BACE1 trafficking and degradation in Alzheimer’s disease

摘要 阿尔茨海默病（Alzheimer's disease，AD）是一种以记忆障碍为特征的进行性神经退行性疾病 和认知能力下降AD的病理学标志之一是存在神经炎斑块， 由与营养不良的神经突密切相关的聚集的淀粉样β（Aβ）核心组成， 小胶质细胞和反应性星形胶质细胞。β位点淀粉样前体蛋白裂解酶（BACE 1）是 Aβ产生的限制酶。BACE 1在神经元中以高水平表达，并定位于神经元中。 突触前末梢此外，BACE 1在Aβ周围的营养不良的突触前末梢中积累 AD患者和AD小鼠模型脑中的斑块。然而，BACE 1的具体机制 在斑块周围营养不良轴突中的积累仍有待澄清。由于BACE 1 mRNA水平的变化 在大多数研究中，翻译后AD脑中BACE 1蛋白的增加并不伴随BACE 1蛋白的增加。 这些机制最可能导致AD中BACE 1升高。我们之前的研究表明， BACE 1通过溶酶体途径降解。此外，我们发现网格蛋白衔接子高尔基体定位， 含γ-ear的ARF结合蛋白3（GGA 3）调节BACE 1溶酶体运输和降解。更 最近，我们发现BACE 1的降解和运输受其泛素化的调控。 因此，我们已经确定了内体相关的去泛素化酶USP 8为阴性。 BACE 1泛素化和降解的调节剂。该建议的中心假设是，GGA 3和 USP 8调节BACE 1轴突运输和神经元中的溶酶体降解及其功能障碍， 在AD中观察到的突触前营养不良神经突中的BACE 1积累。为了支持这一假设，我们 发现死后GGA 3水平降低，并与BACE 1水平呈负相关 AD脑同时伴有caspase-3激活。我们还确定GGA 3是一种半胱天冬酶3底物， 并且在细胞凋亡的细胞模型和中风和创伤性脑损伤的啮齿动物模型中均被耗尽。 我们新的初步数据表明，GGA 3水平随着年龄的增长而下降，而BACE 1水平随着年龄的增长而增加。 AD小鼠模型（5XFAD小鼠）。此外，我们最近的实时成像实验表明，GGA 3 基因缺失导致BACE 1轴突运输的破坏及其在肿胀轴突中的积累， 培养的海马神经元。总之，我们的数据表明，GGA 3耗尽是一个主要的候选人， AD中突触前营养不良神经突中BACE 1积累的潜在机制。因此，我们建议：（1） 确定AD脑源性Aβ种类和家族性阿尔茨海默病（FAD相关） 突变损害小鼠和人iPSC衍生的BACE 1的轴突运输和溶酶体降解 2）确定防止半胱天冬酶介导的GGA 3消耗减少BACE 1的程度 体内斑块周围营养不良性神经突中的积累; 3）确定USP 8消耗的程度 减少营养不良性神经炎中BACE 1的积累，并改善体内Aβ病理和认知缺陷。

ABSTRACT Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by memory impairments and cognitive deterioration. One of the pathological hallmarks of AD is the presence of neuritic plaques, which consist of a core of aggregate amyloid beta (Aβ) closely associated with dystrophic neurites, activated microglia and reactive astrocytes. β-site amyloid precursor protein cleaving enzyme (BACE1) is the rate- limiting enzyme in the production of Aβ. BACE1 is expressed at high levels in neurons and localizes at the presynaptic terminals. Moreover, BACE1 accumulates in dystrophic presynaptic terminals surrounding Aβ plaques in brains of AD patients and mouse models of AD. However, the specific mechanism(s) of BACE1 accumulation in peri-plaque dystrophic axons remains to be clarified. As changes in BACE1 mRNA levels did not accompany BACE1 protein increases in AD brains in the majority of the studies, post-translation mechanisms are most likely responsible for BACE1 elevation in AD. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway. Furthermore, we found that the clathrin adaptor Golgi-localized γ-ear-containing ARF binding protein 3 (GGA3) regulates BACE1 lysosomal trafficking and degradation. More recently, we have discovered that BACE1 degradation and trafficking is regulated by its ubiquitination. Accordingly, we have identified the endosomal-associated deubiquitinating enzyme USP8 as a negative regulator of BACE1 ubiquitination and degradation. The central hypothesis of this proposal is that GGA3 and USP8 regulate BACE1 axonal trafficking and lysosomal degradation in neurons and their dysfunction results in BACE1 accumulation in pre-synaptic dystrophic neurites observed in AD. In support of this hypothesis, we discovered that levels of GGA3 were decreased and inversely correlated with BACE1 levels in post-mortem AD brains concurrently with caspase-3 activation. We also determined that GGA3 is a caspase 3 substrates and is depleted both in cellular models of apoptosis and in rodent models of stroke and traumatic brain injury. Our new preliminary data demonstrated that GGA3 decreases while BACE1 levels increases with age in a mouse model of AD (5XFAD mice). Furthermore, our recent live-imaging experiments revealed that GGA3 genetic deletion results in the disruption of BACE1 axonal trafficking and its accumulation in swollen axons in cultured hippocampal neurons. Altogether our data indicate that GGA3 depletion is a leading candidate mechanism underlying BACE1 accumulation in pre-synaptic dystrophic neurites in AD. Thus we propose 1) to determine the extent to which AD brain-derived Aβ species and Familial Alzheimer's Disease (FAD-linked) mutations impair axonal trafficking and lysosomal degradation of BACE1 in murine and human iPSC-derived neurons; 2) to determine the extent to which preventing caspase-mediated depletion of GGA3 reduces BACE1 accumulation in peri-plaques dystrophic neurites in vivo; 3) to determine the extent to which depletion of USP8 reduces BACE1 accumulation in dystrophic neuritis and ameliorates Aβ pathology and cognitive deficits in vivo.