Terminally differentiated postmitotic cells such as mature neurons and glia are long-lived and must cope with the accumulation of damage over the course of an animal’s lifespan. The mechanisms used by such long-lived cells to deal with aging-related damage are poorly understood. The brain of the fruit fly Drosophila melanogaster is an ideal context to examine this since the fly has a relatively short lifespan and the adult fly brain is nearly entirely postmitotic with well understood development and excellent tools for genetic manipulations.

The adult central nervous system of Drosophila melanogaster comprises ~110,000 cells, most of which are generated in the larval and early pupal stages of development from various progenitor cell types (Truman and Bate, 1988; White and Kankel, 1978). By late metamorphosis, the Drosophila pupal brain is normally completely non-cycling and negative for markers of proliferation such as thymidine analog incorporation and mitotic markers (Awasaki et al., 2008; Pahl et al., 2019; Siegrist et al., 2010).

In the adult, very little neurogenesis and gliogenesis are normally observed (Awasaki et al., 2008; Ito and Hotta, 1992; von Trotha et al., 2009). A population of about 40 adult neural progenitors has been reported in the optic lobe and a population of glial progenitors has been reported in the central brain (Fernández-Hernández et al., 2013; Foo et al., 2017). Upon damage or cell loss, hallmarks of cycling have been shown to be activated, although the overall level of proliferation in the adult brain remains very low (Crocker et al., 2020; Fernandez-Hernandez et al., 2019; Fernández-Hernández et al., 2013; Foo et al., 2017; Li et al., 2020). Thus, the brain of the adult fly is thought to be almost entirely postmitotic with most cells in G0 with a diploid (2C) DNA content. One known exception to this are the cells that constitute the ‘blood-brain barrier’ of Drosophila.

The ‘blood-brain barrier’ in Drosophila is made up of specialised cells called the Sub-perineurial glia (SPGs). These cells are very few in number and achieve growth without cell division by employing variant cell cycles termed endocycles, that involve DNA replication without karyokinesis or cytokinesis, as well as endomitotic cycles that involve DNA replication and karyokinesis without cytokinesis (Unhavaithaya and Orr-Weaver, 2012; Von Stetina et al., 2018). The SPGs undergo these variant cell cycles to increase their size rapidly to sustain the growth of the underlying brain during larval development. The polyploidization of these cells plays an important role in maintaining their epithelial barrier function, although it remains unclear whether these cells continue to endocycle or endomitose in the adult.

Polyploidy can also confer an increased biosynthetic capacity to cells and resistance to DNA damage induced cell death (Edgar and Orr-Weaver, 2001; Lee et al., 2009; Mehrotra et al., 2008; Zhang et al., 2014). Several studies have noted neurons and glia in the adult fly brain with large nuclei (Robinow and White, 1991; Winberg et al., 1992) and in some cases neurons and glia of other insect species in the adult CNS are known to be polyploid (Nordlander and Edwards, 1969). Rare instances of neuronal polyploidy have been reported in vertebrates under normal conditions (Morillo et al., 2010) and even in the CNS of mammals (López-Sánchez and Frade, 2013; Shai et al., 2015).

Polyploidization is employed in response to tissue damage and helps maintain organ size (Cohen et al., 2018; Tamori and Deng, 2013; Losick et al., 2016; Losick et al., 2013). Therefore, polyploidy may be a strategy to deal with damage accumulated with age in the brain, a tissue with very limited cell division potential. Here we show that polyploid cells accumulate in the adult fly brain and that this proportion of polyploidy increases as the animals approach middle-age. We show that multiple types of neurons and glia which are diploid at eclosion which become polyploid specifically in the adult brain. We have found that the optic lobes of the brain contribute to most of the observed polyploidy. We also observe increased DNA damage with age, and show that inducing oxidative stress and exogenous DNA damage can lead to increased levels of polyploidy. We find that polyploid cells in the adult brain are resistant to DNA damage-induced cell death and propose a potentially protective role for polyploidy in neurons and glia in adult Drosophila melanogaster brains.

Cell ploidy often scales with cell size and biosynthetic capacity (Edgar and Orr-Weaver, 2001; Orr-Weaver, 2015). The brain is thought to be a notable exception to this rule, where the size of postmitotic diploid neurons and glia can be highly variable. We wondered whether alterations in ploidy during late development or early adulthood may contribute to the variability in neuronal and glial cell size in the mature Drosophila brain. We therefore developed a sensitive flow cytometry assay to measure DNA content in Drosophila pupal and adult brains. Briefly, this assay involves dissociating brains with a trypsin or collagenase based solution followed by quenching the dissociation and labeling DNA with DyeCycle Violet (Grushko and Buttitta, 2015) in the same tube, to avoid cell loss from washes. Samples are then immediately run live on a flow cytometer for analysis. We employed strict gating parameters to eliminate doublets (Figure 1—figure supplement 1; López-Sánchez et al., 2017b). This assay is sensitive enough to measure DNA content from small subsets of cells (e.g. Mz19-GFP expressing neurons) from individual pupal or adult brains (Figure 1—figure supplement 1D). Using this approach we confirmed that under normal culturing conditions, cell proliferation and DNA replication ceases in the pupal brain after 24 hr into metamorphosis (24 hr APF) (Figure 1—figure supplement 1E), and that only the previously described mushroom body neuroblasts continue to replicate their DNA and divide in late pupa (Siegrist et al., 2010). The brains of newly eclosed adult flies are 98–99% diploid and we, like others, only rarely observe EdU incorporation during the first week of adulthood in wild-type flies under normal conditions but not later in adulthood (Figure 1—figure supplement 1F; Fernández-Hernández et al., 2013; Foo et al., 2017; Kato et al., 2009; Siegrist et al., 2010; von Trotha et al., 2009). We were therefore surprised to find a distinct population of cells with DNA content of 4C and up to >16C appearing in brains of aged animals of various genotypes under normal culture conditions (Figure 1—figure supplement 1G).

