Proteostasis failure and mitochondrial dysfunction contribute to chromosomal instability-induced microcephaly

Domain: biology
Published: 2026-03-12
Editorial outcome: major_revision

Abstract
Mosaic variegated aneuploidy (MVA), a rare human congenital disorder that causes microcephaly, is characterized by extensive abnormalities in chromosome number and results from mutations in genes involved in accurate mitotic chromosome segregation. To characterize the cellular mechanisms underlying this disease, here we generated a Drosophila model of microcephaly caused by the depletion of a single spindle assembly checkpoint (SAC) gene in the neural stem cell (NSC) compartment. We present evidence that loss of stemness - compromised identity and proliferative capacity of NSCs- plays an important role in MVA and results in a reduced number of neurons and glial cells. We show that loss of stemness arises from the accumulation over time of an unbalanced number of gains and losses of more than one chromosome, rather than a direct consequence of chromosomal instability-induced DNA damage or the production of simple aneuploidies. We unravel a contribution of proteostasis failure and mitochondrial dysfunction to the negative impact of complex aneuploidies on stemness, a highly energy demanding cellular state. We identify overexpression of Radical Oxygen Species scavengers, mitochondria chaperones and apoptosis inhibition as genetic interventions capable of dampening the deleterious effects of aneuploidy on brain size.

Introduction
Mosaic variegated aneuploidy (MVA) is characterized by widespread mosaic aneuploidies, involving different chromosomes and tissues. MVA patients can present growth retardation, early childhood cancer, and microcephaly 1–4. MVA is caused by pathogenic mutations in components of the spindle-assembly checkpoint (SAC) and proteins involved in centrosome dynamics during mitosis 5–10. In cells from MVA patients carrying bi-allelic mutations in these genes, defects in chromosome segregation cause chromosomal instability (CIN), which results in the generation of aneuploid karyotypes. In mouse models of MVA-driven microcephaly caused by mutations in SAC genes, a dramatic reduction in the neural stem cells (NSCs), their intermediate progenitors, and post-mitotic neurons is associated with massive cell death 11, which is largely independent of the tumor suppressor gene p53 12. The Drosophila brain, whose development and growth also relies on the proliferative activity of NSCs, has proven to be a valuable model in which to demonstrate a causal relationship between chromosome segregation errors and microcephaly 13–15. By combining changes in centrosome number (amplification or loss) with mutations in SAC genes or by acute targeting of chromosome cohesion, these studies have shown that the resulting brains present extensive abnormalities in chromosome number, a dramatic reduction of the NSC population, and small brain size.

Unfortunately, the underlying cellular and molecular mechanisms leading to NSC loss and microcephaly are largely unidentified.

Here we have generated a fly model of microcephaly caused by depletion of a single SAC gene, as occurs in MVA patients. We present evidence that targeted depletion of the SAC in NSCs induces microcephaly in flies and that loss of NSC stemness—compromised identity and proliferative capacity—plays an important role of the disease and results in a reduced number of neurons and glial cells. We have characterized the response of the developing brain to CIN-induced DNA damage and different types and levels of aneuploidy and have identified the cellular and molecular mechanisms causing CIN-induced loss of NSC stemness. We present evidence that NSCs are highly resistant to DNA damage and that simple aneuploidies, consisting of single chromosome gains, have a mild impact on the identity and proliferative capacity of these cells and on brain size. In contrast, our results indicate that loss of stemness of SAC-depleted NSCs results from the accumulation, through consecutive cell cycles, of complex aneuploidies consisting of an unbalanced number of gains and losses of more than one chromosome. We performed transcriptomic analysis to unveil a strong effect of CIN on cellular homeostasis. Thus, SAC-depleted brains showed a reduction in genes involved in ribosome biogenesis and RNA metabolism, activation of protein quality control mechanisms (autophagy), and reduction in mitochondrial activity. We demonstrate that proteostasis failure—most probably due to aneuploidy-induced gene dosage and proteome imbalance—and mitochondrial dysfunction contribute to the deleterious effects of complex aneuploidies on NSC stemness, a highly energy demanding cellular state. Lastly, we present evidence that genetic interventions aimed at restoring mitochondrial homeostasis and blocking apoptosis can mitigate the negative impact of complex aneuploidies on brain size.

Results
Targeted depletion of SAC genes in neural stem cells induces microcephaly

The central region of the Drosophila larval brain contains neural stem cells (NSCs), which are of embryonic origin and re-enter the cell cycle after a quiescence period in the first instar larval stage (around 32 hours after egg laying, AEL) 16. Among them, we find the type I neuroblasts (NBs), which divide asymmetrically to self-renew and produce ganglion mother cells (GMCs), which will divide once more to give rise to two neurons or glial cells. NBs will differentiate into GMCs or die at the end of larval development, and differentiating neurons and glial cells will build the adult central brain (Fig. 1D). In humans, MVA-driven microcephaly is caused in half of the patients by bi-allelic mutations in single genes with well‐documented roles in spindle assembly checkpoint (SAC) function or correct kinetochore-microtubule attachment 5–10. Our first aim was then to address whether targeted depletion of a single SAC gene could also cause a reduction in larval brain size in flies and whether NBs were the most susceptible cells to CIN. We used the worniu-gal4 driver to induce chronic expression of RNAi forms against the SAC genes bub3 or rod specifically in NBs and to analyze the total number of NBs (labeled by a nuclear GFP/ß-galactosidase fusion protein expressed by the UAS-GFP-nuc-lacZ transgene 17 and by expression of Deadpan, Dpn), GMCs (labeled by the expression of Prospero, Pros), neurons (labeled by the expression of Elav), and glial cells (labeled by the expression of Repo), as well as the size of the brain lobe. We analyzed control brains and brains subjected to chronic SAC depletion in proliferating NBs at four time points of larval development (in second instar at 48 and 62 h AEL, and in third instar at 72 and 96 h AEL). In control flies not subjected to SAC depletion, NB number in the central brain remained constant throughout larval development, and the number of Pros+ cells and the size of the resulting brain increased as a result of the active proliferative capacity of NBs (Fig. 1A, A’). In contrast, in flies subjected to chronic SAC depletion, the number of NBs was gradually reduced (Fig. 1A, A’) and this reduction was highest at later stages of development (Fig. 1A, A’ and Supplementary Fig.1A, A’). We noticed that developmental timing was not affected by SAC depletion (Supplementary Fig. 1C), and that the expression levels of the Bub3 protein were already compromised at early stages (48 h AEL, Supplementary Fig. 1D). The gradual decrease in the number of NBs was also observed upon temporally controlled depletion of the SAC gene using the thermo-sensitive Gal80 system (Supplementary Fig. 1E, E’). In this experimental setup, expression of bub3-RNAi was restricted to the last 24, 48 or 72 hours of development and NB number was quantified at 96 h AEL. Remarkably, at least 48 h of bub3 depletion were required to get a significant effect on NB number (Supplementary Fig. 1E, E’), despite the rapid cell cycles of this population (every 2 h) at these developmental stages 18. We also noticed that activity of the SAC was already compromised after 24h of RNAi expression (Supplementary Fig. 1F). These results point to a delayed response of NBs to CIN, as previously shown 14. Consistent with this delayed response, only a significant reduction in the number of Pros+ cells and in brain size was observed at later stages of larval development upon chronic depletion of the SAC (Fig. 1A, A’, see also Supplementary Fig. 1A’). The size of the brain of those adults that survived to chronic depletion of the SAC was also significantly reduced (Fig. 1E). As expected, the number of neurons and glial cells in developing SAC-depleted brains was also reduced when compared to control brains (Fig. 1B, B’, see also Supplementary Fig. 1A’). These results indicate that the impact of SAC depletion on brain size is a result of a reduced number of GMCs, neurons, and glial cells. To reinforce this notion, we performed lineage tracing of the progeny of control and SAC-depleted NBs using the G-TRACE technique 19. As shown in Fig. 1C, C’, the area covered by the progeny of SAC-depleted NBs was reduced when compared to control samples.

Targeted depletion of SAC genes induces cell cycle arrest in NSCs

We noticed in control brains expressing the UAS-GFP-nuc-lacZ transgene under the control of the worniu-gal4 driver that the GFP protein was not restricted to NBs and was inherited through perdurance of the Gal4 or GFP proteins by their daughter cells, the GMCs (Fig. 1A, lower panels). In contrast, in brains subjected to SAC depletion, the expression of this transgene was very often restricted to NBs and was not observed in the surrounding GMCs, thereby pointing to a failure in the proliferative activity of NBs upon prolonged SAC depletion (see also 13 ). Mitotic activity, monitored with an antibody against a phosphorylated form of histone H3 at serine 10 (PH3) that labels mitotic figures, was first quantified in control and SAC-depleted NBs. As shown in Fig. 2A, A’, mitotic activity was compromised in SAC-depleted NBs of mature larval brains. This reduction in mitotic activity was already visible at early larval stages (Fig. 2B, B’). We also monitored S phase progression by exposing larval brains to 5-ethynyl-2′-deoxyuridine (EdU) for 45 min and subsequently analyzing its incorporation. The number of EdU-positive NBs was also reduced in SAC-depleted brains when compared to control samples (Fig. 2A, A’). Using Fly-Fucci, which relies on fluorochrome-tagged degrons from the Cyclin B (in red) and E2F1 (in green) proteins which are degraded during mitosis or at the onset of the S phase, respectively 20 , we observed that the number of cells not expressing either of these markers and potentially arrested in late G1 were largely increased in SAC-depleted NBs (Fig. 2C, C’). A low percentage of NBs also expressed Dacapo (Dap, Fig. 2D, D’), the ortholog of p21/p27 in flies that negatively regulates the G1-S transition 21. However, co-depletion of Dap did not restore the number of Pros+ cells or the size of the brain (Fig. 2D’), thereby pointing to irreversible cell cycle arrest in SAC-depleted NBs. All these data indicate that reduced proliferation of NBs contribute to CIN-induced microcephaly.

Single aneuploidies have a minor effect on NSC number and proliferative capacity

The observation that the effects of SAC depletion on NB number required at least 48 h (Fig. 1A, A’ and Supplementary Fig. 1E, E’), despite the rapid cell cycles of this population (every 2 h 18), pointed to a delayed response of these cells to aneuploidy 14. Alternatively, this observation might suggest that NB loss and cycle arrest is a result of a high level of CIN-induced aneuploidy or DNA damage accumulated through consecutive cell cycles. We thus analyzed, in this and the following section, the contribution of different types of aneuploidy and DNA damage to the observed phenotypes. In Drosophila, systemic aneuploidies based on trisomies for the X chromosome or the left arm of chromosome II (2L) give rise to mature larvae or pupae that are not able to hatch as adults 22,23. Trisomic larvae were generated by crossing females bearing a compound X-chromosome (consisting of two attached X chromosomes that segregate together) or a compound-2L (consisting of two attached 2L arms that segregate together and two free 2R arms) with wild-type males carrying an ubiquitously expressed GFP transgene on the X or second chromosomes, respectively, thus labeling trisomic female larvae by the expression of the GFP marker. Trisomies for the X and 2L chromosomes were very stable in larval brains, as shown by the presence of the GFP-expressing chromosome in all cells of the central brain (Fig. 3A, B). Based on the developmental delay of trisomic larvae, we quantified the number of NBs and Pros+ cells, as well as brain size throughout the extended larval period and compared these numbers with two types of control euploid larvae, namely those bearing the compound chromosomes and those bearing two unattached chromosomes. The number of NBs was either mildly reduced in the case of X-trisomies (Fig. 3A’) or largely unaffected in the case of 2L-trisomies (Fig. 3B’). Although NB proliferation (monitored by the number of Pros+ cells at different developmental points) was delayed in trisomic animals when compared to the two controls, the final number of Pros+ cells at the end of the larval period was largely unaffected (Fig. 3A, A’, B, B’). A similar trend was observed for brain size (Fig. 3A, A’, B, B’). However, brains trisomic for the X chromosome were still smaller than the control brains at the end of the larval period, pointing to the presence of X chromosome-linked triplosensitive loci affecting the growth of other regions of the brain. All these data indicate that trisomies for the X chromosome and 2L arm have a minor impact on NB viability and proliferative capacity, despite the high number of protein-encoding genes that are potentially unbalanced in trisomics for the X (2196 genes) and 2L chromosomes (2657 genes).

