ADAM 17‐triggered TNF signalling protects the ageing Drosophila retina from lipid droplet‐mediated degeneration
2020; Springer Nature; Volume: 39; Issue: 17 Linguagem: Inglês
10.15252/embj.2020104415
ISSN1460-2075
AutoresSonia Muliyil, Clémence Levet, Stefan Düsterhöft, Iqbal Dulloo, Sally A. Cowley, Matthew Freeman,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle26 July 2020Open Access Transparent process ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet-mediated degeneration Sonia Muliyil Sonia Muliyil orcid.org/0000-0003-1374-9304 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Clémence Levet Clémence Levet Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Stefan Düsterhöft Stefan Düsterhöft orcid.org/0000-0002-6926-136X Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Iqbal Dulloo Iqbal Dulloo Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Sally A Cowley Sally A Cowley orcid.org/0000-0003-0297-6675 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Matthew Freeman Corresponding Author Matthew Freeman [email protected] orcid.org/0000-0003-0410-5451 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Sonia Muliyil Sonia Muliyil orcid.org/0000-0003-1374-9304 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Clémence Levet Clémence Levet Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Stefan Düsterhöft Stefan Düsterhöft orcid.org/0000-0002-6926-136X Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Iqbal Dulloo Iqbal Dulloo Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Sally A Cowley Sally A Cowley orcid.org/0000-0003-0297-6675 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Matthew Freeman Corresponding Author Matthew Freeman [email protected] orcid.org/0000-0003-0410-5451 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Author Information Sonia Muliyil1,2, Clémence Levet1, Stefan Düsterhöft1,3, Iqbal Dulloo1, Sally A Cowley1 and Matthew Freeman *,1 1Sir William Dunn School of Pathology, University of Oxford, Oxford, UK 2Present address: Elsevier, Oxford, UK 3Present address: Institute of Pharmacology and Toxicology, Medical Faculty, RWTH Aachen University, Aachen, Germany *Corresponding author. Tel: +44 01993 704986; E-mail: [email protected] The EMBO Journal (2020)39:e104415https://doi.org/10.15252/embj.2020104415 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Animals have evolved multiple mechanisms to protect themselves from the cumulative effects of age-related cellular damage. Here, we reveal 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. We show that the glial LDs initially buffer the cells against damage caused by glial and neuronally generated reactive oxygen species (ROS), but that in later life the LDs dissipate, leading to the release of toxic peroxidated lipids. Finally, we demonstrate the existence of a conserved pathway in human iPS-derived microglia-like cells, which are central players in neurodegeneration. Overall, we have discovered a pathway mediated by TNF signalling acting not as a trigger of inflammation, but as a cytoprotective factor in the retina. Synopsis The metalloprotease ADAM17 triggers a signalling mechanism that controls lipid droplet formation and cell survival in the Drosophila retina. Absence of Drosophila ADAM17 in retinal glial cells results in abnormal lipid droplet accumulation, elevated peroxidated lipids, and subsequent age-associated degeneration. Loss of TNF, encoded by the eiger gene, or the TNF receptor Grindelwald, causes the same retinal degeneration phenotype. Retinal degeneration in ADAM17 mutants can be rescued by overexpressing lipase in glial but not neuronal cells. Reactive oxygen species generated by neuronal activity contribute to the lipid droplet-mediated retinal degeneration in ADAM17 mutants. In human iPSC microglia-like cells, loss of ADAM17 also results in elevated peroxidated lipids and lipid droplet formation. Introduction Diseases of ageing are often caused by stress-induced cellular degeneration that accumulates over time (Campisi, 2013; Lopez-Otin et al, 2013). The intrinsic causes of such damage are widespread but include toxic build-up of misfolded and aggregated proteins, as well as oxidative stress caused by the production of reactive oxygen species (ROS), which are by-products of metabolic and other physiological activity (Squier, 2001; Davalli et al, 2016; Klaips et al, 2018). Cells have evolved multiple processes to protect themselves against these potentially damaging stresses, including well-characterised systems like the unfolded protein response, endoplasmic reticulum (ER)-associated