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

Lead Research Organisation: UNIVERSITY OF EXETER
Department Name: Institute of Biomed & Clinical Science

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description A multi-omic approach to elucidate novel disease mechanisms and biomarkers for psychosis in Alzheimer's disease
Amount $1,626,428 (USD)
Funding ID R01 AG067015 
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States
Start 12/2019 
End 11/2023
 
Description Charles Wolfson PhD studentship
Amount £60,000 (GBP)
Organisation Charles Wolfson Charitable Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2019 
End 05/2023
 
Description Cutting-edge precision profiling of specific cell populations using a Laser Capture Microdissection (LCM) system
Amount £239,166 (GBP)
Funding ID MR/X013413/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 11/2022 
End 03/2023
 
Description Utilising big data and long-read sequencing approaches to understand APOE (epi)genetics in Alzheimer's disease
Amount £152,854 (GBP)
Organisation Alzheimer's Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2023 
End 10/2027
 
Title Isolating nuclei from cingulate gyrus 
Description We have optimised a protocol from our lab to allow us to isolate nuclei from neurons, oligodendrocytes, microglia and other cell types from cingulate gyrus brain tissue. We are now using this in the project to isolate further cell types to what we originally planned. We have also applied for pump priming funds to allow us to isolate somas from these cell types, allowing a wider range of cell types to be characterised 
Type Of Material Technology assay or reagent 
Year Produced 2023 
Provided To Others? No  
Impact None yet 
 
Title PRS-multi-trait R package 
Description PRS-multi-trait is an R package that focuses on using generalized summary statistics, calculation and prediction of multi-trait polygenic (risk) scores (PGS) to predict the likelihood of an individual developing a particular trait or disease based on their genetic risk factors. The package includes functions for calculating PGS for individuals based on the presence of specific genetic variants that have been associated with the trait or disease in question. PRS-multi-trait is designed to be user-friendly and easy to use, with a range of functions and documentation to help users get started quickly. Whether you are a researcher or practitioner in the field of genetics and genomics, or simply interested in exploring the genetic basis of complex traits and diseases, PRS-Multi-trait can help you generate PGS models and calculate PGS for your cohort. The method is useful for a wide range of applications, including identifying individuals who may be at risk of developing complex diseases such as diabetes or Alzheimer's disease, as well as for predicting scores for more straightforward traits such as height or eye color. 
Type Of Material Computer model/algorithm 
Year Produced 2022 
Provided To Others? Yes  
Impact Ease of analysis for other researchers 
URL https://github.com/Rrtk2/PRS-multi-trait
 
Description Biomarkers to predict individual risk of dementia in Parkinson's disease 
Organisation University of Stavanger
Country Norway 
Sector Academic/University 
PI Contribution This is a Norwegian Research Council FRIMEDBIO project awarded to Prof Alves and Dr Grodem, where we will be generating DNA methylation data in blood and brain samples from the ParkDem study in Norway
Collaborator Contribution They have provided samples for the analysis and we are working together on the analysis
Impact Named as partner on their application to the Norwegian Research Council. Exchange of staff. Invited visit
Start Year 2020
 
Description Novel collaboration with Oslo University Hospital 
Organisation Oslo University Hospital
Country Norway 
Sector Hospitals 
PI Contribution Novel collaboration with Dr. Lasse Pihlstrøm set up from Department of Neurology, Oslo University Hospital, Oslo, Norway for meta-analysis of Lewy Body Dementia cohorts. They have provided data from 200 DLB brain samples (worth ~ £40,000).
Collaborator Contribution Provided data for a meta-analysis
Impact We have applied for a grant (currently under review) from the Michael J Fox Foundation, on which the collaborator is a co-applicant based on this collaboration
Start Year 2022
 
