Glial Phagocytic Receptors Promote Neuronal Loss in Adult Drosophila Brain

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Glial Phagocytic Receptors Promote Neuronal Loss in Adult Drosophila Brain Graphical Abstract

Authors Ketty Hakim-Mishnaevski, Naama Flint-Brodsly, Boris Shklyar, Flonia Levy-Adam, Estee Kurant

Drosophila Adult Brain

Correspondence [email protected]

Glia

Neuron

Elevated expression of phagocytic receptors SIMU and/or Drpr

MFG-E8

Neuronal loss (DA and GABA) Motor dysfunction Shortened lifespan

PS MFG-E8 Drpr SIMU

Highlights d

Increased levels of glial phagocytic receptors induce neuronal loss in adult brains

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Glial overexpression of SIMU or Drpr causes motor decline and early onset death

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Receptor-induced phenotypes are reversed by masking phosphatidylserine

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Glial phagocytic receptor levels must be controlled for proper brain function

Hakim-Mishnaevski et al., 2019, Cell Reports 29, 1438–1448 November 5, 2019 ª 2019 The Authors. https://doi.org/10.1016/j.celrep.2019.09.086

In Brief Neuronal dysfunction correlates with a declined glial phagocytic ability, suggesting that increased glial phagocytosis may prevent neurodegeneration. In contrast, HakimMishnaevski et al. reveal that an elevated expression of phagocytic receptors SIMU and/or Draper in glia causes neuronal loss, motor dysfunction, and a shortened lifespan, demonstrating that glial phagocytosis may harm proper brain function.

Cell Reports

Report Glial Phagocytic Receptors Promote Neuronal Loss in Adult Drosophila Brain Ketty Hakim-Mishnaevski,1 Naama Flint-Brodsly,1 Boris Shklyar,1 Flonia Levy-Adam,2 and Estee Kurant1,2,3,* 1Department

of Human Biology, Faculty of Natural Sciences, University of Haifa, Haifa 34988, Israel of Genetics and Developmental Biology, The Rappaport Family Institute for Research in the Medical Sciences, Faculty of Medicine, Technion - Israel Institute of Technology, Haifa 31096, Israel 3Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2019.09.086 2Department

SUMMARY

Glial phagocytosis is critical for the development and maintenance of the CNS in vertebrates and flies and relies on the function of phagocytic receptors to remove apoptotic cells and debris. Glial phagocytic ability declines with age, which correlates with neuronal dysfunction, suggesting that increased glial phagocytosis may prevent neurodegeneration. Contradicting this hypothesis, we provide experimental evidence showing that an elevated expression of the phagocytic receptors Six-Microns-Under (SIMU) and Draper (Drpr) in adult Drosophila glia leads to a loss of both dopaminergic and GABAergic neurons, accompanied by motor dysfunction and a shortened lifespan. Importantly, this reduction in neuronal number is not linked to neuronal apoptosis, but rather to phosphatidylserine-mediated phagoptosis of live neurons by hyper-phagocytic glia. Altogether, our study reveals that the level of glial phagocytic receptors must be tightly regulated for proper brain function and that neurodegeneration occurs not only by defective, but also excessive glial cell function. INTRODUCTION Glia, the main phagocytes in the developing and mature CNS, perform their phagocytic function by engulfing and degrading dying neurons, excessive neuronal branches, and synapses and by degenerating neuronal axons (Bilimoria and Stevens, 2015; Casano and Peri, 2015; Cronk and Kipnis, 2013; Cunningham et al., 2013; Freeman, 2006; Hanisch and Kettenmann, 2007). The precision of glial phagocytosis mainly relies on the function of phagocytic receptors, which recognize specific signals such as phosphatidylserine (PS) exposed on biological substrates destined for removal (Brown et al., 2015; Neher et al., 2013; Ravichandran, 2011). Previous studies have demonstrated that Drosophila phagocytic glia are functionally and molecularly similar to their vertebrate counterparts (Awasaki et al., 2006; Fuentes-Medel et al.,

2009; Kurant, 2011; Kurant et al., 2008; Logan and Freeman, 2007; MacDonald et al., 2006, 2013; Purice et al., 2017, 2016; Tasdemir-Yilmaz and Freeman, 2014; Watts et al., 2004). Two glial transmembrane phagocytic receptors, Six-Microns-Under (SIMU; a Drosophila homolog of Stabilin-2) (Kim et al., 2012; Kurant et al., 2008; Park et al., 2008) and Draper (Drpr; a Drosophila homolog of MEGF10 and Jedi) (Freeman et al., 2003; Scheib et al., 2012; Wu et al., 2009), play important roles during CNS development and in the maintenance of the adult brain. Loss of simu or drpr leads to impaired removal of apoptotic neurons, remodeling of synapses, and axonal pruning during development, as well as delayed clearance of degenerating axons in the adult brain, ultimately causing neurodegeneration (Awasaki et al., 2006; Etchegaray et al., 2016; Freeman et al., 2003; Fuentes-Medel et al., 2009; Kurant et al., 2008; MacDonald et al., 2006, 2013; Purice et al., 2017, 2016; Ray et al., 2017; Tasdemir-Yilmaz and Freeman, 2014). Moreover, it has been shown that with age, glial phagocytosis of injured axons declines, and this is associated with a reduced expression of Drpr (Purice et al., 2016). Recently, an increased expression of Drpr was reported to rescue amyloid beta toxicity in a Drosophila model of Alzheimer’s disease (Ray et al., 2017). These latest findings suggest that an overexpression of phagocytic receptors promoting glial phagocytosis could play a protective role in the aging brain and prevent neurodegeneration. Contradicting this hypothesis, we provide experimental evidence showing that an elevated expression of the phagocytic receptors Drpr and/or SIMU in adult Drosophila glia leads to a neuronal loss of both dopaminergic (DA) and GABAergic (GABA) neurons, accompanied by motor dysfunction and a shortened lifespan. We further show that the neuronal loss is not the result of neuronal apoptosis, but rather is due to glial phagocytosis of live neurons, a process termed ‘‘phagoptosis.’’ Strikingly, we observed that survival and motility defects of affected flies can be significantly rescued by masking PS through the expression of the milk fat globule-EGF factor 8 (MFG-E8) protein, indicating that phagoptosis of live neurons is mediated via recognition of PS. Surprisingly, we observed that PS masking protects GABA neurons yet does not affect the loss of DA neurons. This suggests that DA neurons are not the main contributors to the motor ability and survival of adult flies. Altogether, our study reveals that though glial phagocytosis is

