Table 1. Genotypic and phenotypic characterisation of investigated individuals.

<p>Table 1. Genotypic and phenotypic characterisation of investigated individuals.</p

Mitochondrial function.

<p>(A) Basal ATP synthesis rates. The assay demonstrated a significant reduction in ATP production in the <i>Parkin</i>-mutant patients (median [IQR]: 39% [23%, 55%]) compared to controls (set to 100%). (B) Basal ATP levels. Quantifying the overall cellular ATP concentration showed significantly lower levels in the mutants (median [IQR]: 69% [58%, 75%]) than in controls (set to 100%). (C) Mitochondrial membrane potential under basal and oxidative stress conditions. Control (median [IQR]: 100% [94%, 115%]) and patient fibroblasts (median [IQR]: 113% [93%, 128%]) with <i>Parkin</i> mutations show similar membrane potential under basal conditions. When the cells were treated with paraquat (shaded boxes), no relevant changes were detected in the controls (median [IQR]: 102% [100%, 118%]). In the <i>Parkin</i> mutants, a significant drop of the membrane potential was observed (median [IQR]: 84% [81%, 103%]). The median, the interquartile range (IQR), the minimum and the maximum value of 6 (A) or 4 (B) independent experimental runs are plotted. In each experimental run the average ATP level in the controls was set to 100%.</p

Protein oxidation under basal and stress conditions.

<p>Oxidation of proteins in <i>Parkin</i>-mutant fibroblasts and controls was determined by means of an OxyBlot. (A) When quantifying the protein oxidation in each individual using an antibody against DNP, the <i>Parkin</i> mutants (median [IQR]: 123% [113%, 136%]) showed significantly higher levels of oxidation than the controls (median [IQR]: 100% [97%, 105%]). After treatment of the cells with paraquat (shaded boxes), the difference in oxidation between mutants (median [IQR]: 131% [96%, 172%]) and controls (median [IQR]: 100% [90%, 102%]) increased, but was no longer significant. Equal protein loading was verified with an antibody against β-actin. Expression ratios of oxidized proteins vs. β-actin were calculated. The median, the interquartile range (IQR), the minimum and the maximum value of the investigated groups of individuals are given. (B) OxyBlot of pooled protein samples before and after paraquat treatment showing a similar trend as identified by individual measurements.</p

Morphology of the mitochondrial network.

<p>(A) Images of the mitochondrial network in control and patient fibroblasts demonstrating similar degrees of branching under basal culturing conditions. (B) After treatment with paraquat, the network was less branched in patients and controls. (C) The degree of mitochondrial branching (form factor) was comparable in patients (median [IQR]: 78% [66%, 90%]) and controls (median [IQR]: 100% [73%, 105%]) under standard cell culturing conditions. When treated with paraquat (shaded boxes), the form factor decreased significantly in the mutant samples (median [IQR]: 46% [43%, 54%]). By contrast, a drop seen in controls (median [IQR]: 70% [32%, 84%]) was not significant. (D) Citrate synthase activity in cell lysates. <i>Parkin</i> mutants (median [IQR]: 183% [125%, 232%]) showed significantly higher citrate synthase activities than controls (median [IQR]: 100% [43%, 101%]), indicative of increased mitochondrial mass per cell in the former. Citrate synthase activity in cell lysates was normalized for protein concentration. The median, the interquartile range (IQR), the minimum and the maximum value of the investigated groups of individuals are shown.</p

Profiling of Parkin-Binding Partners Using Tandem Affinity Purification

<div><p>Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting approximately 1–2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the <i>Parkin</i> gene (<i>PARK2</i>) are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types (HEK293T and SH-SY5Y neuronal cells), we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from <i>Drosophila melanogaster</i>. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function.</p></div

Results from genome-wide analyses of two traits.

<p>Plots show the likelihood ratio test (LRT) and regional, genomic and total heritabilities across the genome from analyses of data from Croatian and Italian populations: a) serum uric acid concentration and b) height. Vertical axis is the LRT and heritability (%) and horizontal axis is window number across the genome. RG h2, WG h2 and total h2 are regional heritability, residual whole genome heritability and total (sum of genomic and regional) heritability, respectively.</p

Comparison of single marker analysis and regional heritability analyses of serum uric acid concentration in a population from Orkney.

<p>-log P values are plotted against position in the genome. Points represent individual markers and circles results from regional heritability analysis with 100 marker windows. Genome-wide significance thresholds are represented by dashed lines (red for single marker analysis, blue for regional heritability analysis). Alternating shades represent the separate chromosomes. Results surpassing the genome-wide significance threshold are solid red for single marker analysis and solid blue for regional heritability analysis.</p

GO biological processes enriched in ParkinTAP and RelatedPD datasets.

<p>GO biological processes enriched in ParkinTAP and RelatedPD datasets.</p

Regional heritability using single and multiple regional relationship matrices (100 SNPs) for Height.

<p>(LRT from all windows were significant at suggestive level).</p>1<p>Likelihood ratio test for regional heritability >0;</p>2<p>Estimated regional heritability in model with that single region and genomic effects;</p>3<p>Minimum heritability for that region from models with sets of 5 regional effects and genomic effect;</p>4<p>Maximum heritability for that region from models with sets of 5 regional effects and genomic effect;</p>5<p>Average heritability for that region over models with sets of 5 regional effects and genomic effect.</p

Summary of results for ParkinTAP candidates passing the first seven selection levels.

<p>Summary of results for ParkinTAP candidates passing the first seven selection levels.</p