InteractiveFly: GeneBrief

grindelwald: Biological Overview | References

BIOLOGICAL OVERVIEW

Disruption of epithelial polarity is a key event in the acquisition of neoplastic growth. JNK signalling is known to play an important part in driving the malignant progression of many epithelial tumours, although the link between loss of polarity and JNK signalling remains elusive. In a Drosophila genome-wide genetic screen designed to identify molecules implicated in neoplastic growth, this study identified grindelwald (grnd; CG10176), a gene encoding a transmembrane protein with homology to members of the tumour necrosis factor receptor (TNFR) superfamily. This study shows that Grnd mediates the pro-apoptotic functions of Eiger (Egr), the unique Drosophila TNF, and that overexpression of an active form of Grnd lacking the extracellular domain is sufficient to activate JNK signalling in vivo. Grnd also promotes the invasiveness of RasV12/scrib-/- tumours through Egr-dependent Matrix metalloprotease-1 (Mmp1) expression. Grnd localizes to the subapical membrane domain with the cell polarity determinant Crumbs (Crb) and couples Crb-induced loss of polarity with JNK activation and neoplastic growth through physical interaction with Veli (also known as Lin-7). Therefore, Grnd represents the first example of a TNFR that integrates signals from both Egr and apical polarity determinants to induce JNK-dependent cell death or tumour growth (Andersen, 2015).

A genome-wide screen was carried to identify molecules that are required for neoplastic growth. The condition used for this screen was the disc-specific knockdown of avalanche, also known as syntaxin 7), a gene encoding a syntaxin that functions in the early step of endocytosis2. avl-RNAi results in ectopic Wingless (Wg) expression, neoplastic disc overgrowth, and a 2-day delay in larva-to-pupa transition. A collection of 10,100 transgenic RNA interference (RNAi) lines were screened for their ability to rescue the pupariation delay, and 121 candidate genes were identified. Interestingly, only eight candidate genes also rescued ectopic Wg expression and neoplastic overgrowth. These included five lines targeting core components of the JNK pathway (Bendless, Tab2, Tak1, Hemipterous and Basket. Using a puckered enhancer trap (puc-lacZ) as a readout for JNK activity, it was confirmed that JNK signalling is highly upregulated in avl-RNAi discs. One of the remaining lines targets CG10176, a gene encoding a transmembrane protein. Reducing expression of CG10176 by using two different RNAi lines was as efficient as tak1 silencing to restore normal Wg pattern and suppresses JNK signalling and neoplastic growth in the avl-RNAi background. Sequence analysis of GC10176 identified a cysteine-rich domain (CRD) in the extracellular part with homology to vertebrate TNFRs harbouring a glycosphingolipid-binding motif (GBM) characteristic of many TNFRs including Fas. CG10176 was named grindelwald (grnd) , after a village at the foot of Eiger, a Swiss mountain that lent its name to the unique Drosophila TNF, Egr. Immunostaining and subcellular fractionation of disc extracts confirmed that Grnd localizes to the membrane. Moreover, co-immunoprecipitation experiments showed that both Grnd full-length and Grnd-intra, a form lacking its extracellular domain, directly associate with Traf2, the most upstream component of the JNK pathway. This interaction is disrupted by a single amino acid substitution within a conserved Traf6-binding motif (human TRAF6 is the closest homologue to Traf2. Overexpression of Grnd-intra, but not full-length Grnd, is sufficient to induce JNK signalling, ectopic Wg expression and apoptosis, and Grnd-intra-induced apoptosis is efficiently suppressed in a hep (JNKK) mutant background, confirming that Grnd acts upstream of the JNK signalling cascade (Andersen, 2015).

