RESEARCH ARTICLE

Neuromuscular degeneration and locomotor deficit in aDrosophila model of mucopolysaccharidosis VII is attenuatedby treatment with resveratrolSudipta Bar, Mohit Prasad* and Rupak Datta*

ABSTRACTMucopolysaccharidosis VII (MPS VII) is a recessively inheritedlysosomal storage disorder caused by β-glucuronidase enzymedeficiency. The disease is characterized by widespread accumulationof non-degraded or partially degraded glycosaminoglycans, leading tocellular and multiple tissue dysfunctions. The patients exhibit diverseclinical symptoms, and eventually succumb to premature death. Theonly possible remedy is the recently approved enzyme replacementtherapy, which is an expensive, invasive and lifelong treatmentprocedure. Small-molecule therapeutics for MPS VII have so farremained elusive primarily due to lack of molecular insights into thedisease pathogenesis and unavailability of a suitable animal model thatcan be used for rapid drug screening. To address these issues, wedeveloped aDrosophilamodel of MPS VII by knocking out theCG2135gene, the fly β-glucuronidase orthologue. The CG2135−/− flyrecapitulated cardinal features of MPS VII, such as reduced lifespan,progressive motor impairment and neuropathological abnormalities.Loss of dopaminergic neurons and muscle degeneration due toextensive apoptosis was implicated as the basis of locomotor deficitin this fly. Such hitherto unknown mechanistic links have considerablyadvanced our understanding of the MPS VII pathophysiology andwarrant leveraging this genetically tractable model for deeper enquiryabout the disease progression. We were also prompted to test whetherphenotypic abnormalities in the CG2135−/− fly can be attenuated byresveratrol, a natural polyphenol with potential health benefits. Indeed,resveratrol treatment significantly amelioratedneuromuscular pathologyand restored normal motor function in theCG2135−/− fly. This intriguingfinding merits further preclinical studies for developing an alternativetherapy for MPS VII.

This article has an associated First Person interviewwith the first authorof the paper.

KEY WORDS: β-glucuronidase, Mucopolysaccharidosis,Neuromuscular degeneration, Resveratrol

INTRODUCTIONMucopolysaccharidosis VII (MPS VII), also known as Slysyndrome, is a recessively inherited lysosomal storage disordercaused by β-glucuronidase (β-GUS) deficiency. Because ofheterogeneity of mutations in the β-GUS gene (GUSB) that resultsin varying extents of enzyme deficiency, MPS VII patients exhibitdiverse clinical symptoms, ultimately leading to premature death inmost cases. Short stature, cognitive disability, skeletal abnormality,motor impairment, hernias, hepatosplenomegaly, hydrops fetalis,and heart and respiratory problems are some of the common clinicalsigns seen inMPSVII. Many of these symptoms are also manifestedin other MPS disorders, suggesting a common pathophysiologicalmechanism for this group of disorders (Montaño et al., 2016;Neufeld and Muenzer, 2014; Sly et al., 1973; Zielonka et al., 2017).Until recently, there was no treatment for MPS VII. However, aclinical trial for enzyme replacement therapy with recombinanthuman β-GUS led to FDA approval of the drug in November, 2017(Fox et al., 2015; Harmatz et al., 2018).

β-GUS is one of the eleven lysosomal hydrolases responsiblefor stepwise degradation of complex polysaccharides,glycosaminoglycans (GAGs) (Neufeld and Muenzer, 2014). Lossof its activity leads to accumulation of engorged lysosomes,containing non-degraded or partially degraded GAGs, in variouscells and tissues of the affected individuals (Irani et al., 1983;Vogler et al., 1994). A fraction of the non-degraded GAGs are alsosecreted into the blood stream and excessive amounts are excreted inurine (Sewell et al., 1982). Although∼50 disease-causingmutationsin the β-GUS gene have been reported to date, the molecular eventsthat lead from enzyme deficiency and GAG storage to multipletissue dysfunction or damage is poorly understood (Tomatsu et al.,2009).

In 1989, Birkenmeier et al. characterized a natural mutant mouselacking β-GUS activity that recapitulates many features of MPS VIIpatients. The mutant male mice were reproductively sterile. Themutant females were fertile but they suffered from insufficientlactation and hence could not nurture their pups (Birkenmeier et al.,1989). Even heterozygous mating produced a less-than-expectednumber of pups because of neonatal death, thus making it extremelydifficult to produce these animals in large quantities (Soper et al.,1999). Despite these challenges, this mutant mouse, which was laterfound to carry a frameshift mutation in the β-GUS gene, became themainstay of MPS VII research (Sands and Birkenmeier, 1993).Among all the affected tissues, brain pathology is relatively wellstudied in theMPSVII mouse model (Heuer et al., 2002; Levy et al.,1996). Although vacuolar storage lesions were uniformlydistributed throughout the brain, signs of neurodegeneration wereonly seen in discrete regions, particularly in the hippocampus andcerebral cortex (Heuer et al., 2002). Upregulation of severalinflammatory genes was observed in the posterior cortex of theReceived 21 August 2018; Accepted 5 October 2018

Department of Biological Sciences, Indian Institute of Science Education andResearch (IISER) Kolkata, Mohanpur 741246, West Bengal, India.

*Authors for correspondence (mohitprasad@iiserkol.ac.in;rupakdatta@iiserkol.ac.in)

S.B., 0000-0001-5591-8749; M.P., 0000-0002-3254-0541; R.D., 0000-0003-1820-9251

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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diseased brain (Richard et al., 2008). Many pro-apoptotic geneswere also found to be transcriptionally activated in the MPS VIImouse brain but, surprisingly, no apoptotic cells could be detectedin those tissues (Heuer et al., 2002; Richard et al., 2008). However,the molecular basis of selective neurodegeneration in MPS VII isstill unclear. Also, how lysosomal storage affects non-neuronaltissues remains to be investigated.In recent times, many neurodegenerative diseases, including

some of the lysosomal storage disorders like Niemann-Pick disease,mucolipidosis type IV and Gaucher disease, have been successfullymodelled in Drosophila (Bonini and Fortini, 2003; Feany andBender, 2000; Huang, 2005; Iijima et al., 2004; Kinghorn et al.,2016; Venkatachalam et al., 2008). These simple, geneticallytractable fly models have led to better understanding of themechanism of these diseases and also have provided platforms fortranslational research. Encouraged by these studies, we decided todevelop a Drosophila model of MPS VII. We first identified thefunctional β-GUS orthologue in Drosophila (annotated as CG2135in the FlyBase; also known as betaGlu) and then generated aCG2135−/− knockout fly. The CG2135−/− flies were viable with noovert phenotype. Crosses between them produced the expectednumber of embryos but they had a somewhat reduced hatching rate,possibly a consequence of a low-penetrance developmental defect.The adult CG2135−/− fly exhibited typical features of MPS VII,including a reduced lifespan, progressive decline in locomotoractivity and abnormal neuropathology. This fly also had increasedaccumulation of storage materials in the brain, which is thepathological hallmarks of lysosomal storage disorders.Neurodegeneration, especially the loss of dopaminergic neurons,and extensive muscle atrophy was implicated as the basis oflocomotor disability in this fly. Interestingly, the neuromuscularpathology as well as the locomotor defect in the CG2135−/− fly wasrestored to a significant extent upon treatment with resveratrol, anatural polyphenol (Baur and Sinclair, 2006). Therefore, in additionto providing mechanistic insights into the pathogenesis of MPS VII,our work uncovered a therapeutic lead for managing this debilitatingdisease. This novel fly model may have a far-reaching impact as itopens up the opportunity for rapid screening of potential drugs thatwould not be feasible with the existing MPS VII mouse (Fernández-Hernández et al., 2016). It also offers an excellent system forconducting genetic screens to gain deeper understanding of thedisease progression (St Johnston, 2002).

