Small molecule combination and application thereof

Abstract

The invention provides a small molecule combination. The small molecule combination comprises SB431542, LDN193189, CHIR99021, DAPT (Dimethylparaben-Amino Protein), Repsox, SAG (Soluble Adenosine Glycol) and Purmorphamine. The invention also provides application of the small molecule combination in preparation of drugs for converting astrocytes into neurons, application of the small molecule combination in preparation of drugs for treating nervous system diseases, and a method for converting astrocytes into neurons. The micromolecule combination provided by the invention can convert astrocytes into inhibitory neurons, and realizes in-situ transdifferentiation effect in a mouse body.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a small molecule combination and application thereof. Background Art

[0002] The central nervous system is a complex neural network system. The disruption of the balance between excitatory neurons and inhibitory neurons is one of the main causes of neurological diseases. Excitatory neurons and inhibitory neurons are two basic neuronal types, which use glutamate and γ-aminobutyric acid (GABA) as the main neurotransmitters of excitatory or inhibitory signals, respectively. In the primate cortex, GABAergic neurons account for about 25-30% of the number of neurons. The GABAergic system is composed of GABA, GABA transporters, GABAergic receptors and GABAergic neurons. GABAergic interneurons regulate the balance and dynamic organization of pyramidal neuron groups, thereby mediating countless information processing flows and output channels [2,3]. The diversity of GABAergic neurons makes it possible to deploy a rich inhibitory control mechanism to form a sophisticated division of labor in the highly subtle spatiotemporal dynamics of cortical circuit calculations.

[0003] A variety of neurodevelopmental and neurodegenerative diseases are associated with abnormal function of GABAergic neurons. Neurological diseases are divided into neurodegenerative diseases, such as Alzheimer's disease (AD), neurodevelopmental diseases, such as autism spectrum disorder (ASD), and movement disorders, such as Parkinson's disease (PD). Alzheimer's disease (AD) is a common progressive neurodegenerative disease characterized by β-amyloid (Aβ) deposition, intraneuronal tauopathy, neurofibrillary tangles and neuronal transmission disorders, as well as loss of synapses and neurons. The pathological characteristics are that cognitive deficits are associated with abnormal neuronal network activity, which may be related to the dysfunction of GABAergic neurons, causing decreased levels of GABA and SST in the brain and cerebrospinal fluid. In addition, upregulation of ApoE4 in the brain produces more neurotoxic ApoE fragments and increases the expression of phosphorylated tau, and induces the loss of GABAergic interneurons. Inhibitory signals play an important role in the generation of brain rhythmic oscillations. Inhibitory defects caused by the loss or low activity of GABAergic interneurons may lead to abnormal neural networks, among which impaired inhibition is a potential mechanism for network overactivity. Studies have shown that overactivity of neural networks is an early event in the pathogenesis of AD and may cause harmful changes in cognitive function. Therefore, abnormalities in GABAergic neurons are closely related to many neurodegenerative diseases. Regeneration of GABAergic neurons can significantly improve the symptoms of related neurodegenerative diseases caused by the loss of GABA neurons. Targeting GABAergic neurons can provide direction for AD treatment.

[0004] Regenerating neurons is a means of making up for the loss of neurons caused by damage to achieve the purpose of treating related diseases. Currently, regarding neuronal regeneration technology, fully differentiated cells can be successfully converted into neurons by overexpressing certain specific transcription factors and microRNA or applying small molecules. It mainly includes two methods: transplanting exogenous embryonic stem cells or pluripotent stem cells into the body to produce neurons (i.e., exogenous transplantation of stem cells therapy) and in situ regeneration of neurons. Embryonic stem cells are subject to huge ethical challenges. Early studies have shown that most treatments using stem cells involve implanting these cells into the body, and stem cell therapy is accompanied by corresponding risks, including tumor formation or abnormal circuit formation that leads to dysfunction. iPS cell transplantation technology is still controversial, functional maturation requires a long culture cycle, and experimental design is complex. In addition, multi-stage and long-term differentiation procedures pose technical challenges in terms of reproducibility and consistency. Gene therapy for regenerating neurons is an alternative method based on pluripotent cell programming or reprogramming, using gene therapy to change cell fate. Terminally differentiated cells can be converted into induced pluripotent stem cells, and the induced pluripotent stem cells can be differentiated into neurons in vitro. Somatic cells can also be directly reprogrammed into neurons using overexpression of transcription factors, miRNA or small molecules.

[0005] iPS induction technology and somatic cell reprogramming technology are developing rapidly, and great progress has been made by reducing transcription factors, changing the way of gene introduction and expanding the type of starting cells. Studies have found that under the action of transcription factors, development-related signal pathways undergo tremendous changes. More and more research is devoted to finding more and better small molecule compounds, combining them to improve induction efficiency, shorten induction time, and increase induction success rate.

[0006] The study found that a variety of small molecules activate related transcription factors and related signaling pathways. Under in vitro culture conditions, chemical drugs can be used to convert fibroblasts into pluripotent stem cells, hepatocytes, cardiomyocytes, neural stem cells, etc. The combination of four small molecules is a small molecule mixture of SB431542, FSK, CHIR99021 and ISX9. It directly converts mouse fibroblasts into neuroblasts and neurons by activating two transcription factors NeuroG2 and NeuroD1, and the chemically induced neurons have the same electrophysiological properties and functional synapses as normal neurons. VCR (VPA, CHIR99021, Repsox) can significantly activate the expression of NeuroG2 and NeuroD1, and can directly convert astrocytes into neurons in vitro. It was found that a group of nine compounds including Puromorphin, CHIR99021, SAG, DAPT, Tzav, VPA, TTNPB, SB431542 and LDN193189 directly reprogram human embryonic astrocytes into neurons in vitro. It was found that a combination of four small molecules can efficiently transdifferentiate human astrocytes into excitatory neurons. Intraperitoneal and in situ injection of small molecule drugs into the hippocampus of mice can cause a significant increase in the amount of doublecortin (DCX) in the dentate gyrus of the hippocampus, promoting the occurrence of neurons.

[0007] Small molecule-related research can induce somatic cells to pluripotent stem cells (iPs) or other terminal differentiated cells from a large number of in vitro cell experiments, thus changing the fate of cells and completing cell reprogramming. Taking advantage of the characteristics of small molecule drugs that are non-immunogenic and rapidly degraded, small molecule drugs are used to treat diseases caused by irreversible cell damage. Under the action of chemical drug small molecules, somatic cells are reprogrammed into missing cells to achieve the effect of treating related diseases. This will be the future direction of somatic cell conversion therapy using small molecules. Chemical small molecule drugs use their own advantages such as fast metabolism and high targeting to convert glial cells into neurons to delay the onset of patients, or a protective effect on background neurons to become a new treatment method. Using small molecule combinations to convert glial cells into neurons in vivo has great potential for future treatment of neurodegenerative diseases. Unlike biological agents delivered by viruses, the stable physical and chemical properties of small molecule drugs make it difficult for them to be quickly decomposed after entering the body, and they are easy to reach the effective site, and do not require complex delivery technology for assistance. Therefore, small molecule drugs are the first choice when they can be replaced. Summary of the invention

[0008] One object of the present invention is to provide a small molecule combination capable of converting astrocytes into neurons in response to the above technical problems to be solved.

[0009] Another object of the present invention is to provide the application of the above small molecule combination.

