Generation and Characterization of Mouse Models of Neuro

小鼠神经模型的生成和表征

• 批准号: 7327026
• 负责人: Huaibin Cai
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7327026
• 关键词: Generation; Characterization; Mouse; Models; Neuro

The Section of Transgenesis (TS) in Laboratory of Neurogenetics (LNG) was devoted to study the pathogenic mechanisms and experimental therapeutics of neurodegenerative diseases by a combination of in vivo mouse modeling and in vitro neurobiology approaches. Our research covers three major neurological diseases: Alzheimer's disease (AD), Parkinson's disease (PD), and Amyotrophic lateral sclerosis (ALS). Project 1. BACE1 is Critical for Cognition A transmembrane aspartyl protease termed beta-site APP cleavage enzyme1 (BACE1) that cleaves the amyloid precursor protein (APP) is required for the generation of amyloid-beta (A-beta) peptides implicated in the pathogenesis of AD. Significantly, deletion of BACE1 in a mouse model of AD, the APPswe; PS1delE9 double transgenic mice prevents both A-beta deposition and age-associated cognitive abnormalities that occur in this model of A-beta amyloidosis. Moreover, the A-beta burden is sensitive to BACE1 dosage in young but not in aged APPswe; PS1delE9 mice, suggesting that A-beta clearance mechanisms in aged animals may be compromised. Although BACE1 null mice do not exhibit overt developmental abnormalities or adult-onset neuropathology, however, these animals do manifest alterations in performance on tests of cognition and emotion. Importantly, the memory deficits occurring in BACE1 knockout mice are prevented in APPswe; PS1delE9; BACE1knockout mice. Our results establish that BACE1-dependent APP processing is critical for cognitive and emotional behaviors, suggesting that future studies should be alert to potential mechanism-based toxicities associated with BACE1 inhibitors designed to ameliorate A-beta amyloidosis in AD. These findings were published in the Journal of Neuroscience last year. Project 2. Activation of protein kinase C modulates BACE1-mediated b-secretase activity Previous studies suggest that activation of protein kinase C (PKC) modulates the b-secretase-mediated cleavage of APP and reduces the production of Ab. The mechanism of PKC-mediated modulation of b-secretase activity, however, remains elusive. We report here that activation of PKC modulated b-secretase activity through either suppressing the accumulation or promoting the translocation of BACE1 protein in a cell type-dependent manner. We found that activation of PKC suppressed the accumulation of BACE1 protein in fibroblasts through an enhancement of intracellular protease activities. In neurons, activation of PKC did not alter the expression level of BACE1, but led to more BACE1 translocated to the cell surface, resulting in a decreased cleavage of APP at the b1 site. Together, Our findings provide novel mechanisms of PKC-mediated modulation of b-secretase activity, suggesting that alteration of the intracellular trafficking of BACE1 may serve as a useful therapeutic strategy to lower the production of Ab in AD. This work is under revision from Neurobiology of Aging. Project 3. Loss Function of Alsin is Not Sufficient to Trigger Major Motor Neuron Degeneration but Predispose Neurons to Oxidative Stress ALS, the most common motor neuron disease, is caused