Reversing BDNF Impairments in Rett Mice with TRPC Channel Activators

描述(由申请人提供)：在细胞和分子水平上了解Rett综合征(RTT)的病理生理学机制，并建立成功的生物检测方法来评估潜在的治疗策略，在这种神经发育障碍的研究道路上处于优先地位。RTT是一种自闭症谱系障碍，是一种毁灭性的儿童残疾，其对个人(全球出生人口比例为1：10,000)、他们的家庭和社会的影响RTT是由编码甲基CpG结合蛋白2(MeCP2)基因的功能缺失突变引起的，MeCP2是一种转录调节因子，与包括神经营养因子BDNF在内的几个基因启动子区域的甲基化CpG位点结合，以及钙离子渗透性非选择性阳离子通道亚单位TRPC3和TRPC6。内源性表达的BDNF对其活性依赖释放的有效性可以通过膜电流和TRPC通道介导的树突状钙信号来监测。初步结果表明，在有症状的MeCP2突变小鼠中，刺激突触前苔藓纤维(MF)在CA3锥体神经元中诱发的TRPC电流和钙信号较小。在MeCP2突变神经元中，由重组BDNF或非水解性DAG类似物(激活TRPC通道)引起的反应也受到损害。一致地，在MeCP2突变的海马区，BDNF和TRPC3的mRNA和蛋白水平都较低。初步实验结果表明，TRPC6通道激活剂金丝桃素能激发膜电流和钙信号，促进CA3区锥体神经元树突棘成熟，与BDNF的作用相似。基于这些初步结果，由于TRPC3和TRPC6形成异源多聚体，我们的假设是，MeCP2突变小鼠通过TRPC3/6通道受损的BDNF信号可以通过选择性TRPC6激活剂金丝桃素逆转两种类似RTT的表型：海马网多动和未成熟的树突棘。我们提出了两个特定的目标：1.测试由BDNF诱发的、由TRPC通道介导的CA3锥体神经元和GABA能中间神经元的膜电流和钙信号在MeCP2突变小鼠中是否受到损害；2.测试金丝桃素治疗是否逆转MeCP2突变小鼠的海马表型，即海马网过度活跃和未成熟的树突棘。将TRPC3/6通道确定为药物干预的新靶点对于临床前试验是必要的，该试验有助于合理治疗RTT和其他与MECP2突变和BDNF信号受损相关的神经发育障碍。

DESCRIPTION (provided by applicant): Understanding the pathophysiological mechanisms of Rett syndrome (RTT) at the cellular and molecular levels, and establishing successful bioassays for evaluation of potential therapeutic strategies take priority in the path of research on this neurodevelopmental disorder. RTT, an autism spectrum disorder, is a devastating childhood disability due to its impact on individuals (1:10,000 births worldwide), their families and society RTT is caused by loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2), a transcriptional regulator that binds to methylated CpG sites in promoter regions of several genes, including the neurotrophin Bdnf, and the Ca2+-permeable non-selective cationic channel subunits Trpc3 and Trpc6. The availability of endogenously expressed BDNF for its activity-dependent release can be monitored with membrane currents and dendritic Ca2+ signals mediated by TRPC channels. Preliminary Results demonstrate that TRPC currents and Ca2+ signals evoked in CA3 pyramidal neurons by stimulation of presynaptic mossy fiber (MF) are smaller in symptomatic Mecp2 mutant mice. Responses evoked by either recombinant BDNF or a non-hydrolyzable DAG analog (to activate TRPC channels) are also impaired in Mecp2 mutant neurons. Consistently, mRNA and protein levels of both BDNF and TRPC3 are lower in Mecp2 mutant hippocampus. Preliminary Results show that the TRPC6 channel activator hyperforin evokes membrane currents and Ca2+ signals, and promotes dendritic spine maturation in CA3 pyramidal neurons, resembling well-known actions of BDNF. Based on these Preliminary Results and since TRPC3 and TRPC6 form heteromultimers, our hypothesis is that impaired BDNF signaling through TRPC3/6 channels in Mecp2 mutant mice can be overcome by treatment with the selective TRPC6 activator hyperforin to reverse two RTT-like phenotypes: hippocampal network hyperactivity and immature dendritic spines. We propose two Specific Aims: 1. Test whether membrane currents and Ca2+ signals evoked by BDNF and mediated by TRPC channels in CA3 pyramidal neurons and GABAergic interneurons are impaired in Mecp2 mutant mice; and 2. Test whether treatment with hyperforin reverses hippocampal phenotypes in Mecp2 mutant mice, i.e. hippocampal network hyperactivity and immature dendritic spines. Identifying TRPC3/6 channels as novel targets for pharmacological intervention is necessary for pre-clinical trials leading to rational treatments for RTT and other neurodevelopmental disorders associated with MECP2 mutations and impaired BDNF signaling.