小热休克蛋白8经NF-κB通路对SOD1突变肌萎缩侧索硬化的保护机制研究

小热休克蛋白8（HSPB8）作为一种分子伴侣，具有促进错误折叠蛋白的自噬清除作用，对MN可能有保护作用，但是在ALS中具体机制不清。SOD1基因是最常见的ALS致病基因之一，我们用前期报道的SOD1G41D与SOD1G41S两个临床表现差异显著的家系，制备患者来源的iPSC细胞系，并用CRISPR/Cas9定点修复突变，将修复前后的iPSC分别定向诱导分化为SPMN、UMN、星形胶质及小胶质细胞，将MNs和胶质细胞分组进行混合培养，通过调控NF-κB通路，观察HSPB8在MNs中和各培养体系中表达变化，观察突变SOD1蛋白表达以及聚集，LC3-Ⅱ/Ⅰ比值，自噬等指标；并在动物模型上进行验证。本项目通过研究HSPB8经NF-κB通路调控对MNs功能的影响，揭示HSPB8对MNs的保护机制，提供HSPB8神经保护作用的新证据，为ALS治疗靶点提供新理论依据。

The abnormal accumulation of misfolded proteins in motor neurons (MN) may be one of the common pathological manifestations of ALS. As a molecular chaperone, small heat shock protein 8 (HspB8) can prevent the abnormal aggregation of misfolded protein and promote its autophagy clearance. HspB8 can be expressed in MN and regulated by nuclear transcription factor NF-κB, but its specific role in ALS is not clear. SOD1 gene is the most common pathogenic gene of familial ALS and we have reported two families of G41D/S with obvious difference in clinical manifestations which were used to prepare iPSC cell lines, and CRISPR/cas9 site-specific repair mutation was used to induce and differentiate iPSC before and after repair into SPMN, UMN, astrocytes and microglia. They were divided into groups for mixed culture. By up-regulation or down-regulation of NF-κB pathway, the changes of HspB8 expression in MN and mixed culture system of each group were observed. The changes of MN expression and aggregation of SOD1 protein were detected, and the LC3-Ⅱ/Ⅰ ratio was measured, the results demonstrate the effects of glial cells on HspB8 expression in neurons, and further we verify in animal model. By observing the regulatory mechanism of NF-κB pathway on HspB8 in MNs, this project reveals the protective mechanism of HspB8 on MNs, which can provide new evidence for neuroprotection of HspB8 and provide theoretical basis for the therapeutic target of ALS.