(2017) developed sensitive genetically encoded calcium indicators (GECIs) targeted to the ER lumen, to examine the role of the ER in Ca 2+ handling and signaling in synaptic activity in mammalian cultured neurons. We therefore need tools to study the physiological functions of axonal and presynaptic ER that depend on its architecture. It is imperative to understand the roles of ER architecture and the consequences of disrupting it, to understand how this might result in a neurodegenerative cascade. Although changes in synaptic structure and function are found in these mutants ( Summerville et al., 2016 Li et al., 2017 Lindhout et al., 2019), the direct physiological consequences of altered ER architecture remain elusive. These studies point to the importance of a continuous and connected tubular ER network throughout axons and presynaptic terminals and suggest disruption to this network as a common thread in HSP pathogenesis. Gene products that shape the tubular ER network are among the most commonly mutated in HSP, and mutation of these genes in model organisms leads to physical disruption of the ER network ( O’Sullivan et al., 2012 Fowler and O’Sullivan, 2016 Summerville et al., 2016 Yalçın et al., 2017 Lindhout et al., 2019). The endoplasmic reticulum (ER) is implicated in these mechanisms, with HSP mutations affecting ER proteins with a variety of roles including lipid metabolism, ER membrane-contact-site function, and ER architecture ( Blackstone, 2018). Despite this heterogeneity, there is evidence for some common mechanisms of cellular pathophysiology. It also shows phenotypic heterogeneity in manifestations such as age of onset and the presence of other symptoms ( Blackstone, 2018). Hereditary spastic paraplegia (HSP) is a genetically heterogeneous disorder, with over 80 loci and 60 genes identified. Our lines are useful additions to a Drosophila Ca 2+ imaging toolkit, to explore the physiological roles of ER, and its pathophysiological roles in HSP and in axon degeneration more broadly. 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. Repetitive nerve stimulation generally showed a transient increase of lumenal Ca 2+ in both the axon and presynaptic terminals. The most effective sensor, ER-GCaMP6-210, had a Ca 2+ affinity close to the expected ER lumenal concentration. 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. We generated GAL4-driven Ca 2+ sensors targeted to the ER lumen, to record ER Ca 2+ fluxes in identified Drosophila neurons.

To investigate how the physiological roles of the ER might be affected by such disruption, we developed tools to interrogate its Ca 2+ signaling function. Mutation of these genes in model organisms can lead to disruption of the ER network. Genes for endoplasmic reticulum (ER)-shaping proteins are among the most commonly mutated in hereditary spastic paraplegia (HSP).

2College of Biological Sciences, University of Minnesota, Minneapolis, MN, United States.

1Department of Genetics, University of Cambridge, Cambridge, United Kingdom.

Oliva 1*, Juan José Pérez-Moreno 1, Jillian O’Shaughnessy 1, Trevor J.

Er lumen positive charged