The Drosophila TNF Egr activates JNK signalling and triggers cell death or proliferation, depending on the cellular context. Therefore tests were performed to see whether Grnd is required for the small-eye phenotype generated by Egr-induced apoptosis in the retinal epithelium (via Egr overexpression). Inhibition of JNK signalling by reducing tak1 or traf2 expression, or by overexpressing puckered, blocks Egr-induced apoptosis and rescues the small-eye phenotype. In contrast to a previous report, RNAi silencing of wengen (wgn) , a gene encoding a presumptive receptor for Egr, does not rescue the small-eye phenotype. Furthermore, the small-eye phenotype is not modified in a wgn-null mutant background, confirming that Wgn is not required for Egr-induced apoptosis in the eye. By contrast, reducing grnd levels partially rescues the Egr-induced small-eye phenotype, producing a 'hanging-eye' phenotype that is not further rescued in a wgn-knockout mutant background. A similar phenotype was previously reported as a result of non-autonomous cell death induced by a diffusible form of Egr. This suggests that Grnd prevents Egr from diffusing outside of its expression domain. Co-immunoprecipitation experiments show that both full-length Grnd and Grnd-extra, a truncated form of Grnd lacking the cytoplasmic domain, associate with Egr through its TNF-homology domain. Although Grnd-extra can bind Egr, it cannot activate JNK signalling. Therefore, it was reasoned that Grnd-extra expression might prevent both cell-autonomous and non-autonomous apoptosis by trapping Egr and preventing its diffusion and binding to endogenous Grnd. Indeed, GMR-Gal4-mediated expression of grnd-extra fully rescues the Egr small-eye phenotype. To confirm that the removal of Grnd induces Egr-mediated non-autonomous cell death, wing disc clones were generated expressing egr alone, egr + tak1 RNAi, or egr + grnd RNAi. As expected, reducing tak1 levels in egr-expressing clones prevents their elimination by apoptosis. Similarly, reducing grnd levels prevents autonomous cell death, but also induces non-autonomous apoptosis. This suggests that Egr, like its mammalian counterpart TNF-α, can be processed into a diffusible form in vivo whose interaction with Grnd limits the potential to act at a distance. Flies carrying homozygous (grnd^Minos/Minos) or transheterozygous (grnd^Minos/Df) combinations of a transposon inserted in the grnd locus express no detectable levels of Grnd protein and are equally resistant to Egr-induced cell death. In addition, grnd^Minos/Minos mutant flies are viable and display no obvious phenotype, suggesting that Grnd, like Egr, participates in a stress response to limit organismal damage. Collectively, these data demonstrate that Grnd is a new Drosophila TNF receptor that mediates most, if not all, Egr-induced apoptosis (Andersen, 2015).

TNFs probably represent a danger signal produced in response to tissue damage to rid the organism of premalignant tissue or to facilitate wound healing. Disc clones mutant for the polarity gene scribbled (scrib) induce an Egr-dependent response resulting in the elimination of scrib mutant cells by JNK-mediated apoptosis. To test the requirement for Grnd in this process, scrib-RNAi and scrib-RNAi + grnd-RNAi clones obtained 72 h after heat shock induction were compared. As expected, scrib-RNAi cells undergo apoptosis and detach from the epithelium. By contrast, scrib-RNAi clones with reduced grnd expression survive, indicating that Grnd is required for Egr-dependent elimination of scrib-RNAi cells. Similar results were obtained by generating scrib mutant clones in the eye disc (Andersen, 2015).

In both mammals and flies, TNFs are double-edged swords that also have the capacity to promote tumorigenesis in specific cellular contexts. Indeed, scrib minus eye disc cells expressing an activated form of Ras (Ras^V12) exhibit a dramatic tumour-like overgrowth and metastatic behaviour, a process that critically relies on Egr. Ras^V12/scrib^-/- metastatic cells show a strong accumulation of Grnd and Mmp1, and invade the ventral nerve cord. Primary tumour cells reach peripheral tissues such as the fat body and the gut, where they form micro-metastases expressing high levels of Grnd. Reducing grnd levels in Ras^V12/scrib^-/- clones is sufficient to restore normal levels of Mmp1 and abolish invasiveness in a way similar to that observed in an egr mutant background. Therefore, Grnd is required for the Egr-induced metastatic behaviour of Ras^V12/scrib^-/- tumorous cells. Similarly, reducing grnd, but not wgn levels, strongly suppresses Mmp1 expression in Ras^V12/dlg-RNAi cells and limits tumour invasion, indicating that Wgn does not have a major role in the progression of these tumours (Andersen, 2015).

