Affiliation: University of Cambridge, Department of Genetics, Cambridge, UK
Keywords: j. endoplasmic reticulum; a. neural degeneration
The Hereditary Spastic Paraplegias (HSPs) are a group of rare, clinically and genetically heterogeneous, inherited neurodegenerative and neurodevelopmental diseases characterised by spasticity and lower limb weaknesses. More than 80 causative genes are known, and some of them imply the importance of endoplasmic reticulum (ER) -a neuron within a neuron- function and morphogenesis in HSPs. These HSP proteins are Spastin (SPG4), Atlastin (SPG3A), Receptor Expression Enhancing Protein 1 (REEP1/SPG31) and Reticulon (SPG12), which share a common feature of one or two intramembrane hairpin domains that can recognise or drive curvature of ER membrane. Proteins of the REEP and reticulon families appear to be responsible for forming most peripheral ER tubules in yeast. In Drosophila, removing these families leads to fewer ER tubules in axons, of wider diameter, although there is no widespread absence of tubules. Therefore, other proteins must be involved in shaping the tubular ER network in Drosophila axons. Another HSP protein with predicted hairpin domains is C19orf12; this is therefore another candidate protein for contributing to shaping the axon ER network. Mutations in this gene are found in patients with autosomal recessive HSP and Neurodegeneration with Brain Iron Accumulation (NBIA). C19orf12 protein colocalises with mitochondria and ER, and with ERmitochondria contacts. Most C19orf12 mutations are found in predicted transmembrane (TM) regions. To investigate possible roles of C19orf12 in ER and mitochondria structure and function, we have generated loss-of-function mutants of the widely expressed Drosophila ortholog of C19orf12, CG3740, using P element excision and CRISPR/cas9. These mutants are homozygous viable, as are quadruple mutants lacking CG3740 and all the widely expressed reticulon and REEP proteins, suggesting that these 4 proteins together are not sufficient for tubular ER formation. Testing of ER and mitochondria morphology in these mutants is in progress. For this purpose, we have also generated flies carrying a split-GFP reporter for ER-mitochondrial sites. To identify additional candidate proteins for ER shaping, we have also performed bioinformatic analyses of proteins shown by proteomic analysis to be enriched on ER tubules. These analyses have identified most of the known proteins with roles in ER shaping, implying that it may also be a good way to identify additional proteins with similar roles.