Serum Response Factor (SRF) regulates motoneuron vulnerability and activity- dependent neuroprotection in Amyotrophic Lateral Sclerosis.

肌萎缩性侧索硬化症（ALS）是一种进行性神经退行性疾病，主要影响运动神经元（MN），特别是脊髓的下运动神经元。最近，在SOD1(G93A) ALS小鼠模型中，神经元活动和放电模式被证明对疾病进展有很强的影响。在这里，化学遗传学上MNs放电升高减缓了疾病负担，而干扰神经元活动加速了疾病标志物的积累（例如自噬诱导）。在这项研究中，我们想分析SRF（血清反应因子）的作用，SRF是一种典型的神经元活动诱导转录因子（TF）。SRF通过生理和病理（如癫痫、急性应激）诱导神经元中的神经元活动，介导直接早期基因（egg）如c-Fos、Egr1和Npas4的神经元活性诱导基因转录。因此，我们假设神经元活动对延缓ALS疾病进展的有益影响是通过激活MNs中srf介导的基因转录来实现的。为了验证这一假设，我们已经建立了条件Srf突变小鼠系，并记录了运动神经元（ChAT-Cre+/-; Srf fl/fl）中Srf的排他耗尽。我们进一步将该小鼠系与已建立的小鼠ALS模型SOD1（G93A）杂交，获得ChAT-Cre+/-；Srf fl / fl;SOD1 (G93A)老鼠。在之前的第一项研究中，我们观察到Srf突变/SOD1小鼠与单独的SOD1小鼠相比，体重减轻和握力下降。有了这些小鼠，我们有两个目标：i) mn限制性SRF消耗是否会使SOD1 ALS模型中的疾病进展恶化？为此，我们分析了几个行为参数（握力、阶梯行走、临床评分、倒网格、开阔视野），并进行了详细的组织学检查，包括自噬标志物、MN数、炎症和突触形成。最后，我们通过激光捕获介导的MNs分离，通过RNA-SEQ鉴定这些小鼠中ALS和srf依赖的转录组。ii) SRF是介导MNs化学遗传诱导的活性依赖性神经保护的下游靶点吗？在SOD1 ALS小鼠模型中，我们使用化学遗传学方法在脊髓中病毒过表达激活或抑制通道（阳离子可渗透的actPSAM或阴离子可渗透的inhPSAM），分别缓解或加重疾病进展。这些实验将在Srf突变体/SOD1小鼠中进行，与SOD1小鼠进行比较。根据我们的假设，SRF在调节神经元的神经元活动中起关键作用，我们预计化学遗传学驱动的神经元激活不能保护SRF耗尽后ALS造成的神经退行性变。综上所述，本项目分析了SRF介导的神经元活性，特别是神经元活性介导的基因转录是否是ALS进展的一个新因素，也可能作为未来潜在的药物靶点来减轻ALS对患者的影响。

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative condition affecting primarily motoneurons (MN), in particular lower motoneurons in the spinal cord. Recently, neuronal activity and firing patterns were shown to have a strong impact on disease progression in murine ALS models such as the SOD1(G93A) ALS mouse model. Here, chemogenetically elevated firing in MNs slowed down the disease burden whereas interference with neuronal activity accelerated accumulation of disease markers (e.g. autophagy induction). In this study we want to analyze the role of SRF (serum response factor), a prototypical neuronal activity-induced transcription factor (TF). SRF mediates neuronal-activity induced gene transcription of immediate early genes (IEGs) such as c-Fos, Egr1 and Npas4 upon physiological and pathological (e.g. epilepsy, acute stress) induction of neuronal activity in neurons. Thus, we hypothesize that the beneficial impact of neuronal activity on delaying ALS disease progression works through activation of SRF-mediated gene transcription in MNs. To test this hypothesis we already established a conditional Srf mutant mouse line and documented exclusive SRF depletion in motoneurons (ChAT-Cre+/-; Srf fl/fl). We further bred this mouse line to the established mouse ALS model SOD1(G93A) to obtain ChAT-Cre+/-;Srf fl/fl;SOD1(G93A) mice. In first previous work we observed additional weight loss and grip strength reduction in Srf mutant/SOD1 mice compared to the SOD1 mice alone.With these mice at hand we follow two objectives: i) Does MN-restricted SRF depletion worsen disease progression in the SOD1 ALS model? For this, we analyze several behavioral parameters (grip strength, ladder walk, clinical score, inverted grid, open field) and perform a detailed histological inspection including markers for autophagy, MN numbers, inflammation and synapse formation. Finally, we perform a laser-capture mediated isolation of MNs to identify the ALS- and SRF-dependent transcriptome in these mice by RNA-SEQ.ii) Is SRF a downstream target in mediating chemogenetically-induced, activity-dependent neuroprotection in MNs? Here we use chemogenetics to virally overexpress activating or inhibitory channels (cation-permeable actPSAM or anion-permeable inhPSAM) in the spinal cord that alleviate or worsen disease progression in the SOD1 ALS mouse model, respectively. These experiments will be performed in the Srf mutant/SOD1 mice in comparison to SOD1 mice only. Based on our hypothesis attributing SRF a pivotal role in mediating neuronal activity in neurons we expect that chemogenetically-driven neuronal activation fails to protect from ALS inflicted neurodegeneration upon SRF depletion.In summary, this project analyzes whether neuronal activity and specifically neuronal-activity mediated gene transcription mediated by SRF is a novel factor in ALS progression that may also serve as a future potential drug target to alleviate the impact of ALS in patients.