In vivo pathway analysis to determine the mechanistic role of susceptibility genes for Parkinson's disease

Lead Research Organisation: University of Sheffield
Department Name: Neurosciences

Abstract

Parkinson's disease (PD) is a common and relentlessly progressive brain disease. Its key symptom, slowness of movement, is caused by the death of nerve cells (neurons) that produce a chemical called dopamine in a part of the brain called the substantia nigra. Currently available therapies are inadequate: Firstly, they treat only some, but not all, of the symptoms of the disease. Secondly, they do not modify the underlying disease processes and therefore fail to slow down disease progression. This failure is partially due to our limited knowledge of the mechanisms that cause PD.
Over the last 20 years, there has been huge progress with the identification of genes and mechanisms which cause PD that is inherited due to faults (mutations) in genes (familial PD). However, only about 5-10% of PD patients in the UK carry such a familial PD mutation. This project aims to elucidate how inherited factors contribute to the risk of the much more common sporadic form of PD.
So-called genome-wide association studies (GWAS) have now identified 24 regions within our genome which contain genes that contribute to the risk of sporadic PD. However, it is not clear how these genes contribute to the risk of PD and, in some cases, which particular gene within such a region is responsible.
Our aim is to study those genes which are most likely to contribute to the risk of PD (from hereon called PD GWA genes). We and others believe that these genes will be involved in 3 distinct mechanisms, namely:
1) The regulation of energy production and energy maintenance (homeostasis) by cellular components called mitochondria.
2) The aggregation and breakdown of proteins by processes called autophagy and cellular components called lysosomes.
3) Inflammation (the cellular response to injury and infection).
We propose to use zebrafish carrying gene defects which will inactivate these PD GWA genes for our studies.
Zebrafish are vertebrates and therefore much more closely related to humans then other lower animal models such as worms or fruit flies. They also offer the opportunity to study the interaction of PD GWA genes with ageing processes. This is important since ageing is the most important risk factor for PD. Zebrafish larvae develop outside the body and are transparent. This makes it relatively easy to study the interaction between neurons and immune cells. Zebrafish are also an excellent animal model for drug discovery. They have already been used for a wide range of other human diseases (including liver, heart and clotting disorders) to elucidate how genes first identified in genome-wide association studies contribute to these disorders.
We and others have already been using zebrafish to study familial PD genes. We now want to use our expertise and the major advantages of zebrafish as a model for human diseases to study the PD GWA genes to better understand how they contribute to the risk of sporadic PD. Using a revolutionary new gene editing strategy called 'CRISPR/Cas', we have already made a range of zebrafish lines which carry mutations in 10 of the most important PD GWA risk genes. This pilot work will greatly accelerate the progress of our proposed research. As part of this, we will be studying the effect of these PD GWA risk genes on so-called 'global gene expression'. This involves quantifying how much the brain is using all of the genes in the genome and thereby getting an insight into which biological systems are over or under active. We will also investigate whether PD GWA risk genes interact with familial PD genes.
Our work will have a strong focus on the identification of "druggable" targets within these systems. In the future, we are planning to undertake a drug screen against these targets. This will hopefully help us to identify promising drugs which can then be taken into clinical trials for patients with PD.

Technical Summary

Genome wide association (GWA) studies have identified 24 risk loci for sporadic Parkinson's disease (PD). However, PD GWA loci often contain more than one gene. Furthermore, for most currently proposed PD GWA genes within the associated PD risk loci, the mechanisms linking them to PD are only tentative.
The aim of this proposal is to undertake a biological validation of plausible PD GWA risk genes in zebrafish (Danio rerio), using a CRISPR/Cas-based, in vivo pathway analysis approach.
Zebrafish offer unprecedented opportunities to rapidly study crucial aspects of PD GWA genes in a well characterised vertebrate model system. Zebrafish share greater sequence homology and are more likely to have functionally conserved mechanisms than other small model organisms. They also provide excellent opportunities to study the effect of GWA genes on a wide range of relevant mechanisms, including the 1. Dopaminergic neuronal activity in vivo, neurotransmitter homeostasis and loss of dopaminergic neurons; 2. Interaction between neuronal and non-neuronal cell death mechanisms (in particular neuroinflammation); 3. Interaction between PD GWA genes and ageing.
We will combine neurophysiological, neurochemical, morphological and functional analyses with comparative RNA-seq based transcriptomic studies. Using the CRISPR/Cas strategy, we have already made stable mutant zebrafish lines for the PD GWA risk genes mccc1, gak, inpp5f, vps13c, gch1, sipa1l2, srebf1, acmsd, stx1b and tmem175 as well as additional stable mutant lines for the familial PD genes PINK1, LRRK2, Parkin and ATP13A2. The immediate availability of these lines will greatly accelerate the progress of this proposal. We will particularly focus on three pathobiological mechanisms, namely mitochondrial function/mitophagy, the autophagy/lysosome system and neuroinflammation. We will also undertake an in vivo gene-gene interaction screen to investigate PD GWA genes for their functional interaction with familial PD genes.

