Function of muscle lim protein (MLP) in neuroprotection and axon regeneration

作为典型的中枢神经系统神经元，视网膜神经节细胞（RGCs）在受损视神经中通常不会再生轴突，而是发生凋亡。然而，晶状体损伤引起的内眼炎症刺激（IS）具有很强的神经保护作用，可使RGCs进入主动再生状态，使其能够在受损视神经中再生轴突。基于微阵列研究，我们确定了再生（由IS诱导）与单纯axocut和naïve RGCs中差异表达的基因，其中一个是肌肉LIM蛋白（MLP）。MLP被认为是肌肉特异性的（骨骼肌，但主要是心脏），参与肌肉形成，但在成年哺乳动物中枢神经系统中没有任何已知的表达。通过Western blot和免疫组化分析，我们发现成熟RGCs视神经损伤后胞浆（包括细胞核）和轴突生长锥中MLP蛋白的表达有明显的诱导作用。当RGCs在额外的IS后进入再生状态时，表达进一步增强。此外，我们在坐骨神经损伤后的背根神经节（DRG）神经元亚群中检测到MLP。短发夹RNA （short hairpin RNA, shRNA）抑制MLP表达可抑制RGCs的神经突生长，初步数据表明，MLP过表达可进一步促进is诱导的视神经轴突再生。在本研究项目中，我们将(i)研究MLP在轴突切除的RGCs、视神经和脊髓再生中的神经保护作用；（ii）通过验证核MLP可能调节轴突损伤后基因表达的假设，确定其潜在的分子机制；（iii）通过鉴定功能相关的神经元相互作用伙伴和MLP的下游信号级联。这些实验将确定MLP在中枢神经系统轴突再生中的未知作用的分子机制，并可能为中枢神经系统修复开辟新的途径。

As typical CNS neurons, retinal ganglion cells (RGCs) do not normally regenerate axons in the injured optic nerve and instead undergo apoptosis. However, inflammatory stimulation (IS) in the inner eye, induced by lens injury, is strongly neuroprotective and transforms RGCs into an active regenerative state, enabling them to regrow axons into the injured optic nerve. Based on a microarray study we identified genes differentially expressed in regenerating (induced by IS) vs. solely axotomized and naïve RGCs, one of which was muscle LIM protein (MLP). MLP has been postulated to be muscle-specific (skeletal muscle, but mainly heart) and involved in myogenesis without any known expression in the adult mammalian CNS. Using Western blot analysis and immunohistochemistry, we found profound induction of MLP protein in the cytosol (including nuclei) and axonal growth cones of mature RGCs upon optic nerve injury. Expression was further enhanced when RGCs entered a regenerative state after additional IS. Moreover, we detected MLP in a subpopulation of dorsal root ganglion (DRG) neurons upon sciatic nerve injury. Inhibition of MLP expression by short hairpin RNA (shRNA) compromised neurite growth of cultured RGCs and preliminary data demonstrate that overexpression of MLP further pro-motes IS-induced axon regeneration in the crushed optic nerve. In this proposed research project, we will (i) investigate the role of MLP in neuroprotection of axotomized RGCs, optic nerve as well as spinal cord regeneration, (ii) we will identify the underlying molecular mechanisms by testing the hypothesis that nuclear MLP might regulate gene expression after axon injury and (iii) by identification of functional relevant neuronal interaction partners and downstream signaling cascades of MLP. These experiments will identify the molecular mechanisms underlying the yet unknown role of MLP in CNS axon regeneration and might open novel approaches for CNS repair.