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

Abstract

The invention provides application of Ascl1 in inducing the transdifferentiation of astrocytes into functional neurons. Specifically, provided is a use of a setaria-cuticle complex homolog-like 1(Ascl1) gene or a protein thereof or a promoter thereof for preparing a pharmaceutical composition for inducing astrocytes to form functional neuronal cells; and/or (ii) for the preparation of a pharmaceutical composition for the treatment of a neurological disease. The method is expected to become an effective method for culturing the neuron cells in vitro and stimulating and generating new neuron cells in an adult, so that the method is widely applied to treatment of nervous system diseases, such as neurodegenerative diseases, central nervous traumatic diseases and the like.

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 particularly relates to a method for inducing the transdifferentiation of astrocytes into functional neuronal cells and application thereof.

Background

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 studies have found that specific transcription factors can directly induce fibroblasts into functional neurons, further indicating that non-neuronal cells can be directly transdifferentiated into neurons. Astrocytes in the cerebral cortex of postnatal mice can be transdifferentiated into glutamatergic or gabaergic neurons in vitro, for example, by overexpressing the transcription factor Neurog2 or Dlx 2. Some studies have also shown that non-neuronal cells can be reprogrammed in vivo into neurons or neural precursor cells. However, it is currently unknown whether astrocytes iN vivo can transdifferentiate into neurons and whether these induced neurons (iN) can integrate into an already existing neural circuit.

At present, the overexpression of the preneurog 2 protein is capable of reprogramming astrocytes cultured in vitro from the cerebral cortex of early-born mice into glutamatergic neurons that can form synaptic connections. Overexpression of the proneuroprotein NeuroD1 allows reprogramming of reactive astrocytes after injury to the cerebral cortex of mice into neurons. However, there is currently no method or pathway by which astrocytes in a normal state can be transdifferentiated 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 an innovative therapeutic approach in the repair of brain function.

Increasing evidence supports the very high heterogeneity of astrocytic lineages. It is envisaged that different sources of astrocytes may influence their transdifferentiation into neurons. In this study, it was found that the single transcription factor Ascl1 can efficiently transdifferentiate astrocytes in the dorsal mesencephalon of postnatal mice into neurons that can form synaptic connections in vitro. In addition, a gene expression system specifically targeting astrocytes in vivo was designed, and a single transcription factor Ascl1 was found to be capable of inducing the transdifferentiation of astrocytes in vivo into functional neurons.

Disclosure of Invention

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

In a first aspect of the invention, there is provided a use of an ashless-cuticular complex homolog-like 1(Ascl1) gene or a protein thereof for (i) preparing a pharmaceutical composition for inducing astrocytes to form functional neuronal cells; and/or (ii) for the preparation of a pharmaceutical composition for the treatment of a neurological disease.

In another preferred embodiment, the Ascl1 gene or the protein thereof is derived from a mammal, preferably from a human, a mouse or a rat.

In another preferred embodiment, the astrocytes include astrocytes in a normal state and in 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 mRNA NCBI Reference Sequence number of the encoding Ascl1 gene is NM-008553.4, and the mRNA Sequence is shown in SEQ ID NO. 2.

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

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

In another preferred embodiment, the functional neurons comprise glutamatergic neurons and/or GABA-ergic neurons.

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

In a second aspect of the invention, an expression vector is provided, wherein the expression vector comprises an Ascl1 protein coding sequence, and the expression vector can be integrated into an astrocyte and can express an exogenous Ascl1 protein in the astrocyte.

In another preferred embodiment, the expression vector comprises a plasmid or a viral vector.

In another preferred embodiment, the viral vector infects astrocytes.

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

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

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

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

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

In another preferred embodiment, the viral vector is prepared as follows:

introducing a polynucleotide sequence having the coding sequence of Ascl1 into a packaging cell of a viral particle, thereby forming said viral vector.

In a third aspect of the invention, there is provided a host cell having a polynucleotide encoding an Ascl1 protein integrated into its chromosome or comprising an expression vector according to the second aspect of the invention.

In another preferred embodiment, the host cell is an astrocyte.

In a fourth aspect of the present invention, there is provided an in vitro non-therapeutic method for transdifferentiating astrocytes into functional neuronal cells, comprising the steps of:

astrocytes were cultured in the presence of exogenous Ascl1 protein, thereby inducing 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 expression of the expression vector of the second aspect of the invention.

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

In a fifth aspect of the present invention, there is provided a functional neuronal cell and/or neuronal cell population transdifferentiated from astrocytes, wherein the functional neuronal cell and/or neuronal cell population is obtained by the method according to the fourth aspect of the present invention, and the functional neuronal cell and/or neuronal cell population has one or more of the following characteristics:

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

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

In another preferred example, in the neural cell population, the neuronal cells do not express markers such as Gfap, S100 β, Acsbg1, Sox2 or Pax 6.

In another preferred embodiment, the non-expression includes substantially non-expression, e.g., at least 60%, 70%, 80%, 90%, or 100% of the neuronal cells do not express markers such as Gfap, S100 β, Acsbg1, Sox2, or Pax 6.

In a sixth aspect of the invention, there is provided a use of a functional neuron according to the fifth aspect of the invention, wherein the functional neuron cell and/or the neuron cell population is/are used for preparing a pharmaceutical composition for treating a neurological disease.

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

In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising (a) an expression vector or an Ascl protein according to the second aspect of the invention, or (B) a functional neuron according to the fifth aspect of the invention; and (C) a pharmaceutically acceptable carrier.

In an eighth aspect of the present invention, there is provided a method of treating a neurological condition, comprising the steps of:

administering to a subject in need thereof a safe and effective amount of a pharmaceutical composition according to the seventh aspect of the invention, thereby treating a neurological disease.

In a ninth aspect of the invention, there is provided a method of (a) screening for a candidate compound for the treatment of a neurological disease; and/or (b) a method of screening for a candidate compound that induces the transdifferentiation of astrocytes into functional neuronal cells, comprising the steps of:

(i) adding a test compound into the cell culture system to serve as a test group, and taking the cell culture system without the test compound as a control group;

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

wherein, when E1 is significantly higher than E0 in the test group, the test compound is indicative of (a) a candidate compound for treating a neurological disease; and/or (b) a candidate compound that induces the transdifferentiation of astrocytes into functional neuronal cells.

In another preferred embodiment, the cells are astrocytes.

In another preferred example, the significantly higher is that E1 is higher than E0, and has statistical difference; preferably, E1 is ≧ 2E 0.

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

(iii) adding a test compound into the astrocyte culture system to serve as a test group, and taking the astrocyte culture system without the test compound as a control group;

(iv) comparing the ratio of astrocytes to functional neurons in the test group, thereby determining whether the test compound is (a) a candidate compound for treating a neurological disease; and/or (b) screening for candidate compounds that induce the transdifferentiation of astrocytes into functional neuronal cells;

wherein, when the ratio of astrocyte to functional neurons T1 in the test group is significantly higher than the ratio T0 in the control group, it is indicative that the test compound is (a) a candidate compound for treating a neurological disease; and/or (b) screening for candidate compounds that induce the transdifferentiation of astrocytes into functional neuronal cells.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIGS. 1a-h show the characterization of isolated and purified astrocytes, with most of the cells expressing the astrocyte marker molecules GFAP and S100 β, a small number of the cells expressing oligodendrocyte marker molecules O4 and CNPase, and a small number of the cells expressing NG2 glial marker molecule NG2, and no detectable expression of the neuronal marker molecule Tuj1 and the stem cell marker molecules Sox2 and Oct 4.

