Cellular pathophysiology of prion-mediated neurodegeneration - a model for understanding protein misfolding disorders

In the 1990s, scientists made a connection between bovine spongiform encephalopathy (BSE or "mad cow disease") and the fatal brain disorder variant Creutzfeldt-Jakob disease (vCJD) in humans. This discovery raised the profile of prion diseases both in the UK and internationally due to the associated public health and economic concerns. Rather than bacteria or viruses, prion diseases are caused by a rogue form of one our own proteins (prion protein), which accumulates in the central nervous system, leading to progressive brain cell malfunction and ultimately brain cell death. In patients, these changes appear as a rapidly advancing dementia, where memory, movement, balance and personality are all affected. In the absence of any effective treatment for prion disease sufferers, there is a clear need to study how the rogue prion protein leads to irreversible brain damage. It is becoming increasingly clear that the mechanisms underlying prion diseases are also observed in more common dementias such as Alzheimer's disease and Parkinson's disease. Our research therefore aims to investigate these similar pathways with the goal of identifying new drug targets for the treatment of all neurodegenerative conditions.

Previous work in our laboratory has identified that the rogue prion protein is able to block the cell's waste-processing centre (a structure known as the proteasome). Under normal conditions, the proteasome removes old or abnormally folded proteins from nerve cells to ensure they do not accumulate to toxic levels. By monitoring mice infected with prions we have observed that this disruption in cellular waste-processing occurs very early in disease progression. This suggests that drugs aimed at preserving this function in nerve cells could be candidates for future prion disease therapy. Our research therefore aims to answer two important questions: firstly, how do rogue prion proteins enter and move round inside the brain cells to disrupt normal waste-processing and secondly, what are the downstream consequences which lead to brain cell damage? The team uses a wide range of methods to answer these questions, including the detailed study of isolated cells and prion-infected laboratory mice. By furthering our understanding of how rogue prion proteins move around inside and damage the brain cells they infect, we hope to design treatments which aim to slow or prevent the onset of clinical symptoms in patients with prion disease and related neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease and Huntington's disease where similar pathways to nerve cell death exist.

Prion diseases are fatal neurodegenerative disorders whose pathogenesis is associated with a conformational rearrangement of the normal cellular PrP (PrPC) to abnormal conformers (PrPSc) which causes neurodegeneration via a poorly defined mechanism. Understanding these mechanisms has key relevance to other neurodegenerative proteinopathies where both prion-like mechanisms and dysfunction of the proteostasis networks have been suggested to be important. My laboratory has demonstrated that dysfunction of the ubiquitin proteasome system (UPS) is an early event in prion disease pathogenesis in vivo, and that beta-sheet rich PrP oligomers potently inhibit proteasome activity in vitro. Work from my last MRC grant yielded a novel cell model where PrP knockdown neuroblastoma cells have been engineered to express an epitope-tagged PrP-chimera (PrP-Ala224MYC). This novel chimera is unique since it supports prion replication, and generates bona fide epitope-tagged PrPSc. We have used this model system to study the earliest events in prion infection. This proposal has five main aims directed at extending the understanding of the cellular pathobiology of prion disease. Our existing PrP-Ala224MYC cell model will be used to map the cellular trafficking pathways of PrPSc with a view to defining how it accesses the cytosol. We will reconstitute neuronal stem cells derived from PrP KO mice with PrP-Ala224MYC, to study prion formation, UPS dysfunction and cytotoxicity in neurons. Detailed biochemical studies will refine our molecular understanding of the inhibitory interaction between misfolded PrP and the UPS. Lentiviral-mediated knockdown of PrP in vivo will determine whether UPS activity can be modulated in neurons as a viable therapeutic target. A proteomic study of the ubiquitinated proteome in prion-diseased brain will identify substrate proteins that accumulate as the disease progresses with the aim of uncovering new pathways that may contribute to cellular toxicity.

Neurodegenerative disease is set to have a serious and long-term impact on UK society, extending from the immeasurable burden on the lives of those affected to the wider social and economic implications of long-term care. The Dementia 2010 report estimated that neurodegenerative disease costs the UK economy £23 billion per year, greater than the cost of cancer (£12 billion/year) and heart disease (£8 billion/year) combined. To limit the inexorable rise in healthcare spending, research into neurodegeneration must uncover the sub-cellular mechanisms of disease to aid the design of rational therapeutics. Due to the irreversibility of neuronal loss, future therapies will need to target the pre-degenerative disease state, which is associated with subtle alterations in cellular function. Our research into the ubiquitin-proteasome system (UPS) has identified it as one of the earliest targets of prion-disease pathology. The proposed research will therefore investigate the relationship between misfolded proteins and the UPS to determine its validity as a therapeutic target. This research is of key interest to the pharmaceutical industry which, despite significant investment, has failed to date to develop effective disease-modifying treatments for patients with neurodegenerative diseases. By guiding the development of treatments for early stage dementia, our findings could help to shift public health policy away from long-term care and towards disease prevention.

Each of the specific research aims outlined in the scientific case for support will promote the development of new and innovative methodologies. For instance, the proposed research will continue to develop our novel engineered PrP chimera by using it to generate post-mitotic primary neuronal cultures which express a tagged PrPSc. Successful generation of this cell line will allow researchers to track the earliest sources of cellular toxicity following protein-misfolding - a key question which currently remains unanswered. Altered trafficking of misfolded proteins has been suggested to be of importance in several neurodegenerative diseases (e.g. Alzheimer's, Huntington's and Parkinson's diseases) and therefore the experiments designed to dissect key trafficking pathways in prion toxicity will have relevance to these more common diseases. Thus despite being rooted in the basic mechanisms of prion disease, the research has the potential to influence academic research groups investigating other neurodegenerative diseases. In vivo models of AD, PD and HD require the expression of human mutant proteins, often with variable recapitulation of the human disease phenotype. In contrast, laboratory mice infected with prions genuinely succumb to prion disease, allowing us to identify which pathological mechanisms may be relevant to disease progression. Advances in understanding prion pathophysiology could therefore have major implications for other protein-misfolding disorders through the elucidation of common cellular mechanisms.

The stimulating research environment at the Institute of Neurology will train and deliver highly skilled researchers with the expertise and knowledge to investigate cellular mechanisms of pathology across a wide spectrum of neurodegenerative conditions. We recognise that there is an increasing demand to communicate research progress to the general public, in particular patients and their families. Through our public communication plans (see Pathways to Impact), we hope to provide reassurance that current research efforts are successfully dissecting complex disease aetiologies to identify new treatment options.