© 2014. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2014) 7, 1365-1378 doi:10.1242/dmm.017137

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ABSTRACTClassic galactosemia (CG) is an autosomal recessive disorderresulting from loss of galactose-1-phosphate uridyltransferase(GALT), which catalyzes conversion of galactose-1-phosphate and uridine diphosphate (UDP)-glucose to glucose-1-phosphate and UDP-galactose, immediately upstream of UDP–N-acetylgalactosamine and UDP–N-acetylglucosamine synthesis.These four UDP-sugars are essential donors for driving the synthesisof glycoproteins and glycolipids, which heavily decorate cell surfacesand extracellular spaces. In addition to acute, potentially lethalneonatal symptoms, maturing individuals with CG develop strikingneurodevelopmental, motor and cognitive impairments. Previousstudies suggest that neurological symptoms are associated withglycosylation defects, with CG recently being described as acongenital disorder of glycosylation (CDG), showing defects in bothN- and O-linked glycans. Here, we characterize behavioral traits,synaptic development and glycosylated synaptomatrix formation in aGALT-deficient Drosophila disease model. Loss of Drosophila GALT(dGALT) greatly impairs coordinated movement and results instructural overelaboration and architectural abnormalities at theneuromuscular junction (NMJ). Dietary galactose and mutation ofgalactokinase (dGALK) or UDP-glucose dehydrogenase (sugarless)genes are identified, respectively, as critical environmental andgenetic modifiers of behavioral and cellular defects. Assaying theNMJ extracellular synaptomatrix with a broad panel of lectin probesreveals profound alterations in dGALT mutants, including depletion ofgalactosyl, N-acetylgalactosamine and fucosylated horseradishperoxidase (HRP) moieties, which are differentially corrected bydGALK co-removal and sugarless overexpression. Synaptogenesisrelies on trans-synaptic signals modulated by this synaptomatrixcarbohydrate environment, and dGALT-null NMJs display strikingchanges in heparan sulfate proteoglycan (HSPG) co-receptor andWnt ligand levels, which are also corrected by dGALK co-removaland sugarless overexpression. These results reveal synaptomatrixglycosylation losses, altered trans-synaptic signaling pathwaycomponents, defective synaptogenesis and impaired coordinatedmovement in a CG neurological disease model.

KEY WORDS: Congenital disorder of glycosylation (CDG),sugarless, Galactokinase, Synaptogenesis, Trans-synapticsignaling, WNT, HSPG, Neuromuscular junction

RESEARCH ARTICLE

Department of Biological Sciences, Kennedy Center for Research on HumanDevelopment, Vanderbilt University, Nashville, TN 37232, USA.

*Author for correspondence (kendal.broadie@vanderbilt.edu)

This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricteduse, distribution and reproduction in any medium provided that the original work is properlyattributed.

Received 9 June 2014; Accepted 12 October 2014

INTRODUCTIONClassic galactosemia (CG; OMIM 230400) results from loss ofgalactose-1-phosphate uridyltransferase (GALT), the secondenzyme in the Leloir pathway, which acts immediatelydownstream of galactokinase (GALK), the initial enzyme(McCorvie and Timson, 2011). GALT maintains the balancebetween uridine diphosphate (UDP)-glucose (glc), -galactose (gal),–N-acetylgalactosamine (GalNac) and –N-acetylglucosamine(GlcNac) (Frey, 1996). Levels of these four UDP-sugars are rate-limiting for the biosynthesis of glycoproteins and proteoglycans(Freeze and Elbein, 2009), which form the foundation of theextracellular synaptomatrix of the synaptic cleft and perisynapticspace (Dani and Broadie, 2012). UDP-glc dehydrogenase isencoded by Drosophila sugarless (sgl) (Häcker et al., 1997;Toyoda et al., 2000), and sgl mutations compromise biosynthesisof the heparan sulfate proteoglycan (HSPG) co-receptor Dally-likeprotein (Dlp), which is known to regulate trans-synaptic signalingof the Wnt protein Wingless (Wg); such signaling drivesneuromuscular junction (NMJ) synaptogenesis (Dani et al., 2012).These studies implicate a core pathway involving GALT, GALKand Sgl in the regulation of HSPG co-receptor control of Wntsignaling during NMJ synapse formation, and indicate thatdisruption of this pathway is a potential causal mechanismunderlying CG neuropathology.