FIG. 2 shows the expression of a neuronal marker molecule by astrocytes after transdifferentiation induced by a lentiviral vector. FIGS. 2a-b show that 10 days after transfection of astrocytes with the control lentiviral vector FUGW, astrocytes do not express the neuronal marker molecule Tuj1, and still maintain glial cell morphology while expressing the astrocyte marker molecule GFAP. FIG. 2c shows that 110 days after infection with lentivirus FUGW-Ascl, most astrocytes expressed the neuronal marker molecule Tuj1, while exhibiting typical neuronal morphology. FIGS. 2d and 2e show that astrocytes also express the marker molecules MAP2 and synapsin I of mature neurons after 121 days of lentiviral FUGW-Ascl infection, respectively. Figure 2f shows a whole cell electrophysiological recording of these neurons. FIG. 2g shows that all GFP positive cells (63 iN total) were able to generate action potentials 30-40 days after lentivirus transfection, and that spontaneous postsynaptic currents were recorded on the vast majority of iN cells (87.3%, 55/63 cells). FIG. 2h-i shows the results of FUW-Ascl1-tdTomato and control virus infection of astrocytes (astrocytes with GFP marker) from hGFAP-GFP mice. Wherein, FIG. 2h shows that after infection with control lentivirus FUW-tdTomato containing only red fluorescent protein, the virus-infected cells still maintained astrocyte morphology and expressed GFAP; FIG. 2i shows that overexpression of Ascl1 with lentivirus induces morphological changes in astrocytes, while expressing Tuj 1. These transdifferentiated neurons still had GFP expression.

FIG. 3 shows transmitter properties of induced cells. Fig. 3a shows that most of the iN cells express GABA, while fig. 3b and fig. 3d show that a fraction of the iN cells express gabaergic neuronal marker molecules GAD67 and VGAT, respectively. FIGS. 3c and e show that control virus infected cells do not express the GABAergic neuron marker molecule VGAT and glutamateThe aminoergic neuron marker molecule VGLUT 2. Figure 3f shows that a portion of the iN cells express glutamatergic neuronal marker molecule VGLUT 2. FIG. 3g shows that a fraction of iN cells (19.4%, 7/36 cells) were able to register self-synapses whose current was completely blocked (3/3 cells) when the AMPA/kainate glutamate receptor antagonist CNQX was added. FIG. 3h shows that a fraction of iN cells (21%, 8/38 cells) were able to register to self-synapse when GABA was added_AThe receptor antagonist bicuculine completely blocked the self-synaptic current (5/5 cells).

FIGS. 4a and 4b show that astrocytes from the dorsal mesencephalon of GAD67-GFP mice were infected with lentivirus FUW-Ascl1-tdTomato and the induced cells were found to express GFP. FIG. 4c shows that the addition of neurons isolated from dorsal mesencephalon of P5-P7 wild-type mice after 10 days of iN cell induction, almost all tdTomato at 29-40 days after lentivirus transfection⁺GFP⁺All of the cells (97%, 37/38 cells) were able to generate action potentials. Fig. 4d shows that spontaneous postsynaptic currents could be recorded on the vast majority of iN cells (89%, 34/38 cells).

FIGS. 5a and 5b show that almost all cells positive for mCherry express Acsbg1, 3 days after postnatal dorsal injection of virus in mice, 12-15 days postnatal, whether control virus AAV-mCherry or viral AAV-Ascl1/mCherry, respectively. FIGS. 5c and 5d show that 3 days after AAV-mCherry infection, the vast majority of mCherry-positive cells were also GFP-positive in both the transgenic mice Aldh1l1-GFP and GFAP-GFP mice, respectively. FIG. 5e shows the induction of GFAP-CreERT2 with 4-hydroxytifen (4-OHT); rosa26-CAG-tdTomato mice express tdTomato, which was found to co-localize with Acsbg 1.

Figure 6 shows that mCherry does not co-exist with NG2 cell marker NG2 in the same cell.

FIG. 7a, a 'FIG. 7d, d' show that after 3-5 days dorsal midbrain in mice 12-15 days after control virus AAV-mCherry and virus AAV-Ascl1/mCherry injections, respectively, the immune co-targeting shows that mCherry is not co-localized with NeuN; FIGS. 7b, b ', 7c, c' show that mCherry did not occur in AAV-mCherry mice 10-14 days and 28-32 days after control virus injection, respectivelyCo-localising with NeuN. FIGS. 7e, e 'and 7f, f' show that in mice injected with viral AAV-Ascl1/mCherry, mCherry gradually co-localizes with NeuN: from 44.2. + -. 12.5% (n ═ 3, counts each of 309-. Fig. 7g shows that 45 days after virus injection, a fraction of the iN cells expressed Gad1, and fig. 7h shows that a fraction of the iN cells also expressed VGLUT 2. Fig. 7i and 7j show that cells isolated from the subventricular zone (SVZ) can produce a large number of neurospheres while cells isolated from the dorsal mesencephalon can not produce neurospheres substantially. FIGS. 7k-7n show induction of GFAP-CreERT2 with 4-OHT for 5 consecutive days (P12-P16); rosa26-CAG-tdTomato mice expressed tdTomato, and it was found that tdTomato still did not co-localize with NeuN after 30 days. Therefore, the slave GFAP⁺Induced iN cells are derived from postnatal astrocytes, not neural precursor cells.

FIGS. 8a-8c show that the cell density of dorsal mesencephalon of mice injected with virus AAV-Ascl1/mCherry is substantially comparable to mice injected with control virus AAV-mCherry. FIGS. 8d-8h show that there was no increase in apoptosis in mice injected with viral AAV-Ascl 1/mCherry.

FIG. 9 shows that mCherry expression was still detectable in the dorsal mesencephalon of mice 155 days after injection of viral AAV-Ascl1/mCherry, and that they were well co-localized with NeuN.

FIG. 10 shows gene expression of murine iN cells injected with viral AAV-Ascl 1/mCherry. Analysis of fluorescent real-time quantitative PCR was performed by sorting mCherry + cells at different time points ( days 4, 10, 30) with flow cytometric sorting. It was found that the expression of the astrocyte marker molecules (Gfap, S100 β and Acsbg1) was gradually decreased, the expression of the neuronal marker molecules (Tuj1, Map2 and NeuN) was gradually increased, and the neuronal precursor marker molecules (Sox2 and Pax6) were not substantially detected.

FIG. 11a shows that in the brain of mice infected with control virus AAV-mCherry, the detected cells were found to have lower impedance, higher resting membrane potential, and failed to deliver action potentials. The results of biocytin (biocytin) remodeling showed that control virus infected cellsHas the typical morphology of astrocytes and is connected to adjacent astrocytes by gap junctions. FIGS. 11b-11e show that in mouse brain slices infected with AAV-Ascl1/mCherry 7-30 days, many cells have inward Na in voltage clamp mode⁺Current and outward K⁺Current, and amplitude increases with increasing time of infection; in the current clamp mode, the detected cells have an increased ability to deliver action potentials, and the morphology of the cells becomes more complex. The results of Biocytin remodeling also found that the cells detected formation of gap junctions was also less. 11f and 11g show that the input resistance of the cells gradually increases, while the resting membrane potential gradually decreases. FIG. 11h shows that Ascl 1-induced iN cells become more and more excitable with increasing infection time, all recorded iN cells were able to deliver action potentials at high frequency (50-220Hz) 30 days after infection, while control virus-infected cells all exhibited astrocyte-like "inactive" status. FIG. 11i shows that high frequency spontaneous postsynaptic currents were detected on all virus-infected cells (23/23) 30 days after AAV-Ascl1/mCherry virus infection. Figure 11j shows further pharmacological experiments demonstrating that iN cells receive both excitatory glutamate input and inhibitory GABA input. FIG. 11k shows iN cells (mCherry) found by double whole cell recording⁺) Neurons with the midbrain canopy (mCherry)^-) Synapse formation and GABA addition_AThe receptor antagonist bicuculine completely blocked synaptic currents induced in neurons of the midbrain roof.

