Application of Dcdc2b in preparation of medicine for preventing or treating amyotrophic lateral sclerosis

Abstract

The invention belongs to the technical field of medicines, and discloses application of Dcdc2b in preparation of a medicine for preventing or treating amyotrophic lateral sclerosis. Compared with a control cell pCI, the transcriptional level and the protein level of the Dcdc2b are remarkably reduced in a TDP43M337V cell. The Dcdc2b with low expression in the TDP43M337V cells can reduce the survival of the cells, so that the number of synapses of the cells is reduced, and the Dcdc2b is prompted to reduce the survival of the TDP43M337V cells by reducing the number of synapses.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the use of Doublecortin domain-containing protein 2b (Dcdc2b) in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis (ALS). Background Technology

[0002] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown etiology that primarily affects motor neurons in the cerebral cortex, brainstem, and spinal cord. Clinical manifestations are mainly progressive skeletal muscle atrophy, weakness, fasciculations, bulbar palsy, and pyramidal tract signs. The disease has an insidious onset, and patients often experience respiratory distress in the later stages. The average survival time is 3-5 years. Therefore, ALS is a disease with a short survival time and poor prognosis. ALS is divided into two types: familial ALS (fALS) and sporadic ALS (sALS). fALS accounts for 5-10% of all ALS cases, while the remaining cases are sporadic. The 43kDa Tar DNA-binding protein (TDP43) mutation accounts for approximately 4% of fALS cases, with M337V being a common TDP43 mutation site. It is noteworthy that TDP43 is a major protein component detected in pathological inclusion bodies in the brain and spinal cord of almost all ALS patients.

[0003] Microtubules are key structures in neurons regulating cell morphology and axonal transport, and motor neurons are particularly sensitive to microtubule damage. Double cortical protein domain protein 2b (Dcdc2b), a member of the double corticin (DCX) gene family, has been shown to bind to microtubules and enhance microtubule polymerization, essential for neuronal migration and differentiation. Furthermore, research has found that impaired axonal transport is an early symptom of amyotrophic lateral sclerosis (ALS) and may be involved in motor neuron degeneration and death. The DCX protein structure can interact with nerve tissue-specific actin-binding protein II (Neurabin II), restoring axonal transport and exerting a neuroprotective effect by increasing synaptic expression. However, whether Dcdc2b can exert a protective effect in ALS by increasing synaptic expression remains unreported.

[0004] The main purpose of this invention is to use TDP43 M337V stably transfected cells as a research model to study whether Dcdc2b can improve related pathological damage in the cell model and the specific mechanism, so as to provide effective drug targets and treatment strategies for the treatment intervention of amyotrophic lateral sclerosis. Summary of the Invention

[0005] The technical problem solved by this invention is the application of the double cortical protein domain protein 2b (Dcdc2b) in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis (ALS), and the application of expression vectors and host cells containing Dcdc2b in the preparation of drugs for the prevention or treatment of ALS.

[0006] To solve the technical problem of this invention, the present invention provides the following technical solution:

[0007] The first aspect of the present invention is to provide the application of the bicortical protein domain protein 2b in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis.

[0008] The amino acid sequence of the double cortical protein domain protein 2b is as described in SEQ ID NO.1 and SEQ ID NO.2 in the sequence listing.

[0009] The second aspect of the present invention is to provide the application of a nucleic acid molecule encoding a double cortical protein domain protein 2b in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis (ALS).

[0010] The sequence of the nucleic acid molecule is the nucleotide sequence described in SEQ ID NO.3-SEQ ID NO.4 of the sequence listing or a complementary nucleotide sequence to the nucleotide sequence described in SEQ ID NO.3-SEQ ID NO.4.

[0011] The third aspect of the present invention is to provide an expression vector containing the nucleic acid molecule described in the second aspect for use in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis (ALS).

[0012] The expression vectors include adeno-associated virus vectors, adenovirus vectors, retrovirus vectors, exosomes, liposome complexes, cationic polymers, chitosan polymers, and inorganic nanoparticles.

[0013] The fourth aspect of the present invention is to provide the use of a host cell containing the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect in the preparation of a drug for the prevention or treatment of amyotrophic lateral sclerosis.

[0014] The host cell is selected from bacteria, yeast, Aspergillus, plant cells, insect cells, or mammalian cells.

[0015] The fifth aspect of the present invention is to provide the use of a double cortical protein domain protein 2b activator and / or enhancer in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis (ALS).

