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方法来评估潜在的治疗策略，是研究这一神经发育障碍的优先事项。RTT是一种自闭症谱系障碍，是一种毁灭性的儿童残疾，因为它对个体（全世界1:10 000新生儿）、他们的家庭和社会都有影响。RTT是由编码甲基CpG结合蛋白2 （MeCP2）的基因的功能丧失突变引起的，MeCP2是一种转录调节因子，与几个基因启动子区域的甲基化CpG位点结合，包括神经营养蛋白Bdnf和Ca2+渗透性非选择性阳离子通道亚基Trpc3和Trpc6。内源性表达BDNF的活性依赖性释放的可用性可以通过膜电流和TRPC通道介导的树突Ca2+信号来监测。初步结果表明，在症状性Mecp2突变小鼠中，刺激突触前苔藓纤维（MF）在CA3锥体神经元中引起的TRPC电流和Ca2+信号更小。重组BDNF或不可水解的DAG类似物（激活TRPC通道）引起的反应在Mecp2突变神经元中也受到损害。在Mecp2突变体中，BDNF和TRPC3的mRNA和蛋白水平均较低。初步结果表明，TRPC6通道激活剂hyperperforin唤起膜电流和Ca2+信号，促进CA3锥体神经元的树突棘成熟，类似于众所周知的BDNF的作用。基于这些初步结果，由于TRPC3和TRPC6形成异聚体，我们的假设是，Mecp2突变小鼠中通过TRPC3/6通道受损的BDNF信号可以通过选择性TRPC6激活剂hyperperforin治疗来克服，从而逆转两种rt样表型：海马网络亢进和未成熟的树突棘。我们提出两个具体目标：1。检测Mecp2突变小鼠CA3锥体神经元和gaba能中间神经元中由BDNF诱发、TRPC通道介导的膜电流和Ca2+信号是否受损；和2。测试hyperperforin治疗是否能逆转Mecp2突变小鼠的海马表型，即海马网络过度活跃和未成熟的树突棘。确定TRPC3/6通道作为药物干预的新靶点对于临床前试验是必要的，从而合理治疗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.