Mitochondrial

This special issue reviews the role coenzyme Q (CoQ) plays in physiological and pathological processes. CoQ is an essential component of cellular lipid membranes and has a central role in the redox reactions occurring at them. CoQ possesses a benzoquinone ring and an isopre-noid side chain with a variable number of units. The number of units differs in different species, e.g. the most common form in humans contains ten units, while it contains only six in yeast. CoQ is instrumental for electron transport


INTRODUCTION
Aging is a complex and gradual process accompanied by a progressive decline in physiological integrity and function. Underlying this physiological decline is the loss of essential cellular functions, concomitant with the accumulation of cellular and molecular damage, such as the 5 increase in DNA mutations and accumulation of misfolded proteins, causing a decline in organellar and cellular function (1, 2). One of the key organ systems in aging is the nervous system. The nervous system is not only impacted by aging, as exemplified by neurodegenerative diseases, but can also modulate the aging process of the entire organism (3). The link between the nervous system and aging is governed by its role as a central regulatory hub, mediating the maintenance of 10 homeostasis in response to fluctuations in the external and internal environments, intracellularly and intercellularly, within and between different tissues. One level of homeostasis that neurons are tasked with maintaining and coordinating across tissues is the homeostatic state of protein folding (proteostasis, protein homeostasis) (4). Proteostasis is one of the key features declining with age, due to the reduced ability to counteract misfolding and aggregation of proteins, a hallmark of many 15 neurodegenerative diseases, such as Huntington's disease (5). Neurons are able to regulate protein homeostasis across tissues for several pathways that are dedicated to protecting different cellular compartments (6,7), such as the mitochondrion (8), with activation of the pathway prolonging lifespan, by coordinating mitochondrial protein homeostasis across tissues. This protective pathway, the unfolded protein response of mitochondria (UPR MT ) (9), is triggered upon insults to 20 the organelle and causes the activation of a coordinated transcriptional response, which includes the upregulation of mitochondrially-targeted chaperones and proteases, modulating cellular processes to alleviate the burden of stress on mitochondria. Interestingly, neurons secrete molecules that allow coordination of the UPR MT across tissues, utilizing dense-core vesicles (DCVs), serotonin, and the WNT-like ligand EGL-20 to trigger a response in peripheral tissues, 25 such as the intestine (10,11).
Historically, much scientific work interrogating the homeostatic roles of the nervous system focused on neurons. While it is clear that glia, the other main cell type of the nervous system, can serve many roles in neuronal development and function, these roles are normally associated with 30 support roles, including regulating cell number, neuronal migration, axon specification and growth, synapse formation and pruning, ion homeostasis, synaptic plasticity, and providing metabolic support for neurons (12,13). However, in recent years, it has become increasingly clear that glial health can impact aging and progression of neurodegenerative diseases, like Alzheimer's Disease (AD) (14). For example, expression of ApoE4, one of the strongest risk factors for AD, 35 specifically in astrocytes resulted in increased neuronal tau aggregation (15). Moreover, hyperactivation of the unfolded protein response of the endoplasmic reticulum (UPR ER ), which drives ER stress resilience, solely in astrocyte-like glial cells resulted in a significant lifespan extension in C. elegans (16). While these studies show the importance of glial function in organismal health, what they lacked is an active function of glia in promoting these beneficial 40 effects. To uncover an active role for glia in stress signaling and longevity, we aimed to determine whether glial cells can sense mitochondrial stress and initiate an organism-wide response to promote mitochondrial stress resilience and longevity. We used multiple genetic methods to activate UPR MT in non-neuronal cells, including cell-type specific application of mitochondrial stress and direct activation of the UPR MT in the absence of stress. Interestingly, we found that 45 regardless of method, activation of UPR MT in a small subset of glial cells, the cephalic sheath (CEPsh) glia, provided robust organismal benefits, including prolonged lifespan and increased resistance to oxidative stress. Perhaps most unique in this model is that UPR MT activation in CEPsh glia promotes neuronal health by alleviating protein aggregation in neurons of a Huntington's disease model. In fact, CEPsh glia directly communicate with neurons through the release of small clear vesicles (SCVs) and relay the coordination to the periphery via downstream neuronal mechanisms. This glia to neuron signal results in induction of the UPR MT in distal tissues, through a cell non-autonomous mechanism, which is dependent on the canonical UPR MT pathway, yet 5 surprisingly distinct from paradigms where UPR MT is directly activated in neurons. Collectively, these results reveal a novel function for CEPsh glia in sensing mitochondrial stress, which initiates a signal to promote protein homeostasis in neurons and ultimately prolongs longevity. Therefore, glial cells serve as one of the upstream mediators of mitochondrial stress and its coordination across the entire organism. 10

