Oliva Megan K, Pérez-Moreno Juan José, O'Shaughnessy Jillian, Wardill Trevor J, O'Kane Cahir J
Department of Genetics, University of Cambridge, Cambridge, United Kingdom.
College of Biological Sciences, University of Minnesota, Minneapolis, MN, United States.
Front Neurosci. 2020 Aug 10;14:816. doi: 10.3389/fnins.2020.00816. eCollection 2020.
Genes for endoplasmic reticulum (ER)-shaping proteins are among the most commonly mutated in hereditary spastic paraplegia (HSP). Mutation of these genes in model organisms can lead to disruption of the ER network. To investigate how the physiological roles of the ER might be affected by such disruption, we developed tools to interrogate its Ca signaling function. We generated GAL4-driven Ca sensors targeted to the ER lumen, to record ER Ca fluxes in identified neurons. Using lines specific for Type Ib or Type Is larval motor neurons, we compared the responses of different lumenal indicators to electrical stimulation, in axons and presynaptic terminals. The most effective sensor, ER-GCaMP6-210, had a Ca affinity close to the expected ER lumenal concentration. Repetitive nerve stimulation generally showed a transient increase of lumenal Ca in both the axon and presynaptic terminals. Mutants lacking neuronal reticulon and REEP proteins, homologs of human HSP proteins, showed a larger ER lumenal evoked response compared to wild type; we propose mechanisms by which this phenotype could lead to neuronal dysfunction or degeneration. Our lines are useful additions to a Ca imaging toolkit, to explore the physiological roles of ER, and its pathophysiological roles in HSP and in axon degeneration more broadly.
内质网（ER）塑形蛋白的基因是遗传性痉挛性截瘫（HSP）中最常发生突变的基因之一。在模式生物中这些基因的突变会导致内质网网络的破坏。为了研究内质网的生理功能是如何受到这种破坏影响的，我们开发了工具来探究其钙信号功能。我们生成了靶向内质网腔的GAL4驱动的钙传感器，以记录特定神经元中的内质网钙通量。利用针对I b型或I s型幼虫运动神经元的品系，我们比较了不同腔内指示剂对轴突和突触前终末电刺激的反应。最有效的传感器ER-GCaMP6-210的钙亲和力接近预期的内质网腔浓度。重复神经刺激通常显示轴突和突触前终末的腔内钙短暂增加。缺乏神经元网织蛋白和REEP蛋白（人类HSP蛋白的同源物）的突变体与野生型相比，显示出更大的内质网腔内诱发反应；我们提出了这种表型可能导致神经元功能障碍或退化的机制。我们的品系是钙成像工具包的有用补充，可用于更广泛地探索内质网的生理功能及其在HSP和轴突退化中的病理生理作用。