RESULTSIdentification of CG2135 as an active β-GUS orthologuein DrosophilaWe first confirmed that an enzymatically active β-GUS isubiquitously expressed in all developmental stages of Drosophilaand in all tissues of the adult fly that were analyzed (Table 1).Simultaneously, we identified a putative β-GUS-encoding gene inthe Drosophila genome, annotated as CG2135 (http://flybase.org).The CG2135 protein was predicted to possess an N-terminalendoplasmic reticulum (ER)-targeting signal peptide, which is

characteristic of all lysosomal hydrolases (Fig. S1) (Kornfeld,1987). Sequence alignment revealed that CG2135 shares >40%identity and >60% similarity with that of the human β-GUS protein.Critical amino acids of the human β-GUS active site as well as mostfrequently mutated residues in MPS VII were conserved in CG2135(Fig. 1) (Hassan et al., 2013). Widespread expression of CG2135mRNAwas confirmed by reverse transcription (RT)-PCR, which isconsistent with a previous microarray-based transcriptome analysis(Fig. S2) (Chintapalli et al., 2007). Next, CG2135 cDNA wascloned, the protein expressed in S2 cells, purified by Ni-affinitychromatography and finally subjected to β-GUS assay. Theauthenticity of CG2135 as a functional β-GUS was confirmed byits specific activity of 3.7×106 units/mg, which is comparable to thepreviously reported activity of human β-GUS. Similar to humanβ-GUS, CG2135 was found to be a thermally stable enzyme, activeover a wide range of pH and with an optimum pH∼5.0 (Fig. S3A-C)(Grubb et al., 2008; Islam et al., 1999).

Generation of the CG2135−/− fly by targeted gene disruptionHaving confirmed CG2135 as an authentic β-GUS orthologue, ournext goal was to generate a CG2135−/− fly. For this, a targetingconstruct was generated consisting of the Gal4 and mini-whitemarkers flanked by 5′ and 3′ genomic regions of CG2135.Homologous recombination between this targeting construct andthe endogenous locus produced the targeted allele, in which a212-bp fragment of the CG2135 coding sequence (including thestart codon) was replaced with the Gal4 and mini-white markers,thus resulting in a genetic deletion as well as a frameshift (Fig. 2A).The targeting event in the CG2135−/− fly was verified by genomicPCR with primers (P15-P16) that bind to the genomic sequenceoutside the targeted region and to the Gal4 gene. No PCR productwas amplified from genomic DNA of the wild-type fly.Additionally, genomic PCR was performed with primers (P17-P18 and P19-P20) that bind inside the targeted region and to theregion which gets deleted after recombination. PCR amplificationwas observed only with genomic DNA from the wild-type fly butnot from the CG2135−/− fly, which harbours the deletion (Fig. 2A,B). Knockout of the CG2135 gene was further confirmed by thecomplete absence of the CG2135 mRNA in the CG2135−/− fly(Fig. 2C). Interestingly, β-GUS activity in the CG2135−/− fly,although reduced to a significant extent, was not totally abolished(Fig. 2D). To check whether this residual β-GUS activity in theCG2135−/− fly is contributed by another β-GUS-like protein, weperformed a blast search of the Drosophila genome with theCG2135 protein sequence. This revealed existence of a gene ofunassigned function (CG15117) that has high amino-acid sequencesimilarity with that of the CG2135. This finding is consistent withan older report, where two chromatographically separable forms ofβ-GUS were shown to exist in Drosophila (Langley et al., 1983).Unlike lysosomal hydrolases, CG15117 lacked the N-terminal ER-targeting signal peptide, suggesting it to be a non-lysosomal enzymethat plays some non-conventional role (Fig. S4A,B). PurifiedCG15117 was found to be six-fold less active than CG2135

Table 1. β-GUS activity in different developmental stages and tissues of the adult fly

Tissue sample

Embryo Larva Pupa Adult fly* Brain‡ Midgut‡ Fat body‡ Muscle‡

β-GUS specific activity (units/mg) 52.9±11.35 23.57±3.6 102.4±1.90 63.1±5.23 73.22±13.8 85.89±31.59 48.26±16.28 12.64±0.44

All specific activity values are the average of at least three independent measurements±s.d.*Whole fly, males and females mixed in equal proportions.‡Tissues from male and female flies mixed in equal proportions.

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(Fig. S5A,B). The CG15117mRNAwas expressed in the wild-typeas well as in the CG2135−/− fly, thereby explaining why a low levelof β-GUS activity could be detected in tissues after completeabolition of CG2135 expression (Fig. S5C).

Shortened lifespan and progressive decline of locomotoractivity in the CG2135−/− flyThe CG2135−/− homozygous flies were viable and did not showany gross abnormalities. Crosses between them produced theexpected number of embryos but they had ∼30% less than normalhatching rate, suggesting that loss of CG2135 resulted in an earlydevelopmental defect with incomplete penetrance (Fig. 3A,B).Compared to the wild-type flies, adult CG2135−/− flies had reducedsurvivability as indicated by their shortened mean and maximumlifespan by 5 and 13 days, respectively (Fig. 3C). The CG2135−/−

flies also exhibited age-dependent impairment of locomotor activityas analyzed by negative geotaxis (climbing) assay (Strauss andHeisenberg, 1993; Venkatachalam et al., 2008). While the adultCG2135−/− flies had almost no climbing disability until the secondweek, their performance worsened from the third week onwards. Atthe fourth week, they exhibited ∼85% decline in climbing abilitycompared to their wild-type counterparts (Fig. 3D). To confirm thatthe observed locomotor defect is a direct consequence of loss ofCG2135 function, we created a transgenic fly overexpressingCG2135 on the CG2135−/− background (Fig. S6). Completerestoration of locomotor function was observed in this fly (Fig. 3E).The features of the CG2135−/− flies reported here are thusreminiscent of the movement disability and reduced lifeexpectancy seen in MPS VII patients (Montaño et al., 2016).

