Testing roles of Hereditary Spastic Paraplegia genes in axonal endoplasmic reticulum modelling in Drosophila

Change log

Hereditary spastic paraplegias (HSPs) are neurodegenerative disorders characterized by progressive lower limb spasticity due to axonal degeneration. Smooth endoplasmic reticulum (ER) forms a longitudinal network within the axon; common mutations in HSP affect proteins of the spastin, atlastin, REEP and reticulon (RTN) families, which possess hydrophobic hairpin domains in the ER membrane to help confer curvature on tubular ER. Drosophila mutants lacking members of RTN and REEP families exhibit partial ER fragmentation in axons. Conversely, yeast mutants lacking all RTNs and REEPs lose most tubular ER, which indicates involvement of other proteins in ER organization in Drosophila.

I aimed to test whether additional HSP or functionally related genes might have roles in the organization of axonal ER in Drosophila. I tested several candidate proteins in a Rtnl1- ReepA- ReepB- background, which already has fewer axon ER tubules, and identified Rab18 and Rab3GAP2 as possible additional smooth ER-modelling proteins. I also characterized Arl6IP1, an ER-localized HSP protein, as a likely ER-modelling protein. Loss of Arl6IP1 had no significant effect on ER distribution in motor axons in wild-type or Rtnl1- ReepA- ReepB- backgrounds and showed no significant effect on ER distribution in class IV multidendritic sensory neurons or tubular ER dynamics. However, loss of Arl6IP1 cause an apparent increase in ER levels in presynaptic NMJs. Therefore, Arl6IP1 might not be playing a direct role in shaping axonal ER in a manner similar to other known ER-shaping proteins but may play a role in regulating levels of presynaptic ER at the NMJs. I also tested effects of a null mutation in atlastin, an ER membrane fusion GTPase, on axonal ER, and observed significantly higher ER labelling intensity and impaired ER dynamics in its absence suggesting that loss of atlastin may lead to an increase in ER tubules in axons accompanied by a loss in ER structural complexity. To improve the toolbox for monitoring axonal ER, I also generated transgenic flies that overexpress a smooth ER marker tagged with a photoconvertible protein, CG9186::mEos4b, as a potential tool for understanding the organisation of axonal ER, using both photoconversion and correlative light and electron microscopy (CLEM) in individual axons. Taken together, my work advances our understanding of how HSP proteins may affect axon ER organisation and dynamics.

O'Kane, Cahir J
Drosophila, hereditary spastic paraplegia, enodplasmic reticulum, axon, cell biology, neuroscience
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
Gates Cambridge Trust