FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W4366086446> ?p ?o ?g. }

• W4366086446 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Modification by sialylated glycans can affect protein functions, underlying mechanisms that control animal development and physiology. Sialylation relies on a dedicated pathway involving evolutionarily conserved enzymes, including CMP-sialic acid synthetase (CSAS) and sialyltransferase (SiaT) that mediate the activation of sialic acid and its transfer onto glycan termini, respectively. In Drosophila, CSAS and DSiaT genes function in the nervous system, affecting neural transmission and excitability. We found that these genes function in different cells: the function of CSAS is restricted to glia, while DSiaT functions in neurons. This partition of the sialylation pathway allows for regulation of neural functions via a glia-mediated control of neural sialylation. The sialylation genes were shown to be required for tolerance to heat and oxidative stress and for maintenance of the normal level of voltage-gated sodium channels. Our results uncovered a unique bipartite sialylation pathway that mediates glia-neuron coupling and regulates neural excitability and stress tolerance. Editor's evaluation This important paper uses Drosophila as a model to study the sialylation pathway and its role in nervous system function. Intriguingly, the authors demonstrate that the final two steps of the sialylation biosynthetic pathway are split across glia (CSAS) and neurons (DSiaT). This compelling finding will interest a broad readership as it identifies a new mode by which glia support neuronal function. https://doi.org/10.7554/eLife.78280.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Protein glycosylation, the most common type of posttranslational modification, plays numerous important biological roles, and regulates molecular and cell interactions in animal development, physiology, and disease (Varki, 2017). The addition of sialic acid (Sia), i.e., sialylation, has prominent effects due to its negative charge, bulky size, and terminal location of Sia on glycan chains. Essential roles of sialylated glycans in cell adhesion, cell signaling, and proliferation have been documented in many studies (Schwarzkopf et al., 2002; Varki, 2007; Varki, 2008). Sia is intimately involved in the function of the nervous system. Mutations in genes that affect sialylation are associated with neurological symptoms in human, including intellectual disability, epilepsy, and ataxia due to defects in sialic acid synthase (N-acetylneuraminic acid synthase [NANS]), sialyltransferases (ST3GAL3 and ST3GAL5), the CMP-Sia transporter (SLC35A1), and CMP-Sia synthase (CMAS) (Hu et al., 2011; Mohamed et al., 2013; Boccuto et al., 2014; van Karnebeek et al., 2016). Polysialylation (PSA) of NCAM, the neural cell adhesion molecule, one of the best studied cases of sialylation in the nervous system, is involved in the regulation of cell interactions during brain development (Schnaar et al., 2014). Non-PSA-type sialylated glycans are ubiquitously present in the vertebrate nervous system, but their functions are not well defined. Increasing evidence implicates these glycans in essential regulation of neuronal signaling. Indeed, N-glycosylation can affect voltage-gated channels in different ways, ranging from modulation of channel gating to protein trafficking, cell surface expression, and recycling/degradation (Cronin et al., 2005; Watanabe et al., 2007; Ednie and Bennett, 2012; Baycin-Hizal et al., 2014; Watanabe et al., 2015; Thayer et al., 2016). Similar effects were shown for several other glycoproteins implicated in synaptic transmission and cell excitability, including neurotransmitter receptors (reviewed in Scott and Panin, 2014). Glycoprotein sialylation defects were also implicated in neurological diseases, such as Angelman syndrome and epilepsy (Isaev et al., 2007; Condon et al., 2013). However, the in vivo functions of sialylation and the mechanisms that regulate this posttranslational modification in the nervous system remain poorly understood. Drosophila has recently emerged as a model to study neural sialylation in vivo, providing advantages of the decreased complexity of the nervous system and the sialylation pathway, while also showing conservation of the main biosynthetic steps of glycosylation (Koles et al., 