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a very important process, it must be tightly regulated for proper brain function. We show that neurodegeneration may involve not only defective, but also excessive glial cell function. RESULTS SIMU Is Not Expressed in Adult Brain Glia SIMU is specifically expressed during Drosophila embryogenesis in all phagocytic cell populations, namely macrophages, glia, and ectoderm (Kurant et al., 2008). Previous northern blot analysis detected simu expression in the heads of 1-day-old flies (Kurant et al., 2008). To further examine the tissue-specific expression of the SIMU protein in adults, we performed immunostaining of cryo-sectioned heads of 1-day-old flies with specific anti-SIMU antibodies. In these experiments, glial cells and macrophages were labeled with GFP using the repoGal4,UAScytGFP and serpentGal4,UAScytGFP markers, respectively (Figure 1). We did not detect any SIMU expression in adult brain glia (Figures 1A–1A%), only in macrophages located in the vicinity of the brain (Figures 1B–1B%). These data indicate that SIMU does not function in adult brain glia, pointing to its specific role in the phagocytosis of apoptotic cells during CNS development. Increased Expression of SIMU in Glia Leads to Neuronal Loss Neurodegeneration in older flies is often associated with a decline in glial cell function, most notably in their ability to phagocytose (Harry, 2013; Koellhoffer et al., 2017; Logan, 2017). Given that SIMU is a potent phagocytic receptor for apoptotic cells, we tested whether its expression in glia facilitated neuronal activity upon aging by improving glial cells’ phagocytic ability. Accordingly, we expressed two independent simu transgenes, UASsimuI and UASsimuII, specifically in glia using the pan-glial Gal4 driver, repoGal4 (repoGal4XUASsimuI/II). Surprisingly, these flies exhibited all the hallmarks of neurodegeneration symptoms, including declined motor activity, reduced survival rates, and a loss of neurons. Transgenic flies expressing SIMU in glia exhibited a strong motor deficit and a shorter lifespan, compared to control flies (Figures 1C and 1D). These phenotypes were

much stronger than those described for a Drosophila Parkinson’s disease model (Feany and Bender, 2000). We next quantified the number of DA neurons labeled with antityrosine hydroxylase (TH) antibodies, which are known to be required for motor ability (Brichta and Greengard, 2014; Figures 1E–1E00 ). Compared to wild-type brains, a significantly lower number of DA neurons was detected in the brains when SIMU was expressed in glia (Figures 1E–1E00 ). To examine an additional type of neurons, which could be affected by SIMU-expressing glial cells, we used specific anti-GABA antibodies to label GABA neurons in adult Drosophila brains. Counting these neurons in a specific area on the dorsal side of the brain (Figures 1F and 1F0 ) revealed a significant reduction in their numbers in the flies expressing SIMU in glia (Figures 1F0 and 1F00 ), compared to control flies (Figures 1F and 1F00 ). These data suggest that GABA neurons were eliminated by SIMU-expressing glia similarly to DA neurons. However, we did not detect any difference in glial cell numbers (glia labeled with anti-REPO antibodies) between wild-type and SIMU-expressing flies (Figures 1G–1G00 ). Taken together, these results suggest that SIMU-specific expression in glia causes a neuronal loss in a non-autonomous manner. Importantly, neuron-specific expression of SIMU using an elavGal4 driver (elavGal4XUASsimuI; Figure S1) did not affect the motor function or lifespan of these flies, which behaved similarly to control flies (Figures 1C and 1D). Moreover, the numbers of DA and GABA neurons were similar to those in control flies (data not shown), indicating that only glia-specific expression of SIMU caused neuronal loss. We thus conclude that the ectopic expression of the phagocytic receptor SIMU in glia does not improve brain health, as we initially hypothesized, but rather promotes neurodegeneration. SIMU Expression in Glia Does Not Induce Neuronal Apoptosis Considering the role of SIMU in eliminating apoptotic cells, we first reasoned that the loss of neurons observed in flies expressing SIMU in glia could be due to an increased number of neurons undergoing apoptosis. To test this, we assessed apoptosis in the brains of adult flies carrying SIMU using anti-cleaved Caspase-3 antibodies. In control brains, no staining was detected, consistent with a low level of apoptosis (Figure 1H). As a positive control

Figure 1. Glial Expression of SIMU Causes Neuronal Loss Not Linked to Neuronal Apoptosis and Accompanied by Impaired Climbing Activity and Shortened Lifespan (A–B%) Cryo-sections (13 mm) of heads from 1-day-old Drosophila females stained with anti-SIMU (red; A, A00 , B, and B00 ) and DAPI (blue; A, A%, B, and B%). Glia are labeled with repoGal4,UAScytGFP (green; A and A0 ) and macrophages with serpentGal4,UAScytGFP (green; B and B0 ). Bar, 50 mm. Arrows depict macrophages. (C and D) Climbing ability (C) and survival rates (D) of flies ectopically expressing simu (two different transgenes) in glia with repoGal4 (repoGal4XUASsimuI or II) and in neurons with elavGal4 (elavGal4XUASsimuI). UASsimu flies served as the control. Error bars indicate the SEM; asterisks indicate statistical significance versus control, as determined by two-way ANOVA. ****p < 0.0001, *p < 0.05; n.s., non-significant. (E–G00 ) Projections from confocal stacks of the posterior part (45 mm) (E–F0 ) or the entire brain (G and G0 ) of whole-mount 12-day-old female brains (after the flies stopped climbing). Bar, 50 mm. (E and E0 ) DA neurons labeled with anti-TH (red), (F and F0 ) GABA neurons labeled with anti-GABA (red), and (G and G0 ) glia labeled with anti-REPO (green) in control (E, F, and G) and repoGal4XUASsimu (E0 , F0 , and G0 ) brains. (E00 , F00 , and G00 ) The mean total numbers of DA neurons (E00 ), GABA neurons located in the designated area (white frame) (F00 ), or glial cells (G00 ) within confocal stacks of the brain, ± SEM, n = 7; asterisks indicate statistical significance versus control, as determined by Students’ t test. ****p < 0.0001, **p < 0.01; n.s., non-significant. (H–H00 ) Projections from confocal stacks of whole-mount adult brains of 12-day-old females stained with anti-cleaved Caspase-3 (red). (H) Wild-type. (H0 ) drpr mutant. (H00 ) repoGal4XUASsimu. Bar, 50 mm. (I) Schematic of truncated UASsimu constructs (J) Survival rates of flies expressing truncated UASsimu constructs. Error bars indicate the SEM; asterisks indicate statistical significance versus repoGal4XUASsimu, as determined by two-way ANOVA. ****p < 0.0001; n.s., non-significant.

