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）的病理生理机制，并建立成功的生物测定法以评估潜在的治疗策略，这是这种神经发育障碍研究的优先方向。RTT是一种自闭症谱系障碍，是一种毁灭性的儿童残疾，由于其对个体（全世界1：10，000出生）、其家庭和社会的影响，RTT是由编码甲基-CpG结合蛋白2（MeCP 2）的基因中的功能缺失突变引起的，甲基-CpG结合蛋白2是一种转录调节因子，其结合几个基因的启动子区域中的甲基化CpG位点，包括神经营养因子BDNF和Ca 2+渗透性非选择性阳离子通道亚基Trpc 3和Trpc 6。内源性表达的BDNF的活性依赖性释放的可用性可以用TRPC通道介导的膜电流和树突状Ca 2+信号来监测。初步结果表明，TRPC电流和Ca 2+信号诱发CA 3锥体神经元突触前苔藓纤维（MF）的刺激是较小的症状Mecp 2突变小鼠。由重组BDNF或不可水解的DAG类似物（以激活TRPC通道）引起的反应也在Mecp 2突变神经元中受损。与此同时，BDNF和TRPC 3的mRNA和蛋白水平在Mecp 2突变海马中均较低。初步结果显示，TRPC 6通道激活剂贯叶金丝桃素引起膜电流和Ca 2+信号，并促进CA 3锥体神经元中树突棘的成熟，类似于BDNF的众所周知的作用。基于这些初步结果，并且由于TRPC 3和TRPC 6形成异多聚体，我们的假设是，Mecp 2突变小鼠中通过TRPC 3/6通道受损的BDNF信号传导可以通过用选择性TRPC 6激活剂贯叶金丝桃素治疗来逆转两种RTT样表型：海马网络过度活跃和未成熟树突棘来克服。我们提出两个具体目标：1。测试Mecp 2突变小鼠中CA 3锥体神经元和GABA能中间神经元中由BDNF诱发并由TRPC通道介导的膜电流和Ca 2+信号是否受损;和2.测试贯叶金丝桃素治疗是否逆转Mecp 2突变小鼠的海马表型，即海马网络过度活跃和未成熟树突棘。将TRPC 3/6通道确定为药物干预的新靶点对于临床前试验是必要的，这些临床前试验导致RTT和其他与MECP 2突变和受损BDNF信号相关的神经发育障碍的合理治疗。

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.