Roles of ER in distal axon pathologies

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.

Endoplasmic reticulum (ER) forms a dynamic network of sheets and tubules. It is central to Ca2+ and lipid homeostasis, and forms close contacts with other organelles. In neurons, it appears continuous through dendrites, cell body and axon, and is therefore termed a "neuron within a neuron" - potentially carrying local, regional, or long-range signals, independent of action potentials or physical transport, up to 1m in humans. An important role for it is supported by the finding that several axon degeneration genes, for the condition Hereditary Spastic Paraplegia (HSP) encode ER modelling proteins. In Drosophila we have developed tools to visualise axon ER, and shown that some ER-modelling proteins can affect the amount, continuity, or tubule dimensions of axon ER.

Our aim is to understand the physiological phenotypes in axons and synapses that result from the structural ER changes that we see in Drosophila HSP mutants - local loss of ER in synapses, local increase in ER in axons, disrupted continuity, and increased tubule diameter. We will test local Ca2+ handling, synaptic transmission, ER-plasma membrane lipid exchange, mitochondrial function, and longer range propagation of Ca2+ flows in cytosol and ER lumen.

By understanding the physiological consequences of ER structural changes for synapses and distal axons in Drosophila, we will be able to build models of the pathophysiology of HSP that can inform further work in both Drosophila and mammalian models. Our findings will also inform models of other distal neuropathies where ER plays a role.

Axon dysfunction is central to some of the most common conditions of ageing. In Alzheimer's disease, axon pathology is an early symptom. In diabetes, it is a major component of morbidity - approximately 60%-70% of patients have neuropathy that can promote injuries that require amputation, and >50% of lower limb amputations are in diabetics (www.cdc.gov). Basic understanding of the axon biology that underpins degeneration is an area of great need and promise, for impacts on understanding and amelioration of disease mechanisms.

Beneficiaries outside the immediate academic field include those with an interest in human axon degeneration, where basic Drosophila biology can generate hypotheses for human disease mechanisms. We were the first to show (Wang et al 2007) that a HSP gene had a role in BMP signaling, and susbequent work (e.g. Tsang et al 2009) showed roles for other HSP genes in BMP signalling in mammalian cells. More recently we were the first to show effects of HSP gene mutations on axon ER organisation in flies. A better understanding of local calcium signaling mechanisms in axons, and how they are affected by ER structural changes, could also lead to plausible therapies to ameliorate dysregulation. Ca2+ signalling has a number of druggable targets, and its downstream pathways in axons and synapses may have others too. Development of plausible therapies would ultimately benefit both patients and pharmaceutical businesses - although this would require more clinically based research for several years beyond the immediate work proposed here.

Research on Drosophila axon maintenance replaces the need for considerable experimentation on protected animal species. For example, Early characterisation of Wallerian degeneration was performed in mice, but Drosophila work has enabled identification of several new genes that protect against degeneration, at least one a druggable NADase, without large numbers of mice. Carrying out the early stages of pathophysiological research on Drosophila will not only replace routine mouse use, but also use of mice that would have been bred to show moderately severe neurological phenotypes.

Another important audience is patients suffering from neurodegenerative diseases, and their families. I have contacts with UK HSP and ALS charities through funding applications and reviewing, have hosted visits by their donors, and contributed to the 2015 and 2018 AGMs of the UK HSP support group, in the process learning more of the experience of affected individuals and their families or carers. Through these and similar contacts, I intend to continue this engagement.