Elucidating the role of DNA methylation in the pathophysiology of lewy body diseases.

The Lewy body (LB) diseases include Parkinson's disease (PD), PD dementia (PDD) and Dementia with Lewy bodies (DLB). The hallmarks of all of these diseases are aggregates of a protein called a-synuclein, forming Lewy bodies (LBs) in specific populations of neurons in the brain. The diseases can also have an overlap in clinical symptoms, which can make diagnosis difficult. These diseases are very complex, with their exact cause still largely unknown, although it is suggested that both genetic and environmental factors can alter a person's risk. Although a number of studies have recently searched for new genes that may make an individual more susceptible to these diseases, most of the identified genetic changes are very common, and only have a very modest effect on increasing one's likelihood of developing disease. It is known that the expression of genes relies not only on a person's specific DNA code (their genome), but can also be altered by an extra level of information called the "epigenome". Epigenetic processes are chemical tags added to the DNA that turn genes on and off and can be influenced by external factors such as the environment in which cells dwell. Major genetic differences between the sufferers of people with LB diseases and unaffected individuals have been hard to identify, therefore scientists have speculated that epigenetic differences are involved in the diseases and could be the major way in which environmental risks can alter the expression of genes. In the case of Alzheimer's disease (AD) we, and others, have recently published some of the first studies showing that epigenetic changes are consistently seen in AD brain samples. We hypothesise that epigenetic changes also play a role in LB diseases and we propose that we can identify epigenetic signatures that can distinguish individuals with LBs based on their diagnosis, genetics, degree of brain pathology, symptoms and, in the case of PD, the presence of dementia. In order to address this we have the following research objectives:

AIM 1: To identify specific epigenetic signatures of different LB diseases in post-mortem brain samples. These can teach us about the underlying biological differences between these diseases.

AIM 2: To identify specific epigenetic signatures that are associated with neuropathological markers, regardless of clinical diagnosis. These can teach us about the biological basis behind the selective vulnerability of particular cell types in the different diseases.

AIM 3: To identify specific epigenetic signatures associated with psychosis, regardless of underlying pathology or clinical diagnosis. This can teach us about the underlying biology driving these symptoms.

AIM 4: To identify specific epigenetic signatures that can distinguish PDD from PD. These can teach us about the underlying neurobiology and risk factors associated with developing dementia.

In order to address our hypotheses we propose to analyse epigenetic changes in the different Lewy body diseases, in two brain regions: the substantia nigra (SN), which is affected in the middle stages of disease, and the prefrontal cortex (PFC), which is affected in the later stages of disease. We will use state-of-the-art cell sorting methods to determine which cell types are driving these changes, before focussing on those altered in neurons. We will then determine which of these genes are specifically altered in neurons containing LBs. Finally, we will demonstrate that epigenetic changes in these genes are causing disease pathology by using cutting-edge genetic editing technology in cell culture. Looking to the future, as epigenetic changes are potentially reversible, these changes could represent novel drug targets.

Parkinson's disease (PD), PD dementia (PDD) and Dementia with Lewy bodies (DLB) are Lewy body (LB) diseases, which are characterised by the cytoplasmic accumulation of a-synuclein in neurons, forming Lewy bodies (LBs). At present we do not know what molecular changes may be driving disease onset and progression and there are currently no disease-modifying treatments for these diseases.

This study will represent the first systematic, epigenome-wide association study (EWAS) of DNA methylation across multiple diseases characterised by synucleinopathy. We propose to analyse DNA methylation patterns using the Illumina EPIC array in 100 PD, 100 PDD, 100 DLB and 100 control donors in the substantia nigra (SN) as this is affected in the middle stages of disease, and the prefrontal cortex (PFC) as this is affected in the later stages of disease. We will identify DNA methylation signatures that can stratify individuals based on their diagnosis, genotype, pathology, symptoms and, in the case of PD, the presence of PDD. We will validate these patterns, and determine their cellular specificity using pyrosequencing and qRT-PCR on nucleic acid extracted from neurons and glia isolated from SN and PFC samples using fluorescence-activated cell sorting (FACS). Loci that appear to be neuronal we will investigate further using laser capture microdissection (LCM) in PFC samples. We will collect LB-bearing neurons and neurons free of pathology and use pyrosequencing to determine which loci are specifically altered in LB-bearing neurons. These loci will be the focus of our functional characterisation experiments, where we propose to use CRISPR/Cas9 technology to alter DNA methylation at these loci, and establish the downstream consequence on cell phenotype. Taken together, we will be able to identify differentially methylated loci that are causal in LB disease pathology, which could ultimately deliver new pharmacological targets, potentially improving the lives of sufferers.

In addition to scientists interested in the aetiology of lewy body diseases, the results of this project have the potential to impact on a number of other beneficiaries. These include patients suffering from the lewy body diseases, the pharmaceutical industry, health service providers and academic groups investigating the causes of other complex disease phenotypes. In the UK more than 850,000 people are living with dementia, with care costs in excess of £26 billion per year. Due to an increasingly ageing population, the number of cases is rising dramatically, with profound socioeconomic consequences as our healthcare systems struggle to cope. Dementia with lewy bodies (DLB) and Parkinson's disease dementia (PDD) account for ~20% of dementia cases and the current treatments temporarily alleviate some symptoms but do not modify the underlying disease process. A better understanding of the underlying mechanisms driving disease onset and progression is required to enable the design of new, more effective medications that modify the disease process. Casual mechanisms in sporadic lewy body diseases have not yet been established to date; we hypothesise that epigenetic dysregulation drives lewy body pathology in these diseases, resulting in neurodegeneration.

Identifying changes in the brain that are causal in disease pathology would be particularly ground-breaking as these would represent viable new targets for designing new, more effective medications. Further, given the dynamic and potentially-reversible nature of the epigenome, the outputs from this research could represent realistic targets for disease-modifying pharmacological interventions. A number of pharmaceutical companies are actively developing "epigenetic-drugs" and could rapidly take advantage of these outputs.