Acute CG neonatal symptoms are alleviated by dietary galactoserestriction (Jumbo-Lucioni et al., 2012), but maturing individualswith CG develop substantial neurodevelopmental, motor andcognitive impairments (Ridel et al., 2005). After >50 years ofresearch, there is still no mechanistic understanding of these chronicneurological symptoms. However, a long-term and extensive bodyof studies documents glycosylation defects in individuals with CG(Haberland et al., 1971; Petry et al., 1991; Charlwood et al., 1998;Liu et al., 2012). Galactose is a major component of complexcarbohydrates in glycoproteins and glycolipids in the nervoussystem, and defective glycosylation impairs neurodevelopment andneurological function (Freeze et al., 2012). In particular, the heavilyglycosylated NMJ synaptomatrix plays crucial roles insynaptogenesis during normal development, and its disruption isimplicated in numerous heritable disease states (Dani and Broadie,2012). For example, glycosylation defects are causal in numerousmuscular dystrophies (MDs) and congenital disorders ofglycosylation (CDGs) that are characterized by severe neurologicalimpairments (Muntoni et al., 2008; Freeze, 2013).

We recently conducted a Drosophila screen of glycogenes viaRNAi knockdown of N/O-linked glycans, glycosaminoglycans,glycosyltransferases and glycan-binding lectins to test the effects onNMJ structure and function (Dani et al., 2012). This screenidentified Drosophila GALT (dGALT) as a potent regulator of NMJarchitecture. We therefore set forth to characterize synapticmorphology and glycosylated synaptomatrix composition in therecently established Drosophila CG disease model (dGALT

Overelaborated synaptic architecture and reduced synaptomatrixglycosylation in a Drosophila classic galactosemia disease modelPatricia Jumbo-Lucioni, William Parkinson and Kendal Broadie*

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deficiency) (Kushner et al., 2010), and to identify glycanmechanisms driving synaptogenic defects. We found that dGALTnulls exhibit a profoundly altered carbohydrate landscape within theNMJ synaptomatrix, accompanied by loss of the HSPG co-receptorDlp and extracellular accumulation of Wg ligand. Crucially,synaptomatrix defects were differentially corrected by dGALK co-removal and sgl overexpression. Consistently, we found that dGALKremoval and sgl overexpression in dGALT mutants corrected boththe motor defects and NMJ architectural abnormalities resultingfrom dGALT loss of function. We conclude that dGALT, dGALK andsgl define a genetic pathway regulating synaptomatrix glycosylationstate to modulate components of a Wnt trans-synaptic signalingpathway, and thereby control NMJ synaptic morphogenesis tosupport coordinated movement.

RESULTSImpaired coordinated movement of dGALT nulls is rescuedby human GALT expressionEvidence amassed over decades from individuals with CGdocuments common movement defects (Waggoner et al., 1990;Schweitzer et al., 1993; Kaufman et al., 1995; Hughes et al., 2009;Rubio-Agusti et al., 2013). Similarly, dGALT-null mutants displaydefects in startle-induced, geo-negative climbing behavior (Ryanet al., 2012). To assay locomotion defects directly, we first assayeddaily motor activity levels in individual animals using theDrosophila Activity Monitoring (DAM) system, which measuresmovement disruption of an infrared beam (Chiu et al., 2010).Adult animals aged 3-5 days were compared between dGALTΔAP2

(null) and dGALTC2 (precise-excision genetic control) flies. Bothgenotypes were entrained to 12:12-hour light:dark cycles for 2days, and then activity counts were recorded for ≥3 days. dGALT-null mutants were significantly (P

horseradish-peroxidase (HRP; presynaptic) and anti-DLG(postsynaptic) in nine genotypes: genetic background control(dGALTC2; precise excision), homozygous-null dGALTΔAP2 anddGALTΔAP2/Df, and ubiquitous UH1-, neuronal elav- and muscle24B-Gal4-driven dGALT-RNAi with their respective controls (UH1-Gal4/+, elav-Gal4/+ and 24B-Gal4/+, respectively). Confocalimages for representative genotypes are shown in Fig. 1C.

dGALT nulls as well as ubiquitous (UH1-Gal4) and tissue-targeted (elav-Gal4 and 24B-Gal4) dGALT-knockdown NMJs allexhibited obvious synaptic structural overelaboration (Fig. 1C,bottom row), compared with matched genetic controls (Fig. 1C, toprow). Synaptic bouton number was significantly increased in all fourmutant conditions (Fig. 1D). The increase in bouton number washighly significant in both dGALT-null conditions (dGALT: 39.4±2.7,n=14, P

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genotypes. Mean EJC amplitudes were not significantly different indGALT nulls compared with controls (419.2±23.9 versus 403.2±17.5nA; P>0.05; data not shown). Similarly, ubiquitous dGALTknockdown animals also displayed similar synaptic strength tocontrols (369.9±19.5 versus 328.6±27.5 nA; P>0.05; data not shown).Although there was a trend towards increased neurotransmission inboth comparisons, neither change was significant. We thereforeconclude that dGALT does not significantly alter neurotransmissionstrength at the NMJ, but rather plays a specific role in controllingsynaptic architecture, which we therefore focused on in subsequentstudies. Because exposure to dietary galactose is a well-recognizedmodifier of acute outcome in CG (Jumbo-Lucioni et al., 2013), wenext characterized locomotion and NMJ structural defects withgalactose dietary supplementation.