Polyploid cells accumulate in the adult Drosophila brain

We performed a systematic time-course to measure accumulation of polyploid cells in the adult brain in isogenic w¹¹¹⁸ male and female flies cultured under standard conditions (Linford et al., 2013). We measured the percentage of polyploid cells in individual brains from the day of eclosion until 56 days (8 weeks) at weekly intervals. Polyploid cells appear as early as 7 days into adulthood, and the proportion of polyploidy continues to rise until animals are 21 days old (Figure 1A). This increase in polyploidy is only observed until week 3, after which the proportion of polyploid cells observed remains variable from animal to animal, but on average, does not increase. We observe similar patterns of polyploidy accumulation in males and females (Figure 1A). To ensure that the polyploidy we observe is not an artefact of one particular strain, we performed similar measurements across the lifespan in other commonly used lab ‘wild-type’ strains Canton-S (Figure 1B) and Oregon-R (Figure 1C). Interestingly, Oregon-R flies show lower levels of polyploidy in the first two weeks than w¹¹¹⁸ and Canton-S suggesting that different genetic backgrounds may influence polyploidy in the brain. We also performed DNA content measurements of brains from the distantly related D.americana which diverged ~50 million years ago and a more closely related species, D.mauritiana, which diverged ~2 million years ago (Figure 1C). While both species show accumulation of polyploidy, it is interesting to note that they show differences in levels of polyploidy.

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We next measured changes in ploidy in the D. melanogaster adult brain over time. We pooled data from multiple animals and binned polyploid cells from w¹¹¹⁸ brains into two categories: cells with 4C (tetraploid) DNA content measured by flow cytometry and cells with >4C DNA content - this includes 8C, 16C and even some 32 C cells (Figure 1D). The majority of the polyploid cells appear to be tetraploid, and the fraction of cells exhibiting >4C DNA content increases during the first week of adulthood, but remains relatively consistent with age.

We next asked whether polyploid cells are located in a specific region of the brain. We dissected the Drosophila central nervous system (CNS) into the central brain, optic lobes, and ventral nerve cord (VNC) and measured levels of polyploidy in each region from day of eclosion to 2 weeks into adulthood (Figure 1E). We found that while there is a low level of polyploidy in the central brain and VNC that increases with age, most of the polyploidy comes from the optic lobes. Strikingly, by 3 weeks, over 20% of the cells in the optic lobes can exhibit polyploidy.

Since the optic lobes contribute to most of the polyploidy observed, we wondered if this phenomenon may be dependent on light. Canton-S animals reared in complete darkness did not show difference in polyploidy compared to age-matched controls raised in regular 12 hr light/12 hr dark cycles (Figure 1—figure supplement 2A). Next, we hypothesized that polyploidy accumulation may depend on proper photoreceptor function. However, glass^60j flies devoid of photoreceptors and pigment cells in compound eyes (Helfrich-Förster et al., 2001) still show polyploidy (Figure 1—figure supplement 2B).

Multiple cell types exhibit adult-onset polyploidy in the brain

To identify which cell types in the brain are becoming polyploid, we used the binary GAL4/UAS system to drive the expression of a nuclear-localised green or red fluorescent protein (nGRP or nRFP) with cell type-specific drivers. We then measured DNA content using Dye-Cycle Violet in the GFP or RFP-positive populations.

We first examined the SPGs, as previous work from the Orr-Weaver lab identified these to be highly polyploid (Unhavaithaya and Orr-Weaver, 2012; Von Stetina et al., 2018). When we used the SPG driver moody-GAL4, we found that the SPGs are highly polyploid (Unhavaithaya and Orr-Weaver, 2012), but contributed to less than 5% the polyploid cells observed in mature adult brains (Figure 2—figure supplement 1A,C). Another class of cells previously shown to be polyploid in some contexts are tracheal cells that carry oxygen to tissues (Zhou et al., 2016). Using the tracheal driver breathless-GAL4, we found that in 10 day old adult brains, tracheal cells comprise less than 3% of all polyploid cells (Figure 2—figure supplement 1B,C). Thus, 90% of the polyploid cells we observe in the brain arise from cell types not previously known to become polyploid.

The adult fly brain is thought to be composed almost entirely of neurons (90% of total population) and glia (10% of total population). First, we asked if neurons become polyploid by using a pan-neuronal driver, nSyb-GAL4 to drive UAS-nGFP (Figure 2A). We found that indeed, by two weeks ~ 5–6% of cells expressing nSyb-GAL4 show >2C DNA content. Similarly, we used the pan-glial driver Repo-GAL4 and found that by 2 weeks ~ 6–7% of glia also become polyploid in the adult brain (Figure 2B). Neurons outnumber glial cells in the fly brain, and we find that the relative proportions of polyploid cells reflect the total ratio of neurons vs. glia in the adult brain (Figure 2C). We next asked whether specific types of neurons or glia show higher levels of polyploidy. We measured the proportion of polyploid vs diploid cells in various classes of neurons (Figure 2D) and glia (Figure 2E) in 7 day old optic lobes. Interestingly, we found that most differentiated cell types we assayed in the optic lobes show some level of polyploidy by one week of age. We conclude that polyploidy arises in multiple neuronal and glial types that are initially diploid upon eclosion and become polyploid after terminal differentiation and specifically during adulthood.

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Most of the polyploidy is not a result of cell fusion

We reasoned that cells in the brain could become polyploid either by re-entering the cell cycle or by undergoing cell fusion (Alvarez-Dolado and Martínez-Losa, 2011; Giordano-Santini et al., 2016; Grendler et al., 2019; Losick et al., 2013; Schoenfelder et al., 2014; Shu et al., 2018; Starnes et al., 2016). To examine whether cell fusion occurs, we used a genetic labeling tool called CoinFLP (Bosch et al., 2015). The CoinFLP genetic cassette contains two overlapping but exclusive Flippase Recombination Target (FRT) sites flanking a stop cassette that can be ‘flipped -out’ using FRT mediated recombination to give rise to cells expressing either a LexGAD driver or a GAL4 driver, which can be used to drive expression of lexA_op-GFP (green) and UAS-RFP (red). In animals heterozygous for CoinFLP, a diploid cell has only one copy of the transgenic cassette which can only be ‘flipped’ to give rise to a cell permanently labeled with either red or green fluorescent proteins, hence the name CoinFLP. If labeling is induced in the brain early during development before eclosion, cells become stochastically and permanently labeled with either red or green fluorescent proteins. If cells fuse in the ageing brain, up to ⅓ of cells undergoing fusion could fuse a red-labeled cell with a green cell and appear yellow. We used a FLP recombinase (flippase) under the control of the eyeless promoter (ey-FLP) to label most of the cells red or green in the optic lobes early in development (Figure 3A–B’) and did not observe any double labeled (yellow) cells in young adult brains or older brains. We also expressed flippase enzyme more broadly under the control of a heat-shock promoter (hs-FLP) and labeled cells using a nuclear GFP or RFP at larval L2-L3 stages (Figure 3C) and measured the number of double-labeled cells in the optic lobes at on the day of eclosion or after aging at 14 days post-eclosion. We never observed more than 10–12 cells per optic lobes exhibiting double-labeling under these ‘early-FLP’ conditions. These double-labeled cells under the larval hs-FLP conditions likely include the SPG cells which become polyploid early in larval development and do not express ey-FLP. We conclude that very little cell fusion occurs in the adult OL, even with age.

