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

Reviewer #1 (Public review):

Summary:

The manuscript discusses the role of phosphorylated ubiquitin (pUb) by PINK1 kinase in neurodegenerative diseases. It reveals that elevated levels of pUb are observed in aged human brains and those affected by Parkinson's disease (PD), as well as in Alzheimer's disease (AD), aging, and ischemic injury. The study shows that increased pUb impairs proteasomal degradation, leading to protein aggregation and neurodegeneration. The authors also demonstrate that PINK1 knockout can mitigate protein aggregation in aging and ischemic mouse brains, as well as in cells treated with a proteasome inhibitor. While this study provided some interesting data, several important points should be addressed before being further considered.

Strengths:

(1) Reveals a novel pathological mechanism of neurodegeneration mediated by pUb, providing a new perspective on understanding neurodegenerative diseases.
(2) The study covers not only a single disease model but also various neurodegenerative diseases such as Alzheimer's disease, aging, and ischemic injury, enhancing the breadth and applicability of the research findings.

Weaknesses:

(1) PINK1 has been reported as a kinase capable of phosphorylating Ubiquitin, hence the expected outcome of increased p-Ub levels upon PINK1 overexpression. Figures 5E-F do not demonstrate a significant increase in Ub levels upon overexpression of PINK1 alone, whereas the evident increase in Ub expression upon overexpression of S65A is apparent. Therefore, the notion that increased Ub phosphorylation leads to protein aggregation in mouse hippocampal neurons is not yet convincingly supported.
(2) The specificity of PINK1 and p-Ub antibodies requires further validation, as a series of literature indicate that the expression of the PINK1 protein is relatively low and difficult to detect under physiological conditions.
(3) In Figure 6, relying solely on Western blot staining and golgi staining under high magnification is insufficient to prove the impact of PINK1 overexpression on neuronal integrity and cognitive function. The authors should supplement their findings with immunostaining results for MAP2 or NeuN to demonstrate whether neuronal cells are affected.
(4) The authors should provide more detailed figure captions to facilitate the understanding of the results depicted in the figures.
(5) While the study proposes that pUb promotes neurodegeneration by affecting proteasomal function, the specific molecular mechanisms and signaling pathways remain to be elucidated.

Reviewer #2 (Public review):

Summary:

The manuscript makes the claim that pUb is elevated in a number of degenerative conditions including Alzheimer's Disease and cerebral ischaemia. Some of this is based on antibody staining which is poorly controlled and difficult to accept at this point. They confirm previous results that a cytosolic form of PINK1 accumulates following proteasome inhibition and that this can be active. Accumulation of pUb is proposed to interfere with proteostasis through inhibition of the proteasome. Much of the data relies on over-expression and there is little support for this reflecting physiological mechanisms.

Weaknesses:

The manuscript is poorly written. I appreciate this may be difficult in a non-native tongue, but felt that many of the problems are organisational. Less data of higher quality, better controls and incision would be preferable. Overall the referencing of past work is lamentable.
Methods are also very poor and difficult to follow.

Until technical issues are addressed I think this would represent an unreliable contribution to the field.

Reviewer #3 (Public review):

Summary:

This study aims to explore the role of phosphorylated ubiquitin (pUb) in proteostasis and its impact on neurodegeneration. By employing a combination of molecular, cellular, and in vivo approaches, the authors demonstrate that elevated pUb levels contribute to both protective and neurotoxic effects, depending on the context. The research integrates proteasomal inhibition, mitochondrial dysfunction, and protein aggregation, providing new insights into the pathology of neurodegenerative diseases.

Strengths:

- The integration of proteomics, molecular biology, and animal models provides comprehensive insights.
- The use of phospho-null and phospho-mimetic ubiquitin mutants elegantly demonstrates the dual effects of pUb.
- Data on behavioral changes and cognitive impairments establish a clear link between cellular mechanisms and functional outcomes.

Weaknesses:

- While the study discusses the reciprocal relationship between proteasomal inhibition and pUb elevation, causality remains partially inferred.
- The role of alternative pathways, such as autophagy, in compensating for proteasomal dysfunction is underexplored.
- The immunofluorescence images in Figure 1A-D lack clarity and transparency. It is not clear whether the images represent human brain tissue, mouse brain tissue, or cultured cells. Additionally, the DAPI staining is not well-defined, making it difficult to discern cell nuclei or staging. To address these issues, lower-magnification images that clearly show the brain region should be provided, along with improved DAPI staining for better visualization. Furthermore, the Results section and Figure legends should explicitly indicate which brain region is being presented. These concerns raise questions about the reliability of the reported pUb levels in AD, which is a critical aspect of the study's findings.
- Figure 4B should also indicate which brain region is being presented.

