Abstract Sensory disabilities have been identified as significant risk factors for dementia but underlying molecular mechanisms are unknown. In different Drosophila models with loss of sensory input, we observe non-autonomous induction of the integrated stress response (ISR) deep in the brain, as indicated by eIF2α^S50 phosphorylation-dependent elevated levels of the ISR effectors ATF4 and XRP1. Unlike during canonical ISR, however, the ATF4 and XRP1 transcription factors are enriched in cytosolic granules that are positive for RNA and the stress granule markers Caprin, FMR1, and p62, and are reversible upon restoration of vision for blind flies. Cytosolic restraint of the ATF4 and XRP1 transcription factors dampens expression of their downstream targets including genes of cell death pathways activated during chronic cellular stress and thus constitutes a chronic stress protective response (CSPR). Cytosolic granules containing both p62 and ATF4 are also evident in the thalamus and hippocampus of mouse models of congenital or degenerative blindness. These data indicate a conserved link between loss of sensory input and curbed stress responses critical for protein quality control in the brain. Subject terms: Visual system, Cellular neuroscience, Stress signalling __________________________________________________________________ Chronic stress responses can be as damaging as the hazards they counteract. Here the authors show how a blindness-induced brain-wide stress response is dampened by sequestration of transcription factors in cytosolic granules. Introduction In addition to genetic causes, potentially modifiable factors significantly contribute to the risk for dementia^[34]1,[35]2. For example, hearing loss in midlife is estimated to contribute to 8.2% of world-wide dementia cases^[36]1 and vision impairment is associated with 1.8% of US dementia cases^[37]2. Together these studies highlight the importance of maintaining sensory input during ageing but mechanistic links between the loss of sensory input and the onset or progression of dementia remain unknown. A common aspect of different neurodegenerative disorders, including dementia, is dysregulated protein homeostasis or proteostasis^[38]3–[39]7. In response to cellular stressors, proteostasis is maintained by a network of interdependent protein quality control mechanisms^[40]8, including the ubiquitin proteasomal system, autophagy and the integrated stress response (ISR)^[41]9. Proteostasis is particularly crucial in brain health because neurons need to be maintained for an entire lifespan, they have intricate morphology which defines their participation in complex neural circuits, and the dynamic regulation of pre and postsynaptic sites imposes localized constrains on their protein turnover^[42]10,[43]11. A prominent stress response is the assembly of neuronal stress granules (SGs). SGs are membraneless organelles in the cytoplasm formed by liquid-liquid phase separation of ribonucleoproteins (RNPs)^[44]12,[45]13. SGs can serve a transient protective function^[46]14, followed by their dissolution once homeostasis is restored^[47]15,[48]16. Under chronic stress, however, persistent SGs accumulate additional markers such as p62 (also known as Sequestosome-1 in mammals or Ref(2)p in Drosophila) and can serve as seeds for the aggregation of proteins related to neurodegenerative diseases^[49]13. Here, we discover that sensory disabilities cell non-autonomously trigger dysregulation of proteostasis. Potential negative effects of chronically elevated ISR are, however, dampened by the sequestration of stress-induced transcription factors into cytosolic RNA granules, which can serve a protective function that we term chronic stress protective response (CSPR). Results Spatial disruption of sensory neuron function causes brain-wide cell non-autonomous proteostasis dysregulation The CarT and BalaT transporters are specifically expressed in photoreceptor neurons and surrounding glia, respectively, and required for histamine neurotransmitter recycling; the corresponding mutant animals are functionally blind^[50]17–[51]20. The resulting inhibition of synaptic activity specifically of photoreceptors triggered proteostasis defects throughout the brain (Fig. [52]1a–d). This was visualized by staining for ATF4 and XRP1, two transcription factors induced by the integrated stress response (ISR)^[53]21. For both, we detected densely stained punctae throughout the brain, in numbers significantly increased compared to wild-type controls (Fig. [54]1b–f). Furthermore, punctae of Ref(2)p/p62, a recently appreciated marker for proteostasis defects^[55]22, were also significantly increased throughout the brains of blind CarT and BalaT mutants but not wild-type controls (Fig. [56]1b”–d”, g). Fig. 1. Sensory disability causes formation of punctae of p62, ATF4 and XRP1 throughout the brain cell non-autonomously. [57]Fig. 1 [58]Open in a new tab a Schematic representation of approach used in (b–g) to disrupt photoreceptor activity by blocking synaptic recycling. (b–b”) Micrographs of control adult head cryosections immunostained for XRP1 (b), ATF4 (b’) and Ref(2)p (b”), quantified in (e–g). (c–d”) Representative adult head cryosections of BalaT (c) and CarT (d) mutants inhibiting histamine neurotransmitter recycling immunostained for XRP1 (c, d), ATF4 (c’, d’), and Ref(2)p punctae (c”, d”). Insets are 3x magnified from the indicated area and adjusted for brightness and contrast to better visualize punctae. Yellow arrows indicate punctae which colocalize. e–g Quantification of immunostained heads as shown in (b–d). Bar graphs show median with interquartile range. Data are pooled from 3 independent sets of experiments (n = 6/control, 11/Balat, 10/CarT[43]). Significance threshold was determined by one-way ANOVA with Bonferroni correction for multiple comparisons. P-values are shown in each graph. h Schematic representation of inhibition of olfaction by olfactory receptor mutants (i–m) and Created in BioRender. Kramer, H. (2024) [59]https://BioRender.com/p69s813. i–j Cryosections of Orco (i) or Or67d (j) adult brains immunostained for XRP1(i, j), ATF4(i’, j’) and Ref(2)p (i”, j”). k–m Quantification of immunostained heads as shown in (i,j). Bar graphs show median with interquartile range of number of punctae per fly brain. Data are pooled from 3 independent experiments (n = 11). Statistical significance threshold was determined by Two-tailed Unpaired t test and P-value is represented in each graph. Scale bars shown in b” and j” are 100 µm and the same for (b–d and i–j). Source data for bar graphs are provided in source data file. Genotypes are listed in Suppl. Table [60]1. To test whether this cell non-autonomous proteostasis dysregulation is specific to visual system impairment, or extends to other sensory impairments, we targeted the olfactory system (Fig. [61]1h). The orco gene encodes a co-receptor necessary for the function of the majority of odorant receptors^[62]23. Interestingly, loss of Orco function