Perturbation of cell polarity is an early hallmark of tumour progression in epithelial cells. In contrast to small patches of polarity-deficient cells, for example, scrib mutant clones, organ compartments or animals fully composed of polarity-deficient cells become refractory to Egr-induced cell death and develop epithelial tumours. The formation of these tumours requires JNK/MAPK signalling, but not Egr, suggesting Egr-independent coupling between loss of polarity and JNK/MAPK-dependent tumour growth. In line with these observations, it was noticed that, in contrast to Grnd, Egr is not required to drive neoplastic growth in avl-RNAi conditions. This suggests that, in addition to its role in promoting Egr-dependent functions, Grnd couples loss of polarity with JNK-dependent growth independently of Egr. Disc immunostainings revealed that Grnd co-localizes with the apical determinant Crb in the marginal zone, apical to the adherens junction protein E-cadherin (E-cad) and the atypical protein kinase C (aPKC). In avl-RNAi discs, Grnd and Crb accumulate in a wider apical domain. Apical accumulation of Crb is proposed to be partly responsible for the neoplastic growth induced by avl knockdown, since overexpression of Crb or a membrane-bound cytoplasmic tail of Crb (Crb-intra) mimics the avl-RNAi phenotype. Therefore whether Grnd might couple the activity of the Crb complex with JNK-mediated neoplastic growth was examined. Indeed, reducing grnd levels, but not wgn, in ectopic crb-intra discs suppresses neoplastic growth as efficiently as inhibiting the activity of the JNK pathway. Notably, Yki activation is not rescued in these conditions, illustrating the ability of Crb-intra to promote growth independently of Grnd by inhibiting Hippo signalling through its FERM-binding motif (FBM). Indeed, neoplastic growth and polarity defects induced by a form of Crb-intra lacking its FBM (CrbΔFBM-intra) are both rescued by Grnd silencing. As expected, the size of ectopic crbΔFBM-intra;grnd-RNAi discs is reduced compared to the size of ectopic crb-intra; grnd-RNAi discs (Andersen, 2015).

Crb, Stardust (Sdt; PALS1 in humans), and Pals1-associated tight junction protein (Patj) make up the core Crb complex, which recruits the adaptor protein Veli (MALS1-3 in humans). In agreement with previous yeast two-hybrid data, this study found that Grnd binds directly and specifically to the PDZ domain of Veli through a membrane-proximal stretch of 28 amino acids in its intracellular domain. Grnd localization is unaffected in crb and veli RNAi mutant clones. However, reducing veli expression rescues the patterning defects and disc morphology of ectopic crb-intra mutant cells, suggesting that Grnd couples Crb activity with JNK signalling through its interaction with Veli. Interestingly, aPKC-dependent activation of JNK signalling also depends on Grnd. aPKC is capable of directly binding and phosphorylating Crb, which is important for Crb function. This suggests that aPKC, either directly or through Crb phosphorylation, activates Grnd-dependent JNK signalling in response to perturbation of apico-basal polarity (Andersen, 2015).

These data are consistent with a model whereby Grnd integrates signals from Egr, the unique fly TNF, and apical polarity determinants to induce JNK-dependent neoplastic growth or apoptosis in a context-dependent manner. Recent work reveals a correlation between mammalian Crb3 expression and tumorigenic potential in mouse kidney epithelial cells. The conserved nature of the Grnd receptor suggests that specific TNFRs might carry out similar functions in vertebrates, in which the link between apical cell polarity and tumour progression remains elusive (Andersen, 2015).

The Drosophila TNF Eiger is an adipokine that acts on insulin-producing cells to mediate nutrient response

Adaptation of organisms to ever-changing nutritional environments relies on sensor tissues and systemic signals. Identification of these signals would help understand the physiological crosstalk between organs contributing to growth and metabolic homeostasis. This study shows that Eiger, the Drosophila TNF-alpha, is a metabolic hormone that mediates nutrient response by remotely acting on insulin-producing cells (IPCs). In the condition of nutrient shortage, a metalloprotease of the TNF-alpha converting enzyme (TACE) family protein CG7908 is active in fat body (adipose-like) cells, allowing the cleavage and release of adipose Eiger in the hemolymph. In the brain IPCs, Eiger activates its receptor Grindelwald, leading to JNK-dependent inhibition of insulin production. Therefore, this study has identified a humoral connexion between the fat body and the brain insulin-producing cells relying on TNF-alpha that mediates adaptive response to nutrient deprivation (Agrawal, 2016).

This study shows that the fly TNF Eiger functions as a metabolic hormone produced by the fat body in response to chronic protein deprivation. Elevated circulating levels of human TNF-α and TNFR are also observed in malnutrition conditions and in catabolic states associated with cachexia induced by sepsis and cancer. Moreover, this study presents evidence that the molecular mechanism leading to decreased insulin expression by TNF-α is conserved in mammalian β cells. Therefore, activation of TNF signaling could be an evolutionary conserved response to undernutrient imbalance, recently highjacked as an adaptive response to continuous nutritional surplus. Drosophila Eiger has so far mostly been implicated in local, autonomous responses, activating JNK signaling in the cells or tissues that express it. In recent studies, however, it has been postulated that Egr can diffuse outside of its expression domain in a paracrine manner. The current data now demonstrate that Egr circulates in the hemolymph and acts remotely, allowing crossorgan communication. This raises questions relative to the mode of transport of Eiger in the hemolymph and its specificity of action on remote target tissues. Indeed, although overexpression of Egr in fat cells leads to a strong increase in Egr levels in the hemolymph, flies show no obvious defects, suggesting that secreted Egr has limited access to peripheral tissues while in the hemolymph. Previous studies have shown that human and mouse TNF-α efficiently cross the blood-brain barrier (BBB) after i.v. injection and are detected in the cerebrospinal fluid in a process requiring the presence of TNF receptors in glial cells. This study shows that hemolymph Eiger can penetrate the brain and access to the insulin-producing cells. It will be important to evaluate in future experiments the mechanisms by which Egr travels in the hemolymph and across the larval BBB (Agrawal, 2016).

