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

描述（由申请人提供）：阿尔茨海默病被预测为未来几十年全球最优先的健康风险之一，但目前没有有效的预防或疾病修饰疗法。该领域的主要焦点是靶向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，脂质修饰酶的遗传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、脂质活化蛋白激酶和脂质磷酸酶都有效地靶向改善该疾病小鼠模型的行为缺陷，表明磷脂调节可能是该疾病表型的关键因素。一种特殊的磷脂，磷酸肌苷（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，在阿尔茨海默病患者的大脑和小鼠模型中都发生了改变。控制磷酸肌苷代谢和磷酸化的酶的活性是广泛相互关联的，代表了一个控制信号脂质水平的易处理的脂质修饰酶家族。具体来说，关键信号脂质磷脂酰肌醇-4,5-二磷酸[P(4,5)P2]，通过用Aβ处理神经元和疾病小鼠模型的突触而改变。为了研究脂质变化对a β的反应，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，可以有效地用于筛选范例。回声状态网络的使用克服了通常用于筛选的其他转化细胞系的局限性，通过表达关键的神经元表型，以及通过可扩展性解离的神经元培养的局限性。ESN形成形态成熟的功能突触，对a β的挑战敏感，导致突触连接的丧失。这些神经元在接受a β治疗后表现出突触后蛋白95 （PDS-95）的显著缺失。我们建议建立一种检测突触/PSD-95缺失的检测方法，并使用RNA干扰来筛选突触维持PI代谢途径的候选组分。由于先前的研究已经确定了几种与AD表型相关的PI修饰酶，我们期望这种全系统的方法将能够确定已知和新的靶标，以改善a β引发的突触丢失，并验证高含量筛选平台。这些研究将支持未来对PI在a β触发缺陷的细胞通路中的作用的研究，并为使用ESN进行更广泛的筛选范例铺平道路。

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 β-peptide (Aβ) 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 Aβ and in synapses of mouse models of the disease. In order to study lipid changes in response to Aβ, 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 Aβ challenge resulting in a loss of synaptic connections. These neurons display a prominent loss of post-synaptic protein 95 (PDS-95) after treatment with Aβ. 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 Aβ-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 Aβ-triggered deficits as well as pave the way for more extensive screening paradigms using ESN.