The presence of a single X chromosome in Drosophila males causes an imbalance between X-linked and autosomal genes, which is solved by the dosage compensation mechanism (Supplementary Fig. 2A). The Male-Specific Lethal complex, which can be visualized in male tissues by the expression of the Msl2 protein (Supplementary Fig. 2B), targets the X chromosome in males, leading to de-compaction of the chromatin fiber and increased gene transcription to a similar level to that observed in autosomal chromosomes. In females, RNA binding protein Sex lethal (Sxl) induces the translational repression of Msl2 (Supplementary Fig. 2A, B, 24). Experimental depletion of Msl2 and Sxl in male and female tissues, respectively, causes an X chromosome-wide gene expression imbalance with respect to autosomes (Supplementary Fig. 2C, 25,26). In contrast to the deleterious effects of these manipulations on the growth and development of epithelial tissues in Drosophila 27, the number of NBs was largely unaffected upon Msl2 depletion in males or Sxl depletion in females (Supplementary Fig. 2D). These results reinforce the conclusion regarding the minor impact of single trisomies and their resulting gene dosage imbalance on NB viability and extend this conclusion to single monosomies (in terms of gene expression) caused by Msl2 depletion in male brains.

DNA damage has a delayed impact on NSC number and proliferative capacity

Lagging chromosomes are frequent in cells with SAC defects and are likely to be damaged 28. Consequently, the DNA damage response (DDR) pathway can be activated 29. Interestingly, SAC-depleted NBs showed increased levels of phosphorylated H2Av (pH2Av, a target of ATM and ATR in flies 30), and activation of p53 (monitored by a GFP activity sensor 31, Fig. 4A). However, depletion of DDR genes [Dp53, ATR (mei41 in flies) or chk1 (grapes in flies)] by RNAi or the use of a dominant negative form of Dp53 (eg. Dp53H159N) did not have a major impact on the number of SAC-depleted NBs, and gave rise only to a modest reduction in the size of CIN brains (Fig. 4B, B’). Depletion of DDR genes alone did not cause any major effect on brain size or NB number (Fig. 4B, B’). To further address the contribution of DNA damage to the observed CIN-induced phenotype, we examined whether DNA damage caused by high doses of ionizing radiation (X-ray, IR, 4500 rads) would phenocopy the CIN-induced reduction in the number of NBs or their progeny, as well as the decrease in brain size. Despite strong activation of the DDR pathway, monitored by increased levels of pH2Av in larval brains 5 h after IR, cell death (monitored with an antibody against the cleaved form of the effector Caspase Dcp1, cDcp1), NB number, and mitotic activity were largely unaffected when compared to control non-irradiated brains (Fig. 4C, C’). In contrast, epithelial cells of the larval wing primordium subjected to a similar dose of IR showed high levels of cDcp1 and low mitotic activity 5 h after exposure (Supplementary Fig. 3A-C). cDcp1 and mitotic activity in the wing primordium were restored to control levels 3 days after IR exposure (Supplementary Fig. 3A-C). However, the number of NBs and Pros+ cells was clearly reduced 3 days after IR exposure, at a time at which pH2Av levels were reduced to those of control brains (Fig. 4C, C’, D, D’, see also 32). The temporal delay observed between H2Av phosphorylation, which marks the presence of double-strand breaks (DSBs), and loss of NBs and reduction in the number of Pros+ cells suggests that these phenotypes are an indirect consequence of the presence of DSBs in the tissue, whereby unrepaired DNA would cause genome rearrangements and aneuploidy. Fluorescence in situ hybridization (FISH) analysis of NBs 3 days after IR to count the number of copies of chromosome II revealed a clear increase in the percentage of NBs losing or gaining at least one chromosome (Fig. 4E, E’, note that fly chromosomes in interphase are closely attached in normal conditions, thus the presence of a single chromosome in control NBs). These data suggest that the delayed impact of DNA damage to the brain phenotype relies on the production of aneuploid karyotypes, and they point to a role of DNA damage in enhancing aneuploidy levels in CIN brains rather than an immediate effect on the viability or proliferative capacity of NBs.

Our experimental data on the minor effects of simple aneuploidies on NB viability and proliferative capacity (Fig. 3 and Supplementary Fig. 4) and on the proposed effects of DNA damage in enhancing the aneuploidy levels of CIN brains (Fig. 4) point to complex aneuploidies, consisting of multiple losses and gains of chromosomes, as those responsible for NB loss, cell cycle arrest, and CIN-induced microcephaly. Consistently, FISH analysis of NBs to count the number of copies of chromosomes II and III revealed an increase throughout larval development in the percentage of NBs losing or gaining at least one chromosome upon chronic induction of CIN (Fig. 4F, F’, see also Supplementary Fig. 1B, B’). Moreover, by using two distinct FISH probes to label chromosomes II and III, we observed that most NBs (18/19) accumulated more than one chromosome and showed an imbalanced number of chromosomes. This finding points to this imbalance as the major contributor of CIN-induced microcephaly (Fig. 4F). These results support the proposal that complex aneuploidies accumulated through consecutive cell cycles and consisting of multiple gains and losses of chromosomes exert a negative impact on NB number and proliferative capacity.

CIN induces a transcriptional response in developing brains

To identify the molecular mechanisms underlying the negative impact of complex aneuploidies on NB number and proliferation, we characterized the transcriptional profile of brains subjected to chronic depletion of the SAC gene bub3 and compared it with that of control brains. Among the most upregulated genes (>1.5 fold, 28 genes, Supplementary Data 1) in CIN brains, we observed the presence of four members of the Heat Shock Protein 70 (Hsp70) superfamily of chaperones (hsp70Ba, hsp70Bb, hsp70Bc, and hsp70Bbb, Fig. 5A), mediating protein folding, and Glutathione S transferase E8, which belongs to a protein family involved in ROS scavenging. Gene set enrichment analysis also pinpointed genes involved in vesicle trafficking, autophagy, mitophagy, and protein folding as classes of genes whose expression profiles were also significantly upregulated in CIN brains (Fig. 5C, D, and Supplementary Fig. 4B, see also Supplementary Data 2 and 3). Among the most downregulated genes (>1.4 fold, 33 genes, Supplementary Data 1) in CIN brains, we found genes encoding transcription factors involved in neuronal or glial specification, including, as expected, prospero (e.g., castor, datilografo, huckebein, bric a brac 1), genes involved in mitochondrial homeostasis (e.g., dj-1beta 33,34) and bub3 (Fig. 5A and Supplementary Data 1). Gene set enrichment analysis also highlighted genes involved in RNA metabolism, nucleolar function, and mitochondrial ribosomes as classes whose expression profiles were also significantly downregulated in CIN brains (Fig. 5C, D, and Supplementary Fig. 4B, see also Supplementary Data 2 and 3). Interestingly, the overall transcriptional profile of the mitochondrial genome was also reduced in these brains (Fig. 5B). The transcriptional response of developing brains to the generation of complex aneuploidies was not reproduced by simple aneuploidies. On one hand, when comparing the changes in gene expression caused by CIN or by trisomies for the X or 2L chromosomes, the observed patterns of upregulation and downregulation showed mild but significant negative correlations (Supplementary Fig. 5A). On other hand, gene set enrichment analysis of brains trisomic for the X or 2L chromosomes did not recapitulate any of the gene classes pinpointed in brains subjected to CIN (Supplementary Fig. 5B). All these results point to a reduction in translation (e.g., ribosome biogenesis) and RNA metabolism, activation of protein quality control mechanisms (autophagy), and repression of mitochondria activity as the major responses of developing brains to complex aneuploidies. We next validated some of these responses by immunochemistry or the use of activity reporters and characterized their functional contribution to the deleterious effects of complex aneuploidies on NBs.

Aneuploidy induces loss of stemness in NSCs

In developing NBs, ribosome biogenesis in the nucleolus relies on the activity of the dMyc proto-oncogene, which contributes to their enlarged cell and nuclear size 35. Consistent with the observed transcriptional reduction in nucleolar protein-encoding genes in CIN brains (Fig. 5C, D), a large proportion of aneuploid NBs lost dMyc expression, showed reduced levels of Fibrilarin (a nucleolar protein involved in RNA processing and regulated by dMyc 36,37), and were small (Fig. 5E, E’). dMyc also plays an important role in conferring NB identity by maintaining cortical polarity, a requirement for the intrinsic capacity of NBs to divide asymmetrically and avoid the nuclear entry of the transcription factor Prospero 35. Consistently, a proportion of aneuploid NBs showed nuclear localization of Prospero (Fig. 5E, E’, see also 13,14), and, most interestingly, aneuploid NBs frequently lost the expression of Dpn or Worniu (Fig. 5E, E’), two transcription factors involved in promoting NB self-renewal through asymmetric cell division and avoiding premature differentiation 38,39. All these observations indicate that the aneuploidy-induced reduction in NB number and proliferative capacity is most probably a result of loss of stemness. Overexpression of dMyc or Worniu did not block the elimination of aneuploid NBs (Fig. 5E’’). Only Worniu, when overexpressed, was able to partially rescue the number of Prospero-positive cells in CIN brains (Fig. 5E’’), most probably through its role in driving NB cell cycle progression 38. Neither did depletion of the pro-differentiation transcription factor Prospero (Pros) rescue the reduction in the number of aneuploid NBs caused by CIN (Fig. 5E’’). Apoptosis has alternatively been proposed to play a role in the reduction in brain size caused by changes in centrosome number and SAC depletion 15. To quantify the levels of apoptosis in NBs subjected to bub3 depletion, we monitored activation of the apoptotic machinery with an antibody that detects the cleaved form of the effector Caspase Dcp1 (cDcp1) and an activity reporter of effector Caspases Dcp1 and Drice that becomes fluorescent upon caspase-driven cleavage (GC3Ai 40) Whereas the percentage of control NBs positive for either of these two markers remained close to zero throughout larval development, a low but significant percentage of NBs subjected to CIN were positively labeled by these markers (Supplementary Fig. 4C, C’’). Inhibition of apoptosis with the baculovirus protein p35, an inhibitor of effector caspases in Drosophila, restored the number of NBs subjected to CIN only at early stages of development. However, the dramatic reduction in NB number in mature brains was still observed (Supplementary Fig. 4C’’, see also 13). All these results indicate that the aneuploidy-induced loss of NB identity and proliferative capacity is a consequence of loss of stemness but not cell death or premature differentiation, as previously proposed 13,15. Consistent with this notion, prospero depletion combined with apoptosis inhibition did not rescue the dramatic reduction in NB number in mature brains subjected to CIN either (Supplementary Fig. 4D, D’).

We then addressed whether the loss of stemness - a highly energy demanding cellular state - observed in highly aneuploid NSCs is caused by proteostasis failure and mitochondrial dysfunction.

