Dysregulation of Cav1.2 by beta amyloid peptide

摘要 β-淀粉样蛋白对Cav1.2的调节失调 L类钙通道是基因转录、神经元兴奋性和突触的关键调节因子 功能。Cav1.2是大脑中普遍存在的LTCC(Hell等人，1993，JCB 123,949-962)。长期增长 通过LTCC的钙离子内流在早期被认为与老年性症状和阿尔茨海默病(AD)有关(例如， 科学272,1017)。我们发现，老年大鼠显著增加了PKA介导的磷酸化 1928年丝氨酸上的Cav1.2(Davare and Hell，2003年，PNA 100,16018-23)，这增加了Cav1.2通道活动 (…钱学森，地狱，2017，Sci Sig 10，eaaf9659)。我们还发现，Cav1.2形成了一个独特的信号复合体，与 B2肾上腺素能受体(B2AR)也包含cAMP介导的所有其他必需的蛋白质 Cav1.2的调节(例如Davare等人，2001，Science 293，98)，使Cav1.2成为b2 AR信号的主要目标。 B-淀粉样多肽1-42(Ab)是阿尔茨海默病的标志。寡聚体ABO刺激b2受体。支持证据来自 单通道记录表明，Abo通过b2受体增加Cav1.2的活性。我们假设这是 增加是由Cav1.2的主要PKA位点S1928的磷酸化介导的，并触发下游事件 最终会导致神经元损伤。目标1是测试Abo通过Cav1.2是否存在Cav1.2失调- 相关的b2AR发生在树突和棘突(钙离子成像)，无论它是否部分地通过增加 Cav1.2的表面插入，以及是否通过药物或在创新的S1928A中阻断该信号 敲入(KI)小鼠减轻ABO神经毒性。目的2是测试ABO-b2 AR-S1928信号是否刺激 钙离子通透性(CP)AMPAR(GluA1同聚体)的表面插入和CP-AMPAR的封闭可减轻ABO 神经毒性。我们将使用SEP标记的GluA1和GluA2的尖端实时成像，并分析田间EPSP 和mEPSC抑制谷氨酸再摄取前后检测突触周围AMPAR活性 太空。目的3将测试ABO-b2 AR-S1928信号在ABO增强长期抑郁中的作用， 而在长时程增强中，这一点被ABO削弱。将我们的S1928A Ki小鼠与高度淀粉样变性小鼠杂交 5xFAD小鼠将显示Abo-b2 AR-S1928信号是否在抗体诱导的Cav1.2功能障碍中起重要作用 活性和突触传递及其可塑性。这个项目的重点是我们假设的早期 触发后续神经毒性事件的ABO(分钟范围)的影响，因为我们需要了解所有 ABO毒性的几个方面。此外，作为早期事件，Abo-b2 AR-S1928信号构成了一个潜在的目标 用于AD的早期药物干预。我们将在某种程度上探索Cav1.2下游的后续事件 通过测试6-48小时的神经毒性是否可以通过Abo-B2AR-受体阻滞剂的阻滞剂来改善 CaV1.2信号和CP-AMPAR在神经元培养中的表达。我们还将探讨阻止此信号在 5xFAD小鼠模型将改善这些小鼠的突触功能。我们的多肽的研究进展 这破坏了b2 AR与Cav1.2的绑定，这是非常创新的，因为它允许专门阻止信号 ABO通过b2AR而不影响涉及b2AR的其他信号通路。

Abstract Dysregulation of Cav1.2 by beta amyloid peptide L-type Ca2+ channels (LTCC) are key regulators of gene transcription, neuronal excitability, and synaptic functions. Cav1.2 is the prevalent LTCC in brain (Hell et al., 1993, JCB 123, 949-962). Chronically increased Ca2+ influx via LTCCs has been implicated early on in senile symptoms and Alzheimer’s disease (AD) (e.g., Science 272, 1017). We found that aged rats have significantly increased PKA-mediated phosphorylation of Cav1.2 on Serine 1928 (Davare and Hell, 2003, PNAS 100, 16018-23), which increases Cav1.2 channel activity (Qian, …, Hell, 2017, Sci Sig 10, eaaf9659). We also found that Cav1.2 forms a unique signaling complex with the b2 adrenergic receptor (b2 AR) that also contains all other proteins necessary for the cAMP-mediated regulation of Cav1.2 (e.g., Davare et al., 2001, Science 293, 98), making Cav1.2 a prime target for b2 AR signaling. b amyloid peptide 1-42 (Ab) is a hallmark of AD. Oligomeric Abo stimulates the b2 AR. Supportive evidence from single channel recording indicates that Abo increase Cav1.2 activity via the b2 AR. We hypothesize that this increase is mediated by phosphorylation of S1928, the main PKA site of Cav1.2, and triggers downstream events that ultimately cause neuronal damage. Aim 1 is to test whether Cav1.2 dysregulation by Abo via Cav1.2- associated b2 AR occurs in dendrites and spines (Ca2+ imaging), whether it is in part mediated by increased surface insertion of Cav1.2, and whether blocking this signaling pharmacologically or in innovative S1928A knock-in (KI) mice alleviates Abo neurotoxicity. Aim 2 is to test whether Abo - b2 AR - S1928 signaling stimulates surface insertion of Ca2+ permeable (CP) AMPARs (GluA1 homomers) and blocking CP-AMPARs alleviates Abo neurotoxicity. We will use cutting edge live imaging of SEP-tagged GluA1 and GluA2 and analyze field EPSPs and mEPSC before and after inhibition of glutamate re-uptake to measure AMPAR activity in the perisynaptic space. Aim 3 will test the role of Abo - b2 AR - S1928 signaling in augmentation of long-term depression by Abo, and in long-term potentiation, which is impaired by Abo. Crossing our S1928A KI mice with highly amyloidogenic 5xFAD mice will show whether Abo - b2 AR - S1928 signaling is important for Ab-induced dysfunction of Cav1.2 activity and synaptic transmission and its plasticity. This project is focused on what we hypothesize is an early effect of Abo (minute range) that triggers subsequent neurotoxic events because we need to understand all aspects of Abo toxicity. Also, as an early event the Abo - b2 AR - S1928 signaling constitutes a potential target for early pharmacological intervention in AD. We will to some degree explore later events downstream of Cav1.2 dysregulation by testing whether neurotoxicity in the 6-48h range can be improved by blockers of Abo - b2AR - CaV1.2 signaling and of CP-AMPAR in neuronal cultures. We will also explore whether blocking this signaling in the 5xFAD mouse model of AD will improve synapse function in these mice. The development of our peptide that disrupts binding of the b2 AR to Cav1.2 is highly innovative because it allows to specifically block signaling by Abo via the b2 AR without affecting other signaling pathways that involve the b2 AR.