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for the antibody staining, drpr-null mutant flies were used, in which apoptotic cells persist for long periods of time (Etchegaray et al., 2016; Figure 1H0 ). In the brains of flies expressing simu in glia (repoGal4XUASsimu), no caspase staining was detected (Figure 1H00 ), indicating no apoptosis in these brains. We conclude that the loss of neurons observed upon the glial expression of SIMU could not be explained by increased levels of neuronal apoptosis. Truncated SIMU Proteins Do Not Promote Neuronal Loss SIMU is a transmembrane receptor that has been implicated solely in phagocytosis. If the observed neuronal loss was not a result of apoptosis, an alternative mechanism underlying this phenotype could be glial phagocytosis of live neurons, a process named phagoptosis (Brown and Neher, 2014; Brown and Vilalta, 2015). To test this possibility, we examined whether SIMU protein variants lacking a phagocytic ability retained the ability to induce neuronal loss. Using repoGal4, we specifically expressed different truncated SIMU proteins in glia (Figure 1I), previously shown as being incapable of performing phagocytosis (Shklyar et al., 2013). Transgenic flies expressing truncated constructs lacking specific domains required for SIMU binding to apoptotic cells (UASsimuDEmi-Nim2) or specific domains involved in SIMU-co-receptor interactions (UASsimuDNim3Nim4) exhibited normal lifespans (Figure 1J). These data indicate that domains essential for the phagocytic ability of SIMU are also required for neuronal loss and suggest that SIMU expression potentiates the phagocytic activity of glia. Restricted Expression of SIMU in Brain Glia of Adults Causes Neuronal Loss To determine the effect of simu in flies that underwent normal development, we conditionally expressed simu in adult brain glia using the temperature-sensitive allele of a Gal80 repressor (tubGal80ts;repoGal4XUASsimu). At 18 C, tubGal80ts is expressed in all embryonic tissues and prevents repoGal4 from inducing simu expression. At 29 C, Gal80 is inactivated, and repoGal4 is able to drive simu expression specifically in adult brain glia (Figures 2A and 2B). To test the dose-dependent effect of SIMU in adult brain glia, we generated transgenic flies expressing one copy

(tubGal80ts;repoGal4XUASsimu) or two copies (tubGal80ts; repoGal4XUASsimuX2) of simu in glia (Figure 2B). The transgenic flies were transferred immediately after eclosion from 18 C to 29 C and examined using the tests described above. Flies expressing one or two copies of simu exhibited significantly shorter lifespans and reduced climbing activity (Figures 2C and 2D), as well as lower numbers of DA and GABA neurons in the adult brains, compared to control flies (Figures 2E and 2F). In all these experiments, neurodegenerative symptoms were more marked in the flies expressing two rather than one copy of simu, which demonstrates that temporarily regulated expression of SIMU caused neuronal loss in a dose-dependent manner. No differences in glial cell numbers were detected between control and simu-expressing brains (Figure 2G), once more demonstrating the non-autonomous effect of SIMU on neuronal survival. Elevated Expression of Drpr in Adult Brain Glia Causes Neuronal Loss Unlike SIMU, which is not expressed in the adult CNS, Drpr is continuously expressed in the brains of adult flies (MacDonald et al., 2006, 2013); Figures 2H and 2I), and its expression is upregulated during axonal injury to promote the clearance of neuronal debris (MacDonald et al., 2006, 2013). To further explore whether any phagocytic receptor can promote neuronal loss, we repeated our experiments described above, this time specifically overexpressing drpr in adult brain glia using the temperature-sensitive Gal80 system (tubGal80ts;repoGal4XUASdrpr) (Figure 2I). Similarly to simu-expressing flies, drpr-overexpressing flies exhibited significantly reduced climbing activity (Figure 2J) and shorter lifespans (Figure 2K), as well as lower numbers of DA and GABA neurons, compared to control flies (Figures 2L and 2M). These data demonstrate that an increased expression of Drpr specifically in adult brain glia causes non-autonomous neuronal loss, again linking glial phagocytosis and neuronal loss in a cause-effect relationship. Concomitant Expression of SIMU and Drpr in Adult Brain Glia Increases Neuronal Loss We next investigated whether SIMU and Drpr cooperate in the induction of neuronal loss. Overexpression of both receptors in

Figure 2. SIMU or Drpr Expression in Adult Brain Glia Causes Neuronal Loss Accompanied by Impaired Climbing Ability and Shortened Lifespan (A) Head cryo-sections (13 mm) of a Drosophila female ectopically expressing SIMU in adult brain glia (tubGal80ts;repoGal4XUASsimu), stained with anti-SIMU (red) and DAPI (blue). Glia are labeled with repoGal4,UAScytGFP (green). Bar, 50 mm. (B) Western blot analysis of extracts from wild-type, simu mutant, tubGal80ts;repoGal4XUASsimu, and tubGal80ts;repoGal4XUASsimuX2 female fly heads with anti-SIMU and anti-actin including low temperature controls at 18 C. (C and D) Climbing ability (C) and survival rates (D) of flies ectopically expressing simu in glia with tubGal80ts;repoGal4 (one or two copies). tubGal80tsXUASsimu served as control. Error bars indicate the SEM; asterisks indicate statistical significance versus control, as determined by two-way ANOVA, ****p < 0.0001. (E–G) The mean total numbers of DA neurons (E), GABA neurons in the designated area (F), or glial cells (G) within confocal stacks of the brain, ± SEM, n = 7; asterisks indicate statistical significance, as determined by two-way ANOVA (E and F) or by Students’ t test (G). ****p < 0.0001, *p < 0.05; n.s., non-significant. (H) Projections from confocal stacks (13 mm) of whole-mount adult brain stained with anti-Drpr (red) and glia labeled with repoGal4,UAScytGFP (green). Bar, 50 mm. (I) Western blot analysis of extracts from wild-type, drpr mutant, and tubGal80ts;repoGal4XUASdrpr (at 18 C and at 29 C) female fly heads with anti-Drpr and antiactin. (J and K) Climbing ability (J) and survival rates (K) of flies with elevated expression of drpr in adult glia. tubGal80tsXUASdrpr flies served as control. Error bars indicate the SEM; asterisks indicate statistical significance versus control, as determined by two-way ANOVA. ****p < 0.0001. (L and M) The mean total numbers of DA neurons (L) and GABA neurons located in the designated area (M) within confocal stacks of the brain, ± SEM, n = 7; asterisks indicate statistical significance versus control, as determined by Students’ t test. ****p < 0.0001,**p < 0.01.

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Figure 3. Concomitant SIMU and Drpr Expression in Adult Brain Glia Increases the Neuronal Loss Caused by Either Receptor Alone (A and B) Climbing ability (A) and survival rates (B) of flies expressing simu (tubGal80ts;repoGal4XUASsimu;UAScytGFP) or drpr (tubGal80ts;repoGal4XUASdrpr; UAScytGFP) or both (tubGal80ts;repoGal4XUASsimu,UASdrpr) in adult brain glia. tubGal80ts;repoGal4XUAScytGFP flies served as the control. Error bars

