Post-synaptic disruption drives motoneuron vulnerability and disease progression in ALS

肌萎缩性侧索硬化症（ALS）中运动神经元（MN）的丢失被认为是由于过度兴奋性引起的兴奋毒性损伤，导致线粒体功能障碍、内质网应激和自噬功能障碍。然而，最近的证据表明，脆弱的MN是低兴奋性的，在变性之前很久就不能放电，恢复放电能力可以减轻疾病负担并延迟去神经控制。MN放电丧失的起源尚不清楚，也不清楚这可能如何导致神经元易损性；然而，众所周知，减少突触输入通过限制神经保护活性依赖的转录程序的表达而降低神经元适应性。我们已经初步证明，突触后结构（特别是那些在Ia突触）显示形态和功能的破坏迹象。特别是在Ia突触，shank1富集的突触后密度是碎片化的，Homer簇和GluR4点严重减少。突触改变是疾病进展减少MN兴奋从而限制活动依赖性神经保护的原因吗？为了解决这一点，我们计划进行基因操作，将选择性地影响突触输入。详细地说，我们将把突变SOD1 ALS小鼠模型与Shank1 KO杂交：在由此产生的双tg小鼠中，兴奋性突触将在疾病过程的早期被选择性地破坏。我们预计会观察到疾病进展的恶化和疾病标志物的出现和积累的加速。同时，我们将研究是否在人类患者和非als对照的脊髓样本中检测到突触紊乱。最后，我们将采用一种治疗策略来恢复突触后密度的完整性：我们将使用AAV载体在突变SOD1小鼠的MN中特异性地过表达PSD95或短Shank1亚型。我们期望验证疾病标记负担的改善和去神经的延迟。在项目结束时，我们将证明突触功能障碍与ALS疾病发病和进展之间的因果关系，最终，我们将证明一种通过恢复突触完整性来治疗MN疾病的新方法。

Loss of motoneurons (MN) in Amyotrophic Lateral Sclerosis (ALS) has been attributed to excitotoxic damage due to hyperexcitability and resulting in mitochondrial dysfunction, ER stress and autophagy dysfunction. However, recent evidence shows that vulnerable MN are hypoexcitable and unable to fire long before degeneration and that restoring firing capabilities reduces disease burden and delays denervation. The origin of the loss of firing in MN is unknown and it is unclear how this may contribute to neuronal vulnerability; nevertheless, it is known that reduced synaptic input decreases neuronal fitness by limiting the expression of neuroprotective activity-dependent transcriptional programs. We have preliminary demonstrated that postsynaptic structures (in particular, those at the Ia synapses) display morphological and functional signs of disruption. In particular, Shank1-enriched post-synaptic densities at Ia synapses are fragmented and Homer clusters and GluR4 puncta are severely reduced. Are synaptic changes a cause of the disease progression reducing MN excitation, and therefore limiting activity-dependent neuroprotection? To address this point, we plan to perform genetic manipulations that will affect selectively only the synaptic inputs. In detail, we are going to cross mutant SOD1 ALS mouse model with a Shank1 KO: in the resulting double-tg mice, excitatory synapses will be selectively disrupted early on in disease course. We anticipate observing a worsening of disease progression and an acceleration of disease markers appearance and accumulation. In parallel, we will investigate if synaptic derangement is detectable in spinal cord samples from human patients and non-ALS controls. Finally, we will put in place a therapeutic strategy to restore the integrity of the post-synaptic density: we will use AAV vectors to overexpress PSD95 or short Shank1 isoforms specifically in the MN of mutant SOD1 mice. We anticipate verifying an improvement in disease markers burden and a delay in denervation. At the end of the project, we will have demonstrated the causal link between synaptic dysfunction and disease onset and progression in ALS and eventually, we could demonstrate a new approach to treat MN disease by restoring synaptic integrity.