Targeting RXRs to enhance autophagy and reduce gliosis in Alzheimer's disease

描述（由申请人提供）：阿尔茨海默病（AD）的特征在于淀粉样蛋白β（A？）的沉积，神经胶质增生和广泛的神经元缺失。然而，最常研究的AD小鼠模型集中于斑块病理学，并且不表现出神经变性。重要的是，神经元死亡是否是淀粉样病变或反应性神经胶质增生的结果尚不清楚。我们将利用Vassar 5XFAD小鼠模型来确定神经元死亡是否主要由神经元内淀粉样前体蛋白（APP）的积累或随后的反应性神经胶质增生引起，其首要目标是验证RXR激动剂贝沙罗汀（Bex）的治疗潜力。作为一种RXR激动剂，Bex影响多种细胞类型（即神经元、小胶质细胞和星形胶质细胞），并可能诱导各种未探索的下游效应，包括自噬。我们选择了一种非常适合研究神经元死亡的小鼠模型，即5XFAD。5XFAD淀粉样蛋白β沉积起源于约1-2个月大的神经元，在4个月时检测到认知缺陷，并且在8个月时观察到相当多的神经元死亡，整个过程中有大量的神经胶质增生。Cramer等人证明了RXR核受体信号传导和ApoE在几种非退行性AD小鼠模型中减少淀粉样蛋白病理和改善认知方面的作用。Bex诱导胆固醇代谢基因ApoE、Abca 1和Abcg 1的转录，并反过来下调促炎基因的表达。使用接受7天Bex治疗的4和8个月5XFAD小鼠获得初步数据。5XFAD小鼠中的Bex治疗增加了ApoE、Abca 1和Abcg 1水平，并减少了可溶性和不溶性淀粉样蛋白种类。重要的是，神经元内的APP/ A？在4个月的5XFAD小鼠中皮质层V和IV中显著降低。这与7天Bex后嗅觉行为的8个月5XFAD改善相关。此外，Bex治疗增加了小胶质细胞与淀粉样斑块的关联。因此，我们推测，长期的贝伐他汀治疗5XFAD小鼠可以防止或减轻神经元死亡，在以后的年龄，通过减少可溶性A？和抑制神经胶质增生。 自噬是Bex去除APP/ A？的一种有吸引力的机制，因为自噬的缺陷与包括AD在内的几种蛋白质错误折叠和神经变性疾病有关。此外，初步的Western分析表明，Bex处理改变了Beclin-1和LC 3 II的蛋白表达，这两种蛋白表达分别对自噬和自噬体形成的起始至关重要。我们的初步数据表明，Bex降低神经元内APP。明确APP去除机制是充分理解和解释Bex治疗的关键。RXR和PPAR异二聚体也通过抑制NF κ B减少炎性基因表达。NFkB的调节对于小胶质细胞（脑的主要免疫效应细胞）的促炎性活化是最关键的。小胶质细胞对A？通过诱导NFkB驱动的基因表达和产生神经毒性炎症介质（即TNF、IL-6和IL-1）以及抑制A？的吞噬作用。体外研究已经确定LXR和PPAR-γ激动剂都下调小胶质细胞中的炎症基因表达，尽管RXR激动剂Bex对小胶质细胞的影响尚未定量。然而，目前尚不清楚神经元死亡是否可归因于淀粉样蛋白β聚集体或随后的炎症反应。总的来说，这些目标将提供关键的洞察力的相对贡献o神经元内APP和反应性神经胶质增生神经元死亡，以及是否Bex可以改善这两种病理通过不同的机制。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is characterized by deposition of amyloid beta (A?), gliosis, and extensive neuronal loss. However, most commonly studied AD mouse models focus on plaque pathology, and do not exhibit neurodegeneration. Importantly, it is unknown if neuronal death is a consequence of amyloid pathology or reactive gliosis. We will utilize the Vassar 5XFAD mouse model to determine if neuronal death is principally caused by accumulation of intraneuronal amyloid precursor protein (APP) or by subsequent reactive gliosis with the overarching goal of validating the therapeutic potential of RXR agonist Bexarotene (Bex). As an RXR agonist, Bex affects multiple cell types (i.e neurons, microglia, and astrocytes) and may induce a variety of unexplored downstream effects including authophagy. We have chosen a mouse model well suited for studying neuronal death, the 5XFAD. 5XFAD amyloid beta deposition originates in neurons around 1-2 months of age, cognitive deficits are detected at 4 months, and considerable neuronal death is observed at 8 months with abundant gliosis throughout. Cramer et al demonstrated the role of RXR nuclear receptor signaling and ApoE in reducing amyloid pathology and improving cognition in several non-degenerative AD mouse models. Bex induces the transcription of cholesterol metabolism genes ApoE, Abca1, and Abcg1, and conversely down regulates proinflammatory gene expression. Preliminary data was obtained using 4 and 8 month 5XFAD mice which received 7 day Bex treatment. Bex treatment in 5XFAD mice increased ApoE, Abca1, and Abcg1 levels and reduced soluble and insoluble amyloid species. Importantly, intraneuronal APP/ A? was significantly reduced in cortical layers V and IV in 4 month 5XFAD mice. This correlated to 8 month 5XFAD improvements in olfaction behavior after 7 day Bex. Furthermore, Bex treatment increased association of microglia with amyloid plaques. Therefore, we hypothesize that long term Bex treatment in 5XFAD mice can prevent or attenuate neuronal death at later ages through reduction in soluble A? and suppression of gliosis. Autophagy is an attractive mechanism for Bex removal of APP/ A?, since defects in autophagy have been implicated in several diseases of protein misfolding and neurodegeneration including AD. Furthermore, preliminary western analysis has indicated that Bex treatment alters protein expression of Beclin-1 and LC3II which are critical for initiation of autophagy and autophagosome formation, respectively. Our preliminary data indicates Bex decreases intraneuronal APP. Defining the mechanism of APP removal is critical for fully understanding and interpreting Bex treatment. RXR and PPAR heterodimers also reduce inflammatory gene expression through suppression of NFkB. The regulation of NFkB is most critical for the proinflammatory activation of microglia, the main immune effector cell of the brain. Microglia respond to A? by induction of NFkB driven gene expression and production of neurotoxic inflammatory mediators (i.e. TNF ¿, IL-6, and IL-1¿) and inhibition of phagocytosis of A?. In vitro studies have established that both LXR and PPAR-¿ agonists downregulate inflammatory gene expression in microglia, though the effects of RXR agonist, Bex, on microglia has not been quantified. However, it is unknown whether neuronal death can be attributed to amyloid beta aggregates or the subsequent inflammatory responses. Collectively, these aims will provide critical insight into the relative contributions o intraneuronal APP and reactive gliosis to neuronal death and whether Bex can ameliorate both pathologies through diverse mechanisms.