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

肌萎缩侧索硬化症（ALS）中运动神经元（MN）的丢失归因于过度兴奋引起的兴奋性毒性损伤，并导致线粒体功能障碍、ER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，并且在变性之前很久就不能发射，并且恢复发射能力可以减少疾病负担并延迟去神经支配。在MN的发射损失的起源是未知的，目前还不清楚这可能有助于神经元的脆弱性;然而，它是已知的，减少突触输入减少神经元的健身通过限制神经保护活性依赖性转录程序的表达。我们已经初步证明，突触后结构（特别是那些在Ia突触）显示破坏的形态和功能的迹象。特别是，在Ia突触的Shank 1-富集突触后密度是片段化的，荷马集群和GluR 4斑点严重减少。突触改变是否是疾病进展的原因，减少MN兴奋，从而限制活动依赖性神经保护？为了解决这一点，我们计划进行遗传操作，只选择性地影响突触输入。详细地说，我们将把突变的SOD 1 ALS小鼠模型与Shank 1 KO交叉：在产生的双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以及疾病标志物出现和积累的加速。与此同时，我们将研究是否在人类患者和非ALS对照的脊髓样品中可检测到突触紊乱。最后，我们将实施一种治疗策略来恢复突触后密度的完整性：我们将使用AAV载体在突变型SOD 1小鼠的MN中特异性过表达PSD 95或短Shank 1亚型。我们预计将验证疾病标志物负担的改善和去神经支配的延迟。在项目结束时，我们将证明ALS中突触功能障碍与疾病发作和进展之间的因果关系，最终，我们可以通过恢复突触完整性来证明治疗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.