Neuropathological abnormalities in the CG2135−/− flySince locomotor activities are primarily controlled by brain andmuscle function, we analyzed brain pathologies inCG2135−/− flies tounderstand the underlying cause of their climbing defect (Flash andHochner, 2005). First, 30-day-old fly brains were stained withLysoTracker Red to detect acidic organelles such as lysosomesand autolysosomes (Chazotte, 2011). Increased abundance ofLysoTracker-positive vesicles was observed in the CG2135−/− flybrain, which is consistent with accumulation of engorged lysosomesseen in patients suffering fromMPS VII and other lysosomal storagedisorders (Fig. 4A-D) (Ferreira and Gahl, 2017; Irani et al., 1983;Vogler et al., 1994). As compared to the age-matched controls, theCG2135−/− flies exhibited elevated levels of ubiquitinated proteins intheir brain (Fig. 4E-I). Interestingly, we also observed a significantincrease in mitochondrial population in the CG2135−/− fly brain(Fig. 4J-M). This abnormal build-up of ubiquitinated proteins andmitochondria might be due to a defect in the lysosome-mediatedcellular clearance machinery that has been reported in manylysosomal storage disorders and other neurodegenerative diseases(Heuer et al., 2002; Osellame and Duchen, 2014; Platt et al., 2012).Examination of Toluidine-Blue-stained brain sections revealedincreased vacuolation in the 30-day-old CG2135−/− flies withrespect to the age-matched controls (Fig. 4N-P). Those vacuolarlesions were mostly seen in the central complex region, which isassociated with locomotor function in flies (Fig. 4O) (Strauss, 2002).The brain dopaminergic system is a critical modulator of locomotionin flies as well as in mammals (Riemensperger et al., 2013; Zhou andPalmiter, 1995). Also, movement disorders such as Parkinson’sdisease and progressive supranuclear palsy are characterized by loss

Fig. 1. Sequence alignment of humanand Drosophila β-GUS. Amino acidsequence alignment of Drosophilaβ-GUS (annotated as CG2135 inFlyBase) and human β-GUS (hβ-GUS;UniProt accession number P08236).The active-site residues of hβ-GUS andmost frequently mutated residues inMPS VII are marked by black and whitetriangles, respectively. An asterisk (*)indicates positions with a single, fullyconserved residue; a colon (:) indicatesconservation between groups ofstrongly similar properties; and a period(.) indicates conservation betweengroups of weakly similar properties.

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of the dopaminergic neurons (Auluck et al., 2002; Murphy et al.,2008). This led us to assess the status of tyrosine-hydroxylase-positive dopaminergic neurons in wild-type and CG2135−/− flybrains. A significant loss of dopaminergic neurons was seen in 30-day-oldCG2135−/− fly brain as compared to the age-matched control(Fig. 4Q-S). This loss might be responsible, at least in part, for thelocomotion defect seen in theCG2135−/− flies. Furthermore, analysisof the retinal architecture revealed a gross defect in ommatidialorganization (appearance of holes between the ommatidia) and

occasional loss of photoreceptors in the 30-day-old CG2135−/− fly.Such signs of retinal degeneration were barely detectable in the age-matched wild-type fly, in which the overall ommatidial architectureand the photoreceptors were well preserved (Fig. 4T,U). The ocularphenotype in the CG2135−/− fly reported here, which mimics thephotoreceptor degeneration earlier seen in the MPS VII mouse, mayexplain why visual impairment is a common occurrence in MPS VIIpatients (Lazarus et al., 1993; Montaño et al., 2016). As expected, theneuropathological abnormalities in the CG2135−/− fly were reverted

Fig. 2. Generation of the CG2135−/− fly.(A) Schematic representation of the linearized CG2135knockout construct containing Gal4 and mini-whitemarkers flanked by 5′ and 3′ genomic regions ofCG2135 with a truncation of a 212-bp fragment. FRTand I-SceI sites are also shown. The targeted alleleafter homologous recombination is shown at thebottom. Primer binding sites are indicated by arrows.(B) Genotype of the wild-type (WT) and the CG2135−/−

fly was verified by genomic PCR. Integration of theGal4 marker gene in the CG2135−/− fly was confirmedby PCR amplification of the 3.7-kb product with P15-P16 primers. Deletion of the genomic fragment in theCG2135−/− fly was verified by the absence of any PCRproducts with P19-P20 and P17-P18 primer sets. The2.5-kb and 144-bp PCR products were amplified in theWT fly, as expected. (C) RT-PCR with CG2135-specific primer sets (P1-P2), showing absence of theCG2135 mRNA in the CG2135−/− fly. RT-PCR withactin amplification (P5-P6) served as control.(D) β-GUS-specific activity in the WT and CG2135−/−

fly. Error bars represent s.e.m. of values from threeindependent experiments. *P≤0.05.

Fig. 3. Phenotypic abnormalities in the CG2135−/− fly. (A) Scatter plot showing the wild-type (WT) and the CG2135−/− fly both produce a similar number ofembryos per day. Line represents the mean value; error bar represents s.e.m. and dots represent individual data point (N>20). (B) Bar graph showing asignificantly reduced hatching percentage of the CG2135−/− embryos compared to the WT (N=150). (C) Survival curve of the WT and the CG2135−/− flies.Mean and maximum lifespan values are provided in the adjacent table. (D) Scatter plot representing climbing index of WT and CG2135−/− flies of different ages(N∼200). (E) Bar graph showing a significant decline in climbing index of the 4-week-old CG2135−/− flies as compared to the age-matched WT flies. Completerestoration of climbing activity was observed in the age-matched rescue fly (N∼200). Error bar represents s.e.m. of values from at least three independentexperiments. ‘ns’ indicates not significant. *P≤0.05, **P≤0.01, ***P≤0.001.

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to a significant extent by transgenic overexpression of the CG2135gene in the homozygous knockout background (Fig. S7).

Muscle degeneration in the CG2135−/− flyApart from neurodegeneration, a defect in muscle could be anotherpathological basis for locomotor disability in CG2135−/− flies.Indeed, histological analysis of the thoracic muscles revealedsignificant loss of muscle fibre integrity in the 30-day-oldCG2135−/− flies but not in the age-matched control flies or in thetransgenic rescue fly (Fig. 5A,B, Fig. S8). TUNEL staining showedextensive apoptosis in muscles of the CG2135−/− flies (Fig. 5C-H).

The percentage of the apoptotic nuclei in muscles of the CG2135−/−

flies was >60% as compared to only ∼1% in the wild-type flies(Fig. 5I). These data indicate that muscle degeneration in theCG2135−/− flies is due to extensive apoptosis of the myocytes,which may contribute to impaired locomotion in these flies.

Resveratrol attenuated neuromuscular degenerationand locomotor disability in the CG2135−/− flyResveratrol, a natural polyphenol, has been shown to exert multiplehealth benefits in a variety of animal models (Baur and Sinclair,2006). This includes, lifespan extension, delaying the age-related

Fig. 4. Neuropathological abnormalities in the CG2135−/− fly. (A-D) LysoTracker (red) and DAPI (blue) staining of 30-day-old fly brains, showing increasedabundance of the LysoTracker-positive vesicles in theCG2135−/− fly compared to the wild type (WT). (E-H) Immunostaining of the 30-day-old fly brains with anti-ubiquitin antibody (red), showing an elevated level of ubiquitinated proteins in the CG2135−/− fly. (I) Bar graph showing a significant increase in mean intensity ofubiquitin staining in the CG2135−/− fly brain. (J-M) MitoTracker (red) and DAPI (blue) staining of the 30-day-old fly brains, showing increased mitochondrialaccumulation in the CG2135−/− fly brain. (N,O) Toluidine-Blue-stained brain sections of the 30-day-old flies, showing increased vacuolation (indicated byarrowheads) in theCG2135−/− fly. (P) The number of vacuoles in theWTandCG2135−/− fly brain sections. (Q,R) Immunostaining of the 30-day-old fly brains withanti-tyrosine-hydroxylase antibody (green), showing loss of dopaminergic neurons in the CG2135−/− fly brain compared to the WT flies. Enlarged view of adopaminergic neuron cell body from the boxed regions of the brain is provided in the inset. (S) Quantitation of dopaminergic neurons in theWT andCG2135−/− flybrains. (T,U) Toluidine-Blue-stained retinal sections showing well-preserved ommatidial architectures in the 30-day-old WT fly, as opposed to defectiveommatidial organization (‘x’ showing the gaps) in the age-matched CG2135−/− fly. Error bar represents s.e.m. of values from at least three independentexperiments. *P≤0.05, **P≤0.01.

