原发性震颤(ET)是一种常见的神经系统疾病,其特征是8-10Hz的震颤。ET的分子机制仍然知之甚少。临床数据表明小脑在疾病病理生理学中的重要性,病理研究表明,浦肯野细胞(PC)会受到损害。我们最近的小脑皮质和PC特异性转录组研究确定了钙(Ca2)信号通路的改变,包括ET中的ryanodine受体1型(RyR1)。RyR1是位于内质网(ER)上的细胞内Ca2释放通道,在小脑中主要在PC中表达。在应力条件下,RyR1经历了几个翻译后修饰(蛋白激酶A[PKA]磷酸化,氧化,亚硝基化),再加上通道稳定结合伴侣calstabin1的耗尽,它们共同表征了“泄漏通道”的生化特征。在这项研究中,我们发现RyR1-S2844位点的PKA磷酸化明显增加,RyR1氧化和亚硝基化增加,和死后ET小脑RyR1复合物中的calstabin1消耗。calstabin1-RyR1结合亲和力的降低与PC的丢失和ET中纤维-PC突触的攀升有关。在对照或帕金森氏病小脑中未发现这种“漏”的RyR1特征。死后小脑的微粒体显示ET中ERCa2+过度渗漏与controls,衰减的通道稳定。我们使用具有RyR1点突变的小鼠模型进一步研究了RyR1在震颤中的作用,该模型模拟了组成型位点特异性PKA磷酸化(RyR1-S2844D)。RyR1-S2844D纯合小鼠在小脑生理记录中出现10Hz动作震颤和强烈的异常振荡活动。RyR1激动剂或拮抗剂的小脑内微输注,分别,RyR1-S2844D小鼠的震颤幅度增加或减少,支持小脑RyR1渗漏对震颤产生的直接作用。用新型RyR1通道稳定化合物治疗RyR1-S2844D小鼠,Rycal,有效抑制小脑振荡活动,抑制震颤,和小脑RyR1-calstabin1结合正常化。这些数据共同支持通过RyR1的压力相关ERCa2泄漏可能有助于震颤病理生理学。
Essential Tremor (ET) is a prevalent neurological disease characterized by an 8-10 Hz action tremor. Molecular mechanisms of ET remain poorly understood. Clinical data suggest the importance of the cerebellum in disease pathophysiology, and pathological studies indicate Purkinje Cells (PCs) incur damage. Our recent cerebellar cortex and PC-specific transcriptome studies identified alterations in calcium (Ca2+) signaling pathways that included ryanodine receptor type 1 (RyR1) in ET. RyR1 is an intracellular Ca2+ release channel located on the Endoplasmic Reticulum (ER), and in cerebellum is predominantly expressed in PCs. Under stress conditions, RyR1 undergoes several post-translational modifications (protein kinase A [PKA] phosphorylation, oxidation, nitrosylation), coupled with depletion of the channel-stabilizing binding partner calstabin1, which collectively characterize a \"leaky channel\" biochemical signature. In this study, we found markedly increased PKA phosphorylation at the
RyR1-S2844 site, increased
RyR1 oxidation and nitrosylation, and calstabin1 depletion from the RyR1 complex in postmortem ET cerebellum. Decreased calstabin1-RyR1-binding affinity correlated with loss of PCs and climbing fiber-PC synapses in ET. This \'leaky\' RyR1 signature was not seen in control or Parkinson\'s disease cerebellum. Microsomes from postmortem cerebellum demonstrated excessive ER Ca2+ leak in ET vs. controls, attenuated by channel stabilization. We further studied the role of
RyR1 in tremor using a mouse model harboring a
RyR1 point mutation that mimics constitutive site-specific PKA phosphorylation (
RyR1-S2844D). RyR1-S2844D homozygous mice develop a 10 Hz action tremor and robust abnormal oscillatory activity in cerebellar physiological recordings. Intra-cerebellar microinfusion of RyR1 agonist or antagonist, respectively, increased or decreased tremor amplitude in
RyR1-S2844D mice, supporting a direct role of cerebellar
RyR1 leakiness for tremor generation. Treating
RyR1-S2844D mice with a novel
RyR1 channel-stabilizing compound, Rycal, effectively dampened cerebellar oscillatory activity, suppressed tremor, and normalized cerebellar RyR1-calstabin1 binding. These data collectively support that stress-associated ER Ca2+ leak via RyR1 may contribute to tremor pathophysiology.