InteractiveFly: GeneBrief

refractory to sigma P: Biological Overview | References

Homeostasis of intestinal epithelia is maintained by coordination of the proper rate of regeneration by stem cell division with the rate of cell loss. Regeneration of host epithelia is normally quiescent upon colonization of commensal bacteria; however, the epithelia often develop dysplasia in a context-dependent manner, the cause and underlying mechanism of which remain unclear. This study shows that in Drosophila intestine, autophagy lowers the sensitivity of differentiated enterocytes to reactive oxygen species (ROS) that are produced in response to commensal bacteria. Autophagy deficiency provokes ROS-dependent excessive regeneration and subsequent epithelial dysplasia and barrier dysfunction. Mechanistically, autophagic substrate Ref(2)P/p62, which co-localizes and physically interacts with Dachs, a Hippo signaling regulator, accumulates upon autophagy deficiency and thus inactivates Hippo signaling, resulting in stem cell over-proliferation non-cell autonomously. These findings uncover a mechanism whereby suppression of undesirable regeneration by autophagy maintains long-term homeostasis of intestinal epithelia (Nagai, 2021).

The epithelium of the gastrointestinal tract is continuously in contact with microorganisms, which cause damage to the epithelial cells. To maintain the tissue integrity and function as a front line of host defense, regulated turnover of the epithelial cells that coordinates the extent of damage is essential and is achieved by eliminating the damaged cells and replenishing them via an appropriate rate of division and differentiation of intestinal stem cells (ISCs). This coordination is required for protecting the epithelial integrity not only from potential pathogens but also from commensal bacteria, toward which ISCs should be quiescent. However, the host-microbe interactions are context dependent, and potentially symbiotic microbes often contribute to the pathogenesis of chronic inflammatory bowel diseases (IBDs), or lead to hyperplasia in a host condition-dependent manner, the underlying mechanism of which is poorly understood (Nagai, 2021).

With its genetic amenability, strong conservation of cellular signaling pathways that regulate immune responses, and relatively simple composition of the enteric microbes, the Drosophila intestine has been an ideal model system for investigating intestinal homeostasis. The Drosophila gut immune system consists of physical and chemical barriers, the former of which includes the barrier integrity of intestinal epithelial cells. Two indispensable chemical barriers, the anti-microbial peptides (AMPs), as well as reactive oxygen species (ROS) produced by dual oxidase (DUOX), control the gut microbiota. The host epithelial cells sense bacteria-derived uracil, which results in the activation of DUOX. In both Drosophila and mammals, ROS production toward the gut lumen is crucial for antagonizing infecting pathogens. Although the ROS damage the host epithelial cells as well, the host activates regenerative responses to eliminate the damaged cells and replenish them by upregulating the proliferation of ISCs. In the Drosophila intestine, upon tissue damage by pathogen infection, the Hippo pathway, an evolutionally conserved signaling pathway, is involved in the production of Upd3, an IL-6 family cytokine, which activates ISC proliferation. It has been proposed that the reason why pathogens activate these responses but commensal bacteria do not is because the uracil excretion from commensal bacteria is much lower than that from pathogenic bacteria, and the ROS generation by DUOX is tightly regulated by the level of uracil. However, some opportunistic pathogens are normally quiescent but provoke chronic responses in a host-dependent manner. It is plausible that even non-pathogenic quiescent enteric bacteria weakly activate DUOX, which provokes the host regenerative responses in susceptible hosts. However, the host system that regulates regenerative responses to ROS that are produced against resident bacteria remains poorly understood (Nagai, 2021).

Macroautophagy, hereafter referred to as autophagy, an intracellular degradation system, has a fundamental role in intestinal homeostasis, as indicated by the association between defects in autophagy and Crohn’s disease (CD), a common type of IBD. Mutations in autophagy-related genes, such as Atg16L1, are genetic risk factors of CD. In addition to the genetic factors, environmental factors such as microorganisms contribute to CD pathogenesis. Recent studies have revealed functions of autophagy genes in ISCs or in enterocytes (ECs) and the involvement of intestinal microbes, but little has been revealed about the molecular mechanisms by which environmental factors interact with the genetic risk factor autophagy deficiency (Nagai, 2021).

This study identified autophagy in gut ECs, differentiated epithelial cells, as a critical system for suppressing undesirable regenerative responses to commensal bacteria to maintain the homeostatic state in the non-infected intestine of Drosophila. ROS produced by gut epithelia in the presence of commensal bacteria provoked chronic responses in autophagy-deficient ECs via the Hippo pathway, which resulted in ISC over-proliferation and loss of barrier integrity of the midgut that led to systemic inflammation and shorter lifespan. The underlying mechanism documented, in which autophagic substrate Ref(2)P, a p62 homolog, together with Dachs, an upstream regulator of the Hippo pathway, were continuously downregulated by autophagy in ECs. These studies elucidate how autophagy in Drosophila gut epithelia ensures gut homeostasis in the non-infected state and have implications about the molecular mechanisms by which enteric microbes affect the pathology of CD caused by autophagy deficiency (Nagai, 2021).

Regenerative responses of the intestinal epithelium are tightly regulated to achieve the homeostatic state of the tissue for maintaining its functions. This regulation is indispensable not only to respond appropriately to variable rates of cell loss upon injury but also to repress excessive regeneration in response to commensal bacteria, a property essential for their long-term co-habitation. Previously reported findings suggested that because of their lower ability to provoke ROS secretion from host epithelial cells, enteric resident bacteria have little harmful effect on the host and consequently have colonizing ability. Using Drosophila intestine as a model system, this study demonstrates that it is a host regulatory mechanism, i.e., autophagy, which avoids overactivation of the regenerative responses to commensal bacteria by suppressing Hippo signaling inactivation that is triggered by the lower amount of ROS. Importantly, while the low-level ROS allow the enteric bacteria to reside in the gut, they continuously stimulate the regeneration without increasing epithelial cell death. We propose that these undesirable chronic responses cause continuous imbalance of the epithelial renewal, which results in a cell-junctional defect that eventually leads to systemic inflammation. The results also provide one mechanistic explanation for why some commensal bacteria that are normally quiescent cause wide and variable effects on the pathology of IBD and a possible regulatory mechanism of cellular signaling mediated by the Ref(2)P/p62 platform (Nagai, 2021).

Both in mammals and Drosophila, regulation of Hippo signaling activity in intestinal epithelia is crucial for maintaining the tissue homeostasis by controlling ISC proliferation. Despite their essential role in proper regeneration of the epithelia after injury, less is known about the triggers that inactivate the Hippo pathway and the underlying mechanism. These analyses showed that in ECs, it is DUOX-derived ROS that activate Dachs, which inactivates Hippo signaling via the Ref(2)P platform and that this platform is quantitatively regulated by autophagy. Given that autophagosomes can target large intracellular components, subcellular targeting by autophagosomes is an effective system for regulating signaling via such multi-molecular signaling platforms. It is noteworthy that because autophagy regulates the ROS-dependent Hippo inactivation by reducing the amount of existing Ref(2)P platforms, it allows the epithelia to respond to increased ROS upon pathogen infection to activate the regenerative responses strongly enough to overcome the autophagic suppression. It is proposed that the autophagic regulation of signaling platforms functions to lower their sensitivity to the triggers, rather than shutting off the signal (Nagai, 2021).

In contrast to the subcellular localization of Dachs protein in epithelia of wing discs, which localizes to the distal sides of cell membranes, Dachs in gut ECs exhibits a punctate shape together with Ref(2)P that localizes close to the cell membrane. Although the oligomerization of Ref(2)P is indispensable for inducing dachs-dependent Hippo pathway inactivation, it remains an open question whether Dachs protein on the Ref(2)P platform is in its active state and how it activates the downstream signaling. Because the direct Yki repressor Wts co-localized to some of the Dachs-Ref(2)P platform, and transient association of Dachs with Wts could inactivate Wts, which results in its destabilization, it is plausible that the Ref(2)P punctate structure is where active Dachs inactivates the Wts for Yki activation (Nagai, 2021).

Hippo pathway core components have been shown to localize to the cell cortex just apical to cellular junctions, which is proposed to be the regulatory site of Hippo pathway activity. Interestingly, the Ref(2)P-Dachs co-localizing puncta was frequently detected at the apical side of ECs, raising the possibility that a Ref(2)P platform functions there in ECs. How the Ref(2)P puncta are formed preferentially at this region is still unknown. Because ROS secreted toward the gut lumen trigger the formation of Ref(2)P puncta, it is possible that apically localized factor(s) sense DUOX-derived ROS or the ROS-mediated damage to induce the formation of a Ref(2)P platform at this site (Nagai, 2021).

One of the unexpected aspects of these findings is that Ref(2)P and Dachs puncta formation are mutually dependent. Besides the PB1 domain, which acts for self-oligomerization, Ref(2)P/p62 contains multiple binding motifs for signaling molecules and thereby functions as a signaling hub (Moscat, 2012). However, it remains obscure whether Ref(2)P/p62 requires the organized structure when it functions as a signaling hub. Intriguingly, self-oligomerized p62 forms a filamentous structure in vitro, implying that p62 oligomer can form an ordered structure. The requirement for self-oligomerization of Ref(2)P for the intestinal disorder, together with the mutual dependency between Ref(2)P and Dachs for the puncta formation, suggests the possibility that Ref(2)P puncta in ECs might have an integrated structure in which Ref(2)P functions as a signaling platform (Nagai, 2021).

In both mammals and Drosophila, barrier dysfunction of the intestine is a hallmark of aging, and aged animals exhibit systemic inflammation. In aged flies, activation of systemic immune responses leads to gut hyperplasia, and this study has demonstrated that defective autophagy in intestinal ECs caused ISC over-proliferation, which eventually accelerated the age-related barrier dysfunction and systemic immune activation. Together, these facts suggest that dysregulation of gut barrier integrity and hyperactivation of systemic immune responses form a vicious cycle, which may result in an exacerbation of the age-related inflammation (Nagai, 2021).

One common feature of CD is altered gut bacterial composition, although it is unknown whether the dysbiosis is the causative mechanism of the inflammation or its consequence. Using Drosophila, this study has found that commensal bacteria are sufficient to disrupt epithelial integrity in non-infected autophagy-deficient intestine. Given the functional conservation of p62 and Ref(2)P, a possible mechanism is proposed whereby inflammation in CD is initiated by the overactivation of p62-mediated signaling in response to commensal bacteria. One candidate signaling pathway is TNF signaling, since autophagy-deficient gut epithelial cells show increased sensitivity to TNFα-induced cell death in an enteric microbe-dependent manner. The Hippo pathway is also a candidate, because YAP (Yki in Drosophila) is reported to be upregulated in colonic biopsies collected from CD patients. Overall, these findings showing the fundamental role of autophagy and its molecular targets in suppressing the host responses to commensal bacteria will help to achieve a better understanding of the causative etiology of CD (Nagai, 2021).

V-ATPase controls tumor growth and autophagy in a Drosophila model of gliomagenesis

Glioblastoma (GBM), a very aggressive and incurable tumor, often results from constitutive activation of EGFR (epidermal growth factor receptor) and of phosphoinositide 3-kinase (PI3K). To understand the role of autophagy in the pathogenesis of glial tumors in vivo, an established Drosophila melanogaster model of glioma was used based on overexpression in larval glial cells of an active human EGFR and of the PI3K homolog Pi3K92E/Dp110. Interestingly, the resulting hyperplastic glia express high levels of key components of the lysosomal-autophagic compartment, including vacuolar-type H(+)-ATPase (V-ATPase) subunits and Ref(2)P (refractory to Sigma P), the Drosophila homolog of SQSTM1/p62. However, cellular clearance of autophagic cargoes appears inhibited upstream of autophagosome formation. Remarkably, downregulation of subunits of V-ATPase, of Pdk1, or of the Tor (Target of rapamycin) complex 1 (TORC1) component raptor prevents overgrowth and normalize ref(2)P levels. In addition, downregulation of the V-ATPase subunit VhaPPA1-1 reduces Akt and Tor-dependent signaling and restores clearance. Consistent with evidence in flies, neurospheres from patients with high V-ATPase subunit expression show inhibition of autophagy. Altogether, these data suggest that autophagy is repressed during glial tumorigenesis and that V-ATPase and MTORC1 components acting at lysosomes could represent therapeutic targets against GBM (Formica, 2021).

Inhibition of Ref(2)P, the Drosophila homologue of the p62/SQSTM1 gene, increases lifespan and leads to a decline in motor function

Sequestosome 1 (p62/SQSTM1) is a multifunctional scaffold/adaptor protein encoded by the p62/SQSTM1 gene with function in cellular homeostasis. Mutations in the p62/SQSTM1 gene have been known to be associated with patients with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Parkinson disease (PD). The aim of the present study was to create a novel model of human neurogenerative disease in Drosophila melanogaster by altering the expression of Ref(2)P, the Drosophila orthologue of the human p62/SQSTM1 gene. Ref(2)P expression was altered in all neurons, the dopaminergic neurons and in the motor neurons, with longevity and locomotor function assessed over time. Inhibition of Ref(2)P resulted in a significantly increased median lifespan in the motor neurons, followed by a severe decline in motor skills. Inhibition of Ref(2)P in the dopaminergic neurons resulted in a significant, but minimal increase in median lifespan, accompanied by a drastic decline in locomotor function. Inhibition of Ref(2)P in the ddc-Gal4-expressing neurons resulted in a significant increase in median lifespan, while dramatically reducing motor function (Hurley, 2021).

Intracellular density of Wolbachia is mediated by host autophagy and the bacterial cytoplasmic incompatibility gene cifB in a cell type-dependent manner in Drosophila melanogaster

Autophagy is an intracellular degradation pathway involved in innate immunity. Pathogenic bacteria have evolved several mechanisms to escape degradation or exploit autophagy to acquire host nutrients. In the case of endosymbionts, which often have commensal or mutualistic interactions with the host, autophagy is not well characterized. This study utilized tissue-specific autophagy mutants to determine if Wolbachia, a vertically transmitted obligate endosymbiont of Drosophila melanogaster, is regulated by autophagy in somatic and germ line cell types. This analysis revealed core autophagy proteins Atg1 and Atg8 and a selective autophagy-specific protein Ref(2)p negatively regulate Wolbachia in the hub, a male gonad somatic cell type. Furthermore, it was determined that the Wolbachia effector protein, CifB, modulates autophagy-Wolbachia interactions, identifying a new host-related pathway which these bacterial proteins interact with. In the female germ line, the cell type necessary for inheritance of Wolbachia through vertical transmission, it was discovered that bulk autophagy mediated by Atg1 and Atg8 positively regulates Wolbachia density, whereas Ref(2)p had no effect. Global metabolomics of fly ovaries deficient in germ line autophagy revealed reduced lipid and carbon metabolism, implicating metabolites from these pathways as positive regulators of Wolbachia. This work provides further understanding of how autophagy affects bacteria in a cell type-dependent manner (Deehan, 2021).