degradation, and reactive oxygen species (ROS) scavenging enzymes (Wellen & Thompson, 2010; Walter & Ron, 2011; Bravo et al, 2013). Recently, lipid droplets (LDs) have begun to be implicated in the machinery of stress protection (Bailey et al, 2015; Van Den Brink et al, 2018). The significance of this role of LDs has been most studied in the fruit fly Drosophila. In central nervous system stem cell niches, elevated ROS levels induce the formation of LDs, which appear to sequester polyunsaturated acyl chains, protecting them from the oxidative chain reactions that generate toxic peroxidated species (Bailey et al, 2015). In another context, however, LDs are part of the cellular damage causing pathway: ROS generated by defective mitochondria in the Drosophila retinal neurons induces the formation of LDs in adjacent glial cells, and these LDs later contribute to glial and neuronal degeneration (Liu et al, 2015). More recent work has illustrated that toxic fatty acids produced by activated neurons in culture can be taken up by astrocytes via endocytosis, where they get subsequently catabolised by mitochondrial beta-oxidation (Ioannou et al, 2019). Although the overall significance of these mechanisms, and how they are integrated, remains to be understood, it is clear that the long-held view of LDs as primarily inert storage organelles is no longer tenable (Olzmann & Carvalho, 2019). Furthermore, beyond their role in regulating cell survival and death, LDs are increasingly found to act in other cellular processes, including acting as platforms for the assembly of viruses, and modulators of cell signalling (Boulant et al, 2008; Cheung et al, 2010; Li et al, 2014; Sandoval et al, 2014; Welte & Gould, 2017). They also form intimate contacts with ER and mitochondria (Schuldiner & Bohnert, 2017; Thiam & Dugail, 2019) and act inside the nucleus (Layerenza et al, 2013; Uzbekov & Roingeard, 2013; Soltysik et al, 2019). In addition to the cellular mechanisms of protection against stresses, there is also protection at the level of the whole organism. This higher order, coordinated protection is primarily mediated by the inflammatory and immune systems (Chen et al, 2018; Ferrucci & Fabbri, 2018; Franceschi et al, 2018; O'Neil et al, 2018). Inflammatory signalling pathways are increasingly understood to have relevance to an extraordinary range of biology, extending far beyond classical inflammation, and including age-related damage. This is highlighted by the ever-growing list of diseases associated with inflammation including, for example, neurodegeneration, multiple sclerosis and other neurological diseases; metabolic pathologies like type 2 diabetes and obesity/metabolic syndrome; and atherosclerosis (Ferrucci & Fabbri, 2018). The signalling molecules associated with these myriad functions are equally diverse, but TNF (tumour necrosis factor) stands out as being a primary trigger of much classical and non-classical inflammatory signalling (Sedger & McDermott, 2014). Like many cytokines and growth factors, TNF is synthesised with a transmembrane (TM) anchor, and its release as an active signal is triggered by the proteolytic cleavage of the extracellular domain from its TM anchor by the "shedding" protease ADAM17 (a disintegrin and metalloproteinase 17; also known as TACE, TNF alpha converting enzyme) (Black et al, 1997). Its function of being the essential trigger of all TNF signalling makes ADAM17 a highly significant enzyme. But in fact its importance is even greater because, in addition to shedding TNF, it is also responsible for the release of many other signalling molecules including EGF (epidermal growth factor) receptor ligands (Sahin et al, 2004; Baumgart et al, 2010; Dang et al, 2013). Consistent with this central role in inflammation and a wide range of other cellular events, ADAM17 has been extensively studied, both from a fundamental biological perspective and also as a drug target. The fruit fly Drosophila, an important model organism for revealing the molecular, cellular and genetic basis of development, is increasingly used to investigate conserved aspects of human physiology and even disease mechanisms. With this motivation, we have investigated in flies the pathophysiological role of ADAM17. We report the first ADAM17 mutation in Drosophila and find that the mutant flies exhibit abnormally high lipid droplet accumulation followed by severe age- and activity-dependent neurodegeneration in the adult retina. These observations have uncovered a new cytoprotective pathway mediated by soluble TNF acting not as a trigger of inflammation, but as