Proteostasis failure and mitochondrial dysfunction contribute to the loss of stemness

As a follow-up of the identification of a general upregulation of genes involved in protein folding, vesicle trafficking and autophagy in CIN brains, by mean of RNAseq, we next monitored the activity of the two main protein quality control mechanisms, namely the ubiquitin-proteasome system (UPS) and autophagy, in aneuploid NBs. To characterize the in vivo activity of the proteasome in these cells, we used CL1-GFP, a fusion protein created by attaching a proteasome degradation signal to GFP 41. Whereas CL1-GFP was rapidly degraded in control NBs and no accumulation was observed, this protein accumulated in aneuploid NBs (Fig. 6A), pointing to a situation of near saturation of the proteasome in these cells. To reinforce this notion, we used Htt25Q, a fusion protein consisting of human Huntingtin with a polyQ stretch containing only 25 glutamines and fused to a Cerulean fluorescent protein 42. Htt25Q does not form aggregates nor does it cause toxicity on its own. However, the presence of protein aggregates in the cell can seed Htt25Q polymerization 43. Whereas Htt25Q showed diffuse expression in control NBs (Fig. 6B) and did not alter the number of NBs (Fig. 6D and Supplementary Fig. 6B), it accumulated in large foci in aneuploid NBs (Fig. 6B) and caused a further reduction in the size of this population (Fig. 6D and Supplementary Fig. 6B). Together with the UPS, autophagy—a pathway responsible for the lysosomal degradation of cellular components—plays an essential role in maintaining protein homeostasis (proteostasis). High levels of autophagy were induced in aneuploid NBs, as visualized by the punctated accumulation of ChAtg8a (a mCherry-tagged form of Atg8a, which becomes membrane-associated under autophagy induction and localizes to the site of autophagosome nucleation 44) in these cells (Fig. 6C). In control brains, small Atg8a-positive puncta were observed only in small cells around the NBs but never in the NBs themselves (Fig. 6C, arrowheads). Interestingly, boosting autophagy (by overexpressing Atg1 or depleting the activity of Tor through Rheb depletion or the use of Tor mutants in heterozygosis) led to a significant rescue of the number of aneuploid NBs in mature CIN brains, without causing any major effect in otherwise control brains (Fig. 6D and Supplementary Fig. 6A). A similar result was obtained by feeding larvae with the TOR inhibitor Rapamycin (Supplementary Fig. 6C). These results indicate that proteostasis failure, most probably due to an imbalance in the proteome caused by aneuploidy, functionally contributes to the reduction in the number of NBs in CIN brains. However, we noticed that none of the genetic interventions aimed at increasing autophagy was able to rescue the number of Pros+ cells or the size of the larval brain (Fig. 6E and Supplementary Fig. 6C). These results suggest that TOR inhibition or non-physiological levels of autophagy might have a negative impact on the proliferative capacity of highly aneuploid NBs.

The transcriptional upregulation of genes involved in mitochondrial disassembly and the transcriptional downregulation of genes encoding mitochondrial ribosome components (Fig. 5C), together with the reduction in overall mitochondrial transcription (Fig. 5B), pointed to a negative impact of aneuploidy on mitochondrial number or function. The developing brain is characterized by NBs strongly enriched by mitochondria surrounding the centrally localized nucleus (Fig. 7A, B). In aneuploid NBs, we observed large accumulations of mitochondria that were not equally distributed around the nucleus (Fig. 7A, B). Using a MitoTimer reporter, which targets the oxidation-sensitive Timer protein to mitochondria 45, we detected a shift of mitochondrial fluorescence toward red in aneuploid NBs (Fig. 7C), which points either to high rates of mitochondrial oxidation or slow rates of mitochondrial turnover. We then used MitoQC 46, where a tandem GFPP-mCherry fusion protein is targeted to the outer mitochondrial membrane, to analyze mitophagy, an important quality control mechanism that removes damaged mitochondria, in CIN brains. MitoQC exploits the pH sensitivity of GFP to enable differential labeling of mitochondria in the acidic microenvironment of the lysosome as a proxy endpoint readout. Only a few small MitoQC-positive puncta, most of them red, marking mitolysosomes, were observed in progeny cells of control brains (Fig. 7D, arrowheads). In contrast, large MitoQC-positive accumulations were observed in aneuploid NBs, and most (if not all) were positive for both colors (Fig. 7D). We support the proposal that near saturation of autophagy compromises mitophagic activity, thus leading to the accumulation of dysfunctional mitochondria in aneuploid NBs. Consistent with this, overexpression of mitochondria-specific chaperones Hsp60 and Hsp60c, or ROS scavengers Sod2 and GTPx-1 was able to significantly rescue the number of aneuploid NBs observed in mature CIN brains, without causing any major effect in otherwise control brains (Fig. 7E and Supplementary Fig. 7A). These genetic interventions also rescued the number of Pros+ cells (Fig. 7E) and, most interestingly, yielded a complete restoration of brain size (Fig. 7G and Supplementary Fig. 7A). The observation that overexpression of ROS scavengers and mitochondrial chaperones in NB cells fully restored brain size, but only partially NB cell counts, points to a role of mitochondrial dysfunction in compromising not only the fitness but also the mitotic activity of highly aneuploid NBs. Alternatively, these genetic interventions might target, through perdurance of the proteins that are overexpressed, the progeny cells thus restoring their fitness. Notably, the inhibition of apoptosis through p35 expression rescued the number of Pros+ cells and resulted in a complete restoration of brain size (Supplementary Fig. 4C’’ and Supplementary Fig. 7B), despite no concomitant rescue of NB numbers. These findings can be explained by inheritance of p35 by progeny cells and points to a role of apoptosis in the elimination of these cells.

Methods
Fly maintenance, husbandry and transgene expression

Strains of Drosophila melanogaster were maintained on standard medium (4% glucose, 55 g/L yeast, 0.65% agar, 28 g/L wheat flour, 4 ml/L propionic acid and 1.1 g/L nipagin) at 25°C in light/dark cycles of 12 h. When not specified, the sex of experimental larvae was not considered relevant to this study and was not determined. The strains used in this study are summarized in Supplementary Data 4. Research in Drosophila is not subject to animal experimentation regulations. All experiments were performed in both sexes, except those altering Sxl or Msl2, which inherently have sex-specific roles and so were treated separately by sex.

Standard induction of CIN and gene expression

Virgin female flies carrying the wor-GAL4 driver and UAS-bub3-i were crossed with male flies carrying the gene of interest for each experiment. Fertilized females were allowed to lay eggs at 25ºC and the resulting larvae were switched to 29oC 3 h after egg laying (AEL) in time point experiments, and 8 h AEL for the rest of the experiments. Brains were dissected after 48, 62, 72 or 96 h AEL. Both female and male larvae were used, except when characterizing Sxl and Msl2, in which females and males were considered separately. UAS-gfp-RNAi was used as a control for those experimental UAS transgenes targeting the corresponding pathways or genes. When using the Gal80ts system, flies and their progeny were maintained at 18ºC to keep GAL4/UAS suppressed, and shifted to 29ºC to inactivate the temperature-sensitive Gal80ts form and activate the GAL4/UAS system.

Pupariation assays

Flies were allowed to lay eggs for 4 h at 25°C after which they were switched to 29ºC. Hatching larvae were counted. Number of pupae was scored always at the same time point and 24h apart. The experiment was performed in four replicates, and the resulting percentage of pupae was calculated with respect to the total number of larvae that hatched (47 larvae in the control group and 35 in experimental group) or the total number of larvae that survived until pupal stages (47 larvae in the control group and 30 in experimental group).

Rapamycin treatment

Flies of interest were crossed and allowed to lay eggs for 12 h at 25ºC. Larvae were then kept for 48 h AEL at 25ºC before being shifted to 29ºC for 24 h. Afterwards, rapamycin was added to reach a final concentration of 40 µM, while controls received DMSO at a final concentration of 0.4%. Larvae were maintained for approximately 48 h more at 29ºC and then dissected.

Colchicine assay

Flies of interest were crossed and allowed to lay eggs for 24 h at 18ºC. Larvae were maintained at 18ºC during development, except for the last 24 h, when they were shifted to 29ºC to inactivate the Gal80ts and thus permit activation of the GAL4/UAS system. Larvae were dissected and incubated with 50 µM colchicine for experimental condition and 0.05% DMSO for controls for 4 h at 25°C in Schneider’s media (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Invitrogen) before immunostaining.

Irradiation

Whole wild-type larvae were X-irradiated with 4500 rads in an YXLON MaxiShot X-ray system. Larval brains and wing discs were dissected and analyzed at the indicated time points after the irradiation.

Chromosome labeling by FISH

Fluorescence in situ DNA hybridization (FISH) was carried out to label the 2nd and 3rd chromosomes in brain cells. Briefly, larval brains were dissected in cold PBS and fixed with 4% paraformaldehyde for 20 min. Fixed tissues were washed with PBS-Tr (PBS1X; 0.3% Triton X-100) and transferred gradually into pre-Hybridization Mixture (pHM) (50% formamide; 4X SSC; 100mM NaH2PO4 pH7; 0.1% Tween 20). 150 ng of AACAC(2nd) -Alexa 488 - (AACAC) ) 53and dodeca (3rd)-Alexa 488-CCCGTACTGGTCCCGTACTGGT 54 chromosome probes were added and probe/chromosomal DNA was denatured by heating the sample to 91°C for 5 min. Hybridization was performed at 37°C overnight. After post-hybridization washes, brains were blocked and incubated overnight at 4°C in PBS-Tr; 10% Normal Goat Serum (NGS) with the primary antibody (anti-DPN). DAPI was used to counterstain DNA. Confocal sections covering the entire nuclei were taken and analyzed using Fiji Software (NIH, USA).

Immunohistochemistry

Larvae of interest were selected, and brains were dissected in phosphate-buffered saline (PBS), fixed for 20 min in 4% formaldehyde in PBS, and stained with antibodies in PBS with 0.3% BSA and 0.2% Triton X-100. Primary and secondary antibodies are summarized in the Supplementary Data 4.

DNA synthesis

Click-iT™ Plus EdU Alexa Fluor™ 647 Imaging Kit from Invitrogen (C10640) was used to measure DNA synthesis (S phase) in proliferating cells, following the manufacturer’s indications. EdU (5-ethynyl-2´-deoxyuridine) provided in the kit is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis. Larval brains were incubated with EdU for 20 min and then permeabilized in PBT of 40 min. The samples were incubated with the Click-iT reaction cocktails for 45 min at room temperature.

Generation of trisomic larvae

To generate trisomic larvae for the X chromosome, C(1)DNX, y virgin females were crossed with GFP-FRT18A males (see Supplementary Data 4). Trisomic X female larvae were identified based on the absence of testes and the presence of GFP. To generate trisomic larvae for the 2L chromosome, C(2L)SH1 virgin females were crossed with Oregon R males (see Supplementary Data 4). This cross produces both monosomic and trisomic embryos for the 2L chromosome. However, monosomic embryos do not survive past embryogenesis, ensuring that all larvae in the progeny are trisomic and available for dissection.