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‘Late-FLP’ can label polyploid cells that arise from cell cycle reentry

By using a modified labeling paradigm, we can also use CoinFLP to label polyploid cells in situ (Figure 4A). Previous work with CoinFLP has shown that inducing ‘flipping’ in cells that are already polyploid results in a fraction of double labeled yellow cells (Bosch et al., 2015). We therefore reasoned that heterozygous CoinFLP brain cells that become polyploid by replicating their genome during adulthood will contain two or more copies of the CoinFLP transgene cassette. If we label cells by activating hs-FLP late in adulthood after polyploidy appears, some polyploid cells may ‘flip’ one copy green and one copy red, appearing yellow. When we induce an adult FLP at one day, before polyploidy occurs, we do not observe any double-labeled cells in the optic lobe (Figure 4B) but when we induce an adult FLP at 30 days post-eclosion, we observe several double labeled cells (Figure 4B’) indicating that these cells have undergone genome replication and contain at least two heterozygous copies of the CoinFLP transgenic cassette. To quantify this, we used an adult ‘late-FLP’ to drive nuclear GFP and RFP, and we observe around 300 double-labeled cells per optic lobes in 14 day old brains (Figure 4C). The presence of double-labelled nuclei in aged optic lobes suggest cells become polyploid by cell cycle re-entry and endoreduplicating DNA.

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We further confirmed that the double-labeled cells visualized by microscopy are polyploid by performing DAPI intensity quantification in high-magnification images (Figure 4—figure supplement 1) and flow cytometry. (Figure 4—figure supplement 2). All CoinFLP double-labeled cells were confirmed to be polyploid by DAPI integrated intensity measurements and 97% of all double-labeled cells show >2C DNA content by FACS (Figure 4—figure supplement 2). CoinFLP double-labeling confirmed several types of glia identifiable by location and shape to become polyploid, such as a subset of cortex glia of the outer (Figure 4D) and inner (Figure 4E) optic chiasm and astrocyte-like glia (Figure 4F) in the medulla of the OL.

To test whether polyploidy in the adult optic lobes is driven by cell cycle re-entry, we used cell-type specific RNA-interference (RNAi) to modulate the DNA replication licensing factors cdc6 and Geminin in postmitotic neurons. Cdc6 is an essential factor for DNA replication licensing that promotes the recruitment of the MCM complex to load the DNA replication complex (Kang et al., 2014), while Geminin is a replication licensing inhibitor that sequesters DNA replication licensing factors to inhibit DNA re-replication (Lutzmann et al., 2006). Using nSyb-GAL4, we expressed UAS-cdc6^RNAi in differentiated neurons which significantly reduced levels of polyploidy by 14d (Figure 4G) from ~25% in control optic lobes to ~14% on optic lobes expressing the RNAi. We next knocked down geminin and found that we increase levels of polyploidy in the optic lobes (Figure 4G). This suggests that a fraction of post-mitotic neurons reactivate DNA replication to become polyploid in the adult fly brain.

DNA damage accumulates in adult optic lobes

To investigate whether transcriptional changes that occur with age may be associated with cell cycle reactivation and polyploidy in the brain, we performed RNA sequencing on three parts of the CNS: optic lobes, central brain and VNC from male and female Canton-S animals at different time points: 1 day, 2 days, 7 days and 21 days post-eclosion. To infer biological processes that are affected with age, gene ontology analysis was performed using GOrilla and redundant terms were filtered using reviGO. The most significant changes observed in the optic lobes at 21d compared to 2d are shown in Figure 5A,B. Among the most significantly upregulated groups of genes are those associated with the cell cycle, DNA damage response and DNA damage repair. The enrichment of up-regulated genes associated with the DNA damage response was also observed in the optic lobes at 7 days (Figure 5—figure supplement 1), but the enrichment and fold-induction of specific genes is stronger at day 21 (Figure 5A). A gene expression signature associated with DNA damage is specific to the optic lobes. However, the most significantly downregulated GO terms in the optic lobes at 21d are shared with the central brain and VNC and include metabolism, transmembrane transport and cellular respiration-associated processes (Figure 5—figure supplement 1).

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To examine whether DNA damage is higher in the OL, we performed immunostaining against the phosphorylated histone 2A variant (pH2AV) in 1d optic lobes and central brain and 21d optic lobes and central brain (Figure 5C–E) from Canton-S male brains. Young brains show very low levels of pH2AV in both the optic lobes and central brain (Figure 5C,C’,E) but older brains show higher levels of pH2AV in the optic lobes compared to the central brain (Figure 5D,D’,E).

To further understand the DNA damage and cell cycle signatures observed with age, we looked at the change in expression of specific genes involved in the DNA damage response and the cell cycle (Figure 5F). Recent work has identified a specific transcriptional response to induced DNA damage in the head that involved a non-canonical role for tumor suppressor protein p53 (Kurtz et al., 2019). This signature was termed head Radiation Induced p53-Dependent or hRIPD. In addition to genes such as FANCI, loki, rad50 and xrp1 which are involved in a canonical, ionising radiation-induced DNA damage response, we also find robust upregulation of hRIPD genes Ku80, Irbp, Cht8, CG3344 and CG4734 in our RNAseq data set at 7d and 21d in optic lobes. However, these genes are not as strongly upregulated in the aged central brain or VNC and p53 itself shows only a small increase in the optic lobes at 21d (Figure 5F). Consistent with cell cycle re-entry in a fraction of cells in the OL, upregulation of cell cycle genes such as myc, cyclin D, orc1 is observed specifically in the optic lobes and increases with age.

