Function of MEF2 in Neuroprotection and Neuro-regeneration Following Stroke

 描述（由申请方提供）：缺血性视神经病变是一种中枢神经系统（CNS）白色卒中，由于视网膜神经节细胞（RGC）轴突损伤和细胞死亡导致永久性失明。尽管这种白色缺血是人类发病的重要来源，但对导致神经元功能障碍和死亡的分子机制仍然知之甚少，并且几乎没有有效的治疗方法来预防或恢复视力丧失。本研究的一个主要目标是确定缺血性损伤后神经元中有缺陷的信号通路，以便合理设计赋予神经保护和神经再生的新治疗方案。肌细胞增强因子2（MEF 2）转录因子家族是神经元中促存活信号传导的重要介质，其由逆行神经营养因子刺激和神经元去极化两者激活。MEF 2A和MEF 2D（在RGC中表达的主要MEF 2同种型）的转录活性通过细胞外信号调节激酶5（ERK 5）催化的磷酸化和钙调磷酸酶（CaN）催化的去磷酸化对不同氨基酸残基的翻译后修饰来调节。MEF 2A/D、ERK 5和CaN都是支架蛋白肌肉A激酶锚定蛋白α（mAKAPα/AKAP 6）的结合伴侣，mAKAPα/AKAP 6是一种主要在视网膜RGC中表达的支架蛋白。在本申请中，我们将测试新的假设，即mAKAPα信号体通过协调MEF 2激活神经保护和轴突生长，在神经元信号转导网络中充当关键节点。在目标1中，我们将使用原代培养物确定mAKAPα信号体对脑源性神经营养因子（BDNF）和去极化刺激的MEF 2A/D翻译后修饰（包括磷酸化和类小泛素化）的调节和功能。在目标2中，使用条件性MEF 2敲除小鼠和光化学诱导的缺血性视神经病变（PCI-ION）模型，我们将测试MEF 2A和MEF 2D转录活性是否是中风中RGC存活所需的，以及玻璃体内注射后神经营养因子（如BDNF）的促存活和再生作用。在目的3中，我们提出通过使用玻璃体内注射腺相关病毒（AAV）基因治疗载体在体内RGC中表达组成型活性MEK 5和MEF 2蛋白来增加ERK 5信号传导和MEF 2活性，从而增强PCI-ION后的神经保护和再生。来自这些实验的数据将推进我们对MEF 2如何在神经元中调节以及区室化信号如何调节神经元存活和再生的基本理解，最终有助于中风和视神经病变的新治疗策略。

 DESCRIPTION (provided by applicant): Ischemic optic neuropathy is a type of central nervous system (CNS) white matter stroke that causes permanent blindness due to retinal ganglion cell (RGC) axon damage and cell death. Although such white matter ischemia comprises an important source of morbidity in humans, the molecular mechanisms contributing to neuronal dysfunction and death remain poorly understood, and there exist few effective therapies to prevent or restore loss of vision. A major goal for this research is to define the signaling pathways defective in neurons following ischemic injury, so that novel therapeutic regimens that confer neuroprotection and neuroregeneration may be rationally designed. The myocyte enhancer factor 2 (MEF2) transcription factor family is an important mediator of pro-survival signaling in neurons that is activated by both retrograde neurotrophin stimulation and neuronal depolarization. Transcriptional activity of MEF2A and MEF2D, the primary MEF2 isoforms expressed in RGCs, is regulated by post-translational modification of distinct amino acid residues by extracellular signal-regulated kinase 5 (ERK5) catalyzed phosphorylation and calcineurin (CaN) phosphatase catalyzed dephosphorylation. MEF2A/D, ERK5, and CaN are all binding partners for the scaffold protein muscle A-kinase anchoring protein α (mAKAPα/AKAP6), a scaffold protein expressed within the retina primarily in RGCs. In this application we will test the novel hypothesis that mAKAPα signalosomes serve as critical nodes in the neuronal signal transduction network by orchestrating MEF2 activation in neuroprotection and axon growth. In Aim 1 using primary cultures, we will determine the regulation and function of MEF2A/D post- translational modifications, including phosphorylation and sumoylation, by mAKAPα signalosomes in response to brain-derived neurotrophic factor (BDNF) and depolarizing stimuli. In Aim 2 using conditional MEF2 knock-out mice and a photochemically-induced ischemic optic neuropathy (PCI-ION) model, we will test whether MEF2A and MEF2D transcriptional activity are required for RGC survival in stroke and for the pro-survival and regenerative effects of neurotrophic factors such as BDNF after intravitreal injection. In Aim 3, we propose to enhance neuroprotection and regeneration following PCI-ION by increasing ERK5 signaling and MEF2 activity using intravitreal injection of adeno-associated virus (AAV) gene therapy vectors to express constitutively active MEK5 and MEF2 proteins in RGCs in vivo. Data from these experiments will advance our basic understanding of how MEF2 is regulated in neurons and how compartmentalized signaling regulates neuronal survival and regeneration, ultimately contributing to new therapeutic strategies for stroke and optic neuropathy.