InteractiveFly: GeneBrief

PNGase-like: Biological Overview | References

It remains unknown how intracellular glycosylation, O-GlcNAcylation, interfaces with cellular components of the extracellular glycosylation machinery, like the cytosolic N-glycanase NGLY1. This study utilized the Drosophila gut and uncovered a pathway in which O-GlcNAcylation cooperates with the NGLY1 homologue PNG1 to regulate proliferation in intestinal stem cells (ISCs) and apoptosis in differentiated enterocytes. Further, the CncC antioxidant signaling pathway and ENGase, an enzyme involved in the processing of free oligosaccharides in the cytosol, interact with O-GlcNAc and PNG1 through regulation of protein aggregates to contribute to gut maintenance. These findings reveal a complex coordinated regulation between O-GlcNAcylation and the cytosolic glycanase PNG1 critical to balancing proliferation and apoptosis to maintain gut homeostasis (Na, 2022).

Intestinal stem cells regulate tissue homeostasis by balancing self-renewal, proliferation, and differentiation all of which are supported by elevated flux through the hexosamine biosynthetic pathway (HBP). Both N-linked glycosylation and intracellular O-GlcNAc modifications are regulated by the HBP pathway in a nutrient-sensing manner. However, how NGLY1 is utilized to control stem cell homeostasis and differentiation in cells remains largely unknown. This is a critical question as patients with NGLY1-deficiency display global developmental delay, movement disorder and growth retardation. Elevation of NGLY1 was observed in patients' tumor samples, suggesting a function in oncogenic signaling. In Drosophila, PNG1 mutants had severe developmental defects and reduced viability, with the surviving adults frequently sterile (Funakoshi, 2010). This study has identified a pathway by which PNG1 regulates ISC homeostasis in vivo. This study shows that PNG1 levels increased in ISC/EBs concomitant with O-GlcNAc. This interaction between PNG1 and O-GlcNAcylation is critical for maintaining normal ISC proliferation and differentiation. Thus, through their mutual regulation, OGT and PNG1 have key roles in both progenitor (ISCs/EBs) and differentiated cells (ECs) contributing to tissue homeostasis. Previous reports indicated that PNG1 null larvae have specific developmental abnormalities in their midgut that contributes to their lethality. Further, intestinal inflammation in Crohn's disease is associated with increased O-GlcNAc modification. A previous study also showed that increased O-GlcNAc promotes gut dysplasia through regulation of DNA damage (Na, 2020). Thus, PNG1 or O-GlcNAc might still be associated with gut dysfunction in a disease context (Na, 2022).

The regulation of O-GlcNAc by PNG1 and the interaction between PNG1 and O-GlcNAc has been implicated previously. In fact, GlcNAc supplementation partially rescued lethality associated with PNG1 knockdown (Funakoshi, 2010). Although the mechanism by which GlcNAc supplementation rescued these mutant flies has not been fully worked out, Gfat1 transcript levels were downregulated in PNG1 knockdown flies (Funakoshi, 2010). Gfat1 is the enzyme that controls the rate limiting step in the HBP to produce UDP-GlcNAc. Thus, PNG1 through regulation of Gfat1 could impact levels of UDP-GlcNAc and ultimately O-GlcNAc. Additionally, it has been hypothesized that the loss of PNG1 could increase the presence of intracellular N-GlcNAc modification, potentially interfering with O-GlcNAc mediated signaling. Therefore, alterations in UDP-GlcNAc levels or presence of intracellular N-GlcNAc upon PNG1-deficiency can interact with O-GlcNAc to regulate stem cell homeostasis (Na, 2022).

Previous reports have shown that Nrf1 undergoes NGLY1-mediated deglycosylation, followed by proteolytic cleavage and translocation into the nucleus as an active transcription factor. Loss of NGLY1 caused Nrf1 dysfunction, as evidenced by an enrichment of deregulated genes encoding proteasome components and proteins involved in oxidation reduction. Proteasome activity can induce an apoptotic cascade that leads to growth arrest and, subsequently, cell death. The current data indicated that PNG1 or OGT knockdown suppressed ISC proliferation, which was rescued by Oltipraz (CncC activation) treatment in ISCs/EBs. Furthermore, there was increased apoptosis in PNG1 or OGT knockdown with treatment compared to non-treated groups. Interestingly, it was found O-GlcNAc-induced intestinal dysplasia was rescued by knockdown of PNG1 in ISCs/EBs through regulation of ROS levels. Similarly, increases in global O-GlcNAcylation in embryos of diabetic mice caused an overproduction of ROS and subsequent oxidative and ER stress. It is known that activation of SKN-1A/Nrf1 also requires deglycosylation by PNG-1/NGLY1 in C. elegans. Further, SKN-1 is O-GlcNAc modified and translocates to the nucleus in ogt-1(ok430)-null worms. Together, these studies all suggest conserved functional connections between O-GlcNAc and Nrf family transcription factors. This study also showed EC-specific OGT or PNG1 knockdown-induced hyperproliferation and cell death was decreased by CncC activation. This data indicated OGT or PNG1 can be regulated by CncC activity in ISCs/EBs and ECs. CncC has high activity within ISCs/EBs of unstressed as well young ISCs and quiescent ISCs but decreases with age and damage. These data indicated that CncC acts to properly balance between signaling and damage responses necessary for tissue homeostasis. CncC activation increased ISC proliferation in ISCs/EBs and decreased ISC proliferation in ECs of OGT or PNG1 knockdown contributing towards tissue homeostasis. Another study showed that inhibition of NGLY1 resulted in Nrf1 being misprocessed, mislocated, and inactive, thus indicating that functional NGLY1 is essential for Nrf1 processing, nuclear translocation, and transcription factor activity (Tomlin, 2017). Therefore, the data suggests that PNG1 and OGT modulated by CncC activation contribute to ISC proliferation and ultimately regulating tissue homeostasis. Nrf2 activation was able to rescue the developmental growth of NGLY1 deficiency in worm and fly models. In cancer-initiating cells, ER stress-dependent (ROS-independent) CncC induction is an event necessary to maintain stemness. The data showed that PNG1 knockdown-induced Poly-UB accumulation and 26S proteasome expression that was rescued by CncC overexpression and chemical activation. Through functioning as a sensor of cytosolic proteasome activity and an activator of aggresomal formation, Nrf2 alleviates cell damages caused by proteasomal stress. Expression of proteasome subunit genes and mitophagy-related genes were broadly enhanced after sulforaphane (Keap1 inhibitor) treatment and pharmacologically induction of Nrf2 promotes mitophagy and ameliorates mitochondrial defect in Ngly1-/- cells. Thus, it is believed that the sensitized background of the OGT or PNG1 mutant provides an environment where CncC activation promotes proliferation to the normal level through regulation of proteasome activity and protein aggregation (Na, 2022).

