Comparison of ∼110-kDa complex formation between wild-type (WT) parkin and PD-linked parkin variants

<p><b>Copyright information:</b></p><p>Taken from "Parkin occurs in a stable, non-covalent, ∼110-kDa complex in brain"</p><p></p><p>The European Journal of Neuroscience 2008;27(2):284-293.</p><p>Published online Jan 2008</p><p>PMCID:PMC2253705.</p><p>© The Authors (2007). Journal Compilation © Federation of European Neuroscience Societies and Blackwell Publishing Ltd</p> COS1 cells were transiently transfected with 70 ng/cm of WT or R256C mutant parkin cDNA, 50 ng/cm of A82E or K161N parkin cDNA, 30 ng/cm of K211N cDNA and 120 ng/cm of R275W cDNA. (A) Transfected COS1 cells were extracted in 1% Triton X-100, followed by glycerol gradient centrifugation of the extracts. Fractions 2–6 of the gradient were analysed by BN-PAGE and parkin immunoblotting to visualize monomer (arrowhead) and ∼110-kDa complex (arrow). (B) In the experiments shown in (A), the amounts of parkin complex and parkin monomer were quantified. The graph represents the amount of parkin complex, expressed as a percentage of the sum of parkin complex and monomer. Asterisks denote significant difference ( < 0.05) from WT. (C and D) In parallel with each of the experiments shown in (A and B), 15 µg of Triton-soluble protein extract was analysed with SDS–PAGE and parkin immunoblotting to compare soluble parkin protein levels between WT and mutant variants. At the low signal intensity of the blots shown in (C), parkin appeared as a doublet due to the presence of an N-terminally truncated parkin species generated through an internal translation initiation site (). No endogenous parkin signal could be observed by SDS–PAGE in untransfected (Untransf.) COS1 cells (C), except after very prolonged film exposures (not shown). The graph in (D) shows SDS–PAGE and Western blot quantification of the Triton-soluble parkin levels. Within each experiment, the level of the parkin mutants was normalized to that of WT parkin. There were no significant differences in soluble parkin levels between WT, A82E, K161N, K211N or R256C ( = 0.96 by one-way )

BN-PAGE reveals a stable, non-covalent, ∼110-kDa parkin complex in brain

<p><b>Copyright information:</b></p><p>Taken from "Parkin occurs in a stable, non-covalent, ∼110-kDa complex in brain"</p><p></p><p>The European Journal of Neuroscience 2008;27(2):284-293.</p><p>Published online Jan 2008</p><p>PMCID:PMC2253705.</p><p>© The Authors (2007). Journal Compilation © Federation of European Neuroscience Societies and Blackwell Publishing Ltd</p> (A and B) Extracts of brain stem and diencephalon from wild-type and parkin knockout (KO) mice were separated into pellet (P) and supernatant (S) fractions, as described in Materials and methods and . P and S fractions were further fractionated by glycerol gradient centrifugation. Twelve P and 12 S fractions (numbered from the top of the glycerol gradient to the bottom) were analysed by BN-PAGE, followed by Western blotting with either the polyclonal anti-parkin antibody CS2132 (A) or the monoclonal anti-parkin antibody PRK109 (B) The CS2132 antibody detected a 450–550-kDa band in fraction S6, which was also found in parkin-null extract (A). By contrast, the PRK109 antibody (B) revealed a band in fraction S5 at a lower molecular weight (indicated by the arrow), which was absent in parkin knockout brain. The PRK109 antibody also showed some minor, non-specific immunoreactivity at higher molecular weights (indicated by the asterisk) in S5 from both wild-type and parkin knockout. (C) The graph shows how BN gels were calibrated based on the relative mobilities of native protein size markers to determine the molecular weight (MW) of the parkin complex. Markers, denoted by black circles, were thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa) and bovine serum albumin (67 kDa). The dotted line indicates the estimated MW of the parkin complex. (D) In the left and middle panels, heating brain fraction S5 at 100 °C in 1.5% sodium dodecyl sulphate (SDS) for 10 min immediately prior to BN-PAGE disrupted the ∼110-kDa parkin complex and led to the appearance of monomeric parkin (indicated by the arrow). In the rightmost panel, approximately 50 ng of purified recombinant parkin was analysed by BN-PAGE and Western blot with PRK109 without heat treatment or addition of SDS, revealing a band (indicated by the arrow) with apparent molecular weight (∼50 kDa) consistent with that of monomeric parkin. (E) Omission of Triton X-100 from the extraction protocol did not change the native molecular mass of the parkin complex from brain or the amount of parkin extracted. Numbers to the left of (A), (B), (D) and (E) indicate MWs of the native protein size markers

