Imbalances in Calcium Homeostasis: Mechanism Behind Ethanol-Related Dendritic Deg

描述(申请人提供)：成人持续饮酒会导致小脑的永久性改变，这种改变会离散地扰乱运动功能，并随着年龄的增长而变得更加明显。随着人口老龄化的持续升级，这是一个主要的健康问题。在衰老大鼠中，慢性酒精摄入会导致浦肯野神经元(PN)树突退化，这一过程肯定会改变小脑功能，因为PN是小脑唯一的输出神经元。与酒精相关的树突退化伴随着树突的其他退行性变化，如PN树突的主要钙存储成分--平滑内质网(SER)的扩张。PN树突内SER的扩张强烈提示树突内钙的增加，这是无数神经元通路的第二信使信号。乙醇相关的SER扩张也提示肌浆网钙ATPase(SERCA)泵可能发生了乙醇相关的改变，该泵在钙信号转导后将钙重新释放到SER管腔中。这项建议的总体目标是表明与酒精相关的细胞内钙水平的增加和SERCA 2b钙ATPase泵的水平和/或功能的下降可能是导致衰老和酒精喂养的大鼠PN树突状变性的最终驱动力。钙网蛋白是一种与SERCA泵相互作用的钙结合蛋白，也将被检测。为了达到这一目标，12个月龄的F344雄性大鼠将喂饲AIN-93M乙醇(EF大鼠)、AIN-93M对照(PF)大鼠或大鼠饲料(CF大鼠)，为期数周。年轻的对照组老鼠也将被包括在分析中。为了测量树突内的钙和特定的树突成分，急性小脑切片将在10、20或40周的乙醇处理后制成，电穿孔以促进Fura-2进入PN，并在切片上进行比率钙测量。治疗10、20或40周后，近端和远端树突状分支、棘突和分支点的SERCA 2b ATPase泵和钙网织蛋白的水平将通过共聚焦显微镜和荧光强度相关性进行评估。SERCA 2b泵的功能将通过以下方法进行评估：当SERCA泵以外的钙隔离系统被阻断时，PN处理大的钙瞬变的能力；通过评估乙醇是否使神经元对thapsigargin诱导的caspase-12和GRP78的表达具有易感性。据预测，慢性酒精摄入将导致细胞内钙离子增加，这将与乙醇导致SERCA 2b泵的功能或表达下降有关。本研究的结果将为进一步研究乙醇对细胞内钙稳态的作用以及导致PN树突状变性的机制提供基础。 与公共健康相关：到2050年，美国老年人口将翻一番，这一增长伴随着老年终生饮酒消费者数量的大幅增加。最近的研究表明，成人持续的适度饮酒会导致基于小脑的运动协调性的永久性改变，以及归因于小脑与额叶的联系的认知活动。永久性缺陷是由这些改变造成的，这些改变随着年龄的增长变得更加普遍，并使我们越来越多的老年人口容易摔倒、事故和认知功能障碍。

DESCRIPTION (provided by applicant): Consistent, albeit moderate alcohol consumption in human adults results in permanent alterations in the cerebellum that discretely disrupt motor functions and become more pronounced with age. This is a major health concern as the aging population continues to escalate. In aging rats, chronic ethanol consumption results in regression of Purkinje neuron (PN) dendrites, a process that surely alters cerebellar function as PN are the sole output neurons from the cerebellum. Ethanol-related dendritic regression is coupled with other degenerative changes in dendrites such as dilation of the smooth endoplasmic reticulum (SER), the major calcium storage component of PN dendrites. Dilation of the SER in PN dendrites is strongly suggestive of increases in intradendritic calcium, a second messenger signal for a myriad of neuronal pathways. Ethanol-related dilation of the SER also suggests that ethanol-related alterations may have occurred in the sarcoplasmic reticulum calcium ATPase (SERCA) pump that resequesters calcium into the SER lumen following calcium signaling. The overall goal of this proposal is to show that ethanol-related increases in intracellular calcium levels and declines in the levels and/ or function of the SERCA 2b calcium ATPase pump may be the driving force that culminates in PN dendritic degeneration in aging and alcohol-fed rats. Calreticulin, a calcium binding protein that interacts with the SERCA pump will also be examined. To achieve this goal, 12 mo old F344 male rats will be fed the AIN-93M ethanol (EF rats), AIN-93M control (PF) rats, or rat chow (CF rats) for a period of weeks. Young control rats will also be included in the analysis. To measure calcium within dendrites and specific dendritic components, acute cerebellar slices will be made following 10, 20, or 40 weeks of ethanol treatment, electroporated to facilitate Fura-2 entry into PN, and ratiometric calcium measures made on the slices. Levels of the SERCA 2b ATPase pump and calreticulin will be assessed with confocal microscopy, and fluorescence intensity correlation after treatment durations of 10, 20 or 40 weeks in proximal and distal dendritic branches, spines, and branchpoints. SERCA 2b pump function will be assessed by observing the ability of the PN to handle large calcium transients when calcium sequestration systems other than the SERCA pump are blocked and by assessing whether ethanol predisposes the neuron to thapsigargin-induced expression of caspase-12.and GRP78 It is predicted that chronic ethanol consumption will result in increases in intracellular calcium ions that will be related to ethanol-related declines in the function or expression of the SERCA 2b pump. Results from this study will provide a basis for future studies of the actions of ethanol on intracellular calcium homeostasis and the mechanisms that result in PN dendritic degeneration. PUBLIC HEALTH RELEVANCE: The elderly U.S. population will double by 2050, an escalation accompanied by a tremendous increase in the number of elderly lifetime alcohol consumers. Recent studies show that consistent, albeit moderate alcohol consumption in human adults results in permanent alterations in cerebellar- based motor coordination and also in the cognitive activities ascribed to the cerebellum's connections with the frontal lobe. Permanent deficits result from these alterations that become more prevalent with age and predispose our increasing elderly population to falls, accidents, and cognitive dysfunctions.