In a previously published paper (Ha, 2020), it was shown that OGT overexpression and OGA knockdown in ISCs/EBs both increased O-GlcNAc levels and induced hyperproliferation of the stem cells, whereas OGT knockdown decreased proliferation. However, in differentiated ECs, OGT overexpression and OGA knockdown phenotypes were similar to the normal gut, whereas OGT knockdown elevated proliferation and cell death. In general, EC death promotes proliferation in order to maintain gut homeostasis. Here, NGLY1 knockdown in ISCs/EBs decreased proliferation and clone size but NGLY1 knockdown in ECs induced hyperproliferation and cell death and importantly decreased O-GlcNAc levels. Thus, the phenotypes of OGT and NGLY1 were similar, demonstrating that maintenance of OGT and NGLY1 protein expression is highly interdependent for the maintenance of tissue homeostasis. It is interesting that the progenitor and differentiated cell types within the gut respond differently to changes in O-GlcNAc. It is possible that a certain level of O-GlcNAcylation is needed to maintain stem cells and promote proliferation and self-renewal, however, differentiated cells that do not have the same energy and growth requirements are not as reliant on high levels of O-GlcNAc. On the other hand, both ISC/EBs and ECs require some level of O-GlcNAc and without OGT there is decreased proliferation in progenitor cells and increased cell death of ECs. There are a few possibilities how NGLY1 and OGT can collaboratively work, however, it is unlikely that they share protein targets. First, a previous publication showed that additional deletion of ENGase, another N-deglycosylating enzyme that leaves a single GlcNAc residue, alleviates some of the lethality of Ngly1-deficient mice. Thus, it is possible with the accumulation of aggregation prone intracellular N-GlcNacylated proteins, there is disruption of normal O-GlcNac signaling. These data also showed increased protein aggregation in OGT or NLGY1 knockdown that was rescued by ENGase knockdown. In addition, MYC-OGT protein levels in OGT overexpression fly guts were decreased by PNG1 knockdown. It is possible that loss of NGLY1 disrupts normal OGT degradation and thus impacts levels global of O-GlcNAcylation (Na, 2022).

This study has shown that ENGase levels increased in PNG1 or OGT knockdown ISCs/EBs and ECs. PNGase is involved in the process of endoplasmic reticulum associated degradation (ERAD), acting as a deglycosylating enzyme that cleaves N-glycans attached to ERAD substrates. The small molecule ENGase inhibitors have potential to treat pathogenesis associated with NGLY1 deficiency. Rabeprazole, a proton pump inhibitor, was identified as a potential ENGase inhibitor. It was demonstrated that the consequences of knockdown of OGT or PNG1 on ISC proliferation and ENGase activity was rescued by Rabeprazole treatment in ISCs/EBs or ECs. The data showed that cell death was elevated in ISCs/EBs-specific PNG1/OGT knockdown with Rabeprazole treatment compared to non-treated groups concomitant with an increase in ISC proliferation. On the other hand, cell death decreased in EC-specific PNG1 knockdown treated with Rabeprazole resulting in a decrease in ISC proliferation. It is known that loss of PNG1 function in cells can cause the accumulation of aberrant proteins in the cytosol and the interruption of ERAD. Further, downregulation of ER stress-related genes has been reported in B-cell-specific OGT mutant mice. The protective effects of O-GlcNAc are not limited to mitochondrial function but also rescue injury caused by ER stress. Therefore, NGLY1/OGT seems to be functionally associated with the ERAD machinery. More recently, using a model ERAD substrate, it was reported that the ablation of Ngly1 causes a disruption in the ERAD process in mouse embryonic fibroblast (MEF) cells. Moreover, lethality of mice bearing a knockout of the Ngly1-gene was partially rescued by the additional deletion of the Engase gene. Interestingly, this study showed that OGA knockdown rescued ENGase levels of PNG1 knockdown ISCs/EBs. Hence, these findings suggest that there is a correlation between OGT/PNG1 and ENGase contributing to tissue maintenance (Na, 2022).