Polyploidy accumulation in neurons is p53-independent

Work in other polyploid tissues in Drosophila has shown that polyploid cells in the salivary gland can tolerate high levels of DNA double-strand breaks and resist apoptosis caused by DNA damage (Hassel et al., 2014; Mehrotra et al., 2008; Qi and Calvi, 2016; Zhang et al., 2014). This is possible because polyploid cells in tissues such as the salivary gland have intrinsically low levels of p53 protein and also suppress the expression of pro-apoptotic genes (Zhang et al., 2014). It has also been shown in various tissues and organisms that DNA damage can induce polyploidization (Bretscher and Fox, 2016; Donovan and Corbo, 2012; Grendler et al., 2019). Since we observe a modest upregulation of p53 as well as a p53-dependent gene expression signature in older optic lobes, we asked if the induction of polyploidy in neurons is p53 dependent. To address this, we overexpressed wildtype (p53^WT) or a dominant-negative allele of p53 (p53^DN) that is unable to bind to DNA and evoke a transcriptional response in neurons using the nSyb-GAL4 driver (Figure 6A). We did not see a significant difference in levels of polyploidy in 7 day old brains with overexpression of either WT or mutant p53, suggesting that accumulation of polyploidy in neurons is p53-independent. We also performed cell death measurement in the brain using flow cytometry. We calculated cell death by measuring proportions of cells incorporating either Propidium Iodide (PI) or Sytox-Green (Figure 6—figure supplement 1). We did not see a significant difference in the proportion of dead cells in overexpression of p53^WT or p53^DN conditions compared to control (Figure 6—figure supplement 1B) consistent with recent work suggesting a non-canonical, non-apoptotic role for p53 in the adult Drosophila brain (Kurtz et al., 2019).

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Acceptance summary:

Polyploidization is hypothesized to promote survival in response to the accumulation of age-related DNA damage. Polyploid and aneuploid cells in the brain have been described before, as is their accumulation during ageing and disease. However, the idea of this being a response to DNA damage is novel. The authors make an effort to associate ploidy changes in adult animals with brain regions and cell types, which is an important unaddressed question in the field. The experiments aiming to demonstrate that polyploid cells in the brain are a consequence of cell cycle re-entry are also of interest.

Decision letter after peer review:

Thank you for submitting your article "Polyploidy in the adult Drosophila brain" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by K VijayRaghavan as the Senior and Reviewing Editor. The following individual involved in review of your submission has agreed to reveal their identity: Grace E Boekhoff-Falk (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The manuscript entitled "Polyploidy in the adult Drosophila brain" by Nandakumar and colleagues describes the age-related accumulation of polyploid cells in the Drosophila brain and proposes this mechanism as a response to DNA damage. Polyploidization is hypothesized to promote survival in response to the accumulation of age-related DNA damage.

Polyploid and aneuploid cells in the brain have been described before, as is their accumulation during aging and disease. However, the idea of this being a response to DNA damage is novel. The authors make an effort to associate ploidy changes in adult animals with brain regions and cell types, which is an important unaddressed question in the field. The experiments aiming to demonstrate that polyploid cells in the brain are a consequence of cell cycle reentry are also of interest. Overall, while the study is of interest, it lacks quantitative follow-up experiments and includes several correlative studies as well as overstatements of many conclusions. These and other concerns are outlined below. The reviews also involve suggestions for experiments. The authors should decide which are best suited to speedily and robustly address the requirements for clear evidence for their assertions, or where discussion of previous work from their labs will suffice. In any event, the experiments are doable in a 2-month period. The authors should make every effort to firmly address the reviewers' concerns so that a decision can be taken by avoiding further revisions. The work will represent a strong contribution to the field if the substantive concurs are well-addressed.

Essential revisions:

1) The study is entirely based on ploidy detection by FACS using a custom assay to measure DNA content; however, this assay has not been benchmarked to determine its sensitivity and specificity. The only validation is correlative with the frequency of EdU positive cells shown in Figure 1—figure supplement 1F. The FACS based method needs to be validated with an independent technique (i.e. FISH, single cell sequencing) in order to strengthen the following statement "We were therefore surprised to find a distinct population of cells with DNA content of 4C and up to >16C appearing in brains of aged animals of various genotypes under normal culture conditions (Figure 1—figure supplement 1G)". We recommend that polyploidy be also shown in a second way such as high resolution imaging and single cell fluorescence measurements of DNA, or using DNA FISH probes.

2) The animals analyzed in this study are 1 week increment up to 8 weeks; the age related increased in polyploidy begins at 7 days and continues to accumulate up to 21 days (week 3) after which reaches a plateau. This age group should be considered middle age and therefore the data do not support the statement in the Abstract "that "polyploidy protects against DNA damage-induced cell death".

3) The COINFLP system applied to demonstrate that cells reenter the cell cycle shows correlative data; green, red and yellow cells should be FACS enriched to measure the frequency of polyploid cells in the yellow fraction. This is important because while it is understood that yellow labelled cells are assumed to be polyploid this has not been formally demonstrated. Likewise, in the silencing experiments (cdc6 and Geminin) yellow cells are assumed polyploid and even labelled as such in Figure 4G but no quantification of DNA content is ever provided.

4) The RNA seq data have not been validated, and again show correlative results.

5). There are three main part to the model presented.

a) DNA damage accumulating with age. This result is not surprising and there are several examples that show this in various tissues, including in brains. We also think that the staining and imaging for pH2AV must be greatly improved. There are several other experiments that could benefit from images, or better images. The authors must look at their experiments and see where this can be added meaningfully or experiments done.

b) "Exogenous" DNA damage leading to increased polyploidy. I think the authors were very careful to use the exogenous damage here as their analysis of ploidy after PQ treatment might not reflect normal polyploidy due to normal levels of stress and age. In addition, their analysis is done in bulk tissue and not on a single cell basis to correlate a cell's DNA damage with its ploidy. It would be interesting to further test the idea that DNA damage induces polyploidy by over-expressing or knocking down a DNA repair factor such as Prp19 and then measuring the proportion of cells that are polyploid. Again, this is experimentally feasible speedily, but if the authors choose not to do this, these must have a cogent and strong reasoning.

c) Polyploidy protecting from cell death. We think this idea is founded in the literature from other cell types, and thus it is a reasonable hypothesis in the fly brain. However, we do not think the experiment presented is sufficient to make this a solid conclusion. A change in the percent of dying cells from 7 to 2.5 (diploid vs polyploid) from cell counts that are already very small and from brains that are pooled does not seem like a reliable experiment. We think additional experiments should be done. For example, the authors could use a GAL4 expressed in a restricted sub-population of cells (ex. SPGs via R54C07-GAL4 which was used in Kremer et al., 2017) to label and directly count the number of cells in the optic lobes via light microscopy (it should be <100 cells per lobe). Then, RNAi against cdc6 and geminin (as in Figure 4G) could be used to manipulate the proportion of cells that are polyploid. Using such an approach would allow the authors to link the proportion of cells that are polyploid to the total number of cells and thus directly show that polyploidy protects the cells from DNA damage-induced cell death.

