CN105535992B - Application of Ascl1 in inducing transdifferentiation of astrocytes into functional neurons - Google Patents

Abstract

The invention provides an application of Ascl1 in inducing the transdifferentiation of astrocytes into functional neurons. Specifically, there is provided a use of aachaete-scute complex homolog-like 1 (achaete-scute complex homolog-like 1, Ascl1) gene or a protein thereof or a promoter thereof, characterized in that (i) for use in preparing a pharmaceutical composition for inducing astrocytes to form functional neuronal cells; and/or (ii) preparing a pharmaceutical composition for treating nervous system diseases. This method is expected to be an effective method for culturing neuronal cells in vitro and stimulating the generation of new neuronal cells in adults, thus being widely used in the treatment of neurological diseases, such as neurodegenerative diseases, central nervous system trauma diseases, and so on.

Description

Application of Ascl1 in inducing transdifferentiation of astrocytes into functional neurons

technical field

The invention belongs to the field of biotechnology and cell therapy, and in particular, the invention relates to a method for inducing transdifferentiation of astrocytes into functional neuron cells and applications thereof.

Background technique

Many transcription factors and chromatin epigenetic modification processes play a very important role in maintaining the stability of the properties of differentiated cells. However, studies of induced pluripotent stem cells (iPS cells) have shown that differentiated cells are not irreversibly locked in their mature state, but can be dedifferentiated by selective overexpression of specific transcription factors.

Recent findings have found that specific transcription factors can directly induce fibroblasts to become functional neurons, further suggesting that non-neuronal cells can be directly transdifferentiated into neurons. For example, astrocytes in the postnatal mouse cerebral cortex can be transdifferentiated into glutamatergic or GABAergic neurons in vitro by overexpressing the transcription factors Neurog2 or Dlx2. Several studies have also shown that non-neuronal cells can be reprogrammed into neurons or neural precursor cells in vivo. However, little is known about whether astrocytes in vivo can transdifferentiate into neurons and whether these induced neurons (iNs) can integrate into pre-existing neural circuits.

At present, overexpression of the pro-neuronal protein Neurog2 can reprogram in vitro cultured astrocytes from early-natal mouse cerebral cortex into glutamatergic neurons that can form synaptic connections. Overexpression of the proneuronal protein NeuroD1 can reprogram reactive astrocytes into neurons after injury in the mouse cerebral cortex. However, there is currently no method or pathway that can transdifferentiate normal astrocytes into functional neurons.

Therefore, there is an urgent need in the art to find a cytokine that can induce the transdifferentiation of mature cells into active neuronal cells, thereby opening up a new therapeutic approach in the restoration of brain function.

Growing evidence supports the very high heterogeneity of the lineage of astrocytes. It can be envisaged that different sources of astrocytes may influence the effect of their transdifferentiation into neurons. In the present study, a single transcription factor, Ascl1, was found to efficiently transdifferentiate postnatal mouse dorsal midbrain astrocytes into neurons that can form synaptic connections in vitro. In addition, a gene expression system that specifically targets astrocytes in vivo was designed, and it was found that a single transcription factor Ascl1 could induce the transdifferentiation of astrocytes into functional neurons in vivo.

SUMMARY OF THE INVENTION

The present invention provides the use of Ascl1 gene or its protein in inducing the transdifferentiation of astrocytes into functional neuron cells, and the application thereof in the treatment of nervous system diseases.

The first aspect of the present invention provides the use of aachaete-scutecomplex homolog-like 1 (Ascl1) gene or its protein, (i) for preparing induced astrocytes A pharmaceutical composition in which the cells form functional neuronal cells; and/or (ii) a pharmaceutical composition for use in the preparation of a neurological disease.

In another preferred embodiment, the Ascl1 gene or its protein is derived from mammals, preferably from humans, mice, and rats.

In another preferred embodiment, the astrocytes include astrocytes in a normal state and a damaged state.

In another preferred example, the GenBank number of the Ascl1 gene is U68534.2, and the protein sequence is shown in SEQ ID NO.:1.

In another preferred example, the NCBI Reference Sequence number of the mRNA encoding the Ascl1 gene is NM_008553.4, and the mRNA sequence is shown in SEQ ID NO.:2.

In another preferred embodiment, the pharmaceutical composition includes an expression vector (FUGW and GFAP-AAV) containing the Ascl1 coding sequence, and a pharmaceutically acceptable vector (GFAP-AAV).

In another preferred embodiment, the astrocytes are derived from the striatum, spinal cord, dorsal midbrain or cerebral cortex, preferably, the astrocytes are derived from the cortex, dorsal midbrain or cerebral cortex brain.

In another preferred example, the functional neurons include glutamatergic neurons and/or γ-aminobutyric acid (GABA) neurons.

In another preferred embodiment, the functional neurons are capable of firing action potentials and forming synaptic connections.

The second aspect of the present invention provides an expression vector, the expression vector contains the Ascl1 protein coding sequence, and the expression vector can be integrated into astrocytes and express exogenous in astrocytes of Ascl1 protein.

In another preferred embodiment, the expression vector includes plasmid and viral vector.

In another preferred embodiment, the viral vector can infect astrocytes.

In another preferred embodiment, the expression vector is an astrocyte-specific expression vector.

In another preferred embodiment, the expression vector GFAP-AAV vector.

In another preferred embodiment, the viral vector includes a lentiviral FUGW vector.

In another preferred example, the expression vector includes the following elements from 5' to 3' in order: GFAP-AAV vector: viral ITR sequence + CMV enhancer + human GFAP promoter + Ascl1 and red fluorescent protein mCherry The coding frame of the + post-transcriptional regulatory element WPRE + viral ITR sequence + the promoter and coding frame of the ampicillin resistance gene

FUGW vector: viral ITR sequence+Ubiquitin promoter promoter+Ascl1 coding frame+IRES sequence+green protein GFP coding frame+post-transcriptional regulatory element WPRE+viral ITR sequence+promoter and coding frame of ampicillin resistance gene

In another preference, the viral vector is prepared as follows:

The viral vector is formed by introducing the polynucleotide sequence having the Ascl1 coding sequence into the packaging cell of the viral particle.

The third aspect of the present invention provides a host cell, wherein the chromosome of the host cell is integrated with a polynucleotide encoding the Ascl1 protein, or the host cell contains the expression vector described in the second aspect of the present invention.

In another preferred embodiment, the host cells are astrocytes.

A fourth aspect of the present invention provides an in vitro non-therapeutic method for transdifferentiating astrocytes into functional neuron cells, comprising the steps of:

In the presence of exogenous Ascl1 protein, astrocytes were cultured to induce astrocytes to form neuronal cells.

In another preferred embodiment, the exogenous Ascl1 protein is an exogenous Ascl1 protein produced by expressing an exogenous Ascl1 coding sequence in the astrocytes.

In another preferred embodiment, the exogenous Ascl1 protein is an exogenous Ascl1 protein produced by expressing an exogenous Ascl1 coding sequence in the astrocytes.

In another preferred embodiment, the exogenous Ascl1 protein is obtained by expressing the expression vector described in the second aspect of the present invention.

In another preferred embodiment, the expression vector is a lentiviral particle.

A fifth aspect of the present invention provides a functional neuron cell and/or neuron cell group transdifferentiated from astrocytes, wherein the functional neuron cell and/or neuron cell group is composed of the first The method described in Sifang is obtained, and the functional neuron cells and/or neuron cell groups have one or more of the following characteristics:

(a) at least 50% of neuronal cells, preferably at least 60%, 70%, 80%, 90%, or 100% of neuronal cells express neuronal markers Tuj1, MAP2, NeuN or Synapsin I;

(b) capable of firing action potentials and capable of forming synaptic connections.

In another preferred example, in the neuronal cell population, the neuronal cells do not express markers such as Gfap, S100β, Acsbg1, Sox2 or Pax6.

In another preferred embodiment, the non-expression includes substantially non-expression, for example, at least 60%, 70%, 80%, 90%, or 100% of neuronal cells do not express Gfap, S100β, Acsbg1, Sox2 or Pax6, etc. landmark.

The sixth aspect of the present invention provides the use of the functional neurons described in the fifth aspect of the present invention, wherein the functional neuron cells and/or neuron cell populations are used to prepare a pharmaceutical composition for treating nervous system diseases.

In another preferred embodiment, the neurological diseases include epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), neuronal death caused by stroke and the like.

The seventh aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition comprising (A) the expression vector or Ascl protein described in the second aspect of the present invention, or (B) the fifth aspect of the present invention. functional neurons; and (C) a pharmaceutically acceptable carrier.

An eighth aspect of the present invention provides a method for treating nervous system diseases, comprising the steps of:

A safe and effective amount of the pharmaceutical composition of the seventh aspect of the present invention is administered to a subject in need, thereby treating nervous system diseases.