2009; Scott and Panin, 2014). The final step in sialylation is mediated by sialyltransferases, enzymes that use CMP-Sia as a sugar donor to attach Sia to glycoconjugates (Figure 1; Varki et al., 2015a). Unlike mammals that have 20 different sialyltransferases, Drosophila possesses a single sialyltransferase, DSiaT, that has significant homology to mammalian ST6Gal enzymes (Koles et al., 2004). The two penultimate steps in the biosynthetic pathway of sialylation are mediated by sialic acid synthase (also known as NANS) and CMP-sialic acid synthetase (CSAS, also known as CMAS), the enzymes that synthesize sialic acid and carry out its activation, respectively (Varki et al., 2015a). These enzymes have been characterized in Drosophila and found to be closely related to their mammalian counterparts (Kim et al., 2002; Viswanathan et al., 2006; Mertsalov et al., 2016). In vivo analyses of DSiaT and CSAS demonstrated that Drosophila sialylation is a tightly regulated process limited to the nervous system and required for normal neural transmission. Mutations in DSiaT and CSAS phenocopy each other, resulting in similar defects in neuronal excitability, causing locomotor and heat-induced paralysis phenotypes, while showing strong interactions with voltage-gated channels (Repnikova et al., 2010; Islam et al., 2013). DSiaT was found to be expressed exclusively in neurons during development and in the adult brain (Repnikova et al., 2010). Intriguingly, although the expression of CSAS has not been characterized in detail, it was noted that its expression appears to be different from that of DSiaT in the embryonic ventral ganglion (Koles et al., 2009), suggesting a possibly unusual relationship between the functions of these genes. Here, we tested the hypothesis that CSAS functions in glial cells, and that the separation of DSiaT and CSAS functions between neurons and glia underlies a novel mechanism of glia-neuron coupling that regulates neuronal function via a bipartite protein sialylation. Figure 1 Download asset Open asset Schematic of the sialylation pathways in vertebrate and Drosophila. In vertebrates, phosphorylated sialic acid is produced by N-acetylneuraminic acid synthase (Neu5Ac-9-P synthase, or NANS) from N-acetyl-mannosamine 6-phosphate (ManNAc-6-P), converted to sialic acid by N-acylneuraminate-9-phosphatase (Neu5Ac-9-P phosphatase, or NANP), and then activated by CMP-sialic acid synthetase (CSAS, also known as CMAS) to become CMP-Sia, the substrate for sialyltransferase enzymes that work in the Golgi and attach sialic acid to termini of glycan chains. While NANS and NANP work in the cytosol, CSAS enzymes normally localize to the nucleus in vertebrate cells, and the transfer of CMP-Sia to the Golgi requires CMP-Sia transporter. In Drosophila, both CSAS and sialyltransferase are localized in the Golgi, and CMP-Sia transporter is not required for sialylation. Biochemical activities of NANS, CSAS, and the Drosophila sialyltransferase (DSiaT) were confirmed in vitro (Kim et al., 2002; Koles et al., 2004; Viswanathan et al., 2006; Mertsalov et al., 2016). Dashed bracket: subcellular localization was not experimentally confirmed. #CMP-Sia transporter was not identified in invertebrates. *NANP was found to be not essential for sialylation (Willems et al., 2019). Glial cells have been recognized as key players in neural regulation (reviewed in Volterra and Meldolesi, 2005; Bosworth and Allen, 2017; Magistretti and Allaman, 2018). Astrocytes participate in synapse formation and synaptic pruning during development, mediate the recycling of neurotransmitters, affect neurons via Ca2+ signaling, and support a number of other essential evolutionarily conserved functions (reviewed in Neniskyte and Gross, 2017; Bittern et al., 2021; Nagai et al., 2021). Studies of Drosophila glia have revealed novel glial functions in vivo (reviewed in Freeman, 2015; Rittschof and Schirmeier, 2018; Bittern et al., 2021). Drosophila astrocytes were found to modulate dopaminergic function through neuromodulatory signaling and activity-regulated Ca2+ increase (Ma et al., 2016). Glial cells were also shown to protect neurons and neuroblasts from oxidative stress and promote the proliferation of neuroblasts in the developing Drosophila brain (Bailey et al., 2015; Liu et al., 2015; Kanai et al., 2018). The metabolic coupling between astrocytes and neurons, which is