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.
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 evaluate 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.
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 evaluate 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.