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Roles of spastic paraplegia proteins in organisation of axonal endoplasmic reticulum

Periodic Reporting for period 1 - ParaplegiaERDros (Roles of spastic paraplegia proteins in organisation of axonal endoplasmic reticulum)

Reporting period: 2016-01-01 to 2017-12-31

"The basic functional and structural unit of nervous system is a specialized cell called a neuron, which consists of a cell body and projections known as dendrites and axons. To innervate targets far from the cell body, axons can travel many orders of magnitude longer than the diameter of the cell body. In some lengthy nerves in humans, axons can extend up to a meter. Various machineries are employed in long axons to maintain their form and function, and defects in these machineries could give rise to neuropathies, such as axon degeneration, paralysis, or numbness, depending on which axons are affected.

How do axons maintain communication along such a distance and avoid dysfunction? Small cellular compartments called organelles supply energy and carry signals for axonal function, and can be transported within the axon. Another membrane-bound tubular organelle, endoplasmic reticulum (ER), can form a continuous network throughout the whole cell and so could conduct local or long-range signals like a ""neuron within a neuron"". However, the physiological function of ER in axons, the mechanisms that form it, and the relationship between its form and function, are all poorly understood.

The importance of ER in axon maintenance is supported by the fact that different mutations affecting ER-shaping proteins can lead to hereditary spastic paraplegia (HSP), a motor neuron disease which features degeneration of corticospinal motor tract – some of the longest axons in the body. Therefore, we aim to understand the specific role of HSP proteins on axonal ER shaping, and the mechanisms underlying the role of ER in axon maintenance and dysfunction. We believe that studies on HSP causative genes which encode ER-localised proteins will shed light on the pathology of HSP disease and the ensuing potential therapy targets. By further investigation of ER morphogenesis and its roles in axon function, we aim to deepen our understanding of how the nervous system works.

Two evolutionarily conserved HSP protein families (the reticulon and REEP families) play a critical role in ER shaping (Voeltz et al., 2006). However, removing them from Drosophila (fruit fly) did not delete all tubular ER in axons as predicted (Yalcin et al., 2017), implying that more proteins function in this process. We therefore performed a genetic screen to find more proteins, by testing for abnormal axonal ER in fly larvae lacking additional HSP proteins. We found roles of a few such HSP proteins on levels and continuity of axonal ER, and we studied one such protein, atlastin, along with reticulon and REEPs.

ER dynamics could be critical for its function. Fluorescent markers make it feasible to visualise axonal ER dynamics in live animals. We recorded axonal ER movement in larvae and found that it was surprisingly dynamic, with frequent forward and backward movement of tubules along axons (Fig 1). We also bleached ER fluorescence markers in normal axons, and in mutant axons lacking specific HSP proteins; in the former we found rapid recovery due to diffusion of fluorescent proteins from nearby ER. In HSP mutants, this recovery was impaired, implying that the ER network was disrupted in these mutants.

ER tubules are structures on the scale of nanometers, which cannot be explored by conventional light microscopy. In collaboration with Dr M Terasaki (University of Connecticut, USA), we performed electron microscopy (EM) on Drosophila larval axons and reconstructed the ER network at ultrastructural level. Our results show that in fly axons ER forms a continuous network with multiple branches and some dead ends, and ER tubules often show proximity to mitochondria or plasma membrane (Fig 2); however in axons lacking reticulon and REEP proteins, tubules were fewer, larger, with more gaps, consistent with reduced membrane curvature.

One clinical feature of HSP is progressive lower limb spasticity and weakness. Although third instar larvae of fruit flies are used for a wide range of analysis, they are not suited for progressive phenotype studies, since they progress immediately to pupation. Therefore, we developed a method to visualise axonal ER in fly adults which live for over 30 days. Fluorescently labelled ER proteins expressed in adult flies reveal labelled motor axons in live undissected adult legs. This now allows us to test HSP mutations for age-dependent or degenerative defects using this system; it also makes large mutant screens feasible since it does not require time-consuming dissection and immunostaining.

Exploitation and dissemination. Further understanding of the roles of HSP proteins might suggest targets or approaches for therapy of axon degeneration diseases, although further research on the physiological consequences of these ER defects is still required. For dissemination, we have published part of our work in a peer-reviewed journal, eLife (Yalcin et al., 2017), and are currently writing a second manuscript on our atlastin work. We have also presented the work at an annual departmental symposium in Cambridge and the European Drosophila Research Conference. Some images from this project were shown as part of a matching game in Cambridge Science Festival, an annual event which makes science accessible to public. We have also posted movies showing reconstructed axonal ER network, and talks that include the work, on the Departmental web page (see URL below).

Voeltz, Prinz, Shibata, Rist and Rapoport (2006). A class of membrane proteins shaping the tubular endoplasmic reticulum. Cell 124, 573-586.

Yalcin, Zhao, Stofanko, O'Sullivan, Kang, Roost, Thomas, Zaessinger, Blard, Patto,, et al. (2017). Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins. eLife 6:e23882.
Scientific advances of the grant include the following
1. We have found several HSP proteins, from the reticulon, REEP and atlastin families, that are required for the continuity of axonal ER, and for its normal size and occurrence.
2. This is the first evidence for roles of these proteins in axonal ER, implying that this organelle is critical for the axon maintenance and function, and that axon degeneration may result in HSP from its disruption.
3. We developed tools and methods for monitoring the dynamics of ER in axons, and found that ER is highly dynamic there. We suggest that its dynamic nature is part of the process that ensures a continuous ER network throughout neurons.
4. We tested the feasibility of using Drosophila as a model to study axon ER biology. Use of Drosophila can allow testing of a large number of genotypes for effects on axonal ER efficiently and economically, without experiments on protected animals.

Further investigation on the roles of HSP proteins will give us a fuller picture of the ER modeling machinery in axons, which will guide us to mechanisms for axon degeneration when these proteins are mutated in humans. An understanding of these mechanisms is a prerequisite for finding therapeutic targets for these and similar axon degeneration diseases. Axonopathies are also common in diabetes, obesity and after chemotherapy, and our work may suggest mechanisms for these axonopathies. Although our findings cannot be transformed into clinical treatment immediately, the accumulation of the knowledge on basic biology of axons in long run are prerequisites for the exploitation and transformation in Pharma industry.
Axon ER labeling is dynamic, and recovers after bleaching. HSP Mutants (right) show some ER gaps.
EM sections of axons with ER (arrows, top); 3D views (bottom). HSP Mutant ER tubules fewer & larger.