Tankyrase (Tnks) transfers poly(ADP-ribose) on substrates. Whereas studies have highlighted the pivotal roles of Tnks in cancer, cherubism, systemic sclerosis, and viral infection, the requirement for Tnks under physiological contexts remains unclear. This study report that the loss of Tnks or its muscle-specific knockdown impairs lifespan, stress tolerance, and energy homeostasis in adult Drosophila. Tnks is a positive regulator in the JNK signaling pathway, and modest alterations in the activity of JNK signaling can strengthen or suppress the Tnks mutant phenotypes. JNK was identified as a direct substrate of Tnks. Although Tnks-dependent poly-ADP-ribosylation is tightly coupled to proteolysis in the proteasome, it was demonstrated that Tnks initiates degradation-independent ubiquitination on two lysine residues of JNK to promote its kinase activity and in vivo functions. This study uncovers a type of posttranslational modification of Tnks substrates and provides insights into Tnks-mediated physiological roles (Li, 2018).

Tankyrase (Tnks) belongs to the poly(ADP-ribose) polymerase (PARP) superfamily, which consists of 17 members in human. PARP-1 is the founding member of the family and has a critical role in the repair of DNA damage. The PARPs are characterized by a structurally similar PARP catalytic domain that successively transfers ADP-ribose from NAD+ onto substrate proteins. This post-translational modification is referred to as poly-ADP-ribosylation, or PARsylation. It has been shown that some PARPs actually catalyze mono-ADP-ribosylation rather than the polymerization of poly(ADP-ribose) chains. Because of this reason, the PARP family members have been renamed as ADP-ribosyltransferases diphtheria toxin-like (ARTDs). Tnks was identified as a telomere-associated protein that binds to the telomere-specific DNA binding protein TRF1. In addition to the PARP domain, Tnks contains two unique domains that distinguish it from other PARPs, including an Ankyrin repeat domain that is involved in the recruitment of substrate and a sterile alpha motif (SAM) that mediates oligomerization. Tnks is evolutionarily conserved in human, mouse, rat, chicken, C. elegans, and Drosophila. The human and mouse genome encodes two Tnks proteins (Tnk1/PARP5A/ARTD5 and Tnk2/PARP5B/ARTD6), whereas there is only one Tnks homolog in Drosophila (Li, 2018).

Tnks catalyzes PARsylation on its substrates and is involved in a variety of cellular processes, such as telomere homeostasis, cell-cycle progression, Wnt/β-catenin signaling, PI3K signaling, Hippo signaling, glucose metabolism, stress granule formation, and proteasome regulation. Various amino acid residues including lysine, arginine, aspartate, glutamate, asparagine, cysteine, phospho-serine, and diphthamide may serve as acceptors for PARsylation. In many cases, proteins modified by Tnks are subsequently poly-ubiquitinated and targeted for proteasomal degradation. For example, Tnks promotes telomere elongation by mediating the degradation of TRF1, a negative regulator of telomere length maintenance. Tnks PARsylates the β-catenin destruction complex component Axin, triggers its degradation, and thereby activates Wnt signaling. Tnks also regulates the stability of centrosomal P4.1-associated protein CPAP to limit centriole elongation during mitosis. On the other hand, the effects of PARsylation on some Tnks substrates, such as the nuclear mitotic apparatus protein NUMA, have not been elucidated, implying that alternative regulation following PARsylation may exist (Li, 2018).

Aberrant Tnks expression or activity has been implicated in a diversity of diseases including cancer, systemic sclerosis, cherubism, Herpes simplex, and Epstein-Barr viral infections. Particularly, the pro-oncogenic role of Tnks in many types of cancer strongly suggests a therapeutic benefit of Tnks inhibition. Whereas a substantial amount of studies have highlighted the important functions of Tnks under pathological conditions, the requirement for Tnks under physiological contexts is largely unexplored (Li, 2018).

This study investigate the physiological functions of Tnks during the adult stage in Drosophila with its mutant alleles and RNAi strains. The loss of Tnks was shown to impair lifespan, stress tolerance, and energy storage in adult flies. Ubiquitous or muscle-specific knockdown of Tnks causes defects similar to those observed in the mutant alleles. It was further shown that Tnks is specifically required for the activity of the c-Jun N-terminal kinase (JNK) and positively regulates the outputs of JNK signaling during organ development. In addition, mild reduction and slight increase in the activity of JNK signaling via genetic manipulations can strengthen and suppress the phenotypes of the Tnks mutants, respectively. Last, the results reveal for the first time that Tnks mediates degradation-independent ubiquitination on its substrate. Drosophila JNK was identified as a direct substrate protein of Tnks. The PARsylation by Tnks triggers K63-linked poly-ubiquitination on JNK, enhancing its kinase activity and maintaining its in vivo functions. Together, this study uncovers that Tnks is a positive regulator of JNK activity, mediates a novel type of post-translational modification, and functions through JNK signaling to affect lifespan, stress resistance, and energy homeostasis in Drosophila (Li, 2018).

PARsylation by Tnks appears to be tightly coupled to poly-ubiquitination and subsequent proteasome-dependent degradation, as observed for known Tnks substrates including Axin, TRF1, PTEN, 3BP2, CPAP, and AMOT family proteins. This study found that Tnks mediated PARsylation and poly-ubiquitination of Drosophila JNK (Bsk), however, without affecting its protein levels. This observation prompted investigation og the form of poly-ubiquitin chain assembled on Bsk in the presence of Tnks. Although all the seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) and the N-terminal methionine residue in the ubiquitin can serve as the attachment site to generate poly-ubiquitin chain, K48-linked and K63-linked poly-ubiquitination are two predominant forms in cells. It is well-established that K48-linked poly-ubiquitination targets substrates to the 26S proteasome and promotes protein degradation, while K63-linked poly-ubiquitin chain performs non-proteolytic functions. Consistently, this study observed that Tnks promoted the assembly of K63-linked poly-ubiquitin chain on Bsk and increased its kinase activity. In contrast, Tnks did not affect K48-linked poly-ubiquitination of Bsk. Although the possibility cannot be completely that these modifications occur on a Bsk partner that tightly binds to Bsk even after stringent washing, this study reveals for the first time that Tnks mediates degradation-independent ubiquitination of its substrate, which may represent a novel type of modification induced by PARsylation. Indeed, it has been reported that PARsylation of some Tnks substrates such as the proteasome regulator PI31 and the mitotic spindle-pole protein NuMA may affect the activities of these proteins. It will be interesting to investigate whether similar modification occurs during these processes (Li, 2018).

JNK is an evolutionarily conserved stress-activated protein kinase (SAPK) and is one of the most versatile stress sensors in metazoans. The JNK signaling pathway adapts growth and metabolism to environmental conditions, and mediates stress tolerance, damage repair, and apoptosis. Therefore, JNK signaling has pivotal roles in regulating homeostasis, longevity, and organ development. This study reports an essential role of Tnks in regulating JNK signaling. As an initial hint, adult Tnks mutants are more sensitive to oxidative stress and to starvation, and live much shorter than the wild-type controls. Although several signaling pathways are known to coordinate these functions in Drosophila, this study observed that the loss of Tnks specifically decreased the activity of Bsk without affecting that of ERK, p38, Akt, and Nrf2 signaling. It was further found that Tnks was required for the outputs of JNK signaling during the development of eye, wing, and thorax. The knockdown of Tnks in the thorax strengthened the thoracic cleft phenotype caused by bsk knockdown. The loss of Tnks, or reducing its gene dosage, suppressed ectopic JNK signaling-induced phenotypes during the formation of compound eye and wing vein. In addition, Tnks overexpression in the wing disc activated puc expression in a bsk-dependent manner. Last, it was observed that bsk with mutations in Tnks-induced ubiquitination sites lacked the ability to rescue the lethality of the bsk^1/2 mutant and displayed impaired activity in regulating stress tolerance, climbing, energy storage, and organ development compared to wild-type bsk. This work thus reveals Tnks as a positive regulator of the JNK signaling pathway (Li, 2018).

Aberrant Tnks expression or activity has been implicated in a variety of disease states including cancer, cherubism, systemic sclerosis, and viral infection. The proposed roles of Tnks in telomere homeostasis, mitosis, and Wnt signaling have made it an attractive drug target in many types of cancer. Tnks is implied in a diversity of additional cellular processes, such as glucose metabolism, stress granule assembly, and proteasome regulation. However, the requirement for Tnks under physiological contexts remains poorly understood. While a double knockout of Tnks1 and Tnk2 is embryonically lethal in mice, Tnks mutant flies do not display any noticeable developmental defects. Although a previous study reported that Tnks-deficient mice exhibited substantially reduced adiposity, the underlying molecular mechanism is unclear. This work has focused on the physiological changes in Tnks mutant flies during the adult stage. It is reported that the loss of Tnks or its knockdown shortens lifespan, decreases climbing ability, reduces resistance to oxidative stress and starvation, and impairs energy storage in adult flies. Through tissue-specific knockdown, it is concluded that the above functions of Tnks are mainly mediated via its activity in the muscle. These physiological functions of Tnks are attributed to its regulation of JNK signaling activity, which is supported by several lines of evidence. First, the loss of Tnks specifically weakens Bsk activity and JNK signaling. Second, mild reduction in JNK signaling activity by removing one copy of bsk gene aggravates the phenotypes in Tnks mutants, whereas slightly elevated JNK signaling largely rescues these phenotypes. Third, this study shows that Tnks triggers PARsylation and K63-linked poly-ubiquitination of Bsk and enhances the kinase activity and in vivo functions of Bsk (Li, 2018).

Developmental pathways must be responsive to the environment. Phosphorylation of eIF2alpha enables a family of stress-sensing kinases to trigger the integrated stress response (ISR), which has pro-survival and developmental consequences. Bone morphogenetic proteins (BMPs) regulate multiple developmental processes in organisms from insects to mammals. This study shows in Drosophila that GCN2 antagonises BMP signalling through direct effects on translation and indirectly via the transcription factor crc (dATF4). Expression of a constitutively active GCN2 or loss of the eIF2alpha phosphatase dPPP1R15 impairs developmental BMP signalling in flies. In cells, inhibition of translation by GCN2 blocks downstream BMP signalling. Moreover, loss of d4E-BP, a target of crc, augments BMP signalling in vitro and rescues tissue development in vivo. These results identify a novel mechanism by which the ISR modulates BMP signalling during development (Malzer, 2018).

Sleep disruptions are quite common in psychological disorders, but the underlying mechanism remains obscure. Wolfram syndrome 1 (WS1) is an autosomal recessive disease mainly characterized by diabetes insipidus/mellitus, neurodegeneration and psychological disorders. It is caused by loss-of function mutations of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes an endoplasmic reticulum (ER)-resident transmembrane protein. Heterozygous mutation carriers do not develop WS1 but exhibit 26-fold higher risk of having psychological disorders. Since WS1 patients display sleep abnormalities, this study aimed to explore the role of WFS1 in sleep regulation so as to help elucidate the cause of sleep disruptions in psychological disorders. It was found in Drosophila that knocking down wfs1 in all neurons and wfs1 mutation lead to reduced sleep and dampened circadian rhythm. These phenotypes are mainly caused by lack of wfs1 in dopamine 2-like receptor (Dop2R) neurons which act to promote wake. Consistently, the influence of wfs1 on sleep is blocked or partially rescued by inhibiting or knocking down the rate-limiting enzyme of dopamine synthesis, suggesting that wfs1 modulates sleep via dopaminergic signaling. Knocking down wfs1 alters the excitability of Dop2R neurons, while genetic interactions reveal that lack of wfs1 reduces sleep via perturbation of ER-mediated calcium homeostasis. Taken together, a role is proposed for wfs1 in modulating the activities of Dop2R neurons by impinging on intracellular calcium homeostasis, and this in turn influences sleep. These findings provide a potential mechanistic insight for pathogenesis of diseases associated with WFS1 mutations (Hao, 2023).

Sleep disruptions are common in individuals with psychiatric disorders, and sleep disturbances are risk factors for future onset of depression. However, the mechanism underlying sleep disruptions in psychiatric disorders are largely unclear. Wolfram Syndrome 1 (WS1) is an autosomal recessive neurodegenerative disease characterized by diabetes insipidus, diabetes mellitus, optic atrophy, deafness and psychiatric abnormalities such as severe depression, psychosis and aggression. It is caused by homozygous (and compound heterozygous) mutation of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes wolframin, an endoplasmic reticulum (ER) resident protein highly expressed in the heart, brain, and pancreas. On the other hand, heterozygous mutation of WFS1 does not lead to WS1 but increase the risk of depression by 26 fold. A study in mice further confirmed that WFS1 mutation is causative for depression. Consistent with the comorbidity of psychiatric conditions and sleep abnormalities, WS1 patients also experience increased sleep problems compared to individuals with type I diabetes and healthy controls. It has been proposed that sleep symptoms can be used as a biomarker of the disease, especially during relatively early stages, but the mechanisms underlying these sleep disturbances are unclear. Considering that heterozygous WFS1 mutation is present in up to 1% of the population and may be a significant cause of psychiatric disorder in the general population, it was decided to investigate the role of wolframin in sleep regulation so as to probe the mechanism underlying sleep disruptions in psychiatric disorders (Hao, 2023).

Although the wolframin protein does not possess distinct functional domains, a number of ex vivo studies in cultured cells demonstrated a role for it in regulating cellular responses to ER stress and calcium homeostasis, as well as ER-mitochondria cross-talk. Mice that lack Wfs1 in pancreatic β cells develop glucose intolerance and insulin deficiency due to enhanced ER stress and apoptosis. Knocking out Wfs1 in layer 2/3 pyramidal neurons of the medial prefrontal cortex in mice results in increased depression-like behaviors in response to acute restraint stress. This is accompanied by hyperactivation of the hypothalamic-pituitary-adrenal axis and altered accumulation of growth and neurotrophic factors, possibly due to defective ER function. A more recent study in Drosophila found that knocking down wfs1 in the nervous system does not increase ER stress, but enhances the susceptibility to oxidative stress-, endotoxicity- and tauopathy-induced behavioral deficits and neurodegeneration (Sakakibara, 2018). Overall, the physiological function of wolframin in vivo, especially in the brain, remains elusive for the most part. This study identified a role for wolframin in regulating sleep and circadian rhythm in flies. Wfs1 deficiency in the dopamine 2-like receptor (Dop2R) neurons leads to reduced sleep, while inhibiting dopamine synthesis blocks the effect of wfs1 on sleep, implying that wfs1 influences sleep via dopaminergic signaling. It was further found that these Dop2R neurons function to promote wakefulness. Depletion of wfs1 alters neural activity, which leads to increased wakefulness and reduced sleep. Consistent with this, it was found that knocking down the ER calcium channel Ryanodine receptor (RyR) or 1,4,5-trisphosphate receptor (Itpr) rescues while knocking down the sarco(endoplasmic)reticulum ATPase SERCA synergistically enhances the short-sleep phenotype caused by wfs1 deficiency, indicating that wolframin regulates sleep by modulating calcium homeostasis. Taken together, these findings provide a potential mechanism for the sleep disruptions associated with WFS1 mutation, and deepen understanding of the functional role of wolframin in the brain (Hao, 2023).

Sleep problems have been reported in WS1 patients. Their scores on Pediatric Sleep Questionnaire are more than 3 times higher than healthy controls and doubled compared to individuals with type I diabetes, indicating that the sleep issues are not merely due to metabolic disruptions. Indeed, this study suggests that the sleep problems in human patients are of neural origin, specifically in the wake-promoting Dop2R neurons. Given that the rebound sleep is not significantly altered in wfs1 depleted flies, it is believed that lack of wfs1 does not shorten sleep duration by impairing the sleep homeostasis system. Instead, wfs1 deficiency leads to excessive wakefulness which in turn results in decreased sleep. Considering that heterozygous WFS1 mutation is present in up to 1% of the population, it would be interesting to examine whether these heterozygous mutations contribute to sleep disruptions in the general population (Hao, 2023).

In mouse, chick, quail and turtle, Wfs1 has been shown to be expressed in brain regions where dopamine receptor Drd1 is expressed. D1-like dopamine receptor binding is increased while striatal dopamine release is decreased in Wfs1-/- mice. The current results also implicate a role for wolframin in dopamine receptor neurons and that lack of wfs1 impacts dopaminergic signaling, as the effects of wfs1 deficiency on both sleep and mushroom body (MB) calcium concentration is blocked by the tyrosine hydroxylase inhibitor AMPT. Both Dop2RGAL4 and GoαGAL4 exhibit prominent expression in the MB, and to be more specific, in the α and β lobes of MB. Previous studies have shown that dopaminergic neurons innervate wake promoting MB neurons, and this study found Dop2R and Goα+ cells to be wake-promoting as well. Therefore, it is suspected that wolframin functions in MB Dop2R/Goα+ neurons to influence sleep. Taken together, these findings suggest an evolutionarily conserved role of wolframin within the dopaminergic system. As this system is also important for sleep/wake regulation in mammals, it is reasonable to suspect that wolframin functions in mammals to modulate sleep by influencing the dopaminergic tone as well (Hao, 2023).

MB neural activity appears to be enhanced in wfs1 deficient flies based on the results obtained using CaLexA and spH reporters. This elevated activity is consistent with behavioral data, as activation of Dop2R/Goα+ cells reduces sleep, similar to the effects of wfs1 deficiency. Moreover, silencing Dop2R neurons rescues the short-sleep phenotype of wfs1 mutants, while over-expressing wfs1 restores the decreased sleep induced by activation of Dop2R neurons. These findings suggest that wolframin functions to suppress the excitability of MB Dop2R neurons, which in turn reduces wakefulness and promotes sleep. Comparable cellular changes have been observed in SERCA mutant flies. Electric stimulation leads to an initial increase followed by prolonged decrease of calcium concentration in mutant motor nerve terminal compared to the control, while action potential firing is increased in the mutants. This series of results underpin the importance of ER calcium homeostasis in determining membrane excitability and thus neural function (Hao, 2023).

GCaMP6 monitoring reveals that wfs1 deficiency selectively reduces fluorescence signal in the MB both under baseline condition and after dopamine treatment, which should reflect a reduction of cytosolic calcium level that is usually associated with decreased excitability. Previous studies have shown that lack of wolframin leads to increased basal calcium level in neural progenitor cells derived from induced pluripotent stem (iPS) cells of WS1 patients and primary rat cortical neurons, but after stimulation the rise of calcium concentration is smaller in Wfs1 deficient neurons, resulting in reduced calcium level compared to controls. Similarly, evoked calcium increase is also diminished in fibroblasts of WS1 patients and MIN6 insulinoma cells with WFS1 knocked down. Notably, wolframin has been shown to bind to calmodulin (CaM) in rat brain, and is capable of binding with calcium/CaM complex in vitro and in transfected cells. This may undermine the validity of using GCaMP to monitor calcium level in wfs1 deficient animals and cells, and could potentially account for the contradictory data acquired using CaLexA vs GCaMP (Hao, 2023).

It is intriguing that in this study wfs1 deficiency appears to selectively impair the function of Dop2R/Goα+ neurons. It has been shown that in the rodent brain Wfs1 is enriched in layer II/III of the cerebral cortex, CA1 field of the hippocampus, central extended amygdala, striatum, and various sensory and motor nuclei in the brainstem. Wfs1 expression starts to appear during late embryonic development in dorsal striatum and amygdala, and the expression quickly expands to other regions of the brain at birth. It is suspect that in flies wfs1 may be enriched in Dop2R/Goα+ cells during a critical developmental period, and that sufficient level of wolframin is required for their maturation and normal functioning in adults. Another possibility is that these cells are particularly susceptible to calcium dyshomeostasis induced by loss of wfs1. Indeed, this is believed to be an important cause of selective dopaminergic neuron loss in Parkinson's Disease, as dopaminergic neurons are unique in their autonomic excitability and selective dependence on calcium channel rather than sodium channel for action potential generation. It is reasoned that Dop2R/Goα+ neurons may also be more sensitive to abnormal intracellular calcium concentration, making them particularly vulnerable to wfs1 deficiency. The pathogenic mechanism underlying the neurodegeneration of WS1 is quite complex, possibly involving brain-wide neurodegenerative processes and neurodevelopmental dis-regulations. The findings of this study provide some evidence supporting a role for altered dopaminergic system during development. Obviously, much more needs to be done to test these hypotheses (Hao, 2023).

The precise role of wolframin in ER calcium handling is not yet clear. It has been shown in human embryonic kidney (HEK) 293 cells that knocking down WFS1 reduces while over-expressing WFS1 increases ER calcium level. The authors concluded that wolframin upregulates ER calcium concentration by increasing the rate of calcium uptake. Consistently, this study found by genetic interaction that knocking down RyR or Itpr (which act to reduce ER calcium level and thus knocking down either one will increase ER calcium level) rescues the short-sleep phenotype caused by wfs1 mutation, while knocking down SERCA (which acts to increase ER calcium level and thus knocking down this gene will reduce ER calcium level) synergistically enhances the short-sleep phenotype. Based on the results of these genetic interactions, it is proposed that lack of wfs1 increases cytosolic calcium while decreasing ER calcium, leading to hyperexcitability of Dop2R neurons and thus reduced sleep. Knocking down RyR or Itpr decreases cytosolic calcium and increases ER calcium, counteracting the influences of wfs1 deficiency and thus rescuing the short-sleep phenotype. On the other hand, knocking down SERCA further increases cytosolic calcium and decreases ER calcium, rendering an enhancement of the short-sleep phenotype. In line with this, study conducted in neural progenitor cells derived from iPS cells of WS1 patients demonstrated that pharmacological inhibition of RyR can prevent cell death caused by WFS1 mutation. In addition, inhibiting the function of IP3R may mitigate ER stress in wolframin deficient cells. One caveat is that SERCA protein level is increased in primary islets isolated from Wfs1 conditional knock-out mice, as well as in MIN6 cells and neuroblastoma cell line with WFS1 knocked down. It is reasoned that this may be a compensatory increase to make up for the reduced ER calcium level due to wolframin deficiency. It is acknowledged that the hypothesis proposed in in the papert is not supported by GCaMP data, which indicates decreased cytosolic calcium level in Dop2R neurons of wfs1 deficient flies. It is suspected that since the sleep phenotype associated with lack of wfs1 is of developmental origin, it is possible there is an initial increase of cytosolic calcium during critical developmental period in wfs1 deficient flies and this influences the function of Dop2R neurons in adults. Clearly, further characterizations need to be done to fully elucidate this issue, and preferably another calcium indicator independent of the GCaMP system should be employed (Hao, 2023).

In conclusion, this study identified a role for wolframin in the wake-promoting Dop2R neurons. wfs1 depletion in these cells lead to impaired calcium homeostasis and altered neural activity, which in turn leads to enhanced wakefulness and reduced sleep. This study may provide some insights for the mechanisms underlying the sleep disruptions in individuals with WFS1 mutation, as well as for the pathogenesis of WS1 (Hao, 2023).

The proper balance of synthesis, folding, modification, and degradation of proteins, also known as protein homeostasis, is vital to cellular health and function. The unfolded protein response (UPR) is activated when the mechanisms maintaining protein homeostasis in the endoplasmic reticulum become overwhelmed. However, prolonged or strong UPR responses can result in elevated inflammation and cellular damage. Previously, it was discovered that the enzyme filamentation induced by cyclic-AMP (Fic) can modulate the UPR response via posttranslational modification of binding immunoglobulin protein (BiP) by AMPylation during homeostasis and deAMPylation during stress. Loss of fic in Drosophila leads to vision defects and altered UPR activation in the fly eye. To investigate the importance of Fic-mediated AMPylation in a mammalian system, a conditional null allele of Fic was induced in mice, and the effect of Fic loss on the exocrine pancreas was characterized. Compared to controls, Fic(-/-) mice exhibit elevated serum markers for pancreatic dysfunction and display enhanced UPR signaling in the exocrine pancreas in response to physiological and pharmacological stress. In addition, both fic^-/- flies and Fic^-/- mice show reduced capacity to recover from damage by stress that triggers the UPR. These findings show that Fic-mediated AMPylation acts as a molecular rheostat that is required to temper the UPR response in the mammalian pancreas during physiological stress. Based on these findings, it is proposed that repeated physiological stress in differentiated tissues requires this rheostat for tissue resilience and continued function over the lifetime of an animal (Casey, 2022).

Protein homeostasis is regulated by proper synthesis, folding, modification, and degradation of proteins and is vital to cellular health. In the endoplasmic reticulum (ER), when the load of unfolded proteins is excessive, the unfolded protein response (UPR) is activated, triggering signaling pathways that result in changes to protein synthesis, modification, and degradation until the load of unfolded proteins is resolved. If the burden of unfolded proteins is prolonged and/or remains high, proapoptotic pathways can be activated. The activation of the UPR is, in part, regulated by the Hsp70 protein chaperone binding immunoglobulin protein (BiP), a protein that binds and helps fold proteins as they pass through the ER checkpoint and into the secretory pathway. Depending on the level of unfolded protein, complex signaling networks are activated and respond in accordance to the severity. Mild to moderate levels of UPR signaling are prominent with cell recovery and cell survival, whereas strong and prolonged UPR signaling leads to apoptosis. These responses are mediated by three distinct ER signaling branches: inositol-requiring enzyme-1α (IRE1α); protein kinase R-like ER kinase; and activating transcription factor 6α (Casey, 2022).

In addition, the UPR stress can be divided into two phases: the adaptive phase and the maladaptive phase. For the adaptive phase, the UPR induction responds to mild to moderate stress and promotes prosurvival and restorative mechanisms to promote ER homeostasis. By contrast, the maladaptive phase is induced by chronic and severe ER stress resulting in the activation of proinflammatory responses and apoptosis. Disruption of ER homeostasis is predicted to play a key role in the integrated stress response and the progression of many neurodegenerative, inflammatory, and metabolic disorders. Elucidating the roles that the UPR plays in modulating ER stress provides potential therapeutic targets to treat or prevent the death of the cells subjected to prolonged ER stress and ameliorate UPR-related degenerative diseases (Casey, 2022).

Previously, the activity of BiP was shown to be regulated by a posttranslational modification (PTM) called AMPylation. AMPylation is a reversible PTM best described as the covalent linkage of adenosine monophosphate (AMP) to the hydroxyl group of a serine, threonine, or tyrosine residue (11). Initially discovered in the 1960s with a nucleotidyl transferase domain, protein AMPylation was rediscovered in 2009 with a filamentation induced by cyclic-AMP (Fic) domain from a bacterial pathogen that is also conserved in eukaryotic organisms. To date, only two AMPylating enzymes have been identified in metazoans: Fic (also known as FicD and HYPE) localizes in the ER, and SelO localizes in the mitochondria (Casey, 2022).

Using Drosophila as an animal model, this study found that Fic is responsible for reversible AMPylation of BiP. During low ER stress or resting cells, Fic AMPylates BiP, thereby creating an inactive pool of BiP in the ER lumen. When ER stress rises, BiP is deAMPylated and returned to an active state. Since this discovery, other laboratories have confirmed that this function is conserved in other metazoans, including Caenorhabditis elegans, rodents, and humans. It has been demonstrated that Fic has dual catalytic activity for both the AMPylation and deAMPylation of metazoan BiP, and this activity changes depending on levels of ER stress (Casey, 2022).

Further studies on the Drosophila model revealed that Fic plays a crucial role in protein homeostasis for metazoans. For Fic null flies (fic^-/-), the gross morphology of the fly eyes appeared normal, albeit they exhibited mild vision defects. When acute physiological ER stress was induced in fly eyes by exposure to continuous light, photoreceptors in wild-type flies, but not in fic^-/- mutants, could adapt. The damaged fic^-/- eyes exhibited severe structural defects in rhabdomeres (rhodopsin-containing membranes), elevated IRE1 activity, and reduced neurotransmission. Flies expressing BiPS366A that were unable to be AMPylated at Ser366 phenocopied the fic^-/- flies, with damaged rhabdomeres and loss of postsynaptic responses for photoreceptors stressed with continuous light. Taken together, these studies support the proposal that having a reserve of inactive AMPylated BiP that can be immediately accessed by deAMPylation allows cells to deal with physiological stress more efficiently (Casey, 2022).

Overall, it is proposed that Fic acts as a rheostat that tempers the cellular response to stress and maintains homeostasis by deAMPylating a reserve pool of modified BiP, thereby increasing levels of active BiP to alleviate mild ER stress. When the rheostat is disrupted, either by the absence of Fic or by a mutation in BiP that hampers its AMPylation, recovery from physiologically stressed cells is hindered, as there is no resource for immediate access to additional BiP pools. In the absence of this pool, more BiP can only be provided by the time-consuming transcription and translation of de novo BiP, coincidently with the triggering of UPR (Casey, 2022).

Based on these findings, it is predicted that Fic is also required for the proper regulation of physiological stress in mammals. To address this hypothesis, a conditional knockout line of Fic was generated in the mouse. As with flies, the fic^-/- animals are viable, fertile, and appear healthy upon initial inspection. However, closer characterization of fic^-/- pancreata revealed altered responses to physiological and pathological stresses, with significant changes in UPR-induced signaling. It is hypothesized that without Fic, the balance and threshold between the adaptive phase and the maladaptive phase of the UPR is shifted in tissues that rely heavily on ER secretory pathway to maintain protein homeostasis. Interestingly, marked resilience was observed in wild-type flies and mice when dealing with repeated stress. By contrast, both fic^-/- flies and fic^-/- mice lack the ability to efficiently recover from these stresses, resulting in damaged eyes and scarred pancreas, respectively. Taken together, these findings support the hypothesis that metazoan Fic plays a critical role by acting as a rheostat for the regulation of the UPR and protein homeostasis, likely to be important for the resilience of terminally differentiated, professional secretory cells that must respond to fluctuating needs of an organ (Casey, 2022).

The UPR is crucial for the maintenance of protein homeostasis during physiological stress of cells with high secretory capacity. When cellular stress levels reach maladaptive levels, recovery from stress becomes challenging due to prolonged attenuated protein synthesis of nonstress-responsive genes and activation of apoptotic machinery. Thus, regulation of the UPR must be tightly coordinated to meet the needs of the tissue. To understand the role of reversible AMPylation of BiP in protein homeostasis in a mammalian system, a conditional knockout of Fic was created in mice. It was speculated that tissues reliant on secretion might be most affected by the deletion of Fic and therefore initial efforts focused on the pancreas. Using both a fast-feeding and a caerulein-induced pancreatic injury model, changes were observed to UPR signaling and physiology of the pancreas suggestive of exocrine pancreas disfunction in fic^-/- mice. Analysis of UPR markers for these experiments revealed changes in the timing and duration of the UPR transcriptional response (Casey, 2022).

Furthermore, analysis of tissue recovery after light-induced or caerulein-induced damage in Drosophila eyes and mouse pancreas, respectively, indicates that loss of Fic reduces recovery from ER stress-associated tissue damage in both animal models. Therefore, the loss of Fic-mediated AMPylated BiP leaves tissues vulnerable to irreversible damage with chronic and repeated stresses (Casey, 2022).

