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性疾病 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 防止NMJ的去神经支配和刺激NMJ的再神经支配将阻止肌肉功能障碍和虚弱， 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内源性制动器， 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， 使异戊烯化酶、法呢基转移酶和I型香叶基香叶基转移酶沉默，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 表达突变体SOD 1（mSOD 1）。这个项目的基本原理是，一旦知道哪个异戊烯化 蛋白质是ALS相关的运动轴突病理学所必需的， 通过异戊二烯化，可以设计用于治疗ALS的新的和创新的策略。因此，以下三个 提出了具体目标。在AIM 1中，我们将确定参与运动轴突病理学的异戊烯基转移酶 通过在Tg mSOD 1小鼠中单独或组合沉默这些酶，然后，我们将比较， 不同时间点，腰椎和膈MN的数量以及行走和行走的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ALS相关运动轴突病理学中最有效的 在ALS的非SOD1模型中的上述鉴定的沉默。在AIM 2中，我们将确定蛋白质的特异性 通过监测行为、电生理学和解剖学参数进行运动轴突病理学的异戊烯化 在促细胞死亡基因Bax缺陷的Tg mSOD 1小鼠中，有和没有异戊二烯化抑制。自从Bax 在这些小鼠中，缺失消除了脊髓MN死亡，但没有消除运动轴突病理学，Tg mSOD 1/Bax-/-动物将 使我们能够确定：（i）运动轴突病理和MN死亡是否由不同的分子调控 程序和（ii）抑制异戊二烯化和Bax不仅延迟了运动缺陷的发生， 延长寿命。在AIM 3中，我们将阐明特定的异戊二烯化蛋白，有助于运动轴突 通过产生MN异戊二烯化蛋白质组，然后使用该信息进行 在ALS样轴突病理学的体外模型中进行功能筛选。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 mSOD 1小鼠中使用与 在AIM 2中。有鉴于此，我们预计，拟议工作的顺利完成将确定 异戊烯化途径及其靶点，有助于ALS中运动轴突病理学。这些发现将有一个 重要的积极影响，因为它们将为预防和治疗干预提供机会 并从根本上推进我们对ALS和相关疾病的机械理解。

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.