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

描述（由申请人提供）：尽管进行了多项临床试验，但仍然没有针对成人发病的神经退行性疾病ALS（肌萎缩侧索硬化症）的有效疗法。其中一个主要原因是，除了家族性ALS的致病基因外， 目标已经过验证。靶点的实例是在疾病进展中起关键作用的酶，其抑制可延缓疾病发作或减缓进展。引人注目的是，即使在患有肌萎缩侧索硬化症（ALS）的晚期患者中，眼球运动和眼球运动也被保留下来，反映了眼内运动神经元和Onuf核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，将有显著的治疗益处。因此，了解耐药机制为确定新靶点提供了一种方法。在初步研究中，我们确定了新的基因表达的ALS敏感，但不是在ALS耐药运动神经元，反之亦然，使用激光捕获显微切割和微阵列分析。其中之一是MMP-9（基质金属蛋白酶-9），一种细胞外酶，其不存在于抗性眼和Onuf核中。我们发现它在不同运动神经元亚群中的表达与它们的脆弱性密切相关。引人注目的是，我们发现ALS模型小鼠中mmp 9基因的失活-其正常寿命约为6个月-导致肌肉去神经延迟>3个月，存活率增加24%。甚至在mmp 9杂合子小鼠中也观察到了显著的益处。因此，MMP-9是ALS中作为潜在治疗靶点的强有力候选者。该项目的总体目标是了解MMP-9触发运动神经元变性的细胞和分子机制，并对阻断这种机制的潜在治疗策略进行初步评估。该提案围绕三个主要问题展开。首先，我们将确定MMP-9触发运动神经元变性的分子机制，重点关注涉及Fas受体和谷氨酸兴奋性毒性的候选途径。其次，我们将研究MMP-9的细胞作用位点，使用不同的管理途径的病毒载体表达mmp 9 shRNA。第三，我们将询问MMP-9的酶活性的抑制是否足以赋予益处，或者是否也涉及已知的非酶促作用模式。总的来说，这些结果应该为ALS运动神经元变性的机制提供新的见解，并为MMP-9作为未来发展的治疗靶点的潜力提供重要的临床前指征。

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.