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adult brain glia (tubGal80ts;repoGal4XUASsimu,UASdrpr) caused much stronger defects in climbing ability (Figure 3A), as well as in survival rates (Figure 3B), compared to control flies and flies expressing either receptor alone (Figures 3A and 3B). The numbers of DA and GABA neurons were also significantly lower than in flies carrying either receptor alone (Figures 3C–3C00 and 3D–3D00 ). This strong phenotype was similar to what was seen in flies expressing two copies of simu in glia (Figures 2C–2F), suggesting an expression-level-dependent effect of glial phagocytic receptors on the elimination of live neurons. Since repoGal4 is a strong pan-glial Gal4 driver that induces a robust expression of SIMU and Drpr in all adult brain glia simultaneously, we next investigated whether a lower and more restricted expression of SIMU and Drpr would also lead to the same phenotype. We replicated our experiments by using Gal4 drivers that are known to be weaker than repoGal4 and restricted to the specific glial cell types, including cortex glia (R54H02Gal4), astrocytes (R86E01-Gal4), and ensheathing glia (R56F03-Gal4) (Kremer et al., 2017). These Gal4 drivers were combined with tubGal80ts to restrict SIMU and Drpr expression to the adult stage. Using the neurodegeneration tests described above, we observed significantly reduced motor ability, lifespan, and neuronal cell numbers in flies expressing SIMU and Drpr together (Figures 3E–3H) in cortex and ensheathing glia, though the phenotypes appeared weaker compared to the pan-glial repoGal4-driven expression (Figures 3A–3D00 ). Moreover, the specific expression of SIMU and Drpr in astrocytes significantly reduced climbing activity and neuronal cell number (Figures 3E, 3G, and 3H) but did not affect the survival rate of the flies (Figure 3F). This interesting result suggests that the cortex and ensheathing glia, but not astrocytes, affect additional types of neurons (not only DA and GABA) that influence lifespan. Interestingly, overexpression of simu solely in different glial cell types slightly but significantly affected climbing ability, survival rate, and DA neuronal cell numbers (Figures S2D–S2F) but did not reduce the number of GABA neurons (Figure S2G). We conclude that the strength of glia-mediated neurodegeneration depends on the expression level of the phagocytic receptor and the subtype of glial cells.

nate stressed neurons exposing PS on their surfaces via phagoptosis (Brown and Neher, 2014; Brown and Vilalta, 2015). Given that SIMU and Drpr bind PS on apoptotic cells (Shklyar et al., 2013; Tung et al., 2013), we speculated that these proteins may also promote neuronal loss by phagoptosis. To test this hypothesis, we used transgenic flies expressing a secreted mammalian molecule, MFG-E8, which specifically binds PS (Hanayama et al., 2002) and prevents its interaction with receptors. When we analyzed the expression of GFP-tagged MFG-E8 (UASmfg-e8-GFP) (Tung et al., 2013) driven by repoGal4 in adult brain glia (tubGal80ts;repoGal4XUASmfg-e8GFP), GFP was detected everywhere (Figures 4A–4A00 ) and explicitly on GABA neurons (Figures 4B0 ,4B00 , 4C0 , and 4C00 ). In addition, we expressed the same UASmfg-e8-GFP construct with the specific cortex glia driver (R54H02-Gal4XUASmfge8-GFP;tubGal80ts) and found a colocalization of GFP with GABA neurons as well (Figure S3). These data demonstrate that GFP-tagged MGF-E8 is distributed throughout the brain, where it can bind PS on neuronal surfaces. When we concomitantly expressed SIMU or Drpr with MFG-E8 in adult brain glia (tubGal80ts;repoGal4XUASsimu;UASmfg-e8 or tubGal80ts; repoGal4XUASdrpr;UASmfg-e8), we found that strikingly, it significantly improves climbing activity (Figures 4D and 4F) and survival rates (Figures 4E and 4G), compared to flies expressing either SIMU or Drpr, suggesting that these phenotypes are PS dependent. Interestingly, MFG-E8 expression did not increase the number of DA neurons (data not shown), proposing that the reduced climbing ability and shorter lifespan may have resulted from the elimination of other neuronal type(s). Consistent with this notion, we found that MFG-E8 prevented the loss of GABA neurons in SIMU- and Drpr-expressing flies (Figures 4H–4N), demonstrating that the loss of these neurons depends on PS and suggesting that a loss of GABA but not DA neurons caused the motor defect. Collectively, our study reveals that an increased expression of phagocytic receptors in glia is sufficient to induce neuronal loss in the absence of apoptosis, and this process involves the exposure of PS on neuronal surfaces.

Masking PS Rescues SIMU-/Drpr-Induced Motor Impairment and Increases Survival Rates In mammals, microglia have been shown to engulf live neurons upon inflammation (Brown and Neher, 2014; Brown and Vilalta, 2015; Vilalta and Brown, 2018). In this context, microglia elimi-

Phagocytic glia play critical roles in the developing and adult CNS, where they clear unneeded and dangerous materials. Consistent with this role, reduced phagocytic ability has been associated with neurodegeneration (Harry, 2013; Koellhoffer et al., 2017; Logan, 2017; Napoli and Neumann, 2009; Salter

DISCUSSION

indicate the SEM; asterisks indicate statistical significance versus control and expression of each receptor alone, as determined by two-way ANOVA. ****p < 0.0001. (C–D00 ) Projections from confocal stacks of the posterior part (45 mm) of whole-mount 14-day-old female brains (after the flies stopped climbing). Bar, 50 mm. (C and C0 ) DA neurons labeled with anti-TH (red) and (D and D0 ) GABA neurons labeled with anti-GABA (green) in (C and D) control and (C0 and D0 ) tubGal80ts;repoGal4XUASsimu,UASdrpr brains. (C" and D") The mean total numbers of (C00 ) DA or (D00 ) GABA neurons located in the designated area (white frame) within confocal stacks of the brain, ± SEM, n = 7; asterisks indicate statistical significance versus control, as determined by two-way ANOVA. ***p < 0.001,*p < 0.05; n.s., non-significant. (E and F) Climbing ability (E) and survival rates (F) of flies expressing simu and drpr concurrently (tubGal80ts;type-specificGal4XUASsimu,UASdrpr) in different types of adult brain glia: cortex glia (R54H02-Gal4), ensheathing glia (R56F03-Gal4), and astrocytes (R86E01-Gal4). UASsimu,UASdrpr;tubGal80tsXw- flies served as the control. Error bars indicate the SEM; asterisks indicate statistical significance versus control, as determined by two-way ANOVA. ****p < 0.0001; n.s., non-significant. (G and H) The mean total numbers of (G) DA or (H) GABA neurons located in the designated area within confocal stacks of the brain, ± SEM, n = 7; asterisks indicate statistical significance versus control, as determined by two-way ANOVA. ***p < 0.001, **p < 0.01, *p < 0.05.

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and Stevens, 2017; von Bernhardi et al., 2015; Wolf et al., 2017). This led us to hypothesize that an increased expression of phagocytic receptors could be beneficial. Contrary to this hypothesis, we observed that an increased phagocytic ability of glia promoted neurodegeneration. As such, we have discovered a mechanism that leads to neuronal loss in the adult brain in which glial phagocytic receptors play a central role. In Drosophila, two phagocytic receptors (SIMU and Drpr) are sufficient for establishing the phagocytic ability of glial cells (Shklyar et al., 2014). Both receptors are expressed during development, when brain tissue remodeling is important; a lack of these receptors causes specific defects (Freeman et al., 2003; Hilu-Dadia et al., 2018; Kurant et al., 2008; McLaughlin et al., 2019; Tasdemir-Yilmaz and Freeman, 2014). They play critical roles in glial phagocytosis and have been shown to recognize PS on apoptotic cells (Shklyar et al., 2013; Tung et al., 2013). While Drpr remains expressed in adult mature brains and can be induced upon injury, the expression of SIMU is turned off under similar conditions. This observation was initially surprising, as we expected that a higher phagocytic ability would correlate with better brain health. Our present study explicates this by showing that increased levels of SIMU in the adult brain glia are deleterious, causing neuronal loss. This demonstrates that the levels of phagocytic receptors must be tightly regulated and eventually reduced after development. As SIMU and Draper are transmembrane receptors implicated solely in phagocytosis, and neuronal loss is observed when they are expressed in glial cells of the adult, we favor the notion that they eliminate neurons by virtue of their phagocytic ability through a process of phagoptosis and not by other ways. Our finding that neurodegeneration can result from excessive glial cell activity is likely not restricted to the fly brain. Studies have shown that in mammals, inflammation-activated glia secrete factors that induce PS exposure on viable neurons, which leads to their phagoptosis by glia (Brown and Vilalta, 2015). In the experiments presented here, the only manipulation we performed was the specific glial expression of SIMU, Drpr, or both receptors. In these cases, neurons did not show any sign of apoptosis. Since the level of PS exposure was not modified when SIMU was expressed in glia (Figure S3), our results further