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phenotypes and prevention of diseases such as cancer or diabetes(Howitz et al., 2003; Jang et al., 1997; Lagouge et al., 2006;Valenzano et al., 2006; Wood et al., 2004). The neuroprotective roleof resveratrol is also well documented (Bastianetto et al., 2015; Kimet al., 2007; Parker et al., 2005). These promising results prompted usto test whether resveratrol can attenuate neuromuscular degenerationand associated locomotor disability seen in the older CG2135−/−

flies. We observed that the 30-day-old CG2135−/− flies, when fedwith resveratrol, had significantlymore dopaminergic neurons in theirbrain as compared to thosemaintained on a standard diet. The numberof dopaminergic neurons in the resveratrol-fed CG2135−/− flies wasalmost similar to that observed in the wild type flies (Fig. 6A-D). The30-day-oldCG2135−/− flies on the resveratrol diet did not exhibit anysigns of apoptosis in the muscles. This is in stark contrast withextensive muscle cell apoptosis seen in the age-matched CG2135−/−

flies reared on standard diet (Fig. 6E-K). Furthermore, suppression ofneuromuscular degeneration in the 30-day-old resveratrol-fedCG2135−/− flies was accompanied by near-complete recovery oftheir climbing ability (Fig. 6L). This suggests that therapeuticadministration of resveratrol can be explored as an alternativeapproach to management of MPS VII.

DISCUSSIONWe report here the generation and characterization of a novel β-GUSknockout (CG2135−/−) fly that closely resembles the MPS VIIdisease phenotypes. The cardinal features of MPS VII, such asreduced lifespan, progressive decline in locomotor activity andvacuolation, as well as accumulation of storage materials in thebrain, were manifested in this fly. The CG2135−/− fly thusrepresents the first invertebrate model of MPS VII and has provento be extremely useful for unravelling mechanistic complexities ofthe disease and exploring therapeutic possibilities.The locomotor deficit in the CG2135−/− fly was quite striking.

This could be attributed to neuromuscular degeneration, particularlythe loss of dopaminergic neurons and muscle cell apoptosis. Theadult fly brain contains eight different clusters of dopaminergicneurons that are involved in different functions (Mao and Davis,2009). Using cluster-specific drivers of α-synuclein expression, theprotocerebral anterior medial (PAM) cluster dopaminergic neuronswere shown to be important for locomotor control in a Drosophila

model of Parkinson’s disease (Riemensperger et al., 2013). Hence,it is likely that selective degeneration of PAM-cluster dopaminergicneurons underlies the locomotor defect of the CG2135−/− fly.Apoptotic muscle degeneration seen in this fly may further worsentheir motor function. A link between muscle cell apoptosis andmovement disability was earlier established in a Drosophila modelof autosomal recessive juvenile parkinsonism by knocking out theparkin gene, which encodes a mitochondrial ubiquitin-proteinligase (Greene et al., 2003). Although the status of dopaminergicneurons or muscle pathology is yet to be studied inMPSVII patientsor in the mouse model, our findings suggest that, in addition toskeletal abnormalities, dopaminergic neuronal loss and musclewasting are major contributing factors for restricted mobility seen inthe majority of these patients (Montaño et al., 2016). Furthercharacterization of the CG2135−/− fly may reveal additionalphenotypes, including some of the complex neurological andmetabolic aspects of this disease.

One major advantage with this Drosophila model of MPS VII isthat it can be used for rapid screening of small molecules to identifynew therapeutic leads (Fernández-Hernández et al., 2016; Giacomottoand Ségalat, 2010). As a proof of this concept, we tested the efficacy ofresveratrol, which has emerged as a promising compound havingpotential health benefits (Bastianetto et al., 2015; Baur and Sinclair,2006; Howitz et al., 2003; Jang et al., 1997; Kim et al., 2007; Lagougeet al., 2006; Parker et al., 2005; Valenzano et al., 2006; Wood et al.,2004). Compelling evidence was presented for resveratrol-mediatedprotection against neuromuscular degeneration and progressivelocomotor disability in the CG2135−/− fly. These results areconsistent with previous reports where resveratrol treatment wasshown to protect against chemically induced depletion ofdopaminergic neurons or muscle wasting (Hori et al., 2011;Okawara et al., 2007; Zhang et al., 2010). However, the mechanismby which resveratrol could rescue the CG2135−/− fly is yet unclear.Antioxidant, anti-inflammatory and autophagy-modulating propertiesof resveratrol arewidely reported (Bastianetto et al., 2015; Hasima andOzpolat, 2014; Hori et al., 2011; Zhang et al., 2010). Also, resveratrol-mediated activation of AMPK has been proposed to control cellularenergy homeostasis and confer neuroprotection (Dasgupta andMilbrandt, 2007). These plausible mechanistic pathways remain tobe explored in our model to establish the mode of action of resveratrol.

Fig. 5. Muscle degeneration in the CG2135−/− fly.(A,B) Hematoxylin and eosin staining of longitudinal sections ofthoracic muscle of 30-day-old flies, showing intact musclestructure in the wild type (WT) fly as opposed to fragmentedmuscle fibres (indicated by arrows) in the CG2135−/− fly.(C-H) TUNEL staining (red) of the thoracic muscles, showingwidespread apoptosis in the 30-day-old CG2135−/− fly. The totalnumber of cells is represented by DAPI (blue). (I) Bar graphshowing that the percentage of TUNEL-positive (apoptotic) cellsin the muscle of the CG2135−/− fly is significantly higher than inthe WT fly. Error bars represent the s.e.m. of values from fourindependent experiments. **P≤0.01.

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Nevertheless, our results provide a strong impetus for further studies toexplore the possible use of resveratrol as an alternative treatmentstrategy for MPS VII.Apart from being a convenient drug screening platform, the

CG2135−/− fly may also be utilized for large-scale geneticscreenings leading to identification of modifiers of the diseasephenotype (St Johnston, 2002). Such carefully designed screenshave earlier led to the identification of genetic suppressors ofpolyglutamine toxicity in flies (Kazemi-Esfarjani and Benzer,2000). Therefore, this novel model of MPS VII, combined with thepower of fly genetics, holds the key to deeper exploration of thedisease mechanism and drug discovery. The findings derived fromthis model may also have broader implications in understanding andmanaging other closely related MPS disorders.

MATERIALS AND METHODSUnless specified otherwise, all reagents were from Sigma-Aldrich. Detailsof all the primers (IDT) are provided in Table S1.