thought to support and modulate neuronal functions in mammals (Magistretti and Allaman, 2018), is apparently conserved in flies. Indeed, Drosophila glial cells can secrete lactate and alanine to fuel neuronal oxidative phosphorylation (Volkenhoff et al., 2015; Liu et al., 2017). In the current work, we described a novel mechanism of glia-neuron coupling mediated by a unique compartmentalization of different steps in the sialylation pathway between glial cells and neurons in the fly nervous system. We explore the regulation of this mechanism and demonstrate its requirement for neural functions. Results Expression of Drosophila CSAS is restricted to glial cells and does not overlap with DSiaT expression Previous studies indicated that CSAS is expressed in the nervous system and functions together with DSiaT in a pathway that affects neural transmission (Islam et al., 2013). However, the expression of CSAS has not been characterized in detail. To determine the expression of CSAS in different cells, we created a LexA reporter construct based on a genomic BAC clone that included the CSAS gene along with a large surrounding genomic region (Venken et al., 2009, see Materials and methods). We modified the BAC-CSAS by replacing part of the CSAS coding region with the sequence encoding LexA::p65 transcription activator (Pfeiffer et al., 2010) using recombineering (Venken et al., 2009) to generate a CSAS-LexA driver. This strategy has been useful to generate reporters with expression patterns that correspond to endogenous genes (Venken et al., 2009). We combined CSAS-LexA with LexAop2-mCD8-GFP and LexAop-GFP.nls reporters to label cell surfaces and nuclei of CSAS-expressing cells, respectively, and analyzed the expression pattern of CSAS at different developmental stages. Double-labeling experiments using Repo as a glial marker revealed that CSAS is expressed in many glial cells in the CNS throughout development and in adult flies (Figure 2A–B); in contrast, no expression was detected in neurons (Figure 2C). This is a surprising result, considering that DSiaT, the enzyme that functions downstream of CSAS in the sialylation pathway, is expressed only in neurons but not in glial cells (Repnikova et al., 2010). To confirm that CSAS-LexA expression recapitulates the endogenous expression of CSAS, we carried two sets of control experiments. First, we introduced the original BAC-CSAS clone as a transgene in flies and combined it with CSAS knockout, which resulted in full rescue of the temperature-sensitive (TS) paralysis phenotype of CSAS mutants (Figure 2—figure supplement 1). This supported the notion that the genomic clone includes all important regulatory elements to induce CSAS in endogenous manner. Second, we used CSAS-LexA to drive the transgenic expression of CSAS cDNA in CSAS mutants. This also fully rescued the TS paralysis, supporting the notion that CSAS-LexA recapitulates the CSAS endogenous expression (Figure 2—figure supplement 2). To further confirm that the expression of CSAS is indeed confined to the cells without DSiaT expression, we carried out double-labeling experiments to visualize CSAS and DSiaT-expressing cells simultaneously. To label DSiaT-expressing cells, we used a transgenic BAC-DSiaT-HA construct carrying a large genomic locus including the DSiaT gene modified with a 3xHA tag sequence to allow immunodetection (see Materials and methods). In agreement with previous studies (Repnikova et al., 2010), the expression of DSiaT-HA was detected only in differentiated neurons labeled by Elav, but not in neural progenitors expressing a neuroblast marker Deadpan (Figure 2—figure supplement 3). Importantly, we observed no overlap between the expression patterns of CSAS and DSiaT (Figure 2D–F). Taken together, these results show that CSAS and DSiaT are expressed in distinct cell populations within the CNS, glial cells, and neurons, respectively. Figure 2 with 3 supplements see all Download asset Open asset CSAS expression is restricted to glial cells and shows no overlap with the expression of DSiaT during development and in the adult brain. (A–A”) CSAS expression (green) is detected in glial cells (Repo, red) of the developing ventral ganglion during late embryonic stages. Arrows indicate examples of glial cells with CSAS expression. A” is the overlay of green (A) and red (A’) channels. (B–B’’’) CSAS expression (green) is