Glucocerebrosidase reduces the spread of protein aggregation in a Drosophila melanogaster model of neurodegeneration by regulating proteins trafficked by extracellular vesicles

Abnormal protein aggregation within neurons is a key pathologic feature of Parkinson's disease (PD). The spread of brain protein aggregates is associated with clinical disease progression, but how this occurs remains unclear. Mutations in glucosidase, beta acid 1 (GBA), which encodes glucocerebrosidase (GCase), are the most penetrant common genetic risk factor for PD and dementia with Lewy bodies and associate with faster disease progression. To explore how GBA mutations influence pathogenesis, a Drosophila model of GBA deficiency (Gba1b) was created that manifests neurodegeneration and accelerated protein aggregation. Proteomic analysis of Gba1b mutants revealed dysregulation of proteins involved in extracellular vesicle (EV) biology, and altered protein composition of EVs was found from Gba1b mutants. Accordingly, it was hypothesized that GBA may influence pathogenic protein aggregate spread via EVs. It was found that accumulation of ubiquitinated proteins and Ref(2)P, Drosophila homologue of mammalian p62, were reduced in muscle and brain tissue of Gba1b flies by ectopic expression of wildtype GCase in muscle. Neuronal GCase expression also rescued protein aggregation both cell-autonomously in brain and non-cell-autonomously in muscle. Muscle-specific GBA expression reduced the elevated levels of EV-intrinsic proteins and Ref(2)P found in EVs from Gba1b flies. Perturbing EV biogenesis through neutral sphingomyelinase (nSMase), an enzyme important for EV release and ceramide metabolism, enhanced protein aggregation when knocked down in muscle, but did not modify Gba1b mutant protein aggregation when knocked down in neurons. Lipidomic analysis of nSMase knockdown on ceramide and glucosylceramide levels suggested that Gba1b mutant protein aggregation may depend on relative depletion of specific ceramide species often enriched in EVs. Finally, ectopically expressed GCase was identified within isolated EVs. Together, these findings suggest that GCase deficiency promotes accelerated protein aggregate spread between cells and tissues via dysregulated EVs, and EV-mediated trafficking of GCase may partially account for the reduction in aggregate spread (Jewett, 2021).

Many genetic influences of PD have now been identified, and much work has been focused on how these genes lead to protein aggregation through mechanisms such as protein misfolding and autophagy defects. However, none of these genes have been implicated in cell-to-cell spread of pathogenic protein aggregates, which closely correlates with clinical disease progression. Proteomic analysis and non-cell-autonomous rescue of protein aggregation in Gba1b mutants has led to the hypothesize that GBA mutations may influence the rate of propagation of protein aggregates between neurons. This work suggests a link between GBA mutations and faster spread of intracellular protein aggregates via a novel EV-mediated mechanism, possibly explaining the recent clinical finding that GBA mutations accelerate the progression of clinical disease. Using a Drosophila model of GBA deficiency that manifests accelerated protein aggregation, this study found that expressing WT GCase in specific tissues of a GBA-deficient fly can not only rescue protein aggregation cell-autonomously and in distant tissues, but also rescue alterations in protein cargo observed in EVs isolated from Gba1b mutant hemolymph. Interestingly, ectopically expressed WT GCase itself was found within EVs of GBA-deficient flies, suggesting that the non-cell-autonomous rescue due to GCase expression is mediated by both reduction in aggregated proteins in EVs and trafficking of GCase via EVs to distant cells and tissues. Perturbing EV biogenesis through decreased expression of ESCRT-independent nSMase affected protein aggregation in local tissues in a tissue-dependent manner, and further decreased a subset of Cer species already reduced in Gba1b mutants. Interestingly, this subset of Cer species is known to be enriched in EV membranes. Together, these findings suggest that mutations in GBA result in the accelerated spread of protein aggregates through changes in cellular lipid composition and dysregulation of proteins trafficked by EVs (Jewett, 2021).

Although the model of GBA mutations promoting spread of protein aggregates via EVs is novel, the idea that proteostasis can be maintained in a non-cell-autonomous fashion is well supported in the literature. For example, in C. elegans, misfolded α-synuclein accumulating in endo-lysosomal vesicles was found to be transmitted from muscle to the hypodermis, a nearby tissue, for degradation. It is possible that a non-cell-autonomous mechanism is necessary because certain tissues may be more efficient in reducing protein aggregation. This has been previously described, where overexpression of FOXO in Drosophila muscle decreased aging-related protein aggregates in muscle as well as brain and other distant tissues, but FOXO overexpression in adipose tissue was unable to prevent protein aggregation in muscle. In the current model, overexpressing dGba1b in Drosophila muscle or neuronal tissue prevented accumulation of protein aggregates throughout the organism, however overexpression of WT GCase in midgut and fat body did not significantly reduce protein aggregation in the brain. These discrepancies could be due to tissue-specific biogenesis of EVs, which could depend on factors such as metabolic rate or endovesicular trafficking. Although dGba1b is expressed in all tissues, a second homologue of human GBA1, dGba1a, is expressed only in the midgut. Gba1b mutants retain ~25% expression of dGba1a. Deficiency of dGba1a was found to extend lifespan and does not result in significant accumulation of GlcCer, suggesting that there can be significant tissue-specific differences in function for GCase that could influence EV biogenesis (Jewett, 2021).

The unexpected results from perturbation of EV biogenesis suggest that the EV-mediated regulation of protein aggregation is tissue-specific and complex. Because an increase in EV-intrinsic proteins and alteration of protein cargo were observed in Gba1b mutants (Thomas, 2018), it is anticipated that genetic perturbations decreasing the biogenesis of EVs might rescue protein aggregation non-cell-autonomously by reducing the production of dysregulated EVs. However, decreased expression of ESCRT-independent nSMase in muscle did not rescue protein aggregation in heads, suggesting that a tissue-specific decrease in biogenesis of dysregulated EVs is not sufficient to reduce protein aggregation in the rest of the organism, and the cargo of EVs may need to be corrected to reduce spread of protein aggregation. In contrast, decreased expression of nSMase in the nervous system had no effect on protein aggregation in the head. This difference in outcome in perturbation of EV biogenesis in muscle and neurons could be due to cell-specific compensatory mechanisms or intrinsic metabolic demands and solicits further investigation (Jewett, 2021).

A possible explanation for why decreased muscle expression of nSMase enhanced cell-autonomous protein aggregation and EV protein cargo alterations is that both GCase and nSMase enzymatically produce Cer. If GCase-deficient phenotypes are dependent on a relative reduction in Cer, decreased nSMase expression could exacerbate Gba1b mutant phenotypes. Indeed, lipidomic analysis of alterations in Cer metabolism due to nSMase knockdown revealed a further decrease in a subset of Cer species that were already significantly decreased in Gba1b mutants compared to controls. The further reduction in Cer species due to nSMase knockdown correlates with enhancement of cell-autonomous protein aggregation and EV cargo alterations, suggesting that accelerated protein aggregation in Gba1b mutants is mediated by Cer deficiency rather than GlcCer accumulation, as nSMase knockdown had a much more modest effect on the significantly increased levels of GlcCer species in Gba1b mutants compared to controls (Jewett, 2021).

Cer has been implicated in the composition and biogenesis of EVs, and nSMase knockdown further altered EV cargo in Gba1b mutants, suggesting that decreased Cer levels may directly influence EV biogenesis in Gba1b mutants. However, Cer species were not globally decreased, suggesting that the regulation of Cer metabolism is complex and may be more dependent on specific Cer species. Interestingly, only 1 of the 9 Cer species significantly increased in Gba1b mutants versus controls had a monounsaturated fatty acyl group, while all 5 of the Cer species significantly decreased in Gba1b mutants versus controls had a monounsaturated fatty acyl group, suggesting GBA influences the metabolism of specific subset of Cer species that may be implicated in Gba1b mutant phenotypes. This subset of Cer species is enriched in species with long chain monounsaturated fatty acyl chains. Interestingly, lipids with monounsaturated fatty acyl groups are an abundant component in mammalian exosome membranes. Investigating the alterations in lipid composition of EVs resulting from GCase deficiency and nSMase knockdown will be important in elucidating the role of Cer metabolism in Gba1b mutant phenotypes (Jewett, 2021).

This work suggests that GCase deficiency influences EV biogenesis to promote faster propagation of pathogenic protein aggregates throughout the tissues of an organism, which may be a compensatory response to cell-autonomous lysosomal stress. In the initial characterization of the Drosophila GBA-deficient model, accelerated insoluble ubiquitinated protein aggregates, accumulation of Ref(2)P, and oligomerization of ectopically expressed human α-synuclein was found in Gba1b mutants, suggesting an impairment in lysosomal degradation. A similar GBA-deficient Drosophila model also found evidence of lysosomal dysfunction, including enlarged lysosomes in GBA-deficient brains. However, proteomic analysis of Gba1b mutants did not support a profound impairment in autophagy, but instead suggested dysregulation of EVs with altered protein cargo that could be suppressed locally with knockdown of genes encoding ESCRT machinery important for EV biogenesis. Based on these results, it is believed that the initial observations of increased insoluble ubiquitinated proteins and Ref(2)P in Gba1b mutants are due to lysosomal stress. One possible explanation for the proteomic findings is that there may be a compensatory increase in EV biogenesis and packaging of autophagy substrates within EVs for discard outside of the cell in Gba1b mutants. Such an increase may have prevented detection of defects in autophagy. Upregulation of EV biogenesis may be cell-autonomously neuroprotective in the setting of lysosomal stress, particularly in aggregation-prone neurodegenerative diseases such as PD. It was recently demonstrated in a human neuronal cell culture model of PD that inhibiting macroautophagy protects against α-synuclein-induced cell death by promoting the release of α-synuclein-containing EVs. However, it remains possible that upregulating EV biogenesis may relieve lysosomal stress within cells containing aggregate-prone proteins, while simultaneously promoting the spread of protein aggregates between cells and throughout the organism (Jewett, 2021).

This work suggests a novel mechanism for GBA in reducing the spread of pathogenic protein aggregation from cell-to-cell via regulation of EV protein cargo, but many key questions remain. To better understand the progression of neurodegenerative diseases, it is important to uncover the mechanisms by which GCase deficiency alters EV protein content and biogenesis, identify the specific changes in EVs facilitating propagation of pathogenic protein aggregates, and determine how these changes influence recipient cells internalizing dysregulated EVs. GCase is a critical enzyme in ceramide metabolism, hydrolyzing glucosylceramide into glucose and ceramide. Ceramides are a key component of EV membranes, and alterations in ceramide metabolism due to GCase deficiency may directly influence EV biogenesis and protein cargo trafficked via EVs. Further studies using this Drosophila model and mammalian cell culture models should better elucidate how GCase deficiency alters the protein cargo of EVs to induce propagation of pathogenic protein aggregates, as well as whether endogenous GCase is enzymatically functional when trafficked to distant tissues via EVs. Understanding this mechanism could have broad implications in understanding the pathogenesis of aggregate-prone neurodegenerative diseases and reveal new therapeutic targets to slow or halt disease progression (Jewett, 2021).

Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover

The inability to remove protein aggregates in post-mitotic cells such as muscles or neurons is a cellular hallmark of aging cells and is a key factor in the initiation and progression of protein misfolding diseases. While protein aggregate disorders share common features, the molecular level events that culminate in abnormal protein accumulation cannot be explained by a single mechanism. This study shows that loss of the serine/threonine kinase NUAK causes cellular degeneration resulting from the incomplete clearance of protein aggregates in Drosophila larval muscles. In NUAK mutant muscles, regions that lack the myofibrillar proteins F-actin and Myosin heavy chain (MHC) instead contain damaged organelles and the accumulation of select proteins, including Filamin (Fil) (Cheerio) and CryAB. NUAK biochemically and genetically interacts with the cochaperone Starvin (Stv), the ortholog of mammalian Bcl-2-associated athanogene 3 (BAG3). Consistent with a known role for the co-chaperone BAG3 and the Heat shock cognate 71 kDa (HSC70)/HSPA8 ATPase in the autophagic clearance of proteins, RNA interference (RNAi) of Drosophila Stv, Hsc70-4, or autophagy-related 8a (Atg8a) all exhibit muscle degeneration and muscle contraction defects that phenocopy NUAK mutants. It was further demonstrated that Fil/Cheerio is a target of NUAK kinase activity and abnormally accumulates upon loss of the BAG3-Hsc70-4 complex. In addition, Ubiquitin (Ub), Ref(2)P/p62, and Atg8a are increased in regions of protein aggregation, consistent with a block in autophagy upon loss of NUAK. Collectively, these results establish a novel role for NUAK with the Stv-Hsc70-4 complex in the autophagic clearance of proteins that may eventually lead to treatment options for protein aggregate diseases (Brooks, 2020).

Proteins must fold into an intrinsic three dimensional structure to perform distinct cellular functions. Denatured or misfolded proteins can be refolded by chaperones or are subject to degradation by the ubiquitin-proteasome system (UPS) and/or the autophagosome-lysosome pathway (ALP). The accumulation of misfolded proteins upon genetic mutation or decreased chaperone function causes protein aggregates that are not effectively cleared by the UPS or the ALP. Environmental insults or aging may exacerbate this accumulation of misfolded proteins, resulting in disease and eventual cell death (Brooks, 2020).

A specialized autophagy pathway, termed chaperone-assisted selective autophagy (CASA), has been verified in both Drosophila and mammalian systems. The CASA complex includes BAG3 in concert with the chaperones HSC70/HSPA8 (HSP70 family), HSPB8 (small HSP family), and the ubiquitin (Ub) ligase CHIP/STUB1. CASA regulates the removal and degradation of Fil from the Z-disc in striated muscle or actin stress fibers in non-muscle cells. The N-terminal actin-binding domain (ABD) in Fil is followed by multiple immunoglobulin (Ig)-like repeats which bind numerous proteins to link the internal cytoskeleton to the sarcolemma. Tension exerted by contractile muscle tissue requires continuous folding and refolding of individual Ig-like domains in Fil, eventually damaging the ability of the protein to sense and transmit mechanical strain (Arndt, 2010; Razinia, 2012). The BAG3-HSC70 protein complex binds to the mechanosensor region (MSR) of Fil and upon detection of protein damage, CHIP ensures the addition of polyubiquitin (polyUb) moieties. Rather than promoting delivery to the proteasome, these Ub chains instead recruit the autophagic Ub adapter protein p62/SQSTM1. p62 interacts with Atg8a/LC3 to induce autophagophore formation and the subsequent clearance of Fil through lysosomal degradation. Fil aggregates and a block in autophagosome-lysosome fusion are present in lysosomal associated membrane protein 2 (LAMP2)-deficient muscles, thus linking impaired autophagy to abnormal protein deposits (Brooks, 2020).

