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

描述（由申请人提供）：预计阿尔茨海默病将是未来几十年最优先的全球健康风险之一，但目前没有有效的预防或疾病改善疗法。该领域的主要焦点是减少突触毒性淀粉样蛋白β肽（A β），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已被有效地靶向用于改善该疾病的小鼠模型中的行为缺陷，表明磷脂调节可能是该疾病表型的关键因素。已知对细胞和神经元信号传导至关重要的一类特定磷脂，即磷脂酰肌醇（PI），在历史上和最近的工作中已显示在受AD影响的患者大脑以及小鼠模型中发生改变。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，关键的信号脂质磷脂酰肌醇-4，5-二磷酸[P（4，5）P2]通过用A β处理神经元和该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，需要生理学相关的神经元模型。小鼠胚胎干细胞衍生的神经元（ESN）是适合于中高通量平台的可扩展的神经元模型，并且可以有效地用于筛选范例。ESN的使用克服了通常用于通过表达关键神经元表型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通过可扩展性的局限性。ESN形成形态成熟和功能性突触，这些突触对A β刺激敏感，导致突触连接的丧失。这些神经元在A β处理后表现出突触后蛋白95（PDS-95）的显著丢失。我们建议建立并验证一种检测突触/PSD-95丢失的方法，并使用RNA干扰来筛选用于突触维持的PI代谢途径的候选组分。由于先前的研究已经确定了与AD表型相关的几种PI修饰酶，我们预计这种全系统的方法将能够确定改善A β触发的突触丢失的已知和新靶点，并验证高含量筛选平台。这些研究将支持未来的调查， PI在A β触发缺陷的细胞通路中的作用，以及为使用ESN进行更广泛的筛选模式铺平道路。

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 �-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.