Activation of the UPR MT in glia elicits beneficial effects for the entire organism
To activate the UPR MT in glial cells, we leveraged the ability of JMJD-1.2 (JumonjiC (JmjC)domain-containing protein), a histone demethylase, to induce a robust UPR MT in the absence of 15 stress (17). Specifically, we overexpressed jmjd-1.2 under several established glial promoters (18), targeting most glial cells, or glial sub-types using specific promoters. The activation of UPR MT in most glial cells resulted in a mild lifespan extension ( Fig. 1A and fig. S1A). Interestingly, we found that overexpressing jmjd-1.2 in either amphid sheath and phasmid sheath glia (fig-1p), or the four CEPsh glia (hlh-17p) alone was not only sufficient to prolong lifespan but had a more 20 profound effect than overexpressing jmjd-1.2 in most glial cells simultaneously (Fig. 1, B and C). On the cellular level, the activation of the UPR MT in all different glial subtypes was also able to trigger the activation of the UPR MT in distal, intestinal cells, as can be observed by the activation of a fluorescent reporter under the regulation of a mitochondrial chaperone promoter (hsp-6p::GFP) (Fig. 1, D and E). Similar to lifespan extensions, overexpression of jmjd-1.2 in CEPsh 25 glia had the most profound effect on distal UPR MT activation. We confirm that this is a beneficial, functional activation of UPR MT as these animals also show increased resistance to paraquat ( Fig.  1F and fig. S1G), a drug that increases superoxide levels mainly in the mitochondria (19). In fact, animals with glial jmjd-1.2 overexpression exhibit a similar increase in paraquat resistance to animals with daf-2 knockdown, a condition with one of the highest recorded lifespan extensions 30 (20), primarily through oxidative stress resistance (21).
The most profound peripheral UPR MT activation and lifespan extension was observed when overexpressing jmjd-1.2 in CEPsh glia, the four astrocyte-like cells that associate with sensory organs and extend processes that wrap around the nerve ring, the major neuropil of C. elegans 35 (12). Therefore, we focused our efforts on this specific glial subtype (heretofore referred to as glial jmjd-1.2). To determine whether CEPsh glia can elicit non-autonomous signals in direct response to stress rather than solely via ectopic overexpression of UPR MT activators, we induced mitochondrial stress specifically in CEPsh glia in two different ways. First, we use expression of a mitochondrially-targeted KillerRed, which generates highly cytotoxic reactive oxygen species 40 when fluorescently activated. KillerRed can be used to specifically induce a localized acute oxidative stress in the mitochondria by localizing the fluorophore to the mitochondrial matrix and exposing animals to the excitation spectrum of KillerRed (green light) (22). We also induced mitochondrial stress by overexpressing the mitochondrial-binding polyglutamine tract Q40 (10). The introduction of mitochondrial stress in CEPsh glia using both methods resulted in a similar 45 activation of UPR MT in peripheral tissue as jmjd-1.2 overexpression ( fig. S1, B to D). Furthermore, we verified our results using an alternative CEPsh glia-specific promoter recently identified using a single-cell RNA-seq dataset ( fig. S1E) (23). Lastly, the distal activation by CEPsh glia was dependent on intact glial cells, as ablating the cells using the mutant vab-3 (24) abrogated the effect ( fig. S1F). Taken together, these data indicate that not only can CEPsh glia communicate mitochondrial stress in a non-autonomous manner, but they can directly sense mitochondrial stress. Importantly, these studies also highlight the utility of the jmjd-1.2 overexpression line as a viable mimetic for non-autonomous communication downstream of mitochondrial stress in glial cells 5 without the caveats of the potentially detrimental effects of mitochondrial dysfunction in neural cells (10).
We sought to further decipher the underlying mechanisms of the cell non-autonomous activation triggered by CEPsh glia. First, we tested whether the UPR MT transcriptional program mediated by 10 DVE-1/UBL-5 is also induced upon glial UPR MT induction (25,26). DVE-1 is a transcription factor, acting with UBL-5, and mediating UPR MT to alleviate stress in the mitochondria (25). We found that inducing the UPR MT in CEPsh glia caused an increase in the DVE-1::GFP signal both in the head and the intestinal region of the worm (Fig. 2, A and B). Using the fluorescent reporter hsp-6p::GFP, we also found that the activation of the distal UPR MT in response to glial signals 15 depends on three different transcriptional programs of the UPR MT : ATFS-1, UBL-5/DVE-1, and LIN-65/MET-2 (27) (Fig. 2, C to E). Intact UPR MT was also required for the longevity phenotype, as knocking down the transcription factor ATFS-1, which suppressed UPR MT activation, also abrogated the lifespan extension of the glial jmjd-1.2 animals (Fig. 2F). Collectively, these results indicate that the canonical components of the UPR MT pathway are required for the peripheral 20 response of cell non-autonomous communication from CEPsh glia.