High-galactose diet phenocopies dGALT-mutant defectsDietary galactose is a well-recognized modifier of CG patient acuteoutcome (Jumbo-Lucioni et al., 2012). However, there is controversyregarding galactose restriction in preventing chronic complications(Hughes et al., 2009; Jumbo-Lucioni et al., 2012), particularly inpreventing CG neurological symptoms, and whether supplementation

or restriction are important environmental modifiers of diseaseoutcome. We therefore next assayed whether galactose feeding in theDrosophila CG model modifies movement and NMJ phenotypes.Genetic background control (dGALTC2) and homozygous-null(dGALTΔAP2) animals were raised on either (1) normal molasses-basedfood containing negligible galactose levels (Kushner et al., 2010; Jewet al., 1990), or (2) 200 mM galactose-supplemented food. WanderingL3 larvae were collected to first characterize movement (Fig. 2A). Wefound that galactose feeding severely compromised the ability ofcontrols to display coordinated motor activity. In rollover assays,controls raised in galactose-enriched food took as much time asdGALT nulls raised in the same conditions to complete the movement(33.0±4 s, n=27 versus 31.1±4.2 s, n=21, respectively), and ~120%more time than control animals fed galactose-free food (n=24,P

homozygous-dGALT-null larvae raised in the absence of galactose,displayed supernumerary boutons (28.4±1.4, n=13; 35.0±2.1, n=12;35.1±1.5, n=18, respectively; Fig. 2B,C); these values were allsignificantly greater than those for control animals raised ingalactose-free conditions (23.7±1.2 boutons, n=21; P

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et al., 2013). More specifically, the HSPG Dlp has been identifiedas a mediator of synapse formation (Johnson et al., 2006; Van Vactoret al., 2006). At the Drosophila NMJ, Dlp acts as a co-receptor ofthe Wnt protein Wg to limit extracellular Wg availability andpresentation, regulating NMJ development (Dani et al., 2012;Friedman et al., 2013). Importantly, this mechanism is linked to theLeloir pathway via UDP-glc dehydrogenase (Fig. 1A), whose lossreduces Dlp biosynthesis (Binari et al., 1997; Toyoda et al., 2000).We therefore hypothesized that loss of dGALT would alter Dlpexpression to misregulate Wg ligand abundance, with the change inWg trans-synaptic signaling providing a mechanistic basis for NMJsynaptogenesis defects in the CG disease model. To test thishypothesis, we probed NMJs from wandering L3 larvae with anti-Dlp and -Wg, using anti-HRP as the synaptic marker. Data andrepresentative images are shown in Fig. 4.

In controls, Dlp expression overlapped that of the HRP synapticmarker throughout most of the NMJ, extending slightly beyond theHRP boundaries (Fig. 4A). Compared with the strong Dlpexpression in controls (dGALTC2, n=20), dGALT-null NMJs(dGALTΔAP2, n=5) showed a >50% reduction in Dlp levels(normalized control: 1.0±0.10; dGALTΔAP2: 0.45±0.06; P50% increase in Wgligand levels (control: 1.0±0.07, n=23; dGALTΔAP2: 1.55±0.20,n=23), a significant elevation compared with the genetic control(P

Interacting Genes (STRING) (Szklarczyk et al., 2011) database toidentify candidate dGALT-interacting gene products (Fig. 5A). Weidentified dGALK as a highly associated dGALT interactor. This isnot surprising, because dGALK is directly upstream of dGALT inthe Leloir pathway (Fig. 1A). Moreover, it is commonly conjecturedthat gal-1-P accumulation is central to the symptoms of individualswith CG (Gitzelmann, 1995; Webb et al., 2003; Tang et al., 2010;Cangemi et al., 2012), suggesting that dGALK co-removal shouldameliorate dGALT-null phenotypes in our CG disease model. To testthis hypothesis, we generated dGALT; dGALK double-null mutants,and then retested all of the behavioral, NMJ architecture andglycosylated synaptomatrix phenotypes described above. Asummary of these analyses is shown in Figs 5 and 6.