[0016] In the first to fifth aspects of the above technical solutions, the amyotrophic lateral sclerosis (ALS) includes axonal loss in ALS, as well as central nervous system diseases, peripheral nerve diseases, Parkinson's disease, and glaucoma related to axonal loss.

[0017] Beneficial technical effects:

[0018] This invention provides the application of Dcdc2b as a novel target and therapeutic strategy for amyotrophic lateral sclerosis (ALS). Dcdc2b exerts a protective effect in ALS cell models, specifically by reducing the survival rate of TDP43 M337V cells, decreasing the expression level of the synapse-associated protein synaptophysin, and increasing the expression level of the synapse-associated protein Neurabin II, thereby increasing intracellular synapse content. Dcdc2b protein shows promise as a potential therapeutic target for ALS. Attached Figure Description

[0019] Figure 1 Dcdc2b was significantly downregulated in TDP43 M337V cells. A: Dcdc2b transcriptional level in TDP43 M337V cells, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. B and C: Dcdc2b protein level in TDP43 M337V cells, with GAPDH as an internal control.

[0020] Figure 2 Low expression of Dcdc2b in TDP43 M337V cells resulted in decreased cell survival. The effect of 72 hours of low expression of Dcdc2b protein on cell survival in TDP43 M337V cells was investigated.

[0021] Figure 3 Low expression of Dcdc2b can reduce synaptic content in TDP43 M337V cells. A and B: Effect of 72 hours of low expression of Dcdc2b protein in TDP43 M337V cells on Neurabin II protein levels, with GAPDH as an internal control. B and C: Effect of 72 hours of low expression of Dcdc2b protein in TDP43M337V cells on Synaptophysin protein levels, with GAPDH as an internal control. Detailed Implementation

[0022] Example 1. Dcdc2b was significantly downregulated in TDP43 M337V cells.

[0023] This embodiment uses qRCR and Western Blot to detect the transcription and protein levels of Dcdc2b in TDP43 M337V cells.

[0024] 1. Cells

[0025] The NSC-34 cell line was purchased from Shanghai Hongshun Biotechnology Co., Ltd.

[0026] The TDP43 M337V stably transfected cell line and the pCI empty vector control cell line were constructed in our laboratory.

[0027] 2. Methods

[0028] 2.1 qPCR

[0029] 2.1.1 RNA Extraction

[0030] (1) Taking the cell sample in the 6-well plate as an example, discard the culture medium, wash the cells once with PBS, add 1 mL of Trizol RNA extraction reagent, lyse at room temperature for 2 min, and then transfer the cell lysate to a 1.5 mL EP tube.

[0031] (2) Take the above lysis buffer and add 0.2 mL of RNA Extraction Agent to each 1 mL of Trizol RNA lysis buffer. Vortex vigorously for 30 seconds to mix the liquid thoroughly (at the beginning of this step, pay attention to using pipette tips and centrifuge tubes without DNase / RNase).

[0032] (3) Vortex for 5 min, centrifuge at 10000×g for 15 min (4℃), carefully aspirate the upper aqueous phase into a 1.5 mL EP tube, add an equal volume of isopropanol, and mix by inverting the tube.

[0033] (4) Let stand at room temperature for 10 min, then centrifuge at 10000×g for 10 min (4℃).

[0034] (5) Discard the supernatant and add 1 mL of pre-cooled 75% ethanol. Gently tap the tube wall to allow the precipitate to float. Centrifuge at 10000×g for 5 min (4℃).

[0035] (6) Remove the supernatant, leaving the white precipitate. Leave the tube open and let it stand at room temperature to dry until the white precipitate becomes colorless and transparent.

[0036] (7) Add an appropriate amount of DNase / RNase-free ddH2O to dissolve the precipitate, and detect the RNA concentration using an enzyme-linked immunosorbent assay (ELISA) reader.

[0037] 2.1.2 Quantitative Real-Time PCR

[0038] Perform qPCR reactions according to the kit instructions. Data were evaluated using relative quantification to assess changes in the transcriptional level of the target gene, calculated according to the following formula:

[0039] ΔCt = Ct(target gene) - Ct(internal reference gene)

[0040] ΔΔCt=ΔCt(test group)-ΔCt(control group) Fold change=2-ΔΔCt

[0041] 2.2 Western Blot

[0042] 2.2.1 Cell Sample Preparation

[0043] Taking a six-well plate as an example, add 200 μL of RIPA lysis buffer (containing protease inhibitor) to each well. Sonicate in an ice-water bath (6% power, sonicate for 3 seconds, pause for 3 seconds). Centrifuge the lysis buffer at 4°C and 12000 rpm for 30 minutes, collect the supernatant, repeat the centrifugation once, and collect the supernatant for protein quantification. Protein quantification is mainly based on Pierce... TM Follow the instructions in the BCA protein quantification kit. Dilute the sample to the same concentration according to the protein quantification results, add loading buffer, boil at 100°C for 5 minutes, and then collect the sample.