Activation of UPR MT in glia rewires multiple cellular processes
To examine more global changes that occur upon activation of the UPR MT in CEPsh glia, we profiled gene expression changes using whole-animal bulk RNA-seq. We observed many changes 25 in gene expression in glial jmjd-1.2 animals with 547 significantly upregulated genes and 413 downregulated genes compared to a wild-type control (Fig. 3A). As expected, gene expression changes show large similarities to other UPR MT paradigms including electron transport chain (ETC) inhibition via cox-5B RNAi (28) or whole-animal overexpression of jmjd-1.2 (17) (Fig.  3B). Indeed, animals with glial jmjd-1.2 overexpression showed an overall increase in UPR MT 30 genes (Fig. 3, C and D), while other stress pathways did not show a significant difference. Moreover, we observed a decrease in the expression of genes involved in translation and ribosome biology (Fig. 3C), a characteristic of stress responses meant to alleviate the protein burden on the organelle (29). We also observed an overrepresentation of genes involved in the response to oxidative stress, with ~40% of the transcriptional response overlapping with the response to 35 paraquat (Fig. 3E), consistent with our data demonstrating that animals with jmjd-1.2 induction in CEPsh glia exhibit increased resilience to oxidative stress.
Further enrichment analysis of the upregulated genes for different gene groups revealed an overrepresentation of genes involved in organellar processes, chromatin-related pathways, 40 metabolic processes, and stress pathways (Fig. 3F). To validate a physiological outcome of the increase in genes involved in stress response and protein homeostasis, we measured the capacity of animals to clear aggregation-prone proteins. Specifically, we measured the aggregation of a polyglutamine (polyQ) tract in intestinal cells (30), which showed a reduction in aggregation upon UPR MT activation in glia both at mid-age (day 5, Fig. 3, G and H) and in early adulthood when 45 exposed to hypertonic stress (31) (fig. S2A). In addition, our gene expression analysis suggested that there is also a rewiring of metabolic pathways, including lipid-related genes, which we validated directly by observing an increase in lipid droplet levels and lipid content in the intestine using both the neutral lipid dye BODIPY ( fig. S2, B and C) and the lipid droplet marker DHS-3::GFP ( Fig. 5I and fig. S2, D and E) (32). These data, collectively with intestinal imaging of hps-6p::GFP, polyQ, and lipid content suggests that glial jmjd-1.2 triggers a transcriptional program across the animal, which impacts UPR MT , protein homeostasis, and lipid levels.. 5

Glia utilize small clear vesicles to communicate with other tissues
The coordination of a whole animal response upon UPR MT activation in CEPsh glia likely requires the transmission of a specific signal(s). The transfer of information between neurons and from neurons relies on distinct secretion pathways, with different encapsulated cargo. Previously, we found that activating the UPR MT in neurons can trigger the UPR MT in the intestine, utilizing the 10 secretion of dense core vesicles (DCVs) (8,10,11), a pathway utilized for the secretion of polypeptide hormones and neuropeptides. These are synthesized as precursors and packaged into dense-core vesicles, processed, and depend on the Ca2+-dependent activator UNC-31/CAPS protein for secretion (33)(34)(35). In contrast, neurotransmitters, which are generally packaged in small clear vesicles (SCVs), and their release is dependent UNC-13/Munc13 (36), were not found to be 15 involved in the activation of UPR MT by neurons (10).
We examined whether the cell non-autonomous activation from CEPsh glia depends on the exocytosis of DCVs and/or SCVs, by mutating components important for their exocytosis, unc-31 and unc-13, respectively. We found that the activation of UPR MT in intestinal cells depends on 20 both functional UNC-13 and UNC-31, and on neuropeptide processing via EGL-3 as visualized by the loss of intestinal hsp-6p::GFP induction in these mutants (Fig. 4, A and B). We also tested for another known signaling molecule required for non-autonomous communication of mitochondrial stress from neurons, the WNT signal EGL-20/WNT5A (11). Similar to neuronal signaling, we find that egl-20 is also required for the distal activation of hsp-6p::GFP initiated 25 from glia ( fig. S3). Taken together, our results indicate that glial jmjd-1.2 activation shares similar genetic requirements in secretion pathways with that of neuronal UPR MT activation with the additional requirement of SCVs (unc-13). The requirement of SCVs is perhaps the most surprising since it clearly differentiates glial signaling from neuronal signaling, as SCVs were previously shown to be dispensable for the non-autonomous communication from neurons. We therefore 30 hypothesize that glia communicate to neurons via unc-13 (SCVs) and neurons communicate to the periphery via unc-31 (DCVs) signaling.
To directly test this hypothesis and uncover in which tissues (glia vs neurons) unc-13 and unc-31 participate in the UPR MT signaling, we knocked out unc-13 and unc-31 in a tissue-specific manner 35 using the FLP/FRT system (37). This system allows deleting a portion of a gene by expressing the flipase, FLP D5, in specific tissues of interest (CEPsh glia vs pan-neuronal) in combination with an allele of the gene of interest (unc-13 or unc-31) to which two copies of the FRT sequence have been introduced using CRISPR/Cas9 genome editing ( Fig. 5A and fig. S4, A to C). The resulting animals have spatially distinct genotypes, with a defect in the secretion of SCVs or DCVs 40 exclusively in one tissue. Strikingly, we observed that knocking out unc-13 specifically in glia, or unc-31 specifically in neurons, was able to alleviate the activation of the UPR MT in the intestine (Fig. 5, B to E). Moreover, rescuing unc-13 specifically in neurons, under the snb-1 promoter, was not able to restore the activation of UPR MT in the periphery ( fig. S4D). Finally, we found that knocking out unc-13 specifically in glia suppressed the lifespan extension of glial xbp-1s animals, 45 whereas knockout of unc-31 in glia had no effect (S5). These results, together with our previous work on neuronal activation of the UPR MT , indicate a linear model, in which glia secrete a molecule by SCVs (unc-13), that is perceived by neurons, which then relay the information to the periphery via DCVs (unc-31). Importantly, glial communication to neurons through UNC-13 is necessary for the lifespan extension found in these animals.

