Identification of new components in Parkinson's Disease signalling pathways

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

Abstract

Our knowledge of the molecular events leading to Parkinson‘s disease (PD) is being greatly enhanced by the study of familial forms of the disease. Professor Wood‘s laboratory has identified a gene (PINK1) which when mutated causes nerve cell death and ultimately PD. Moreover, point mutations in a second gene called HtrA2 have also recently been identified as a susceptibility factor for PD. One of the first questions which arise is; do these two molecules interact either together or in the same pathway or process to produce PD? We have very recently established that they not only interact in the same pathway but that PINK1 regulates the activity of HtrA2. However much more work needs to be done to understand these processes more fully. This application focuses on this pathway and uses a range of molecular biological and cellular techniques to identify other proteins involved in this process. We plan to assess how these proteins talk to each other and how important this crosstalk is in the development of the disease. To this end we have already developed the core resources of appropriate cell models and have identified human brain tissue carrying abnormalities in the PINK1 gene. Moreover, we will have access to mice and flies in which HtrA2 and PINK1 genes have been disrupted.

If we can indeed discover more of the pathway to cell dysfunction and death, then this in turn provides more options for therapeutic intervention.

Technical Summary

The identification of genes responsible for mendelian forms of Parkinson‘s disease (PD) has transformed our understanding of the molecular pathogenesis of nigral neurodegeneration. Whether these mendelian genes interact, and which signalling pathways are disrupted in PD remain unknown. The aim of this project is to characterise a molecular pathway that appears to be of major significance in PD.

Mutations in the PINK1 gene are known to cause PD. PINK1 is a mitochondrial putative kinase which exerts a neuroprotective function. Mutations in HtrA2, a mitochondrial serine protease, have also been identified in patients with PD. Targeted deletion of HtrA2 in mice causes mitochondrial dysfunction leading to a neurodegenerative disorder with parkinsonian features. We have shown for the first time that HtrA2 interacts with PINK1 and both are components of the same stress-sensing pathway. Phosphorylation of HtrA2 takes place in a PINK1 dependent manner and HtrA2 phosphorylation is decreased in brains of PD patients carrying PINK1 mutations. We hypothesise that PINK1-dependent phosphorylation of HtrA2 may modulate its proteolytic activity, thereby contributing to increased resistance of neurons to mitochondrial stress.

A range of techniques will be employed in a variety of in vitro and in vivo models to dissect the HtrA2/PINK1 pathway. The project has four major objectives:
- To determine the functional effect of specific PD-associated PINK1 mutations on HtrA2 phosphorylation. This will be studied using human post-mortem brain with PINK1 mutations, and PINK1 knockout mouse cells re-expressing mutant forms of PINK1.
- To identify other members of the HtrA2/PINK1 signalling pathway. Expression of a tandem affinity purification (TAP)-tagged version of HtrA2 in HEK293 cells has enabled the co-purification of PINK1 as well as other putative interacting partners. A combination of phospho-proteomic approaches and biological assays using RNAi technology will be used to aluate the relevance of other interactors within the same pathway.
- To elucidate the functional interaction between the HtrA2/PINK1 pathway and other mendelian genes, in particular parkin. The HtrA2, PINK1 and parkin knockout flies and mice will be used to explore the proposed functional link between PINK1 and parkin.
- To investigate whether mutations in other members of this pathway are responsible for familial PD. The IoN has over 100 familial PD cases with as yet unidentified gene defects. Functional data emerging from these studies will be used in a candidate gene discovery approach to identify new mendelian forms of PD.

Characterisation of this signalling pathway will have a major impact on our understanding of the pathogenesis of sporadic PD, and will ultimately drive target discovery for developing new therapies in PD.

Publications


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Burchell VS (2010) Targeting mitochondrial dysfunction in neurodegenerative disease: Part II. in Expert opinion on therapeutic targets
Burchell VS (2010) Targeting mitochondrial dysfunction in neurodegenerative disease: Part I. in Expert opinion on therapeutic targets
Deas E (2011) Mitophagy and Parkinson's disease: the PINK1-parkin link. in Biochimica et biophysica acta
Deas E (2009) PINK1 function in health and disease. in EMBO molecular medicine
Devine MJ (2011) Parkinson's disease and cancer: two wars, one front. in Nature reviews. Cancer
 
Description Identification of new components in HtrA2/PINK1 signallimg pathways/MRC/BRT studentship for Victoria Burchell
Amount £91,389 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 09/2008 
End 09/2011
 
Description Investigating the functioinal significance between PINK1 and mortalin - two genes implicated in Parkinson's disease
Amount £9,058,020 (GBP)
Organisation Parkinson's UK (Parkinson's Disease Society) 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 03/2011 
End 03/2014
 
Description UCL Central Research Committee (Phosphorylation of HtrA2 by CDK5- Implications in Parkinson's Disease.)
Amount £65,600 (GBP)
Organisation University College London (UCL) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 04/2008 
End 04/2010
 
Description Understanding LRRK2 biology grant: Investigating signaling dysfunction linked to LRRK2
Amount $390,000 (USD)
Organisation Michael J Fox Foundation 
Sector Charity/Non Profit
Country United States of America
Start 10/2010 
End 10/2012
 
