High-resolution imaging of presynaptic ER networks in Atlastin mutants

The endoplasmic reticulum (ER) is a continuous organelle that extends to the periphery of neurons and regulates many neuronal functions including neurite outgrowth, neurotransmission, and synaptic plasticity. Mutations in proteins that control ER shape are linked to the neurodegenerative disorder Hereditary Spastic Paraplegia (HSP). However, the ultrastructure and dynamics of the neuronal ER have been under-investigated, particularly at presynaptic terminals. Here we developed new super-resolution and live imaging methods in D. melanogaster larval motor neurons to investigate ER structure at presynaptic terminals from wild-type animals, and in null mutants of the HSP gene Atlastin. Previous studies indicated diffuse localization of an ER lumen marker at Atlastin mutant presynaptic terminals, which was attributed to ER fragmentation. By contrast, we found using an ER membrane marker that the ER in Atlastin mutants formed robust networks. Further, our high-resolution imaging results suggest that overexpression of luminal ER proteins in Atlastin mutants causes their progressive displacement to the cytosol at synapses, perhaps due to proteostatic stress and/or changes in ER membrane integrity. Remarkably, these luminal ER proteins remain correctly localized in cell bodies, axons, and other cell types such as body wall muscles, suggesting that ER tubules at synapses have unique structural and functional characteristics. This displacement phenotype has not been reported in numerous studies of Atlastin in non-neuronal cells, emphasizing the importance of conducting experiments in neurons when investigating the mechanisms leading to upper motor neuron dysfunction in HSP.