a trophic survival factor for retinal glial cells. We have also shown that inhibition of ADAM17 in human iPSC-derived microglia-like cell lines leads to the same cellular phenomena as seen in fly retinas—lipid droplet accumulation, ROS production and generation of peroxidated lipids—suggesting that ADAM17 may have an evolutionarily conserved cytoprotective function. Results Loss of ADAM17 in glial cells triggers age-dependent degeneration To investigate the full range of its pathophysiological functions, we made Drosophila null mutants of ADAM17 using CRISPR/Cas9. The knockout flies did not display any obvious defects during development and were viable as adults. They did, however, have reduced lifespan, indicating potential physiological defects. We pursued this possibility by more detailed analysis of the adult ADAM17 mutant (ADAM17−/−) retina, a tissue widely used for studying age-related degeneration of the nervous system (Morante & Desplan, 2005). The retina of the Drosophila compound eye comprises ommatidial units of eight photoreceptors, each containing a light-collecting organelle called the rhabdomere, that project axons into the brain. Photoreceptor cell bodies are surrounded by pigmented glial cells (PGCs) believed to provide metabolic support to the neurons (Edwards & Meinertzhagen, 2010; Liu et al, 2015). ADAM17−/− retinas exhibited extensive degeneration of both photoreceptor neurons and the surrounding PCGs in 5-week-old flies (Fig 1A–C and L). The same result was seen with a heterozygous mutant allele in combination with a chromosome deficiency that includes the ADAM17 locus (ADAM17−/Df) (Fig 1D and L). This supports the conclusion that the degeneration was indeed caused by the loss of ADAM17. To further confirm the role of ADAM17 in the degeneration phenotype, we analysed the siRNA knockdown of ADAM17 using a retinal specific driver, GMR-GAL4. This too showed glial and neuronal degeneration (Fig 1E, F and L). Figure 1. Glia-specific loss of ADAM17 results in age-associated retinal degeneration A. Diagram of a tangential (left) and horizontal (right) section of the adult Drosophila retina, with photoreceptors highlighted in violet and the pigmented glial cells (PGCs) in grey. PR: photoreceptor; R: rhabdomere; CB: cell body. B–K. Transmission electron microscopy (TEM) images of 5-week-old adult retinas showing the overall structure of groups of ommatidia in (B) wild type; (C) ADAM17−/− mutant; (D) ADAM17−/Deficiency; (E, F) RNAi against ADAM17 and control (lacZ), expressed throughout the retina; (G, H) RNAi against ADAM17 and control (lacZ), expressed in the neurons; (I, J) RNAi against ADAM17 and control (lacZ), expressed in the pigmented glial cells; and (K) a kuz−/− mutant. L. Quantitation of the percentage of normal, abnormal and missing rhabdomeres from TEM images for the genotypes above, expressed as a stacked bar chart; n = 180 ommatidia from 3 different fly retinas for each. M. Whole-mount retina stained with anti-ADAM17 (green) and phalloidin to mark the photoreceptor membranes (red). N. Line intensity profiles delineating expression of ADAM17 and phalloidin across individual ommatidia, n = 10 fly retinas. Data information: Scale bars: 10 μm. Download figure Download PowerPoint An ADAM17-specific antibody revealed that expression of the enzyme is strongly enriched in the PGCs that surround the photoreceptor neurons (Fig 1M and N). This expression pattern hinted that ADAM17 function may be required specifically in glial cells, so we tested this idea by cell-type specific knockdown. ADAM17 siRNA, expressed specifically in neurons with elav-GAL4, produced no phenotype (Fig 1G, H and L, and EV1L). In sharp contrast, glial-specific knockdown with a sparkling-GAL4 driver caused severe and characteristic degeneration (Fig 1I, J and L, and EV1L). We confirmed that ADAM17 is not needed in neurons with another neuron-specific GAL4 driver, Rh1-GAL4, which drives expression specifically in photoreceptors (Fig EV1A, B, K and L). The specific requirement of ADAM17 in glia was further demonstrated by showing that glial expression of ADAM17 was sufficient to rescue the degeneration phenotype of the mutant (Fig EV1C, D and K). We also investigated the specificity of the phenotype by examining fly mutants of the closely related ADAM metalloprotease, Kuzbanian, the Drosophila orthologue of ADAM10 (Qi et al, 1999). kuz−/− retinas showed no signs of degeneration (Fig 1K and L). Click here to expand this figure. Figure EV1. Loss of ADAM17 in PGCs induces age-dependent degeneration A–J. TEM images of adult retinas; (A) overexpressing