Molecular Neuropathology and Mechanisms of BACE1 Elevation in Alzheimer's Disease

 描述（由申请方提供）：BACE 1是启动Ablast产生的β-分泌酶，是AD的主要治疗靶点。抑制BACE 1酶活性的药物正在进行AD的临床试验，但这些药物的安全性和有效性尚不清楚。最近的研究表明，BACE 1抑制可能导致多种神经系统副作用。因此，至关重要的是开发替代的治疗策略，减少BACE 1裂解APP，而不损害基本的BACE 1功能。我们已经表明，在APP转基因小鼠和AD脑中，整体BACE 1蛋白水平显著升高。升高的BACE 1集中在营养不良的轴突和淀粉样斑块周围的终末内，并与BACE 1切割的APP片段和A β 42的产生增加有关。我们还发现，A β 42导致静息[Ca 2 +]i增加和神经元中微管破坏。我们假设一种前馈机制，其中斑块相关的AAF导致轴突营养不良，BACE 1积累，并加速AAF的产生，驱动AD进展。阐明营养不良性BACE 1升高的分子和细胞机制可能导致新的AD治疗策略，以使BACE 1水平正常化并减少斑块周围的ApoA产生，同时保留BACE 1活性以实现基本功能和减轻副作用。我们假设，AAF诱导的Ca 2+流入斑块周围轴突导致微管破坏，受损的轴突运输，BACE 1积累，轴突营养不良，加速AAF生成和淀粉样蛋白负荷。我们的初步数据表明，黄芪通过Ca 2+选择性通道提高初级神经元的静息[Ca 2 +]i。此外，黄芪处理的原代神经元的轴突表现出破坏的微管和受损的BACE 1轴突运输。5XFAD小鼠中的斑块周围营养不良轴突也显示出升高的静息[Ca 2 +]i和破坏的微管。使用Ca 2+通道抑制剂或shRNA-AAV，我们将在体外和体内鉴定介导腺苷酸诱导的神经元中Ca 2+内流的通道（目的1）。此外，使用Ca 2+通道抑制剂或shRNA-AAV，我们将降低腺苷酸诱导的静息[Ca 2 +]i升高，阻断微管和运动蛋白破坏，改善轴突运输，并降低BACE 1升高（目的2）。我们还将通过将Ablast处理的原代神经元和5XFAD小鼠暴露于微管稳定剂埃博霉素D来挽救BACE 1升高和轴突运输，并确定Ablast诱导的BACE 1升高是否是tau蛋白非依赖性的（目的2）。最后，我们将通过以下方式确定BACE 1升高是否加速了AAF生成和淀粉样蛋白进展：1）AAF处理的原代神经元的35 S-代谢标记以测量从头AAF生成，2）体内AAF微透析以分析斑块周围区域中的AAF生成，3）AAF时间戳标记以分析5XFAD小鼠中个体斑块生长和营养不良性神经突形成的速率（目的3）。这些实验将为减少斑块周围BACE 1升高的治疗策略提供概念证明，作为直接抑制BACE 1酶活性的更安全的替代方案。

 DESCRIPTION (provided by applicant): BACE1 is the ß-secretase enzyme that initiates Aß production and is a prime therapeutic target for AD. Drugs that inhibit BACE1 enzyme activity are in clinical trials for AD, however the safety and efficacy of these agents are unknown. Recent studies suggest that BACE1 inhibition may cause multiple neurological side effects. Thus, it is crucial to develop alternative therapeutic strategies that reduce BACE1 cleavage of APP without impairing essential BACE1 functions. We have shown that global BACE1 protein levels are markedly elevated in APP transgenic mouse and AD brains. Elevated BACE1 is concentrated within dystrophic axons and terminals surrounding amyloid plaques and is associated with increased generation of BACE1- cleaved APP fragments and Aß42. We also find that Aß42 causes increased resting [Ca2+]i and microtubule disruption in neurons. We hypothesize a feed-forward mechanism in which plaque-associated Aß causes axonal dystrophy, BACE1 accumulation, and accelerated Aß generation that drives AD progression. Elucidating the molecular and cellular mechanisms of dystrophic BACE1 elevation could lead to novel AD therapeutic strategies to normalize BACE1 levels and reduce peri-plaque Aß production, yet preserve BACE1 activity for essential functions and side effect mitigation. We hypothesize that Aß-induced Ca2+ influx into peri-plaque axons causes microtubule disruption, impaired axon transport, BACE1 accumulation, axonal dystrophy, and accelerated Aß generation and amyloid load. Our preliminary data show that Aß elevates resting [Ca2+]i in primary neurons via Ca2+-selective channels. Moreover, axons of Aß-treated primary neurons exhibit disrupted microtubules and impaired BACE1 axon transport. Peri-plaque dystrophic axons in 5XFAD mice also show elevated resting [Ca2+]i and disrupted microtubules. Using Ca2+ channel inhibitors or shRNA-AAVs, we will identify the channel(s) that mediates Aß-induced Ca2+ influx in neurons in vitro and in vivo (Aim 1). Additionally, using Ca2+ channel inhibitors or shRNA-AAVs, we will decrease Aß-induced elevated resting [Ca2+]i, block microtubule and motor protein disruption, improve axon transport, and reduce BACE1 elevation (Aim 2). We will also rescue BACE1 elevation and axon transport by exposing Aß-treated primary neurons and 5XFAD mice to the microtubule stabilizing agent Epothilone D and determine whether Aß-induced BACE1 elevation is tau-independent (Aim 2). Finally, we will determine whether BACE1 elevation accelerates Aß generation and amyloid progression by performing 1) 35S-metabolic labeling of Aß-treated primary neurons to measure de novo Aß production, 2) in vivo Aß microdialysis to analyze Aß production in peri-plaque regions, 3) multicolor Aß time-stamp labeling to analyze the rate of individual plaque growth and dystrophic neurite formation in 5XFAD mice (Aim 3). These experiments will provide proof of concept for therapeutic strategies to reduce peri-plaque BACE1 elevation as a safer alternative to direct inhibition of BACE1 enzyme activity.