This study shows that an important aspect of Egr secretion relies on its shedding from the membrane by the convertase enzyme TACE. Drosophila TACE was recently shown to be required for Egr function in a tumor model using activated ras (Chabu, 2014). This study shows that transcription of TACE, but not egr, is induced in fat cells under low-protein diet (LPD) and that adipose TACE activity is critical for metabolic adaptation to low protein. Moreover, a genetic link was identified between TACE expression and TORC1, the main amino acid sensor in fat cells. Interestingly, REPTOR and REPTOR-BP, two transcription factors that are responsible for most of the transcription response to TORC1 inhibition, are not required for TACE expression, suggesting that an alternative mechanism is required for TACE induction in response to TORC1 inhibition following exposure to LPD. Vertebrate TACE/ADAM17 acts on a small number of cytokines and growth factors including TNF-α and several membrane receptors (Menghini, 2013). Mice deficient in TACE function are lean and resistant to high-fat-diet-induced obesity and diabetes type 2, a range of phenotypes that could be linked to TNF-α shedding defects. In mammals, TACE activity is controlled through balanced expression of the Tissue Inhibitor of Metalloprotease 3 (TIMP3). Although a TIMP3 homolog exists in Drosophila, there is no indication that it participates in modulating Drosophila TACE activity in addition to TACE transcriptional activation observed in LPD (Agrawal, 2016).

Circulating Eiger produced by adipose cells remotely acts on the brain neurosecretory cells that produce insulin (IPCs), leading to general body growth inhibition. This correlates with specific expression of the TNF receptor Grindelwald in the IPCs. Indeed, knocking down Grnd in these neurons mimics the effect of knocking down Egr in the fat body. No effect is observed upon knocking down the other fly TNFR Wengen in IPCs, indicating that Grnd mediates Egr metabolic action and that specific targeting of TNF signaling to the IPCs is the consequence of localized Grnd expression. As a consequence of Grnd activation, JNK signaling is elevated in the IPCs of animals raised on a LPD. TNF signaling is not required in the IPCs for the retention of Dilp2 observed upon acute protein starvation. By contrast, activation of JNK in larval IPCs leads to reduced expression of Dilp2 and Dilp5, two major circulating Dilps. Strikingly, this is reminiscent of the role described for JNK in vertebrate pancreatic β cells. Indeed, JNK inhibitors increase insulin expression in Langerhans islets from obese mice, suggesting that JNK represses expression of the insulin gene. These results are also in line with the present finding that TNF-α inhibits INS1 and INS2 gene transcription from Min6 cells and mouse islets (Agrawal, 2016).

In conclusion, this work unravels an anciently conserved mechanism by which TNF signaling mediates direct response to low nutrient through a fat-brain crossorgan communication leading to the modulation of growth. Other signals contributing to reduction of insulin signaling in condition of nutrient shortage have recently been identified in Drosophila. Systemic Hedgehog is produced by gut cells in response to low nutrients and targets both fat cells and ecdysone-producing cells to adapt larval growth to nutrient shortage. A hormone called Limostatin produced by the corpora cardiaca in low-glucose condition acts directly on adult insulin-producing cells to block insulin secretion. These findings together with the present work indicate that a variety of signals allow the integration of different physiological contexts for the proper control of insulin production (Agrawal, 2016).

Drosophila tumor suppressor gene prevents tonic TNF signaling through receptor N-glycosylation

Drosophila tumor suppressor genes have revealed molecular pathways that control tissue growth, but mechanisms that regulate mitogenic signaling are far from understood. This study reports that the Drosophila TSG tumorous imaginal discs (tid), whose phenotypes were previously attributed to mutations in a DnaJ-like chaperone, are in fact driven by the loss of the N-linked glycosylation pathway component ALG3. tid/alg3 imaginal discs display tissue growth and architecture defects that share characteristics of both neoplastic and hyperplastic mutants. Tumorous growth is driven by inhibited Hippo signaling, induced by excess Jun N-terminal kinase (JNK) activity. Ectopic JNK activation is caused by aberrant glycosylation of a single protein, the fly tumor necrosis factor (TNF) receptor homolog, Grindelwald, which results in increased binding to the continually circulating TNF. These results suggest that N-linked glycosylation sets the threshold of TNF receptor signaling by modifying ligand-receptor interactions and that cells may alter this modification to respond appropriately to physiological cues (de Vreede, 2018).

