Mechanisms of Axon Pathology in ALS

肌萎缩侧索硬化症（ALS）是一种致命性麻痹性疾病，其特征是神经肌肉接头（NMJ） 脊髓运动神经元（MN）死亡和肌肉无力之前的去神经支配。我们假设 预防 NMJ 的去神经支配和刺激 NMJ 的神经支配将阻止肌肉功能障碍和肌无力 ALS，从而改善患者的生活质量，并可能延长生存期。在此，我们试图证明 据报道，蛋白质异戊二烯化是轴突生长的一个关键因素。 ALS 相关运动轴突病理学的决定因素。为了支持这一目标，我们的试点工作表明， 沉默异戊二烯化酶、法尼基转移酶和香叶基香叶基转移酶 I 型，或独特地 沉默香叶基香叶基转移酶 II 型，减轻转基因 (Tg) 小鼠的 NMJ 去神经支配 表达突变型 SOD1 (mSOD1)。该项目的基本原理是，一旦知道哪个异戊二烯化 蛋白质对于 ALS 相关运动轴突病理学至关重要，并且异戊烯基转移酶催化其 异戊二烯化可以设计新的创新策略来治疗 ALS。因此，以下三 提出了具体目标。在 AIM 1 中，我们将鉴定参与运动轴突病理学的异戊二烯基转移酶 通过在 Tg mSOD1 小鼠中单独或组合沉默这些酶，然后，我们将进行比较 不同时间点、腰椎和膈肌 MN 的数量以及动态和运动的 NMJ 神经支配 呼吸肌分别对生活质量和寿命至关重要。我们还将展示 通过评估最有效的方法，了解 ALS 相关运动轴突病理学中蛋白质异戊二烯化的一般性质 上文在 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 通过监测行为、电生理和解剖参数来进行运动轴突病理学的异戊二烯化 在缺乏促细胞死亡基因 Bax 的 Tg mSOD1 小鼠中，有或没有异戊二烯化抑制。自从巴克斯 缺失消除了脊髓 MN 死亡，但没有消除这些小鼠的运动轴突病理学，Tg mSOD1/Bax–/– 动物将 使我们能够确定：（i）运动轴突病理学和 MN 死亡是否由不同的分子控制 (ii) 抑制异戊二烯化和 Bax 不仅可以延迟运动缺陷的发生，而且还可以 延长寿命。在 AIM 3 中，我们将阐明有助于运动轴突的特定异戊烯化蛋白 通过生成 MN 异戊二烯化蛋白质组来进行病理学分析，然后使用此信息执行丢失- ALS 样轴突病理学体外模型中的功能筛选。最后，那些沉默的 MN 异戊二烯化 体外减轻轴突表型的蛋白质将在 Tg mSOD1 小鼠中使用与 在 AIM 2 中。鉴于上述情况，我们预计拟议工作的成功完成将确定 异戊二烯化途径及其靶标有助于 ALS 运动轴突病理学。这些发现将有 重要的积极影响，因为它们将为预防和治疗干预提供机会 并且从根本上增进我们对 ALS 及相关疾病的机制理解。

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.