Author response:

Public Reviews:
Reviewer #1 (Public review):

Summary:

The manuscript discusses the role of phosphorylated ubiquitin (pUb) by PINK1 kinase in neurodegenerative diseases. It reveals that elevated levels of pUb are observed in aged human brains and those affected by Parkinson's disease (PD), as well as in Alzheimer's disease (AD), aging, and ischemic injury. The study shows that increased pUb impairs proteasomal degradation, leading to protein aggregation and neurodegeneration. The authors also demonstrate that PINK1 knockout can mitigate protein aggregation in aging and ischemic mouse brains, as well as in cells treated with a proteasome inhibitor. While this study provided some interesting data, several important points should be addressed before being further considered.

Strengths:

(1) Reveals a novel pathological mechanism of neurodegeneration mediated by pUb, providing a new perspective on understanding neurodegenerative diseases.

(2) The study covers not only a single disease model but also various neurodegenerative diseases such as Alzheimer's disease, aging, and ischemic injury, enhancing the breadth and applicability of the research findings.

Weaknesses:

(1) PINK1 has been reported as a kinase capable of phosphorylating Ubiquitin, hence the expected outcome of increased p-Ub levels upon PINK1 overexpression. Figures 5E-F do not demonstrate a significant increase in Ub levels upon overexpression of PINK1 alone, whereas the evident increase in Ub expression upon overexpression of S65A is apparent. Therefore, the notion that increased Ub phosphorylation leads to protein aggregation in mouse hippocampal neurons is not yet convincingly supported.

Indeed, overexpression of sPINK1* alone caused little change in Ub levels in the soluble fraction (Figure 5E), which is expected. Ub in the soluble fraction is in a relatively stable, buffered state. However, overexpression of sPINK1* resulted in an increase in Ub levels in the insoluble fraction, indicating protein aggregation. The molecular weight of Ub in the insoluble fraction was predominantly below 70 kDa, implying that phosphorylation inhibits Ub chain elongation.

To further examine this, we used the Ub/S65A mutant to antagonize Ub phosphorylation, and found that the aggregation at low molecular weight was significantly reduced, indicating a partial restoration of proteasomal activity. The increase in Ub levels in both the soluble and insoluble fractions likely results from the high rate of ubiquitination driven by the elevated levels of Ub. Notably, the overexpressed Ub/S65A was detected in the Western blot using the wild-type Ub antibody, which accounts for the apparently increased Ub level.

When overexpressing Ub/S65E, we again saw an increase in Ub levels in the insoluble fraction (but no increase in the soluble fraction), with low molecular weight bands even more prominent than those observed with sPINK1* transfection. These findings collectively support the conclusion that sPINK1* promotes protein aggregation through Ub phosphorylation.

(2) The specificity of PINK1 and p-Ub antibodies requires further validation, as a series of literature indicate that the expression of the PINK1 protein is relatively low and difficult to detect under physiological conditions.

We acknowledge the challenges in achieving optimal specificity for commercially available and custom-generated antibodies targeting PINK1 and pUb, particularly given the low endogenous levels of these proteins under physiological conditions. Despite these limitations, we observed robust immunofluorescent staining for PINK1 (Figures 1A, 1C, and 1G) and pUb (Figures 1B, 1D, and 1G) in human brain samples from Alzheimer's disease (AD) patients, as well as in mouse brains from models of AD and cerebral ischemia. The significant elevation of PINK1 and pUb under these pathological conditions likely accounts for the clear visualization. To validate antibody specificity, we have included images from pink1-/- mice as negative controls in the revised manuscript (Figure 1C and 1D, third panel).

In addition, we detected a significant increase in pUb levels in aged mouse brains compared to young ones (Figures 1E and 1F). Notably, in pink1-/- mice, pUb levels remained unchanged between young and aged groups, despite some background signal, further supporting the conclusion that pUb accumulation during aging is PINK1-dependent.

In HEK293 cells, pink1-/- cells served as a negative control for PINK1 (Figure 2B and 2C) and for pUb (Figure 2D and 2E). While the Western blot using the pUb antibody displayed some nonspecific background, pUb levels in pink1-/- cells remained unchanged across all MG132 treatment conditions (Figures 2D and 2E), further attesting the reliability of our findings.