A ninth aspect of the present invention provides a method for (a) screening candidate compounds for the treatment of neurological diseases; and/or (b) screening for candidate compounds inducing the transdifferentiation of astrocytes into functional neuronal cells, wherein It is characterized in that it includes the steps:

(i) adding the test compound to the cell culture system as the test group, and using the cell culture system without the test compound as the control group;

(ii) comparing the expression and/or activity E1 of the Ascl1 gene or its protein in the test group with the expression and/or activity E0 in the control group;

Wherein, when E1 in the test group is significantly higher than E0, it indicates that the tested compound is (a) a candidate compound for the treatment of neurological diseases; and/or (b) induces the transdifferentiation of astrocytes into functional neuron cells. candidate compounds.

In another preferred embodiment, the cells are astrocytes.

In another preferred example, the said significantly higher means that E1 is higher than E0, and there is a statistical difference; preferably, E1≥2E0.

In another preferred embodiment, the method further comprises the steps:

(iii) adding the test compound to the astrocyte culture system as the test group, and using the astrocyte culture system without the test compound as the control group;

(iv) comparing the proportion of astrocytes transformed into functional neurons in the test group to determine whether the test compound is (a) a candidate compound for the treatment of neurological diseases; and/or (b) screening for inducing astrocytes Candidate compounds for the transdifferentiation of glial cells into functional neuronal cells;

Wherein, when the ratio T1 of astrocytes transformed into functional neurons in the test group is significantly higher than the ratio T0 in the control group, it indicates that the test compound is (a) a candidate compound for the treatment of neurological diseases; and/or (b) Screening of candidate compounds for inducing transdifferentiation of astrocytes into functional neuronal cells.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1a-h shows the identification of the properties of the isolated and purified astrocytes. Most of the cells express astrocyte marker molecules GFAP and S100β, and a small number of cells express oligodendrocyte marker molecules O4 and S100β. CNPase, a small amount of cells expressed NG2, a marker molecule of glial cells, NG2, but no expression of neuron marker molecule Tuj1 and stem cell marker molecules Sox2 and Oct4 was detected.

Figure 2 shows the expression of neuronal marker molecules in astrocytes after transdifferentiation induced by lentiviral vector. Figure 2a-b shows that 10 days after astrocytes were transfected with the control lentiviral vector FUGW, astrocytes did not express the neuronal marker molecule Tuj1, but still maintained the shape of glial cells and expressed astrocyte markers Molecular GFAP. Figure 2c shows that after 10 days of infection with lentivirus FUGW-Ascl1, most of the astrocytes expressed Tuj1, a marker molecule of neurons, and presented typical neuronal morphology. Figure 2d and Figure 2e show that 21 days after infection with lentivirus FUGW-Ascl1, astrocytes also expressed mature neuron marker molecules MAP2 and synapsin I, respectively. Figure 2f shows whole-cell electrophysiological recordings from these neurons. Figure 2g shows that 30-40 days after lentiviral transfection, all GFP-positive cells (63 cells in total) were able to generate action potentials, and the vast majority of iN cells (87.3%, 55/63 cells) were able to record to spontaneous postsynaptic currents. Figures 2h-i are the results of FUW-Ascl1-tdTomato and control virus infection of astrocytes (astrocytes with GFP labeling) from hGFAP-GFP mice. Among them, Figure 2h shows that after infection with the control lentivirus FUW-tdTomato containing only red fluorescent protein, the virus-infected cells still maintain the morphology of astrocytes and express GFAP; Figure 2i shows that overexpression of Ascl1 with lentivirus can induce Astrocytes were morphologically altered and Tuj1 was expressed. These transdifferentiated neurons still expressed GFP.

Figure 3 shows the transmitter properties of the induced cells. Figure 3a shows that most iN cells express GABA, while Figure 3b and Figure 3d show that some iN cells express GABAergic neuron marker molecules GAD67 and VGAT, respectively. Figures 3c and e show that control virus-infected cells do not express GABAergic neuron marker molecule VGAT and glutamatergic neuron marker molecule VGLUT2, respectively. Figure 3f shows that some iN cells express the glutamatergic neuron marker molecule VGLUT2. Figure 3g shows that some iN cells (19.4%, 7/36 cells) were able to record from self-synapses, and when CNQX, an antagonist of AMPA/kainate glutamate receptors, was added, the self-synaptic currents were completely blocked ( 3/3 cells). Figure 3h shows that some iN cells (21%, 8/38 cells) could record from self-synapses, and when bicuculline, a GABA _A receptor antagonist, was added, the self-synaptic currents were completely blocked (5/5 cells). cell).

Figure 4a and Figure 4b show that astrocytes taken from the dorsal midbrain of GAD67–GFP mice were infected with lentivirus FUW-Ascl1-tdTomato and the induced cells were found to express GFP. Figure 4c shows that iN cells were co-cultured 10 days after induction with neurons isolated from the dorsal midbrain of P5-P7 wild-type mice, and almost all tdTomato ⁺ GFP ⁺ cells (97 %, 37/38 cells) were able to generate action potentials. Figure 4d shows that the vast majority of iN cells (89%, 34/38 cells) could record spontaneous postsynaptic currents.

Figure 5a and Figure 5b show that almost all mCherry-positive cells express Acsbg1 3 days after virus injection in the dorsal midbrain of mice at postnatal days 12-15, regardless of whether they were injected with the control virus AAV-mCherry or the virus AAV-Ascl1/mCherry. Figure 5c and Figure 5d show that, after 3 days of AAV-mCherry infection, the vast majority of mCherry-positive cells were also GFP-positive cells in two transgenic mice, Aldh1l1-GFP mice and GFAP-GFP mice, respectively. Figure 5e shows that GFAP-CreERT2 was induced with 4-hydroxytamoxifen (4-OHT); Rosa26-CAG-tdTomato mice expressed tdTomato, and tdTomato was found to co-localize with Acsbg1.

Figure 6 shows that mCherry does not coexist in the same cell with NG2, a marker for NG2 cells.

Figure 7a, a' Figure 7d, d' show that 3-5 days after injection of control virus AAV-mCherry and virus AAV-Ascl1/mCherry in the dorsal midbrain of mice 12-15 days after birth, immunoco-labeling showed that mCherry was No colocalization with NeuN; Figures 7b,b', 7c,c' show that none of mCherry colocalized with NeuN in mice with AAV-mCherry 10-14 days and 28-32 days after injection of control virus, respectively. Fig. 7e,e' and Fig. 7f,f' show that in mice injected with virus AAV-Ascl1/mCherry, mCherry gradually co-localized with NeuN: 44.2±12.5% from 10-14 days after virus injection (n=3, each 309-436 cells per count) to 93.1 ± 1.7% (n=3, 412-557 cells per count) to 28-32 days after virus injection. Figure 7g shows that 45 days after virus injection, some iN cells express Gad1, and Figure 7h shows that some iN cells also express VGLUT2. Figures 7i and 7j show that cells isolated from the subventricular zone (SVZ) can produce large numbers of neurospheres whereas cells isolated from the dorsal midbrain are essentially incapable of producing neurospheres. Figures 7k-7n show that GFAP-CreERT2; Rosa26-CAG-tdTomato mice were induced with 4-OHT for 5 consecutive days (P12-P16) expressing tdTomato and found that tdTomato still did not co-localize with NeuN after 30 days. Thus, iN cells induced from GFAP ⁺ were derived from postnatal astrocytes, not neural precursor cells.

Figures 8a-8c show that the cell density in the dorsal midbrain of mice injected with virus AAV-Ascl1/mCherry was substantially comparable to mice injected with control virus AAV-mCherry. Figures 8d-8h show that apoptosis is not increased in mice injected with the virus AAV-Ascl1/mCherry.

Figure 9 shows that mCherry expression can still be detected in the dorsal midbrain of mice 155 days after virus AAV-Ascl1/mCherry injection, and they co-localize well with NeuN.

Figure 10 shows the gene expression of mouse iN cells injected with virus AAV-Ascl1/mCherry. Analysis by real-time quantitative PCR was performed by sorting mCherry+ cells at different time points (days 4, 10, 30) by flow cytometry. It was found that the expression of astrocyte marker molecules (Gfap, S100β and Acsbg1) gradually decreased, the expression of neural cell marker molecules (Tuj1, Map2 and NeuN) gradually increased, and the neural precursor cell marker molecules (Sox2 and Pax6) Basically no expression can be detected.