6) A criticism of the work as presented is that the vast majority of the polyploid cells are tetraploid, yet the authors do not address the possibility that these cells simply have replicated their DNA prior to dividing (i.e. are transiently polyploid) as versus remaining indefinitely polyploid. Earlier work from other laboratories suggests that this unlikely, and the manuscript would be improved by a discussion of the possibilities.

7) The authors state that up to 20% of the cells in the optic lobes are polyploid. However, later in the text (and the figure legends) the number is 25%. Please be consistent.

8) In the Discussion, the authors propose that polyploidization may permit cells to maintain contacts when some of their neighbors die. This hypothesis also was proposed by Unhavaithaya and Orr-Weaver who should be cited. The BBB example is particularly compelling b/c the contacts create the barrier.

9) The authors state “It is also possible that we see higher levels of DNA damage foci in the optic lobes because they have more polyploid cells” but do not offer an explanation for why this could be biologically relevant.

10) Also, the authors state “We see an upregulation of cell cycle-associated genes specifically in the optic lobes with age. The transcription of cell cycle genes and genes involved in the DNA damage response and repair are intimately coordinated and can be controlled by the same factors.” Please complete the argument – namely that upregulation of DNA damage response and cell cycle genes may share an upstream regulatory mechanism.

11) The authors state “Whether cell cycle re-entry is a cause or a consequence of neurodegeneration has been difficult to test since both are associated with age and damage.” An earlier paper would seem to be relevant and could be cited: Mutations in String/CDC25 inhibit cell cycle re-entry and neurodegeneration in a Drosophila model of Ataxia-telangiectasia. Rimkus et al., 2008.

12) In Figure 1, there is an intriguing dip in the % polyploidy after day 21 in males and day 28 in females. What are the authors' thoughts about this? Could the initial wave of polyploidization result in death and then be followed by a wave of polyploidization that results in persistent, stress-resistant cells?

13) In Figure 3, the # of double-labeled cells from early flp significantly increases from Day 1 to Day 14 – doesn't this suggest there is some cell fusion? Also, image B' is from a 30 day animal, while the quantitation in C is from 14-day animals. What does the quantitation look like at 30 days? Does the number of yellow cells increase further?

14) For the data presented in Figure 4, the labeling of 300 cells/optic lobe is impressive. However, if there are ~20,000 cells/optic lobe, then the 300 cells would be only ~1-2% of the total, not the estimated 25% that you measure as >4N. Can you explain the discrepancy? (BTW, we think lineage tracing methods such as Permatwin under-report dividing cells in the adult brain).

15) For Figure 6, it would be interesting to pulse the flies with paraquat, then chase a few days without paraquat before assessing ploidy. What if the polyploid cells are en route to dying? This seems unlikely for UV irradiated flies, b/c the irradiation was done 5 days prior to dissection. However, in Figure 5D, the fact that the control and paraquat-treated lines cross at 21 days seems to support the idea that some of the polyploid cells in the paraquat-treated animals die and/or divide. Please comment.

16) In Figure 6—figure supplement 1. It appears that pH2AV levels are elevated in all neurons in the paraquat treated brains. However, only a fraction of these die. Why might this be?

Essential revisions:

1) The study is entirely based on ploidy detection by FACS using a custom assay to measure DNA content; however, this assay has not been benchmarked to determine its sensitivity and specificity. The only validation is correlative with the frequency of EdU positive cells shown in Figure 1—figure supplement 1F. The FACS based method needs to be validated with an independent technique (i.e. FISH, single cell sequencing) in order to strengthen the following statement "We were therefore surprised to find a distinct population of cells with DNA content of 4C and up to >16C appearing in brains of aged animals of various genotypes under normal culture conditions (Figure 1—figure supplement 1G)". We recommend that polyploidy be also shown in a second way such as high resolution imaging and single cell fluorescence measurements of DNA, or using DNA FISH probes.

We agree this is an important issue.

Several years ago we attempted to resolve chromosome copy numbers using FISH on the Drosophila brain. We found this to be impossible using high resolution confocal microscopy, due to constitutive somatic homologue pairing (Joyce et al., 2012; McKee, 2004; Metz, 1916; Stevens, 1908). We therefore attempted to resolve homologues using STORM super-resolution microscopy and large FISH probes (~10kb). However we could not image deeper than the most superficial layer of cells using STORM. Despite several months of work, we were ultimately unable to apply FISH to conclusively resolve chromosome copy number in adult brains.

Image-based quantification of Dapi nuclear intensity is another common way to measure ploidy in tissues, but this is most accurate in flat epithelial tissues, where individual nuclei can be resolved and segmented for intensity measurements. This was extremely challenging in the adult brain, where many cells are closely overlapping in all three dimensions. However we agree confirming polyploidy with another method is an important issue, so we revisited this approach. This time we used nuclear lamin staining and nuclear Coin-FLP labeling to aid in manually resolving individual nuclei for Dapi measurements. In this revision, we present new high magnification confocal images of optic lobes from CoinFLP “late-FLP” animals where we induced labelling 24h prior to dissection and labeling with anti-lamin and Dapi. We used the lamin staining to mark nuclear boundaries and Dapi intensity to measure ploidy (normalized integrated intensity). We manually measured over 100 cells and found that the CoinFLP double positive or “yellow” cells always have a greater than diploid DNA content. This also agrees with new FACS data we have added on CoinFLP cells to address point #3. However it should be clarified that due to the stochastic nature of CoinFLP labeling, polyploid cells can also be single-labeled (e.g. green/green or red/red) or unlabeled (did not flip), which are also included in our ploidy measurements (Described in detail in point #14). This new data can be found in Figure 4—figure supplements 1 and 2.

2) The animals analyzed in this study are 1 week increment up to 8 weeks; the age related increased in polyploidy begins at 7 days and continues to accumulate up to 21 days (week 3) after which reaches a plateau. This age group should be considered middle age and therefore the data do not support the statement in the Abstract "that "polyploidy protects against DNA damage-induced cell death".