Tumorigenesis is ultimately driven by dysregulated cellular signaling that promotes unchecked proliferation. Proliferation-regulating signaling pathways in animals are therefore normally under tight control, to prevent aberrant growth. The primary mechanism of signaling regulation is limited availability of ligand, although levels of receptor can also be regulated, as can receptor availability on the plasma membrane or even its polarized localization. A full understanding of the mechanisms that limit mitogenic signaling is an important goal of both basic biology and cancer research (de Vreede, 2018).

Major insight into growth regulation has arisen from research in model organisms such as Drosophila melanogaster. For instance, Drosophila studies revealed key steps of receptor tyrosine kinase signaling and uncovered the phenomenon of cell competition. Additional insight into growth regulatory mechanisms has come from the analysis of fly tumor suppressor genes (TSGs). Disruption of a single fly TSG is sufficient to cause overproliferation in epithelial organs of the larva called imaginal discs. Initial genetic screens identified several classes of fly TSGs. The neoplastic TSGs (discs large, lethal giant larvae, and scribble) revealed an intimate link between cell polarity and cell proliferation control, a principle also relevant to human cancers. The hyperplastic TSGs, including hippo, warts, and salvador, uncovered the novel Hippo (Hpo) signal transduction pathway, which is now recognized as a conserved growth control mechanism. Even less prominent Drosophila TSGs such as lethal giant discs have demonstrated important biological concepts (de Vreede, 2018).

One classic Drosophila TSG that remains understudied is tumorous imaginal discs (tid). Imaginal discs of tid homozygous larvae develop into overgrown masses. Genetic mapping and cytogenetic analyses attributed this phenotype to loss of a conserved molecular chaperone of the DnaJ family. Evidence for a tumor-suppressive role for a mammalian homolog, hTid-1, has been presented. However, the exact molecular mechanism through which tid could regulate cell and tissue proliferation remains mysteriou (de Vreede, 2018 and references therein).

This study reports that the tid gene was cloned incorrectly. Aberrant cell proliferation in the Drosophila mutant arises not from disruptions to the DnaJ homolog but rather to an adjacent gene that encodes the mannosyltransferase ALG3, involved in N-linked glycosylation. Overgrowth in tid/ALG3 mutants is caused by mis-glycosylation of a single transmembrane protein, the Drosophila tumor necrosis factor (TNF) receptor homolog Grindelwald, which results in downstream activation of Jun N-terminal kinase (JNK) and inactivation of the growth-suppressing Hpo pathway. The results suggest that this post-translational modification modulates ligand-receptor affinity in the TNF receptor (TNFR) pathway and thus provides a regulatory mechanism setting a dynamic threshold for JNK-mediated stress signaling and growth control (de Vreede, 2018).

This study has shown that mutations in the classic Drosophila TSG tumorous imaginal discs (tid) disrupt the ALG3 homolog CG4084, altering the lipid-linked biosynthetic pathway that generates oligosaccharides for protein N-linked glycosylation. Although altered glycosylation affects many proteins and can induce a unfolded protein response (UPR), this study finds that the growth control phenotype of Alg3 can be ascribed to a single target and a single mechanism. This target is the Drosophila TNFR homolog, whose proper modification at a single extracellular site is required to prevent inappropriate TNF binding, subsequent JNK activation, and downstream Yki-driven overproliferation. It is postulated that N-glycosylation can act as a mechanism to modulate JNK signaling in response to cellular stresses (de Vreede, 2018).

The alg3 mutations were originally identified for their overgrowth phenotype in imaginal discs. Like most other Drosophila TSGs, this phenotype is caused by changes in Hpo-regulated Yki activation, but alg3 mutants differ in both upstream regulation and downstream targets. Mutations in core Hpo signaling components result in rapid proliferation of disc cells, while the slow growth of alg3 mutant tissue resembles that of the neoplastic TSGs. Nonetheless, the STAT pathway, which is a major mitogenic effector in neoplastic mutants, is not elevated in alg3 tissue. Upstream, JNK-dependent Yki activity is seen in both alg3 and neoplastic mutants. However, JNK activation in neoplastic mutants has been suggested to occur either through ligand-independent Grnd activation caused by alteration to apicobasal polarity or through Grnd-independent mechanisms. In alg3 mutants, polarity is intact and overgrowth entirely relies on a Grnd-Egr axis, specifically the increased sensitivity of misglycosylated Grnd for endocrine Egr. Thus, TNFR signaling induced by altered N-glycosylation seems to define distinct consequences for downstream Hpo-mediated growth control (de Vreede, 2018).