present in the majority of glial cells (Repo, red) in the CNS at larval stages. (B’–B’’’) are zoomed-in images of a brain region outlined in B, B’’’ is overlay of B’ and B”. (C–C’’’) CSAS expression (green) is not detected in neurons (Elav, red) in the CNS at larval stages. Arrows and arrowheads indicate examples of cells with CSAS and Elav expression, respectively. (C’–C’’’) are zoomed-in images of a ventral ganglion region outlined in C, C’’’ is overlay of C’ and C”. (D–D’’’) CSAS expression (green) is not detected in the CNS cells expressing DSiaT (red) at larval stages. Arrows and arrowheads indicate examples of cells with CSAS and DSiaT expression, respectively. (D’–D’’’) are zoomed-in images of a ventral ganglion region outlined in D, D’’’ is overlay of D’ and D”. (E) CSAS (green) is expressed throughout the adult brain, including glial cells in the optic lobes (OL), around the antennal lobe (AL), and the sub esophageal zone (SEZ), but CSAS expression is not detected in DSiaT-expressing neurons (red). (F–F’’’) Zoomed-in images of the antennal lobe region indicated by a dashed circle in E. CSAS-expressing cells produce processes surrounding the soma of DSiaT-expressing projection neurons (arrow), enveloping the antennal lobe (filled arrowhead), and sending fine projections inside the glomeruli (empty arrowheads). Brp staining (blue) labels neuropil in E–F. (A) Embryonic stage 17, lateral view, anterior is left, ventral is up; (B) third instar larval stage, anterior is top-right; (C) first instar larval stage, anterior is top-right; (D) third instar larval stage, anterior is left; (E–F) adult brain, frontal view. Scale bars: 10 μm (A, C’, D’), 100 μm (B, E), 20 μm (B’, F), 50 μm (C–D). CSAS expression was visualized using CSAS-LexA driver-induced expression of GFP with membrane (mCD8-GFP) or nuclear localization (GFPnls) tags. Images were acquired using confocal microscopy. CSAS is required in glial cells, but not in neurons To investigate the cell-specific requirement of CSAS in the nervous system, we carried out rescue experiments using UAS-GAL4 ectopic expression system (Brand et al., 1994). CSAS function was shown to be required for normal neural transmission, while CSAS mutations cause locomotor defects and TS paralysis phenotype (Islam et al., 2013). Using cell-specific GAL4 drivers, we induced the transgenic expression of UAS-CSAS in CSAS homozygous mutants and assayed them for TS paralysis. Glial-specific expression of transgenic CSAS using drivers expressed in all glial cells (Repo-Gal4), ensheathing (Mz709-Gal4), astrocyte-like (dEAAT1-Gal4), neuropile ensheathing glia (R56F03-Gal4), or subperineurial glia (Gli-Gal4) could fully rescue the phenotype of CSAS mutants (Auld et al., 1995; Ito et al., 1995; Sepp et al., 2001; Rival et al., 2004; Doherty et al., 2009; Kremer et al., 2017), while the expression in neurons using a pan-neuronal driver (C155-GAL4) or other neuronal drivers broadly expressed in the nervous system (Mj85b-Gal4, 1407-Gal4) (Lin and Goodman, 1994; Dubnau et al., 2001; Kraft et al., 2016) did not result in rescue (Figure 3A, Figure 3—figure supplements 1–2). Interestingly, perineurial driver (R85G01-Gal4, Kremer et al., 2017) could partially rescue the phenotype, even though perineurial glia is separated from the brain by a tightly sealed layer of subperineurial cells maintaining the blood-brain barrier, and thus perineurial cells are not well poised to provide CMP-Sia for brain functions. This partial rescue is potentially explained by the fact that R85G01-Gal4 was found to be also expressed in a small number of cortex and astrocyte-like glial cells (Weiss et al., 2022). Taken together, our results demonstrated that CSAS expression in glial cells, but not in neurons, is sufficient to restore neural function in CSAS mutants. Figure 3 with 2 supplements see all Download asset Open asset CMP-sialic acid synthetase (CSAS) is required in glial cells, but not in neurons, for normal neural functions. (A) Rescue of TS paralysis phenotype of CSAS mutants using UAS-Gal4 system. CSAS21 (null) and CSASMi (strong loss-of-function) mutant alleles were used in homozygous and heteroallelic combinations. The expression of transgenic UAS-CSAS construct was induced using a panel of cell-specific Gal4 drivers. A pan-neuronal Gal4 driver (C155) or drivers broadly expressed in the CNS neurons (1407 and Mj85b) did