Drosophila NUAK encodes for a conserved serine/threonine kinase that is homologous to the mammalian kinases NUAK1/ARK5 and NUAK2/SNARK. These proteins comprise a family of twelve AMP-activated protein kinase (AMPK)-related kinases (NUAK1 and 2, BRSK 1 and 2, QIK, QSK, SIK, MARK 1-4, and MELK) that share a conserved N-terminal kinase domain activated by the upstream liver kinase B1 (LKB1). NUAK1 and NUAK2 proteins are broadly expressed, but enriched in cardiac and skeletal muscle. Muscle contraction and LKB1 phosphorylation can activate both NUAK proteins. NUAK2 activity is additionally stimulated by oxidative stress, AMP, and glucose deprivation in various cell types. Interestingly, NUAK2 expression increases during muscle differentiation and in response to stress or in aging muscle tissue, whereas dominant-negative (DN)-NUAK2 induces atrophy. Homozygous NUAK1 KO mice are embryonic lethal and <10% of NUAK2 homozygotes survive, precluding analysis of post-embryonic contributions. Because of this embryonic lethality, conditional NUAK1 KO mice were generated to examine muscle function . However, no change was observed in muscle mass or fiber size between control or muscle-specific NUAK1 KO mice, likely due to functional redundancy (Brooks, 2020).

The presence of single NUAK orthologs in worms (Unc-82) or flies (NUAK/CG43143) allows for the study of NUAK protein function without compensation from additional family members that may mask cellular roles. unc-82 associates with Paramyosin and likely Myosin B to promote proper myofilament assembly in C. elegans. The kinase domain in Drosophila NUAK shares 61% identity and 80% similarity to human NUAK1 and NUAK2. In flies, RNAi knockdown of NUAK phenocopies weak Lkb1 defects in regulating cell polarity during ommatidial formation and actin cone formation in spermatogenesis. NUAK kinase targets or additional functions in other tissues have not been reported (Brooks, 2020).

This study identified Drosophila NUAK as a key regulator of autophagic protein clearance in muscle tissue. NUAK physically interacts with and phosphorylates Fil [encoded by Drosophila cheerio (cher)]. NUAK also genetically and biochemically interacts with the Stv-Hsc-70-4 complex and Stv overexpression is sufficient to rescue NUAK-mediated muscle deterioration. The identification of Fil as a cargo protein that abnormally accumulates in muscle tissue deficient for NUAK, Stv, Hsc70-4, and Atg8a links protein aggregation to defects in autophagic disposal (Brooks, 2020).

Prior to this study, few substrates of NUAK kinase activity had been uncovered. One of these is Myosin phosphatase targeting-1 (MYPT1), a regulatory subunit of myosin light-chain phosphatase. Two Drosophila regulatory subunits, MYPT75D and Myosin binding subunit (Mbs), were tested in Stv NUAK sensitized genetic assay and no protein aggregation and/or muscle degeneration was observed. While negative, this data nevertheless argues that this family of phosphatases likely does not function with NUAK in muscle tissue. Since the mammalian NUAK1-MYPT1 interaction was identified in vitro and further validated in HEK293 cells, NUAK likely has cell and tissue-specific targets that regulate diverse biological outputs (Brooks, 2020).

Based upon the discovery of Fil as a novel NUAK substrate, two scenarios are envisioned that are not mutually exclusive to explain the molecular function of NUAK in preventing protein aggregation. First, the increase in sarcomere number upon muscle-specific NUAK RNAi suggests that at least one role of NUAK may be to negatively regulate the addition of proteins (such as Fil) into sarcomeres. This data is consistent with studies that show C. elegans unc-82 regulates myofilament assembly. Notably, one key feature of the misincorporated proteins in unc-82 mutants is their inclusion into aggregate-like structures, similar to the accumulation of Fil and CryAB in NUAK-/- muscles. An additional, or alternative possibility, is that NUAK phosphorylates unfolded or 'damaged' Fil for removal from the sarcomere, thereby triggering the Stv-Hsc70-4 complex to promote autophagic turnover. Thus, proteins such as Fil that fail to get incorporated into sarcomeres and/or sustain damage due to repeated rounds of tension-induced muscle contraction, may destabilize myofilament architecture and trigger abnormal protein (Brooks, 2020).

In both contractile muscle tissue and in adherent cells subjected to mechanical force, BAG3 acts as a hub to coordinate Fil-induced tension-sensing and autophagosome formation. The MSR of Fil is comprised of Ig repeats whose conformational transitions between open and closed states dictate differential protein-protein interactions and biological outputs. While the chaperones Hsc70/HSPA8 and HSPB8 weakly bind to the MSR of Fil, this biochemical interaction is greatly enhanced in the presence of BAG3. Interestingly, BAG3 interacts with Ig repeats 19-21 in the MSR, while the selected interaction domain of NUAK with Fil comprises Ig repeats 15-18. These data suggest that NUAK and Stv each bind to a separate region of the MSR in Fil (Brooks, 2020).

It remains to be determined if NUAK-mediated phosphorylation is a prerequisite for the removal of damaged Fil protein by BAG3. The rescue results suggest that this phosphorylation event is not required as Stv overexpression alleviates protein aggregation and muscle degeneration upon a loss of NUAK. An alternative possibility is that this excess Stv protein is present in sufficient amounts to interact with Fil and overcome the necessity for phosphorylation by NUAK. The inability of NUAK overexpression to restore muscle defects due to knockdown of Stv, Hsc70-4, or Atg8a suggests that NUAK functions upstream or parallel to this pathway. It seems likely that NUAK has additional target substrates for kinase activity that may regulate autophagic protein clearance in muscle tissue (Brooks, 2020).

Recent studies demonstrate that increased autophagic degradation of Fil by BAG3 also induces fil transcription as a compensatory mechanism to ensure steady-state Fil levels. Thus, whether loss of NUAK or Stv alters gene expression upon a block in protein clearance was investigated. While the mRNA levels of cher, CryAB, Hsc70-4, or Atg8a were not altered in NUAK or stv mutants, there was a large increase in p62 transcripts. Thus, this increase in p62 mRNA synthesis may contribute to the elevated p62 protein levels observed upon loss of NUAK or Stv as multiple stress conditions increase p62 transcription, including proteasome inhibition, starvation and atrophic muscle conditions. Data that support a role for an autophagic block include the localization of p62 and Atg8a to regions of protein aggregation (Brooks, 2020).

A model for NUAK is proposed that incorporates these new findings with existing roles for BAG3. Fil and CryAB are physically associated at the Z-disc in Drosophila larval muscle. The phosphorylation of Fil by NUAK may control the incorporation of Fil into the Z-disc during myofibril assembly and/or may be required for the disposal of damaged Fil protein. BAG3 and chaperones such as Hsc70/HSPA8 are thought to monitor the MSR of Fil to detect force-induced damage and to promote the addition of K63-linked polyUb chains. Recruitment of the ubiquitin autophagic adapter p62/SQSTM1 induces autophagosome initiation through the accumulation of Atg8a. Eventual fusion of these autophagosomes with lysosomes promotes protein client complex destruction (Brooks, 2020).

Upon loss of NUAK, excess Fil protein that fails to be incorporated into the Z-disc and/or is damaged due to tension-induced muscle contraction begins to accumulate near the Z-disc. The presence of CryAB in Fil-like aggregates may be due to the normal association of CryAB with Fil at the Z-disc, either to monitor Fil protein damage, or to prevent protein aggregation. It is interesting that while both Fil and CryAB contain actin-binding domains, these associations are lost in NUAK-/- muscle tissue as F-actin is displaced from regions of Fil-CryAB accumulation. At this point it cannot be determined if NUAK preferentially binds to the short (~90kD) and/or long (~240 kD) Fil isoforms since the mapped interaction domains (Ig domains 15-18) are present in both isoforms (Brooks, 2020).

In the initial stages of aggregate formation, nearly all Fil puncta are decorated with Ub. It is hypothesized that the observed decrease in Ub-Fil colocalization in large regions of aggregate formation may be due to intrinsic properties of aggregation-prone proteins whereby protein misfolding triggers aggregation of Fil with itself and other proteins. The accumulation of p62 and circular structures that stain positive for Atg8a in regions of Fil accumulation demonstrate that the autophagosome machinery is recruited to BAG3-client complexes. The absence of lysosomes in these aggregate regions suggest that either fusion and/or transport to sites of degradation are compromised (Brooks, 2020).

CASA-mediated autophagy via the BAG3-client complex includes Hsc70-4/HSPA8, HSPB8, and the E3 ligase CHIP/STUB1, the latter of which ubiquitinates Fil for the subsequent recruitment of p62 to initiate autophagosome formation. However, fibroblasts deficient for CHIP are not defective in autophagy and mice or flies lacking CHIP/STUB1 are viable. A failure to enhance protein aggregation defects upon CHIP RNAi knockdown in the sensitized NUAK+/- or stv+/- backgrounds suggests that additional Ub ligases cooperate with the Stv/BAG3 complex to remove damaged proteins. Future studies will also determine which Drosophila protein is the equivalent of HSPB8 since no genetic interactions were observed with putative CG14207 or Hsp67Bc RNAi lines. This negative data does not rule out the possibility that protein levels are not reduced enough to see phenotypes upon RNAi induction or possible functional redundancy exists between CG14207 and Hsp67Bc (Brooks, 2020).

An interesting hallmark of protein aggregate diseases is the accumulation of specific proteins in affected cells or tissues. Thus, proteins susceptible to aggregation in vivo may possess specific structural characteristics or shared biological functions. This latter feature is evident in a group of protein aggregate diseases termed myofibrillar myopathies (MFM). Laser microdissection of aggregates from normal or affected muscles reveal specificity in the types of proteins that accumulate in patients afflicted with MFMs. Common proteins present in these aggregates include Filamin C (FILC), αB-crystallin (CRYAB), BAG3, and Desmin (DES), among others. The inability of MFM patients to clear these aggregates results in myofibrillar degeneration and a decline in muscle function. Interestingly, mutations in Drosophila NUAK phenocopy both structural and functional deficits observed in MFM patients, including Fil and CryAB accumulation, muscle degeneration, and locomotor defects. The discovery of cellular degeneration and protein aggregation in muscle tissue upon loss of the single fly NUAK ortholog highlights the power of Drosophila as a model. Future studies will focus on identifying kinase targets of NUAK and defining additional proteins that function in NUAK and stv-mediated autophagy for the eventual development of therapeutic targets to treat MFMs and other protein aggregate diseases (Brooks, 2020).

TFEB/Mitf links impaired nuclear import to autophagolysosomal dysfunction in C9-ALS

Disrupted nucleocytoplasmic transport (NCT) has been implicated in neurodegenerative disease pathogenesis; however, the mechanisms by which disrupted NCT causes neurodegeneration remain unclear. A Drosophila screen identified ref(2)P/p62, a key regulator of autophagy, as a potent suppressor of neurodegeneration caused by the GGGGCC hexanucleotide repeat expansion (G4C2 HRE) in C9orf72 that causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). p62 is increased and forms ubiquitinated aggregates due to decreased autophagic cargo degradation. Immunofluorescence and electron microscopy of Drosophila tissues demonstrate an accumulation of lysosome-like organelles that precedes neurodegeneration. These phenotypes are partially caused by cytoplasmic mislocalization of Mitf/TFEB, a key transcriptional regulator of autophagolysosomal function. Additionally, TFEB is mislocalized and downregulated in human cells expressing GGGGCC repeats and in C9-ALS patient motor cortex. These data suggest that the C9orf72-HRE impairs Mitf/TFEB nuclear import, thereby disrupting autophagy and exacerbating proteostasis defects in C9-ALS/FTD (Cunningham, 2020).

Protective mechanism of Wnt4 gene on Parkinson's disease (PD) transgenic Drosophila

The main pathological change of Parkinson's disease (PD) is progressive degeneration and necrosis of dopaminergic neurons in the midbrain, forming a Lewy body in many of the remaining neurons. Studies have found that in transgenic Drosophila, mutations in the PTEN-inducible kinase 1 (PINK1) gene may cause indirect flight muscle defects in Drosophila, and mitochondrial structural dysfunction as well. In this study, Wnt4 gene overexpression and knockdown were performed in PINK1 mutant PD transgenic Drosophila, and the protective effect of Wnt4 gene on PD transgenic Drosophila and its possible mechanism were explored. The Wnt4 gene was screened in the previous experiment; And by using the PD transgenic Drosophila model of the MHC-Gal4/UAS system, the PINK1 gene could be specifically activated in the Drosophila muscle tissue. In PINK1 mutation transgenic fruit flies, the Wnt4 gene to study its implication on PD transgenic fruit flies' wing normality and flight ability. Overexpression of Wnt4 gene significantly reduced abnormality rate of PD transgenic Drosophila and improved its flight ability, and then, increased ATP concentration, enhanced mitochondrial membrane potential and normalized mitochondrial morphology were found. All of these findings suggested Wnt4 gene may have a protective effect on PD transgenic fruit flies. Furthermore, in Wnt4 gene overexpression PD transgenic Drosophila, down-regulation autophagy and apoptosis-related proteins Ref(2)P, Pro-Caspase3, and up-regulation of Beclin1, Atg8a, Bcl2 protein were confirmed by Western Blotting. The results imply that the restoring of mitochondrial function though Wnt4 gene overexpression in the PINK1 mutant transgenic Drosophila may be related to autophagy and/or apoptosis (Wu, 2019).

Upregulation of the autophagy adaptor p62/SQSTM1 prolongs health and lifespan in middle-aged Drosophila

Autophagy, a lysosomal degradation pathway, plays crucial roles in health and disease. p62/SQSTM1 (hereafter p62) is an autophagy adaptor protein that can shuttle ubiquitinated cargo for autophagic degradation. This study shows that upregulating the Drosophila p62 homolog ref(2)P/dp62, starting in midlife, delays the onset of pathology and prolongs healthy lifespan. Midlife induction of dp62 improves proteostasis, in aged flies, in an autophagy-dependent manner. Previous studies have reported that p62 plays a role in mediating the clearance of dysfunctional mitochondria via mitophagy. However, the causal relationships between p62 expression, mitochondrial homeostasis, and aging remain largely unexplored. This study shows that upregulating dp62, in midlife, promotes mitochondrial fission, facilitates mitophagy, and improves mitochondrial function in aged flies. Finally, this study shows that mitochondrial fission is required for the anti-aging effects of midlife dp62 induction. These findings indicate that p62 represents a potential therapeutic target to counteract aging and prolong health in aged mammals (Aparicio, 2019).