It has been proposed that inactivating PTMs on BiP could provide a mechanism by which rapidly changing physiological fluctuations of the ER stress can be nimbly regulated. AMPylation of BiP by Fic allows for a pool of inactive chaperone to remain in the ER without deleterious consequences to protein folding that might otherwise be hindered in the presence of excess chaperone. Previous studies indicate that the pool of inactivated BiP is significant in various cell types, ~40% in fasted pancreas and over 50% in unstressed Drosophila S2 cells. This inactive pool of BiP can then be readily activated to address increasing loads of unfolded proteins in cells with rapidly fluctuating demands on protein synthesis and secretion, such as the pancreas, while tempering the activation of the UPR (Casey, 2022).

It is predicted that Fic provides a necessary level of regulation of the UPR to properly adjust protein homeostasis in tissue with frequent physiological ER stress. By keeping a readily accessible pool of inactive BiP, cells can provide a nimble response to ER stress through a short burst of UPR activation. Rapid deAMPylation of BiP results in additional active chaperone much faster than what can be accomplished by new protein synthesis. This results in smaller, more moderate pulses of UPR signaling under repeated physiological stresses, keeping the cell in the beneficial adaptive phase of the UPR. As Fic is a UPR-responsive gene, it is possible that as these stresses are repeated, a larger pool of inactive BiP may be generated over time, resulting in a more robust rapid response to unfolded proteins in the ER in these adapted tissues (Casey, 2022).

Cells without Fic regulation of BiP lack this pool of chaperone on standby, resulting in prolonged and elevated UPR signaling, as a delayed response requires more chaperone via transcription and translation to accommodate the increased physiological stress. This is supported by qPCR analysis of UPR-responsive genes in fic^-/- mice under both physiological and pharmacological stresses. Repetitive stress in fic^-/- tissues would result in amplified UPR, leading to progression into the maladaptive phase of the UPR and tissue damage over the lifetime of the tissue. The data point to evidence of this in the exocrine pancreas, where elevated UPR signaling and serum amylase indicate functional disruption. As elevated serum amylase is one of the first key clinical indicators of pancreas dysfunction, it is suspected that additional and continued physiological stresses to fic^-/- tissue will lead to increased prevalence of disease (Casey, 2022).

The pancreas primarily comprises terminally differentiated professional secretory cells with limited regenerative capability. Therefore, the pancreas must employ mechanisms to ensure resilience to repetitive stress in order to last and properly function for the lifetime of the animal. This study provides evidence that Fic provides one such mechanism through the moderation of the UPR during physiological stress. Similarly, a wild-type fly eye has the capacity to recover from the physiological stress of continuous light. In the absence of the Fic rheostat, the fic^-/- eyes are challenged over time and lose the potential to regenerate rhabdomere integrity. Analogously, e more scarring was observed in the injured fic^-/- pancreas (Casey, 2022).

Many studies to date have used tissue culture cell lines as a model to study the UPR in which a chemical stress is applied to cells resulting in a very strong, and frequently irreversible, induction of the UPR. Under these conditions, tissue culture cells respond in basically two ways, cell death or replication, allowing for new cells to overcome the stress. These options are far from optimal for differentiated cells within a tissue where cellular function needs to be maintained for survival of the organ and/or animal. It is predicted that many subtleties of UPR regulation will only be apparent under such physiological stresses in the context of specific tissues. Thus, it is not surprising that studies with tissue culture models have only exhibited very subtle differences in activation of UPR in the absence of Fic. Systems in an animal that use cells with high regenerative capacity and shorter life spans may not require Fic mediation of the UPR, as turnover and replenishment with new cells will bypass the need of rheostat. This is consistent with observations by other groups with a Fic deletion model. In sum, it is predicted that terminally differentiated postmitotic cells will be principally reliant upon the Fic-mediated rheostat to maintain a healthy response to continuing physiological stress over an animal's lifetime (Casey, 2022).

Whereas this study focuses on this one tissue only, it is speculated that other tissues with professional secretory, terminally differentiated cells that must adapt to fluctuating stress will be similarly affected in the fic^-/- mouse. It is proposed the presence of Fic rheostat allows for tempering of the UPR response by maintaining a window for reversible UPR response that is critical for maintenance of protein homeostasis. The importance of this window has been highlighted with the treatment of UPR stress with the pharmacological agent ISRIB (integrated stress response inhibitor) where it is only observed to be efficacious during the adaptive phase of UPR. Future studies with ISRIB and fic^-/- mice will be useful for understanding the importance of the Fic-mediated rheostat and treatment of disease (Casey, 2022).

For the health of an animal, it is critical to maintain resilience in terminally differentiated cells during repeated physiological stress to prevent disease. It is predicted that Fic regulation of the UPR will play a role in mitigating the deleterious effects of UPR activation in a variety of tissues with UPR-associated diseases, including retinal degeneration, atherosclerosis, metabolic syndrome, and various neurodegenerative disorders. Future studies will focus on the identification of tissues in which Fic plays a role in the regulation of the UPR and the physiological consequences of the absence of Fic-mediated regulation of the UPR (Casey, 2022).

Cell competition allows winner cells to eliminate less fit loser cells in tissues. In Minute cell competition, cells with a heterozygous mutation in ribosome genes, such as RpS3(+/-) cells, are eliminated by wild-type cells. How cells are primed as losers is partially understood and it has been proposed that reduced translation underpins the loser status of ribosome mutant, or Minute, cells. Using Drosophila this study shows that reduced translation does not cause cell competition. Instead, proteotoxic stress was identified as the underlying cause of the loser status for Minute competition and competition induced by mahjong, an unrelated loser gene. RpS3^+/- cells exhibit reduced autophagic and proteasomal flux, accumulate protein aggregates and can be rescued from competition by improving their proteostasis. Conversely, inducing proteotoxic stress is sufficient to turn otherwise wild-type cells into losers. Thus, it is proposed that tissues may preserve their health through a proteostasis-based mechanism of cell competition and cell selection (Baumgartner, 2021).

This work shows that single copy loss of ribosome genes leads to major defects in cellular proteostasis. Heterozygosity of ribosome genes in humans leads to genetic disorders collectively known as ribosomopathies, characterized by severe malformations and pathologies. The mechanisms through which ribosomal mutations lead to these defects are only partially understood. This work suggests that proteotoxic stress may be an underlying cause for some such defects and that they might be improved by drugs that promote proteostasis, such as the FDA-approved compound rapamycin that was used in this study (Baumgartner, 2021).

This work shows that proteotoxic stress is sufficient to confer the loser status. This finding broadens the scope of cell competition and suggests it may be an active mechanism in physiological and pathological contexts characterized by proteotoxic stress. This may help explain the competitive elimination of neurons in Drosophila models of neurodegenerative diseases. It may be especially relevant to cancer, where proteotoxic stress is often observed. These findings suggest that cancer cells might represent concealed losers that have escaped proteotoxic stress-induced cell competition through masking mutations. Understanding how Minute mutations and proteotoxic stress lead to cell competition may help unmask the loser status in cancer cells in ways that could be exploited therapeutically (Baumgartner, 2021).

Healthy proteostasis is a driver of organism fitness and contributes to organism longevity, whereas impaired proteostasis is associated with aging and with age-related pathologies. It is proposed that tissues preserve their health and youth through a proteostasis-based mechanism of cell elimination. By measuring cell fitness on the basis of proteostasis and converting it into the loser status through the activation of the oxidative stress response, proteostasis-based cell competition could act as a general mechanism of cell selection in adult homeostasis. How proteotoxic stress induces the loser status remains to be established (Baumgartner, 2021).

The endoplasmic reticulum (ER) is the site where most membrane and secretory proteins undergo folding and maturation. This organelle contains an elaborate network of chaperones, redox buffers, and signaling mediators, which work together to maintain ER homeostasis. When the amount of misfolded or nascent proteins exceeds the folding capacity of a given cell, the ER initiates a gene expression regulatory program that is referred to as the Unfolded Protein Response (UPR) (Brown, 2021).

The ER also represents an important nexus between protein folding and oxidative stress. The ER maintains an oxidizing environment for the formation of intra- and intermolecular disulfide bonds that contribute to the oxidative folding of client proteins. A product of this reaction is hydrogen peroxide, and excessive protein misfolding in the ER can cause the accumulation of reactive oxygen species (ROS). Consistently, genes involved in redox homeostasis are induced in response to ER stress (Brown, 2021).

In metazoans, there are three evolutionarily conserved branches of the UPR initiated by the ER transmembrane proteins IRE1, PERK (PKR-like ER Kinase, also known as Pancreatic ER Kinase (PEK)), and ATF6. The best studied downstream effectors of IRE1 and PERK signaling are the bZIP family transcription factors XBP1 and ATF4, respectively. Once activated in response to ER stress, these transcription factors induce the expression of genes involved in ER quality control, antioxidant response, and amino acid transport. The Drosophila genome encodes mediators of all three branches of the UPR, and the roles of the IRE1-XBP1 and PERK-ATF4 branches in Drosophila development and tissue homeostasis have been established (Brown, 2021).

The PERK branch of UPR draws considerable interest in part because its abnormal regulation underlies many metabolic and neurodegenerative diseases. Stress-activated PERK is best known to initiate downstream signaling by phospho-inhibiting the translation initiation factor eIF2α. While most mRNA translation becomes attenuated under these conditions, ATF4 protein synthesis increases to mediate a signaling response. Such ATF4 induction requires ATF4's regulatory 5' leader sequence that has an upstream Open Reading Frame (uORF) that overlaps with the main ORF in a different reading frame. This overlapping uORF interferes with the main ORF translation in unstressed cells. (Harding, 2000; Kang, 2015; Vattem, 2004). But eIF2α phosphorylation causes the scanning ribosomes to bypass this uORF, ultimately allowing the translation of the main ORF assisted by the noncanonical translation initiation factors eIF2D and DENR. The literature also reports PERK effectors that may be independent of ATF4. These include a small number of factors that are translationally induced in parallel to ATF4 in stressed mammalian cells. Compared to the ATF4 axis, the roles of these ATF4-independent PERK effectors remain poorly understood (Brown, 2021).

This study reports that a previously uncharacterized UPR signaling axis is required for the expression of the most significantly induced UPR targets in the larval eye disc of Drosophila melanogaster. Specifically, glutathione-S-transferases (gstDs) were among the most significantly induced UPR target genes in Drosophila. It was further shown that such gstD induction was dependent on Perk, but did not require crc, the Drosophila ortholog of ATF4. Instead, this response required Xrp1, which encodes a bZIP transcription factor with no previously established connections to the UPR. Together, these findings suggest that PERK-Xrp1 forms a previously unrecognized signaling axis that mediates the induction of the most highly upregulated UPR targets in Drosophila (Brown, 2021).

This study reports that ER stress activates a previously unrecognized UPR axis mediated by PERK and Xrp1. Specifically, it was shown that gstD family genes are among the most highly induced UPR targets in Drosophila, and that such induction requires Perk, one of the three established ER stress sensors in metazoans. Surprisingly, the induction of gstD genes in this context did not require crc, the ATF4 ortholog. Instead, it was found that a poorly characterized transcription factor Xrp1 is induced downstream of Perk to promote the expression of gstDs and other antioxidant genes (Brown, 2021).

These findings are surprising given that ATF4 is considered a major effector of PERK-mediated transcription response. ATF4 was the first PERK downstream transcription factor to be identified in part based on the similarity of its regulatory mechanisms with that of yeast GCN4. But more recent studies have shown there could be parallel effectors downstream of PERK activation. The functional significance of these alternative factors had remained poorly understood. This study has led to the conclusion that an ATF4-independent branch of PERK signaling is required for the expression of the most highly induced UPR target in Drosophila (Brown, 2021).

As a potential mediator of this ATF4-independent PERK signaling, cncC was first considered as a prime candidate for a few reasons: cncC is an established regulator of gstD-GFP induction, and previous studies had reported that Nrf2 is activated by PERK in cultured mammalian cells and in zebrafish. However, the results reported in this study do not support the simple idea that gstD-GFP is induced by CncC, which in turn is activated by PERK. Specifically, it was found that the loss of Perk blocked gstD-GFP induction in this experimental setup, but the loss of cncC did not. While Nrf2/CncC clearly regulates antioxidant gene expression in response to paraquat, the results indicate that PERK mediates an independent antioxidant response in Drosophila (Brown, 2021).

The data indicates that this ATF4-independent PERK signaling response requires the AT-hook bZIP transcription factor Xrp1. Several pieces of evidence support the idea that Xrp1 is translationally induced, analogous to the mechanism reported for ATF4 induction. First, RNA-seq and qRT-PCR results indicate that Xrp1 transcript levels do not change significantly in Rh1G69D expressing eye discs. These results argue against the idea that Xrp1 is induced at the transcriptional level. Second, it was found that PERK's kinase domain is required for Xrp1 protein induction. Third, knockdown of gadd34 (Protein phosphatase 1 regulatory subunit 15), which increases phospho-eIF2α levels downstream of Perk, is sufficient to induce Xrp1 protein and gstD-GFP expression. Finally, this study find that Xrp1's 5' leader has a uORF that overlaps with the main ORF, similar to what is found in ATF4's regulatory 5' leader sequence. Moreover, Xrp1's uORF2 encodes a peptide sequence that is phylogenetically conserved in other Drosophila species. High-sequence conservation at the peptide level enhances confidence that uORF2 is a peptide coding sequence (Brown, 2021).

Xrp1 is known to respond to ionizing radiation, motor neuron-degeneration in a Drosophila model for amyotrophic lateral sclerosis (ALS), and in cell competition caused by Minute mutations that cause haplo-insufficiency of ribosomal protein genes. Interestingly, two recent studies reported that these Minute cells induce gstD-GFP, and also show signs of proteotoxic stress as evidenced by enhanced eIF2α phosphorylation (Baumgartner, 2021; Recasens-Alvarez, 2021). Although these studies did not examine the relationships between Xrp1, gstD-GFP and eIF2α kinases such as Perk, the current findings make it plausible that the PERK-Xrp1 signaling axis regulates cell competition caused by Minute mutations (Brown, 2021).

Despite the rising levels of interest in Xrp1 as a stress response factor, the identity of its mammalian equivalent remains unresolved. Xrp1 is well conserved in the Dipteran insects, but neither NCBI Blast searches nor Hidden Markov Model-based analyses identify clear orthologs in other orders. Such evolutionary divergence is not unprecedented in UPR signaling: GCN4 is considered a yeast equivalent of ATF4, but they are not the closest homologs in terms of their peptide sequences. Likewise, the yeast equivalent of XBP1 (IRE1 effector, not to be confused with Xrp1 in this study) is Hac1, but there is little sequence conservation between the two genes. Yet, the UPR signaling mechanisms are considered to be conserved due to the shared regulatory mechanisms. Along these lines, mammalian cells may have functional equivalents of Xrp1. Among the candidate equivalent factors those with regulatory 5' leader sequences that respond to eIF2α phosphorylation were considered. Based on the emerging roles of Xrp1 in Drosophila models of human diseases, it is speculated that those ATF4-independent PERK signaling effectors may play more significant roles in diseases associated with UPR than had been generally assumed (Brown, 2021).

It is noted that genes encoding cytoplasmic glutathione S-transferases (GSTs) such as gstD1 and gstD9 are among the most prominently induced UPR targets in the eye imaginal disc-based gene expression profiling analysis. Previous studies also reported these as ER stress-inducible genes in Drosophila S2 cells. GSTs are cytoplasmic proteins that participate in the detoxification of harmful, often lipophilic intracellular compounds damaged by ROS. These enzymes catalyze the formation of water-soluble glutathione conjugates that can be more easily eliminated from the cell. It is noteworthy that ROS is generated as a byproduct of Ero-1-mediated oxidative protein folding, and such ROS generation increases when mutant proteins undergo repeated futile cycles of protein oxidation. Therefore, it is speculated that cytoplasmic GSTs evolved as UPR targets as they have the ability to detoxify lipid peroxides or oxidized ER proteins that increase in response to ER stress (Brown, 2021).

In conclusion, these findings support the idea that an ATF4-independent branch of PERK signaling mediates the expression of the most highly induced UPR targets in eye disc cells. This axis of the UPR requires Xrp1, a gene that had not previously been associated with ER stress response. The identification of this new axis of UPR signaling may pave the way for a better mechanistic understanding of various physiological and pathological processes associated with abnormal UPR signaling in metazoans (Brown, 2021).

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the endoplasmic reticulum (ER) exit sites under the stress of amino acid starvation. This study addresses which signaling pathways cause Sec body formation and find that two pathways are critical. The first is the activation of the salt-inducible kinases (SIKs; SIK2 and SIK3) by Na+ stress, which, when it is strong, is sufficient. The second is activation of IRE1 and PERK (also known as PEK in flies) downstream of ER stress induced by the absence of amino acids, which needs to be combined with moderate salt stress to induce Sec body formation. SIK, and IRE1 and PERK activation appear to potentiate each other through the stimulation of the unfolded protein response, a key parameter in Sec body formation. This work shows the role of SIKs in phase transition and re-enforces the role of IRE1 and PERK as a metabolic sensor for the level of circulating amino acids and salt (Zhang, 2021).

Cell compartmentalization is not only mediated by membrane-bound organelles. It also relies on non-membrane bound biomolecular condensates (so-called membraneless organelles) that populate the nucleus and the cytoplasm (Zhang, 2021).

The formation of membraneless organelles has been shown to occur through phase separation, which can be driven by stress (such as ER, oxidative, proteostatic or nutrient stress), resulting in the formation of stress assemblies. Those are mesoscale coalescence of specific and defined components that phase separate. For instance, nutrient stress leads to the formation of many biocondensates. Most of them are RNA based, such as stress granules and P-bodies, but some are not. This is the case for glucose-starved yeast where metabolic enzymes foci and proteasome storage granules form, as well as Drosophila S2 cells that form Sec bodies under conditions of amino acid starvation (Zhang, 2021).

Sec bodies are related to the inhibition of protein secretion in the early secretory pathway. The early secretory pathway comprises the endoplasmic reticulum (ER), where newly synthesized proteins destined to the plasma membrane and the extracellular medium are synthesized. Proteins exit the ER at the ER exit sites (ERES) to reach the Golgi. The ERES are characterized by the concentration of COPI-coated vesicles whose formation requires six proteins, including Sec12 and Sar1, the inner coat proteins Sec23 and Sec24, and the outer coat proteins Sec13 and Sec31 (Gomez-Navarro, 2016). In addition, a larger hydrophilic protein called Sec16, has been identified as a key regulator of the ERES organization and COPII vesicle budding. Many additional lines of evidence support the role of Sec16 in optimizing COPII-coated vesicle formation and export from the ER (Zhang, 2021).

Upon the stress of amino acid starvation in Krebs Ringer bicarbonate buffer (KRB), the ERES of Drosophila S2 cells are remodeled into large round non-membrane bound phase-separated Sec bodies. They are typically observed by immunofluorescence after staining of endogenous Sec16, Sec23 and expressed Sec24-GFP. Importantly, Sec bodies are very quickly resolved upon stress relief (addition of growth medium). Finally, they appear to protect the components of the ERES from degradation and they help cells to survive under conditions of amino acid shortage (Zhang, 2021).

Phase separation has been shown to be driven by specific components, the so-called drivers, either RNAs or proteins harboring structural features that become exposed or modified under certain conditions. In the case of Sec bodies, Sec24AB and Sec16 have been shown to drive Sec body coalescence in a manner that depends on a small stretch of 44 residues in Sec16 and on the mono-ADP-ribosylation enzyme by PARP16. This illustrates the critical role of post-translational modifications in phase separation (Zhang, 2021).

In parallel, changes in cytoplasmic biophysical properties have also been shown to be important in phase separation, such as a drop of cytoplasmic pH within minutes, without post-translational modifications (Zhang, 2021).

This study sought out to (1) identify the pathways elicited in S2 cells upon incubation in the starvation medium KRB that lead to Sec body formation, and (2) to assess whether changes in the cytoplasmic biophysical properties play a role in the phase transition leading to Sec body formation. Amino acid starvation in KRB is shown to stimulate ER stress and activation of two downstream kinases, IRE1 and PERK (also known as PEK in flies) leading to the stimulation of the unfolded protein response (UPR). However, the sole activation of the IRE1 and PERK does not lead to Sec body formation. To form Sec bodies in KRB, IRE1 and PERK activation needs to be combined with a moderate salt stress. Accordingly, KRB incubation is faithfully mimicked by cell incubation with dithiothreitol (DTT) and addition of 100 mM NaCl. Interestingly, a high-salt stress addition of 150 mM NaCl, which activates the salt-inducible kinases (SIKs; SIK2 and SIK3), is sufficient to efficiently drive Sec body formation. Importantly, it was found that a decrease in the cytoplasmic ATP concentration, a general RNA degradation and the stimulation of the UPR are factors strongly correlated to Sec body formation (Zhang, 2021).

This study shows that the Sec bodies that form in Drosophila S2 cells incubated in KRB are fully recapitulated by activation of SIKs, IRE1 and PERK (through SCH100 plus DTT), leading to the activation of a downstream UPR. Strikingly, the strong activation of SIKs in (SCH150) also induces the UPR and leads to Sec body formation. The resulting structures in each condition appear to be similar in size and number, and their formation is reversible. Whether their content is strictly similar has not been addressed in this study (Zhang, 2021).

Taken together, the results show that Sec body formation requires the stimulation of two main signaling pathways. The first is the salt stress pathway (addition of 150 mM NaCl), which activates the SIKs in a necessary and sufficient manner. It also does not lead to a change in the cytoplasmic ATP concentration. It does induce RNA degradation and it stimulates the UPR in an unexpected manner, given that PERK and IRE1 inhibitors do not alter SCH150 driven Sec body formation (Zhang, 2021).

The second pathway is the activation of IRE1 and PERK (but not ATF6), downstream of ER stress, which is partly induced by the absence of amino acids in KRB. Activation of either IRE1 or PERK is necessary but not sufficient. To form Sec bodies, this activation needs to be combined with a moderate salt stress. It is proposed that IRE1 and PERK activation combined with SIK activation occur in KRB, which is recapitulated by SCH100 plus DTT. This is associated with a decrease in the cytoplasmic concentration of ATP, with RNA degradation and with a stimulation of the UPR (Zhang, 2021).

Interestingly, both strong salt stress (SCH150) and KRB lead to the activation of the UPR (measured by the increase in Bip protein level), leading to the possibility that SIKs, IRE1 and PERK interact with and/or activate, each other. Either IRE1 and/or PERK activate the SIKs, or SIK activation activates IRE1 and/or PERK. This still needs to be refined. The prominent role of salt stress and SIKs in remodeling the cytoplasm Strong salt stress is induced by a 4-fold increase of Na+ in the medium combined with bicarbonate. This triggers an increase of Na+ in the cytoplasm that activates one or more SIK (as shown by the phosphorylation of the SIK target HDAC4). Accordingly, SIK inhibition decreases Sec body formation (Zhang, 2021).

Keeping intracellular Na+ as near as possible to physiological concentrations (5 mM) is critical for cellular life, and the cell spends 40% of its available ATP to extrude Na+ against K+ with the NaK ATPase. It is therefore not surprising that Na+ stress would elicit a cytoprotective response, such as prominent as Sec body formation (and stress granule formation in mammalian cells). This will need to be further elucidated, as many organisms and tissues are subjected to increased circulating Na+. This study shows, however, that it is not equivalent to an osmotic shock and that this addition of salt does not lead to a decrease in a cell volume. In contrast to P-bodies in yeast, osmotic stress does not induce Sec bodies. Interestingly, Na+/salt stress has recently been shown to induce the biogenesis of the lysosomal pathway (i.e. more endo/lysosomes as well as an increase in its activity) via TFEB and TOR (Zhang, 2021).

Increased Na+ activates the intracellular Na+-sensor network revolving around the SIKs. The SIKs belong to the family of AMPKs, and have been shown to be part of a nutrient-sensing mechanism so far revolving around glucose and unbalance of the ATP-to-ADP ratio. In mammals, there are three genes encoding SIK (SIK1-SIK3) but only 2 in Drosophila. Drosophila SIK2 is the ortholog of human SIK1 and SIK2, and has been shown to have a link to the fly Hippo pathway, possibly linking nutrient to growth. Drosophila SIK3 is required for glucose sensing in the fly. Which SIK is involved in Sec body formation has not been clarified, as overexpression of each SIK individually has not proven enough to trigger Sec body formation, even when combined to some excess salt (SCH84 or SCH100). However, at least two SIKs appear to change their intracellular localization in KRB, that is, SIK2 and the long SIK3 isoform, which appear to cluster near the plasma membrane and localize to the nuclear envelope. The role of SIKs in the formation of stress assemblies (here the Sec bodies) appears important and novel, and needs to be investigated further. Other members of the AMPK family do not appear to be involved and changing the ratio ADP-to-ATP did not alter Sec body formation in the SIC system (Zhang, 2021).

Although a high salt stress is sufficient to trigger Sec body formation, the Sec body formation observed during incubation in the amino acid starvation buffer KRB elicits another pathway, the ER stress pathway, leading to the activation of both IRE1 and PERK. Indeed, KRB-induced Sec body formation is entirely mimicked by a moderate salt stress (SCH100) combined with activation of IRE1 and PERK induced by DTT (SCH100 plus DTT) (Zhang, 2021).

Surprisingly, this study found that salt stress as well as KRB induces the UPR, which this study found is a common downstream event in all conditions inducing Sec body formation. How salt stress activates the UPR and the exact role of IRE1 and PERK is still not fully understood. It does not appear to be via SIKs, as HG does not modulate the UPR, yet strongly reduces Sec body formation. Conversely, IRE1 and PERK inhibitors do not influence SCH150-induced Sec body formation, so the exact link between IRE1, PERK, SIKs and the UPR remains to be further investigated (Zhang, 2021).

How UPR stimulation induces Sec body formation is not completely understood. It could occur through the clustering of IRE1 and PERK, two membrane kinases, and them forming a template in the plane of the ER membrane. In this regard, UPR stimulation has been linked to the MARylation enzyme Drosophila PARP16, which is also a transmembrane protein of the ER that undergoes a remodeling in KRB. What other roles are played by events downstream of the UPR, for example, Bip upregulation itself, and cytoplasmic changes, remains to be elucidated in detail. One interesting aspect is whether the activation of UPR might affect biophysical properties, membrane association dynamics, and conformation and post-translational modifications of Sec16 and COPII subunits, which would lead to their enhanced phase separation properties under stress (Zhang, 2021).

Lowering the cytoplasmic ATP concentration is one parameter that induces Sec body formation The lowering of the cytoplasmic ATP concentration occurs in KRB and SCH100 plus DTT. Forcing this lowering or preventing it has a strong incidence on Sec body formation. This finding is consistent with the hydrotropic property of ATP, that is, it being able to prevent phase separation in the cytoplasm. At least in vitro, addition of 8 mM ATP dissolves or prevents the phase separation of purified FUS and TAF15. This is a concentration matching physiological range of ATP level in mammalian cells (1-10 mM) and that is also compatible with the S2 cell ATP concentration (1.7 mM) (Zhang, 2021).

One possibility to explain the decrease of ATP concentration is the activation of kinases (among them, IRE1, PERK and SIK) that would consume the ATP pool. However, it is proposed that the decrease in the intracellular ATP concentration is upstream of the kinase activation. Indeed, incubating cells in KRB induces a ROS shock (as this study showed experimentally), which could damage mitochondrial respiration, resulting, in turn, in ER stress, and UPR activation. Taken together, although this study unraveled a strong causality between low ATP and Sec body formation, the noticeable exception of such formation in SCH150 suggests other possibilities (Zhang, 2021).

In conclusion, this work illustrates the complexity of amino acid starvation, the number of pathways it activates and how they interact with each other, as well as the different cellular cytoplasmic biophysical parameters it affects. It is proposed that the formation of Sec bodies depends on the activation of signaling pathways leading to activation of SIKs, IRE1 and PERK, altogether leading to the activation of the UPR, one of the common features of all pathways leading to Sec body formation (Zhang, 2021).

A decline in protein homeostasis (proteostasis) has been proposed as a hallmark of aging. Somatic stem cells (SCs) uniquely maintain their proteostatic capacity through mechanisms that remain incompletely understood. This study describes and characterizes a 'proteostatic checkpoint' in Drosophila intestinal SCs (ISCs). Following a breakdown of proteostasis, ISCs coordinate cell cycle arrest with protein aggregate clearance by Atg8-mediated activation of the Nrf2-like transcription factor cap-n-collar C (CncC). CncC induces the cell cycle inhibitor Dacapo and proteolytic genes. The capacity to engage this checkpoint is lost in ISCs from aging flies, and it can be restored by treating flies with an Nrf2 activator, or by over-expression of CncC or Atg8a. This limits age-related intestinal barrier dysfunction and can result in lifespan extension. These findings identify a new mechanism by which somatic SCs preserve proteostasis, and highlight potential intervention strategies to maintain regenerative homeostasis (Rodriguez-Fernandez, 2019).

Protein Homeostasis (Proteostasis) encompasses the balance between protein synthesis, folding, re-folding and degradation, and is essential for the long-term preservation of cell and tissue function. It is achieved and regulated by a network of biological pathways that coordinate protein synthesis with degradation and cellular folding capacity in changing environmental conditions. This balance is perturbed in aging systems, likely as a consequence of elevated oxidative and metabolic stress, changes in protein turnover rates, decline in the protein degradation machinery, and changes in proteostatic control mechanisms. The resulting accumulation of misfolded and aggregated proteins is widely observed in aging tissues, and is characteristic of age-related diseases like Alzheimer's and Parkinson's disease. The age-related decline in proteostasis is especially pertinent in long-lived differentiated cells, which have to balance the turnover and production of long-lived aggregation-prone proteins over a timespan of years or decades. But it also affects the biology of somatic stem cells (SCs), whose unique quality-control mechanisms to preserve proteostasis are important for stemness and pluripotency (Rodriguez-Fernandez, 2019).

Common mechanisms to surveil, protect from, and respond to proteotoxic stress are the heat shock response (HSR) and the organelle-specific unfolded protein response (UPR). When activated, both stress pathways lead to the upregulation of molecular chaperones that are critical for the refolding of damaged proteins and for avoiding the accumulation of toxic aggregates. If changes to the proteome are irreversible, misfolded proteins are degraded by the proteasome or by autophagy. While all cells are capable of activating these stress response pathways, SCs deal with proteotoxic stress in a specific and state-dependent manner. Embryonic SCs (ESCs) exhibit a unique pattern of chaperone expression and elevated 19S proteasome activity, characteristics that decline upon differentiation. ESCs share elevated expression of specific chaperones (e.g., HspA5, HspA8) and co-chaperones (e.g., Hop) with mesenchymal SCs (MSCs) and neuronal SCs (NSCs), and elevated macroautophagy (hereafter referred to as autophagy) with hematopoietic SCs (HSCs), MSCs, dermal, and epidermal SCs. Defective autophagy contributes to HSC aging. It has further been proposed that SCs can resolve proteostatic stress by asymmetric segregation of damaged proteins, a concept first described in yeast (Rodriguez-Fernandez, 2019).

