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 Ca2+ signaling function. We generated GAL4-driven Ca2+ sensors targeted to the ER lumen, to record ER Ca2+ fluxes in identified Drosophila neurons. Using GAL4 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 Ca2+ affinity close to the expected ER lumenal concentration. Repetitive nerve stimulation generally showed a transient increase of lumenal Ca2+ 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 Drosophila Ca2+ imaging toolkit, to explore the physiological roles of ER, and its pathophysiological roles in HSP and in axon degeneration more broadly.

中文翻译：

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果蝇内质网管腔指标揭示 HSP 相关突变对内质网钙动态的影响


内质网（ER）塑造蛋白基因是遗传性痉挛性截瘫（HSP）中最常见的突变之一。模式生物中这些基因的突变可能导致 ER 网络的破坏。为了研究 ER 的生理作用如何受到这种破坏的影响，我们开发了工具来探究其 Ca2+ 信号传导功能。我们生成了针对 ER 腔的 GAL4 驱动的 Ca2+ 传感器，以记录已识别的果蝇神经元中的 ER Ca2+ 通量。使用 Ib 型或 Is 型幼虫运动神经元特异性的 GAL4 系，我们比较了轴突和突触前末梢中不同管腔指标对电刺激的反应。最有效的传感器 ER-GCaMP6-210 的 Ca2+ 亲和力接近预期的 ER 腔浓度。重复神经刺激通常显示轴突和突触前末端的腔内 Ca2+ 短暂增加。与野生型相比，缺乏神经元网织和 REEP 蛋白（人类 HSP 蛋白的同源物）的突变体表现出更大的 ER 腔诱发反应；我们提出了这种表型可能导致神经元功能障碍或变性的机制。我们的细胞系是果蝇 Ca2+ 成像工具包的有用补充，可更广泛地探索 ER 的生理作用及其在 HSP 和轴突变性中的病理生理作用。