Drosophila strains and maintenancew1118, actin 5C Gal4, FLP, I-SceI and balancer flies were all obtained fromthe Bloomington Drosophila Stock Center at Indiana University, USA. Flieswere maintained at 25°C, at controlled density, with 12 h day-night cycle ona standard cornmeal agar medium. Wherever indicated, 400 µM resveratrol(Calbiochem) was added in the fly food.

Cell cultureS2 cells (kindly provided by Dr Sankar Maiti, IISER Kolkata, India) werecultured at 28°C in Schneider’s medium supplemented with 10% fetalbovine serum (FBS; Gibco), 2 mM L-glutamine, 100 U/ml penicillin and100 μg/ml streptomycin.

β-GUS assayAsdescribed previously, β-GUS activitywasmeasured fluorometrically using4-methylumbelliferyl β-D-glucuronide as substrate (Glaser and Sly, 1973).

Briefly, tissues/cells were lysed by homogenization/sonication and the assaywas performed at 37°C for varying times using 100 µl of the substrate solutioncontaining 10 mM 4-methylumbelliferyl β-D-glucuronide and 1 mg/ml BSAin 0.1 M acetate buffer, pH 4.8. The reaction was stopped by adding glycine-carbonate buffer, pH 10.5, following which the product was quantifiedfluorometrically with respect to a standard curve generated using knownconcentrations of 4-methylumbelliferone. The β-GUS activity wasnormalized to protein concentration, determined by Lowry’s method(Lowry et al., 1951).

RNA isolation and RT-PCRTotal RNA from tissues was isolated using TRIzol reagent (Thermo FisherScientific). DNA contaminants were removed by DNase I (Invitrogen)treatment. DNA-free RNA (1 µg) was reverse transcribed using a cDNAsynthesis kit (Invitrogen). The cDNA was used as a template for the PCRreactions with specific primers.

Cloning, expression and protein purificationCoding regions of the CG2135 and CG15117 genes were PCR amplifiedfrom thewhole fly cDNA using gene-specific primer sets P7/P8 and P9/P10,and cloned in the pMT-puro vector (Addgene). Designing of the reverseprimers ensured in-frame addition of a hexa-histidine coding sequence at theC-terminal end of the cDNAs. The clones were verified by sequencing,following which the constructs were transfected in S2 cells by calciumphosphate method (Sambrook and Russell, 2016). Stably transfected S2cells were selected in puromycin-containing medium. For protein induction,the cells were grown for 96 h in serum-free medium in the presence of500 µM CuSO4. Since a substantial amount of the overexpressed proteinwas secreted out of the cell (as indicated by β-GUS assay), a total of 300 mlof the conditioned media was collected in batches and used for proteinpurification. The media was subjected to ammonium sulfate precipitation inthree stages: 0-30%, 30-65% and 65-80%. For both CG2135 and CG15117,maximum β-GUS activity was detected in the 30-65% pellet fraction, whichwas dissolved in a minimum volume of 50 mM sodium phosphate buffer(pH 8.0) containing 10 mM imidazole and 300 mM sodium chloride. Thesolutions were extensively dialyzed against the same buffer and therecombinant enzymes were purified to homogeneity by Ni-NTA (Qiagen)

Fig. 6. Attenuation of neuromuscular degeneration and locomotor deficits in the CG2135−/− fly by resveratrol treatment. (A-C) Immunostaining of the 30-day-old fly brainswith anti-tyrosine-hydroxylase antibody (green), showing the numberof dopaminergic neurons in 30-day-oldWTandCG2135−/− flies fed on normaldiet as opposed to the age-matched CG2135−/− fly fed with resveratrol. Enlarged view of a dopaminergic neuron cell body from the boxed regions of thebrain is provided in the insets. (D) Bar graph represents the number of dopaminergic neuron cell bodies in the fly brains. (E-J) TUNEL staining (red) of the thoracicmuscles, showing widespreadmyocyte apoptosis in theCG2135−/− fly but not in theWT fly or resveratrol-fedCG2135−/− fly. The total number of nuclei is marked byDAPI staining (blue). (K) Percentage of TUNEL-positive nuclei (apoptotic cells) in the 30-day-old WT and the CG2135−/− fly fed on normal diet as opposed tothe age-matchedCG2135−/− fly fedwith resveratrol. (L) Climbing index of 30-day-oldWTand theCG2135−/− flies fed on normal diet as opposed to the age-matchedCG2135−/− fly fed with resveratrol (N∼200). Error bar represents s.e.m. of values from >three independent experiments. *P≤0.05, **P≤0.01, ***P≤0.001.

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affinity chromatography, following the manufacturer’s suggested protocol.Protein purity was confirmed by SDS-PAGE followed by silver staining.

Generation of CG2135 knockout and rescue fliesThe CG2135−/− fly was generated by ends-out gene targeting (Rong andGolic, 2000). For this, 5′ and 3′ untranslated regions (UTRs) of the CG2135gene (corresponding to the 3R:31771758-31774848 and the 3R:31768557–31771547genomic regions of the Drosophila melanogaster genome,respectively) was PCR amplified using P11/P12 and P13/P14 primer sets.The amplified 5′ and 3′ UTRs were respectively subcloned into Not1 andKpnI/BamHI restriction sites of the pw35Gal4 vector (a kind gift from DrCraig Montell, USCB, USA). The targeting construct was verified bysequencing and then used to generate a donor transgenic fly (in w1118

background) by germline transformation (embryo microinjection serviceprovided by C-CAMP, Bangalore, India). For targeting of the CG2135gene, the donor transgenic fly was crossed with the w[1118];P{ry[+t7.2]=70FLP}23 P{v[+t1.8]=70I-SceI}4A/TM3,Sb[1] fly(Bloomington stock number 6935). The larval progenies were heat-shocked at 37°C for 1 h for two subsequent days to induce FLP andI-SceI expression. The targeting construct was excised from the genome as acircular DNA by FLP recombinase, which was subsequently linearized byI-SceI endonuclease. The highly recombinogenic linearized targetingconstruct can, in principle, disrupt the CG2135 gene in somatic as well asgerm cells by replacing a 212-bp coding sequence with the targetingconstruct containing mini-white and Gal4 markers. Flies in which suchhomologous recombination has happened were selected based on a red-white mosaic eye pattern (Maggert et al., 2008). Those flies wereindividually mated to w1118; Pin/Cyo;TM2, Ubx[130] e[s]/TM6B,e[1]Tb[1] balancer fly. Red-eyed progenies were selected and backcrossed to thesame balancer fly to obtain the CG2135−/− fly. Balancer and marker fromthe second chromosome were replaced with wild chromosome by usingw1118; +/+; TM2, Ubx[130] e[s]/TM6B,e[1] Tb[1] to get pure w1118; +/+;CG2135−/− flies. For generation of the rescue fly, CG2135 cDNAwas PCRamplified using the P7/P21 primer set and subcloned into the XhoI site of thepUAST vector (DGRC). The clone was verified by sequencing. Theconstruct was microinjected in w1118 embryos and progeny was selected onthe basis of the red eye colour marker. The fly containing the construct in thesecond chromosome was selected by chromosomal mapping. The actin 5CGal4 driver was used to drive theUAS-CG2135 in rescue flies. Genotypes ofthe knockout and rescue flies were confirmed by genomic PCR withappropriate primer sets.

