PKC inhibition-mediated spinal cord regeneration

描述(申请人提供)：成年哺乳动物中枢神经系统(CMS)中受损轴突的再生能力被认为是成熟神经元固有再生能力下降和损伤部位局部抑制环境共同作用的结果。成年中枢神经系统的大部分抑制活性似乎来自两种中枢神经系统特有的神经胶质细胞类型，即少突胶质细胞和星形胶质细胞。虽然硫酸软骨素蛋白多糖(CSPGs)被认为是星形胶质细胞来源的胶质瘢痕的一类主要抑制物，但一些髓鞘相关分子是少突胶质细胞的主要抑制活性的原因。我们最近的数据表明，蛋白激酶C(PKC)的传统异构体是介导髓鞘成分和CSPG抑制活性的通路中的关键信号成分。值得注意的是，鞘内注入PKC抑制剂Go6976到C3背侧半横断部位，由独特的VibraknifeTM装置制成，促进了损伤部位内外上升背柱(DC)的强大轴突再生，从而导致功能再生。我们的数据还表明，抑制PKC可能对红核脊髓束(RST)的再生有影响。然而，目前尚不清楚这些再生轴突是否能够到达它们最初的目标并形成突触连接。出乎意料的是，在这个模型中，相同的PKC抑制剂并没有促进皮质脊髓束(CST)的再生。这些结果为我们提供了一个独特的机会来研究不同的中枢神经系统通路对再生的要求是否不同，以及再生轴突在受损的成年中枢神经系统中的行为。在这一应用中，我们将探讨PKC抑制介导的轴突再生和功能恢复在C3背侧半横断后的作用和机制，目的1将决定由PKC抑制诱导的DC轴突再生后的解剖再生、再神经支配、躯体组织和功能。目的2确定在同一损伤模型中，抑制PKC是否也能诱导另一条下行通路RST的再生，以测试其是否对轴突再生有广泛的影响。目的3探索C3背侧半切术后最大化再生和功能恢复的组合策略。具体地说，我们将PKC抑制与三种有希望的策略相结合：1)增强受损神经元的内在再生能力，2)为神经营养因子再生的轴突提供趋化作用，3)去除沉积在胶质瘢痕中的CSPG。我们将检查联合策略是否会在体外导致抑制底物上更强的轴突生长，以及在体内更大的轴突再生和功能恢复。总之，这些研究可能使我们了解PKC介导的轴突抑制不同中枢神经系统通路的潜在机制，并为设计有效的治疗策略以鼓励不同类型的轴突在损伤的成年脊髓中再生提供新的见解。

DESCRIPTION (provided by applicant): Inability of lesioned axons in the adult mammalian central nervous system (CMS) to regenerate has been hypothesized to result from both a decrease in intrinsic regenerative capacity of mature neurons and a local inhibitory environment at the site of injury. It appears that the majority of inhibitory activities in the adult CNS are derived from two CNS-specific glial cell types, the oligodendrocyte and astrocyte. While chondroitin sulfate proteoglycans (CSPGs) have been implicated as a major class of inhibitors in astrocyte-derived glial scar, several myelin-associated molecules account for the major inhibitory activities of the oligodendrocytes. Our recent data demonstrated that conventional isoforms of protein kinase C (PKC) are key signaling components in the pathways that mediate the inhibitory activities of both myelin components and CSPGs. Strikingly, intrathecal infusion of a PKC inhibitor, Go6976, into the site of C3 dorsal hemisection, made by a unique VibraknifeTM device, promoted robust axonal regeneration of the ascending dorsal column (DC) across and beyond the lesion site which led to functional reinnervation. Our data also indicates that PKC inhibition may have an effect on regeneration of the rubrospinal tract (RST). It remains unclear, however, whether these regenerating axons are able to reach their original targets and form synaptic connections. Unexpectedly, the same PKC inhibitor did not promote the regeneration of the corticospinal tract (CST) in this model. These results provide us with a unique opportunity to investigate whether different CNS pathways differ in their requirements for regeneration and how regenerating axons behave in the injured adult CNS. In this application, we will explore the role and mechanism of PKC inhibition-mediated axonal regeneration and functional recovery after the C3 dorsal hemisection with the following three (3) Specific Aims: Aim 1 will determine anatomical regeneration, reinnervation, somatotopic organization and function following DC axonal regeneration elicited by PKC inhibition. Aim 2 will determine whether PKC inhibition also induces regeneration of another descending pathway, the RST, in the same lesion model to test whether it has a broad effect on axonal regeneration. Aim 3 will explore combinatorial strategies to maximize regeneration and functional recovery after C3 dorsal hemisection. Specifically, we will combine the PKC inhibition with three promising strategies to: 1) enhance injured neuron's intrinsic regenerative capacity, 2) provide chemotropism for regenerating axons with neurotrophins, and 3) remove CSPGs deposited within the glial scar. We will examine whether the combination strategies will result in stronger neurite outgrowth on inhibitory substrates in vitro and greater axonal regeneration and functional recovery in vivo. Collectively, these studies may allow us to understand mechanisms underlying PKC-mediated axonal inhibition of different CNS pathways and provide new insights into designing effective therapeutic strategies to encourage axonal regeneration of different types of axons in the injured adult spinal cord.