(3) In Figure 6, relying solely on Western blot staining and Golgi staining under high magnification is insufficient to prove the impact of PINK1 overexpression on neuronal integrity and cognitive function. The authors should supplement their findings with immunostaining results for MAP2 or NeuN to demonstrate whether neuronal cells are affected.

Thank you for raising this important point. We included NeuN immunofluorescent staining in Figure 5—figure supplement 2 of the original manuscript. The results demonstrate a significant loss of NeuN-positive cells in the hippocampus following Ub/S65E overexpression, while no apparent change in NeuN-positive cells was observed with sPINK1* transfection alone. These findings provide evidence of neuronal loss in response to Ub/S65E, further supporting the impact of pUb elevation on neuronal integrity.

While we did not perform MAP2 immunostaining, we included complementary analyses to assess neuronal integrity. Specifically, we performed Western blotting to determine MAP2 protein levels and used Golgi staining to study neuronal morphology and synaptic structure in greater detail. These analyses revealed that overexpression of sPINK1* or Ub/S65E decreased MAP2 levels and caused damage to synaptic structures (Figures 6F and 6H). Importantly, the deleterious effects of sPINK1* overexpression could be rescued by co-expression of Ub/S65A, further underscoring the role of pUb in mediating these changes.

Together, our NeuN immunostaining, MAP2 analysis, and Golgi staining provide strong evidence for the impact of PINK1 overexpression and pUb elevation on neuronal integrity and synaptic health. We believe these complementary approaches sufficiently address the reviewer’s concern and highlight the pathological consequences of elevated pUb levels.

(4) The authors should provide more detailed figure captions to facilitate the understanding of the results depicted in the figures.

Figure captions will be updated with more details in the revised manuscript.

(5) While the study proposes that pUb promotes neurodegeneration by affecting proteasomal function, the specific molecular mechanisms and signaling pathways remain to be elucidated.

The specific molecular mechanisms and signaling pathways through which pUb promotes neurodegeneration are likely multifaceted and interconnected. Mitochondrial dysfunction appears to be a central contributor to neurodegeneration following sPINK1* overexpression. This is supported by (1) an observed increase in full-length PINK1, indicative of impaired mitochondrial quality control, and (2) proteomic data revealing enhanced mitophagy at 30 days post-transfection and substantial mitochondrial injury by 70 days post-transfection. The progressive damage to mitochondria caused by protein aggregates can cause further neuronal injury and degeneration.

In addition, reduced proteasomal activity may result in the accumulation of inhibitory proteins that are normally degraded by the ubiquitin-proteasome system. Our proteomics analysis identified a >54-fold increase in CamK2n1 (UniProt ID: Q6QWF9), an endogenous inhibitor of CaMKII activation, following sPINK1* overexpression. This is particularly significant because the accumulation of CamK2n1 could suppress CaMKII activation and, subsequently, inhibit the CREB signaling pathway (illustrated below). As CREB is essential for synaptic plasticity and neuronal survival, its inhibition may further amplify neurodegenerative processes.

While our study identifies proteasomal dysfunction and mitochondrial damage as key initial triggers, downstream effects—such as disruptions in signaling pathways like CaMKII-CREB—likely contribute to a broader cascade of pathological events. These findings highlight the complexity of pUb-mediated neurodegeneration and suggest that further exploration of downstream mechanisms is necessary to fully elucidate the pathways involved.

We plan to include the proteomics data, in the revised manuscript, of mouse brain tissues at 30 days and 70 days post-transfection, to further highlight this downstream effect upon proteasomal dysfunction.

Author response image 1.

Reviewer #2 (Public review):

Summary:

The manuscript makes the claim that pUb is elevated in a number of degenerative conditions including Alzheimer's Disease and cerebral ischemia. Some of this is based on antibody staining which is poorly controlled and difficult to accept at this point. They confirm previous results that a cytosolic form of PINK1 accumulates following proteasome inhibition and that this can be active. Accumulation of pUb is proposed to interfere with proteostasis through inhibition of the proteasome. Much of the data relies on over-expression and there is little support for this reflecting physiological mechanisms.

Weaknesses:

The manuscript is poorly written. I appreciate this may be difficult in a non-native tongue, but felt that many of the problems are organisational. Less data of higher quality, better controls and incision would be preferable. Overall the referencing of past work is lamentable.

Methods are also very poor and difficult to follow.
Until technical issues are addressed I think this would represent an unreliable contribution to the field.

