Roles of reticulon proteins in neurodegenerative diseases

描述(由申请人提供)：网状/Nogo(RTN/Nogo)蛋白是神经和神经退行性疾病的关键因素。在我们之前的资金周期中，我们研究了神经元RTN3在BACE1蛋白水解酶活性负调控中的潜在作用。我们证明，RTN3在小鼠体内的表达增加，减少了大脑皮质区域的淀粉样蛋白沉积。然而，RTN3表达的增加也促进了RTN3免疫反应性营养不良神经元(RIDN)在海马区的形成，RIDN的出现损害了海马区的突触功能。这些结果表明，RTN3在神经炎斑块的形成中可能扮演两个相反的角色，在斑块中淀粉样蛋白沉积通常被各种营养不良的神经突起(包括RIDN)和反应性胶质细胞包围。在这项提案中，我们的目标是研究如何将这两个看似对立的角色分开。我们假设躯体树突状RTN3调节BACE1的活性，聚集的轴突RTN3促进营养不良神经突起的形成。我们将利用本实验室建立的四个动物模型(TG-RTN3小鼠、Tet-off可诱导启动子下的RTN3转基因小鼠、RTN1和RTN3 KO小鼠)来验证这一假说，具体目的如下：1)研究RTN蛋白水平对RIDNS形成的影响；2)探讨RIDNS的病理生理后果；3)确定RTN蛋白在体内神经性斑块形成中的作用。此外，我们还发现RIDN自然存在于老年小鼠的大脑中。为了进一步了解它们在老年人脑中的发生，我们将解决另一个具体目标，即识别和表征人脑中的RIDN。至少将使用来自两个不同来源的尸检大脑来解决这一特定目标。我们将确定RIDN是否发生在老年人的大脑中，以及这种情况在AD大脑中是否明显更常见。上述研究的结果将提供进一步的证据，表明RIDN代表了老年和AD脑中营养不良神经突起的早期阶段，减少RIDN是一个重要的新药靶点，目的是改善老年人和AD患者的认知功能。 公共卫生相关性：阿尔茨海默病(AD)患者大脑中的两个已知病理特征是细胞外神经炎(老年性)斑块和神经元内神经原纤维缠结。神经性斑块主要由淀粉样物沉积组成，常与营养不良的轴突和反应性胶质细胞有关。我们的研究集中在淀粉样蛋白沉积和营养不良神经突起的调节形成上。具体地说，我们在上一个资金周期中研究了网状蛋白(Rtns)在AD发病中的作用，并证明了rtns作为BACE1的负调制子发挥作用，BACE1是一种关键酶，在b-分泌酶位置裂解淀粉样前体蛋白(APP)，促进淀粉样沉积中的主要成分AB的释放。我们进一步证明了调节BACE1的主要RTN是神经元网状3(RTN3)。然而，我们也发现，RTN3表达的增加可以促进RTN3免疫反应性营养不良神经元(RIDN)的形成，RIDN主要以丰富的形式存在于AD脑中的淀粉样沉积周围。这些对比结果表明，RTN3在神经炎斑块的形成中具有双重作用。在这项提议中，我们的目标是研究这两种对立的影响是如何分离的。我们的中心假设是RTN3在轴突中的过度聚集导致RIDN的形成，而体突胶质细胞中的单体RTN3调节BACE1的活性。为了验证这一假说，我们将进行一系列相辅相成的体外和体内实验，主要目的有四个：1)研究RTN蛋白水平和聚集对RIDN形成的影响；2)探讨RIDN的病理生理学后果；3)确定RTN蛋白在体内神经学斑块形成中的作用；4)鉴定和表征人脑中的RIDN。因此，这些研究将有助于利用四种不同的动物模型揭示RTN3在淀粉样蛋白沉积和营养不良神经突起形成中的作用，也将有助于我们理解RIDN在人脑中的发生。从这一系列研究中获得的知识将确定rtns(特别是rtn3)在营养不良神经突起形成中的重要性，并将有助于开发治疗药物和策略，以抑制与rtn3聚集相关的神经性斑块的形成。

DESCRIPTION (provided by applicant): Reticulon/Nogo (RTN/Nogo) proteins are critical factors in neurological and neurodegenerative disorders. In our previous funding cycle, we investigated potential roles of neuronal RTN3 in the negative modulation of BACE1 proteolytic activity. We demonstrated that increased expression of RTN3 in mice reduced amyloid deposition in cortical brain regions. However, this increased expression of RTN3 also facilitated the formation of RTN3 immunoreactive dystrophic neurites (RIDNs) in the hippocampus, and the occurrence of RIDNs impaired hippocampal synaptic function. These results suggest that RTN3 can play two opposing roles in the formation of neuritic plaques, in which amyloid deposits are often surrounded by various dystrophic neurites (including RIDNs) and reactive glial cells. In this proposal, we aim to investigate how these two seemingly opposing roles can be dissociated. We hypothesize that somatodendritic RTN3 modulates BACE1 activity, and that aggregated axonal RTN3 facilitates the formation of dystrophic neurites. We will perform a set of experiments utilizing four animal models (Tg-RTN3 mice, RTN3 transgenic mice under Tet-Off inducible promoter, RTN1 and RTN3 KO mice) generated in our lab to test this hypothesis, specifically addressing the following specific aims: 1) To investigate the effect of RTN protein levels on the formation RIDNs; 2) To explore the pathophysiological consequence of RIDNs; 3) To determine the in vivo role of RTN proteins in the formation of neuritic plaques. Moreover, we have found that RIDNs naturally occur in the