also triggered brain-wide punctae of XRP1 (Fig. [63]1i, k), ATF4 (Fig. [64]1i’, l) and Ref(2)p (Fig. [65]1i”, m). By contrast, no punctae were observed in mutants for Or67d (Fig. [66]1j, k–m), which encodes a receptor specific for the Drosophila sex pheromone 11-cis-vaccenyl acetate^[67]24. Together, our data demonstrate that severe disruption of different sensory functions cell non-autonomously induces ISR effectors brain wide. Visual circuit activity reversibly regulates punctae formation in the brain To test whether loss of synaptic activity in the visual circuit is sufficient to trigger punctae formation in the brain, we blocked photoreceptor synaptic output by expression of dominant-negative Shibire (Shi^ts1) or Tetanus Toxin Light chain (TTL) under control of a photoreceptor-specific driver thereby inhibiting photoreceptor synaptic vesicle recycling or fusion, respectively (Fig. [68]2a). The resulting loss of photoreceptor synaptic output caused brain-wide punctae formation of XRP1 (Fig. [69]2c–e), ATF4 (Fig. [70]2c’, d’, f) and Ref(2)p (Fig. [71]2c”, d”, g) compared to control (Fig. [72]2b, e–g). Fig. 2. Visual circuit activity-dependent punctae formation in the brain. [73]Fig. 2 [74]Open in a new tab a Schematic representation of approach used in Fig. 2 (b–g) to disrupt photoreceptor activity by blocking synaptic release. b–b” Adult head cryosections of Quas-Shi^ts1 control flies lacking any driver (b) immunostained for XRP1 (b), ATF4 (b’), and Ref(2)p (b”). c–c” Representative adult head cryosections of flies expressing Shi^ts1 in photoreceptors immunostained for XRP1 (c), ATF4 (c’), and Ref(2)p (c”). d–d” Cryosections of adult fly heads expressing Tetanus Toxin Light chain (TTL) in photoreceptors to block synaptic release immunostained for XRP1(d), ATF4 (d’), and Ref(2)p (d’‘). e–g Quantification of punctae immunostained for XRP1(n = 16/control, 11/Shi^ts, 14/TTL), ATF4 (f) n = 17/control, 11/Shi^ts, 20/TTL) and Ref(2)p (g) n = 17/control, 11/Shi^ts, 17/TTL) in indicated genotypes. Bar graphs show a median with an interquartile range. Data are pooled from 3 independent sets of experiments. Significance threshold was determined by one-way ANOVA with Bonferroni correction for multiple comparisons. P-values are shown in each graph. h Schematic representing UAS-dTRPA1 expression-based approach to hyperactivate lamina neurons and disrupt visual circuits in (i–q). i–m Micrographs of adult heads expressing UAS-dTRPA1 under control of ort-Gal4 (i–k) or L4-Gal4 (l, m) drivers maintained at 21 °C (i–i”, l–l”), activated at 29 °C (j–j”, m–m”) or recovered from 29 °C at 21 °C (k–k”) immunostained for XRP1, (i–m, quantified in n) ATF4 (i’–m’, quantified in o) and Ref(2)p (i”–m” quantified in p) revealed reversible induction of brain-wide punctae by ort-Gal4-driven dTRPA1 expression at elevated temperature, but not by L4-Gal4 driven expression. n–p Quantification of punctae in immunostained heads as shown in (i–k). Bar graphs show median with interquartile range of number of punctae (n = 10/XRP1, 9/ATF4 and 10/Ref(2)p. Data are pooled from 2 independent sets of experiments. Significance threshold was determined by one-way ANOVA with Bonferroni correction for multiple comparisons and P-value is represented in each graph. q Functional expression of dTRPA1 by Ort-Gal4 and L4-Gal4 was indicated by altered sleep profiles for the indicated genotypes when dTRPA was active at 29 °C. Arrowheads indicate time of temperature shift, (n = 40 each genotype, experiments are done 3 time independently). Scale bars for b-d” and i-m are 100 µm, shown in (b” and m”) respectively. Source data for bar graphs are provided in source data file. Genotypes are listed in Suppl. Table [75]1. Photoreceptor synapses release histamine as an inhibitory neurotransmitter and therefore loss of photoreceptor synaptic output may hyperactivate downstream lamina neurons in the visual circuit^[76]25. To mimic this situation in vivo, we used a thermogenetic approach: hyperactivation of lamina neurons by the temperature-sensitive dTRPA1 channel (Fig. [77]2h). To express UAS-dTrpA1 specifically in synaptic targets of photoreceptors we used the ort-Gal4 driver which is expressed in multiple classes of lamina neurons that receive direct input from photoreceptors. Activation of dTRPA1 at 29 °C in these lamina neurons significantly increased brain-wide ATF4, XRP1 and Ref(2)p punctae compared to flies kept at 21 °C (Fig. [78]2i, j, n–p). Moreover, three days after shifting these flies from 29 °C back to 21 °C, the numbers of brain-wide punctae for ATF4, XRP1 and Ref(2)p were significantly reduced (Fig. [79]2k, n–p) indicating that blindness-induced punctae are reversible, although with a significant delay. Unlike Ort-positive neurons, L4 lamina neurons do not receive direct input from photoreceptors^[80]26. L4 hyperactivation by dTRPA1 expression at 29 °C did not cause elevated punctae levels in brains, which were indistinguishable from those maintained at 21 °C (Fig. [81]2l-m). This indicates that ATF4/XRP1 punctae formation is not a generic response to neuronal hyperactivation or elevated temperature. L4-expressed dTRPA1 is functional, nevertheless, as indicated by the altered sleep behaviors triggered by the shift to 29 °C for Ort- and L4-expressed dTrpA1 compared to the control lacking a Gal4 driver (Fig. [82]2q). Together, these data suggest that blindness-induced punctae formation in the brain is reversible and can be triggered by hyperactivation of a specific subset of neurons within the visual circuit. Blindness-induced RNP granules require the ISR and are present in neurons and glia To further test the hypothesis that blindness cell non-autonomously dysregulates brain proteostasis, we used norpA mutants (Fig. [83]3a). The norpA gene encodes Phospholipase-c beta which catalyzes an early step in the phototransduction pathway^[84]27; norpA mutants have been extensively used to study functional consequences of blindness in flies^[85]28. In norpA brains, punctae of ATF4, XRP1 and p62 were prevalent (Fig. [86]3b, d–f). Fig. 3. Blindness-induced XRP1 and ATF4 expression depends on eIF2α^S50 phosphorylation. [87]Fig. 3 [88]Open in a new tab a Schematic representation of norpA-based disruption of photoreceptor activity in (b–f). b–c Representative images of adult heads of norpA (b) or norpA rescued by Rh1-Gal4 driven NorpA (c) immunostained for XRP1 (b, c), ATF4 (b’, c’) and Ref(2)p (b”, c”). d–f Quantification of immunostained heads as shown in (b, c). Bar graphs show median with interquartile range of number of punctae per fly brain. Data are pooled from 3 independent experiments [n = 15 for (d), 9,5 for (e), 16, 10 for (f)]. Statistical significance threshold was determined by Two-tailed Unpaired t test and P-value is represented in each graph. g Schematic representation of blocked ISR by phospho-inert eIF2α^S50A mutant in (h–n). h Western blot showing total eIF2α and pS50-eIF2α levels from head lysates of indicated genotypes. For GCN^ACT only eyes were dissected and used for