Investigation of the mitochondrial functions of proteins genetically associated with Parkinson's Disease

Parkinson’s disease (PD) is a common neurodegenerative disorder which usually occurs sporadically, but in 5-10% of cases is genetically inherited. Many of the causative mutations underlying these familial forms have been identified, and studying the functions of the proteins encoded by these genes has highlighted several common pathogenic mechanisms. In particular, mitochondrial dysfunction has been shown to play a major role in the pathogenesis of both familial and sporadic forms of the disease. This thesis investigates the importance of the proteins encoded by these PD-associated genes in mitochondrial function, focusing on two proteins in detail. Firstly, live cell imaging techniques were used to investigate the mitochondrial physiology of cells derived from HtrA2 knockout mice, an animal model in which the loss of a mitochondrial protein gives rise to severe neurodegenerative phenotype. Similar approaches were then applied to an RNAi screen to investigate the effects of other PD-associated genes on mitochondrial function, while a separate study specifically investigated the putative mitochondrial localisation and function of the PD-associated protein Fbxo7. Results from these studies revealed that HtrA2 has an important role in maintaining the function of the ATP synthase, as HtrA2 deficient cells exhibited a severe uncoupling combined with an increase in proton translocation through the ATP synthase but a reduction in ATP synthesis. Furthermore, Fbxo7, a protein with no reported link to the mitochondria, was found to partially localise to the mitochondria under basal conditions and to further accumulate on depolarised mitochondria. Further work indicated that this protein interacts with two other PD proteins, PINK1 and Parkin, and together with these proteins functions in a previously described pathway to mediate the selective autophagic clearance of damaged mitochondria. These results contribute to our understanding of the functions of these proteins and further emphasise the relevance of mitochondrial dysfunction in PD pathogenesis

Cell metabolism affects selective vulnerability in PINK1-associated Parkinson's disease

Mitochondrial dysfunction plays a primary role in the pathogenesis of Parkinson's disease (PD), particularly in autosomal recessive forms of the disease caused by mutations encoding PINK1. Although mitochondrial pathology can be demonstrated in many cell types, it is neurons that bear the brunt of cell death in PD. We studied the mitochondrial physiology of neurons and muscle cells with loss of function of the nuclear encoded mitochondrial protein PINK1. PINK1 is widely expressed in many types of tissues, but deficiency selectively induces death in neurons. We report here that the same genetic defect results in opposing phenotypes in different cell types, depending on the metabolic properties of the cell. Thus, PINK1-deficient myocytes exhibit high basal mitochondrial membrane potential (Δψm), whereas PINK1-deficient neurons have been shown to exhibit a low Δψm. PINK1 deficiency induces impaired respiration in both cell types, with a concomitant increase in glycolytic activity. We demonstrate that the high glycolytic capacity in myocytes compared with neurons enables them to produce more ATP and, therefore, compensates for the metabolic defects induced by PINK1 deficiency. Furthermore, the high Δψm generated in PINK1 knockout (KO) muscle mitochondria enables them to buffer cytosolic Ca2+ fluxes, rendering them resistant to Ca2+ stress effectively. Conversely, PINK1 KO neurons were previously shown to develop mitochondrial Ca2+ overload and Ca2+-induced mitochondrial depolarisation. Prevention of Ca2+ dysregulation in myocytes might therefore account for the sparing of these cells in PD

High Throughput Microplate Respiratory Measurements Using Minimal Quantities Of Isolated Mitochondria

Recently developed technologies have enabled multi-well measurement of O2 consumption, facilitating the rate of mitochondrial research, particularly regarding the mechanism of action of drugs and proteins that modulate metabolism. Among these technologies, the Seahorse XF24 Analyzer was designed for use with intact cells attached in a monolayer to a multi-well tissue culture plate. In order to have a high throughput assay system in which both energy demand and substrate availability can be tightly controlled, we have developed a protocol to expand the application of the XF24 Analyzer to include isolated mitochondria. Acquisition of optimal rates requires assay conditions that are unexpectedly distinct from those of conventional polarography. The optimized conditions, derived from experiments with isolated mouse liver mitochondria, allow multi-well assessment of rates of respiration and proton production by mitochondria attached to the bottom of the XF assay plate, and require extremely small quantities of material (1–10 µg of mitochondrial protein per well). Sequential measurement of basal, State 3, State 4, and uncoupler-stimulated respiration can be made in each well through additions of reagents from the injection ports. We describe optimization and validation of this technique using isolated mouse liver and rat heart mitochondria, and apply the approach to discover that inclusion of phosphatase inhibitors in the preparation of the heart mitochondria results in a specific decrease in rates of Complex I-dependent respiration. We believe this new technique will be particularly useful for drug screening and for generating previously unobtainable respiratory data on small mitochondrial samples

Dopamine Induced Neurodegeneration in a PINK1 Model of Parkinson's Disease

Parkinson's disease is a common neurodegenerative disease characterised by progressive loss of dopaminergic neurons, leading to dopamine depletion in the striatum. Mutations in the PINK1 gene cause an autosomal recessive form of Parkinson's disease. Loss of PINK1 function causes mitochondrial dysfunction, increased reactive oxygen species production and calcium dysregulation, which increases susceptibility to neuronal death in Parkinson's disease. The basis of neuronal vulnerability to dopamine in Parkinson's disease is not well understood

LRRK2 at the interface of autophagosomes, endosomes and lysosomes

Over the past 20 years, substantial progress has been made in identifying the underlying genetics of Parkinson’s disease (PD). Of the known genes, LRRK2 is a major genetic contributor to PD. However, the exact function of LRRK2 remains to be elucidated. In this review, we discuss how familial forms of PD have led us to hypothesize that alterations in endomembrane trafficking play a role in the pathobiology of PD. We will discuss the major observations that have been made to elucidate the role of LRRK2 in particular, including LRRK2 animal models and high-throughput proteomics approaches. Taken together, these studies strongly support a role of LRRK2 in vesicular dynamics. We also propose that targeting these pathways may not only be beneficial for developing therapeutics for LRRK2-driven PD, but also for other familial and sporadic cases