suggest that basal levels of PS exposed on live neurons can interact with phagocytic receptors on glia. Thus, the phagocytosis of neurons is not only determined by the level of PS exposure on the neuronal surface, but also by the level of phagocytic receptors on glial cells. This statement is further corroborated by the milder neurodegeneration phenotypes that we observed when SIMU or SIMU and Drpr expression was restricted to specific glial cell types using weaker and restricted Gal4 drivers. It is striking that reducing PS exposure by expressing MFG-E8 was sufficient to revert the neurodegeneration phenotype. We observed that PS masking by MFG-E8 significantly improved both climbing activity and survival rates, as well as prevented the loss of GABA neurons. This clearly demonstrates the role of PS exposure in neuronal loss. However, DA neurons were not rescued by PS masking, suggesting that distinct types of neurons present different sensitivities to phagoptosis. In neurodegenerative disorders, it remains elusive why different types of neurons are highly vulnerable to degeneration in different diseases (Brichta and Greengard, 2014). Based on our data, we speculate that DA neurons expose higher amounts of PS on their surfaces, compared to other types of neurons. Therefore, masking PS with a given amount of MFG-E8 can rescue the elimination of other types of neurons, such as GABA neurons, but would be insufficient to rescue the loss of DA neurons. Our experiments using the glial-type-specific Gal4 drivers further verify this speculation, since GABA neurons were less affected when the receptors were overexpressed in specific glial cell types, demonstrating that they are less sensitive to phagoptosis than DA neurons. The improved climbing activity of flies expressing both SIMU or Drpr and MFG-E8 with a reduced number of DA neurons, as compared to control flies, suggests that DA neurons are not entirely responsible for climbing ability, thus opening new directions in understanding the molecular mechanisms underlying the neuronal control of motility in adult flies. Increased expression of the phagocytic receptor TREM2 in mice microglia and astrocytes has been implicated in Alzheimer’s disease (AD) (Frank et al., 2008). Similar TREM2 overexpression was observed in post-mortem temporal cortices from AD patients (Lue et al., 2014). However, a cause-effect relationship between glial TREM2 expression and neuronal pathology

Figure 4. Glia-Derived MFG-E8 Rescues Climbing Ability, Survival Rates, and Loss of GABA Neurons in Flies Overexpressing SIMU or Drpr in Adult Brain Glia (A–C00 ) Apotome single-slice images of whole-mount female brains of flies expressing GFP-tagged human MFG-E8 (tubGal80ts;repoGalX:UASmfg-e8-GFP). MFG-E8 is labeled with anti-GFP. Bar, 50 mm. (A0 and A00 ) Glial nuclei are labeled with anti-REPO (red). (B0 , B00 , C0 , and C00 ) GABA neurons are labeled with anti-GABA (red). Arrows depict GFP staining on GABA neurons. (D and E) Climbing ability (D) and survival rates (E) of flies expressing UASsimu (tubGal80ts;repoGal4XUASsimu;UAScyGFP), UASmfg-e8-GFP (tubGal80ts; repoGal4XUASmfg-e8-GFP), and UASsimu with UASmfg-e8-GFP (tubGal80ts;repoGal4XUASsimu;UASmfg-e8-GFP) in adult brain glia. tubGal80ts; repoGal4XUAScytGFP flies served as the control. (F and G) Climbing ability (F) and survival rates (G) of flies expressing UASdrpr (UAScytGFP;repoGal4XUASdrpr;tubGal80ts), UASmfg-e8-GFP (tubGal80ts; repoGal4XUASmfg-e8-GFP), and UASdrpr with UASmfg-e8-GFP (UASmfg-e8-GFP;repoGal4XUASdrpr;tubGal80ts) in adult brain glia. tubGal80ts; repoGal4XUAScytGFP flies served as the control. Error bars indicate the SEM; asterisks indicate statistical significance versus expression of each receptor alone, or MFG-e8-GFP alone, as determined by two-way ANOVA. ****p < 0.0001, **p < 0.001; n.s., non-significant. (H–M) Projections from Apotome stacks of the posterior part of whole-mount female brains (after the flies stopped climbing). GABA neurons labeled with anti-GABA (red). (H) tubGal80ts;repoGal4XUAScytGFP as control, (I) tubGal80ts;repoGal4XUASmfg-e8-GFP;UAScytGFP, (J) tubGal80ts; repoGal4XUAScytGFP;UASsimu, (K) tubGal80ts;repoGal4XUASmfg-e8-GFP;UASsimu, (L) UAScytGFP;repoGal4XUASdrpr;tubGal80ts, and (M) UASmfg-e8GFP;repoGal4XUASdrpr;tubGal80ts. (N) The mean total numbers of GABA neurons located in the designated area (white frame) within Apotome stacks of the brain, ± SEM, n = 7; statistical significance is determined by two-way ANOVA. *p < 0.05.

1446 Cell Reports 29, 1438–1448, November 5, 2019

has yet to be established. Our study provides experimental evidence for a causative role of glial phagocytosis in neuronal loss, which is instigated by the increased expression of phagocytic receptors. This gain-of-function of glial phagocytic receptors resulting from their abnormal expression must be considered a risk factor in neurodegenerative disorders. Many genetic and environmental parameters can elicit an increase in the level of phagocytic receptors on glial cells. Chromosomal translocations affecting phagocytic receptor genes are predisposed to induce increased expression, as observed for oncogenes in cancer. Alternatively, the elevated expression of receptors induced upon microbial infection or in response to inflammatory signals associated with autoimmunity could cause glia-mediated neurodegenerative diseases. Further assessments of phagocytic receptor levels in healthy individuals before the onset of neurodegenerative disease symptoms may help in early diagnosis and treatment of such disorders. The fly system, allowing easy manipulation of phagocytic receptor levels, can be of great use for uncovering molecular mechanisms of glia-induced neurodegeneration.

Draft, E.K.; Writing – Review & Editing, E.K.; Funding Acquisition, K.H.-M. and E.K.; Supervision, K.H.-M. and E.K.

STAR+METHODS

Brown, G.C., and Neher, J.J. (2014). Microglial phagocytosis of live neurons. Nat. Rev. Neurosci. 15, 209–216.