not induce rescue, while the expression of UAS-CSAS by glial-specific drivers, including Repo, Gli, and Mz709 (expressed in nearly all glial cells, ensheathing glial cells, and subperineural glia, respectively), rescued the phenotype. Analyses of control mutant genotypes (UAS-CSAS without driver, and driver-only mutant genotypes) confirmed the specificity of the rescue results (Figure 3—figure supplement 1). The expression of CSAS induced by C155 was confirmed using immunostaining (Figure 3—figure supplement 2). At least 19 files (5-day-old females) were assayed for each genotype. (B) Locomotor phenotype of CSAS mutants rescued using UAS-Gal4 system. The expression of UAS-CSAS induced in neurons by C155 driver did not rescue the phenotype, while the glial-specific expression driven by Repo-Gal4 resulted in full rescue. Mutant genotypes with UAS-CSAS alone or drivers alone were used as controls, and they did not show rescue. At least 20 females were assayed for each genotype. (C) Rescue of neuromuscular excitatory junction potential (EJP) defect of CSAS mutants using UAS-Gal4 system. The reduced EJP phenotype was rescued by glial-specific expression of UAS-CSAS induced by Repo-Gal4. The expression of UAS-CSAS in motoneurons using C164-Gal4 did not result in rescue. Representative EJP traces are shown on the right. EJPs were evoked in 0.5 mM Ca2 and analyzed at muscle 6/7 neuromuscular junctions (NMJs) of third instar larvae (see Materials and methods for details). 6-9 larvae were assyed for each genotype. (D) Cell-specific RNAi-mediated knockdown reveals that CSAS is required in glial cells. UAS-CSAS-RNAi was induced in glial cells by Repo-Gal4, which resulted in TS paralyses phenotype. The expression of UAS-CSAS-RNAi in neurons induced by C155-Gal4 did not cause the phenotype. To potentiate the effect of CSAS-RNAi, the knockdown experiments were performed in the genetic background with co-expression of UAS-dcr2 and heterozygous for CSAS21 mutant allele (^, genotypes with matching genetic background including UAS-dcr2 and CSAS21/+). At least 20 females were assayed for each genotype (all data points represent different flies). In all panels: error bars are SEM; one-way ANOVA with post hoc Tukey test was used for statistical analyses; *** p<0.001; ns, no significant difference (p>0.05). See Supplementary file 1 for detailed genotype information. Figure 3—source data 1 Source data for Figure 3A. https://cdn.elifesciences.org/articles/78280/elife-78280-fig3-data1-v2.xlsx Download elife-78280-fig3-data1-v2.xlsx Figure 3—source data 2 Source data for Figure 3B. https://cdn.elifesciences.org/articles/78280/elife-78280-fig3-data2-v2.xlsx Download elife-78280-fig3-data2-v2.xlsx Figure 3—source data 3 Source data for Figure 3C. https://cdn.elifesciences.org/articles/78280/elife-78280-fig3-data3-v2.xlsx Download elife-78280-fig3-data3-v2.xlsx Figure 3—source data 4 Source data for Figure 3D. https://cdn.elifesciences.org/articles/78280/elife-78280-fig3-data4-v2.xlsx Download elife-78280-fig3-data4-v2.xlsx Sialylation mutants have locomotion defects, such as an inability to promptly right themselves after falling upside down (Repnikova et al., 2010; Islam et al., 2013). Expression of UAS-CSAS in glial cells using Repo-Gal4 rescued this locomotion phenotype of CSAS mutants, while expression in neurons using C155-Gal4 did not result in rescue (Figure 3B). To assess the requirement of CSAS in synaptic transmission, we examined the function of motor neurons using electrophysiological assays at the neuromuscular junctions (NMJs). In sialylation mutants, larval motoneurons exhibit defects in excitability associated with a pronounced decrease of excitatory junction potentials (EJP) at NMJs (Repnikova et al., 2010; Islam et al., 2013). We analyzed EJPs in CSAS mutant third instar larvae and assessed the cell-specific requirement of CSAS by transgenic rescue. We used Repo-Gal4 and C164-Gal4 (Choi et al., 2004) drivers to induce the expression of UAS-CSAS in glial cells and motoneurons of CSAS mutants, respectively. CSAS expression in glial cells was sufficient to restore normal EJPs, however, CSAS expressed in motoneurons did not rescue neurotransmission defects (Figure 3C). These results provide compelling evidence that CSAS normally functions in glial cells but not in neurons, consistent with the results of behavioral assays. We also examined