[0010] In order to achieve the above objectives, the present invention provides a small molecule combination, which includes SB431542, LDN193189, CHIR99021, DAPT, Repsox, SAG and Purmorphamine.

[0011] On the other hand, the present invention also provides use of the small molecule combination for preparing a drug for converting astrocytes into neurons.

[0012] Preferably, the neurons are inhibitory neurons.

[0013] On the other hand, the present invention also provides the use of the small molecule combination for preparing a drug for causing astrocytes to transdifferentiate into neurons in situ.

[0014] On the other hand, the present invention also provides the use of the small molecule combination for preparing a drug for treating nervous system diseases.

[0015] Preferably, the nervous system disease includes but is not limited to neurodegenerative diseases, neural damage, neuroaging, neurodevelopmental diseases or movement disorders.

[0016] Preferably, the nervous system disease includes but is not limited to any one of Alzheimer's disease, stroke, amyotrophic lateral sclerosis, glioma, Parkinson's disease, epilepsy and Huntington's disease.

[0017] In another aspect, the present invention also provides a method for converting astrocytes into neurons, characterized by comprising administering the small molecule combination.

[0018] Preferably, in the method, the combination of SB431542, LDN193189, CHIR99021, DAPT and Repsox is administered first, and then the combination of SB431542, LDN193189, CHIR99021, DAPT, SAG and Purmorphamine is administered.

[0019] The small molecule combination of the present invention can transform astrocytes into inhibitory neurons and achieve an in situ transdifferentiation effect in mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the experimental technical route for using small molecules to transdifferentiate astrocytes into inhibitory neurons.

[0021] Figure 2The components of rat primary cells are shown. (A) Schematic diagram of obtaining primary cells. Cortical cells of rats 3-5 days after birth were taken, and primary astrocytes were isolated for cell culture research. (B) Expression and proportion of markers of astrocytes (GFAP, S100β, SOX9), neurons (DCX, Tuj), neural precursor cells (SOX2, Nestin), microglia (Iba1) and oligodendrocytes (Olig2) in untreated primary cells (scale bar: 20μm). (C) Statistical analysis of the proportion of each type of cell in primary cells. Among them, astrocytes (GFAP, S100β, SOX9) accounted for 95%, neurons (DCX, Tuj, MAP2, NeuN) accounted for 1%, microglia accounted for 1% (Iba1), and no expression of oligodendrocytes (Olig2) and stem cells (SOX2, Nestin) was found. (D) Statistical analysis: There was no significant difference in the amount of neuronal marker MAP2 between the control group and the “core drug” subgroup treatment group (nsP>0.05, n=3baches).

[0022] Figure 3 The results show the screening of small molecule combinations to convert primary astrocytes into neurons. (A) Cell culture using small molecule combinations to convert primary astrocytes into neurons at different stages. (B) The bright field and immunofluorescence staining results of the control and experimental groups were analyzed after 9 days of treatment. The cells in the control group were flattened into a glial cell state. Immunofluorescence staining analysis found that almost no immature neurons Tuj1 were found + Cells. The translucent protrusions of the experimental group cells are in the neuronal state, and the immunofluorescence staining is Tuj1 + Immature neuronal precursor cells (green). (C) Primary astrocytes treated with 1% DMSO were used as the control group. The astrocytes treated with small molecules were used as the experimental group. The neurons transformed from the 15-day differentiation treatment had axonal elongation (microtubule protein MAP2, green) under the action of neurotrophic factors and expressed the neuronal marker NeuN (red). (D) Quantitative analysis showed that the transformation efficiency (Tuj1 + / DAPI) reached 34.55±6.92%. The control group had a small number of Tuj1+ cells, about 0.37±0.65% (n=3baches, **P<0.01). (E) Quantitative analysis showed that the transformation efficiency (MAP2 + / field) was 89.86±5.41. + The number of cells in each field of view was approximately 3.34±1.72 (20× field of view, n=3baches, ****P<0.0001).

[0023] Figure 4 The figure shows retroviral verification of astrocyte conversion into neurons. (A) Schematic diagram of retroviral labeling, primary cells infected with RV-GFP and treated with small molecules. (B) Retroviral labeled cells astrocytes (S100β, red, GFAP, purple, scale bar: 20μm) (C) GFP-positive cells show Tuj1 after 9 days of small molecule treatment + (GFP + Tuj1 + ) (Scale bar: 20 μm) (D) Quantitative analysis showed that the majority of cells infected with the retrovirus were astrocytes, with 87.81 ± 6.76% expressing GFP. + GFAP + , 47.94±2.79% were GFP + S100β + There are no other types of glial cells. (E) Quantitative analysis shows that retroviral labeling shows GFP + After 9 days of cell small molecule treatment, the transformation efficiency was 51.2±9.36% (****P<0.0001, n=3baches).

[0024] Figure 5 The process of tracking cell morphological changes under bright field is shown.

[0025] Figure 6 The effect of each small molecule screened on the transdifferentiation efficiency is shown. (A) After subtracting one small molecule from the small molecule combination, the expression of tubulin MAP2 shown by immunostaining (scale bar: 20 μm). (B) Quantitative analysis found that subtracting any small molecule would cause a decrease in transformation efficiency. Compared with all small molecule groups, there was no significant difference after subtracting DAPT, and the transformation efficiency decreased significantly after subtracting any other small molecule, especially after subtracting RepSOX and CHIR193189 (n=3baches, ns, P>0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0026] Figure 7 The neuronal subtypes induced by small molecule-induced astrocyte transformation are shown. (A) The neurons induced by small molecule-induced astrocyte transformation are almost all inhibitory neurons (GABA + 、GAD67 + , red), while the excitatory neuron marker VGlut2 (red), dopamine neuron marker DAT (red), and motor neuron marker ChAT (red) were all negative (scale bar: 20 μm). (B) Quantitative analysis showed that 99% of the neurons induced by small molecules to transform into astrocytes were GABAergic neurons, and no other types of neurons were detected.

[0027] Figure 8 The subtypes of GABAergic neurons induced by small molecule-induced astrocyte transformation are shown. (A) The subtypes of GABAergic neurons induced by small molecule-induced astrocyte transformation partially showed PV and CR positive, and negative for CB, SST, NOS, and NPY (scale bar: 20 μm). (B) Quantitative analysis of the proportion of GABAergic neuron subtypes showed that 62.7% were CR positive, 23.3% were PV positive, and CB, SST, NOS, and NPY were not detected.

[0028] Fig. 9 The electrophysiological signal activities of neurons converted by patch clamp recording are shown. (A) Patch clamp recording of cells in the control group and small molecule treatment group under bright field. (B) Representative action potential pattern diagrams of neurons converted by the control group and experimental group. (C) Quantitative analysis of the frequency of action potential discharge in the control group and experimental group (n = 12 cells per group). (D) Representative sodium-potassium current pattern diagrams of neurons converted by the control group and experimental group. (E) Quantitative analysis of the changes in the amplitude of sodium-potassium current in the control group and experimental group (n = 12 cells per group). (FH) Quantitative analysis of the changes in the excitatory spontaneous postsynaptic current in the control group and experimental group (*P<0.05, ****P<0.0001; n = 12 cells per group). (IK) Quantitative analysis of the changes in the inhibitory spontaneous postsynaptic current in the control group and experimental group (***P<0.001, ****P<0.0001; n = 12 cells per group).

