中间丝蛋白(IF)家族及其相关MicroRNA在脑出血后神经轴突再生中的作用研究

Stroke is a widely speread pathology with currently no effective treatment for any symptom. The severe disability resulting from spastic hemiplegia of intracerebral hemorrhage (ICH) is due to white matter damage confined to the centrum semiovale, internal capsule or pons. Rehabilitation after ICH mostly relies on the axon remodeling and synapse plasticity in those lesion areas. To achieve CNS regeneration, we can learn from CNS development of a fetus and successful nerve regeneration of PNS, which depends on the activation of intrinsic growth capacity of surviving neurons and existence of a local permissive environment consisted of glial cells. .Nerve cell-related intermediate filament proteins (IFs), the most abundant of the structural cytoskeletal proteins of neurons, axons, and gliocytes, once considered to play rather passive roles within the axon, are now known to be highly dynamic structures, undergoing significant changes in subunit composition in both developing and adult neurons that influence both the morphology and physiology of axons. Recent evidences have shown that this tight regulation depends critically on post-transcriptionally regulated changes in IFs mRNA transport, translation and stability. Post-transcriptional mechanisms modulate increases in IF gene transcription during axon regeneration to yield the final pattern of IF protein expression. To switch gene expression on and off in injured neurons, and surrounding gliocytes, transcription factors and their networks should be carefully orchestrated according to the regeneration program. They are the so-called "intrinsic power of axonal growth" and "permissive environment". There is an increasing repertoire of candidate transcription factors induced by nerve injury, including proteins and MicroRNAs. Some of them potentiate the survival and axonal regeneration of damaged neurons in vivo; however, our knowledge of transcriptional events in injured neurons is still limited. .Recent studies and our preliminary outputs revealed that in different stages of axon regeneration after ICH injury, some specific IFs unregulated, and noncoding MicroRNAs may play an essential role in the regeneration program. In this project, we propose to study the function and underlying mechanisms of IFs and IFs related MicroRNAs in the axonal regeneration of ICH. We will investigate the dynamic regulation of nerve cell IFs after collagenase induced ICH in vivo. The coordinate fibre regrowth and functions will be evaluated on the stable established assessing platform including fibre tracing and evoked potential examination. Based on the animal ICH model and neuron/astrocyte co-culture system, efficiencies of three candidate MicroRNAs in promoting axonal regeneration by post-transcriptional regulation of IFs will be examined in vivo and in vitro..A comprehensive understanding of the nuclear events of IFs in injured CNS neurons, will provide clues to clinical interventions for successful nerve regeneration.

原发性脑出血多因损伤皮层下白质神经损伤而导致伤残，严重影响患者的生活质量和社会功能。脑出血后神经康复的关键在于轴突再生和突触重建。促进中枢损伤后神经再生，目前尚无有效方法。如何重启中枢神经元再生能力和平衡中枢神经系统胶质细胞微环境，是研究的热点。神经细胞中间丝蛋白（IFs）在神经发育过程中通过一系列分布和构象的动态变化，决定着神经形态和功能的发展方向。IFs蛋白的转录后机制，是调控IFs蛋白表达动态变化和功能网形成的可能途径。MicroRNAs作为一种转录因子，可能决定着转录调控的方向。我们通过建立脑出血动物模型和神经元/星形胶质细胞共培养模型，观察中枢轴突损伤后体内IFs的动态变化与纤维损伤、再生的关系，以及神经细胞IFs相关的MicroRNAs在体内外环境对IFs表达的调控作用，对神经轴突再生和功能重建的影响，拟建立一个稳定的轴突再生形态、功能检测平台，探索IFs蛋白与神经再生的关系。

原发性脑出血多因损伤皮层下白质神经损伤而导致伤残，严重影响患者的生活质量和社会功能。脑出血后神经康复的关键在于轴突再生和突触重建。MicroRNA作为一种调控细胞发育生长的因子，在神经系统中的作用已有很多报道。中间丝 （intermediate filaments, IFs）是神经元的中间丝蛋白，在脊椎动.物和哺乳动物的轴突中以结构性细胞骨架多聚体的形式大量表达。IFs家族根据氨基酸序列不同可分为6个亚型。其中与神经元和轴突生长相关的有IV型（alpha-internexin, neurofilament）和VI型（Nestin）。在轴突生长初期，中枢神经系统神经元主要表达neurofilament-M（NF-M），之后是NF-L和NF-M。 .目前已经报道一些microRNA 在三种NF亚型mRNA上有结合位点。MiR-32和miR-367在NF-L和NF-M两个亚型有共同结合位点，但调控机制不清楚。MiR-125b在大鼠神经元和星形胶质细胞中均有表达，已有报道MiR-182在神经发育和损伤神经再生上发挥功能。. .过去的一年，我们筛选了多个microRNA，包括miR-367，miR-32，miR-125b和miR-182，发现miR-182促进神经元轴突生长，并建立了小鼠脑出血模型和行为学功能评价系统。在后续的试验中，我们进一步发现miR-182可以通过促进中间丝蛋白-L表达，显著促进神经轴突生长和树突分支，进而确认PTEN/AKT通路参与这一过程。在功能测试试验中，我们发现miR-182显著改善小鼠脑出血疾病症状，并促进皮质脊髓束神经轴突再生和中间丝的表达。这项研究成果预示，miR-182可以作为脑出血神经轴突再生的靶标。