RESEARCH ARTICLE

Neurodegeneration and locomotor dysfunction in Drosophilascarlet mutantsPatrick C. Cunningham1, Katherine Waldeck2, Barry Ganetzky2 and Daniel T. Babcock1,*

ABSTRACTParkinson’s disease (PD) is characterized by the loss of dopaminergicneurons, resulting in progressive locomotor dysfunction. Identificationof genes required for the maintenance of these neurons should help toidentify potential therapeutic targets. However, little is known regardingthe factors that render dopaminergic neurons selectively vulnerable toPD. Here, we show that Drosophila melanogaster scarlet mutantsexhibit an age-dependent progressive loss of dopaminergic neurons,along with subsequent locomotor defects and a shortened lifespan.Knockdown of Scarlet specifically within dopaminergic neurons issufficient to produce this neurodegeneration, demonstrating a uniquerole for Scarlet beyond its well-characterized role in eye pigmentation.Both genetic and pharmacological manipulation of the kynureninepathway rescued loss of dopaminergic neurons by promoting synthesisof the free radical scavenger kynurenic acid (KYNA) and limitingthe production of the free radical generator 3-hydroxykynurenine(3-HK). Finally, we show that expression of wild-type Scarlet isneuroprotective in a model of PD, suggesting that manipulatingkynurenine metabolism may be a potential therapeutic option intreating PD.

This article has an associated First Person interview with the firstauthor of the paper.

KEY WORDS: Dopaminergic neuron, Parkinson’s disease,Kynurenine

INTRODUCTIONMost neurodegenerative diseases are characterized by the loss ofselectively vulnerable populations of neurons in the central nervoussystem. The neurons rendered most vulnerable in Parkinson’sdisease (PD) are dopaminergic neurons in the substantia nigra.However, the factors that render these neurons particularlyvulnerable in this disease are poorly understood. To betterunderstand the genes responsible for this selective vulnerability,we conducted a screen to identify mutants that display a progressiveloss of dopaminergic neurons.Drosophila models of PD have proven to be useful for

uncovering the cellular and molecular mechanisms responsible forthe loss of dopaminergic neurons (Feany and Bender, 2000). Theseinclude studies involving transgenic expression of α-Synuclein(Auluck et al., 2002), Leucine-rich repeat kinase 2 (LRRK2) (Liu

et al., 2008), and mutations in PINK1 and parkin (Clark et al., 2006;Park et al., 2006), both of which are well conserved in Drosophila.Dopaminergic neurons within the Drosophila brain are organizedinto eight well-defined clusters (Mao and Davis, 2009), allowing foreasy identification and quantification of neuronal loss (Barone andBohmann, 2013). Moreover, the progressive loss of dopaminergicneurons results in defects in locomotor function, analogous towhat is seen in PD patients (Babcock et al., 2015; Feany andBender, 2000).

Previous studies have demonstrated that Drosophila eye colormutants display a variety of phenotypes that are independent ofeye pigmentation. For example, both deep-orange and carnationmutants are defective in late endosomal biogenesis, lysosomaldelivery, SNARE-mediated trafficking to lysosomes or programmedautophagy (Akbar et al., 2009; Lindmo et al., 2006; Sevrioukov et al.,1999; Sriram et al., 2003). It has also been shown that white andbrown mutants enhance the severity of neurodegeneration in atauopathy model (Ambegaokar and Jackson, 2010). White mutantsalso show courtship behavioral changes with higher sexual arousal inmales (Krstic et al., 2013). The cardinal mutant is associatedwith neurodegeneration leading to age-dependent memory loss andsynaptic pathology (Savvateeva et al., 2000). Finally, the mutationscinnabar and vermilion are protective in a model of Huntington’sdisease (Campesan et al., 2011).

Here, we demonstrate the neurodegeneration of dopaminergicneurons in scarlet mutants. Scarlet encodes an ABC transporter(Ewart andHowells, 1998) responsible for the transport ofmetabolitessuch as 3-hydroxykynurenine (3-HK) across the membrane ofpigment granules, leading to the development of brown eyepigments (Mackenzie et al., 2000). Scarlet mutants, which aredefective in this transport, exhibit a bright red eye color (Mackenzieet al., 2000). We show that scarletmutants display locomotor defectsas well as a shortened lifespan. We also show that manipulation ofthe kynurenine pathway can rescue this neurodegeneration. Finally,we describe a neuroprotective role of the Scarlet protein that ismediated via inhibiting α-Synuclein-mediated toxicity in a PDmodel.

