Role of MMP-9 in selective motor neuron degeneration in ALS

描述(申请人提供)：尽管进行了多项临床试验，但仍然没有有效的治疗成人起病的神经退行性疾病ALS(肌萎缩侧索硬化症)。其中一个主要原因是，除了家族性肌萎缩侧索硬化症的致病基因外，没有治疗方法 目标已被确认。靶标的例子是在疾病进展中发挥关键作用的酶，其抑制作用可以延缓疾病的发生或减缓疾病的进展。引人注目的是，即使在肌萎缩侧索硬化症(ALS)的晚期患者中，眼球运动和可控性也得到了保护，反映了动眼神经和Onuf核中的运动神经元对疾病过程的几乎完全抵抗。如果有可能将这种抵抗力的一小部分赋予通常脆弱的脊髓运动神经元，将会有显著的治疗益处。因此，了解耐药性的机制为定义新的目标提供了一种方法。在初步研究中，我们利用激光捕获显微解剖和微阵列分析，发现了在ALS敏感的运动神经元中表达的新基因，但在ALS抵抗的运动神经元中不表达，反之亦然。其中之一是基质金属蛋白酶-9，这是一种细胞外酶，在耐药动眼神经和Onuf核中缺失。我们发现它在不同的运动神经元亚群中的表达与其易损性密切相关。引人注目的是，我们发现，在ALS模型小鼠中，MMP9基因的失活--其正常寿命约为6个月--会导致肌肉失神经延迟3个月，存活率提高24%。即使在MMP9杂合子的小鼠身上也观察到了显著的益处。因此，基质金属蛋白酶-9是ALS潜在的治疗靶点。该项目的总体目标是了解基质金属蛋白酶-9触发运动神经元变性的细胞和分子机制，并对阻止这种退化的潜在治疗策略提供初步评估。该提案围绕三个主要问题展开。首先，我们将确定基质金属蛋白酶-9触发运动神经元变性的分子机制(S)，重点是涉及Fas受体和谷氨酸兴奋毒性的候选途径。其次，我们将使用表达MMP9 shRNA的病毒载体的不同给药途径来研究基质金属蛋白酶-9的细胞作用部位。第三，我们将询问抑制基质金属蛋白酶-9的酶活性是否足以带来益处，或者是否已知的非酶作用模式也被牵连。总体而言，这些结果将为ALS运动神经元变性的机制提供新的见解，并为基质金属蛋白酶-9作为未来开发的治疗靶点的潜力提供重要的临床前迹象。

DESCRIPTION (provided by applicant): Despite multiple clinical trials, there is still no effective therapy for the adult-onset neurodegenerative disease ALS (amyotrophic lateral sclerosis). One major reason for this is that, aside from the genes that are causal in familial ALS, no therapeutic targets have been validated. Examples of targets would be enzymes that play a critical role in disease progression and whose inhibition retards disease onset or slows progression. Strikingly, even in late-stage patients with amyotrophic lateral sclerosis (ALS), eye movement and continence are preserved, reflecting the near-complete resistance of motor neurons in oculomotor and Onuf's nuclei to the disease process. If it were possible to confer even a fraction of this resistance upon the normally vulnerable spinal motor neurons, there would be significant therapeutic benefit. Understanding the mechanisms of resistance therefore provides a method for defining new targets. In preliminary studies, we identified novel genes expressed in ALS-susceptible but not in ALS-resistant motor neurons, or vice versa, using laser-capture microdissection and microarray analysis. One of these is MMP-9 (matrix metalloproteinase-9), an extracellular enzyme which is absent from resistant oculomotor and Onuf's nuclei. We showed that its expression in different motor neuron subsets is tightly correlated with their vulnerability. Strikingly, we find that inactivation of the mmp9 gene in ALS model mice - whose normal lifespan is ~6 months - leads to a >3-month delay in muscle denervation and a 24% increase in survival. Significant benefit was observed even in mice that were heterozygotes for mmp9. MMP-9 is therefore a strong candidate as a potential therapeutic target in ALS. The overall goal of the proposed project is to understand the cellular and molecular mechanisms through which MMP-9 triggers motor neuron degeneration and to provide initial evaluation of potential therapeutic strategies to block this. The proposal is structured around three main questions. First, we will determine the molecular mechanism(s) through which MMP-9 triggers motor neuron degeneration, focusing on candidate pathways involving the Fas receptor and glutamate excitotoxicity. Second, we will investigate the cellular site of action of MMP-9, using different routes of administration of viral vectors expressing mmp9 shRNA. Third, we will ask whether inhibition of the enzymatic activity of MMP-9 is sufficient to confer benefit, or whether is known non-enzymatic modes of action are also implicated. Overall, the results should provide novel insights into the mechanisms of motor neuron degeneration in ALS and important preclinical indications as to the potential of MMP-9 as a therapeutic target for future development.