lacZ in neurons; (B) RNAi for ADAM17 in neurons; (C) ADAM17−/− mutant; (D) ADAM17−/− mutant overexpressing WT-ADAM17 under control of the spa-Gal4 driver (spa>ADAM17) in PGCs; (E, G, I) wild type; or (F, H, J) ADAM17−/− mutant retinas at 2 weeks (E, F), 3 weeks (G, H) and 4 weeks (I, J). K. Quantitation of the numbers of normal, abnormal and missing rhabdomeres from the TEM images corresponding to the genotypes mentioned above; n = 180 ommatidia from 3 different fly retinas for each. L. qPCR measurements of knockdown efficiency of ADAM17 with sparkling, elav or Rh1 GAL4s; n = 3 independent biological replicates with 3 technical replicates for each experiment. n = 3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median, and the square is the mean. Data information: All data were quantified for significance using Student's t test. ***P < 0.001. Scale bars: 10 μm. Download figure Download PowerPoint The degeneration we observed was progressive with age. Ommatidia in the retinas of 1-day-old ADAM17 mutant flies were mostly intact (Fig 2A and B), although they did display enlarged PGCs, indicating some abnormalities. Retinas of 2-, 3- and 4-week-old flies showed progressively more severe phenotypes, with only few identifiable neurons or glia seen in ADAM17 mutants from about 3 weeks of age (Fig EV1E–K). Collectively, these observations imply that ADAM17 in the glial cells of the adult retina protects both neurons and glia from age-dependent degeneration. Figure 2. Abnormal LD accumulation in young adult retinas upon a loss of ADAM17 in glial cells A–C. TEM images of 1-day-old adult retinas showing the overall structure of clusters of ommatidia (A1, B1, C1), or a single ommatidium (A2, B2, C2) in wild type (A1, A2), ADAM17−/− mutant (B1, B2) or ADAM17−/Df (C1, C2). Red arrows show lipid droplets in PGCs, and green shading highlights PGCs. D–J. Fluorescent images of 1-day-old fly retinas stained with BODIPY (green) and FM dye (red) to mark lipid droplets and the photoreceptor membranes, respectively; (D) wild type; (E) ADAM17−/− mutant; (F) ADAM17−/Deficiency; (G) knockdown of ADAM17 throughout the retina; (H) knockdown in glial cells; (I) knockdown in neurons; and (J) kuz−/−; n = 10 fly retinas. K, L. Quantitation of the BODIPY signal shown as integrated total lipid and normalised LD size of lipid droplets for the genotypes in (D–I). The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values, respectively, the central band is the median, and the square is the mean. Data were analysed using the Kruskal–Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***P < 0.001, **P < 0.01. M, N. Q-PCR analysis of mRNA transcripts of lipogenic genes -Acetyl CoA carboxylase (ACC) and Fatty Acid Synthase (FASN) from heads of wild-type and ADAM17−/− mutants; n = 4 and n = 3 biological replicates for M and N, respectively, with 3 technical replicates for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values, respectively, the central band is the median, and the square is the mean. Data were quantified for significance using Student's t test. ***P < 0.001, **P < 0.01. Data information: Scale bars for A2, B2 and C2: 2 μm and 10 μm for all other panels. Download figure Download PowerPoint Retinal degeneration of ADAM17 mutant flies is associated with LD accumulation To investigate the fundamental cause of the retinal degeneration, we looked in more detail for the earliest detectable phenotype. We saw no visible exterior eye defects, suggesting that there was no defect in eye development (Appendix Fig S1A and B). However, ultrastructural investigation by transmission electron microscopy revealed that PGCs of 1-day-old ADAM17−/− and ADAM17−/Df flies were enlarged and had an accumulation of lipid droplet-like structures; these were rare in wild-type retinas (Fig 2A–C). To confirm the identity of these structures, we labelled 1-day-old retinas with BODIPY 493/503 and FM 4-64FX dyes, which, respectively, label neutral lipids and cellular membranes. There was a striking accumulation of BODIPY-positive LDs in both ADAM17−/− and ADAM17−/Df retinas (Fig 2D–F and K). In order to quantify and compare lipid accumulation between conditions, we used image analysis to measure LD number, size and a combined measure of "integrated total lipid" (see Materials and Methods). In comparison with wild type, we observed a clear increase in the average size of LDs in ADAM17−/− mutants (Fig 2L), and an even more pronounced increase in integrated total lipid (Fig 2K). To strengthen the identification of these structures as LDs, we co-stained wild-type and ADAM17−/− adult