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1.  PINK1-Parkin Pathway Activity Is Regulated by Degradation of PINK1 in the Mitochondrial Matrix 
PLoS Genetics  2014;10(5):e1004279.
Loss-of-function mutations in PINK1, which encodes a mitochondrially targeted serine/threonine kinase, result in an early-onset heritable form of Parkinson's disease. Previous work has shown that PINK1 is constitutively degraded in healthy cells, but selectively accumulates on the surface of depolarized mitochondria, thereby initiating their autophagic degradation. Although PINK1 is known to be a cleavage target of several mitochondrial proteases, whether these proteases account for the constitutive degradation of PINK1 in healthy mitochondria remains unclear. To explore the mechanism by which PINK1 is degraded, we performed a screen for mitochondrial proteases that influence PINK1 abundance in the fruit fly Drosophila melanogaster. We found that genetic perturbations targeting the matrix-localized protease Lon caused dramatic accumulation of processed PINK1 species in several mitochondrial compartments, including the matrix. Knockdown of Lon did not decrease mitochondrial membrane potential or trigger activation of the mitochondrial unfolded protein stress response (UPRmt), indicating that PINK1 accumulation in Lon-deficient animals is not a secondary consequence of mitochondrial depolarization or the UPRmt. Moreover, the influence of Lon on PINK1 abundance was highly specific, as Lon inactivation had little or no effect on the abundance of other mitochondrial proteins. Further studies indicated that the processed forms of PINK1 that accumulate upon Lon inactivation are capable of activating the PINK1-Parkin pathway in vivo. Our findings thus suggest that Lon plays an essential role in regulating the PINK1-Parkin pathway by promoting the degradation of PINK1 in the matrix of healthy mitochondria.
Author Summary
Mitochondria are essential organelles that provide most of the cell's energy and perform many other critical functions. The gradual accumulation of defective mitochondria is thought to play a role in aging and in diseases of the nervous system, including Parkinson's disease. The selective elimination of defective mitochondria is therefore a vital task for the cell, and the protein PINK1 was recently identified as a critical player in this process. PINK1 accumulates on the surface of mitochondria after they are damaged, starting a process that leads ultimately to the elimination of defective mitochondria. Previous work indicated that PINK1 does not accumulate on healthy mitochondria because it is rapidly degraded. However, it was unclear exactly how and where this degradation occurred. Our work shows that Lon protease promotes the degradation of PINK1 in the mitochondrial matrix. This finding provides new insight into the mechanisms of mitochondrial quality control, and reveals a potential strategy for treating the many diseases associated with the accumulation of defective mitochondria.
doi:10.1371/journal.pgen.1004279
PMCID: PMC4038460  PMID: 24874806
2.  VCP is essential for mitochondrial quality control by PINK1/Parkin and this function is impaired by VCP mutations 
Neuron  2013;78(1):65-80.
Mutations in VCP cause multisystem degeneration impacting the nervous system, muscle, and/or bone. Patients may present with ALS, Parkinsonism, frontotemporal dementia, myopathy, Paget’s disease or a combination of these. The disease mechanism is unknown. We developed a Drosophila model of VCP mutation-dependent degeneration. The phenotype is reminiscent of PINK1 and parkin mutants, including a pronounced mitochondrial defect. Indeed, VCP interacts genetically with the PINK1/parkin pathway in vivo. Paradoxically, VCP complements PINK1 deficiency but not parkin deficiency. The basis of this paradox is resolved by mechanistic studies in vitro showing that VCP recruitment to damaged mitochondria requires Parkin-mediated ubiquitination of mitochondrial targets. VCP recruitment coincides temporally with mitochondrial fission, and VCP is required for proteasome-dependent degradation of Mitofusins in vitro and in vivo. Further, VCP and its adaptor Npl4/Ufd1 are required for clearance of damaged mitochondria via the PINK1/Parkin pathway, and this is impaired by pathogenic mutations in VCP.
doi:10.1016/j.neuron.2013.02.029
PMCID: PMC3683300  PMID: 23498974
3.  Surveillance mammography for detecting ipsilateral breast tumour recurrence and metachronous contralateral breast cancer: a systematic review 
European Radiology  2011;21(12):2484-2491.
Objectives
To determine the diagnostic accuracy of surveillance mammography for detecting ipsilateral breast tumour recurrence and metachronous contralateral breast cancer in women previously treated for primary breast cancer.
Methods
A systematic review of surveillance mammography compared with ultrasound, magnetic resonance imaging (MRI), specialist-led clinical examination or unstructured primary care follow-up, using histopathological assessment for test positives and follow-up for test negatives as the reference standard.
