Investigating the Neuronal Signals Initiating Synapse Loss in Aging and Alzheimer's Disease

摘要：迟发性阿尔茨海默病(LOAD)的靶点为联想皮质，引起深刻 痴呆症和现有的治疗不会改变疾病的进展。负荷的最大风险因素是 年事已高，但尚不清楚为什么衰老的联合皮质容易退化。认知 损伤与背外侧前额叶联合皮质(DlPFC)突触丢失密切相关， 服从更高层次的认知。小鼠模型已经表明，小胶质细胞可以通过 与神经元上的分子“标签”相互作用，包括补体(C1q)和磷脂酰丝氨酸(PS)。 然而，目前还不清楚神经元内部的上游变化会触发这些分子的产生，而且 这些机制是否在老化的dlPFC中被激活。这项拟议的研究将利用老年人 用机械体外实验建立恒河猴模型，以确定其神经内机制 导致衰老相关皮质中的突触丢失。衰老恒河猴的dlPFC扩大 并自然发展为认知缺陷、斑块和缠结、补体C1q表达和区域特异性 突触丢失。老年猕猴还会发展出一种异常的线粒体表型，称为“线粒体-on-a”. 字符串“(MOAS)，在人类负载中可见。我推测MOAS可能是由慢性钙离子引起的。 线粒体超载，产生参与突触移除的分子，包括激活的C1q，PS， 和Caspase3。dlPFC中的突触特别容易受到钙离子失调的影响，因为它们表达cAMP- 蛋白激酶A(PKA)信号通过兰尼定受体2(RyR2)和 三磷酸肌醇受体1型(IP3R1)。这一过程是由磷酸二酯酶PDE4D调节的 年轻的大脑，随着年龄的增长而消失。我推测细胞内钙离子持续升高 导致线粒体钙超载，并随着年龄的增长诱发MOAS，导致 表达介导附近小胶质细胞移除突触的分子(C1q、PS和裂解的caspase3)。 AIMS 1和2将利用高分辨率免疫电子显微镜(EM)和3D EM重建来 阐明MOAS和(AIM 1)已知的介导突触移除的分子之间的相互作用，以及 (目的2)青年猕猴与老年猕猴dlPFC中钙离子失衡的标志物。初步数据显示， MOA优先与C1q相关，在PDE4D表达为 缺席。目标3将使用原代小鼠皮质神经元培养，免疫荧光，超分辨 显微镜和生物化学，在体外模拟钙离子失调，并测试慢性升高的钙离子 水平可以诱导MOAS表型，并产生介导突触移除的分子。相关位置 体外研究结果将在猕猴组织中进行交叉验证。识别导致神经内事件 脆弱的老化皮质中的突触丢失将提供关于年龄增长如何有助于 加载发病机制，并帮助确定早期治疗干预的潜在靶点。

Abstract: Late-onset Alzheimer’s disease (LOAD) targets the association cortices to cause profound dementia, and available treatments do not alter disease progression. The greatest risk factor for LOAD is advanced age, yet it is unknown why the aging association cortices are vulnerable to degeneration. Cognitive impairment tightly correlates with synapse loss in the dorsolateral prefrontal association cortex (dlPFC), which subserves higher-order cognition. Mouse models have shown that microglia can remove synapses by interacting with molecular “tags” on neurons, including complement (C1q) and phosphatidylserine (PS). However, it is unclear what upstream changes within neurons trigger the generation of these molecules, and whether these mechanisms are activated in the aging dlPFC. The proposed research will utilize an aged rhesus macaque model with mechanistic in vitro experiments, to identify the intraneuronal mechanisms that contribute to synapse loss in the aging association cortex. Aging rhesus macaques have an expanded dlPFC and naturally develop cognitive deficits, plaques and tangles, complement C1q expression, and region-specific synapse loss. Aged macaques also develop an abnormal mitochondrial phenotype termed “Mitochondria-on-a- string” (MOAS) that is seen in human LOAD. I hypothesize that MOAS may arise from chronic calcium (Ca2+) overload of mitochondria, generating molecules participating in synapse removal, including activated C1q, PS, and Caspase 3. Synapses in dlPFC are especially vulnerable to Ca2+ dysregulation as they express cAMP- protein kinase A (PKA) signaling to magnify internal Ca2+ release through ryanodine receptor 2 (RyR2) and inositol triphosphate receptor type 1 (IP3R1). This process is regulated by the phosphodiesterase PDE4D in young brain, which is lost with advancing age. I hypothesize that sustained elevations in cytosolic calcium leads to Ca2+ overload of mitochondria and the induction of MOAS with advancing age, leading to the expression of molecules (C1q, PS, and cleaved caspase 3) that mediate synapse removal by nearby microglia. Aims 1 and 2 will utilize high resolution immuno-Electron Microscopy (EM) and 3D EM reconstruction to elucidate the interactions between MOAS, and (Aim 1) molecules known to mediate synapse removal, and (Aim 2) markers of Ca2+ dysregulation in the dlPFC of young vs. aged macaques. Preliminary data indicate that MOAS preferentially associate with C1q and are more frequent under conditions when PDE4D expression is absent. Aim 3 will use primary murine cortical neuron cultures, immunofluorescence, super-resolution microscopy, and biochemistry to model Ca2+ dysregulation in vitro and test whether chronically elevated Ca2+ levels can induce the MOAS phenotype and generate the molecules mediating synapse removal. Relevant in vitro findings will be cross-validated in the macaque tissue. Identification of the intraneuronal events that lead to synapse loss in the vulnerable aging cortex will provide key insights into how advancing age contributes to LOAD pathogenesis, and help identify potential targets for early therapeutic interventions.