PINK1 Defect Causes Mitochondrial Dysfunction, Proteasomal Deficit and α-Synuclein Aggregation in Cell Culture Models of Parkinson's Disease

Mutations in PTEN induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson's disease (PD), PARK6. Here, we report that PINK1 exists as a dimer in mitochondrial protein complexes that co-migrate with respiratory chain complexes in sucrose gradients. PARK6 related mutations do not affect this dimerization and its associated complexes. Using in vitro cell culture systems, we found that mutant PINK1 or PINK1 knock-down caused deficits in mitochondrial respiration and ATP synthesis. Furthermore, proteasome function is impaired with a loss of PINK1. Importantly, these deficits are accompanied by increased α-synclein aggregation. Our results indicate that it will be important to delineate the relationship between mitochondrial functional deficits, proteasome dysfunction and α-synclein aggregation

Proteasome function is impaired by mutant or loss of PINK1.

<p>Proteasome activity was measured from SH-SY5Y cells expressing mutant PINK1 (A), and PC12 cells expressing siRNA against PINK1 (B, C, D). A) Fluorescence of fluorogenic proteasome substrate Suc-LLVY-AMC (Calbiochem) is positively correlated with proteasome function. No statistically significant changes were detected in proteasome activity between control SH-SY5Y cells and the cells expressing wild type PINK1 (n = 8, p = 0.484, paired student t test). There was a statistically significant decrease of proteasome activity in the SH-SY5Y cells expressing L347P-PINK1 (23% reduction, n = 7, p = 0.018, paired student t test) or in SH-SY5Y cells expressing E417G-PINK1 (19.4% reduction, n = 8, p = 0.012, paired student t test) compared to cells expressing wild type PINK1. MG132, a proteasome inhibitor, was used as a negative control. The bottom panel is a Western analysis of the above samples with the 20S α subunit Ab for normalization. B) Proteasome activity was measured in 20 µg of cell lysate isolated from wild type control PC12 cells (open diamond) or SiPINK1-4 PC12 cell line (filled circle) for 60 min after 30 min incubation. Wild type PC12 cells lysate treated with MG132 (filled triangle) was used as a negative control. The result revealed that the kinetic of proteasome activity monitored over 60 min was markedly decreased in the cells with reduced PINK1. The bottom panel is a Western analysis of the above samples with the 20S α subunit antibody for normalization. C) Histographic presentation for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004597#pone-0004597-g007" target="_blank">Figure 7B</a>. The reduction of PINK1 by siRNA impairs the proteasome activity (31.8% reduction, n = 8, p = 0.01, ANOVA). Experiments were repeated with SiPINK1-2 PC12 cell line, and consistent results were obtained (data not shown). D) PINK1 mediated proteasome activity deficit confirmed by another independent method in the HeLa cells. Compared to control (CFP-de transfection), siRNA against PINK1 (siPINK1) knocked down PINK1 and led to a sigfinicant inhibition of CFP degradation (p = 0.0001, ANOVA) to an extent similar to direct proteasome inhibition by MG132 (p = 0.0014, ANOVA). A scrambled siRNA (siSCR) had no effect (p = 0.876, ANOVA). The RNAi sequences are: GAGAGGUCCAAGCAACUA TT and CCUGGUCGACUACCCUGAU TT.</p

Proteasome function is impaired by reduction of ATP.

<p>Proteasome function is ATP dependent. Fluorescent CFP was fused to degron, a signaling peptide that directs its protein to proteasome for degradation. An increase of fluorescence (open circle) indicates a reduction of proteasome function. ATP production was inhibited by 2-deoxyglucose (2DG), and ATP content was measured with the ATP Assay Kit (Calbiochem) for luminescence (filled circle). A) Increasing dosages of 2DG caused a decrease in ATP production (filled circle) and enhanced proteasome inhibition (open circle). B) Compared to non-treated cells in DMEM, there is a significant proteasome inhibition by 6 mM 2DG (p = 0.0004, ANOVA) and the proteasome inhibitor MG132 (p = 0.0001, ANOVA).</p

PINK1 constructs and their expressions.