Loss of protein homeostasis (proteostasis) and mitochondrial dysfunction are two cellular hallmarks of aging, each of which has been proposed to contribute to age-related health decline. Therefore, identifying interventions that could improve proteostasis and/or mitochondrial function when targeted to aged animals could lead to treatments to forestall disease and promote healthy aging. Macroautophagy, hereafter autophagy, is a degradation pathway that plays key roles in development, tissue homeostasis, and disease pathogenesis. In this process, cellular materials (referred to as autophagic cargo) are sequestered by double-membrane vesicles known as autophagosomes (APs) and delivered to the lysosome for degradation. In recent years, autophagy, and more specifically a requirement for autophagy-related genes, has been implicated in genetic, dietary, and pharmacological interventions that extend lifespan in model organisms. These findings support the idea that autophagy induction plays a causal role in these prolongevity paradigms. In addition, constitutively increasing basal levels of autophagy, by directly manipulating autophagy-related genes, has been reported to promote longevity in diverse species including mice. Importantly, however, while autophagy induction is generally considered to be cytoprotective, it has also been linked to cell death and disease pathogenesis. Therefore, it is likely that in certain physiological contexts, autophagy can contribute to pathophysiology and, thereby, limit lifespan. Indeed, recent work has shown that inhibition of autophagy-related genes, in post-reproductive C. elegans, can prolong lifespan and health span, leading to the proposal that autophagy switches to a harmful role in aged animals. Hence, fundamental questions remain unanswered regarding the mechanism(s) by which age-related modulation of autophagy impacts organismal health and lifespan. Critically, there is a relative lack of understanding of how to modulate autophagy in aged animals to improve tissue homeostasis and prolong health (Aparicio, 2019).

Autophagy receptors designate substrate specificity through the recognition of specific cargo, including protein aggregates (aggrephagy), mitochondria (mitophagy), and pathogens (xenophagy). Critically, however, an understanding of the role of autophagy cargo receptors in aging and lifespan determination remains elusive. p62 (also known as Sequestosome 1) is a prototypic autophagy adaptor that possesses a C-terminal ubiquitin-binding domain and a short LC3-interacting region responsible for LC3/ATG8 interaction, allowing recruitment of ubiquitinated cargo into nascent APs. Studies in Drosophila melanogaster and mice have shown that p62 is required for the aggregation of ubiquitinated proteins and their autophagic clearance. In addition, p62 has been shown to play a role in the PINK1/Parkin pathway of mitophagy. A key step in mitophagy involves the recruitment of Parkin, an E3 ubiquitin ligase, from the cytosol to a dysfunctional mitochondrion. Once there, Parkin ubiquitinates outer mitochondrial membrane proteins and induces mitophagy. p62 accumulates on damaged mitochondria and can recognize Parkin-mediated, poly-ubiquitinated chains (Geisler, 2010). The question of whether p62 is required for mitophagy remains controversial with some studies in mammalian cells reporting that p62 is required for Parkin-mediated mitophagy, but not others. Consistent with a key role for p62 in mitophagy, Refractory to Sigma P, ref(2)P, the single Drosophila ortholog of p62 has been shown to play an essential role in promoting mitophagy (de Castro, 2013). Studies in both flies and mice have shown that genetic inactivation of p62 leads to early-onset mitochondrial dysfunction, neurodegeneration, and reduced lifespan (de Castro, 2013; Kwon, 2012; Ramesh Babu, 2008). However, the consequences of increasing p62 expression, in aging animals, on mitochondrial homeostasis, proteotoxicity, and organismal health are not known (Aparicio, 2019).

The proposed roles of p62 in the autophagic clearance of protein aggregates (aggrephagy) and dysfunctional mitochondria (mitophagy) led examination of whether p62 could modulate tissue and/or organismal aging. To do so, the impact of upregulation of the Drosophila p62 homolog ref(2)P/dp62 (de Castro, 2013) was examined at different stages of adulthood. Importantly, midlife induction of dp62 was shown to improves markers of health and prolongs lifespan. Short-term, midlife dp62 induction improves proteostasis in aged muscle. Critically, the ability of midlife dp62 induction to improve proteostasis and prolong lifespan is dependent upon autophagy-related genes. In addition, midlife dp62 induction promotes mitophagy and improves markers of mitochondrial function in aged flies. The process of mitophagy is intimately linked to mitochondrial fission/fusion processes. This study shows that inhibiting mitochondrial fission, via expression of a dominant-negative Dynamin-related protein 1 (Drp1) transgene, impairs dp62-mediated improvements in mitochondrial function and longevity. Furthermore, knockdown of parkin abrogates dp62-mediated longevity. Together, these findings indicate that activating dp62 expression, in midlife, is an effective approach to improve proteostasis and mitochondrial function and, thereby, prolong healthy lifespan (Aparicio, 2019).

Numerous lines of evidence indicate that aging is linked to alterations in the activity of the autophagy pathway. However, the underlying mechanisms that lead to these changes and the causal relationships between altered autophagic activity and age-related health decline remain subject to speculation. Critically, the question of whether increasing the expression of autophagy-related genes in aged animals can slow tissue aging and/or promote longevity remains largely unexplored. This study used the fruit fly Drosophila as a model organism to address the question of whether p62, a prototypic autophagy cargo receptor, can modulate tissue and/or organismal aging. Using an inducible gene expression system, it was shown that upregulation of dp62 from midlife onward leads to a significant increase in fly lifespan. Furthermore, induction of dp62 in middle-aged animals improves several markers of organismal health and delays age-onset pathology. Induction of dp62 in young flies did not produce a prolongevity effect, indicating that dp62 expression levels are not limiting for health in early life. These findings reveal that p62 represents a therapeutic target to counteract aging and, thereby, prolong health span in aged animals. It is interesting to note that an 8-fold induction of dp62 mRNA levels was associated with lifespan extension (Aparicio, 2019).

As P62 is a multifunctional protein that serves as a signaling hub for a myriad of cellular processes including amino acid sensing, immunity, and the oxidative response, there exist several potential candidate mechanisms that could underlie the beneficial effects of midlife dp62 induction. Critically, this study shows that the anti-aging effects of midlife dp62 induction, both at the tissue and organismal level, are dependent upon autophagy-related genes. More specifically, it was show that midlife dp62 induction reduces proteotoxicity in aged muscles and promotes longevity in an Atg1-dependent manner. Moreover, midlife dp62 induction leads to a shift toward mitochondrial fission and improves mitochondrial function in an Atg1-dependent fashion. To better understand the importance of mitochondrial fission in dp62-mediated longevity, this study set out to simultaneously inhibit mitochondrial fission and upregulate dp62 in midlife. Consistent with the idea that mitochondrial fission is important in facilitating mitophagy in aged animals, inhibiting mitochondrial fission abrogates the beneficial effects of midlife dp62 induction on mitochondrial function and longevity. Studies in yeast have shown that upon mitophagy induction, Dnm1 (yeast Drp1 ortholog) is recruited to the degrading mitochondria via the scaffold protein Atg11 to induce fission. Future work could focus on elucidating the molecular mechanisms by which increased dp62 expression promotes mitochondrial fission in aged animals. In addition, it was shown that dp62-mediated longevity requires parkin, a key component of the mitophagy pathway. These findings, therefore, indicate that the selective clearance of mitochondria, via mitophagy, is key to midlife dp62-mediated longevity (Aparicio, 2019).

The autophagy pathway represents an attractive therapeutic target to promote healthy aging in humans. However, the question of when and how to manipulate autophagy in aging mammals, in order to prolong health, is not understood. A recent study reported that a gain-of-function mutation in a core autophagy gene, Becn1, can extend mammalian lifespan. However, it is not clear whether targeting approaches of this kind to aged mammals can promote longevity. Recent findings, in C. elegans, have shown that inhibiting genes involved in early stages of autophagy in aged animals can prolong lifespan. As a result, it has been proposed that dysfunctional autophagy in aged animals, linked to blockage of autophagy at a late stage, may contribute to age-onset health decline. Hence, it is possible that interventions that induce early stages of autophagy, including AP formation, may not promote health when targeted to aged animals. In contrast, the current findings suggest that midlife up-regulation of the autophagy adaptor protein, p62, can promote the autophagic clearance of protein aggregates and mitochondria in aged animals. Hence, increasing p62 expression by pharmacological means, in midlife, may be an effective approach to prolong health span in mammals (Aparicio, 2019).

Generation and characterization of germline-specific autophagy and mitochondrial reactive oxygen species reporters in Drosophila

Oogenesis is a fundamental process that forms the egg and, is crucial for the transmission of genetic information to the next generation. Drosophila oogenesis has been used extensively as a genetically tractable model to study organogenesis, niche-germline stem cell communication, and more recently reproductive aging including germline stem cell (GSC) aging. Autophagy, a lysosome-mediated degradation process, is implicated in gametogenesis and aging. However, there is a lack of genetic tools to study autophagy in the context of gametogenesis and GSC aging. This study describes the generation of three transgenic lines mcherry-Atg8a, GFP-Ref(2)P and mito-roGFP2-Orp1 (an H2O2 sensor) that are specifically expressed in the germline compartment including GSCs during Drosophila oogenesis. These transgenes are expressed from the nanos promoter and present a better alternative to UASp mediated overexpression of transgenes. These fluorescent reporters can be used to monitor and quantify autophagy, and the production of reactive oxygen species during oogenesis. These reporters provide a valuable tool that can be utilized in designing genetic screens to identify novel regulators of autophagy and redox homeostasis during oogenesis (Nilangekar, 2019).

The lysosomal membrane protein LAMP2A promotes autophagic flux and prevents SNCA-induced Parkinson disease-like symptoms in the Drosophila brain

The autophagy-lysosome pathway plays a fundamental role in the clearance of aggregated proteins and protection against cellular stress and neurodegenerative conditions. Alterations in autophagy processes, including macroautophagy and chaperone-mediated autophagy (CMA), have been described in Parkinson disease (PD). CMA is a selective autophagic process that depends on LAMP2A (Lysosomal associated membrane protein 2A), a mammal and bird-specific membrane glycoprotein that translocates cytosolic proteins containing a KFERQ-like peptide motif across the lysosomal membrane. Drosophila reportedly lack CMA and use endosomal microautophagy (eMI) as an alternative selective autophagic process. This study reports that neuronal expression of human LAMP2A protected Drosophila against starvation and oxidative stress, and delayed locomotor decline in aging flies without extending their lifespan. LAMP2A also prevented the progressive locomotor and oxidative defects induced by neuronal expression of PD-associated human SNCA (synuclein alpha) with alanine-to-proline mutation at position 30 (SNCA(A30P)). LAMP2A expression stimulated selective autophagy in the adult brain and not in the larval fat body. Noteworthy, neurally expressed LAMP2A markedly upregulated levels of Drosophila Atg5, a key macroautophagy initiation protein, and of the Atg5-containing complex, and that it increased the density of Atg8a/LC3-positive puncta, which reflects the formation of autophagosomes. Furthermore, LAMP2A efficiently prevented accumulation of the autophagy defect marker Ref(2)P/p62 in the adult brain under acute oxidative stress. These results indicate that LAMP2A can promote autophagosome formation and potentiate autophagic flux in the Drosophila brain, leading to enhanced stress resistance and neuroprotection (Issa, 2018).

Glial Draper rescues Abeta toxicity in a Drosophila model of Alzheimer's Disease

Pathological hallmarks of Alzheimer's disease (AD) include amyloid-beta (Abeta) plaques, neurofibrillary tangles, and reactive gliosis. Glial cells offer protection against AD by engulfing extracellular Abeta peptides, but the repertoire of molecules required for glial recognition and destruction of Abeta are still unclear. This study shows that the highly conserved glial engulfment receptor Draper/MEGF10 provides neuroprotection in an AD model of Drosophila (both sexes). Neuronal expression of human Abeta42^arc in adult flies results in robust Abeta accumulation, neurodegeneration, locomotor dysfunction, and reduced lifespan. Notably, all of these phenotypes are more severe in draper mutant animals, while enhanced expression of glial Draper reverses Abeta accumulation, as well as behavioral phenotypes. Stat92E, c-Jun N-terminal Kinase (JNK)/AP-1 signaling, and expression of matrix metalloproteinase-1 (Mmp1) are activated downstream of Draper in glia in response to Abeta42^arc exposure. Furthermore, Abeta42-induced upregulation of the phagolysosomal markers Atg8 and p62 was notably reduced in draper mutant flies. Based on these findings, it is proposed that glia clear neurotoxic Abeta peptides in the AD model Drosophila brain through a Draper/STAT92E/JNK cascade that may be coupled to protein degradation pathways such as autophagy or more traditional phagolysosomal destruction methods (Ray, 2017).

Increasing autophagy and blocking Nrf2 suppress laminopathy-induced age-dependent cardiac dysfunction and shortened lifespan

Mutations in the human LMNA gene cause a collection of diseases known as laminopathies. These include myocardial diseases that exhibit age-dependent penetrance of dysrhythmias and heart failure. The LMNA gene encodes A-type lamins, intermediate filaments that support nuclear structure and organize the genome. Mechanisms by which mutant lamins cause age-dependent heart defects are not well understood. This study modeled human disease-causing mutations in the Drosophila Lamin C gene and expressed mutant Lamin C exclusively in the heart. This resulted in progressive cardiac dysfunction, loss of adipose tissue homeostasis, and a shortened adult lifespan. Within cardiac cells, mutant Lamin C aggregated in the cytoplasm, the CncC(Nrf2)/Keap1 redox sensing pathway was activated, mitochondria exhibited abnormal morphology, and the autophagy cargo receptor Ref2(P)/p62 was upregulated. Simultaneous over-expression of the autophagy kinase Atg1 gene and an RNAi against CncC eliminated the cytoplasmic protein aggregates, restored cardiac function, and lengthened lifespan. These data suggest that simultaneously increasing rates of autophagy and blocking the Nrf2/Keap1 pathway are a potential therapeutic strategy for cardiac laminopathies (Bhide, 2018).

Mutations in the human LMNA gene are associated with a collection of diseases called laminopathies in which the most common manifestation is progressive cardiac disease. This study has generated Drosophila melanogaster models of age-dependent cardiac dysfunction. In these models, mutations synonymous with those causing disease in humans were introduced into Drosophila LamC. Cardiac-specific expression of mutant LamC resulted in (1) cardiac contractility, conduction, and physiological defects, (2) abnormal nuclear envelope morphology, (3) cytoplasmic LamC aggregation, (4) nuclear enrichment of the redox transcriptional regulator CncC (mammalian Nrf2), (5) and upregulation of autophagy cargo receptor Ref(2)P (mammalian p62). These cardiac defects were enhanced with age and accompanied by increased adipose tissue in the adult fat bodies and a shortened lifespan (Bhide, 2018).

