Life Sciences and Medicine Overview: Neurotransformation Therapy Helps "Grow Elegantly" - PTB Downgrades Transform Astrogliosinto Dopamine Neurons.

With the advent of global aging era, "aging gracefully" may become a major pursuit of human beings.however, many aging related diseases hinder the realization of this pursuit.for example, Parkinson's disease, which ranks first in the list of "human health killers" in the middle-aged and elderly, brings a lot of pain to many patients.many researchers are trying to find a treatment for Parkinson's disease.based on the characteristics of progressive loss of dopamine (DA) neurons in substantia nigra compacta in Parkinson's disease, the current mainstream treatment strategy is to find key molecules to prevent the degenerative changes of primary neurons as soon as possible, or to build new neuronal connections to replace the lost neurons.on June 24, 2020, nature published a cover article, reversing a model of Parkinson's disease with in situ converted nigral nerves.this paper reflects the key and difficult points, breakthrough points and most commonly used research methods in the field of nerve regeneration. Background 1.1 for weight programming, astrocytes have the following advantages: sufficient number and proliferation in the event of injury, and high plasticity. The results of previous studies in this experimental group showed that the down-regulation of PTB expression could effectively promote the transdifferentiation of mouse and human fibroblasts into functional neurons. However, the inactivation of NPTB (PTB homologue, only expressed in nerve cells) can promote the maturation of functional neurons.the theoretical models of PTB – miR-124 – restloop 1 and NPTB – miR-9 – brn2loop) 2 were established. Objective: To study and compare the expression of key molecules in the transformation and maturation cycle of astrocytes, neurons and fibroblasts.method & amp; Results: in vitro culture of astrocytes from young individuals (mice and human), and the expression of PTB was reduced by shRNA interference. RT-qPCR showed that the level of miR-124 was low (similar to fibroblasts), and the level of miR-9 was high (similar to neurons); the level of Brn2 in astrocytes was high, similar to neurons; the level of PTB was higher in astrocytes The level of NPTB increased first and then decreased.conclusion: for rat and human astrocytes, the transformation cycle is similar to that of fibroblasts, while the mature cycle is similar to neurons. Therefore, only knocking down PTB in astrocytes can transform them into neurons.2.2 Objective: To explore whether astrocyte transformed neurons have physiological functions.method & amp; Results: (1) lentivirus could decrease the expression of PTB in astrocytes, and 50% - 80% of the cells showed neuron morphology after 4 weeks, and the positive results of TuJ1 and MAP2 were obtained by immunohistochemistry staining. In addition, RNA sequencing analysis showed that the genes related to astrocytes were inhibited in the process of transformation Neuron related genes were induced. In addition, the expression of many DA neuron specific genes in astrocytes derived from midbrain was significantly increased.(2) the voltage-gated sodium and potassium currents, as well as repetitive action potentials, can be recorded by patch clamp.when co cultured with freshly isolated rat astrocytes, the transformed neurons could record spontaneous postsynaptic currents with variable frequencies of glutamatergic and GABAergic activities.conclusion: astrocyte transformed neurons have the basic morphology and electrical activity of neurons, and can also form synaptic connections with surrounding cells.2.3 Objective: To explore the in vivo transformation effect and specificity of reduced expression of PTB in astrocytes, and to pay attention to the axonal growth dynamics of newly transformed neurons.methods & amp; results: (1) AAV containing CRE dependent shptb and RFP sequences was injected into the substantia nigra of GFAP CRE mice, and then immunohistochemical staining was performed at different time points of virus infection expression and cell transformation.the results showed that the proportion of positive results of multiple mature neuron markers increased gradually, while that of glial cells decreased.