While this study has not tested biochemical affinities directly, the data are consistent with a model where TNF-binding properties are directly regulated by glycosylation of TNFR. Partial or complete removal of the glycan at N63, within the ligand-binding domain of Grnd, leads to an increase of bound Egr, indicating that N-glycosylation normally limits Grnd engagement and downstream signaling. In Drosophila larvae, Egr is continuously transcribed in the fat body for secretion into the hemolymph, bathing Grnd-expressing tissues, including imaginal discs and IPCs in ligand. The results suggest that proper N-glycosylation of Grnd sets a threshold that prevents tonic signaling in these and other tissues under normal circumstances. This raises the intriguing possibility that cell-autonomous changes in N-glycosylation, perhaps induced by stress inputs, could modulate ligand affinity, allowing a rapid and local response to this endocrine signal under different physiological conditions (de Vreede, 2018).

The modulation of Grnd ligand binding suggested here echoes the regulation of Notch by the glycosyltransferase Fringe. However, the obligate role of Alg3 in all N-glycan synthesis is fundamentally distinct from Fringe's substrate-specific elaboration of a particular O-glycan. In the case of Notch, the specific sugar residues added by Fringe alter receptor selectivity for one ligand over another. Since either aberrant or absent Grnd N-glycosylation results in increased ligand binding and ectopic signaling, evidence for specific glycan structures in modulating the ligand-receptor interface does not currently exist. Whether the glycan could provide a simple steric obstacle to ligand binding or may regulate it through more complex interactions will await structural studies (de Vreede, 2018).

Grnd shows strong homology to vertebrate TNFR family members in its extracellular TNF-binding domain, although downstream signaling in the fly acts mainly through JNK, in contrast to mammalian homologs that also signal through nuclear factor κB (NF-κB), p38, and caspases. Among the 29 mammalian TNFR superfamily members, at least seven have predicted N-glycosylation sites in their extracellular domains. Several of these sites have been studied, and their proposed roles vary from promoting signaling to inhibiting it or being functionally neutral. The current results motivate analyses of the receptors BCMA and DR4, which are closely related to Grnd and whose predicted N-glycosylation sites each lie in an analogous location within the ligand-binding domain (de Vreede, 2018 and references therein).

The data presented above, which highlight a new mechanism for restraining TNF signaling, hint at pathogenic mechanisms for several human diseases. Altered glycosylation is emerging as a frequent hallmark of cancer, in which JNK signaling is increasingly implicated. Moreover, mutations in the extracellular domain of human TNFR1, including predicted N-glycosylation sites, can cause the autoinflammatory disease TRAPS (TNFR-associated periodic syndrome). Because the erroneous activation of Grnd in alg3 mutants is akin to an autoinflammatory response, defective N-glycosylation could be an additional mechanism for hyperactive TNFR1 signaling. Finally, mutations in N-glycosylation pathway enzymes including Alg3 result in recessive genetic diseases called type I congenital disorders of glycosylation (CDG-I). CDG patients exhibit a variety of poorly characterized symptoms associated with multiple organs, and the etiology of CDG is largely unknown. The finding of altered inflammatory TNFR/JNK signaling in analogous fly mutants provides a new avenue to investigate (de Vreede, 2018).

ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet-mediated degeneration

Animals have evolved multiple mechanisms to protect themselves from the cumulative effects of age-related cellular damage. This study revealed an unexpected link between the TNF (tumour necrosis factor) inflammatory pathway, triggered by the metalloprotease ADAM17/TACE, and a lipid droplet (LD)-mediated mechanism of protecting retinal cells from age-related degeneration. Loss of ADAM17, TNF and the TNF receptor Grindelwald in pigmented glial cells of the Drosophila retina leads to age-related degeneration of both glia and neurons, preceded by an abnormal accumulation of glial LDs. The glial LDs initially buffer the cells against damage caused by glial and neuronally generated reactive oxygen species (ROS), but in later life the LDs dissipate, leading to the release of toxic peroxidated lipids. Finally, this study demonstrates the existence of a conserved pathway in human iPS-derived microglia-like cells, which are central players in neurodegeneration. Overall, this study has discovered a pathway mediated by TNF signalling acting not as a trigger of inflammation, but as a cytoprotective factor in the retina (Muliyil, 2020).