FIGS. 12a, a 'and 12e, e' show that the immune co-targeting of mCherry did not co-localize with NeuN, whether in the midbrain of adult mice injected with control virus AAV-mCherry or AAV-Ascl1/mCherry, respectively. Fig. 12b, b 'and fig. 12c, c' show that mCherry is not substantially co-localized with NeuN, either 16 days or 38 days after virus injection, respectively. The electrophysiological experiments in FIG. 12d show that cells infected with the control virus AAV-mCherry have typical astrocytic properties. FIGS. 12f, 12f 'and FIGS. 12g, 12 g' show that in mice injected with viral AAV-Ascl1/mCherry, mCherry gradually co-localizes with NeuN from 63.5 + -3.1% at 16 days to 92.1 + -1.5% at 38 days. FIG. 12h shows that after 15-21 days of infection, most of the iN cells (9/10) had inward and outward currents iN voltage clamp mode and were able to deliver action potentials iN viral AAV-Ascl1/mCherry infected cells. Fig. 12i shows that spontaneous post-synaptic current was recorded on most of the iN cells (8/10). FIG. 12j shows GFP infection with control plasmid AAV-FLEX-NLSGFP⁺Cells hardly express NeuN. FIG. 12k shows AAV-FLEX-Ascl1/GFP infected GFP after 28 days of infection⁺The vast majority of cells express NeuN.

FIG. 13 shows AAV-FLEX-NLSGFP infected GFP⁺The vast majority of cells express Acsbg 1.

FIG. 14a shows that 3 days after AAV-mCherry virus injection, the majority of mCherry sites of brain injury in the dorsal aspect of adult mice⁺The cells express GFAP. FIG. 14b shows mCherry 30 days after viral infection⁺The cells still rarely express NeuN. FIG. 14c shows mCherry 3 days after AAV-Ascl1/mCherry virus infection⁺Most cells express NeuN. FIG. 14d shows AAV-Ascl1/mCherry virus infected mCherry⁺Cells have greater membrane resistance and a more depolarized resting membrane potential after 30 days. FIG. 14e shows that the cell is unable to deliver action potentials. FIGS. 14f-h show that all cells recorded (17/17) were able to release multiple action potentials and receive spontaneous excitatory and inhibitory synaptic afferents.

FIG. 15 shows the majority of mCherry sites of dorsal midbrain injury in adult mice 3 days after AAV-mCherry virus injection⁺Cells hardly express NeuN.

FIGS. 16a-d show that mCherry is hardly expressed in striatal neurons (NeuN)⁺) Microglial cell (IBA 1)⁺) Oligodendrocyte (Olig 2)⁺) And NG2 cell (NG 2)⁺) In (1). FIG. 16e, f shows mCherry at about 96%⁺The cells express the astrocyte marker Glutamine Synthetase (GS). FIG. 16g shows mCherry 30 days after AAV-mCherry virus injection⁺The cells express GS. FIG. 16h shows AAV-Ascl1/mCherry virus infected mCherry⁺Most cells no longer express GS. FIG. 16i, j, l shows that AAV-mCherry virus-infected cells have a relative high percentage of cells after 30 daysSmall membrane resistance (in 2.9 ± 1.0M Ω, n ═ 7), more hyperpolarized membrane potential, while failing to deliver action potentials. 16k shows mCherry infected with AAV-Ascl 1/mChery virus⁺Most (15/16) of the cells are able to detect inward and outward currents in voltage clamp mode. Figure 16m shows that most (12/16) of these cells were able to record spontaneous excitatory and inhibitory postsynaptic currents.

FIGS. 17a, a' show mCherry 30 days after AAV-mCherry virus injection⁺Cells hardly express NeuN. FIG. 17b, b' AAV-Ascl1/mCherry virus infected mCherry⁺The cells express NeuN.

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

FIGS. 19a, b show that at 7 days after virus injection, the immune co-targeting showed that mCherry was barely co-localized with BrdU in mice injected with control virus AAV-mCherry or AAV-Ascl 1/mCherry. FIG. 19c, d shows that at 15 days after virus injection, the immune co-targeting showed that mCherry was barely co-localized with Ki67 in mice injected with control virus AAV-mCherry or AAV-Ascl 1/mCherry. FIG. 19e, f shows that at 30 days after virus injection, the immune co-targeting showed that mCherry still did not co-localize with BrdU in mice injected with control virus AAV-mCherry or AAV-Ascl 1/mCherry. FIG. 19g shows that at 30 days post virus injection, in mice injected with AAV-Ascl1/mCherry, the immune co-targeting showed that mCherry did not co-localize with Ki 67.

FIGS. 20a, b show that at 7 days after virus injection, immune co-labeling showed that mCherry was barely co-localized with the oligodendrocyte marker GST-pi in the midbrain of mice injected with control virus AAV-mCherry or AAV-Ascl 1/mCherry. FIG. 20c, d shows that at 30 days after virus injection, immune co-targeting showed little co-localization of mCherry with oligodendrocyte marker Olig2 in the brains of mice injected with control virus AAV-mCherry or AAV-Ascl 1/mCherry. FIGS. 20e-h show that at 30 days after virus injection, in the striatum and cortex of mice injected with control virus AAV-mCherry or AAV-Ascl1/mCherry, immune co-targeting showed that mCherry rarely co-localized with oligodendrocyte marker Olig 2.

FIG. 21A, B shows the firing patterns of dorsal mesencephalon neurons in wild-type (P42-P70) and Gad67-GFP (P51-P55) mice, respectively. Fig. 21C, D shows the neuronal firing patterns induced from young and adult mice, respectively. Figure 21E shows the categorical statistics of firing patterns of dorsal cranial neurons in the four mice described above.

FIGS. 22a, a ', b' show the expression of GFAP (reactive astrocyte marker) and IBA1 (microglial marker) around the mouse injection site 7 days after AAV injection with glass electrodes. FIGS. 22c, c ', d' show expression of GFAP and IBA1 around the mouse injection site 7 days after AAV injection with a 31G needle.

Detailed Description

The present inventors have made extensive and intensive studies and, for the first time, have unexpectedly found that overexpression of Ascl1 gene or its protein can effectively induce transdifferentiation of astrocytes into neuronal cells having normal electrophysiological functions, and that such transdifferentiation effect can be exerted on astrocytes in normal or injured forms both in vivo and in vitro. In addition, the inventors also experimentally confirmed that astrocytes from different site sources (dorsal midbrain, striatum and cerebral cortex) were able to differentiate into neuronal cells in the presence of Ascl 1. Therefore, the method is expected to become an effective method for culturing the neuron cells in vitro and stimulating and generating new neuron cells in an adult, thereby being widely applied to the treatment of nervous system diseases, such as neurodegenerative diseases, central nervous traumatic diseases and the like. On the basis of this, the present invention has been completed.

Cladophora glabrata complex homologue-like 1(Ascl1) gene or protein or promoter thereof

The cladophora-free complex homolog-like 1 gene or the protein thereof, Ascl, achaete-scar complexhomolog-like 1, bHLH class transcription factor. U68534.2 in Ascl1GenBank, the protein sequence is shown in SEQ ID NO. 1;

its NCBI Reference Sequence is NM-008553.4; the mRNA sequence is shown as SEQ ID NO. 2.