First, co-removal of dGALK fully corrected the behavioralmovement defects of dGALT nulls (Fig. 5C). Null dGALT larvaeagain showed a slowed movement time (and movement wasuncoordinated) compared with genetic controls (control: 17.2±1.0 s,n=37; dGALTΔAP2: 24.3±2.1 s, n=32), which was fully restored tothe wild-type condition in double mutants (dGALT; dGALK:12.2±2.2 s, n=20), a highly significant improvement (P

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towards wild-type values in double mutants (0.87±0.13, n≥9,P

dGALK activity. Taken together, these results demonstrate thatdGALK is a key genetic modifier in the Drosophila CG diseasemodel, raising the intriguing possibility that inhibition of GALKactivity in patients might similarly modulate CG neurologicalsymptoms. Because sgl is essential for Dlp biosynthesis (Häcker etal., 1997), we next tested whether increasing Dlp biosynthesis viasgl overexpression could similarly prevent defective NMJsynaptogenesis and impaired movement.

Overexpression of sgl prevents the movement and NMJdefects in dGALT nullsOur STRING analysis (Szklarczyk et al., 2011) revealed theDrosophila sgl gene product to be strongly associated with dGALT(Fig. 5A). The sgl gene encodes UDP-glc dehydrogenase, whichmakes UDP-glucuronate from UDP-glc (Fig. 1A), an activity that isessential for the biosynthesis of proteoglycans (Häcker et al., 1997).Importantly, sgl mutants have a compromised production of theHSPG co-receptor Dlp (Haerry et al., 1997), which in turn regulatesthe Wg ligand driving Drosophila NMJ synaptogenesis (Dani et al.,2012), as discussed above (see Fig. 4). We therefore hypothesizedthat overexpressing sgl should prevent dGALT mutant phenotypesby antagonistically increasing Dlp levels, thus acting to limit Wg

abundance to properly control NMJ morphogenesis and coordinatedmovement behavior. To test this hypothesis, we made transgenicanimals overexpressing sgl (Monnier et al., 2002) in the dGALT-nullbackground (dGALTΔAP2/dGALTΔAP2; P{Mae-UAS.6.11}sglUY771/UH1-Gal4) to assay the movement behavior, NMJ architecture,glycosylated synaptomatrix and Wnt pathway phenotypes describedabove. A summary of these data is shown in Fig. 8.

UH1-Gal4 driven UAS-sgl overexpression in dGALT nullsstrongly restored coordinated movement (Fig. 8B). Loss of dGALTactivity nearly doubled rollover time compared with matchedcontrols (control: 13.7±0.9 s, n=54; dGALTΔAP2; UH1-Gal4/+:26.1±2.3 s, n=39, P

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36±1.7 boutons, n=7), concomitant overexpression of sglsignificantly decreased bouton number (dGALT; UAS-sgl/UH1-Gal4: 28.9±1.5 boutons, n=24; Fig. 8A,C). Synaptic bouton numberincreased >30% with sgl overexpression, compared with matchedcontrol (UH1-Gal4/+: 24.5±2.0, n=12; UAS-sgl/UH1: 31.6±1.1,n=18; P=0.0009; Fig. 8C). The overexpression of sgl in dGALTnulls similarly restored interbouton distance to wild-type levels(control: 0.86±0.10 μm; dGALT: 0.46±0.12 μm; dGALT; UAS-sgl/UH1-Gal4: 0.83±0.09; Fig. 8D).

To test whether sgl overexpression modifies glycosylatedsynaptomatrix composition, antibody and lectin glycan labelingwere assayed. Whereas sgl overexpression alone did not impactlevels of HRP compared with driver-alone controls (UAS-sgl/UH1-Gal4: 1.21±0.02, n=7; UH1-Gal4/+: 1±0.09, n=6; Fig. 8E), loss offucosylated HRP glycans in dGALT nulls was fully prevented by sgloverexpression (n=14; Fig. 8E), a highly significant improvementcompared with single mutants (dGALT; UH1-Gal4/+: n=10,P