(2) from 3 to 12 weeks after virus injection, the number of RFP positive cells transformed into DA neurons (co labeled with th labeled DA neurons) gradually increased to about 1 / 3, and these transformed cells also co labeled with other DA neuron specific markers. The electrophysiological records showed that the electrical properties of these transformed neurons were similar to those of mature DA neurons after 12 weeks of injection. However, the transformed cells of astrocytes in cortex and striatum were hardly co labeled with th.RT qPCR analysis showed that the regional specificity of this transformation may be related to the fact that there are transcription factors rich in DA neurons with higher basal expression level in astrocytes of midbrain region, and these factors are more active during transformation.(3) after 12 weeks of virus injection, fibers of newly transformed neurons were observed in the substantia nigra striatum tract and several brain regions, and the fibers projecting across the brain regions were TH positive, especially in the caudate nucleus. In addition, there were synaptic structures in the caudate nucleus.conclusion: shptb mediated astrocytes in the midbrain DA neurons are more efficient and can gradually transform into new functional mature DA neurons over time, and the specificity of this region is related to the local microenvironment and the intracellular molecular network.2.4 Objective: To explore whether the transformed new DA neurons can reconstruct the damaged substantia nigra striatum pathway and restore the Da level targeted to the brain area in the pathological model of PD, and reverse the disease-related motor performance.methods & amp; results: (1) in the unilateral PD model induced by 6-OHDA injection, at 10-12 weeks after injection of AAV shptb, the number of TH positive cells in midbrain and the TH positive fibers in striatum increased significantly, and the number of newly transformed DA neurons and fibers accounted for about 1 / 3 of the uninjured side. (2) high performance liquid chromatography (HPLC) and carbon fiber microelectrode assay showed that the Da level of the injured side recovered to 65% of that of the uninjured side.(3) drug induced rotation test and spontaneous motor activity test showed that the behavioral recovery effect of shptb treatment was better than that of 1-month-old mice.conclusion: AAV shptb is enough to induce endogenous mesencephalic astrocytes into new DA neurons to reconstruct the substantia nigra striatum pathway, recover DA release level to a large extent, and restore motor performance to a certain extent, which has a certain age-related relationship.2.5 Objective: to prove whether the recovery of motor function really stems from the newly transformed DA neuron method & amp; Results: after the motor behavior of PD model mice was improved, CNO, a specific ligand drug, was used to activate these receptors to inhibit the function of newly differentiated neurons, and abnormal behavior was observed again. After CNO was metabolized, the motor behavior was improved.conclusion: the efficacy of these motor behaviors is attributed to the new neurons transformed from astrocytes. Objective: to verify the efficacy of transdifferentiation by antisense oligonucleotides (ASO). Methods: in vitro and in vivo experiments, Aso targeting PTB mediated the degradation of PTB mRNA. The results of molecular marker staining, electrophysiological recording and behavioral test were consistent with those of shptb method.references 1. Xue y, Ouyang K, Huang J, et al. Directconversion of fibroblasts to nerves by reprogramming PTB regulated micrornapractices. Cell. 2013; 152 (1-2): 82-96 doi:10.1016/j.cell.2012.11.0452.Xue, Y., Qian, H., Hu, J. et al. Sequentialregulatory loops as key gatekeepers for neuronal reprogramming in humancells.NatNeurosci 19,Qian, H., Kang, X., Hu, J. et al, Nurr1 and Lmx1a reprogrammed mouse and human fibroblasts into dopaminergic neurons. caiazzo, M., dell'anno, M., dvoretskova, e. et al. Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 476, 224 – 227 (2011). The CRISPR casrx method can also knock down PTB and efficiently transdifferentiate glial cells into functional neurons to treat neurological diseases. Zhou H, Su J, Hu X, et al. Glia-to-NeuronConversion by CRISPR-CasRx Alleviates Symptoms of Neurological Disease inMice. Cell. 2020; 181(3):590-603.e16. doi:10.1016/j.cell.2020.03.024ASNChinaEND Special statement of life science and medicine overview series: the above contents (including pictures or videos) are provided by the author. ASN only provides information release channel. If there is infringement, please contact the backstage to delete. 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