Results
Nine studies met our inclusion criteria. Variations in study comparisons precluded meta-analysis. For routine ipsilateral breast tumour detection, surveillance mammography sensitivity ranged from 64–67% and specificity ranged from 85–97%. For MRI, sensitivity ranged from 86–100% and specificity was 93%. For non-routine ipsilateral breast tumour detection, sensitivity and specificity for surveillance mammography ranged from 50–83% and 57–75% and for MRI 93–100% and 88–96%. For routine metachronous contralateral breast cancer detection, one study reported sensitivity of 67% and specificity of 50% for both surveillance mammography and MRI.
Conclusion
Although mammography is associated with high sensitivity and specificity, MRI is the most accurate test for detecting ipsilateral breast tumour recurrence and metachronous contralateral breast cancer in women previously treated for primary breast cancer. Results should be interpreted with caution because of the limited evidence base.
Key Points
• Surveillance mammography is associated with high sensitivity and specificity
• Findings suggest that MRI is the most accurate test for detecting further breast cancer
• Robust conclusions cannot be made due to the limited evidence base
• Further research comparing surveillance mammography and other diagnostic tests is required
doi:10.1007/s00330-011-2226-z
PMCID: PMC3217137  PMID: 21833567
Mammography; Surveillance; Diagnostic accuracy; Neoplasm recurrence, local; Neoplasm, second primary
4.  Using the theory of planned behaviour as a process evaluation tool in randomised trials of knowledge translation strategies: A case study from UK primary care 
Background
Randomised trials of knowledge translation strategies for professional behaviour change can provide robust estimates of effectiveness, but offer little insight into the causal mechanisms by which any change is produced. To illustrate the applicability of causal methods within randomised trials, we undertook a theory-based process evaluation study within an implementation trial to explore whether the cognitions of primary care doctors' predicted their test requesting behaviours and, secondly, whether the trial results were mediated by the theoretical constructs.
Methods
The process evaluation comprised a cross-sectional questionnaire survey of a random 50% sample of the randomised groups of primary care practices in Grampian (NHS Grampian), UK, who took part in a trial of the effect of enhanced feedback and brief educational reminders on test requesting behaviour. The process evaluation was based upon the Theory of Planned Behaviour and focussed on three of the test requesting behaviours that were targeted in the trial -- ferritin, follicle stimulating hormone (FSH), and Helicobacter Pylori serology (HPS).
Results
The questionnaire was completed by 131 primary care doctors (56%) from 42 (98%) of the sampled practices. Behavioural intention, attitude, and subjective norm were highly correlated for all the tests. There was no evidence that perceived behavioural control was correlated with any of the other measures. Simple linear regression analysis of the rate of test requests on minimum behavioural intentions had R2 of 11.1%, 12.5%, and 0.1% for ferritin, FSH, and HPS requesting, respectively. Mediational analysis showed that the trial results for ferritin and FSH were partially mediated (between 23% and 78% mediation) through intentions. The HPS trial result was not mediated through intention.
Conclusions
This study demonstrated that a theory-based process evaluation can provide useful information on causal mechanisms that aid not only interpretation of the trial but also inform future evaluations and intervention development.
doi:10.1186/1748-5908-5-71
PMCID: PMC2959079  PMID: 20920277
5.  The Mitochondrial Fusion-Promoting Factor Mitofusin Is a Substrate of the PINK1/Parkin Pathway 
PLoS ONE  2010;5(4):e10054.
Loss-of-function mutations in the PINK1 or parkin genes result in recessive heritable forms of parkinsonism. Genetic studies of Drosophila orthologs of PINK1 and parkin indicate that PINK1, a mitochondrially targeted serine/threonine kinase, acts upstream of Parkin, a cytosolic ubiquitin-protein ligase, to promote mitochondrial fragmentation, although the molecular mechanisms by which the PINK1/Parkin pathway promotes mitochondrial fragmentation are unknown. We tested the hypothesis that PINK1 and Parkin promote mitochondrial fragmentation by targeting core components of the mitochondrial morphogenesis machinery for ubiquitination. We report that the steady-state abundance of the mitochondrial fusion-promoting factor Mitofusin (dMfn) is inversely correlated with the activity of PINK1 and Parkin in Drosophila. We further report that dMfn is ubiquitinated in a PINK1- and Parkin-dependent fashion and that dMfn co-immunoprecipitates with Parkin. By contrast, perturbations of PINK1 or Parkin did not influence the steady-state abundance of the mitochondrial fission-promoting factor Drp1 or the mitochondrial fusion-promoting factor Opa1, or the subcellular distribution of Drp1. Our findings suggest that dMfn is a direct substrate of the PINK1/Parkin pathway and that the mitochondrial morphological alterations and tissue degeneration phenotypes that derive from mutations in PINK1 and parkin result at least in part from reduced ubiquitin-mediated turnover of dMfn.
doi:10.1371/journal.pone.0010054
PMCID: PMC2850930  PMID: 20383334

Results 1-6 (6)