To understand the mechanistic basis of cardiolaminopathy and identify genetic suppressors, advantage was taken of powerful genetic tools available in Drosophila. The presence of cytoplasmic LamC aggregates prompted a determination of whether increasing autophagy would suppress the cardiac defects. Cardiac-specific upregulation of autophagy (Atg1 OE) suppressed G489V-induced cardiac defects. Consistent with this, decreased autophagy due to expression of Atg1 DN resulted in enhanced deterioration of G489V-induced cardiac dysfunction. Interestingly, cardiac-specific Atg5 OE and Atg8a OE, two factors that also promote autophagy, showed little to no suppression of G489V-induced heart dysfunction, suggesting that Atg1 might be rate limiting in this context. These findings are consistent with studies in mouse laminopathy models in which rapamycin and temsirolimus had beneficial effects on heart and skeletal muscle through inhibition of AKT/mTOR signaling. These findings are depicted in a model (see Model for the interactions between the autophagy and CncC/Keap1 signaling pathway in mutant lamin-induced cardiac disease) in which cytoplasmic aggregation of mutant LamC results in upregulation of p62, which in turn inhibits autophagy via activation of TOR and inactivation of AMPK. AMPK inactivation leads to the activation of PI3K/Akt/mTOR pathway and inhibition of autophagy Atg1 OE promoted clearance of the LamC aggregates and restored proteostasis in these Drosophila models. Thus, the data suggest that mutant LamC reduces autophagy, resulting in impairment of cellular proteostasis that leads to cardiac dysfunction (Bhide, 2018).

Cardiac-specific expression of mutant LamC altered CncC subcellular localization. Previously, Drosophila larval body wall muscles expressing G489V were shown to experience reductive stress, an atypical redox state characterized by high levels of reduced glutathione and NADPH, and upregulation CncC target genes (Dialynas, 2015). Cardiac-specific CncC RNAi in the wild-type LamC background did not produce major cardiac defects. Consistent with this, Nrf2 deficiency in mice does not compromise cardiac and skeletal muscle performance. Cardiac-specific CncC RNAi suppressed G489V-induced cardiac dysfunction and reduced cytoplasmic LamC aggregation, but not R205W-induced defects. However, cardiac-specific RNAi against CncC did not affect G489V-induced adipose tissue accumulation and lifespan shortening. Similar to the nuclear enrichment of CncC in hearts expressing G489V, human muscle biopsy tissue from an individual with a point mutation in the LMNA gene that results in G449V (analogous to Drosophila G489V) showed nuclear enrichment of Nrf2. Disruption of Nrf2/Keap1 signaling has also been reported for Hutchinson-Gilford progeria, an early-onset aging disease caused by mutations in LMNA. In this case, however, the thickened nuclear lamina traps Nrf2 at the nuclear envelope that results in a failure to activate Nrf2 target genes, leading to oxidative stress. In these studies, CncC nuclear enrichment was observed; however, a redox imbalance was not readily observed at the three-time points investigated. This might indicate that there is a window of time in disease progression in which redox imbalance occurs and that mechanisms are in place to re-establish homeostasis (Bhide, 2018).

It has been postulated that there is cross-talk between autophagy and Nrf2/Keap1 signaling. This was tested by manipulating autophagy and CncC (Nrf2) alone and in combination. CncC RNAi suppressed the cardiac defects caused by G489V, but not the lipid accumulation and lifespan shortening, suggesting the latter two phenotypes are not specifically due to loss of cardiac function. In contrast, Atg1 OE suppressed the cardiac and adipose tissue defects and lengthened the lifespan. The double treatment (simultaneous Atg1 OE and RNAi knockdown of CncC) gave the most robust suppression of the mutant phenotypes and completely restored the lifespan. Interestingly, Atg1 DN and RNAi knockdown of CncC simultaneously did not further deteriorate or improve the mutant phenotypes. Taken together, these data suggest that autophagy plays a key role in suppression of the G498V-induced phenotypes and that knockdown on CncC enhances this suppression (Bhide, 2018).

These findings support a model whereby autophagy and Nrf2 signaling are central to cardiac health. It is proposed that cytoplasmic aggregation of LamC increases levels of Ref(2)P (p62), which competitively binds to Keap1, resulting in CncC (Nrf2) translocation to the nucleus. Inside the nucleus, Nrf2 regulates genes involved in detoxification. Continued expression of antioxidant genes results in the disruption of redox homeostasis, defective mitochondria, and dysregulation of energy homeostasis/energy sensor such as AMPK and its downstream targets. Simultaneously, upregulation of Ref(2)P (p62) causes inhibition of autophagy via activation of TOR, which leads to the inactivation of AMPK. AMPK inactivation in combination with activation of the TOR pathway causes cellular and metabolic stress that leads to cardiomyopathy. In support of this model, transcriptomics data from muscle tissue of an individual with muscular dystrophy expressing Lamin A/C G449V (analogous to Drosophila G489V) showed (1) upregulation of transcripts from Nrf2 target genes, (2) upregulation of genes encoding subunits of the mTOR complex, and (3) downregulation of AMPK, further demonstrating relevance of the Drosophila model for providing insights on human pathology (Bhide, 2018).

Drosophila Vps13 Is required for protein homeostasis in the brain

Chorea-Acanthocytosis is a rare, neurodegenerative disorder characterized by progressive loss of locomotor and cognitive function. It is caused by loss of function mutations in the Vacuolar Protein Sorting 13A (VPS13A) gene. This study characterized a Drosophila Vps13 mutant line. The data suggest that Vps13 is a peripheral membrane protein located to endosomal membranes and enriched in the fly head. Vps13 mutant flies showed a shortened life span and age associated neurodegeneration. Vps13 mutant flies were sensitive to proteotoxic stress and accumulated ubiquitylated proteins. Levels of Ref(2)P, the Drosophila orthologue of p62, were increased and protein aggregates accumulated in the central nervous system. Overexpression of the human Vps13A protein in the mutant flies partly rescued apparent phenotypes. This suggests a functional conservation of human VPS13A and Drosophila Vps13. The results demonstrate that Vps13 is essential to maintain protein homeostasis in the larval and adult Drosophila brain. Drosophila Vps13 mutants are suitable to investigate the function of Vps13 in the brain, to identify genetic enhancers and suppressors and to screen for potential therapeutic targets for Chorea-Acanthocytosis (Vonk, 2017).

Drosophila Clueless is involved in Parkin-dependent mitophagy by promoting VCP-mediated Marf degradation

PINK1/Parkin-mediated mitochondrial quality control (MQC) requires valosin-containing protein (VCP)-dependent Mitofusin/Marf degradation to prevent damaged organelles from fusing with the healthy mitochondrial pool, facilitating mitochondrial clearance by autophagy. Drosophila clueless (clu) was found to interact genetically with PINK1 and parkin to regulate mitochondrial clustering in germ cells. However, whether Clu acts in MQC has not been investigated. This study shows that overexpression of Drosophila Clu complements PINK1, but not parkin, mutant muscles. Loss of clu leads to the recruitment of Parkin, VCP/p97, p62/Ref(2)P and Atg8a to depolarized swollen mitochondria. However, clearance of damaged mitochondria is impeded. This paradox is resolved by the findings that excessive mitochondrial fission or inhibition of fusion alleviates mitochondrial defects and impaired mitophagy caused by clu depletion. Furthermore, Clu is upstream of and binds to VCP in vivo and promotes VCP-dependent Marf degradation in vitro. Marf accumulates in whole muscle lysates of clu-deficient flies and is destabilized upon Clu overexpression. Thus, Clu is essential for mitochondrial homeostasis and functions in concert with Parkin and VCP for Marf degradation to promote damaged mitochondrial clearance (Wang, 2016).

Myopathic lamin mutations cause reductive stress and activate the nrf2/keap-1 pathway

Mutations in the human LMNA gene cause muscular dystrophy by mechanisms that are incompletely understood. The LMNA gene encodes A-type lamins, intermediate filaments that form a network underlying the inner nuclear membrane, providing structural support for the nucleus and organizing the genome. To better understand the pathogenesis caused by mutant lamins, a structural and functional analysis was performed on LMNA missense mutations identified in muscular dystrophy patients. These mutations perturb the tertiary structure of the conserved A-type lamin Ig-fold domain. To identify the effects of these structural perturbations on lamin function, these mutations were modeled in Drosophila Lamin C, and the mutant lamins were expressed in muscle. The structural perturbations had minimal dominant effects on nuclear stiffness, suggesting that the muscle pathology was not accompanied by major structural disruption of the peripheral nuclear lamina. However, subtle alterations in the lamina network and subnuclear reorganization of lamins remain possible. Affected muscles had cytoplasmic aggregation of lamins and additional nuclear envelope proteins. Transcription profiling revealed upregulation of many Nrf2 target genes. Nrf2 is normally sequestered in the cytoplasm by Keap-1. Under oxidative stress Nrf2 dissociates from Keap-1, translocates into the nucleus, and activates gene expression. Unexpectedly, biochemical analyses revealed high levels of reducing agents, indicative of reductive stress. The accumulation of cytoplasmic lamin aggregates correlated with elevated levels of the autophagy adaptor p62/SQSTM1, which also binds Keap-1, abrogating Nrf2 cytoplasmic sequestration, allowing Nrf2 nuclear translocation and target gene activation. Elevated p62/SQSTM1 and nuclear enrichment of Nrf2 were identified in muscle biopsies from the corresponding muscular dystrophy patients, validating the disease relevance of the Drosophila model. Thus, novel connections were made between mutant lamins and the Nrf2 signaling pathway, suggesting new avenues of therapeutic intervention that include regulation of protein folding and metabolism, as well as maintenance of redox homoeostasis (Dialynas, 2015).

p62/sequestosome-1, Autophagy-related Gene 8, and autophagy in Drosophila are regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), independent of transcription factor TFEB

The selective autophagy receptor p62/sequestosome 1 (SQSTM1) interacts directly with LC3 and is involved in oxidative stress signaling in two ways in mammals. First, p62 is transcriptionally induced upon oxidative stress by the NF-E2-related factor 2 (NRF2) by direct binding to an antioxidant response element (ARE) in the p62 promoter. Secondly, p62 accumulation, occurring when autophagy is impaired, lead to increased p62 binding to the NRF2 inhibitor KEAP1 resulting in reduced proteasomal turnover of NRF2. This gives chronic oxidative stress signaling through a feed forward loop. This study shows that the Drosophila p62/SQSTM1 orthologue, Ref(2)P, interacts directly with Atg8a via a LC3-interacting region (LIR) motif, supporting a role for Ref(2)P in selective autophagy. The ref(2)P promoter also contains a functional ARE that is directly bound by the NRF2 orthologue, CncC which can induce ref(2)P expression along with the oxidative stress associated gene gstD1. However, distinct from the situation in mammals, Ref(2)P does not interact directly with DmKeap1 via a KEAP1-interacting region (KIR) motif. Neither does ectopically expressed Ref(2)P, nor autophagy deficiency, activate the oxidative stress response. Instead, DmAtg8a interacts directly with DmKeap1, and DmKeap1 is removed upon programmed autophagy in Drosophila gut cells. Strikingly, CncC induced increased Atg8a levels and autophagy independent of TFEB/MitF in fat body and larval gut tissues. Thus, these results extend the intimate relationship between oxidative stress sensing NRF2/CncC transcription factors and autophagy, and suggests that NRF2/CncC may regulate autophagic activity in other organisms too (Jain, 2015).

Autophagy is a catabolic process where an isolation membrane engulfs part of the cytoplasm to create a double-membrane vesicle called the autophagosome, which fuses with lysosomes and leads to degradation of their contents. Selective autophagy receptors bind to cargo and dock onto the forming phagophore through a direct interaction with ATG8 family proteins, enabling delivery and autophagic degradation of the cargo. Human p62/sequestosome 1 (hereafter named p62) interacts with LC3 and ubiquitin, is a selective autophagic substrate, and is the first identified cargo receptor for autophagic degradation of ubiquitinated targets. When autophagy is abolished in the liver of Atg7 conditional knock-out mice, p62 accumulates in aggregates, and antioxidant proteins and phase II detoxification enzymes are strongly induced. p62 is induced by various stressors both at the mRNA and protein levels, and this p62 induction is inhibited in cells from Nrf2 knock-out mice. Several groups have reported that p62 competes with NRF2 for binding to KEAP1, resulting in stabilization of NRF2, whereas KEAP1 is sequestered into p62 bodies and subsequently degraded by autophagy. It was also recently shown that phosphorylation of the KEAP1-interacting region (KIR) motif of p62 enhanced binding to KEAP1. It has been reported earlier that NRF2 bound to an ARE site in the p62 promoter and induced p62 expression upon oxidative stress. Hence, it was not possible to conclude that p62 is involved in establishing a positive feedback loop inducing its own expression and prolonged NRF2 response under stress conditions (Jain, 2015).

D. melanogaster ref(2)P is the orthologue of mammalian p62 and was first characterized as a modifier of σ virus multiplication. Ref(2)P has been reported to be a major component of protein aggregates in flies defective in autophagy or with impaired proteasomal function and in fly models of neurodegenerative diseases. However, it is not known if Ref(2)P binds directly to DmAtg8 via a functional LIR motif (Jain, 2015).

This study shows that Ref(2)P interacts with DmAtg8a in vitro and in vivo through a LIR motif and that this is necessary for autophagic degradation of Ref(2)P. ref(2)P is a transcriptional target of CncC and contains a CncC-responsive ARE in its promoter. However, Ref(2)P does not bind directly to DmKeap1 via a KIR motif, as found for mammalian p62 and KEAP1. Consequently, ectopically expressed Ref(2)P does not induce the oxidative stress response in fly tissues. Very interestingly, this study found CncC induces atg8a and stimulates autophagy in the fat body and larval gut. Hence, the positive feedback loop between p62 and Nrf2 seen in mammals is not present in D. melanogaster. However, CncC can induce ref(2)P, atg8a, and autophagy (Jain, 2015).

Ref(2)P, the single p62 orthologue in D. melanogaster, is an established signaling adapter. Similar to p62, Ref(2)P accumulates with ubiquitin-containing protein aggregates in the brain of autophagy-deficient and neurodegenerative mutants of Drosophila. Ref(2)P is involved in maintenance of the viable mitochondria pool by acting downstream of Pink1 and Parkin, where Ref(2)P recycles excessive unfolded proteins via autophagy (Pimenta de Castro, 2012). This indicates a role for Ref(2)P as an autophagy receptor, similar to mammalian p62. Moreover, a recent report suggests that Ref(2)P is a selective autophagy substrate, but direct binding of Ref(2)P to DmAtg8a has not been shown. This study found that Ref(2)P interacts with DmAtg8a in a LC3-interacting region (LIR)-dependent manner. The Ref(2)P LIR also fulfills the requirement for a canonical LIR motif. The functional importance of the LIR motif in Ref(2)P was demonstrated by its requirement for accumulation of Ref(2)P in acidic vesicles and subsequent autophagic degradation (Jain, 2015).