Detailed methods are provided in the online version of this paper and include the following:

Brown, G.C., and Vilalta, A. (2015). How microglia kill neurons. Brain Res. 1628, 288–297.

d d d d

d d

KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS B Fly Strains METHOD DETAILS B Immunohistochemistry B Imaging and Quantification Procedure B Western Blot Analysis B Cryo-sections B Behavioral Assays QUANTIFICATION AND STATISTICAL ANALYSIS B Statistical Analysis DATA AND CODE AVAILABILITY

DECLARATION OF INTERESTS The authors declare no competing interests. Received: November 21, 2018 Revised: July 30, 2019 Accepted: September 27, 2019 Published: November 5, 2019 REFERENCES Awasaki, T., Tatsumi, R., Takahashi, K., Arai, K., Nakanishi, Y., Ueda, R., and Ito, K. (2006). Essential role of the apoptotic cell engulfment genes draper and ced-6 in programmed axon pruning during Drosophila metamorphosis. Neuron 50, 855–867. Bilimoria, P.M., and Stevens, B. (2015). Microglia function during brain development: New insights from animal models. Brain Res. 1617, 7–17. Brichta, L., and Greengard, P. (2014). Molecular determinants of selective dopaminergic vulnerability in Parkinson’s disease: an update. Front. Neuroanat. 8, 152.

Brown, G.C., Vilalta, A., and Fricker, M. (2015). Phagoptosis - Cell Death By Phagocytosis - Plays Central Roles in Physiology, Host Defense and Pathology. Curr. Mol. Med. 15, 842–851. Casano, A.M., and Peri, F. (2015). Microglia: multitasking specialists of the brain. Dev. Cell 32, 469–477. Cronk, J.C., and Kipnis, J. (2013). Microglia - the brain’s busy bees. F1000Prime Rep. 5, 53. Cunningham, C.L., Martı´nez-Cerden˜o, V., and Noctor, S.C. (2013). Microglia regulate the number of neural precursor cells in the developing cerebral cortex. J. Neurosci. 33, 4216–4233. Etchegaray, J.I., Elguero, E.J., Tran, J.A., Sinatra, V., Feany, M.B., and McCall, K. (2016). Defective Phagocytic Corpse Processing Results in Neurodegeneration and Can Be Rescued by TORC1 Activation. J. Neurosci. 36, 3170–3183. Feany, M.B., and Bender, W.W. (2000). A Drosophila model of Parkinson’s disease. Nature 404, 394–398.

SUPPLEMENTAL INFORMATION

Frank, S., Burbach, G.J., Bonin, M., Walter, M., Streit, W., Bechmann, I., and Deller, T. (2008). TREM2 is upregulated in amyloid plaque-associated microglia in aged APP23 transgenic mice. Glia 56, 1438–1447.

Supplemental Information can be found online at https://doi.org/10.1016/j. celrep.2019.09.086.

Freeman, M.R. (2006). Sculpting the nervous system: glial control of neuronal development. Curr. Opin. Neurobiol. 16, 119–125.

ACKNOWLEDGMENTS

Freeman, M.R., Delrow, J., Kim, J., Johnson, E., and Doe, C.Q. (2003). Unwrapping glial biology: Gcm target genes regulating glial development, diversification, and function. Neuron 38, 567–580.

We thank B. Jones, M. Freeman, O. Schuldiner, Y. Nakanishi, U. Gaul, the Developmental Studies Hybridoma Bank, and the Bloomington Stock Center for generously providing fly strains and antibodies. We thank M. Choder and T. Schultheiss for comments on the manuscript and members of the Kurant laboratory for constructive criticism and support. We also thank E. Suss-Toby and L. Liba at the Interdepartmental Bioimaging facility for excellent technical support. We gratefully acknowledge financial support from the Israel Science Foundation (grant 1872/15).

Fuentes-Medel, Y., Logan, M.A., Ashley, J., Ataman, B., Budnik, V., and Freeman, M.R. (2009). Glia and muscle sculpt neuromuscular arbors by engulfing destabilized synaptic boutons and shed presynaptic debris. PLoS Biol. 7, e1000184. Hanayama, R., Tanaka, M., Miwa, K., Shinohara, A., Iwamatsu, A., and Nagata, S. (2002). Identification of a factor that links apoptotic cells to phagocytes. Nature 417, 182–187.

AUTHOR CONTRIBUTIONS

Hanisch, U.K., and Kettenmann, H. (2007). Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat. Neurosci. 10, 1387– 1394.

Conceptualization, K.H.-M. and E.K.; Methodology, K.H.-M., N.F.-B., B.S., F.L.-A., and E.K.; Investigation, K.H.-M., N.F.-B., and E.K.; Writing – Original

Harry, G.J. (2013). Microglia during development and aging. Pharmacol. Ther. 139, 313–326.

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Hilu-Dadia, R., Hakim-Mishnaevski, K., Levy-Adam, F., and Kurant, E. (2018). Draper-mediated JNK signaling is required for glial phagocytosis of apoptotic neurons during Drosophila metamorphosis. Glia 66, 1520–1532.

Purice, M.D., Speese, S.D., and Logan, M.A. (2016). Delayed glial clearance of degenerating axons in aged Drosophila is due to reduced PI3K/Draper activity. Nat. Commun. 7, 12871.

Kim, S., Park, S.Y., Kim, S.Y., Bae, D.J., Pyo, J.H., Hong, M., and Kim, I.S. (2012). Cross talk between engulfment receptors stabilin-2 and integrin avb5 orchestrates engulfment of phosphatidylserine-exposed erythrocytes. Mol. Cell. Biol. 32, 2698–2708.

€nzel, E.J., Pope, B.J., Park, D.J., Speese, S.D., and Purice, M.D., Ray, A., Mu Logan, M.A. (2017). A novel Drosophila injury model reveals severed axons are cleared through a Draper/MMP-1 signaling cascade. eLife 6, e23611.

Koellhoffer, E.C., McCullough, L.D., and Ritzel, R.M. (2017). Old Maids: Aging and Its Impact on Microglia Function. Int. J. Mol. Sci. 18, E769. Kremer, M.C., Jung, C., Batelli, S., Rubin, G.M., and Gaul, U. (2017). The glia of the adult Drosophila nervous system. Glia 65, 606–638. Kurant, E. (2011). Keeping the CNS clear: glial phagocytic functions in Drosophila. Glia 59, 1304–1311. Kurant, E., Axelrod, S., Leaman, D., and Gaul, U. (2008). Six-microns-under acts upstream of Draper in the glial phagocytosis of apoptotic neurons. Cell 133, 498–509. Logan, M.A. (2017). Glial contributions to neuronal health and disease: new insights from Drosophila. Curr. Opin. Neurobiol. 47, 162–167. Logan, M.A., and Freeman, M.R. (2007). The scoop on the fly brain: glial engulfment functions in Drosophila. Neuron Glia Biol. 3, 63–74.