the cell-specific requirement of CSAS by downregulating its function in different cells. To this end, we knocked down CSAS specifically in glial cells or neurons by expressing UAS-CSAS-RNAi using Repo-Gal4 or C155-Gal4, respectively. To potentiate the RNAi-mediated knockdown, we co-expressed UAS-CSAS-RNAi with UAS-Dcr-2 (Dietzl et al., 2007) and used a genetic background that was heterozygous for a CSAS deletion allele (Islam et al., 2013). The knockdown of CSAS in glial cells resulted in the TS paralysis phenotype similar to that of CSAS null mutants. No paralysis was induced by knocking down CSAS in neurons (Figure 3D). Taken together, our data show that CSAS is necessary and sufficient in glial cells to support normal neural functions. DSiaT is required in neurons Previous studies using an endogenously expressed tagged version of DSiaT demonstrated that DSiaT could be detected in neurons but not in glial cells (Repnikova et al., 2010). However, whether DSiaT is required specifically in neurons was not examined. Although this question can be in principle clarified by a rescue strategy using UAS-Gal4 system, this approach has been hampered by the ‘leaking’ expression of UAS-DSiaT that was able to rescue the DSiaT mutant phenotypes without the presence of a Gal4 driver. As an alternative approach, we investigated the cell-specific requirement of DSiaT by RNAi-mediated knockdown. To increase the efficiency of knockdown, we carried out the knockdown in heterozygotes for a DSiaT null allele, DSiaTS23/+, which did not show the TS paralysis phenotype themselves. When DSiaT was downregulated by the expression of UAS-DSiaT-RNAi in neurons using C155-GAL4, the flies became paralytic at elevated temperature, showing the TS paralysis, a phenotype that recapitulated that of DSiaT null mutants. In contrast, DSiaT knockdown in glial cells did not cause the mutant phenotype (Figure 4). These results show that DSiaT function is required in neurons, consistent with the expression pattern of DSiaT. Figure 4 Download asset Open asset Cell-specific knockdown revealed that DSiaT is required in neurons. UAS-DSiaT-RNAi was induced in neurons by C155-Gal4, which resulted in temperature-sensitive (TS) paralyses phenotype. The expression of UAS-DSiaT-RNAi in glial cells by Repo-Gal4 did not produce the phenotype. To potentiate the effect of RNAi, knockdown was carried out in the genetic background heterozygous for DSiaT mutant allele (DSiaTS23/+) and flies were reared at 29°C. 20-28 five-day-old female flies were assayed for each genotype. #, genotypes with matching genetic background heterozygous for DSiaTS23. Error bars are SEM; one-way ANOVA with post hoc Tukey test was used for statistical analyses; *** p<0.001; ns, no significant difference (p>0.05).See Supplementary file 1 for detailed genotypes. Figure 4—source data 1 Source data for Figure 4. https://cdn.elifesciences.org/articles/78280/elife-78280-fig4-data1-v2.xlsx Download elife-78280-fig4-data1-v2.xlsx CSAS is required for the biosynthesis of CMP-Sia in Drosophila, while both CSAS and DSiaT are necessary for the production of sialylated N-glycans in vivo Genetic and phenotypic analyses previously demonstrated that CSAS and DSiaT genes work in the same functional pathway affecting neural transmission (Repnikova et al., 2010; Islam et al., 2013). Although the biochemical activities of their protein products were characterized in vitro (Koles et al., 2004; Mertsalov et al., 2016), the roles of these genes in sialylation were not examined in vivo. Considering the unusual separation of CSAS and DSiaT expression patterns at the cellular level, we decided to test their requirements for the biosynthesis of sialylated glycans in vivo. First, we analyzed the production of CMP-Sia in wild-type flies and CSAS mutants by a liquid chromatography-mass spectrometry approaches (see Materials and methods). A prominent peak corresponding to CMP-Sia was detected in wild-type flies as shown before (van Scherpenzeel et al., 2021), while no CMP-Sia was found in CSAS mutants. Transgenic rescue using UAS-GAL4 system resulted in the restoration of CMP-Sia biosynthesis in the mutants (Figure 5A–B). These results revealed that the production of CMP-Sia in Drosophila specifically requires CSAS activity. Second, we examined N-glycans in CSAS and DSiaT mutants by mass spectrometry. Sialylated glycans are present in