Activation of UPR MT in glia improves neuronal protein homeostasis
Considering our model whereby activation of glial UPR MT results in a glia-to-neuron signal, we 5 next questioned whether glial UPR MT is able to drive improved protein homeostasis in neurons. Indeed, our data and previous studies (10) showed that non-autonomous UPR MT signals can promote protein homeostasis in the target tissues. Moreover, the importance of protein homeostasis in neurons is best illustrated by neurodegenerative diseases, in which protein aggregation occurs (5), of which glial health has been suggested as a critical factor in maintenance of neuronal fitness 10 (14). Thus, we employed the well-established Huntington's disease model in C. elegans, in which a 40-repeat polyglutamine tract (Q40) is expressed in all neuronal cells. Interestingly, Q40 is able to bind directly to mitochondria, and has been shown to also affect mitochondria directly in the same tissue (10,38). We activated the UPR MT in glia and measured a battery of phenotypes associated with the neuronal HD model. Notably, activating UPR MT in glia was able to rescue the 15 thrashing and chemotaxis defects observed in neuronal Q40 animals (Fig. 6, A and B and fig. S6).
The polyglutamine model is also useful for directly measuring protein aggregation, as the Q40 tract is tagged with a YFP. We imaged worms and observed decreased aggregation of the protein in late age (Fig. 6, D and E and fig. S6C). We further verified the aggregation using a filter-trap 20 assay (39), where we observed less aggregation when activating the UPR MT in glia (Fig. 6C). Next, we tested whether these effects are dependent on SCVs. Indeed, we find that intact UNC-13 signaling was required for alleviating the aggregation in neurons, such that mutants of unc-13 show failure of decreasing Q40 aggregation in neurons when overexpressing jmjd-1.2 in CEPsh glia (Fig. 6, D and E). Finally, we asked whether this protection by glia is a general attribute of CEPsh 25 glia, by activating other protein homeostasis pathways in CEPsh glia and testing their ability to improve protein homeostasis in neurons. To this end, we employed our previous model of activating the UPR ER in CEPsh glia, using an overexpression of the spliced version of the transcription factor xbp-1s (16). Interestingly, we were not able to observe any beneficial effects (Fig. S6D), highlighting that the ability of glia to improve neuronal protein homeostasis is unique 30 to the UPR MT .