retinas with BODIPY and lysotracker to check that they were not in fact lysosomal. There was no co-localisation between the two markers in either of the two genotypes (Pearson's correlation coefficient < 0.02; Appendix Fig S1C–E). We also assayed the levels of lipid storage droplet-2 (LSD2), a protein known to associate with lipid droplets, through Western blot (Welte et al, 2005). Consistent with our conclusion, LSD2 was upregulated in ADAM17−/− head lysates when compared to wild type (Appendix Fig S1F and G). LD accumulation was also caused by knockdown of ADAM17 with Actin-GAL4, which drives expression in both neurons and glial cells (Fig 2G, K and L). As with the degeneration phenotype, knockdown of ADAM17 specifically in PGCs, but not in neurons, also led to LD accumulation (Fig 2H, I, K and L). Also consistent with the degeneration phenotype, we showed that glial-specific expression of ADAM17 rescued the LD phenotype of the ADAM17−/− mutant (Fig EV2A–E) and that loss of the ADAM10 homologue, Kuzbanian, caused no accumulation of LDs in the retina (Fig 2J–L). Click here to expand this figure. Figure EV2. Rescue of LD phenotype with WT-ADAM17 and expression of lipogenic genes with a knockdown of ADAM17 using different drivers A–C. Fluorescent images of 1-day-old retinas, stained with BODIPY (green) and FM dye (red); (A) ADAM17−/− mutant; (B) glial-specific overexpression of ADAM17 in ADAM17−/− mutant; or (C) glial-specific overexpression of ADAM17 in a wild-type background. Scale bars: 10 μm. D, E. Quantitation of BODIPY staining (shown in A–C) depicted as (D) integrated total lipid and (E) normalised lipid droplet size; n = 10 for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values, respectively, the central band is the median, and the square is the mean. F, G. qPCR measurements of lipogenic transcripts of ACC and FASN across different knockdowns of ADAM17 with sparkling, elav or Rh1 GAL4s; n = 3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values, respectively, the central band is the median, and the square is the mean. Data information: All data were quantified for significance using Student's t test. ***P < 0.001. Download figure Download PowerPoint Increase in LD amount and size was accompanied by increased expression of lipogenic genes. We compared by qPCR the transcript levels of acetyl coA carboxylase (ACC) and fatty acid synthase 1 (FASN1), both enzymes in the lipid biosynthetic pathway. Both genes were upregulated in ADAM17−/− fly head lysates (Fig 2M and N). Consistent with all other data, when knocking down ADAM17 specifically in neurons or glia, we found that lipid synthesis genes only responded to glial ADAM17 loss (Fig EV2F and G). We also looked for defects earlier in retinal development. There was no upregulation of lipogenic gene transcripts, or in the numbers of LDs, in the larval brain, or eye or wing imaginal discs of ADAM17−/− mutants (Fig EV3A–H). Similarly, we observed no difference in total LD numbers between wild-type and ADAM17−/− pupal retinas (Fig EV3I–K). Furthermore, there was a sharp rise in ADAM17 transcript and protein levels between 40 h pupae and 1-day-old adults (Fig EV3L and M). Together, these results strongly suggest that the defects associated with a loss of ADAM17 arise in adulthood (or possibly very late pupal stages), rather than being caused by earlier developmental defects. Click here to expand this figure. Figure EV3. Loss of ADAM17 does not affect LD in larval and pupal tissues A–F. Fluorescent images of either wild-type or ADAM17−/− mutant larval (A, B) brain; (C, D) eye imaginal disc; and (E, F) wing imaginal disc, stained with BODIPY (green) and FM dye (red). G. Quantitation of BODIPY staining (shown in A–F) depicted as integrated total lipid; n = 10 for each genotype. The box end points represent the maximum and minimum values, respectively, the central band is the median, and the square is the mean. Data were analysed using the Kruskal–Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. H. FASN mRNA transcript levels measured by qPCR in wild-type and ADAM17−/− larvae; n = 4 biological replicates, with 3 technical replicates for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values, respectively, the central band is the median, and the square is the mean. I, J. Fluorescent images of either wild-type (I) or ADAM17−/− (J) mutant pupal (I, J) retinas stained with BODIPY (green) and FM dye (red). K. Quantitation of BODIPY staining (shown in A–F) depicted as integrated total lipid; n = 10 for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values, respectively, the