[0029] Fig.10 The results of in situ injection of small molecule drugs into the mouse cortex show the transformation effect. (A) Schematic diagram of in situ injection of small molecule combination into the cortex, and samples were taken and analyzed after 9 and 21 days. (B) Representative images of cortical expression in the control group and experimental group. (C) Representative high-magnification images of the control group and experimental group, with a small number of transformed neuron-like cells (GFP) in the experimental group + NeuN + , *Indicates where green and red are co-labeled, scale bar: 20 μm).

[0030] Fig.11 The results show the conversion effect of small molecule drugs injected in situ into the striatum of mice. (A) Schematic diagram of the in situ injection of small molecule combination into the striatum, and the sample was collected and analyzed 11 days later. (B) Representative image of DCX expression in the striatum of the control group, and the red dotted area is the damaged area (scale bar: 100μm). (C) High-magnification image of the damaged edge area, there is no immature neuron DCX expression around the damage (red, scale bar: 20μm). (D) Quantitative analysis found that the cells labeled with green fluorescent protein are mainly astrocytes (GFAP+). (E) Quantitative analysis of DCX in the damaged area of ​​the experimental group +NeuN + Number (ns, P = 0.609, n = 3 animals).

[0031] Fig.12 The results show the conversion effect of small molecule drugs injected in situ into the mouse striatum. (A) Representative images of DCX expression in the striatum of the experimental group (SMs), the red dotted area is the damaged area (scale bar: 100 μm). (B) High-magnification DCX expression image of the striatum of the experimental group (red, scale bar: 20 μm). (C) High-magnification GFP expression in the striatum of the experimental group + and DCX + Co-expression images (scale bar: 50 μm).

[0032] Fig.13 The transformative effect of in situ injection of small molecule drugs into the mouse hippocampus is shown. (A) Schematic diagram of in situ injection of small molecule combinations into the hippocampus, and samples were collected and analyzed 21 days later. (B) Representative images of DCX expression in the hippocampal region of the control group and the experimental group. The drug was injected on the left side, and the right side was not treated. The left hippocampal DG region of the experimental group highly expressed DCX compared with the control group and the untreated right side (scale bar: 100μm). (C) Representative images of DCX expression in the striatum of the control group (scale bar: 100μm). (D) Small molecules in the experimental group converted astrocytes into neuron-like cells (GFP+NeuN+, scale bar: 20μm). (E) Quantitative analysis showed that the number of DCX in the experimental group was significantly increased compared with the control group (*, P<0.05, n=3 animals).

[0033] Fig.14 The transformation effect of small molecule drugs injected into the retro-orbital vein of mice is shown. (A) Schematic diagram of the process of injecting small molecule combination into the retro-orbital vein, and sampling and analysis are performed 10 days later. (B) Representative images of the cortex of the experimental and control groups were selected. The virus-infected cells in the control group were glial cells, and some glial cells in the experimental group were transformed into neuron-like cells (circled parts indicate partially transformed cells, scale bar: 200μm). (C) High-magnification image shows the transformation of GFP + NeuN + The neuron part is inhibitory interneuron (scale bar: 20 μm). (D) Quantitative analysis of the conversion efficiency of the control group and the experimental group showed that the conversion efficiency of the experimental group was significantly higher than that of the control group (ns, P=0.08, n=3 animals).

[0034] Fig.15The results show the transformation effect of small molecule drugs injected into the orbit of transgenic mice. (A) Schematic diagram of gene expression in transgenic mice. (B) Quantitative analysis showed that S100β accounted for 92.96% of RFP-positive cells, GFAP-positive cells accounted for about 77.23%, and no microglia were detected. (C) Schematic diagram of the process of small molecule drugs injected into the orbit of transgenic mice. (D) Sagittal cross-section of brain tissue expressing red fluorescent protein 7 days later (scale bar: 1000μm).

[0035] Fig.16 The results of the drug administration to each brain region of transgenic mice are shown. (A) Cortical astrocytes are transformed into neurons (scale bar: 20 μm). (B) Striatal astrocytes are transformed into neurons (scale bar: 20 μm). (C) Hippocampal astrocytes are transformed into neurons (scale bar: 20 μm).

[0036] Fig.17 The results show the transformation effect after administration to the spinal cord of transgenic mice. (A) Astrocytes in the cervical, thoracic and lumbar segments of the spinal cord were transformed into neurons (left 10× field of view, scale bar: 100 μm, right 40× field of view, scale bar: 20 μm). DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. If it is not specifically pointed out, the reagents, culture media, experimental instruments, and biomaterials used in the present invention are all conventional reagents, culture media, experimental instruments, and biomaterials known to those skilled in the art.

[0038] 1. Reagents and Materials

[0039] The components of the cell culture medium are shown in Table 1.

[0040] Table 1: Cell culture medium components

[0041]

[0042]

[0043] The small molecule compounds are shown in Table 2.

[0044] Table 2: Small molecule compounds

[0045] name Use concentration company Part Number SB431542 2.5mM Selleek S1067 LDN193189 0.25mM Selleck S7507 CHIR99021 1.5mM Selleck S1263 DAPT 2.5mM Selleck S2215 Repsox 1mM Selleck S7223 SAG 1mM Selleck S7779 Purmorphamine 1mM Selleck S3042

[0046] Other common reagents are shown in Table 3.

[0047] Table 3: Common reagents

[0048]

[0049]

[0050] The antibodies used are listed in Table 4.

[0051] Table 4: Antibodies

[0052]

[0053]

[0054] The viruses used are listed in Table 5.

[0055] Table 5: Viruses

[0056] name company Working titer Retrovirus - <![CDATA[1×10 8 GC / mL]]> AAV9-GFAP-1.6-GFP Paizhen Biotechnology <![CDATA[1×10 11 GC / mL]]> AAVPHP.eB-GFAP-104-GFP Paizhen Biotechnology <![CDATA[1×10 13 GC / mL]]>

[0057] 2. Conventional reagent configuration

[0058] (1) Cell culture medium: Place the sterilized culture medium reagent in a biosafety cabinet, add the cell culture medium formula reagent in sequence and mix well, then place in a 4°C refrigerator. Take out some culture medium and heat it in a water bath before use. The cell culture medium should not be stored for more than two weeks.

[0059] (2) Phosphate buffered saline (PBS): Pour the phosphate powder into a clean beaker, add a small amount of deionized water until it is completely dissolved, add the solution volume to 2L, and continue stirring until the solution is clear. Dispense into glass bottles for storage.

[0060] (3) 4% paraformaldehyde solution (PFA): Preheat 400 ml of deionized water to 80°C, weigh 80 g of PFA powder and pour it into a beaker, pour the preheated deionized water into the beaker and stir. Add 0.1 Mol / L NaOH solution to the solution until the pH is close to neutral. Continue heating and stirring until the solution is transparent, add 500 ml of 0.2 Mol / L Pb solution, continue heating and stirring until the solution is clear. Add the solution volume to 2 L, filter with filter paper, and store in a refrigerator.

