Organisation and Roles of Axonal Endoplasmic Reticulum

Animal and human movement depends on the ability of nerve cells to carry signals along narrow projections known as axons, which in humans can extend as much as a metre from the centre of the cell, the cell body - and even several metres in giraffes or whales. If the cell body has the size of a lecture theatre in Cambridge, the axon is like a corridor reaching out of it as far as Edinburgh or Paris, allowing transport of materials and communication.

Maintaining the structure and function of long axons requires a lot of engineering. The importance of this is reflected in problems that occur when it goes wrong - diseases with effects such as axon degeneration, paralysis, or lack of sensation. Some of these preferentially affect long axons, or the ends of axons furthest from the cell body - suggesting impairment in communication or transport, that the parts of axons furthest from the cell body are most susceptible to.

How does communication along axons maintain their form and function? Axons have many internal organelles that are bounded by membranes, whose transport and organisation contributes to the engineering and communication to maintain longer axons. Some of them are transported along tracks called microtubules, and carry materials and signals forward and back along axons. Another membrane-bound structure within axons are tubules known as smooth endoplasmic reticulum (ER), which run lengthwise along the axon. Due to their length and continuity, and the ensuing potential to conduct signals for long distances within neurons, they have been likened to a "neuron within a neuron". However, the mechanisms that form them, their function in axons, and the relationship between their form and function, are poorly understood.

Mechanisms to maintain function and integrity of long axons can be revealed by indentifying the genetic causes of axon degeneration. In support of a role for ER in this, the disease Hereditary Spastic Paraplegia (HSP), characterised by degeneration of longer spinal motor axons, is often caused by mutations in proteins with roles in modeling ER membrane. These proteins insert in one face of the membrane, thus curving it, and some have additional roles such as fusing tubules into a network, or severing microtubules (tracks along which organelles are transported). In yeast, removing two groups of these proteins even deletes nearly all tubular ER.

We aim to understand the mechanisms that govern the architecture of ER in axons, and the importance of this for axon function. We use the fruitfly Drosophila, given the ease of generating mutant and transgenic flies that lack particular proteins or express altered forms of them, and the availability of reagents and methods to study nerve cells, including axons. Some mutations in Drosophila HSP genes even have similar phenotypes to human HSP: larvae whose anterior (controlled by shorter axons) moves normally, but whose posterior (controlled by longer axons) cannot. We have also shown that a Drosophila HSP protein is important for shaping ER in non-neuronal cells, and for normal amounts of ER in longer axons - the first direct evidence that HSP genes affect ER in longer axons, and implying that axonal ER is important for axon survival.

We will therefore examine the detailed organisation of tubular ER in axons, using high resolution electron microscopy. We will develop ways to visualise ER in live animals, in single axons, and in high resolution electron microscopy, and will use these to assess in detail the roles of a range of proteins on the presence, extent, morphology and network organisation of axonal ER. We will also assess how far axonal ER contributes to signaling in axons by release of an important signaling molecule, calcium ions, and how this signaling depends on ER form as determined by HSP and related proteins. In the longer term, our approach can illuminate roles of ER in axonal injury and long-range signaling in neurons.

Axons have long been recognised to have tubular smooth endoplasmic reticulum (ER), seen by electron microscopy to run along axons longitudinally. The length and continuity of ER, from dendrites through cell body and axons to presynaptic terminals, has led it to be likened to a "neuron-within-a-neuron", a membrane system that can potentially conduct local or long-range signals through neurons, independently of action potentials, but faster than microtubule-based transport, which would take several days to transport cargoes physically along the longest human axons.

Little is understood of how this characteristic form of axonal ER arises, and the relationship between its form and function. However, clues are emerging, for example from the identification of proteins that model ER membrane as causative for many cases of Hereditary Spastic Paraplegia (HSP), in which there is preferential degeneration of longer spinal cord motor axons. This suggests both a role for these proteins in modeling of axonal ER, as well as a requirement for axonal ER in maintenance of longer axons.

In support of this model, we recently showed a role for a Drosophila HSP protein in organisation of the distal parts of longer motor axons, including levels of a smooth ER marker. Here we develop this model further, by trying to understand axonal ER organisation at light microscopy and ultrastructural level, and in single axons. We will use these approaches to identify proteins that are responsible for ER localisation, morphology and network formation in axons, both membrane proteins that may be responsible for modeling ER membrane, and motor proteins that may be responsible for transporting it. We will also develop tools for monitoring ER calcium release in axons, and use these and other tools to follow calcium release in axons, and the requirements for smooth ER organisation in this process.

While this work is basic in nature and is not primarily aimed at understanding specific disease mechanisms, basic axon biology fits squarely within the BBSRC strategic priority of Basic Bioscience Underpinning Health. 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 lead to injuries that require amputation, and >50% of lower limb amputations in the US occur among persons with diabetes (www.cdc.gov). Basic understanding of the axon biology that underpins mechanisms of degeneration is an area of great need and promise in making impacts on both the understanding of disease mechanisms, and for their amelioration.

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. For example, we were the first to show (Wang et al 2007, highlighted in the BBSRC 2007 Annual Report) that a HSP gene had a role in BMP signaling, and susbequent work from ourselves and others (e.g. Tsang et al 2009) showed roles for a number of HSP genes in this signaling pathway. The variety of roles of BMP signaling means that great care is needed before using it as a therapeutic target, and good mouse models of most HSP diseases are still lacking, but as these models emerge, BMP signaling deserves exploration as a therapeutic target, e.g. via localised delivery of agonists or antagonists. A better understanding of local calcium signaling mechanisms in axons, or other consequences of ER defects, could also lead to plausible therapies to ameliorate dysregulation of this, in the range of diseases with axonal dysfunction. 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.

Basic research on Drosophila axon biology also replaces the need for some experimentation on protected animal species. A precedent for this is that the early characterisation of Wallerian degeneration was performed in mice (much of it actually in a BBSRC Institute, in Babraham, by Dr. Michael Coleman), but use of Drosophila has enabled identification of several new genes that protect against degeneration, without the need for large numbers of mice. Likewise, our proposal allows testing of a large number of genotypes for effects on axonal ER in flies, an undertaking that would otherwise require some hundreds of mice.