Visual impairment cell non-autonomously dysregulates brain-wide proteostasis.

Loss of hearing or vision has been identified as a significant risk factor for dementia but underlying molecular mechanisms are unknown. In different Drosophila models of blindness, we observe non-autonomous induction of stress granules in the brain and their reversal upon restoration of vision. Stress granules include cytosolic condensates of p62, ATF4 and XRP1. This cytosolic restraint of the ATF4 and XRP1 transcription factors dampens expression of their downstream targets during cellular stress. Cytosolic condensates of p62 and ATF4 were also evident in the thalamus and hippocampus of mouse models of congenital or degenerative blindness. These data indicate conservation of the link between loss of sensory input and dysregulation of stress responses critical for protein quality control in the brain.

Allnighter pseudokinase-mediated feedback links proteostasis and sleep in Drosophila.

In nervous systems, retrograde signals are key for organizing circuit activity and maintaining neuronal homeostasis. We identify the conserved Allnighter (Aln) pseudokinase as a cell non-autonomous regulator of proteostasis responses necessary for normal sleep and structural plasticity of Drosophila photoreceptors. In aln mutants exposed to extended ambient light, proteostasis is dysregulated and photoreceptors develop striking, but reversible, dysmorphology. The aln gene is widely expressed in different neurons, but not photoreceptors. However, secreted Aln protein is retrogradely endocytosed by photoreceptors. Inhibition of photoreceptor synaptic release reduces Aln levels in lamina neurons, consistent with secreted Aln acting in a feedback loop. In addition, aln mutants exhibit reduced night time sleep, providing a molecular link between dysregulated proteostasis and sleep, two characteristics of ageing and neurodegenerative diseases.

In situ Dephosphorylation Assay with Recombinant Nil Phosphatase.

The activity of numerous autophagy-related proteins depends on their phosphorylation status, which places importance on understanding the responsible kinases and phosphatases. Great progress has been made in identifying kinases regulating autophagy, but much less is known about the phosphatases counteracting their function. Genetic screens and modern proteomic approaches provide powerful tools to identify candidate phosphatases, but further experiments are required to assign direct roles for candidates. We have devised a novel protocol to test the role of purified phosphatases in dephosphorylating specific targets in situ . This approach has the potential to visualize context-specific differences in target dephosphorylation that are not easily detected by lysate-based approaches such as Western blots. Graphical abstract.

Fic-mediated AMPylation tempers the unfolded protein response during physiological stress.

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, we 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, we generated a conditional null allele of Fic in mice and characterized the effect of Fic loss on the exocrine pancreas. 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, we propose that repeated physiological stress in differentiated tissues requires this rheostat for tissue resilience and continued function over the lifetime of an animal.

STING controls energy stress-induced autophagy and energy metabolism via STX17.

The stimulator of interferon genes (STING) plays a critical role in innate immunity. Emerging evidence suggests that STING is important for DNA or cGAMP-induced non-canonical autophagy, which is independent of a large part of canonical autophagy machineries. Here, we report that, in the absence of STING, energy stress-induced autophagy is upregulated rather than downregulated. Depletion of STING in Drosophila fat cells enhances basal- and starvation-induced autophagic flux. During acute exercise, STING knockout mice show increased autophagy flux, exercise endurance, and altered glucose metabolism. Mechanistically, these observations could be explained by the STING-STX17 interaction. STING physically interacts with STX17, a SNARE that is essential for autophagosome biogenesis and autophagosome-lysosome fusion. Energy crisis and TBK1-mediated phosphorylation both disrupt the STING-STX17 interaction, allow different pools of STX17 to translocate to phagophores and mature autophagosomes, and promote autophagic flux. Taken together, we demonstrate a heretofore unexpected function of STING in energy stress-induced autophagy through spatial regulation of autophagic SNARE STX17.

A phosphoswitch at acinus-serine437 controls autophagic responses to cadmium exposure and neurodegenerative stress.

Neuronal health depends on quality control functions of autophagy, but mechanisms regulating neuronal autophagy are poorly understood. Previously, we showed that in Drosophila starvation-independent quality control autophagy is regulated by acinus (acn) and the Cdk5-dependent phosphorylation of its serine437 (Nandi et al., 2017). Here, we identify the phosphatase that counterbalances this activity and provides for the dynamic nature of acinus-serine437 (acn-S437) phosphorylation. A genetic screen identified six phosphatases that genetically interacted with an acn gain-of-function model. Among these, loss of function of only one, the PPM-type phosphatase Nil (CG6036), enhanced pS437-acn levels. Cdk5-dependent phosphorylation of acn-S437 in nil1 animals elevates neuronal autophagy and reduces the accumulation of polyQ proteins in a Drosophila Huntington's disease model. Consistent with previous findings that Cd2+ inhibits PPM-type phosphatases, Cd2+ exposure elevated acn-S437 phosphorylation which was necessary for increased neuronal autophagy and protection against Cd2+-induced cytotoxicity. Together, our data establish the acn-S437 phosphoswitch as critical integrator of multiple stress signals regulating neuronal autophagy.