Figure 11a shows that in the mouse brain slices infected with the control virus AAV-mCherry, the detected cells have lower impedance, higher resting membrane potential, and cannot fire action potentials. Results of biocytin remodeling revealed that control virus-infected cells had the typical morphology of astrocytes and were connected to neighboring astrocytes by gap junctions. Figures 11b-11e show that in mouse brain slices infected with virus AAV-Ascl1/mCherry for 7-30 days, in voltage-clamp mode, many cells have inward Na ⁺ currents and outward K ⁺ currents, and the amplitudes vary with infection time In the current-clamp mode, the ability of the detected cells to fire action potentials also increased, and the cell morphology became more complex. The results of Biocyte remodeling also found that the detected cells also formed fewer gap junctions. 11f and 11g show a gradual increase in the input resistance of the cell, while a gradual decrease in the resting membrane potential. Figure 11h shows that Ascl1-induced iN cells became more and more excited with the prolongation of infection time, and all recorded iN cells were able to fire action potentials at high frequency (50-220 Hz) 30 days after infection, while the control virus-infected cells exhibited Similar to the "inactive" state of astrocytes. Figure 11i shows that 30 days after AAV-Ascl1/mCherry virus infection, high-frequency spontaneous postsynaptic currents were found to be detected in all virus-infected cells (23/23). Figure 11j shows further pharmacological experiments showing that iN cells received both excitatory glutamate input and inhibitory GABA input. Figure 11k shows that by double whole-cell recording, iN cells (mCherry ⁺ ) can form synaptic connections with neurons in the midbrain tectum (mCherry ^- ), and when bicuculline, a GABA _A receptor antagonist, is added to the midbrain tectum The synaptic current evoked in the neuron is completely blocked.

Figures 12a,a' and Figures 12e,e' show that neither in the midbrain of adult mice injected with the control virus AAV-mCherry or AAV-Ascl1/mCherry, respectively, co-immunolabeling showed that mCherry did not co-localize with NeuN. Figures 12b,b' and Figures 12c,c' show that mCherry does not substantially co-localize with NeuN either 16 days or 38 days after virus injection, respectively. Electrophysiological experiments in Figure 12d show that cells infected with the control virus AAV-mCherry have typical astrocytic properties. Figures 12f, 12f' and Figures 12g, 12g' show that in mice injected with the virus AAV-Ascl1/mCherry, mCherry gradually co-localized with NeuN, from 63.5±3.1% at 16 days to 92.1±1.5% at 38 days. Figure 12h shows that the majority of iN cells (9/10) had inward and outward currents in voltage-clamp mode and were able to fire action potentials 15-21 days after infection of cells infected with virus AAV-Ascl1/mCherry. Figure 12i shows that spontaneous postsynaptic currents can be recorded in most iN cells (8/10). Figure 12j shows that GFP ⁺ cells infected with the control plasmid AAV-FLEX-NLSGFP hardly express NeuN. Figure 12k shows that AAV-FLEX-Ascl1/GFP infected GFP ⁺ cells overwhelmingly expressed NeuN after 28 days of infection.

Figure 13 shows that AAV-FLEX-NLSGFP infected GFP ⁺ cells overwhelmingly express Acsbg1.

Figure 14a shows that 3 days after AAV-mCherry virus injection, the majority of mCherry ⁺ cells at the site of dorsal midbrain injury in adult mice express GFAP. Figure 14b shows that 30 days after virus infection, mCherry ⁺ cells still rarely express NeuN. Figure 14c shows that mCherry ⁺ cells mostly express NeuN 3 days after AAV-Ascl1/mCherry virus infection. Figure 14d shows that AAV-Ascl1/mCherry virus-infected mCherry ⁺ cells had greater membrane resistance and more depolarized resting membrane potential after 30 days. Figure 14e shows that the cells were unable to fire action potentials. Figures 14f-h show that all recorded cells (17/17) were able to fire multiple action potentials and receive spontaneous excitatory and inhibitory synaptic afferents.

Figure 15 shows that 3 days after AAV-mCherry virus injection, the majority of mCherry ⁺ cells at the site of dorsal midbrain injury in adult mice hardly express NeuN.

Figures 16a-d show that mCherry is hardly expressed in striatal neurons (NeuN ⁺ ), microglia (IBA1 ⁺ ), oligodendrocytes (Olig2 ⁺ ) and NG2 cells (NG2 ⁺ ). Figures 16e,f show that approximately 96% of mCherry ⁺ cells express glutamine synthetase (GS), a marker molecule of astrocytes. Figure 16g shows mCherry ⁺ cells express GS 30 days after AAV-mCherry virus injection. Figure 16h shows that the majority of mCherry ⁺ cells infected with AAV-Ascl1/mCherry virus no longer express GS. Figure 16i,j,l show that AAV-mCherry virus-infected cells had a relatively small membrane resistance (2.9 ± 1.0 MΩ, n = 7), a more hyperpolarized membrane potential, and were unable to fire after 30 days potential. 16k shows that most (15/16) of mCherry ⁺ cells infected with AAV-Ascl1/mCherry virus can detect both inward and outward currents in voltage-clamp mode. Figure 16m shows that spontaneous excitatory and inhibitory postsynaptic currents could be recorded in most (12/16) of these cells.

Figures 17a, a' show that 30 days after AAV-mCherry virus injection, mCherry ⁺ cells hardly express NeuN. Figure 17b, b'AAV-Ascl1/mCherry virus-infected mCherry ⁺ cells express NeuN.

Figure 18a shows that AAV-mCherry-infected adult mouse cortical cells (mCherry ⁺ ) rarely express NeuN 30 days after virus injection, while 18b shows that AAV-Ascl1/mCherry virus-infected cortical mCherry ⁺ cells overwhelmingly express NeuN . Figure 18c shows that cells infected with AAV-Ascl1/mCherry virus for 30 days have larger membrane resistance and a more depolarized resting membrane potential, and Figure 18d,f show that cells infected with control virus AAV-mCherry for 30 days still exhibit Similar membrane properties to astrocytes. Figure 18e,f shows that in 30 cells infected with AAV-Ascl1/mCherry virus, all recorded cells (10/10) were able to fire action potentials. Figure 18g shows that spontaneous excitatory and inhibitory postsynaptic currents could be recorded in these cells (10/10).

Figure 19a,b shows that 7 days after virus injection, in mice injected with the control virus AAV-mCherry or AAV-Ascl1/mCherry, immunoco-labeling showed that mCherry hardly co-localized with BrdU. Figures 19c,d show that 15 days after virus injection, in mice injected with the control virus AAV-mCherry or AAV-Ascl1/mCherry, immunoco-labeling showed that mCherry hardly co-localized with Ki67. Figure 19e,f show that 30 days after virus injection, in mice injected with the control virus AAV-mCherry or AAV-Ascl1/mCherry, immunoco-labeling showed that mCherry still did not co-localize with BrdU. Figure 19g shows that 30 days after virus injection, in mice injected with AAV-Ascl1/mCherry, immunoco-labeling showed that mCherry did not co-localize with Ki67.

Figure 20a,b shows that 7 days after virus injection, in the midbrain of mice injected with the control virus AAV-mCherry or AAV-Ascl1/mCherry, immunoco-labeling showed that mCherry hardly interacted with the oligodendrocyte marker GST- π colocalization. Figure 20c,d show that 30 days after virus injection, in the midbrain of mice injected with the control virus AAV-mCherry or AAV-Ascl1/mCherry, immunoco-labeling showed that mCherry was rarely co-labeled with Olig2, a marker of oligodendrocytes. position. Figure 20e-h shows that 30 days after virus injection, in the striatum and cortex of mice injected with the control virus AAV-mCherry or AAV-Ascl1/mCherry, immunoco-labeling showed that mCherry was rarely associated with the hallmarks of oligodendrocytes Olig2 colocalization.

Figures 21A and B show the firing patterns of dorsal midbrain neurons in wild-type mice (P42-P70) and Gad67-GFP (P51-P55) mice, respectively. Figures 21C, D show neuronal firing patterns induced from juvenile and adult mice, respectively. Figure 21E shows the classification statistics of the firing patterns of the above four mouse dorsal midbrain neurons.

Figure 22a, a', b, b' show the expression of GFAP (reactive astrocyte marker) and IBA1 (microglia marker) around the injection site in mice 7 days after AAV injection with glass electrodes Happening. Figures 22c, c', d, and d' show the expression of GFAP and IBA1 around the injection site in mice 7 days after AAV injection with a 31G needle.

Detailed ways

After extensive and in-depth research, the inventors unexpectedly discovered for the first time that overexpression of the Ascl1 gene or its protein can effectively induce transdifferentiation of astrocytes into neuronal cells with normal electrophysiological functions, and in vivo and in vitro , has such a transdifferentiation effect on astrocytes in normal or damaged form. In addition, the inventors have also confirmed through experiments that astrocytes from different parts (dorsal midbrain, striatum and cerebral cortex) can differentiate into neurons in the presence of Ascl1. Therefore, this method is expected to be an effective method for culturing neuronal cells in vitro and stimulating the generation of new neuronal cells in adults, thereby being widely used in the treatment of neurological diseases, such as neurodegenerative diseases, central nervous system trauma diseases and so on. On this basis, the present invention has been completed.