(1) Antibody specificity and detection under pathological conditions

We acknowledge the limitations of commercially available antibodies for detecting PINK1 and pUb. Despite these challenges, our findings demonstrate a significant increase in PINK1 and pUb levels under pathological conditions, such as Alzheimer's disease (AD) and ischemia. Additionally, we observed an increase in pUb level during brain aging, further highlighting its relevance in this particular physiological process. To ensure reliable quantification of PINK1 and pUb levels, we used pink1-/- mice and HEK293 cells as negative controls. For example, PINK1 levels were extremely low in control cells but increased dramatically after 2 hours of oxygen-glucose deprivation (OGD) and 6 hours of reperfusion (Figure 1H). Together, these controls validate that the observed elevations in PINK1 and pUb are specific and linked to pathological or certain physiological conditions.

(2) Overexpression as a model for pathological conditions

To investigate whether the inhibitory effects of sPINK1* on the ubiquitin-proteasome system (UPS) are dependent on its kinase activity, we utilized a kinase-dead version of sPINK1* as a negative control. Since PINK1 has multiple substrates, we further explored whether its effects on UPS inhibition were mediated specifically by ubiquitin phosphorylation. For this, we used Ub/S65A (a phospho-null mutant) to antagonize Ub phosphorylation by sPINK1*, and Ub/S65E (a phospho-mimetic mutant) to mimic phosphorylated Ub. These well-defined controls ensured the robustness of our conclusions.

While overexpression does not perfectly replicate physiological conditions, it serves as a valuable model for studying pathological scenarios such as neurodegeneration and brain aging, where pUb levels are known to increase. For example, we observed a 30.4% increase in pUb levels in aged mouse brains compared to young brains (Figure 1F). Similarly, in our sPINK1* overexpression model, pUb levels increased by 43.8% and 59.9% at 30- and 70-days post-transfection, respectively, compared to controls (Figures 5A and 5C). Notably, co-expression of sPINK1* with Ub/S65A almost entirely prevented sPINK1* accumulation (Figure 5B), indicating that an active UPS can efficiently degrade sPINK1*. Collectively, these findings show that sPINK1* accumulation inhibits UPS activity, a defect that can be rescued by the phospho-null Ub mutant. Thus, this overexpression model closely mimics pathological conditions and offers valuable insights into pUb-mediated proteasomal dysfunction.

(3) Organization of the manuscript

We believe the structure of the manuscript is justified and systematically addresses the key aspects of the study in a logic flow:

(a) Evidence for the increase of PINK1 and pUb in multiple pathological and physiological conditions.

(b) Identification of the sources and consequences of sPINK1 and pUb elevation.

(c) Mechanistic insights into how pUb inhibits UPS-mediated degradation.

(d) Validation of these findings using pink1-/- mice and cells.

(e) Evidence of the reciprocal relationship between proteasomal inhibition and pUb elevation, culminating in neurodegeneration.

(f) Demonstration of elevated pUb levels and protein aggregation in the hippocampus following sPINK1* overexpression, supported by proteomic analyses, behavioral tests, Western blotting, and Golgi staining.

Thus, this organization provides a clear and cohesive narrative, culminating in the demonstration that sPINK1* overexpression induces hippocampal neuron degeneration.

(4) Revisions to writing, referencing, and methodology

We will improve the clarity and flow of the manuscript, add more references to properly acknowledge prior work, and incorporate additional details into the Methods section to enhance readability and reproducibility. These improvements should address the organizational and technical concerns raised, while strengthen the overall quality of the manuscript.

Reviewer #3 (Public review):

Summary:

This study aims to explore the role of phosphorylated ubiquitin (pUb) in proteostasis and its impact on neurodegeneration. By employing a combination of molecular, cellular, and in vivo approaches, the authors demonstrate that elevated pUb levels contribute to both protective and neurotoxic effects, depending on the context. The research integrates proteasomal inhibition, mitochondrial dysfunction, and protein aggregation, providing new insights into the pathology of neurodegenerative diseases.

Strengths:

- The integration of proteomics, molecular biology, and animal models provides comprehensive insights.

- The use of phospho-null and phospho-mimetic ubiquitin mutants elegantly demonstrates the dual effects of pUb.

- Data on behavioral changes and cognitive impairments establish a clear link between cellular mechanisms and functional outcomes.

Weaknesses:

- While the study discusses the reciprocal relationship between proteasomal inhibition and pUb elevation, causality remains partially inferred.

