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Chaperoning Drp1 mediated fission in neurons

Reference	BB/L02294X/1
Principal Investigator / Supervisor	Professor Paul Chapple
Co-Investigators / Co-Supervisors
Institution	Queen Mary University of London
Department	William Harvey Research Institute
Funding type	Research
Value (£)	324,057
Status	Completed
Type	Research Grant
Start date	28/10/2014
End date	27/10/2017
Duration	36 months

Abstract

Cells have quality control mechanisms that selectively remove damaged mitochondrial proteins or parts of the mitochondrial network. At the organelle level fission, mediated by dynamin-related protein-1 (Drp1), is critical to the segregation and degradation of dysfunctional mitochondria. Fission is also required for division of the mitochondrial network into transportable units. Neurons distribute mitochondria to match high-energy demands at sites away from the cell body. Thus perturbation of mitochondrial fission is detrimental to neuronal survival. Molecular chaperones are key modulators of protein quality control, folding and complex assembly. The ataxia protein sacsin has a modular structure with domains linking it to chaperone systems. These include a J domain, indicating sacsin functions with an Hsp70 partner. We have localised sacsin to mitochondria and shown that it interacts with Drp1. Loss of sacsin leads to a more interconnected and functionally impaired mitochondrial network. Furthermore, mitochondria accumulate in the soma of sacsin knockdown neurons. These data indicate a role for sacsin in mitochondrial dynamics. This is supported by evidence that loss of sacsin reduces Drp1 recruitment to mitochondria. Moreover, Drp1 facilitates peroxisome fission and in agreement with sacsin functioning in the same pathway as Drp1 changes in peroxisome incidence occurs in sacsin null cells. Cumulatively these findings are consistent with sacsin bringing chaperone action to Drp1 mediated fission. Defining the consequences of loss of sacsin for Drp1 recruitment, stabilisation and activation at sites of fission will test this hypothesis. This will be done in conjunction with identifying how sacsin functions with the Hsp70 machinery and dynactin-6, a protein that preliminary data suggests is part of a sacsin:Drp1 complex. This research will expand knowledge of Drp1 mediated fission and may identify a novel role for chaperones in mitochondrial regulation.

Summary

Problems with mitochondria, the cellular 'power plants', have recently been linked to multiple neurodegenerative diseases. Cells need energy to function and mitochondria convert energy into a form that can fuel cellular processes. Because of this and other important cellular roles healthy mitochondria are essential. In cells, mitochondria undergo continuous cycles of division (fission) and fusion that regulate their organisation into networks. This dynamic nature is key to regulating mitochondrial function, maintaining healthy mitochondria and removing damaged parts of the mitochondrial network. This is particularly important in neurons because if mitochondria are the wrong size they cannot be effectively transported along thin branching dendrites. Indeed, there is significant evidence that structural and functional abnormalities in mitochondria are involved in ageing and age-related neurodegenerative diseases, such as Alzheimer's, Parkinson's, Huntington's and amyotrophic lateral sclerosis. Given this context it is essential that we understand the mechanisms that regulate mitochondrial fission in neurons. The principle driver of this process has been identified as dynamin related protein-1 (Drp1). For fission to occur Drp1 must be recruited to future sites of mitochondrial division where it polymerises into spirals that act to constrict and pinch off parts of the network. In mammalian cells the recruitment and activation of Drp1 is not fully understood. We have identified an interaction between Drp1 and another protein called sacsin. Mutations in sacsin cause the inherited neurodegenerative disease Autosomal Spastic Ataxia of Charlevoix Saguenay (ARSACS). Defects in mitochondrial dynamics and function are features of ARSACS with loss of sacsin causing mitochondrial networks to become more interconnected. Moreover, cells lacking sacsin had a reduced incidence of Drp1 associated with their mitochondria even after fission is induced. Drp1 also functions in biogenesis of another organelle, the peroxisomes. Consistent with sacsin functioning in a common pathway with Drp1 we observed loss of sacsin correlates with a decrease in peroxisome number. Sacsin is one of the largest proteins ever identified and contains a number of regions with homology to other proteins of known function. These include domains that are found in molecular chaperone and cochaperone proteins. Chaperones and their cochaperone partners work together in the folding of other proteins and assembly of protein complexes. The presence of a J-domain in sacsin indicates that it functions with an Hsp70 protein, potentially recruiting Hsp70 action to a specific cellular role. Together these data suggest a requirement for chaperone activity in Drp1 mediated fission. To comprehend how Drp1 mediated fission is modulated by sacsin we looked for additional proteins that interact with sacsin. This identified dynactin-6, which has been suggested to play a role in mitochondrial biogenesis and is part of a complex involved in transport of Drp1 to mitochondria. We found dynactin-6 and Drp1 also interact and that reducing dynactin-6 levels in cells lead to a more interconnected mitochondrial network while increasing Drp1 levels caused mitochondrial fragmentation. In combination these data are consistent with Drp1 mediated fission being modulated by a novel protein complex that utilises Hsp70 chaperone action. We hypothesise that sacsin represents the core component or scaffold of such a complex, which is required for Drp1 to be fully active in neurons. The research outlined in this proposal will test these concepts through a comprehensive series of experiments designed to identify how sacsin and dynactin-6 function in Drp1 mediated fission. The main outcome of this research will be the elucidation of the cellular mechanisms controlling mitochondrial fission and the requirement for molecular chaperone action.

Impact Summary

This is a basic science project that will have the generation of new knowledge and scientific advancement as its most significant immediate impacts. The research will deliver a comprehensive analysis of the function of the molecular chaperone domain protein sacsin and its interacting partners in mitochondrial dynamics. It will test our hypothesis that sacsin represents the central component of a molecular chaperone complex involved in regulation of mitochondrial fission. Conventional academic publication in high quality open access journals and presentation at conferences will be the main methods of ensuring impact within the research community. Our previous research on the cellular function of sacsin has been disseminated by these means and a key objective of this work will be to publish results in years 2 and 3. The potential long-term medical and scientific opportunities are good, as manipulation of molecular chaperone systems and cellular protein homeostasis networks is becoming recognised as a strategy for intervention in human diseases and in particular neurodegenerations. The manipulation of mitochondrial dynamics is another potential therapeutic target for some diseases and it is therefore important to fully understand the biology of these systems. For example, pharmacological inhibition of mitochondrial fission has been reported to protect the heart against ischemia/reperfusion injury. Moreover, it has been hypothesised that controlling mitochondrial oxidant damage may slow the ageing process, again indicating the potential impact of understanding systems involved in quality control of mitochondria. Therefore, beyond academic beneficiaries, this research may ultimately lead to societal and economic impacts related to the development of novel disease treatment. Any commercial potential would be maximised by utilising the QMUL Innovation and Enterprise Unit and BBSRC commercialisation and development opportunities. Regular meetings with representatives fromthe QMUL Innovation and Enterprise Unit will be scheduled to identify such opportunities. This project will also have impact in the area of training and skills development for the named postdoctoral research assistant that will work on the project.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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