Elevated pUb levels are widespread across neurodegenerative conditions.

(A,B) Double immunofluorescence staining showing the distribution of PINK1 and Aβ (A), and pUb and Aβ (B), within the cingulate gyrus brain region of AD patients compared to similar brain regions from age-matched controls. Detailed donor information is provided in Supplementary Table 1. (C, D) Double immunofluorescence staining of PINK1 and Aβ (C), and pUb and Aβ (D) in the brains of wildtype, pink1-/-, and APP/PS1 transgenic mice. The images were taken for the neocortex of APP/PS1, wildtype, and pink1-/- mice. (E) Double immunofluorescence staining for pUb and the neuronal marker NeuN in the neocortex of young and aged brains of both wildtype and pink1-/- mice. The images were taken for the layer III-IV of neocortex. (F) Western blot analysis of pUb levels in the cortex of 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 brain regions are shown in Figure 1—table supplementary 1. (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. (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.

Ubiquitin phosphorylation by sPINK1 affects proteasomal activity in HEK293 cells.

(A) Representative Western blot showing the levels of PINK1 following the administration of CCCP, O/A, or MG132. The CCCP was treated for 12 hours, O/A was treated for 2 hours, and the MG132 was treated for 8 hours. (B) Western blot analysis showing the concentration-dependent effect of MG132 (0-5 µM) over an 8-hour period on PINK1 level. N=3; *P < 0.05, ***P < 0.001, compared to 0 µM MG132, one-way ANOVA. (C) Western blot analysis showing the time-dependent effect of 5 µM MG132 on PINK1 levels in 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 0-24 hours with 5 µM MG132 treatment. N=3; *P < 0.05, compared to 0 hours, one-way ANOVA. (F) Representative Western blot image showing the levels of pUb and PINK1 at 24 hours post-transfection with different PINK1 constructs: sPINK1* (PINK1/F101M/L102-L581), sPINK1*-KD (kinase-dead sPINK1* with additional K219A/D362A/D384A mutations), and UbGG-sPINK1 (a short-lived version of native sPINK1 with an appended N-terminal Ub). (G) Representative immunofluorescence images of ubiquitin staining in cells treated with 5µM MG132 for 8 hours or transfected with different sPINK1 constructs, highlighting differences in ubiquitin localization and aggregation. The white arrows indicate positively transfected cells. (H) Representative Western blot image of ubiquitin in the insoluble protein fraction of cells. The cells were collected at 8 hours after treatment with 5 µM MG132, or collected at 24 hours after transfection with different sPINK1 constructs. (I) Western blot analysis showing GFP degradation in HEK293 cells transfected with Ub-R-GFP. Cells were harvested at 8 hours after 5 µM MG132 treatment, or 24 hours following sPINK1* or sPINK1*-KD transfection. N=3; *P < 0.05, ***P < 0.001, compared to the control, one-way ANOVA. (J) Immunofluorescence staining of ubiquitin illustrating how sPINK1 over-expression impacts on proteasomal and autophagic degradation. Puromycin blocks protein synthesis at the translation elongation stage, leading to the production of truncated proteins; BALA (bafilomycin A1, a v-ATPase inhibitor) blocks the degradation of autophagic cargo by inhibiting autophagosome-lysosome fusion, as illustrated in the left panel. Puromycin (5 µg/ml) was applied for 2 hours before harvesting with or without the treatment of 0.1 µM BALA. The white arrows indicate positively transfected cells.

Ubiquitin phosphorylation inhibits both ubiquitin chain elongation and ubiquitin proteasome interactions.

