Biochemistry and Chemical Biology

Elevated Ubiquitin Phosphorylation by PINK1 Contributes to Proteasomal Impairment and Promotes Neurodegeneration

Cong Chen
Tong-Yao Gao
Hua-Wei Yi
Yi Zhang
Tong Wang
Zhi-Lin Lou
Tao-Feng Wei
Yun-Bi Lu
Ting-Ting Li
Chun Tang author has email address
Wei-Ping Zhang author has email address

Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou, China
The First People’s Hospital of Jingzhou, First Affiliated Hospital of Yangtze University, Jingzhou, China
Department of Biomedical Informatics, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China
Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Center for Quantitate Biology, Center for Life Science, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
Zhejiang Province Key Laboratory of Mental Disorder’s Management, Zhejiang University School of Medicine, Hangzhou, China

https://doi.org/10.7554/eLife.103945.1

Open access
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Figures and data

Elevated pUb levels in neurodegenerative conditions.
(A, B) Double immunofluorescence staining showing the distribution of PINK1 and Aβ in (A), and pUb and Aβ in (B), within the brains of AD patients compared to age-matched controls. Detailed donor demographics are available in Supplementary Table 1.
(C, D) Double immunofluorescence staining of PINK1 and Aβ (C), and pUb and Aβ (D) in the brains of wildtype and APP/PS1 transgenic mice.
(E) Double immunofluorescence staining for pUb and the neuronal marker NeuN in young and aged brains of both wildtype and pink1-/- mice.
(F) Western blot analysis of pUb levels in young (Y) and aged (A) wildtype and pink1-/- mouse brains, quantitatively comparing protein levels across ages and genotypes. *P<0.05, **P<0.01 for comparisons with young wildtype mice; ^###P<0.001 for comparisons with aged wildtype mice, one-way ANOVA.
(G) Immunofluorescence staining for PINK1 and pUb in the contralateral and penumbra of mouse brains subjected to MCAO for 2 hours followed by 24 hours of reperfusion. Locations of the analyzed regions are indicated in Figure S1.
(H) Western blot analysis of PINK1 and pUb levels in HEK293 cells subjected to OGD for 2 hours, followed by reperfusion at 0, 6, and 12 hours, to study changes over time.
(I) Western blot analysis of ubiquitin in the insoluble fraction of HEK293 cells post 2-hour OGD and subsequent 0, 6, 12 hours of reperfusion.

Impact of sPINK1-mediated Ub phosphorylation on proteasomal activity in HEK293 cells.
(A) Representative Western blot showing levels of PINK1 following the administration with CCCP, O/A, and MG132.
(B) Western blot analysis detailing the concentration-dependent effects of MG132 (0-5 µM) over 8 hours on PINK1 levels. N=3; *P < 0.05, ***P < 0.001, compared to 0 µM MG132, one-way ANOVA.
(C) Western blot analysis showing the time-dependent effects of 5 µM MG132 on PINK1 levels over 0-24 hours. N=4; *P < 0.05, ***P < 0.001, compared to 0 hours, one-way ANOVA.
(D) Western blot analysis of pUb levels under a concentration gradient of MG132 (0-5 µM) for 8 hours. N=3; *P < 0.05, compared to 0 µM MG132, one-way ANOVA.
(E) Western blot analysis of pUb levels over a time course of 24 hours with 5 µM MG132 treatment. N=3; *P < 0.05, compared to 0 hours, one-way ANOVA.
(F) Western blot analysis showing levels of pUb and PINK1 following transfection with various PINK1 constructs: sPINK1* (PINK1/F101M/L102-L581), sPINK1*-KD (kinase-dead sPINK1* with additional K219A/D362A/D384A mutations), and Ub^GG-sPINK1 (a short-lived version of native sPINK1 with an appended N-terminal Ub).
(G) Representative immunofluorescence images of Ub staining in cells treated with MG132 or transfected with various sPINK1 constructs, highlighting differences in ubiquitin localization and aggregation. The white arrows indicate positively transfected cells.
(H) Western blot analysis of Ub in the insoluble fraction of cell lysates following treatment with 5 µM MG132 or after transfection with different sPINK1 constructs.
(I) Immunofluorescence staining of Ub differentiating the roles of sPINK1 over-expression on proteasomal and autophagic degradation of puromycin increased unfolded proteins. The left panel illustrates the experimental strategy. Puromycin (5 µg/ml) was applied 2 hours before harvesting with or without 0.1 µM BALA (an autophagy inhibitor). The white arrows indicate positively transfected cells.
(J) Western blot analysis showing GFP degradation in HEK293 cells, with the top panel illustrating the Ub-GFP construct, a model substrate to evaluate proteasomal degradation efficiency. N=3; *P < 0.05, ***P < 0.001, compared to the control group, one-way ANOVA.