<p>A) A schematic depiction of PINK1 constructs. Full length wild type, L347P-, or E417G- PINK1 tagged with Flag, V5, or GFP are indicated. Several truncated PINK1 tagged with Flag or V5 are also depicted. M stands for mitochondrial targeting sequence. B) Confirmation of the expression of the above constructs in HEK 293 cells. HEK293 cells were transfected by various PINK1 constructs, and their lysates were analyzed by Western blots with Flag antibody (Left panel), V5 antibody (the middle panel) or GFP antibody (the right Panel). Lane 1–10 are lysates from cells transfected by plasmids with the same numbering as shown in A). The lysates from the cells transfected with the empty cloning vector without PINK1 insert were used as controls (labeled as C). The results demonstrated that the expression of all constructs yielded recombinant PINK1 proteins with expected molecular weights.</p

Recombinant and endogenous PINK1 are associated with protein complexes in mitochondria.

<p>In all experiments, adenoviruses of PINK1-Flag, L347P-PINK1-Flag, E417G-PINK1-Flag or Del 245 PINK1-Flag were infected into SH-SY5Y cells. A) Western blot analysis of PINK1 sub-cellular distributions with anti-Flag Ab. The two forms of PINK1 (a 64 kD full length protein and a truncated form of 50 kD, presumably a proteolytic product) are present in mitochondria and cytosol. The L347P, E417G or Del 245 mutant PINK1 did not affect this distribution. S: cytosolic fraction; M: mitochondrial fraction. B) PINK1 is associated with protein complexes. Mitochondrial proteins were sub-fractionated by 15% to 35% discontinuous sucrose gradient, from which fractions 1–10 were collected from top (lighter proteins) to bottom (heavier proteins or complexes). They were subjected to SDS-PAGE, and Western analyses with anti-Flag Ab for PINK1 (the top three panels); anti-39 kD protein Ab for complex I (4^th panel); anti-70 kD protein Ab for complex II (5^th panel); anti-core 2 Ab for complex III (6^th panel); and anti-cox I Ab for complex IV (7^th panel). No PINK1 was observed in lane 1 and 2, the fractions that contained proteins of the sizes for monomeric PINK1. Instead, PINK1 was associated with protein complexes ranging from 130–900 kD, which co-migrated with ETC complexes. The L347P and E417G mutations did not affect the PINK1 association and distribution of these complexes. More importantly, anti-human PINK1antibodies (Novus) detected endogenous PINK1 in SH-SY5Y cells with similar distribution along the sucrose gradient (bottom panel).</p

Loss of PINK1 impairs OXPHOS function in PC12 cells with reduced PINK1 expression.