Description Understanding Parkinson's disease, lessons from biology/Wellcome/MRC neurodegenerative disease strategic awards
Amount £5,300,000 (GBP)
Organisation The Wellcome Trust Ltd 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 09/2010 
End 09/2015
 
Title Human patient fibroblasts 
Description Homozygous Parkin fibroblasts 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2011 
Provided To Others? Yes  
Impact work in progress 
 
Title PINK1, parkin cell lines 
Description PINK1 and parkin knockdown SH-SY5Y cells SH-SY5Y cells over-expressing PINK1 and parkin Parkinson's disease mutants 
Type Of Material Cell line 
Provided To Others? No  
Impact see publications 
 
Title Phospho-Ser400 HtrA2 antibody 
Description Rabbit polyclonal antibody Application: Western blot 
Type Of Material Antibody 
Year Produced 2010 
Provided To Others? Yes  
Impact Fitzgerald JC, Camprubi MD, Dunn L, Wu HC, Ip NY, Kruger R, Martins LM, Wood NW, Plun-Favreau H. Cell Death Differ. 2011 Jun 24. 
 
Description Investigating signaling pathway dysfunction linked to LRRK2 
Organisation University College London (UCL)
Department UCL Institute of Neurology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Design of the experiments
Collaborator Contribution Contribution to experiments
Impact PMID: 27731364 PMID: 24211199 PMID: 23916833
Start Year 2009
 
Description PDUK: Understanding Parkinson's disease, lessons from biology 
Organisation Medical Research Council (MRC)
Department MRC Centre for Developmental and Biomedical Genetics
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Investigating the functions of the new Parkinson's disease genes
Collaborator Contribution Experimental workExperimental work
Impact PMID: 22739987 PMID: 23498975 PMID: 23933751 PMID: 26893374 PMID: 27689878
Start Year 2010
 
Description PDUK: Understanding Parkinson's disease, lessons from biology 
Organisation Medical Research Council (MRC)
Department MRC Protein Phosphorylation Unit
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Investigating the functions of the new Parkinson's disease genes
Collaborator Contribution Experimental workExperimental work
Impact PMID: 22739987 PMID: 23498975 PMID: 23933751 PMID: 26893374 PMID: 27689878
Start Year 2010
 
Description PDUK: Understanding Parkinson's disease, lessons from biology 
Organisation University College London (UCL)
Department UCL Institute of Neurology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Investigating the functions of the new Parkinson's disease genes
Collaborator Contribution Experimental workExperimental work
Impact PMID: 22739987 PMID: 23498975 PMID: 23933751 PMID: 26893374 PMID: 27689878
Start Year 2010
 
Description The Parkinson's disease-linked proteins Fbxo7 and Parkin interact to mediate mitophagy 
Organisation University of Cambridge
Department Department of Genetics
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Compelling evidence indicates that two autosomal recessive Parkinson's disease genes, PINK1 (PARK6) and Parkin (PARK2), cooperate to mediate the autophagic clearance of damaged mitochondria (mitophagy). Mutations in the F-box domain-containing protein Fbxo7 (encoded by PARK15) also cause early-onset autosomal recessive Parkinson's disease, by an unknown mechanism. Here we show that Fbxo7 participates in mitochondrial maintenance through direct interaction with PINK1 and Parkin and acts in Parkin-mediated mitophagy. Cells with reduced Fbxo7 expression showed deficiencies in translocation of Parkin to mitochondria, ubiquitination of mitofusin 1 and mitophagy. In Drosophila, ectopic overexpression of Fbxo7 rescued loss of Parkin, supporting a functional relationship between the two proteins. Parkinson's disease-causing mutations in Fbxo7 interfered with this process, emphasizing the importance of mitochondrial dysfunction in Parkinson's disease pathogenesis.
Collaborator Contribution In vivo work in flies with Sheffield university (Alex Whitworth) Biochemistry with Cambridge university (Heike Laman)
Impact PMID: 23933751 PMID: 27689878
Start Year 2011
 
Description The Parkinson's disease-linked proteins Fbxo7 and Parkin interact to mediate mitophagy 
Organisation University of Sheffield
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Compelling evidence indicates that two autosomal recessive Parkinson's disease genes, PINK1 (PARK6) and Parkin (PARK2), cooperate to mediate the autophagic clearance of damaged mitochondria (mitophagy). Mutations in the F-box domain-containing protein Fbxo7 (encoded by PARK15) also cause early-onset autosomal recessive Parkinson's disease, by an unknown mechanism. Here we show that Fbxo7 participates in mitochondrial maintenance through direct interaction with PINK1 and Parkin and acts in Parkin-mediated mitophagy. Cells with reduced Fbxo7 expression showed deficiencies in translocation of Parkin to mitochondria, ubiquitination of mitofusin 1 and mitophagy. In Drosophila, ectopic overexpression of Fbxo7 rescued loss of Parkin, supporting a functional relationship between the two proteins. Parkinson's disease-causing mutations in Fbxo7 interfered with this process, emphasizing the importance of mitochondrial dysfunction in Parkinson's disease pathogenesis.
Collaborator Contribution In vivo work in flies with Sheffield university (Alex Whitworth) Biochemistry with Cambridge university (Heike Laman)
Impact PMID: 23933751 PMID: 27689878
Start Year 2011