blots. The uncropped blot is provided in the source data file. i Quantification of pS50-eIF2α signal, normalized to total eIF2α signal ⊡ Bar graphs show median with interquartile range. n = 3 independent biological replicates. Significance threshold was determined by one-way ANOVA with Bonferroni correction for multiple comparisons. j Representative image of eIF2α^S50A (k) or norpA, eIF2α^S50A (i) adult heads immunostained for XRP1 (h, i), ATF4 (h’, i’) and Ref(2)p (h”, i”). l–n Quantification of immunostained brain only as shown in (h–i). The visual system was excluded from analysis because the eIF2α^S50A allele expresses strong 3xP3 DsRed-based fluorescence in the eye. Bar graphs show median with interquartile range of number of punctae per fly brain. Data are pooled from 2 independent experiments [n = 5/norpA, 10/norpA-eIF2α^SA, 10/eIF2α^SA for (l–m) and n = 10 for all genotypes in (n)]. Statistical significance threshold was determined by one-way ANOVA with Bonferroni correction for multiple comparisons. P-values are shown in each graph. Scale bars in (c” and k”) are 100 µm and same for (b–c and j–k). Source data for bar graphs and uncropped blot are provided in the source data file. Genotypes are listed in Suppl. Table [89]1. Importantly, the brain-wide presence of ATF4, XRP1 and Ref(2)p punctae was effectively suppressed (Fig. [90]3c) when vision in norpA mutants was restored by photoreceptor-specific expression of NorpA under control of the Rh1 rhodopsin promoter^[91]29. The number of punctae of ATF4, XRP1 and Ref(2)p in these rescued flies was reduced to near wild-type levels (Fig. [92]3c, d–f, compare to Figs. [93]1b, e–g; [94]2b, e–g). Despite the elevated ATF4 and XRP1 protein levels, RTqPCR detected no increase in the level of transcripts for either transcription factor (Suppl. Figure [95]1a), suggesting their levels may be enhanced by elevated translation. Phosphorylation of eIF2α (serine-50 in Drosophila) is a necessary step in the ISR-induced elevated translation of ISR effectors^[96]30–[97]32. To test whether elevated ATF4 and XRP1 levels in norpA mutants depend on activation of the ISR (Fig. [98]3g), we combined the norpA mutant with the eIF2α^S50A allele that abolishes the selective translation of ISR effectors^[99]32,[100]33 (Fig. [101]3g). Loss of S50-eIF2α phosphorylation in this allele was confirmed using western blots (Fig. [102]3h) using head lysates from eIF2α^S50A or eIF2α^S50A, norpA flies compared to those from wild type or norpA mutants, or flies overexpressing an activated form of GCN2, an eIF2α kinase^[103]34. Consistent with blindness inducing ISR deep in the brain, norpA, eIF2α^S50A double mutants exhibited low levels of ATF4 and XRP1 expression in the brain (Fig. [104]3k, l–n), indistinguishable from wild-type (Fig. [105]1b, e–g) and rescued norpA mutants (Fig. [106]3c, d–f). Together these data indicate a brain-wide elevated ISR in blind norpA flies. As part of canonical ISR activation, elevated levels of ATF4 and XRP1 enter the nucleus and drive a transcriptional program that aids in restoring cellular homeostasis^[107]21. By contrast, in brains of blind flies, ATF4 and XRP1 accumulate in punctae (Fig. [108]4a) that resemble cytosolic SGs, typically assembled by liquid-liquid phase separation of ribonucleoproteins^[109]13. Within these largely spherical granules (Suppl. Figure [110]2f), ATF4 closely colocalized with XRP1 and partially with Ref(2)p (Fig. [111]1c, d; Fig. [112]4b, and Airyscan images in Suppl. Figure [113]2a–e). These granules were not associated with nuclei (Fig. [114]4a). Instead, nuclear XRP1 was significantly reduced in brains of norpA flies (Suppl. Figure [115]1b). Consistent with the presence of XRP1 in RNP granules in norpA brains, we observed co-localization with the RNA-binding proteins Caprin and dFMR1 (Fig. [116]4c–e) as well as polyA RNA (Fig. [117]4m-o), which are markers of SGs^[118]35,[119]36. Fig. 4. ATF4, XRP1 and p62 form cytosolic RNP granules in neurons and glia. [120]Fig. 4 [121]Open in a new tab a–a”’ Immunostaining of cryosectioned norpA brains show colocalization and cytosolic restraint of XRP1 and ATF4. XRP1 (a) and ATF4 (a’) colocalize in cytosolic granules outside of nuclei marked by DNA (a”, a”‘). Yellow box in (a”’) is to show cytosolic location of ATF4 and XRP1. b Quantification of colocalization of ATF4 with XRP1 and ATF4 with Ref(2)p (images used for quantification of ATF4 to p62 colocalization are shown in Fig.[122]1c; n = 6,5). Bar graphs in (b) show median with interquartile range of percentage of colocalization. Scale bar for a-a”’ is 10 µm shown in (a). c–e Cryosections of norpA (c-d) or w^1118 control (e) brains stained for XRP1 (c–e), Caprin (c’-e’) and dFMR1 (c”–e”). Representative images shown from two independent experiments. (d) High-magnification images show partial colocalization of XRP1, Caprin, and dFMR1 as indicated by yellow arrowheads. Scale bar in (d) is 10 µm. Scale bar shown in (e”) is 100 µm for in (c and e) is and shown in (e”). f, g Cryosections of norpA; mCherry-Rin fly brain immunostained for XRP1 (f), mCherry (f’) and DNA (f”). Merged image shown in (f”’). (g’–g”’) High resolution details show exclusion of mCherry-Rin from XRP1-positive granules. Micrographs shown (i–l) are representative of 3 independent experiments each with 5 biological repeats. h–h”’ Cryosections of norpA brains immunostained for XRP1 (h), endogenous RIN protein (h’) and DNA (h”). Merged image in (h’”) shows exclusion of Rin protein from XRP1-positive granules. The micrographs shown are representative of 3 independent experiments, each with 5 biological repeats. (i–l) Cryosections of heads of norpA flies expressing UAS-mCD8-RFP under control of the glial repo-Gal4 driver stained for XRP1 (i, l), RFP (i’, l’), and neuronal ELAV protein (i”, l”); merged images (i”’, l”’). Magnified images (j, k) show XRP1-positive granules within glia and neurons (white arrowheads in k). Micrographs shown are representative of 2 independent experiments each with 5 biological repeats. (m-o) mRNA-FISH-IF, cryosections of adult brain either w^1118 control or norpA showing XRP1 (m, n), mRNA (m’, n’), DNA (m”, n”). Merge 3D image showing mRNA and XRP1 granules in (o), XRP1 granules are shown transparent to highlight sequestration of mRNA (yellow). Micrographs shown in (m–n) are representative of 3 independent experiments each with 5 biological repeats. Arrow in (n) indicates mRNA and XRP1 colocalization. Scale bar in (d) is 10 µm and in (o) is 1 µm. Scale bars in (g”, i”, and l”)are 5 µm. Source data for bar graphs are provided in the source data file. Genotypes are listed in Suppl. Table [123]1. Rasputin (Rin) is the Drosophila ortholog of G3BP which is commonly associated with SGs^[124]36–[125]39. Interestingly, XRP1/ATF4-positive granules in norpA brains were negative for Rin, as indicated by mCherry-tagged Rin (Fig. [126]4f, g) and by staining with anti-Rin antibodies (Fig. [127]4h) of norpA brains. Similarly, Rin did not localize to transiently induced ATF4/XRP1 granules in