Atheinoscalen complex homolog-like 1 (Ascl1) gene or its protein or its promoter

No setae-scute complex homolog-like 1 gene or its protein, Ascl, achaete-scute complexhomolog-like 1, bHLH class of transcription factors. Ascl1GenBank:U68534.2, its protein sequence is as shown in SEQ ID NO.:1;

Its NCBI Reference Sequence: NM_008553.4; the mRNA sequence is shown in SEQ ID NO.:2.

The promoter of Ascl1 is not particularly limited, and can be any substance that promotes the expression and/or activity of the Ascl1 gene or its protein, such as small-molecule compounds and stimulatory miRNAs. Those skilled in the art can screen Ascl1 promoters according to existing databases. It should be understood that, based on the transdifferentiation induction effect of Ascl1 on astrocytes disclosed in the present invention, those skilled in the art can reasonably predict that any substance that has a promoting effect on Ascl1 will induce transdifferentiation of astrocytes. effect.

astrocytes

Astrocytes are the most abundant type of cells in the mammalian brain. They perform many functions, including biochemical support (such as forming the blood-brain barrier), providing nutrients to neurons, maintaining extracellular ion homeostasis, and participating in repair and scarring following brain and spinal cord injury. According to the content of glial filaments and the shape of cell processes, astrocytes can be divided into two types: fibrous astrocytes are mostly distributed in the white matter of the brain and spinal cord, with elongated processes and fewer branches. , the cytoplasm contains a lot of glial filaments; protoplasmic astrocytes (protoplasmic astrocytes) are mostly distributed in the gray matter, with thick and short cell processes and many branches.

The astrocytes that can be used in the present invention are not particularly limited, including various astrocytes derived from the central nervous system of mammals, such as those derived from the striatum, spinal cord, dorsal midbrain or cerebral cortex, preferably , originating from the dorsal midbrain or cerebral cortex.

In the present invention, astrocytes from various sources have higher induction and transformation efficiency, among which the induction efficiency of cells derived from cerebral cortex is the highest, followed by the dorsal midbrain.

Typically, astrocytes are specific markers for GFAP, and astrocytes in gray matter have relatively low GFAP expression but express Acsbg1 and GS. And when induced by the method of the present invention, these astrocytes exhibit neuronal cell-specific markers such as Tuj1, MAP2 and synapsin I.

functional neuron

As used herein, the term "functional neuron" refers to neuronal cells with normal neuronal electrophysiological activity, including GABA, transdifferentiated from astrocytes in the presence of exogenous Ascl1 gene or protein Neuron cells and glutamatergic neuron cells.

Typically, the functional neuronal cells have the following properties:

(a) Expression of neuronal markers Tuj1, MAP2 and Synapsin I;

(b) capable of firing action potentials and capable of forming synaptic connections.

Expression vector

The expression vector that can be used in the present invention is not particularly limited, and can be any expression vector containing the Ascl1 protein coding sequence that can integrate into astrocytes and express exogenous Ascl1 protein. For example, a viral vector can be any viral vector that can utilize the characteristics of a virus to transmit its genome and bring genetic material into other cells for infection. Can occur in whole in vivo or in cell culture. Including lentiviral vector, adenoviral vector, herpes virus vector, poxvirus vector.

In the present invention, a preferred expression vector is a lentiviral vector. For example, the cDNA of the mouse Ascl1 gene was cloned into the lentiviral expression vector FUGW-IRES-EGFP by conventional PCR technology to obtain FUGW-Ascl1, and the sequence encoding GFP was replaced with other fluorescent proteins, such as tdTomato, to form FUW-Ascl1 - tdTomato vector.

In one embodiment of the present invention, a method for packaging Ascl1 lentiviral vectors can be performed according to conventional methods, preferably, "Production and purification of lentiviral vectors" (Tiscornia, G., Singer, O. & Verma, Packaging of lentiviral vectors was performed by the method described in I.M..Nat.Protoc. 1, 241-245 (2006)).

Induction method

The present invention also provides methods for inducing transdifferentiation of astrocytes into functional neuronal cells in vitro and in vivo, respectively.

In vitro, the method includes the steps of: infecting astrocytes with an Ascl1 vector (eg, lentivirus), and maintaining the infected cells in culture for at least 10 days, more preferably, more than 20 days, so that the astrocytes are cultured transformed into mature neuronal cells.

In vivo, the Ascl1-containing vector can be administered (eg, injected) to a desired site of the subject containing astrocytes, such as the dorsal midbrain, striatum, or cerebral cortex. Typically, such administration allows injection into undamaged and damaged nervous system tissue to induce transdifferentiation of astrocytes in specific parts of the nervous system.

Pharmaceutical composition and mode of administration

The present invention also provides a composition useful for inducing astrocytes to form functional neurons. The pharmaceutical composition of the present invention can also treat or prevent neurodegenerative diseases, traumatic diseases of the nervous system, and the like.

The pharmaceutical composition of the present invention includes the above-mentioned expression vector (eg virus particle) of the present invention, or the exogenous Ascl1 protein itself, and a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention usually contains 10 ⁷ -10 ¹² PFU/ml lentivirus or AAV virus particles, preferably 10 ⁸ -10 ¹² PFU/ml lentivirus or AAV virus particles, more preferably 10 ⁹ -10 ¹² PFU/ml of lentivirus or AAV virus particles.

"Pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent and includes various excipients and diluents.

The term refers to pharmaceutical carriers which are not themselves essential active ingredients and which are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the compositions may contain liquids such as water, saline, buffers. In addition, auxiliary substances such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. The carrier may also contain cell transfection reagents.

Generally, the pharmaceutical composition of the present invention can be obtained after mixing the expression vector (lentiviral particle) and a pharmaceutically acceptable carrier.

The mode of administration of the composition of the present invention is not particularly limited, and representative examples include (but are not limited to): intravenous injection, subcutaneous injection, brain injection, and the like.

application

The Ascl1 of the present invention can be used to prepare and induce astrocytes to generate functional neurons, so that the newly induced neurons can be applied to various diseases related to the reduction of the number of neurons, cell decline, apoptosis or neuron function decline. Wherein, the nervous system-related diseases include epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), neuronal death caused by stroke, and the like.

Beneficial effects of the present invention

In the present invention, a single transcription factor Ascl1 can transdifferentiate the astrocytes in the dorsal midbrain into functional neurons in vitro. Using a GFAP-AAV vector specifically expressed in astrocytes, Ascl1 was able to transdifferentiate adult mouse dorsal midbrain, striatum, and somatosensory cortex astrocytes into functional neurons. These induced neurons gradually mature, take on neuronal morphology and express neuronal marker molecules, and these neurons are able to fire action potentials, receive synaptic afferents from other neurons, and release neurotransmitters and other Neurons make synaptic connections. Therefore, this method is expected to be an effective method for culturing neuronal cells in vitro and stimulating the generation of new neuronal cells in adults, thereby being widely used in the treatment of neurological diseases, such as neurodegenerative diseases, central nervous system trauma diseases and so on.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the following examples, usually according to normal conditions, such as people such as Sambrook, molecular cloning: conditions described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or according to manufacturer the proposed conditions. Percentages and parts are weight percentages and parts unless otherwise specified.

general approach

astrocyte culture

For the preparation of astrocytes, refer to "Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue" (McCarthy, K.D. & deVellis, J.J. Cell Biol. 85, 890-902 (1980)). The dorsal midbrain of postnatal day 5-7 mice or adult mice was removed and digested with 0.25% trypsin for 15 min. The blown cells were cultured in DMEM/F12 solution containing 10% serum for 7-9 days. After shaking to remove oligodendrocytes, the resulting cells are astrocytes.

Immunochromic

The immunochromatization of cultured cells refers to "Direct conversion of fibroblasts to functionalneurons by defined factors" (Vierbuchen, T. et al. Nature 463, 1035-1041 (2010)). Immunochromatization of tissue sections combined with in situ hybridization and immunofluorescence Color double-labeling experiments were performed according to published methods. The primary antibodies used for immunochromatography include: mouse anti-GFAP (Millipore, 1:1,000), rabbit-GFAP (DAKO, 1:1,000), mouse anti-Tuj1 (Covance, 1:500), mouse anti-Map2 (Sigma, 1:500), rabbit anti-GFP (Invitrogen, 1:1,000), chick anti-GFP (Invitrogen, 1:1,000), mouse anti-NeuN (Millipore, 1:100), rabbit anti-Synapsin I ( Millipore, 1:1,000), rabbit anti-GABA (Sigma, 1:3,000), rabbit anti-GAD67 (Millipore, 1:200), guinea pig anti-VGAT (Synaptic Systems, 1:200), rabbit anti-Dsred ( Clontech, 1:500), mouse anti-Dsred (Santa Cruz, 1:100), rabbit anti-VGLUT1 (Synaptic Systems, 1:500), guinea piganti-VGLUT2 (Frontier Institute Co., 1:400), rabbit anti -Acsbg1 (Abcam, 1:100), mouse anti-glutamine synthetase (610518, BD Biosciences, 1:200), rabbit anti-Sox2 (Millipore, 1:500), mouse anti-S100β (Sigma, 1:1,000), rabbit anti-EAAT1(Abcam,1:500),rabbit anti-NG2(Millipore,1:200),rabbit anti-Iba1(Wako,1:500),mouse anti-CNPase(Abcam,1:500),mouse anti -O4 (Millipore, 1:500), mouse anti-Ascl1 (BD Biosciences, 1:200), rabbit anti-Sox2 (Millipore, 1:500), rabbit anti-Olig2 (AB9610, Millipore, 1:500), rabbit anti-DC X(ab77450, Abcam, 1:500).