While these studies reveal unique proteostatic capacity and regulation in SCs, how the proteostatic machinery is linked to SC activity and regenerative capacity, and how specific proteostatic mechanisms in somatic SCs ensure that tissue homeostasis is preserved in the long term, remains to be established. Drosophila intestinal stem cells (ISCs) are an excellent model system to address these questions. ISCs constitute the vast majority of mitotically competent cells in the intestinal epithelium of the fly, regenerating all differentiated cell types in response to tissue damage. Advances made by numerous groups have uncovered many of the signaling pathways regulating ISC proliferation and self-renewal. In aging flies, the intestinal epithelium becomes dysfunctional, exhibiting hyperplasia and mis-differentiation of ISCs and daughter cells. This age-related loss of homeostasis is associated with inflammatory conditions that are characterized by commensal dysbiosis, chronic innate immune activation, and increased oxidative stress. It further seems to be associated with a loss of proteostatic capacity in ISCs, as illustrated by the constitutive activation of the unfolded protein response of the endoplasmic reticulum (UPR-ER), which results in elevated oxidative stress, and constitutive activation of JNK and PERK kinases. Accordingly, reducing PERK expression in ISCs is sufficient to promote homeostasis and extend lifespan (Rodriguez-Fernandez, 2019).

ISCs of old flies also exhibit chronic inactivation of the Nrf2 homologue CncC. CncC and Nrf2 are considered master regulators of the antioxidant response, and are negatively regulated by the ubiquitin ligase Keap1. In both flies and mice, this pathway controls SC proliferation and epithelial homeostasis. It is regulated in a complex and cell-type specific manner. Canonically, Nrf2 dissociates from Keap1 in response to oxidative stress and accumulates in the nucleus, inducing the expression of antioxidant genes. Drosophila ISCs, in turn, exhibit a 'reverse stress response' that results in CncC inactivation in response to oxidative stress. This response is required for stress-induced ISC proliferation, including in response to excessive ER stress, and is likely mediated by a JNK/Fos/Keap1 pathway (Rodriguez-Fernandez, 2019).

The Nrf2 pathway has also been linked to proteostatic control: 'Non-canonical' activation of Nrf2 by proteostatic stress as a consequence of an association between Keap1 and the autophagy scaffold protein p62 has been described in mammals. A similar non-canonical activation of Nrf2 has been described in Drosophila, where CncC activation is a consequence of the interaction of Keap1 with Atg8a, the fly homologue of the autophagy protein LC3. Nrf2/CncC activation induces proteostatic gene expression, including of p62 in mammalian cells and of p62/Ref2P and LC3/Atg8a in flies. Nrf2 is further a central transcriptional regulator of the proteasome in both Drosophila and mammals. Whether and how Nrf2 also influences proteostatic gene expression in somatic SCs remains unclear (Rodriguez-Fernandez, 2019).

This study shows that Drosophila CncC links cell cycle control with proteostatic responses in ISCs via the accumulation of dacapo, a p21 cell cycle inhibitor homologue, as well as the transcriptional activation of genes encoding proteases and proteasome subunits. This study establish that this program constitutes a transient 'proteostatic checkpoint', which allows clearance of protein aggregates before cell cycle activity is resumed. In old flies, this checkpoint is impaired and can be reactivated with a CncC activator (Rodriguez-Fernandez, 2019).

The central role of Nrf2/CncC in the proteostatic checkpoint is consistent with its previously described and evolutionarily conserved influence on longevity and tissue homeostasis, and is likely to be conserved in mammalian SC populations, as Nrf2 has for example been shown to influence proliferative activity, self-renewal and differentiation in tracheal basal cells. It may be unique to somatic SCs, however, as CncC or Nrf2-mediated inhibition of cell proliferation is not observed during development (such as in imaginal discs) or in other dividing cells. Assessing the existence of an Nrf2-induced proteostatic checkpoint in mammalian SC populations will be an important future endeavor (Rodriguez-Fernandez, 2019).

Mechanistically, the results support a model in which the presence of protein aggregates activates CncC through Atg8a-mediated sequestration of Keap1. In mammals, Nrf2 activation can also be achieved through the interaction of Keap1 with the Atg8a homologue LC3 and p62, and ref2p/p62 contributes to the degradation of polyQ aggregates in Drosophila, suggesting that a conserved Atg8a/p62/Keap1 interaction may be involved in the activation of the proteostatic checkpoint. The activation of CncC after cytosolic proteostatic stress described in this study thus differs mechanistically and in its consequence from the regulation of CncC after other types of protein stress in ISCs: in response to unfolded protein stress in the ER, CncC is specifically inactivated by a ROS/JNK-mediated signaling pathway. This mechanism allows ISC proliferation to be increased in response to tissue damage, but can also contribute to the loss of tissue homeostasis in aging conditions. The activation of CncC after cytosolic protein stress, in turn, allows arresting ISC proliferation during protein aggregate clearance. The distinct responses of ISCs to cytosolic or ER-localized proteostatic stress has interesting implications for understanding of the maintenance of tissue homeostasis. While the XBP1-mediated UPR-ER allows the expansion of the ER and the induction of ER chaperones to deal with a high load of unfolded proteins in the ER, it also stimulates ISC proliferation through oxidative stress and the activation of PERK and JNK. It is tempting to speculate that the sequestration of unfolded proteins within the ER allows ISCs to proceed through mitosis without the possibility of major misregulation, while the presence of cytosolic protein aggregates may be a unique danger to the viability of the cell and its daughters. It seems likely that constitutive activation of autophagy and proteasome pathways during the clearance of cytosolic aggregates is incompatible with the need for intricate regulation of these same pathways during the cell cycle in proliferating ISCs. It will be of interest to explore this hypothesis further in the future (Rodriguez-Fernandez, 2019).

The data suggest that the coordination of cell cycle arrest and aggregate clearance is achieved by the simultaneous induction of the cell cycle inhibitor Dacapo and a battery of genes encoding proteins involved in proteolysis. While it was possible to detect dacapo transcript expression in ISCs by fluorescent in situ hybridization at 24h after HttQ138 expression, it remains unclear whether Dacapo is induced directly by CncC or via the action of a CncC target gene. It is surprising that transcriptional induction of autophagy genes in was not seen in a RNAseq experiment, but it is possible that this is due to the fact that only one timepoint was sampled after induction of protein aggregates. Since the transcriptional response of autophagy genes is likely very dynamic, a more time-resolved transcriptome analysis during aggregate formation and clearance may have captured such a response (Rodriguez-Fernandez, 2019).

It is further notable that dap deficient ISC clones exhibit a significantly higher aggregate load in these experiments than wild-type ISC clones. This suggests that the induction of proteolytic genes and of cell cycle regulators is not only coincidentally linked by CncC, but that aggregate clearance and the cell cycle arrest mediated by Dacapo need to be tightly coordinated for effective ISC proteostasis. It will be interesting to explore the mechanism of this requirement in the future. It is tempting to speculate that, as the elimination of protein aggregates requires an increase in proteasome activity, and proteasome activity can influence cell cycle timing, cell cycle inhibition is a critical safeguard against de-regulation of normal cell cycle progression (Rodriguez-Fernandez, 2019).

The data suggest that Atg8a induction in ISCs experiencing proteostatic stress may serve a dual purpose: sustained activation of the proteostatic checkpoint as well as increased autophagy flux. This dual role is distinct from other autophagy components like Atg1, since Atg1 over-expression, an efficient way of promoting autophagy in Drosophila cells, counteracts the checkpoint rather than promoting it. Exploring the relative kinetics of Atg8a and Atg1 induction in ISCs after proteostatic stress is likely to provide deeper mechanistic insight into the regulation of the proteostatic checkpoint (Rodriguez-Fernandez, 2019).

Critically, the proteostatic checkpoint is reversible. Based on the current data and previous studies, it is proposed that upon clearance of aggregates, the Keap1/Atg8a interaction is decreased, thus releasing Keap1 to inhibit CncC. Lineage-tracing studies show that this allows re-activation of ISC proliferation and recovery of normal regenerative responses (Rodriguez-Fernandez, 2019).

The loss of proteostatic checkpoint efficiency in ISCs of old guts is likely a consequence of the age-related inactivation of CncC in these cells (possibly caused by chronic oxidative stres. Accordingly, reactivating Nrf2/CncC in the gut by overexpressing CncC is sufficient to restore epithelial homeostasis in the intestine of old flies, and this study found that exposing animals to Otipraz intermittently late in life promotes epithelial barrier function and extends lifespan (Rodriguez-Fernandez, 2019).

Since Nrf2/CncC and other components required for the proteostatic checkpoint are conserved across species, it is anticipated that the current findings will be relevant to homeostatic preservation of adult SCs in vertebrates. Supporting this view, mammalian Cdkn1a (p21) has been described as an Nrf2 target gene. Transient activation of Nrf2 may thus be a viable intervention strategy to improve proteostasis and maintain regenerative capacity in high-turnover tissues of aging individuals (Rodriguez-Fernandez, 2019).

Disturbances in the homeostasis of endoplasmic reticulum (ER) referred to as ER stress is involved in a variety of human diseases. ER stress activates unfolded protein response (UPR), a cellular mechanism the purpose of which is to restore ER homeostasis. Previous studies show that Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is an important novel component in the regulation of UPR. In vertebrates, MANF is upregulated by ER stress and protects cells against ER stress-induced cell death. Biochemical studies have revealed an interaction between mammalian MANF and GRP78, the major ER chaperone promoting protein folding. This study discovered that the upregulation of MANF expression in response to drug-induced ER stress is conserved between Drosophila and mammals. Additionally, by using a genetic in vivo approach genetic interactions was found between Drosophila Manf and genes encoding for Drosophila homologues of GRP78, PERK and XBP1, the key components of UPR. These data suggest a role for Manf in the regulation of Drosophila UPR (Lindstrom, 2016).

The accumulation of unfolded or misfolded proteins causes disturbances in endoplasmic reticulum (ER) homeostasis, a phenomenon referred to as ER stress. ER stress in turn activates the unfolded protein response (UPR). In order to overcome ER stress, UPR leads to attenuation of protein synthesis, enhancement of degradation of unfolded proteins, and activation of specific signalling cascades. These events aim to reduce the overall protein load in the ER and to enhance the protein folding capacity by selective transcription of chaperones. UPR is activated through three signalling cascades by ER transmembrane sensor proteins PERK (PRKR-like endoplasmic reticulum kinase), IRE1 (inositol requiring enzyme 1), and ATF6 (activating transcription factor 6). All of these three proteins are maintained inactive in normal cellular status by binding to the major ER chaperone GRP78/BiP (Glucose-regulated protein 78/Binding immunoglobulin protein). Upon ER stress, GRP78 is dissociated from the sensor proteins which are subsequently activated. The most ancient of these signalling cascades is mediated by IRE1, the sole branch of UPR identified in Saccharomyces cerevisiae. IRE1 has kinase activity and endoribonuclease activity needed for degradation of mRNAs in order to relieve the protein synthesis load. IRE1 is also responsible for the unconventional splicing and thus activation of transcription factor XBP1 (X-box Binding Protein-1), a positive regulator of ER chaperone and other UPR related gene expression. Activated PERK attenuates overall protein synthesis through phosphorylating and thus inhibiting EIF2α (eukaryotic translation initiation factor 2, subunit 1 α). However, the decreased activation of EIF2α results in an upregulated translation of specific target mRNAs including ATF4 (activating transcription factor 4). The third signalling pathway is mediated through ATF6, a transcription factor activated by its cleavage and translocation to the nucleus (Lindstrom, 2016).

In Drosophila, both IRE1- and PERK-mediated UPR signalling cascades are conserved. The amino acid sequence of the Drosophila homologue of ATF6 is highly similar to its mammalian counterpart, but experimental evidence for its involvement in Drosophila UPR is lacking. Similar to mammals, the expression of Drosophila homologue of GRP78, Hsc3 (Heat shock protein cognate 3), is upregulated upon induced ER stress in Xbp1-dependent manner but no biochemical data are available to show its association with ER stress sensor proteins (Lindstrom, 2016).

The MANF/CDNF family of neurotrophic factors was first characterized based on its trophic function on dopaminergic neurons in vitro and in vivo. When injected into the brain, recombinant mammalian MANF (Mesencephalic Astrocyte-derived Neurotrophic Factor) and CDNF (Cerebral Dopamine Neurotrophic Factor) protect and repair dopaminergic neurons in toxin-induced rodent models of Parkinson's disease (PD) in vivo. The sole Drosophila homologue, DmManf, is expressed in and secreted from glial cells and supports the dopaminergic system in non-cell-autonomous manner. The role of MANF as an extracellular trophic factor is further supported by the evidence that mammalian MANF is protective against ischemic injury in both neurons and cardiomyocytes. However, the biology of MANF is not thoroughly understood. Intriguingly, MANF localizes to the ER and has a protective role against ER stress in vitro and in vivo. Additionally, mammalian MANF binds GRP78 in Ca2+-dependent manner in vitro and this binding may regulate MANF secretion. MANF can be retained in the ER by its C-terminal signal sequence, RTDL in human and RSEL in Drosophila. Experimental evidence suggests that mammalian MANF interacts with KDEL-R [KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor] and that the C-terminal RTDL sequence of MANF is responsible for this interaction. The relevance of KDEL-R as a mediator of the functions of MANF has not been explored in vivo, yet. Recently, MANF was also shown to regulate the expression of ER-resident protein CRELD2 (Lindstrom, 2016).

Both in vivo and in vitro studies have shown that MANF is upregulated after chemically induced ER stress and by misfolded mutant proteins accumulating in the ER. Mammalian MANF expression is activated upon ER stress by ATF6 and XBP1 through an ER stress response element II found in the promoter region of MANF. Based on a knockout mouse model, MANF was found to be essential for the survival of pancreatic β-cells and its loss resulted in severe diabetes due to reduction of beta cell mass and activation of UPR in the pancreatic islets. The protective role against 6-OHDA induced and ischemic neuronal damage has been suggested to rise from the ER-related functions of MANF as these processes have been shown to induce ER stress (Lindstrom, 2016).

In Drosophila, the loss of DmManf is associated with upregulated expression of genes involved in UPR (Palgi, 2012). Additionally, the overexpression of DmManf resulted in downregulation of several UPR-related genes (Palgi, 2012). This study shows that, similar to mammalian MANF, the expression of DmManf is induced in response to ER stress in vitro. Further, transgenic approaches for gene silencing in vivo were applied to reveal genetic interactions between DmManf and genes with known functions in the maintenance of ER homeostasis and in UPR (Lindstrom, 2016).

Increasing evidence indicates that ER stress and UPR play a major role in variety of human diseases including diabetes mellitus and neurodegenerative disorders. MANF is a secreted protein, but also localizes to the ER and has a role in mammalian UPR. This study examined the role of DmManf in UPR in the Drosophila model. Upregulation of MANF mRNA expression by ER stress-inducing agents was shown to be conserved in Drosophila S2 cells. Additionally, genetic interaction between DmManf and genes known to function in the ER and UPR were shown (see A simplified presentation of UPR and genetic interactions discovered for Drosophila Manf; Lindstrom, 2016).

One of the interacting partners was Hsc3, the Drosophila homologue of mammalian chaperone GRP78. The silencing of Hsc3 in the wing resulted in an abnormal wing phenotype in wild type background. This wing phenotype was stronger in DmManf-overexpressing background. In cultured mammalian cells MANF has been shown to bind GRP78 in Ca2+-dependent manner and the loss of interaction between mammalian MANF and GRP78 was associated with increased secretion of MANF. In line, the knockdown of Hsc3 could lead to increased secretion of DmManf and lead to deprivation of intracellular DmManf. In a previous study, it was noticed that the deletion of ER retention signal RSEL increased the secretion of DmManf in S2 cells and decreased its functionality in rescue experiments in vivo. Based on the physical interaction found between mammalian MANF and GRP78, the simultaneous overexpression of DmManf and knockdown of Hsc3 could also result in the abundant DmManf binding the residual Hsc3 and preventing other important cellular functions of Hsc3. Alternatively, the loss of Hsc3 could lead to decreased protein folding capacity in the ER and activation of UPR. The vast amount of DmManf protein could exhaust this already disturbed cellular state (Lindstrom, 2016).

In previous studies, mammalian MANF has been suggested to have chaperone-like functions, e.g. by binding unfolded proteins in vitro but the putative chaperone activity remains unconfirmed. The major ER chaperone Hsc3 and DmManf clearly have distinct roles as either the overexpression or the loss of one could not complement for the loss of the other. However, the current study indicates that the interaction between MANF and GRP78 (Glembotski, 2012) is conserved. In future, the functional significance of this intriguing interaction deserves to be addressed in detail (Lindstrom, 2016).

DmManf genetically interacted with PEK/PERK, an ER stress sensor protein. Similar to the silencing of Hsc3, simultaneous overexpression of DmManf worsened the phenotypes observed in PEK knockdown flies. Previous studies have indicated functional conservation of PERK in Drosophila and mammals. The Drosophila homologue to ATF4, the downstream target of activated PERK and selectively upregulated by UPR, showed no genetic interaction with DmManf in this study. It was previously shown that the abolishment of both zygotic and maternal DmManf resulted in increased phosphorylation of eIF2α, another molecular marker used for detecting ER stress. This study abolished only the zygotic DmManf while maternal DmManf was still present. The loss of zygotic DmManf alone did not induce UPR when evaluated by other readouts, i.e. increased Hsc3 mRNA level and splicing of Xbp1. Although the zygotic DmManf^Δ96 mutant larvae show only low amount of persisting maternal DmManf mRNA and protein, it could be sufficient to prevent the induction of UPRl (Lindstrom, 2016).

Additionally, a genetic interaction was discovered between DmManf and Xbp1, a transcription factor mainly responsible for the regulation of UPR-induced genes. Upon UPR, the mRNA of Xbp1 is spliced by IRE1 and translated into a transcriptional activator of chaperone expression in response to the increased protein folding demand. According to previous studies, the spliced form of Xbp1 could mediate the UPR-induced upregulation of MANF in mammals. MANF has been suggested to have protective role against ER stress. During normal development, ER stress is detected in the secretory cells and the silencing of Xbp1 disturbs this developmental ER stress. Both mammalian and Drosophila MANF has been shown to have especially high expression levels in secretory tissues. Overexpression of DmManf increased Xbp1 mRNA level but the knockdown of Xbp1 did not affect DmManf expression levels. Also, the mRNA levels of Hsc3 were not upregulated in Xbp1 knockdown larvae. This could indicate the lack of transcriptional activation of DmManf and Hsc3 expression by Xbp1s in Xbp1-knockdown larvae. Therefore, knockdown of Xbp1 could compromise the regulation of DmManf expression in the developmental ER stress and deteriorate its function in the secretory cells (Lindstrom, 2016).

ERAD is a cellular process aiming to clear out the unfolded and misfolded proteins from the ER. According to previous transcriptome analysis, sip3 was downregulated in DmManf^Δ96 mutant larvae (Palgi, 2012). This study also found a genetic interaction between DmManf and sip3. Sip3 encodes a homologue to mammalian ER resident E3 ubiquitin ligase synoviolin/HRD1 with specific function in ERAD. Mammalian MANF is upregulated by ERSE-II (ER stress response element II) found in its promoter region. Interestingly, ERSE-II is also found in ERAD-related components HERPUD1 (homocysteine-inducible, ER stress-inducible, ubiquitin-like domain member 1, also known as HERP) and VIMP (VCP-interacting membrane protein, also known as selenoprotein S). ERSE-II has been hypothesized to regulate the protein quality control and degradation of misfolded proteins during ER stress suggesting that MANF could also have a role in these functions (Lindstrom, 2016).

Surprisingly, overexpression of DmManf led to enhanced phenotypes in flies of which a UPR-related gene was knocked down. Thus far, overexpression of DmManf with any GAL4 driver tested has never resulted in a detectable phenotype or altered viability. According to a previous microarray analysis, DmManf overexpression led to downregulation of UPR-related genes. This suggests that the overexpression of DmManf would disturb UPR signalling. Hypothetically, wild type background cells would be able to deal with the increased DmManf expression and the subsequent downregulation of UPR-related genes whereas the additional knockdown of an important component of UPR, e.g. Hsc3, PEK or Xbp1, could compromise the cell homeostasis (Lindstrom, 2016).

An alternative explanation for these observations in interaction studies between UPR genes and DmManf would be that DmManf is actually a substrate for UPR. Then, the abundant expression of DmManf by UAS/GAL4 would rather model the effects of increased overall protein synthesis in ER than indicate specific ER-related functions for DmManf. DmManf enters the secretory pathway and its ectopic expression may cause stress to the protein folding machinery in the ER. Although the Xbp1 mRNA level was increased, the expression of Hsc3 was not altered indicating that overexpression of DmManf induces mild UPR. However, no similar effects were seen with overexpression of membrane-directed GFP suggesting that the observed phenomena were specific for DmManf (Lindstrom, 2016).

The previous microarray study found that the total loss of DmManf is associated with upregulated expression of genes involved in UPR. However, in the current study the mRNA levels of Hsc3 and Xbp1 were mildly decreased in DmManf mutant larvae. In the previous study, transcriptome analysis was done from the embryonic DmManf mutants lacking both maternal and zygotic DmManf. In the current study, RNA was collected from zygotic DmManf mutants with the persisting maternal DmManf mRNA and protein. The maternal DmManf is apparently sufficient to prevent induction of UPR and upregulation of UPR related genes (Lindstrom, 2016).

This work provides evidence for the contribution of DmManf in Drosophila UPR. Further biochemical studies on the interaction between DmManf and UPR genes in Drosophila are needed to elucidate the details of this process (Lindstrom, 2016).

Eukaryotic cells respond to stress caused by the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum by activating the intracellular signaling pathways referred to as the unfolded protein response (UPR). In metazoans, UPR consists of three parallel branches, each characterized by its stress sensor protein, IRE1, ATF6, and PERK, respectively. In Drosophila, IRE1/XBP1 pathway is considered to function as a major branch of UPR; however, its physiological roles during the normal development and homeostasis remain poorly understood. To visualize IRE1/XBP1 activity in fly tissues under normal physiological conditions, previously reported XBP1 stress sensing systems, were modified based on the recent reports regarding the unconventional splicing of XBP1/HAC1 mRNA. The improved XBP1 stress sensing system allowed detection of new IRE1/XBP1 activities in the brain, gut, Malpighian tubules, and trachea of third instar larvae and in the adult male reproductive organ. Specifically, in the larval brain, IRE1/XBP1 activity was detected exclusively in glia, although previous reports have largely focused on IRE1/XBP1 activity in neurons. Unexpected glial IRE1/XBP1 activity may provide novel insights into the brain homeostasis regulated by the UPR (Sone, 2013).

Inadequate sensitivity of existing XBP1 stress sensing systems can be overcome by improving the efficiency of unconventional splicing of xbp1 mRNA. Recent reports regarding the cellular localization of XBP1/HAC1 mRNA during its splicing allowed construction of a highly sensitive HG stress indicator that can visualize the activation of IRE1/XBP1 pathway at the third instar larval stage during normal Drosophila development. Several types of cells in the organs where IRE1/XBP1 activity was detected are known for having high secretory capacity (Sone, 2013).

In the larval brain, significant IRE1/XBP1 activity was found in glial cells. While glia had been originally thought to function as the structural support cells in the nervous system, it has been revealed that they play several important roles in the development and homeostasis of the nervous system. In Drosophila, glial cells are classified into three classes (surface-, cortex-, and neuropil-associated glia), each of which is subdivided further morphologically. Whether IRE1/XBP1 active glia is restricted to only a subtype of those glia, or more broadly, is currently under investigation (Sone, 2013).

In mammals, oligodendrocytes in the central nerve system and Schwann cells in the peripheral nerve system myelinate axons by producing a large amount of myelin membrane proteins, cholesterol, and membrane lipids through the secretory pathway. Recent reports suggested that ER stress in myelinating cells is important in the pathogenesis of various disorders of myelin. Neuropil glia and peripheral glia in Drosophila are the counterparts of oligodendrocytes and Schwann cells, respectively. Therefore, these cells are the candidates that show constitutive IRE1/XBP1 activity. Although Drosophila glia do not generate myelin sheaths, they form multi-layered membrane sheaths around neurons that are morphologically similar to the myelin sheaths in mammals. Thus, it is possible that the IRE1/XBP1 active glia protect neurons from their deterioration through this ensheathment, thereby contributing to brain homeostasis. Further studies are expected to be informative as to the pathological significance of IRE1/XBP1 functions in human glia (Sone, 2013).

IRE1/XBP1 pathway does not appear to be active in neuron. However, the possibility of neuronal IRE1/XBP1 activation in the brain was not excluded. In fact, slight neuronal IRE1/XBP1 activity was occasionally observed in the ventral nerve cord during repeated experiments. In this study, it is concluded that in the third instar larval brain, the IRE1/XBP1 pathway is predominantly activated in glia while the activation is not detectable in neurons (Sone, 2013).

The importance of IRE1/XBP1 activity in the gut has already been studied in Caenorhabditis elegans and mammals. Intra-tissue distribution of IRE1/XBP1 activity was detected in the proventriculus region of the gut. In the larval midgut and hindgut, an irregular distribution of IRE1/XBP1 active cells was observed. These were not entero-endocrine cells, as they did not colocalize with anti-Prospero antibody that marks those cells. Secretory intestinal cells in the midgut other than entero-endocrine cells including the intestinal stem cells are possible candidates for these IRE1/XBP1 active cells (Sone, 2013).

IRE1/XBP1 activity in the fly Malpighian tubules (analogous to the kidney in mammals) was also unexpected. The activity was detected throughout the organ, but not all of the cells were IRE1/XBP1 active. Although the Malpighian tubules are attached at the junction of the midgut and the hindgut, they are morphologically and functionally independent from both of them. Identification of the IRE1/XBP1 active cells in the gut and the Malpighian tubules might reflect a shared physiological function of both organs. One possible shared function may be the selective uptake of the essential molecules, including several metal ions, from the contents passing through those organs. IRE1/XBP1 pathway might regulate the function of some transporter channels in these organs. Drosophila Malpighian tubules are expected to be one of the models for the mammalian diabetic kidney diseases that are associated with UPR activation (Sone, 2013).

In this study, IRE1/XBP1 activity was also detected in the trachea. Previous reports suggest its relevance to glial IRE1/XBP1 activity. One of them showed that tracheal development in Drosophila brain was controlled by signals from glia. According to the report, the branches of cerebral trachea grow around the neuropile. If IRE1/XBP1 active glia were neuropile-associated glia, assessing IRE1/XBP1 activity at neuropile-associated glia is likely reveal the shared physiological function of IRE1/XBP1 pathway between brain and trachea. The other report, using embryonic trachea, indicated that the proper combination of secretory activity and endocytotic activity was importaAnt for the maturation of trachea as an airway. In tracheal maturation, Sar1, one of the core COPII proteins, was required for the secretion of protein, the luminal matrix assembly, and the following expansion of tube diameter to avoid the clogging of protein, while Rab5, the small GTPase that regulates the early stage of endocytosis, was required for the clearance of deposited materials in the lumen. It can be predicted that, even in larval trachea, IRE1/XBP1 pathway plays a crucial role in tracheal maturation by supplying the properly folded proteins to the transport machinery. In that case, in view of second instar larval lethality of xbp1^-/- hypomorph mutant, it could also be hypothesized that the tracheal maturation/maintenance is still important for larval lethality, in addition to its importance for the embryonic development (Sone, 2013).

IRE1/XBP1 activity in the salivary gland has already been reported in a previous study. The salivary gland is commonly used for the determination of the subcellular localization of the protein in Drosophila cells due to its morphological features. The nuclear localization of HG indicator, XBP1-EGFP molecule, was clearly indicated. In addition, weak IRE1/XBP1 activity was detected in the fat body; it was attached to the salivary gland. Generally, the Drosophila fat body, which is equivalent to mammalian adipose tissue, functions as the organ for energy/lipid storage and is distributed throughout the larval body (Sone, 2013).

In addition to the larval tissues, IRE1/XBP1 activity was analyzed in the adult male reproductive organs. Though a previous RT-PCR suggested the activity in the testis, the areas where IRE1/XBP1 activity was detected were the accessory glands and a limited area of the testis close to the testicular duct. In the accessory gland, seminal fluid containing several hormones, which facilitate reproductive traits such as sperm transfer, sperm storage, female receptivity, ovulation, and oogenesis, are produced and secreted. There are two morphologically distinct secretory cell types in Drosophila accessory gland. Ninety-six percent of the secretory cells are categorized as main cells and the others are secondary cells. Based on the intra-tissue distribution of IRE1/XBP1 active cells in the accessory gland, the active cells are likely to be main cells. Since each of these cell types expresses a unique set of genes, the confirmation of IRE1/XBP1 active cell type is expected to allow ingnarrow down the proteins related to IRE1/XBP1 activity. IRE1/XBP1 pathway is likely to function, to some extent, in maintaining proper fertility (Sone, 2013).

On the other hand, a possibility is considered that the EGFP signal detected in each organ might not necessarily reflect the unconventional splicing of xbp1-EGFP mRNA. Higher concentrations of the spliced xbp1-EGFP mRNA and resulting XBP1-EGFP in the cells induced by the Gal4/UAS system might cause the artifactual EGFP signal. The possibility was excluded that the EGFP signal in this study was detected independently of the unconventional splicing, based on the results in this study and the following reasoning (Sone, 2013).

There are two possible molecular mechanisms that cause the artifactual EGFP signal which is not derived from the unconventional splicing of xbp1-EGFP mRNA. One is the generation of EGFP or abnormal EGFP fusion proteins, resulting from translation initiation at the start codon of the EGFP coding sequence or at ATG codons coding Met residues in XBP1(s), respectively. The other is the proteolytic digestion of XBP1-EGFP fusion protein at the junction of XBP1 and EGFP portions. Both of these are prone to happen upon overexpression of fusion proteins in cells. In particular, the proteolytic digestion is often observed in the overexpression of GST fusion protein in Escherichia coli (Sone, 2013).

In this system, there is no nuclear localization signal (NLS) on the EGFP molecule. In contrast, xbp1 gene carries a NLS coding sequence located upstream of the unconventional splice site. There are a total of 11 ATG codons that code the Met residues of XBP1(s) molecule. Eight of the ATG codons are located downstream of NLS coding sequence. Therefore, due to the lack of NLS, both EGFP and the EGFP fusion proteins using these eight ATG codons as start codons should diffuse all over the cell upon their synthesis, if they were generated. EGFP signal was detected exclusively in the nucleus in the salivary gland, which is often used for the analysis of the cellular localization of the proteins in Drosophila. Hence, it is not reasonable to conclude that either EGFP or the possible eight EGFP fusion proteins above were expressed in the cells. Only the EGFP fusion proteins that use the other three ATG codons that are upstream of the NLS as start codons should be synthesized upon unconventional splicing and localized in the nucleus. The estimated molecular weights of those fusion proteins are 73.7, 74.3, and 80.0 kDa, respectively. Only a significant single band that represented the intact XBP1-EGFP (80.0 kDa) was detected in the S2 cell extract, in which XBP1-EGFP was overexpressed through the Gal4/UAS system. Therefore, it is concluded that the EGFP fusion protein synthesized in this study was the intact XBP1-EGFP (Sone, 2013).