response to Ort-gal4 driven dTRPA1 expression in lamina neurons (Suppl. Figure [128]3). In norpA brains, XRP1-positive RNP granules were present in neurons, marked by elav staining, and in glia marked by repo-Gal4 driven mCD8::RFP (Fig. [129]4i–l). To further explore the nature of these granules, we tested whether they are responsive to mitoxantrone, a member of a class of drugs that has been used to inhibit SG formation downstream of eIF2a phosphorylation^[130]40. In norpA brains, we observed significant reduction of XRP1-stained RNP granules in norpA brains after feeding of 10 or 25 µM mitoxantrone (Fig. [131]5a–d). Parallel to this decrease, we observed elevated staining for ubiquitin (Fig. [132]5a’–c’, e) and Ref(2)p (Fig. [133]5a”–c’, f) suggesting that reduction of RNP granules at least in part may reflect degradation of their content by proteasome or autophagy, consistent with previous observations of SG disassembly^[134]16,[135]41. In addition, we observe restoration of nuclear localization of the XRP1 transcription factor (Fig. [136]5g–i). This may reflect release of XRP1 previously sequestered in the granules or newly translated protein no longer targeted to granules in the presence of mitoxantrone. Fig. 5. Pharmacologically dissolving cytosolic XRP1 granules results in increased nuclear XRP1. [137]Fig. 5 [138]Open in a new tab a–a”’) Immunostaining of cryosectioned norpA heads of no drug control brains show (a) XRP1 (a’) FK2 (a”) Ref(2)p and (a”’) Hoechst. b–c Adult head sections of flies feed two different concentrations of Mitoxantrone (a) 10 µM and (b) 25 µM added with blue dye in food. b, c XRP1, (b’, c’) FK2 (b”, c”) Ref (2)p and (b”’, c”’) Hoechst. Insets are magnified from the indicated area and adjusted for brightness and contrast to better visualize granules. Scale bar shown in (c”’) is 100 µm for (a–c). d–f Quantifications of punctae of XRP1 (d, n = 13, 6, 6 for Mitoxantrone 0, 10 and 25 µM respectively), FK2 (e, n = 6,6,7 for Mitoxantrone 0, 10 and 25 µM, respectively) and Ref (2)p (f, n = 12, 6, 12 for Mitoxantrone 0, 10 and 25 µM respectively). Bar graphs show median with interquartile range of number of punctae only quantified from brain excluding visual system. Data pooled from 2 independent experiments and significance threshold was determined by one-way ANOVA with Tukey’s multiple comparisons test. P-values are shown in each graph. g–h Immunostaining of cryosectioned norpA (g) XRP1, (g’) Hoechst for no drug control and (h) XRP1, (h’) Hoechst for Mitoxantrone 25 µM feed flies. White circles show nuclear staining. Scale bar in (h”’) is 5 µm. i Quantification of nuclear XRP1 with and without drug. Bar graphs show median with interquartile range of nuclear fluorescence IntDen (n = 66, 87 nuclei, for Mitoxantrone 0 and 25 µm respectively from 7 brains). Significance threshold was determined by Two-tailed Mann-Whitney U test as non-parametric test. Source data for bar graphs are provided in the source data file. Together, these results indicate that blindness and the resulting lack of photoreceptor synaptic activity triggers the accumulation of ATF4 and XRP1 in RNP granules lacking Rin protein in both neurons and glia throughout the brain. Sequestration of ATF4 and XRP1 in RNP granules dampens canonical responses to stress To test whether RNP granule-mediated sequestration of ATF4 and XRP1, two key effectors of the ISR, affects the response to cellular stress, we used a transcriptional reporter expressing LacZ under control of the Thor promoter which is responsive to ATF4 activity^[139]21. To induce cellular stress, we fed flies tunicamycin (TM) at a concentration known to induce the ISR^[140]42. Whereas wild-type brains displayed significant TM-induced enhancement of Thor-lacZ expression in both dorsal and ventral clusters of neuronal cell bodies (Fig. [141]6a–c, f), the number of Thor-lacZ expressing cells was significantly lower in TM-fed norpA brains (Fig. [142]6d–f). Similar changes were observed for GstD-GFP, a reporter for XRP1 activity^[143]21. In sections of TM-fed norpA brains, cells with prominent XRP1-positive RNP granules exhibited negligible activation of the GstD-GFP reporter, in contrast to cells lacking RNP granules (indicated by dashed lines in Suppl. Figure [144]4a–f). Western analysis of whole-head lysates revealed TM-induced increase of GstD-GFP expression in wild-type flies (Suppl. Figure [145]4g, h). Compared to wild-type flies exposed to vehicle-control only, corresponding norpA flies show elevated level of GFP but failed to further increase GFP expression upon TM feeding (Suppl. Figure [146]4g, h). Fig. 6. Sequestration of ATF4 and XRP1 in RNP granules dampens their transcriptional activity. [147]Fig. 6 [148]Open in a new tab a Schematic of fly brain pointing to dorsal (d) and ventral (v) regions imaged in panels (b–e). b–e Adult brain cryosections of Thor-lacZ (b, c) or norpA;Thor-lacZ (d, e) brains from flies fed DMSO (0.02%) as vehicle control (VC) or tunicamycin (TM, 12 µM) as indicated and stained for LacZ, XRP1 and DNA. Scale bar in b^d is 25 µm. f Quantification of the number of cells with elevated Thor-lacZ staining as exemplified in panels (b–e; n = 12/WT and norpA VC, 5/WT TM, 8/norpA TM). Bar graphs show median with interquartile range. Data are pooled from 3 independent sets of experiments. Significance threshold was determined by two-way ANOVA with Bonferroni correction for multiple comparisons. P-values are shown in each graph. g Volcano plot derived from RNAseq data (n = 3). Inset depicts target genes of the transcription factors XRP1 (magenta) and ATF4 (green). For complete list of genes and relative expression levels see Suppl. Data [149]1. h Bar graphs show enrichment ratios of KEGG categories discovered by over-representation analyzes of differentially expressed genes (DEGs) more highly expressed in w^1118 (blue) or norpA (red). i Heat map of the top 100 DEGs found in the three independent RNAseq datasets. Red indicates high and blue low expression. j Model depicting ATF4/XRP1 sequestration into granules as a chronic stress protective response (CSPR). Created in BioRender. Kramer, H. (2024) [150]https://BioRender.com/o16n195 Source data for bar graphs are provided in the source data file. Genotypes are listed in Suppl. Table [151]1. To systematically test the effects of altered expression of the ATF4 and XRP1 transcription factors on their target genes, we performed RNAseq on heads from 3-5-day old norpA and wild-type flies. In norpA heads, 2268 transcripts were differentially expressed (false discovery rate < 0.01), with 735 of these down-regulated in norpA heads (Fig. [152]6g–i, Suppl. Data [153]1). That includes the NorpA transcript, consistent with its nonsense-mediated decay in the norpA^P24 frameshift allele. KEGG pathway enrichment analysis indicates a reduction of transcripts related to phototransduction, consistent with the loss of photoreceptor cells due to retinal degeneration in norpA mutants^[154]43. Strong downregulation is also observed for Neprilysin6 (Nep6) and Neprilysin-like20 (Nepl20), two members of a family of secreted metallopeptidases linked to different degenerative diseases^[155]44. Specific functions for Nep6 or Nepl20 have not yet been identified. A major transcript upregulated in norpA brains encodes Sordd2, the Drosophila homolog of the ERAD E3 ubiquitin ligase RNF185 (Fig. [156]6g). Interestingly, loss of Sordd2 suppresses retinal degeneration in a Drosophila model of retinitis pigmentosa^[157]45. Other enriched transcripts point to changes in metabolic pathways including pyruvate metabolism, citrate cycle, glycolysis and carbon metabolism (Fig. [158]6h), whereas transcripts related to one-carbon pools, seleno-compounds and glutathione metabolism were downregulated. Consistently with the RT-qPCR data (Suppl. Figure [159]1), mRNA levels of ATF4 and XRP1 were not affected by blindness in our RNAseq datasets. Importantly, the target genes of these ISR effectors did not change either (Fig. [160]6g, Suppl. Data [161]1–[162]3), despite the elevated ATF4 and XRP1 protein levels. To test whether this dysregulation of the ISR may be linked to neurodegeneration in the brain, we measured the number of vacuoles in two-week old brains^[163]46. Compared to photoreceptor-rescued norpA;Rh1>NorpA flies, norpA mutants displayed strong degeneration of the retina as previously reported^[164]43. While there was a significant increase in brain vacuoles (Suppl. Figure [165]5 a–c) that increase was most prominent in the lamina and medulla layers of the visual system (green arrowheads in Suppl. Figure [166]5b) and less so in the central brain. To further test behavioral long-term consequences, we tested locomotion using a climbing assay^[167]47. We did not find a significant reduction of climbing ability of norpA mutants compared to wild type or norpA, eIF2α^S50A double mutants (Suppl. Fig, [168]5d). Thus, despite the high levels of ATF4 and XRP1in the norpA brains, we do not observe significant neurodegenerative consequences. Altogether, these data suggest that the sequestration of ATF4 and XRP1 in RNP granules compromises their canonical transcriptional function and dysregulates stress responses. Under conditions of sensory quiescence, as tested here by loss of vision or olfaction, minimizing the deleterious effects of chronic ATF4 and XRP1 transcription by their sequestration into RNP granules (Fig. [169]6j) amounts to a beneficial effect that we call “chronic stress protective response” (CSPR). Blindness induced non-autonomous proteostasis dysregulation in brain is conserved in mice The visual systems of Drosophila and mammals share conserved design principles and mechanisms of synaptogenesis and neuronal circuit formation^[170]48. We wondered whether this similarity extends to the blindness-induced dysregulation of proteostasis in the brain. In the thalamus and hippocampus of congenital blind Vsx2 mice^[171]49,[172]50, we observe increased punctae of p62 (Fig. [173]7a–d,e,i) and significantly elevated ATF4 levels (Fig. [174]7f–h), especially in the cytosolic staining of ATF4 (Fig. [175]7h”) when compared to wild-type 129S1/SvlmJ controls (Fig. [176]6g”). Fig. 7. Brain-wide visual impairment-induced granules are evolutionarily conserved in mice. [177]Fig. 7 [178]Open in a new tab a–i Section of thalamus (a, b, g, h) quantified in (e) n = 45-32/4, f, 11/4) and hippocampus (c,d) quantified in (i) of adult, congenitally blind Vsx2 mice (b, d, h) and age-matched 129S1/SvlmJ controls (a, c, g) stained for p62 (a–d) or ATF4 (g–h). Magnified, merged images highlight increased cytosolic staining for p62 (b”–d”) and ATF4 (g”, h”) in Vsx2 brains. h” Dotted green line indicates cytosolic content. e–f, i Bar graphs show median with interquartile range of integrated density. Data are pooled from 4 independent biological replicates. Significance threshold was determined by Mann-Whitney Two-tailed test and P-values shown in each graph. (Number of images quantified in (e): n = 45 from 4 B6 mice, n = 32 from 4 Vsx2 mice; for (f): n = 11 from 4 B6 mice, n = 12 from 4 Vsx2 mice; for (i): n = 17 from B6 mice, n = 13 from Vsx2 mice). j–s Sections from 10-week-old B6/J control (j, l) and degenerative Rho^P23H (k, m) brains stained for p62 (j–m) and ATF4 (j’–m’). Regions shown are thalamus (j, k,)quantified in (t, u) and hippocampus (l, m, quantified in (v, w). n–s Elevated cytosolic staining is revealed in magnified, merged images showing p62/DNA (n, o) or ATF4/DNA (p, q) in thalamus and p62/DNA (r,s) in hippocampus. t–w Bar graphs show median with interquartile range of integrated density. t, u Number of images quantified: n = 85 from 4 B6 mice, and n = 111 from 4 Rho^P23H mice for p62, n = 78 from 4 B6 mice, and n = 89 from 4 Rho^P23H mice for ATF4, (v, w) n = 55/ from 4 B6 mice and n = 84 from 4 Rho^P23H mice for p62, n = 56 from 4 B6 mice and n = 81 from 4 Rho^P23H mice for ATF4). Data are pooled from 4 independent biological replicates. Significance threshold was determined by Two-tailed Mann-Whitney test and P-values shown in each graph. Scale bars in (b’, d, h’ and k”) is 50 µm for (a–d, g, h, and j–m). Scale bars in (g”, n and r) is 5 µm in (g, h, n–s). Scale bar in (b”) is 8 µm in (a”, b”). Scale bar in c” is 10 µm in c”,d”. Source data for bar graphs are provided in the source data file. Genotypes are listed in Supplementary Table [179]1. To test whether similar consequences arise from retinal degeneration, we used Rho^P23H mice that model the most common rhodopsin mutant in humans linked to retinal degeneration^[180]51. In 10-week-old homozygous Rho^P23H mice, we detected increased levels of cytosolic ATF4 and p62 punctae in the thalamus and hippocampus when compared to C57BL/6 J (B6/J) wild-type controls (Fig. [181]7j–w; Suppl. Figure [182]6). Together, these data indicate a link between visual impairment and dysregulated brain proteostasis that is conserved from flies to mammals. Discussion Once neurons are stressed, activation of the ISR as well as the formation of stress granules are both double-edged swords. ISR-mediated eIF2α phosphorylation lightens the load of unfolded proteins by reducing general translation and selectively elevates ATF4 levels thereby promoting expression of a range of transcripts that aid in cellular stress adaptation^[183]9,[184]52. Upon persistent activation, however, the induced expression of CHOP in mammals and XRP1 in Drosophila complicates the outcomes, as their transcriptional targets not only promote stress recovery but also can induce cell death^[185]21,[186]52–[187]56. Similarly, SGs can serve a transient protective function by limiting the translation of a subset of mRNAs and confining caspases^[188]12,[189]14,[190]37, but their persistent presence can also promote neurodegeneration^[191]13,[192]57. Under condition of sensory quiescence, in flies with a loss of vision or olfaction, we observe a merger of these two stress responses with the two ISR-inducible transcription factors ATF4 and XRP1 being highly enriched within RNP granules. This confinement