Drosophila at extremely low levels (Aoki et al., 2007; Koles et al., 2007). We decided to focus our analyses on third instar larval brains because CSAS and DSiaT show prominent expression during late larval stages (Figure 1B–D and Repnikova et al., 2010; Islam et al., 2013). We found N-glycan profiles were dominated by high- and pauci-mannose glycans in all genotypes, with hybrid and complex structures representing a small portion of the total N-glycome (Figure 5C–D), consistent with previous studies that analyzed N-glycans produced in embryos and adult heads (Aoki et al., 2007; Koles et al., 2007). Sialylated structures were detected in wild-type larval brains, but were not detected in CSAS or DSiaT mutants (Figure 5C–D). These results demonstrated that CSAS and DSiaT are essential for the biosynthesis of sialylated N-glycans in vivo, and that each of these genes plays a non-redundant role in this pathway. Figure 5 Download asset Open asset Analysis of CMP-Sia and sialylated glycans in Drosophila. (A) Quantification of CMP-Sia using LC-MS/MS by normalized peak area (see Materials and methods). CMP-Sia was detected in wild-type flies (WT) but not in CSAS mutants (CSAS21/21). Transgenic expression of UAS-CSAS in glial cells of CSAS mutants by Repo-Gal4 (Repo>CSAS CSAS21/21, a rescue genotype) could significantly restore the level of CMP-Sia. ND, not detected (signal/noise ratio <1). Data were obtained from three biological repliacates per genotype, each including 100 adult flies (50 males plus 50 females) analyzed in three technical repeats. Error bars are SEM; one-way ANOVA with post hoc Tukey test was used for statistical analyses; ***, *, differences with p<0.001 and p<0.05, respectively. (B) Typical examples of normalized CMP-Sia signal intensity traces for wild-type, CSAS mutant, and rescue genotypes, as well as CMP-Sia standard. (C) Summary of glycomic analyses of N-linked glycans in wild-type Drosophila, CSAS, and DSiaT mutants. The N-glycome of third instar larval brains was analyzed. No sialylated N-glycans were detected in the mutants. Samples from wild-type and mutant genotypes were analyzed in parallel using the glycomic protocol described in Materials and methods. n, number of replicates. 1Most abundant glycan detected in wild-type is shown as representative. ND, not detected. Graphical representation and description of structures are according to the accepted glycan nomenclature (Aoki et al., 2007; Varki et al., 2015b). See extended table of N-glycan species identified by glycomic analyses in Figure 5—source data 3. (D) Example of fragmentation of a sialylated N-glycan extracted from wild-type larvae. MS/MS fragmentation of the doubly charged, permethylated ion at m/z=1003 (m+Na)2+ reveals signature ions consistent with loss of charge (m/z=1983), loss of sialic acid (m/z=608, 1330, 1307), as well as cross-ring fragmentation and loss of reducing terminal residues. The fragmentation pattern confirms the presence of the depicted sialylated structure. Similar fragmentation was not detected in DSiaT or CSAS mutants. Figure 5—source data 1 Source data for Figure 5A. https://cdn.elifesciences.org/articles/78280/elife-78280-fig5-data1-v2.xlsx Download elife-78280-" @default.
• W4366086446 created "2023-04-19" @default.
• W4366086446 creator A5006151284 @default.
• W4366086446 creator A5016397939 @default.
• W4366086446 creator A5022176742 @default.
• W4366086446 creator A5026995692 @default.
• W4366086446 creator A5028585628 @default.
• W4366086446 creator A5040327186 @default.
• W4366086446 creator A5042228418 @default.
• W4366086446 creator A5043688508 @default.
• W4366086446 creator A5045177221 @default.
• W4366086446 creator A5053216777 @default.
• W4366086446 creator A5064738584 @default.
• W4366086446 creator A5071303454 @default.
• W4366086446 creator A5090914265 @default.
• W4366086446 creator A5091754764 @default.
• W4366086446 date "2023-02-27" @default.
• W4366086446 modified "2023-09-26" @default.
• W4366086446 title "Author response: Glia-neuron coupling via a bipartite sialylation pathway promotes neural transmission and stress tolerance in Drosophila" @default.
• W4366086446 doi "https://doi.org/10.7554/elife.78280.sa2" @default.
• W4366086446 hasPublicationYear "2023" @default.
• W4366086446 type Work @default.
• W4366086446 citedByCount "0" @default.
• W4366086446 crossrefType "peer-review" @default.