elderly mouse brain. To further understand their occurrence in elderly human brains, we will address an additional specific aim, to identify and characterize RIDNs in human brains. Postmortem brains from at least two different sources will be used to address this specific aim. We will determine whether RIDNs occur in elderly human brains and whether this occurrence is significantly more frequent in AD brains. The results from the above studies will provide further evidence that RIDNs represent an early stage of dystrophic neurites in elderly and AD brains and that reducing RIDNs is an important novel drug target with the aim of improving cognitive function in the elderly population and AD patients. PUBLIC HEALTH RELEVANCE: Extracellular neuritic (senile) plaques and intraneuronal neurofibrillary tangles are two known pathological hallmarks in brains of patients with Alzheimer's disease (AD). Neuritic plaques are mainly composed of amyloid deposits that are frequently associated with dystrophic neurites and reactive glial cells. Our research is focused on the regulated formation of amyloid deposition and dystrophic neurites. Specifically, we investigated the role of reticulon proteins (RTNs) in AD pathogenesis in the previous funding cycle, and demonstrated that RTNs function as negative modulators of BACE1, a critical enzyme that cleaves amyloid precursor protein (APP) at the b-secretase site, contributing to the release of AB, the major component in amyloid deposits. We further demonstrated that the primary RTN that modulates BACE1 is neuronal reticulon 3 (RTN3). However, we also showed that increased expression of RTN3 can facilitate the formation of RTN3 immunoreactive dystrophic neurites (RIDNs), which are present in an abundant form primarily surrounding amyloid deposits in AD brains. These contrasting results suggest that RTN3 has a dual role in the formation of neuritic plaques. In this proposal, we aim to investigate how these two opposing effects can be dissociated. Our central hypothesis is that excessive aggregation of RTN3 in axons leads to the formation of RIDNs, whereas monomeric RTN3 in somoatodendrites modulates BACE1 activity. To test this hypothesis, we will perform a series of complimentary in vitro and in vivo experiments with four major aims: 1) To investigate the effects of RTN protein levels and aggregation on the formation RIDNs; 2) To explore the pathophysiological consequences of RIDNs; 3) To determine the in vivo role of RTN proteins in the formation of neuritic plaques; 4) To identify and characterize RIDNs in human brains. Therefore, these studies will help reveal the role of RTN3 in the formation of amyloid deposition and dystrophic neurites using four different animal models, and will also contribute to our understanding of the occurrence of RIDNs in human brains. The knowledge gained from this series of studies will determine the importance of RTNs (specifically RTN3) in the formation of dystrophic neurites and will be useful in the development of therapeutic agents and strategies to inhibit the formation of neuritic plaques that are associated with RTN3 aggregation.