Additionally, there are several ATG codons that are located upstream of the unconventional splice site and are in frame with EGFP coding sequence on unspliced xbp1 mRNA. However, inside the 23 bp of unconventional-spliced fragment, there is a TGA stop codon that is also in frame with EGFP coding sequence. Even if the translation initiated from these start codons, the synthesis of these products should be terminated at this TGA stop codon before the ribosome would reach the EGFP coding region (Sone, 2013).

Regarding the proteolytic digestion of XBP1-EGFP fusion protein, the resulting EGFP should diffuse all over the cell upon its synthesis due to the lack of NLS. Therefore, the possibility of the proteolytic digestion is also excluded based on the same reasoning as above. Taken together, it is concluded that the detected EGFP signal in this study exclusively reflected the occurrence of unconventional splicing of xbp1-EGFP mRNA (Sone, 2013).

Endoplasmic reticulum (ER) stress results from an imbalance between the load of proteins entering the secretory pathway and the ability of the ER to fold and process them. The response to ER stress is mediated by a collection of signaling pathways termed the unfolded protein response (UPR), which plays important roles in development and disease. This study shows that in Drosophila melanogaster S2 cells, ER stress induces a coordinated change in the expression of genes involved in carbon metabolism. Genes encoding enzymes that carry out glycolysis were up-regulated, whereas genes encoding proteins in the TCA cycle and respiratory chain complexes were down-regulated. The UPR transcription factor Atf4 was necessary for the up-regulation of glycolytic enzymes and Lactate dehydrogenase (Ldh). Furthermore, Atf4 binding motifs in promoters for these genes could partially account for their regulation during ER stress. Finally, flies up-regulated Ldh and produced more lactate when subjected to ER stress. Together these results suggest that Atf4 mediates a shift from a metabolism based on oxidative phosphorylation to one more heavily reliant on glycolysis, reminiscent of aerobic glycolysis or the Warburg effect observed in cancer and other proliferative cells (Lee, 2015).

As the flux of proteins through the ER varies considerably among cell types and in different conditions, cells maintain a balance between the load on the ER and its protein folding capacity. However, a number of biochemical, physiological, and pathological stimuli can disrupt this balance, resulting in ER stress. To re-establish ER homeostasis, the unfolded protein response is activated. This network of pathways up-regulates genes encoding ER-specific chaperones and other proteins involved in protein secretion, while also attenuating protein translation and degrading certain ER-associated mRNAs. The UPR is broadly conserved across eukaryotes and is essential for normal development in several model organisms, particularly for professional secretory cells, where it is thought to be important for the establishment and maintenance of high levels of protein secretion . It is also induced during many metabolic conditions including diabetes, hyperlipidemia, and inflammation, and has been implicated in various cancers, especially in the growth of large tumors that rely on an effective response to hypoxia (Lee, 2015).

The UPR is carried out by three main signaling branches. One of these is initiated by the ER transmembrane protein Inositol-requiring enzyme 1 (Ire1). When activated by ER stress, the cytosolic endoribonuclease domain of Ire1 cleaves the mRNA encoding the transcription factor Xbp1, thereby initiating an unconventional splicing event that produces the mRNA template encoding a highly active form of Xbp1. Ire1 also cleaves other mRNAs associated with the ER membrane, through a pathway that is particularly active in Drosophila cells and that may reduce the load on the ER. A second sensor of ER stress, Activating transcription factor 6 (Atf6), is activated by proteolysis, which releases it from the ER membrane and allows it to travel to the nucleus and regulate gene expression. Finally, Protein kinase RNA (PKR)- like Pancreatic ER kinase (Perk) (see Drosophila pancreatic eIF-2α kinase or Pek) phosphorylates eukaryotic initiation factor 2 alpha, leading to a general attenuation of protein synthesis as well as the translational up-regulation of certain mRNAs that contain upstream open reading frames (ORFs) in their 5' untranslated regions. Activating transcription factor 4 (Atf4)/Cryptocephal is among those proteins that are up-regulated translationally during ER stress, and regulates genes involved in protein secretion as well as amino acid import and resistance to oxidative stress (Lee, 2015).

In addition to its direct effects on the protein secretory pathway, the UPR influences several other cellular pathways including apoptosis, inflammation, and lipid synthesis. Furthermore, the UPR (particularly the Perk/Atf4 branch) appears to have close ties to mitochondrial function. For example, knockout of Mitofusin 2, a key mitochondrial fusion protein, activates Perk, leading to enhanced reactive oxygen species (ROS) production and reduced respiration. Atf4 also increases expression of Parkin, which mediates degradation of damaged mitochondria, protecting cells from ER stress-induced mitochondrial damage. Despite clear links between ER stress and mitochondria, the mechanistic relationship between the UPR and mitochondrial metabolism is not well-understood (Lee, 2015).

This study reports that the UPR in Drosophila S2 cells triggers a coordinated change in the expression of genes involved in carbon metabolism. The metabolism of glucose as an energy source produces pyruvate, which can then enter the mitochondria and the tricarboxylic acid (TCA) cycle to produce reducing equivalents for oxidative phosphorylation (OXPHOS). For most cells in normal conditions, the majority of ATP is produced through OXPHOS. However, in hypoxic conditions when OXPHOS is limited, cells rely heavily on glycolysis to compensate for the decrease in ATP production, and convert the excess pyruvate to lactate, which then leaves the cel. This shift from OXPHOS to glycolysis is seen in a variety of cancers even when cells have access to oxygen, an effect known as aerobic glycolysis or the Warburg effect, and is thought to be a hallmark of cancer cells. Aerobic glycolysis is also becoming increasingly recognized as a metabolic signature of other cell types as well, including stem cells and activated immune cells (Lee, 2015).

In Drosophila, the Estrogen-related receptor (dERR) is the only transcription factor known to regulate glycolytic genes (Li et al. 2013). Its activity is temporally regulated during mid-embryogenesis to support aerobic glycolysis during larval growth (Tennessen, 2011). Moreover, a recent study found that glycolytic gene expression under hypoxic conditions in larvae is partially dependent on dERR (Li, 2013). This study shows that the UPR transcription factor Atf4 also regulates glycolytic genes, contributing to a broad regulation of metabolic gene expression during ER stress that is reminiscent of the Warburg effect (Lee, 2015).

This study has shown that Drosophila S2 cells subjected to ER stress up-regulate glycolytic genes and Ldh and down-regulate genes involved in the TCA cycle and respiratory chain complex. Furthermore, Atf4 is responsible for the up-regulation of glycolytic genes and Ldh. How TCA cycle and respiratory chain complex genes are down-regulated during ER stress requires further investigation, although the lack of effect of Atf4 depletion suggests that these are not regulated as an indirect consequence of glycolysis up-regulation (Lee, 2015).

Despite a highly coordinated change in gene expression for metabolic genes during ER stress, this study did not detect any changes in actual metabolism in S2 cells. Because these cells have been in culture for decades and have likely been selected for rapid proliferation, it is possible that they are already undergoing some version of aerobic glycolysis, such that the underlying gene regulation during ER stress is preserved but any metabolic changes are masked. Others have also noted that S2 cells are resistant to hypoxia, and do not produce more lactate except in extreme conditions. The increase in lactate observed through in vivo studies in flies subjected to ER stress, however, suggests that in a more physiological setting, the gene expression changes shown here do mediate a metabolic shift toward aerobic glycolysis (Lee, 2015).

Up-regulation of glycolytic genes during ER stress has not been observed in genome-wide studies of mammalian cells. However, several lines of evidence suggest that mammalian cells subjected to ER stress may undergo a glycolytic shift. For example, a recent study examining human gliomas found coordinated up-regulation of UPR targets and glycolysis, which correlated with poor patient prognosis; and both ER stress and overexpresson of Perk have been shown to reduce mitochondrial respiration in cultured mammalian cells (Lee, 2015).

The link between ER stress and metabolism can be rationalized by the need to generate building blocks for biosynthesis of glycoproteins and lipids. Early intermediates of glycolysis are necessary for production of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), an important donor molecule for N-glycosylation of proteins in the ER. Both fructose-6-phosphate and dihydroxyacetone phosphate are also required for synthesis of glycolipids. An increased flux through glycolysis may therefore be important to support the increased production of glycerophospholipids and glycoproteins that are associated with the UPR. In support of this view, glucose deprivation or inhibition of glycolysis with 2-deoxy-D-glucose induces the UPR, which contributes to cell death, especially in cancer cells, and this effect can be rescued by UDP-GlcNAc. The hexosamine biosynthetic pathway generating UDP-GlcNAc is also directly activated by Xbp1, and stimulates cardioprotection during ischemia/reperfusion injury and increases longevity in worms (Lee, 2015).

A second, non-mutually exclusive explanation for a shift to glycolysis during ER stress is the need to limit production of ROS. Along with mitochondrial respiration, protein folding in the ER is one of the main sources of ROS, which are produced by the normal process of disulfide bond-coupled folding. If allowed to accumulate, these ROS can cause oxidative stress and damage to cells, eventually leading to apoptosis. Several studies have confirmed that ROS are produced during ER stress, when protein folding is inefficient and more rounds of oxidation and reduction are required to fold proteins. Limiting other sources of oxidative stress, such as by down-regulating the TCA cycle and thereby restricting the flux through OXPHOS (the main source of ROS in the mitochondria), may be a way to mitigate the damage and allow cells to recover more effectively (Lee, 2015).

Finally, the advantage of the Warburg effect for tumor growth may arise from the increased rate of ATP production by glycolysis compared to OXPHOS, despite its lower efficiency of conversion. By analogy, a metabolic shift during ER stress could rapidly supply ATP necessary for protein folding and processing. Indeed, cancer cells showing elevated levels of ENTPD5, an ER UDPase, promotes aerobic glycolysis to increase ATP for protein N-glycosylation and refolding (Lee, 2015).

Overall, these results identify Atf4 as a transcriptional regulator of glycolysis during ER stress. As Atf4 is expressed throughout fly development, it may regulate glycolysis in other situations as well: notably, Atf4 mutant flies are lean and have reduced circulating carbohydrates, suggesting a role in metabolism. Furthermore, because the Perk-Atf4 branch of UPR is activated during hypoxia, it will be interesting to see whether Atf4 contributes to regulation of glycolysis in other developmental, physiological (hypoxia), or pathological process during which glycolysis regulated. More broadly, since the UPR is activated in many types of cancer, its ability to regulate glucose metabolism may play a contributing role in the Warburg effect (Lee, 2015).

p53 gene family members in humans and other organisms encode a large number of protein isoforms whose functions are largely undefined. Using Drosophila as a model, it was found that a p53B isoform is expressed predominantly in the germline where it colocalizes with p53A into subnuclear bodies. It is only p53A, however, that mediates the apoptotic response to ionizing radiation in the germline and soma. In contrast, p53A and p53B are both required for the normal repair of meiotic DNA breaks, an activity that is more crucial when meiotic recombination is defective. In oocytes with persistent DNA breaks p53A is also required to activate a meiotic pachytene checkpoint. These findings indicate that Drosophila p53 isoforms have DNA lesion and cell type-specific functions, with parallels to the functions of mammalian p53 family members in the genotoxic stress response and oocyte quality control (Chakravarti, 2022).

The Drosophila melanogaster genome has a single p53 family member. Similar to human p53 (TP53), it has a C terminal oligomerization domain (OD), a central DNA-binding domain (DBD) and an N terminal transcriptional activation domain (TAD), and functions as a tetrameric transcription factor. This single p53 gene expresses four mRNAs that encode three different protein isoforms. A 44 kD p53A protein isoform was the first to be identified and is the most well characterized. Later RNA-Seq and other approaches revealed that alternative promoter usage and RNA splicing results in a 56 kD p53B protein isoform, which differs from p53A by a 110 amino acid longer N-terminal TAD that is encoded by a unique p53B 5' exon. Because the p53A isoform differs from p53B by a shorter N terminus, p53A is also known as ΔNp53. A p53C transcript starts at a different promoter than p53A but is predicted to encode the same 44 kD protein. A short p53E mRNA isoform is predicted to encode a protein of 38 kD that contains the DNA-binding domain but lacks the longer N-terminal TADs of p53A and p53B (Chakravarti, 2022).

Like its human ortholog, Drosophila p53 regulates apoptosis in response to genotoxic stress and mediates other stress responses and developmental processes. To promote apoptosis, p53 induces transcription of several proapoptotic genes at one locus called H99. Early analyses of p53 function in apoptosis focused on the p53A isoform because the others had yet to be discovered. Using BAC rescue transgenes that were mutant for either p53A or p53B, previous work showed that in larval tissues it is the shorter p53A, and not p53B, that is both necessary and sufficient for the apoptotic response to DNA damage caused by ionizing radiation. In contrast, when each isoform was overexpressed, p53B was much more potent than p53A at inducing proapoptotic gene transcription and the programmed cell death response, likely because of the longer p53B TAD. Other evidence suggests that p53B may regulate tissue regeneration and has a redundant function with p53A to regulate autophagy in response to oxidative stress. It is largely unknown, however, why the Drosophila genome encodes a separate p53B isoform and what its array of functions are (Chakravarti, 2022).

The p53 gene family is ancient with orthologs found in the genomes of multiple eukaryotes, including single-celled Choanozoans, which are thought to be the ancestors of multicellular animals. Evidence suggests that the ancestral function of the p53 gene family was in the germline, with later evolution of tumor suppressor functions in the soma. In mammals, p63 mediates a meiotic pachytene checkpoint arrest in response to DNA damage or chromosome defects, and also induces apoptosis of a large number of oocytes with persistent defects, thereby enforcing an oocyte quality control. It has been shown that in the Drosophila germline p53 regulates stem cell divisions, responds to programmed meiotic DNA breaks, and represses mobile elements. This study has uncovered that the Drosophila p53A and p53B isoforms have overlapping and distinct functions during oogenesis to protect genome integrity and mediate the meiotic pachytene checkpoint arrest, with parallels to the germline function of mammalian p53 family members in oocyte quality control (Chakravarti, 2022).

This study found that the Drosophila p53B protein isoform is more highly expressed in the germline where it colocalizes with a shorter p53A isoform in subnuclear bodies. Despite this p53B germline expression, it is the p53A isoform that was necessary and sufficient for the apoptotic response to IR in both the germline and soma. Although apoptosis is repressed in meiotic oocytes and endocycling nurse cells, it was found that both p53 isoforms are required in these cells for the timely repair of meiotic DNA breaks. The role of the p53 isoforms in DNA repair was cell type specific, with p53B playing the most prominent role in the nurse cells, whereas both p53B and p53A were required in the oocyte. The data has also uncovered a requirement for the Drosophila p53A isoform in the meiotic pachytene checkpoint response to unrepaired DNA breaks. Overall, these data suggest that Drosophila p53 isoforms have evolved overlapping and distinct functions to mediate different responses to different types of DNA damage in different cell types. These findings are relevant to understanding the evolution of p53 isoforms, and have revealed interesting parallels to the function of mammalian p53 family members in oocyte quality control (Chakravarti, 2022).

p53 isoforms colocalized to subnuclear bodies in the Drosophila male and female germline. This finding is consistent with a previous study that reported p53 bodies in the Drosophila male germline, although that study did not examine individual isoforms. It is deemed likely that these p53 bodies form by phase separation, an hypothesis that remains to be formally tested. Drosophila p53 subnuclear bodies are reminiscent of human p53 protein localization to subnuclear PML bodies. Evidence suggests that trafficking of human p53 protein through PML bodies mediates p53 post-translational modification and function, although the relationship between nuclear trafficking and the functions of different p53 isoforms has not been fully evaluated. Similarly, a decline was observed in abundance of p53B within p53 bodies in germarium region 2a, followed by a restoration of p53B within bodies in region 3. This fluctuation of p53B in bodies temporally correlates with the onset of meiotic DNA breaks in region 2a and their repair in regions 2b - 3. These observations are consistent with the idea that nuclear trafficking of p53B out of bodies may mediate its response to meiotic breaks, although it is also possible that p53B is degraded and rapidly resynthesized during this 24 hr period. Future analysis of Drosophila p53 bodies will help to define how p53 isoform trafficking mediates the response to genotoxic and other stresses (Chakravarti, 2022).

TUNEL labeling indicated that p53A is necessary and sufficient for apoptosis in both the germline and soma. IR induced apoptosis to a similar frequency in p53+ (A+B+) wild type and p53B^41.5 (A+B-) mutants, whereas the frequency of apoptosis in p53A^2.3 (A-B+) mutants was equivalent to that of p53^5A-1-4 (A-B-) null and unirradiated controls. Consistent with this, hid-GFP reporter expression was not induced by IR in the p53^5A-1-4 (A-B-) null mutant, whereas IR-induced hid-GFP expression in the p53B^41.5 (A+B-) mutant was equivalent to p53⁺ (A+B+) wild type, indicating that the p53A isoform is required for the transcriptional response to IR-induced DNA breaks. It is interesting to note that while germline cystocytes in germarium region one apoptosed after IR, their ancestor GSCs and descendent meiotic cells did not. The observed IR-induced expression of the hid-GFP promoter reporter in GSCs is consistent with previous evidence that apoptosis is repressed in these stem cells downstream of hid transcription by the miRNA bantam. How meiotic cells repress apoptosis is not known, although it is crucial that they do so because they have programmed DNA breaks. Together, these data suggest that p53A is necessary and sufficient for induction of proapoptotic gene expression and apoptosis in response to IR-induced DNA breaks in the soma and germline (Chakravarti, 2022).

While this manuscript was in preparation, it was reported that p53A and p53B both participate in the apoptotic response to IR in the ovary (Park, 2019). That study used the GAL4/ UAS system to express either p53A or p53B rescue transgenes in a p53 null background. In contrast, this study created and analyzed loss-of-function, isoform-specific alleles at the endogenous p53 locus, which is believed to more accurately reflect the physiological function of p53 isoforms. The conclusion, therefore, is favored that it is the p53A isoform that has the primary function of mediating the apoptotic response to IR in the soma and germline (Chakravarti, 2022).

In the absence of IR, there was a lower but detectable hid-GFP expression at the onset of meiosis in germarium region 2. This region 2 expression was dependent on p53 and formation of meiotic breaks by Mei-W68, consistent with previous reports that used a rpr-GFP reporter to show that p53 responds to meiotic DNA breaks. This low level of hid-GFP expression in region two without IR was similar between p53⁺ (A+B+) wild type and p53^B41.5 (A+B-) mutants, suggesting that the p53A transcription factor activity responds to meiotic DNA breaks. The results for the p53A^2.3 (A-B+) mutant were not informative, however, because in that mutant hid-GFP expression was constitutively higher than wild type beginning in early region 1 of the germarium. γ-H2Av labeling was not observed before late region 1/ region 2 a indicating that this low-level activity of p53B is not a response to DNA breaks. While further experiments are required to define the mechanism, a cogent hypothesis is that in the absence of the p53A subunit p53B homotetramers have somewhat higher basal activity. This hypothesis is consistent with previous evidence that the p53B isoform with a longer transactivation domain is a much stronger transcription factor than p53A, and that p53A and p53B can form heterocomplexes. It is also consistent with evidence that the shorter p53 isoforms in humans and other organisms repress the transcriptional activity of longer isoforms in heterotetramers. It is important to note, however, that while hid expression was higher in the p53A mutants than in wild type, it was not associated with apoptosis. Overall, while the hid-GFP reporter evidence suggests that p53A responds to meiotic DNA breaks, it is unclear whether this low-level activation of p53A transcription factor activity is related to its role in meiotic DNA break repair or checkpoint activation, which is discussed further below (Chakravarti, 2022).

The evidence suggests that both p53 isoforms are required for the timely repair of meiotic DNA breaks in the Drosophila female germline. p53 null and isoform-specific mutants had a persistent germline DNA break phenotype that was dependent on the creation of double-strand DNA breaks by Mei-W68. Further consistent with a role in meiotic DNA break repair, p53 mutants had an increased number of cells with γ-H2Av foci beginning in germarium stage 2a, the time when Mei-W68 induces programmed meiotic DNA breaks. Moreover, the number of persistent breaks per cell was higher in oocyte and adjacent nurse cell, the presumptive pro-oocyte, which are known to have more meiotic breaks. This p53 DNA break mutant phenotype is similar to that of okra (RAD54L) and other genes required for meiotic break repair and was enhanced in okra; p53 double mutants. It was previously shown using p53 null alleles that p53 also protects the germline genome by restraining mobile element activity, but this study did not evaluate whether one or both of the p53 isoforms are required for this function. Overall, the current data strongly suggest that both p53 isoforms have an important role in the repair of meiotic DNA breaks (Chakravarti, 2022).

This analysis also revealed that p53 isoforms have overlapping and distinct requirements for meiotic break repair in different cell types. Both p53A and p53B were required in the oocyte, whereas p53B played the more prominent role in nurse cells, even though nurse cells express both p53A and p53B isoforms. This differential requirement for p53 isoforms may reflect differences in how meiotic breaks are repaired in nurse cells versus oocytes. While it is not known whether DNA repair pathways differ between nurse cells and oocytes, evidence suggests that the creation of meiotic breaks does, with breaks in pro-oocytes but not pro-nurse cells depending on previous SC formation. Important questions motivated by the current results are how distinct responses to DNA damage in different cells are determined by different types of DNA lesions, checkpoint signaling and repair pathways, and p53 isoform structure (Chakravarti, 2022).

The consequences of p53 null and isoform-specific alleles for oogenesis were also similar to okra mutants in that they caused reduced female fertility and defects in eggshell patterning and synthesis. Previous evidence suggested that defective meiotic DNA break repair causes these maternal effect phenotypes in part through disrupting patterning signals from the oocyte to somatic follicle cells. The maternal effect on egg hatch rates, however, was much more severe in the okra mutants, which were completely female sterile, consistent with previous studies. Thus, although the p53 and okra null mutants had similar levels of germline DNA damage, the severity of their maternal-effect on egg patterning and embryo viability differ, suggesting that some of their pleiotropic effects on oogenesis are distinct. Together, the results indicate that defects in repair of meiotic DNA breaks in both p53 and okra mutant females negatively impact embryo patterning and female fertility (Chakravarti, 2022).

The requirement for Drosophila p53 in the repair of meiotic DNA breaks is consistent with evidence from other organisms that p53 has both indirect and direct roles in DNA repair. It is known that Drosophila p53 and specific isoforms of human p53 induce the expression of genes that are required for different types of DNA repair. p53 also acts locally at DNA breaks in a variety of organisms, including humans, where it can mediate the choice between HR versus non-homologous end joining (NHEJ) repair. In fact, it has been shown that human p53 directly associates with RAD54 at DNA breaks to regulate HR repair, consistent with the finding that p53; okra (RAD54L) double mutants have severe DNA repair defects. Moreover, the C. elegans p53 ortholog CED-4 localizes to DNA breaks to promote HR and inhibit NHEJ repair in the germline. Although the hid-GFP reporter indicated that meiotic DNA breaks induce a low level of p53A transcription factor activity, Hid has no known role in DNA repair, and it remains unknown whether p53-regulated expression of DNA repair genes is required for the timely repair of meiotic DNA breaks. It is deemed likely that the persistent DNA damage that was observed in the germline of Drosophila p53 mutants may, in part, reflect a local requirement for p53 protein isoforms to regulate meiotic DNA repair. Important remaining questions include whether different p53 isoforms participate indirectly in DNA repair by inducing transcription and directly at DNA breaks to influence the choice among different DNA repair pathways (Chakravarti, 2022).

This study has also uncovered a requirement for Drosophila p53 in the meiotic pachytene checkpoint. This function was isoform-specific, with p53A, but not p53B, being required for full checkpoint activation in oocytes with persistent DNA breaks. The failure to engage the pachytene checkpoint in the majority of okra; p53A^2.3 double mutant oocytes is more striking given that these cells had more severe DNA repair defects than the okra single mutants that strongly engaged the checkpoint. While the pachytene arrest was compromised to similar extents in okra; p53 null and okra; p53A^2.3 mutants, some egg chambers in both genotypes did engage a pachytene arrest. This observation suggests that there are p53-independent mechanisms that also activate the checkpoint, perhaps in response to secondary defects in chromosome structure, which are known to independently trigger the pachytene checkpoint in flies and mammals. Moreover, although the pachytene checkpoint was strongly compromised in the p53 null and p53A mutant alleles, it did not suppress okra female sterility, suggesting that other mechanisms ensure that oocytes with excess DNA damage do not contribute to future generations. Altogether, the results indicate that p53A is required for both DNA repair and full pachytene checkpoint activation in the oocytes (Chakravarti, 2022).

Evidence suggests that the ancient function of the p53 family was of a p63-like protein in the germline. Consistent with this, the findings in Drosophila have parallels to mammals where the TAp63α isoform and p53 mediate a meiotic pachytene checkpoint arrest, and the apoptosis of millions of oocytes that have persistent defects. The current evidence suggests that the different isoforms of the sole p53 gene in Drosophila may subsume the functions of vertebrate p53 and p63 paralogs to protect genome integrity and mediate the pachytene arrest. Unlike p53 and p63 in mammals, however, Drosophila p53 does not trigger apoptosis of defective oocytes. Instead, the activation of the pachytene checkpoint disrupts egg patterning, resulting in inviable embryos that do not contribute to future generations. Thus, in both Drosophila and mammals, the p53 gene family participates in an oocyte quality control system that protects the integrity of the transmitted genome (Chakravarti, 2022).

Unlike the mammalian brain, Drosophila melanogaster can tolerate several hours of hypoxia without any tissue injury by entering a protective coma known as spreading depression. However, when oxygen is reintroduced, there is an increased production of reactive oxygen species (ROS) that causes oxidative damage. Methionine sulfoxide reductase (MSR) acts to restore functionality to oxidized methionine residues. The present study characterized in vivo effects of MSR deficiency on hypoxia tolerance throughout the lifespan of Drosophila. Flies subjected to sudden hypoxia that lacked MSR activity exhibited a longer recovery time and a reduced ability to survive hypoxic/re-oxygenation stress as they approached senescence. However, when hypoxia was induced slowly, MSR deficient flies recovered significantly quicker throughout their entire adult lifespan. In addition, the wildtype and MSR deficient flies had nearly 100% survival rates throughout their lifespan. Neuroprotective signaling mediated by decreased apoptotic pathway activation, as well as gene reprogramming and metabolic downregulation are possible reasons for why MSR deficient flies have faster recovery time and a higher survival rate upon slow induction of spreading depression. These data are the first to suggest important roles of MSR and longevity pathways in hypoxia tolerance exhibited by Drosophila (Suthakaran, 2021).

Cellular and systemic responses to low oxygen levels are principally mediated by Hypoxia Inducible Factors (HIFs), a family of evolutionary conserved heterodimeric transcription factors, whose alpha- and beta-subunits belong to the bHLH-PAS family. In normoxia, HIFalpha is hydroxylated by specific prolyl-4-hydroxylases, targeting it for proteasomal degradation, while in hypoxia the activity of these hydroxylases decreases due to low oxygen availability, leading to HIFalpha accumulation and expression of HIF target genes. To identify microRNAs required for maximal HIF activity, an overexpression screen was conducted in Drosophila melanogaster, evaluating the induction of a HIF transcriptional reporter. miR-190 overexpression was found to enhanced HIF-dependent biological responses, including terminal sprouting of the tracheal system, while in miR-190 loss of function embryos the hypoxic response was impaired. In hypoxic conditions, miR-190 expression was upregulated and required for induction of HIF target genes by directly inhibiting the HIF prolyl-4-hydroxylase Fatiga. Thus, miR-190 is a novel regulator of the hypoxia response that represses the oxygen sensor Fatiga, leading to HIFalpha stabilization and enhancement of hypoxic responses (De Lella Ezcurra,, 2016).

Facilitating fructose-driven metabolism exerts a protective effect on anoxic stress in Drosophila

Hypoxic stress is linked to various cardiovascular disorders (e.g., stroke, myocardial infarction), mediated, at least in part, by a reduction in ATP synthesis. Fructose-driven glycolysis is proposed as an alternative pathway capable of sustaining ATP production even under anoxic conditions. This study tested the hypothesis that facilitating fructose-driven metabolism exerts a protective effect against anoxic stress in Drosophila. Genetically modified flies with the human fructose transporter (GluT5) and ketohexokinase (KHK) genes downstream of upstream activating sequence (UAS) were constructed. The GAL4-UAS system was confirmed to: i) increase the expression of GluT5 and KHK in a tissue-specific and a time-dependent manner (i.e., whole flies [with Act5c-gene switch GAL4 driver], neurons [with elav-gene switch GAL4 driver]) and ii) reduce mortality of flies when placed under anoxic stress. Taken together, these data suggest that increasing fructose metabolism may be a clinically relevant approach to minimize hypoxia-induced cellular damage (Kim, 2020).

The tracheal system of Drosophila is an interconnected network of gas-filled epithelial tubes that develops during embryogenesis and functions as the main gas-exchange organ in the larva. Larval tracheal cells respond to hypoxia by activating a program of branching and growth driven by HIF-1α/sima-dependent expression of the breathless (btl) FGF receptor. By contrast, the ability of the developing embryonic tracheal system to respond to hypoxia and integrate hard-wired branching programs with sima-driven tracheal remodeling is not well understood. This study shows that embryonic tracheal cells utilize the conserved ubiquitin ligase (von Hippel-Lindau) (dVHL) to control the HIF-1 α/sima hypoxia response pathway, and two distinct phases of tracheal development with differing hypoxia sensitivities and outcomes were identified: a relatively hypoxia-resistant 'early' phase during which Sima activity conflicts with normal branching and stunts migration, and a relatively hypoxia-sensitive 'late' phase during which the tracheal system uses the dVHL/sima/btl pathway to drive increased branching and growth. Mutations in the archipelago (ago) gene, which antagonizes btl transcription, re-sensitize early embryos to hypoxia, indicating that their relative resistance can be reversed by elevating activity of the btl promoter. These findings reveal a second type of tracheal hypoxic response in which Sima activation conflicts with developmental tracheogenesis, and identify the dVHL and ago ubiquitin ligases as key determinants of hypoxia sensitivity in tracheal cells. The identification of an early stage of tracheal development that is vulnerable to hypoxia is an important addition to models of the invertebrate hypoxic response (Mortimer, 2009).