[0061] 3. Cell Experiment Methods

[0062] (1) Isolation of primary cells

[0063] Primary astrocytes were isolated from the cerebral cortex of rats that were 3-5 days old. After the young rats were anesthetized with low temperature, their heads were disinfected with iodine and 75% alcohol, and then their heads were cut off and placed in a dish. The skull and meninges were carefully peeled off to expose the brain parenchyma. The brain tissue was removed and placed in a pre-cooled HBSS solution. The cortex was carefully picked up and cut into small pieces of 1 mm3 with scissors. Then, 5 mL of 0.25% trypsin and 1% DNaseⅠ were used for digestion at 37°C for 15 minutes, shaking once in the middle. Finally, 5 mL of astrocyte culture medium was used to terminate the digestion. After standing, the supernatant was collected, and the remaining tissue blocks were taken out and placed in another cooled culture medium, blown, stood, and the supernatant was taken. Repeat the above steps and the obtained supernatant was centrifuged at 800 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended and centrifuged at 10×10 6 Culture in a 10 mm culture dish. After 30 minutes, transfer the supernatant to a 75 cm 2 The culture flasks were cultured in a 37°C, 5% CO2 + 95% air incubator. The medium was completely replaced every 3 days. When the cells completely covered the bottom of the culture flask, it was placed on a shaker at 280 rpm for 18 hours. The oligodendrocytes were suspended in the culture medium, the culture medium was discarded, and the oligodendrocytes and astrocytes were separated.

[0064] (2) Primary cell culture

[0065] The primary isolated astrocytes were used as P1. They were frozen in liquid nitrogen and thawed to yield approximately 3 × 10 cells per culture dish. 5 Astrocytes of 10 cells were used as P2. After 3-4 days in the glial cell culture medium, the cells completed division, the culture medium was discarded, and the cell debris and residual culture medium were washed with 5mL DPBS. 2ml of 0.05% trypsin was added and placed in a 37℃ incubator for 3min. After the culture dish was shaken and the cells were completely detached, 2ml of astrocyte culture medium was added to terminate the digestion. The cell suspension was collected and centrifuged at 200G for 5min, the supernatant was discarded, and 1ml of culture medium was added to resuspend the cells. The volume was subcultured at 1:3 as P3. Next, the same operation was used to subculture at a volume of 1:2 as P4. After centrifugation, the P4 cells were resuspended with cell banker2, and 500μl of each tube was placed in a cell cryopreservation tube. The P4 cells were cooled in a gradient at -80℃ refrigerator and stored in a -80℃ refrigerator for a short period of time as the same batch of processed cells. When used, the cells were thawed and subcultured to P5 and then used for this study.

[0066] (3) Retrovirus tracking

[0067] In order to achieve the purpose of fluorescently labeling astrocytes, retrovirus was used to infect dividing astrocytes. Retrovirus (RV) was prepared by transfecting the GP293 cell line with a plasmid expressing GFP through a lipofectamine 40000 (PEI40000) reagent. After 24 hours, the culture medium was collected and replaced with fresh culture medium. The culture medium was collected once after 48 hours and 72 hours respectively. After low-speed centrifugation, the supernatant was filtered with a 0.45um PES filter membrane. The filtered culture medium was centrifuged at 20000rpm for 2 hours using a vacuum ultracentrifuge. The supernatant was discarded and the virus particles were resuspended in a PBS solution containing 0.1% serum. Finally, the GP293T cell line was plated on a 96-well plate, and the collected virus stock solution was titered by gradient dilution. The final packaged retrovirus titer was 1×108GC / ml.

[0068] After 24 h of plating astrocytes in a 24-well plate, approximately 2 × 10 4 The virus infection amount per cell is 2.5×10 4 Virus particles. About 1 μL of virus stock solution was added to each well, and then the Zeiss inverted fluorescence microscope was used to continuously photograph the same position every 24 hours for 15 days during the small molecule treatment process. The morphology of RV-CAG-GFP-labeled astrocytes was monitored over time.

[0069] (4) Small molecule chemical drug induction process

[0070] The slides for culturing primary astrocytes were pre-incubated with 500ul 0.1mg / mL poly-lysine (PDL) overnight. After filtering with a 0.22 micron filter, the slides were washed three times with deionized water and dried. 20,000 cells were resuspended in 50μL of astrocyte culture medium, and 50ul of cell suspension was covered with the slides pre-treated with 0.1mg / mL PDL, and placed in a 37°C, 5% CO2 incubator for 45 minutes. When the cells adhered to the wall and grew, 500ul of astrocyte culture medium (DMEM / F12, 2% B27, 10% FBS, 1% P / S, glucose (volume ratio 1:300)) was added to complete the cell plating. When the cells reached 90% confluence, half of the N2 culture medium was replaced. The experimental group used N2 medium (DMEM / F12, 1% P / S, 1% N2) as the basis, and added the small molecule combination R-SP (SB431542, LDN193189, CHIR99021, DAPT, Repsox, SAG and Purmorphamine) as the small molecule medium. The control group used N2 medium as the basis and added 1% DMSO solvent. The small molecule medium SM1 (containing SB431542 (5μM), LDN193189 (0.25μM), CHIR99021 (1.5μM), DAPT (5μM) and Repsox (1μM)) was fully replaced every other day (D0). On the second day (D2), half of the medium was replaced with SM2 (containing SB431542 (5 μM), LDN193189 (0.25 μM), CHIR99021 (1.5 μM), DAPT (5 μM), SAG (1 μM) and Purmorphmine (1 μM)). Two days later (D5), half of the medium was replaced with SM2. After three days of continuous culture (D9), the astrocytes gradually became convex and rounded and transformed into neuron-like cells, and the neuronal differentiation medium (NDM) was replaced.

[0071] (DMEM / F12, N2 (2%), B27 (1%), FBS (0.5%), Y-27632 (1uM), vitamin C (5ug / mL), BDNF (brain-derived nerve growth factor) (10ng / mL), NT3 (neurotrophic factor 3) (10ng / mL), IGF-1 (20ng / mL), FudR (5-fluorodeoxyuridine) (0.5uM), uridine (0.5uM), GDNF (glial cell-derived neurotrophic factor) (10ng / ml)) and continue to culture. In the differentiation medium, the cell protrusions continue to elongate and transform into mature neurons. Immunofluorescence staining analysis and electrophysiological function analysis were performed on the 14th day and the 21st day, respectively. The schematic diagram of the dressing process at different stages of cell culture using a combination of small molecules to convert primary astrocytes into neurons is shown in the figure. Figure 3 As shown in A.

[0072] (5) Cell immunofluorescence staining and imaging

[0073] After the cultured cell plate is taken out, the culture medium is removed, PBS solution is added to the plate and placed on a shaker (the shaker speed is 75 rpm, the time is 5 min, and the temperature is room temperature for all incubation states). After washing, the solution is removed and 300ul PFA solution is added for incubation for 15 min. Then PBS is added for washing three times. Then PBST (0.1% Triton X-100 is added to the PBS solution) is added for incubation for 10 min. After washing three times with PBS solution, 3% bovine serum albumin (BSA) solution is added for blocking and incubation for 30 min. Primary antibody is added to the 3% BSA solution, the blocking solution is removed and the primary antibody solution is added for incubation at 4°C overnight. After the primary antibody plate is taken out at 4°C and allowed to stand and rewarm to room temperature, PBST (0.2% Tween-20 is added to the PBS solution) is added for washing three times, and then the secondary antibody and DAPI (nuclear dye) are added for incubation in the dark for 45 min. Then PBST is added for washing twice, and PBS is added for washing twice. After the slide is reversed and placed on a slide containing anti-fading mounting medium to complete cell immunofluorescence staining. The immunofluorescence staining results were imaged by Zeiss upright fluorescence microscope and Zeiss laser confocal microscope at high magnification.

