Transcription profiling of mouse brain and cerebellum from adult mutant stg, tg, and lh mice, which display the ataxic and epileptic phenotypes vs. wild type litter-mate controls

Inherited mutations of calcium ion channels exhibit neurological defects, such as epilepsy, ataxia, and migraine, and these phenotypes are shared among humans and mouse models. Absence epilepsy and ataxic phenotypes are present in the calcium channelopathy mutants stargazer (stg-gamma2 subunit), tottering (tg-alpha1 subunit), and lethargic (lh-beta4 subunit). These mutations of high-voltage-activated (HVA) calcium channel subunits initiate increases in membrane excitability of low-voltage-activated (LVA) calcium channels in thalamic neurons, thus enhancing LVA currents. Elevated LVA currents, produced from T-type calcium channels, induce rhythmic thalamocortical burst firing and spike-wave seizures. The changes in gene expression originating from the different mutations result in similar T-type channel function; however, the network of gene modifications causing altered molecular plasticity and the emergence of pathological phenotypes remain unknown. We would like to understand how loss of 3 different subunits of a calcium ion channel differentially alter gene expression in the brain. Characterizing the gene expression profiles of the mutant stg, tg, and lh mice will provide evidence of the epileptic and ataxic mechanisms, which may identify potential therapeutic targets. We will compare the gene expression profiles among adult mutant stg, tg, and lh mice, which display the ataxic and epileptic phenotypes, along with their wildtype, litter-mate controls, in the cerebellum and brain (without cerebellum). We propose that the neuronal dysfunction associated with the ataxic and epileptic phenotypes develops from a common network of interacting gene alterations within the mutant stg, tg, and lh brain. The changes dictated by the initial mutations leading to the ataxic and epileptic phenotypes are hypothesized to be localized to the cerebellum and the remaining areas of the brain, including the thalamus and cortex, respectively. Adult mutant stg, tg, and lh mice, between 2 and 5 months of age, along with wildtype, litter-mate controls, will be sacrificed. Two brain regions responsible for two phenotypes will be examined. The cerebellum (ataxia) will be dissected from remainder of the forebrain (generalized epilepsy). Each tissue set will be collected in triplicate (3 separate mice per set) to account for biological variance, and total RNA isolated via standard Trizol procedure (Invitrogen) and purified with the RNeasy cleanup kit (Qiagen). RNA samples will be stored at -80 C until sent for analysis using the Affymetrix GeneChip Mouse Genome 430 2.0 whole genome array.钙离子通道的遗传性突变可引发癫痫、共济失调、偏头痛等神经功能缺陷，此类表型在人类与小鼠模型中均有出现。在钙通道病突变体stargazer（stg-γ2亚基）、tottering（tg-α1亚基）和lethargic（lh-β4亚基）中，可观察到失神性癫痫与共济失调表型。这类高电压激活（high-voltage-activated, HVA）钙通道亚基的突变，会导致丘脑神经元内低电压激活（low-voltage-activated, LVA）钙通道的膜兴奋性升高，进而增强LVA电流。由T型钙通道介导的LVA电流升高，会诱发节律性丘脑皮层爆发式放电与棘波癫痫发作。不同突变引发的基因表达变化最终可产生相似的T型通道功能异常，但介导分子可塑性改变与病理表型出现的基因修饰网络仍未明确。 本研究旨在阐明钙离子通道的3种不同亚基缺失如何差异性地改变大脑内的基因表达。对stg、tg、lh突变小鼠的基因表达谱进行表征，可为癫痫与共济失调的发病机制提供实验依据，有望识别潜在的治疗靶点。我们将对表现出共济失调与癫痫表型的成年stg、tg、lh突变小鼠，及其野生型同窝对照小鼠，分别在小脑与全脑（去除小脑）中开展基因表达谱的比较分析。我们推测，与共济失调和癫痫表型相关的神经元功能障碍，源于stg、tg、lh突变小鼠大脑内一组共同的相互作用基因改变网络。引发共济失调与癫痫表型的初始突变所带来的变化，可分别定位于小脑（对应共济失调表型）以及包括丘脑、皮层在内的大脑其余区域（对应全身性癫痫表型）。 本研究将对2至5月龄的成年stg、tg、lh突变小鼠及其野生型同窝对照小鼠实施安乐死。选取与两种表型相关的两个脑区进行检测：分离小脑（对应共济失调）与前脑其余部分（对应全身性癫痫）。每个组织样本设置3次生物学重复（每组3只独立小鼠）以抵消生物学变异，随后通过标准Trizol法（Invitrogen）提取总RNA，并使用RNeasy纯化试剂盒（Qiagen）进行纯化。RNA样品将保存于-80℃，直至送至Affymetrix GeneChip小鼠基因组430 2.0全基因组芯片进行分析。