<p>Normal respiration is impaired in the PC12 cell lines with PINK1 knocked-down by RNAi. A) PINK1 mRNA is significantly reduced in two stable cell lines expressing PINK1 siRNA. Compared to the wild type control, there is an 81.7% and 91% reduction in PINK1 mRNA in SiPINK1-2 and SiPINK1-4 cell line. PINK1 mRNA is significantly reduced in two stable cell lines expressing PINK1 siRNA. The level of PINK1 mRNA is normalized to GAPDH. B) Oxygen consumption is significantly reduced in both SiPINK1-2 (22.4% reduction, n = 10, p<0.05, ANOVA) and SiPINK1-4 (33.1% reduction, n = 11, p<0.01, ANOVA) cell lines compared to that of control cells. SiPINK1-2: 46.38±3.5SE; SiPINK1-4: 39.96±1.93SE; control cell: 59.70±2.1SE. C) The respiratory deficit in SiPINK1-4 cells can be partially rescued by wild type (n = 7, p = 0.008, student T test) but not E417G-PINK1 (n = 4, p = 0.76, student T test) or Del 245 PINK1 (n = 3, p = 0.1, student T test). Control: 60 ±3.38SE; SiPINK1-4: 37±2.32SE; SiPINK1-4/wt-PINK1: 47.9±2.39SE; SiPINK1/E417G: 38.53±3.35SE; SiPINK1/Del 245: 28.63±4.75SE. D) With glutamate/malate as substrates, ATP synthesis rate was significantly reduced in both SiPINK1-2 (41.3% reduction, n = 7, p<0.01, ANOVA) and SiPINK1-4 (29.8% reduction, n = 8, p<0.01, ANOVA) cell lines compared to that of control cell. SiPINK1-2: 8.73±0.99SE; SiPINK1-4:10.59±0.69SE; control cells: 14.883±0.78SE. E) Western analysis of the samples used for the rescued experiment shown in (C) with Tim 23 Ab. The result demonstrates that equal amount of mitochondria is present in all the samples subject to respiration experiment. F) Equal amount of mitochondria were used for the experiments shown in B and D as demonstrated by identical Tim 23 in all the samples.</p

Mutant PINK1 impairs mitochondrial respiration and ATP synthesis in cultured SH-SY5Y cells.

<p>A) Oxygraphic measurement of SH-SY5Y cells infected with adenovirus of wild type (WT) PINK1, L347P-PINK1, and Del 245 PINK1. Normal respiration was measured with pyruvate as a substrate in non-infected SH-SY5Y control (C), cells infected with WT, L347P, E417G or Del 245 PINK1. A statistically significant deficit in oxygen consumption was detected in cells expressing L347P-PINK1 compared to wild type PINK1 (37.7% reduction; n = 3, p<0.01, ANOVA) or to non-infected control cells (39.3% reduction; n = 3, p<0.01, ANOVA). In cells expressing E417G-PINK1, oxygen consumption was significantly reduced compared to cells expressing wild type PINK1 (23.1% reduction, n = 3, p<0.05, ANOVA) or to non-infected cells (25.2% reduction, n = 3, p<0.05, ANOVA). There is no significant difference in respiration changes between cells expressing WT-PINK1 and non-infected control cells or between cells expressing WT-PINK1 and Del 245-PINK1. B) Measurements of ATP synthesis with malate/pyruvate as substrates in cultured SH-SY5Y cells. A statistically significant deficit in ATP synthesis was detected in cells expressing L347P-PINK1 compared to wild type PINK1 (23% reduction; n = 15, p = 0.001, Student t test) or to non-infected control cells (30% reduction; n = 5, p = 0.012, student t test). In cells expressing E417G-PINK1, ATP synthesis was significantly reduced compared to cells expressing wild type PINK1 (19.7% reduction, n = 12, p = 0.001, student t test) or to non-infected cells (27.4% reduction, n = 12, p = 0.011, student t test). There is no significant difference in ATP synthesis between cells expressing WT-PINK1 and non-infected control cells (n = 5, p = 0.646, student t test) or between cells expressing WT-PINK1 and Del 245-PINK1 (n = 5, p = 0.849, student t test). C) As controls, lysates used for respiration and ATP synthesis were subsequently subjected to Western analysis with Flag Ab (top panel) and Tim 23 Ab (bottom panel). Results indicate that equal expression of various recombinant PINK1 and equal amount of mitochondria were used for all the experiments.</p

Dimerization of PINK1 via the kinase domain.

