Polo-like-kinase-2-dependent α-Synuclein Serine-129 Phosphorylation: a Physiological RoleDuring Synaptic Activity

摘要/摘要 帕金森病(PD)、路易体痴呆(DLB)、多系统萎缩(MSA)和某些 阿尔茨海默病(AD)是一种以病变为特征的脑部疾病。 富含α-突触核蛋白)(αS)。无法获得治疗疾病的方法，部分原因是缺乏 洞察原生αS的活力是如何变得异常的。 我们的长期目标是了解αS的生物学 αS.αS的正常生理状态和功能的保存/重建策略 路易小体/突起通常在丝氨酸-129(PS129)上被磷酸化，参与其中的激酶(S)已被 被认为是潜在的药物靶标。然而，pS129也可能在突触上具有正常的生理作用。 我们的观察结果表明，pS129是由神经活动可逆地诱导的。我们在这项应用中的总体目标是- 阳离子的作用是(I)确定调节pS129活性的突触机制，以及(Ii)确定pS129的活性。 α的pS129的Vance在突触中发挥作用，S。我们的中心假设是Polo-like kinase2(PLK2)磷光体- 根据突触活动，使αS的丝氨酸-129位碱化，从而微调αS的功能。这样做的理由是 项目是，了解正常突触αS的磷酸化可能为发育提供新的见解 研究了维护αS动态平衡、纠正αS失衡和量化签名αS的策略。我们 提出以下具体目标：1)找出控制可逆性αS pS129的机制(S)。 罗纳尔活动。2)确定可逆性pS129在微调αS功能中的作用。在第一个目标下，初级啮齿动物 皮层神经元培养将被用来确认PLK2是在神经元活动过程中介导pS129的激酶 并确定α、S和PLK2中可能触发pS129的分子变化。关键发现将在小鼠身上得到证实 海马片和在丰富环境下的小鼠模型。在第二个目标中，我们将研究这些影响 PS129在体外和体内对αS生物学功能的影响。最重要的是，我们将表征S129磷酸- 功能缺陷(S129A)和拟态(S129D)敲入小鼠模型，特别关注多巴胺 释放，使用在苏尔泽实验室建立的方法。这项拟议的研究具有创新性，因为它聚焦于 关于动态αS S129在αS的正常位置(成熟神经元的突触)的磷酸化，认为是突触 活性作为一个重要参数，识别关键蛋白质，解决功能后果，并扩展细胞 培养结果到活体。尽管pS129已经得到了广泛的研究，但以前的大多数工作都集中在它在 病理性沉积。这一贡献将是巨大的，因为它有望提供新的、范例-- 将视角转移到正常的αS突触生物学上。证实了αS的一种改性通常与- 疾病是正常发生的，了解疾病是如何发生的，为什么会发生，是了解疾病的重要一步- αS对健康和疾病的深刻看法，对药物和生物标记物开发的重大影响。这部作品 致力于“阿尔茨海默病相关痴呆症的建议”中概述的研究重点- 恩斯“。它侧重于解决AD相关痴呆症(ADRD)的优先事项，特别是DLB和PD痴呆症。

SUMMARY/ABSTRACT Parkinson’s disease (PD), dementia with Lewy bodies (DLB), multiple-system atrophy (MSA) and certain forms of Alzheimer’s disease (AD) are ‘synucleinopathies’ - brain diseases characterized by lesions (Lewy bod- ies/Lewy neurites) rich in α-synuclein (αS). Disease-modifying treatments are not available, in part due to a lack of insight into how native αS dynamics becomes aberrant. Our long-term goal is to understand αS biology in detail and to develop strategies to preserve/reestablish the normal physiological state and function of αS. αS in Lewy bodies/neurites is often phosphorylated on serine-129 (pS129), and the kinase(s) involved have been discussed as potential drug targets. However, pS129 may also have normal physiological role at synapses sup- ported by our observation that pS129 is reversibly induced by neural activity. Our overall objectives in this appli- cation are to (i) identify the synaptic mechanisms by which activity regulates pS129, and (ii) determine the rele- vance of pS129 for αS function at the synapse. Our central hypothesis is that polo-like kinase 2 (Plk2) phosphor- ylates αS at serine-129 in response to synaptic activity, thereby fine-tuning αS function. The rationale for this project is that understanding normal synaptic αS phosphorylation is likely to offer new insight for the development of strategies to preserve αS homeostasis, correct αS imbalance and quantify signatures of αS pathology. We propose the following specific aims: 1) Identify the mechanism(s) that govern reversible αS pS129 during neu- ronal activity. 2) Identify the role of reversible pS129 in fine-tuning αS function. Under the first aim, primary rodent cortical neuron cultures will be used to confirm Plk2 as the kinase that mediates pS129 during neuronal activity and to identify molecular changes in αS and Plk2 that may trigger pS129. Key findings will be confirmed in mouse hippocampal slices and in a mouse model of enriched environment. In the second aim, we will study the effects of pS129 on αS biology functionally in vitro and in vivo. Most importantly, we will characterize S129 phospho- deficient (S129A) and -mimicking (S129D) knock-in mouse models functionally with a special focus on dopamine release, using established methods in the Sulzer lab. The proposed research is innovative, because it focuses on dynamic αS S129 phosphorylation at αS’s normal locale (the synapse of mature neurons), considers synaptic activity as an important parameter, identifies key proteins, addresses functional consequences, and extends cell culture findings to in vivo. Whereas pS129 has been widely studied, most previous work focused on its role in pathological deposits. The contribution will be significant because it is expected to provide novel, paradigm- shifting insight into normal αS biology at the synapse. Corroborating that an αS modification commonly associ- ated with disease occurs normally, and understanding how and why, is an important step towards a comprehen- sive view of αS in health and disease with major implications for drug and biomarker development. This work pursues research priorities outlined in “Recommendations of the Alzheimer's disease-related dementias confer- ence”. It focuses on priorities that address AD-related dementias (ADRD), specifically DLB and PD dementia.