Neuroprotective role of RAB3B in rodent models of Parkinson's disease

描述（由申请人提供）：相对神经元脆弱性是神经退行性疾病的一个普遍但知之甚少的特征。在帕金森病（PD）中，黑质（SN; A9）中的多巴胺能（DA）神经元特别脆弱，而腹侧被盖区（VTA; A10）内的相邻DA神经元基本上幸免。我们以前已经证明，这些DA神经元之间的分子差异有助于他们的差异脆弱性1，并可用于防止体内神经退行性病变2。在这里，我们建议研究的单体GTdR蛋白RAB 3B是高度富集的VTA（A10）相比，SN（A9）在大鼠和人类中脑。RAB 3B定位于神经元中的突触囊泡，并且已知通过调节突触囊泡运输过程（包括在突触末端的对接、融合和再循环）来调节神经递质胞吐。RAB 3B还激活磷酸肌醇-3-激酶（PI 3 K），其磷酸化并激活促存活激酶Akt。这些功能是特别感兴趣的，因为，首先，囊泡运输和突触功能障碍的破坏强烈地涉及由α-突触核蛋白诱导的毒性，α-突触核蛋白是一种在PD中聚集的蛋白质，并且与疾病有遗传联系;第二，Akt信号传导先前已显示出保护免受氧化应激，这一过程被认为在PD发病机制中起重要作用，并在各种毒性PD模型中得到利用。我们的初步数据通过证明：1）RAB 3B增加了体外多巴胺稳态关键调节因子以外的突触蛋白水平; 2）RAB 3B增加了体外和体内多巴胺含量; 3）RAB 3B在体外保护免受模拟PD方面的毒素，包括氧化应激物6-羟基多巴胺（6-OHDA）和蛋白酶体抑制剂MG-132; 4）大鼠腺相关病毒-2（AAV 2）中DA神经元损失之前的显著早期变化突触核蛋白病模型包括突触蛋白的改变。基于RAB 3B的天然生物学功能和我们的初步数据，我们假设RAB 3B有助于降低PD中VTA（A10）DA神经元的脆弱性，并且过表达RAB 3B将保护体内脆弱的SN（A9）DA神经元。通过利用AAV 2系统，我们建议描绘RAB 3B表达在大鼠SN体内的生物学效应，特别强调突触功能的关键过程，包括囊泡运输和神经递质的释放和储存。然后，我们将测试体内RAB 3表达是否在两种互补的啮齿动物PD模型中保护SN（A9）神经元：AAV 2介导的1-突触核蛋白过表达和6-OHDA诱导的氧化应激损伤。参考文献1. Chung，C.Y.，中脑多巴胺能神经元的细胞类型特异性基因表达揭示了参与其脆弱性和保护的分子。《分子遗传学》14，1709-1725（2005）。2. Chung，C.Y.，Koprich，J.B.，Endo，S. & Isacson，O.一种内源性丝氨酸/苏氨酸蛋白磷酸酶抑制剂，G-底物，降低帕金森病模型的脆弱性。J. Neurosci 27，8314-8323（2007）。在帕金森病（PD）中，黑质（A9）中的多巴胺能（DA）神经元比腹侧被盖区（A10）中紧邻的DA神经元更容易受到影响，这些神经元群体之间的这种差异可能有助于设计新的PD治疗和神经保护策略。RAB 3B是一种在A10 DA神经元中高度表达的分子，其在调节突触囊泡运输（PD中中断的过程）中的功能使其成为令人兴奋的候选神经保护剂。我们的目标是在两种不同的帕金森病啮齿动物模型中表征RAB 3B的神经保护作用。

DESCRIPTION (provided by applicant): Relative neuronal vulnerability is a universal yet poorly understood feature of neurodegenerative diseases. In Parkinson's disease (PD), dopaminergic (DA) neurons in the substantia nigra (SN; A9) are particularly vulnerable, whereas adjacent DA neurons within the ventral tegmental area (VTA; A10) are essentially spared. We have previously demonstrated that molecular differences between these DA neurons contribute to their differential vulnerability 1 and can be utilized to protect against neurodegeneration in vivo 2. Here we propose to study the monomeric GTPase protein RAB3B that is highly enriched in VTA (A10) compared to SN (A9) both in rat and human midbrain. RAB3B is localized to synaptic vesicles in neurons and is known to regulate neurotransmitter exocytosis by regulating synaptic vesicle trafficking processes including docking, fusion, and recycling at the synaptic terminals. RAB3B also activates phosphoinositide-3-kinase (PI3K) that phosphorylates and activates the pro-survival kinase Akt. These functions are of particular interest because, first, disruption of vesicle trafficking and synaptic dysfunction have been strongly implicated in toxicity induced by a-synuclein, a protein that aggregates in PD and that has been genetically linked to the disease; second, Akt signaling has previously been shown to protect against oxidative stress, a process thought to play an important role in PD pathogenesis and that is exploited in a variety of toxic PD models. Our preliminary data strengthen the rationale for our grant proposal by demonstrating that: 1) RAB3B increases the levels of synaptic proteins in addition to key regulators of dopamine homeostasis in vitro; 2) RAB3B increases dopamine content in vitro and in vivo; 3) RAB3B protects against toxins that simulate aspects of PD in vitro, including the oxidative stressor 6-hydroxydopamine (6-OHDA) and the proteasome inhibitor MG-132; 4) a prominent early change preceding DA neuron loss in a rat adeno-associated virus-2 (AAV2) synucleinopathy model includes alteration of synaptic proteins. On the basis of the native biological function of RAB3B and our preliminary data, we hypothesize that RAB3B contributes to the reduced vulnerability of VTA (A10) DA neurons in PD and that overexpressing RAB3B will protect vulnerable SN (A9) DA neurons in vivo. By utilizing the AAV2 system, we propose to delineate the biological effects of RAB3B expression in the rat SN in vivo, with a particular emphasis on processes critical for synaptic function including vesicular transport and neurotransmitter release and storage. We will then test whether RAB3 expression in vivo protects SN (A9) neurons in two complementary rodent models of PD: AAV2-mediated 1-synuclein overexpression and a 6-OHDA induced oxidative stress lesion. References 1. Chung, C.Y., et al. Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection. Hum Mol Genet 14, 1709-1725 (2005). 2. Chung, C.Y., Koprich, J.B., Endo, S. & Isacson, O. An endogenous serine/threonine protein phosphatase inhibitor, G-substrate, reduces vulnerability in models of Parkinson's disease. J. Neurosci 27, 8314-8323 (2007). PUBLIC HEALTH RELEVANCE In Parkinson's disease (PD), dopaminergic (DA) neurons in the substantia nigra (A9) are considerably more susceptible than the immediately adjacent DA neurons in the ventral tegmental area (A10) and this differences between these neuronal populations may be useful in designing novel therapeutic and neuroprotective strategies for PD. RAB3B is a molecule more highly expressed in A10 DA neurons and its function in regulating synaptic vesicle trafficking, a process disrupted in PD, makes it an exciting candidate neuroprotective agent. Our goal in this grant is to characterize the neuroprotective role of RAB3B in two different rodent models of Parkinson's disease.