Hypersensitivity of Vps33B mutant flies to non-pathogenic infections is dictated by aberrant activation of p38b MAP kinase.

Loss of the arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome-linked Vps33B protein results in exaggerated inflammatory responses upon activation of receptors of the innate immune system in both vertebrates and flies. However, little is known about the signaling elements downstream of these receptors that are critical for the hypersensitivity of Vps33B mutants. Here, we show that p38b MAP kinase contributes to the enhanced inflammatory responses in flies lacking Vps33B. Loss of p38b mitogen-activated protein kinase (MAPK) reduces enhanced inflammatory responses and prolongs the survival of infected Vps33B deficient flies. The function of p38 MAPK is not limited to its proinflammatory effects downstream of the PGRP-LC receptor as p38 also modulates endosomal trafficking of PGRP-LC and phagocytosis of bacteria. Expression of constitutively active p38b MAPK, but not dominant negative p38b MAPK enhances accumulation of endocytosed PGRP-LC receptors or phagocytosed bacteria within cells. Moreover, p38 MAPK is required for induction of macropinocytosis, an alternate pathway for the downregulation of immune receptors. Together, our data indicate that p38 MAPK activates multiple pathways that can contribute to the dysregulation of innate immune signaling in ARC syndrome.

Yorkie Growth-Promoting Activity Is Limited by Atg1-Mediated Phosphorylation.

The expression of multiple growth-promoting genes is coordinated by the transcriptional co-activator Yorkie with its major regulatory input provided by the Hippo-Warts kinase cascade. Here, we identify Atg1/ULK1-mediated phosphorylation of Yorkie as an additional inhibitory input independent of the Hippo-Warts pathway. Two serine residues in Yorkie, S74 and S97, are Atg1/ULK1 consensus target sites and are phosphorylated by ULK1 in vitro, thereby preventing its binding to Scalloped. In vivo, gain of function of Atg1, or its activator Acinus, caused elevated Yorkie phosphorylation and inhibited Yorkie's growth-promoting activity. Loss of function of Atg1 or Acinus raised expression of Yorkie target genes and increased tissue size. Unlike Atg1's role in autophagy, Atg1-mediated phosphorylation of Yorkie does not require Atg13. Atg1 is activated by starvation and other cellular stressors and therefore can impose temporary stress-induced constraints on the growth-promoting gene networks under the control of Hippo-Yorkie signaling.

Autophagy Keeps the Balance in Tissue Homeostasis.

Gut cells are exposed to diverse insults that necessitate their replacement from a stem cell pool balancing differentiation and proliferation. In this issue of Developmental Cell, Zhang and colleagues (2019) show autophagy-mediated regulation of EGFR signaling cell autonomously controls intestinal stem cell proliferation, with implications for human colorectal cancer development.

Drosophila p53 directs nonapoptotic programs in postmitotic tissue.

TP53 is the most frequently mutated gene in human cancers, and despite intensive research efforts, genome-scale studies of p53 function in whole animal models are rare. The need for such in vivo studies is underscored by recent challenges to established paradigms, indicating that unappreciated p53 functions contribute to cancer prevention. Here we leveraged the Drosophila system to interrogate p53 function in a postmitotic context. In the developing embryo, p53 robustly activates important apoptotic genes in response to radiation-induced DNA damage. We recently showed that a p53 enhancer (p53RErpr) near the cell death gene reaper forms chromatin contacts and enables p53 target activation across long genomic distances. Interestingly, we found that this canonical p53 apoptotic program fails to activate in adult heads. Moreover, this failure to exhibit apoptotic responses was not associated with altered chromatin contacts. Instead, we determined that p53 does not occupy the p53RErpr enhancer in this postmitotic tissue as it does in embryos. Through comparative RNA-seq and chromatin immunoprecipitation-seq studies of developing and postmitotic tissues, we further determined that p53 regulates distinct transcriptional programs in adult heads, including DNA repair, metabolism, and proteolysis genes. Strikingly, in the postmitotic context, p53-binding landscapes were poorly correlated with nearby transcriptional effects, raising the possibility that p53 enhancers could be generally acting through long distances.

The glial sodium-potassium-2-chloride cotransporter is required for synaptic transmission in the Drosophila visual system.