We thank the reviewers for this comment. We do agree that 7day animals cannot be considered “old” and we have changed the Abstract to read “in the adult brain” instead of “in the ageing brain”. We have also made a much clearer distinction in this revision about polyploidy in the early adult vs. aged brains (see Results section, Figure 6 and Discussion). Our RNA sequencing shows that 7day old optic lobes already exhibit a DNA damage-associated gene expression signature (Figure 5—figure supplement 1), suggesting that even early adult animals are coping with accumulating DNA damage and this is an issue we discuss further in this revision. This is similar to defects in proteostasis associated with aging that are already detectable during early adulthood, as described for C. elegans (Morimoto, 2020)

3) The COINFLP system applied to demonstrate that cells reenter the cell cycle shows correlative data; green, red and yellow cells should be FACS enriched to measure the frequency of polyploid cells in the yellow fraction. This is important because while it is understood that yellow labelled cells are assumed to be polyploid this has not been formally demonstrated. Likewise, in the silencing experiments (cdc6 and Geminin) yellow cells are assumed polyploid and even labelled as such in Figure 4G but no quantification of DNA content is ever provided.

We agree that showing that the CoinFLP “yellow” cells are polyploid via FACS will greatly strengthen our study. To address this we have performed additional FACS and have added a supplemental figure showing >97% of all CoinFLP double-labelled cells display >2C DNA content (Figure 4—figure supplement 2). We also provide verification of these FACS results with direct DAPI intensity measurements from confocal images (please see response to point #1 and Figure 4—figure supplement 1).

With regard to the knockdown experiments, we apologise for the lack of clarity. The changes in ploidy for the cdc6 and geminin experiments were measured by flow cytometry, not CoinFLP. We knocked down the respective genes in neurons (which are ~90% of cells in the brain) using the driver nSybGAL4 and measured changes in the % of cells showing >2C DNA content by flow cytometry. (Figure 4G) We have clarified this in the figure legend as well as the text.

4) The RNA seq data have not been validated, and again show correlative results.

We thank the reviewer for encouraging us to provide additional information on the RNAseq dataset to strengthen the paper. There are two commonly used types of validation, 1. Comparison to an external dataset to verify sample quality/comparisons and 2. Individual gene fold-change confirmation by another method – most commonly qRT-PCR. Over the past 3 years we have anecdotally found that qRT-PCR is actually less reproducible and accurate for measuring gene expression across 3-6 biological replicates than RNAseq (e.g. data within (Flegel et al., 2016) ). This has been explored more thoroughly by others, which have also come to the conclusion that RNAseq with 3 or more replicates is more reproducible and accurate to measure gene expression change than qRT-PCR, especially when no accurate, unchanged reference gene is available for normalization, as is the case for brain aging. (See for examples: (Griffith et al., 2010;Pombo et al., 2017;Zhang et al., 2019)) We therefore focused on ensuring the accuracy of our comparisons by using external datasets to confirm that our RNAseq correctly identifies previously published age-specific gene expression changes, region-specific expression and sex-specific gene expression patterns. See new Figure 5—figure supplement 2 and Supplementary file 3 and references therein.

5) There are three main part to the model presented.

a) DNA damage accumulating with age. This result is not surprising and there are several examples that show this in various tissues, including in brains. We also think that the staining and imaging for pH2AV must be greatly improved. There are several other experiments that could benefit from images, or better images. The authors must look at their experiments and see where this can be added meaningfully or experiments done.

We have included better representative images of pH2AV in Figure 5C through D’. We have also included more and higher resolution images of CoinFLP labeled cells in Figure 4—figure supplement 1. Please note that ELAV immmuno-fluorescence in older brains does not look as strongly nuclear as in young brains and appears as subnuclear puncta. This has also been observed in work from other labs studying the fly brain at ages beyond 1 week. (Frost et al., 2016).

b) "Exogenous" DNA damage leading to increased polyploidy. I think the authors were very careful to use the exogenous damage here as their analysis of ploidy after PQ treatment might not reflect normal polyploidy due to normal levels of stress and age. In addition, their analysis is done in bulk tissue and not on a single cell basis to correlate a cell's DNA damage with its ploidy. It would be interesting to further test the idea that DNA damage induces polyploidy by over-expressing or knocking down a DNA repair factor such as Prp19 and then measuring the proportion of cells that are polyploid. Again, this is experimentally feasible speedily, but if the authors choose not to do this, these must have a cogent and strong reasoning.

We agree with the reviewers’ comment and we are keen to perform additional experiments to address this. However we received the reviewers comments a few weeks before our research operations shut down due to COVID-19. Our lab has only partially re-opened under a part-time shift schedule as of July 1st and we have finally acquired several lines to attempt to address this issue. These lines include UAS-Prp19 from Dr.Thomas Flatt’s lab, and other lines from various stock centers to knock down key players in the DNA damage response that show changes with age such as Irbp, Ku80, xrp1, rad51b, lig3, mus308 and xpd. We plan to knock these factors down in neurons as well as glia to interrogate which DNA damage pathways may play a role in inducing polyploidy. Since these experiments require 1-3 week age adult animals, we have written to the editor that a realistic timeline for these experiments is an additional 2-3 months. We would like to propose to perform these experiments and submit our findings as a preprint linked to this manuscript. We suspect DNA damage leads to increased polyploidy non-autonomously, however at this time we cannot rule out cell-autonomous effects. In this revision we also provide additional data addressing this issue (see response to point 5C and response to point #9).

c) Polyploidy protecting from cell death. We think this idea is founded in the literature from other cell types, and thus it is a reasonable hypothesis in the fly brain. However, we do not think the experiment presented is sufficient to make this a solid conclusion. A change in the percent of dying cells from 7 to 2.5 (diploid vs polyploid) from cell counts that are already very small and from brains that are pooled does not seem like a reliable experiment. We think additional experiments should be done. For example, the authors could use a GAL4 expressed in a restricted sub-population of cells (ex. SPGs via R54C07-GAL4 which was used in Kremer et al., 2017) to label and directly count the number of cells in the optic lobes via light microscopy (it should be <100 cells per lobe). Then, RNAi against cdc6 and geminin (as in Figure 4G) could be used to manipulate the proportion of cells that are polyploid. Using such an approach would allow the authors to link the proportion of cells that are polyploid to the total number of cells and thus directly show that polyploidy protects the cells from DNA damage-induced cell death.

