Final Report Summary - DYNAMITO (The analysis of mitochondrial dynamics in ageing and neurodegeneration)
Mitochondria are central to the life and death of neurons. They provide the cellular energy required by these cells and protect them by buffering potentially lethal levels of cytoplasmic calcium. At the same time mitochondria produce much of the molecules that cause cellular damage and contain a lethal arsenal of apoptotic cell death machinery. These organelles require exquisite maintenance processes to keep them intact and prevent potentially catastrophic disruption. Failure in mitochondrial homeostasis is strongly linked to neurodegeneration.
Previous work has revealed that two genes linked to parkinsonism, PINK1 and parkin, act in common pathway to mediate the autophagic destruction of dysfunctional mitochondria as part of a quality control process. A major aim of this project was to analyse the mechanisms of PINK1/Parkin-mediated mitophagy in an in vivo setting. To achieve this, we have used the powerful genetic tools available for Drosophila. Using genetic approaches, we have have found that Fbxo7, mutations in which cause early onset parkinsonism, promotes mitochondrial homeostasis by participating in PINK1/Parkin-mediated mitophagy.
Cells with reduced Fbxo7 expression showed deficiencies in translocation of Parkin to mitochondria, 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. We also found a strong genetic link between Parkin and another PD gene vps35, providing the foundation for others to further this link showing a molecular interaction.
We have also conducted cell-based RNAi screens to find modulators of mitochondrial homeostasis. We have found that the SREBF1, a master regulator of lipogenesis and a risk factor for Parkinson’s disease, also plays a role in mitophagy. These findings further underscore the contribution of mitochondrial quality control in familial and sporadic Parkinson’s disease. In a separate study we screened for kinases and phosphatases that phenocopied loss of PINK1 we identified was the Complex I subunit NDUFA10. We demonstrated a genetic interaction between NDUFA10 and PINK1, and provided a mechanistic link to observations that PINK1 mutation causes altered Complex I activity.
Another major theme of this project was to analyse the impact of Parkinson’s disease genes on mitochondrial dynamics, and how mitochondrial dynamics impacts on neuronal survival. Axonal transport of mitochondria is known to be perturbed in many neurodegenerative diseases, so we investigated axonal transport deficits in models of Parkinson’s disease. We found that LRRK2 containing pathogenic Roc-COR domain mutations (R1441C, Y1699C) preferentially associates with deacetylated microtubules, and inhibits axonal transport in primary neurons and in Drosophila, causing locomotor deficits in vivo. In vitro, increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase alphaTAT1 prevents association of mutant LRRK2 with microtubules, and the deacetylase inhibitor trichostatin A (TSA) restores axonal transport. In vivo knockdown of the deacetylases HDAC6 and Sirt2, or administration of TSA rescues both axonal transport and locomotor behavior. Thus, these results revealed a pathogenic mechanism and a potential intervention for Parkinson’s disease.
Extending this methodology, we have also interrogated the extent to which axonal transport defects exist in models of ALS/FTD. We have found that some form of axonal transport deficit, whether of mitochondria or other cellular components, is a common feature in models of this disease. This places axonal transport as an important consideration for understanding and tackling this disease.
Previous work has revealed that two genes linked to parkinsonism, PINK1 and parkin, act in common pathway to mediate the autophagic destruction of dysfunctional mitochondria as part of a quality control process. A major aim of this project was to analyse the mechanisms of PINK1/Parkin-mediated mitophagy in an in vivo setting. To achieve this, we have used the powerful genetic tools available for Drosophila. Using genetic approaches, we have have found that Fbxo7, mutations in which cause early onset parkinsonism, promotes mitochondrial homeostasis by participating in PINK1/Parkin-mediated mitophagy.
Cells with reduced Fbxo7 expression showed deficiencies in translocation of Parkin to mitochondria, 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. We also found a strong genetic link between Parkin and another PD gene vps35, providing the foundation for others to further this link showing a molecular interaction.
We have also conducted cell-based RNAi screens to find modulators of mitochondrial homeostasis. We have found that the SREBF1, a master regulator of lipogenesis and a risk factor for Parkinson’s disease, also plays a role in mitophagy. These findings further underscore the contribution of mitochondrial quality control in familial and sporadic Parkinson’s disease. In a separate study we screened for kinases and phosphatases that phenocopied loss of PINK1 we identified was the Complex I subunit NDUFA10. We demonstrated a genetic interaction between NDUFA10 and PINK1, and provided a mechanistic link to observations that PINK1 mutation causes altered Complex I activity.
Another major theme of this project was to analyse the impact of Parkinson’s disease genes on mitochondrial dynamics, and how mitochondrial dynamics impacts on neuronal survival. Axonal transport of mitochondria is known to be perturbed in many neurodegenerative diseases, so we investigated axonal transport deficits in models of Parkinson’s disease. We found that LRRK2 containing pathogenic Roc-COR domain mutations (R1441C, Y1699C) preferentially associates with deacetylated microtubules, and inhibits axonal transport in primary neurons and in Drosophila, causing locomotor deficits in vivo. In vitro, increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase alphaTAT1 prevents association of mutant LRRK2 with microtubules, and the deacetylase inhibitor trichostatin A (TSA) restores axonal transport. In vivo knockdown of the deacetylases HDAC6 and Sirt2, or administration of TSA rescues both axonal transport and locomotor behavior. Thus, these results revealed a pathogenic mechanism and a potential intervention for Parkinson’s disease.
Extending this methodology, we have also interrogated the extent to which axonal transport defects exist in models of ALS/FTD. We have found that some form of axonal transport deficit, whether of mitochondria or other cellular components, is a common feature in models of this disease. This places axonal transport as an important consideration for understanding and tackling this disease.