Planned Impact

127,000 people are affected by PD in the UK alone (www.parkinsons.org.uk); its worldwide prevalence is predicted to double from 2005 to 2030 (Dorsey ER et al, Neurology. 2007; 68:384-6). Currently available treatment is only symptomatic, often only partially effective and can be associated with side-effects, in particular in the latter stages of the disease. The lack of disease-modifying treatment which would slow down or even halt disease progression is at least partially due to our still limited understanding of the mechanisms leading to PD. It has been estimated that any neuroprotective treatment that slowed disease progression by 20% could result in monetary benefits of £38,217 (or £47,815 including lost income) per patient (Johnson SJ et al, Mov Disord. 2013;28(3):319-26). Precise identification of PD risk genes, especially if combined with functional characterisation, network analysis and proposed druggable targets at crucial functional hubs, will be of major interest to other PD researchers and the pharmaceutical industry. Zebrafish are ideally suited for in vivo high throughput screens: several drugs identified in such screens have now been taken into clinical trials (MacRae and Peterson, Nat Rev Drug Discovery 2015; 14:721-731). We will use our previous experience in drug discovery to engage with both PD charities and other appropriate funding organisations (e.g. MRC-DPFS) as well as drug companies at the earliest opportunity to explore the possibility of undertaking an in vivo drug screen subsequent to the identification of robust, "druggable" targets. The strong focus of our work on the common sporadic form of PD will make this proposal particularly relevant to patients with PD as well as their families and carers.

Publications

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Chhabria K (2020) The effect of hyperglycemia on neurovascular coupling and cerebrovascular patterning in zebrafish. in Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism

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Larbalestier H (2022) GCH1 Deficiency Activates Brain Innate Immune Response and Impairs Tyrosine Hydroxylase Homeostasis. in The Journal of neuroscience : the official journal of the Society for Neuroscience

 
Description The effect of klotho deficiency on alpha-synuclein toxicity in a transgenic zebrafish model of Parkinson's disease
Amount £120,000 (GBP)
Organisation University of Sheffield 
Sector Academic/University
Country United Kingdom
Start 09/2019 
End 09/2022
 
Title gch1 mutant zebrafish line 
Description First lower vertebrate model of gch1 deficiency - heterozygous gch1 mutations are genetic risk factors for Parkinson's disease 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2021 
Provided To Others? Yes  
Impact Our work demonstrates that gch1 deficiency does not directly cause loss of dopaminergic neurons 
 
Description Functional analysis of microglia in PD mutant zebrafish 
Organisation University of Edinburgh
Department Edinburgh Neuroscience
Country United Kingdom 
Sector Academic/University 
PI Contribution We functionally characterized PD mutant zebrafish models and identified marked transcriptional changes indicating an important role of inflammation
Collaborator Contribution Our partners at the University of Edinburgh provided advice and hosted a PhD student in their lab. The student has now established these challenging methods in our own lab. The Edinburgh group continues to provide advice.
Impact None yet
Start Year 2018
 
Description LRRK2 kinase activity in lrrk2 mutant zebrafish 
Organisation University of Dundee
Department Biomedical Research Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution My group has made lrrk2 mutant zebrafish lines, applying CRISPR/Cas technology.
Collaborator Contribution Prof Dario Alessi's group is currently investigating whether these lrrk2 mutant zebrafish have altered kinase activity
Impact Experiments ongoing
Start Year 2022
 
Description Microglial activation in zebrafish models of PD 
Organisation University of Pittsburgh
Country United States 
Sector Academic/University 
PI Contribution we supplied our collaborator with mutant zebrafish
Collaborator Contribution Our collaborator Dr Ed Burton investigated microglial activation in these mutant zebrafish lines
Impact on-going but we hope to publish a scientific manuscript describing the results soon
Start Year 2016
 
Description Neurochemical analysis of neurotransmitters in zebrafish brains 
Organisation University of Helsinki
Country Finland 
Sector Academic/University 
PI Contribution we provide our collaborators with larval and adult tissue of zebrafish mutant lines
Collaborator Contribution Our collaborators undertake technically challenging quantification of dopamine, serotonine and their metabolites
Impact No publications or other outputs yet
Start Year 2018
 
Description Neurochemical analysis of zebrafish brains 
Organisation University College London
Department Institute of Neurology
Country United Kingdom 
Sector Academic/University 
PI Contribution we supplied mutant zebrafish material
Collaborator Contribution Prof Heales and his team undertook the neurotransmitter analysis
Impact No outputs yet but we're hoping to publish the results soon
Start Year 2016
 
Description collaboration with Prof Sherif El-Khamisy, University of Sheffield 
Organisation University of Sheffield
Department Department of Biomedical Science
Country United Kingdom 
Sector Academic/University 
PI Contribution We have developed considerable expertise in the use of zebrafish to study PD-related mechanisms.
Collaborator Contribution Prof El-Khamisy is an international expert in DNA repair. He and his team have contributed with expertise, experimental protocols, general advice on deficient DNA repair and its possible links with PD.
Impact No output yet
Start Year 2020
 
Description Gretchen Amphlett Lecture, Cambridge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact lecture and discussion about disease stratification in PD
Year(s) Of Engagement Activity 2018
 
Description Parkinson's Academy, Sheffield 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact excellent small meeting, focussed on increasing research engagement
Year(s) Of Engagement Activity 2018
 
Description Regional (Y/H) Parkinson's UK Research Update and engagement meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact talk to regional audience about our SITraN-based research activities
Year(s) Of Engagement Activity 2018
 
Description Talk at the SY/Humber younger PD patient support group annual meeting, Leeds, October 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact I gave a talk at the annual meeting of the South Yorkshire/Humber P-UK regional support group for younger PwP about our drug discovery work and drug trial. The talk was well received and met with considerable interest.
Year(s) Of Engagement Activity 2019