Promoters of Ascl1 are not particularly limited and may be any substance that promotes expression and/or activity of Ascl1 gene or its protein, such as small molecule compounds, promoting mirnas. The skilled person can screen the Ascl1 promoters from existing databases. It will be appreciated that, based on the transdifferentiation-inducing effects of Ascl1 on astrocytes as disclosed herein, one skilled in the art can reasonably predict that any substance having a promoting effect on Ascl1 will have a transdifferentiation-inducing effect on astrocytes.

Astrocytes

Astrocytes are the largest group of cells in the brain of mammals. They perform a number of functions, including biochemical support (e.g., forming the blood-brain barrier), providing nutrition to neurons, maintaining extracellular ionic balance, and participating in repair and scarring following brain and spinal cord injury. Astrocytes can be classified into two types according to the content of glial filaments and the shape of the apophysis: fibrous astrocytes (fibro astrocytes) are mostly distributed in the white matter of the brain and spinal cord, have slender processes and fewer branches, and contain a large amount of glial filaments in the cytoplasm; protoplasmic astrocytes (protoplasmic astrocytes) are mostly distributed in gray matter, and have coarse and short cell processes and many branches.

Astrocytes useful in the present invention are not particularly limited, and include various astrocytes of mammalian central nervous system origin, for example, derived from the striatum, spinal cord, dorsal mesencephalon or cerebral cortex, preferably, dorsal mesencephalon or cerebral cortex.

In the present invention, the astrocytes from each source have high induction transformation efficiency, wherein the cells from the cerebral cortex have the highest induction efficiency, and the dorsal mesencephalon is the second place.

Typically, the specific marker for astrocytes is GFAP, with astrocytes in gray matter expressing relatively low GFAP, but expressing Acsbg1 and GS. When induced by the methods of the invention, however, these astrocytes display markers specific for neuronal cells, such as Tuj1, MAP2 and synapsin I.

Functional neuron

As used herein, the term "functional neuron" refers to a neuronal cell with normal neuronal electrophysiological activity, which is transdifferentiated from astrocytes in the presence of an exogenous asci 1 gene or protein, including gabaergic neuronal cells and glutamatergic neuronal cells.

Typically, the functional neuronal cells have the following properties:

(a) markers for neurons Tuj1, MAP2 and Synapsin I;

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

Expression vector

The expression vector that can be used in the present invention is not particularly limited, and may be any expression vector that can be integrated into astrocytes and express a foreign Ascl1 protein, containing the coding sequence of Ascl1 protein. For example, a viral vector, which may be any viral vector capable of infecting a virus by bringing genetic material into other cells using the characteristics of the virus to transport its genome. Can be found in whole living organisms or in cell culture. Including lentiviral vectors, adenoviral vectors, herpesvirus vectors, and poxvirus vectors.

In the present invention, a preferred expression vector is a lentiviral vector. For example, the FUW-Ascl1-tdTomato vector is constructed by cloning cDNA of mouse Ascl1 gene into a lentiviral expression vector FUGW-IRES-EGFP using a conventional PCR technique to obtain FUGW-Ascl1, and replacing a sequence encoding GFP with another fluorescent protein, such as tdTomato.

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

Induction method

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

In vitro, the method comprises the steps of: astrocytes are infected with an Ascl1 vector (e.g., lentivirus), and the infected cells are maintained in culture for at least 10 days, more preferably, 20 days or more, thereby converting astrocytes into mature neuronal cells.

In vivo, a vector comprising Ascl1 may be administered (e.g., injected) to a site in a subject in need thereof containing astrocytes, such as the dorsal midbrain, striatum or cerebral cortex. Typically, such administration will involve injection of both undamaged and damaged nervous system tissue to induce transdifferentiation of astrocytes in specific sites of the nervous system.

Pharmaceutical composition and mode of administration

The 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 comprises the above-described expression vector of the present invention (e.g., a viral particle), or the exogenous Ascl1 protein itself, and a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention generally contains 10⁷-10¹²PFU/ml of lentivirus or AAV viral particles, preferably 10⁸-10¹²PFU/ml of lentivirus or AAV viral particles, more preferably 10⁹-10¹²PFU/ml of lentivirus or AAV viral particles.

"pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the composition may comprise 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 vector may also contain a cell transfection reagent.

In general, the pharmaceutical composition of the present invention can be obtained by 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, etc.

Applications of

The Ascl1 can be used for preparing functional neurons for inducing astrocytes, so that the newly induced neurons can be applied to various diseases related to neuron quantity reduction, cell decline, apoptosis or neuron function reduction. Wherein, the related diseases of the nervous system comprise epilepsy, Alzheimer Disease (AD), Parkinson Disease (PD), neuron death caused by stroke and the like.

The invention has the beneficial effects

The invention can transdifferentiate astrocytes on the dorsal mesencephalon into functional neurons by using a single transcription factor Ascl1 in vitro. Using a GFAP-AAV vector specifically expressed in astrocytes, Ascl1 was able to transdifferentiate adult mouse dorsal mesencephalon, striatum, and somatosensory cortex astrocytes into functional neurons. These induced neurons gradually mature, take on the form of neurons and express their marker molecules, and these neurons are able to fire action potentials, and are able to accept synaptic afferents from other neurons and release neurotransmitters to establish synaptic connections with other neurons. Therefore, the method is expected to become an effective method for culturing the neuron cells in vitro and stimulating and generating new neuron cells in an adult, thereby being widely applied to the treatment of nervous system diseases, such as neurodegenerative diseases, central nervous traumatic diseases and the like.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

General procedure

Astrocyte culture

Astrocytes are prepared by reference to "Preparation of partial agar and dendritic cell cultures from cellular tissue" (McCarthy, K.D. & DeVellis, J.J.cell biol.85,890-902 (1980)). Dorsal midbrain of mice or adult mice 5-7 days after birth was removed and digested with 0.25% pancreatin for 15 minutes. The blown-off cells were cultured in a DMEM/F12 medium containing 10% serum for 7 to 9 days. And (3) removing the oligodendrocyte by shaking to obtain the astrocyte.

Immunochromatographic development

The immunocolouring of cultured cells is carried out by referring to "Direct conversion of microorganisms to functional neurons by defined factors" (Vierbuchen, T.et al. Nature 463, 1035-. Primary antibodies for immunocolorimetry include: mouse anti-GFAP (Millipore,1:1,000), rabbitt-GFAP (DAKO,1:1,000), mouse anti-Tuj1 (Coonce, 1:500), mouse anti-Map2(Sigma,1:500), rabbitanti-GFP (Invitrogen,1:1,000), chip anti-GFP (Invitrogen,1:1,000), mouse anti-NeuN (Millipore,1:100), rabbitanti-Synapsin I (Millipore,1:1,000), rabbitanti-GABA (Sigma,1:3,000), rabbitanti-GAD 67(Millipore,1:200), guineabit pig anti-VG (synoptic VG, 1:200), mouse anti-GAD67(Millipore,1: 1), Sansuti-Gal LUT (Sansut-5: 5), Sansuti-Gal-1: 100), Rubbiti-Galilei-Gal-1: 100, Sansuti-1: 5-Xanti-S1, Sansuti-5-Gal, Sansuti-G (Sansut, 1:1, D-G-, BD Biosciences,1:200), rabbitant-Sox 2(Millipore,1:500), mobbitant-S100 beta (Sigma,1:1,000), rabbitant-EAAT 1(Abcam,1:500), rabbitant-NG 2(Millipore,1:200), rabbitant-Iba 1(Wako,1:500), mobbitant-CNPase (Abcam,1:500), mobustant-O4 (Millipore,1:500), mobustant-Ascl 1(BD Biosciences,1:200), rabbitant-Sox 2(Millipore,1:500), rabbitant-Olig 2(AB9610, 1:500), and rabbitant-Sox 77450 (Abcam,1: 500).