FITC-, Cy3- and Cy5-conjugated secondary antibodies were purchased from Jackson Immunoresearch. Alexa-350-, Alexa-488- and Alexa-546-conjugated secondary antibodies were purchased from Invitrogen.

Stereotactic injection of AAV virus

AAV virus was performed with reference to mouse brain atlas. After virus injection, the dorsal midbrain, striatum, and cerebral cortex were collected at different time points for immunochromatization or brain slice recording. In preparing the injured dorsal midbrain model, virus injection was done with a 5 ml syringe and a 31G needle.

Flow cytometric sorting and quantitative RT-PCR

Cells expressing mCherry were sorted by flow cytometry. Total RNA of cells was extracted, cDNA was synthesized, and then detected by real-time quantitative PCR. GAPDH was used as an internal reference for gene expression levels.

Example 1 Plasmid construction and virus infection

The cDNA of mouse Ascl1 gene was cloned into lentiviral expression vector FUGW-IRES-EGFP to obtain FUGW-Ascl1. Substituting tdTomato for GFP in the FUGW-Ascl1 plasmid yielded FUW-Ascl1-tdTomato. Empty lentiviral expression vectors FUGW and FUW-tdTomato were used as controls, respectively. For the packaging of lentivirus, refer to the document "Production and purification of lentiviral vectors" (Tiscornia, G., Singer, O. & Verma, I.M. Nat. Protoc. 1, 241-245 (2006)).

Lentivirus was added 24 hours after astrocytes were plated and cultured, and the medium was changed 24 hours after infection: DMEM/F12, B27, Glutamax and penicillin/streptomycin. After infection 6-7, brain-derived neurotrophic factor (BDNF; PeproTech, 20 ng/ml) was added to the medium every three days.

To prepare the GFAP-AAV vector, the CMV promoter in the AAV-FLEX-Arch-GFP plasmid (Addgene) was replaced with the hGFAP promoter (2.2 kb). AAV-mCherry plasmid was obtained after inserting mCherry. The AAV-Ascl1/mCherry plasmid was obtained after Ascl1 was cloned into AAV-mCherry. Ascl1 was cloned into AAV-FLEX-Arch-GFP to obtain AAV-FLEX-Ascl1/GFP. AAV-mCherry and AAV-FLEX-NLSGFP served as controls. NLS is a nuclear localization signal, and its sequence is: VPKKKRKVEA.

Example 2 Ascl1 transdifferentiates dorsal midbrain astrocytes into neurons in vitro

First, astrocytes from the dorsal midbrain of mice 5-7 days after birth (P5-P7) were isolated and purified. The properties of these glial cells were verified by examining molecular markers of different cell types (Figure 1). The vast majority of cells expressed astrocyte marker molecules GFAP and S100β, a small number of cells expressed oligodendrocyte marker molecules O4 and CNPase, and a small number of cells expressed NG2 glial cell marker molecule NG2, which was not detected. Expression of neuron marker molecule Tuj1 and stem cell marker molecules Sox2 and Oct4.

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2.1 Transformed astrocytes express mature neuron marker molecules

10 days after astrocytes were transfected with the control lentiviral vector FUGW, astrocytes did not express the marker molecule Tuj1 of neurons (Fig. 2a), but still maintained the shape of glial cells and expressed the marker molecules of astrocytes. GFAP (Fig. 2b). In contrast, 10 days after infection with lentivirus FUGW-Ascl1, most of the astrocytes expressed the neuronal marker molecule Tuj1 and exhibited typical neuronal morphology (76.8±6.4%, n=3, 348–384 counts per count). GFP ⁺ cells, Figure 2c). After 21 days of infection with lentivirus FUGW-Ascl1, astrocytes also expressed mature neuron marker molecules MAP2 (Fig. 2d) and synapsin I (Fig. 2e).

2.2 Electrophysiological characteristics test showed that the transformed cells were functional cells

To examine whether neurons transdifferentiated from Ascl1 (induced neuronal (iN) cells) possess neuronal electrophysiological properties, whole-cell recordings were performed on these cells (Fig. 2f).

The results showed that all GFP-positive cells (63 in total) were able to generate action potentials 30-40 days after lentiviral transfection (Fig. 2g). Meanwhile, spontaneous postsynaptic currents were recorded on the vast majority of iN cells (87.3%, 55/63 cells) (Fig. 2g), indicating that these neurons can form functional synapses.

2.3 Verify that the induced neurons are derived from astrocytes

To further verify that the induced neurons were derived from astrocytes, astrocytes (tagged with GFP) from hGFAP-GFP mice were infected with lentivirus FUW-Ascl1-tdTomato. The Ascl1-induced cells changed their morphology and expressed Tuj1, while still expressing GFP (Fig. 2i), while after infection with the control lentivirus FUW-tdTomato containing only red fluorescent protein, the virus-infected cells still maintained astrocytes The morphology of cytoplasmic cells and expression of GFAP (Fig. 2h). This indicated that the induced neurons were derived from astrocytes.

2.4 Detection of transmitter properties of iN cells by immunochromatography

The experiment found that most iN cells expressed GABA (74.9±4.5%, n=3, 153-228 GFP ⁺ Tuj1 ⁺ cells were counted each time, Figure 3a), and some iN cells expressed GABAergic neuron marker molecule GAD67 (Figure 3a). 3b) and VGAT (Fig. 3d). In addition, some iN cells were found to express the glutamatergic neuron marker molecule VGLUT2 (10.8±3.8%, n=3, 134-280 GFP ⁺ Tuj1 ⁺ cells were counted each time, Figure 3f). This indicates that iN cells contain inhibitory neurons as well as excitatory neurons. To further demonstrate this electrophysiologically, iN cells were examined for their autapse.

It was found that some iN cells (19.4%, 7/36 cells) could record from self-synapses (Fig. 3g), and when CNQX, an antagonist of AMPA/kainate glutamate receptors, was added, the self-synaptic currents were completely suppressed. Block (3/3 cells). This indicates that iN cells contain glutamatergic neurons.

To verify the presence of GABA release in iN cells, astrocytes taken from the dorsal midbrain of GAD67–GFP mice were infected with lentivirus FUW-Ascl1-tdTomato (Fig. 4a), and the induced cells were found to express GFP ( Figure 4b), indicating that they may be GABAergic neurons.

Neurons isolated from the dorsal midbrain of P5-P7 wild-type mice were co-cultured 10 days after iN cell induction, and 29-40 days after lentiviral transfection, almost all tdTomato ⁺ GFP ⁺ cells (97%, 37/ 38 cells) were able to generate action potentials (Fig. 4c). Meanwhile, spontaneous postsynaptic currents were recorded on the vast majority of iN cells (89%, 34/38 cells) (Fig. 4d). In addition, some iN cells (21%, 8/38 cells) could record from self-synapses, and when bicuculline, a GABA _A receptor antagonist, was added, the self-synaptic currents were completely blocked (5/5 cells) (Fig. 3h), which indicated that iN cells contained GABAergic neurons.

The results show that astrocytes in the early postnatal dorsal midbrain can be reprogrammed into functional glutamatergic or GABAergic neurons in vitro.

Example 3 GFAP-AAV vector efficiently infects dorsal midbrain astrocytes in vivo

To explore the possibility of reprogramming astrocytes into neurons in vivo, a recombinant adeno-associated virus (AAV) vector containing red fluorescent protein (mCherry) driven by hGFAP promoter was constructed. The virus was injected into the lateral tectum of P12-P15 wild-type mice, and mCherry expression was detectable by immunostaining three days later.

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3. The co-labeling of 1mCherry and astrocyte marker molecule Acsbg1 showed that after 3 days of virus injection, regardless of the control virus AAV-mCherry (96.1±0.7%, n=3, 220-326 cells were counted each time; Fig. 5a), or viral AAV-Ascl1/mCherry (93.1±2.7%, n=3, 130-280 cells per count; Figure 5b), almost all mCherry-positive cells express Acsbg1 (representative cells are astrocytes) ). In addition, immunoco-labeling of mCherry and the neuronal marker molecule NeuN indicated that mCherry was not expressed in neurons (Fig. 7a, a', d, d'). It was also found that mCherry did not coexist in the same cells as NG2, a marker for NG2 cells (Fig. 6).

3.2 To further determine the specificity of the GFAP-AAV vector, two transgenic mice GFAP-GFP and Aldh1l1-GFP expressing GFP specifically in astrocytes were used.

