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

描述（由申请人提供）：相对神经元易感性是神经退行性疾病的一个普遍但鲜为人知的特征。在帕金森病（PD）中，黑质（SN; A9）内的多巴胺能（DA）神经元特别脆弱，而邻近的腹侧被盖区（VTA; A10）内的DA神经元基本不受影响。我们之前已经证明，这些DA神经元之间的分子差异导致了它们不同的易感性1，并可用于防止体内神经退行性变2。本研究拟研究大鼠和人中脑VTA （A10）中比SN （A9）中高富集的GTPase蛋白RAB3B单体。RAB3B定位于神经元突触囊泡，已知通过调节突触囊泡运输过程，包括突触末端的对接、融合和再循环，来调节神经递质胞外分泌。RAB3B还能激活磷酸化并激活促生存激酶Akt的磷酸肌醇激酶（PI3K）。这些功能特别令人感兴趣，因为，首先，囊泡运输的破坏和突触功能障碍与a-突触核蛋白引起的毒性密切相关，a-突触核蛋白是PD中聚集的一种蛋白质，与该疾病有遗传联系；其次，Akt信号传导先前已被证明可以防止氧化应激，这一过程被认为在PD发病机制中发挥重要作用，并在多种毒性PD模型中得到利用。我们的初步数据通过证明：1)RAB3B在体外增加突触蛋白水平，以及多巴胺稳态的关键调节因子；2) RAB3B在体外和体内均能增加多巴胺含量；3) RAB3B在体外对模拟PD方面的毒素有保护作用，包括氧化应激源6-羟基多巴胺（6-OHDA）和蛋白酶体抑制剂MG-132；4)在大鼠腺相关病毒-2 （AAV2）突触核蛋白病模型中，DA神经元丢失前的显著早期变化包括突触蛋白的改变。根据RAB3B的天然生物学功能和我们的初步数据，我们假设RAB3B有助于降低PD中VTA (A10) DA神经元的易损性，过表达RAB3B可以在体内保护SN (A9) DA神经元的易损性。利用AAV2系统，我们拟描述RAB3B在大鼠SN体内表达的生物学效应，特别强调对突触功能至关重要的过程，包括囊泡运输和神经递质释放和储存。然后，我们将在两种互补的PD啮齿动物模型中检测RAB3的体内表达是否能保护SN （A9）神经元：aav2介导的1-突触核蛋白过表达和6-OHDA诱导的氧化应激损伤。引用1。Chung, c.y.，等。中脑多巴胺能神经元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。植物学通报，2004,17(5):444 - 444。2. 钟春英，Koprich， J.B, Endo， S.和Isacson， O.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易感性。中国生物医学工程学报，2009,32(2):444 - 444。在帕金森病（PD）中，黑质（A9）中的多巴胺能（DA）神经元比紧邻的腹侧被盖区（A10）中的多巴胺能神经元更容易受到影响，这些神经元群之间的差异可能有助于设计新的PD治疗和神经保护策略。RAB3B是一种在A10 DA神经元中高度表达的分子，其调节突触囊泡运输的功能使其成为令人兴奋的候选神经保护剂。突触囊泡运输是PD中中断的一个过程。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RAB3B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.