(Bosch, 2006). However, our recent Drosophila screen ofglycogenes identified a role of dGALT in NMJ morphologicalsynaptogenesis (Dani et al., 2012). In light of previous reports inrodent (Audouard et al., 2012) and Drosophila (Feiguin et al., 2009)providing compelling evidence associating deficient movementbehaviors with NMJ structural abnormalities, we set forth tocharacterize NMJ synaptic architecture under conditions of completeand targeted loss of dGALT activity. These studies reveal elevatedsynaptic growth and structural overelaboration, albeit without achange in basal NMJ transmission strength. These findings areconsistent with previous studies showing that rollover movementdefects occur independently of synaptic transmission defects (Bodilyet al., 2001), and suggest that defective NMJ architecture impairsthe muscle control that is required to coordinate the sequence ofmotor outputs to optimally produce a bilateral coordinatedmovement (twist-and-roll behavior). These NMJ structural defectsare also consistent with our earlier work showing that loss ofspecific N-glycans (Dani et al., 2012), or the whole cassette ofcomplex/branched N-glycans (Parkinson et al., 2013), removesconstraints on NMJ growth and structural elaboration. We concludethat glycans play a primarily inhibitory role in modulating synapsemorphogenesis.

Major controversies surround the benefits of a galactose-restricted diet in the CG disease state (Jumbo-Lucioni et al., 2012),particularly in alleviating chronic neurological symptoms (Bosch,2006). We therefore tested impacts of galactose in the diet onmovement and NMJ structure in our Drosophila model. A high-galactose diet did not impact either the impaired movement oroverelaborated NMJ architecture of dGALT mutants, consistentwith earlier reports that dGALT long-term phenotypes areindependent of dietary galactose (Ryan et al., 2012). Strikingly,however, wild-type animals fed a high-galactose diet phenocopiedmany dGALT-mutant phenotypes, displaying slowed coordinatedmovement and grossly overelaborated NMJs with supernumeraryboutons over decreased interbouton distances. Previous studies onexperimental models of galactosemia in genetically wild-typeanimals (Cui et al., 2006; Wei et al., 2005; Long et al., 2007)demonstrate that exposure to high levels of galactose leads toneurodegeneration and cognitive disability, some of the chroniccomplications reported in CG. Increased dietary galactosedecreases the UDP-glc:UDP-gal ratio (Gibson et al., 1995; Gibsonet al., 1996). Because glycosylation depends upon UDP-sugaravailability, NMJ architectural defects in both wild type overfedgalactose and dGALT nulls might result from disrupted UDP-sugarbalance and consequently impaired glycosylation. Drosophilalarvae have been shown to accumulate significantly higher levelsof gal-1-P on a high-galactose diet (Kushner et al., 2010), whichhas been shown to directly impact UDP-sugar balance (Lai et al.,2003). In contrast to the Drosophila CG model, the viability ofmice deficient in GALT activity and reared on a high-galactosediet is reportedly unaffected, despite the accumulation of very highgal-1-P levels (Leslie et al., 1996; Ning et al., 2001). A theory toexplain this discrepancy is that very low levels of aldose reductaseactivity in mice prevent the accumulation of galactitol, a toxicgalactose intermediate. A newly established GALT-null mousemodel (Tang et al., 2014) reveals reduced viability and abnormalcellular changes in the brain as a result of galactose exposure, butfails to inform whether such changes persist under galactoserestriction. Our findings in galactose-fed dGALK mutants suggestthat gal-1-P accumulation is a primary determinant of thebehavioral deficits characterizing dGALT mutants, and wild-typeanimals fed a high galactose diet.

Glycan-binding lectins have long been used to define theextracellular glycan landscape at the NMJ synapse (Ohtsubo andMarth, 2006; Martin and Freeze, 2003; Scott et al., 1988). NulldGALT NMJs displayed striking glycosylation defects, includingsubstantial reductions in galactosyl, N-acetylgalactosamine andfucosylated HRP moieties. Differences between lectin probes arisefrom different binding specificity. ECL and PNA lectins both bindterminal galactose, whereas PNA binds preferentially Gal(β-1,3)-GalNAc and ECL binds Gal(β1,4)-GlcNAc. Similarly, WFArecognizes terminating N-acetylgalactosamine (α/β-linked togalactose) and VVA binds preferentially to Tn-antigen (i.e. a singleα-GalNAc residue linked to serine or threonine). Earlier studiesexamining the molecular basis of neural anti-HRP staining inDrosophila have demonstrated specific recognition of core α1,3-fucosylated glycoproteins (Fabini et al., 2001). Galactose-containingglycans have key synaptogenesis roles (Tai and Zipser, 1999), andglycosylation losses in rodents (Lowe and Marth, 2003) and specificloss of HRP epitopes in Drosophila (Baas et al., 2011) have bothbeen independently linked to motility abnormalities. However,although other CDG models also display evidence of overelaboratedmotor neuron architectures (Cline et al., 2012), the correlation withloss of HRP epitopes is less clear. Two previous studies have showna reduction of HRP expression with underelaborated DrosophilaNMJ structure (Rendić et al., 2010; Baas et al., 2011), in contrast toour results, whereas a recent study from our lab demonstrated HRPloss accompanied by increased synaptic growth and structuraloverelaboration (Parkinson et al., 2013), as in the current study.Such complicated carbohydrate-mediated tuning of NMJsynaptogenesis might explain the correction of the synapticarchitectural overelaboration in dGALT nulls with dGALK co-removal or sgl overexpression, despite the differential andcomplementary correction of synaptomatrix glycosylation losses.

