CALRETININ CONTAINING NEURONS AND EXCITOTOXIC INJURY

钙视网膜蛋白（CR）是一种钙结合蛋白，存在于 脑神经元的亚群，如黑质（SN）和 腹侧被盖区我们最近证明了神经毒性 兴奋性氨基胍诱导的中脑神经元原代培养物 酸（EAA）对含酪氨酸羟化酶（TH）的 神经元而不是含钙视蛋白的神经元（Isaacs等，1996年）。 我们正在调查其他神经元培养物（皮质， 丘脑和嗅球），以确定这一明显的 神经保护状态可以推广到其他神经元细胞类型 也含有CR。 因为CR和相关蛋白质已经与 在SN的神经保护功能，我们决定治疗中脑 用神经递质的前体L-DOPA培养神经元 多巴胺和一种用于治疗帕金森病的药物。我们发现- 含有神经元的细胞对低水平的L-DOPA非常敏感 治疗5天后，超过60%的细胞丢失。含CR的神经元， 相比之下，只有轻微的影响。然而，最令人感兴趣的是 当CR与TH共定位于多巴胺能神经元时， 神经元对L-DOPA的有害作用免疫。 为了 为了确定CR单独是否具有神经保护功能， 由于其钙结合能力，我们转染了PC 12细胞， 用编码由绿色 荧光蛋白和CR。 通过这种方式，我们能够监测 荧光细胞（也含有CR）的形态和数量 在血清剥夺和钙离子载体治疗后， 使体内钙含量上升到兴奋性中毒水平 我们没有发现 来自CR存在的神经保护证据， 处理条件 我们认为要么是钙离子的流入 转染PC 12细胞后， 通过CR提供细胞保护或CR需要特定的 靶向蛋白质，以便有效地存在于SN细胞中 而在转染的PC 12细胞中不存在。 或者，CR无相关性 神经保护，只是巧合地与保护联系在一起， 黑质我们建议进一步分析CR的响应- 含有细胞（内源性或转染的）的各种处理 来研究这些关键问题

Calretinin (CR) is a calcium binding protein that is found in subpopulations of brain neurons such as the substantia nigra (SN) and the ventral tegmental area. We recently demonstrated that the neurotoxicity induced in primary neuronal mesencephalic cultures by excitatory amino acids (EAA) was selective for tyrosine hydroxylase (TH)-containing neurons rather than calretinin-containing neurons (Isaacs et al., 1996). We are in the process of surveying other neuronal cultures (cortex, thalamus and olfactory bulb) to determine if this apparent neuroprotective status can be generalized to other neuronal cell types which also contain CR. Because CR and related proteins have been linked to neuroprotective functions in the SN, we decided to treat mesencephalic cultured neurons with L-DOPA, the precursor to the neurotransmitter dopamine, and a drug used to treat Parkinson's disease. We found that TH- containing neurons were very sensitive to low levels of L-DOPA in culture and over 60% were lost after a five day treatment. CR-containing neurons, in contrast were only marginally affected. Of most interest, however, was that when CR was colocalized with TH in dopaminergic neurons, these neurons were immune to the deleterious effects of L-DOPA. In an attempt to determine whether CR alone was serving a neuroprotective function in neurons due to its calcium binding capacity, we transfected PC12 cells with a plasmid which coded for a fusion protein consisting of green fluorescent protein and CR. In this manner we were able to monitor the morphology and number of the fluorescent cells (which also contained CR) after serum deprivation and treatment with calcium ionophores which would raise internal calcium levels to excitotoxic levels. We found no evidence of neuroprotection derived from the presence of CR in either treatment condition. We would suggest that either the calcium influx following calcium ionophore treatment of transfected PC 12 cells was too drastic to offer the cells protection via CR or that CR requires specific target proteins in order to be effective which are present in SN cells but not in transfected PC12 cells. Alternatively, CR has no relevance to neuroprotection and is only coincidentally linked with protection in the substantia nigra. We propose to further analyze the response of CR- containing cells (endogenous or transfected) to a variety of treatments to investigate these critical questions.