DISCUSSION
In this study, we discovered a role for a small subset of glial cells in sensing mitochondrial stress and signaling a beneficial cell-non-autonomous communication to neurons. This signal drives a 35 coordinated change in mitochondrial protein homeostasis across tissues, via a relay-cellular pathway mediated by neurons. We found that communication from glia occurs as a two-step process: the astrocyte-like CEPsh glial cells secrete a signal via SCVs, and subsequently, neurons relay the signal to peripheral tissues by utilizing DCVs, and the WNT ligand EGL-20 (Fig. 6F). 40 We found that different subpopulations of glial cells can regulate longevity and coordinate the communication of mitochondrial proteotoxic stress across the tissues of the animal. We were most struck by the CEPsh glia, a cell type that includes only four individual cells, that resulted in multiple physiological benefits, including increased lifespan and stress resistance, with full dependency on the functionality of the regulators involved in the UPR MT . Our efforts to uncover 45 and disentangle the requirement of genes involved in intercellular signaling revealed that CEPsh glia utilize small clear vesicles to signal the mitochondrial proteotoxic stress. Indeed, mammalian astrocytes were suggested to possibly express Munc13, in cultured and freshly isolated astrocytes (40,41), as similarly observed in RNA-seq data from isolated CEPsh glia in nematodes (Fig. S6E) (42), and our findings may provide the context in which these vesicles are utilized.
Our dissection of the functional importance of different genes in communicating the UPR MT from glia to the intestine suggests that the signal is mediated by neurons. We were thus inspired to test 5 whether the internal state of neurons themselves is altered upon receiving the UPR MT from glia. Interestingly, we found that glial UPR MT can promote protein homeostasis in neurons and protect them from the detrimental effects of polyQ expression. Conventionally, neurons have been at the center of studying neurodegenerative diseases and glial cells, such as astrocytes, have been implicated in neurogenerative diseases, mostly by ablation experiments showing that glial cells 10 can exacerbate disease pathology (13). Thus, whether the loss of glial cells directly caused disease phenotypes was not understood, and most studies concluded that the breakdown of neuronal function was the true culprit. However, our study supports a redefining role of astrocytes from simply supporting neuronal cells to playing an active role in sensing stress and communicating a beneficial signal to neurons, which can then mediate a whole-organism response to stress. These 15 findings have potentially huge ramifications for the study of neurodegenerative diseases, as it shifts attention to glial cells and asks the question of whether glial cells should be the true target for potential therapies in neurodegenerative disorders. Indeed, increased activity of astrocytes identified by those with higher levels of the glutamate transporter, GLT-1, was found to preserve cognitive function in patients with Alzheimer's (43). 20 An important question raised by our study is whether CEPsh glia can uniquely sense mitochondrial stress. Our data show that activation of the UPR MT , but not the UPR ER , in CEPsh glia resulted in increased neuronal protein homeostasis. There are several plausible explanations for this: first, it is possible that a distinct glial population is required for communicating ER stress to neurons, 25 whereas CEPsh glia can only communicate ER stress directly to the periphery. Indeed, other studies have shown that neuronal cells differ in their capacity to sense and signal distinct types of UPR ER signals (44), and it is entirely possible that additional subsets of glia can signal ER stress, and that only a specific subtype can signal directly to neurons.Another possibility is that mitochondrial stress signals are uniquely able to activate protein homeostasis machinery adept at 30 clearing polyQ aggregates, whereas other stress signals may activate protein homeostasis machinery for other forms of protein dysfunction. Indeed, previous studies have shown that polyQ aggregates can bind mitochondria and induce the UPR MT (10), suggesting there are organellespecific effects of certain classes of aggregates. Finally, it is entirely possible that glial UPR ER signals do not involve communication with neurons, neither to promote neuronal health nor to 35 utilize neurons as the intermediate signal to the periphery. Indeed, glial UPR ER signals were independent of neuronal signaling and unc-31 rescue solely in glial cells were able to drive nonautonomous activation of UPR ER in glial xbp-1s animals (16). In comparison of our data with the previously published study, it seems likely that for UPR ER signals, glial cells can communicate directly to the periphery via DCVs, while for UPR MT signals, glia first communicate to neurons 40 through SCVs, then neurons communicate to the periphery through DCVs. Thus, by bypassing neurons, it is possible that glial UPR ER fails to improve neuronal proteostasis. Perhaps the most important lesson from these studies is that the same glial cells -CEPsh glia in this casecan utilize completely divergent signaling mechanisms based on which type of stress is sensed. 45 Our data whereby SCV release (unc-13), but not DCVs (unc-31) by glial cells is required for the glial UPR MT signal adds further evidence that signaling of stress is context dependent. Glial signaling of UPR ER , in contrast to UPR MT , required DCV release (unc-31), but not SCVs (unc-13) (16). Moreover, glial UPR ER signaling was seemingly independent of neurons, whereby our data suggests that UPR MT signals follow a glia to neuron to periphery signature. Taken together, these studies suggest that similar to neurons, glia have the capacity to sense and signal various different types of stress and utilize divergent pathways to communicate with the periphery based on the type of stress. While seemingly far 'fetched, a recent study has shown that neurons can elicit entirely 5 different downstream responses in the periphery solely based on which neurotransmitter is utilized. Specifically, dopaminergic and serotonergic circuitsboth experiencing identical stress in the form of UPR ER activationhas the potential to induce lipid remodeling or protein homeostatic responses in the periphery, despite the origin of the signal, xbp-1s, being identical (44). Thus, it is entirely possible that glia can also initiate different signaling paradigms using unique 10 "gliotransmitters" based on the type of stress that is being sensed. Moreover, it is also possible that certain glial subtypes benefit from UPR MT activation, while others do not: specifically, we found that pan-glial jmjd-1.2 overexpression resulted in a much milder lifespan extension compared to jmjd-1.2 overexpression in CEPsh glia alone. Overexpression of jmjd-1.2 may have negative consequences in some glial cells, which negate any positive benefits from other glial subtypes. 15 Indeed, in UPR ER activation, xbp-1s overexpression in muscle cells resulted in decreased lifespan, suggesting that hyperactivation of stress responses can be detrimental in some instances (6). An alternative hypothesis is that glial UPR MT signatures can only be transmitted via CEPsh glia, and while the ptr-10 promoter includes these neurons, expression in CEPsh glia via the prt-10 promoter is much more limited in comparison to the hlh-17 promoter. Overall, these differences do highlight 20 that on an organismal level, the capacity to compartmentalize whole organism-stress responses by utilizing divergent signaling methods would be useful to avoid upregulating unnecessaryor even detrimentalpathways irrelevant to the stress that is currently being experienced.
Finally, we found that glia have the ability to improve protein homeostasis not only on the 25 physiological level (thrashing, chemotaxis) but also on the molecular level, measuring the aggregating protein itself. Similarly, recent work in mice found that indeed astrocytes, through activation of the JAK2-STAT3 pathway, are able to clear protein aggregates in neurons in a mutant huntingtin mouse model (45), highlighting our findings of a UNC-13-related improvement of protein homeostasis in the glial-neuron axis. Of note, UNC-13/Munc13 has previously been linked 30 also to another neurodegenerative disease, Amyotrophic Lateral Sclerosis (ALS). Genome-wide association studies linked UNC13A to ALS as mutations associated with higher susceptibility and shorter survival (46)(47)(48) in individuals with ALS, a link that is evolutionarily conserved in C. elegans (49). Whether the involvement of UNC-13 in ALS stems from its glial functions remains to be explored. 35 In summary, our work not only highlights the importance of glial cells in communicating mitochondrial proteotoxic stress, and coordinating between tissues, but also mechanistically mapped SCVs as the secretion pathway utilized from these cells. Our study places CEPsh glia as upstream regulators of coordination of stress responses and underlines their role as a major 40 signaling hub that can affect cellular and organismal homeostasis. In combination with our findings on protein aggregation in neurons, our results underscore the importance on examining the roles and mechanisms used by glial cells to regulate protein homeostasis within the nervous system and in the periphery. Advancing our knowledge of how glial cells regulate protein homeostasis in the context of mitochondrial stress will be critical in ongoing efforts to understand 45 the glia-neuron communication axis, in neurodegenerative diseases, and in aging.

