Understanding Neural Development--Use of Mouse Mutations

癫痫模型与钙通道功能 P/Q型电压依赖性钙通道CACNA1A突变主要导致人类遗传性偏头痛、发作性共济失调和小脑萎缩，并导致小鼠隐性遗传性共济失调、发作性运动障碍、小脑萎缩和失神癫痫。这些物种差异的基础和致病机制(S)尚不清楚。为了解决这个问题并确定在活体中所需的P/Q功能，我们通过基因打靶创建了一个胚系Cacna1a零突变(命名为Cacna1aFcrtm1)。空小鼠以一种特定的模式发展成肌张力障碍和迟发性小脑变性。这表明P/Q功能是小脑神经元生存所必需的。纯合子缺失小鼠完全缺乏P/Q通道活性，也缺乏w-CTX-MVIIC受体，表明P/Q通道活性由单个基因编码。P/Q零颗粒细胞的L和N型电流密度增加。Cacna1aFcrtm1/+杂合子小鼠的表型正常，尽管电流密度降低了50%，这表明电流密度降低本身不足以导致自发突变的小鼠出现共济失调和癫痫发作。 有假说认为R型钙电流是由Cacna1e基因的表达引起的。为了验证这一假设，我们研究了Cacna1e通道亚单位缺失的小鼠的电压依赖性钙通道的特性。在培养的野生型小鼠小脑颗粒神经元中应用欧米茄毒素GVIA、欧米伽毒素IVA和尼莫地平可抑制全细胞电流的成分，留下幅度约为总电流的30%的“残余”R电流。该R电流的一小部分可被Cacna 1e选择性毒素SNX-482抑制，表明该R电流是由Cacna 1e的表达引起的。然而，SNX-482不能抑制大部分R电流。Cacna1e基因敲除小鼠缺乏颗粒细胞R电流的SNX-482敏感部分。我们还从野生型小鼠中鉴定了背根节(DRG)神经元的一个亚群，它们表达R电流的SNX-482敏感成分。然而，与颗粒细胞一样，大部分DRG R电流不被SNX-482阻断。我们从这些实验中得出结论，存在一个R电流分量，它是由Cacna 1e钙通道亚基表达的，但大多数R电流肯定是由其他钙通道α亚基的表达引起的。 耳聋模型 Waltzer(V)基因的突变会导致耳聋和前庭功能障碍，这是由于内耳内神经上皮细胞的退化所致。我们发现Waltzer编码一种新的钙粘附素(CDH23)，它与果蝇脂肪蛋白关系最密切。预测vj等位基因中的单核苷酸缺失和v等位基因中的单核苷酸插入会截断N末端附近的每个蛋白质，并产生功能性零等位基因。原位杂交分析表明，CDH23在内耳的感觉毛细胞中表达，它被认为是立体纤毛交联的关键分子。此外，CDH23在椭圆球囊孔、团状管和Reissner膜中也有表达，提示CDH23也可能参与维持内淋巴的离子组成。最后，最近描述了人类CDH23的两个基因座DFNB12和USH1D的突变，这两个基因座会导致非综合征性耳聋，从而将Waltzer确定为人类听力损失的小鼠模型。 神经退行性变模型 共济失调(AXJ)突变纯合子小鼠在2-3周大时出现严重震颤，随后后肢瘫痪，并在6周大时死亡。最近，我们发现AXJ编码泛素特异性蛋白水解酶14(Usp14)。USPS包括一个半胱氨酸蛋白酶的大家族，这些半胱氨酸蛋白酶专门裂解泛素结合物。尽管USP14可以切割泛素标记的蛋白质，但它不能处理多泛素，而多泛素被认为与帕金森病(PD)、脊髓小脑性共济失调1型(SCA1)和股薄型轴突营养不良(GAD)的蛋白质聚集体有关。因此，USP14的生理底物可能含有一个短的泛素侧链，它的去除可能调节蛋白质定位和蛋白质活性等过程。在AXJ小鼠中，由于在Usp14的第5内含子中插入了一个脑池内A粒子(IAP)，Usp14的表达发生了显著的变化。原位杂交研究表明，Usp14在成人中枢神经系统(CNS)内的各种神经元中都有表达，包括大脑皮层、海马、浦肯野细胞和小脑深部核。然而，与人类的PD和SCA1或小鼠的GAD等神经退行性疾病不同，AXJ CNS中没有可检测到的泛素化蛋白聚集体，也没有神经细胞丢失。相反，我们发现了AXJ CNS中的突触连接缺陷，这表明USP可能是调节突触活动的重要因素，可能是通过神经递质释放。

Epilepsy Models and Calcium Channel Function P/Q-type voltage-dependent calcium channel CACNA1A mutations cause dominantly inherited migraine, episodic ataxia, and cerebellar atrophy in humans and cause recessively inherited ataxia, episodic dyskinesia, cerebellar atrophy, and absence epilepsy in mice. The basis of these species differences and the disease mechanism(s) are not understood. To address this question and to identify required P/Q function in vivo, we created a germline Cacna1a null mutation (designated Cacna1aFcrtm1) by gene targeting. Null mice develop dystonia and late-onset cerebellar degeneration in a specific pattern. This indicates a requirement for P/Q function for survival in a subset of cerebellar neurons. Homozygous null mice completely lack P/Q-type channel activity, and they also lack w-CTx-MVIIC receptors, indicating that a single gene encodes P/Q channel activity. An increase of L- and N-type current densities is detected in P/Q-null granule cells. Heterozygous Cacna1aFcrtm1/+ mice are phenotypically normal, despite having a 50% reduction in current density, indicating that reduced current density is not itself sufficient to cause the pathophysiology of spontaneous mouse mutants with ataxia and seizures. It has been hypothesized that R-type Ca currents result from the expression of the Cacna1e gene. To test this hypothesis, we examined the properties of voltage-dependent Ca channels in mice in which the Cacna1e channel subunit had been deleted. Application of omega-conotoxin GVIA, omega-agatoxin IVA, and nimodipine to cultured cerebellar granule neurons from wild-type mice inhibited components of the whole-cell Ba current, leaving a "residual" R current with an amplitude of approximately 30% of the