[0074] (6) Whole-cell patch clamp recording

[0075] Remove the glass slide with cell adhesion and growth from the well plate and place it in a 50mm culture dish. Remove the cell culture medium, add artificial cerebrospinal fluid and perform oxygenation to ensure cell activity. First, select the neuron-like cells to be recorded under a microscope. Embed the electrode into the cell membrane with an electrode resistance of 5-12 megohms, and record the action potential induced by the cell under the stimulation of gradient current. Then perform cell membrane voltage detection. Record the spontaneous postsynaptic current of the cell at a voltage of -70mV. Record the cell sodium-potassium current under gradient voltage stimulation. All cell electrophysiological data were collected using pClamp10.4, and all data were analyzed using Clampfit.

[0076] 4. Experimental Animals Experimental Methods

[0077] (1) Experimental animals

[0078] The experimental animals C57BL / 6J mice used in the present invention were purchased from Guangzhou Yaokang Biotechnology Co., Ltd. The Aldh1l1×creERT2 transgenic mice were donated by Yao Maojin's research group at South China University of Technology. The mouse strain is from the Jackson Laboratory, the strain number is 031008, the strain name is B6N.FVB (Aldh1l1-creERT2) 1Khakh / J, and the strain background is derived from C57BL / 6N. All experimental design principles have been approved by the Experimental Animal Ethics Committee of Jinan University and are in line with animal experimental ethics. The experimental design complies with the "3R principle". The experimental animals are kept in the SPF-level barrier system of the Animal Management Center of Jinan University. The living environment temperature is 25°C, the ambient humidity is 40%-60%, and a 12h rhythmic light cycle is given. After leaving the SPF barrier system, the experimental animals are kept in the clean-level barrier system of the Animal Management Center of Jinan University. The ethical approval numbers for this study were 20211216-05, 20220506-010, and 20220719-03.

[0079] (2) Stereotaxic brain injection

[0080] Select adult male and female mice aged 6-8 weeks and anesthetize them with 0.25% tribromoethanol solution (20ml / kg). Observe the stable breathing of the mice, no reaction when touching the whiskers, and no leg extension reaction after pinching the fingers to confirm that they are in anesthesia. Place the mice prone on the operating table, and use a sterile surgical blade to cut the skin of the head to expose the skull after disinfecting the surface of the skin with iodine. Fix the mice in the mouse adapter and adjust them to the appropriate position under the microscope field of view. Use the bregma as the origin, and use an electric drill to drill a small hole in the mouse skull at the target coordinate site (AP: +1.0mm; ML: +2.0mm; DV: -3.0mm) to expose the brain substance, and inject the virus to be injected into the target brain area at a speed of 100nL / min. After the injection is completed, stop the needle for 8min until the virus is completely diffused. Complete the virus injection.

[0081] (3) Acupuncture injury model

[0082] Select adult male and female mice aged 6-8 weeks and inject 0.25% tribromoethanol solution (20mL / kg) intraperitoneally for anesthesia. Observe the stable breathing of the mice, no reaction when touching the whiskers, and no leg extension reaction after pinching the fingers to confirm that they are in anesthesia. Place the mice prone on the operating table, and use a sterile surgical blade to cut the head skin to expose the skull after disinfecting the skin surface with iodine. Fix the mice in the mouse adapter and adjust them to the appropriate position under the microscope field of view. The bregma is determined as the origin. Use an electric drill to drill a circular hole with a diameter of more than 1mm in the mouse skull at the target coordinate site (AP: +1.0mm; ML: +2.0mm; DV: -3.0mm) to expose the brain parenchyma. Replace the circular needle with a diameter of 1mm and poke it into the brain parenchyma from the hole. Stop the needle for 3min to form a physical acupuncture injury model for the brain parenchyma.

[0083] (4) Orbital injection

[0084] After the mouse is anesthetized with isoflurane, it is placed flat on the operating table. The drug solution to be injected is drawn with an insulin needle. During the anesthesia of the mouse, one eye is selected, the eye is tilted to bulge outward, the needle is tilted and inserted into the retro-orbital vein from the corner of the eye, and the solution is slowly injected into the retro-orbital vein. After the injection is completed, the mouse stops inhaling isoflurane gas. When the drug is administered continuously every day, the left and right eyes are alternately administered.

[0085] (5) Tissue sampling

[0086] After the mice were anesthetized by intraperitoneal injection of 0.25% tribromoethanol solution (20 mL / kg), the mice were observed to breathe steadily, and there was no reaction when the whiskers were touched. After pinching the fingers, there was no reaction to stretching the legs, which confirmed that they were in an anesthetized state. The mouse's limbs were fixed on the operating table, and the chest was cut open with surgical scissors to expose the heart. The left atrium was pierced with a needle and perfused with normal saline. When the mouse's limbs turned white and the liver turned from bright red to gray, PFA solution was perfused until the mouse's tail was lifted. The perfusion was completed after continuous perfusion until there was no reaction, and the mouse brain tissue was taken to complete the sampling.

[0087] (6) Tissue Immunofluorescence Staining

[0088] After obtaining the tissue, embed the tissue with OCT embedding agent and freeze it in a -80℃ refrigerator. The frozen microtome slices the tissue continuously and cut it into brain slices with a thickness of 30μm. When staining, take a group of brain slices and wash them three times with PBS. Add PBST (0.3% Triton X-100 added to PBS solution) containing 3% BSA blocking solution and incubate for 60min. Add the primary antibody to the blocking solution, absorb the blocking solution and add the primary antibody solution to incubate overnight at 4℃ (some antibodies are incubated for 2 days). After taking out the primary antibody plate at 4℃ and letting it stand to warm up to room temperature, add PBST (0.2% Tween-20 added to PBS solution) to wash three times, then add the secondary antibody and DAPI (nuclear dye) to incubate in the dark for 60min, then add PBST to wash twice, then add PBS to wash twice, then stick the brain slice to the slide, wait for the surface water to evaporate, add a small amount of anti-quenching sealing agent, and then seal it with a cover glass, and finally seal the cover glass with nail polish.

[0089] (7) Mouse genotype identification

[0090] One week after the birth of the mouse, the tail tissue of the mouse was taken and marked, DNA was first extracted from the tissue, and then the target gene was transferred by PCR. The obtained product was imaged by agarose gel electrophoresis to screen the positive target mice.

[0091] 5. Statistical analysis methods

[0092] The software used for data statistics in this article is Graphpad Prisn6. All sample data are displayed as mean or mean ± standard deviation (Mean ± SD). The t-test analysis was used for pairwise comparison of samples, and One-Way ANOVA analysis was used when the number of samples exceeded 3 groups. P < 0.05 was considered significant, and the statistically significant differences were marked in the figures.

[0093] 6. Experimental Design

[0094] Common neurodegenerative diseases of the nervous system cause neuronal loss. Therefore, restoring the number of non-renewable neurons is of great significance for the treatment of neurodegenerative diseases. Many studies have shown that neurons can be regenerated by inducing stem cell differentiation, mRNA, knocking down genes, overexpressing transcription factors, and chemical small molecules. As a key component of neurons in the brain, inhibitory neurons can be converted into inhibitory neurons using small molecules. First, the small molecule combination screened for human cells was tried on primary mouse cells, and then the Sonic Heghog signaling pathway inhibitor was added to explore whether astrocytes can be converted into inhibitory neurons. Then, the small molecules screened by in vitro cell experiments were injected into mice in various ways, and administration methods such as in situ injection and orbital injection were tried. It is expected that the small molecule combination screened at the cell level under the acupuncture injury model can achieve in situ conversion effect in animal brain tissue.

