a high level of CRYAB expression (Figure 4b); CRYAB was most abundantly expressed in exosomes derived from oligodendrocytes and microglia (Figure 4e); compared with the control group (n=6), the level of CRYAB in exosomes from the plasma of epilepsy patients (n=6) was significantly increased (Figures f and g).

[0131] Example 7: Preparation of a mouse model of epilepsy treated with CRYAB

[0132] Kamarinic acid (KA) is known to be widely used to induce acute brain epilepsy seizures and is often used to construct mouse epilepsy models.

[0133] 1. Grouping of the CRYAB treatment experiment for epilepsy. The experiment consisted of three groups: the CRYAB treatment group, the KA treatment group, and the control group. In both the CRYAB treatment group and the KA treatment group, epilepsy was induced by intraperitoneal injection of KA. The CRYAB treatment group received a pre-injection of CRYAB before the KA injection, while the KA treatment group received a pre-injection of normal saline before the KA injection. The control group received two intraperitoneal injections of normal saline.

[0134] 2. Male C57BL / 6 mice (Speford Biotechnology Co., Ltd.) aged 8 weeks were used in the experiment. One week before the modeling began, electrode sockets were installed on the mice. The electrode sockets were welded with 4 screws and fixed with dental cement.

[0135] 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 mouse behavior, and use a three-dimensional behavior video monitoring system (animal behavior acquisition and analysis system (3D) BA-DC01, Shenzhen Yiwan Life Technology Co., Ltd.) to record mouse seizures and score them according to the Racine classification.

[0136] 4. Mice in the CRYAB treatment group were injected intraperitoneally with 1 mg / kg CRYAB, while mice in the KA treatment group and the control group were injected with an equal volume of physiological saline. The behavior of the mice was observed and recorded for 30 minutes.

[0137] 5. Mice in the CRYAB treatment group and KA treatment group were injected intraperitoneally with 25 mg / kg KA, while the control group was injected with an equal volume of saline. The epileptic behavior of the mice was observed and recorded for 120 min.

[0138] 6. After recording, the mice were housed alone. 72 hours later, the mice were anesthetized by injecting 80 mg / kg sodium pentobarbital, perfused with PBS and PFA and the whole brain was removed, or fresh brain tissue was taken after anesthesia.

[0139] 7. Record the epileptic state of each mouse, categorized into grades 1 to 5: 1. Rigidity; 2. Continuous head nodding; 3. Twitching of the upper body and forelimbs; 4. Generalized convulsions, standing; 5. Generalized convulsions, loss of balance, jumping, supine. Epileptic behaviors of grade 4 or higher are classified as grand mal seizures. Record the number and duration of grand mal seizures for each mouse, and record the duration and frequency of epileptic waves observed in the computer. Calculate the average value for each group as the standard for judging the severity of the epileptic seizure.

[0140] 8. Compared with the KA treatment group, the CRYAB treatment group showed a significant reduction in the number of grade 4 or higher grand mal seizures, indicating that pre-injection of CRYAB had an anti-epileptic effect in this acute epilepsy model.

[0141] The video monitoring system recorded the behavior of the mice, as shown in Figures 5a and 5c. The original EEG recordings of mice with epileptic status scores of 2, 3, 4, and 5 are shown in Figure 5b; the behavioral video recordings show that facial clonic movements, head nodding, and monolimb clonic movements in mice are grade 2 / 3 epileptic seizures. Note that the head is where the EEG recording electrodes are implanted, as shown in Figure 5c.

[0142] 9. After KA administration to establish an epileptic mouse model, immunofluorescence staining was performed, followed by image analysis. Immunofluorescence staining (green) of CRYAB in the corpus callosum and cortex of mice after KA modeling showed an increase in CRYAB-positive nerve fiber markers compared to the control group, as shown in Figure 5d; A1-A3, B1-B3, and C1-C3 are high-power images of the areas shown in the box in the left image. Statistical analysis showed an increase in CRYAB-co-expressing cells with olig2 and Iba1 in the hippocampus of epileptic mice, as shown in Figure 5e.