RNAseq library preparation

The samples used for this study were late third instar (L3) larvae from Drosophila (96 h AEL). The genotypes of control larvae were (i) w; worniu-gal4, uas-gfp-RNAi; (ii) C(1)DX/Y; and (iii)C(2L)SH1, while the genotypes of the experimental larvae were (i) w; worniu-gal4, uas-bub3-RNAi; (ii) C(1)DX/X (trisomic for the X chromosome) and (iii) C(2L)SH1/+ (trisomic for the 2L chromosome). Three independent biological replicates were performed on different days, with three brains dissected per genotype and replicate. All larvae used in these experiments were female. The brains were collected in 45 µl of lysis buffer containing 20 mM DTT, 10 mM Tris-HCl pH 7.4, 0.5% SDS, and 0.5µg/µl proteinase K, incubated at 65 ºC for 15 min, and immediately frozen until processing. RNA isolation and library preparation were done at the IRB Barcelona Functional Genomics Core Facility. RNA was treated with DNAse I and purified using magnetic beads (RNAClean XP, Beckman Coulter). RNA quantity was measured with the Qubit RNA HS Assay kit (Invitrogen), and integrity was assessed with the Bioanalyzer 2100 RNA Pico assay (Agilent). Poly-A mRNA was purified from 150 ng of total RNA using the kit NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). Dual-indexed cDNA libraries were generated using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs). All libraries underwent 13 min of RNA fragmentation and 13 cycles of PCR amplification. The final libraries were quantified using the Qubit dsDNA HS assay (Invitrogen) and subjected to quality control using the Tapestation HS D5000 assay (Agilent). An average size of 380 bp was confirmed. An equimolar pool was prepared with all the libraries for SE50 sequencing on a NextSeq 2000 (Illumina), and at least 26 million reads were obtained for each library.

Data processing

Adapters from reads were removed using trim galore (v.0.6.7) (https://github.com/FelixKrueger/TrimGalore) with default parameters. Resulting reads were aligned to the dm6 D. melanogaster genome version using STAR (v.2.7.10a) 55. The count matrix was generated in R (https://www.R-project.org/) through the function "featureCounts" of the Rsubread package 56.

Differential expression

Differential expression was performed using the DESeq2 package 57. The function lfcShrink was used to attenuate the influence of low counts in fold changes (FC Shrunk in figures). For plotting purposes and generating principal components, the count matrix was normalized using the "vst" function. In the case of trisomic brains, normalization and DEG analysis was performed chromosome by chromosome.

Gene set enrichment analysis

Gene set enrichment analysis was performed using the roastgsa package 58. GO and KEGG pathways were downloaded using the org.Dm.eg.db package.

Comparison of DEGs and NES between genotypes

A linear model was fitted to the log2 fold changes of CIN and trisomies. R squared and association p-value are reported. The regression line and the corresponding equation was added to the plot.

Confocal microscopy

Samples were analyzed using the following confocal microscopes: LSM 780 Zeiss and LSM880 Zeiss fitted with a Fast Airyscan module (Carl Zeiss). Z-stacks were acquired using either the laser scanning confocal mode or the High-Resolution mode (Airyscan). Image acquisition was done with 40x and 63x oil lenses. The most representative image is shown in all experiments.

Brain size and cell quantification

Number of NBs was quantified manually using Fiji Software (NIH, USA) according to Dpn staining and nuclear size (>7µm). Only the NBs located in the ventral part of the central brain were taken into consideration. Size of the whole brain lobe, represented as area, was measured manually using Fiji Software on Z stacks obtained using a Zeiss LSM780 confocal microscope at 40x oil immersion objective. The numbers of progeny cells (GMCs, neurons and glia) were determined using IMARIS Software with the ‘Spots’ tool, according to Pros, Elav or Repo staining, respectively. Areas of G-TRACE and Ph2Av were measured with IMARIS software using the ’Surface’ tool. UAS-gfp-RNAi was used as a control for those experimental UAS transgenes targeting the corresponding pathways or genes.

Statistical analysis and reproducibility

Statistical analysis was performed by unpaired equal-variance two-tail Student’s t-test when comparing the difference in means between two groups. We used a one-way ANOVA to compare three or more groups within the same experiment. In contrast, a two-way ANOVA was employed when there were multiple comparisons across different groups at various time points. Results for pairwise comparisons were adjusted by multiple contrasts using Tukey/Šídák correction, depending on the experiment (details in figures and Supplementary Data 4). Differences were considered significant when p values were less than 0.0001 (****), 0.001 (***), 0.01 (**), or 0.05 (*). All genotypes included in each histogram or scatter plot were subjected to the same experimental conditions (temperature and time of transgene induction) and analyzed in parallel. Mean values and standard deviations were calculated and the corresponding statistical analysis and graphical representations were carried out with GraphPad Prism 10 statistical software. All quantifications were carried out in at least two independent replicates per genotype. Representative micrographs shown in all figures are based on at least 10 different brains per genotype and experimental condition.

