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

Summary of the performance of wild-type and <i>parkin</i> knockout (<i>parkin</i>^−/−) mice on behavioral tests evaluating olfactory, emotional and motor functions.

<p>Data are expressed as mean ± s.e.m. of 9–11 animals per group. Statistical analysis revealed absence of significant differences between wild-type and <i>parkin</i>^−/− mice in the performance of such behavioral test.</p><p>Summary of the performance of wild-type and <i>parkin</i> knockout (<i>parkin</i>^−/−) mice on behavioral tests evaluating olfactory, emotional and motor functions.</p

Effects of <i>parkin</i> genetic deletion on hippocampal synaptic transmission and plasticity processes.

<p>(A) Input-output curve of field excitatory post-synaptic potentials (fEPSP, measured as their slope) recorded in Schaffer fibers-CA1 pyramid synapses of hippocampal slices from wild-type and <i>parkin</i>^−/− mice. (B) Paired pulse facilitation (PPF), measured as the ratio of fEPSPs' slope ratio between the second and first paired stimuli (P2/P1 ratio) with different interpulse intervals (25, 50, 100, 200 and 400 ms) recorded in hippocampal slices from wild-type and <i>parkin</i>^−/− mice. (C) Modification of fEPSPs' slope, presented as % of baseline, before and after θ-burst stimulation in slices from wild-type and <i>parkin</i>^−/− mice. (D) Potentiation of fEPSPs' slope after the θ-burst stimulation, presentage as % increase of baseline fEPSPs' slope, in slices from wild-type and <i>parkin</i>^−/− mice <i>*P</i><0.05 compared to wild-type control group. Data are mean ± s.e.m. of n = 4 per group.</p

Effects of <i>parkin</i> genetic deletion on the spatial memory performance.

<p>(A) total investigation time by wild-type and <i>parkin</i>^−/− mice during the training session. (B) Recognition index of wild-type and <i>parkin</i>^−/− mice during the test session. <i>*P</i><0.05 compared to the hypothetical 50% (random investigation). <i>^#P</i><0.05 compared to the wild-type control group. Data are mean ± s.e.m. of n = 9–10 per group.</p

Effects of <i>parkin</i> genetic deletion on locomotor activity and habituation.

<p>(A) total distance traveled by wild-type and <i>parkin</i>^−/− mice at days 1 and 2 of analysis in an open field arena. <i>*P</i><0.05 compared to day 1, <i>^#P</i><0.05 compared to day 2 of wild-type group using a Newman-Keuls post-hoc test. (B) Pattern of locomotion at day 1 (divided in blocks of 5 min) of wild-type and <i>parkin</i>^−/− mice. (C) Pattern of locomotion at day 2 (divided in blocks of 5 min) of wild-type and <i>parkin</i>^−/− mice. Data are mean ± s.e.m. of n = 9–10 per group.</p

The inner mitochondrial membrane potential is normal in midbrain and striatum of <i>PARK2</i>^−/− mice.

<p>Dark circles = <i>PARK2^−/−</i>, white circles = wild type; ΔΨm = inner mitochondrial membrane potential expressed as mV after transformation of the fluorescent rhodamine 123 signal using the Nernst equation, as explained in the methods section; A = ΔΨm in the presence of glutamate+malate; B = A+1 mM ADP; C = B+1 µM oligomycin; D = C+1 µM cccp; E = C+2 µM cccp; F = C+4 µM cccp;G = C+8 µM cccp. The data were obtained in parallel to the analysis of respiration; two animals, one per genotype, were analyzed the same day; the data obtained from seven 9-month-old mice and six 24-month-old ones of each genotype, they are expressed as mean and SD.</p

Respiration was similar in brain mitochondrial preparations from 24-month-old <i>PARK2^−/−</i> and wild type mice examined with standard oxygraphy.

<p>(<b>A</b>) Representative experiment with a crude mitochondrial pellet from striatum. Blue curve = oxygen (O₂) concentration expressed as nmol/mL; red curve = rate of oxygen consumption expressed as nmoles O₂/mL and min; ADP = addition of 400 µM ADP; OM = addition of 1 µg/mL oligomycin, a complex V inhibitor; KCN = addition of 1 mM KCN (non-respiratory oxygen consumption); (<b>B, C, D</b>) Bars showing state 3 respiration (respiration in the presence of substrates +ADP – non-respiratory oxygen consumption) and state 4 respiration (respiration in the presence of oligomycin – non-respiratory oxygen consumption) on complex I substrates (glutamate+malate); black bars = <i>PARK2^−/−</i>, white bars = wild type; data are expressed as nmoles O₂/mL and mg proteins and are shown as means ± SD; two animals, one per genotype, were analyzed the same day; the numbers between brackets indicate the number of individual animals of each genotype; (B) results from crude mitochondrial pellets from striatum (n = 6), (C) crude mitochondrial pellets from cortex (n = 7) and (D) purified mitochondria from whole brain (n = 2).</p

High resolution respirometry reveals a respiration defect in <i>PARK2^−/−</i> mice.

<p>(<b>A</b>) Representative experiment with striatal post-nuclear supernatant. Blue curve = oxygen concentration (O₂) expressed as nmol/mL; red curve = rate of oxygen consumption expressed as nmoles O₂/mL and min; successive additions: ADP = 1 mM ADP in the medium with 10 mM glutamate+5 mM malate; OM = 1 µg/mL oligomycin, cccp = successive additions of 1.25 µM (up to the maximal respiration rate independent from ATP synthase capacity); KCN = 1 mM KCN (non-respiratory oxygen consumption); (<b>B, C, D</b>) Bars showing state 3 respiration, state 4 respiration and respiratory reserve on complex I substrates (glutamate+malate) in post-nuclear supernatants from (B) striatum, (C) midbrain and (D) liver of 24- and 9-month-old <i>PARK2^−/−</i> (black bars) and wild type mice (white bars); all data are expressed as nmoles O₂/mL and mg proteins and shown as means ± SD; two animals, one per genotype, were analyzed the same day; the data were obtained from 6 to 8 individual animals of each genotype. * <i>p</i><0.05 using Mann and Whitney test. In liver, state 3 respiration rate and respiratory reserve significantly decreased with age in both wild type mice (p = 0.011 and <0.001 when comparing state 3 respiration and respiratory reserve respectively between 9 and 24 months of age using Mann and Whitney test) and in <i>PARK2^−/−</i> mice (p = 0.019 and <0.001 when comparing state 3 respiration and respiratory reserve respectively between 9 and 24 months of age using Mann and Whitney test).</p

Effects of <i>parkin</i> genetic deletion on spatial recognition memory performance.

<p>(A and D) Total number of entries during the training and the test sessions (respectively) by wild-type and <i>parkin</i>^−/− mice. (B and E) Percentage of arms' entries during the training and test sessions (respectively); <i>*P</i><0.05 compared to the hypothetical value of 33% (random entries). (C and F) Percentage of time spent in each arm during the training and test sessions (respectively) by wild-type and <i>parkin</i>^−/− mice; <i>*P</i><0.05 compared to the hypothetical value of 33% (random time). Data are mean ± s.e.m. of n = 9–10 per group.</p