Taken together, these findings implicate O-GlcNAc and PNG1 as key regulators of tissue maintenance. PNG1 can impact stem cell homeostasis through regulation of O-GlcNAc both in ISCs/EBs or ECs. Of significance is the finding that PNG1 and OGT phenotypes are rescued by modulating CncC and ENGase activity in ISCs/EBs or ECs. Thus, these findings reveal that nutrient-driven glycosylation contribute towards control of ISC and progenitor cell proliferation and EC cell death via regulation of CncC and ENGase. This study provides a platform for future designs of interventions in which changes in O-GlcNAc can be utilized as a therapeutic for stem-cell-derived diseases like cancer. This study also presents a molecular mechanism and unexpected pathway that can be targeted for treating NGLY1-deificient patients (Na, 2022).

Tissue-specific regulation of BMP signaling by Drosophila N-glycanase 1

Mutations in the human N-glycanase 1 (NGLY1) cause a rare, multisystem congenital disorder with global developmental delay. However, the mechanisms by which NGLY1 and its homologs regulate embryonic development are not known. This study shows that Drosophila Pngl encodes an N-glycanase and exhibits a high degree of functional conservation with human NGLY1. Loss of Pngl results in developmental midgut defects reminiscent of midgut-specific loss of BMP signaling. Pngl mutant larvae also exhibit a severe midgut clearance defect, which cannot be fully explained by impaired BMP signaling. Genetic experiments indicate that Pngl is primarily required in the mesoderm during Drosophila development. Loss of Pngl results in a severe decrease in the level of Dpp homodimers and abolishes BMP autoregulation in the visceral mesoderm mediated by Dpp and Tkv homodimers. Thus, these studies uncover a novel mechanism for the tissue-specific regulation of an evolutionarily conserved signaling pathway by an N-glycanase enzyme (Galeone, 2017).

The broad phenotypes of children affected with NGLY1 deficiency and the semi-lethality of Pngl^-/- flies (Funakoshi, 2010) indicate that NGLY1 plays important roles during animal development. However, the N-glycanase function has not been linked to any developmental signaling pathway. This study reports that fly Pngl regulates BMP signaling during embryonic midgut development without affecting BMP signaling in ectodermal and head regions of the embryo. The data indicate that Pngl is not required in the midgut endoderm to receive the BMP signal, but rather is required in the visceral mesoderm (VM) to send the BMP signal. It has previously been shown that BMP signaling uses a paracrine/autocrine loop in the VM to sustain and increase the expression of Dpp in PS3 and PS7 of embryonic VM. This loop is proposed to ensure that the level of BMP ligands in the VM is high enough to induce signaling in the endoderm and to specify gastric caeca, the second midgut constriction and the acid zone. Several lines of evidence indicate that the BMP autoregulation mediated by the para-autocrine loop in the VM is the step which is impaired in Pngl-deficient embryos. First, Pngl is not required for the initial, Ubx-dependent expression of dpp. In fact, even a 50% decrease in the expression of dpp in the visceral mesoderm of dpp^s2/+ animals does not impair BMP autoactivation and midgut development. Second, despite expressing Dpp at early stages, BMP signaling is not activated in Pngl^-/- VM, as evidenced by the lack of pMad staining. Third, overexpression of Dpp-GFP in the mesoderm is able to induce BMP signaling in the endoderm in Pngl^-/- embryos. Lastly, bypassing the para-autocrine loop by transgenic expression of a constitutively active BMP receptor in the mesoderm results in restoration of BMP signaling in PS3 and PS7 regions of the endoderm and in partial rescue of lethality in Pngl^-/- embryos (Galeone, 2017).

In the BMP para-autocrine loop, VM cells both secrete the BMP ligand and respond to it. Therefore, theoretically, Pngl might play a critical role in sending the BMP signal, receiving the BMP signal, or both. Although the data do not allow exclusion of any of these possibilities, based on the following observations, a scenario is favored in which Pngl is required in VM cells to send the Dpp signal not to receive it: (1) Pngl is not required to receive the BMP signal in the endoderm; (2) Loss of Pngl and Pngl KD result in a dramatic decrease in the level of Dpp homodimers and the Dpp-positive puncta; (3) Expression of a constitutively active form of Tkv in the mesoderm is able to restore midgut pMad staining in embryos and the copper cell region in the adult midgut, and partially rescue the lethality of Pngl^-/- animals; (4) Loss of Pngl almost fully suppresses the aberrant BMP signaling caused by mesodermal overexpression of Dpp-GFP (Galeone, 2017).