Only the longest Cnc isoform, CncC, contains the Keap1-interacting DLG and ETGE motifs. Homology with the Neh2 domain of NRF2 suggests CncC to be the direct homologue of NRF2. Similar to NRF2, CncC is thought to interact with DmKeap1, and the activity of CncC is inhibited by DmKeap1. This study tested all three isoforms of Cnc (A, B, and C) for binding to DmKeap1, and only CncC interacted. The binding is mediated by the conserved ETGE motif. It has been reported both in humans and rodents that p62 binds directly to Keap1 using an ETGE-like motif, and this interaction positively regulates Nrf2 by blocking the interaction between Keap1 and Nrf2. A simple protein-protein interaction map involving p62/Ref(2)P, KEAP1/DmKeap1, NRF2/CncC, and ATG8/DmAtg8a highlights the major differences and similarities between humans and D. melanogaster. Surprisingly, no direct interaction was found between Ref(2)P and DmKeap1 in Drosophila. This was supported by reporter gene assays where Ref(2)P did not activate its own promoter, as p62 does in mammals. Recently, Ref(2)P and DmKeap1 were reported to be co-immunoprecipitated from Drosophila cells. This study found this to be mediated by the UBA domain of Ref(2)P, which probably recognized ubiquitinated DmKeap1. Hence, the direct KIR-mediated interaction between p62 and Keap1 evolved with the vertebrates, consistent with the lack of a KIR motif in p62 orthologues in non-chordate metazoans (Jain, 2015).

CncC has a central role in regulation of xenobiotic response, cellular stress, and electrophilic stress. CncC significantly induced the ref(2)P promoter, CncB gave no induction, and CncA had a significant negative effect. This is most probably due to the lack of the N-terminal transactivation domain found in CncC. Overexpressed CncC is a proteasome substrate, not detectable under normal conditions, but is stabilized by inhibition or depletion of proteasome subunit S5a. Consistently, it was not possible to easily detect overexpressed CncC by Western blot in cultured cells unless proteasomal degradation was inhibited. However, CncA and CncB were very well expressed under similar conditions. This is interesting, because only CncC has the DLG and ETGE motifs for Keap1 binding. This indicates that DmKeap1 may work as an E3 ligase adaptor protein to recruit CncC for its proteasomal degradation as found for mammalian Keap1. The current data suggest that CncA and -B may compete with CncC for DNA binding to the ARE on the ref(2)P promoter. This indicates a role for the CncA and CncB isoforms as competitive repressors of CncC under non-stress conditions, to maintain homeostasis in the D. melanogaster antioxidant defense system. This is in analogy to p65 (truncated isoform of Nrf1) and a caspase-cleaved form of Nrf2, which are bothreported to act as transcriptional repressors in vertebrates (Jain, 2015).

Consistent with the results from cell culture experiments, CncC overexpression induced ref(2)P and the target gene gstD in hindgut, wing discs, and fat bodies of D. melanogaster. These in vivo results strongly support the finding that ref(2)P is a target gene for CncC. Interestingly, overexpression of Ref(2)P did not induce an oxidative stress response, at least not as measured by gstD-GFP expression in hindgut and wing discs. This correlates with the lack of a ETGE-like motif in Ref(2)P and confirms the absence of a ref(2)P-mediated positive feedback loop in D. melanogaster (Jain, 2015).

Surprisingly, this study found a direct interaction between DmKeap1 and DmAtg8a. The canonical LIR-LDS interaction is dependent on both the N-terminal part (residues 1-28) and the C-terminal part (residues 30-125) of LC3B. However, the mode of the DmKeap1-DmAtg8a interaction seems different because DmKeap1 could bind to the N-terminal 71 amino acids of DmAtg8a(1-71). It has not been possible to map any motif mediating this non-canonical interaction. However, the interaction of DmKeap1 with DmAtg8a is interesting, both because DmKeap1 appears to recognize a different binding surface in DmAtg8a than Ref(2)P and other LIR-containing proteins and because the interaction might have an important role for DmAtg8a-mediated autophagic degradation of DmKeap1 during Drosophila development. Recent studies show mammalian Keap1 to be degraded by autophagy under nutritional starvation and oxidative stress in a p62-dependent manner, whereas degradation of mammalian Keap1 by basal autophagy has not been clearly demonstrated. Consistent with this, no degradation of DmKeap1 by basal autophagy was observed in Drosophila, but DmKeap1 was degraded under programmed autophagy during Drosophila development. The significance of the DmAtg8a-DmKeap1 interaction for the degradation of DmKeap1 by autophagy remains to be tested. The idea is favored that autophagic degradation of DmKeap1 depends on a co-recruitment of autophagy receptors like Ref(2)P, and this is strongly supported by the finding that Ref(2)P interacts with ubiquitinated DmKeap1 in cell culture. Possibly, a combined binding of Ref(2)P and DmKeap1 to DmAtg8 may help to increase the local concentration of DmAtg8 to secure an efficient encapsulation of the aggregate. However, further work is needed to reveal the underlying mechanism and significance of this interaction (Jain, 2015).

The finding that there is no positive feedback loop between CncC and Ref(2)P in flies was quite unexpected. However, the introduction of a KIR motif in p62 homologs during evolution of the most primitive fish, the amphioxus, suggests that the interdependent role of p62 and NRF2 in oxidative stress regulation developed early in vertebrate evolution. A related type of positive feedback loop has been reported for mammalian p62 and NFκB, predicting a putative cross-talk between NRF2 and NFκB pathways. Both gain of function mutations in NRF2 and loss of function mutations in Keap1 have been identified in human cancers and are believed to contribute to cancer cell survival and stress resistance upon cancer treatment. This study has found that loss of Keap1 or CncC gain of function induces Atg8a up-regulation and autophagy. These results are thought provoking, because autophagy, like NRF2 gain of function, has been shown to prevent initial tumor development. Once established, however, autophagy promotes cancer cell survival during stress conditions and cancer treatment. Under which physiological settings CncC-mediated control of autophagy may function remains an open question. The most obvious possibility is that CncC controls autophagy in response to reactive oxygen species. Previous studies have established that Drosophila utilizes a TRAF6/Atg9/Jun N-terminal kinase (JNK) stress pathway to activate autophagy upon oxidative stress provoked by hydrogen peroxide feeding. In concordance with those studies, this study found no evidence that CncC activity is required for autophagy induced upon hydrogen peroxide feeding. It remains possible that more subtle and physiological conditions of reactive oxygen species formation during aging, mitochondrial dysfunction, or oncogene-induced stress may enlist CncC in stress coping mechanisms involving autophagy. It will be interesting to pursue potential roles of CncC in in vivo cancer models (Jain, 2015).

Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 in Drosophila

Phagophore-derived autophagosomes deliver cytoplasmic material to lysosomes for degradation and reuse. Autophagy mediated by the incompletely characterized actions of Atg proteins is involved in numerous physiological and pathological settings including stress resistance, immunity, aging, cancer, and neurodegenerative diseases. This study characterized tg17/FIP200A, the Drosophila ortholog of mammalian RB1CC1/FIP200, a proposed functional equivalent of yeast Atg17. Atg17 disruption inhibits basal, starvation-induced and developmental autophagy, and interferes with the programmed elimination of larval salivary glands and midgut during metamorphosis. Upon starvation, Atg17-positive structures appear at aggregates of the selective cargo Ref(2)P/p62 near lysosomes. This location may be similar to the perivacuolar PAS (phagophore assembly site) described in yeast. Drosophila Atg17 is a member of the Atg1 kinase complex as in mammals, and it binds to the other subunits including Atg1, Atg13 and Atg101 (C12orf44 in humans, 9430023L20Rik in mice and RGD1359310 in rats). Atg17 is required for the kinase activity of endogenous Atg1 in vivo, as loss of Atg17 prevents the Atg1-dependent shift of endogenous Atg13 to hyperphosphorylated forms, and also blocks punctate Atg1 localization during starvation. Finally, it was found that Atg1 overexpression induces autophagy and reduces cell size in Atg17-null mutant fat body cells, and that overexpression of Atg17 promotes endogenous Atg13 phosphorylation and enhances autophagy in an Atg1-dependent manner in the fat body. A model is proposed according to which the relative activity of Atg1, estimated by the ratio of hyper- to hypophosphorylated Atg13, contributes to setting low (basal) vs. high (starvation-induced) autophagy levels in Drosophila (Nagy, 2014).

The putative HORMA domain protein Atg101 dimerizes and is required for starvation-induced and selective autophagy in Drosophila

The large-scale turnover of intracellular material including organelles is achieved by autophagy-mediated degradation in lysosomes. Initiation of autophagy is controlled by a protein kinase complex consisting of an Atg1-family kinase, Atg13, FIP200/Atg17, and the metazoan-specific subunit Atg101. This study show that loss of Atg101 impairs both starvation-induced and basal autophagy in Drosophila. This leads to accumulation of protein aggregates containing the selective autophagy cargo ref(2)P/p62. Mapping experiments suggest that Atg101 binds to the N-terminal HORMA domain of Atg13 and may also interact with two unstructured regions of Atg1. Another HORMA domain-containing protein, Mad2, forms a conformational homodimer. Drosophila Atg101 also dimerizes, and it is predicted to fold into a HORMA domain. Atg101 interacts with ref(2)P as well, similar to Atg13, Atg8a, Atg16, Atg18, Keap1, and RagC, a known regulator of Tor kinase which coordinates cell growth and autophagy. These results raise the possibility that the interactions and dimerization of the putative HORMA domain protein Atg101 play critical roles in starvation-induced autophagy and proteostasis, by promoting the formation of protein aggregate-containing autophagosomes (Hegedus, 2014).

Paternal mitochondrial destruction after fertilization is mediated by a common endocytic and autophagic pathway in Drosophila

Almost all animals contain mitochondria of maternal origin only, but the exact mechanisms underlying this phenomenon are still vague. This study investigated the fate of Drosophila paternal mitochondria after fertilization. The sperm mitochondrial derivative (MD) is rapidly eliminated in a stereotypical process dubbed paternal mitochondrial destruction (PMD). PMD is initiated by a network of vesicles resembling multivesicular bodies and displaying common features of the endocytic and autophagic pathways. These vesicles associate with the sperm tail and mediate the disintegration of its plasma membrane. Subsequently, the MD separates from the axoneme and breaks into smaller fragments, which are then sequestered by autophagosomes for degradation in lysosomes. Evidence is provided for the involvement of the ubiquitin pathway and the autophagy receptor p62 in this process. Finally, it was shown that the ubiquitin ligase Parkin is not involved in PMD, implying a divergence from the autophagic pathway of damaged mitochondria (Politi, 2014).

Drosophila ref(2)P is required for the parkin-mediated suppression of mitochondrial dysfunction in pink1 mutants

Autophagy is a critical regulator of organellar homeostasis, particularly of mitochondria. Upon the loss of membrane potential, dysfunctional mitochondria are selectively removed by autophagy through recruitment of the E3 ligase Parkin by the PTEN-induced kinase 1 (PINK1) and subsequent ubiquitination of mitochondrial membrane proteins. Mammalian sequestrome-1 (p62/SQSTM1) is an autophagy adaptor, which has been proposed to shuttle ubiquitinated cargo for autophagic degradation downstream of Parkin. This study shows that loss of Ref(2)P, the Drosophila orthologue of mammalian P62, results in abnormalities, including mitochondrial defects and an accumulation of mitochondrial DNA with heteroplasmic mutations, correlated with locomotor defects. Furthermore, expression of Ref(2)P is able to ameliorate the defects caused by loss of Pink1, and this depends on the presence of functional Parkin. Finally, both the PB1 and UBA domains of Ref(2)P are crucial for mitochondrial clustering. It is concluded that Ref(2)P is a crucial downstream effector of a pathway involving Pink1 and Parkin and is responsible for the maintenance of a viable pool of cellular mitochondria by promoting their aggregation and autophagic clearance (de Castro, 2013).

Mitochondrial dysfunction has been strongly associated with different neurodegenerative diseases, such as PD. Cells within complex multicellular organisms have developed quality-control mechanisms to cope with the many challenges that are constantly imposed on mitochondria and to suppress the accumulation of dysfunctional organelles. This study provides evidence that the Drosophila orthologue of p62, Ref(2)P is an important component of the Pink1/Parkin quality-control pathway (de Castro, 2013).

ref(2)P mutant flies exhibited several pathological and functional phenotypes reminiscent of those observed in the pink1 or parkin mutants. They exhibited the following: mitochondrial abnormalities of the sperm cells; defective locomotor activity; and a shorter lifespan. These defective phenotypes are more profound in the ref(2)P mutants lacking the UBA domain, suggesting that the ability of Ref(2)P to bind ubiquitinated targets is required for mitochondrial integrity and function. Despite the observed mitochondrial defects in the ref(2)P^od2 and ref(2)P^od3 mutants, no global alterations were seen in mitochondrial mass or function, suggesting that mitochondrial dysfunction in the ref(2)P mutants is not as pronounced as that observed in pink1 or parkin mutant flies. The observed decrease in motor performance from an early age suggests that any mitochondrial defects in ref(2)P mutants might preferentially affect tissues with high energetic demand such as the skeletal muscles and spermatids. It is therefore possible that such defects are undetectable in the respirometry assays, as these are performed using mitochondria derived from whole flies. There have been a number of reports of mtDNA point mutations associated with neurodegenerative diseases such as PD. This study shows that defects in ref(2)P, the single Drosophila P62 orthologue, result in an increase in mtDNA heteroplasmy. It is therefore conceivable that defects in mitophagy might contribute to neurodegenerative diseases such as PD by increasing the load of deleterious mtDNA mutations, leading eventually to increased mitochondrial dysfunction and an impairment of neuronal function (de Castro, 2013).

ref(2)P mutants exhibited a marked sensitivity to rotenone, an organic pesticide that directly targets respiration by inhibiting mitochondrial complex I. These mutants also showed a lower sensitivity to paraquat, an herbicide widely used in agriculture that has been linked to PD. Paraquat increases oxidative stress, whereas rotenone causes mitochondrial dysfunction; however, both processes are linked and both pesticides affect these mechanisms. Paraquat does not directly target mitochondria. In cells, it undergoes redox cycling in vivo to produce superoxide-free radicals that can damage not only these organelles but also other cellular components. It was therefore reasoned that, within this context, the Ref(2)P-dependent mitophagy might be particularly important in suppressing damage caused by PD-linked toxins whose mechanism of action directly targets mitochondrial function such as rotenone (de Castro, 2013).