[0044] 2.2.2 SDS-PAGE electrophoresis

[0045] Taking a 10% separating gel as an example, add each component according to the following formula, add the separating gel to the gap between the thick and thin glass plates, and seal with anhydrous ethanol. After the separating gel solidifies at room temperature for 0.5-1 hour, discard the anhydrous ethanol and use filter paper to absorb the residual anhydrous ethanol. Add each component of the 5% stacking gel according to the formula, and add it on top of the separating gel. Immediately insert a clean comb. The stacking gel solidifies in about 15-20 minutes.

[0046]

[0047] Place the gel in a vertical electrophoresis tank and add an appropriate amount of electrophoresis buffer. After removing the comb, load the samples and protein markers in sequence and start electrophoresis at a constant voltage of 80V. When the bromophenol blue enters the separating gel, increase the voltage to 120V. Stop electrophoresis when the bromophenol blue is close to the bottom.

[0048] 2.2.3 Transfer of film

[0049] Prepare a 0.22μm PVDF membrane of the same size as the gel block, immerse it in anhydrous methanol to activate it, soak the filter paper and sponge pad in the transfer buffer, and place the gel and membrane in the following order: black transfer clip - sponge pad - 3 sheets of filter paper - gel - membrane - 3 sheets of filter paper - sponge pad - white transfer clip. Remove air bubbles, clamp tightly and place in the transfer tank, and electrotransfer at a constant current of 200mA for 1.5h.

[0050] 2.2.4 Antigen-antibody reaction

[0051] After the transfer was completed, the PVDF membrane was blocked with TBST containing 5% skim milk powder and incubated on a shaker at room temperature for 2 hours. The PVDF membrane was then cut according to the molecular weight of the target protein and placed in an incubation box. The corresponding antibody was added and the membrane was incubated overnight at 4°C on a shaker. The next day, the primary antibody was aspirated and the membrane was washed with TBST at room temperature for 5 minutes, for a total of 5 times. Horseradish peroxidase-labeled secondary antibody was added and the membrane was incubated on a shaker at room temperature for 1 hour. The secondary antibody was aspirated and the membrane was washed with TBST at room temperature for 5 minutes, for a total of 5 times.

[0052] 2.2.5 Colorimetric Reaction

[0053] Place the PVDF membrane on the development tray and add Immobilon Western HRP substrate (solution A:solution B) to the protein side of the membrane.

[0054] The protein bands were luminescent (mixed at a 1:1 ratio) and imaged using a GE ImageQuant LAS 500 biomolecular imager. The results were then analyzed using ImageJ software to determine the grayscale of the protein bands in the images.

[0055] 2.3 Data Statistical Analysis

[0056] Data are presented as mean ± standard error (mean ± SD), and statistical analysis was performed using Student's t-test. "#" indicates comparison with pCI cells, where #P < 0.05, ##P < 0.01, and ###P < 0.001.

[0057] 3. Results

[0058] Compared with control cells pCI, the transcriptional and protein levels of Dcdc2b in TDP43 M337V cells were significantly decreased.

[0059] The results of this embodiment show that, compared with pCI cells, the Dcdc2b transcription level in TDP43 M337V cells ( Figure 1 -A and B) and protein levels decreased significantly ( Figure 1 -C).

[0060] Example 2. Low expression of Dcdc2b can reduce the survival of TDP43 M337V stably transfected cells.

[0061] This embodiment found that, compared with TDP43 M337V cells without TDP43 knockdown, the survival of TDP43 M337V cells with Dcdc2b knockdown decreased.

[0062] 1. Cells and siRNA

[0063] The TDP43 M337V stably transfected cell line was constructed in our laboratory; NC and siRNA were purchased from Gemma Gene Co., Ltd.

[0064] 2. Methods

[0065] 2.1 MTT assay for cell viability

[0066] TDP43 M337V cells were seeded in six-well plates. When the cells reached 50%-60% confluence, the procedure was performed according to the jetPRIME instructions. Transfection reagent was prepared as follows: For a six-well plate, 6 μL of siRNA was added to 200 μL of buffer, vortexed for 10 seconds, centrifuged for 1 second, then 4 μL of regent was added, vortexed for 1 second, centrifuged for 1 second, and incubated at room temperature for 10 minutes to obtain the prepared transfection reagent. The prepared transfection reagent was then added dropwise to the cells and gently shaken to mix thoroughly.