Whole larval protein extracts from Pngl-deficient animals show an increase in the level of the monomeric forms of Dpp (full-length and a cleavage product) and a simultaneous decrease in the bands corresponding in size to Dpp dimers. Moreover, Pngl^-/- embryos show a decrease in Dpp-positive puncta both in the mesoderm, where signaling is impaired, and in the ectoderm, where signaling is not impaired. Together, these observations indicate that the effect of loss of Pngl on the Dpp protein itself is not limited to the mesoderm. Indeed, protein extracts from Pngl-deficient midgut and carcass (without midgut) both show a decrease in Dpp dimer levels. This suggests that either Pngl regulates BMP signaling by affecting Dpp dimer levels in other larval tissues not identified yet, or that Dpp dimers are only important in the midgut and although they are decreased elsewhere, Dpp-Gbb heterodimers compensate for the lack of Dpp dimers in most other tissues. Regardless, it is proposed that loss of BMP signaling in Pngl mutant midguts results from a requirement for Dpp homodimers in the para-autocrine autoregulatory loop present in the visceral mesoderm (Galeone, 2017).

BMP ligands can signal both as homodimers and as heterodimers. In vitro and in vivo studies have shown that in general, BMP heterodimers have stronger bioactivity than their homodimers counterparts. In some cases, the homodimers induce weak to moderate signaling, and in other cases they either do not elicit signaling or even play an antagonistic role. Stronger activity of BMP heterodimers can at least in part be explained by differential affinities of individual BMP ligands for different BMP receptors, combined with stronger signal transduction by heterodimeric type I receptors compared to homodimers of each type I receptor. For example, in Drosophila, Dpp has a higher affinity for Tkv, whereas the other two ligands-Gbb and Scw-have a higher affinity for Sax. A similar receptor-ligand binding preference has been observed among the vertebrate orthologs. In the embryonic dorsal midline and the wing imaginal disc, Dpp/Scw and Dpp/Gbb heterodimers induce high levels of signaling, respectively, through Tkv/Sax heterodimers. Comparison of the gbb mutant phenotypes in the midgut with those caused by Pngl loss and by dpp KD indicates that Dpp homodimers are the only productive form of ligand in PS7. Moreover, mesodermal KD of tkv severely decreases BMP signaling in PS7, but mesodermal KD of sax not only does not decrease pMad staining in PS7, but also results in an expansion of pMad expression domain in the PS7 region, similar to gbb mutant embryos. Together, these observations strongly support the notion that the BMP autoregulatory loop in the VM, which is essential for the activation of BMP signaling in the endoderm, relies solely on Dpp and Tkv homodimers, and therefore is impaired in Pngl mutants due to the severe decrease in the level of Dpp homodimers in these animals (Galeone, 2017).

Vertebrate and invertebrate BMP proteins and other members of the TGFβ superfamily each harbor several N-linked glycosylation sites, which have been shown to be glycosylated in many cases. Various functional roles have been ascribed to N-glycans on these ligands, including enhancing receptor binding of BMP6, keeping the TGFβ1 ligand in a latent state, and promoting inhibin (α

/β) heterodimer formation at the expense of activin (β/β) homodimer formation. Accordingly, given the significant increase in Dpp monomeric forms and the simultaneous decrease in Dpp dimers upon loss of Pngl, it is possible that Pngl removes one or more N-glycans from Dpp and thereby promotes the formation or the stability of Dpp homodimers. Whether the regulation of Dpp by Pngl is direct or mediated via other proteins will remain to be explored (Galeone, 2017).

In agreement with a previous report, the current data suggest that the lethality of Pngl mutants cannot be fully explained by shortening of the gastric caeca and impairment of BMP signaling in midgut development. Pngl KD with mesodermal drivers leads to a higher degree of lethality compared to dpp KD with the same drivers. Moreover, how^24B > Pngl^RNAi animals show ~70% lethality, even though they do not have gastric caeca defects. Finally, restoring BMP signaling in the midgut by expressing tkvCA only recues the lethality in ~30% of Pngl^-/- animals. Phenotypic analysis of Pngl mutants combined with rescue and KD experiments suggest that a failure to properly empty the gut before puparium formation contributes to lethality in these animals. The molecular mechanisms for the food accumulation phenotype and other potential Pngl^-/- phenotypes contributing to lethality are still under investigation (Galeone, 2017).

In summary, this work indicates that the fly Pngl is an evolutionarily conserved N-glycanase enzyme necessary to sustain BMP autoactivation in the VM mediated by para-autocrine activity of Dpp homodimers through Tkv homodimers. Although it cannot be. excluded that Pngl plays important roles in other cell types as well, the data indicate that Pngl is primarily required in the mesoderm during midgut development and its loss results in Dpp-dependent and Dpp-independent midgut defects. Given the reports on potential para-autocrine functions of mammalian Dpp homologs and prominent human pathologies associated with dysregulated BMP signaling in ophthalmic, gastrointestinal and musculoskeletal systems, tissue-specific alterations in BMP signaling might contribute to some of the NGLY1 deficiency phenotypes including retinal abnormalities, delayed bone age and osteopenia, small feet and hands, and chronic constipation. Finally, understanding the mechanisms underlying the food accumulation phenotype in Pngl-/- larvae might shed light on the pathophysiology of chronic constipation in NGLY1 deficiency patients (Galeone, 2017).