The development and survival of an organism are dependent on its ability to adapt to changing environmental conditions. Responses to some environmental changes, for example in nutrient availability, temperature, or oxygen concentration, involve alterations in patterns of gene expression that allow the organism to survive periods of environmental stress. In metazoan cells, the cellular response to reduced oxygen is mediated primarily by the HIF (hypoxia inducible factor) family of transcription factors, which are heterodimers composed of α and β subunits belonging to the bHLH Per-ARNT-Sim (bHLH-PAS) protein family. The HIF-1 αβ heterodimer is the primary oxygen-responsive HIF in mammalian cells and binds to a specific DNA sequence termed hypoxia response element (HRE) present in the promoters of target genes involved in energy metabolism, angiogenesis, erythropoiesis, and autophagy. HIF-1 activity is inhibited under normoxic conditions by two hydroxylase enzymes that use dioxygen as a substrate for catalysis to hydroxylate specific proline or aspartate residues in the HIF-1α subunit. These modifications limit HIF-1 activity by either reducing HIF-1α levels or inhibiting its ability to activate HRE-containing target promoters. One of these inhibitory mechanisms involves the 2-oxoglutarate/Fe(II)-dependent HIF-1 prolyl hydroxylase (HPH), which attaches a hydroxyl group onto each of two conserved proline residues in the oxygen-dependent degradation domain (ODD) of mammalian HIF-1α. These modifications create a binding site in the HIF-1α ODD for the Von Hippel-Lindau (VHL) protein, the substrate adaptor component of a ubiquitin ligase that subsequently polyubiquitinates HIF-1α and targets it for degradation by the proteasome. This degradation mechanism operates constitutively in normoxia and is epistatic to otherwise wide spread expression of HIF-1α mRNA. HIF-1α protein is also modified by a second oxygen-dependent hydroxylase termed Factor Inhibiting HIF (FIH) that hydroxylates an asparagine residue in the HIF-1α C-terminal activation domain. This blocks interaction with the CBP/p300 transcriptional co-factor and thus further restricts expression of HIF-1 responsive genes. These parallel O₂-dependent hydroxylation mechanisms by HPH and FIH ensure that HIF-1α levels and activity remain low in normoxic conditions. However as oxygen levels become limiting in the cellular environment, rates of hydroxylation decline and HIF-1α is rapidly stabilized in a form that dimerizes with HIF-1β, translocates to the nucleus, and promotes transcription of HRE-containing target genes (Mortimer, 2009).

Evidence suggests that invertebrate homologs of HIF-1 are also regulated in response to changes in oxygen availability. In the fruit fly Drosophila melanogaster, the HPH homolog fatiga (fga) has been shown to genetically antagonize the HIF-1α homolog similar (sima) during development. The Drosophila VHL homolog dVHL has also been shown to be capable of binding to human HIF-1α and stimulating its proteasomal turnover in vitro. In addition, the Drosophila genome encodes a well-characterized HIF-1β homolog tango (tgo), and two potential FIH homologs (CG13902 and CG10133; Berkeley Drosophila Genome Project) that have yet to be analyzed functionally. Spatiotemporal analysis of sima activation using sima-dependent hypoxia-reporter transgenes has shown that exposure to an acute hypoxic stress induces Sima most strongly in cells of the larval and embryonic tracheal system, while induction of reporter activity in other tissues requires more chronic exposure to low oxygen. The larval tracheal system is composed of an interconnected network of polarized, epithelial tubes that duct gases through the organism. As the trachea acts as the primary gas-exchange organ in the larva, it is thus a logical site of hypoxia sensitivity. During larval stages, specific cells within the tracheal system called 'terminal cells' respond to hypoxia by initiating new branching and growth that results in the extension of fine, unicellular, gas-filled tubes toward hypoxic tissues in a manner somewhat analogous to mammalian angiogenesis . Studies have shown that sima and its upstream antagonist fga function within terminal cells to regulate this process. sima is necessary for terminal cell branching in hypoxia and its ectopic activation, by either transgenic overexpression or loss of fga, is sufficient to induce excess branching even in normoxia. These phenotypes have been linked to the ability of sima to promote expression of the breathless (btl) gene, which encodes an FGF receptor that is activated by the branchless (bnl) FGF ligand. This receptor/ligand pair is known to act via a downstream MAP-kinase signaling cascade to promote cell motility and tubular morphogenesis in a variety of systems. Excessive activation of this pathway within tracheal cells by transgenic expression of btl is sufficient to drive excess branching. Reciprocally, misexpression of the bnl ligand in certain peripheral tissues is sufficient to attract excess terminal cell branching. Indeed production of secreted factors such as Bnl may be a significant part of the physiologic mechanism by which hypoxic cells attract new tracheal growth. Sima-driven induction of btl in conditions of hypoxia thus allows larval terminal cells to enter what has been termed an 'active searching' mode in which they are hyper-sensitized to signals emanating from nearby hypoxic non-tracheal cells (Mortimer, 2009 and references therein).

The role of the btl/bnl pathway in tracheal development is not restricted to hypoxia-induced branching of larval terminal cells. It also plays a critical, earlier role in the initial development of the embryonic tracheal system from the tracheal placodes, groups of post-mitotic ectodermal cells distributed along either side of the embryo that undergo a process of invagination, polarization, directed migration, and fusion to create a network of primary and secondary tracheal branches . btl and bnl are each required for this process via a mechanism in which restricted expression of bnl in cells outside the tracheal placode represents a directional cue for the migration of btl-expressing cells within the placode. Accordingly, btl expression is normally highest in pre-migratory and migratory embryonic fusion cells. In contrast to the larval hypoxic response, sima does not appear to be required for morphogenesis of the embryonic tracheal system. Rather, developmentally programmed signals in the embryo dictate a stereotyped pattern of btl and bnl expression that leads to a similarly stereotyped pattern of primary and secondary tracheal branches. The btl/bnl pathway thus responds to developmental signals to drive a fixed pattern of branching in the embryo, while in the subsequent larval stage it responds to hypoxia-dependent sima activity to facilitate the homeostatic growth of larval terminal cells and tracheal remodeling (Mortimer, 2009 and references therein).

Under normal circumstances, developing Drosophila tissues do not begin to experience hypoxia until the first larval stage, when organismal growth and movement begin to consume more oxygen than can be provided by passive diffusion alone. As a consequence, the first hypoxic challenge normally occurs after the btl/bnl-dependent elaboration of the primary and secondary embryonic branches is complete. Thus, the ability of the larval tracheal system to drive new branching and remodeling via sima and btl represents the response of a developed 'mature' tracheal system to reduced oxygen availability. By contrast the effect of hypoxia on embryonic tracheal development, which requires tight spatiotemporal control of Btl signaling to pattern the tracheal network, is not as well understood. Given that the trachea does not function as a gas-exchange organ until after fluid is cleared from the tubes at embryonic stage 17, it may be that the transcriptional response of embryonic tracheal cells to hypoxia leads to mainly metabolic changes rather than to a btl-driven program of tubulogenesis and remodeling. However, if the embryonic tracheal system does utilize the sima pathway to induce hypoxia-dependent changes in btl gene transcription, then hypoxic exposure of embryos might be predicted to produce a situation of competing developmental and homeostatic inputs that converge on the btl/bnl pathway. The ability of tracheal cells to integrate such signals may then determine whether or not the embryonic tracheal system is able to adapt to oxygen stress, or whether embryonic tracheal development represents a sensitive period during which the organism's ability to respond to changes in oxygen levels is inherently limited by a pre-programmed pattern of developmental gene expression (Mortimer, 2009).

This study shows that the embryonic tracheal system utilizes the dVHL/sima pathway to respond to hypoxia, but that the type and severity of resulting phenotypes depend on the developmental stage of exposure. Hypoxic challenge while embryonic tracheal cells are responding to developmentally programmed btl/bnl migration signals disrupts tracheal development and results in fragmented and unfused tracheal metameres. In contrast, hypoxic challenge at a somewhat later embryonic stage after fusion is complete results in overgrowth of the primary tracheal branches and the production of extra secondary branches. Interestingly, it was found that the threshold of hypoxia required to induce tracheal phenotypes in the early embryo is higher than that required to induce excess branching phenotypes in later embryonic stages, indicating that tracheal patterning events in the embryo are relatively resistant to hypoxia. Genetic analysis indicates that both types of hypoxic tracheal phenotypes -- stunting and overgrowth -- require sima and can be phenocopied in normoxia by reducing expression of the HIF-1α ubiquitin ligase gene dVHL specifically within tracheal cells. Moreover, it was found that reduced dVHL expression in the larval trachea leads to excess terminal cell branching in a manner quite similar to that observed in fga mutants. Molecular and genetic data indicate that excess btl transcription is a major cause of hypoxia-induced tracheal phenotypes. Consistent with this, mutations in the archipelago (ago) gene, which antagonizes btl transcription in tracheal fusion cells, synergize strongly with dVHL inactivation to disrupt tracheal migration and branching. Interestingly, ago mutations also lower the threshold of hypoxia required to elicit tracheal phenotypes in the 'early' embryo, suggesting that the relative activity of the btl promoter can affect hypoxic sensitivity. These findings show that the dVHL/sima pathway plays an important role in tracheal development, and identify two distinct phases of embryonic development that show different phenotypic outcomes of activating this pathway: an early phase during which sima activity conflicts with developmental control of tracheal branching and migration, and a later phase during which the tracheal system uses the dVHL/sima/btl pathway to adapt to hypoxia by increasing its future capacity to deliver oxygen to target tissues (Mortimer, 2009).

Hypoxia-induced remodeling of tracheal terminal cells represents the response of a developed larval tracheal system to reduced levels of O₂ in the environment. By contrast, the response of the developing embryonic tracheal system to systemic hypoxia has not been as well characterized. In light of the observation that embryonic tracheal cells display hypoxia-induced activation of a Sima-reporter) and that sima promotes btl expression in larval tracheal cells, embryonic exposure to hypoxia may thus produce a situation in which hard-wired btl/bnl patterning signals in the embryo come into conflict with the type of sima/btl-driven plasticity of tracheal cell branching seen in the larva. This study examined the effect of hypoxia on embryonic tracheal branching and migration. It was found that hypoxia has dramatic effects on the patterns of morphogenesis of the primary and secondary tracheal branches. Surprisingly, varying the timing and severity of hypoxic challenge is able to shift the outcome from severely stunted tracheal branching to excess branch number and enhanced branch growth. Genetic and molecular data indicate that both classes of phenotypes, stunting and overgrowth, involve regulation of sima activity and btl transcription by dVHL, and that the effects of hypoxia on tracheal development can be mimicked in normoxia by tracheal-specific knockdown of dVHL. This observation confirms a central role for dVHL in restricting the hypoxic response in vivo, and identifies a role for dVHL as a required inhibitor of sima and btl during normal tracheogenesis (Mortimer, 2009).

Since Trh and Sima/HIF-1α share a similar consensus DNA binding site, it is likely that the tracheal phenotypes elicited by either hypoxia or dVHL knockdown are to some degree the product of a combined 'Trh/Sima-like' transcriptional activity in tracheal cells. This conclusion is supported both by the general phenotypic similarity (i.e. migration and overgrowth defects) between hypoxia/dVHL knockdown and trh overexpression, by the modest ability of trh alleles to suppress dVHLⁱ phenotypes, and by the previously demonstrated overlap of transcriptional activity between Trh and human HIF-1α. Indeed, Trh is well-established as a required activator of developmental btl expression. However, because the excess Btl activity that occurs in hypoxia or in the absence of dVHL occurs independently of a change in Trh expression, it thus appears to be mediated largely by increased sima activity (Mortimer, 2009).

This analysis suggests that there are two distinct developmental 'windows' of embryogenesis during which hypoxia has opposite effects on tracheal branching. The first corresponds to a period immediately before and during primary branch migration that is relatively insensitive to hypoxia. Embryos in this stage show a minimal response to 1% O₂, but show a nearly complete arrest of migration in 0.5% O₂. Interestingly, a prior study found that similarly staged embryos (stage 11) respond to complete anoxia by prolonged developmental arrest, from which they can emerge and resume normal development. These somewhat paradoxical results -- that acute hypoxia is more detrimental to development than chronic anoxia -- might be explained by the observation that chronic exposure to low O₂ induces Sima activity throughout the embryo while acute exposure activates Sima only in tracheal cells. The former scenario may result in coordinated developmental and metabolic arrest throughout the organism, while in the latter scenario developmental patterns of gene expression in non-tracheal cells may proceed such that tracheal cells emerging from an 'early' hypoxic response find an embryonic environment in which developmentally hard-wired migratory signals emanating from non-tracheal cells have ceased (Mortimer, 2009).

The second type of tracheal response occurs during a later 'window' of embryogenesis after btl/bnl-driven primary and secondary branch migration and fusion are largely complete. It involves sinuous overgrowth of the primary and secondary branches, and duplication of secondary branches. As in the 'early' response, 'late' hypoxic phenotypes are controlled by the dVHL/sima pathway, yet unlike the 'early' response, these phenotypes occur at high penetrance even at 1% O₂. Thus the 'late' embryonic tracheal system is relatively sensitized to hypoxia and responds with increased branching in a manner similar to larval terminal cells. Indeed, much as larval branching increases with decreasing O₂ levels, it was observed that dorsal trunk growth in the late embryo is graded to the degree of hypoxia. The mechanism underlying the differential sensitivity of the 'early' and 'late' tracheal system may be quite complex. However, it was found that tracheogenesis can be sensitized to hypoxia by reducing activity of ago, a ubiquitin ligase component that restricts btl transcription in tracheal cells via its role in degrading the Trh transcription factor. Increasing transcriptional input on the btl promoter thus appears to sensitize 'early' tracheal cells to hypoxia. As Sima also controls btl transcription, one explanation of the difference in sensitivity between different embryonic stages may thus lie in differences in the activation state of the btl promoter. If so then the activity of the endogenous btl regulatory network may be an important determinant of the threshold of hypoxia required to elicit changes in tracheal architecture (Mortimer, 2009).

An organism can have its hypoxic response triggered in two ways, either by systemic exposure of the whole organism to a reduced O₂ environment or by localized hypoxia produced by increased O₂ consumption in metabolically active tissues. Data from this study and others suggests there may be distinctions between these two triggers. Exposing larvae or embryos to a systemic pulse of hypoxia results in a 'btl-centric' response specifically in tracheal cells. Outside of an 'early' vulnerable period which corresponds to embryonic branch migration and fusion, elevated Btl activity in embryonic tracheal cells promotes branch duplications and overgrowth similar to that seen in larvae. By contrast, tracheal growth induced by localized hypoxia in the larva has been suggested to involve a 'bnl-centric' model in which the hypoxic tissue secretes Bnl and recruits new tracheal branching. Whether this type of mechanism operates in embryos, or whether embryos ever experience localized hypoxia in non-tracheal cells, has not been established (Mortimer, 2009).

tHE data indicate that dVHL is a central player in the hypoxic response pathway in embryonic and larval tracheal cells. A prior study found that injection of dVHL dsRNA into syncytial embryos disrupted normal tracheogenesis, but was technically limited in its ability to conduct a detailed analysis of dVHL function in development and homeostasis. The current study found that dVHL knockdown specifically in tracheal cells mimics the effect of systemic hypoxia on embryonic tracheal architecture and larval terminal cell branching. dVHL knockdown thus phenocopies loss of the HPH gene fga, which normally functions to target Sima to the dVHL ubiquitin ligase in normoxia. Moreover, all phenotypes that result from reduced dVHL expression can be rescued by reducing sima activity, suggesting that Sima is the major target of dVHL in the tracheal system. These data support a model in which dVHL, fga, and sima function as part of a conserved VHL/HPH/HIF-1α pathway to control tracheal morphogenesis in embryos and larvae. The btl receptor appears to be an important target of this pathway in embryonic (this study) and larval tracheal cells. Knockdown of dVHL elevates btl transcription in embryonic placodes and tracheal branches, and removal of a copy of the gene effectively suppresses dVHL tracheal phenotypes. Reciprocally, overexpression of wild type btl in embryonic tracheal cells can produce migration defects and sinuous overgrowth, while expression of a constitutively active btl chimera (btl^λ) also leads to primary branch stunting and duplication of secondary branches. Interestingly, pupal lethality associated with tracheal-specific knockdown of dVHL is not sensitive to the dose of btl, but is dependent on sima. Thus the dVHL/sima pathway may have btl independent effects on tracheal cells in later stages of development (Mortimer, 2009).

The Hypoxia Inducible Factor (HIF) mediates cellular adaptations to low oxygen. Prolyl-4-hydroxylases are oxygen sensors that hydroxylate the HIF alpha-subunit (Similar in Drosophila), promoting its proteasomal degradation in normoxia. Three HIF-prolyl hydroxylases, encoded by independent genes, PHD1, PHD2, and PHD3, occur in mammals. PHD2, the longest PHD isoform includes a MYND domain, whose biochemical function is unclear. PHD2 and PHD3 genes are induced in hypoxia to shut down HIF dependent transcription upon reoxygenation, while expression of PHD1 is oxygen-independent. The physiologic significance of the diversity of the PHD oxygen sensors is intriguing. This study has analyzed the Drosophila PHD locus, fatiga, which encodes 3 isoforms, FgaA, FgaB and FgaC that are originated through a combination of alternative initiation of transcription and alternative splicing. FgaA includes a MYND domain and is homologous to PHD2, while FgaB and FgaC are shorter isoforms most similar to PHD3. Through a combination of genetic experiments in vivo and molecular analyses in cell culture, it was shown that that fgaB but not fgaA is induced in hypoxia, in a Sima-dependent manner, through a HIF-Responsive Element localized in the first intron of fgaA. The regulatory capacity of FgaB is stronger than that of FgaA, as complete reversion of fga loss-of-function phenotypes is observed upon transgenic expression of the former, and only partial rescue occurs after expression of the latter. It is concluded that diversity of PHD isoforms is a conserved feature in evolution. As in mammals, there are hypoxia-inducible and non-inducible Drosophila PHDs, and a fly isoform including a MYND domain co-exists with isoforms lacking this domain. These results suggest that the isoform devoid of a MYND domain has stronger regulatory capacity than that including this domain (Acevedo, 2010).

In response to oxygen deprivation cells, tissues and whole organisms induce the expression of a wide range of genes that tend to restore energy homeostasis. Hypoxic gene induction is mainly mediated by the Hypoxia Inducible Factor (HIF), a heterodimeric α/β transcription factor composed of two basic-Helix-Loop-Helix-PAS (bHLH-PAS) subunits. Whereas the HIFβ subunit is constitutive, HIFα is tightly regulated by oxygen levels through various mechanisms that include protein stability, transcription coactivator recruitment and subcellular localization. The molecular mechanism that controls HIFα protein stability has been characterized in detail: In normoxia, HIFα is ubiquitinated and degraded at the 26S proteasome, while in hypoxia the protein is stabilized. HIFα ubiquitination in nomoxia is mediated by the Von Hippel Lindau (VHL) tumor suppressor factor which is the substrate recognition subunit of a multimeric E3 ubiquitin ligase complex. Physical interaction between VHL and HIFα requires hydroxylation of 2 key prolyl residues in the HIFα sequence (P402 and P564 in human HIF-1α), which is catalyzed by specific prolyl-4-hydroxylases, named PHD1- PHD2 and PHD3. These enzymes are members of the Fe (II) and 2-oxoglutarate dependent dioxygenase superfamily that utilizes O₂ as a co-substrate for catalysis. Under hypoxia, PHD hydroxylase activity is reduced, HIFα escapes hydroxylation and proteolysis, leading to HIF nuclear accumulation and transcriptional induction of target genes. HIF-dependent transcription involves direct binding to Hypoxia Response Elements (HREs) that are characterized by an invariant 5′CGTG 3′ core consensus. Interestingly, a negative feed back loop, limiting HIFα activity in chronic hypoxia or upon re-oxygenation has been reported: PHD2 and PHD3 mRNAs are induced by low oxygen in a HIF-dependent manner to shut-down HIF activity; PHD1 transcription is oxygen-independent (Acevedo, 2010 and references therein).

The occurrence of three mammalian PHD isoforms encoded by three independent genes (PHD 1, PHD2 and PHD3) has opened the question of how each of these enzymes contributes to HIF regulation. It has been shown that all three PHDs can hydroxylate HIFα in vitro, and that upon over-expression, they can all suppress HRE-reporter induction. Cell culture analysis revealed that, PHD2 has a dominant role in controlling HIF-1α in normoxia, while PHD3 is important for regulating HIF in hypoxia or upon re-oxygenation. Furthermore, in vivo studies showed that PHD2, but not PHD1 or PHD3 knockout mice, exhibit enhanced angiogenesis and erythropoiesis, whereas PHD1 knockout mice display metabolic differences under ischemic conditions (Acevedo, 2010 and references therein).

Previous work has led to the identification of a hypoxia response system in Drosophila that is homologous to mammalian HIF, in which the bHLH-PAS protein Similar (Sima), and the prolyl-4-hydroxylase Fatiga (Fga) are the homologues of HIFα and PHDs, respectively. sima null mutant individuals are unable to carry out transcriptional responses to hypoxia, although they are fully viable in normoxia. fga loss-of-function alleles showed different levels of Sima accumulation in normoxia, as well as tracheal defects and lethality at different developmental stages. Interestingly, sima loss-of-function mutations rescued viability and tracheal defects of fatiga mutants, suggesting that Sima protein over-accumulation accounts for these phenotypes (Acevedo, 2010 and references therein).

This study describes a characterization of the single fatiga locus. The locus encodes three Fatiga variants, FgaA, FgaB and FgaC that originate from a combination of alternative transcription initiation and alternative mRNA splicing. FgaA includes a MYND domain, so it is homologous to PHD2, while both FgaB and FgaC are shorter isoforms that lack the MYND domain, and are similar to PHD3. Expression pattern of FgaA and FgaB were analyzed, as well as their transcriptional induction in hypoxia. Whereas FgaA expression remains constant and relatively low throughout the life cycle, FgaB is strongly upregulated in adult stages. FgaB but not FgaA is induced in hypoxia in a Sima dependent manner, both in cell culture and in vivo. Cell culture studies revealed that an HRE lying 759 to 756 base pairs upstream of the FgaB transcription initiation site accounts for FgaB induction in hypoxia. Finally, the ability of FgaA and FgaB to shut down Sima-dependent gene expression was explored; although the two isoforms are active, the regulatory capacity of FgaB is clearly stronger than that of FgaA (Acevedo, 2010).

Three PHD variants occur in mammals, and one single PHD gene, named EGL9, has been reported in Caenorabditis elegans. In Drosophila, previous studies on Fatiga, the Drosophila PHD homologous gene, have focused on its role in the regulation of Sima protein abundance and CyclinD-dependent cellular growth. In these functional studies, however, the occurrence of diverse Fga isoforms has not been addressed. This study has analyzed the fatiga locus, revealing that three different PHD isoforms occur in the fruit fly, which are generated through a combination of alternative splicing and alternative initiation of transcription. One of the isoforms, FgaA, includes a MYND domain, so it is homologous to mammalian PHD2, and the other two isoforms, FgaB and FgaC, lack a MYND domain, and are similar to PHD3. Thus, the diversity of PHD isoforms, including or not a MYND domain, seems to be an ancestral condition in evolution maintained in phylogenetically distant phyla such as insects and mammals. The occurrence of a single PHD isoform including a MYND domain in C. elegans might be due to evolutionary loss of shorter PHD variants (Acevedo, 2010).

In mammals PHD2 and PHD3, but not PHD1 mRNAs, are HIF-inducible. This work has shown that FgaB, but not FgaA, is hypoxia-inducible, and that this induction depends on Drosophila HIF/Sima. A HIF Responsive Element (HRE) that mediates hypoxic transcriptional activation of fgaB mRNAs is localized at the position –759 to –756 with respect to the transcription initiation site of fgaB. Most HREs of hypoxia inducible genes of various organisms localize at their 5' regulatory region no more than 1 Kb upstream to the transcription initiation site. The identified HRE upstream to the fgaB open reading frame adjusts to this general rule. Due to the structure of the fga locus, the 5' regulatory region of fgaB lies in the large (8630 bp) first intron of fgaA (Acevedo, 2010).

Sequence conservation of the HRE lying upstream of Drosophila fgaB transcription initiation site and the mammalian PHD3 HRE -- localized in its first intron -- is remarkable, and extends beyond CGTG HRE invariant core. Fourteen out of 17 nucleotides around the fgaB HRE (CTGGGCTACGTGAGCAT) are conserved in the PHD3 regulatory region. This observation supports the notion that oxygen-dependent induction of PHD isoforms is important for adaptation of organisms to changing oxygen conditions (Acevedo, 2010).

The fact that a single Drosophila PHD locus encodes different isoforms that parallel two of the mammalian PHD variants encoded by independent genes is remarkable, and argues in favor that a combination of PHDs including or not a MYND domain is functionally relevant. The role of the MYND domain in HIF prolyl-4-hydroxylases is intriguing. Although PHD2 is the most abundant mammalian isoform and hence, has a dominant role in controlling HIFα in normoxia, PHD3 has been reported to have stronger intrinsic hydroxylation capacity than PHD2, which includes the MYND domain. Consistent with this, the MYND domain has been proposed to mediate inhibition of PHD2 hydroxylase activity, as deletion of this domain led to increased activity of the enzyme. Supporting the notion of the MYND domain provoking reduction of PHD regulatory capacity, it has been shown that direct interaction of the peptidyl cis/trans isomerase FKBP38 with the MYND domain of PHD2 negatively regulates PHD2 protein stability. FKBP38 does not interact with the hydroxylase isoforms PHD1 or PHD3, which lack the MYND domain. Some reports, however, weigh in favor of a model of a MYND domain enhancing PHD negative regulation of HIF, as PHD2 but not PHD1 or PHD3 have the capacity to inhibit HIF transcriptional activity through a hydroxylation-independent mechanism. Consistent with this, proteins including a MYND domain have been reported to mediate transcriptional inhibition of other transcription factors, so it is conceivable that transcription inhibitory capacity is a general feature of this domain. Thus, it is still unclear as whether the MYND domain increases or decreases the regulatory capacity of PHDs. The results in Drosophila support the latter possibility, as the PHD isoform that lacks the MYND domain has stronger regulatory capacity than the isoform that includes this domain. Detailed biochemical and functional studies are required to define the precise role of this protein domain in transcriptional responses to hypoxia (Acevedo, 2010).

Neurons regulate ionic fluxes across their plasma membrane to maintain their excitable properties under varying environmental conditions. However, the mechanisms that regulate ion channels abundance remain poorly understood. This study shows that pickpocket 29 (ppk29), a gene that encodes a Drosophila degenerin/epithelial sodium channel (DEG/ENaC), regulates neuronal excitability via a protein-independent mechanism. The mRNA 3'UTR of ppk29 affects neuronal firing rates and associated heat-induced seizures by acting as a natural antisense transcript (NAT) that regulates the neuronal mRNA levels of seizure (sei), the Drosophila homolog of the human Ether-a-go-go Related Gene (hERG) potassium channel. The regulatory impact of ppk29 mRNA on sei is independent of the sodium channel it encodes. Thus, these studies reveal a novel mRNA dependent mechanism for the regulation of neuronal excitability that is independent of protein-coding capacity (Zheng, 2014).

Activating transcription factor (ATF)-2 is a member of the ATF/cAMP response element-binding protein family of transcription factors, and its trans-activating capacity is enhanced by stress-activated protein kinases such as c-Jun NH₂-terminal kinase (JNK) and p38. However, little is known about the in vivo roles played by ATF-2. Identified here is the Drosophila homologue of ATF-2 (dATF-2) consisting of 381 amino acids. In response to UV irradiation and osmotic stress, Drosophila p38 (dp38), but not JNK, phosphorylates dATF-2 and enhances dATF-2-dependent transcription. Consistent with this, injection of dATF-2 double-stranded RNA (dsRNA) into embryos did not induce the dorsal closure defects that are commonly observed in the Drosophila JNK mutant. Furthermore, expression of the dominant-negative dp38 enhanced the aberrant wing phenotype caused by expression of a dominant-negative dATF-2. Similar genetic interactions between dATF-2 and the dMEKK1-dp38 signaling pathway also were observed in the osmotic stress-induced lethality of embryos. Loss of dATF-2 in Drosophila S2 cells by using dsRNA abrogated the induction of 40% of the osmotic stress-induced genes, including multiple immune response-related genes. This indicates that dATF-2 is a major transcriptional factor in stress-induced transcription. Thus, dATF-2 is critical for the p38-mediated stress response (Sano, 2005).

The activating transcription factor/cAMP response element-binding protein (ATF/CREB) family of proteins bears a DNA-binding domain consisting of a cluster of basic amino acids and a leucine zipper that together form the so-called b-ZIP structure. These proteins can form homodimers or heterodimers by binding via their leucine zipper motifs, after which they can bind to the cyclical AMP response element (CRE: 5'-TGACGTCA-3') via their basic region. The two major subgroups of the ATF/CREB family proteins are CREB and ATF-2. The CREB subgroup includes CREB and cAMP response element modulator (CREM), whereas the ATF-2 subgroup contains ATF-2, ATFa (recently also called ATF-7), and CRE-BPa. When the Ser-133 residue of CREB is phosphorylated by cAMP-dependent protein kinase, CREB can bind to the transcriptional coactivator CREB-binding protein (CBP), which greatly stimulates the trans-activating capacity of CREB. The trans-activating capacity of ATF-2, on the other hand, is enhanced by the phosphorylation of its Thr-69 and Thr-71 residue by stress-activated protein kinases (SAPKs) such as c-Jun NH₂-terminal kinase (JNK) and p38 (Gupta, 1995; Livingstone, 1995; van Dam, 1995). SAPKs are activated by various extracellular stress such as UV, osmotic stress, and inflammatory cytokines. All three members of the ATF-2 subgroup bear the trans-activation domain in their N-terminal region: this domain consists of two subdomains, namely, the N-terminal subdomain containing the well known zinc finger motif and the C-terminal subdomain containing the SAPK phosphorylation sites. The latter subdomain has a highly flexible and disordered structure. Although the coactivator CBP binds to the protein surface of b-ZIP domain of ATF-2 (Sano, 1998), the cofactor that binds to the N-terminal activation domain of ATF-2 remains unknown (Sano, 2005).

The physiological roles played by ATF-2 have been analyzed by using mutant mice. Null Atf-2 mutant mice die shortly after birth and display symptoms of severe respiratory distress and have lungs filled with meconium (Maekawa, 1999). In the mutant embryos, hypoxia occurs, which may lead to strong gasping respiration with the consequent aspiration of the amniotic fluid containing meconium. This is due to the impaired development of cytotrophoblast cells in the placenta that in turn is caused by decreased levels of expression of the platelet-derived growth factor receptor alpha. In addition, another Atf-2 mutant mouse, which expresses only a fragment of ATF-2, exhibits lowered postnatal viability and growth, a defect in endochondrial ossification, and reduced numbers of cerebellar Purkinje cells (Reimold, 1996). However, the physiological roles played by the other ATF-2 family proteins remain unknown (Sano, 2005).

