Synaptic & intrinsic motoneuron excitability in ALS excitotoxicity

描述(申请人提供)：肌萎缩侧索硬化症(ALS)是一种成人起病的神经退行性疾病，主要针对运动神经元。在20%的家族性肌萎缩侧索硬化症(FAL)患者中，存在超氧化物歧化酶(SOD1)的特异性突变。带有突变的人SOD1的小鼠表现出与人类ALS患者相同的病理和表型症状，因此被广泛用作FALS的动物模型。然而，SOD1在确定运动神经元死亡中所起的作用仍不清楚。一个主要的假说是，由于运动神经元的过度兴奋导致细胞内钙超载而引起的兴奋性毒性，导致运动神经元选择性死亡，因为运动神经元缓冲钙的能力很低。这一建议旨在确定野生型和SOD1运动神经元之间内在和突触运动神经元兴奋性变化的相对贡献。此外，FDA批准的治疗ALS的唯一药物利鲁唑对固有运动神经元和突触运动神经元兴奋性的影响也将得到评估。这项建议的目的将通过使用胚胎培养的运动神经元、新生脊髓切片和一种新的体外骶髓准备来实现。这些制剂的使用将使野生型和SOD1小鼠之间的运动神经元兴奋性得到有效和完整的表征。通过在发育和成年期的不同时间点进行细胞内记录，可以在体外进行骶髓制备，从而评估固有的和突触运动神经元的兴奋性。它还使我们能够在治疗水平上和在多个时间段内给予利鲁唑，以广泛地表征随着时间的推移利鲁唑的效果。新生儿脊髓切片制备允许通过使用全细胞膜片钳配置直接沐浴药物抑制特定离子通道来评估内在与突触运动神经元的兴奋性。此外，利鲁唑对运动神经元的直接和即时影响也可以确定。胚胎培养的运动神经元为了解出生前存在的运动神经元异常提供了一个窗口，也为我们提供了一个没有突触输入的准备。在这种制剂中，可以分离利鲁唑多次给药对运动神经元内在特性的影响。为了开发新的药物治疗方法，需要确定肌萎缩侧索硬化症运动神经元变性的原因(S)，了解运动神经元功能的差异或变化将导致这些未来药物的特定靶点。此外，运动神经元功能的早期可检测变化可能使预防性治疗能够显著延缓甚至阻止疾病的发展。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease which specifically targets motoneurons. In 20% of familial ALS (fALS) cases, there is a specific mutation in superoxide dismutase (SOD1). Mice with mutated human SOD1, display the same pathological and phenotypical symptoms in human ALS patients and are therefore widely used as an animal model of fALS. However, the role SOD1 plays in determining motoneuron death remains unclear. A leading hypothesis is that excitotoxicity caused by an overload of calcium into the cell due to motoneuron hyperexcitability causes selective motoneuron death because motoneurons have a low capacity to buffer calcium. This proposal aims to determine the relative contribution of changes in intrinsic and synaptic motoneuron excitability between wild type and SOD1 motoneurons. Furthermore, the effect of Riluzole, the only FDA approved drug for ALS, on both intrinsic and synaptic motoneuron excitability will also be assessed. The aims of this proposal will be accomplished through the use of embryonic cultured motoneurons, neonatal spinal cord slices, and a new in vitro sacral cord preparation. The use of these preparations will allow an efficient and complete characterization of motoneuron excitability between wild type and SOD1 mice. The in vitro sacral cord preparation allows intrinsic and synaptic motoneuron excitability to be assessed through intracellular recordings at time points throughout development and adulthood. It also enables us to administer Riluzole at therapeutic levels and in multiple time courses to extensively characterize the effects of Riluzole over time. The neonatal spinal cord slice preparation allows intrinsic versus synaptic motoneuron excitability to be assessed using direct bath application of drugs to inhibit specific ion channels using a whole cell patch clamp configuration. Also, the direct and immediate effects of Riluzole on motoneurons can be determined. The embryonic cultured motoneurons provide a window into motoneuron abnormalities present before birth and also give us a preparation where no synaptic inputs are present. In this preparation the effects of Riluzole administered over multiple time courses on the intrinsic properties of motoneurons can be isolated. The cause(s) of motoneuron degeneration in ALS needs to be determined in order to develop novel drugs treatments and understanding differences or changes in motoneuron function will lead to specific targets for these future drugs. Furthermore, early detectable changes in motoneuron function may enable the use of pre-emptive therapies that can significantly delay or even arrest the development of the disease.