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)的特征是淀粉样β蛋白(A？)沉积，胶质增生，以及广泛的神经元丢失。然而，大多数通常研究的AD小鼠模型侧重于斑块病理，而不表现出神经退行性变。重要的是，尚不清楚神经元死亡是淀粉样蛋白病理还是反应性胶质细胞增生症的结果。我们将利用Vassar 5XFAD小鼠模型来确定神经元死亡是由神经元内淀粉样前体蛋白(APP)的积累还是随后的反应性胶质增生引起的，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)，并可能诱导包括自噬在内的各种未知的下游效应。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β蛋白沉积始于1-2月龄左右的神经元，4个月龄时出现认知障碍，8个月龄时观察到大量神经元死亡，并伴有丰富的胶质细胞增生。Cramer等人在几个非退行性AD小鼠模型中证明了RXR核受体信号和ApoE在减少淀粉样蛋白病变和改善认知方面的作用。Bex诱导胆固醇代谢基因ApoE、ABCA1和Abcg1的转录，反过来下调促炎基因的表达。用4月龄和8月龄的5XFAD小鼠进行7天Bex治疗，获得初步数据。在5XFAD小鼠中，Bex治疗增加了ApoE、ABCA1和Abcg1的水平，减少了可溶性和不溶性淀粉样蛋白的种类。重要的是，神经元内APP/A？4个月龄5XFAD小鼠大脑皮层V层和IV层明显减少。这与7天后8个月5XFAD改善嗅觉行为有关。此外，Bex治疗增加了小胶质细胞与淀粉样斑块的联系。因此，我们假设长期Bex治疗5XFAD小鼠可以通过减少可溶性A？和抑制神经胶质细胞增多症。自噬是APP/A？Bex去除的一种有吸引力的机制，因为自噬缺陷与包括AD在内的蛋白质错误折叠和神经变性等多种疾病有关。此外，初步的Western分析表明，Bex处理改变了Beclin-1和LC3II的蛋白表达，这两个蛋白分别是启动自噬和自噬小体形成的关键。我们的初步数据显示，Bex减少了神经元内APP。明确APP的去除机制是充分理解和解释Bex治疗的关键。RXR和PPAR异源二聚体也通过抑制NFkB减少炎症基因的表达。NFkB的调节对于脑内主要免疫效应细胞--小胶质细胞的致炎激活至关重要。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(即肿瘤坏死因子、白介素6和白介素1)，抑制A？体外研究已经证实，LXR和PPAR激动剂都下调小胶质细胞中炎症基因的表达，尽管RXR激动剂Bex对小胶质细胞的影响尚未量化。然而，尚不清楚神经元死亡是否可归因于淀粉样β聚集体或随后的炎症反应。总之，这些目标将提供关键的洞察力，以了解神经元内APP和反应性胶质细胞增生对神经元死亡的相对贡献，以及Bex是否可以通过不同的机制改善这两种病理。

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.