The Drosophila Ncc69 gene encodes a Na+-K+-2Cl--cotransporter (NKCC) that is critical for regulating intra- and extracellular ionic conditions in different tissues. Here, we show that the Ncc69 transporter is necessary for fly vision and that its expression is required non-autonomously in glia to maintain visual synaptic transmission. Flies mutant for Ncc69 exhibit normal photoreceptor depolarization in response to a light pulse but lack the ON and OFF-transients characteristic of postsynaptic responses of lamina neurons, indicating a failure in synaptic transmission. We also find that synaptic transmission requires the Ncc69 regulatory kinases WNK and Fray in glia. The ERG phenotype is associated with a defect in the recycling of the histamine neurotransmitter. Ncc69 mutants exhibit higher levels of the transport metabolite carcinine in lamina cartridges, with its accumulation most intense in the extracellular space. Our work reveals a novel role of glial NKCC transporters in synaptic transmission, possibly through regulating extracellular ionic conditions.

Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration.

In response to environmental, developmental, and pathological stressors, cells engage homeostatic pathways to maintain their function. Among these pathways, the Unfolded Protein Response protects cells from the accumulation of misfolded proteins in the ER. Depending on ER stress levels, the ER-resident Fic protein catalyzes AMPylation or de-AMPylation of BiP, the major ER chaperone and regulator of the Unfolded Protein Response. This work elucidates the importance of the reversible AMPylation of BiP in maintaining the Drosophila visual system in response to stress. After 72 hr of constant light, photoreceptors of fic-null and AMPylation-resistant BiPT366A mutants, but not wild-type flies, display loss of synaptic function, disintegration of rhabdomeres, and excessive activation of ER stress reporters. Strikingly, this phenotype is reversible: photoreceptors regain their structure and function within 72 hr once returned to a standard light:dark cycle. These findings show that Fic-mediated AMPylation of BiP is required for neurons to adapt to transient stress demands.

Cdk5-mediated Acn/Acinus phosphorylation regulates basal autophagy independently of metabolic stress.

In neurons, autophagy counteracts consequences of aging. It is therefore of interest how basal rates of macroautophagy/autophagy can be controlled independently of metabolic stress. We recently investigated the regulation of basal, starvation-independent autophagy by Acn/Acinus, a multifunctional nuclear protein with proposed roles in apoptosis, alternative RNA splicing, and basal autophagy. We found that Acn is stabilized by phosphorylation of the conserved serine 437. The phosphomimetic AcnS437D mutation causes no overt developmental phenotypes, but significantly elevates levels of basal autophagy and extends life spans. An RNAi screen identified Cdk5 as a kinase targeting S437, a role confirmed by gain- and loss-of-function mutants of Cdk5 or its obligatory cofactor Cdk5r1/p35. Flies lacking Cdk5 function display reduced basal autophagy and a shortened life span. Both of these phenotypes are suppressed by the phosphomimetic AcnS437D mutation, indicating that phosphorylating serine 437 of Acn, and thereby maintaining basal levels of autophagy, is critical for Cdk5's function in maintaining neuronal health.

Intracellular Chloride and Scaffold Protein Mo25 Cooperatively Regulate Transepithelial Ion Transport through WNK Signaling in the Malpighian Tubule.

Background With No Lysine kinase (WNK) signaling regulates mammalian renal epithelial ion transport to maintain electrolyte and BP homeostasis. Our previous studies showed a conserved role for WNK in the regulation of transepithelial ion transport in the Drosophila Malpighian tubule.Methods Using in vitro assays and transgenic Drosophila lines, we examined two potential WNK regulators, chloride ion and the scaffold protein mouse protein 25 (Mo25), in the stimulation of transepithelial ion flux.ResultsIn vitro, autophosphorylation of purified Drosophila WNK decreased as chloride concentration increased. In conditions in which tubule intracellular chloride concentration decreased from 30 to 15 mM as measured using a transgenic sensor, Drosophila WNK activity acutely increased. Drosophila WNK activity in tubules also increased or decreased when bath potassium concentration decreased or increased, respectively. However, a mutation that reduces chloride sensitivity of Drosophila WNK failed to alter transepithelial ion transport in 30 mM chloride. We, therefore, examined a role for Mo25. In in vitro kinase assays, Drosophila Mo25 enhanced the activity of the Drosophila WNK downstream kinase Fray, the fly homolog of mammalian Ste20-related proline/alanine-rich kinase (SPAK), and oxidative stress-responsive 1 protein (OSR1). Knockdown of Drosophila Mo25 in the Malpighian tubule decreased transepithelial ion flux under stimulated but not basal conditions. Finally, whereas overexpression of wild-type Drosophila WNK, with or without Drosophila Mo25, did not affect transepithelial ion transport, Drosophila Mo25 overexpressed with chloride-insensitive Drosophila WNK increased ion flux.Conclusions Cooperative interactions between chloride and Mo25 regulate WNK signaling in a transporting renal epithelium.