FITC-, Cy 3-and Cy 5-conjugated secondary antibodies were purchased from Jackson Immunoresearch, Alexa-350-, Alexa-488-and Alexa-546-conjugated secondary antibodies from Invitrogen.

Stereotactic injection of AAV

AAV viruses were performed with reference to mouse brain maps. After injection of the virus, the midbrain, striatum, and cerebral cortex of the back were collected at various time points for immuno-visualization or brain slice recording. In preparing a dorsal mesencephalon model of the lesion, injection of the virus was accomplished using a 5 ml syringe and a 31G needle.

Flow cytometric sorting and quantitative RT-PCR

Cells expressing mCherry were sorted by flow cytometry. Extracting total RNA of cells, synthesizing cDNA, and detecting real-time quantitative PCR. GAPDH serves as an internal control for gene expression levels.

Example 1 plasmid construction and viral infection

The cDNA of the mouse Ascl1 gene is cloned into a lentiviral expression vector FUGW-IRES-EGFP to obtain FUGW-Ascl 1. The replacement of GFP in the FUGW-Ascl1 plasmid with tdTomato resulted in FUW-Ascl 1-tdTomato. The empty lentiviral expression vectors, FUGW and FUW-tdTomato, were used as controls, respectively. Lentivirus packaging is described in "Production and characterization of viral vectors" (Tiscornia, G., Singer, O. & Verma, I.M. Nat. Protoc.1,241-245 (2006)).

After 24 hours of astrocyte plating, lentivirus was added and the medium was changed 24 hours after infection: DMEM/F12, B27, Glutamax and penicillin/streptomycin. Brain-derived neurotrophic factor (BDNF; PeproTech, 20ng/ml) was added to the medium every three days after infection 6-7.

To prepare a GFAP-AAV vector, the CMV promoter in the AAV-FLEX-Arch-GFP plasmid (Addgene) was replaced with the hGFAP promoter (2.2 kb). Inserting mCherry to obtain AAV-mCherry plasmid. Cloning of Ascl1 into AAV-mCherry yielded AAV-Ascl1/mCherry plasmid. Cloning of Ascl1 into AAV-FLEX-Arch-GFP yielded AAV-FLEX-Ascl 1/GFP. AAV-mCherry was compared to AAV-FLEX-NLSGFP. NLS is nuclear localization signal, the sequence of which is: VPKKKRKVEA are provided.

Example 2 Ascl1 willIn vitroTransdifferentiation of dorsal mesencephalic astrocytes into neurons

Astrocytes of the dorsal mesencephalon of mice 5-7 days after birth (P5-P7) were first isolated and purified. The properties of these glial cells were verified by examining molecular markers of different cell types (fig. 1). The vast majority of cells expressed the astrocyte marker molecules GFAP and S100 β, a small number of cells expressed the oligodendrocyte marker molecules O4 and CNPase, a small number of cells expressed the NG2 glial marker molecule NG2, and no detectable expression of the neuronal marker molecule Tuj1 and the stem cell marker molecules Sox2 and Oct 4.

Results

2.1 expression of mature neuronal marker molecules by transformed astrocytes

10 days after transfection of the astrocytes with the control lentiviral vector FUGW, the astrocytes did not express the neuronal marker molecule Tuj1 (FIG. 2a), and still maintained glial cell morphology while expressing the astrocyte marker molecule GFAP (FIG. 2 b). In contrast, after 110 days of infection with the lentivirus FUGW-Ascl, most astrocytes expressed the neuronal marker molecule Tuj1, while exhibiting typical neuronal morphology (76.8. + -. 6.4%, n. sub.3, 348-GFP counts each⁺Cells, fig. 2 c). After 121 days of lentiviral FUGW-Ascl infection, astrocytes also expressed the marker molecules MAP2 (FIG. 2d) and synapsin I (FIG. 2e) of mature neurons.

2.2 electrophysiological property detection showed that the transformed cells were all functional cells

To examine whether the neurons (i) cells transdifferentiated by Ascl1 have the electrophysiological properties of neurons, whole-cell recordings were performed on these cells (fig. 2 f).

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

2.3 validation that induced neurons are derived from astrocytes

To further verify that the induced neurons were derived from astrocytes, astrocytes (with a GFP marker) from hGFAP-GFP mice were infected with lentivirus FUW-Ascl 1-tdTomato. Cells induced by Ascl1 exhibited morphological changes and expressed Tuj1 while still expressing GFP (fig. 2i), whereas after infection with control lentivirus FUW-tdTomato containing only red fluorescent protein, virus-infected cells still maintained astrocyte morphology and expressed GFAP (fig. 2 h). This indicates that the induced neurons were derived from astrocytes.

2.4 detection of transmitter Properties of iN cells by Immunochromatographic assay

It was found experimentally that most of the iN cells expressed GABA (74.9. + -. 4.5%, n ═ 3, 153 + 228 GFP cells per count⁺Tuj1⁺Cells, fig. 3a), wherein part of the iN cells express the gabaergic neuronal marker molecules GAD67 (fig. 3b) and VGAT (fig. 3 d). iN addition, a fraction of the iN cells were found to express the glutamatergic neuronal marker molecule VGLUT2 (10.8. + -. 3.8%, n ═ 3, 134 and 280 GFP counts per time⁺Tuj1⁺Cells, fig. 3 f). This indicates that the iN cells contain inhibitory neurons as well as excitatory neurons. To further demonstrate this electrophysiologically, self-synaptic (autapse) status of the iN cells was examined.

It was found that some iN cells (19.4%, 7/36 cells) were able to register self-synapses (FIG. 3g) whose current was completely blocked (3/3 cells) when the antagonist CNQX, AMPA/kainate glutamate receptor, was added. This indicates that the iN cells contain glutamatergic neurons.

To verify whether GABA release was present iN the iN cells, astrocytes from the dorsal mesencephalon of GAD67-GFP mice were infected with lentivirus FUW-Ascl1-tdTomato (fig. 4a) and the induced cells were found to express GFP (fig. 4b), suggesting that they may be gabaergic neurons.

The iN cells were co-cultured 10 days after induction with neurons isolated from dorsal mesencephalon of P5-P7 wild-type mice, and almost all of tdTomato 29-40 days after lentiviral transfection⁺GFP⁺All of the cells (97%, 37/38 cells) produced action potentials (FIG. 4 c). At the same time, spontaneous postsynaptic currents were recorded on the vast majority of iN cells (89%, 34/38 cells) (fig. 4 d). iN addition, some iN cells (21%, 8/38 cells) were able to register self-synapses when GABA was added_AThe receptor antagonist bicuculine completely blocked the self-synaptic current (5/5 cells) (FIG. 3h), indicating that the iN cells contain GABAergic neurons.

The results indicate that astrocytes of the dorsal mesencephalon in the early postnatal period can be reprogrammed in vitro to functional glutamatergic or gabaergic neurons.

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

To explore the possibility of reprogramming astrocytes into neurons in vivo, recombinant adeno-associated virus (AAV) vectors containing a red fluorescent protein (mCherry) driven by the hGFAP promoter were constructed. The virus was injected into one side of the cap of a wild type mouse from P12-P15, and the expression of mCherry was detected by immunostaining three days later.

