An altered cell-specific subcellular distribution of translesion synthesis DNA polymerase kappa (POLK) in aging neurons

Reviewer #1 (Public review):

Summary:

Abdelmageed et al. investigate age-related changes in the subcellular localization of DNA polymerase kappa (POLK) in the brains of mice. POLK has been actively investigated for its role in translesion DNA synthesis and involvement in other DNA repair pathways in proliferating cells, very little is known about POLK in a tissue-specific context, let alone in post-mitotic cells. The authors investigated POLK subcellular distribution in the brains of young, middle-aged, and old mice via immunoblotting of fractioned tissue extracts and immunofluorescence (IF). Immunoblotting revealed a progressive decrease in the abundance of nuclear POLK, while cytoplasmic POLK levels concomitantly increased. Similar findings were present when IF was performed on brain sections. Further, IF studies of the cingulate cortex (Cg1), the motor cortex (M1, M2), and the somatosensory (S1) cortical regions all showed an age-related decline in nuclear POLK. Nuclear speckles of POLK decrease in each region, meanwhile, the number of cytoplasmic POLK granules decreases in all four regions, but granule size is increasing. The authors report similar findings for REV1, another Y-family DNA polymerase.

The authors then investigate the colocalization of POLK with other DNA damage response (DDR) proteins in either pyramidal neurons or inhibitory interneurons. At 18 months of age, DNA damage marker gH2AX demonstrated colocalization with nuclear POLK, while strong colocalization of POLK and 8-oxo-dG was present in geriatric mice. The authors find that cytoplasmic POLK granules colocalize with stress granule marker G3BP1, suggesting that the accumulated POLK ends up in the lysosome.

Brain regions were further stained to identify POLK patterns in NeuN+ neurons, GABAergic neurons, and other non-neuronal cell types present in the cortex. Microglia associated with pyramidal neurons or inhibitory interneurons were found to have a higher abundance of cytoplasmic POLK. The authors also report that POLK localization can be regulated by neuronal activity induced by Kainic acid treatment. Lastly, the authors suggest that POLK could serve as an aging clock for brain tissue, but POLK deserves further characterization and correlation to functional changes before being considered as a biomarker.

Strengths:

Investigation of TLS polymerases in specific tissues and in post-mitotic cells is largely understudied. The potential changes in sub-cellular localization of POLK and potentially other TLS polymerases open up many questions about DNA repair and damage tolerance in the brain and how it can change with age.

Weaknesses:

The work is quite novel and interesting, and the authors do suggest some potentially interesting roles for POLK in the brain, but these are in and of themselves a bit speculative. The majority of the findings of this paper draw upon findings from POLK antibody and its presumed specificity for POLK. However, this antibody has not been fully validated and needs further work. Further validation experiments using Polk-deficient or knocked-down cells to investigate antibody specificity for both immunoblotting and immunofluorescence should be performed. More mechanistic investigation is needed before POLK could be considered as a brain aging clock.

Reviewer #2 (Public review):

Summary:

Abdelmageed et al., demonstrate POLK expression in nervous tissue and focus mainly on neurons. Here they describe an exciting age-dependent change in POLK subcellular localization, from the nucleus in young tissue to the cytoplasm in old tissue. They argue that the cytosolic POLK is associated with stress granules. They also investigate the cell-type specific expression of POLK, and quantitate expression changes induced by cell-autonomous (activity) and cell nonautonomous (microglia) factors.

I think it is an interesting report but requires a few more experiments to support their findings in the latter half of the paper. Additionally, a more mechanistic understanding of the pathways regulating POLK dynamics between the nucleus and cytosol, what is POLK doing in the cytosol, and what is it interacting with; would greatly increase the impact of this report. However, additional mechanistic experiments are mostly not needed to support much of the currently presented results, again, it would simply increase the impact.

Reviewer #3 (Public review):

Summary:

In this study, the authors show that DNA polymerase kappa POLK relocalizes in the cytoplasm as granules with age in mice. The reduction of nuclear POLK in old brains is congruent with an increase in DNA damage markers. The cytoplasmic granules colocalize with stress granules and endo-lysosome. The study proposes that protein localization of POLK could be used to determine the biological age of brain tissue sections.

Strengths:

Very few studies focus on the POLK protein in the peripheral nervous system (PNS). The microscopy approach used here is also very relevant: it allows the authors to highlight a radical change in POLK localization (nuclear versus cytoplasmic) depending on the age of the neurons.