[0095] Experimental research technology route Figure 1 shown.

[0096] 7. Experimental Results

[0097] (1) Analysis of the composition of primary cells

[0098] Astrocytes were isolated and purified from the cortex of 3-5 day old rats. Figure 2 , A), primary cells have strong proliferation and stem cell characteristics. In order to avoid the influence of the stem cell characteristics of primary cells, they were continuously subcultured in DMEM / F12 medium containing 10% fetal bovine serum, so that the primary cells continued to proliferate and divide to differentiate to the terminal state. Primary isolated astrocytes were used as the first generation, and they were used in this study after continuous subculture to the fifth generation.

[0099] Immunofluorescence staining was used to explore the composition of primary astrocytes. The cell types contained in the cells were analyzed, among which glial cell markers GFAP and S100β accounted for about 95%, microglial cell marker Iba1 accounted for about 1%, and in addition to glial cells, neuron-related markers MAP2 and NeuN accounted for about 1%, and immature neuron marker Tuj1, indicating that primary cells contain a small number of neurons. Oligodendrocyte marker Olig2 and stem cell markers SOX2 and Nestin ( Figure 2 , B). From the composition of the isolated primary cells, most of them are astrocytes, and a very small part contains other types of glial cells, so the primary astrocytes used in this study were obtained.

[0100] (2) Screening of small molecule drugs to convert astrocytes into neurons

[0101] Previous studies have shown that four small molecules (SB431542, LDN193189, CHIR99021 and DAPT, also known as "coredrug") can efficiently transdifferentiate astrocytes into neurons. In this process, the TGFβ, BMP, GSK3 and Notch signaling pathways are regulated, and most of the neurons that are finally converted are excitatory neurons (78%). The types of brain neurons are mainly composed of excitatory neurons and inhibitory neurons. In order to achieve the transdifferentiation of astrocytes into inhibitory neurons using chemical drugs, it is necessary to target the origin of inhibitory neurons. Cortical GABAergic neurons originate from the ganglionic eminence on the ventral side of the telencephalon. Different types of GABAergic neurons are generated in different areas of the eminence and at different times, and the signaling pathway sonic hedgehog (SHH) is related to them. The present invention selects SHH signaling pathway activators SAG and Purmorphamine as potential candidate small molecule compounds to test the effect of the SHH signaling pathway.

[0102] Unlike human embryonic astrocytes, the four small molecule combination "core drug" could not complete the transdifferentiation of astrocytes into neurons in the rat primary astrocyte system ( Figure 2 , D). One is the difference between humans and rats in species, and the other is the difference in cell culture systems in different laboratories. In addition, there are also differences in the transdifferentiation process between primary cells and cell lines. Therefore, the present invention chooses to screen new small molecule drugs in the culture system of rat primary astrocytes to achieve transdifferentiation.

[0103] After hundreds of attempts, it was found that adding the ALK5 inhibitor Repsox in the TGFβ signaling pathway to the original four small molecules can cause changes in astrocyte morphology (data not shown), but ultimately cannot be converted into neurons. At the same time, it was found that adding SM1 (containing SB431542 (5μM), LDN193189 (0.25μM), CHIR99021 (1.5μM), DAPT (5μM) and Repsox (1μM), that is, the ratio of SB431542, LDN193189, CHIR99021, DAPT and Repsox is 20:1:6:20:4.), and then adding the small molecule combination SM2 (containing SB4 31542 (5μM), LDN193189 (0.25μM), CHIR99021 (1.5μM), DAPT (5μM) SAG (1μM), Purmorphmin (1μM), that is, the ratio of SB431542, LDN193189, CHIR99021, DAPT to SAG, Purmorphmin is 20:1:6:20:4:4. ), after 9 days of treatment, a large number of DCX + Phenotype of immature neurons ( Figure 3 , B), the conversion efficiency was 34.55±6.92%. In the control group without small molecule treatment, only 0.37±0.65% DCX positive cells were detected. This may be due to the small number of neuronal precursor cells in primary cells and the production of ( Figure 3 , D). Quantitative analysis showed that the transformation efficiency (MAP2 + / field) was 89.86±5.41, and the control group had a small amount of MAP2 + The number of cells per field of view was approximately 3.34±1.72 ( Figure 3 , E). After two weeks of incubation in differentiation medium (NDM, Neural Differential Medium), neurons transformed with small molecules became MAP2 with longer axons and more mature neurons. + NeuN + Co-labeled neurons ( Figure 3 , C), while the control group produced only a small number of neurons. Therefore, after screening, a chemical combination was successfully obtained that can successfully convert primary rat astrocytes into neurons.

[0104] (3) Retroviral verification of astrocytes into neurons

[0105] In order to verify that the small molecule combination drug induces the transdifferentiation of astrocytes into neurons, retrovirus (RV, retrovirus, RV-GFAP-GFP) with green fluorescent marker was selected to label the cells. Retrovirus infects dividing cells but not non-dividing cells. Therefore, retrovirus labels dividing primary astrocytes but not non-dividing neurons. After the cells were seeded on the slide for 24 hours, 2.5×10 per cell were added. 4 GC / ml to infect cells in culture ( Figure 4 , A). 87.81±6.76% of the cells infected with the retrovirus were astrocyte GFP + GFAP + Almost no other cell types (GFP + Iba1 + )( Figure 4 , B; Figure 4 , D). After 9 days of small molecule treatment, 51.2±9.36% of retrovirus-infected astrocytes were found to be transformed into Tuj1-positive immature neurons ( Figure 4 , C; Figure 4 , E). This proves that the primary astrocytes obtained are transformed into neurons under the action of small chemical molecules.

[0106] (4) Astrocyte to neuron transformation process

[0107] When observing the process of astrocyte transdifferentiation induced by small molecules into neurons under a microscope, it was found that the cell morphology changed significantly during the transdifferentiation process, from the flat glial cell morphology to gradually bulging and rounding into the neuronal morphology. In order to track the transdifferentiation process of astrocytes into neurons, a Zeiss inverted fluorescence microscope was used to record the transformation process of astrocytes into neurons under bright field at each key time point.

[0108] It was observed from the continuous shooting process that compared with the control group, the morphology of the experimental group changed dramatically under the action of small molecules. Starting from the second day, the cytoskeleton began to shrink significantly, and the cell morphology did not change significantly in the next two days. However, starting from the fifth day, cell bulges with transparent edges began to appear in the field of vision, and astrocytes began to transform into neuron-like cells. From the sixth day, the number of cells in the bulging immature neuron state gradually increased, reaching a peak on the ninth day. The cell morphology changed from the initial flat state to a bulging morphology with transparent edges. After replacing the differentiation medium, the cell protrusions gradually elongated, the cell body became transparent, and it tended to be more like a mature neuron morphology ( Figure 5 , A). Therefore, from a morphological point of view, the cells gradually shrink and become round from the flat glial cell state, the processes gradually elongate, and the astrocytes transform into neurons.

