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

视网膜神经节细胞（retinal ganglion cells，RGC）作为典型的中枢神经系统神经元，在正常情况下不能再生视神经损伤后的轴突，而是发生凋亡。然而，由透镜损伤诱导的内眼中的炎性刺激（IS）具有强烈的神经保护作用，并将RGCs转化为主动再生状态，使其能够将轴突再生到受损的视神经中。基于微阵列研究，我们鉴定了在再生（由IS诱导）与单独轴突切断和幼稚RGC中差异表达的基因，其中之一是肌肉LIM蛋白（MLP）。MLP被认为是肌肉特异性的（骨骼肌，但主要是心脏），并参与肌肉发生，在成年哺乳动物CNS中没有任何已知的表达。使用Western印迹分析和免疫组化，我们发现深刻的诱导MLP蛋白质的胞浆（包括核）和轴突生长锥的成熟RGCs视神经损伤后。当额外的IS后RGCs进入再生状态时，表达进一步增强。此外，我们检测到MLP在背根神经节（DRG）神经元的亚群坐骨神经损伤后。通过短发夹RNA（shRNA）抑制MLP表达损害了培养的RGCs的轴突生长，并且初步数据表明MLP的过表达进一步促进了IS诱导的压碎视神经中的轴突再生。在这个拟议的研究项目中，我们将（i）研究MLP在轴突切断的RGCs，视神经以及脊髓再生的神经保护作用，（ii）我们将通过测试核MLP可能调节轴突损伤后基因表达的假设来确定潜在的分子机制，以及（iii）通过识别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CNS轴突再生中未知作用的分子机制，并可能为CNS修复开辟新途径。

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.