Ravichandran, K.S. (2011). Beginnings of a good apoptotic meal: the find-me and eat-me signaling pathways. Immunity 35, 445–455. Ray, A., Speese, S.D., and Logan, M.A. (2017). Glial Draper Rescues Ab Toxicity in a Drosophila Model of Alzheimer’s Disease. J. Neurosci. 37, 11881–11893. Salter, M.W., and Stevens, B. (2017). Microglia emerge as central players in brain disease. Nat. Med. 23, 1018–1027. Scheib, J.L., Sullivan, C.S., and Carter, B.D. (2012). Jedi-1 and MEGF10 signal engulfment of apoptotic neurons through the tyrosine kinase Syk. J. Neurosci. 32, 13022–13031. Shklover, J., Mishnaevski, K., Levy-Adam, F., and Kurant, E. (2015). JNK pathway activation is able to synchronize neuronal death and glial phagocytosis in Drosophila. Cell Death Dis. 6, e1649. Shklyar, B., Levy-Adam, F., Mishnaevski, K., and Kurant, E. (2013). Caspase activity is required for engulfment of apoptotic cells. Mol. Cell. Biol. 33, 3191–3201.

Lue, L.F., Schmitz, C.T., Serrano, G., Sue, L.I., Beach, T.G., and Walker, D.G. (2014). TREM2 Protein Expression Changes Correlate with Alzheimer’s Disease Neurodegenerative Pathologies in Post-Mortem Temporal Cortices. Brain Pathol. 25, 469–480.

Shklyar, B., Sellman, Y., Shklover, J., Mishnaevski, K., Levy-Adam, F., and Kurant, E. (2014). Developmental regulation of glial cell phagocytic function during Drosophila embryogenesis. Dev. Biol. 393, 255–269.

MacDonald, J.M., Beach, M.G., Porpiglia, E., Sheehan, A.E., Watts, R.J., and Freeman, M.R. (2006). The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons. Neuron 50, 869–881.

Tasdemir-Yilmaz, O.E., and Freeman, M.R. (2014). Astrocytes engage unique molecular programs to engulf pruned neuronal debris from distinct subsets of neurons. Genes Dev. 28, 20–33.

MacDonald, J.M., Doherty, J., Hackett, R., and Freeman, M.R. (2013). The c-Jun kinase signaling cascade promotes glial engulfment activity through activation of draper and phagocytic function. Cell Death Differ. 20, 1140–1148.

Tung, T.T., Nagaosa, K., Fujita, Y., Kita, A., Mori, H., Okada, R., Nonaka, S., and Nakanishi, Y. (2013). Phosphatidylserine recognition and induction of apoptotic cell clearance by Drosophila engulfment receptor Draper. J. Biochem. 153, 483–491.

McLaughlin, C.N., Perry-Richardson, J.J., Coutinho-Budd, J.C., and Broihier, H.T. (2019). Dying Neurons Utilize Innate Immune Signaling to Prime Glia for Phagocytosis during Development. Dev Cell 48, 506–522.e506. Napoli, I., and Neumann, H. (2009). Microglial clearance function in health and disease. Neuroscience 158, 1030–1038. Na¨ssel, D.R., and Elekes, K. (1992). Aminergic neurons in the brain of blowflies and Drosophila: dopamine- and tyrosine hydroxylase-immunoreactive neurons and their relationship with putative histaminergic neurons. Cell Tissue Res. 267, 147–167. Neher, J.J., Emmrich, J.V., Fricker, M., Mander, P.K., The´ry, C., and Brown, G.C. (2013). Phagocytosis executes delayed neuronal death after focal brain ischemia. Proc. Natl. Acad. Sci. USA 110, E4098–E4107. Park, S.Y., Jung, M.Y., Kim, H.J., Lee, S.J., Kim, S.Y., Lee, B.H., Kwon, T.H., Park, R.W., and Kim, I.S. (2008). Rapid cell corpse clearance by stabilin-2, a membrane phosphatidylserine receptor. Cell Death Differ. 15, 192–201.

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Vilalta, A., and Brown, G.C. (2018). Neurophagy, the phagocytosis of live neurons and synapses by glia, contributes to brain development and disease. FEBS J. 285, 3566–3575.. von Bernhardi, R., Eugenı´n-von Bernhardi, L., and Eugenı´n, J. (2015). Microglial cell dysregulation in brain aging and neurodegeneration. Front. Aging Neurosci. 7, 124. Watts, R.J., Schuldiner, O., Perrino, J., Larsen, C., and Luo, L. (2004). Glia engulf degenerating axons during developmental axon pruning. Curr. Biol. 14, 678–684. Wolf, S.A., Boddeke, H.W., and Kettenmann, H. (2017). Microglia in Physiology and Disease. Annu. Rev. Physiol. 79, 619–643. Wu, H.H., Bellmunt, E., Scheib, J.L., Venegas, V., Burkert, C., Reichardt, L.F., Zhou, Z., Farin˜as, I., and Carter, B.D. (2009). Glial precursors clear sensory neuron corpses during development via Jedi-1, an engulfment receptor. Nat. Neurosci. 12, 1534–1541.

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Rabbit polyclonal anti-SIMU

Shklyar et al., 2013

N/A

Rabbit polyclonal anti-cleaved caspase-3

Cell Signaling

Cat# 9661; RRID:AB_2341188

Rabbit polyclonal anti-GABA

Sigma-Aldrich

Cat# A2052; RRID:AB_477652

Guinea pig polyclonal anti-SIMU

Shklyar et al., 2013

N/A

Guinea pig polyclonal anti-Drpr

Shklover et al., 2015

N/A

Mouse monoclonal anti-Drpr

DSHB

Cat# 5D14-S; RRID:AB_2618105

Mouse monoclonal anti-TH

Millipore

Cat# MAB318; RRID:AB_2313764

Mouse monoclonal anti-GFP

Roche

Cat# 11814460001; RRID:AB_390913

Mouse monoclonal anti-Actin

MP Biomedicals

Cat# 69100; RRID:AB_10683472

Mouse monoclonal anti-REPO

DSHB

Cat# 8D12; RRID:AB_528448

Alexa Fluor 488 conjugated affinity pure Donkey Anti-mouse IgG (H+L)

Jackson ImmunoResearch Labs

Cat# 715-545-151; RRID:AB_2341099

CyTH3 conjugated affinity pure Donkey Anti-Rabbit IgG (H+L)

Jackson ImmunoResearch Labs

Cat# 711-165-152; RRID:AB_2307443

CyTH3 conjugated affinity pure Donkey Anti-Guinea pig IgG (H+L)

Jackson ImmunoResearch Labs

Cat# 706-165-148; RRID:AB_2340460

DAPI

Sigma-Aldrich

Cat# D9542

Peroxidase-conjugated AffiniPure Goat Anti-Guinea Pig IgG (H+L)

Jackson ImmunoResearch Labs

Cat# 106-035-003; RRID:AB_2337402

Peroxidase-conjugated AffiniPure Donkey Anti-Mouse IgG (H+L)