[0109] (5) Screening the effects of various small molecules on transdifferentiation efficiency

[0110] Each small molecule plays a crucial role in the process of inducing the transformation of astrocytes into neurons by a combination of small molecules. Studies have shown that the selected small molecules all play a role in various key pathways. CHIR99021 is a selective glycogen synthase kinase 3 (GSK-3) inhibitor that activates the Wnt / β-catenin pathway in human and mouse stem cells. DAPT inhibits γ-secretase and Notch signaling and is used to promote neuronal differentiation of ES cells. LDN193189 is an inhibitor of bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3, used to inhibit the development of mesoderm and endoderm. SB431542 inhibits TGF-β type I receptors ALK4, ALK5 and ALK7. SAG and Purmorphamine inhibit the Hedgehog signaling pathway.

[0111] In order to intuitively show the role of each small molecule and its corresponding signal pathway in the transformation process in the screened small molecule compound combination, compared with the entire small molecule combination, the conversion efficiency will be reduced if any small molecule is reduced ( Figure 6 , A), indicating that each small molecule plays a vital role in the conversion process. After subtracting the Notch pathway inhibitor DAPT, although the conversion efficiency decreased, it did not reach a significant statistical significance. When any of the remaining small molecules were subtracted, the conversion efficiency decreased significantly. Statistical results showed that there was a significant decrease when the Hedgehog signaling pathway Purmorphamine and the TGF-β signaling pathway inhibitor SB431542 were subtracted. When CHIR99021, Repsox and SAG were subtracted, it was found that the degree of decrease in conversion efficiency was even greater. It was found that the decrease in conversion efficiency after subtracting RepSOX was more significant than that of SB431542 ( Figure 6 , B). At the same time, it was tested whether a single small molecule could convert astrocytes into neurons. The results showed that it was difficult to convert astrocytes into neurons by inhibiting or activating the signaling pathway with a single small molecule (results not shown).

[0112] (6) Analysis of small molecules that convert astrocytes into neuronal subtypes

[0113] (A) Neuronal subtypes induced by small molecules

[0114] Analysis of the types of neurons transformed from astrocytes induced by small molecules revealed that almost all neurons transformed by small molecule combinations were GABAergic neurons, with no excitatory neurons (VGlut2) or other types of neurons, such as dopaminergic neurons (DAT) and motor neurons (ChAT) ( Figure 7). As an important component of neurons, GABAergic neurons inhibit the transmission of neural signals and regulate the balance of neural signals. When inhibitory neurons are dysfunctional or missing, a large amount of neurotransmitters released by excitatory neurons are transmitted step by step, and abnormal electrical signals in some areas of the brain induce local epilepsy. Therefore, inhibitory neurons distributed in various brain regions play an indispensable role.

[0115] (B) Subtype subdivision of GABAergic neurons in response to small molecule-induced astrocyte conversion

[0116] The GABAergic interneurons induced by small molecules were further subdivided and tested, including calbindin (CB), calretinin (CR), somatostanin (SST), parvalbumin (PV), NOS and neuropeptide Y (NPY). + Interneurons accounted for the majority, accounting for about 62.7%. Some PVs were also detected. + Interneurons, about 23.3%. In addition to PV + and CR + No other types of interneurons were detected ( Figure 8 ). This indicates that most of the GABAergic neurons induced by chemical drugs are CR + and PV + Interneurons. Studies have shown that interneurons expressing parvalbumin (PV) may also play a key role in many brain diseases, including complex psychiatric disorders such as epilepsy and schizophrenia. PV interneurons may become an important therapeutic target in the future. CR is a calcium-binding protein that is mainly expressed by GABAergic interneurons in the hippocampus. Impairment of the CR interneuron population in the epileptic hippocampus may be related to the sprouting of axons of excitatory pathways and enhanced principal cell innervation.

[0117] (7) Patch clamp whole-cell recording of converted neurons has electrophysiological functions

[0118] Neurons are an important part of the nervous system, and the connection between neurons is a crucial link in transmitting information. Electrical signals are received by dendrites, transmitted to the cell body, and then transmitted downward by axons to the next level of neurons, thus completing the transmission of electrical signals. Primary cultured astrocytes are transdifferentiated into GABAergic neurons under the induction of small molecule compounds. The functionality of these newborn neurons is detected by whole-cell recording with patch clamp.

[0119] The morphological results of immunofluorescence staining of neuronal microtubule-associated protein (MAP2) were used as a reference, and the time point after 30 days of cell culture was used as the key time point for detection. Whole-cell patch clamp recording of neurons transformed from astrocytes treated with small molecules was able to fire action potentials ( Fig. 9 , A, B), while the control group had no action release ( Fig. 9 , C), the neurons transformed from astrocytes treated with small molecules in the experimental group can also produce Na + Current and K + Current( Fig. 9 , D), and spontaneous postsynaptic currents ( Fig. 9 , F, I). Compared with the control group, the potassium current in the experimental group was significantly lower than that in the control group, while no sodium current was detected in the control group ( Fig. 9 , E), indicating that the cells collected from the control group were glial cells, and the cells collected from the experimental group were neurons. More importantly, there was no spontaneous postsynaptic current in the control group ( Fig. 9 , FG, IJ), while in the experimental group, both the excitatory spontaneous postsynaptic current ( Fig. 9 , GH; G: 0.045 ± 0.051 Hz; H: 6.04 ± 3.83 pA) or inhibitory spontaneous postsynaptic current ( Fig. 9 , JK; J: 0.35 ± 0.28 Hz; K: 42.24 ± 16.11 pA) were significantly higher than those in the control group, indicating that there were synaptic connections between neurons. It can be said that the neurons derived from astrocytes transdifferentiated by small molecules are functional.

[0120] (8) In situ transdifferentiation of R-SP by small molecule combination

[0121] (A) In situ injection of small molecule combination R-SP into mouse cortex

[0122] In order to achieve in situ transdifferentiation in vivo, we attempted to inject the newly selected small molecule combination in situ. In the virus titer gradient experiment, low-titer virus (1E+11) infected astrocytes did not leak into neurons (data not shown). We chose to activate astrocytes in the acupuncture injury model, and pre-labeled background astrocytes with low-titer adeno-associated virus AAV9-GFAP-1.6-GFP. After the virus was fully expressed in astrocytes, we injected the newly selected small molecule combination R-SP in situ twice into the cortex of C57BL / 6J mice ( Fig.10 , A). After 9 days, it was found that both the control group and the experimental group had severe cortical damage ( Fig.10 , B), a small amount of GFP was found at the edge of the injury + NeuN + Neuron-like cells ( Fig.10, C). The number of converted neurons after 21 days did not increase significantly compared with that after 9 days. This may be due to the limited effect of a single administration, the physical damage caused by acupuncture and the irreversible damage of DMSO solvent to brain parenchyma cells, and the metabolism of a small amount of chemical drugs in the brain parenchyma in a short time, and the drug's continued effect is limited. The R-SP combination screened at the cellular level has the effect of converting astrocytes into neurons in the mouse cortex.

[0123] (B) In situ injection of small molecule combination R-SP into mouse striatum

[0124] The small molecule combination R-SP was injected into the intact striatum and hippocampus in the same manner as above. Fig.11 , A). Seven days after the injection of the small molecule combination R-SP, it was found that the neurons near the injection of solvent or small molecule drugs in the control group and the experimental group were lost ( Fig.11 , B, red dotted area), causing strong immunogenicity and activation of astrocytes. When the small molecule combination R-SP was injected into the cortex, only a small amount of GFP appeared. + NeuN + Neuron-like cells, but no similar transformation was found in the striatum. In the control group, no cells expressing immature neuron DCX appeared near the injection of solvent, while in the small molecule group, cells positive for immature neuron marker DCX appeared near the injection of small molecule drugs ( Fig.12 , A, the red dotted line is the drug-administered area), the cell morphology has grown longer synapses, tending to the morphology of mature neurons ( Fig.12 , A, B). Some DCX+ cells and GFP+ cells were co-labeled in different regions of the striatum ( Fig.12 , C), indicating that the small molecule drug combination injected into the striatum converted green fluorescent protein-labeled astrocytes into immature neurons, which in turn may continue to grow in the direction of mature neurons in the brain parenchyma.