Acknowledgments:
We are grateful to Larry Joe, Melissa Sanchez, Dr. Tslil Ast, Dr. Naama Aviram, Dr. Ranit Kedmi, and all members of the Dillin lab for technical support and sharing of reagents and equipment.  Competing interests: All authors of the manuscript declare that they have no competing interests.

Data and materials availability:
All data required to evaluate the conclusions in this manuscript are available within the manuscript and Supplementary Materials. All strains synthesized in this manuscript are derivatives of N2 or other strains from CGC and are either made available on CGC or available upon request to the lead contact. Plasmids cloned to generate the used strains are 30 available upon request. RNA-seq datasets supporting the conclusions of this article are available in the NCBI Sequence Read Archives repository, accession number BioProject PRJNA858426.

Strains and maintenance
All C. elegans strains are derivatives of the Bristol N2 wild-type strain from the Caenorhabditis Genetics Center (CGC) and are listed in Table 1. All worms were grown at 15° to 20°C on NGM 5 (nematode growth media) agar plates, fed with OP50 E. coli B strain as a food source, for general maintenance, and handled as previously described (50). For all experiments, worms were switched to HT115 E. coli K12 strain after synchronization using bleaching. Worms were grown for at least 3 generations at 20°C on OP50 prior to synchronization to acclimate to the temperature. HT115 bacteria were carrying the pL4440 empty vector control or expressing double-stranded RNA 10 containing the sequence against a target gene, as specified in the text. RNAi clones against atfs-1 and daf-2 were acquired from the Vidal RNAi library (51) and verified using Sanger Sequencing. All experiments were performed on age-matched animals synchronized using a standard bleaching protocol, to Day 1 of adulthood, or aged to later ages, as specified. Synchronization was achieved by washing animals fed with OP50 E. coli with M9 solution (22 mM KH2PO4 monobasic, 42.3 15 mM Na2HPO4, 85.6 mM NaCl, and 1 mM MgSO4), bleached using a solution of 1.8% sodium hypochlorite and 0.375 M KOH diluted in DDW, for 4-5 minutes, until all carcasses were digested. Intact eggs were then washed 4× with M9 solution, and intact eggs were verified under the microscope after seeding. 20 Strains were generated by cloning the cDNA of interest under the relevant promoter, using Gibson Assembly (52). The cDNAs of jmjd-1.2 and the polyglutamine Q40 were amplified from pCM407 (17) and pJKD101 (10), respectively. The promoters ptr-10p, fig-1p, and hlh-17p were sub-cloned from pAF12, pAF5 and pAF6 (16), respectively. Synthetic DNA of tbb-2 3'UTR and mfsd-13.2 promoter was synthesized by Twist Bioscience. Wild type N2 strain worms were injected with the 25 construct of interest, along with a myo-2p::tdtomato or unc-122p::RFP co-injection marker. Worms were selected under a fluorescent microscope and then integrated by UV irradiation. Integrated lines were backcrossed 8 times to a wild-type N2 strain. For same-orientation FRT insertions; unc-13 between exon 18 and 19 and between exon 20 and 21, and for unc-31, insertions between exon 1 and 2, and 2 and 3 were made using CRISPR/Cas9 by SUNY biotech, and 30 successful cutting was verified using PCR and gel electrophoresis, followed by sequencing (see Figure S5A).

Microscopy and of UPR MT reporters
Imaging of hsp-6p::GFP and DVE-1::GFP fluorescent reporters was done as previously described 35 (53). Briefly, animals were grown on standard RNAi plates from hatch at 20°C until day 1 of adulthood. Animals were picked under a standard dissection microscope with white light at random to avoid biased sampling. Then, animals were anesthetized using a 10-15ul drop of 100 mM sodium azide (NaN 3 ) on NGM plates with no bacteria and aligned to have the same orientation. Images were captured on a Leica M250FA stereoscope equipped with a Hamamatsu 40 ORCA-ER camera driven by LAS-X software, or using an Echo Revolve R4 microscope equipped with an Olympus 4x Plan Fluorite NA 0.13 objective lens, a standard Olympus FITC filter (ex 470/40; em 525/50; DM 560). Brightfield and fluorescent images were acquired, with exposure time and laser intensity matched within experiments. Each micrograph contained 10 individual worms and was independently replicated at least three times.

Large-particle flow cytometry
To quantify fluorescent reporters, flow cytometry using a Union Biometrica bioSorter (cat #250-5000-000) was done as previously described (53). Briefly, staged worms were washed off plates using M9, allowed to settle by gravity, and washed once with M9 to separate from eggs. The signal was collected for time of flight (length) and extinction (thickness) of animals, along with the GFP 5 and RFP. Data were collected gating for size (time of flight [TOF] and extinction) to exclude eggs. Data are represented as an integrated intensity of fluorescence normalized to the size of the animal using the integrated GFP output and dividing by the extinction and time of flight. All data that exceed the measurement capacity of the PMT, calculated as a signal of 65355, are considered saturated and are censored from the calculation. For spatial profiles, the complete profiles were 10 extracted, and worms were aligned according to their myo-2p::tdtomato (red head) signal using MATLAB (MathWorks), and binned into 100 bins to account for differences in animal length (n>50). Then, the average profile and SEM was calculated on binned profiles. For hsp-6p::GFP worms, which do not harbor a myo-2p::tdtomato co-injection marker, the profiles were aligned according to the GFP signal, with the highest peak of signal defined as the posterior intestine. 15

