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

描述（由申请人提供）：成人持续适度饮酒会导致小脑的永久性改变，这种改变会离散地破坏运动功能，并随着年龄的增长而变得更加明显。随着人口老龄化不断升级，这是一个主要的健康问题。在衰老的大鼠中，慢性乙醇消耗导致浦肯野神经元（PN）树突的退化，这一过程肯定会改变小脑的功能，因为PN是小脑唯一的输出神经元。乙醇相关的树突退化与树突的其他退行性变化相结合，如光滑内质网（SER）的扩张，SER是PN树突的主要钙储存成分。PN树突中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扩张也表明，肌浆网钙atp酶（SERCA）泵可能发生了与乙醇相关的改变，该泵在钙信号传导后将钙释放到SER管腔中。该提案的总体目标是表明，酒精相关的细胞内钙水平升高和SERCA 2b钙atp酶泵水平和/或功能下降可能是衰老和酒精喂养大鼠PN树突变性的最终驱动力。钙网蛋白，一种与SERCA泵相互作用的钙结合蛋白也将被检查。为了实现这一目标，12月龄的F344雄性大鼠将被喂食AIN-93M乙醇（EF大鼠）、AIN-93M对照（PF大鼠）或大鼠饲料（CF大鼠）一段时间。年轻的对照大鼠也将被纳入分析。为了测量树突和特定树突成分中的钙，将在乙醇处理10、20或40周后制作急性小脑切片，电穿孔以促进Fura-2进入PN，并在切片上进行比例钙测量。在治疗10周、20周或40周后，用共聚焦显微镜评估近端和远端树突分支、脊柱和分支点的SERCA 2b atp酶泵和钙调蛋白水平和荧光强度相关性。SERCA 2b泵的功能将通过观察当除SERCA泵外的钙隔离系统被阻断时PN处理大钙瞬态的能力，以及通过评估乙醇是否使神经元倾向于thapsigargin诱导的caspase-12表达来评估。据预测，慢性乙醇消耗会导致细胞内钙离子增加，这与乙醇相关的SERCA 2b泵功能或表达下降有关。本研究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树突变性的机制提供基础。

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.