After 3 days of AAV-mCherry infection, it was found that in both transgenic Aldh1l1-GFP mice (98.7±1.0%, n=3, 426-475 cells counted each, Figure 5c) and GFAP-GFP mice (93.5 ±1.4%, n=3, 123-186 cells were counted each time, in Figure 5d), the vast majority of mCherry-positive cells were also GFP-positive cells.

In addition, GFAP-CreERT2 was induced with 4-hydroxytamoxifen (4-OHT); Rosa26-CAG-tdTomato mice expressed tdTomato, and tdTomato was found to co-localize with Acsbg1 (93.8±1.6%, n=3, 169-177 cells per count) , Figure 5e).

Therefore, the GFAP promoter-driven AAV vector can specifically direct the expression of foreign genes in astrocytes in P12-P15 mice.

Example 4 Ascl1 converts juvenile mouse dorsal midbrain astrocytes into neurons in vivo

The virus AAV-mCherry or AAV-Ascl1/mCherry was injected into the lateral tectum of P12-P15 wild-type mice, and then brain tissue samples were collected at several different time points.

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4.1 Compared with the control, injection of Ascl1-containing virus gradually revealed the co-localization of mCherry and NeuN

3-5 days after virus injection, either the control virus AAV-mCherry (3.4±0.2%, n=3, 472-489 cells counted each; Fig. 7a, a'), or the virus AAV-Ascl1/mCherry (4.5±2.3%, n=3, 279-419 cells per count; Fig. 7d,d'), immunoco-labeling showed that none of mCherry co-localized with NeuN.

But from 44.2±12.5% (n=3, 309-436 cells per count) at 10-14 days after virus injection (Fig. 7e,e') to 93.1±1.7% (n=3) at 28-32 days after virus injection , 412-557 cells per count) (Fig. 7f,f'), mCherry gradually colocalized with NeuN in mice injected with the virus AAV-Ascl1/mCherry.

However, in mice injected with the control virus AAV-mCherry, both 10-14 days after virus injection (4.0±0.5%, n=3, 325-487 cells per count; Fig. 7b,b'), Also after 28-32 days (3.9±0.4%, n=3, 389-515 cells per count; Fig. 7c,c'), neither mCherry co-localized with NeuN.

4.2 Injection of Ascl1-containing virus did not increase apoptosis of neuronal cells

The results of Nissl staining showed that the cell density of the dorsal midbrain of mice injected with virus AAV-Ascl1/mCherry was basically comparable to that of mice injected with control virus AAV-mCherry (Figure 8a-8c). The results of TUNEL staining showed that apoptosis was not increased in mice injected with virus AAV-Ascl1/mCherry (Fig. 8d-8h).

Expression of mCherry was still detectable in the dorsal midbrain of mice 155 days after virus AAV-Ascl1/mCherry injection, and they colocalized well with NeuN (Figure 9). This shows that iN cells can survive for a long time in vivo.

4.3 Detection of transmitter properties of iN cells generated in vivo

The transmitter properties of iN cells generated in vivo were further examined. 45 days after virus injection, some iN cells were found to express Gad1 (13.2±4.2%, n=3, 57-180 cells per count; Figure 7g), while some iN cells expressed VGLUT2 (6.5±2.2%, n= 3, 48-118 cells were counted each time; Figure 7h).

This indicated that iN cells generated in vivo contained glutamatergic neurons and GABAergic neurons.

4.4 Induced cells are derived from astrocytes, not neural precursors

In order to detect whether there are neural stem cells in the dorsal midbrain of P12-P15 wild-type mice, the cells in vivo were isolated and cultured into spheroids. Cells isolated from the subventricular zone (SVZ) could produce a large number of neurospheres (364.9 ± 53.5 neurospheres/well (six-well plate), n=3, 333-426 neurospheres per count; Figure 7i). Whereas cells isolated from the dorsal midbrain were essentially incapable of producing neurospheres (0.8 ± 0.2 neurospheres/well (six-well plate), n = 3, 0-1 neurosphere per count; Figure 7j). In addition, to explore whether P12GFAP ⁺ cells could generate neurons at a late stage, GFAP-CreERT2 was induced with 4-OHT for 5 consecutive days (P12-P16; Rosa26-CAG-tdTomato mice expressed tdTomato, and it was found that tdTomato still did not interact with tdTomato after 30 days). NeuN co-localized (Figures 7k-7n).

These results suggest that iN cells induced from GFAP ⁺ are derived from postnatal astrocytes, rather than neural precursor cells.

Example 5 Electrophysiological properties of iN cells in vivo

To examine the electrophysiological properties of iN cells in vivo, whole-cell recordings were performed on acute brain slices at different time points after virus injection. Infected cells were identified by mCherry expression.

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5.1 Infection of cells containing Ascl1 virus can generate action potentials

In the mouse brain slices infected with the virus AAV-Ascl1/mCherry for 7-30 days, it was found that many cells had inward Na ⁺ current and outward K ⁺ current in voltage-clamp mode, and the amplitude increased with the increase of infection time. (Figures 11b-11e). Correspondingly, the ability of the detected cells to fire action potentials was also enhanced in current-clamp mode (Figures 11b-11e). Further, the morphology of the cells became more complex, and the results of biocytin remodeling also found that the detected cells formed fewer gap junctions (Figures 11b-11e). At the same time, the cell's input resistance gradually increased, while the resting membrane potential gradually decreased (Figures 11f and 11g). These results all indicate that the function of iN cells induced by transcription factor Ascl1 in vivo gradually matures.

However, in the mouse brain slices infected with the control virus AAV-mCherry, the detected cells were found to have lower impedance (1.88±0.77MΩ, n=9), higher resting membrane potential (-79.21±0.37mV, n=14) and could not fire action potentials (Fig. 11a). Meanwhile, biocytin remodeling results showed that control virus-infected cells had a typical morphology of astrocytes and were connected to neighboring astrocytes by gap junctions (Fig. 11a). These results indicate that the control virus, AAV-mCherry, is specifically expressed in astrocytes in vivo, while it does not alter the physiological properties of astrocytes.

5.2 Ascl1-induced iN is more prone to action potentials with prolonged infection, and the generated currents can be blocked by GABA _A receptor antagonists

According to the different current and voltage response modes, iN cells are divided into 4 groups: non-active cells (non-active), cells with inward current but unable to fire action potentials (inward), cells capable of firing single action potentials (sAP) and A cell capable of firing multiple action potentials (mAP).

The results showed that Ascl1-induced iN cells became more and more excited with the prolongation of infection time, and all recorded iN cells were able to fire action potentials at high frequency (50-220 Hz) 30 days after infection (Fig. 11h). In contrast, control virus-infected cells all exhibited an "inactive" state similar to astrocytes (Fig. 11h). It was further observed that there were spontaneous postsynaptic currents in iN cells, and the number of iN cells with spontaneous postsynaptic currents increased with the prolongation of infection time. After 30 days of AAV-Ascl1/mCherry virus infection, high-frequency spontaneous postsynaptic currents were found to be detected in all virus-infected cells (23/23) (Fig. 11i).

Further pharmacological experiments showed that iN cells received both excitatory glutamate input and inhibitory GABA input (Fig. 11j). Finally, iN cells (mCherry ⁺ ) formed synaptic connections with neurons in the midbrain tectum (mCherry ^- ) by double whole-cell recording (Fig. 11k). Synaptic currents evoked in neurons of the midbrain tectum were completely blocked when the GABA _A receptor antagonist bicuculline was added (Fig. 11k). This suggests that iN cells can establish GABAergic synaptic connections with surrounding neurons and integrate into existing neural circuits in vivo.

Example 6 Ascl1 transdifferentiates adult dorsal midbrain astrocytes into neurons in vivo

This experiment further investigated whether adult mouse astrocytes could be reprogrammed into neurons. The virus AAV-mCherry or AAV-Ascl1/mCherry was injected into wild-type mice at P60 and showed whether mChrrey colocalized with NeuN.

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6.1 Astrocyte-induced iN cells from adult mice can form functional synapses in vivo

In mice injected with the virus AAV-Ascl1/mCherry, mCherry gradually co-localized with NeuN, from 63.5 ± 3.1% (n = 3, 131-266 cells per count) at 16 days (Figures 12f and 12f') to 38 Day 92.1 ± 1.5% (n=3, 152-216 cells per count) (Figures 12g and 12g'). Electrophysiological recordings showed that the majority of iN cells (9/10) had inward and outward currents in voltage-clamp mode and were able to fire action potentials 15-21 days after infection in cells infected with the virus AAV-Ascl1/mCherry (Fig. 12h). At the same time, spontaneous postsynaptic currents were recorded in the majority of iN cells (8/10) (Fig. 12i). This suggests that astrocyte-induced iN cells from adult mice can form functional synapses in vivo.