We thank the reviewers for suggesting this experiment. Using a driver for SPG may not be ideal because the SPGs become polyploid very early in development, and their endocycles and endomitoses are critical for the growth of the brain in the larval stages (Unhavaithaya and Orr-Weaver, 2012). Since these cells will already be highly polyploid at eclosion, we will not be able to manipulate their ploidy specifically in the adult brain. We would instead like to manipulate the ploidy of a cell type that is diploid during development and becomes polyploid specifically in the adult, such as the glia of the optic chiasm. We have finally obtained lines to allow us to manipulate ploidy in these cells, and we propose to include our findings in a preprint linked to this manuscript.

We have also added new data that addresses this question in another way. To show that polyploid cells are protected from cell death induced by DNA damage, we performed an experiment similar to that shown in Figure 6G. We induced DNA damage in flies 21d old (a time when the OL shows high levels of polyploidy). We then performed a timecourse to identify the window where cells show high levels of death in response to UV. We see very high levels of cell death (>15% of cells incorporate PI) 24h post UV exposure. When we measured the ploidy of the dying cells, we see that while ~24% of diploid cells undergo cell death, only ~3% of the polyploid cells do. This much more convincingly shows that polyploid cells are protected from DNA damageinduced cell death. This data is now included in Figure 6.

With regards to pooling brains, we understand that it does not give us information about individual brains, but since the proportion of polyploid cells that are dying are so low, we needed to pool brains to ensure that our statistics are robust.

6) A criticism of the work as presented is that the vast majority of the polyploid cells are tetraploid, yet the authors do not address the possibility that these cells simply have replicated their DNA prior to dividing (i.e. are transiently polyploid) as versus remaining indefinitely polyploid. Earlier work from other laboratories suggests that this unlikely, and the manuscript would be improved by a discussion of the possibilities.

We have considered the possibility that the cells with 4C DNA content are simply in G2 and poised to undergo mitosis. However, we have stained for PH3 to look for mitosis in nearly 100 adult brains at different ages and have never observed a convincing mitotic event. We have also stained for G2 markers (e.g. CycA protein) many times at many ages and we have never resolved any convincing, reproducible staining. Importantly, most of the polyploid cells are neurons. We know from other experiments in the developing brain that pushing a neuron into mitosis is catastrophic and results in neuronal death. It is possible that a low level of mitoses occur that we have missed due to rapid cell death. We address this further in the revised Discussion.

7) The authors state that up to 20% of the cells in the optic lobes are polyploid. However, later in the text (and the figure legends) the number is 25%. Please be consistent.

Thank you to the reviewers for catching this. We have made the change in the text to be more consistent.

8) In the Discussion, the authors propose that polyploidization may permit cells to maintain contacts when some of their neighbors die. This hypothesis also was proposed by Unhavaithaya and Orr-Weaver who should be cited. The BBB example is particularly compelling b/c the contacts create the barrier.

Thank you for this helpful comment, we have included this in the revised text.

9) The authors state “It is also possible that we see higher levels of DNA damage foci in the optic lobes because they have more polyploid cells” but do not offer an explanation for why this could be biologically relevant.

We have removed this statement. During the original manuscript drafting we considered the possibility that polyploid cells accumulate DNA damage without dying, and therefore may over time, ultimately acquire more DBSs than diploid cells thereby biasing the pH2Av staining pattern. To test whether this is true, we measured pH2Av fluorescence and Dapi integrated intensity to examine the correlation of ploidy and number of DSBs. We found no correlation between ploidy and pH2Av staining (See Author response image 1), so we have removed this comment.

Author response image 1

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10) Also, the authors state “We see an upregulation of cell cycle-associated genes specifically in the optic lobes with age. The transcription of cell cycle genes and genes involved in the DNA damage response and repair are intimately coordinated and can be controlled by the same factors.” Please complete the argument – namely that upregulation of DNA damage response and cell cycle genes may share an upstream regulatory mechanism.

Thank you for this helpful comment, we have included this in the revised text.

11) The authors state “Whether cell cycle re-entry is a cause or a consequence of neurodegeneration has been difficult to test since both are associated with age and damage.” An earlier paper would seem to be relevant and could be cited: Mutations in String/CDC25 inhibit cell cycle re-entry and neurodegeneration in a Drosophila model of Ataxia-telangiectasia. Rimkus et al., 2008.

Thank you for pointing this out to us. We have included this citation in our Discussion.

12) In Figure 1, there is an intriguing dip in the % polyploidy after day 21 in males and day 28 in females. What are the authors' thoughts about this? Could the initial wave of polyploidization result in death and then be followed by a wave of polyploidization that results in persistent, stress-resistant cells?

This is an interesting point. We looked closely and we do not observe any significant difference in cell death between 21 and 28 days in males or 28 and 35 days in females (Figure 6). In both cases, most of the cells that die are diploid, so we do not have evidence for a wave of cell death eliminating polyploid cells. Although it is possible that it is transient and rapid, and therefore missed in our timecourse.

Another possibility is that there is a wave of mitosis, reducing tetraploid cells to diploid. We have examined this time window for mitoses using PH3 staining and have never observed any mitoses at this age. However, it is again possible that we have missed a transient wave of mitosis. If this is the case, we would expect higher ploidies (e.g. above tetraploid) to remain or increase, while tetraploid cells decrease. We examined the data more carefully to see if this occurs. When we look at male or female Canton-S after 28 days we see both tetraploid and >4N cells dip, suggesting the dip in polyploidy cannot be solely explained by a wave of mitosis resolving tetraploid cells (Figure 1D shows data for males). We do not have an explanation for this dip after 28 days, but a more detailed analysis of alternative modes of cell division or a finer timecourse analysis of cell death at these ages is needed.

13) In Figure 3, the # of double-labeled cells from early flp significantly increases from Day 1 to Day 14 – doesn't this suggest there is some cell fusion? Also, image B' is from a 30 day animal, while the quantitation in C is from 14-day animals. What does the quantitation look like at 30 days? Does the number of yellow cells increase further?

Thank you for pointing this out. Although we believe the majority of these yellow cells from the early flip are sub-perineurial glia we do not rule out a very low level of cell fusion. However, the level of fusion does not account for the vast majority of the polyploid cells we observe in later stages. We have changed our language to clarify this point.