NMJ synaptogenesis requires bidirectional trans-synapticsignaling via secreted glycoprotein ligands (Carbonetto andLindenbaum, 1995; Dani et al., 2012; Collins and DiAntonio, 2007).Such signals must necessarily traverse the heavily glycosylatedsynaptomatrix, and we have shown that the glycan state of thisextracellular environment is crucially important for enabling andshaping trans-synaptic signaling during NMJ synaptogenesis(Rohrbough and Broadie, 2010; Rushton et al., 2012; Parkinson etal., 2013). This synaptomatrix glycosylation state is stronglycompromised in our CG disease model, suggesting that signalingshould be similarly altered. Consistently, dGALT mutants exhibitedelevated Wnt Wg ligand, the best-characterized trans-synaptic signalat the Drosophila NMJ (Kamimura et al., 2013; Miech et al., 2008)The HSPG Dlp acts as a Wg co-receptor, regulating bothextracellular distribution and signaling (Han et al., 2005; Kirkpatricket al., 2004), and we have recently established that Dlp limits Wgtrans-synaptic signaling at the Drosophila NMJ (Dani et al., 2012;Friedman et al., 2013). Consistently, dGALT nulls exhibited a sharpdecrease in Dlp levels, providing a mechanism for Wgoverexpression. Wg overexpression in turn is well known toincrease NMJ bouton formation (Packard et al., 2002; Ataman et al.,2008), providing a mechanism to explain the supernumeraryboutons characterizing our CG disease model. Importantly, defectsin trans-synaptic Wg co-receptor and ligand levels are rescued withtransgenic expression of hGALT, showing functional conservation.

We identify two key dGALT genetic interactors: (1) dGALK,upstream in the Leloir pathway, and (2) sgl, a downstream UDP-glcdehydrogenase. GALK catalyzes galactose phosphorylation to gal-1-P, whose accumulation is linked to CG symptoms (Pesce andBodourian, 1982; Cangemi et al., 2012), although recent studies

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have challenged this conclusion (Jumbo-Lucioni et al., 2013;Jumbo-Lucioni et al., 2014). The current work shows that dGALKco-removal in dGALT nulls restores normal movement behavior,NMJ architecture and synaptomatrix galactosylation. Evidence insupport of this from individuals with CG shows that pathologicalaccumulation of gal-1-P directly reduces both UDP-glc and UDP-gal bioavailability in GALT deficiency (Lai et al., 2003). Similarly,earlier studies have shown that elimination of GALK activity viaGALK pharmacological inhibitors in CG patient cells (Tang et al.,2010) or GALK deletion in yeast (De-Souza et al., 2014) preventsgal-1-P accumulation, relieves galactose toxicity and restores UDP-sugar balance (Lai et al., 2003).

Sgl is required for the synthesis of the HSPG co-receptor Dlp(Toyoda et al., 2000; Haerry et al., 1997), which modulates Wgsignaling, as described above. Consistently, sgl mutants recapitulateWg phenotypes (Superina et al., 2014; Haerry et al., 1997). Thisestablished interaction strongly supports findings here showing thatsgl is a strong genetic modifier in our CG disease model. Moreover,sgl overexpression by itself phenocopies loss of dGALT, and sglgain-of-function in dGALT mutants restores NMJ architecture, Wgtrans-synaptic signaling components and motor behavior output. Inwild type, increased UDP-glc dehydrogenase activity can augmenthyaluronan production, glycosaminoglycan release and extracellularmatrix elaboration (Clarkin et al., 2011), and thus impactglycosylated synaptomatrix composition to account for negativeoutcomes of sgl overexpression. Conversely, overproduction ofextracellular glycans is predicted to be beneficial under conditionsof a depleted glycosylated synaptomatrix, as occurs in dGALT nulls.Furthermore, Dlp and Wg levels are both strongly corrected towardcontrol values by dGALK co-removal and sgl overexpression geneticinterventions. These findings strengthen the argument that the Wntsignaling pathway plays a crucial role in the pathogenesis ofneurological complications in CG. Taken together, these resultssuggest that both GALK inhibition and UDP-glc dehydrogenaseactivation might combat neurological symptoms in individuals withCG.