Lifespan analysis
Lifespan analyses were performed as previously described (53). All animals were grown at 20°C on HT115 E. coli, with 80-120 animals used per condition and scored every day or every other day, starting from day 1 of adulthood. Animals were moved away from progeny onto fresh plates for the first 5-7 days until progeny were no longer visible. Animals with bagging, vulval 20 explosions, or other age-unrelated deaths were censored and removed from quantification. At least two independent replicates per condition. For lifespans where FUDR is used, 100 µL of a 10mg/mL FUDR solution (dissolved in M9 solution) was added on top of the bacterial lawn 24 hours prior to animals being moved onto the plate. Animals were moved onto FUDR containing plates at Day 1 of adulthood to eliminate progeny. In all instances where FUDR is used, at least 1 25 biological replicate is performed in the absence of FUDR to ensure that there are no artifacts from using this sterilization technique. Analyses were done using Prism 8 (GraphPad). P-values were calculated using the log-rank (Mantel-Cox) method.

Paraquat resistance assay
Resistance to oxidative stress generated by exposure to 100mM paraquat (Sigma-Aldrich 36541) 30 (19) was done as previously described (53), with three biological replicates per condition. Briefly, fresh paraquat was prepared in M9 solution, and aliquoted into a flat-bottom 96-well plate, with 5 animals per well, with 12 wells per strain (n=60). Every two hours, animals were scored for death in each well. For all paraquat assays, daf-2 RNAi is used as a positive control as daf-2 animals exhibit significant resistance to oxidative stress using this assay (53). 35

RNA sequencing and analysis
Animals were synchronized using a standard bleaching protocol and all RNA collection was performed at day 1 of adulthood fed with HT115 bacteria. A total of 1000-2000 Day 1 animals were harvested using M9, and animals were pelleted by centrifugation. M9 was subsequently aspirated and replaced with TRIzol solution. Worms were freeze-thawed 3× with liquid nitrogen, 40 and a ~30-s vortexing was performed before each refreeze. After the final thaw, chloroform was added at a 1:5 ratio (chloroform:TRIzol), and aqueous separation of RNA was performed via centrifugation in a heavy gel phase-lock tube (VWR, 10847--802). The aqueous phase was collected, mixed with isopropanol at a 1:1 ratio, and then RNA purification was performed using a QIAGEN RNeasy Kit as per the manufacturer's directions. Library preparation was performed using Kapa Biosystems mRNA Hyper Prep Kit. Sequencing was performed at the Vincent J. 5 Coates Genomic Sequencing Core at the University of California, Berkeley using an Illumina NovaSeq SP SR100. For N2 control and hlh-17p::jmjd-1.2, two or three biological replicates were done, respectively. Reads were aligned using HISAT2 (Version 2.2.1) (54) with WBcel235 as the reference genome, quantified using featureCounts (55), and DEG were calculated using DESeq2 (56). Gene groups for analyses: mitochondrial UPR were defined as previously annotated by Soo 10 et al. (57), ER-UPR (GO:0030968), HSR (GO:0009408), and translation (GO:0006412) genes were defined using Gene Ontology (58,59). Comparison to other gene expression collections was done using WormExp (60). Gene enrichment was done using gProfiler (61) and GOrilla (62), for significantly changing genes (P<0.05). Plots were generated using MATLAB (MathWorks). 15

BODIPY staining
Neutral lipid measurements was done using BODIPY 409/503 staining as previously described (32), with three biological replicates. Briefly, worms were synchronized by bleaching, grown on EV, and harvested at L4. Worms were washed three times in M9 buffer. Then 4% PFA was added and incubated for 15 min to fixate the samples. Next, the sample was frozen in liquid nitrogen, and 20 immediately thawed (one time), followed by three washes in 1X PBS. Staining was carried out by incubating worms in 500ul of 1ug/ml BODIPY 493/503 for 1 HR at RT shaking, in the dark. After incubation, worms were washed with M9 three times to remove BODIPY. Worms were then left O/N at 4°C, in shaking, in 1X PBS. Finally, worms were imaged using a fluorescent microscope, or sorted using COPASS BioSort (see relevant section).

Q40 aggregation
Worms were bleached, and eggs grown to day 1 of adulthood on HT115 bacteria. Worms were aged to Day 3 of adulthood, and then imaged using fluorescent microscopy (see relevant section). Q40::YFP was expressed in neurons or intestine using promoters rgef-1p or vha-6p 30 respectively. In addition, the number of aggregates was counted in each worm, with each experimental group composing at least 100 worms. For osmotic stress, worms at day 1 of adulthood were moved to a plate with 500 mM NaCl and imaged after 4 hours of incubation. Three biological replicates were done. Thrashing assay 35 Thrashing of worms was measured using the WormLab (MBF Bioscience) system. Worms were grown to day 1 of adulthood, washed off plates using M9 buffer, and then allowed to settle by gravity in an Eppendorf tube, washed once, and then moved to an empty 6mm plate (without NGM). Worms were video recorded, in three biological replicates, and their thrashing (body waves) were measured using WormLab 2.0 software. The size of each worm (length and width 40 output from WormLab) to gate for worm events, and thrashing was averaged across the gated population.