In Drosophila, three members of the mitogen-activated protein kinase (MAPK) protein family have been identified: Rolled (Erk homologue), dJNK (JNK homologue, also called Basket), and dp38a and dp38b (p38 homologue). Rolled mediates various receptor tyrosine kinase signals in the process of tracheal elaboration, cell proliferation, mesodermal patterning, R7 photoreceptor cell differentiation, and differentiation of terminal embryonic structures. In contrast, the pathway containing Hemipterous (Hep; MAPK kinase [MAPKK] homologue), dJNK, and Drosophila Jun (dJun) is involved in dorsal closure during embryo development. All mutants of this pathway exhibit the dorsal open phenotype and a decreased level of the expression of Decapentaplegic (Dpp), a secretory ligand belonging to the transforming growth factor (TGF)-β superfamily, in leading edge cells. With regard to the dp38s, they are phosphorylated by various stresses, including UV, lipopolysaccharide (LPS), and osmotic stress. The phenotype resulting from the ectopic expression of the dominant negative (DN) dp38b in the wing imaginal disc indicates that dp38b functions downstream of thickvein (Tkv), a type I receptor of the Dpp ligand, in wing morphogenesis (Sano, 2005).

To determine the in vivo function of ATF-2, the Drosophila ATF-2 homologue (dATF-2) has been identified and characterized. dATF-2 is directly phosphorylated by dp38b but not by dJNK. Moreover, genetic analyses indicated that dATF-2 acts in the dp38 signaling pathway. In addition, DNA array analysis demonstrated that dATF-2 is a major transcriptional activator of osmotic stress-inducible genes (Sano, 2005).

The amino acid sequences of the b-Zip domain and the region containing the p38/JNK phosphorylation sites of mammalian ATF-2 are well conserved in dATF-2. However, dATF-2 lacks the N-terminal zinc finger domain that is conserved in the three members of the mammalian ATF-2 family (ATF-2, CRE-BPa, and ATF-a). The N-terminal zinc finger motif and the adjacent region that contains the p38/JNK phosphorylation sites in the mammalian ATF-2 together act as the transcriptional activation domain (Matsuda, 1991). Therefore, the mediators that regulate the transcriptional activation of mammalian ATF-2 and dATF-2 may have different characteristics (Sano, 2005).

Extracellular stress such as UV or osmotic stress induces the dp38-induced phosphorylation of dATF-2 at Thr-59 and Thr-61 and this increases the trans-activation capacity of dATF-2. Although mammalian ATF-2 is well known to be phosphorylated not only by p38 but also by JNK, this study found that dJNK neither directly phosphorylated dATF-2 nor enhanced dATF-2-dependent transcription. Furthermore, transgenic embryos expressing DN-dATF-2 or dATF-2 dsRNA did not clearly reveal the dorsal-open phenotype that is common to the Hep, Bsk, dJun, and dFos mutants. The entire amino acid sequence of JNK1 shares 65% identity with dJNK, and the ~50-amino acid stretch within the N-terminal domain of mammalian ATF-2 that contains the phosphorylation sites is also well conserved in dATF-2 (59% identity). Therefore, it is surprising that dJNK cannot phosphorylate dATF-2, unlike what is observed for mammalian JNK and ATF-2. Furthermore, it was found that although dATF-2 is phosphorylated only by dp38, dJun is phosphorylated by both dp38 and dJun. In contrast, mammalian ATF-2 is phosphorylated by both p38 and JNK, whereas Jun is phosphorylated only by JNK. It is worth noting that ATFa is not phosphorylated by JNK (De Graeve, 1999). This may raise the possibility that a regulation mechanism of dATF-2 resembles to that of ATFa, and that an ancestral ATF-2/CRE-BPa gene were derived from a duplicated ATFa-like gene. The relationship between SAPKs and transcription factors in Drosophila and mammals may be useful in understanding how the stress-inducible gene expression system is established during evolution (Sano, 2005).

The GAL4-dATF-2 fusions containing the N-terminal 150 amino acids had a stronger activity than those containing the N-terminal 274 amino acids, indicating that the region between amino acids 150 and 274 has a negative effect on the activation domain of dATF-2. In the case of vertebrate ATF-2, the b-ZIP DBD suppresses the ATF-2 activation domain via intramolecular interaction (Li, 1996). This difference may suggest that the mechanism by which the C-terminal region suppresses the activation domain is different between vertebrate ATF-2 and dATF-2. It is interesting whether the region between amino acids 150 and 274 of dATF-2 affects the stability or conformation of dATF-2 protein. Wild-type dATF-2 stimulated the luciferase expression from the CRE-containing promoter under nonstimulated condition. Because the alanine mutants of Thr-59 and Thr-61 dramatically decreased this trans-activating capacity of dATF-2, phosphorylation of these residues seems to be essential for trans-activating capacity of dATF-2. These results suggest the possibility that the Thr-59 and Thr-61 residues are phosphorylated at low levels even under nonstimulated condition. This could be due to the low levels of TNF-α or IL-1 involved in serum. Alternatively, other kinase(s) also may phosphorylate these residues, because vertebrate ATF-2 is activated by Raf-MEK-ERK pathway (Ouwens, 2002) via phosphorylation of Thr-71 (Sano, 2005).

Using two different assay systems, this study has demonstrated at the genetic level that dATF-2 acts in the dp38 signaling pathway. First, it was shown that expression of DN-dp38b enhances the aberrant wing phenotype caused by DN-dATF-2. It has been reported previously that dp38b acts downstream of the Dpp receptor Tkv, because DN-dp38b expressed in the wing imaginal disc causes a phenotype resemble to the mutant of dpp, a Drosophila homologue of mammalian bone morphogenetic protein/TGF-β/activin superfamily. Therefore, dATF-2 may functions in the Dpp signaling pathway. This may be consistent with the finding that mammalian ATF-2 is phosphorylated by TGF-β signaling via TAK1 and p38, and it then directly binds to the Smad3/4 complex to synergistically activate transcription with Smad3/4 (Sano, 1999). This study also demonstrated that DN-dp38b coexpression enhances the sensitivity of embryos expressing DN-dATF-2 to high osmolarity. Thus, dATF-2 acts in the dp38 signaling pathway, at least in wing pattern formation and the response to osmotic stress. However, no oocyte defects were observed in the transgenic flies expressing DN-dATF-2, although the dp38 MAPK pathway is known to be required during oogenesis for asymmetric egg development. Thus, dATF-2 may function only in some specific events that are regulated by the dp38 signaling pathway (Sano, 2005).

DNA array analysis indicated that ~40% of the genes that are induced by osmotic stress are also regulated by dATF-2, indicating that dATF-2 is a major inducer of osmotic stress-inducible gene expression. These genes encode cell surface and cuticle proteins, transporters, and receptors, and various endopeptidases. It is not surprising that osmotic stress may increase the production of cell surface proteins, including some receptors. In addition, the endopeptidases may be produced because high osmolarity may increase the denaturation of proteins, which must then be degraded by the cell. The dATF-2 target genes also include seven immune response genes, namely, several encoding antimicrobial peptides and one encoding a peptidoglycan recognition protein, which binds to the peptidoglycans of bacterial cell walls and triggers immune responses. LPS has been shown to increase the kinase activity of dp38. Consequently, dp38-phosphorylated dATF-2 may directly induce these immune response-related genes. However, it also has been shown that overexpression of dp38 inhibits the expression of immune response genes. This could be explained by the possibility that dp38 overexpression may inhibit the p38 signaling pathway by activating negative feedback regulatory mechanisms, such as the p38α-induced decrease of MKK6 mRNA stability in mammalian cells. In Drosophila, Gram positive bacteria and fungi predominantly induce the Toll signaling pathway to activate genes such as Drosomycin, whereas Gram negative bacteria activate the Imd pathway to activate genes such as Diptericin. DNA array analysis indicated that both Drosomycin and Diptericin are regulated by dATF-2, which suggests that dATF-2 may be a component of both the Toll and Imd pathways. Further analyses of dATF-2 will most likely enhance understanding of the molecular mechanisms involved in the Drosophila immune system (Sano, 2005).

Lipid droplets (LDs) are ubiquitous organelles that facilitate neutral lipid storage in cells, including energy-dense triglycerides. They are found in all investigated metazoan embryos where they are thought to provide energy for development. Intriguingly, early embryos of diverse metazoan species asymmetrically allocate LDs amongst cellular lineages, a process which can involve massive intracellular redistribution of LDs. However, the biological reason for asymmetric lineage allocation is unknown. To address this issue, the Drosophila embryo was used where the cytoskeletal mechanisms that drive allocation are well characterized. Allocation were disrupted by two different means: Loss of the LD protein Jabba results in LDs adhering inappropriately to glycogen granules; loss of Klar alters the activities of the microtubule motors that move LDs. Both mutants cause the same dramatic change in LD tissue inheritance, shifting allocation of the majority of LDs to the yolk cell instead of the incipient epithelium. Embryos with such mislocalized LDs do not fully consume their LDs and are delayed in hatching. Through use of a dPLIN2 mutant, which appropriately localizes a smaller pool of LDs, it was found that failed LD transport and a smaller LD pool affect embryogenesis in a similar manner. Embryos of all three mutants display overlapping changes in their transcriptome and proteome, suggesting that lipid deprivation results in a shared embryonic response and a widespread change in metabolism. Excitingly, this study found abundant changes related to redox homeostasis, with many proteins related to glutathione metabolism upregulated. LD deprived embryos have an increase in peroxidized lipids and rely on increased utilization of glutathione-related proteins for survival. Thus, embryos are apparently able to mount a beneficial response upon lipid stress, rewiring their metabolism to survive. In summary, this study demonstrated that early embryos allocate LDs into specific lineages for subsequent optimal utilization, thus protecting against oxidative stress and ensuring punctual development (Kilwein, 2023).

Activation of IRE1, PERK and salt-inducible kinases leads to Sec body formation in Drosophila S2 cells

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the endoplasmic reticulum (ER) exit sites under the stress of amino acid starvation. This study addresses which signaling pathways cause Sec body formation and find that two pathways are critical. The first is the activation of the salt-inducible kinases (SIKs; SIK2 and SIK3) by Na+ stress, which, when it is strong, is sufficient. The second is activation of IRE1 and PERK (also known as PEK in flies) downstream of ER stress induced by the absence of amino acids, which needs to be combined with moderate salt stress to induce Sec body formation. SIK, and IRE1 and PERK activation appear to potentiate each other through the stimulation of the unfolded protein response, a key parameter in Sec body formation. This work shows the role of SIKs in phase transition and re-enforces the role of IRE1 and PERK as a metabolic sensor for the level of circulating amino acids and salt (Zhang, 2021).

Does local adaptation along a latitudinal cline shape plastic responses to combined thermal and nutritional stress

Thermal and nutritional stress are commonly experienced by animals. This will become increasingly so with climate change. Whether populations can plastically respond to such changes will determine their survival. Plasticity can vary among populations depending on the extent of environmental heterogeneity. However, theory conflicts as to whether environmental heterogeneity should increase or decrease plasticity. Using three locally adapted populations of Drosophila melanogaster sampled from a latitudinal gradient, thus study investigated whether plastic responses to combinations of nutrition and temperature increase or decrease with latitude for four traits: egg-adult viability, egg-adult development time, and two body size traits. Employing nutritional geometry, larvae were reared on 25 diets varying in protein and carbohydrate content at two temperatures: 18° and 25°C. Plasticity varied among traits and across the three populations. Viability was highly canalized in all three populations. The tropical population showed the least plasticity for development time, the sub-tropical showed the highest plasticity for wing area, and the temperate population showed the highest plasticity for femur length. No evidence was found of latitudinal plasticity gradients in either direction. These data highlight that differences in thermal variation and resource predictability experienced by populations along a latitudinal cline are not sufficient to predict their plasticity (Chakraborty, 2020).

4E-BP is a target of the GCN2-ATF4 pathway during Drosophila development and aging

Reduced amino acid availability attenuates mRNA translation in cells and helps to extend lifespan in model organisms. The amino acid deprivation-activated kinase GCN2 mediates this response in part by phosphorylating eIF2α. In addition, the cap-dependent translational inhibitor 4E-BP (Thor) is transcriptionally induced to extend lifespan in Drosophila melanogaster, but through an unclear mechanism. This study shows that GCN2 and its downstream transcription factor, ATF4 (Cryptocephal), mediate 4E-BP induction, and GCN2 is required for lifespan extension in response to dietary restriction of amino acids. The 4E-BP intron contains ATF4-binding sites that not only respond to stress but also show inherent ATF4 activity during normal development. Analysis of the newly synthesized proteome through metabolic labeling combined with click chemistry shows that certain stress-responsive proteins are resistant to inhibition by 4E-BP, and gcn2 mutant flies have reduced levels of stress-responsive protein synthesis. These results indicate that GCN2 and ATF4 are important regulators of 4E-BP transcription during normal development and aging (Kang, 2016).

Salt-Inducible kinase 3 provides sugar tolerance by regulating NADPH/NADP+ redox balance

Nutrient-sensing pathways respond to changes in the levels of macronutrients, such as sugars, lipids, or amino acids, and regulate metabolic pathways to maintain organismal homeostasis. Consequently, nutrient sensing provides animals with the metabolic flexibility necessary for enduring temporal fluctuations in nutrient intake. Recent studies have shown that an animal's ability to survive on a high-sugar diet is determined by sugar-responsive gene regulation. It remains to be elucidated whether other levels of metabolic control, such as post-translational regulation of metabolic enzymes, also contribute to organismal sugar tolerance. Furthermore, the sugar-regulated metabolic pathways contributing to sugar tolerance remain insufficiently characterized. This study identified Salt-inducible kinase 3 (SIK3), a member of the AMP-activated protein kinase (AMPK)-related kinase family, as a key determinant of Drosophila sugar tolerance. SIK3 allows sugar-feeding animals to increase the reductive capacity of nicotinamide adenine dinucleotide phosphate (NADPH/NADP+). NADPH mediates the reduction of the intracellular antioxidant glutathione, which is essential for survival on a high-sugar diet. SIK3 controls NADP+ reduction by phosphorylating and activating Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway. SIK3 gene expression is regulated by the sugar-regulated transcription factor complex Mondo-Mlx, which was previously identified as a key determinant of sugar tolerance. SIK3 converges with Mondo-Mlx in sugar-induced activation of G6PD, and simultaneous inhibition of SIK3 and Mondo-Mlx leads to strong synergistic lethality on a sugar-containing diet. In conclusion, SIK3 cooperates with Mondo-Mlx to maintain organismal sugar tolerance through the regulation of NADPH/NADP+ redox balance (Teesalu, 2017).

A search for new genes essential for sugar tolerance resulted in the identification of Salt-inducible kinase 3 (SIK3; CG42856). The salt-inducible kinases (SIKs) belong to the family of the AMP-activated protein kinase (AMPK)-related kinases, and they are emerging as key regulators of energy metabolism and. Although SIK^Δ null mutants were previously denoted to display an early larval lethal phenotype, nearly 50% of them developed to pupal stage on a low-sugar diet (LSD). In contrast, on a high-sugar diet (HSD), the development of SIK^Δ larvae was strikingly impaired, leading to almost complete larval lethality. Similarly to the mutants, animals with ubiquitous knockdown of SIK3 by RNAi were highly sugar intolerant. Furthermore, SIK3 knockdown larvae survived poorly on a sugar-only diet. HSD reduced food intake in general, but there was no significant difference between control and SIK^Δ mutant animals on an HSD. Earlier findings of reduced lipid levels in SIK3-deficient animals were confirmed but several additional metabolic phenotypes were also discovered. While circulating glucose remained unchanged, the SIK^Δ mutants displayed elevated levels of circulating trehalose. High levels of lactate and sorbitol, two glucose-derived metabolites, also implied that glucose metabolism was disturbed in SIK3-deficient animals. Moreover, SIK^Δ mutants displayed hemolymph acidification, a phenotype observed earlier in mutants of Activin encoding dawdle with impaired glucose metabolism. In conclusion, the data suggest that SIK3 is a key determinant of sugar tolerance and that its role in metabolic regulation in vivo is significantly broader than previously anticipated (Teesalu, 2017).

Similarly to SIK^Δ mutants, mlx mutants display sugar intolerance and high circulating trehalose levels, as well as reduced triacylglycerol (TAG) levels. Moreover, mlx mutants also displayed high circulating sorbitol levels and low hemolymph pH. These phenotypic similarities led to an exploration of the possible functional relationship between SIK3 and Mondo-Mlx. Interestingly, the expression of SIK3 was downregulated in mlx mutants during all larval stages. The Mondo-Mlx complex is most highly expressed in the fat body and in the gut and renal (Malpighian) tubules. Consistently, the mRNA expression of SIK3 was found to be Mlx dependent in all of these tissues. To test the possible sugar-dependent regulation of SIK3, first-instar Drosophila larvae were fed with an LSD versus an HSD for 16 hr and SIK3 expression was modestly, but significantly, elevated on an HSD. mlx mutants displayed no elevation of SIK3 expression in response to dietary sugar. To explore whether SIK3 is a direct target of Mondo-Mlx, the SIK3 promoter region was examined for putative Mondo-Mlx binding sites, i.e., carbohydrate response elements (ChoREs; consensus CACGTGnnnnnCACGTG). A putative ChoRE, was found which was conserved among Drosophilae. Chromatin immunoprecipitation (ChIP) in S2 cells revealed a moderate, but significant, enrichment of Mlx on the SIK3 promoter region, and the Mlx binding was increased on high glucose. In conclusion, these results show that SIK3 gene expression is regulated by Mondo-Mlx, and the phenotypic similarities further suggest functional interplay between SIK3 and Mondo-Mlx on metabolic regulation (Teesalu, 2017).

It was observed earlier that the pentose phosphate pathway (PPP) is transcriptionally regulated by Mondo-Mlx and that PPP activity is essential for sugar tolerance and maintaining TAG levels. The phenotypic similarities of SIK3 and mlx mutants led to a hypothesis that SIK3 might also regulate PPP activity. Indeed, co-immunoprecipitation uncovered a physical interaction between SIK3 and glucose-6-phosphate dehydrogenase (G6PD; encoded by Zwischenferment; Zw), the rate-limiting enzyme of the PPP. To analyze G6PD phosphorylation, phosphate-binding tag (Phos-tag) SDS-PAGE was used. Co-expression of SIK3 induced several slow-migrating bands of G6PD, which were confirmed to be phosphorylated forms by alkaline phosphatase treatment. An in vitro kinase assay to detect the activity of SIK3 co-purified with G6PD provided further evidence of SIK3-mediated phosphorylation of G6PD (Teesalu, 2017).

To identity the phosphorylation sites of SIK3, mass spectrometric analysis of G6PD, which was affinity purified from S2 cells, was used. In total, eight high-confidence phosphorylation sites were detected, and six of them were only present upon SIK3 co-expression. These six sites may be both directly and indirectly regulated by SIK3. Since SIK3 is a serine/threonine kinase, phosphorylation of Y384 is most likely mediated by another kinase, possibly following the priming phosphorylation by SIK3. Transgenic flies of wild-type (WT) G6PD and the mutant form were generated with the six SIK3-dependent phosphorylation sites mutated into corresponding non-phosphorylatable amino acids (6xP-mut). An in vitro assay to measure G6PD enzyme activity from larval lysates revealed that WT G6PD activity was increased upon sugar feeding, while the activity of the phospho-deficient mutant was not. This was consistent with the idea that SIK3-mediated phosphorylation activates G6PD upon sugar feeding. Endogenous G6PD activity in control larvae was also elevated in response to an HSD, but this increase was not observed in SIK3 mutants or in SIK3 RNAi animals. Knockdown of G6PD served as a positive control. In accordance with Zw and SIK3 being transcriptional targets of Mondo-Mlx, an impaired sugar-induced activation of G6PD was observed in mlx mutants. However, unlike mlx mutants, SIK3 mutants did not display reduced Zw mRNA expression, which supports the idea that SIK3 regulates G6PD activity post-translationally. Furthermore, knockdown of G6PD led to elevated circulating trehalose levels, in addition to sugar intolerance and low TAG levels reported earlier (Teesalu, 2017).

The data implied that SIK3 synergizes with Mondo-Mlx to control G6PD activity. Thus, it was plausible that mondo-mlx and SIK3 interact genetically. To test this, SIK3 and mondo (encoding the essential interaction partner of Mlx) by were depleted RNAi and the development of the animals was monitored. Strikingly, ubiquitous double knockdown of Mondo and SIK3 caused a strong synthetic phenotype, leading to larval growth impairment and lethality on moderate levels (5%) of dietary sucrose. Furthermore, the SIK3, mlx double mutants displayed synergistic lethality on a sugar-only diet (Teesalu, 2017).

Since the oxidative branch of the pentose phosphate pathway is crucial in generating reductive power in the form of NADPH, it was predicted that the regulation of NADPH/NADP+ balance might be deregulated in the SIK3 mutant animals. This was the case, since the NADPH/NADP+ ratio was significantly elevated in HSD-fed control animals, but such an increase was not observed in SIK3 mutants. Similar results were obtained with mlx mutants. The reducing equivalents of NADPH are necessary for counteracting oxidative stress through the glutathione (GSH) redox couple (GSH/GSH disulfide, GSH/GSSG). In agreement with a low NADPH/NADP+ ratio, the GSH/GSSG ratio was reduced in SIK3 mutants on an HSD, as well as upon G6PD knockdown. Moreover, feeding larvae with reduced glutathione partially rescued the pupariation of SIK^Δ mutants on a sugar-containing diet (Teesalu, 2017).

Drosophila genome lacks glutathione reductase, and the glutathione reduction is mediated through reduced thioredoxin. Loss-of-function of thioredoxin reductase-1, an enzyme that uses NADPH to reduce thioredoxin (and, consequently, GSH), led to significantly impaired sugar tolerance. Glutathione prevents oxidative damage of cellular biomolecules, including peroxidation of lipids. Consistent with the low GSH/GSSG ratio, the levels of lipid peroxides were significantly elevated in sugar-feeding SIK3 mutants. Furthermore, depletion of glutathione peroxidase PHGPx, a GSH-dependent enzyme involved in counteracting lipid peroxidation, led to sugar intolerance. This further corroborated the role of oxidative stress prevention in sugar tolerance (Teesalu, 2017).

This study has shown that SIK3-deficient Drosophila larvae display lethality on an HSD and thus that SIK3 is a critical mediator of sugar tolerance. While SIK3 was earlier shown to control Drosophila lipid catabolism and tissue growth, this study provides evidence for SIK3-mediated control of glucose metabolism and NADPH redox balance, thereby significantly broadening the known in vivo role of SIK3. Earlier studies have shown that Drosophila SIK3 regulates metabolism via phosphorylation of the transcriptional cofactor HDAC4 and tissue growth by phosphorylating Salvador, a component of the Hippo signaling pathway. This study observed that SIK3 forms a complex with G6PD and controls its activity by phosphorylation. Loss of SIK3-dependent phosphorylation sites prevented post-translational activation of G6PD upon sugar feeding, demonstrating the functional relevance of SIK3-mediated G6PD phosphorylation in vivo (Teesalu, 2017).

Earlier studies in mammalian cells and rats have shown G6PD to be phosphorylated by protein kinase A, which inhibits G6PD activity. It is perhaps not surprising that SIK3 and protein kinase A (PKA) might be counteracting each other on G6PD regulation since, in cAMP-response-element-binding protein (CREB)-mediated transcription, SIK family members and PKA also mediate opposing activities. PKA-mediated phosphorylation activates CREB, while SIK family members inhibit the cofactor of CREB, CRTC (CREB-regulated transcription coactivator). Furthermore, PKA phosphorylates and inhibits Drosophila SIK3, while SIK3 is activated by insulin-mediated phosphorylation. This study revealed an additional layer of SIK3 regulation by observing that SIK3 gene expression is reduced in mlx mutants. A binding site for Mlx was identified in the SIK3 promoter, suggesting that SIK3 is a direct Mondo-Mlx target, although indirect mechanisms cannot be ruled out. Given the relatively modest increase of SIK3 expression on an HSD, it is also likely that post-translational mechanisms are involved in the sugar-induced activation of SIK3. It was recently shown that Mondo-Mlx transcriptionally activates the pentose phosphate pathway, including the G6PD-encoding gene Zw. Thus, Mondo-Mlx and SIK3 appear to form a regulatory circuit, which converges on the control of G6PD. Such dual regulation through gene expression and phosphorylation is likely to increase the dynamic range of G6PD activation upon sugar feeding and thereby extend the range of tolerated dietary sugar. Indeed, simultaneous RNAi-mediated inhibition of SIK3 and Mondo-Mlx had devastating consequences, leading to early larval lethality on moderate (5%) sugar levels. It will be interesting to learn whether the convergent control via gene expression and phosphorylation will also involve other sugar-regulated genes (Teesalu, 2017).

One of the key findings of this study is the dynamic control of NADPH-GSH reductive capacity in response to sugar feeding and its importance on sugar tolerance. Larvae lacking SIK3 were unable to elevate their NADPH/NADP+ ratio and displayed signs of oxidative stress on an HSD. Inhibition of glutathione reduction by RNAi against thioredoxin reductase-1 conferred animals intolerant to an HSD, while having no impact on animals on an LSD, and the feeding of glutathione increased the survival of SIK3 mutants specifically on a sugar-containing diet. This study, together with earlier findings, supports a model where sugar-sensing pathways synchronously coordinate the activities of several pathways that mediate safe elimination and storage of the excess carbon skeletons provided by dietary sugars. This includes activation of glycolytic and lipogenic gene expression programs, as well as an increase of NADPH reductive capacity through G6PD activation. The need for elevated GSH reductive capacity on HSD might stem from the challenge posed by reactive metabolic intermediates, such as methylglyoxal, formed during high glycolytic activity. On the other hand, de novo lipogenesis requires a high degree of NADPH, which would impair the proper function of the GSH-mediated prevention of oxidative stress, unless the generation of reductive capacity is simultaneously increased. Future studies will elucidate whether other pathways regulating NADPH/NADP+ balance contribute to sugar tolerance (Teesalu, 2017).

Reactive oxygen species (ROS) have been extensively studied as damaging agents associated with ageing and neurodegenerative conditions. Their role in the nervous system under non-pathological conditions has remained poorly understood. Working with the Drosophila larval locomotor network, this study showed that in neurons ROS act as obligate signals required for neuronal activity-dependent structural plasticity, of both pre- and postsynaptic terminals. ROS signaling is also necessary for maintaining evoked synaptic transmission at the neuromuscular junction, and for activity-regulated homeostatic adjustment of motor network output, as measured by larval crawling behavior. The highly conserved Parkinson's disease-linked protein DJ-1β was identified as a redox sensor in neurons where it regulates structural plasticity, in part via modulation of the PTEN-PI3 Kinase pathway. This study provides a new conceptual framework of neuronal ROS as second messengers required for neuronal plasticity and for network tuning, whose dysregulation in the ageing brain and under neurodegenerative conditions may contribute to synaptic dysfunction (Oswald, 2018).

This study set out to investigate potential roles for ROS in the nervous system under non-pathological conditions, which are much less well understood. The brain is arguably the most energy demanding organ and mitochondrial oxidative phosphorylation is a major source of ROS. It was therefore asked whether neurons might utilize mitochondrial metabolic ROS as feedback signals to mediate activity-regulated changes. As an experimental model the motor system was used of the fruitfly larva, Drosophila melanogaster allowing access to uniquely identifiable motoneurons in the ventral nerve cord and their specific body wall target muscles. An experimental paradigm was established for studying activity-regulated structural adjustments across an identified motoneuron, quantifying changes at both pre- and postsynaptic terminals. Thermogenetic neuronal over-activation leads to the generation of ROS at presynaptic terminals, and ROS signaling is necessary and sufficient for the activity-regulated structural adjustments. As a cellular ROS sensor the conserved redox sensitive protein DJ-1β, a homologue of vertebrate DJ-1 (PARK7), was identified and the phosphatase and tensin homolog (PTEN) and PI3kinase were identified as downstream effectors of activity-ROS-mediated structural plasticity. ROS signaling is also required for maintaining constancy of evoked transmission at the neuromuscular junction (NMJ) with a separate ROS pathway regulating the amplitude of spontaneous vesicle release events. Behaviourally, ROS signaling is required for the motor network to adjust homeostatically to return to a set crawling speed following prolonged overactivation (Oswald, 2018).

Building on previous work that had shown oxidative stress as inducing NMJ growth (Milton, 2011), this study identified ROS as obligatory signals for activity-regulated structural plasticity. It was further shown that ROS are also sufficient to bring about structural changes at synaptic terminals that largely mimic those induced by neuronal overactivation. A mitochondrially targeted ROS reporter suggests a positive correlation between levels of neuronal activity and ROS generated in mitochondria, potentially as a byproduct of increased ATP metabolism or triggered by mitochondrial calcium influx. Although this study did not specifically investigate the nature of the active ROS in this context, three lines of evidence suggest that H2O2, generated by the dismutation of O2-, is the principal signaling species. First, under conditions of neuronal overactivation (but not control levels of activity) over-expression of the O2- to H2O2 converting enzyme SOD2 potentiated structural plasticity phenotypes. Second, over-expression of the H2O2 scavenger Catalase efficiently counter-acts all activity-induced changes that were quantified, at both postsynaptic dendritic and presynaptic NMJ terminals. Third, over-expression of the H2O2 generator Duox in motoneurons is sufficient to induce NMJ bouton phenotypes that mimic overactivation. In addition to mitochondria, other sources of ROS include several oxidases, notably NADPH oxidases. These have been implicated during nervous system development in the regulation of axon growth and synaptic plasticity. NADPH oxidases can be regulated by NMDA receptor stimulation and activity-associated pathways, including calcium, Protein kinases C and A and calcium/calmodulin-dependent kinase II (CamKII). The precise sources of activity-regulated ROS, potentially for distinct roles in plasticity, will be interesting to investigate (Oswald, 2018).

This study demonstrated that ROS are necessary for activity-dependent structural plasticity of Drosophila motoneurons, at both their postsynaptic dendrites in the CNS and presynaptic NMJs in the periphery. The mechanisms by which ROS intersect with other known plasticity pathways now need to be investigated. Among well documented signaling pathways regulating synaptic plasticity, are Wnts, BMPs, PKA, CREB and the immediate early gene transcription factor AP-1. ROS signaling could be synergistic with other neuronal plasticity pathways, potentially integrating metabolic feedback. Indeed, ROS modulate BMP signaling in cultured sympathetic neurons and Wnt pathways in non-neuronal cells. Biochemically, ROS are well known regulators of kinase pathways via oxidation-mediated inhibition of phosphatases. Redox modifications also regulate the activity of the immediate early genes Jun and Fos, which are required for LTP in vertebrates, and in Drosophila for activity-dependent plasticity of motoneurons, both at the NMJ and central dendrites. It was therefore hypothesized that ROS may provide neuronal activity-regulated modulation of multiple canonical synaptic plasticity pathways (Oswald, 2018).