Data availability

Materials generated for the study are available from the corresponding author on request. The raw and processed RNAseq data have been deposited in the GEO database under accession numbers GSE290206 (CIN data) and GSE301548 (trisomies data). Source data are provided with this paper in Source Data File.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study investigates the cellular and molecular mechanisms driving microcephaly in mosaic variegated aneuploidy (MVA), a rare congenital disorder caused by defects in the spindle assembly checkpoint (SAC) that leads to widespread chromosomal instability (CIN). A fly model was used to mimic MVA by selectively depleting the SAC gene bub3 in neural stem cells (NSCs). The authors propose that the microcephaly is driven by a loss of NSC stemness, not by apoptosis or premature differentiation. This loss of stemness is largely driven through the deleterious effects of complex aneuploidies (multiple chromosome gains/losses) that accumulate over time—simple aneuploidies and DNA damage alone had minimal impact. Further transcriptomic analysis revealed downregulation of ribosome biogenesis, RNA metabolism, and mitochondrial gene expression. Upregulated genes included those involved in autophagy, proteostasis pathways, and stress response genes. Accordingly, aneuploid NSCs exhibited impaired proteasome function and accumulation of protein aggregates, autophagy saturation, and mitochondrial dysfunction including oxidative stress & defective mitophagy. Genetic rescue experiments, including activation of autophagy and overexpression of mitochondrial chaperones and ROS scavengers, could partially restore NSC populations.\nOverall, the data presented in this paper are of high quality and the use of the fly as model system is warranted. The findings largely mirror a previous study by the group, which evaluated proteostasis failure and mitochondrial dysfunction in response to aneuploidy/CIN in the fly wing imaginal disc, which is a simple epithelial tissue whose developmental process is distinct from the brain (Joy et al. 2021). While I find the results of this study to be very intriguing and of interest to the field, we have a few comments/concerns:\n1. How confident are the authors that the phenotypes reported here for knockdown of Bub3 are an accurate reflection of SAC inactivation, and not another role for Bub3 in mitosis that leads to the CIN that was observed? This is an important clarification, especially for extrapolating these results to mammals (particularly human clinical phenotypes). This needs to be addressed explicitly, considering: 1) the SAC is not essential for viability or fertility under normal developmental contexts in the fly as reported for Mad2 (Buffin et al. 2007), another SAC gene part of the same MCC complex as Bub3. 2) Previous work in the brain of Mad2 mutants showed little to no impact on central brain NB numbers (Poulton et al. 2018).\n2. The level of rescue observed in Figs. 6&7 upon genetic intervention of proteostasis, autophagy, ROS scavenging, etc, while statistically significant, is limited in practical terms, especially for the relevance to the microcephaly phenotype. This could be strengthened by providing additional quantification on brain size in these experiments, rather than just NBs/lobe which may not accurately reflect brain size (as shown in Fig. 3A’). In this case, highlighting the rescue size data as a “percent increase”, compared to the genotyped matched controls would be a better metric to help readers assess the levels by which these pathways actually contribute to the MVA microcephaly phenotype, as opposed to focusing on the statistics alone.\n3. Considering the points raised in #2, should the additional brain size analysis prove to show a low level of brain size rescue upon genetic intervention (<30% increase in size compared to controls), we would suggest rewording the title and dampening statements in the manuscript which might otherwise give the reader the wrong impression regarding the role of proteostasis failure and mito dysfunction in the MVA microcephaly phenotype.\nMinor Comments:\n• Do Bub3 KD animals show normal larval development timing, or are they delayed? This could be useful information for interpreting the results for the timepoints shown in Fig. 1A’.\n• It would also be interesting to quantify GMC numbers as well as the numbers of postmitotic populations of neurons and glia in Rod KD animals to determine whether they are also reduced as compared to wild-type levels at the temporal endpoint (96 hours) like Bub3.\n• The single aneuploidy data shown in Fig. 3 for the X chromosome is fascinating and provides a good example of why NB#/lobe & GMC#/lobe is not the most accurate reflection of overall brain size. Do the authors have any idea what these ‘triplosensitive’ X loci might be, and perhaps more importantly, what cellular properties might be affected in these brains? Is it cell size? Cell packing density in the tissue? Something else entirely?\n• In Fig. 4E’, F’, the case for complex aneuploidies would be strengthened by providing a similar graphical analysis for Chromosome 3.\n• It might be more appropriate to measure brain size in adults, after the neurogenic period has completed, to ensure consistency with clinical diagnoses in humans. This could become more relevant depending on the developmental timing experiments mentioned above.\n• Please check the labeling of subpanels in Figure 6 (A-F) and ensure consistency with manuscript text and the figure legend.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "Mosaic variegated aneuploidy (MVA) is a rare human congenital disease caused by bi-allelic mutations in genes (BUB1B, CEP57 and TRIP13) that play a crucial role in ensuring chromosome segregation during cell division. Patients exhibit widespread mosaic aneuploidies, involving different chromosomes and tissues, which cause a range of developmental problems, including microcephaly. Here, González-Blanco et al. generated a Drosophila model of MVA-driven microcephaly to gain insight into the mechanisms underlying neural stem cell (NSC) loss in this disease. The authors present evidence that targeted depletion of bub3 and rod genes (involved in accurate chromosome segregation) in NSCs induces microcephaly and loss of type I neuroblasts (NBs) in larval stages. The authors claim that is the accumulation of complex aneuploidy during consecutive cycles of NB cell division that ultimately accounts for microcephaly, rather than DNA damage or simple aneuploidies. Transcriptome analysis of larval brain lobes unveiled proteostasis failure and mitochondrial dysfunction as major contributors of the deleterious effect of complex aneuploidies in NBs. The experimental approaches are elegant and widely explore the advantages of the Drosophila genetic model, by resorting to tissue-specific gene deletion (or overexpression) as well as numerous in vivo functional reporters. Materials and Methods are well described, and statistical analyses are adequate. I am supportive of the manuscript’s publication provided the authors address the issues raised below in order to strengthen their conclusions and improve the quality of the manuscript. Questions are not ordered by their relevance.\nMajor issues\n1. The authors should provide rationale on their choice to deplete the bub3 and rod genes instead of, for instance, bub1b.\n2. In my opinion, the authors should reinforce data on rod depletion (Fig. S1B is minimal to extrapolate conclusions for both bub3 and rod depletions, considering these genes have different roles in chromosome segregation). For instance, show that complex aneuploidies also accumulate in rod depletion.\n3. I would like to see at an early stage in the manuscript, perhaps following Fig. 1 experimental layout, clear measurement of apoptosis and aneuploidy levels along 48h, 72h and 96h.\n4. It would be useful if the authors mention the timepoint AEL at which late embryo quiescent NBs start dividing in the larval CNS (24h?). Does this account for the onset of NB loss only at 48h? Do complex aneuploidies only arise at 48h?\n5. Fig. 4F: relevant data, that as I mentioned, should come earlier on the manuscript. Aneuploidy levels, notwithstanding the difficulty of measuring it, need dedicated analysis in this manuscript to support the authors’ conclusions. I noticed both chromosomes II and III were labelled by FISH. However, only chromosome II was quantified in Fig. 4F’. To conclude about complex aneuploidy, it is important to integrate both chromosome II and III quantification. Also, to exclude polyploidy.\n6. Regarding the transcriptomic data, I consider it would be important to include another experimental condition to confirm for specific pathways driven by complex aneuploidy in NBs. The bulk RNA-seq brings up differences due to changes in cell subpopulations (besides NBs, reduced number of GMCs and neurons). A non-MVA microcephaly model, or a single trisomy model, could help to strengthen the authors’ conclusions.\n7. Fig. 5E: relevant data to support loss of stemness. However, I recommend the authors to correlate dMyc loss with NB size or Fbl (are dMyc- cells smaller?); also, it would be useful to correlate Dpn with Wor and Pros.\n8. Although in Fig. 5E the authors observe increased % of small NBs in bub3-i (likely dMyc- and Fbl low), in Fig. 6 it appears that NBs are actually large due to proteotoxic stress. Please elucidate on this.\nMinor issues\n1. The authors should adjust the figures so that the order of panels/graphs - A, B, C,… - fits with the order of appearance in the text (e.g. Fig. 1D is mentioned before Fig. 1A; Fig. S1B is mentioned before Fig. S1A). Also, in Fig. 6, the lettering A-D is different from Figs. 1-5 where A’, B’, C’… was used to match graphs with corresponding panels. Be consistent across all figures.\n2. Please make sure to clearly explain/distinguish the experimental approaches used in Fig. 1A and Fig. S1A. In Fig. 1A, bub3 depletion starts in the embryo, and larval brain lobes were observed at different timepoints (so please consider revising text in lines 94-96 page 5 for clarity). In Fig. S1A, bub3 depletion started at different timepoints, and brain larval lobes were then all analyzed at 96h. Both figures are important and should be compared in more detail as it serves to highlight that is the perdurance of CIN, rather than the developmental stage it starts, that leads to microcephaly (e.g. 72h in Fig. 1A’ is similar to 72h in Fig. S1A’).\n3. Line 220-221, page 9: consider revising the subtitle to “CIN brains shown transcriptional changes in genes involved in protein control and translation”.\n4. Fig. 5E’: ‘small (<7 mm)’ should read ‘small (<7 um)’.\n5. Fig. 5E, middle panel: please show also Pros staining in black&white.\n6. Line 266, page 11: typo in reference Schott et al. 2017.\n7. Lines 296-297, page 12: mention to Fig. 6E but Fig. 6 only shows A-D.\n8. Line 508, page 29: ‘sq-Mito-YFP’ should read ‘sqh-Mito-YFP’.", "reviewer_4": "In their manuscript, the authors investigate a Drosophila model of aneuploidy-mediated microcephaly, due to defects in a single spindle assembly checkpoint (SAC) gene bub3 (or rod). The authors find that RNAi-mediated depletion of SAC genes specifically in neuroblasts (NBs) of the developing brain (Worniu-GAL4) causes progressive loss of NBs. They conduct a wide range of experiments to study genetic interactions with pathways involved in apoptosis, differentiation, autophagy/proteostasis, and mitochondrial quality control and oxidative stress.\nThe manuscript is well written and the figures are well-presented, appropriate and illustrate the findings clearly. The experiments seem mostly well-conducted, and the findings are interesting. My main concern is that it is not entirely clear to me how much the paper adds beyond what has been shown by others before (for example: PMID: 26568519, PMID: 26573328 and PMID: 30794535). The dissection of the downstream pathways that contribute to the phenotypes are valuable, but some of the interactions (eg related to autophagy in Figure 6D or mitochondrial dysfunction in Figure 7E) are rather subtle, and some have been seen before in other systems (eg PMID: 26404941).\nI would be supportive of publishing this manuscript, but have several major concerns that remain to be addressed, and a few minor concerns that are listed below.\nMajor concerns:\n- Throughout, the authors make many interpretations and statements that seem not always strongly supported by experiments. This is an issue throughout the manuscript, but my main concern (and example) for this is that mitochondrial dysfunction is mentioned as a pivotal contribution in the title, while the data for this are rather scarce, and the rescue experiments subtle at best.\n- A major argument from the authors rests on the fact that NB phenotypes only appear after a delay, interpreted as relative tolerance of NBs to aneuploidy (eg line 102 or line 136). However, all experiments are RNAi-mediated, and the authors do not measure SAC levels after RNAi expression. KD efficiency should be quantified, both at the tissue level (qRT-PCR, WB or AB staining) and within the NBs itself (reporter, ISH or AB staining).\n- The authors compare the impact of irradiation on NBs vs wing disc epithelium (Figure 4). However, they do not consider the contribution of cell competition, which is likely to contribute to cell death within an epithelium. NBs are not exposed to this competition, as they are separated from each other by glial cells and do not directly interact. This might explain the delayed apoptosis as well, and should either be addressed experimentally by the authors, or their interpretation revisited accordingly.\n- The ‘loss of stemness’, as discussed in the abstract, is, apart from the decreased proliferation and loss of NBs due to apoptosis, probably based on the data in Figure 5, where some NB markers are lost in a percentage of the cells. However, a major concern is that a subset of cells, as they become aneuploid, will either have gained a transgene, or lost a marker gene. If the authors really want to address stemness, they could for example assess presence of the genetic locus in these cells, and then show absent expression from this locus. Or otherwise adjust their interpretation.\n- Although the authors provide all the quantifications and statistical analyses in a supplementary table, which is commendable, it is not clear to me how many separate biological replicates (i.e. separate crosses) were conducted to obtain the numbers of animals analysed. In particular for the subtle changes seen in Figures 6 and 7, using only single transgenes, it would be good to be sure that these data come from different actual biological replicate experiments, and were quantified blinded.\nMinor comments:\n- The authors use too many abbreviations throughout their manuscript, which makes it sometimes hard to follow.\n- It seems unexpected that the NBs do not undergo mitotic stalling, but instead delayed G1-S progression. It might be relevant for the authors to briefly comment on this?\n- Figure 6A: the expression of bub3 seems almost not decreased upon RNAi? Is this expected?"