central band is the median, and the square is the mean. L. Comparison of mRNA levels of ADAM17 between pupae and 1-day-old wild-type adults. n = 3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values, respectively, the central band is the median, and the square is the mean. M. Whole-mount pupal retina stained with anti-ADAM17 (green) and FM dye to mark the photoreceptor membranes (red); n = 10 fly retinas. Data information: Data were quantified for significance using Student's t test. ***P < 0.001. Scale bars: 10 μm. Download figure Download PowerPoint In summary, glial LD accumulation and upregulation of lipogenic genes in ADAM17−/− mutants mirrored, but preceded, cell degeneration in all experimental contexts we tested. Disrupting LD accumulation rescues degeneration in ADAM17−/− mutants There is a growing link between LD accumulation and the onset of neuronal degeneration (see, for example, Liu et al, 2015; Van Den Brink et al, 2018). When we measured the temporal pattern of LD accumulation in ADAM17−/− adult retinas, we observed a sharp decline in the numbers of LDs with age, starting at around 1 week, with their almost complete disappearance by about 2 weeks after eclosion (Appendix Fig S2A–H). This corresponds to the time when degeneration in the ADAM17−/− retinas was becoming prominent (Fig EV1E, F and K). To explore a potential functional link between LDs and cell degeneration, we asked whether degeneration depends on prior LD accumulation. We expressed the LD-associated lipase, Brummer (Bmm), a homologue of human adipocyte triglyceride lipase, either in PGCs or neurons using the same drivers as above, which are expressed from early in development. In PGCs, this continuously elevated lipase activity led to a striking reduction of LD accumulation in ADAM17−/− retinas (Fig 3A–C and E–G). In contrast, lipase expressed specifically in neurons had no effect on the LD accumulation phenotype of ADAM17 loss (Fig 3D–G). Consistent with these results, upon ageing, the flies in which LD accumulation was prevented by glial lipase expression showed almost complete rescue of retinal degeneration; there was much less rescue of degeneration when lipase was overexpressed in neurons (Fig 3H–L). We conclude that prior LD accumulation in PGCs is causally linked to the degeneration induced by loss of ADAM17 in the adult retina; also that the primary source of the abnormally accumulating lipids in these mutants are the glial cells themselves. Figure 3. Overexpressing lipase in ADAM17−/− mutant retinas rescues both lipid droplet accumulation and retinal degeneration A–D. Fluorescent images of 1-day-old retinas, stained with BODIPY (green) and FM dye (red) to stain lipid droplets and the photoreceptor membranes, respectively; (A) wild type; (B) ADAM17−/− mutant; (C) overexpression of lipase in glial cells of ADAM17−/− mutant; (D) overexpression of lipase in neurons of ADAM17−/− mutant. E–G. Quantitation of the BODIPY signal shown as (E) LD numbers; (F) normalised LD size; (G) integrated total lipid, for the genotypes mentioned above, n = 10 for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values, respectively, the central band is the median, the square is the mean, and the diamond an outlier. H–K. TEM images of 5-week-old adult retinas corresponding to (H) wild type; (I) ADAM17−/− mutant; (J) glial overexpression of lipase in wild-type glial cells; (K) neuronal overexpression of lipase in the ADAM17−/−mutant. L. Quantitation of the percentage of normal, abnormal and missing rhabdomeres observed in the TEM images for the genotypes mentioned above, presented as a stacked graph plot; n = 180 ommatidia from 3 different fly retinas for each. Data information: Data were quantified for significance using Student's t test. ***P < 0.001, **P < 0.01. Scale bars: 10 μm. Download figure Download PowerPoint Drosophila ADAM17 has metalloprotease activity Is Drosophila ADAM17 an active protease like its mammalian counterpart? It has been predicted to be, but there has been no direct demonstration of its ability to cleave substrates. Alignment of Drosophila and human ADAM17 revealed that the N-terminal pro-domain is very different between species and that fly ADAM17 has a shorter C-terminus compared to its human counterpart (Appendix Fig S3A). Conversely, two essential functional domains, the catalytic site and the CANDIS juxtamembrane domain, are well conserved (Appendix Fig S3A), suggesting that the Drosophila enzyme may also have metalloprotease activity. To test this, we co-expressed Drosop
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