The conclusions of the study are strong. Several types of neurones are compared, the colocalization with several proteins from the NHEJ and BER repair pathways is tested, and microscopy images are systematically quantified.

Weaknesses:

The authors do not discuss the physical nature of POLK granules. There is a large field of research dedicated to the nature and function of condensates: in particular numerous studies have shown that some condensates but not all exhibit liquid-like properties (https://www.nature.com/articles/nrm.2017.7, https://pubmed.ncbi.nlm.nih.gov/33510441/ https://www.mdpi.com/2073-4425/13/10/1846). The change of physical properties of condensates is particularly important in cells undergoing stress and during aging. The authors should discuss this literature.

Author response:

We sincerely thank all the reviewers for their enthusiasm and positive feedback, which has encouraged us to delve deeper into this research. As this is the first report of POLK in the brain using a longitudinal normative aging model, our primary aim was to establish the observational and phenomenological aspects. We agree with the reviewers that more detailed molecular, biochemical, and cellular studies are essential to elucidate underlying mechanisms. However, as noted by some reviewers, these investigations, while they will raise the impact, may fall outside the scope of the current report. Indeed, many of these lines of investigation are currently ongoing. Below, we provide our provisional responses to individual reviewer comments.

Response to Reviewer #1:

a) Concern over POLK antibody characterization in mice:

We performed knocking down of POLK by siRNA in mice cortical primary neuronal culture (Fig S1C). In the revised version, we will provide a more detailed characterization of POLK antibodies in mouse cells.

b) More mechanistic investigation is needed before POLK could be considered as a brain aging clock:

We sincerely appreciate the valuable suggestion. In our ongoing work exploring the mechanisms of POLK in postmitotic neurons, preliminary findings using siPOLK indicate an upregulation of senescence markers along with a reduction in DNA repair synthesis (manuscript in preparation). We will reference this companion manuscript in the revised version and are pleased to share these data with the reviewers for their consideration.

Response to Reviewer #2:
a) Concern on more mechanistic understanding of the pathways regulating POLK dynamics between the nucleus and cytosol:

We sincerely appreciate the reviewer’s enthusiasm and valuable guidance in helping us better understand the mechanism of nuclear-cytoplasmic POLK dynamics. Previously, we developed a modified aniPOND (accelerated native isolation of proteins on nascent DNA) protocol, which we termed iPoKD-MS (isolation of proteins on Pol kappa synthesized DNA followed by mass spectrometry), to capture proteins bound to nascent DNA synthesized by POLK in human cell lines (bioRxiv https://www.biorxiv.org/content/10.1101/2022.10.27.513845v3). In this dataset, we identified potential candidates that may regulate nuclear/cytoplasmic POLK dynamics. These candidates are currently undergoing validation in human cell lines, and we are preparing a manuscript on these findings. Among these, some candidates, including previously identified proteins such as exportin and importin (Temprine et al., 2020, PMID: 32345725), are being explored further as potential POLK nuclear/cytoplasmic shuttles. We are also conducting tests on these candidates in mouse cortical primary neurons to assess their role in POLK dynamics. In the revised version of the manuscript, we will include a discussion of our current understanding and outline our planned studies.

b) Question on “… what is POLK doing in the cytosol, and what is it interacting with …”:

Our data so far indicate that POLK accumulates in stress granules and lysosomes. We are very grateful for the reviewer’s insightful suggestions and will make every effort to incorporate them in the revised manuscript. Currently, we are characterizing POLK accumulation in the cytoplasm using additional lysosomal markers, as recommended by the reviewer. If these experiments prove challenging in mouse brain tissues, we plan to investigate them in primary neuron cultures. We are hopeful to include these findings in the revised version. Additionally, we have optimized the POLK antibody for immunoprecipitation from nuclear and cytoplasmic fractions of mouse brain tissue. These findings, which are beyond the scope of the current study, will be reported in a separate manuscript.

Response to Reviewer #3:

We highly appreciate the reviewer bringing up the context of biomolecular condensates. Our iPoKD-MS data referenced above suggests candidates from various biomolecular condensates that we are currently investigating. We are currently investigating by subcellular fractionation the presence of POLK in different biomolecular condensates that will be fully reported in future publications. We appreciate the reviewer providing important literature that will be cited and potential biomolecular condensates will be discussed in the revised version.