Screening lipid modifying enzymes to ameliorate A-beta triggered synaptic loss

筛选脂质修饰酶以改善 A-β 引发的突触损失

• 批准号: 8874327
• 负责人: Laura Beth Johnson McIntire
• 金额: $ 20万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8874327
• 关键词: 1-Phosphatidylinositol 4-Kinase; Affect; Aftercare; Alzheimer' s Disease; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Animal Model; Antibodies; Behavioral; Benchmarking; Biological Assay; Brain; Catabolism; Cell Survival; Cell physiology; Central Nervous System Diseases; Clinical Trials; Cognitive deficits; Critical Pathways; Custom; Cyclic AMP-Responsive DNA-Binding Protein; Defect; Dendritic Spines; Detection; Development; Disease; Disease model; Down Syndrome; Enzymes; Family; Functional disorder; Future; Genes; Genetic; Health; In Vitro; Investigation; Libraries; Lipase; Lipids; Maintenance; Mediator of activation protein; Memory; Mental Retardation; Metabolic Pathway; Metabolism; Methods; Modeling; Mus; Mutation; Neurons; Pathology; Pathway interactions; Patients; Phenotype; Phosphatidylinositols; Phospholipase; Phospholipase C; Phospholipids; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Process; Protein Kinase; Proteins; RNA Interference; Reporting; Research; Resistance; Resources; Risk; Role; Secondary to; Signal Transduction; Staging; Stem cells; Synapses; System; Testing; Therapeutic; Toxic effect; Transformed Cell Line; Validation; Viral; Virus Diseases; Work; aging population; amyloid peptide; base; cell type; cellular targeting; clinical investigation; density; disease phenotype; drug discovery; embryonic stem cell; enzyme activity; global health; hippocampal pyramidal neuron; immunoreactivity; in vivo Model; knock-down; loss of function; miniaturize; mouse model; neurotransmission; novel; particle; peptide A; pre-clinical; prevent; prophylactic; response; screening; small hairpin RNA; synaptic function; synaptojanin; therapeutic target; trafficking; transcription factor

DESCRIPTION (provided by applicant): Alzheimer's disease is predicted to be one of the highest priority global health risks in coming decades, yet there are currently no effective prophylactic or disease modifying therapies. The primary focus in the field has been to target reduction of the synaptotoxic amyloid �-peptide (A�) however, recent late-stage clinical trials have proven disappointing. Therefore, new strategies and cellular targets are required. Recent work has found that genetic alteration of lipid modifying enzymes can ameliorate behavioral and synaptic defects in mouse models of the disease. Phospholipases, lipid kinases, lipid activated protein kinases and a lipid phosphatase have all effectively been targeted for ameliorating behavioral deficits in mouse models of the disease indicating that phospholipid modulation may be a key factor of the disease phenotype. A specific class of phospholipids, phosphoinositides (PI), known to be critical for cell and neuronal signaling, has been shown historically and in recent work to be altered in AD affected patient brain as well as in mouse models. The activities of enzymes that control phosphoinositide metabolism and phosphorylation are extensively interconnected and represent a family of tractable lipid modifying enzymes which control levels of signaling lipids. Specifically, the critical signaling lipid phosphatidylinositol-4,5-bisphosphae [P(4,5)P2], is altered by treatment of neurons with A� and in synapses of mouse models of the disease. In order to study lipid changes in response to A�, a physiologically relevant neuronal model is required. Mouse embryonic stem cell derived neurons (ESN) are a scalable neuronal model amenable to mid to high throughput platforms and can be used effectively for screening paradigms. Use of ESN overcomes the limitations of other transformed cell lines commonly used for screening by expression of critical neuronal phenotypes as well as the limitation of dissociated neuronal cultures by scalability. ESN form morphologically mature and functional synapses which are sensitive to A� challenge resulting in a loss of synaptic connections. These neurons display a prominent loss of post-synaptic protein 95 (PDS-95) after treatment with A�. We propose to establish and miniaturize an assay to detect synapse/PSD-95 loss and use RNA interference to screen candidate components of the PI metabolic pathway for synapse maintenance. Since previous research has identified several PI modifying enzymes in connection with AD phenotypes, we expect that this system-wide approach will be able to identify both known and novel targets for amelioration of A�-triggered synaptic loss as well as validate the high content screening platform. These studies will support future investigations into the role of PI in cellular pathways underlying A�-triggered deficits as well as pave the way for more extensive screening paradigms using ESN.

描述（由申请人提供）：阿尔茨海默病预计将成为未来几十年最优先的全球健康风险之一，但目前尚无有效的预防或疾病缓解疗法。该领域的主要关注点是减少突触毒性淀粉样β-肽（Aβ），然而，最近的后期临床试验证明令人失望。因此，需要新的策略和细胞靶点。最近的研究发现，脂质修饰酶的基因改变可以改善该疾病小鼠模型的行为和突触缺陷。磷脂酶、脂质激酶、脂质激活蛋白激酶和脂质磷酸酶都已有效地改善小鼠疾病模型的行为缺陷，表明磷脂调节可能是该疾病表型的关键因素。一类特定的磷脂，即磷酸肌醇 (PI)，已知对细胞和神经元信号传导至关重要，历史上和最近的研究表明，在 AD 患者大脑以及小鼠模型中，其会发生改变。控制磷酸肌醇代谢和磷酸化的酶的活性广泛地相互关联，并且代表了控制信号脂质水平的易于处理的脂质修饰酶家族。具体来说，通过用 A� 治疗神经元和该疾病小鼠模型的突触，关键信号脂质磷脂酰肌醇-4,5-双磷酸 [P(4,5)P2] 会发生改变。为了研究 A� 引起的脂质变化，需要一个生理相关的神经元模型。小鼠胚胎干细胞衍生神经元 (ESN) 是一种可扩展的神经元模型，适用于中高通量平台，可有效用于筛选范例。 ESN 的使用克服了通常用于通过关键神经元表型表达进行筛选的其他转化细胞系的局限性，以及解离神经元培养物在可扩展性方面的局限性。 ESN 形成形态成熟且功能齐全的突触，这些突触对 A� 挑战敏感，导致突触连接丧失。在用 A� 治疗后，这些神经元表现出突触后蛋白 95 (PDS-95) 的显着损失。我们建议建立并小型化一种检测突触/PSD-95损失的方法，并使用RNA干扰来筛选用于突触维持的PI代谢途径的候选成分。由于之前的研究已经确定了几种与 AD 表型相关的 PI 修饰酶，我们预计这种全系统方法将能够确定改善 Aβ 触发的突触损失的已知和新靶标，并验证高内涵筛选平台。这些研究将支持未来的调查 PI 在 Aβ 触发缺陷的细胞通路中的作用，并为使用 ESN 进行更广泛的筛查范例铺平道路。