Defective lysosomal membrane fission mediates axonal lysosome accumulation in dystrophic neurites in Alzheimer's disease.

阿尔茨海默病(AD)损害老年人的独立性，并与巨大的社会 医疗保健和照顾者成本的负担。迫切需要一种有效的治疗方法来遏制这种疾病的浪潮 痴呆症的持续流行和不断增长。最近的许多临床试验都试图降低β的水平- 大脑中的淀粉样蛋白，通常是通过激活免疫系统来促进清除，但这些试验中没有一项 已经取得了成功。我们打算用一种不同的方式来探索这个问题--我们发现神经元 β-淀粉样蛋白沉积周围的轴突肿胀，充满异常的溶酶体，这些溶酶体缺乏 蛋白质降解酶。因为溶酶体对蛋白质的动态平衡至关重要，所以我们假设 这些异常的溶酶体导致神经退行性变，如果它们的功能能够被挽救，它可能 改善大脑功能。我们将通过关注一种新的基因PLD3来研究这一假说，该基因已被发现 会增加AD的风险。在我们的初步工作中，我们发现PLD3在这些基础上得到了极大的丰富 溶酶体异常和脑内PLD3水平与β-淀粉样蛋白负荷和 人类队列中的认知退化。大脑PLD3水平也与记忆呈负相关 在小鼠模型中的表现。我们发现PLD3在酸性条件下起着磷脂酶D的作用 这是溶酶体膜分裂所必需的。我们建议评估在多大程度上 在小鼠中观察到的溶酶体功能障碍在人类组织中存在，并充分评估 PLD3伴神经元溶酶体病理。然后我们将确定编码变体是否报告给 AD风险通过转染含有PLD3的质粒(已经被 突变以复制这些编码变体)，并观察其对溶酶体膜分裂和对PLD3的影响 酶活性。我们将确定有缺陷的神经元溶酶体分裂是否会影响认知和AD 通过将遗传性AD的5xFAD模型与图4的条件基因敲除相结合，使用 Cre在大脑神经元中选择性表达。图4是PIKfyve复合体的组件，图4的丢失 导致有缺陷的溶酶体分裂。重要的是，PIKfyve复合体也调节PLD3的加工和 改变PLD3活性。我们将评估这只小鼠的学习和记忆能力，并确定是否存在缺陷 溶酶体分裂影响β-淀粉样变性和营养不良的轴突。这些研究将帮助我们理解这两个 该蛋白在阿尔茨海默病发病中的作用及溶酶体膜融合在认知衰老中的作用 和AD。平行训练计划将支持我在模型动物发育和行为能力方面的训练 表型，发展溶酶体神经生物学的先进技术，完善衰老的临床管理 相关疾病，所有这些都是我职业发展的关键步骤。总而言之，杰出的 机构环境、资源和跨学科指导团队具有广泛的互补性 专业领域将加速我获得向独立过渡所需的专业知识。

Alzheimer’s disease (AD) compromises the independence of aging adults and is associated with huge societal burden in healthcare and caregiver costs. An effective treatment is urgently needed to stem the tide of the ongoing and growing epidemic of dementia. Many recent clinical trials have attempted to reduce the level of β- amyloid in the brain, typically by activating the immune system to promote clearance, yet none of these trials has been successful. We propose to explore this problem in a different way – we discovered that neuronal axons around deposits of β-amyloid are swollen and filled with abnormal lysosomes which are deficient in protein-degrading enzymes. Because lysosomes are critical for protein homeostasis, we hypothesize that these abnormal lysosomes contribute to neurodegeneration and if their function could be rescued, it could improve brain function. We will study this hypothesis by focusing on a novel gene, PLD3, which was identified as contributing to AD risk. In our preliminary work, we discovered that PLD3 is robustly enriched on these abnormal lysosomes and brain PLD3 levels correlated inversely with both β-amyloid burden and the rate of cognitive deterioration in a human cohort. Brain PLD3 levels also correlated inversely with memory performance in a mouse model. We discovered that PLD3 functions as a phospholipase D in acidic environments and is necessary for lysosomal membrane fission. We propose to evaluate to what degree the lysosome dysfunction observed in mice is present in human tissue and to fully evaluate the association of PLD3 with neuronal lysosomal pathology. We will then determine whether the coding variants reported to confer AD-risk impact the function of PLD3 by transfecting plasmids containing PLD3 (which have been mutated to copy these coding variants) and observing the effect on lysosomal membrane fission and on PLD3 enzyme activity. We will determine whether defective neuronal lysosomal fission impacts cognition and AD neuropathology in vivo by crossing the 5xFAD model of genetic AD with a conditional knock-out of Fig4 using a Cre selectively expressed in cerebral neurons. Fig4 is a component of the PIKfyve complex and loss of Fig4 leads to defective lysosomal fission. Importantly, the PIKfyve complex also regulates PLD3 processing and alters PLD3 activity. We will evaluate learning and memory in this mouse and determine whether defective lysosomal fission impacts β-amyloidosis and dystrophic neurites. These studies will help us understand both how this protein functions in the setting of AD and the role of lysosome membrane fusion in cognitive aging and AD. The parallel training plan will support my training in model animal development and behavioral phenotyping, developing advanced skills in lyosomal neurobiology and refine clinical management of aging related diseases, all of which are critical steps for my career development. Collectively, the outstanding institutional environment, resources, and interdisciplinary mentorship team with broad and complementary areas of expertise will accelerate my acquisition of the necessary expertise to transition to independence.