by a selective loss of motor neurons in the central nervous system. Mutations in the ALS2 gene have been linked to one form of autosomal recessive juvenile onset ALS (ALS2). To investigate the pathogenic mechanisms of ALS2, we generated ALS2 knockout (ALS2-/-) mice. While ALS2-/- mice lacked obvious developmental abnormalities, they exhibited age-dependent deficits in motor coordination and motor learning. Moreover, ALS2-/- mice showed a higher anxiety response in the open field and elevated plus maze tasks. Although they failed to recapitulate clinical or neuropathological phenotypesconsistent with motor neuron disease by 20 months of age, ALS2-/- mice or primary cultured neurons derived from these mice were more susceptible to oxidative stress compared to wild type controls. These observations suggest that loss of ALS2 function is insufficient to cause major motor deficits or motor neuron degeneration in a mouse model, but predisposes neurons to oxidative stress. These data were published in Journal of Neuroscience this August. Project 4. ALS2-Deficiency Leads to Neuronal Degeneration in Amyotrophic Lateral Sclerosis through Altered AMPA Receptor Trafficking To further understand the function of alsin that is encoded by the full-length ALS2 gene, we screened proteins interacting with alsin. Here we report that alsin interacted with glutamate receptor interacting protein 1 (GRIP1) both in vitro and in vivo, and co-localized with GRIP1 in neurons. In support of the physiological interaction between alsin and GRIP1, the subcellular distribution of GRIP1 was altered in ALS2-/- spinal motor neurons, which correlates with a significant reduction of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptor subunit 2 (GluR2) at the synaptic/cell surface of ALS2-/- neurons. The decrease of calcium-impermeable GluR2-containing AMPA receptors at the cell/synaptic surface rendered ALS2-/- neurons more susceptible to glutamate receptor-mediated neurotoxicity. Our findings reveal a novel function of alsin in AMPA receptor trafficking and provide a novel pathogenic link between ALS2-deficiency and motor neuron degeneration, suggesting a protective role of alsin in maintaining the survival of motor neurons. This work is under review from Journal of Neuroscience. Project 5. Deficiency in ALS2 gene does not affect the motor neuron degeneration in SOD1G93A transgenic mice Previous in vitro studies suggest that over-expression of ALS2 protects cells from mutant Cu/Zn superoxide dismutase (SOD1)-induced cytotoxicity. To test whether ALS2 plays a protective role against mutant SOD1-mediated motor neuron degeneration in vivo, we examined the progression of motor neuron disease in SOD1G93A mice on an ALS2 null background. Our data suggest that deficiency in the ALS2 gene does not affect the pathogenesis of SOD1G93A mice. These data were published in Neurobiology of Aging this September. Project 6. A Novel Function of Alsin in Regulating Endosome Degradation that is up-regulated in a Mouse Model of Amyotrophic Lateral Sclerosis Alsin is a newly defined guanine-nucleotide-exchange factor of Rab5 family small GTPase. Dysfunction of alsin has been linked to one form of juvenile onset recessive familial ALS (ALS2). However, how loss of alsin affects Rab5-mediated endocytosis is unclear. Here we report that the