Lifespan analysisTotal 120newlyeclosed flieswere used for lifespan analysis.Males and femaleswere separated and 20 flies were placed per vial. The flies were transferred tofresh vials every alternate day and the number of dead flies was recorded.Meanlifespan was calculated from the survivorship curve that represents percentsurvival with increasing age. Maximum lifespan was calculated as average ageof the top 10% long-lived flies (Lushchak et al., 2012).

Climbing assayThe climbing assay was performed as described earlier (Strauss andHeisenberg, 1993; Venkatachalam et al., 2008). For each set, 20-25 flieswere acclimatized for 24 h and then transferred to a 50 ml graduatedcylinder. The flies were gently tapped to the bottom, following which theyimmediately tend to climb up because of their negative geotactic behaviour.The climbing index was calculated as the percentage of flies that could climbup to the 25 ml mark within 15 s. Each experiment was performed severaltimes with more than 100 flies.

Egg laying and hatching assaysFreshly eclosed healthy male and female flies were kept separated for 3 daysto attain maturity, following which they were mated at a 1:1 male:femaleratio. As soon as the first batch of embryos emerged, the female flies wereplaced in separate wells with fly food. After 24 h, the flies were discardedand the number of embryos laid per day was counted. Hatching assay wasperformed in a 35 mm dish with fly food, each containing 50 freshly

collected embryos. A moist paper was kept at the side of each plate toprevent drying. The number of unhatched embryos was counted after 48 h.

Tissue processing, staining and imagingFor brain histology, the tissues were fixed in 2.5% glutaraldehyde for 4 hfollowed by post-fixation in 2% osmium tetraoxide for 2 h. The tissues werethen subjected to ethanol dehydration and embedded in Epon. Semi-thintransverse sections (0.5 µm) were prepared and were stained with 0.1%Toluidine Blue. For examination of the retinas, whole fly heads weresimilarly fixed with glutaraldehyde–osmium-tetroxide and embedded inEpon. Tangential retinal sections (0.5 µm) were stained with Toluidine Blue.The thoracic muscles were fixed with 4% paraformaldehyde overnight. Thefixed tissues were dehydrated in increasing concentrations of ethanol,infiltrated with paraffin wax, followed by embedding in paraffin moulds.Finally, thick sections (4 µm) were prepared and stained with haematoxylinand eosin. All tissue sections were analyzed by light microscopy. For whole-mount immunohistochemistry, the fly brains were dissected in S2 mediasupplemented with 15% FBS. Immediately after dissection, the tissueswere fixed in 4% paraformaldehyde for 30 min and immunostaining wasperformed as described previously (Wu and Luo, 2006). Briefly, the tissueswere incubated overnight with primary antibody at 4°C followed by washingthree times. The following primary antibodies were used at the indicateddilutions: rabbit anti-tyrosine hydroxylase (Merck Millipore; 1:400) orrabbit anti-ubiquitin (Cell Signaling; 1:200). Anti-rabbit Alexa-Fluor-488 oranti-mouse Alexa-Fluor-568 goat secondary antibodies (Molecular Probes;1:500, 3 h incubation at room temperature) were used for detection.LysoTracker Red (100 nM) and MitoTracker Red (250 nM) staining wasdone in live brain tissues following the manufacturer’s (both from ThermoFisher Scientific) instructions. After staining, the tissues were mounted inVectashield (Vector Laboratories) mounting medium and fluorescenceimaging was performed with Apotome.2 microscope (Carl Zeiss). A total of9-12 image tiles were taken using the motorized stage where images of 5slices of 0.32 μm were present in each tile. The images were reconstructedby the microscope’s own software (ZEN) and analyzed. Mean fluorescenceintensity in the Drosophilawhole-brain image was calculated using ImageJsoftware. TUNEL staining of muscle sections was performed as per themanufacturer’s (Roche) instructions.

Statistical analysisStatistical analyses were performed by paired two-tailed Student’s t-testusing GraphPad Prism. The results were expressed as the mean±s.e.m. fromat least three independent experiments. P-values ≤0.05 were considered asstatistically significant as indicated by asterisks: *P≤0.05, **P≤0.01 and***P≤0.001.

AcknowledgementsThe authors sincerely thank Raymond Meade, Ritabrata Ghosh and SomnathHalder for their expert technical assistance. The authors also thank William S. Sly,Jayasri Das Sarma, Sankar Maiti and Piyali Mukherjee for their constructivesuggestions and for providing various reagents.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: M.P., R.D.; Methodology: S.B., M.P., R.D.; Formal analysis: S.B.,M.P., R.D.; Investigation: S.B.; Resources: M.P., R.D.; Writing - original draft: R.D.;Writing - review & editing: S.B., M.P., R.D.; Project administration: R.D.; Fundingacquisition: M.P., R.D.

FundingThis research was supported by the Department of Biotechnology, Ministry ofScience and Technology, Government of India grants BT/PR6423/GBD/27/437/2012 and BT/RLF/Re-entry/34/2010 to R.D., and BT/HRD/35/02/2009 to M.P. S.B.was supported by the University Grants Commission fellowship.

Supplementary informationSupplementary information available online athttp://dmm.biologists.org/lookup/doi/10.1242/dmm.036954.supplemental

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ReferencesAuluck, P. K., Chan, H. Y. E., Trojanowski, J. Q., Lee, V. M. Y. and Bonini, N. M.(2002). Chaperone suppression of alpha -Synuclein toxicity in a Drosophila modelfor Parkinson’s disease. Science (80-.) 295, 865-868.

Bastianetto, S., Menard, C. and Quirion, R. (2015). Neuroprotective action ofresveratrol. Biochim. Biophys. Acta - Mol. Basis Dis. 1852, 1195-1201.

Baur, J. A. and Sinclair, D. A. (2006). Therapeutic potential of resveratrol: the invivo evidence. Nat. Rev. Drug Discov. 5, 493-506.

Birkenmeier, E. H., Davisson, M. T., Beamer, W. G., Ganschow, R. E., Vogler,C. A., Gwynn, B., Lyford, K. A., Maltais, L. M. and Wawrzyniak, C. J. (1989).Murine mucopolysaccharidosis type VII. Characterization of a mouse with beta-glucuronidase deficiency. J. Clin. Invest. 83, 1258-1266.

Bonini, N. M. and Fortini, M. E. (2003). Human neurodegenerative diseasemodeling using Drosophila. Annu. Rev. Neurosci. 26, 627-656.

Chazotte, B. (2011). Labeling lysosomes in live cells with LysoTracker. Cold SpringHarb. Protoc. 2011, .

Chintapalli, V. R., Wang, J. and Dow, J. A. T. (2007). Using FlyAtlas to identifybetter Drosophila melanogaster models of human disease. Nat. Genet. 39,715-720.

Dasgupta, B. and Milbrandt, J. (2007). Resveratrol stimulates AMP kinase activityin neurons. Proc. Natl. Acad. Sci. USA 104, 7217-7222.

Feany, M. B. and Bender, W. W. (2000). A Drosophila model of Parkinson’ sdisease. Nature 404, 394-398.

Fernandez-Hernandez, I., Scheenaard, E., Pollarolo, G. and Gonzalez, C.(2016). The translational relevance of Drosophila in drug discovery. EMBO Rep.17, 471-472.