RESULTSProgressive loss of dopaminergic neurons in scarletmutantsTo identify novel genes associated with degeneration ofdopaminergic neurons, we screened through a collection ofmutants previously identified as having progressive vacuolarpathology in the brain (Palladino et al., 2002). We specificallyexamined degeneration of dopaminergic neurons located in theprotocerebral posterior lateral 1 (PPL1) cluster within the centralbrain. In parkin mutants there is significant neurodegenerationwithin the PPL1 cluster that is not observed in other DA neuronclusters (Whitworth et al., 2005). In wild-type (WT) flies there arebetween 12±0.3 PPL1 neurons (mean±s.e.m.) per cluster (Fig. 1A).We identified several uncharacterized mutants, referred to asM1–M4 that displayed progressive loss of dopaminergic neurons.Received 19 February 2018; Accepted 14 August 2018

1Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015,USA. 2Laboratory of Genetics, University of Wisconsin-Madison, Madison,WI 53706, USA.

*Author for correspondence (dab416@lehigh.edu)

D.T.B., 0000-0002-8102-9133

1

© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs216697. doi:10.1242/jcs.216697

Journal

ofCe

llScience

http://jcs.biologists.org/lookup/doi/10.1242/jcs.224295http://jcs.biologists.org/lookup/doi/10.1242/jcs.224295mailto:dab416@lehigh.eduhttp://orcid.org/0000-0002-8102-9133

When raised at 29°C, these mutants lost nearly one third of theirPPL1 neurons by day 21 (Fig. 1B). Because all of these mutantshad a scarlet phenotype, we tested whether the scarlet mutationitself is responsible for this neurodegeneration. Interestingly, wefound that st1 flies also had a loss of dopaminergic neurons(Fig. 1B), demonstrating that a scarlet mutation itself promotesdegeneration of dopaminergic neurons. Lines M1, M2, M3 andM4 also failed to compliment the st1 allele, suggesting thatthis loss of dopaminergic neurons is due to the scarlet mutation(Fig. S1A).To test whether the decreased number of neurons is due to

neurodegeneration rather than improper development, we alsoanalyzed dopaminergic neurons in these mutants at an earlier age.On day 3, scarlet flies showed no significant difference in thenumber of dopaminergic neurons compared to WT controls(Fig. 1C–G). By day 21, however, this number had decreased toan average of 8.5 neurons per cluster. This result suggests that thereis an age-dependent progressive loss of dopaminergic neurons inscarlet mutants. To distinguish between a loss of dopaminergic

neurons versus lower levels of tyrosine hydroxylase, we droveexpression of a nuclear red fluorescent protein (UAS-RedStinger) indopaminergic neurons using the TH-Gal4 driver. We used thesetransgenes to determine the number of PPL1 neurons in a WT andscarlet mutant background. We found a progressive loss ofRedStinger+ neurons over time in scarlet mutants compared tocontrols (Fig. S1B–F). These results suggest that there is aprogressive loss of dopaminergic neurons in scarlet mutants.Dopaminergic neuron loss appears to be a temperature-sensitivephenotype of st1 mutants, as flies raised at 25° do not losedopaminergic neurons, even by day 42 (Fig. S1G).

To verify that st1 is responsible for the loss of dopaminergicneurons, we also examined st1 heterozygous with a deficiency thatuncovers this locus (Ryder et al., 2007), and found that these fliesalso exhibited loss of dopaminergic neurons (Fig. 1H–J). Theseflies had difficulty living to day 21 and displayed an earlier onsetof neurodegeneration by day 18. These results demonstrate thatloss of scarlet function is sufficient to promote degeneration ofdopaminergic neurons.