This study focused on three aspects of synaptic terminal plasticity: dendritic arbor size in the CNS, and bouton and active zone numbers at the NMJ. These were used as phenotypic indicators for activity-regulated changes. By working with identified motoneurons adaptations could be observed across the entire neuron, relating adjustments of postsynaptic dendritic input terminals in the CNS to changes of the presynaptic output terminals at the NMJ in the periphery. For the aCC motoneuron, the degree of neuronal overactivation correlates with changes in synaptic terminal growth: notably reductions of dendritic arbor size centrally and of active zones at the NMJ. Interestingly, presynaptic active zone numbers did not show a linear response profile. Within a certain range low-level activity increases lead to more active zones, associated with potentiation; however, with stronger overactivation active zone number decrease. Reduction of active zones, as was observed at the NMJ, and of Brp levels by increased activation was previously also reported in photoreceptor terminals of the Drosophila adult visual system. At a finer level of resolution it will be interesting to determine how these activity-ROS-mediated structural changes might change active zone cytomatrix composition, which can impact on transmission properties, such as vesicle release probability (Oswald, 2018).

Previous work found that in these motoneurons dendritic length correlates with the number of input synapses and with synaptic drive) Therefore, the negative correlation between the degree of overactivation and the reduction in central dendritic arbors is tentatively interpreted as compensatory. In agreement, it was found that blockade of activity-induced structural adjustment targeted to the motoneurons prevents behavioral adaptation normally seen after prolonged overactivation. Less clear is if and how overactivation-induced structural changes at the NMJ might be adaptive. Unlike many central synapses that facilitate graded analogue computation, the NMJ is a highly specialized synapse with a large safety factor and intricate mechanisms that ensure constancy of evoked transmission in essentially digital format. Rearing larvae at 29°C (which acutely increases motor activity) leads to more active zones at the NMJ and potentiated transmission, yet these larvae crawl at the same default speed as other larvae reared at 25°C (control) or 32°C with reduced numbers of active zones. This suggests that at least with regard to regulating crawling speed, plasticity mechanisms probably operate at the network level, rather than transmission properties of the NMJ. Indeed, recordings of transmission at the NMJ, and those reported by others, show homeostatic maintenance of eEJP amplitude irrespective of changes in bouton and active zone number. Though in this study focus was placed on anatomical changes, these structural adjustments are expected to be linked to, and probably preceded by compensatory changes in neuronal excitability that have been documented (Oswald, 2018).

The observations of activity-regulated adjustments of both dendritic arbor size and NMJ structure give the impression of processes coordinated across the entire neuron. If this was the case, it could be mediated by transcriptional changes, potentially via immediate early genes (AP-1), which are involved in activity and ROS-induced structural changes at the NMJ and motoneuron dendrites (Oswald, 2018).

In neurons the highly conserved protein DJ-1β; is critical for both structural and physiological changes in response to activity-generated ROS. In neurons DJ-1β might act as a redox sensor for activity-generated ROS. In agreement with this idea, DJ-1β has been shown to be oxidized by H2O2 at the conserved cysteine residue C106 (C104 in Drosophila). Oxidation of DJ-1 leads to changes in DJ-1 function, including translocation from the cytoplasm to the mitochondrial matrix, aiding protection against oxidative damage and maintenance of ATP levels. The ability of motoneurons to respond to increased activation is potently sensitive to DJ-1β dosage. It is also blocked by expression of mutant DJ-1βC104A that is non-oxidisable on the conserved Cys104. These observations suggest that DJ-1β is critical to ROS sensing in neurons. They also predict that cell type-specific DJ-1β levels, and associated DJ-1β reducing mechanisms, could contribute to setting cell type-specific sensitivity thresholds to neuronal activity (Oswald, 2018).

The data suggest that DJ-1β could potentially be part of a signaling hub. At the NMJ, this might mediate plasticity across a range, from the addition of active zones associated with potentiation to, following stronger overactivation, the reduction of active zones. This study identified disinhibition of PI3Kinase signaling as one DJ-1β downstream pathway, a well-studied intermediate in metabolic pathways and a known regulator of synaptic terminal growth, including active zone addition. However, with stronger overactivation DJ-1β might engage additional downstream effectors that reduce active zone addition or maintenance, potentially promoting active zone disassembly. While at the presynaptic NMJ PI3Kinase disinhibition explains activity-regulated changes in bouton addition, different DJ-1β effectors likely operate in the somato-dendritic compartment, which responds to overactivation with reduced growth and possibly pruning. Thus, sub-cellular compartmentalization of the activity-ROS-DJ-1β signaling axis could produce distinct plasticity responses in pre- versus post-synaptic terminals (Oswald, 2018).

Previous studies demonstrated a requirement for ROS for LTP and found learning defects in animal models with reduced NADPH oxidase activity, suggesting that synaptic ROS signaling might be a conserved feature of communication in the nervous system. Sharp electrode recordings from muscle DA1 revealed three interesting aspects. First, that changing ROS signaling in the presynaptic motoneuron under normal activity conditions does not obviously impact on NMJ transmission. Second, quenching of presynaptic ROS by expression of Catalase under overactivation conditions led to a significant decrease in eEJP amplitude and concomitantly reduced quantal content. This shows that upon chronic neuronal overactivation ROS signaling is critically required in the presynaptic motoneuron for maintaining eEJP amplitude by increasing vesicle release at the NMJ. This could be achieved by increasing vesicle release probability, which would counteract the reduction in active zone number following a period of neuronal overactivation. In this context it is interesting that components of the presynaptic release machinery, including SNAP25, are thought to be directly modulated by ROS, while others, such as Complexin, might be indirectly affected, for example via ROS-mediated inhibition of phosphatases leading disinhibition of kinase activity. Third, this study found that overactivation of motoneurons leads to reduced mEJP amplitude, also recently reported by others (Yeates, 2017). Curiously, mEJP amplitude, unlike eEJP amplitude, is regulated by DJ-1β, but is not impacted on by artificially increased cytoplasmic levels of the H2O2 scavenger Catalase. How it is that under conditions of neuronal overactivation eEJP and mEJP amplitudes are differentially sensitive to cytoplasmic Catalase versus DJ-1β oxidation is unclear, though it marks these two processes as distinct. One possibility is that cytoplasmic Catalase changes the local redox status, which could directly affect the properties of the presynaptic active zone cytomatrix. In contrast, mEJP amplitude regulation might be indirect and cell non-autonomous, via modulation of glutamate receptors in the postsynaptic target muscle (Oswald, 2018).

Thus, several distinct ROS responsive pathways appear to operate at the NMJ. Structural adjustments in terms of synaptic terminal growth and synapse number are mediated by mechanisms sensitive to DJ-1β oxidation, potentially regulated via local reducing systems, including Catalase. In addition and distinct from these structural changes, at least in part, are the ROS-regulated adjustments in synaptic transmission that show different ROS sensitivities, one maintaining quantal content of evoked transmission while the other reduces mEJP amplitude when neuronal activity goes up. It is conceivable that spatially distinct sources of ROS, for example mitochondria versus membrane localized NADPH oxidases, with different temporal dynamics could potentially mediate such differences in ROS sensitivities at the NMJ (Oswald, 2018).

Experiments exploring the potential behavioral relevance of activity-regulated structural plasticity demonstrated that network drive is regulated by ambient temperature. Acute elevation in ambient temperature produces faster crawling, while acute temperature reduction has the opposite effect. In contrast, with chronic temperature manipulations, larval crawling returns to its default speed (approx. 0.65-0.72 mm/sec). This adaptation to chronic manipulations might overall be energetically more favorable. It also allows larvae to retain a dynamic range of responses to relative changes in ambient temperature (i.e., speeding up or slowing down) (Oswald, 2018).

Where in the locomotor network these adjustments take place remains to be worked out. It is reasonable to assume that proprioceptive sensory neurons, and potentially also central recurrent connections, provide feedback information that facilitates homeostatic adjustment of network output. This study's manipulations of the glutamatergic motoneurons show these cells are clearly important. For example, cell type-specific overactivation of the glutamatergic motoneurons (via dTrpA1) on the one hand, and blockade of activity-induced structural adjustment (by mis-expression of non-oxidizable DJ-1βC104A) on the other demonstrated that ROS-DJ-1β-mediated processes that were showed important for structural adjustment are also required for implementing homeostatic tuning of locomotor network output. The capacity of motoneurons as important elements in shaping motor network output, might be explicable in that these neurons constitute the final integrators on which all pre-motor inputs converge (Oswald, 2018).

In conclusion, this study identified ROS in neurons as novel signals that are critical for activity-induced structural plasticity\. ROS levels regulated by neuronal activity have the potential for operating as metabolic feedback signals. The conserved redox-sensitive protein DJ-1β was further as important to neuronal ROS sensing, and the PTEN/PI3Kinase synaptic growth pathway was identified as a downstream effector pathway for NMJ growth in response to neuronal overactivation. These findings suggest that in the nervous system ROS operate as feedback signals that inform cells about their activity levels. The observation that ROS are important signals for homeostatic processes explains why ROS buffering is comparatively low in neurons. This view also shines a new light on the potential impact of ROS dysregulation with age or under neurodegenerative conditions, potentially interfering with neuronal adaptive adjustments and thereby contributing to network malfunction and synapse loss (Oswald, 2018).

Downregulation of oxidative stress-mediated glial innate immune response suppresses seizures in a fly epilepsy model

Previous work has shown that mutations in prickle (pk) cause myoclonic-like seizures and ataxia in Drosophila, similar to what is observed in humans carrying mutations in orthologous PRICKLE genes. This study shows that pk mutant brains show elevated, sustained neuronal cell death that correlates with increasing seizure penetrance, as well as an upregulation of mitochondrial oxidative stress and innate immune response (IIR) genes. Moreover, flies exhibiting more robust seizures show increased levels of IIR-associated target gene expression suggesting they may be linked. Genetic knockdown in glia of either arm of the IIR (Immune Deficiency [Imd] or Toll) leads to a reduction in neuronal death, which in turn suppresses seizure activity, with oxidative stress acting upstream of IIR. These data provide direct genetic evidence that oxidative stress in combination with glial-mediated IIR leads to progression of an epilepsy disorder (Nukala, 2023).

Canavalia ensiformis lectin induced oxidative stress mediate both toxicity and genotoxicity in Drosophila melanogaster

Mannose/glucose-binding lectin from Canavalia ensiformis seeds (Concanavalin A - ConA) has several biological applications, such as mitogenic and antitumor activity. However, most of the mechanisms involved in the in vivo toxicity of ConA are not well known. In this study, the Drosophila melanogaster model was used to assess the toxicity and genotoxicity of different concentrations of native ConA (4.4, 17.5 and 70 microg/mL) in inhibited and denatured forms of ConA. The data show that native ConA affected: the survival, in the order of 30.6 %, and the locomotor performance of the flies; reduced cell viability to levels below 50 % (4.4 and 17.5 microg/mL); reduced nitric oxide levels; caused lipid peroxidation and increased protein and non-protein thiol content. In the Comet assay, native ConA (17.5 and 70 microg/mL) caused DNA damage higher than 50 %. In contrast, treatments with inhibited and denatured ConA did not affect oxidative stress markers and did not cause DNA damage. It is believed that protein-carbohydrate interactions between ConA and carbohydrates of the plasma membrane are probably the major events involved in these activities, suggesting that native ConA activates mechanisms that induce oxidative stress and consequently DNA damage (de Oliveira Dos Santos, 2022).

Melatonin Increases Life Span, Restores the Locomotor Activity, and Reduces Lipid Peroxidation (LPO) in Transgenic Knockdown Parkin Drosophila melanogaster Exposed to Paraquat or Paraquat/Iron

Parkinson's disease (PD) is a complex progressive neurodegenerative disorder involving impairment of bodily movement caused by the specific destruction of dopaminergic (DAergic) neurons. Mounting evidence suggests that PD might be triggered by an interplay between environmental neurotoxicants (e.g., paraquat, PQ), heavy metals (e.g., iron), and gene alterations (e.g., PARKIN gene). Unfortunately, there are no therapies currently available that protect, slow, delay, or prevent the progression of PD. Melatonin (Mel, N-acetyl-5-methoxy tryptamine) is a natural hormone with pleiotropic functions including receptor-independent pathways which might be useful in the treatment of PD. Therefore, as a chemical molecule, it has been shown that Mel prolonged the lifespan and locomotor activity, and reduced lipid peroxidation (LPO) in wild-type Canton-S flies exposed to PQ, suggesting antioxidant and neuroprotective properties. However, it is not yet known whether Mel can protect or prevent the genetic model parkin deficient in flies against oxidative stress (OS) stimuli. This study shows that Mel (0.5, 1, 3 mM) significantly extends the life span and locomotor activity of TH > parkin-RNAi/ + Drosophila melanogaster flies (>15 days) compared to untreated flies. Knock-down (K-D) parkin flies treated with PQ (1 mM) or PQ (1 mM)/iron (1 mM) significantly diminished the survival index and climbing abilities (e.g., 50% of flies were dead and locomotor impairment by days 4 and 3, respectively). Remarkably, Mel reverted the noxious effect of PQ or PQ/iron combination in K-D parkin. Indeed, Mel protects TH > parkin-RNAi/ + Drosophila melanogaster flies against PQ- or PQ/iron-induced diminish survival, locomotor impairment, and LPO (e.g., 50% of flies were death and locomotor impairment by days 6 and 9, respectively). Similarly, Mel prevented K-D parkin flies against both PQ and PQ/iron. Taken together, these findings suggest that Mel can be safely used as an antioxidant and neuroprotectant agent against OS-stimuli in selective individuals at risk to suffer early-onset Parkinsonism and PD (Ortega-Arellano, 2021).

Metabolomics provide new insights into mechanisms of Wolbachia-induced paternal defects in Drosophila melanogaster

Wolbachia is a group of intracellular symbiotic bacteria that widely infect arthropods and nematodes. Wolbachia infection can regulate host reproduction with the most common phenotype in insects being cytoplasmic incompatibility (CI), which results in embryonic lethality when uninfected eggs were fertilized with sperm from infected males. This suggests that CI-induced defects are mainly in paternal side. However, whether Wolbachia-induced metabolic changes play a role in the mechanism of paternal-linked defects in embryonic development is not known. Untargeted metabolomics revealed 414 potential differential metabolites between Wolbachia-infected and uninfected 1-day-old (1d) male flies. Most of the differential metabolites were significantly up-regulated due to Wolbachia infection. Wolbachia infection was shown to result in an increased energy expenditure of the host by regulating glycometabolism and fatty acid catabolism, which was compensated by increased food uptake. Furthermore, overexpressing two acyl-CoA catabolism related genes, Dbi (coding for diazepam-binding inhibitor) or Mcad (coding for medium-chain acyl-CoA dehydrogenase), ubiquitously or specially in testes caused significantly decreased paternal-effect egg hatch rate. Oxidative stress and abnormal mitochondria induced by Wolbachia infection disrupted the formation of sperm nebenkern. These findings provide new insights into mechanisms of Wolbachia-induced paternal defects from metabolic phenotypes (Zhang, 2021).

Overexpression of SIRT3 Suppresses Oxidative Stress-induced Neurotoxicity and Mitochondrial Dysfunction in Dopaminergic Neuronal Cells

Sirtuin 3 (SIRT3), a well-known mitochondrial deacetylase, is involved in mitochondrial function and metabolism under various stress conditions. This study found that the expression of SIRT3 was markedly increased by oxidative stress in dopaminergic neuronal cells. In addition, SIRT3 overexpression enhanced mitochondrial activity in differentiated SH-SY5Y cells. SIRT3 overexpression was shown to attenuated rotenoneor H(2)O(2)-induced toxicity in differentiated SH-SY5Y cells (human dopaminergic cell line). It wa further found that knockdown of SIRT3 enhanced rotenone- or H(2)O(2)-induced toxicity in differentiated SH-SY5Y cells. Moreover, overexpression of SIRT3 mitigated cell death caused by LPS/IFN-γ stimulation in astrocytes. The rotenone treatment was found to increase the level of SIRT3 in Drosophila brain. Downregulation of sirt2 (Drosophila homologue of SIRT3) significantly accelerated the rotenone-induced toxicity in flies. Taken together, these findings suggest that the overexpression of SIRT3 mitigates oxidative stress-induced cell death and mitochondrial dysfunction in dopaminergic neurons and astrocytes (Lee, 2021).

Natural variation in the transcriptional response of Drosophila melanogaster to oxidative stress

Broadly distributed species must cope with diverse and changing environmental conditions, including various forms of stress. Cosmopolitan populations of Drosophila melanogaster are more tolerant to oxidative stress than those from the species' ancestral range in sub-Saharan Africa, and the degree of tolerance is associated with an insertion/deletion polymorphism in the 3' untranslated region of the Metallothionein A (MtnA) gene that varies clinally in frequency. Oxidative stress tolerance and the transcriptional response to oxidative stress were examined in cosmopolitan and sub-Saharan African populations of D. melanogaster, including paired samples with allelic differences at the MtnA locus. The effect of the MtnA polymorphism on oxidative stress tolerance was found to be dependent on the genomic background, with the deletion allele increasing tolerance only in a northern, temperate population. Genes that were differentially expressed under oxidative stress included MtnA and other metallothioneins, as well as those involved in glutathione metabolism and other genes known to be part of the oxidative stress response or the general stress response. A gene coexpression analysis revealed further genes and pathways that respond to oxidative stress including those involved in additional metabolic processes, autophagy, and apoptosis. There was a significant overlap among the genes induced by oxidative and cold stress, which suggests a shared response pathway to these two stresses. Interestingly, the MtnA deletion was associated with consistent changes in the expression of many genes across all genomic backgrounds, regardless of the expression level of the MtnA gene itself. It is hypothesize that this is an indirect effect driven by the loss of microRNA binding sites within the MtnA 3' untranslated region (Ramnarine, 2021).

The metabolome as a link in the genotype-phenotype map for peroxide resistance in the fruit fly, Drosophila melanogaster

This study used metabolomics to explore the nature of genetic variation for hydrogen peroxide (H(2)O(2)) resistance in the sequenced inbred Drosophila Genetic Reference Panel (DGRP). First genetic variation was studied for H(2)O(2) resistance in 179 DGRP lines and along with identifying the insulin signaling modulator u-shaped and several regulators of feeding behavior, it is estimated that a substantial amount of phenotypic variation can be explained by a polygenic model of genetic variation. Then a portion of the aqueous metabolome was profiled in subsets of eight 'high resistance' lines and eight 'low resistance' lines. These lines were used to represent collections of genotypes that were either resistant or sensitive to the stressor, effectively modeling a discrete trait. Across the range of genotypes in both populations, flies exhibited surprising consistency in their metabolomic signature of resistance. Importantly, the resistance phenotype of these flies was more easily distinguished by their metabolome profiles than by their genotypes. Furthermore, a metabolic response was found to H(2)O(2) in sensitive, but not in resistant genotypes. Metabolomic data further implicated at least two pathways, glycogen and folate metabolism, as determinants of sensitivity to H(2)O(2). A confounding effect was found of feeding behavior on assays involving supplemented food. This work suggests that the metabolome can be a point of convergence for genetic variation influencing complex traits, and can efficiently elucidate mechanisms underlying trait variation (Harrison, 2020).

Mitochondrial stress causes neuronal dysfunction via an ATF4-dependent increase in L-2-hydroxyglutarate

Mitochondrial stress contributes to a range of neurological diseases. Mitonuclear signaling pathways triggered by mitochondrial stress remodel cellular physiology and metabolism. How these signaling mechanisms contribute to neuronal dysfunction and disease is poorly understood. This study finds that mitochondrial stress in neurons activates the transcription factor ATF4 as part of the endoplasmic reticulum unfolded protein response (UPR) in Drosophila. ATF4 activation reprograms nuclear gene expression and contributes to neuronal dysfunction. Mitochondrial stress causes an ATF4-dependent increase in the level of the metabolite L-2-hydroxyglutarate (L-2-HG) in the Drosophila brain. Reducing L-2-HG levels directly, by overexpressing L-2-HG dehydrogenase, improves neurological function. Modulation of L-2-HG levels by mitochondrial stress signaling therefore regulates neuronal function (Hunt, 2019).

Drosophila SLC22 Orthologs Related to OATs, OCTs, and OCTNs Regulate Development and Responsiveness to Oxidative Stress

The SLC22 family of transporters play a major role in regulating homeostasis by transporting small organic molecules such as metabolites, signaling molecules, and antioxidants. Evolutionary analysis of Drosophila melanogaster putative SLC22 orthologs reveals that, while many of the 25 SLC22 fruit fly orthologs do not fall within previously established SLC22 subclades, at least four members appear orthologous to mammalian SLC22 members (SLC22A16:CG6356, SLC22A15:CG7458, CG7442 and SLC22A18:CG3168). This study functionally evaluated the role of SLC22 transporters in Drosophila melanogaster by knocking down 14 of these genes. Three putative SLC22 ortholog knockdowns-CG3168, CG6356, and CG7442/SLC22A-did not undergo eclosion and were lethal at the pupa stage, indicating the developmental importance of these genes. Additionally, knocking down four SLC22 members increased resistance to oxidative stress via paraquat testing. Consistent with recent evidence that SLC22 is central to a Remote Sensing and Signaling Network (RSSN) involved in signaling and metabolism, these phenotypes support a key role for SLC22 in handling reactive oxygen species (Engelhart, 2020).

Protein kinase D is dispensable for development and survival of Drosophila melanogaster Members of the Protein Kinase D (PKD) family are involved in numerous cellular processes in mammals, including cell survival after oxidative stress, polarized transport of Golgi vesicles, as well as cell migration and invasion. PKD proteins belong to the PKC/CAMK class of serine/threonine kinases, and transmit diacylglycerol-regulated signals. Whereas three PKD isoforms are known in mammals, Drosophila melanogaster contains a single PKD homologue. Previous analyses using overexpression and RNAi studies indicated likewise multi-facetted roles for Drosophila PKD, including the regulation of secretory transport and actin-cytoskeletal dynamics. This study generated PKD null alleles that are homozygous viable without apparent phenotype. They largely match control flies regarding fertility, developmental timing and weight. Males, but not females, are slightly shorter lived and starvation sensitive. Furthermore, migration of pole cells in embryos and border cells in oocytes appears normal. PKD mutants tolerate heat, cold and osmotic stress like the control but are sensitive to oxidative stress, conforming to the described role for mammalian PKDs. A candidate screen to identify functionally redundant kinases uncovered genetic interactions of PKD with Pkcdelta, sqa and Drak mutants, further supporting the role of PKD in oxidative stress response, and suggesting its involvement in starvation induced autophagy and regulation of cytoskeletal dynamics. Overall, PKD appears dispensable for fly development and survival presumably due to redundancy, but influences environmental responses (Maier, 2019).

Alkaline ceramidase mediates the oxidative stress response in Drosophila melanogaster through Sphingosine

Alkaline ceramidase (Dacer) in Drosophila melanogaster was demonstrated to be resistant to paraquat-induced oxidative stress. However, the underlying mechanism for this resistance remained unclear. This study has shown that sphingosine feeding triggered the accumulation of hydrogen peroxide (H2O2). Dacer-deficient D. melanogaster (Dacer mutant) has higher catalase (CAT) activity and CAT transcription level, leading to higher resistance to oxidative stress induced by paraquat. By performing a quantitative proteomic analysis, this study identified 79 differentially expressed proteins in comparing Dacer mutant to wild type. Three oxidoreductases, including two cytochrome P450 (CG3050, CG9438) and an oxoglutarate/iron-dependent dioxygenase (CG17807), were most significantly upregulated in Dacer mutant. It is presumed that altered antioxidative activity in Dacer mutant might be responsible for increased oxidative stress resistance. This work provides a novel insight into the oxidative antistress response in D. melanogaster (Zhang, 2019).

Modification by sialylated glycans can affect protein functions, underlying mechanisms that control animal development and physiology. Sialylation relies on a dedicated pathway involving evolutionarily conserved enzymes, including CMP-sialic acid synthetase (CSAS) and sialyltransferase (SiaT) that mediate the activation of sialic acid and its transfer onto glycan termini, respectively. In Drosophila, CSAS and DSiaT genes function in the nervous system, affecting neural transmission and excitability. These genes were found to function in different cells: the function of CSAS is restricted to glia, while DSiaT functions in neurons. This partition of the sialylation pathway allows for regulation of neural functions via a glia-mediated control of neural sialylation. The sialylation genes were shown to be required for tolerance to heat and oxidative stress and for maintenance of the normal level of voltage-gated sodium channels. The results uncovered a unique bipartite sialylation pathway that mediates glia-neuron coupling and regulates neural excitability and stress tolerance (Scott, 2023).

Protein glycosylation, the most common type of posttranslational modification, plays numerous important biological roles, and regulates molecular and cell interactions in animal development, physiology, and disease. The addition of sialic acid (Sia), i.e., sialylation, has prominent effects due to its negative charge, bulky size, and terminal location of Sia on glycan chains. Essential roles of sialylated glycans in cell adhesion, cell signaling, and proliferation have been documented in many studies. Sia is intimately involved in the function of the nervous system. Mutations in genes that affect sialylation are associated with neurological symptoms in human, including intellectual disability, epilepsy, and ataxia due to defects in sialic acid synthase (N-acetylneuraminic acid synthase [NANS]), sialyltransferases (ST3GAL3 and ST3GAL5), the CMP-Sia transporter (SLC35A1), and CMP-Sia synthase (CMAS). Polysialylation (PSA) of NCAM, the neural cell adhesion molecule, one of the best studied cases of sialylation in the nervous system, is involved in the regulation of cell interactions during brain development. Non-PSA-type sialylated glycans are ubiquitously present in the vertebrate nervous system, but their functions are not well defined. Increasing evidence implicates these glycans in essential regulation of neuronal signaling. Indeed, N-glycosylation can affect voltage-gated channels in different ways, ranging from modulation of channel gating to protein trafficking, cell surface expression, and recycling/degradation. Similar effects were shown for several other glycoproteins implicated in synaptic transmission and cell excitability, including neurotransmitter receptors. Glycoprotein sialylation defects were also implicated in neurological diseases, such as Angelman syndrome and epilepsy. However, the in vivo functions of sialylation and the mechanisms that regulate this posttranslational modification in the nervous system remain poorly understood (Scott, 2023).

Drosophila has recently emerged as a model to study neural sialylation in vivo, providing advantages of the decreased complexity of the nervous system and the sialylation pathway, while also showing conservation of the main biosynthetic steps of glycosylation (Koles, 2009; Scott, 2014). The final step in sialylation is mediated by sialyltransferases, enzymes that use CMP-Sia as a sugar donor to attach Sia to glycoconjugates (see Schematic of the sialylation pathways in vertebrate and Drosophila. Unlike mammals that have 20 different sialyltransferases, Drosophila possesses a single sialyltransferase, DSiaT, that has significant homology to mammalian ST6Gal enzymes. The two penultimate steps in the biosynthetic pathway of sialylation are mediated by sialic acid synthase (also known as NANS) and CMP-sialic acid synthetase (CSAS, also known as CMAS), the enzymes that synthesize sialic acid and carry out its activation, respectively. These enzymes have been characterized in Drosophila and found to be closely related to their mammalian counterparts. In vivo analyses of DSiaT and CSAS demonstrated that Drosophila sialylation is a tightly regulated process limited to the nervous system and required for normal neural transmission. Mutations in DSiaT and CSAS phenocopy each other, resulting in similar defects in neuronal excitability, causing locomotor and heat-induced paralysis phenotypes, while showing strong interactions with voltage-gated channels (Repnikova, 2010; Islam, 2013). DSiaT was found to be expressed exclusively in neurons during development and in the adult brain (Repnikova, 2010). Intriguingly, although the expression of CSAS has not been characterized in detail, it was noted that its expression appears to be different from that of DSiaT in the embryonic ventral ganglion (Koles, 2009), suggesting a possibly unusual relationship between the functions of these genes. This study tested the hypothesis that CSAS functions in glial cells, and that the separation of DSiaT and CSAS functions between neurons and glia underlies a novel mechanism of glia-neuron coupling that regulates neuronal function via a bipartite protein sialylation (Scott, 2023).

In vertebrates, phosphorylated sialic acid is produced by N-acetylneuraminic acid synthase (Neu5Ac-9-P synthase, or NANS) from N-acetyl-mannosamine 6-phosphate (ManNAc-6-P), converted to sialic acid (Scott, 2023).

Glial cells have been recognized as key players in neural regulation. Astrocytes participate in synapse formation and synaptic pruning during development, mediate the recycling of neurotransmitters, affect neurons via Ca2+ signaling, and support a number of other essential evolutionarily conserved functions. Studies of Drosophila glia have revealed novel glial functions in vivo. Drosophila astrocytes were found to modulate dopaminergic function through neuromodulatory signaling and activity-regulated Ca2+ increase. Glial cells were also shown to protect neurons and neuroblasts from oxidative stress and promote the proliferation of neuroblasts in the developing Drosophila brain. The metabolic coupling between astrocytes and neurons, which is thought to support and modulate neuronal functions in mammals, is apparently conserved in flies. Indeed, Drosophila glial cells can secrete lactate and alanine to fuel neuronal oxidative phosphorylation. In the current work, a novel mechanism id described of glia-neuron coupling mediated by a unique compartmentalization of different steps in the sialylation pathway between glial cells and neurons in the fly nervous system. This study explored the regulation of this mechanism and demonstrate its requirement for neural functions (Scott, 2023).

Paternal environmental factors can epigenetically influence gene expressions in offspring. This study demonstrates that restraint stress, an experimental model for strong psychological stress, to fathers affects the epigenome, transcriptome, and metabolome of offspring in a MEKK1-dATF2 pathway-dependent manner in Drosophila melanogaster. Genes involved in amino acid metabolism are upregulated by paternal restraint stress, while genes involved in glycolysis and the tricarboxylic acid (TCA) cycle are downregulated. The effects of paternal restraint stress are also confirmed by metabolome analysis. dATF-2 is highly expressed in testicular germ cells, and restraint stress also induces p38 activation in the testes. Restraint stress induces Unpaired 3 (Upd3), a Drosophila homolog of Interleukin 6 (IL-6). Moreover, paternal overexpression of upd3 in somatic cells disrupts heterochromatin in offspring but not in offspring from dATF-2 mutant fathers. These results indicate that paternal restraint stress affects metabolism in offspring via inheritance of dATF-2-dependent epigenetic changes (Seong, 2020).

This study indicates that paternal restraint stress induces epigenetic status of offsprings via a p38-MEKK1-dATF-2 pathway and restraint stress-induced upd3 might have a role in transmission of the stress information from somatic to germline cells. Notably, it was observed that paternal restraint stress reduces energy metabolism activity in offspring and sensitizes to rotenone toxicity (Seong, 2020).

Restraint stress information, received by the central nervous system (CNS) and/or other sensing tissues, promotes expression of upd3 in peripheral somatic tissues. In germ cells, accumulated humoral upd3 activates the JAK/STAT pathway, which subsequently activates the p38-dATF-2 pathway. Phosphorylated dATF-2 may be released from the promotor regions of its target genes, resulting in decreased H3K9me2 levels. Epigenetic marks may be retained in mature sperm and inherited by offspring. After fertilization, histone marks may act as inheritable epigenetic memory and regulate gene expression. It was observed that genes involved in the one-carbon metabolic pathway were upregulated in offspring from the paternal restraint stress-exposed fathers, while genes involved in the respiratory metabolic pathway were downregulated. It is assumed that paternal restraint stress-induced- and dATF-2-mediated upregulation of the one-carbon cycle induces downregulation of respiratory metabolism due to a trade-off relationship between these two metabolic processes (Seong, 2020).