Results

3.1 Immunomacription of mCherry with the astrocyte marker molecule Acsbg1 showed that almost all cells positive for mChery expressed Acsbg1 (representative cells were astrocytes) 3 days after virus injection, either control virus AAV-mChery (96.1. + -. 0.7%, n ═ 3, counting 326 cells at 220-. Furthermore, immunological co-labeling of mCherry and the neuronal marker molecule NeuN indicated that mCherry is not expressed in neurons (fig. 7a, a ', d, d'). Further, mCherry was found not to coexist in the same cell as NG2, a marker of NG2 cells (fig. 6).

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

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

Additionally, GFAP-CreERT2 was induced with 4-hydroxyformazan (4-OHT); rosa26-CAG-tdTomato mice expressed tdTomato, and tdTomato was found to co-localize with Acsbg1 (93.8 ± 1.6%, n ═ 3, 169 cells per count-.

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

Example 4 Ascl1 in vivo conversion of brain astrocytes in the dorsal side of young mice into neurons

Viral AAV-mCherry or AAV-Ascl1/mCherry was injected into one side cap of wild type mice P12-P15, and brain tissue samples were collected at several different time points.

Results

4.1 injection of Ascl1 containing virus gradually revealed co-localization of mCherry and NeuN compared to controls

The immune co-targeting showed that mCherry was not co-localized with NeuN in mice injected with either control virus AAV-mCherry (3.4. + -. 0.2%, n ═ 3, counts 472 + 489 cells per time; FIG. 7a, a '), or virus AAV-Ascl 1/mChery (4.5. + -. 2.3%, n ═ 3, counts 279 + 419 cells per time; FIG. 7d, d') 3-5 days after virus injection.

However, from 44.2. + -. 12.5% (n ═ 3, counts 309-.

However, mCherry did not co-localize with NeuN in mice injected with the control virus AAV-mCherry, either 10-14 days (4.0 + -0.5%, n-3, 325 + 487 cells per count; FIG. 7b, b ') or 28-32 days (3.9 + -0.4%, n-3, 389 + 515 cells per count; FIG. 7c, c') after virus injection.

4.2 injection of Ascl 1-containing Virus did not increase apoptosis of neuronal cells

The Nie's visualization results showed that the cell density of dorsal mesencephalon of mice injected with AAV-Ascl1/mCherry was substantially equivalent to that of mice injected with control virus AAV-mCherry (FIGS. 8a-8 c). TUNEL staining showed no increase in apoptosis in mice injected with viral AAV-Ascl1/mCherry (FIGS. 8d-8 h).

mCherry expression was still detectable in the dorsal mesencephalon of mice 155 days after injection of viral AAV-Ascl1/mCherry, and they were well co-localized with NeuN (FIG. 9). This indicates that the iN cells can survive for a longer period of time iN vivo.

4.3 transmitter Property detection of iN vivo generated iN cells

Transmitter properties of iN vivo produced iN iN cells were further examined. 45 days after virus injection, a fraction of the iN cells were found to express Gad1 (13.2. + -. 4.2%, n ═ 3, 57-180 cells counted each; FIG. 7g), while a fraction of the iN cells expressed VGLUT2 (6.5. + -. 2.2%, n ═ 3, 48-118 cells counted each; FIG. 7 h).

This indicates that iN vivo generated iN iN cells contain glutamatergic and GABAergic neurons.

4.4 induced cells are derived from astrocytes, not neural precursor cells

To examine the presence of neural stem cells in the dorsal mesencephalon of wild-type mice of P12-P15, in vivo cells were isolated and subjected to a spherulite culture experiment. Cells isolated from the subventricular zone (SVZ) produced large numbers of neurospheres (364.9 ± 53.5 neurospheres per well (six well plate), n ═ 3, 333-426 neurospheres per count; fig. 7 i). While cells isolated from the dorsal mesencephalon are essentially unable to give rise toNeurospheres (0.8 ± 0.2 neurospheres/well (six well plate), n ═ 3, 0-1 neurospheres counted each time; fig. 7 j). In addition, in order to search for P12GFAP⁺Whether the cells of (a) are capable of producing neurons in the late stage, GFAP-CreERT2 is induced by 4-OHT for 5 consecutive days (P12-P16); rosa26-CAG-tdTomato mice expressed tdTomato, and it was found that tdTomato still did not co-localize with NeuN after 30 days (FIGS. 7k-7 n).

These results indicate that the slave GFAP⁺Induced iN cells are derived from postnatal astrocytes, not neural precursor cells.

EXAMPLE 5 electrophysiological Properties of iN cells iN vivo

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

Results

5.1 infection of cells containing Ascl1 Virus produces action potentials

In mice brains infected with AAV-Ascl1/mCherry 7-30 days, many cells were found to have inward Na in the voltage clamp mode⁺Current and outward K⁺The current, and the amplitude, increased with increasing infection time (FIGS. 11b-11 e). Correspondingly, the detected cells also have an increased ability to deliver action potentials in the current clamp mode (FIGS. 11b-11 e). Further, the morphology of the cells became more complex and the results of biocytin remodeling also found that fewer gap junctions were formed by the detected cells (FIGS. 11b-11 e). At the same time, the input resistance of the cells gradually increased, while the resting membrane potential gradually decreased (FIGS. 11f and 11 g). These results all indicate that the iN vivo induction of the transcription factor Ascl1 gradually matures the iN cell function.

However, in the brain of mice infected with the control virus AAV-mCherry, the cells tested were found to have lower impedance (1.88 ± 0.77 Μ Ω, n ═ 9), higher resting membrane potential (-79.21 ± 0.37mV, n ═ 14), and failed to deliver action potentials (fig. 11 a). Meanwhile, the result of biocytin (biocytin) remodeling showed that the control virus-infected cells had the typical morphology of astrocytes and were connected to the neighboring astrocytes by gap junctions (fig. 11 a). 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 Ascl 1-induced iN is more likely to generate action potential with longer infection time, and the generated current can be GABA_ABlockade by receptor antagonists

The iN cells were divided into 4 groups according to the current and voltage response patterns: non-active cells (non-active), cells with an inward current but no action potential (inward), cells capable of delivering a single action potential (sAP) and cells capable of delivering multiple action potentials (maps).

The results show that the iN cells induced by Ascl1 become more and more excitable with increasing infection time, and that all recorded iN cells were able to deliver action potentials at high frequency (50-220Hz) 30 days after infection (fig. 11 h). Whereas the control virus infected cells all exhibited an astrocyte-like "inactive" state (FIG. 11 h). It was further observed that spontaneous post-synaptic currents were present iN the iN cells, the more iN the iN cells there were spontaneous post-synaptic currents as the infection time was extended. High frequency, spontaneous postsynaptic currents were detected in all virus-infected cells (23/23) 30 days after AAV-Ascl1/mCherry virus infection (FIG. 11 i).

Further pharmacological experiments showed that the iN cells received both excitatory glutamate input and inhibitory GABA input (fig. 11 j). Finally, it was found by double whole cell recording that iN cells (mCherry)⁺) Neurons with the midbrain canopy (mCherry)^-) Synaptic connections may be formed (fig. 11 k). Adding GABA_AThe receptor antagonist bicuculine completely blocked synaptic currents induced in neurons of the midbrain roof (fig. 11 k). This suggests that the iN cells are able to establish gabaergic synaptic connections with surrounding neurons and integrate into existing neural circuits iN vivo.

Example 6 Ascl1 transdifferentiation of adult dorsal mesencephalon astrocytes into neurons in vivo

This experiment further investigated whether adult mice can reprogram astrocytes into neurons. Viral AAV-mCherry or AAV-Ascl1/mCherry was injected into wild type mice of P60 and showed whether mChrry co-localized with NeuN.