}, "author_response": "MVA in humans is caused by pathogenic mutations in components of the spindle-assembly checkpoint (SAC) and proteins involved in centrosome dynamics during mitosis. Thus, the common factor in all these patients is CIN in brains. One of the main goals of our work is to provide a mechanistic understanding of how CIN induces MVA-driven microcephaly. We and others (e.g. Basto, Peifer and Oliveira’s labs) have used Drosophila to produce CIN in the brain and analyze whether this has an effect on brain development. CIN has been produced with RNAi forms of the SAC genes bub3 and rod (this work), by mutations in bub3 (PMID: 26573328), by combining changes in centrosome number (amplification or loss) with mutations in mad2 (PMID: 26573328, PMID: 28351851) or by acute targeting of chromosome cohesion (PMID: 30794535). In all these cases, a reduction in the number of neural stem cells was observed. All these results indicate that CIN is highly deleterious in brain development. Whether CIN is produced by a SAC independent role of bub3 appears to be out of the scope of this work but is somehow questioned by the fact that depletion of another SAC gene (rod, see Figure S1A) has also an impact on the number of neural stem cells and on brain size. We have indeed added new experimental data in Figure S1 to reinforce our observations on the impact of rod depletion by quantifying the number of Prospero, Repo and Elav positive cells and brain size (data included in Figure S1A’).\nWe appreciate reviewer’s comment on the role of Mad2 and the SAC in Drosophila development. The experimental observation of the viable phenotype of mad2 mutations in flies was used by Buffin et al 2007 to claim that the SAC is not required for Drosophila development. However, work by the Sunkel lab (PMID:17182852) showed that, in contrast to vertebrates, Drosophila Mad2 does not play a fundamental role in SAC functioning and that Mad2 has a conserved role in the mitotic clock. This group elegantly showed that if mitosis is experimentally delayed in mad2 mutant cells, the SAC is fully functional. This report also concluded that mad2 mutants are viable and fertile since reduction of the mitotic timing does not affect cell division in flies during normal development. A few years later, it was demonstrated in PMID: 20421417 that, upon SAC activation in Drosophila, it is BubR1 (and NOT Mad2) the one that recruits Cdc20 to the kinetochore. All these results, together with the fact that mutations in bona fide SAC genes (bub1, bubR1, rod, bub3) have been reported by several groups to lead to lethality in Drosophila, indicate that the SAC is required for Drosophila development.\nWe have provided the suggested additional quantification on brain size. We are very pleased to say that brain size (and progeny numbers) can be fully rescued by restoring mitochondria homeostasis (with ROS scavengers or mitochondria chaperones) or blocking apoptosis (with p35). Data are included as Figures 7G and S1C’’) and discussed as follows in the ms:\nData on mitochondria homeostasis (pg 14):\n“……overexpression of mitochondria-specific chaperones Hsp60 and Hsp60c, or ROS scavengers Sod2 and GTPx-1 ….also rescued the number of Prospero-positive cells (Figure 7E) and, most interestingly, yielded a complete restoration of brain size (Figure 7G). The observation that overexpression of ROS scavengers and mitochondrial chaperones in NB cells fully restored brain size, but only partially NB cell counts, points to a role of mitochondrial dysfunction in compromising not only the fitness but also the mitotic activity of highly aneuploid neuroblasts. Alternatively, these genetic interventions might target, through perdurance of the proteins that are overexpressed, the progeny cells thus restoring their fitness.”\nData on apoptosis inhibition (pg 14):\n“Notably, the inhibition of apoptosis through p35 expression also resulted in a complete restoration of brain size and GMC number (Figure S4C’), despite no concomitant rescue of NB numbers. These findings can be explained by inheritance of p35 by progeny cells and points to a role of apoptosis in the elimination of these cells”\nBy contrast, we noticed that autophagy induction, which was able to partially rescue NB number, did not rescue progeny cell number or brain size. Data are included in Figure 6E and discussed in pg 13 as follows;\n“We noticed that none of the genetic interventions aimed at increasing autophagy was able to rescue the number of Prospero-positive cells or the size of the larval brain (Figure 6E and Figure S6C). These results suggest that TOR inhibition or non-physiological levels of autophagy might have a negative impact on the proliferative capacity of highly aneuploid NBs.”\nThe new data have also led to minor changes (labeled in red in the ms) in the Abstract, Introduction, and Discussion and have led us to conclude the paper as follows (pg 16):\n“Taken together, our data indicate that CIN-induced microcephaly stems, on one hand, from the generation, over time and through consecutive cell cycles, of NSCs carrying highly complex aneuploid karyotypes. Complex aneuploidies compromise the highly energy-demanding stemness of NSCs – their stem cell identity and proliferative capacity- by causing proteostasis failure and mitochondrial dysfunction. On other hand, the ultimate effect of CIN on brain size also stems from the apoptotic elimination of progeny cells, most probably as a direct consequence of aneuploidy.”\nSince the rescues of brain size caused by the expression of ROS scavengers, mitochondria chaperons and p35 was complete, we rather prefer to keep the brain size numbers rather than the percentages. We hope this reviewer will agree with our decision.\nAlthough the new data on brain size reinforces the pivotal role of mitochondria dysfunction in CIN-induced microcephaly by impacting on NB and progeny cells, we really appreciate Reviewer’s suggestion and have included the following changes to avoid any potential overstatement.\n- We have reworded the title of our article: “Proteostasis failure and mitochondrial dysfunction contribute to chromosomal instability-induced microcephaly”.\n- We have rephrased the following statements in the abstract (“We unravel a contribution of proteostasis failure and mitochondrial dysfunction to the negative impact of complex aneuploidies on stemness, a highly energy demanding cellular state”) and introduction (“…proteostasis failure….and mitochondrial dysfunction contribute to the deleterious effects of complex aneuploidies on NSC stemness…”) and the title of the last section of the results (“Proteostasis failure and mitochondrial dysfunction contribute to the loss of stemness”).\nAs stated above, we have included minor changes (labeled in red in the ms) in the Abstract, Introduction and Discussion sections to conclude that both loss of stemness and elimination of progeny cells by apoptosis contribute to MVA-driven microcephaly.\nWe have introduced the suggested data in Figure S1C where it is shown that developmental timing is indeed not affected by SAC depletion in NBs. Data are referenced in pg 5 as follows:\n“We noticed that developmental timing was not affected by SAC depletion (Figure S1C).”\nAs suggested, we have now included the numbers of GMCs, neurons and glia in rod-depleted brains as Figure S1A’. Data are referenced in pg 5 as follows (underlined):\n“…only a significant reduction in GMC number and brain size was observed at later stages of larval development upon chronic depletion of the SAC (Figure 1A, A’, see also Figure S1A’). As expected, the number of neurons and glial cells in SAC-depleted brains was also reduced when compared to control brains (Figure 1B, B’, see also Figure S1A’).’’\nWe fully agree with this conclusion, especially after realizing that expression of ROS scavengers, mitochondria chaperons and p35 were able to fully rescue brain lobe size (but partially NB number), by impacting on NB and progeny cells.\nWe agree these are very interesting questions, but they might need an extraordinarily amount of work to be addressed. As such, we think these questions might be out of the scope of the ms.\nAs suggested, we have now provided a graphical analysis for Chromosome 3.\nSAC depletion in NBs results in a highly lethal phenotype in adults (as also shown by Raquel Oliveira’s lab, PMID: 30794535, and our own results). Most individuals die either before reaching adulthood or shortly after eclosion, which is why we focused our analysis exclusively on larval stages. Nevertheless, we were still able to dissect some individuals that reached adulthood, and their brain size was also reduced upon CIN induction in NBs compared to control brains. These data are included as Figure 1E and explained in the text as follows: “The size of the brain of those adults that survived to chronic depletion of the SAC was also significantly reduced (Figure 1E).” We monitored the genetic rescues of brain size only in larval stages due to the low number of surviving adults.\nWe would like to point out that MVA-driven microcephaly can be already observed in fetal stages in humans as reported in PMID: 25696020 PMID: 32884756, thus reinforcing our analysis on the impact of CIN on the size of the developing brain during larval stages in flies.\nAs indicated above, developmental timing is not affected by SAC depletion. Thus, the negative effect of SAC depletion on larval brain size is not a trivial consequence of developmental delay and/or growth retardation. The new data on adult brains reinforce this conclusion.\nLabeling has been corrected in the text and in the legend to Figure 6.\nWe appreciate reviewer’s interest and feedback on the paper. The report has contributed to increase the quality and clarity of the paper.\nIt is certainly true that it was in 2004 when the first mutations associated to MVA in humans were shown to affect the bub1b gene. However, afterwards mutations in other components of the SAC and proteins involved in centrosome dynamics during mitosis (eg. CEP57, bub1, trip13, cenatac, mad2, cep192) have been genetically linked to MVA in human patients. Thus, the common factor in all these patients is CIN in brains. One of the main goals of our work is to provide a mechanistic understanding of how CIN induces MVA-driven microcephaly. We and others (e.g. Basto, Peifer and Oliveira’s labs) have used Drosophila to produce CIN in the brain with a large variety of genetic tools and analyze whether this has an effect on brain development. CIN has been produced with RNAi forms of the SAC genes bub3 and rod (this work), by mutations in bub3 (PMID: 26573328), by combining changes in centrosome number (amplification or loss) with mutations in mad2 (PMID: 26573328, PMID: 28351851) or by acute targeting of chromosome cohesion (PMID: 30794535). In all these cases, brain size was reduced and this reduction was accompanied by a depletion in the number of neural stem cells. Thus, bub3 or rod depletion has been used in our work as two efficient tools to produce CIN in the developing fly brain. Why not bub1b? Most probably, because this tool was not in our hands when we initiated the project.\nIn order to complement the data on rod depletion, on one hand we have now included quantification of chromosome II in rod-depleted NBs. Data are included as Figure S1B, B’ and referenced in the paper in pg 9 as follows (underlined):\n“Consistently, FISH analysis of NBs to count the number of copies of chromosomes II and III upon chronic induction of CIN revealed an increase in the percentage of NBs losing or gaining at least one chromosome (Figure 4F, F’, see also Figure S1B, B’).”\nOn the other hand, we have also included the quantification of Prospero positive cells, neurons and glia in rod-depleted brains. Data are referenced in pg 5 as follows (underlined):\n“…only a significant reduction in GMC number and brain size was observed at later stages of larval development upon chronic depletion of the SAC (Figure 1A, A’, see also Figure S1A’). As expected, the number of neurons and glial cells in SAC-depleted brains was also reduced when compared to control brains (Figure 1B, B’, see also Figure S1A’).!\nAs suggested, we have now included measurement of apoptosis at 48h, 72h and 96h AEL of developing brains subjected to chronic SAC depletion in Figure S4C’. As indicated in the text (pg. 11):”Whereas the percentage of control NBs positive for either of these two markers remained close to zero throughout larval development, a low but significant percentage of NBs subjected to CIN were positively labeled by these markers (Figure S4C, C’’).”\nSimilarly, we have also measured aneuploidy levels at an early time point (e.g. 48h AEL). Data are now included in Figure 4F’ and referenced in the paper (pg. 9) as follows: “…. FISH analysis of NBs to count the number of copies of chromosomes II and III revealed an increase throughout larval development in the percentage of NBs losing or gaining at least one chromosome upon chronic induction of CIN (Figure 4F, F’, see also Figure S1B, B’)””\nOur data on the steady increase in aneuploidy levels overtime (upon chronic depletion of the SAC) is in agreement with Mirkovic et al. (2019). In this paper, authors induced acute cohesin loss (well known to induce CIN) and observed “a significant number of NBs proliferating for several days and displaying a tendency for chromosome accumulation over time,” as demonstrated by their quantification of CID signal. Consistently, their FISH experiments confirmed this trend, as they stated in their ms: “The FISH profiles confirmed the propensity for chromosome accumulation over time’’.\nQuiescent NBs start dividing in first instar, around 8 hours after larva hatching (which corresponds to 32 hours AEL. As suggested, this information is now included in pg 4 as follows (underlined): “….. neural stem cells (NSCs)….. re-enter the cell cycle after a quiescence period in the first instar larval stage (around 32 hours after egg laying, AEL)”.\nIn larvae exposed to chronic expression of bub3-RNAi under the control of the wor-gal4 driver, expression of the RNAi starts already at embryo stages. Thus, in this experimental condition NBs proliferate during embryogenesis and after re-entering the cell cycle at 32 h AEL, thus explaining the subtle although significant impact on NB number at 48h AEL shown in Fig 1A’.\nIn the Gal80ts experiments, it has been reported by several groups that more than 8 hours upon the temperature shift are required to block Gal80ts functioning, drive RNAI expression and target gene expression (in this case bub3), thus explaining our observations that temporally control expression of the bub3-RNAi required more than 24 hours to get an impact on NB number (Fig. S1E’).\nAs now shown in Figure 4F’, the subtle impact on NB number at 48h AEL shown in Figure 1A’ is accompanied by a subtle increase in the number of NBs with more than 2 chromosomes II. It is certainly expected that the complete loss of any of the chromosomes (which is not compatible with cell viability) might already contribute to NB loss at this early stages but not be quantified by our FISH analysis.\nIn order to maintain the linear flow of the paper, we have rather preferred to discard first the obvious candidates (DNA damage and simple aneuploidies in Figures 3 and 4) as the underlying causes of the phenotype (described in Figures 1 and 2) and then to suggest the presence of complex aneuploidies (based on the delayed response of NBs shown in Figure 1 and S1) and confirm it in Figure 4F by doing FISH with the probes labeling two chromosomes.\nAs suggested, we have now provided a graphical analysis for Chromosome 3 in Figure 4F’.