A conserved role for AMP-activated protein kinase in NGLY1 deficiency

Mutations in human N-glycanase 1 (NGLY1) cause the first known congenital disorder of deglycosylation (CDDG). Patients with this rare disease, which is also known as NGLY1 deficiency, exhibit global developmental delay and other phenotypes including neuropathy, movement disorder, and constipation. NGLY1 is known to regulate proteasomal and mitophagy gene expression through activation of a transcription factor called "nuclear factor erythroid 2-like 1" (NFE2L1). Loss of NGLY1 has also been shown to impair energy metabolism, but the molecular basis for this phenotype and its in vivo consequences are not well understood. Using a combination of genetic studies, imaging, and biochemical assays, this study reports that loss of NGLY1 in the visceral muscle of the Drosophila larval intestine results in a severe reduction in the level of AMP-activated protein kinase alpha (AMPKalpha), leading to energy metabolism defects, impaired gut peristalsis, failure to empty the gut, and animal lethality. Ngly1-/- mouse embryonic fibroblasts and NGLY1 deficiency patient fibroblasts also show reduced AMPKalpha levels. Moreover, pharmacological activation of AMPK signaling significantly suppressed the energy metabolism defects in these cells. Importantly, the reduced AMPKalpha level and impaired energy metabolism observed in NGLY1 deficiency models are not caused by the loss of NFE2L1 activity. Taken together, these observations identify reduced AMPK signaling as a conserved mediator of energy metabolism defects in NGLY1 deficiency and suggest AMPK signaling as a therapeutic target in this disease (Han, 2020).

The cytoplasmic enzyme N-glycanase 1 (NGLY1) catalyzes the removal of N-linked glycans from glycoproteins and is thought to operate as part of the endoplasmic reticulum-associated degradation (ERAD) pathway. Recessive mutations in human NGLY1 result in a genetic disorder with various phenotypes including developmental delay, seizures, hypo-/alacrima, elevated liver enzymes, diminished deep tendon reflexes, muscle weakness, orthopedic manifestations, and chronic constipation. This disease is a congenital disorder of deglycosylation (OMIM # 615273) and is commonly referred to as NGLY1 deficiency. NGLY1 and its homologs in model organisms have been shown to regulate the proteasomal gene expression by deglycosylating a transcription factor called "nuclear factor erythroid 2-like 1" (NFE2L1; also called NRF1; SKN-1 in worms). However, the extent to which this proteasomal defect contributes to NGLY1 deficiency phenotypes in human patients and developmental abnormalities observed in Ngly1-mutant animal models remains to be determined. It is worth mentioning that NFE2L1 and its paralog NFE2L2 have also been called NRF1 and NRF2. However, since NRF1 is the official symbol for a distinct protein called nuclear respiratory factor 1 in mammals, we will use NFE2L1 and NFE2L2 in this study (Han, 2020).

Studies in tissue samples from NGLY1 deficiency patients, patient fibroblasts, Ngly1^-/- mouse embryonic fibroblasts (MEFs) and C. elegans mutants for the NGLY1 homolog (png-1^-/-) have shown structural and functional abnormalities in mitochondria. Moreover, Ngly1^-/- MEFs fail to properly clear damaged mitochondria via mitophagy. Given that a number of NGLY1 deficiency phenotypes like developmental delay, neuropathy, muscle weakness, and seizures are also observed in mitochondrial disorders, they are considered to be one of the differential diagnoses in patients suspected of having NGLY1 deficiency. Therefore, although a specific phenotype in human NGLY1 deficiency patients and Ngly1-mutant animals is yet to be directly linked to energy homeostasis defects, these studies suggest that mitochondrial abnormalities might contribute to some aspects of NGLY1 deficiency pathophysiology, and that improving energy homeostasis might be beneficial in this patient population (Han, 2020).

The Drosophila genome encodes a single NGLY1 homolog called PNGase-like or Pngl. Under regular culture conditions, Pngl-null (Pngl^-/-) Drosophila show a significant developmental delay and ~99% lethality. We have previously reported that 80%-85% of the lethality observed in Pngl^-/- animals can be rescued by transgenic expression of human NGLY1 in the mesoderm [16]. Around 20-30% of the lethality can be explained by a tissue-specific requirement for Pngl in the visceral mesoderm to promote bone morphogenetic protein (BMP) signaling mediated by the fly BMP protein Decapentaplegic or Dpp [16]. Pngl^-/- larvae also show a food accumulation phenotype (severe failure in gut clearance) that cannot be explained by the loss of BMP signaling [16]. However, the molecular basis of gut clearance defects and their contribution to the lethality observed in Pngl^-/- animals remained to be identified. Moreover, it was not clear whether at a mechanistic level the regulation of Drosophila gut clearance by Pngl has any parallels in mammals (Han, 2020).

This study shows that the food accumulation phenotype in Pngl^-/- larvae is caused by reduced mesodermal expression of AMP-activated protein kinase α subunit (AMPKα), which encodes the catalytic subunit for a major energy sensor in the cells. The midgut in Pngl^-/- larvae exhibits abnormal mitochondrial cristae, reduced ATP content and increased oxidative stress, all of which can be improved upon restoring AMPKα levels in the mesoderm. Importantly, both Ngly1^-/- MEFs and fibroblasts from NGLY1 deficiency patients show a similar reduction in AMPKα1 and AMPKα2 expression. Moreover, pharmacological enhancement of AMPK signaling rescues the impaired energy homeostasis in both model systems. Importantly, the reduction in AMPKα level cannot be explained by impaired proteasomal gene expression in NGLY1-deficient models. Our work identifies reduced AMPKα expression as an evolutionarily conserved mechanism contributing to the energy homeostasis defects in NGLY1-deficient animals and suggests AMPK signaling as a potential therapeutic target in NGLY1 deficiency patients (Han, 2020).