It is noted that expression of ref(2)P is capable of suppressing pink1 but not parkin mutant phenotypes. This finding indicates that Ref(2)P exerts a protective effect downstream of Pink1 but requires the presence of functional Parkin. This is compatible with a model in which Ref(2)P recognises mitochondrial substrates ubiquitinated by Parkin and therefore, in the absence of Parkin, is incapable of recognising ubiquitin-decorated mitochondria and targeting them for autophagy. Parkin failed to restore the mitochondrial function of pink1 mutant flies in the absence of functional Ref(2)P, supporting the notion that Ref(2)P is a critical downstream effector of Parkin (de Castro, 2013).

Mutations in ref(2)P suppressed mitochondrial aggregation in the pink1 mutant flies. In mammalian cells, p62 has been suggested to mediate the aggregation of dysfunctional mitochondria into tight clusters, thereby minimising the surface area exposed to other cellular components. This study shows that this function of p62 is conserved in Drosophila and that Ref(2)P coordinates mitochondrial clustering through its PB1 and UBA domains. Reducing the surface area of impaired mitochondria within the cell by mitochondrial clustering may help minimise the uptake of respiratory substrates and limit the spread of mitochondrial ROS to other cellular compartments. Additionally, the clustering of the dysfunctional mitochondria could be beneficial to subcellular compartments with high-energy requirements, such as neuronal synapses, by preventing damaged mitochondria from being transported at the expense of bioenergetically active mitochondria. Alternatively, as p62 clustering of ubiquitinated substrates has been shown to cause cell death in the absence of its autophagic degradation, it is possible that, in Drosophila, Ref(2)P functions as a sensor of defective mitophagy quality control, triggering cell death when the removal of Parkin-ubiquitinated mitochondria is insufficient. This scenario could explain the suppression of the pink1 mutant phenotypes by ref(2)P expression (de Castro, 2013).

Finally, both Parkin and p62 are important regulators of mitophagy. Parkin is responsible for the autophagic elimination of damaged mitochondrial units. p62, on the other hand, binds directly to the autophagy protein LC3 and is believed to serve as an autophagy receptor for ubiquitinated protein aggregates as well as peroxisomes and intracellular bacteria. The data provide robust genetic evidence that inhibiting autophagy through mutations in Drosophila atg1 prevents both Parkin and Ref(2)P from exerting their protective effects on mitochondria (de Castro, 2013).

These data indicate that enhancing the autophagy pathway improved mitochondrial function in a Drosophila model of mitochondrial dysfunction, suggesting this pathway and clearance of damaged mitochondria as a potential therapeutic target in PD pathogenesis. This opens a promising avenue of exploring the role of autophagy-inducing agents in the prevention and treatment of neurodegenerative diseases, such as PD associated with mitochondrial dysfunction (de Castro, 2013).

The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins

Initially described as a nonspecific degradation process induced upon starvation, autophagy is now known also to be involved in the degradation of specific ubiquitinated substrates such as mitochondria, bacteria and aggregated proteins, ensuring crucial functions in cell physiology and immunity. This study reports that the deubiquitinating enzyme USP36 controls selective autophagy activation in Drosophila and in human cells. dUsp36 loss of function autonomously inhibits cell growth while activating autophagy. Despite the phenotypic similarity, dUSP36 is not part of the TOR signaling pathway. Autophagy induced by dUsp36 loss of function depends on p62/SQSTM1, an adaptor for delivering cargo marked by polyubiquitin to autophagosomes. Consistent with p62 requirement, dUsp36 mutant cells display nuclear aggregates of ubiquitinated proteins, including Histone H2B, and cytoplasmic ubiquitinated proteins; the latter are eliminated by autophagy. Importantly, USP36 function in p62-dependent selective autophagy is conserved in human cells. This work identifies a novel, crucial role for a deubiquitinating enzyme in selective autophagy (Taillebourg, 2012).

p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects

Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. This study shows that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique it was shown that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. These findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells (Bartlett, 2011).

The PI 3-kinase regulator Vps15 is required for autophagic clearance of protein aggregates

Autophagy is involved in cellular clearance of aggregate-prone proteins, thereby having a cytoprotective function. Studies in yeast have shown that the PI 3-kinase Vps34 and its regulatory protein kinase Vps15 are important for autophagy, but the possible involvement of these proteins in autophagy in a multicellular animal has not been addressed genetically. This study created a Drosophila deletion mutant of vps15 and investigated its role in autophagy and aggregate clearance. Homozygous δvps15 Drosophila died at the early L3 larval stage. Using GFP-Atg8a as an autophagic marker, fluorescence microscopy was employed to demonstrate that fat bodies of wild type Drosophila larvae accumulated autophagic structures upon starvation whereas δvps15 fat bodies showed no such response. Likewise, electron microscopy revealed starvation-induced autophagy in gut cells from wild type but not δvps15 larvae. Fluorescence microscopy showed that δvps15 mutant tissues accumulated profiles that were positive for ubiquitin and Ref(2)P, the Drosophila homolog of the sequestosome marker SQSTM1/p62. Biochemical fractionation and Western blotting showed that these structures were partially detergent insoluble, and immuno-electron microscopy further demonstrated the presence of Ref(2)P positive membrane free protein aggregates. These results provide the first genetic evidence for a function of Vps15 in autophagy in multicellular organisms and suggest that the Vps15- containing PI 3-kinase complex may play an important role in clearance of protein aggregates (Lindmo, 2008).

Studies of the involvement of specific PI3Ks in autophagy in higher organisms such as Drosophila and mammals by pharmacological PI3K inhibitors have been complicated by the fact that these animals express multiple classes of PI3Ks that may have opposing roles. It has been found that class I PI3K represses autophagy during the early larval stages in Drosophila, and that its downregulation in response to ecdysone signaling triggers developmental autophagy. The present study sought to clarify the possible involvement of class III PI3K in autophagy and aggregate clearance by generating a Drosophila mutant in which the gene for the regulatory Vps15 subunit was deleted. The δvps15 mutant larvae turned out to be defective for starvation induced autophagy. Importantly, vps15 mutant animals accumulated detergent-soluble and -insoluble structures that are likely to represent endosomes and sequestosomes, respectively. This provides evidence for the involvement of Vps15 in autophagy and aggregate clearance in metazoans (Lindmo, 2008).

The only PI3K in S. cerevisae, Vps34, can participate in two distinct protein complexes; one consisting of Vps34, Vps15, Vps30 and Vps38 that functions in vacuolar protein sorting and one consisting of Vps34, Vps15, Vps30 and Atg14 that functions in autophagy (Kihara, 2001). So far, no metazoan homolog of Atg14 has been reported, whereas metazoan homologs of Vps34, Vps15 and Vps30 are known. Of these, the Vps30 homolog, Beclin-1, an interactor of the antiapoptotic proteins Bcl-2 and Bcl-XL, has been most studied for its role in autophagy in metazoans. Overexpression of Beclin-1 in MCF7 breast carcinoma cells promotes autophagy and inhibits cell proliferation, whereas its depletion promotes apoptosis. The possible role of Beclin-1 in aggregate clearance has not been investigated, nor have metazoan Vps34 and Vps15 been studied in this context. It was therefore considered important to study whether the metazoan Vps34-Vps15 subcomplex is required for autophagy and aggregate clearance. Because of the availability of appropriate Drosophila FRT strains, a specific deletion of the vps15 gene was generated in Drosophila. The inhibition of starvation-induced autophagy in gut and fat body tissues of δvps15 larvae demonstrates the importance of the Vps15 for autophagy in metazoans. Most importantly, the accumulation of protein aggregates in the δvps15 mutants shows that this complex is critically required for normal clearance of such aggregates (Lindmo, 2008).

The polyubiquitin binding p62 protein accumulates strongly on ubiquitin-positive protein aggregates and serves as a reporter for such structures. Protein aggregates are not formed in p62/Ref(2)P mutants and the fact that p62 binds directly to the mammalian Atg8 homolog LC3 and recruits it to ubiquitin-positive aggregates suggests that p62 may serve to mark the protein aggregates for autophagic degradation. The present report used antibodies against conjugated ubiquitin and the Drosophila homolog of p62, Ref(2)P, as a marker for protein aggregates. Although Ref(2)P was originally identified as a factor involved in male fertility and sigma virus replication, it contains all the structural hallmarks of a p62 ortholog, including the PB1, ZZ and UBA domains. Interestingly, δvps15 Drosophila larvae accumulated numerous Ref(2)P-positive structures, indicative of impaired metabolism of protein aggregates. Consistent with this, the δvps15 mutants also accumulated ubiquitin-positive structures. Because depletion of certain proteins involved in endocytic trafficking causes the accumulation of ubiquitinated membrane proteins in early endosomes, some of the ubiquitin- and Ref(2)P positive profiles might correspond to endosomes. This is supported by the finding that a fraction of the ubiquitin- and Ref(2)P-positive structures could be solubilized in Tx. Because confocal and electron microscopy indicated that Ref(2)P is preferentially found in membrane-free structures in δvps15 mutants, an alternative explanation for the partial Tx solubility of ubiquitin- and Ref(2)P-positive structures may be that smaller accumulations of aggregating proteins are Tx soluble. In any case, a substantial fraction of the ubiquitin- and Ref(2)P positive structures that accumulated in δvps15 mutants were Tx insoluble, strongly suggesting that protein aggregates accumulate in the absence of Vps15. The ultrastructural appearance of these aggregates has striking resemblance to Ref(2)P positive structures found in neuronal tissue of atg8 mutant flies. In both cases, accumulation of vesicular structures surrounding a densely labeled matrix was observed. This might indicate that either the recruitment of autophagic membranes onto or their functional elongation around protein aggregates is dependent on both Atg8 function and PI3K class III activity (Lindmo, 2008).

In conclusion, this study has shown that the PI3K class III co-activator, Vps15, is required for autophagy in Drosophila. δvps15 mutant tissues accumulate Tx-insoluble ubiquitin and Ref(2)P positive structures, indicating a role of Vps15 in autophagic clearance of aggregate-prone proteins. Given that enhanced autophagy can inhibit aggregate-induced neurodegeneration in Huntington models, neuronal-specific stimulation of the Vps34-Vps15 complex might provide a prospective strategy for developing drugs against neurodegenerative diseases (Lindmo, 2008).

Genetic inactivation of p62 leads to accumulation of hyperphosphorylated tau and neurodegeneration

The signaling adapter p62 plays a coordinating role in mediating phosphorylation and ubiquitin-dependent trafficking of interacting proteins. However, there is little known about the physiologic role of this protein in brain. This study reports age-dependent constitutive activation of glycogen synthase kinase 3beta, protein kinase B, mitogen-activated protein kinase, and c-Jun-N-terminal kinase in adult p62(-/-) mice resulting in hyperphosphorylated tau, neurofibrillary tangles, and neurodegeneration. Biochemical fractionation of p62(-/-) brain led to recovery of aggregated K63-ubiquitinated tau. Loss of p62 was manifested by increased anxiety, depression, loss of working memory, and reduced serum brain-derived neurotrophic factor levels. These findings reveal a novel role for p62 as a chaperone that regulates tau solubility thereby preventing tau aggregation. This study provides a clear demonstration of an Alzheimer-like phenotype in a mouse model in the absence of expression of human genes carrying mutations in amyloid-beta protein precursor, presenilin, or tau. Thus, these findings provide new insight into manifestation of sporadic Alzheimer disease and the impact of obesity (Ramesh Babu, 2008).

The Drosophila atypical protein kinase C-ref(2)p complex constitutes a conserved module for signaling in the toll pathway

Recent results have demonstrated the critical role of the mammalian p62-atypical protein kinase C (aPKC) complex in the activation of NF-kappaB in response to different stimuli. Using the RNA interference technique on Schneider cells it has been shown that Drosophila aPKC (DaPKC) is required for the stimulation of the Toll-signaling pathway, which activates the NF-kappaB homologs Dif and Dorsal. However, DaPKC does not appear to be important for the other Drosophila NF-kappaB signaling cascade, which activates the NF-kappaB homolog Relish in response to lipopolysaccharides. Interestingly, DaPKC functions downstream of the nuclear translocation of Dorsal or Dif, controlling the transcriptional activity of the Drosomycin promoter. The Drosophila Ref(2)P protein is the homolog of mammalian p62, since it binds to DaPKC: its overexpression is sufficient to activate the Drosomycin but not the Attacin promoter, and its depletion severely impairs Toll signaling. Collectively, these results demonstrate the conservation of the p62-aPKC complex for the control of innate immunity signal transduction in Drosophila melanogaster (Avila, 2002).

Drosophila represents an ideal system in which to determine the primary role of the aPKCs in NF-kappaB signal transduction because it encodes only one aPKC isoform. According to the data presented in this study, aPKC is selectively required for the innate immune Toll-signaling pathway, acting downstream of the translocation of Dorsal and Dif and playing a critical role in the induction (a typical NF-kappaB-dependent process) of the antimicrobial peptide gene for Drosomycin. Therefore, it can be argued that the primary role of the aPKCs, particularly that of zetaPKC in higher eukaryotic cells, is to somehow control the transcriptional activity of NF-kappaB through a still not completely understood mechanism that most likely involves the direct phosphorylation of RelA and Dif. Interestingly, in Drosophila it is well documented that the phosphorylation of Dorsal is required not only for its transcriptional activity but also for its nuclear translocation. In Drosophila aPKC-depleted cells, a strong inhibition of Dorsal or Dif nuclear translocation is not observed, suggesting that the role of Drosophila aPKC is independent of the previously characterized role for Dorsal phosphorylation in regulating nuclear translocation. Based on experiments in mammalian systems, which demonstrate that p65 transcriptional activity must be stimulated by phosphorylation, it is possible that the residues that control the transcriptional activities of both Dorsal and Dif are different from those controlling the nuclear import of the protein. It is also possible that Drosophila aPKC-mediated phosphorylation has a subtle, yet important, role in the nuclear translocation of Dif and/or Dorsal. Future studies will address this important issue (Avila, 2002).

These studies also demonstrate that Ref(2)P is most likely the functional homolog of p62 in Drosophila. Like p62, Ref(2)P interacts physically with the aPKCs. Therefore, it appears that the p62-aPKC signaling module, like the Par/aPKC complex, is highly conserved. Importantly, a functional role of Ref(2)P in Toll signaling is demonstrated. Thus, the ectopic expression of Ref(2)P is capable by itself of activating the Drosomycin promoter. More interestingly, its depletion severely impairs the Toll pathway (Drosomycin induction) but not the LPS pathway (Attacin induction). Thus, the Ref(2)P/DaPKC complex is critical for Toll signaling (Avila, 2002).