\nAs suggested, we have now analyzed the transcriptional response of larval brains to trisomies for the X and 2L chromosome. Data are included as Figure S5 and discussed in the text (pg 10) as follows: “The transcriptional response of developing brains to the generation of complex aneuploidies was not reproduced by simple aneuploidies. On one hand, when examining the changes in gene expression caused by CIN or by trisomies for the X or 2L chromosomes, the observed patterns of upregulation and downregulation did not show a significant correlation (Figure S5A). On other hand, gene set enrichment analysis of brains trisomic for the X or 2L chromosomes did not identify any of the gene classes pinpointed in brains subjected to CIN (Figure S5B).”\nThese results certainly contribute to strengthening our conclusions on “ a reduction in translation (e.g., ribosome biogenesis) and RNA metabolism, activation of protein quality control mechanisms (autophagy), and repression of mitochondria activity as the major responses of developing brains to complex aneuploidies. “\nReviewer should take into consideration that SAC depletion induces a variety of aneuploid karyotypes, most cells tending to accumulate chromosomes and some losing chromosomes, and that both types of aneuploidies are expected to have an impact on nuclear size. Consequently, NB nuclear size is highly heterogenous (loss of stemness making nuclei smaller, accumulation of chromosomes making them bigger), and any correlation between dMyc/Fbl expression and nuclear size will be affected by the number of chromosomes. Thus, smaller nuclei might be a consequence of loss of dMyc/Fbl expression, loss of chromosomes or both. Similarly, big nuclei (as a result of gains of chromosomes) might lose dMyc and Fbl (as a result of loss of stemness).\nHaving said that, we believe that the data shown in Figure 5E clearly indicate loss of stemness measured as a significant percentage of NBs reducing their nuclear and nucleolar size and losing dMyc expression (and the two other stemness markers Wor and Dpn).\nThis is indeed what is shown in Figure 5E’, the % of NBs (Dpn+) that are either Wor (-) or Pros (+), and the %NBs (Wor+) that are Dpn-. This is now better indicated in the figure. Of course, those NBs that lose Dpn and Wor cannot be identified as NBs. We believe these data clearly indicate loss of stemness measured as an increased tendency to lose stemness markers Wor and Dpn.\nAs indicated in the previous point, reviewer should take into consideration that nuclear size is highly heterogenous among NBs subjected to CIN (as a consequence of changes in chromosome number and loss of stemness). This explains the increase in the % of small NBs reported in Figure 5 E’, and the presence of some NBs with big nuclei (as a result of the accumulation overtime of chromosomes).\nIn Figure 6, there are a few NBs that are big but not all the selected examples (labeled now by white arrows) have bigger nuclei (labeled by Dpn). Original dashed lines labeling cell contours in this figure were intended to facilitate the reader seeing the correlation between the expression of Dpn (to label NBs) and the proteotoxic marker. Whereas the accumulation of Atg8, CL1-GFP and Htt25Q-Cerulean has facilitated us to delimit the cytoplasm of those NBs that are aneuploid, we are not able to delimit the cellular contour of control NBs (which might be bigger than the dashed line) as these cells do not accumulate these markers. This type of labeling might have given the impression that aneuploid NBs are bigger, which is not the case. In order to avoid confusion, we have substituted the dashed lines labeling cell contours by arrows to point to the NB nuclei.\nAs suggested, we have reformatted Figure S1 to fit the order of panels to their references in the text.\nIn Figure 1-5, each of the A’ to D` includes the quantification of the corresponding A-D panels, thus facilitating the interpretation of the figure (eg. A’ is the quantification of A panel). In Figures 6 and 7, this is not the case as panels 6D and 7E are showing the quantification of Figures S5 and S6.\nWe appreciate reviewer’s comment. As suggested, we have rephrased this section as follows (underlined text in pg 5) in order to make clear the two experimental settings:\n“….We analyzed control brains and brains subjected to chronic SAC depletion in proliferating NBs at four time points of larval development (in second instar at 48 and 62 h AEL, and in third instar at 72 and 96 h AEL). In control flies not subjected to SAC depletion, NB number in the central brain remained constant throughout larval development, and the number of GMCs and the size of the resulting brain increased as a result of the active proliferative capacity of NBs (Figure 1A, A’). In contrast, in flies subjected to chronic SAC depletion, the number of NBs was gradually reduced (Figure 1A, A’) and this reduction was highest at later stages of development (Figure 1A, A’ and Figure S1A, A’). We noticed that developmental timing was not affected by SAC depletion (Figure S1C), and that the expression levels of the Bub3 protein were already compromised at early stages (48 h AEL, Figure S1D). The gradual decrease in the number of NBs was also observed upon temporally controlled depletion of the SAC gene using the thermo-sensitive Gal80 system (Figure S1E, E’). In this experimental setup, expression of bub3-RNAi was restricted to the last 24, 48 or 72 hours of development and NB number was quantified at 96 h AEL. Remarkably, at least 48 h of bub3 depletion were required to get a significant effect on NB number (Figure S1E, E’), despite the rapid cell cycles of this population (every 2 h) at these developmental stages 18. We also noticed that activity of the SAC was already compromised after 24h of RNAi expression (Figure S1F).These results point to a delayed response of NBs to CIN, as previously shown 14. .\"\nWe have changed the title to a more neutral one at this point of the ms: “CIN induces a transcriptional response in developing brains””, as transcriptional changes in protein quality control mechanisms and protein translation are interpreted in the two following sections.\nThis typo has been corrected.\nIn Figure 5E, each column is devoted to summarize a message. Whereas the third column is devoted to show an example of Pros and Dpn colocalization in aneuploid NBs (and B/W panels of Pros and Dpn are shown), the middle column is devoted to show the lack of co-expression of the two stem cell transcription factors Wor and Dpn (and B/W panels of Wor and Dpn are shown). Including the B/W Pros staining in this middle column might distract the reader.\nThe reference format has been corrected.\nThe mistakes in the references to the non-existing panel 6E have been corrected.\nThe typo has been corrected.\nWe appreciate reviewer’s question on novelty of our paper. There are three publications (PMID: 28351851, PMID: 26573328 and PMID: 30794535) that showed (as indicated in the Introduction of our paper, pg 3) that “The Drosophila brain, whose development and growth also relies on the proliferative activity of NSCs, has proven to be a valuable model in which to demonstrate a causal relationship between chromosome segregation errors and microcephaly”. They certainly come to the conclusion that CIN induces loss of NSCs and impact on their proliferative capacity. Unfortunately, two of these three publications propose completely different mechanisms to explain how CIN induces NSC loss: premature differentiation (PMID: 26573328) and cell death (PMID: 30794535). The third paper (PMID: 28351851) unravels a delayed response of NSCs to aneuploidy (when compared to epithelial cells) but did not identify the underlying mechanism of this delay.\nHow much does our paper add beyond what has been shown by these three publications?\nI will then enumerate our main findings:\n(1) We rule out any functional role of cell death and premature differentiation to the CIN-induced loss of NSC, and point to loss of stemness (compromised identity and proliferative capacity) as the underlying cause.\n(2) We rule out any functional contribution of CIN-induced DNA damage or simple aneuploidies to the CIN-induced loss of NSCs.\n(3) We give a mechanistic explanation of the delayed response of NSCs to CIN. Thus, CIN-induced loss of NSCs is a consequence of the accumulation, overtime and through consecutive cell cycles, of an unbalanced number of gains and losses of more than one chromosome.\n(4) We unraveled, by transcriptional profiling of CIN brains, a strong effect on cellular homeostasis, including a reduction in genes involved in ribosome biogenesis and RNA metabolism, activation of protein quality control mechanisms (autophagy), and repression of mitochondrial activity.\n(5) We demonstrate with the use of reporters and functional genetics that proteostasis failure and mitochondrial dysfunction are major contributors to the deleterious effects of complex aneuploidies on NSC.\n(6) We identify overexpression of Radical Oxygen Species scavengers, mitochondria chaperones and apoptosis inhibition as genetic interventions capable of dampening the deleterious effects of aneuploidy on brain size. We thus conclude that both loss of stemness and elimination of progeny cells by apoptosis contribute to MVA-driven microcephaly\nWe agree with this reviewer that genetic interventions aimed at restoring proteostais or mitochondria homeostasis were able to rescue, although not completely, the number of NSCs. This was already discussed in the Discussion as follows (pg 16):” We noticed that the number of aneuploid NBs was not fully rescued by any of these genetic interventions, most probably as a result of the highly pleiotropic effects of aneuploidy on cell physiology and on the fact that the complete loss of chromosomes is incompatible with cell viability.”\nHowever, we are happy to say that we have now provided quantification on brain size (as suggested by Reviewer 1) upon expression of ROS scavengers, mitochondria chaperones and p35 to show that any of these genetic interventions was able to fully rescue brain size (Figures 7G and S1C’’). Data are discussed as follows in the ms:\nData on mitochondria homeostasis (pg 14):\n“……overexpression of mitochondria-specific chaperones Hsp60 and Hsp60c, or ROS scavengers Sod2 and GTPx-1 ….also rescued the number of Prospero-positive cells (Figure 7E) and, most interestingly, yielded a complete restoration of brain size (Figure 7G). The observation that overexpression of ROS scavengers and mitochondrial chaperones in NB cells fully restored brain size, but only partially NB cell counts, points to a role of mitochondrial dysfunction in compromising not only the fitness but also the mitotic activity of highly aneuploid neuroblasts. Alternatively, these genetic interventions might target, through perdurance of the proteins that are overexpressed, the progeny cells thus restoring their fitness.”\nData on apoptosis inhibition (pg 14):\n“Notably, the inhibition of apoptosis through p35 expression also resulted in a complete restoration of brain size and partial GMC (Pros+ cells) number (Figure S4C’), despite no concomitant rescue of NB numbers. These findings can be explained by inheritance of p35 by progeny cells and points to a role of apoptosis in the elimination of these cells”\nBy contrast, we noticed that autophagy induction, which was able to partially rescue NB number, did not rescue progeny cell number or brain size. Data are included in Figure 6E and discussed in pg 13 as follows;\n“We noticed that none of the genetic interventions aimed at increasing autophagy was able to rescue the number of Prospero-positive cells or the size of the larval brain (Figure 6E and Figure S6C). These results suggest that TOR inhibition or non-physiological levels of autophagy might have a negative impact on the proliferative capacity of highly aneuploid NBs.”\nAll these new data have also led to minor changes (labeled in red in the ms) in the Abstract, Introduction, and Discussion and have led us to conclude the paper as follows (pg 16):\n“Taken together, our data indicate that CIN-induced microcephaly stems, on one hand, from the generation, over time and through consecutive cell cycles, of NSCs carrying highly complex aneuploid karyotypes. Complex aneuploidies compromise the highly energy-demanding stemness of NSCs – their stem cell identity and proliferative capacity- by causing proteostasis failure and mitochondrial dysfunction. On other hand, the ultimate effect of CIN on brain size also stems from the apoptotic elimination of progeny cells, most probably as a direct consequence of aneuploidy.””\nIt is certainly true that the impact of aneuploidy on proteostasis is not new as stated in the Discussion section of our paper (pg 14) as follows:\n“…..the major responses of developing brains to CIN-induced aneuploidy included …. activation of protein quality control mechanisms (autophagy), and repression of mitochondrial activity. These transcriptomic changes are consistent with the conserved impact of aneuploidy on proteostasis (PMID: 22968442, PMID: 26404941, PMID: 17702937) …… Thus, in yeast and human cells, aneuploidy leads to the production of an unbalanced proteome, which causes proteotoxic stress and activation of the major quality control mechanisms, including autophagy.”\nThe effects of aneuploidy on cellular physiology are also well known to be highly pleiotropoic as it induces different types of stresses (mitotic, replicative, metabolic, etc). The novelty provided by model organisms like Drosophila relies on the use of functional genetics to identify genetic interventions able to ameliorate some of these stresses and to reduce the negative impact of aneuploidy on the functioning of certain cellular types (NSCs in this case) and its potential translational effect to disease (MVA in this case).\nAs stated above, we have now provided quantification on brain size (as suggested by Reviewer 1) upon expression of ROS scavengers, mitochondria chaperones and p35 to show that any of these genetic interventions was able to fully rescue brain size.\nThese new data on brain size reinforces the pivotal role of mitochondria dysfunction in CIN-induced microcephaly by impacting on NB and progeny cells.\nHowever, we really appreciate Reviewer’s suggestion and have included the following changes to avoid any potential overstatement:\n- We have reworded the title of our article: “Proteostasis failure and mitochondrial dysfunction contribute to chromosomal instability-induced microcephaly”.\n- We have rephrased the following statements in the abstract (“We unravel a contribution of proteostasis failure and mitochondrial dysfunction to the negative impact of complex aneuploidies on stemness, a highly energy demanding cellular state”) and introduction (“…proteostasis failure….and mitochondrial dysfunction contribute to the deleterious effects of complex aneuploidies on NSC stemness…”) and the title of the last section of the results (“Proteostasis failure and mitochondrial dysfunction contributes to the loss of stemness”).\nWe appreciate reviewer’s comment and we have addressed this issue by adding two new experimental data.\nFirst, we have confirmed knockdown efficiency by immunostaining with Bub3 antibodies. As shown in Figure S1D, Bub3 protein levels were already reduced at early stages (48h AEL) in NBs subjected to chronic bub3-RNAi expression. This is now stated in the ms (underlined text in pg 5): “….in flies subjected to chronic SAC depletion, the number of NBs was gradually reduced (Figure 1A, A’) and this reduction was highest at later stages of development (Figure 1A, A’ and Figure S1A, A’). We noticed … that the expression levels of the Bub3 protein were already compromised at early stages (48 h AEL, Figure S1D).”