HDAC6: a target for regeneration following injury in the nervous system

描述（由申请人提供）：脊髓损伤是影响全球数百万人的问题。再生神经科学的一个主流假设是，脊髓损伤患者由于神经损伤而遭受永久性功能缺陷和瘫痪，但也因为中枢神经系统轴突再生和恢复失去的神经元连接的能力有限。根据这一方案，再生失败的主要原因是成年神经元再生受损轴突的内在能力有限，以及受损脊髓的生长环境不利。已经确定了几个阻碍损伤区域轴突再生的关键分子，包括髓鞘来源的生长抑制蛋白，如髓鞘相关糖蛋白（MAG）和反应性星形胶质细胞产生的硫酸软骨素蛋白聚糖（CSPGs）。髓磷脂和星形胶质细胞衍生的抑制信号最终汇聚到肌动蛋白和微管细胞骨架上，影响它们的稳定性、动力学和指导轴突生长的能力。我们发现，在体外实验中，使用药物抑制剂和敲低方法特异性靶向神经元中的组蛋白去乙酰化酶6 (HDAC6)，可以克服MAG或CSPGs对轴突生长的抑制作用。利用从神经元细胞体中分离轴突的微流体室，我们已经确定轴突的局部过程介导了这种作用。与此一致的是，我们发现抑制或敲低HDAC6不会增加组蛋白乙酰化（pan-HDAC抑制剂针对多种HDAC同工酶的典型功能），并且在转录抑制剂存在的情况下可以恢复生长。HDAC6的主要非核功能是1-微管蛋白赖氨酸40的去乙酰化，进而调节微管动力学。考虑到微管在轴突生长中的作用，我们假设HDAC6通过1-微管蛋白去乙酰化和微管不稳定介导细胞对髓磷脂和星形胶质细胞来源的抑制信号的反应。在本应用的具体目的中，我们将研究HDAC6以及1-微管蛋白乙酰化酶Elp3在生长抑制轴突中的功能。我们将研究它们的活性在多大程度上调节1-微管蛋白的乙酰化水平，以及1-微管蛋白去乙酰化在微管不稳定和轴突再生失败中的作用。我们还将测试HDAC6是否在体内轴突再生失败中起作用，以及通过抑制HDAC增加1-微管蛋白乙酰化是否能促进脊髓损伤后轴突再生。

DESCRIPTION (provided by applicant): Spinal cord Injury is a problem affecting millions worldwide. A dominant hypothesis in regenerative neuroscience is that patients with spinal cord injury suffer from permanent functional deficits and paralysis because of neural damage, but also because of a limited capacity of CNS axons to regenerate and restore lost neuronal connectivity. According to this scheme, a primary cause of failed regeneration is a limited intrinsic ability of adult neurons to regrow injured axons and the growth-hostile environment of the damaged cord. Several critical molecules that impede axon regeneration in the injury territory have been identified and include myelin-derived growth-inhibitory proteins such as myelin associated-glycoprotein (MAG) and reactive astrocyte-produced chondroitin sulfate proteoglycans (CSPGs). Myelin and astrocyte-derived inhibitory signals ultimately converge on the actin and microtubule cytoskeleton, affecting their stability, dynamics, and ability to direct axonal growth. We have found that specifically targeting histone deacetylase 6 (HDAC6) in neurons, using both pharmacological inhibitors and knockdown methods, can overcome the inhibitory effects of MAG or CSPGs to axon growth, in vitro. Using microfluidic chambers that isolate axons from the neuronal cell bodies we have determined that local processes in the axon mediate this effect. Consistent with this, we have found that HDAC6 inhibition or knockdown does not increase histone acetylation (a canonical function of pan-HDAC inhibitors that target multiple HDAC isozymes) and that recovery of growth can occur in the presence of a transcriptional inhibitor. A primary, and non-nuclear, function of HDAC6 is the deacetylation of 1-tubulin lysine 40 and, in turn, the modulation of microtubule dynamics. Given the role of the microtubule in axon growth, we hypothesize that HDAC6 plays a role in mediating a cell's response to myelin and astrocyte derived inhibitory signals via 1- tubulin deacetylation and microtubule destabilization. In the Specific Aims of this Application, we will examine the function of HDAC6, as well as the 1-tubulin acetylating enzyme, Elp3, in growth-inhibited axons. We will examine the extent to which their activities modulate the acetylation level of 1-tubulin and the role of 1-tubulin deacetylation in microtubule destabilization and axonal regeneration failure. We also will test whether HDAC6 plays a role in axon regeneration failure in vivo and whether increasing 1-tubulin acetylation by HDAC inhibition enhances axonal regeneration after spinal cord injury. PUBLIC HEALTH RELEVANCE: Disability following spinal cord injury is attributed, in part, to a limited ability of central nervous system axons to regenerate. Contributing to this inability is the injury environment, where a number of growth-inhibitory proteins are known to be present (e.g., myelin associated-glycoprotein and chondroitin sulfate proteoglycans). Strategies that promote axons to re-grow in their presence are needed. Signals from these growth-inhibitory proteins ultimately converge on the actin and microtubule cytoskeleton, affecting their stability, dynamics, and ability to direct axonal growth. We have found that inhibiting histone deacetylase-6 (HDAC6), an enzyme responsible for the modulation of microtubules, appears to be an excellent, non-toxic therapeutic strategy for promoting regeneration in cell culture models. The proposed studies in this application will further define the mechanisms of HDAC6 inhibition-mediated axonal regeneration, as well as test its therapeutic efficacy in pre- clinical rodent models of spinal cord injury. Our findings in this project could lead to new treatments for spinal cord injury and other central nervous system injuries and disease.