counteracts their elevated levels as we observe little change in the expression of ATF4 and XRP1 target genes in blind flies, especially in cells with prominent ATF4/XRP1-positive RNP granules. Such restrained ISR activation is beneficial in the absence of exogenous stressors, and we propose to call it chronic stress protective response (CSPR). However, the resulting limited induction of their target genes under stress conditions, as we observe in TM-fed flies, constitutes a vulnerability. Indeed, links between dysregulated ISR and neurodegenerative diseases are well established^[193]3,[194]9,[195]32,[196]58. Consistent with this notion, dysregulated proteostasis is a hallmark of neurodegenerative diseases^[197]3,[198]5, typically triggered by interactions with aggregation-prone proteins encoded by diseases-specific risk genes, such as Amyloid-β, Tau, α-Synuclein, Huntingtin or SOD1^[199]59. Our data in fly brains show that persistently altered circuit activity due to loss of sensory input can dysregulate proteostasis and thereby reduce stress resistance and contribute to the risk of dementia in ageing. Elevated cytosolic ATF4 and p62 condensates in the brains of mouse models of retinal degeneration (Rho^P23H) and congenital blindness (Vsx2) suggest an underappreciated role of the connection between the loss of sensory input and non-autonomous proteostasis regulation with consequences for the progression of neurodegenerative diseases. The ATF4 and XRP1 containing RNP granules share several features with stress granules. This includes their induction by S50-eIF2alpha phosphorylation, the key integrator of the ISR^[200]57,[201]60, their reversibility upon relieve of cellular stress and the presence of mRNAs and RNA binding proteins such as FMR1 and Caprin^[202]61. Furthermore, both are dissolved in the presence of mitoxantrone, a compound that has been used to counteract stress granule assembly downstream of eIF2alpha phosphorylation^[203]40. In sensory quiescence-induced RNP granules we could, however, not detect G3BP/Rin which is considered a hallmark of stress granules^[204]37,[205]38. Furthermore, the persistent presence of conventional stress granules appears to contribute to neurodegeneration^[206]13. By contrast, CSPR-associated RNP granules dampen the potential negative effects of persistent ATF4 and XRP1 activation, thereby protecting neurons. A further understanding of overlapping and distinguishing features between conventional stress granules and CSPR-associated RNP granules will require a more detailed analysis of the latter’s biogenesis and components. Methods Ethics statement This study does not conduct any human experiments. All animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research and were all approved by the Animal Care and Use Committee at University of Texas Southwestern Medical Center (APN#2019-102840). Fly rearing conditions Flies used in this study were reared on standard molasses fly food, under room temperature conditions. For tunicamycin treatments, 1% sucrose solution in 1.3% agarose was used at 25 °C. All flies were aged 3–5 days and placed in 5 cm diameter vials containing normal food, with no more than 30 flies. Head dissections were performed between 1 PM and 4 PM. For temperature sensitive experiments, temperature used is indicted in corresponding figures. The Balat^Δ-Gal4 allele was generated by replacing the Balat coding sequences with Gal4 using CRISPR/Cas9 and confirmed by sequencing. Detailed analysis of the allele will be published elsewhere. Mouse lines and husbandry B6.129S6(Cg)-Rho^tm1.1Kpal/J mice, herein referred to as Rho^P23H mice, and 129S1/Sv-Vsx2^or-J/J mice, herein referred to as Vsx2 mice, were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) (RRID:IMSR_JAX:017628; RRID: IMSR_JAX:000395). Age-matched C57BL/6 J (B6/J) mice (RRID:IMSR_JAX:000664) or age-matched 129S1/SvlmJ mice (RRID:IMSR_JAX002448) were used as experimental controls. Mice were bred and maintained at the facilities of the University of Texas Southwestern Medical Center. Animals were kept on a light–dark cycle (12 h–12 h) at 40–60% humidity. Food and water were available ad libitum throughout the experiment and all brain samples were collected between the hours of 1 PM and 4 PM. No sex specific differences were expected for this study, but both male and female mice were used for each study. Data from N = 3 female Vsx2 mice and their respective controls, and N = 4 male RhoP23H mice and their respective controls are shown in the figures to disaggregate by sex and represent images from both sexes. Immunohistochemistry Fly heads were dissected as described previously^[207]62. Briefly, after proboscis removal, heads were immediately transferred to ice-cold 3% glyoxal solution pH 4.0^[208]63 for one hour and washed overnight in 25% (wt/vol) sucrose in phosphate buffer (pH 7.4), embedded in Optimal Cutting Temperature compound (EMS, Hatfield, PA) and quickly frozen in liquid nitrogen and sectioned at 20-μm thickness on a cryostat microtome (CM 1950, Leica Microsystems, Wetzlar, Germany). Sections were washed three times (PBS with 0.1% triton X100, PBST), blocked (10% NGS), and probed with primary antibody diluted in 5% NGS solution. Antibody dilutions are listed in the Key Biological Reagents Table. Images were taken with a 20x NA-0.8 or 40X NA0.95 WD 0.17–0.25 mm FOV 25 mm Air Objective or an oil-immersion 63× NA-1.4 lens on an inverted confocal microscope (LSM710 or LSM880 with Airyscan, Carl Zeiss or Nikon AXR confocal). For each genotype, immunohistochemistry experiments were performed in three biological replicas with independent sets of flies, using identical acquisition settings. Experimenters were blinded to sample identity before acquiring images or before quantification as appropriate. Polyclonal antibodies against the p62 peptide PRTEDPVTTPRSTQ^[209]64 were raised in rabbits and affinity purified (Genemed Synthesis, Inc). For collection of mouse brains for immunohistochemistry, mice were anesthetized by intraperitoneal injection of 0.2 mL/10 g body weight of 1 mL of 100 mg/mL ketamine with 0.1 mL of 20 mg/mL xylazine diluted in 8.9 mL 1X PBS. Mice were then transcardially perfused with 1X PBS, followed by 10% formalin. Brains were removed and fixed in 10% formalin at 4˚C overnight. Brains were then embedded in 4% low-gelling temperature agarose (Sigma Aldrich) and 150 µm serial sections were collected into 1X PBS using the Leica VT1000S vibrating blade microtome. For IHC of mouse brain, sections were gently transferred to fresh 24 well plate and washed five times with PBST. Sections were blocked with 10%NGS for 2 hrs and probed with primary antibodies diluted in 5% NGS solution. After three PBST washes, secondary antibody incubation and three more PBST washes, sections were float-mounted on ProbeOn Plus microscope slides (Fisher Scientific). Antibody dilutions are listed in the Key Biological Reagents Table. Images were taken with a 20x NA-0.8 or an oil-immersion 63× NA-1.4 lens on an inverted confocal