Ferreira, C. R. and Gahl, W. A. (2017). Lysosomal storage diseases. Transl. Sci.Rare Dis. 2, 1-71.

Flash, T. and Hochner, B. (2005). Motor primitives in vertebrates and invertebrates.Curr. Opin. Neurobiol. 15, 660-666.

Fox, J. E., Volpe, L., Bullaro, J., Kakkis, E. D. and Sly, W. S. (2015). First humantreatment with investigational rhGUS enzyme replacement therapy in anadvanced stage MPS VII patient. Mol. Genet. Metab. 114, 203-208.

Giacomotto, J. and Segalat, L. (2010). High-throughput screening and smallanimal models, where are we? Br. J. Pharmacol. 160, 204-216.

Glaser, J. H. and Sly, W. S. (1973). Beta-glucuronidase deficiencymucopolysaccharidosis: methods for enzymatic diagnosis. J. Lab. Clin. Med.82, 969-977.

Greene, J. C., Whitworth, A. J., Kuo, I., Andrews, L. A., Feany, M. B. andPallanck, L. J. (2003). Mitochondrial pathology and apoptotic muscledegeneration in Drosophila parkin mutants. Proc. Natl. Acad. Sci. 100,4078-4083.

Grubb, J. H., Vogler, C., Levy, B., Galvin, N., Tan, Y. and Sly, W. S. (2008).Chemically modified -glucuronidase crosses blood-brain barrier and clearsneuronal storage in murine mucopolysaccharidosis VII. Proc. Natl. Acad. Sci.105, 2616-2621.

Harmatz, P., Whitley, C. B., Wang, R. Y., Bauer, M., Song, W., Haller, C. andKakkis, E. (2018). A novel Blind Start study design to investigate vestronidase alfafor mucopolysaccharidosis VII, an ultra-rare genetic disease. Mol. Genet. Metab.123, 488-494.

Hasima, N. and Ozpolat, B. (2014). Regulation of autophagy by polyphenoliccompounds as a potential therapeutic strategy for cancer. Cell Death Dis. 5,e1509-e1509.

Hassan, M. I., Waheed, A., Grubb, J. H., Klei, H. E., Korolev, S. and Sly, W. S.(2013). High resolution crystal structure of human β-glucuronidase revealsstructural basis of lysosome targeting. PLoS One 8, e79687.

Heuer, G. G., Passini, M. A., Jiang, K., Parente, M. K., Lee, V. M.-Y., Trojanowski,J. Q. and Wolfe, J. H. (2002). Selective neurodegeneration in murinemucopolysaccharidosis VII is progressive and reversible. Ann. Neurol. 52,762-770.

Hori, Y. S., Kuno, A., Hosoda, R., Tanno,M., Miura, T., Shimamoto, K. andHorio,Y. (2011). Resveratrol ameliorates muscular pathology in the dystrophic mdxmouse, a model for duchenne muscular dystrophy. J. Pharmacol. Exp. Ther. 338,784-794.

Howitz, K. T., Bitterman, K. J., Cohen, H. Y., Lamming, D. W., Lavu, S., Wood,J. G., Zipkin, R. E., Chung, P., Kisielewski, A., Zhang, L.-L. et al. (2003). Smallmolecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature425, 191-196.

Huang, X. (2005). A Drosophila model of the Niemann-Pick type C lysosomestorage disease: dnpc1a is required for molting and sterol homeostasis.Development 132, 5115-5124.

Iijima, K., Liu, H. P., Chiang, A.-S., Hearn, S. A., Konsolaki, M. and Zhong, Y.(2004). Dissecting the pathological effects of human A beta 40 and A beta 42 inDrosophila: a potential model for Alzheimer’s disease. Proc. Natl. Acad. Sci. USA101, 6623-6628.

Irani, D., Kim, H.-S., El-Hibri, H., Dutton, R. V., Beaudet, A. and Armstrong, D.(1983). Postmortem observations on beta-glucuronidase deficiency presenting ashydrops fetalis. Ann. Neurol. 14, 486-490.

Islam, M. R., Tomatsu, S., Shah, G. N., Grubb, J. H., Jain, S. and Sly, W. S.(1999). Active site residues of human beta-glucuronidase. Evidence for Glu(540)

as the nucleophile and Glu(451) as the acid-base residue. J. Biol. Chem. 274,23451-23455.

Jang, M., Cai, L., Udeani, G. O., Slowing, K. V., Thomas, C. F., Beecher, C. W.,Fong, H. H., Farnsworth, N. R., Kinghorn, A. D., Mehta, R. G. et al. (1997).Cancer chemopreventive activity of resveratrol, a natural product derived fromgrapes. Science 275, 218-220.

Kazemi-Esfarjani, P. andBenzer, S. (2000). Genetic suppression of polyglutaminetoxicity in Drosophila. Science 287, 1837-1840.

Kim, D., Nguyen, M. D., Dobbin, M. M., Fischer, A., Sananbenesi, F., Rodgers,J. T., Delalle, I., Baur, J. A., Sui, G., Armour, S. M. et al. (2007). SIRT1deacetylase protects against neurodegeneration in models for Alzheimer’sdisease and amyotrophic lateral sclerosis. EMBO J. 26, 3169-3179.

Kinghorn, K. J., Gronke, S., Castillo-Quan, J. I., Woodling, N. S., Li, L., Sirka, E.,Gegg, M., Mills, K., Hardy, J., Bjedov, I. et al. (2016). A Drosophila model ofneuronopathic gaucher disease demonstrates lysosomal-autophagic defects andaltered mTOR signalling and is functionally rescued by rapamycin. J. Neurosci.36, 11654-11670.

Kornfeld, S. (1987). Trafficking of lysosomal enzymes. FASEB J. 1, 462-468.Lagouge, M., Argmann, C., Gerhart-Hines, Z., Meziane, H., Lerin, C., Daussin,

F., Messadeq, N., Milne, J., Lambert, P., Elliott, P. et al. (2006). Resveratrolimproves mitochondrial function and protects against metabolic disease byactivating SIRT1 and PGC-1α. Cell 127, 1109-1122.

Langley, S. D., Wilson, S. D., Gross, A. S., Warner, C. K. and Finnerty, V. (1983).A genetic variant of beta-glucuronidase in Drosophila melanogaster. J. Biol.Chem. 258, 7416-7424.

Lazarus, H. S., Sly, W. S., Kyle, J. W. and Hageman, G. S. (1993). Photoreceptordegeneration and altered distribution of interphotoreceptor matrix proteoglycansin the Mucopolysaccharidosis VII mouse. Exp. Eye Res. 56, 531-541.

Levy, B., Galvin, N., Vogler, C., Birkenmeier, E. H. and Sly, W. S. (1996).Neuropathology of murine mucopolysaccharidosis type VII. Acta Neuropathol. 92,562-568.

Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Proteinmeasurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.

Lushchak, O. V., Gospodaryov, D. V., Rovenko, B. M., Glovyak, A. D.,Yurkevych, I. S., Klyuba, V. P., Shcherbij, M. V. and Lushchak, V. I. (2012).Balance between macronutrients affects life span and functional senescence infruit fly Drosophila melanogaster. Journals Gerontol. Ser. A Biol. Sci. Med. Sci.67A, 118-125.