Results

6.1 astrocyte-induced iN cells from adult mice form functional synapses iN vivo

In mice injected with viral AAV-asci 1/mCherry, mCherry was gradually co-localized with NeuN from 63.5 ± 3.1% on 16 days (n ═ 3, counting 266 cells each time) (fig. 12f and 12f ') to 92.1 ± 1.5% on 38 days (n ═ 3, counting 216 cells each time) (fig. 12g and 12 g'). Electrophysiological recordings indicated that 15-21 days after infection with viral AAV-Ascl1/mCherry infected cells, most of the iN cells (9/10) had inward and outward currents iN voltage clamp mode and were able to deliver action potentials (FIG. 12 h). At the same time, spontaneous post-synaptic currents were recorded on most of the iN cells (8/10) (fig. 12 i). This indicates that the iN cells induced by astrocytes iN adult mice can form functional synapses iN vivo.

In contrast, the immune co-labeling showed that mCherry did not co-localize with NeuN 5 days after injection, whether in mice injected with control virus AAV-mCherry (4.2. + -. 1.4%, n ═ 3, counting 182 + 216 cells per time; FIG. 12a, a ') or AAV-Ascl1/mCherry (5.6. + -. 1.6%, n ═ 3, counting 151 + 335 cells per time; FIG. 12e, e'). Subsequently, experiments found that in mice injected with the control virus AAV-mCherry, mCherry was not substantially co-localized with NeuN, either 16 days after virus injection (6.7. + -. 3.6%, n ═ 3, counting 312 cells per time; FIG. 12b, b ') or 38 days after virus injection (3.7. + -. 1.2%, n ═ 3, counting 144 cells per time; FIG. 12c, c'). Electrophysiological experiments showed that cells infected with the control virus AAV-mCherry had typical astrocytic properties (FIG. 12 d).

It can be seen that Ascl1 transdifferentiates adult mouse dorsal mesencephalic astrocytes into functional neurons in vivo.

6.2 Cre-dependent expression of Ascl1 also transdifferentiate adult dorsal mesencephalon astrocytes into neurons

AAV viruses whose expression was induced by Cre recombinase were prepared according to conventional techniques: AAV-FLEX-NLSGFP and AAV-FLEX-Ascl 1/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.

AAV-FLEX-Ascl 1/GFP-infected GFP after 28 days of infection⁺The majority of cells expressed NeuN (90.1. + -. 2.1%, n ═ 3, 170 cells counted 126-. GFP infection of AAV-FLEX-NLSGFP⁺The vast majority of cells expressed Acsbg1 (94.8. + -. 1.7%, n ═ 3, 233-.

Whereas GFP + cells infected with the control plasmid AAV-FLEX-NLSGFP expressed almost no NeuN (2.9. + -. 1.1%, n. sup.3, 121-181 cells per count; FIG. 12j),

thus, expression of Cre-dependent Ascl1 may also transdifferentiate adult dorsal mesencephalic astrocytes into neurons.

6.3 injured mesencephalon astrocytes (reactive cells) can transdifferentiate into functional neurons

The dorsal cranial stab model in adult mice was created by injecting AAV virus AAV-mCherry or AAV-Ascl1/mCherry with a needle.

After 3 days of AAV-mCherry virus injection, the majority of mCherry sites were injured⁺Cells (92.8 ± 1.2%, n ═ 3, counts 60-117 cells per time; fig. 14a) expressed GFAP, while hardly any NeuN (2.4 ± 1.3%, n ═ 3, counts 69-107 cells per time; fig. 15). 30 days after viral infection, mCherry⁺The cells still expressed little NeuN (2.5 ± 1.2%, n ═ 3, 78-82 cells counted each time; fig. 14 b). AAV-mCherry virus infected cells had a relatively small membrane resistance (in 5.3 ± 1.9M Ω, n ═ 6) after 30 days, more hyperpolarized membrane potentials (-81.2 ± 1.7mV, n ═ 5) (fig. 14d), while failing to deliver action potentials (fig. 14 e).

Immunofluorescence showed mCherry 30 days after AAV-Ascl1/mCherry virus infection⁺Most of the cells expressed NeuN (54.2 ± 6.9%, n ═ 3, 114-. AAV-asci 1/mCherry virus infected mCherry⁺Cells had larger membranes after 30 daysResistance (424.7 ± 88.7M Ω, n ═ 17) and more depolarized resting membrane potential (-61.2 ± 1.6mV, n ═ 17) (fig. 14 d). At the same time, all recorded cells (17/17) were able to issue multiple action potentials (fig. 14f, g) and receive spontaneous excitatory and inhibitory synaptic afferents.

These results indicate that injured mesencephalic astrocytes can transdifferentiate into functional neurons.

Example 7 Ascl1 transdifferentiation of adult mouse striatal astrocytes into neurons in vivo

To investigate whether the transdifferentiation of astrocytes into neurons by Ascl1 was regiospecific, it was further examined whether striatal astrocytes of adult mice could be reprogrammed to neurons. Viral AAV-mCherry or AAV-Ascl1/mCherry was injected into the striatum of adult wild type mice (P60).

Immunostaining showed approximately 96% mCherry⁺The cells expressed the marker molecule Glutamine Synthetase (GS) for astrocytes (fig. 16e, f). Whereas mCherry is hardly expressed in neurons (NeuN)⁺) Microglial cell (IBA 1)⁺) Oligodendrocyte (Olig 2)⁺) And NG2 cell (NG 2)⁺) (FIGS. 16a-d, f).

To determine mCherry after AAV infection⁺Cell properties, three immunostaining for mCherry, GS and NeuN was performed. The results indicate that AAV-Ascl1/mCherry virus infected mCherry⁺Most of the cells no longer expressed GS (FIG. 16h), but NeuN (64.4. + -. 3.4%, n. sup.3, 129 cells counted 119-. And after 30 days of AAV-mCherry virus injection, mCherry⁺The cells expressed GS (FIG. 16g) and hardly NeuN (3.2. + -. 2.1%, n. sup.3, 140 cells per count 104-. This suggests that astrocytes transdifferentiate into neurons.

To further investigate whether the induced neurons were functional, further electrophysiological analyses were performed. Experiments show that AAV-Ascl1/mCherry virus infected mCherry⁺The majority of cells were able to detect inward and outward currents in voltage-clamp mode (15/16) (FIG. 16k), and in current-clamp mode (1)3/16) can issue action potentials (fig. 16k, l). In addition, spontaneous excitatory and inhibitory postsynaptic currents could be recorded in most of these cells (12/16) (FIG. 16 m). However, AAV-mCherry virus infected cells had a relatively small membrane resistance (in 2.9 ± 1.0M Ω, n ═ 7), more hyperpolarized membrane potential (-79.0 ± 0.3mV, n ═ 7) after 30 days (fig. 16i), while no action potential could be issued (fig. 16j, l). This indicates that astrocytes of the striatum of adult mice are capable of transdifferentiating into functional neurons.

Example 8 Ascl1 transdifferentiation of adult mouse cortical astrocytes into neurons in vivo

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

AAV-Ascl1/mCherry virus infected cortical mCherry was found experimentally 30 days after virus injection⁺The cells expressed NeuN for the most part (93.9 ± 1.2%, n ═ 3, 147 cells counted 132-. AAV-mCherry infected cortical cells (mCherry)⁺) NeuN was rarely expressed (2.6. + -. 0.8%, n. sup.3, each count of 120-133 cells; fig. 18 a).

