Characterisation of a novel cell model for Huntington's disease: insights into pathogenesis

Lead Research Organisation: University College London
Department Name: Institute of Neurology


Huntington's disease is a devastating neurological condition caused by a mutation in the gene encoding Huntingtin protein. It affects 4-10 people per 100,000 population. Patients inherit a copy of the mutated gene from an affected parent, and normally start to show symptoms of the disease at around 40 years of age. Symptoms include excessive uncontrolled movements of the body, and psychiatric problems, and death occurs 15-20 years from onset. Successive generations are affected at progressively earlier ages.

The responsible gene and the causative mutation were discovered in 1993, but there remain many unanswered questions about the exact way in which this mutation leads to degeneration of neurons in the brain. In order to better understand the process, we need cellular models of the disease that we can test in the lab. I am in the process of creating a cell model of Huntington's disease, using a new technique to insert the mutated gene into human neural stem cells. These are cells that can be matured into many different types of neurons, including medium spiny neurons that are the type of cell most severely affected in Huntington's disease. This is particularly useful as I will then have a model which is:
(a) of a human cell,
(b) of the cell type most affected in Huntington's disease and
(c) carries one copy of the mutated gene, just like the actual disease state.

I am then planning to observe the effect of the mutation on the functioning of these cells, looking at effects on lifespan and resistance to cell stressors such as oxygen deprivation or exposure to toxins. Normal neurons form long, branching "arms" that make contact with other neurons in order to transmit electrical messages throughout the brain. I will observe the effect of the mutation on the shape and functioning of the cells, using a combination of microscopy and electrical studies.

I will also use the model to gain a clearer idea of how the mutated protein (made from the mutated gene) functions differently to the normal protein and how this might lead to dysfunction and death of the neuron. Using real time, live-imaging microscopy techniques I will visualize how the mutated protein is processed and moves around the cell, and how this is different from normal.

Ultimately, I hope to contribute to a better understanding of the pathway linking the gene mutation to neuronal dysfunction and subsequent symptoms in humans. This will enable design of drugs that can block this process from happening, and potentially prevent or cure this disease in the future.

Technical Summary

Huntington's disease (HD) is a fatal, autosomal dominant inherited condition caused by a CAG repeat expansion in exon 1 of the gene encoding Huntingtin (Htt) protein. We aim to create a cell model of Huntington's disease using two different human neural stem cell (hNSC) lines and knocking the mutated exon 1 with various CAG repeat lengths (50, 80 and 120 repeats) into one of the Htt gene alleles to create heterozygous cell lines. This will be achieved using the rAAV vector, which triggers homologous recombination into the native gene locus, thus creating a model that is far more representative of the diseased cell. The two hNSC lines differentiate into dopaminergic and GABA-ergic neurons, a proportion of them specifically forming the medium spiny neurons which are preferentially destroyed in HD.

We will then use this model to characterise the effect that the mutated gene has on the phenotype of the neurons. My hypothesis is that mutant HTT will cause CAG length dependent cellular dysfunction in MSNs. This will be tested using LDH and ATP assays, as well as the Cellomics fluorescence based cytotoxicity bioapplication. I will also use electrophysiology and quantitative PCR to assess synaptic function and transcriptional dysregulation, which are known problems in HD.

The other hypothesis I wish to test is that wild type and mutant HTT have different cellular trafficking pathways, which impacts on cell function. This will be tested using immunofluorescence and confocal laser microscopy in conjunction with an existing panel of anti-HTT antibodies to different HTT epitopes. I will also use live cell imaging techniques to study the effect of mutant HTT on axonal transport.

Planned Impact

Huntington's disease is a devastating neurological condition that affects 4-10 people per 100, 000 population. It is an autosomal dominant inherited condition, with typical age of onset of symptoms at 40 years of age, although with successive generations this becomes progressively younger. Over just a few years, patients develop excessive body movements, psychiatric problems and cognitive dysfunction - they are no longer able to continue working, and ultimately require full time care, often for years. The disease leads to death after around 15 years.

It is of utmost importance that high quality research is carried out into this disease, in order to find treatments that can prevent or cure it. The impact on society of losing people of working age as well as the costs of care, are high - both in terms of economic and social burden. Patient's partners and relatives often have to stop working themselves in order to become full time carers. In addition to this, the disease causes significant emotional trauma, as affected parents have usually already had children and have a fifty percent chance of having passed the gene mutation on to them. In addition, they may have already witnessed the disease claiming the lives of their own parents.

Since the introduction of genetic testing for this condition, we are able to identify people who will get the disease before they actually develop any symptoms. This provides us with an opportunity to potentially prevent it's occurrence. The first step of this is to fully understand the way in which the gene mutation leads to neuronal degeneration and disease - this is a process which starts with basic science research in a lab, and where I hope my own research will make the biggest impact.

The ultimate hope is that researchers will then be able to design therapies that can stop the mutation from having these effects. This would then save HD patients from a slow decline into death, and allow them to continue as productive members of society. Their families would be spared the devastation of watching this happen, and would also be freed of their caregiver duties allowing them also to remain in work. The timescales of translating basic science research into potential therapies is many years, but good communication and the increased methods of dissemination of findings can speed up the process.

In our ageing society, neurodegenerative diseases are sadly affecting a greater proportion of people, and so it is vital that this area of research continues to be prioritised in terms of funding. Increasing public awareness of these conditions, as described in my communications plan, is required in order to achieve this. A better understanding of neuronal degeneration in Huntington's disease may impact on other diseases in which protein folding is implicated, such as Alzheimer's disease and other dementias. These conditions are responsible for a large number of those requiring residential and nursing home care, which is a significant and ever increasing cost for the tax-payer. If we can better understand the process by which neurons degenerate then we may be able to find better treatments for these conditions also, thus benefitting both the patients, the economy and society at large.


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