The discovery of recessive NGLY1 mutations in patients with a multi-system developmental disorder has prompted a series of studies on the consequences of loss of this enzyme in several cellular and animal model systems. Perhaps the most-studied cellular process downstream of NGLY1 is the NFE2L1-mediated regulation of the proteasomal gene expression, which depends on NGLY1 in both invertebrates and mammals. In addition, work in Drosophila, MEFs and mouse embryos has identified the Drosophila Dpp and its mammalian homolog BMP4 as direct, biologically relevant targets of Pngl/NGLY1 and has shown that impaired BMP signaling in the visceral mesoderm of Pngl^-/- larvae results in specific developmental abnormalities in their midgut and contributes to their lethality. The current data indicate that loss of N-glycanase 1 also results in a significant reduction in the level of AMPKα mRNA in the fly larval midgut, MEFs, and human patient fibroblasts, accompanied by reduced AMPKα and pAMPKα protein levels in all three models. It has been previously reported that in addition to the tissue-specific loss of BMP signaling in the midgut, Pngl^-/- larvae exhibit a food accumulation phenotype (failure to empty the gut) that cannot be explained by loss of BMP signaling. This study now shows that this phenotype is associated with impaired energy homeostasis in the midgut, and that restoring AMPKα expression rescues the food accumulation phenotype and allows 40-45% of Pngl^-/- larvae to reach adulthood, compared to ~1% without rescue. In addition, AMPKα RNAi in the mesoderm recapitulates the gut clearance and energy homeostasis phenotypes of Pngl^-/- larvae. Moreover, pharmacological activation of AMPK signaling significantly improves the energy homeostasis defects observed in NGLY1-deficient MEFs and patient fibroblasts. Together, these observations suggest that reduced AMPK signaling underlies some of the phenotypes observed in NGLY1-deficient models. It is worth noting that in Ngly1^-/- MEFs and patient fibroblasts, but not in Drosophila visceral mesoderm, the pAMPKα/AMPKα ratio was also decreased compared to controls. These observations suggest that in addition to reduced AMPKα levels, NGLY1-deficient mammalian cells might also be defective in AMPKα phosphorylation (activation) (Han, 2020).

Loss of N-glycanase 1 in worms, MEFs, and fibroblasts and muscle biopsies from NGLY1 deficiency patients results in functional abnormalities in mitochondria, including a reduction in oxidative phosphorylation, basal OCR and maximal OCR, and an increase in oxidative stress. Moreover, Ngly1^-/- MEFs showed mitochondrial fragmentation. In agreement with these observations, this study found that mitochondria in the midgut visceral muscles of Pngl^-/- larvae exhibit abnormal cristae structure, accompanied by reduced ATP levels and increased oxidative stress. Importantly, increasing the AMPKα expression level in Pngl^-/- animals partially rescued the mitochondrial morphology in the visceral muscle, significantly reduced the level of reactive oxygen species, and fully restored the ATP levels in the midgut. Moreover, pharmacological activation of AMPKα reduced reactive oxygen species and increased ATP levels in MEFs, and restored the basal and maximal OCR in fibroblasts from NGLY1 deficiency patients. Together, these observations indicate that enhancing AMPK signaling improves mitochondrial energy metabolism in several N-glycanase 1-deficient contexts (Han, 2020).

Given the impairment of NFE2L1-mediated gene regulation in all N-glycanase 1-deficient animal models tested so far, it was hypothesized that the reduction in AMPKα levels observed in the current models might result from the loss of NFE2L1's transcriptional activity. However, Nfe2l1^-/- and wild-type MEFs showed comparable levels of AMPKα and pAMPKα, despite the impaired proteasome bounce-back response in Nfe2l1^-/- MEFs. Moreover, although treatment with the NFE2L2 activator SFN increased the expression of proteasomal genes in the midguts of Pngl^-/- larvae and in Ngly1^-/- MEFs, it failed to restore normal AMPKα levels in these models. Lastly, treating wild-type MEFs with a proteasome inhibitor did not affect AMPKα and pAMPKα levels. Together, these data indicate that the reduced AMPKα observed in NGLY1-deficient contexts is not caused by loss of NFE2L1 activity or impaired proteasome function. The mitochondrial fragmentation phenotype observed in Ngly1^-/- MEFs was shown to primarily result from impaired mitophagy due to loss of NFE2L1 activity and was significantly improved upon treating the cells with SFN. However, SFN treatment failed to suppress the energy metabolism defects in Ngly1^-/- MEFs, even though it rescued the mitophagy gene expression in these cells. Finally, adding SFN to the fly food did not lead to any rescue of the AMPKα-dependent food accumulation and lethality in Pngl^-/- larvae. Taken together, these data suggest that the impairment of energy metabolism observed upon loss of NGLY1 is caused by reduced AMPKα activity and is independent of NFE2L1-related defects in proteasome and mitophagy (Han, 2020).