This paper reports a previously unrecognised role of ADAM17 and TNF in protecting Drosophila retinal cells from age- and activity-related degeneration. Loss of ADAM17 and TNF signalling in retinal glial cells causes an abnormal accumulation of LDs in young glial cells. These LDs disperse by about 2 weeks after eclosion (middle age for flies), and their loss coincides with the onset of severe glial and neuronal cell death. By 4 weeks of age, no intact glia or neurons remain. Cell death depends on neuronal activity: retinal degeneration and, to a lesser extent, LD accumulation are rescued in flies reared fully in the dark. LD accumulation does not merely precede, but is actually responsible for subsequent degeneration, because preventing the accumulation of LDs fully rescues cell death. The data indicate that Eiger/TNF released by ADAM17 acts specifically through the Grindelwald TNF receptor. Loss of ADAM17-mediated TNF signalling also leads to elevated production of mitochondrial ROS in glial cells, causing activation of the JNK pathway and elevated lipogenic gene expression. Together, these changes trigger cell death through the production of toxic peroxidated lipids. Importantly, toxicity is also contributed to by ROS generated by normal activity of neighbouring neurons. Finally, this study shows that a similar signalling module is conserved in mammalian cells: when ADAM17 is inhibited in human iPSC-derived microglial-like cells, the same series of events is seen: LD accumulation, elevated mitochondrial ROS and high levels of toxic peroxidated lipids (Muliyil, 2020).

It is proposed that TNF is an autocrine trophic factor that protects retinal pigmented glial cells from age-related cumulative damage caused by the ROS that are normal by-products of neuronal activity. This ADAM17/TNF protection system is located specifically in retinal glial cells, but its role is to protect both glia and neighbouring neurons. In the absence of this TNF cytoprotective pathway, severe early-onset retinal neurodegeneration is seen. The data imply that cells die by being overwhelmed by toxic peroxidated lipids when abnormal accumulations of LDs disperse. This occurs in Drosophila middle age, when LDs stop accumulating and begin to disperse, triggering the cytotoxic phase of the ADAM17-/- phenotype. It is important to emphasise that despite the ADAM17/TNF protection system being located specifically in retinal glial cells, there is neuronal involvement. Not only does TNF indirectly protect against neurodegeneration, but photoreceptor neurons are also significant sources of the ROS that generate the toxic peroxidated lipids in glia. More generally, this work provides a model for investigating more widely the functional links between ageing, cellular stress, lipid droplet accumulation and neurodegeneration. Indeed, in the light of the discovery that the pathway discovered in Drosophila is conserved in human microglia-like cells, it is significant that lipid droplets have been reported to accumulate in human microglia, cells that are increasingly prominent in the pathology of Alzheimer's disease and other neurodegenerative conditions (Muliyil, 2020).

In a Drosophila model of neuronal mitochondrionopathies, abnormal neuronal ROS production led to elevated neuronal lipid production, followed by transfer of the lipids to the PGCs, where LDs accumulated. In that work, lipase expression in neurons suppressed LD accumulation; in contrast, suppression was only observed when lipase was expressed in PGCs, not neurons, suggesting that in the case of ADAM17 mutants, the primary source of accumulating lipids is the glial cells. Despite not being able to detect a role for neuronal lipid production in ADAM17-/- mutants, it was found that photoreceptor neurons are significant sources of the ROS that generate the toxic peroxidated lipids in glia. Despite these differences between this work and what has been previously reported, a growing body of work points to a close coupling between ROS, the JNK pathway, lipid droplets and cellular degeneration, a relationship conserved in mammals. This study did not investigate the involvement of SREBP in mediating LD accumulation caused by ADAM17 loss, but its well-established connection with stress-induced and JNK-mediated lipid synthesis suggests that it is a likely additional shared component of this conserved regulatory axis (Muliyil, 2020).