Rab5-mediated endosome trafficking is severely altered in neurons derived from alsin knockout (ALS2-/-) mice. We found that the Rab5-mediated endosome fusion was significantly increased in ALS2-/- neurons, resulting in an obvious decrease of endosome motility and increase of endosomes conversed to lysosomes in these neurons. Consequently, a significant increase of endosome/lysosome-dependent degradation of internalized cargo proteins such as glutamate receptors was observed in ALS2-/- neurons. Together, our data reveal a novel function of alsin in endosome trafficking, suggesting that the increased endosome degradation in ALS2-/- neurons underlies the pathogenic mechanism of ALS2 and related motor neuron diseases. This work is ready to submit to PNAS.

神经遗传学实验室（LNG）转基因研究室（TS）致力于通过体内小鼠模型和体外神经生物学方法相结合来研究神经退行性疾病的发病机制和实验治疗。我们的研究涵盖三种主要的神经系统疾病：阿尔茨海默病（AD）、帕金森病（PD）和肌萎缩侧索硬化症（ALS）。 项目 1. BACE1 对于认知至关重要 一种跨膜天冬氨酰蛋白酶，称为 β 位点 APP 裂解酶 1 (BACE1)，可裂解淀粉样前体蛋白 (APP)，是生成与 AD 发病机制有关的淀粉样β (A-β) 肽所必需的。值得注意的是，AD 小鼠模型 APPswe 中 BACE1 的缺失； PS1delE9 双转基因小鼠可预防 A-β 淀粉样变性模型中发生的 A-β 沉积和与年龄相关的认知异常。此外，年轻 APPswe 中的 A-β 负荷对 BACE1 剂量敏感，但老年 APPswe 中则不然； PS1delE9 小鼠，表明老年动物的 A-β 清除机制可能受到损害。尽管 BACE1 缺失小鼠没有表现出明显的发育异常或成年发病的神经病理学，但这些动物在认知和情感测试中确实表现出表现改变。重要的是，APPswe 可以预防 BACE1 敲除小鼠中发生的记忆缺陷； PS1delE9； BACE1 基因敲除小鼠。我们的结果表明，BACE1 依赖性 APP 处理对于认知和情绪行为至关重要，这表明未来的研究应警惕与旨在改善 AD 中 A-β 淀粉样变性的 BACE1 抑制剂相关的潜在机制毒性。这些发现去年发表在《神经科学杂志》上。 项目2.蛋白激酶C的激活调节BACE1介导的β分泌酶活性 先前的研究表明，蛋白激酶 C (PKC) 的激活可调节 b 分泌酶介导的 APP 裂解并减少 Ab 的产生。然而，PKC 介导的 b 分泌酶活性调节机制仍不清楚。我们在此报告，PKC 的激活通过以细胞类型依赖性方式抑制 BACE1 蛋白的积累或促进其易位来调节 b 分泌酶活性。我们发现 PKC 的激活通过增强细胞内蛋白酶活性来抑制成纤维细胞中 BACE1 蛋白的积累。在神经元中，PKC 的激活不会改变 BACE1 的表达水平，但会导致更多的 BACE1 易位到细胞表面，从而导致 b1 位点 APP 的裂解减少。总之，我们的研究结果提供了 PKC 介导的 b 分泌酶活性调节的新机制，表明改变 BACE1 的细胞内运输可能作为降低 AD 中抗体产生的有效治疗策略。这项工作正在由衰老神经生物学进行修订。 项目 3. Alsin 的损失函数不足以引发主要运动神经元变性，但会使神经元容易遭受氧化应激 ALS 是最常见的运动神经元疾病，是由中枢神经系统中运动神经元的选择性丧失引起的。 ALS2 基因突变与一种常染色体隐性青少年发病 ALS (ALS2) 相关。为了研究 ALS2 的致病机制，我们培育了 ALS2 敲除 (ALS2-/-) 小鼠。虽然 ALS2-/- 小鼠缺乏明显的发育异常，但它们在运动协调和运动学习方面表现出年龄依赖性缺陷。此外，ALS2-/- 小鼠在旷场和高架十字迷宫任务中表现出更高的焦虑反应。尽管它们在 20 个月大时未能重现与运动神经元疾病一致的临床或神经病理表型，但与野生型对照相比，ALS2-/- 小鼠或源自这些小鼠的原代培养神经元更容易受到氧化应激的影响。这些观察结果表明，ALS2 功能的丧失不足以在小鼠模型中引起主要运动缺陷或运动神经元变性，但会使神经元容易受到氧化应激。这些数据发表在今年八月的《神经科学杂志》上。 项目 4. ALS2 缺陷通过改变 AMPA 受体贩运导致肌萎缩侧索硬化症的神经元变性 为了进一步了解全长 ALS2 基因编码的 alsin 的功能，我们筛选了与 alsin 相互作用的蛋白质。在这里，我们报道了 alsin 在体外和体内与谷氨酸受体相互作用蛋白 1 (GRIP1) 相互作用，并与神经元中的 GRIP1 共定位。为了支持 alsin 和 GRIP1 之间的生理相互作用，GRIP1 在 ALS2-/- 脊髓运动神经元中的亚细胞分布发生了改变，这与 α-氨基-3-羟基-5-甲基-4-异恶唑丙酸酯的显着减少相关。 ALS2-/- 神经元突触/细胞表面的 AMPA）型谷氨酸受体亚基 2 (GluR2)。细胞/突触表面不透钙的含有 GluR2 的 AMPA 受体的减少使得 ALS2-/- 神经元更容易受到谷氨酸受体介导的神经毒性的影响。我们的研究结果揭示了 alsin 在 AMPA 受体运输中的新功能，并提供了 ALS2 缺陷和运动神经元变性之间的新致病联系，表明 alsin 在维持运动神经元存活中具有保护作用。这项工作正在接受《神经科学杂志》的审查。 项目5. ALS2基因缺陷不影响SOD1G93A转基因小鼠运动神经元变性 先前的体外研究表明，ALS2 的过度表达可以保护细胞免受突变铜/锌超氧化物歧化酶 (SOD1) 诱导的细胞毒性。为了测试 ALS2 是否对体内突变 SOD1 介导的运动神经元变性发挥保护作用，我们检查了 ALS2 无效背景下 SOD1G93A 小鼠运动神经元疾病的进展。我们的数据表明 ALS2 基因缺陷不会影响 SOD1G93A 小鼠的发病机制。这些数据发表在今年九月的《衰老神经生物学》杂志上。 项目 6. Alsin 调节内体降解的新功能，该功能在肌萎缩侧索硬化症小鼠模型中上调 Alsin 是 Rab5 家族小 GTPase 中新定义的鸟嘌呤核苷酸交换因子。 alsin 功能障碍与一种青少年发病的隐性家族性 ALS (ALS2) 相关。然而，alsin 的缺失如何影响 Rab5 介导的内吞作用尚不清楚。在这里，我们报告说，在来自 alsin 敲除 (ALS2-/-) 小鼠的神经元中，Rab5 介导的内体运输发生了严重改变。我们发现ALS2-/-神经元中Rab5介导的内体融合显着增加，导致这些神经元内内体运动明显降低，内体转化为溶酶体的数量增加。因此，在 ALS2-/- 神经元中观察到内化货物蛋白（例如谷氨酸受体）的内体/溶酶体依赖性降解显着增加。总之，我们的数据揭示了 alsin 在内体运输中的新功能，表明 ALS2-/- 神经元中内体降解的增加是 ALS2 和相关运动神经元疾病的致病机制的基础。这项工作已准备好提交给 PNAS。