Stress-induced Cdk5 activity enhances cytoprotective basal autophagy in Drosophila melanogaster by phosphorylating acinus at serine437.

Cdk5 is a post-mitotic kinase with complex roles in maintaining neuronal health. The various mechanisms by which Cdk5 inhibits and promotes neurodegeneration are still poorly understood. Here, we show that in Drosophila melanogaster Cdk5 regulates basal autophagy, a key mechanism suppressing neurodegeneration. In a targeted screen, Cdk5 genetically interacted with Acinus (Acn), a primarily nuclear protein, which promotes starvation-independent, basal autophagy. Loss of Cdk5, or its required cofactor p35, reduces S437-Acn phosphorylation, whereas Cdk5 gain-of-function increases pS437-Acn levels. The phospho-mimetic S437D mutation stabilizes Acn and promotes basal autophagy. In p35 mutants, basal autophagy and lifespan are reduced, but restored to near wild-type levels in the presence of stabilized AcnS437D. Expression of aggregation-prone polyQ-containing proteins or the Amyloid-ß42 peptide, but not alpha-Synuclein, enhances Cdk5-dependent phosphorylation of S437-Acn. Our data indicate that Cdk5 is required to maintain the protective role of basal autophagy in the initial responses to a subset of neurodegenerative challenges.

Fic-mediated deAMPylation is not dependent on homodimerization and rescues toxic AMPylation in flies.

Protein chaperones play a critical role in proteostasis. The activity of the major endoplasmic reticulum chaperone BiP (GRP78) is regulated by Fic-mediated AMPylation during resting states. By contrast, during times of stress, BiP is deAMPylated. Here, we show that excessive AMPylation by a constitutively active FicE247G mutant is lethal in Drosophila This lethality is cell-autonomous, as directed expression of the mutant FicE247G to the fly eye does not kill the fly but rather results in a rough and reduced eye. Lethality and eye phenotypes are rescued by the deAMPylation activity of wild-type Fic. Consistent with Fic acting as a deAMPylation enzyme, its activity was both time- and concentration-dependent. Furthermore, Fic deAMPylation activity was sufficient to suppress the AMPylation activity mediated by the constitutively active FicE247G mutant in Drosophila S2 lysates. Further, we show that the dual enzymatic activity of Fic is, in part, regulated by Fic dimerization, as loss of this dimerization increases AMPylation and reduces deAMPylation of BiP.

Isolation and Infection of Drosophila Primary Hemocytes.

Phagocytosis of invading pathogens and their subsequent clearance in lysosomes is important for organismal fitness. We have devised the following protocol to extract phagocytic hemocytes from wild-type and mutant Drosophila larvae and infect the isolated hemocytes with GFP-labeled E. coli to measure the rate of phagocytosis and degradation within individual hemocytes over time.

Escherichia coli Infection of Drosophila.

Following septic insults, healthy insects, just like vertebrates, mount a complex immune response to contain and destroy pathogens. The failure to efficiently clear bacterial infections in immuno-compromised fly mutants leads to higher mortality rates which provide a powerful indicator for genes with important roles in innate immunity. The following protocol is designed to reproducibly inject a known amount of non-pathogenic E. coli into otherwise sterile flies and to measure the survival of flies after infection. The protocol can be easily adapted to different types of bacteria.

ARC Syndrome-Linked Vps33B Protein Is Required for Inflammatory Endosomal Maturation and Signal Termination.

Toll-like receptors (TLRs) and other pattern-recognition receptors (PRRs) sense microbial ligands and initiate signaling to induce inflammatory responses. Although the quality of inflammatory responses is influenced by internalization of TLRs, the role of endosomal maturation in clearing receptors and terminating inflammatory responses is not well understood. Here, we report that Drosophila and mammalian Vps33B proteins play critical roles in the maturation of phagosomes and endosomes following microbial recognition. Vps33B was necessary for clearance of endosomes containing internalized PRRs, failure of which resulted in enhanced signaling and expression of inflammatory mediators. Lack of Vps33B had no effect on trafficking of endosomes containing non-microbial cargo. These findings indicate that Vps33B function is critical for determining the fate of signaling endosomes formed following PRR activation. Exaggerated inflammatory responses dictated by persistence of receptors in aberrant endosomal compartments could therefore contribute to symptoms of ARC syndrome, a disease linked to loss of Vps33B.