microscope (LSM880 with Airyscan, Carl Zeiss). mRNA-FISH-Immunofluorescence Fly heads were dissected and cryosectioned as described above in immunohistochemistry section. Head sections on slides were sequentially washed with 100% cold methanol for 10 minutes, 70% cold ethanol for 10 minutes and 1 M Tris, pH 8.0 for 5 minutes. After Tris removal, hybridization buffer [50 % Formamide, 2× SSC prepared from stock solution of 20× SSC, 10 % w/v dextran sulfate (Mw > 500,000), 1 mg/ml yeast t-RNA]  + 5’-labeled Cy3-Oligo-dT(30) at 1 ng/μl was added. Hybridization was carried out at 37 °C for 1 hrs in a humidified slide warming chamber. After hybridization, samples were washed once with 4 × SSC and then once with 2 × SSC 10 mins each at 37 °C. Samples were incubated with primary antibodies (Guinea pig anti- XRP1 1: 500) for 2 hrs at 37 °C. Primary antibodies were removed, and sections washed 3 times with 2 × SSCT (2 × SSC + 0.1% Triton-X-100) 10 mins each. Secondary antibodies (Goat anti- Guineapig 647, 1:500) and Hoechst 1:4000 were incubated for 1 h at room temperature, and washed 3 times with 2 × SSCT 10 mins each. Images were taken with an oil-immersion 63× NA–1.4 lens on an inverted confocal microscope (LSM880 with Airyscan, Carl Zeiss). 3D image showing mRNA within XRP1 granules was prepared using Imaris 10.1 (Oxford Instruments) software. Quantification of fluorescence staining Fluorescence images were quantified using ImageJ (NIH) adapting previous methods^[210]65 and Imaris 9.5 (Oxford Instruments) software was used for punctate quantification and colocalization analysis. For each antibody, a threshold was determined, removing the lowest signal in control samples (to reduce variation from low level background signals). This same threshold was applied to a mask created for every image in a batch of staining. For the quantification of ATF4 and p62 in mouse brains, within 1-µm optical slices, regions were selected manually and assigned as Regions of Interest. The integrated pixel intensity per unit area was measured within this selected area. For the quantification of ATF4 and XRP1 in fly brain, Imaris software was used to quantify punctae in respective images in 3D by creating surface Ref(2)p was quantified by spot counting, regions were selected manually and assigned as Regions of Interest. The same Regions of Interest are used for each experimental condition. For Thor-LacZ, images were AI denoised using Nikon NIS-Elements. Denoised images were used for quantification, wild-type TM-fed images were used to set the threshold for LacZ^+ cells, the same threshold was used to count cells in other groups. H&E staining and quantification To assess neurodegeneration, fly heads were dissected and after proboscis removal fixed in 4% PFA for 48 hrs, embedded in paraffin and 5 µm sections were cut followed by H&E staining as described^[211]46. Images were taken with a 20X objective using a Nikon Fi3 color Camera (NA0.75 WD 1.0 mm FOV 25 mm). Following blinding for genotype and treatment, images were quantified using ImageJ; after thresholding of the ROI, vacuoles in the brain structure were counted with a cutoff of holes more than 5 µm in size^[212]46. RNAseq Total RNA was extracted from ~100 µL fly heads (male and female) using TRIzol (Invitrogen) and the manufacturers recommended protocol. RNA samples were subjected to quality control before and after the cDNA libraries were prepared using TruSeq Stranded mRNA Library Prep Kit from Illumina. cDNA samples were normalized, pooled, then run using a P2 flow cell on an Illumina NextSeq 2000. FastQC (v0.11.2)^[213]66 was used to check quality of reads before at least 31 million unique reads each from three w^1118 and three norpA samples were mapped to the Drosophila reference genome using STAR(v2.5.3a)^[214]67. FeatureCounts^[215]68 was used to generate read counts while edgeR^[216]69 generated the differential expression analysis. The RNAseq raw reads can be found at NCBI BioProject under accession number PRJNA1068665. qPCR RNA was extracted from 50 heads using Trizol (Invitrogen) according to the manufacturers recommended protocol. Reverse transcription of 2 µg of RNA was done using a high-capacity cDNA reverse transcription kit (AppliedBiosystems). qPCR was performed using Quant Studio 6 Pro Real-Time PCR System (ThermoFisher) using fast SYBR green master mix (Applied Biosystems). Primers used are listed in Suppl. Data [217]4. Gene ontology analysis Two differentially expressed gene lists (w^1118 > norpA and w^1118 < norpA) both with a cutoff of FDR < 0.01 were generated for further gene ontology analysis. Over representation analysis with an FDR < 0.05 cutoff for KEGG pathways was done using WebGestalt ([218]https://www.webgestalt.org)^[219]70 with the Drosophila genome as a reference. Western blot For western blotting, 3-5 days aged 10 adult fly heads were homogenized in 100 µl lysis buffer (10% SDS, 6 M urea, and 50 mM Tris-HCl, pH 6.8) and then kept rotating for 30 min at 4 °C followed by boiling at 95 °C, for 2 min. Lysates were spun for 10 min at 15,000xg, supernatant was separated. Before loading on gel 1 mM DTT was added in samples followed by boiling at 95 °C, for 2 min, 5 µl (1/2 heads) of lysates were separated on 4–20 % gradient SDS-PAGE, transferred to nitrocellulose membrane, blocked with 3% non-fat dried milk dissolved in wash buffer (20 mM Tris-HCl pH 7.5, 5.5 mM NaCl and 0.1 % Tween-20) and probed in primary antibodies in following dilutions; rabbit anti-Hook (1:5000), Mouse anti-GFP clone B2 (1: 250), Rabbit anti-pS50-eIF2α (1:5000), mouse anti-eIF2α (1:1000) and incubated overnight at 4 °C on rotator. Blots were washed 3 times 10 mins each with wash buffer. Bound antibodies were detected and quantified using IR-dye labeled secondary antibodies (1:15,000) and the Odyssey scanner (LI-COR Biosciences). Pre-stained molecular weight markers (HX Stable) were obtained from UBP-Bio. Drug feeding For inducing ER stress, fly food was prepared by adding tunicamycin (TM) at a final concentration of 12 µM. TM was added in 1% sucrose solution with1.3% low melting agarose at temperature 40 °C. Flies aged 3 to 5 days were transferred to food containing either TM or DMSO as vehicle control (VC) and placed in the 12 h:12 h LD chambers at 25 °C for 23 hrs. After completion of the treatment, flies were used for western blot or immunofluorescence. To dissolve granules, we used Mitoxantrone at 2 different concentrations 10 and 25 µM. Mitoxantrone was dissolved in water to make stock of 5 mM, and required volume was added to the standard molasses fly food containing 1% blue dye (FD&C Blue #1). Flies aged 3 to 5 days were transferred to food containing drug and after 3 days heads were processed for cryosectioning and IHC as described above in IHC section. Measuring sleep Sleep was measured as described previously^[220]62. Briefly, 3–5 days old male flies were placed inside a 65 ×3 mm glass tube with standard molasses fly food. Tubes were placed in the Ethoscope arena^[221]71. Ethoscopes were placed