CN-119639671-B - Application of Olig2 in astrocyte transdifferentiation into functional neurons

Abstract

The invention belongs to the technical field of biology, and particularly discloses application of Olig2 in astrocyte transdifferentiation into functional neurons, and the application provided by the invention realizes overexpression of a neurogenesis promoting transcription factor and knocking down of an induction blocking factor, which are expected to become effective methods for improving transdifferentiation efficiency and generating more new neurons, thereby being widely applied to treatment of various nervous system diseases involving neuron loss, such as neurodegenerative diseases, traumatic diseases of the central nervous system and the like.

Inventors

• WU ZHENG
• CHEN GONG
• Lai Chuying
• HOU KAIYU

Assignees

• 暨南大学

Dates

Publication Date

20260508

Application Date

20241213

Claims (2)

1. Use of Olig2 for promoting the transdifferentiation of astrocytes into functional neurons expressing NeuN, characterized in that the transdifferentiation of astrocytes into neurons expressing NeuN is promoted by the combined expression of transcription factor Ngn2 and shRNA targeting Olig2, the shRNA having the sequences SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7.
2. 2. A composition for promoting the transdifferentiation of astrocytes into neurons, comprising: (a) A vector for coding a transcription factor Ngn2, wherein the cDNA sequence of the vector is SEQ ID NO. 1; (b) The shRNA sequences of the shRNA codes for Olig2, and are SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, wherein the vectors in (a) and (b) are adeno-associated virus AAV9.

Description

Application of Olig2 in astrocyte transdifferentiation into functional neurons Technical Field The invention belongs to the technical field of biology, and particularly relates to application of Olig2 in astrocyte transdifferentiation into functional neurons. Background The central in-situ glial cell transdifferentiation (Glia-to-Neuron Conversion, gtN) to neurons is to utilize glial cells in the central nervous system as precursor cells to transdifferentiate the glial cells into functional neurons (also called glial cells transdifferentiation in situ), thereby realizing in-situ nerve regeneration and showing great potential application value for the intervention treatment of central injury and neurodegenerative diseases. Astrocytes (Astrocyte) are a type of glial cell in the central nervous system that perform a variety of important physiological functions in the brain, such as nutritional and mechanical support, blood brain barrier formation, synaptic modulation, etc. The presence of heterogeneity in astrocytes from different brain regions is an ideal precursor cell for in situ nerve regeneration. It has been found that in situ astrocyte transdifferentiation can be directly initiated using bHLH (basicHelix-Loop-Helix) family nerve transcription Factors Ascl1, neurog2 and neuroD1 as pioneering Factors (Pioneer Factors). Astrocytes, however, also present some inherent suppressive factors against their fate shift, stabilize and guarantee their cellular properties to avoid spontaneous conversion of astrocytes into neurons, and even into other cell types, to maintain homeostasis of different cell types within the central nervous system. Therefore, the previous research is relatively lack of research on how astrocytes resist the transdifferentiation mechanism by focusing on the development of astrocyte in-situ transdifferentiation driving factors, and the discovery of new transdifferentiation factors promoting astrocyte in-situ transdifferentiation can provide theoretical basis for developing more efficient in-situ transdifferentiation technology. Disclosure of Invention The present invention finds that Olig2, which is predominantly present in cells of the oligodendrocyte lineage in the adult mammalian brain cortex, is significantly up-regulated in astrocytes that overexpress a transcription factor of the neurobhlh family. Olig2 binds to and blocks the expression of genes associated with neural development in astrocytes, and the transdifferentiation of astrocytes into neurons can be promoted by knocking down Olig2 expression. One aspect of the invention relates to the use of Olig2 in the transdifferentiation of astrocytes into functional neurons, whereby knocking down Olig2 promotes the transdifferentiation of astrocytes into functional neurons. As a second aspect of the invention, the use of a vector for knocking down Olig2 for promoting the transdifferentiation of astrocytes into functional neurons, characterized in that the vector is a drug, a cell and/or a virus, and the Olig2 cDNA sequence is SEQ ID NO. 4. As a third aspect of the present invention, the use of Olig2 for promoting overexpression of a transcription factor, characterized in that said transcription factor is Ngn2, ascl1 and NeuroD1, the cDNA sequence of Ngn2, ascl1 and NeuroD1 is SEQ ID NO 1-3, and the Olig2 cDNA sequence is SEQ ID NO 4. As a fourth aspect of the invention, a knock-down virus for knocking down Olig2, wherein the knock-down virus targets a mouse, the knock-down virus comprises shRNA sequence and 9-type adeno-associated virus, and the shRNA sequence is SEQ ID NO. 5-7. As a fifth aspect of the present invention, a knock-down virus for knocking down Olig2 is constructed by cloning three shRNA sequences of a target mouse Olig2 gene in series into an adeno-associated virus type 9 (AAV 9) expression vector, and activating shRNA expression with an RNA polymerase III promoter U6, and activating fluorescent protein GFP expression with a broad-spectrum promoter CAG. As a sixth aspect of the present invention, a mouse model for knocking down Olig2, which is obtained by injecting the knock down virus provided in the fourth aspect of the present invention. According to the invention, when the population change of cerebral cortex cells in early transdifferentiation is studied, the number of Olig2 positive cells is unexpectedly found to be obviously increased, and through detection, the increase of Olig2 is caused by the up-regulation of Olig2 on the expression of astrocytes over-expressing bHLH transcription factors. Based on the experimental results, the influence of Olig2 on the Ngn 2-mediated transdifferentiation process is further analyzed, and the fact that Olig2 knockdown can remarkably promote the transdifferentiation of astrocytes into neurons is found, and the transdifferentiated neurons have electrophysiological functions through patch clamp demonstration. Through single cell sequencing, the molecular mechanism of the Olig2 knockou