The results presented here also demonstrate that, similar to the p62-TRAF6 connection in mammals, Ref(2)P and Drosophila TRAF2 physically and functionally interact. Together with the results demonstrating that Drosophila aPKC and Ref(2)P are essential for a downstream event in the Toll-signaling pathway, this suggests that a putative Ref(2)P/aPKC/TRAF2 complex might function in the signal-induced stimulation of Dif or Dorsal transcriptional activity. In this regard, it is noteworthy that recent results suggest that TRAF6, in addition to its role in IKK recruitment and activation, may also be involved in the control of RelA transcriptional activity. However, the role of Drosophila TRAF2 in Toll signaling requires further investigation, since the effect of inhibiting (or mutating) TRAF2 has not yet been reported. Further studies will also address the precise mechanism whereby aPKC controls the Toll pathway. The data presented here clearly establish the conserved role of the homolog of the p62/aPKC cassette in NF-kappaB signaling in Drosophila (Avila, 2002).

Molecular analysis of ref(2)P, a Drosophila gene implicated in sigma rhabdovirus multiplication and necessary for male fertility

The ref(2)P gene of Drosophila melanogaster is implicated in sigma rhabdovirus multiplication. A permissive allele was cloned and sequenced. The structural gene (3.1 kbp) is divided into three exons. The mRNAs are heterogeneous in size. They differ only in the 5' end of the first exon. The sequence upstream of the short mRNAs contains classical promoter elements. No TATA and CAAT boxes are appropriately positioned upstream of the initiation sites of the long mRNAs, but several repeats, palindromic sequences and inverted CAAT boxes are present. These observations, together with the tissue-dependent distribution of short and long transcripts, support the hypothesis of the existence of at least two classes of genuine initiation sites. The long size of the untranslated leader RNA region suggests a control of gene expression at the translation level. The same translation product of 599 amino acids (76.3 kd) is predicted for all mRNAs, but the in vitro translation product migrates in SDS-PAGE with a higher apparent mol. wt (115-125 kd). The putative ref(2)P protein contains internal repeats, PEST regions which may be signals for protein degradation, and interesting structural motifs such as zinc finger and amphiphilic helices. These later motifs could be mitochondrial pre-sequences. The degeneration of mitochondria is observed in the spermatids of sterile male flies homozygous for the loss-of-function alleles. The amino acid sequence of the ref(2)P product shows no homology with any known protein from the data banks (Dezelee, 1989).

Search PubMed for articles about Drosophila ref(2P)

Aparicio, R., Rana, A. and Walker, D. W. (2019). Upregulation of the autophagy adaptor p62/SQSTM1 prolongs health and lifespan in middle-aged Drosophila. Cell Rep 28(4): 1029-1040. PubMed ID: 31340141

Avila, A, Silverman, N, Diaz-Meco, M. T. and Moscat, J. (2002). The Drosophila atypical protein kinase C-ref(2)p complex constitutes a conserved module for signaling in the toll pathway. Mol. Cell Biol. 22(24): 8787-95. 12446795

Bartlett, B. J., Isakson, P., Lewerenz, J., Sanchez, H., Kotzebue, R. W., Cumming, R. C., Harris, G. L., Nezis, I. P., Schubert, D. R., Simonsen, A. and Finley, K. D. (2011). p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects. Autophagy 7(6): 572-583. PubMed ID: 21325881

Bhide, S., Trujillo, A. S., O'Connor, M. T., Young, G. H., Cryderman, D. E., Chandran, S., Nikravesh, M., Wallrath, L. L. and Melkani, G. C. (2018). Increasing autophagy and blocking Nrf2 suppress laminopathy-induced age-dependent cardiac dysfunction and shortened lifespan. Aging Cell: e12747. PubMed ID: 29575479

Brooks, D., Naeem, F., Stetsiv, M., Goetting, S. C., Bawa, S., Green, N., Clark, C., Bashirullah, A. and Geisbrecht, E. R. (2020). Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover. PLoS Genet 16(4): e1008700. PubMed ID: 32320396

Carroll, B., Otten, E. G., Manni, D., Stefanatos, R., Menzies, F. M., Smith, G. R., Jurk, D., Kenneth, N., Wilkinson, S., Passos, J. F., Attems, J., Veal, E. A., Teyssou, E., Seilhean, D., Millecamps, S., Eskelinen, E. L., Bronowska, A. K., Rubinsztein, D. C., Sanz, A. and Korolchuk, V. I. (2018). Oxidation of SQSTM1/p62 mediates the link between redox state and protein homeostasis. Nat Commun 9(1): 256. PubMed ID: 29343728

Chang, S., Kim, J. H. and Shin, J. (2002). p62 forms a ternary complex with PKCzeta and PAR-4 and antagonizes PAR-4-induced PKCzeta inhibition. FEBS Lett. 510(1-2): 57-61. 11755531

Cunningham, K. M., Maulding, K., Ruan, K., Senturk, M., Grima, J. C., Sung, H., Zuo, Z., Song, H., Gao, J., Dubey, S., Rothstein, J. D., Zhang, K., Bellen, H. J. and Lloyd, T. E. (2020). TFEB/Mitf links impaired nuclear import to autophagolysosomal dysfunction in C9-ALS. Elife 9. PubMed ID: 33300868

de Castro, I. P., Costa, A. C., Celardo, I., Tufi, R., Dinsdale, D., Loh, S. H. and Martins, L. M. (2013). Drosophila ref(2)P is required for the parkin-mediated suppression of mitochondrial dysfunction in pink1 mutants. Cell Death Dis 4: e873. PubMed ID: 24157867

Deehan, M., Lin, W., Blum, B., Emili, A. and Frydman, H. (2021). Intracellular density of Wolbachia is mediated by host autophagy and the bacterial cytoplasmic incompatibility gene cifB in a cell type-dependent manner in Drosophila melanogaster. mBio 12(1). PubMed ID: 33436431

Deng, Z., Lim, J., Wang, Q., Purtell, K., Wu, S., Palomo, G. M., Tan, H., Manfredi, G., Zhao, Y., Peng, J., Hu, B., Chen, S. and Yue, Z. (2020). ALS-FTLD-linked mutations of SQSTM1/p62 disrupt selective autophagy and NFE2L2/NRF2 anti-oxidative stress pathway. Autophagy 16(5): 917-931. PubMed ID: 31362587

Dezelee, S., Bras, F., Contamine, D., Lopez-Ferber, M., Segretain, D. and Teninges, D. (1989). Molecular analysis of ref(2)P, a Drosophila gene implicated in sigma rhabdovirus multiplication and necessary for male fertility. EMBO J 8(11): 3437-3446. PubMed ID: 2510997

Dialynas, G., Shrestha, O. K., Ponce, J. M., Zwerger, M., Thiemann, D. A., Young, G. H., Moore, S. A., Yu, L., Lammerding, J. and Wallrath, L. L. (2015). Myopathic lamin mutations cause reductive stress and activate the nrf2/keap-1 pathway. PLoS Genet 11(5): e1005231. PubMed ID: 25996830

Formica, M., Storaci, A. M., Bertolini, I., Carminati, F., Knaevelsrud, H., Vaira, V. and Vaccari, T. (2021). V-ATPase controls tumor growth and autophagy in a Drosophila model of gliomagenesis. Autophagy: 1-11. PubMed ID: 33978540

Geisler, S., Holmstrom, K. M., Skujat, D., Fiesel, F. C., Rothfuss, O. C., Kahle, P. J. and Springer, W. (2010). PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat Cell Biol 12(2): 119-131. PubMed ID: 20098416

Hegedus, K., Nagy, P., Gaspari, Z. and Juhasz, G. (2014). The putative HORMA domain protein Atg101 dimerizes and is required for starvation-induced and selective autophagy in Drosophila. Biomed Res Int 2014: 470482. PubMed ID: 24895579

Hurley, E. P. and Staveley, B. E. (2021). Inhibition of Ref(2)P, the Drosophila homologue of the p62/SQSTM1 gene, increases lifespan and leads to a decline in motor function. BMC Res Notes 14(1): 53. PubMed ID: 33557921

Issa, A. R., Sun, J., Petitgas, C., Mesquita, A., Dulac, A., Robin, M., Mollereau, B., Jenny, A., Cherif-Zahar, B. and Birman, S. (2018). The lysosomal membrane protein LAMP2A promotes autophagic flux and prevents SNCA-induced Parkinson disease-like symptoms in the Drosophila brain. Autophagy. PubMed ID: 29989488

Jain, A., Rusten, T. E., Katheder, N., Elvenes, J., Bruun, J. A., Sjottem, E., Lamark, T. and Johansen, T. (2015). p62/sequestosome-1, Autophagy-related Gene 8, and autophagy in Drosophila are regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), independent of transcription factor TFEB. J Biol Chem 290: 14945-14962. PubMed ID: 25931115

Jewett, K. A., Thomas, R. E., Phan, C. Q., Lin, B., Milstein, G., Yu, S., Bettcher, L. F., Neto, F. C., Djukovic, D., Raftery, D., Pallanck, L. J. and Davis, M. Y.(2021). Glucocerebrosidase reduces the spread of protein aggregation in a Drosophila melanogaster model of neurodegeneration by regulating proteins trafficked by extracellular vesicles. PLoS Genet 17(2): e1008859. PubMed ID: 33539341

Kwon, J., Han, E., Bui, C. B., Shin, W., Lee, J., Lee, S., Choi, Y. B., Lee, A. H., Lee, K. H., Park, C., Obin, M. S., Park, S. K., Seo, Y. J., Oh, G. T., Lee, H. W. and Shin, J. (2012). Assurance of mitochondrial integrity and mammalian longevity by the p62-Keap1-Nrf2-Nqo1 cascade. EMBO Rep 13(2): 150-156. PubMed ID: 22222206

Lindmo, K., et al. (2008). The PI 3-kinase regulator Vps15 is required for autophagic clearance of protein aggregates. Autophagy 4(4): 500-6. PubMed ID: 18326940

Moscat, J. and Diaz-Meco, M. T. (2012). p62: a versatile multitasker takes on cancer. Trends Biochem Sci 37(6): 230-236. PubMed ID: 22424619

Nagai, H., Tatara, H., Tanaka-Furuhashi, K., Kurata, S. and Yano, T. (2021). Homeostatic regulation of ROS-triggered Hippo-Yki pathway via autophagic clearance of Ref(2)P/p62 in the Drosophila intestine. Dev Cell 56(1): 81-94. PubMed ID: 33400912

Nagy, P., Karpati, M., Varga, A., Pircs, K., Venkei, Z., Takats, S., Varga, K., Erdi, B., Hegedus, K. and Juhasz, G. (2014). Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 in Drosophila. Autophagy 10. PubMed ID: 24419107

Nilangekar, K., Murmu, N., Sahu, G. and Shravage, B. V. (2019). Generation and characterization of germline-specific autophagy and mitochondrial reactive oxygen species reporters in Drosophila. Front Cell Dev Biol 7: 47. PubMed ID: 31001531
Politi, Y., Gal, L., Kalifa, Y., Ravid, L., Elazar, Z. and Arama, E. (2014). Paternal mitochondrial destruction after fertilization is mediated by a common endocytic and autophagic pathway in Drosophila. Dev Cell 29: 305-320. PubMed ID: 24823375

Ramesh Babu, J., Lamar Seibenhener, M., Peng, J., Strom, A. L., Kemppainen, R., Cox, N., Zhu, H., Wooten, M. C., Diaz-Meco, M. T., Moscat, J. and Wooten, M. W. (2008). Genetic inactivation of p62 leads to accumulation of hyperphosphorylated tau and neurodegeneration. J Neurochem 106(1): 107-120. PubMed ID: 18346206

Ray, A., Speese, S. D. and Logan, M. A. (2017). Glial Draper rescues Abeta toxicity in a Drosophila model of Alzheimer's Disease. J Neurosci 37(49):11881-11893. PubMed ID: 29109235

Salazar, G., Cullen, A., Huang, J., Zhao, Y., Serino, A., Hilenski, L., Patrushev, N., Forouzandeh, F. and Hwang, H. S. (2020). SQSTM1/p62 and PPARGC1A/PGC-1alpha at the interface of autophagy and vascular senescence. Autophagy 16(6): 1092-1110. PubMed ID: 31441382

Samuels, I. S., Seibenhener, M. L., Neidigh, K. B. and Wooten M. W. (2001). Nerve growth factor stimulates the interaction of ZIP/p62 with atypical protein kinase C and targets endosomal localization: evidence for regulation of nerve growth factor-induced differentiation. J. Cell Biochem. 82(3): 452-66. 11500922

Sanchez, P., De Carcer, G., Sandoval, I. V., Moscat, J. and Diaz-Meco, M. T. (1998). Localization of atypical protein kinase C isoforms into lysosome-targeted endosomes through interaction with p62. Mol. Cell Biol. 18(5): 3069-80. 9566925

Sanz, L., Sanchez, P., Lallena, M. J., Diaz-Meco, M. T., and Moscat, J. (1999). The interaction of p62 with RIP links the atypical PKCs to NF-kappaB activation. EMBO J. 18: 3044-3053. 10356400

Sanz, L., Diaz-Meco, M. T, Nakano, H. and Moscat, J. (2000). The atypical PKC-interacting protein p62 channels NF-kappaB activation by the IL-1-TRAF6 pathway. EMBO J. 19(7): 1576-86. 10747026

Taillebourg, E., Gregoire, I., Viargues, P., Jacomin, A. C., Thevenon, D., Faure, M. and Fauvarque, M. O. (2012). The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins. Autophagy 8(5): 767-779. PubMed ID: 22622177

Vonk, J. J., et al. (2017). Drosophila Vps13 Is required for protein homeostasis in the brain. PLoS One 12(1): e0170106. PubMed ID: 28107480

Wang, Z. H., Clark, C. and Geisbrecht, E. R. (2016). Drosophila Clueless is involved in Parkin-dependent mitophagy by promoting VCP-mediated Marf degradation. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26931463

Wilson, M. I., et al. (2003). PB1 domain-mediated heterodimerization in NADPH oxidase and signaling complexes of atypical protein kinase C with Par6 and p62. Molec. Cell 12: 39-50. 12887891

Wooten, M. W., et al. (2001). The atypical protein kinase C-interacting protein p62 is a scaffold for NF-kappaB activation by nerve growth factor. J. Biol. Chem. 276(11): 7709-12. 11244088

Wu, W., Han, Y., Fan, X., Li, Q. and Sun, L. (2019). Protective mechanism of Wnt4 gene on Parkinson's disease (PD) transgenic Drosophila. Int J Neurosci 129(7): 703-714. PubMed ID: 30526176

date revised: 18 June 2021

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