total Ba current. A minor portion of this R current was inhibited by the Cacna1e-selective toxin SNX-482, indicating that it resulted from the expression of Cacna1e. However, the majority of the R current was not inhibited by SNX-482. The SNX-482-sensitive portion of the granule cell R current was absent from Cacna1e knockout mice. We also identified a subpopulation of dorsal root ganglion (DRG) neurons from wild-type mice that expressed an SNX-482-sensitive component of the R current. However, as with granule cells, most of the DRG R current was not blocked by SNX-482. We conclude from these experiments that there exists a component of the R current that results from the expression of the Cacna1e Ca channel subunit but that the majority of R currents must result from the expression of other Ca channel alpha subunits. Deafness Models Mutations at the waltzer (v) locus result in deafness and vestibular dysfunction due to degeneration of the neuroepithelium within the inner ear. We showed that waltzer encodes a novel cadherin (Cdh23), which is most closely related to the Drosophila fat protein. A single nucleotide deletion in the vJ allele and a single nucleotide insertion in the v allele are predicted to truncate each protein near the N-terminus and produce a functional null allele. In situ hybridization analysis showed that Cdh23 is expressed in the sensory hair cells of the inner ear, where it has been suggested to be a molecule critical for crosslinking of the stereocilia. In addition, Cdh23 is expressed in the utriculo-saccular foramen, the ductus reuniens, and Reissner's membrane, suggesting that Cdh23 may also be involved in maintaining the ionic composition of the endolymph. Finally, mutations in human CDH23 have recently been described for two loci, DFNB12 and USH1D, which cause nonsyndromic deafness, identifying waltzer as a mouse model for human hearing loss. Neural Degeneration Models Mice homozygous for the ataxia (axJ) mutation develop severe tremors by 2-3 weeks of age, followed by hind limb paralysis and death by 6 weeks of age. Recently, we have shown that axJ encodes ubiquitin-specific protease 14 (Usp14). USPs comprise a large family of cysteine proteases that specifically cleave ubiquitin conjugates. Although Usp14 can cleave a ubiquitin-tagged protein, it is unable to process polyubiquitin, which is believed to be associated with the protein aggregates in Parkinson's disease (PD), spinocerebellar ataxia type 1 (SCA1), and gracile axonal dystrophy (GAD). The physiological substrate of Usp14 may therefore contain a short ubiquitin side chain, the removal of which may regulate processes such as protein localization and protein activity. Expression of Usp14 is significantly altered in axJ mice due to the insertion of an intracisternal-A particle (IAP) into intron 5 of Usp14. In situ hybridization studies show that Usp14 is expressed in a variety of neurons within the adult central nervous system (CNS), including the cerebral cortex, hippocampus, Purkinje cells, and deep cerebellar nuclei. However, unlike neurodegenerative disorders such as PD and SCA1 in humans or GAD in mice, there are no detectable ubiquitinated-protein aggregates nor is there neuronal cell loss in the axJ CNS. Instead, we identify synaptic connection defects in the axJ CNS, suggesting that USPs may be important factors in regulating the activity of synapses, possibly through neurotransmitter release.