Mechanisms of Axon Pathology in ALS

肌萎缩侧索硬化症(ALS)是一种以神经肌肉接头(NMJ)为特征的致死性麻痹疾病 脊髓运动神经元(MN)死亡和肌肉无力之前的失神经。我们假设 防止去神经支配和刺激NMJ的再神经支配将阻止肌肉功能障碍和无力 ALS，从而改善患者的生活质量，并有可能延长生存期。在此，我们试图证明 据报道，这种蛋白质预烯基化起到了抑制轴突生长的内源性刹车作用，这是一个关键 肌萎缩侧索硬化症相关运动神经轴突病理的决定因素。为了支持这一目标，我们的试点工作表明， 使戊烯基化酶、法尼基转移酶和香叶基香叶基转移酶-I型沉默，或唯一地 沉默香叶基香叶基转移酶II型，减轻转基因(TG)小鼠的NMJ失神经 表达突变型SOD1(MSOD1)。这个项目的基本原理是，一旦知道是哪种异丙苯 蛋白质是肌萎缩侧索硬化症相关运动神经轴突病理所必需的，以及哪些异丙烯基转移酶催化它们的 对于肌萎缩侧索硬化症的治疗，可以设计新的和创新的策略。因此，以下三个 提出了具体的目标。在目标1中，我们将确定与运动轴突病理有关的异丙烯基转移酶 通过在转基因mSOD1小鼠中单独或联合沉默这些酶，然后，我们将在 观察不同时点腰、膈MN数目及NMJ神经支配的变化。 呼吸肌分别对生活质量和寿命至关重要。我们还将演示 通过评估最有效的方法研究ALS相关运动轴突病理学中蛋白质预烯基化的一般性质 上面在ALS的非SOD1模型中发现了沉默。在AIM 2中，我们将确定蛋白质的特异性 监测行为学、电生理学和解剖学参数对运动轴突病理学的影响 在缺乏促细胞死亡基因Bax的TG mSOD1小鼠中，存在和不存在预烯基化抑制。自BAX以来 在这些小鼠中，缺失可以消除脊髓MN的死亡，但不能消除运动轴突的病理，Tg mSOD1/Bax-/-动物将 使我们能够确定：(I)运动性轴突病理和MN死亡是否由不同的分子控制 程序和(Ii)抑制预烯基化和Bax不仅延迟运动障碍的发生，而且还 延长寿命。在AIM 3中，我们将阐明对运动轴突起有贡献的特定的前烯基化蛋白 通过产生MN预烯基化的蛋白质组来进行病理学，然后使用这些信息来执行- 肌萎缩侧索硬化症样轴突病理体外模型的功能筛选。最后，那些沉默的MN发生了苯基化 在体外减轻轴突表型的蛋白质将在Tg mSOD1小鼠身上得到验证，使用的测试与 目标2。有鉴于此，我们预计拟议工作的成功完成将确定 肌萎缩侧索硬化症运动轴突发病的前烯基化途径及其靶点。这些发现将会有一个 重要的积极影响，因为它们将为预防和治疗干预提供机会 从根本上提高我们对肌萎缩侧索硬化症和相关疾病的机械性理解。

Amyotrophic lateral sclerosis (ALS) is a fatal paralytic disease characterized by neuromuscular junction (NMJ) denervation that precedes spinal motor neuron (MN) death and muscle weakness. We hypothesize that preventing denervation and stimulating reinnervation of NMJs will thwart muscle dysfunction and weakness in ALS, hence improving the patient's quality of life and, likely extending survival. Herein, we seek to demonstrate that protein prenylation, which was reported to operate as an endogenous brake on axonal growth, is a key determinant of ALS-related motor axon pathology. In support of this goal, our pilot work shows that dually silencing the prenylation enzymes, farnesyl transferase and geranylgeranyl transferase type-I, or uniquely silencing geranylgeranyl transferase type-II, mitigates NMJ denervation in the transgenic (Tg) mouse expressing mutant SOD1 (mSOD1). The rationale for this project is that, once it is known which prenylated proteins are essential for ALS-related motor axon pathology and which prenyl transferases catalyze their prenylation, new and innovative strategies can be devised for the treatment of ALS. Thus, the following three specific aims are proposed. In AIM 1, we will identify the prenyl transferase involved in motor axon pathology by silencing these enzymes individually or in combination in Tg mSOD1 mice and then, we will compare, at different time points, the number of lumbar and phrenic MNs and the NMJ innervation of ambulatory and respiratory muscles that are critical to the quality of life and lifespan, respectively. We will also demonstrate the generic nature of protein prenylation in ALS-related motor axon pathology by assessing the most effective silencing identified above in a non-SOD1 model of ALS. In AIM 2, we will ascertain the specificity of protein prenylation for motor axon pathology by monitoring behavioral, electrophysiological and anatomical parameters in Tg mSOD1 mice deficient in the pro-cell death gene Bax with and without prenylation inhibition. Since Bax deletion abrogates spinal MN death but not motor axon pathology in these mice, Tg mSOD1/Bax–/– animals will enable us to determine whether: (i) motor axon pathology and MN death are governed by distinct molecular programs and (ii) inhibition of both prenylation and Bax not only delays the onset of motor deficit but also extends lifespan. In AIM 3, we will elucidate the specific prenylated proteins that contribute to motor axon pathology by generating the MN prenylated proteome and then, use this information to perform a loss-of- function screening in an in vitro model of ALS-like axon pathology. Lastly, those silenced MN prenylated proteins that mitigate the axon phenotype in vitro will be validated in Tg mSOD1 mice using the same tests as in AIM 2. In light of the above, we expect that the successful completion of the proposed work will identify the prenylation pathway and its targets that contribute to motor axon pathology in ALS. These findings will have an important positive impact in that they will provide opportunities for preventive and therapeutic interventions and, fundamentally, advance our mechanistic understanding of ALS and related disorders.