\nSecond, we have measured the activity of the SAC upon temporally controlled depletion of bub3. As shown in Figure S1F, the SAC was already compromised after 24h of RNAi expression but a significant effect on NB number required at least 48 h of RNAi expression. This is now stated in the ms (underlined text in pg 5): “The gradual decrease in the number of NBs was also observed upon temporally controlled depletion of the SAC gene using the thermo-sensitive Gal80 system (Figure S1E, E’). In this experimental setup, expression of bub3-RNAi was restricted to the last 24, 48 or 72 hours of development and NB number was quantified at 96 h AEL. Remarkably, at least 48 h of bub3 depletion were required to get a significant effect on NB number (Figure S1E, E’), despite the rapid cell cycles of this population (every 2 h) at these developmental stages 18. We also noticed that activity of the SAC was already compromised after 24h of RNAi expression (Figure S1F). These results point to a delayed response of NBs to CIN, as previously shown 14.\nWe would like to point out that we observed a clear reduction in bub3 RNA levels in our RNA seq experiments of whole brains. Notably, the RNA-seq experiment was performed on whole brains, while bub3 downregulation was specifically induced only in NBs. This means that only a fraction of the total brain cells had bub3 downregulated, and yet we still detected a significant decrease in bub3 levels, highlighting the sensitivity and robustness of this RNAi line.\nInterestingly, Poulton et al. (2017) showed that the timing at which differences in brain size (as well as the cell death they observed) become significant is during mid/third instar. In embryos and early larvae, they did not detect any phenotype. Importantly, they used mutants (mad2, sas4), not RNAi. This observation fits very well with our phenotype and allows us to rule out the possibility that the progressive worsening we observe could be due to hypothetical changes in bub3-RNAi levels, since authors using mutants report the same temporal pattern as we do. Likewise, Mirkovic et al. (2019) also observed a progressive deterioration when acutely depleting cohesin. These published findings therefore strongly support our interpretation, in addition to the experiments we have performed.\nWe present evidence that exposure to a high doses of ionizing radiation (IR) induces in wing discs an immediate cellular response 5 hours later: cell cycle arrest in G2 and cell death (Figure S3). By contrast, the same exposure did not cause any effect on proliferating NBs (Figure 4C). Since this response is immediate, cell competition (a cell-to-cell interaction process that requires a certain amount of time to monitor differences in fitness and modulates growth and survival rates) is expected to play no role in this process.\nWhen analyzing the effects of IR 72h after the exposure, the number of NBs was slightly reduced and cell death was observed in wing discs. In the two cases, generation of highly aneuploid karyotypes (as a consequence of initial DNA damage and induction of rearrangements though cell proliferation of those DNA regions not properly repair) is thought to be the underlying cause (as proposed for wing discs in PMID: 19364807 and PMID: 24632609, and for NBs in Figure 4F of the present work). Whether cell competition contributes, after a long period of time after exposure to IR, to the elimination of aneuploid cells in the wing disc but not in the brain remains to be addressed but it seems to be out of context of this paper.\nWe appreciate reviewer’s comment but we have been extremely meticulous in this section by choosing only those stem cell markers whose detection relies on antibodies and not transgenes or reporters. Since complete loss of a chromosome is incompatible with NB viability (as reported in PMID: 30794535), those NBs that remain in the brain upon SAC depletion have at least one copy of each of the genes used as stem cell marker (eg. Dpn, Wor, dMyc, Fib).\nWe have carried six independent genetic experiments to address the potential role of proteostais failure and mitochondria dysfunction to the loss of aneuploid NBs and the reduction in brain size.\nIn the case of autophagy, four independent experimental settings (atg1 overexpression, Rheb depletion, mTOR heterozygosity and Rapamycin treatment) were used to address the potential contribution of proteostais failure. As shown in Figure 6D,E and Figure S6C, all these experimental settings were able to rescue the number of NBs but not the brain size. The failure to rescue brain size was reinforced by the quantification of GMCs in Figure 6E.\nIn the case of mitochondria dysfunction, two independent experimental settings (overexpression of ROS scavengers or mitochondria chaperones) were used. As shown in Figure 7E, G, these experimental settings were able to rescue the number of NBs and the brain size. The rescue of the brain size was reinforced by the quantification of GMCs in Figure 7E.\nAs stated in line 668 of M&M, “Quantifications were carried out in at least two independent replicates per genotype.”\nWe have stopped using two abbreviations (DCM: Dosage Compensation Mechanism, and MSLc: Male-Specific Lethal complex) that were used only twice. We kept the following abbreviations SAC, CIN, MVA, NSC/NB, GMC and DDR, as they are used very frequently throughout the manuscript.\nSAC depletion induces aneuploidy and we present evidence that highly aneuploid NBs show reduced proliferative activity (pH3 and EdU incorporation) and appear to be arrested in G1/S stage (according to FlyFucci). Based on the fact that the experimental setting to induce aneuploid NBs is based on depletion of the SAC, one might not expect aneuploid NBs to undergo mitotic stalling.\nYes, the mild reduction in the expression levels of bub3 is indeed expected as the transcriptional profile was done with total brains, and bub3-RNAi expression was restricted to the NSCs by the wor-gal4 driver. As indicated above, we have now confirmed reduction in Bub3 protein levels in NBs subjected to chronic bub3-RNAi expression. As stated in the ms (pg 5): “We noticed that … the expression levels of the Bub3 protein were already compromised at early stages (48 h AEL, Figure S1D).”\nWe appreciate the kind words and Reviewer’s support of publication of our ms.\nWe appreciate Reviewer’s support of publication of our ms.\nWe appreciate Reviewer’s summary on the revision process and Reviewer’s support of publication of our ms. We also thank this reviewer for all the feedback, which has greatly improved the quality and clarity of the ms.\nStatistics for Figures S1C top and bottom are the same: two-way ANOVA (Šídák's multiple comparisons test). We have corrected the reference to this panel in the legend by removing the word “bottom”, which was in the text by mistake. We apologize for this small mistake.\nThis is true for some cases (some panels of Figure S1 or S4 are referenced later in the text), but this is because all data related to rod depletion (Fig S1) and apoptosis (Fig S4) are summarized in these figures (for consistency) but used in different contexts.\nWe agree that NB number show a statistically significant reduction already at 48h AEL whereas the GMCs (pros+) number only decreases (in a statistically significant manner) at 96h AEL. However, we noticed consistent (although not significant) reduction in GMC (pros+) number at 48 and 72 h AEL. We, thus, rather prefer not to speculate on loss of self-renewal (and symmetric division) of NBs with only these data, as this might compromise attention of the reader to the general message of the figure.\nThe G-Trace experiment in >bub3-I was carried out only in late brains to reinforce the notion that the impact of chronic depletion of the SAC on brain size is a result of a reduced number of GMCs, neurons, and glial cells during whole larval development. These numbers have been generated in two independent experimental settings (chronic depletion of the SAC and temporally-regulated depletion of the SAC) and in the two cases, the results are the same. So, we do not think the suggested experiment might add much.\nIn the lower panels of Figure 1A, we show selected examples of NBs where GFP or Gal4 is restricted to NBs and not inherited to the progeny already at 62 h AEL. Whereas these data suggest that the proliferative capacity of NBs subjected to CIN starts to be compromised at early stages, these cases might be the exceptional case at this early time point of development as it is not reflected at the level of the whole population of Pros+ cells yet as shown in Figure 1A’ (although a tendency is observed at 72 h AEL). We think that commenting on these small details might compromise attention of the reader to the general message of the figure.\nCorrected.\nAs suggested, the sentence in line 143 has been changed as follows: “All these data indicate that reduced proliferation of NBs contribute to CIN-induced microcephaly.”\nAs suggested, we have reduced the tone of the sentence in line 229 as follows: “These results support the proposal that complex aneuploidies accumulated through consecutive cell cycles and consisting of multiple gains and losses of chromosomes exert a negative impact on NB number and proliferative capacity.”\nWe have rephrased the sentence in line 407 as shown below to propose a role of aneuploidy in the loss of progeny cells by apoptosis. Whether this is consequence of complex or simple aneuploidies is difficult to conclude as we did not quantify aneuploidy levels in these cells (a difficult task, by the way):\n“To our surprise, inhibition of apoptosis through p35 expression also resulted in a complete restoration of brain size and partial rescue of progeny cell number, despite no concomitant rescue of NB numbers, pointing to a role of apoptosis in the elimination of progeny cells most probably as a consequence of the generation of aneuploid karyotypes”\nCorrected as shown in point 8 above (“NB number” instead of “NB viability”).\nCorrected as follows (“NB number” instead of “NB survival”).: “To identify the molecular mechanisms underlying the negative impact of complex aneuploidies on NB number and proliferation….”\nIn the original sentence: “Consistently, a proportion of aneuploid NBs showed nuclear localization of Prospero [Figure 5E, E’, see also 13,14], and, most interestingly, these NBs also lost the expression of Dpn or Worniu (Figure 5E, E’),….”\n“these NBs” were referred to “aneuploid NBs” and not to those “aneuploid NBs showing nuclear localization of Prospero”.\nWe have changed the sentence to avoid confusion:\n“Consistently, a proportion of aneuploid NBs showed nuclear localization of Prospero [Figure 5E, E’, see also 13,14], and, most interestingly, aneuploid NBs frequently lost the expression of Dpn or Worniu (Figure 5E, E’),”\nAs seen in the responses of the three other reviewers to our revised version of the ms, they are supporting our ms. Thus, reviewers 1 and 2 conclude that “authors have done a commendable job addressing all my concerns” and Reviewer 3 concludes that “the authors substantially addressed my major concerns, which overall contributed to an improved quality of the manuscript as well as to strengthen the conclusions of this study”. The concerns on “loss of stemness” and “mitochondria dysfunction” chapters are addressed in the responses below.\nWe fully agree with this reviewer that brain size was unexpected to be rescued to that extent based on the fact that NSC number was not fully rescued. These results, which are very interesting by themselves, point to existing homeostatic mechanisms aimed at restoring brain size in a situation of NSC depletion. Whether these homeostatic mechanisms rely on the ability of remaining NSCs (or even progeny cells (GMCs)) to over proliferate in order give rise to more neurons and glia remains to be elucidated. We have discussed this important point at the end of the discussion (lines 410-414) as follows: “The experimental observations that these genetic interventions rescue brain size without fully rescuing NSC numbers point to existing homeostatic mechanisms aimed at restoring brain size in a situation of NSC depletion. Whether these homeostatic mechanisms rely on the ability of progeny cells (GMCs) to over proliferate in order to restore the final number of neurons and glial cells remains to be elucidated.”\nWe have also included the suggested images of brains in Figure S7.\nWe suspect this reviewer is pointing to NSC number and not proliferation, since brain size is rescued when mitochondrial homeostasis is restored or apoptosis is blocked, suggesting that these two genetic interventions rescue NSC proliferation and/or progeny survival.\nWe prefer to keep the last sentence of the Introduction as it is, where we emphasize the positive results of the paper as follows: “Lastly, we present evidence that genetic interventions aimed at restoring mitochondrial homeostasis and blocking apoptosis can mitigate the negative impact of complex aneuploidies on brain size.”\nIt is in the Discussion section where we have thoroughly discussed why some genetic interventions were unable to rescue NSC number and/or brain size (as shown in the previous point).\nWe appreciate reviewer’s comment on this issue, and as such, we have been very cautious in describing stemness as “a highly energy demanding cellular state” in several places in the text.\nOur interpretation of the results is summarized as follows at the end of the discussion:\n“Complex aneuploidies compromise the highly energy-demanding stemness of NSCs – their stem cell identity and proliferative capacity- by causing proteostasis failure and mitochondrial dysfunction.“ In this scenario, proteostasis failure and mitochondrial dysfunction are expected to be highly pleotropic in compromising the ability if NSCs to maintain dMyc, Wor, Deadpan expression by interfering with the mechanisms underlying their sustained expression, thus causing their loss in a salt and pepper manner. In other terms, we do not believe there is activation of a master regulator that turns off the expression of all stem cell markers at once and that loss of stem cell markers in a salt and pepper manner is a consequence of the strong metabolic insult caused by aneuploidy and its impact on the proteome and the mitochondria.\nWe have used these adverbs only in three cases to highlight the three main messages of this work:\nLine 108 (“Remarkably, at least 48 h of bub3 depletion were required to get a significant effect on NB number (Figure S1E, E’), despite the rapid cell cycles of this population (every 2 h) at these developmental stages 18. “) to highlight that gradual accumulation of aneuploidies is indeed needed to negatively impact on NB number.\nLine 270 (“and, most interestingly, aneuploid NBs frequently lost the expression of Dpn or Worniu (Figure 5E, E’), two transcription factors involved in promoting NB self-renewal through asymmetric cell division and avoiding premature differentiation 38,39) to highlight loss of stemness of aneuploid NSCs.\nLine 350 (“These genetic interventions also rescued the number of Pros+ cells (Figure 7E) and, most interestingly, yielded a complete restoration of brain size (Figure 7G).” to highlight that mitochondria dysfunction contributes to the chromosomal instability-induced microcephaly.\nWe appreciate Reviewer’s support of publication of our ms and thank this reviewer for all the feedback, which has greatly improved the quality and clarity of the ms."}]

Decision letter