The stress response, orchestrated by the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system via stress hormones (adrenaline, noradrenaline, and cortisol), plays a key role in physiology and in several diseases including depression. Accumulated evidence has also implicated inflammatory cytokines in depression. A peripheral infection can induce sickness behaviors, including depression-like behavior, which is caused by the immune-to-brain communication via proinflammatory cytokines produced from innate immune cells. Previous meta-analytic reports indicate that depressed patients possess significantly higher concentrations of IL-6. It was also reported that restraint stress activates the HPA axis and induces higher levels of IL-6 in a rodent model. Furthermore, IL-6 knockout mice exhibit resistance to stress-induced depression-like behaviors, suggesting that IL-6 plays a key role in eliciting depression. The present results suggest that upd3, the Drosophila homolog of IL-6, is also induced by restraint stress. Studies on the inflammatory status of the human HMC-1 cell line showed that adrenaline enhances IL-6, IL-8, and IL-13 production, mediated in part by the p38 signaling pathway. The Drosophila model of myeloproliferative neoplasm indicates that p38 pathway contributes to feed-forward loop in JAK pathway by regulating upd3 gene expression. In Drosophila, restraint stress-dependent upd-3 induction in peripheral tissue may also be mediated by the p38 pathway in response to stress hormones such as adrenaline, as occurs in mammalian systems (Seong, 2020).

The present study showed that upd3 has a role in transmitting restraint stress information to germ cells. It was observed that overexpression of upd3 in neuronal somatic tissues affected the heterochromatic status of offspring in a dATF-2 dependent manner. These results indicate that humoral Upd3, secreted from somatic cells, may be able to affect the epigenetic status of germ cells via the dATF-2 pathway. Moreover, p38 can be activated in response to several extracellular factors, such as TNF, IL-1, growth factors, insulin, salt concentration, and reactive oxygen species (ROS), either directly or indirectly. Thus, humoral components can act as key players to regulate the epigenetic status of germline cells. Upd3, a JAK ligand, is expressed in somatic gonads and activates the JAK/STAT pathway in male germ cells suggesting that testicular germ cells are responsive to the restraint stress-induced increase in Upd3. The activation of p38 by JAK signaling has been demonstrated in the Drosophila immune system, as well as in various vertebrate systems59. Somatic tissue-specific overexpression of upd3 clearly alters the heterochromatin status in the next generation. dATF-2 is highly expressed in germ cells in testes, and expression of TotA, a known upd3 signal target gene, is regulated by the MEKK1-dATF-2 pathway. Therefore, restraint stress-induced humoral upd3 in somatic tissues may control the epigenetic status of germ cells via the p38-dATF-2 signaling pathway in Drosophila. Interestingly, restraint stress-dependent activation of p38 in testes was mainly induced during the latter stages of restraint stress treatment for 10 h. A relatively long time may therefore be necessary to accumulate sufficient humoral upd3 for activation of p38 in germ cells (Seong, 2020).

In restraint stress experiments, animals may also receive mechanical stress, and it may be difficult to completely differentiate this from restraint stress. The relationship between restraint stress and mechanical stress is poorly understood. Mechanical stress induces different sets of cytokines in different types of tissues and IL-6 is one of the major components induced by mechanical stress in mammals. In Drosophila, a recent report showed that forceps squeezing of larvae induces upd3 gene expression. IL-6 induction by either restraint stress or mechanical stress may therefore reflect a common mechanism for sensing and communicating stress information between restraint stress in the nervous system and mechanical stress in the peripheral tissues (Seong, 2020).

In Drosophila, the transit time from spermatagonial stem cells to mature sperm available for insemination at 2 °C is ~260 h. During spermatogenesis, transcription mainly occurs in spermatagonial stem cells and spermatocytes. Herein, when fathers were exposed to restraint stress only once, only the first brood, but not successive broods, exhibited paternal restraint stress-induced heterochromatin disruption. However, exposure to restraint stress three times induced heterochromatin disruption not only in the first brood but also in successive broods. These results suggest that single restraint stress exposure induces heterochromatin disruption only in spermatocytes, whereas repeated restraint stress exposure also causes heterochromatin disruption in spermatagonial stem cells. Interestingly, it has been indicated that long-term memory is required for the maintained effect of the maternal transmission of ethanol preference after 24 h of wasp exposure. There is a possibility that the establishment of neuronal memory by restraint stress may be important for the stable transmission of the epigenetic status in germ cells. Further analysis is needed to elucidate the mechanistic aspect of maintenance of epigenetic change induced by restraint stress in germ cells (Seong, 2020).

Increasing evidence indicates that the paternal environment affects the transcriptome and epigenetic status of their offsprings in several animal models. Although the mechanism of paternal inheritance of epigenetic status remains unclear, some evidence suggests that histone modification is an important player. A recent study indicated that histone replacement-completed sperm retain histone H3 mainly on specific promoter regions in mice. In Drosophila, epiallelic status of H3K27me3 can be dominantly transmitted to the progeny. Drosophila paternal Cid/Cenp-A, a centromere histone protein, can be transmitted to progeny embryos. Similarly, human spermatozoa retain and transmit nucleosomes with constitutive heterochromatin containing H3K9me3 to embryos. A previous report showed that dATF-2 is required for the establishment and maintenance of heterochromatin by regulating the H3K9me2 level, and heat shock stress induces dATF-2 phosphorylation by p38 and its release from heterochromatin, and disrupts the heterochromatin status in germ cells. This causes heterochromatin disruption, which is inherited by the next generation. Based on these observations, it is likely that restraint stress disrupts heterochromatin by decreasing H3K9me2 levels in testicular germ cells, and this is transmitted to offspring. Via a similar mechanism, restraint stress may reduce H3K9me2 on dATF-2 target genes, and this may be transmitted to offspring to modulate metabolism. However, it was not possible to analyze dATF-2 target genes in testicular germ cells by ChIP because anti-dATF-2 antibodies worked only on embryo chromatin but not testis chromatin. This could be due to the presence of non-specific proteins that bind to anti-dATF-2 antibodies in testes. To detect epigenetic effect of paternal restraint stress, restraint stress-treated w1118 males were crossed with wm4 females. The level of histone H3K9me2 was observed to be slightly but significantly decreased in the w gene locus of wm4 offsprings from restraint stress-treated fathers. Although the X chromosome harboring the wm4 gene in the male offspring was derived from the unstressed female, paternal restraint stress increased w expression in male offspring, which is reminiscent of paramutation. It has been reported that paternal stress can influence epigenetic status on maternally supplied X chromosome using the wm4 strain. A previous report also indicated that quantitative content of heterochromatin on the Y chromosome affects PEV phenotypes of wm4 flies. Physical pairing between the heterochromatic regions of X chromosomes and other chromosomes, or the trans-action of some molecules, such as noncoding RNA, may induce partial disruption of the heterochromatin on the X chromosome. Small noncoding RNAs have been also reported as a factor of transgenerational transmission of paternal phenotypes in several model animals. Noncoding RNAs in sperm may also influence the transgenerational effect by paternal restraint stress. Further analysis is required to elucidate the detailed mechanism by which restraint stress affects metabolism in offspring via dATF-2 in testicular germ cells (Seong, 2020).

Consistent with a previous report on mice, the present study indicates that paternal restraint stress affects metabolism in offspring, albeit differently, possibly due to differences among species and experimental conditions. In the present study, paternal restraint stress enhanced the expression of genes involved in one-carbon metabolism, while paternal restraint stress decreased the expression of genes involved in glycolysis and the TCA cycle in the previous work on mice. Restraint stress disrupts heterochromatin possibly by decreasing the H3K9me2 level, which is thought to directly enhance the transcription of dATF-2 target genes. Therefore, downregulation of genes by paternal restraint stress, such as glycolysis-related genes, may occur indirectly via upregulation of one-carbon metabolism-associated genes. The phosphoglycerate dehydrogenase (PHGDH) gene is important in balancing glycolysis and one-carbon metabolism, and its amplification in some cancer cells causes the diversion of a relatively large amount of glycolytic carbon into serine and glycine metabolism. The upregulation of one-carbon metabolism genes, including PHGDH, enhances the levels of glutathione and taurine, which are major factors for reducing ROS during detoxification. ROS stabilizes the transcription factor hypoxia-inducible factor-1α (HIF-1α), which activates genes related to glycolysis. Therefore, one possibility is that indirect downregulation of glycolysis genes may be due to decreased activity of HIF-1 following the lowering of ROS levels by paternal restraint stress (Seong, 2020).

Multiple studies have shown that psychological stress affects metabolism, and metabolism conversely modulates the response to psychological stress. The incidence of both depression and diabetes increases with age, and inflammatory cytokines play a key role in eliciting both diseases. Growing evidence indicates that depression and type 2 diabetes share biological origins, particularly overactivation of a cytokine-mediated inflammatory response, potentially through dysregulation of the HPA axis. Thus, psychological stress and metabolism are tightly connected, and this study indicates that psychological stress modulates metabolism in offspring through intergenerational inheritance via sperm. Some previous evidence showed that diet composition may attenuate stress-induced symptoms, and the present results, including precise gene expression changes induced by paternal restraint stress, may contribute to the development of useful foods or supplements with therapeutic benefits (Seong, 2020).

Cold stress is a critical environmental challenge that affects an organism's fitness-related traits. In Drosophila, increased resistance to specific environmental stress may lead to increased resistance to other kinds of stress. The present study aimed to understand whether increased cold stress resistance in Drosophila melanogaster can facilitate their ability to tolerate other environmental stresses. For this study, successfully selected replicate populations of D. melanogaster against cold shock and their control population were used. The present work investigated egg viability and mating frequency with and without heat and cold shock conditions in the selected and their control populations. Resistance to cold shock, heat shock, desiccation, starvation, and survival post-challenge with Staphylococcus succinus subsp. succinus PK-1 were also examined in the selected and their control populations. After cold-shock treatment, it was found a 1.25 times increase in egg viability and a 1.57 times increase in mating frequency in the selected populations compared to control populations. Moreover, more males (0.87 times) and females (1.66 times) of the selected populations survived under cold shock conditions relative to their controls. After being subjected to heat shock, the selected population's egg viability and mating frequency increased by 0.30 times and 0.57 times, respectively, compared to control populations. Additionally, more selected males (0.31 times) and females (0.98 times) survived under heat shock conditions compared to the control populations. Desiccation resistance slightly increased in the females of the selected populations relative to their control, but no change was observed in the case of males. Starvation resistance decreased in males and females of the selected populations compared to their controls. These findings suggest that the increased resistance to cold shock correlates with increased tolerance to heat stress, but this evolved resistance comes at a cost, with decreased tolerance to starvation (Singh, 2022).

Responses to Developmental Temperature Fluctuation in Life History Traits of Five Drosophila Species (Diptera: Drosophilidae) from Different Thermal Niches

Temperature has profound effects on biochemical processes as suggested by the extensive variation in performance of organisms across temperatures. Nonetheless, the use of fluctuating temperature (FT) regimes in laboratory experiments compared to constant temperature (CT) regimes is still mainly applied in studies of model organisms. This study investigated how two amplitudes of developmental temperature fluctuation (22.5/27.5 °C and 20/30 °C, 12/12 h) affected several fitness-related traits in five Drosophila species with markedly different thermal resistance. Egg-to-adult viability did not change much with temperature except in the cold-adapted D. immigrans. Developmental time increased with FT among all species compared to the same mean CT. The impact of FT on wing size was quite diverse among species. Whereas wing size decreased quasi-linearly with CT in all species, there were large qualitative differences with FT. Changes in wing aspect ratio due to FT were large compared to the other traits and presumably a consequence of thermal stress. These results demonstrate that species of the same genus but with different thermal resistance can show substantial differences in responses to fluctuating developmental temperatures not predictable by constant developmental temperatures. Testing multiple traits facilitated the interpretation of responses to FT in a broader context (Manenti, 2021).

The importance of pre- and postcopulatory sexual selection promoting adaptation to increasing temperatures

Global temperatures are increasing rapidly affecting species globally. Understanding if and how different species can adapt fast enough to keep up with increasing temperatures is of vital importance. One mechanism that can accelerate adaptation and promote evolutionary rescue is sexual selection. Two different mechanisms by which sexual selection can facilitate adaptation are pre- and postcopulatory sexual selection. However, the relative effects of these different forms of sexual selection in promoting adaptation are unknown. This study presents the results from an experimental study in which we exposed fruit flies Drosophila melanogaster to either no mate choice or 1 of 2 different sexual selection regimes (pre- and postcopulatory sexual selection) for 6 generations, under different thermal regimes. Populations showed evidence of thermal adaptation under precopulatory sexual selection, but this effect was not detected in the postcopulatory sexual selection and the no choice mating regime. This study further demonstrates that sexual dimorphism decreased when flies evolved under increasing temperatures, consistent with recent theory predicting more sexually concordant selection under environmental stress. These results suggest an important role for precopulatory sexual selection in promoting thermal adaptation and evolutionary rescue (Gomez-Llano, 2021).

Interactions between developmental and adult acclimation have distinct consequences for heat tolerance and heat stress recovery

Developmental and adult thermal acclimation can have distinct, even opposite, effects on adult heat resistance in ectotherms. Yet, their relative contribution to heat-hardiness of ectotherms remains unclear despite the broad ecological implications thereof. Furthermore, the deterministic relationship between heat knockdown and recovery from heat stress is poorly understood but significant for establishing causal links between climate variability and population dynamics. Using Drosophila melanogaster in a full-factorial experimental design, this study assessed the heat tolerance of flies in static stress assays and documented how developmental and adult acclimation interact with a distinct pattern to promote survival to heat stress in adults. Warmer adult acclimation was shown to be the initial factor enhancing survival to constant stressful high temperatures in flies, but also that the interaction between adult and developmental acclimation becomes gradually more important to ensure survival as the stress persists. This provides an important framework revealing the dynamic interplay between these two forms of acclimation that ultimately enhance thermal tolerance as a function of stress duration. Furthermore, by investigating recovery rates post-stress, it was also shown that the process of heat-hardening and recovery post-heat knockdown are likely to be based on set of (at least partially) divergent mechanisms. This could bear ecological significance as a trade-off may exist between increasing thermal tolerance and maximizing recovery rates post-stress, constraining population responses when exposed to variable and stressful climatic conditions (Willot, 2021).

Phenotypic Responses to and Genetic Architecture of Sterility Following Exposure to Sub-Lethal Temperature During Development Thermal tolerance range, based on temperatures that result in incapacitating effects, influences species' distributions and has been used to predict species' response to increasing temperature. Reproductive performance may also be negatively affected at less extreme temperatures, but such sublethal heat-induced sterility has been relatively ignored in studies addressing the potential effects of, and ability of species' to respond to, predicted climate warming. The few studies examining the link between increased temperature and reproductive performance typically focus on adults, although effects can vary between life history stages. This study assessed how sublethal heat stress during development impacted subsequent adult fertility and its plasticity, both of which can provide the raw material for evolutionary responses to increased temperature. Phenotypic and genetic variation was quantified in fertility of Drosophila melanogaster reared at standardized densities in three temperatures (25, 27, and 29°C) from a set of lines of the Drosophila Genetic Reference Panel (DGRP). Little phenotypic variation was found at the two lower temperatures with more variation at the highest temperature and for plasticity. Males were more affected than females. Despite reasonably large broad-sense heritabilities, a genome-wide association study found little evidence for additive genetic variance and no genetic variants were robustly linked with reproductive performance at specific temperatures or for phenotypic plasticity. Results on heat-induced male sterility with other DGRP results on relevant fitness traits were measured after abiotic stress, and an association was found between male susceptibility to sterility and male lifespan reduction following oxidative stress. The results suggest that sublethal stress during development has profound negative consequences on male adult reproduction, but despite phenotypic variation in a population for this response, there is limited evolutionary potential, either through adaptation to a specific developmental temperature or plasticity in response to developmental heat-induced sterility (Zwoinska, 2020).

ALecheta, M. C., Awde, D. N., O'Leary, T. S., Unfried, L. N., Jacobs, N. A., Whitlock, M. H., McCabe, E., Powers, B., Bora, K., Waters, J. S., Axen, H. J., Frietze, S., Lockwood, B. L., Teets, N. M. and Cahan, S. H. (2020). Integrating GWAS and Transcriptomics to Identify the Molecular Underpinnings of Thermal Stress Responses in Drosophila melanogaster. Front Genet 11: 658. PubMed ID: 32655626

Integrating GWAS and Transcriptomics to Identify the Molecular Underpinnings of Thermal Stress Responses in Drosophila melanogaster

Thermal tolerance depends on both the ability to dynamically adjust to a thermal stress and preparatory developmental processes that enhance thermal resistance. This study used a combination of Genome Wide Association mapping (GWAS) and transcriptomic profiling to characterize whether genes associated with thermal tolerance are primarily involved in dynamic stress responses or preparatory processes that influence physiological condition at the time of thermal stress. To test the hypotheses, the critical thermal minimum (CT(min)) and critical thermal maximum (CT(max)) were measured of 100 lines of the Drosophila Genetic Reference Panel (DGRP), and GWAS was used to identify loci that explain variation in thermal limits. Greater variation was observed in lower thermal limits, with CT(min) ranging from 1.81 to 8.60°C, while CT(max) ranged from 38.74 to 40.64°C. 151 and 99 distinct genes associated with CT(min) and CT(max), respectively, were identified, and there was strong support that these genes are involved in both dynamic responses to thermal stress and preparatory processes that increase thermal resistance. Many of the genes identified by GWAS were involved in the direct transcriptional response to thermal stress, and overall GWAS candidates were more likely to be differentially expressed than other genes. Further, several GWAS candidates were regulatory genes that may participate in the regulation of stress responses, and gene ontologies related to development and morphogenesis were enriched, suggesting many of these genes influence thermal tolerance through effects on development and physiological status. Overall, these results suggest that thermal tolerance alleles can influence both dynamic plastic responses to thermal stress and preparatory processes that improve thermal resistance. These results also have utility for directly comparing GWAS and transcriptomic approaches for identifying candidate genes associated with thermal tolerance (ALecheta, 2020).

El-Saadi, M. I., Ritchie, M. W., Davis, H. E. and MacMillan, H. A. (2020). Warm periods in repeated cold stresses protect Drosophila against ionoregulatory collapse, chilling injury, and reproductive deficits. J Insect Physiol 123: 104055. PubMed ID: 32380094

Warm periods in repeated cold stresses protect Drosophila against ionoregulatory collapse, chilling injury, and reproductive deficits

During cold stress, chill susceptible insects like Drosophila melanogaster suffer from a loss of ion and water balance, and the current model of recovery from chilling posits that re-establishment of ion homeostasis begins upon return to a warm environment, but that it takes minutes to hours for an insect to fully restore homeostasis. Following this ionoregulatory model of chill coma recovery, it is predicted that the longer the duration of the warm periods between cold stresses, the better a fly will endure a subsequent chill coma event and the more likely they will be to survive. It was also predicted, however, that this recovery may lead to reduced fecundity, possibly due to allocation of energy reserves away from reproduction. In this study, female D.melanogaster were treated to a long continuous cold stress (25 h at 0 °C), or experienced the same total time in the cold with repeated short (15 min), or long (120 min) breaks at 22 °C. Warm periods in general improved survival outcomes, and individuals that recovered for more time in between cold periods had significantly lower rates of injury, faster recovery from chill coma, and produced greater, rather than fewer, offspring. These improvements in chill tolerance were associated with mitigation of ionoregulatory collapse, as flies that experienced either short or long warm periods better maintained low hemolymph [K(+)]. Thus, warm periods that interrupt cold periods improve cold tolerance and fertility in D. melanogaster females relative to a single sustained cold stress, potentially because this time allows for recovery of ion and water homeostasis (El-Saadi, 2020).

Simoes, P., Santos, M. A., Carromeu-Santos, A., Quina, A. S., Santos, M. and Matos, M. (2020). Beneficial developmental acclimation in reproductive performance under cold but not heat stress. J Therm Biol 90: 102580. PubMed ID: 32479384

Beneficial developmental acclimation in reproductive performance under cold but not heat stress Thermal plasticity can help organisms coping with climate change. This study analysed how laboratory populations of the ectotherm species Drosophila subobscura, originally from two distinct latitudes and evolving for several generations in a stable thermal environment (18°C), respond plastically to new thermal challenges. Adult performance (fecundity traits as a fitness proxy) was measured of the experimental populations when exposed to five thermal regimes, three with the same temperature during development and adulthood (15-15°C, 18-18°C, 25-25°C), and two where flies developed at 18°C and were exposed, during adulthood, to either 15°C or 25°C. This study testd whether (1) flies undergo stress at the two more extreme temperatures; (2) development at a given temperature enhances adult performance at such temperature (i.e. acclimation), and (3) populations with different biogeographical history show plasticity differences. The findings show (1) an optimal performance at 18°C only if flies were subjected to the same temperature as juveniles and adults; (2) the occurrence of developmental acclimation at lower temperatures; (3) detrimental effects of higher developmental temperature on adult performance; and (4) a minor impact of historical background on thermal response. This study indicates that thermal plasticity during development may have a limited role in helping adults cope with warmer - though not colder - temperatures, with a potential negative impact on population persistence under climate change. It also emphasizes the importance of analysing the impact of temperature on all stages of the life cycle to better characterize the thermal limits (Simoes, 2020).

Jorgensen, L. B., Robertson, R. M. and Overgaard, J. (2020). Neural dysfunction correlates with heat coma and CT(max) in Drosophila but does not set the boundaries for heat stress survival. J Exp Biol. PubMed ID: 32434804

Neural dysfunction correlates with heat coma and CT(max) in Drosophila but does not set the boundaries for heat stress survival

When heated, insects lose coordinated movement followed by the onset of heat coma (CT(max)). This study examined the function of the central nervous system (CNS) in five species of Drosophila with different heat tolerances, while they were exposed to either constant high temperature or a gradual increasing temperature (ramp). Tolerant species were able to preserve CNS function at higher temperatures and for longer durations than sensitive species and similar differences were found for the behavioural indices (loss of coordination and onset of heat coma). Furthermore, the timing and temperature (constant and ramp exposure, respectively) for loss of coordination or complete coma coincided with the occurrence of spreading depolarisation (SD) events in the CNS. These SD events disrupt neurological function and silence the CNS suggesting that CNS failure is the primary cause of impaired coordination and heat coma. Heat mortality occurs soon after heat coma in insects and to examine if CNS failure could also be the proximal cause of heat death, selective heating of the head (CNS) and abdomen (visceral tissues) was tested. When comparing the temperature causing 50% mortality (LT(50)) of each body part to that of the whole animal, it was found that the head was not particularly heat sensitive compared to the abdomen. Accordingly, it is unlikely that nervous failure is the principal/proximate cause of heat mortality in Drosophila (Jorgensen, 2020).

A biphasic locomotor response to acute unsignaled high temperature exposure in Drosophila

Unsignaled stress can have profound effects on animal behavior. While most investigation of stress-effects on behavior follows chronic exposures, less is understood about acute exposures and potential after-effects. This study examined walking activity in Drosophila following acute exposure to high temperature or electric shock. Compared to initial walking activity, flies first increase walking with exposure to high temperatures then have a strong reduction in activity. These effects are related to the intensity of the high temperature and number of exposures. The reduction in walking activity following high temperature and electric shock exposures survives context changes and lasts at least five hours. Reduction in the function of the biogenic amines octopamine / tyramine and serotonin both strongly blunt the increase in locomotor activity with high temperature exposure. However, neither set of biogenic amines alter the long lasting depression in walking activity after exposure (Ostrowski, 2018).

Acevedo, J. M., Centanin, L., Dekanty, A. and Wappner, P. (2010). Oxygen sensing in Drosophila: multiple isoforms of the prolyl hydroxylase fatiga have different capacity to regulate HIFalpha/Sima. PLoS One 5(8): e12390. PubMed ID: 20811646

Baumgartner, M. E., Dinan, M. P., Langton, P. F., Kucinski, I. and Piddini, E. (2021). Proteotoxic stress is a driver of the loser status and cell competition. Nat Cell Biol 23(2): 136-146. PubMed ID: 33495633

Brown, B., Mitra, S., Roach, F. D., Vasudevan, D. and Ryoo, H. D. (2021). The transcription factor Xrp1 is required for PERK-mediated antioxidant gene induction in Drosophila. Elife 10. PubMed ID: 34605405

Casey, A. K., Gray, H. F., Chimalapati, S., Hernandez, G., Moehlman, A. T., Stewart, N., Fields, H. A., Gulen, B., Servage, K. A., Stefanius, K., Blevins, A., Evers, B. M., Kramer, H. and Orth, K. (2022). Fic-mediated AMPylation tempers the unfolded protein response during physiological stress. Proc Natl Acad Sci U S A 119(32): e2208317119. PubMed ID: 35914137

Centanin, L., et al. (2008). Cell autonomy of HIF effects in Drosophila: tracheal cells sense hypoxia and induce terminal branch sprouting. Dev. Cell 14(4): 547-58. PubMed Citation: 18410730

Chakraborty, A., Sgro, C. M. and Mirth, C. K. (2020). Does local adaptation along a latitudinal cline shape plastic responses to combined thermal and nutritional stress?. Evolution 74(9): 2073-2087. PubMed ID: 33616935

Chakravarti, A., Thirimanne, H. N., Brown, S. and Calvi, B. R. (2022). Drosophila p53 isoforms have overlapping and distinct functions in germline genome integrity and oocyte quality control. Elife 11. PubMed ID: 35023826

de Oliveira Dos Santos, A. M., Duarte, A. E., Costa, A. R., da Silva, A. A., Rohde, C., Silva, D. G., de Amorim, E. M., da Cruz Santos, M. H., Pereira, M. G., Depra, M., de Santana, S. L., da Silva Valente, V. L. and Teixeira, C. S. (2022). Canavalia ensiformis lectin induced oxidative stress mediate both toxicity and genotoxicity in Drosophila melanogaster. Int J Biol Macromol 222(Pt B): 2823-2832. PubMed ID: 36228819

Engelhart, D. C., Azad, P., Ali, S., Granados, J. C., Haddad, G. G. and Nigam, S. K. (2020). Drosophila SLC22 Orthologs Related to OATs, OCTs, and OCTNs Regulate Development and Responsiveness to Oxidative Stress. Int J Mol Sci 21(6). PubMed ID: 32183456

Gomez-Llano, M., Scott, E. and Svensson, E. I. (2021). The importance of pre- and postcopulatory sexual selection promoting adaptation to increasing temperatures. Curr Zool 67(3): 321-327. PubMed ID: 34616924

Hao, H., Song, L. and Zhang, L. (2023). Wolfram syndrome 1 regulates sleep in dopamine receptor neurons by modulating calcium homeostasis. PLoS Genet 19(7): e1010827. PubMed ID: 37399203

Harrison, B. R., Wang, L., Gajda, E., Hoffman, E. V., Chung, B. Y., Pletcher, S. D., Raftery, D. and Promislow, D. E. L. (2020). The metabolome as a link in the genotype-phenotype map for peroxide resistance in the fruit fly, Drosophila melanogaster. BMC Genomics 21(1): 341. PubMed ID: 32366330

Hunt, R. J., Granat, L., McElroy, G. S., Ranganathan, R., Chandel, N. S. and Bateman, J. M. (2019). Mitochondrial stress causes neuronal dysfunction via an ATF4-dependent increase in L-2-hydroxyglutarate. J Cell Biol. PubMed ID: 31645461

Kilwein, M. D., Dao, T. K. and Welte, M. A. (2023). Drosophila embryos allocate lipid droplets to specific lineages to ensure punctual development and redox homeostasis. PLoS Genet 19(8): e1010875. PubMed ID: 37578970

Kang, M. J., Vasudevan, D., Kang, K., Kim, K., Park, J. E., Zhang, N., Zeng, X., Neubert, T. A., Marr, M. T., and Don Ryoo, H. (2016). 4E-BP is a target of the GCN2-ATF4 pathway during Drosophila development and aging. J Cell Biol. PubMed ID: 27979906

Kim, H., Kim, M. and Kim, M. S. (2020). Facilitating fructose-driven metabolism exerts a protective effect on anoxic stress in Drosophila. Insect Mol Biol. PubMed ID: 32920918

Lee, S., Jeon, Y. M., Jo, M. and Kim, H. J. (2021). Overexpression of SIRT3 Suppresses Oxidative Stress-induced Neurotoxicity and Mitochondrial Dysfunction in Dopaminergic Neuronal Cells. Exp Neurobiol 30(5): 341-355. PubMed ID: 34737239

Li, P., Huang, P., Li, X., Yin, D., Ma, Z., Wang, H. and Song, H. (2018). Tankyrase mediates K63-linked ubiquitination of JNK to confer stress tolerance and influence lifespan in Drosophila. Cell Rep 25(2): 437-448. PubMed ID: 30304683

Lindstrom, R., Lindholm, P., Kallijarvi, J., Palgi, M., Saarma, M. and Heino, T. I. (2016). Exploring the Conserved role of MANF in the unfolded protein response in Drosophila melanogaster. PLoS One 11: e0151550. PubMed ID: 26975047

Maier, D., Nagel, A. C., Kelp, A. and Preiss, A. (2019). Protein kinase D is dispensable for development and survival of Drosophila melanogaster. G3 (Bethesda). PubMed ID: 31142547

Malzer, E., Dominicus, C. S., Chambers, J. E., Dickens, J. A., Mookerjee, S. and Marciniak, S. J. (2018). The integrated stress response regulates BMP signalling through effects on translation. BMC Biol 16(1): 34. PubMed ID: 29609607

Manenti, T., Kjærsgaard, A., Schou, T. M., Pertoldi, C., Moghadam, N. N. and Loeschcke, V. (2021). Responses to Developmental Temperature Fluctuation in Life History Traits of Five Drosophila Species (Diptera: Drosophilidae) from Different Thermal Niches. Insects 12(10). PubMed ID: 34680694

Milton, V. J. and Sweeney, S. T. (2012). Oxidative stress in synapse development and function. Dev Neurobiol 72(1): 100-110. PubMed ID: 21793225

Mortimer, N. T. and Moberg, K. H. (2009). Regulation of Drosophila embryonic tracheogenesis by dVHL and hypoxia. Dev. Biol. 329(2): 294-305. PubMed Citation: 19285057

Nukala, K. M., Lilienthal, A. J., Lye, S. H., Bassuk, A. G., Chtarbanova, S. and Manak, J. R. (2023). Downregulation of oxidative stress-mediated glial innate immune response suppresses seizures in a fly epilepsy model. Cell Rep 42(1): 112004. PubMed ID: 36641750

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