A previous report and current data demonstrate that the loss of BMP signaling observed in Pngl^-/- midguts does not cause the food accumulation phenotype in these animals. Moreover, the data presented in the current study indicate that loss of NFE2L1 activation cannot explain the reduced AMPKα expression in NGLY1-deficient models. These data suggest that NGLY1 regulates AMPKα levels independently of BMP and NFE2L1 pathways. The mechanisms that regulate the level of AMPKα mRNA are not well understood. MicroRNA 148b (miR-148b) and miR-301a are reported to negatively regulate AMPKα levels in pancreatic cancer and osteosarcoma cell lines, respectively. A recent study has shown that TP63 is recruited to the AMPKα1 regulatory region and directly activates AMPKα1 transcription in human mammary gland cells. However,neither TP63 nor the above-mentioned microRNAs have been linked to NGLY1 so far. In fact, transcription factors and other proteins involved in mRNA stability are not N-glycosylated and therefore cannot be direct targets of NGLY1 (with NFE2L1 as a rare exception). How does then loss of NGLY1 lead to reduced AMPKα levels? Ngly1^-/- MEFs have been shown to accumulate cytosolic aggregates of a model ERAD substrate harboring N-linked N-acetylglucosamine monosaccharides (N-GlcNAc), a type of glycan normally not seen in the cytosol. Accumulation of proteins harboring N-GlcNAc can potentially interfere with the function of O-GlcNAc, which is the major type of glycosylation on nucleocytoplasmic proteins and regulates various cellular processes including transcription and mitochondrial activity. Therefore, one possibility is that NGLY1 regulates AMPKα level indirectly through impaired O-GlcNAc signaling. Another possibility is that NGLY1 is involved in the quality control of an N-glycosylated cell surface receptor that is upstream of AMPKα transcription. The molecular mechanism through which NGLY1 regulates AMPKα levels remains to be determined (Han, 2020).

Transcriptome and functional analysis in a Drosophila model of NGLY1 deficiency provides insight into therapeutic approaches

Autosomal recessive loss-of-function mutations in N-glycanase 1 (NGLY1) cause NGLY1 deficiency, the only known human disease of deglycosylation. Patients present with developmental delay, movement disorder, seizures, liver dysfunction and alacrima. NGLY1 is a conserved cytoplasmic component of the Endoplasmic Reticulum Associated Degradation (ERAD) pathway. ERAD clears misfolded proteins from the ER lumen. However, it is unclear how loss of NGLY1 function impacts ERAD and other cellular processes and results in the constellation of problems associated with NGLY1 deficiency. To understand how loss of NGLY1 contributes to disease, a Drosophila model of NGLY1 deficiency was developed. Loss of NGLY1 function resulted in developmental delay and lethality. RNAseq was used to determine which processes are misregulated in the absence of NGLY1. Transcriptome analysis showed no evidence of ER stress upon NGLY1 knockdown. However, loss of NGLY1 resulted in a strong signature of NRF1 dysfunction among downregulated genes, as evidenced by an enrichment of genes encoding proteasome components and proteins involved in oxidation-reduction. A number of transcriptome changes also suggested potential therapeutic interventions, including dysregulation of GlcNAc synthesis and upregulation of the heat shock response. Increasing the function of both pathways rescues lethality. Together, transcriptome analysis in a Drosophila model of NGLY1 deficiency provides insight into potential therapeutic approaches (Owings, 2018).

A Drosophila screen identifies NKCC1 as a modifier of NGLY1 deficiency

N-Glycanase 1 (NGLY1) is a cytoplasmic deglycosylating enzyme. Loss-of-function mutations in the NGLY1 gene cause NGLY1 deficiency, which is characterized by developmental delay, seizures, and a lack of sweat and tears. To model the phenotypic variability observed among patients, a Drosophila model of NGLY1 deficiency was crossed onto a panel of genetically diverse strains. The resulting progeny showed a phenotypic spectrum from 0 to 100% lethality. Association analysis on the lethality phenotype, as well as an evolutionary rate covariation analysis, generated lists of modifying genes, providing insight into NGLY1 function and disease. The top association hit was Ncc69 (human NKCC1/2), a conserved ion transporter. Analyses in NGLY1-/- mouse cells demonstrated that NKCC1 has an altered average molecular weight and reduced function. The misregulation of this ion transporter may explain the observed defects in secretory epithelium function in NGLY1 deficiency patients (Talsnees, 2020).

Defects in the Neuroendocrine Axis Contribute to Global Development Delay in a Drosophila Model of NGLY1 Deficiency

N-glycanase 1 (NGLY1) Deficiency is a rare monogenic multi-system disorder first described in 2014. NGLY1 is evolutionarily conserved in model organisms. A natural history study and chemical-modifier screen were conducted on the Drosophila melanogaster NGLY1 homolog, Pngl. A new fly model of NGLY1 Deficiency was generated, engineered with a nonsense mutation in Pngl at codon 420 that results in a truncation of the C-terminal carbohydrate-binding PAW domain. Homozygous mutant animals exhibit global development delay, pupal lethality and small body size as adults. A 96-well-plate, image-based, quantitative assay of Drosophila larval size was developed for use in a screen of the 2,650-member Microsource Spectrum compound library of FDA approved drugs, bioactive tool compounds, and natural products. This study found that the cholesterol-derived ecdysteroid molting hormone 20-hydroxyecdysone (20E) partially rescued the global developmental delay in mutant homozygotes. Targeted expression of a human NGLY1 transgene to tissues involved in ecdysteroidogenesis, e.g., prothoracic gland, also partially rescues global developmental delay in mutant homozygotes. Finally, the proteasome inhibitor bortezomib is a potent enhancer of global developmental delay in the fly model, evidence of a defective proteasome "bounce-back" response that is also observed in nematode and cellular models of NGLY1 Deficiency. Together, these results demonstrate the therapeutic relevance of a new fly model of NGLY1 Deficiency for drug discovery and gene modifier screens (Rodriguez, 2018).