The reciprocal cycle between proteasomal inhibition and pUb elevation can be initiated by various factors that impair proteasomal activity. These factors include Aβ accumulation, ATP depletion, reduced expression of proteasome components, and covalent modifications of proteasomal subunits—all well-established contributors to the progressive decline in proteasome function. Once initiated, this cycle would become self-perpetuating, with the accumulation of sPINK1 and pUb driving a feedback loop of deteriorating proteasomal activity.

In the current study, this reciprocal relationship between sPINK1/pUb elevation and proteasomal dysfunction is depicted in Figure 4A. Our results demonstrate that increased sPINK1 or PINK1 levels, such as through overexpression, can initiate this cycle. Crucially, co-expression of Ub/S65A effectively rescues the cells from this cycle, highlighting the pivotal role of pUb in driving proteasomal inhibition and establishing causality in this relationship. At the animal level, pink1 knockout could prevent protein aggregation upon aging and cerebral ischemia (Figures 1E and 1G).

Mitochondrial injury is a likely source of elevated PINK1 and pUb levels. A recent study showed that efficient mitophagy is necessary to prevent pUb accumulation (bioRxiv 2023.02.14.528378), suggesting that mitochondrial damage can trigger this cycle. In another study (bioRxiv 2024.07.03.601901), the authors found that mitochondrial damage could enhance PINK1 transcription, further increasing cytoplasmic PINK1 levels and exacerbating the cycle.

- The role of alternative pathways, such as autophagy, in compensating for proteasomal dysfunction is underexplored.

Elevated sPINK1 has been reported to enhance autophagy (Autophagy 2016, 12: 632-647), potentially compensating for the impaired UPS. One mechanism involves the phosphorylation of p62 by sPINK1, which enhances autophagy activity. In our study, we did observe increased autophagic activity upon sPINK1* overexpression, as shown in Figure 2I (middle panel, without BALA). This increased autophagy may help degrade ubiquitinated proteins induced by puromycin, partially compensating for the proteasomal dysfunction.

This compensation might explain why protein aggregation only increased slightly, though statistically significant, at 70 days post sPINK1* transfection (Figure 5F). Additionally, we observed a slight, though statistically insignificant, increase in LC3II levels in the hippocampus of mouse brains at 70 days post sPINK1* transfection (Figure 5—figure supplement 6), further supporting the notion of autophagy activation.

However, while autophagy may provide some compensation, its effect is likely limited. Autophagy and UPS differ significantly in their roles and mechanisms of degradation. Autophagy is a bulk degradation pathway that is generally non-selective, targeting long-lived proteins, damaged organelles, and intracellular pathogens. In contrast, the UPS is highly selective, primarily degrading short-lived regulatory proteins, misfolded proteins, and proteins tagged for degradation.

Together, we found that sPINK1* overexpression enhanced autophagy-mediated protein degradation while simultaneously impairing UPS-mediated degradation. This suggests that while autophagy may provide partial compensation for proteasomal dysfunction, it is not sufficient to fully counterbalance the selective degradation functions of the UPS.

- The immunofluorescence images in Figure 1A-D lack clarity and transparency. It is not clear whether the images represent human brain tissue, mouse brain tissue, or cultured cells. Additionally, the DAPI staining is not well-defined, making it difficult to discern cell nuclei or staging. To address these issues, lower-magnification images that clearly show the brain region should be provided, along with improved DAPI staining for better visualization. Furthermore, the Results section and Figure legends should explicitly indicate which brain region is being presented. These concerns raise questions about the reliability of the reported pUb levels in AD, which is a critical aspect of the study's findings.

We will include low-magnification images in the supplementary figures of the revised manuscript to provide a broader context for the immunofluorescence data presented in Figure 1. DAPI staining at higher magnifications will also be provided to improve visualization of cell nuclei and overall tissue structure. Additionally, we will indicate the brain regions examined in the corresponding figure legends, and incorporate more details in the Results section to provide clearer descriptions of the samples and brain regions analyzed.

The human brain samples presented in Figure 1 are from the cingulate gyrus region of Alzheimer's disease (AD) patients. Our analysis revealed that PINK1 is primarily localized within cell bodies, while pUb is more abundant around Aβ plaques, likely in nerve terminals. These additional clarifications and supplementary figures should provide greater transparency and improve the reliability of our findings.

- Figure 4B should also indicate which brain region is being presented.

The images were taken for layer III-IV in the neocortex of mouse brains, which information will be incorporated in the figure legend of the revised manuscript.