[0125] (C) In situ injection of small molecule combination R-SP into mouse hippocampus

[0126] The small molecule combination R-SP of the present invention was injected unilaterally into the CA1 region of the hippocampus ( Fig.13 , A). The injection of small molecules caused a significant increase in the number of DCX in the DG region of the hippocampus ( Fig.13 , B left, right as untreated control). Astrocytes pre-labeled with AAV9-GFAP-1.6-GFP in the CA1 region of the hippocampus. By injecting the small molecule solution twice. It was observed in the CA1 region that they were transformed into neuron-like cells under the action of small molecules ( Fig.13 , C). In vivo chemical translation of small molecule drugs into neurons in the hippocampus region.

[0127] The above results suggest that in situ injection of small molecules into the cortex, striatum and hippocampus may achieve the transdifferentiation of astrocytes into neurons. In order to achieve high-efficiency conversion, it can be achieved by increasing the drug concentration and the frequency of administration.

[0128] (9) Whole-brain delivery of small molecules by retro-orbital intravenous injection

[0129] The retro-orbital intravenous administration of drugs in mice is to transport small molecule drugs through the retro-orbital venous sinus of mice, cross the blood-brain barrier and finally enter the brain tissue to exert its drug effect. The duration of administration can be controlled by adjusting the drug concentration and frequency. To this end, under the acupuncture injury model, the damage range covers the cortex and striatum, and the adeno-associated virus serotype AAV9-PHP.eb-GFAP-104-GFP that can cross the blood-brain barrier is used to infect astrocytes throughout the brain. The virus is injected into the brain through the retro-orbital vein through the blood to infect astrocytes, so that the background astrocytes are labeled with green fluorescent protein. The small molecule combination R-SP is based on the concentration used for cell screening, and the 100× concentration is also injected into the brain through the blood-brain barrier by orbital injection for 8 consecutive days ( Fig.14 , A). After circulating in the cerebrospinal fluid, the drug's efficacy was also greatly diluted. After one week of continuous injection, it was found that some converted neurons were evenly distributed throughout the cortex of the experimental group of mice, and no obvious conversion effect was found in other brain areas ( Fig.14 , B). It is speculated that the cortex has a higher conversion efficiency because the astrocytes used are also from the cortex, and some of the converted cells are GABAergic neurons ( Fig.14 , C). However, the orbital injection method is extremely unstable and has individual differences among mice ( Fig.14 , D).

[0130] (10) Transgenic mouse system verifies astrocytes transdifferentiation into neurons

[0131] In the normal mouse acupuncture injury model, astrocytes were transdifferentiated into neurons by a combination of chemical small molecule drugs, both by in situ injection of drugs and by retro-orbital intravenous injection of small molecule drugs. The "gold standard" transgenic mouse Aldh1l1-creERT2×Ai14 strain was used. This strain of mice was injected with tamoxifen to activate endogenous astrocytes to express red fluorescent protein ( Fig.15 , A). Immunofluorescence staining revealed that almost all cells expressing endogenous red fluorescent protein were astrocytes, among which RFP + GFAP + About 92.96%, RFP + S1OOβ +About 77.23% of the cases had no RFP detected + Iba1 + Cells and RFP co-labeled with the neuronal marker NeuN ( Fig.15 , B). This indicates that most of the cells that only express endogenous red fluorescent protein are astrocytes.

[0132] Using the same treatment as ordinary mice, the molecular drug was delivered to the brain by orbital injection under the acupuncture injury model ( Fig.15 , C, D), thereby achieving the transformation of astrocytes into neurons. Neuronal loss caused by physical damage caused by acupuncture can be repaired to a certain extent by regenerating neurons under the action of small molecule drugs. Looking at various brain regions after small molecule treatment, it was found that RFP transformed from astrocytes was found in the cortex, striatum and hippocampus 7 days after small molecule administration. + NeuN + Co-expressing neurons ( Fig.16 , AC). The transformed neurons distributed in various brain regions provide feasibility for future application in whole-brain neuroregeneration diseases. In addition to a small number of astrocytes in brain tissue transformed into neurons, the transformed neurons (RFP) can be found in the cervical (C), thoracic (T), and lumbar (L) segments of the mouse spinal cord. + NeuN + , Fig.16 , AC), among the transformed neurons there are motor neuron-like cells ( Fig.17 , * indicates the position). No glial cell transformation into neurons was found in the control group (PBS + equal amount of DMSO solvent) + and NeuN + Regeneration of motor neurons is of great significance for spinal cord injury. Currently, patients lose their mobility due to loss of motor neurons caused by spinal cord injury and neurodegenerative diseases such as lateral sclerosis. Regeneration of motor neurons may provide a possible treatment for such diseases.

[0133] The above demonstrates the conversion of brain (cortex, striatum and hippocampus) and spinal cord astrocytes into neurons by orbital injection of small molecules.

[0134] During the small molecule treatment process, it was also found that the expression of NeuroD1 and NGN2 was not detected during the transcription process, while the transcription factors Ascl1 and Olig2 were significantly upregulated. Transcriptome analysis found that during the transdifferentiation process, neurotrophic factors and complex nutrients caused significant changes in the transcriptome of astrocytes. After removing these influencing factors, under the synergistic effect of small molecules, TGFβ, JAK-STAT, and Wnt signaling pathways were downregulated, and the Hedgehog signaling pathway was upregulated. The Notch signaling pathway showed a trend of first downregulation and then upregulation, and returned to the initial expression level after the withdrawal of small molecules.

[0135] In summary, the chemical small molecule combination used in the present invention can transform astrocytes into functional inhibitory neurons, and can transform astrocytes into neurons both in vitro and in vivo.

Claims

1. A small molecule combination, characterized in that Including SB431542, LDN193189, CHIR99021, DAPT, Repsox, SAG and Purmorphamine.

2. Use of the small molecule combination according to claim 1 for preparing a drug for converting astrocytes into neurons.

3. The use according to claim 2, characterized in that: The neurons are inhibitory neurons.

4. Use of the small molecule combination according to claim 1 for preparing a drug for in situ transdifferentiation of astrocytes into neurons.

5. Use of the small molecule combination according to claim 1 for preparing a drug for treating nervous system diseases.

6. The use according to claim 5, characterized in that The nervous system disease is a neurodegenerative disease, neural injury, neuroaging, neurodevelopmental disease or movement disorder.

7. The use according to claim 5, characterized in that The nervous system disease is any one of Alzheimer's disease, stroke, amyotrophic lateral sclerosis, glioma, Parkinson's disease, epilepsy and Huntington's disease.

8. A method for converting astrocytes into neurons, characterized in that Comprising administration of the small molecule combination of claim 1.

9. The method according to claim 8, characterized in that The combination of SB431542, LDN193189, CHIR99021, DAPT and Repsox was administered first, followed by the combination of SB431542, LDN193189, CHIR99021, DAPT, SAG and Purmorphamine.

Images