In conclusion, the results presented here are the first to revealNMJ glycosylation losses, synaptic architecture defects andconcomitant changes in trans-synaptic Wnt co-receptor and ligandlevels in a CG disease model. These findings suggest a model inwhich loss of GALT activity triggers gal-1-P accumulation, whichsubsequently limits UDP-sugar bioavailability. In this model, UDP-sugar deficits trigger changes in NMJ glycosylated synaptomatrixcomposition, including levels of GPI-anchored HSPG Dlp, anessential co-receptor for a Wnt ligand during NMJ synaptogenesis.Loss of HSPG regulation results in failure to control Wg levels,causing differential trans-synaptic Wnt signaling to promote excessgrowth and overelaborated architectural complexity. Co-removal ofdGALK, and sgl overexpression, both are strong genetic modifiersof outcome in this CG model. We propose that similar defectsunderlie well-characterized glycosylation defects and movementdisorders in the human CG disease state, and might account forneurological pathogenesis characterizing a wide array of relatedCDG disease states, which will be the subject of our futureinvestigations.

MATERIALS AND METHODSDrosophila geneticsFig. 1A shows the Leloir pathway, listing enzyme names and Drosophila CGnumbers of genes targeted in this study. A dGALT imprecise-excision null,dGALTΔAP2, with undetectable enzyme activity, and a precise-excisioncontrol, dGALTC2, with normal enzyme activity, were previously described

(Kushner et al., 2010). The genomic deficiency Df(2L)BSC187 crossed todGALTΔAP2 was used as a second heterozygous-null condition. Ubiquitous(UH1) and tissue-specific (neuronal elav and muscle 24B) Gal4 drivers wereused to drive dGALT-RNAi (v10025; P{KK102974}VIE-260B, ViennaDrosophila RNAi Center). Gal4 drivers alone (Gal4/+) crossed to w1118 weregenetic controls. A dGALK imprecise-excision null, dGALKΔEXC9, withundetectable enzyme activity, was generated by mobilizing a P-elementinsertion (EY03791) in the 5′-UTR of CG5068. UAS-sglUY771 was used tooverexpress sgl (Monnier et al., 2002). dGALT, dGALK and sgl alleles werecombined using standard genetic techniques to generate double mutants.Single and double mutants were reared at 25°C on standard molasses-basedfood. To test effects of a high-galactose diet, food was supplemented with200 mM galactose.

Behavioral assaysAll behavioral experiments were carried out on at least eight individualanimals for each genotype. Locomotor activity was assayed in adult malesusing the Drosophila Activity Monitoring (DAM) system (Trikinetics,Waltham, MA). Starting 1 day after eclosion, animals were entrained to a12-hour light/dark cycle for 2 days before data recording, with activitycounts per hour recorded for 3 days (Chiu et al., 2010). Coordinated larvalmovement was assayed in male wandering third instars (L3) using therollover assay described previously (Bodily et al., 2001; Pan et al., 2008).Animals were placed individually on a room temperature (RT) 1% agar plateand allowed to acclimate for 2 minutes. The L3 animal was then rolled toan inverted position as defined by the ventral midline. Once released, a timerwas used to measure the time the animal took to completely right, as definedby the dorsal midline. Three consecutive measurements were recorded foreach animal and averaged to produce one data point. Data were analyzed byStudent’s t-test for pairwise comparisons, and ANOVA tests for all data setsof ≥three comparisons.