Maggert, K. A., Gong, W. J. and Golic, K. G. (2008). Methods for homologousrecombination in Drosophila. In Methods in Molecular Biology (ed. C. Dahmann),vol 420. Humana Press.

Mao, Z. and Davis, R. L. (2009). Eight different types of dopaminergic neuronsinnervate the Drosophila mushroom body neuropil: anatomical and physiologicalheterogeneity. Front. Neural Circuits 3, 5.

Montan o, A. M., Lock-Hock, N., Steiner, R. D., Graham, B. H., Szlago, M.,Greenstein, R., Pineda, M., Gonzalez-Meneses, A., Çoker, M., Bartholomew,D. et al. (2016). Clinical course of sly syndrome (mucopolysaccharidosis type VII).J. Med. Genet. 53, 403-418.

Murphy, K. E., Karaconji, T., Hardman, C. D. and Halliday, G. M. (2008).Excessive dopamine neuron loss in progressive supranuclear palsy.Mov. Disord.23, 607-610.

Neufeld, E. F. and Muenzer, J. (2014). The mucopolysaccharidoses. In The OnlineMetabolic and Molecular Bases of Inherited Disease, (eds. D. Valle, A. L.Beaudet, B. Vogelstein, K. W. Kinzler, S. E. Antonarakis, A. Ballabio, K. Gibson,G. Mitchell). New York, NY: McGraw-Hill.

Okawara, M., Katsuki, H., Kurimoto, E., Shibata, H., Kume, T. and Akaike, A.(2007). Resveratrol protects dopaminergic neurons in midbrain slice culture frommultiple insults. Biochem. Pharmacol. 73, 550-560.

Osellame, L. D. and Duchen, M. R. (2014). Quality control gone wrong:mitochondria, lysosomal storage disorders and neurodegeneration.Br. J. Pharmacol. 171, 1958-1972.

Parker, J. A., Arango, M., Abderrahmane, S., Lambert, E., Tourette, C., Catoire,H. and Neri, C. (2005). Resveratrol rescues mutant polyglutamine cytotoxicity innematode and mammalian neurons. Nat. Genet. 37, 349-350.

Platt, F. M., Boland, B. and van der Spoel, A. C. (2012). The cell biology ofdisease: lysosomal storage disorders: the cellular impact of lysosomaldysfunction. J. Cell Biol. 199, 723-734.

Richard, M., Arfi, A., Rhinn, H., Gandolphe, C. and Scherman, D. (2008).Identification of new markers for neurodegeneration process in the mouse modelof sly disease as revealed by expression profiling of selected genes. J. Neurosci.Res. 86, 3285-3294.

Riemensperger, T., Issa, A.-R., Pech, U., Coulom, H., Nguyên, M.-V., Cassar, M.,Jacquet, M., Fiala, A. and Birman, S. (2013). A single dopamine pathwayunderlies progressive locomotor deficits in a Drosophila model of Parkinsondisease. Cell Rep. 5, 952-960.

Rong, Y. S. and Golic, K. G. (2000). Gene targeting by homologous recombinationin Drosophila. Science 288, 2013-2018.

Sambrook, J. and Russell, D. W. (2006). Calcium-phosphate-mediatedtransfection of eukaryotic cells with plasmid DNAs. Cold Spring Harb. Protoc.2006, pdb.prot3871.

9

RESEARCH ARTICLE Disease Models & Mechanisms (2018) 11, dmm036954. doi:10.1242/dmm.036954

Disea

seModels&Mechan

isms

Sands, M. S. and Birkenmeier, E. H. (1993). A single-base-pair deletion in thebeta-glucuronidase gene accounts for the phenotype of murinemucopolysaccharidosis type VII. Proc. Natl. Acad. Sci. USA 90, 6567-6571.

Sewell, A. C., Gehler, J., Mittermaier, G. and Meyer, E. (1982).Mucopolysaccharidosis type VII (beta-glucuronidase deficiency): a report of anew case and a survey of those in the literature. Clin. Genet. 21, 366-373.

Sly, W. S., Quinton, B. A., McAlister, W. H. and Rimoin, D. L. (1973). Betaglucuronidase deficiency: report of clinical, radiologic, and biochemical features ofa new mucopolysaccharidosis. J. Pediatr. 82, 249-257.

Soper, B. W., Pung, A. W., Vogler, C. A., Grubb, J. H., Sly,W. S. and Barker, J. E.(1999). Enzyme replacement therapy improves reproductive performance inmucopolysaccharidosis Type VII Mice but does not prevent postnatal losses.Pediatr. Res. 45, 180-186.

St Johnston, D. (2002). The art and design of genetic screens: Drosophilamelanogaster. Nat. Rev. Genet. 3, 176-188.

Strauss, R. (2002). The central complex and the genetic dissection of locomotorbehaviour. Curr. Opin. Neurobiol. 12, 633-638.

Strauss, R. and Heisenberg, M. (1993). A higher control center of locomotorbehavior in the Drosophila brain. J. Neurosci. 13, 1852-1861.

Tomatsu, S., Montan o, A. M., Dung, V. C., Grubb, J. H. and Sly, W. S. (2009).Mutations and polymorphisms in GUSB gene in mucopolysaccharidosis VII (SlySyndrome). Hum. Mutat. 30, 511-519.

Valenzano, D. R., Terzibasi, E., Genade, T., Cattaneo, A., Domenici, L. andCellerino, A. (2006). Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate. Curr. Biol. 16, 296-300.

Venkatachalam, K., Long, A. A., Elsaesser, R., Nikolaeva, D., Broadie, K. andMontell, C. (2008). Motor deficit in a Drosophila model of mucolipidosis Type IVdue to defective clearance of apoptotic cells. Cell 135, 838-851.

Vogler, C., Levy, B., Kyle, J. W., Sly, W. S., Williamson, J. and Whyte, M. P.(1994). Mucopolysaccharidosis VII: postmortem biochemical and pathologicalfindings in a young adult with beta-glucuronidase deficiency. Mod. Pathol. 7,132-137.

Wood, J. G., Rogina, B., Lavu, S., Howitz, K., Helfand, S. L., Tatar, M. andSinclair, D. (2004). Sirtuin activators mimic caloric restriction and delay ageing inmetazoans. Nature 430, 686-689.

Wu, J. S. and Luo, L. (2006). A protocol for dissecting Drosophila melanogasterbrains for live imaging or immunostaining. Nat. Protoc. 1, 2110-2115.

Zhang, F., Shi, J.-S., Zhou, H., Wilson, B., Hong, J.-S. and Gao, H.-M. (2010).Resveratrol protects Dopamine neurons against lipopolysaccharide-inducedneurotoxicity through its anti-inflammatory actions. Mol. Pharmacol. 78, 466-477.

Zhou, Q.-Y. and Palmiter, R. D. (1995). Dopamine-deficient mice are severelyhypoactive, adipsic, and aphagic. Cell 83, 1197-1209.

Zielonka, M., Garbade, S. F., Kolker, S., Hoffmann, G. F. and Ries, M. (2017).Quantitative clinical characteristics of 53 patients with MPS VII: a cross-sectionalanalysis. Genet. Med. 19, 983-988.

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