It has become clear that LDs are much more than simply passive storage vessels for cellular lipids; they have multiple regulatory functions. Indeed, although this study highlighted a developing picture of an LD/ROS-dependent trigger of cell death, in other contexts LDs have protective functions against oxidative damage, both in flies and mammals. This may occur by providing an environment that shields fatty acids from peroxidation by ROS and/or by sequestering toxic peroxidated lipids. Although this superficially appears to contradict the theme of LD/ROS toxicity, it is important to recall that in LD-related cell death is not simultaneous with LD accumulation. In fact, degeneration temporally correlates with the dispersal of LDs in middle age, rather than their earlier accumulation. Together, the strands of evidence from several studies suggest that it is the combination of elevated ROS and the dispersal of abnormally high quantities of lipids from previously accumulated LDs that trigger death. This suggests that cells die by being overwhelmed by toxic peroxidated lipids when abnormal accumulations of LDs break down in the presence of high levels of ROS. Experiments with Brummer lipase are consistent with this idea: the Brummer lipase was expressed from early in development, thereby preventing abnormal LD accumulation, and this protected against cell death. This sequence of events implies the existence of a metabolic switch, when LDs stop accumulating and begin to disperse, triggering the toxic phase of ADAM17 loss. It will be interesting in the future and may provide insights into the normal ageing process, to understand the molecular mechanism of this age- and/or activity-dependent change (Muliyil, 2020).

ADAM17 is one of the most important shedding enzymes in humans, responsible for the proteolytic release of a vast array of cell surface signals, receptors and other proteins. Because of its role in signalling by both TNF and ligands of the EGF receptor, it has been the focus of major pharmaceutical efforts, with a view to treating inflammatory diseases and cancer. It is therefore surprising that it has been very little studied in Drosophila. This is the first report of Drosophila ADAM17 mutants. This study also confirmed for the first time that Drosophila ADAM17 is indeed an active metalloprotease, able to shed cell surface proteins including the Drosophila TNF homologue Eiger. The only other description of Drosophila ADAM17 function is mechanistically consistent with the data, despite relating to a different physiological context. In that case, ADAM17 was shown to cause the release of soluble TNF from the fat body so that it can act as a long range adipokine (Agrawal, 2016). Other ADAM17 substrates in different developmental or physiological contexts cannot be ruled out, although the relatively subtle phenotype of null mutant flies implies that ADAM17 does not have essential functions that lead to obvious defects when mutated. Moreover, wdevelopmental defects or LD accumulation were not observed in any neuronal or non-neuronal ADAM17-/- larval tissues, suggesting that the mechanism reported in this study is both age and tissue specific (Muliyil, 2020).

Although TNF is sometimes viewed as a specific cell death-promoting signal, and the pathways by which it activates caspase-induced apoptosis have been studied extensively in flies and mammals, the response to TNF is in fact very diverse, depending on the biological context. Indeed, its most well-studied role in mammals is as the primary inflammatory cytokine, released by macrophages and other immune cells, and triggering the release of other cytokines, acting as a chemoattractant, stimulating phagocytosis, and promoting other inflammatory responses. However, TNF has not previously been shown to have trophic activity, protecting cells in the nervous system from stress-induced damage, although a link with the Nrf2/Keap1 redox pathway in cardiomyocytes provides an interesting parallel (Muliyil, 2020).

In conclusion, this work highlights three important biological concepts. The first is to identify a new function for the ADAM17/TNF pathway in a cytoprotective role that protects Drosophila retinal cells against age- and activity-dependent degeneration. This contrasts with its well-established roles in inflammation and cell death. Secondly, the existence is highlighted of a glia-centric cellular pathway by which the breakdown of accumulated LDs and ROS together participate in promoting stress-induced and age-related cell death. Finally, this study has shown that the core phenomenon of the ADAM17 protease, acting to regulate the homeostatic relationship between ROS and LD biosynthesis, is conserved in human microglial cells, which themselves are intimately involved in neuroprotection (Muliyil, 2020).

Search PubMed for articles about Drosophila Grindelwald

Agrawal, N., Delanoue, R., Mauri, A., Basco, D., Pasco, M., Thorens, B. and Leopold, P. (2016). The Drosophila TNF Eiger is an adipokine that acts on insulin-producing cells to mediate nutrient response. Cell Metab 23: 675-684. PubMed ID: 27076079

Andersen, D. S., Colombani, J., Palmerini, V., Chakrabandhu, K., Boone, E., Rothlisberger, M., Toggweiler, J., Basler, K., Mapelli, M., Hueber, A. O. and Leopold, P. (2015). The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth. Nature. PubMed ID: 25874673

de Vreede, G., Morrison, H. A., Houser, A. M., Boileau, R. M., Andersen, D., Colombani, J. and Bilder, D. (2018). A Drosophila tumor suppressor gene prevents tonic TNF signaling through receptor N-glycosylation. Dev Cell 45(5): 595-605 PubMed ID: 29870719

Muliyil, S., Levet, C., Dusterhoft, S., Dulloo, I., Cowley, S. A. and Freeman, M. (2020). ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet-mediated degeneration. Embo J: e104415. PubMed ID: 32715522

date revised: 5 August 2023

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