Immunocytochemistry imagingImmunocytochemistry confocal imaging was performed as describedpreviously (Rushton et al., 2012). L3 larvae were dissected in physiologicalsaline consisting of 128 mM NaCl, 2 mM KCl, 4 mM MgCl2, 0.2 mMCaCl2, 70 mM sucrose, 5 mM trehalose and 5 mM HEPES (pH 7.1).Preparations were fixed in 4% paraformaldehyde for 10 minutes at RT, andthen either processed with detergent (PBTX: PBS+1% BSA+0.2% TritonX-100) for cell-permeabilized studies, or detergent-free (PBS with 1% BSA)for non-permeabilized studies. Primary antibodies used included: Alexa-Fluor-488 goat anti-horseradish peroxidase (HRP, 1:200; Jackson Labs);mouse anti-Fasciclin-II [FASII, 1:10; Developmental Studies HybridomaBank (DSHB), University of Iowa]; mouse anti-Discs-large (DLG, 1:250;DSHB); mouse anti-Dally-like-protein (Dlp, 1:4; DSHB) and mouse anti-Wingless (Wg, 1:2; DSHB). Conjugated lectins used included: Peanutagglutinin (PNA, 1:250), Wisteria floribunda (WFA-Fitc, 1:250), Erythrinacristagalli lectin (ECL, 1:250), Vicia villosa agglutinin (VVA, 1:250),Soybean agglutinin (SBA, 1:250) and Wheat germ agglutinin (WGA,1:250), all from Vector Labs. Secondary antibodies used included: Alexa-Fluor-555 donkey anti-mouse (1:300) and streptavidin Alexa-Fluor-488conjugate (1:250), both from Invitrogen. Primary antibodies and lectins wereincubated at 4°C overnight. Secondary antibodies were incubated at RT for2 hours. Preparations were mounted in Fluoromount G (ElectronMicroscopy Sciences).

All mutant and control larvae were dissected, labeled and imaged inparallel. z-stacks were taken with a Zeiss LSM 510 META confocal using40/63× oil-immersion objectives, with sections starting immediately aboveand ending immediately below the NMJ. Expression analyses of conjugatedlectins and anti-HRP were performed using NIH ImageJ software with thethreshold function outlining lectin- or HRP-labeled NMJ within FasII-defined synaptic regions. Anti-Dlp and anti-Wg expression analyses werequantified within HRP-defined synaptic regions. Fluorescence wasnormalized to control values from the same experiment with all imagingparameters kept constant between compared genotypes. For structuralanalyses, preparations were double-labeled with anti-HRP and anti-DLG,with counts made at muscle 4 in segments A2/3 on the right and left sides.Data were averaged for each animal or hemisegment to produce one data

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point for expression and structural analyses, respectively. For structuralquantification, a bouton was defined as an axon varicosity >1 μm inminimum diameter, and ≥two boutons on one axon defined an NMJ branch.NMJ cumulative length was measured by combining branch lengths, andinter-bouton distances obtained as an average from single branches with ≥sixboutons.

Electrophysiology recordingTwo-electrode voltage-clamp (TEVC) electrophysiology was performed asdescribed previously (Rohrbough and Broadie, 2002; Parkinson et al., 2013).Briefly, L3 larvae secured with 3M Vetbond tissue adhesive to sylgard-coatedglass coverslips were cut longitudinally along the dorsal midline, internalorgans removed and larval bodywall glued down. Peripheral nerves were cutat the ventral nerve cord (VNC). Recordings made at 18°C in physiologicalsaline (see above) from preparations imaged using a Zeiss Axioskopmicroscope with 40× water-immersion objective. Muscle 6 in segments A2/3impaled with two 3 M KCl-filled electrodes (~15 MΩ resistance) wasclamped (−60 mV) using an Axoclamp-2B amplifier. A glass suction electrodeon severed motor nerves stimulated at 0.5 Hz with 0.5 ms suprathresholdstimuli (Grass S88 stimulator). Excitatory junction current (EJC) recordsfiltered at 2 kHz (Clampex) were analyzed with Clampfit software.

Statistical analysesBehavioral, functional, structural and fluorescence intensity data wereaveraged per genotype, calculated as a fold-change relative to the meancontrol value from the same experiment. Unpaired two-tailed t-tests withWelch correction were applied for normally distributed datasets withunequal standard deviations. For data not normally distributed, pairwisecomparisons were done with non-parametric Mann-Whitney tests. One-wayanalysis of variance (ANOVA) was used for parametric multiplecomparisons with Tukey-Kramer post-tests. For nonparametric multiplecomparisons, Kruskal-Wallis tests were applied with Dunn’s multiplecomparisons. All statistical analyses were performed using GraphPad InStatversion 3.0 (GraphPad Software). Significance in figures is presented asP

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Loss of dGALT causes striking structural defects at the NMJFig.€1. LossHigh-galactose diet phenocopies dGALT-mutant defectsFig.€2. High-galactosedGALT activity shapes the glycosylated synaptomatrix composition of the NMJdGALT mutants display changes in Wnt trans-synaptic signaling pathway componentsCo-removal of dGALK prevents the movement and NMJ defects inFig.€3. LossFig.€4. NullFig.€5. Co-removalFig.€6. Co-removalOverexpression of sgl prevents the movement and NMJ defects inFig.€7. RestorationFig.€8. sglBehavioral assaysImmunocytochemistry imagingElectrophysiology recordingStatistical analyses