Impact of ubiquitin phosphorylation on chain elongation and proteasomal association.
(A) Coomassie blue staining showing Ub chain formation using different Ub variants as building blocks: wildtype Ub, phosphorylated Ub (pUb), phospho-null Ub/S65A, and phospho-mimic Ub/S65E.
(B) Analysis the transfection and knockout of PINK1 on K48-linked Ub chain formation in HEK293 cells treated with 10 µM MG132. Cells were transfected with FLAG-tagged Ub/48K. Additionally, pink1-/- cells were transfected with either full-length PINK1 or the sPINK1* variant.
(C) In vitro proteasomal degradation assay of GFP modified with K48-linked ubiquitin chains (K48-polyUb-GFP) versus with phosphorylated K48-linked ubiquitin chains (pK48-polyUb-GFP). N=3; **P<0.01, ***P<0.001 compared to respective controls without added proteasome; ^#P<0.05, ^##P<0.01, ^###P<0.001 compared to K48-polyUb-GFP with proteasome, two-way ANOVA.
(D) Representative TIRF microscopy images showing the binding of K48-polyUb-GFP (left) and pK48-polyUb-GFP (right) to surface-immobilized proteasomes, visualized as bright puncta.
(E) Quantitative analysis of puncta density from TIRF images, with N=4; ***P<0.001 compared to K48-polyUb-GFP, using a paired t-test.
(F, H) Representative fluorescence traces of a single punctum for K48-polyUb-GFP (F) and pK48-polyUb-GFP (H), detailing the dynamic interactions at the proteasome surface.
(G, I) Analysis of GFP fluorescence dwell time for K48-polyUb-GFP (G) and pK48-polyUb-GFP (I). Data are binned and modeled with a single exponential decay curve (red line), illustrating differences in substrate-proteasome interaction durations.

pink1 knockout mitigates protein aggregation under proteasomal inhibition.
(A) Schematic representation illustrating the reciprocal feed-forward cycle among proteasomal inhibition, and the elevation of sPINK1 and pUb levels.
(B) Immunofluorescence staining of Ub in the brains of young and aged wildtype and pink1-/- mice.
(C) Western blot analysis quantifying Ub in the insoluble protein fraction from young and aged mouse brains. Statistical analysis includes N=4; **P<0.01, one-way ANOVA.
(D) Western blot analysis of Ub in the insoluble protein fraction of wildtype and pink1-/- mouse brains subjected to MCAO for 2 hours followed by 24 hours of reperfusion. Comparison includes the sham-operated group (same procedure without occlusion). Locations of the analyzed regions (contralateral and ipsilateral) are indicated in Figure 1—figure supplementary 1.
(E) Western blot analysis of Ub levels in the insoluble fraction following treatment with 0-5 µM MG132 for 8 hours. N=3; ***P<0.001 compared to 0 µM MG132; ^##P<0.01 compared with wildtype cells, one-way ANOVA.
(F) Western blot analysis of Ub levels in the insoluble fraction following treatment with 5 µM MG132 over a 0–24-hour period. N=4; *P<0.05, **P<0.01, ***P<0.001 compared to 0 µM MG132; ^##P<0.01 compared with wildtype cells, one-way ANOVA.
(G) Western blot analysis of LC3 levels following treatment with 0-5 µM MG132 for 8 hours. N=4; *P<0.05 compared to 0 µM MG132, one-way ANOVA.
(H) Western blot analysis of LC3 levels after 5 µM MG132 treatment over 0-24 hours. N=3; *P<0.05 compared to 0 µM MG132, one-way ANOVA.

Elevated pUb levels induce protein aggregation in mouse hippocampal neurons.
(A) Western blot analysis showing levels of PINK1 and pUb in mouse hippocampus at 30-days post-transfection. N=4; **P<0.01 compared with control, one-way ANOVA.
(B) Western blot analysis of PINK1 in the mouse hippocampus at 70-days post-transfection, illustrating changes in expression over time. N=4; **P<0.001 compared with control; ^###P<0.001 compared with sPINK1, one-way ANOVA.
(C) Western blot analysis of pUb in the mouse hippocampus at 70-days post-transfection, highlighting the persistence of phosphorylation changes. N=5; *P<0.05 compared with control, one-way ANOVA.
(D) Representative immunofluorescence images depicting ubiquitin staining in the CA1 region of the mouse hippocampus at 70-days post-transfection.
(E) Western blot analysis of ubiquitin in the soluble fraction of hippocampal lysates at 70-days post-transfection. N=7; *P<0.05 compared with control, one-way ANOVA.
(F) Western blot analysis of Ub in the insoluble fraction of hippocampal lysates at 70-days post-transfection. Quantitative analysis includes total protein and specifically proteins smaller than 70 kDa. N=7; *P<0.05, **P<0.001 compared with control, one-way ANOVA.

Elevated pUb levels induce neuronal injury in mouse brains.
(A, B) Novel object recognition test showing the sniff number to objects during training (A) and testing (B) phases. N=10; ***P<0.001 compared with the sniff number toward A1 object during training, paired t-test. ^#P<0.05, one-way ANOVA.
(C, D) Fear conditioning tests evaluating contextual (C) and cued (D) memory. N=10; ***P<0.001 compared with Box A (contextual) or absence of tone (cued), paired t-test. ^#P<0.05, ^##P<0.01, ^###P<0.001, one-way ANOVA.
(E-G) Western blot analysis measuring levels of mitochondrial, dendritic, and synaptic markers: Tom20 (E), MAP2 (F), and PSD95 (G) in the mouse hippocampus. N=7; *P<0.05, **P<0.01, ***P<0.001 compared with control, one-way ANOVA.
(H) Golgi staining used to assess hippocampal neuronal spines and dendrites. The left panel shows representative images; the right panel provides a statistical analysis of spine density on hippocampal neuron dendrites from 3 mice. ***P<0.001 compared with control, one-way ANOVA.

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