Altered white matter tracts in patient subgroups compared to controls.

<p>Tracts identified with probabilistic tractography having increased MD in patient subgroups compared to controls are shown. The red tract connects the left caudate with the contralateral ventral putamen. The blue tract connects the left caudate with the ipsilateral ventral putamen. Both tracts showed increased MD in both patient subgroups compared to controls. The yellow tract connects the right inferior parietal lobule with the right M1 and the green tract connects the right inferior parietal lobule with the right premotor cortex. Both tracts showed increased MD in TD compared to PIGD and controls. Part A shows two sagittal and one axial brain slices where the identified tracts are overlain on the mean FA skeleton. As the yellow and green tract overlap for a large part, only the yellow tract is visible. Part B shows glass brains with 3D representations of the identified tracts.</p

TBSS results for PIGD patients compared to controls.

<p>Red clusters represent white matter areas with significantly (TFCE-corrected p < 0.05) decreased FA in PIGD patients compared to controls. No such areas were found for TD compared to controls. One representative coronal, sagittal and axial slice are shown with the TBSS results overlain on the mean FA skeleton (in grey).</p

FA and MD alterations in white matter tracts between PIGD, TD and controls.

<p>FA and MD alterations in white matter tracts between PIGD, TD and controls.</p

Subject characteristics.

<p>Subject characteristics.</p

Automatization task in the large-amplitude condition.

<p>Mean and standard errors are displayed, corrected for MAM-16. (*) p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Movement parameter trade-off in health and disease.

<p>Dotted arrows indicate the possible interaction between disease-related changes and compensatory change in priorities.</p

Validity and reliability of a new tool to evaluate handwriting difficulties in Parkinson’s disease

<div><p>Background</p><p>Handwriting in Parkinson’s disease (PD) features specific abnormalities which are difficult to assess in clinical practice since no specific tool for evaluation of spontaneous movement is currently available.</p><p>Objective</p><p>This study aims to validate the ‘Systematic Screening of Handwriting Difficulties’ (SOS-test) in patients with PD.</p><p>Methods</p><p>Handwriting performance of 87 patients and 26 healthy age-matched controls was examined using the SOS-test. Sixty-seven patients were tested a second time within a period of one month. Participants were asked to copy as much as possible of a text within 5 minutes with the instruction to write as neatly and quickly as in daily life. Writing speed (letters in 5 minutes), size (mm) and quality of handwriting were compared. Correlation analysis was performed between SOS outcomes and other fine motor skill measurements and disease characteristics. Intrarater, interrater and test-retest reliability were assessed using the intraclass correlation coefficient (ICC) and Spearman correlation coefficient.</p><p>Results</p><p>Patients with PD had a smaller (p = 0.043) and slower (p<0.001) handwriting and showed worse writing quality (p = 0.031) compared to controls. The outcomes of the SOS-test significantly correlated with fine motor skill performance and disease duration and severity. Furthermore, the test showed excellent intrarater, interrater and test-retest reliability (ICC > 0.769 for both groups).</p><p>Conclusion</p><p>The SOS-test is a short and effective tool to detect handwriting problems in PD with excellent reliability. It can therefore be recommended as a clinical instrument for standardized screening of handwriting deficits in PD.</p></div

Correlation analysis.

<p>Correlation analysis.</p