[0143] Example 8: Western Blot detection of inflammatory factor levels in mouse brain tissue samples

[0144] 1. Take mouse hippocampal tissue frozen at -80℃, add RIPA lysis buffer at a ratio of 250μL per 10mg of tissue, grind the tissue twice at 65Hz for 30s, and let it stand at 4℃ for 1h.

[0145] 2. Centrifuge the sample at 10000xg for 5 min at 4℃, and take the clear supernatant into a new tube for protein concentration determination.

[0146] 3. Prepare BCA working solution by mixing BCA reagent solutions A, B, and C in a ratio of 25:24:1.

[0147] 4. Add 20 μL of protein solution or standard, 80 μL of PBS, and 100 μL of BCA working solution to a 96-well plate, incubate at 37°C for 30 min, and measure the absorption at 595 nm using a microplate reader.

[0148] 5. Convert the sample concentration. Using the lowest concentration sample as the standard, dilute each sample to the same concentration using RIPA lysis buffer. Add an equal volume of 2X loading buffer and incubate in a boiling water bath for 6 minutes.

[0149] 6. Prepare an SDS-PAGE plate with 10% separating gel + 5% stacking gel. Load 10 μL of sample into each well and perform electrophoresis at 120V for 80 min.

[0150] 7. Using a PVDF membrane with a specification of 0.44 μm, perform electrophoresis at a constant current of 280 mA for 2 h in a buffer solution containing 20% ​​methanol.

[0151] 8.5% skim milk powder sealed at room temperature for 1 hour.

[0152] 9. Incubate different inflammatory factor antibodies in different bands overnight at 4°C. Wash the bands three times with TBST for 10 minutes each time.

[0153] 10. Incubate with HRP-labeled secondary antibody, 1:2000, at room temperature for 2 hours. Wash the bands three times with TBST, 10 minutes each time.

[0154] 11. Expose and develop colors, and calculate the gray values ​​of each band and the relative expression of the protein.

[0155] 12. Band exposure results showed that the levels of some inflammatory factors were reduced in the mouse tissue samples of the CRYAB treatment group, suggesting that pre-injection of CRYAB may play a role in reducing inflammation levels during KA-induced epilepsy.

[0156] The results showed that CRYAB administration significantly inhibited KA-induced seizure behavior in epileptic mice (Figure 6a); Western blotting revealed the expression of representative inflammatory factor proteins (Figure 6b); statistical analysis showed that CRYAB administration significantly reduced the expression of KA-induced inflammatory factors (NFkB, TNF-α, TLR4) (Figure 6c). Therefore, it can be demonstrated that CRYAB exerts its anti-epileptic effect by intervening in inflammatory factor signaling pathways.

[0157] Example 9: Increased cellular expression of CRYAB induced by chemogenetic stimulation

[0158] hM3Dq, modified from the human muscarinic acetylcholine receptor M3 (hM3D), is a DREADD artificial receptor that responds only to clozapine-N-oxide (CNO) and is no longer activated by acetylcholine. CNO acts on cells expressing hM3Dq, causing depolarization and enhancing cellular excitability. In some neurons, CNO binds to hM3D, activating the Gq protein-coupled phosphatase PLCβ, leading to the degradation of phosphatidylinositol 4,5-bisphosphate (PIP2). This opens the KCNQ outward potassium ion channel, which was previously closed by PIP2, resulting in cell membrane depolarization and the formation of an action potential. Cryab-cre mice were constructed, and the constructed pAAV-hSyn-DIO-hM3Dq-mCherry virus was injected bilaterally into the hippocampus of these mice. 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 is specifically expressed as hM3Dq, which can be specifically activated after CNO injection. Chemogeneous excitation increases CRYAB cellular expression. Figures 7a-h show that after CNO injection to activate hM3Dq, CRYAB cellular expression in the entire hippocampus is significantly increased. Figures 7i-p show 40μm frozen sections of brain tissue from experimental mice after CNO injection to activate hM3Dq. CRYAB and c-fos (a protein with early gene expression, characterizing changes in neuronal activity) were fluorescently stained, with saline as a control. The results showed that the expression of CRYAB and c-fos was significantly increased in the CA1, CA3, and DG regions of the hippocampus (Figure 7).