Yet further electrophysiological analysis found that AAV-asci 1/mCherry virus infected 30 cells had large membrane resistance (163.3 ± 35.9M Ω, n ═ 10) and more depolarized resting membrane potential (-67 ± 2.2mV, n ═ 8) (fig. 18c) and all recorded cells (10/10) were able to release action potentials (fig. 18e, f). Also, spontaneous excitatory and inhibitory postsynaptic currents could be recorded in these cells (10/10) (FIG. 18 g). In contrast, cells infected with the control virus AAV-mCherry for 30 days still exhibited membrane properties similar to astrocytes (membrane resistance, 2.3. + -. 0.5 M.OMEGA., n. sup.8; resting membrane potential, -78.8. + -. 0.8mV, n. sup.7; FIG. 18 c; failure to deliver action potential; FIG. 18d, f).

This indicates that Ascl1 can transdifferentiate astrocytes in the cerebral cortex of adult mice into functional neurons.

8.1 to further determine whether Ascl1 induced astrocytes as neurons through the proliferative phase, intraperitoneal injections of BrdU were performed continuously on days 3-7 and 3-30 after virus injection to label proliferating cells.

At 7 days post injection, the immune co-labeling showed little co-localization of mCherry with BrdU, whether in mice injected with control virus AAV-mCherry (2.2. + -. 0.5%, n ═ 3, counting 325 cells per time; FIG. 19a) or AAV-Ascl1/mCherry (1.6. + -. 0.3%, n ═ 3, counting 213 cells per time; FIG. 19 b). Immunization co-labeling showed that mCherry was still barely co-localized with BrdU, whether in mice injected with control virus AAV-mCherry (4.3. + -. 1.2%, n ═ 3, counting 230 + 363 cells each; FIG. 19e) or AAV-Ascl1/mCherry (1.6. + -. 1.0%, n ═ 3, counting 204 + 290 cells each; FIG. 19f) 30 days after injection. Meanwhile, in mice injected with virus AAV-Ascl1/mCherry, mCherry co-localized with NeuN, whereas in mice injected with control virus AAV-mCherry, mCherry did not co-localized with NeuN (FIG. 19e, f). It was further found that the immunological co-targeting showed little co-localization of mCherry with Ki67, 15 days after virus injection, whether in mice injected with control virus AAV-mCherry (1.0. + -. 0.6%, n ═ 3, counting 246 cells at a time; FIG. 19c) or AAV-Ascl1/mCherry (0.9. + -. 0.3%, n ═ 3, counting 276 cells at a time; FIG. 19 d). 30 days after injection, the immune co-targeting showed that mCherry remained barely co-localized with Ki67, whether in mice injected with control virus AAV-mCherry or AAV-Ascl1/mCherry (0.5. + -. 0.1%, n ═ 3, counting 171-.

These results indicate that Ascl 1-induced reprogramming in vivo did not go through the proliferative phase.

8.3 this example also investigated whether overexpression of Ascl1 could convert astrocytes into oligodendrocytes. At 7 days after injection, the immuno-co-labeling showed that mCherry was hardly co-localized with the oligodendrocyte marker GST- π, whether in mice injected with control virus AAV-mCherry (0.4. + -. 0.1%, n ═ 3, counting 237-303 cells per time; FIG. 20a) or AAV-Ascl1/mCherry (0.4. + -. 0.3%, n ═ 3, counting 219-338 cells per time; FIG. 20 b). Immune co-labeling showed that mCherry was hardly co-localized with the other oligodendrocyte marker Olig2, 30 days after injection, whether in the brains of mice injected with control virus AAV-mCherry (2.8 ± 2.2%, n ═ 3, counting 308-393 cells per time; fig. 20c) or AAV-Ascl1/mCherry (3.3 ± 0.4%, n ═ 3, counting 278-327 cells per time; fig. 20 d). At the same time, mCherry co-localized with NeuN in mice injected with viral AAV-Ascl1/mCherry (FIG. 20 d). These results indicate that, when Ascl1 was overexpressed in the midbrain, astrocytes transdifferentiated into neurons instead of oligodendrocytes. At the same time, mCherry was also found to be hardly co-localized with Olig2 in the striatum of the injected virus, whether in brain tissue injected with control virus AAV-mCherry (3.2. + -. 1.2%, n ═ 3, 156 cells counted once; FIG. 20e) or AAV-Ascl 1/mChery (4.4. + -. 1.7%, n ═ 3, 137-. Furthermore, mCherry hardly co-localized with Olig2 in cortical brain tissue injected with either control virus AAV-mCherry (3.1. + -. 1.4%, n ═ 3, counting 124-.

These results indicate that by over-expressing Ascl1 in the striatum and cortex, astrocytes are also transdifferentiated into neurons rather than oligodendrocytes.

Example 9 electrophysiological Properties of transdifferentiated neurons and endogenous neurons on the dorsal cranial side

To investigate whether transdifferentiated neurons and endogenous brain neurons have similar properties, this example further compared the electrophysiological properties of transdifferentiated neurons and endogenous neurons on the dorsal mesencephalon.

Whole-cell recordings of the dorsal mesencephalon of acute brain sections of wild-type mice (P42-P70) and Gad67-GFP (P51-P55) mice revealed neuronal impedances of 489.1 ± 131.1 Μ Ω (n ═ 21, wild-type mice) and 326.0 ± 31.9 Μ Ω (n ═ 17, Gad67-GFP mice), respectively, and resting membrane potentials of-57.6 ± 2.0mV (n ═ 19, wild-type mice) and-57.1 ± 1.9mV (n ═ 15, Gad67-GFP mice), respectively (fig. 21A, B). These results are similar to neurons induced from young 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 potential, -61.0 ± 1.2, n ═ 6) (fig. 21C, D).

Furthermore, neurons on the dorsal cranial side in wild mice are classified into five major types based on neuron-specific firing patterns. It was found that most of the neurons induced from both the young as well as the adult mice (P12-P15: 95.6%, 22/23; P60: 100%, 9/9) could be classified into the firing patterns of normal neurons in these wild type mice and Gad67-GFP mice (FIG. 21C, D, E). Furthermore, some induced neurons (P12-P15, 30-49 days: 82.6%, 19/23; P60, 15-21 days: 77.8%, 7/9) exhibited the same firing pattern as Gad67-GFP mouse neurons, suggesting that they are likely GABAergic neurons.

Taken together, these results indicate that neurons transdifferentiated from young as well as adult mice have similar electrophysiological properties as endogenous mesencephalic neurons.

Furthermore, in most experiments involving stereotactic injection of AAV viruses, injection was performed using a glass electrode with a diameter of 18-20 microns, while injection was performed in a lesion model using a 31G needle with a diameter of about 260 microns. To compare the difference in injury between these two injection conditions, immunofluorescence experiments were performed in mice 7 days after the midbrain injection, and the expression of GFAP (reactive astrocyte marker) and IBA1 (microglia marker) around the injection site was examined. 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 31G needle-injected mice (glass electrode: 14.2. + -. 2.2, n ═ 3, FIG. 22a, a '; 31G needle: 113.6. + -. 15.7, n ═ 3; FIG. 22c, c'). Also, the number of IBA1 positive microglia around the site of injection of the glass electrode injected mice was significantly less than that of 31G needle injected mice (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 also observed, with significantly more GFAP or IBA1 positive glial cells (GFAP: 84.1 ± 12.1; IBA 1: 57.3 ± 7.5; n ═ 3 mice) surrounding the site of needle injection than glass electrode injected mice (GFAP: 18.3 ± 0.6; IBA 1: 18.4 ± 2.9; n ═ 3 mice). These results indicate that AAV injected with a 31G needle caused greater damage to brain tissue than glass electrode injections.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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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