Fig. 1. Progressive loss of dopaminergic neurons in scarlet mutants. (A) Diagram illustrating the location of dopaminergic neurons in the posteriorregion of the brain. Protocerebral posterior lateral 1 (PPL1) clusters are outlined. (B) Comparing the number of PPL1 dopaminergic neurons in WT flies tothose in scarletmutants as well as other mutants generated in a scarlet background. (C) Number of dopaminergic neurons in PPL1 clusters in bothWTand scarletmutant flies at either 3 or 21 days when raised at 29°C. (D–G) Representative images of PPL1 clusters of dopaminergic neurons for control and scarlet mutantsat day 3 (D3) and day 21 (D21). (H–I) Representative images of PPL1 clusters of dopaminergic neurons for a scarlet mutant heterozygous with a deficiency[Df; DF(3L)ED4606] at day 3 and day 21. (J) Number of dopaminergic neurons per PPL1 cluster in control, scarlet mutants and scarlet mutant heterozygouswith a deficiency. Dopaminergic neurons are stained with anti-tyrosine hydroxylase. Black bars in B, C, and J represent the mean values for each condition.***P

Scarlet flies display a shortened lifespan and locomotordysfunctionTo further characterize the phenotype of scarlet mutants, we alsoperformed lifespan analysis. We found that WT flies have amedian lifespan of ∼40 days when maintained at 29°. By contrast,scarlet flies have a lifespan that is 30% shorter, with a medianlifespan of 27 days (Fig. 2A). Scarlet mutants over the deficiencyhave an even shorter median lifespan of 17 days (Fig. S2B).Because loss of dopaminergic neurons is often associated withlocomotor defects (Feany and Bender, 2000), we examinedwhether scarlet flies also display motor impairment in a climbingassay (Barone and Bohmann, 2013). WT flies are very active atday 4 and only slightly less so by day 18. In contrast, scarlet fliesperformed fairly well on day 4, but showed a substantial decreasein climbing ability by day 11 with an even further decrease byday 18 (Fig. 2B). The climbing defect of scarlet over deficiencyflies was even more severe, emerging as soon as day 4 (Fig. 2B).These results suggest that the loss of dopaminergic neurons inscarlet mutants is associated with locomotor defects and ashortened lifespan.

Other eye color mutants do not show loss of dopaminergicneuronsRecent studies have demonstrated that several eye color mutantsare involved in processes unrelated to eye pigmentation (Akbaret al., 2009; Campesan et al., 2011; Krstic et al., 2013; Lindmo et al.,2006; Savvateeva et al., 2000; Sevrioukov et al., 1999; Sriram et al.,2003). To test whether the loss of dopaminergic neurons is specificto scarlet, we also examined other mutations affecting eye color.All eye color mutants tested, including bw1,w1118, cn1, v1, and ry506,showed a normal phenotype for dopaminergic neurons, demonstratingthat degeneration of dopaminergic neurons is specific to mutation ofscarlet (Fig. 2C).

To test whether loss of dopaminergic neurons in scarlet mutantsis cell autonomous, we knocked down expression of scarlet withinspecific cell types through RNAi (Dietzl et al., 2007). We used TH-Gal4 (Friggi-Grelin et al., 2003) to drive expression specificallywithin dopaminergic neurons, and we also used Repo-Gal4 (Seppet al., 2001) to specifically target glial cells. When scarlet wasknocked down in glial cells, we observed no change in the numberof dopaminergic neurons (Fig. 2D,F–H). However, when scarlet

Fig. 2. Shortened lifespan and locomotor dysfunction in scarlet mutants. (A) Lifespan analysis of both control (WT) and scarlet mutant flies maintainedat 29°C. (B) Climbing index of control, scarlet mutants, scarlet heterozygous with a deficiency (Df ), and scarlet mutants expressing the Scarlet transgene(TH>St::venus) in dopaminergic neurons maintained at 29°C. Error bars represent s.e.m. (C) Number of dopaminergic neurons in PPL1 clusters in controlflies and in various eye color mutants. Flies were maintained for 21 days at 29°C. (D–J) Number of dopaminergic neurons in PPL1 clusters with representativeimages for control and RNAi knockdown of Scarlet in dopaminergic neurons using TH-Gal4 (TH>stIR) and in glial cells using Repo-Gal4 (Repo>stIR) alongwith scarlet mutant flies expressing the Scarlet transgene in dopaminergic neurons. Flies were raised for 21 days at 29°C. Black bars in C and D representthe mean values for each genotype. *P