(A) Coomassie blue staining showing ubiquitin chain formation/elongation using different ubiquitin variants as building blocks: wildtype ubiquitin, phosphorylated ubiquitin (pUb), phospho-null Ub/S65A, and phospho-mimic Ub/S65E. (B) Western blot showing the formation of K48-linked ubiquitin chain in wildtype (WT) and pink1-/- HEK293 cells without or with PINK1 and sPINK1* transfection. Cells were transfected with FLAG-tagged Ub/48K to form K48-linked ubiquitin chain only, and 10 µM MG132 was applied at 12 hours post-transfection to prevent the degradation of ubiquitinated substrates. (C) Western blot analysis of in vitro proteasomal degradation of GFP modified with K48-linked ubiquitin chains (K48-polyUb-GFP) and GFP modified 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 single-molecule association of K48-polyUb-GFP (left) and pK48-polyUb-GFP (right) to the surface-immobilized proteasomes, visualized as bright puncta. Details of TIRF single-molecule visualization is shown in Figure 3—figure supplementary 3. (E) Quantitative analysis of puncta density from TIRF images comparing the number of puncta of K48-polyUb-GFP and pK48-polyUb-GFP associated with surface-immobilized proteasomes. N=4;***P<0.001 compared to K48-polyUb-GFP, using a paired t-test. (F, H) Representative fluorescence time traces of a single punctum for proteasomal bind K48-polyUb-GFP (F) and pK48-polyUb-GFP (H). More representative figures of single-molecule fluorescence are shown in in Figure 3—figure supplementary 4. (G, I) Analysis of GFP fluorescence dwell time for K48-polyUb-GFP (G) and pK48-polyUb-GFP (I) associated with the proteasome. Data were binned and modeled with a single exponential decay curve (red line).

pink1 knockout mitigates protein aggregation upon proteasomal inhibition.

(A) Western blot analysis of ubiquitin levels in the insoluble protein fraction of HEK293 cells following the 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. (B) Western blot analysis of ubiquitin levels in the insoluble protein fraction of HEK293 cells following the treatment with 5 µM MG132 over 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. (C) Western blot analysis of LC3 levels in HEK293 cells following the treatment with 0-5 µM MG132 for 8 hours. N=4; *P<0.05 compared to 0 µM MG132, one-way ANOVA. (D) Western blot analysis of LC3 levels in HEK293 cells following the treatment with 5 µM MG132 treatment over 24 hours. N=3; *P<0.05 compared to 0 µM MG132, one-way ANOVA. (E) Immunofluorescence staining of ubiquitin taken from layer III-IV in the neocortex of brains from young and aged wildtype and pink1-/-mice. (F) Western blot analysis quantifying ubiquitin in the insoluble protein fraction of the cortex from young and aged mouse brains. N=4; **P<0.01, one-way ANOVA. (G) Western blot analysis of ubiquitin 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.

Elevated pUb levels induce protein aggregation in mouse hippocampal neurons.

(A) Western blot analysis showing the 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. 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. N=5; *P<0.05 compared with control, one-way ANOVA. (D) Representative immunofluorescence images depicting ubiquitin staining in the CA1 neurons of the mouse hippocampus at 70-days post-transfection. (E) Western blot analysis of ubiquitin in the soluble protein 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 ubiquitin in the insoluble protein fraction of hippocampal lysates at 70- days post-transfection. Quantitative analysis was conducted for total protein at all molecular weight and for proteins with molecular weight <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 at 70 days post-transfection.

(A) Novel object recognition test showing the number of sniffs toward two distinct objects in the training phase. N=10. (B) Novel object recognition test showing the number of sniffs toward the old object (A1) and the novel object (B) in the testing phase. N=10; ***P<0.001 compared with the sniff number toward A1 object, paired t-test. #P<0.05, one-way ANOVA. (C, D) The percentage of freezing time in fear conditioning tests for the evaluation of contextual (C) and cued (D) memory. For contextual memory, the mice were put in the box and received foot shock. For cued memory, the mice were put in a new box, and the same tone accompanying the foot shock was applied. 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 analyses to assess 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 to assess the number of neuronal spines in hippocampal neurons. The left panel shows representative images, and the right panel provides a statistical analysis of spine density on hippocampal neuron dendrites. N=11-20 dendrites from 3 mice for each group. ***P<0.001 compared with control, one-way ANOVA.

Elevated pUb levels inhibit CamKII-CREB signaling in mouse hippocampal neurons.

(A) An illustration of the inhibitory effect on CamK2n1 in the CamKII-CREB1 signaling pathway. (B-D) Immunofluorescence staining of CamK2n1 (B), pCamKII (C), and pCREB1 (D) in the CA1 region of hippocampus of mice at 70 days post-transfection. (E) Western blot analysis of pCREB1 in mouse hippocampi. N=8, independent t-test. (F and G) The immunofluorescence staining of BDNF (F) and ERK (G) in the CA1 region of mouse hippocampus.