Chemotaxis assay
Animals were synchronized by bleaching, and assay was conducted at day 1 of adulthood as 45 previously described. Briefly, animals were raised on EV bacteria until day 1 of adulthood and washed three times using M9 buffer by gravity settling. Chemotaxis assay plates were prepared: 100mm NGM plates, with 1uL attractant (Benzaldehyde 1:200, Diacetyl 1:1000) and 1uL diluent (EtOH) on opposite sides, with 1uL of sodium azide in the same location, to immobilize worms once reaching their target. In equal distance, 30-50 worms were dispended and scored after 60 minutes. Chemotaxis index was calculated by I = (# worms at attractant at 60' -# worms at diluent at 60') / total number of worms. Assays were repeated at least 3 times, and significance was assessed by one-way ANOVA with Tukey's multiple comparisons test. 5 Filter trap retardation assay Worms grown to day 1 of adulthood were washed off the plate using M9, harvested and flashfrozen in liquid nitrogen. Then, animals were suspended in lysis buffer (100 mM Hepes pH 7.4, 300 mM NaCl, 2 mM EDTA, 2% Triton X-100, with EDTA-free protease inhibitor cocktail 10 (Roche). Next, animals were homogenized using a Precellys Tissue Homogenizer, with glass and zirconium beads (2mm). Lysates were centrifuged (8000g for 5 minutes at 4 °C), supernatant moved and protein quantified using BCA Protein Assay kit (ThermoFisher, 23225). Protein samples were applied on to cellulose acetate membrane with 0. (E) Integrated fluorescence intensity is measured across an entire worm using a large-particle biosorter and normalized to size as described in Materials and Methods. Fold change was calculated normalized to a control (hsp-6p::GFP) population (n>300 per group). One-way analysis 5 of variance (ANOVA) Tukey's multiple comparisons test, **P< 0.01, ****P< 0.0001. See also Figure S1G. (F) Survival of animals expressing glial jmjd-1.2 (red) under paraquat stress, as compared to a control population (black). Day 1 adult animals were exposed to 100 mM paraquat in M9 solution and scored every 2 hours for survival. Worms fed with daf-2 RNAi were used as a positive control (green) (n=60 per group), see also Figure S1H.     10 and plotted in (E). One-way analysis of variance (ANOVA) Tukey's multiple comparisons test. n.s. not significant, ***P < 0.001, ****P < 0.0001. tissues. CEPsh glia utilize SCVs upon UPR MT activation to signal to neurons, which reduce protein aggregation and utilize DCVs, neuropeptide processing, and a WNT-ligand to drive protein homeostasis and metabolic changes in the periphery.   Figure 2A). L4 worms were irradiated with 1 min of light (543 nm excitation filter), and imaged after 24 hours, and quantified (right). unpaired Student's t test, ****P < 0.0001. (C) Same as S1A, for worms carrying an extrachromosomal array of the polyglutamine tract Q40 under the hlh-17 promoter. (D) Worms 10 over-expressing jmjd-1.2 under hlh-17p were analyzed using a biosorter, and their spatial profiles aligned (left, see methods). The 30% most posterior part of the worm was defined as the posterior intestine, and the integral of the signal over the region was calculated (right). Unpaired Student's t test, ***P < 0.001. (E) Same as S1A, for worms over-expressing jmjd-1.2 from a promoter identified as specific for CEPsh glia (23), mfsd-13.2p (C27H5.4) (left), and its quantification using 15 biosorter (right). unpaired Student's t test, ****P < 0.0001. (F) As figure S1A, for hlh-17p::jmjd-1.2 worms in combination with a mutation in the CEPsh glia lineage mutation in vab-3 (24), and its quantification using biosorter (right). (G) Percentage survival of worm exposed to paraquat was calculated by integrating the area under the curve, and the change in resistance was normalized by the average integral of control worms (N2), set as 100%. One-way analysis of variance (ANOVA) Tukey's multiple comparisons test, **P < 0.01, ***P < 0.001, ****P < 0.0001. and D5 of adulthood. D1 adult animals normally do not show aggregation of Q40::YFP as it is mostly soluble under normal, healthy proteostatic environments, but at D5, some aggregates are visible(B) BODIPY straining at D1 of adulthood and its quantification using worm biosorter (C). Imaging of DHS-3::GFP as in Figure 3 and number of lipid droplets were quantified using Fiji (E). Unpaired Student's t test, *P < 0.05, ****P < 0.0001.    Figure 6B, towards diacetyl. (C) Same as in Figure 6D, for worms on D1 of adulthood. (D) As in Figure 6D, for 5 worms expressing the ER-UPR transcription factor xbp-1s under the hlh-17 promoter (16). Oneway analysis of variance (ANOVA) Tukey's multiple comparisons test, n.s. non-significant, *P < 0.05, ****P < 0.0001.  Table S1. DEGs in hlh-17p::jmjd-1.2 animals. Table S2. Enrichment of upregulated genes using gProfiler. Table S3. GO enrichment for DEGs. 5