<p>In all experiments, PINK1 with different tags were co-transfected in pairs into HEK293 cells. A tag was used for immunoprecipitation (IP), and Ab against another tag was used for Western blots (WB). Results were confirmed in COS and HeLa cells (data not shown). A) Wild type PINK1 form dimers. Left lanes in all three panels: lysate from cells without transfection as negative controls; right lanes in all panels: lysate from cells co-transfected with a pair of PINK1 constructs. In the left and middle panels, PINK1-Flag and PINK1-V5 were co-transfected. Anti-Flag Ab could co-IP PINK1-V5 (left panel) and vice versa (middle panel), indicating that PINK1-Flag and PINK1-V5 form a dimer. PINK1 dimerization is confirmed when PINK1-GFP and PINK1-V5 were co-transfected, anti-GFP Ab could co-IP PINK1-V5 (right panel). B) Wild type and mutant PINK1 form homo-dimers via the kinase domain. Lysates from cells expressing PINK1-V5 and PINK1_1-245-Flag (lane 1), PINK1-Flag and PINK1_1-509-V5 (lane 2), or PINK1-Flag and PINK1_1-525-V5 were isolated (lane 3), and subject to Western analysis with V5 antibody (middle panel, input control) or Flag antibody (bottom panel, input control) to confirm the expression of expected tagged recombinant protein. These lysates were then immunoprecipitated with mouse anti-V5 Ab, and subjected to Western analyses with rabbit anti-Flag Ab (upper panel; Co-IP). PINK1_1-245-Flag abolished dimerization (lane 1), whereas PINK1_1-525-V5 and PINK1_1-509-V5 could dimerize normally (lanes 2 and 3). Thus amino acid residues 246–509 are necessary for dimerization. L347P and E417G mutations did not disrupt the PINK1-PINK1 interaction (lane 5 and 6). C) Mutant PINK1 can also form hetero-dimers with wild type PINK. Lysates from cells expressing 1) PINK1-V5, 2) PINK1-V5 and PINK1-Flag, 3) PINK1-V5 and PINK1-L347P-Flag, 4) PINK1-V5 and PINK1-E417G-Flag were isolated and immunoprecipitated with rabbit anti-Flag Ab. The IP and Co-IP fractions were then subjected to Western analyses with mouse anti-V5 Ab (upper panel, Co-IP) or mouse anti-Flag Ab (lower panel, control IP). Wild type PINK1 form dimers (lane 2), and the disease-causing PINK1 mutations did not affect the dimerization (lanes 3, 4).</p

PINK1 auto-phosphorylation is impaired by L347P and E417G mutations.

<p>PINK1 protein is auto-phosphorylated in the cells expressing wild type PINK1 and the auto-phosphorylation was decreased in cells expressing L347P- or E417G-PINK1. Lysates from the SH-SY5Y cells expressing wild type-PINK1-Flag (1^st and 2^nd panels), L347P-PINK1-Flag (3^rd and 4^th panels) or E417G- PINK1-Flag (5^th and 6^th panels) were subjected to 2-dimensional gel electrophoresis, followed by Western analyses using anti-Flag Ab. Of the same protein, the more phosphorylated ones migrated to the more acidic end (left hand) of the isoelectric focusing (IEF) gel, while less or non-phosphorylated ones migrated to the more basic end (right hand) of IEF. 1^st panel: Wild type PINK1 along the pH gradient of the IEF. Spot 1, 2 (64 kD), and 3 (50 kD) migrated to the acidic end, and several others (open arrows) to the basic end. 2^nd panel: A control of wild type PINK1 proteins treated with alkaline phosphotase (AP). While the spots on the basic end still remained in the same region of the gel as in the 1^st panel, spot 1, 2, and 3 were missing. This indicated that these 3 spots were phospho-PINK1 in 1^st panel, and were de-phosphorylated in the 2^nd panel. The difference between spot 1 and 2 reflected the degree of phosphorylation on PINK1. In cells expressing L347P or E417G-PINK1, spot 1, 2 and 3 were greatly reduced, indicating reduced auto-phosphorylation on L347P- or E417G-PINK1 (3^rd and 5^th panels). The 4^th and 6^th panels are controls (treated with AP) for the 3^rd and 5^th panels respectively, similar to the 2^nd panel.</p