• W4366086446 hasAuthorship W4366086446A5006151284 @default.
• W4366086446 hasAuthorship W4366086446A5016397939 @default.
• W4366086446 hasAuthorship W4366086446A5022176742 @default.
• W4366086446 hasAuthorship W4366086446A5026995692 @default.
• W4366086446 hasAuthorship W4366086446A5028585628 @default.
• W4366086446 hasAuthorship W4366086446A5040327186 @default.
• W4366086446 hasAuthorship W4366086446A5042228418 @default.
• W4366086446 hasAuthorship W4366086446A5043688508 @default.
• W4366086446 hasAuthorship W4366086446A5045177221 @default.
• W4366086446 hasAuthorship W4366086446A5053216777 @default.
• W4366086446 hasAuthorship W4366086446A5064738584 @default.
• W4366086446 hasAuthorship W4366086446A5071303454 @default.
• W4366086446 hasAuthorship W4366086446A5090914265 @default.
• W4366086446 hasAuthorship W4366086446A5091754764 @default.
• W4366086446 hasBestOaLocation W43660864461 @default.
• W4366086446 hasConcept C104317684 @default.
• W4366086446 hasConcept C127413603 @default.
• W4366086446 hasConcept C131584629 @default.
• W4366086446 hasConcept C132525143 @default.
• W4366086446 hasConcept C138885662 @default.
• W4366086446 hasConcept C169760540 @default.
• W4366086446 hasConcept C197657726 @default.
• W4366086446 hasConcept C21036866 @default.
• W4366086446 hasConcept C2776041557 @default.
• W4366086446 hasConcept C41008148 @default.
• W4366086446 hasConcept C41895202 @default.
• W4366086446 hasConcept C54355233 @default.
• W4366086446 hasConcept C761482 @default.
• W4366086446 hasConcept C76155785 @default.
• W4366086446 hasConcept C78519656 @default.
• W4366086446 hasConcept C80444323 @default.
• W4366086446 hasConcept C86803240 @default.
• W4366086446 hasConcept C95444343 @default.
• W4366086446 hasConceptScore W4366086446C104317684 @default.
• W4366086446 hasConceptScore W4366086446C127413603 @default.
• W4366086446 hasConceptScore W4366086446C131584629 @default.
• W4366086446 hasConceptScore W4366086446C132525143 @default.
• W4366086446 hasConceptScore W4366086446C138885662 @default.
• W4366086446 hasConceptScore W4366086446C169760540 @default.
• W4366086446 hasConceptScore W4366086446C197657726 @default.
• W4366086446 hasConceptScore W4366086446C21036866 @default.
• W4366086446 hasConceptScore W4366086446C2776041557 @default.
• W4366086446 hasConceptScore W4366086446C41008148 @default.
• W4366086446 hasConceptScore W4366086446C41895202 @default.
• W4366086446 hasConceptScore W4366086446C54355233 @default.
• W4366086446 hasConceptScore W4366086446C761482 @default.
• W4366086446 hasConceptScore W4366086446C76155785 @default.
• W4366086446 hasConceptScore W4366086446C78519656 @default.
• W4366086446 hasConceptScore W4366086446C80444323 @default.
• W4366086446 hasConceptScore W4366086446C86803240 @default.
• W4366086446 hasConceptScore W4366086446C95444343 @default.
• W4366086446 hasLocation W43660864461 @default.
• W4366086446 hasOpenAccess W4366086446 @default.
• W4366086446 hasPrimaryLocation W43660864461 @default.
• W4366086446 hasRelatedWork W1995460088 @default.
• W4366086446 hasRelatedWork W2143275805 @default.
• W4366086446 hasRelatedWork W2215223126 @default.
• W4366086446 hasRelatedWork W2315136132 @default.
• W4366086446 hasRelatedWork W2998957234 @default.
• W4366086446 hasRelatedWork W3096893558 @default.
• W4366086446 hasRelatedWork W4232017260 @default.
• W4366086446 hasRelatedWork W4236915199 @default.
• W4366086446 hasRelatedWork W4242174748 @default.
• W4366086446 hasRelatedWork W4249304790 @default.
• W4366086446 isParatext "false" @default.
• W4366086446 isRetracted "false" @default.
• W4366086446 workType "peer-review" @default.