[0159] Example 10: ROC and DCA analysis of CRYAB as a diagnostic biomarker

[0160] 1. CRYAB testing in patients with glioma and epilepsy

[0161] Plasma was collected from patients with glioma (including those with and without preoperative epilepsy), patients with epilepsy, healthy controls, and patients with refractory epilepsy. The circulating level of CRYAB in the plasma was measured using the method described in Example 6.

[0162] 2. ROC curve analysis and decision curve analysis

[0163] The receiver operating characteristic (ROC) curve analysis in Figure 8A shows an AUC value of 0.9135 > 0.9, indicating high diagnostic accuracy. Therefore, CRYAB concentration has strong diagnostic ability in differentiating gliomas with and without preoperative epilepsy. The ROC curve analysis in Figure 8B shows an AUC value of 0.9474 > 0.9, indicating high diagnostic accuracy. Therefore, CRYAB concentration has strong diagnostic ability in differentiating epilepsy from healthy controls. The ROC curve analysis in Figure 8C shows an AUC value of 0.5, indicating no diagnostic value. Therefore, CRYAB concentration has no diagnostic ability in differentiating refractory epilepsy from epileptic controls.

[0164] The decision curve analysis (DCA) in Figure 8D shows the clinical benefit of reducing plasma circulating CRYAB levels in predicting glioma patients with or without preoperative epilepsy; therefore, reducing plasma circulating CRYAB levels provides a larger net benefit in predicting glioma with preoperative epilepsy. The decision curve analysis in Figure 8E shows the clinical benefit of reducing plasma circulating CRYAB levels in predicting epilepsy; therefore, reducing circulating CRYAB levels provides a larger net benefit in predicting epilepsy. The decision curve in Figure 8F shows that plasma circulating CRYAB levels do not provide an additional net benefit in predicting refractory epilepsy.

[0165] References

[0166] 1. Asadi-Pooya AA, Brigo F, Lattanzi S, Blumcke I (2023) Adult epilepsy. Lancet 402:412–424.

[0167] 2. Hogan RE(2020) Epilepsy as a Disease of White Matter. Epilepsy Curr 21:27–29.

[0168] 3. Bonetto G, Belin D, Káradóttir RT (2021) Myelin: A gatekeeper of activity-dependent circuit plasticity? Science 374:eaba6905.

[0169] 4. Xin W, Chan JR (2020) Myelin plasticity: sculpting circuits in learning and memory. Nat Rev Neurosci 21:682–694.

[0170] 5.Hayashi J,Carver JA(2020)The multifaceted nature of αB-crystallin.Cell Stress Chaperones 25:639–654.

[0171] sequence list

[0172] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of CRYAB or a detection reagent for CRYAB in the manufacture of a reagent or a kit for diagnosing an epileptic encephalopathy, the amino acid sequence of CRYAB being represented by SEQ ID NO: 1 and the nucleotide sequence of CRYAB being represented by SEQ ID NO:

2.

2. Use of CRYAB or a detection reagent for CRYAB in the manufacture of a reagent or a kit for predicting the onset of an epileptic encephalopathy, the amino acid sequence of CRYAB being represented by SEQ ID NO: 1 and the nucleotide sequence of CRYAB being represented by SEQ ID NO:

2.

3. The use of claim 1 or 2, further comprising detecting the expression of CRYAB or CRYAB in a biological sample selected from the group consisting of blood, serum, plasma, saliva, cerebrospinal fluid, urine, feces or a tissue sample.

4. The use of claim 1 or 2, further comprising detecting the expression of CRYAB or CRYAB in oligodendrocytes or in exosomes of oligodendrocyte origin.

5. Use of CRYAB, a CRYAB derivative or CRYAB exosomes in the manufacture of a medicament or a kit for preventing, alleviating or treating an epileptic encephalopathy, the amino acid sequence of CRYAB being represented by SEQ ID NO: 1 and the nucleotide sequence of CRYAB being represented by SEQ ID NO:

2.

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

7. The use of any one of claims 1, 2 or 5, wherein the epileptic encephalopathy refers to a brain disease with an epileptic feature, which is manifested by a seizure and / or a non-seizure.

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