Understanding axonopathy by defining physiological and pathological functions of the microtubule severing protein spastin at membrane traffic pathways

This proposal focuses on a protein called spastin. Spastin is present in all of the cells of the body and is important for three main reasons, i) inherited gene mutations affecting spastin cause a type of hereditary motor neuron disease called hereditary spastic paraplegia, ii) spastin has been implicated in the pathology of Alzheimer's dementia, and iii) spastin carries out functions that control cellular pathways found in many different cell types and so knowing how it works is of importance in understanding the basic biology of cells.

In this work we aim to determine as fully as possible what the normal functions of the spastin protein are, and to understand how abnormality of these functions leads to hereditary spastic paraplegia. We will build on our existing knowledge of spastin. Human cells are bounded by a lipid membrane and contain many different membrane-bound intracellular compartments (called organelles) that are connected by transport pathways. Cargoes are transported in membrane-bound vesicles or tubules from one organelle to another over tracks called microtubules. We know that a main function of spastin is to cut microtubules, and when spastin is abnormal this does not happen. This failure to cut microtubules has several consequences- it results in some cellular organelles having an abnormal shape, and in the inefficient transport of some cargoes. We believe that this may be because spastin controls how well membrane transport tubules are released from the parent organelle.

To achieve our aims we will carry out the following experiments:

i) Although we know that failure of microtubule severing results in abnormal release of transport tubules, we do not know how microtubule severing promotes their release. We will examine this using high powered microscopy approaches to visualise the process in living cells.

ii) We will examine how failure of these transport and shaping processes affects the known functions of the organelles affected. For example, one of the organelles involved is called the endosome, which can be thought of as a sorting centre within the cell. Here, decisions are made as to whether cargo proteins like membrane receptors are sent for degradation, or are recycled back to the cell surface, where they can sense the cellular environment. We will therefore find out which cell surface receptors are affected in cells with abnormal spastin.

iii) We will also determine which of the functional abnormalities that we discover in ii) are likely to be involved in causing the hereditary spastic paraplegia disease. We will do this by examining neurons cultured in Petri dishes. When neurons have abnormality of spastin, they develop swollen sections, and we will test whether these can be prevented by treatments to normalise the particular functional abnormalities that we find. For example, some membrane receptors control signaling pathways that influence how neurons grow. If we find that such a receptor is controlled by spastin, we will examine whether inhibiting or activating, with appropriate drugs, the signaling pathway that it controls is effective at preventing the neuronal abnormalities. This will be a first step in finding treatments for hereditary spastic paraplegia caused by abnormality of spastin.

Overall, these experiments will identify what spastin does in cells, how it does it, and how this translates to pathology in neurons. This level of detailed understanding will likely give rise to rational treatment approaches for hereditary spastic paraplegia, and perhaps other similar neurological condiditons.

We focus on the microtubule severing protein spastin. Mutations in the spastin gene are the most common cause of hereditary spastic paraplegia, in which axons of motor neurons selectively degenerate. Spastin has also been implicated in the pathogenesis of Alzheimer dementia. Spastin is recruited to endosomes and the endoplasmic reticulum, where it controls functionally important membrane modeling events. This led us to propose a pathological model for spastin-HSP in which lack of spastin-mediated microtubule severing causes failure of microtubule-motor dependent membrane modeling steps, which results in abnormality of membrane functions (e.g. traffic of key membrane receptors) that are critical for axonal health.

We will test each part of this hypothesis by answering the following questions:

i) Does microtubule severing increase dynein loading on microtubules by generating new microtubule plus ends, so providing power for membrane modeling? We will focus on endosomes. Our results indicate that spastin is required for fission of recycling tubules from the endosomal body. Using TIRF and spinning disc microscopy in living cells, we will examine the spatial and temporal relationship between microtubule severing, microtubule plus-end generation, dynein motor recruitment to the microtubule, and tubule fission.

ii) What are the functional consequences of failure of spastin-mediated membrane modeling and how do these cause axonopathy? We will examine specific endosomal trafficking steps that are strong candidates to be regulated by spastin. We will also use an unbiased quantitiative SILAC proteomics approach to identify the full range of cell surface receptors regulated by spastin. We will examine the pathological relevance of functional abnormalities identified, prioritising for study those that are candidates to impact on axonal function, by testing whether they are involved in generating axonal swellings in primary neurons derived from our spastin-HSP mice.

Who will benefit from this research and how?

This project focuses on understanding the cell biology and axonal pathology associated with spastin. For this reason the most immediate beneficiaries (within 3 years) will be national and international researchers working in related areas, as described in the "Academic Beneficiaries" section.

In the medium-longer term (5-15 years), understanding the molecular and cellular processes that control axonal maintenance and degeneration will have wider implications for health and wealth. The research may provide the foundation to identify new therapeutic strategies for axonal degenerative diseases. As well as the >5000 people in the UK and >500,000 people worldwide who suffer from HSP, this could be of benefit to the millions of patients who have common neurological disorders that include axonopathy as a feature; for example, patients with some subtypes of multiple sclerosis have a morphologically identical axonopathy to that seen in HSP. In addition, spastin has recently been implicated in the pathogenesis of Alzheimer's dementia and so understanding its functional roles could have implications for treating this very common disease. In these cases our work could be of commercial interest and benefit to UK-based pharmaceutical companies, with whom we would actively seek collaborations. In this we will be aided by the University of Cambridge Office for Translational Research and by Cambridge Enterprise.

Our work may be of relevance to the "healthy" general public. As well as the neurodegenerative diseases mentioned above, axonal degeneration is an important feature of declining neurological function in normal ageing. Our work will elucidate molecular factors that are critical in keeping axons alive and which would be strong candidates to be affected by the ageing process, and which perhaps could be targeted by therapeutics to slow the process. The fact that spastin-HSP shows age-dependant penetrance indicates that there is an interplay between onset of the disease and age.

This proposal will also develop the scientific, management and communications skills of the post-doctoral researcher employed. She will receive training in a variety of laboratory skills that cut across several related disciplines, including cutting edge microscopy on living and fixed cells, proteomics, and work with mouse models of disease. In addition, the researcher will receive further training and experience in writing scientific papers, presenting work at scientific meetings and helping to run a scientific group. Thus the project will contribute to building UK scientific capacity and a highly trained workforce.