Roles of ER in distal axon pathologies

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Our movement depends on the ability of nerve cells to carry signals along narrow projections known as axons, which in humans can extend a metre from the centre of the cell, the cell body. The challenge of maintaining longer axons is shown by symptoms when they go wrong - axon degeneration, paralysis, or lack of sensation. These occurs in genetic conditions like Hereditary Spastic Paraplegia (HSP), in which degeneration of longer spinal cord motor axons selectively paralyses the lower body, or acquired conditions like diabetic or chemotherapy-induced neuropathy, in which peripheral neuron disease can cause pain or numbness of body extremities. However, the physiological processes that make distal axons vulnerable in these conditions are not known; our goal is to answer this question. We focus on genetic conditions, since genes give us clues about the affected mechanisms, and insights from genetics often apply to acquired conditions. We study a structure called endoplasmic reticulum (ER), consisting of hollow tubules that run lengthwise through the axon, and fuse and split from each other to form a network. Due to their length and continuity, and their potential to carry signals for long distances, they have been termed a "neuron within a neuron". Many HSP mutations that disrupt proteins that help model ER, for example by inserting in one face of the ER membrane and curving it to make tubules: this suggests an important role for the ER network in maintaining axon function and survival.Axonal ER is an underexplored structure and we have only recently developed tools to visualize and see defects in it. We see that removing some HSP membrane-curving proteins, in fruitfly mutants, disurupts the ER network in motor axons, e.g. altered levels of ER tubules, larger tubules, or interrupted continuity; loss of ER continuity is an attractive model for distal axon conditions, since the probability of a gap somewhere along the axon increases with distance from the cell body. It has been easier to make these advances in fruitflies than with human or mammalian neurons, and fruitflies also allow us to work with axons in an intact organism. ER tubules store calcium ions, and can either release calcium into the surrounding cytoplasm as a local transient signal, or remove excess calcium from there, thus maintaining low levels of calcium in cytoplasm. We therefore focus mainly on the consequences of ER structural changes in fly HSP mutants, for local and long-range signalling by calcium flows. We have four main aims, that together should uncover the consequences of altering the amount or presence of ER, its continuity, or its tubule diameter; by analysing mainly (not exclusively) distal axons or presynaptic terminals, we hope to learn more of the mechanisms that affect the regions furthest from the cell body. First we examine how mutations in the fly homolog of a commonly mutated HSP gene, atlastin, affect continuity of axonal ER. Second we examine the consequences of ER depletion for local calcium handling, synaptic transmission, and on lipid transfer between ER and plasma membrane, using another HSP mutant that lacks ER in some synaptic branches. This work will test whether these defects could be plausible factors in pathology. Third, we will test the consequences of ER loss or discontinuity on the properties of mitochondria - organelles that provide cells with usable energy, whose activity is controlled by release of calcium from ER, and which can be harmful if uncontrolled. Finally, we will test the consequences of ER abnormalities for the spread of calcium signals in axons, since longer axons could be more vulnerable to impaired spread of a signal than shorter ones. Together these analyses will provide models for the disease mechanisms of HSP, that can be tested in mammalian or human neurons as our previous fly findings were. They can underpin rational approaches to therapy of HSPs or other diseases of distal axons.