Description Novel collaboration with the University of Nottingham 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We have established a collaboration with Prof Anto P R Rajmani and team at the University of Nottingham, where we are providing RNA from our cohort for their analyses, maximising the use of the tissue we received.
Collaborator Contribution They will be profiling the RNA using sequencing
Impact Prof Rajmani has applied for an ARUK pilot grant on which we are co-applicants
Start Year 2021
 
Title PRS-multi-trait R package 
Description PRS-multi-trait is an R package that focuses on using generalized summary statistics, calculation and prediction of multi-trait polygenic (risk) scores (PGS) to predict the likelihood of an individual developing a particular trait or disease based on their genetic risk factors. The package includes functions for calculating PGS for individuals based on the presence of specific genetic variants that have been associated with the trait or disease in question. PRS-multi-trait is designed to be user-friendly and easy to use, with a range of functions and documentation to help users get started quickly. Whether you are a researcher or practitioner in the field of genetics and genomics, or simply interested in exploring the genetic basis of complex traits and diseases, PRS-Multi-trait can help you generate PGS models and calculate PGS for your cohort. The method is useful for a wide range of applications, including identifying individuals who may be at risk of developing complex diseases such as diabetes or Alzheimer's disease, as well as for predicting scores for more straightforward traits such as height or eye color. 
Type Of Technology Webtool/Application 
Year Produced 2022 
Impact Ease of analysis for other researchers 
 
Description AAIC Annual Conference 2022 (in person) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact A talk and two poster on the research findings were presented at AAIC 2022 in San Diego in July 2022
Year(s) Of Engagement Activity 2022
 
Description ADPD Annual Conference 2021, Online 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Poster presentation at ADPD Annual Conference 2021, Online
Year(s) Of Engagement Activity 2021
 
Description ADPD Annual Conference 2022 (in person) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Two talks and one poster on the research findings were presented at ADPD 2022 in Barcelona in March 2022
Year(s) Of Engagement Activity 2022
 
Description ADPD Annual Conference 2023 (in person) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Two oral presentations on the research findings have been selected as oral presentations to be presented at ADPD 2023 in Barcelona in March 2023
Year(s) Of Engagement Activity 2023
 
Description ARUK Annual Conference 2022 (in person) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact A talk and a poster were presented at the ARUK national conference in Brighton in March 2022
Year(s) Of Engagement Activity 2022
 
Description ARUK Annual Conference 2023 (in person) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact One flashtalk and three posters on the research findings were presented in the ARUK conference in Aberdeen in March 2023
Year(s) Of Engagement Activity 2023
 
Description Alzheimer's Research UK Annual Conference 2020, Online 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Poster presentation on the project at the ARUK 2020 (online) conference
Year(s) Of Engagement Activity 2020
 
Description Alzheimer's Research UK Annual Conference 2021, Online 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Poster presentation at the Alzheimer's Research UK Annual Conference 2020, Online
Year(s) Of Engagement Activity 2021
 
Description CRISPR and Beyond Conference 2022 (in person) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact A talk was given on the research findings at the CRISPR and Beyond Conference in June 2022
Year(s) Of Engagement Activity 2022
 
Description Epigenomics of Common Disease (ECD) Annual Conference 2019, Hinxton, Cambridge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Poster presentation at Epigenomics of Common Disease (ECD) Annual Conference 2020, at Welcome Campus, Hinxton, Cambridge
Year(s) Of Engagement Activity 2019
 
Description Epigenomics of Common Disease (ECD) Annual Conference 2020, Online 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Poster presentation ECD Annual Conference 2020, Online
Year(s) Of Engagement Activity 2020
 
Description Pathways to Industry Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Industry/Business
Results and Impact After successfully bidding for event funding we held a "Pathways to Industry" workshop to bring together academics who are interested in translating their research, and working with industry.
Year(s) Of Engagement Activity 2023
 
Description Pint of Science 2023 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact We will be speaking at the Pint of Science Festival in 2023 about our epigenetic editing work as part of this MRC grant
Year(s) Of Engagement Activity 2023