[0067] Eight hours later, the cells in the six-well plate were digested, and the cells were seeded into 96-well plates at a density of 1*10^5. After incubation in an incubator for 64 hours, 10 μL of MTT was added to each well. After incubation in an incubator for another 4 hours, 100 μL of triple solution was added to each well. After dissolution at 37°C overnight, the absorbance value was measured at 570 nm.

[0068] 2.2 Data Statistical Analysis

[0069] Data are presented as mean ± standard deviation (mean ± SD). Statistical analysis was performed using Student's t-test. "#" indicates the change in survival of Dcdc2b-knockdown TDP43 M337V cells compared to unknockdown TDP43 M337V cells. # P<0.05, ## P<0.005.

[0070] 3. Results

[0071] Low expression of Dcdc2b in TDP43 M337V cells reduces cell survival.

[0072] The results of this embodiment show that, compared with TDP43 M337V cells without TDP43 knockdown, the survival of TDP43 M337V cells with Dcdc2b knockdown is reduced. Figure 2() "#" indicates the difference between cells without TDP43 M337V knockout and cells without knockout, where #P<0.05, ##P<0.01, and ###P<0.001.

[0073] Example 3. Low expression of Dcdc2b in TDP43 M337V cells can reduce the number of synapses.

[0074] 1. Cells and siRNA

[0075] The TDP43 M337V stable transfected cell line was constructed in our laboratory; the negative control NC and Dcdc2b siRNA were purchased from Gemma Gene Co., Ltd.

[0076] 2. Methods

[0077] See section 2.2 of Example 1 for details.

[0078] 3. Data Statistical Analysis

[0079] Data are presented as mean ± standard error (mean ± SD), and statistical analysis was performed using Student's t-test. "#" indicates a difference between cells without TDP43 M337V knockdown, where #P < 0.05, ##P < 0.01, and ###P < 0.001.

[0080] 4. Results

[0081] Low expression of Dcdc2b can promote a decrease in synaptic content in TDP43 M337V cells.

[0082] The results of this embodiment show that, compared with TDP43 M337V cells that normally express Dcdc2b, TDP43 M337V cells with low Dcdc2b expression have significantly increased Neurabin II protein levels and significantly decreased Synaptophysin protein levels. Figure 3 -A,B,C), reduce the intracellular synaptic content of TDP43 M337V cells.

[0083] The above embodiments are merely illustrative of the technical concept and features of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. Application of the double cortical protein domain protein 2b in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis.

2. The application according to claim 1, characterized in that, The amino acid sequence of the bidermal protein domain protein 2b is the amino acid sequence shown in SEQ ID NO.1 and SEQ ID NO.2 in the sequence listing.

3. The application of a nucleic acid molecule encoding a double cortical protein domain protein 2b in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis (ALS).

4. The application according to claim 3, characterized in that, The sequence of the nucleic acid molecule is the nucleotide sequence described in SEQ ID NO.3-SEQ ID NO.4 of the sequence listing or a complementary nucleotide sequence to the nucleotide sequence described in SEQ ID NO.3-SEQ ID NO.

4.

5. The use of an expression vector containing the nucleic acid molecule of any one of claims 3-4 in the preparation of a drug for the prevention or treatment of amyotrophic lateral sclerosis (ALS).

6. The application according to claim 5, characterized in that, The expression vectors include adeno-associated virus vectors, adenovirus vectors, retrovirus vectors, exosomes, liposome complexes, cationic polymers, chitosan polymers, and inorganic nanoparticles.

7. The use of a host cell containing the expression vector according to any one of claims 5-6 in the preparation of a drug for the prevention or treatment of amyotrophic lateral sclerosis (ALS).

8. The application according to claim 7, characterized in that, The host cell is selected from bacteria, yeast, Aspergillus, plant cells, insect cells, or mammalian cells.

9. Application of activators and / or enhancers of the double cortical protein domain protein 2b in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis.

10. The application according to any one of claims 1-9, characterized in that, The amyotrophic lateral sclerosis (ALS) mentioned includes axonal loss in ALS, as well as central nervous system diseases, peripheral nerve diseases, Parkinson's disease, and glaucoma associated with axonal loss.

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