in a 21 °C incubator with 300 Lux light at a 12 h:12 h LD cycle and after allowing flies to acclimatize for one day, their movement was recorded. On the third day, the temperature was shifted to 29 °C to activate the temperature-sensitive dTRPA channel. After 3 days at 29 °C, the temperature was shifted back to 21 °C for recovery. Ethoscope data were analyzed using rethomics^[222]72. All codes used in the sleep analysis are available in github ([223]https://github.com/mz27ethio/Ethoscope-Kramerlab.git). Statistics & reproducibility No statistical method was used to predetermine sample size. No data were excluded from analysis; except in Fig. [224]5i, there 3 outlayers from each group were removed after identification by ROUT analysis with Q = 1% using the Graphpad Prism software (10.3.1). The experiments were not randomized. Micrographs were blinded prior to quantification. Statistical significance of results was analyzed using Prism-GraphPad10. Anderson-Darling or Shapiro-Wilk test were used to assess the normality assumption for continuous parameters. Afterwards, skewed data were transformed on log scale followed by similar normality assessment. Independent t-test was used to compare normally distributed parameters between two groups whereas Mann-Whitney U test served as non-parametric test. For comparing groups of three or more, a one-way analysis of variance followed by Bonferroni correction (a multiple comparisons test) was used for normally distributed sample parameters. A two-way analysis of variance followed by Bonferroni’s test was used for multiple comparisons to identify the individual pairwise comparisons to separate the effects of treatment and genetic backgrounds. Skewed parameters were compared using Kruskal-Wallis test followed by Dunn’s test for multiple comparison test. Bar graphs resulting from these analyses demonstrate a median with an interquartile range. P values smaller than 0.05 were considered as statistically significant, and values are indicated in the corresponding graphs. Key Biological Reagents Table Reagent type (species) or resource Designation Source or reference Identifiers Additional information genetic reagent (D. melanogaster) QUAS-Shi^ts Bloomington Drosophila Stock Center [225]BDSC:30013; FBgn0003392; RRID:BDSC_30013 genetic reagent (D. melanogaster) QUAS-TTL Bloomington Drosophila Stock Center [226]BDSC: 91808; FBgn0016753; RRID:BDSC_91808 genetic reagent (D. melanogaster) UAS-dTRPA1 Bloomington Drosophila Stock Center BDSC: 26264 [227]FBti0114502 [228]RRID:BDSC_26264 genetic reagent (D. melanogaster) w ^1118 Bloomington Drosophila Stock Center [229]BDSC:3605; FBti0131930; RRID:BDSC_3605 genetic reagent (D. melanogaster) Balat[Δ-Gal4] this study genetic reagent (D. melanogaster) w[*] norpA[P24]; P{w + =ninaE-norpA.W}2 Bloomington Drosophila Stock Center [230]BDSC: 52276; FBst0052276 RRID:BDSC_52276 genetic reagent (D. melanogaster) w [*] norpA[P24] Bloomington Drosophila Stock Center BDSC: 9048 FBst0009048 RRID:BDSC_9048 genetic reagent (D. melanogaster) repo-Gal4 Bloomington Drosophila Stock Center BDSC: 7415 FBst0009048 RRID:BDSC_7415 genetic reagent (D. melanogaster) UAS-mCD8::RFP Bloomington Drosophila Stock Center BDSC: 32218 FBst0009048 RRID:BDSC_32218 genetic reagent (D. melanogaster) CarT[43] ^[231]19 FBgn0032879 genetic reagent (D. melanogaster) CarT[HA-T2A-QF2] ^[232]62 [233]FBgn0032879 genetic reagent (D. melanogaster) L4-Gal4 Bloomington Drosophila Stock Center [234]BDSC: 49883 RRID:BDSC_49883 genetic reagent (D. melanogaster) Ort-Gal4 Bloomington Drosophila Stock Center [235]BDSC: 56517 RRID:BDSC_56517 genetic reagent (D. melanogaster) Thor-lacZ ^[236]73 Gift from Dr. Don Ryoo, NYU genetic reagent (D. melanogaster) GstD-GFP ^[237]74 Gift from Dr. Don Ryoo, NYU genetic reagent (D. melanogaster Or67[Δ-Gal4] Gift from Dr. Dean Smith, UTSW genetic reagent (D. melanogaster Orco (Or83b) Gift from Dr. Dean Smith, UTSW genetic reagent (D. melanogaster w[*], eIF2α^S50A-3xP3DsRed ^[238]33 genetic reagent (D. melanogaster w[*], mCherry-Rin ^[239]39 Gift from Dr. Paul Taylor, St. Jude Children’s Research Hospital genetic reagent (D. melanogaster w[*], uas-GCN2[ACT] ^[240]34 antibody anti-GFP (Chicken polyclonal) ThermoFisher Scientific [241]ThermoFisher Scientific: A10262; RRID: AB_2534023 1:500 IHC antibody anti-GFP (Mouse monoclonal, B2) Santa Cruz [242]Santa Cruz Anti-GFP Antibody (B-2): sc-9996 1:250 WB antibody anti-P62/SQSTM1 (rabbit polyclonal) [243]Proteintech Group, Inc Catalog Number: 18420-1-AP 1:200 IHC antibody Anti-Ref (2)p (rabbit polyclonal) This paper 1:3000 IHC antibody Alexa 488- or 568- or 647 secondaries Alexa Fluor 568 Goat anti-mouse Alexa Fluor 488 Goat anti-mouse Alexa Fluor 647 Goat anti-mouse Alexa Fluor 488Goat anti-rabbit Alexa Fluor 568 Goat anti-rabbit Alexa Fluor 647 Goat anti-rabbit Alexa Fluor 647 Goat anti-guinea pig Alexa Fluor 568 Goat anti-guinea pig Alexa Fluor 488 Goat anti-guinea pig Alexa Fluor 668 Goat anti-rat Alexa Fluor 488 Goat anti-chicken ThermoFisher [244]thermofisher fluorescent-secondary-antibodies 1:500 IHC antibody IRDye 800CW Goat anti mouse IRDye 700DX Goat anti mouse IRDye 800CW Goat anti Rabbit IRDye 700DX Goat anti Rabbit LICOR Biosciences [245]https://www.licor.com/bio/reagents/new-reagent=category=irdye-seco ndary-antibodies 1:20,000 WB antibody anti-RFP (Rabbit polyclonal) Rockland [246]Rockland:600-401-379; RRID:AB_2209751 1:500 IHC antibody anti-dFMR1(Rabbit polyclonal) Developmental Studies Hybridoma Bank RRID: AB_528252 1:500 IHC antibody anti-Rin (Rabbit polyclonal) ^[247]75 Gift from Dr. Elizabeth R Gavis 1:500 IHC antibody anti-Caprin (Rabbit polyclonal) ^[248]76 Gift from Dr.Ophelia Papoulas UT Austin, 1:500 IHC antibody anti-XRP1 (Guinea Pig polyclonal) ^[249]21 Gift from Dr. Don Ryoo, NYU 1:1000 IHC antibody anti-ATF4 (mouse monoclonal) [250]Proteintech Group, Inc Catalog Number: 60035-1-Ig 1:500 IHC antibody Anti-Crc (ATF4 rat polyclonal) ^[251]77 Gift from Dr. Joseph Bateman, King’s College 1:2000 IHC antibody Anti-Repo (mouse monoclonal) Developmental Studies Hybridoma Bank RRID:AB_528448 1:500 IHC antibody Anti-ELAV (Rat monoclonal) Developmental Studies Hybridoma Bank RRID:AB_528217 1:500 IHC antibody Mouse anti-eIF2α Cell Signaling mAb#2103 (L57A5) 1:1000 WB antibody Rabbit anti-pS50-eIF2α Abcam Recombinant Monoclonal eIF2S1 phospho S51 antibody, Ab32157, [E93] 1:5000 WB [252]Open in a new tab Reporting summary Further information on research design is available in the [253]Nature Portfolio Reporting Summary linked to this article. Supplementary information [254]Supplementary Information^ (3.2MB, pdf) [255]41467_2024_55576_MOESM2_ESM.docx^ (12.8KB, docx) Description of Additional Supplementary Information [256]Supplementary Data 1^ (715.4KB, xlsx) [257]Supplementary Data 2^ (9.6KB, xlsx) [258]Supplementary Data 3^ (9.8KB, xlsx) [259]Supplementary Data 4^ (9.3KB, xlsx) [260]Reporting Summary^ (95.1KB, pdf) [261]Transparent Peer Review file^ (785.5KB, pdf) Source data [262]Source Data^ (1MB, xlsx) Acknowledgements