The representative image in Figure 4B shows CoinFLP “late-FLP” labelled OLs from 30 days from the original CoinFLP lines which express membrane tagged UAS-RFP and LexA_op-GFP. The membrane GFP and RFP posed two issues for quantifications; 1. We encountered membrane staining that was difficult to discern due to cell wrapping and 2. Like (Bosch et al., 2015) we observed unequal labeling, leading to far more LexA_op labelled cells than GAL4 labelled cells, resulting in far fewer RFP positive cells, compounded by the RFP signal being weaker than the GFP fluorescence. To overcome this, we generated a fly line in our lab that had nuclear-localized fluorescent proteins for better identification of polyploid nuclei and switched the colors of the lexA and Gal4 expressing cells (LexA_op-RFP_nls and UAS-GFP_nls). This greatly improved our ability to resolve double labeled cells (Fiugre 5—figure supplement 1). We performed our quantification in 14 day animals because older brains frequently have aggregates of fluorescent proteins that show up as puncta in images (see Author response image 2). We think this is because RFP accumulation over time eventually becomes toxic to cells. We have performed flow cytometry on 21d Coin-FLP labeled optic lobes and indeed we see an increase of polyploid, double-labeled CoinFLP cells from 4.4% (14d) to 7% (21d). We did not include this data since we only had one replicate and we have not performed additional quantifications at later ages due to the lab Covid-19 shut down.

Author response image 2

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14) For the data presented in Figure 4, the labeling of 300 cells/optic lobe is impressive. However, if there are ~20,000 cells/optic lobe, then the 300 cells would be only ~1-2% of the total, not the estimated 25% that you measure as >4N. Can you explain the discrepancy? (BTW, we think lineage tracing methods such as Permatwin under-report dividing cells in the adult brain).

We thank the reviewer for encouraging us to explain the calculations more thoroughly. We do agree that the number of 300 per lobe is lower than expected and we think that this is, in part, because we were very conservative in our quantification and identification of double labelled cells in images. However, the numbers actually are roughly in line with what we observe via imaging and flow cytometry when the stochastic nature and biases of CoinFLP labeling are taken into account.

Here is a sample calculation to explain: If we assume 100% of cells “flip” to label with CoinFLP (which we know based on FACS and imaging we are not approaching 100%) the flipping of LexA (red) : GAL4 (green) ratio is biased 4:1 (Bosch et al., 2015). Thus if flipping is complete, we would expect 80% of diploid cells to label red and ~20% to label green. If (for simplicity) we assume that all the polyploid cells are tetraploid, and that 20% of the cells in the OL (20% of 30,000 = 6000 cells) are tetraploid, 4% of all polyploid cells will label green/green (probability of green is 0.2 therefore 0.2* 0.2=0.04*100), 64% red/red (probability of red is 0.8 thus, 0.8*0.8=0.64*100) and 32% will label green/red or red/green and appear yellow or “double-labelled”. The expectation would be then, that 1,920 cells will label yellow per optic lobe under complete 100% flipping conditions. However, as mentioned above, with our heat-shock protocol we do not label 100% of cells. The amount of flipping is variable from animal to animal and we estimate that in our samples with the lowest flipping we flip about ⅓ of cells and with our strongest flipping we label about ¾ cells. If we label ~50% of cells, we would expect about 900 yellow cells per optic lobe, which is much closer to what we observe for CoinFLP by FACS and in our imaging (Fiugre 5—figure supplement 1 and 2). We have tabulated some of these numbers comparing CoinFLP quantification by imaging and by FACS in Author response table 1 to clarify.

15) For Figure 6, it would be interesting to pulse the flies with paraquat, then chase a few days without paraquat before assessing ploidy. What if the polyploid cells are en route to dying? This seems unlikely for UV irradiated flies, b/c the irradiation was done 5 days prior to dissection. However, in Figure 5D, the fact that the control and paraquat-treated lines cross at 21 days seems to support the idea that some of the polyploid cells in the paraquat-treated animals die and/or divide. Please comment.

This would be an interesting experiment to perform because we think that the animals cultured on low dose of PQ for an extended period of time may be exhibiting adaptations to chronic oxidative stress. In Figure 6D, we do observe a slightly lower % of polyploidy at 21d than at 14d, but this is not statistically significantly different from either of those two timepoints. We interpret the results of this experiment as flies treated with PQ reaching “peak” polyploidy levels earlier than their untreated counterparts (at 7d instead of 21 or 28). We also see (Figure 6—figure supplement 1) that there is no significant difference in % of cell death between control and PQ treated animals. However, in all time points (21d shown below), most of the dying cells are diploid.

16) In Figure 6—figure supplement 1. It appears that pH2AV levels are elevated in all neurons in the paraquat treated brains. However, only a fraction of these die. Why might this be?

This is in interesting observation and there are two parts to address. The first is specific to our paraquat treatment. The dose is intentionally low to mimic chronic oxidative stress with age and this might be inducing a low “below threshold” level of DNA damage in neurons. The second part to address is that this is widely observed with DNA damage – where damage occurs in many cells, but only some cells of a tissue die while others can be protected from death, despite sustaining high levels of DNA damage. Work from other labs has shown that not all the cells exposed to DNA damage undergo cell death and that various factors such as cell type, cell signaling etc. can influence this (Jaklevic and Su, 2004; Verghese and Su, 2017). Finally, recent work from the Abrams lab (DOI: 10.1091/mbc.E18-12-0791) has shown that high levels of DNA damage to the postmitotic adult fly brain does not induce cell death with the canonical kinetics established in embryos or wings. Our data also agrees with this and instead of death at 1-8h post damage (which they examine) we observe very delayed kinetics (16-24h post damage, see new data in Figure 6) with death resolving after 24h, despite high levels of DNA damage persisting. The Abrams work attributes this lack of cell death to noncanonical functions of p53 in the adult brain (which undoubtedly is true), but here we show there actually is an acute death response in the adult brain, at time later than what they examined. We believe this response is likely p53independent. There is a whole area of work to be done here to resolve how postmitotic tissues signal and cope with DNA damage, which we hope to pursue in the future.

Author details

1. Shyama Nandakumar

   Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0624-3452

2. Olga Grushko

   Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, United States

   Contribution

   Conceptualization, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

3. Laura A Buttitta

   Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Investigation, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   buttitta@umich.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5064-0650

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