In contrast, 5 days after injection, either control virus AAV-mCherry (4.2±1.4%, n=3, 182-216 cells counted per time; Fig. 12a, a') or AAV-Ascl1/mCherry ( 5.6±1.6%, n=3, 151-335 cells per count; immunoco-labeling in mice in Figure 12e,e') showed that mCherry did not co-localize with NeuN. Subsequently, it was found that in mice injected with the control virus AAV-mCherry, either 16 days after virus injection (6.7±3.6%, n=3, 236-312 cells per count; Fig. 12b,b') or 38 Days later (3.7±1.2%, n=3, 118-144 cells per count; Figure 12c,c'), mCherry did not substantially co-localize with NeuN. Electrophysiological experiments showed that cells infected with the control virus AAV-mCherry had typical astrocyte properties (Fig. 12d).

Thus, Ascl1 can transdifferentiate adult mouse dorsal midbrain astrocytes into functional neurons in vivo.

6.2 Cre-dependent Ascl1 expression can also transdifferentiate adult dorsal midbrain astrocytes into neurons

According to conventional techniques, Cre recombinase-inducible AAV viruses were prepared: AAV-FLEX-NLSGFP and AAV-FLEX-Ascl1/GFP. The AAV vector contains a FLEX sequence capable of responding to Cre recombinase. These adenoviruses were injected into the dorsal midbrain of adult Aldh1l1-Cre transgenic mice.

After 28 days of infection, AAV-FLEX-Ascl1/GFP infected GFP ⁺ cells overwhelmingly expressed NeuN (90.1±2.1%, n=3, 126-170 cells per count; Figure 12k). In addition, AAV-FLEX-NLSGFP-infected GFP ⁺ cells overwhelmingly expressed Acsbg1 (94.8±1.7%, n=3, 233-268 cells per time; Figure 13), suggesting that Cre recombinase is in astrocytes specific expression.

While GFP+ cells infected with the control plasmid AAV-FLEX-NLSGFP hardly expressed NeuN (2.9±1.1%, n=3, 121-181 cells per count; Figure 12j),

Thus, Cre-dependent Ascl1 expression can also transdifferentiate adult dorsal midbrain astrocytes into neurons.

6.3 Injured midbrain astrocytes (reactive cells) can transdifferentiate into functional neurons

A stab wound model was created in the dorsal midbrain of adult mice by needle injection of the AAV virus AAV-mCherry or AAV-Ascl1/mCherry.

At 3 days after AAV-mCherry virus injection, the majority of mCherry ⁺ cells (92.8±1.2%, n=3, 60-117 cells per count; Figure 14a) at the lesion site expressed GFAP and almost none of NeuN (2.4±1.2%). 1.3%, n=3, 69-107 cells per count; Figure 15). After 30 days of viral infection, mCherry ⁺ cells still rarely expressed NeuN (2.5±1.2%, n=3, 78-82 cells per count; Figure 14b). AAV-mCherry virus-infected cells had a relatively small membrane resistance (5.3 ± 1.9 MΩ, n = 6) and more hyperpolarized membrane potential (-81.2 ± 1.7 mV, n = 5) after 30 days (Fig. 14d), while unable to fire action potentials (Fig. 14e).

Immunofluorescence showed that mCherry ⁺ cells 30 days after AAV-Ascl1/mCherry virus infection mostly expressed NeuN (54.2±6.9%, n=3, 114-142 cells per count; Figure 14c). AAV-Ascl1/mCherry virus-infected mCherry ⁺ cells had larger membrane resistance (424.7 ± 88.7 MΩ, n = 17) and more depolarized resting membrane potential (-61.2 ± 1.6 mV, n = 17) after 30 days 17) (Fig. 14d). At the same time, all recorded cells (17/17) were able to fire multiple action potentials (Fig. 14f,g) and receive spontaneous excitatory and inhibitory synaptic afferents.

These results suggest that damaged midbrain astrocytes can be transdifferentiated into functional neurons.

Example 7 Ascl1 transdifferentiates adult mouse striatal astrocytes into neurons in vivo

To investigate whether the transdifferentiation of astrocytes into neurons by Ascl1 is region-specific, we further examined whether striatal astrocytes from adult mice could be reprogrammed into neurons. The virus AAV-mCherry or AAV-Ascl1/mCherry was injected into the striatum of adult wild-type mice (P60).

Immunostaining indicated that approximately 96% of mCherry ⁺ cells expressed glutamine synthase (GS), a marker molecule of astrocytes (Fig. 16e,f). In contrast, mCherry was hardly expressed in neurons (NeuN ⁺ ), microglia (IBA1 ⁺ ), oligodendrocytes (Olig2 ⁺ ) and NG2 cells (NG2 ⁺ ) ( FIG. 16 a-d , f ).

To determine the nature of mCherry ⁺ cells following AAV virus infection, immunotriple staining for mCherry, GS, and NeuN was performed. The results showed that the majority of mCherry ⁺ cells infected with AAV-Ascl1/mCherry virus no longer expressed GS (Fig. 16h), but NeuN (64.4±3.4%, n=3, 119-129 cells per count; Fig. 17b ) ). Whereas 30 days after AAV-mCherry virus injection, mCherry ⁺ cells expressed GS (Fig. 16g) and hardly NeuN (3.2±2.1%, n=3, 104-140 cells per count; Fig. 17a). This suggests that astrocytes transdifferentiate into neurons.

To further investigate whether the induced neurons were functional, further electrophysiological analyses were performed. Experiments showed that AAV-Ascl1/mCherry virus-infected mCherry ⁺ cells detected inward and outward currents in most (15/16) of voltage-clamp mode (Fig. 16k), and most (13/16) in current-clamp mode 16) Ability to fire action potentials (Fig. 16k,l). Furthermore, spontaneous excitatory and inhibitory postsynaptic currents were recorded in the majority (12/16) of these cells (Fig. 16m). However, AAV-mCherry virus-infected cells had a relatively small membrane resistance (2.9 ± 1.0 MΩ, n = 7), a more hyperpolarized membrane potential (-79.0 ± 0.3 mV, n = 7) after 30 days ( Fig. 16i), while action potentials could not be fired (Fig. 16j,l). This suggests that astrocytes in the adult mouse striatum can transdifferentiate into functional neurons.

Example 8 Ascl1 transdifferentiates adult mouse cortical astrocytes into neurons in vivo

8.1 This example investigates whether Ascl1 can transdifferentiate astrocytes in adult mouse cortex into neurons. Viral AAV-mCherry or AAV-Ascl1/mCherry were injected into the somatosensory cortex of adult wild-type mice (P60).

Thirty days after virus injection, it was found that the vast majority of cortical mCherry ⁺ cells infected with AAV-Ascl1/mCherry virus expressed NeuN (93.9±1.2%, n=3, 132-147 cells per count; Figure 18b). In contrast, AAV-mCherry-infected cortical cells (mCherry ⁺ ) rarely expressed NeuN (2.6±0.8%, n=3, 120-133 cells per count; Figure 18a).

Further electrophysiological analysis found that cells infected with AAV-Ascl1/mCherry virus had a larger membrane resistance (163.3±35.9MΩ, n=10) and a more depolarized resting membrane potential (-67±2.2mV) , n=8) (Fig. 18c), and all recorded cells (10/10) were able to fire action potentials (Fig. 18e,f). Likewise, spontaneous excitatory and inhibitory postsynaptic currents could be recorded in these cells (10/10) (Fig. 18g). In contrast, cells infected with the control virus AAV-mCherry for 30 days still exhibited membrane properties similar to those of astrocytes (membrane resistance, 2.3 ± 0.5 MΩ, n = 8; resting membrane potential, -78.8 ± 0.8 mV, n=7, Fig. 18c; unable to fire action potentials, Fig. 18d,f).

This suggests that Ascl1 can transdifferentiate astrocytes in the adult mouse cerebral cortex into functional neurons.

8.1 In order to further determine whether Ascl1 induced astrocytes to be neurons or not through the proliferation stage, BrdU was continuously injected intraperitoneally to label the proliferating cells on days 3-7 and 3-30 after virus injection.

Seven days after injection, either control virus AAV-mCherry (2.2±0.5%, n=3, 325-410 cells per count; Figure 19a) or AAV-Ascl1/mCherry (1.6±0.3%, n=3) 3, 213-307 cells were counted each time; in mice of Figure 19b), immunoco-labeling showed that mCherry hardly co-localized with BrdU. Thirty days after injection, either the control virus AAV-mCherry (4.3±1.2%, n=3, 230-363 cells per count; Figure 19e) or AAV-Ascl1/mCherry (1.6±1.0%, n=3) or AAV-Ascl1/mCherry (1.6±1.0%, n= 3, 204-290 cells per count; Figure 19f) in mice, immunoco-labeling showed that mCherry still barely co-localized with BrdU. Meanwhile, mCherry colocalized with NeuN in mice injected with the virus AAV-Ascl1/mCherry, whereas mCherry did not colocalize with NeuN in mice injected with the control virus AAV-mCherry (Fig. 19e,f). It was further found that 15 days after virus injection, either control virus AAV-mCherry (1.0 ± 0.6%, n = 3, 192-246 cells per count; Figure 19c) or AAV-Ascl1/mCherry (0.9 ± 0.3 %, n=3, 185-276 cells per count; in mice of Figure 19d), immunoco-labeling showed that mCherry hardly co-localized with Ki67. Co-immunolabeling was performed 30 days after injection in mice injected with either the control virus AAV-mCherry or AAV-Ascl1/mCherry (0.5±0.1%, n=3, 171-248 cells per count; Figure 19g). showed that mCherry still barely colocalized with Ki67.