Jackson ImmunoResearch Labs

Cat# 715-035-151; RRID:AB_2340771

Antibodies

Experimental Models: Organisms/Strains D. melanogaster: repo-Gal4

B. Jones

D. melanogaster: UAScytGFP; w[*]; P{w[+mC] = UAS-GFP.S65T}Myo31DF[T2]

Bloomington Drosophila Stock Center

BDSC:1521; RRID:BDSC_1521

D. melanogaster: tubP-Gal80; w[*]; sna[Sco]/CyO; P{w[+mC] = tubP-GAL80[ts]}ncd[GAL80ts-7]

Bloomington Drosophila Stock Center

BDSC:7018; RRID:BDSC_7018

D. melanogaster: tubP-Gal80; w[*]; P{w[+mC] = tubP-GAL80[ts]}20; TM2/TM6B, Tb[1]

Bloomington Drosophila Stock Center

BDSC:7019; RRID:BDSC_7019

D. melanogaster: UASsimu

Shklyar et al., 2013

N/A

D. melanogaster: UASdrpr

Laboratory of M. Freeman

N/A

D. melanogaster: elav-Gal4

Laboratory of O. Schuldiner

N/A

D. melanogaster: UASmgf-e8-GFP

(Laboratory of Y. Nakanishi; Tung et al., 2013)

N/A

D. melanogaster: R54H02-Gal4

Laboratory of U. Gaul (Kremer et al., 2017)

N/A

D. melanogaster: R86E01-Gal4

Laboratory of U. Gaul (Kremer et al., 2017)

N/A

D. melanogaster: R56F03-Gal4

Laboratory of U. Gaul (Kremer et al., 2017)

N/A

AxioVision

ZEISS

https://zeiss.com

IMARIS

Bitplane

https://imaris.oxinst.com/packages

Prism 8.1.2

GraphPad

https://www.graphpad.com/

Software and Algorithms

LEAD CONTACT AND MATERIALS AVAILABILITY This study did not generate new unique reagents. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Estee Kurant ([email protected]).

Cell Reports 29, 1438–1448.e1–e3, November 5, 2019 e1

EXPERIMENTAL MODEL AND SUBJECT DETAILS Fly Strains The following fly strains were used in this work: repoGal4 (B. Jones), UAScytGFP (#1521; Bloomington), UASsimu (Shklyar et al., 2013), UASdrpr (M. Freeman), tubGal80ts (#7019; Bloomington), elavGal4 (O. Schuldiner), UASmgf-e8-GFP (Y. Nakanishi), R54H02-Gal4, R56F03-Gal4, R86E01-Gal4 (U. Gaul; Kremer et al., 2017). For adult-specific expression of simu, drpr or mfg-e8GFP in pan-glial (repoGal4) or type-specific cortex glia (R54H02-Gal4), ensheathing glia (R56F03-Gal4) and astrocytes (R86E01Gal4) manner tubGal80ts;repoGal4XUASsimu, tubGal80ts;repoGal4XUASdrpr, tubGal80ts;repoGal4XUASmfg-e8-GFP;UASsimu, UASdrpr;tubGal80tsXUASmfg-e8-GFP;repoGal4, tubGal80ts;repoGal4XUASsimu,UASdrpr, UASsimu;tubGal80Xtype-specificGal4s or UASsimu,UASdrpr;tubGal80Xtype-specificGal4s, UASmfg-e8-GFP;cortexGal4, UASmfg-e8-GFP;cortexGal4XUASsimu crosses were maintained at 18 C. Adult flies were transferred to 29 C immediately after eclosion. METHOD DETAILS Immunohistochemistry For immunohistochemistry, whole brains were dissected, fixed, and stained according to standard procedures. The following antibodies were used (at the dilutions listed): rabbit anti-SIMU (Shklyar et al., 2013), mouse anti-Drpr (1:100; Developmental Studies Hybridoma Bank), mouse anti-REPO (1:10; Developmental Studies Hybridoma Bank), mouse anti-TH (1:500; Millipore), rabbit anticleaved Caspase-3 (1:100; Cell Signaling), rabbit anti-GABA (1:200; Sigma) and mouse anti-GFP (1:100; Roche). Fluorescent Cy3 or 488 secondary antibodies from Jackson ImmunoResearch were used at 1:250 dilution. Dako medium was used for mounting. For immunohistochemistry and quantification of REPO-positive glia and DA and GABA neurons, flies were dissected after they stopped climbing. Therefore, flies of appropriate ages were selected in the different experiments. Imaging and Quantification Procedure All images were acquired on a Zeiss LSM 700 confocal microscope using a Plan-Apochromat 20x/0.8 M27 lens or on a Zeiss Axio Observer microscope equipped with an Apotome system using the AxioVision software. Image analysis was performed using Zeiss ZEN and Imaris (Bitplane) software. To quantitate the number of REPO-positive glia or DA and GABA neurons, confocal or Apotome stacks were acquired from entire adult brains (glia) or the posterior part of adult brains (DA and GABA neurons), as this region is more assessable (Na¨ssel and Elekes, 1992). Western Blot Analysis Drosophila female head extracts were prepared in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, supplemented with a mixture of protease inhibitors (Roche), using a hand homogenizer. Protein concentration was determined (Bradford reagent, Bio-Rad), and 100 mg protein were separated by SDS-PAGE. After electrophoresis, proteins were transferred to a PVDF membrane (Bio-Rad) and probed with guinea pig anti-SIMU (1:10000) (Shklyar et al., 2013) or guinea pig anti-Drpr (1:2000) (Shklover et al., 2015) or mouse anti-Actin (1:20000) (MP Biomedicals) followed by HRP-conjugated secondary antibodies (Jackson ImmunoResearch). Antibody binding was revealed using an enhanced chemiluminescent substrate (Bio-Rad). Cryo-sections Drosophila female heads were fixed in 4% paraformaldehyde for 3 hours. Fixed heads were oriented perpendicularly in relation to their long axis, embedded in 7.5% gelatin and 15% sucrose in PBS, and sectioned at 10 mm intervals with a Leica CM 1900 cryostat. Behavioral Assays Climbing and survival assays were performed as previously described, with slight modifications (Feany and Bender, 2000). For each genotype, triplicates of ten flies per vial were tested. Flies were collected and transferred to new vials every 2–3 days. The number of dead flies was recorded every day. For climbing, flies were gently tapped to the bottom, and then given 10 s to climb a height of 7 cm. QUANTIFICATION AND STATISTICAL ANALYSIS Statistical Analysis Each experiment was repeated independently a minimum of three times, error bars represent the standard error of replicate experiments. Statistical significance of climbing, survival, and cell numbers data was calculated with Two-way ANOVA followed

e2 Cell Reports 29, 1438–1448.e1–e3, November 5, 2019

by Dunnett’s multiple comparisons test using GraphPad Prism version 8.1.2 for Windows, GraphPad Software, San Diego, California USA, https://www.graphpad.com. P values of < 0.05 = *, < 0.005 = **, < 0.0005 = ***, < 0.0001 = **** were considered significant. P values are indicated in figure legends. DATA AND CODE AVAILABILITY This study did not generate any unique datasets or code.

Cell Reports 29, 1438–1448.e1–e3, November 5, 2019 e3