Evidence for an essential deglycosylation-independent activity of PNGase in Drosophila melanogaster

Peptide:N-glycanase (PNGase) is an enzyme which releases N-linked glycans from glycopeptides/glycoproteins. This enzyme plays a role in the ER-associated degradation (ERAD) pathway in yeast and mice, but the biological importance of this activity remains unknown. This study characterized the ortholog of cytoplasmic PNGases, PNGase-like (Pngl), in Drosophila melanogaster. Pngl was found to have a molecular weight of approximately 74K and was mainly localized in the cytosol. Pngl lacks a CXXC motif that is critical for enzymatic activity in other species and accordingly did not appear to possess PNGase activity, though it still retains carbohydrate-binding activity. Microdeletions were generated in the Pngl locus in order to investigate the functional importance of this protein in vivo. Elimination of Pngl led to a serious developmental delay or arrest during the larval and pupal stages, and surviving mutant adult males and females were frequently sterile. Most importantly, these phenotypes were rescued by ubiquitous expression of Pngl, clearly indicating that those phenotypic consequences were indeed due to the lack of functional Pngl. Interestingly, a putative "catalytic-inactive" mutant could not rescue the growth-delay phenotype, indicating that a biochemical activity of this protein is important for its biological function. This study has shown that Pngl is inevitable for the proper developmental transition and the biochemical properties other than deglycosylation activity is important for its biological function (Funakoshi, 2010).

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Funakoshi, Y., Negishi, Y., Gergen, J. P., Seino, J., Ishii, K., Lennarz, W. J., Matsuo, I., Ito, Y., Taniguchi, N. and Suzuki, T. (2010). Evidence for an essential deglycosylation-independent activity of PNGase in Drosophila melanogaster. PLoS One 5(5): e10545. PubMed ID: 20479940

Galeone, A., Han, S. Y., Huang, C., Hosomi, A., Suzuki, T. and Jafar-Nejad, H. (2017). Tissue-specific regulation of BMP signaling by Drosophila N-glycanase 1. Elife 6. PubMed ID: 28826503

Han, S. Y., Pandey, A., Moore, T., Galeone, A., Duraine, L., Cowan, T. M. and Jafar-Nejad, H. (2020). A conserved role for AMP-activated protein kinase in NGLY1 deficiency. PLoS Genet 16(12): e1009258. PubMed ID: 33315951

Na, H. J., Akan, I., Abramowitz, L. K. and Hanover, J. A. (2020). Nutrient-Driven O-GlcNAcylation Controls DNA Damage Repair Signaling and Stem/Progenitor Cell Homeostasis. Cell Rep 31(6): 107632. PubMed ID: 32402277

Na, H. J., Abramowitz, L. K. and Hanover, J. A. (2022). Cytosolic O-GlcNAcylation and PNG1 maintain Drosophila gut homeostasis by regulating proliferation and apoptosis. PLoS Genet 18(3): e1010128. PubMed ID: 35294432

Owings, K. G., Lowry, J. B., Bi, Y., Might, M. and Chow, C. Y. (2018). Transcriptome and functional analysis in a Drosophila model of NGLY1 deficiency provides insight into therapeutic approaches. Hum Mol Genet 27(6): 1055-1066. PubMed ID: 29346549

Rodriguez, T. P., Mast, J. D., Hartl, T., Lee, T., Sand, P. and Perlstein, E. O. (2018). Defects in the Neuroendocrine Axis Contribute to Global Development Delay in a Drosophila Model of NGLY1 Deficiency. G3 (Bethesda) 8(7): 2193-2204. PubMed ID: 29735526

Talsness, D. M., Owings, K. G., Coelho, E., Mercenne, G., Pleinis, J. M., Partha, R., Hope, K. A., Zuberi, A. R., Clark, N. L., Lutz, C. M., Rodan, A. R. and Chow, C. Y. (2020). A Drosophila screen identifies NKCC1 as a modifier of NGLY1 deficiency. Elife 9. PubMed ID: 33315011

Tomlin, F. M., Gerling-Driessen, U. I. M., Liu, Y. C., Flynn, R. A., Vangala, J. R., Lentz, C. S., Clauder-Muenster, S., Jakob, P., Mueller, W. F., Ordonez-Rueda, D., Paulsen, M., Matsui, N., Foley, D., Rafalko, A., Suzuki, T., Bogyo, M., Steinmetz, L. M., Radhakrishnan, S. K. and Bertozzi, C. R. (2017). Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity. ACS Cent Sci 3(11): 1143-1155. PubMed ID: 29202016

date revised: 7 April, 2023

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