These results suggest that Ascl1-induced in vivo reprogramming does not go through the proliferative phase.

8.3 This example also investigates whether Ascl1 overexpression can convert astrocytes into oligodendrocytes. Seven days after injection, 237-303 cells were counted per injection with either control virus AAV-mCherry (0.4 ± 0.1%, n=3; Figure 20a) or AAV-Ascl1/mCherry (0.4 ± 0.3%, n= 3, 219-338 cells were counted each time; in the mouse midbrain of Figure 20b), immunoco-labeling showed that mCherry hardly co-localized with GST-π, a marker of oligodendrocytes. Thirty days after injection, either the control virus AAV-mCherry (2.8±2.2%, n=3, 308-393 cells counted each; Figure 20c) or AAV-Ascl1/mCherry (3.3±0.4%, n=3) were injected 3, 278-327 cells were counted each time; Figure 20d) mouse midbrain, immunoco-labeling showed that mCherry hardly co-localized with Olig2, another marker of oligodendrocytes. Meanwhile, in mice injected with the virus AAV-Ascl1/mCherry, mCherry colocalized with NeuN (Fig. 20d). These results suggest that overexpression of Ascl1 in the midbrain transdifferentiates astrocytes into neurons rather than oligodendrocytes. At the same time, mCherry was also found to hardly co-localize with Olig2 in the virus-injected striatum, regardless of whether it was injected with the control virus AAV-mCherry (3.2 ± 1.2%, n = 3, 156-181 cells per count; Figure 20e ) or AAV-Ascl1/mCherry (4.4±1.7%, n=3, 137-199 cells per count; Figure 20f). Further, either control virus AAV-mCherry (3.1±1.4%, n=3, 124-197 cells counted each; Figure 20g) or AAV-Ascl1/mCherry (3.2±1.4%, n=3, each 119-145 cells were counted; in the cortical brain tissue of Fig. 20h), mCherry hardly co-localized with Olig2.

These results suggest that overexpression of Ascl1 in the striatum and cortex also transdifferentiates astrocytes into neurons rather than oligodendrocytes.

Example 9 Electrophysiological properties of transdifferentiated and endogenous neurons in the dorsal midbrain

In order to investigate whether transdifferentiated neurons and endogenous brain neurons have similar properties, this example further compares the electrophysiological properties of transdifferentiated neurons and endogenous neurons in the dorsal midbrain.

Whole-cell recordings from the dorsal midbrain of acute brain slices from wild-type mice (P42-P70) and Gad67-GFP (P51-P55) mice revealed a neuronal impedance of 489.1 ± 131.1 MΩ, respectively (n = 21 , wild-type mice) and 326.0 ± 31.9 MΩ (n = 17, Gad67-GFP mice), the resting membrane potentials were -57.6 ± 2.0 mV (n = 19, wild-type mice) and -57.1 ± 1.9 mV, respectively (n=15, Gad67-GFP mice) (Fig. 21A,B). These results are consistent with those from juvenile mice (impedance, 177.3±16.6, n=23; resting membrane potential, -61.9±1.0, n=8) and adult mice (impedance, 240.0±81.9, n=9; resting membrane Potentials, -61.0±1.2, n=6) (FIG. 21C, D) induced similar neurons.

Furthermore, neurons in the dorsal midbrain of wild mice were classified into five main types based on neuron-specific firing patterns. found that the vast majority of neurons induced from juvenile mice as well as from adult mice (P12-P15: 95.6%, 22/23; P60: 100%, 9/9) could be classified into these wild-type mice and Gad67 -GFP mouse firing patterns in normal neurons (Fig. 21C,D,E). In addition, some induced neurons (P12-P15, 30-49 days: 82.6%, 19/23; P60, 15-21 days: 77.8%, 7/9) exhibited the same performance as Gad67-GFP mouse neurons firing patterns, suggesting that they may be GABAergic neurons.

Taken together, these results demonstrate that neurons transdifferentiated from juvenile as well as adult mice have similar electrophysiological properties to endogenous midbrain neurons.

Furthermore, in most experiments involving stereotaxic injection of AAV virus, glass electrodes with a diameter of 18-20 μm were used for injection, whereas injections were performed with a 31G needle with a diameter of about 260 μm in the injury model. To compare the difference in damage between these two injection conditions, immunofluorescence experiments were performed on mice 7 days after midbrain injection to detect GFAP (reactive astrocyte marker) and IBA1 (microglia marker) around the injection site. ) expression. The results showed that the GFAP-positive reactive astrocytes around the injection site of the glass electrode-injected mice were significantly less than those of the mice injected with the 31G needle (glass electrode: 14.2±2.2, n=3, Fig. 22a, a'; 31G Needle: 113.6±15.7, n=3; Fig. 22c, c'). Moreover, the number of IBA1-positive microglia around the injection site of the mice injected with the glass electrode was significantly less than that of the mice injected with the 31G needle (glass electrode: 24.3±5.4, n=3, Fig. 22b, b′; 31G needle: 74.3± 12.0, n=3; Fig. 22d, d'). In the cortex, a similar situation was observed, with significantly more GFAP- or IBA1-positive glial cells surrounding the site of needle injection (GFAP: 84.1±12.1; IBA1: 57.3±7.5; n=3 mice) than glass electrode injections mice (GFAP: 18.3±0.6; IBA1: 18.4±2.9; n=3 mice). These results suggest that AAV injection with a 31G needle caused greater damage to brain tissue than glass electrode injection.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (9)

1. Use of an ashaped-cutis squamosa complex homolog-like 1(Ascl1) gene or a protein thereof or a promoter thereof for the preparation of a pharmaceutical composition for inducing astrocytes to form functional neuronal cells;

wherein the astrocytes are derived from the striatum, spinal cord, dorsal midbrain or cerebral cortex;

wherein the GenBank number of the Ascl1 gene is U68534.2, and the protein sequence is shown as SEQ ID No. 1;

and, the functional neurons comprise glutamatergic and/or GABA-ergic neurons; and said functional neuron is capable of firing action potentials and forming synaptic connections;

the pharmaceutical composition comprises an expression vector, wherein the expression vector contains an Ascl1 protein coding sequence, can be integrated into astrocytes and expresses exogenous Ascl1 protein in the astrocytes, and is an adeno-associated virus (AAV) vector;

wherein the CMV promoter is replaced by the hGFAP promoter in the expression vector.

2. The use of claim 1, wherein said astrocytes are derived from the cortex or dorsal mesencephalon.

3. The use according to claim 1, wherein the astrocytes comprise astrocytes in a normal state and in a damaged state.

4. The use according to claim 1, wherein the mRNA encoding the Ascl1 gene has NCBIReference Sequence number NM-008553.4 and the mRNA Sequence is shown as SEQ ID No. 2.

5. The use of claim 1, wherein said pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

6. The use according to claim 1, wherein the astrocytes are of striatum origin.

7. An in vitro non-therapeutic method of transdifferentiating astrocytes into functional neuronal cells comprising the steps of:

culturing astrocytes in the presence of exogenous Ascl1 protein, thereby inducing astrocytes to form neuronal cells;

wherein the astrocytes are derived from the striatum, spinal cord, dorsal midbrain or cerebral cortex;

wherein the GenBank number of the Ascl1 gene is U68534.2, and the protein sequence is shown as SEQ ID No. 1;

and, the functional neurons comprise glutamatergic and/or GABA-ergic neurons; and said functional neuron is capable of firing action potentials and forming synaptic connections;

and, the exogenous Ascl1 protein is obtained by an expression vector, the expression vector contains an Ascl1 protein coding sequence, the expression vector can be integrated into astrocytes and expresses exogenous Ascl1 protein in the astrocytes, and the expression vector is an adeno-associated virus AAV vector;

wherein the CMV promoter is replaced by the hGFAP promoter in the expression vector.

8. The method of claim 7, wherein the functional neuronal cell has one or more of the following characteristics:

(a) a marker for expressing neurons Tuj1, MAP2, NeuN or Synapsin I;

(b) action potentials can be issued and synaptic connections can be formed.

9. The method of claim 8, wherein the functional neuronal cell does not express a Gfap, S100 β, Acsbg1, Sox2, or Pax6 marker.

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