was knocked down in dopaminergic neurons, we observed a loss ofneurons in the PPL1 cluster (Fig. 2D,E,G,I). These results suggestthat scarlet is working cell autonomously in the degeneration ofdopaminergic neurons. To determine whether expression of Scarletspecifically in dopaminergic neurons can rescue the degenerationseen in scarlet mutants, we used TH-Gal4 to drive expression ofUAS-Scarlet::venus in a scarlet mutant background as well asin controls. We found that expression of the Scarlet transgene indopaminergic neurons was sufficient to rescue neuronal loss inscarlet mutants (Fig. 2D,J). We also found that dopaminergicneuron expression of Scarlet rescues the progressive climbing defectin scarlet mutants (Fig. 2B), suggesting that dopaminergic neuronexpression of Scarlet is necessary and sufficient for theseconditions. Interestingly, expression of the Scarlet transgene indopaminergic neurons did not have a significant impact on lifespan(Fig. S2B), suggesting that the role of Scarlet in longevity requiresmore than expression in dopaminergic neurons.

Manipulation of the kynurenine pathway reveals aneuroprotective function of kynurenic acid in scarletmutant fliesPrevious studies have shown that Drosophila eye color mutationscan modify the outcomes in various models of neurodegenerativediseases. For example, white and brown mutations exacerbateneurodegeneration seen in a Tau model (Ambegaokar and Jackson,2010). More recently, mutation of eye color proteins that act asenzymes in kynurenine metabolism have been shown to be protectivein a model of Huntington’s disease (Campesan et al., 2011).

Manipulation of the kynurenine metabolic pathway can result inthe accumulation of metabolites such as kynurenic acid (KYNA), afree radical scavenger (Lugo-Huitron et al., 2011), as well as3-hydroxykynurenine (3-HK), a free radical generator (Wang et al.,2012b) (Fig. 3A). The kynurenine pathway is well conservedbetween humans and invertebrates, and the oxidative stress 3-HKimposes, specifically on mitochondria, is involved in numerousneurodegenerative diseases (Sas et al., 2007; Tan et al., 2012). Forexample, PD patients show increased levels of 3-HK and lowerlevels of KYNA (Ogawa et al., 1992). In mammals, 3-HK isconverted into quinolinic acid (QUIN). In PD, QUIN upregulatesNMDA-R activity in DA neurons, which causes mitochondrialoveractivation leading to oxidative stress and ultimately apoptosis.KYNA is thought to be protective in PD by acting to downregulateNMDA-R activity, and therefore preventing DA neuron apoptosis(Tan et al., 2012). Increasing the levels of KYNA whilesimultaneously reducing the levels of QUIN in a rat brain hasbeen shown to be neuroprotective in a PD model (Miranda et al.,1997). InDrosophila, cinnabar encodes the homolog of kynurenine3-monooxygenase (KMO), the enzyme needed to convertkynurenine into 3-HK. The abnormal eye color in scarlet mutantsis due in large part to the inability to take up 3-HK into pigmentgranules, where it can be processed into xanthommatin, thus resultingin a buildup of 3-HK (Mackenzie et al., 2000). Consequently, weexamined whether 3-HK accumulation could be responsible for theneurodegeneration of dopaminergic neurons in scarlet.

We hypothesized that if the neurodegeneration in scarletmutantsis due to accumulation of 3-HK, then inhibiting 3-HK synthesis

Fig. 3. Altering kynurenine metabolism prevents dopaminergic neuron loss in scarlet mutants. (A) Diagram of the kynurenine pathway illustrating therelationship between kynurenine and its metabolites. Eye color mutants involved in this pathway are highlighted in red. The proposed role for Scarlet is highlighted inblue. (B–F) Number of dopaminergic neurons in PPL1 clusters in control (WT) flies, and cinnabar, scarlet, and cinnabar; scarlet double-mutant flies. Flies weremaintained for 21 days at 29°C. (G–K)Number of dopaminergic neurons in PPL1 clusters in control flies, control flies raised onKYNA-supplementedmedium, scarletflies and scarlet flies raised on KYNA-supplemented medium. Flies were maintained for 21 days at 29°C. Dopaminergic neurons are stained with anti-tyrosineantibody. Black bars in B and G represent the mean values for each condition. ***P

should rescue this phenotype. To test this hypothesis, we generatedcinnabar, scarlet (cn;st) double mutants and analyzed them for lossof dopaminergic neurons. We found that there was no loss ofdopaminergic neurons in cinnabarmutants alone. Moreover, loss ofdopaminergic neurons in scarlet mutants (Fig. 3B–F) was rescuedin cn;st double mutants, suggesting that preventing 3-HKaccumulation is neuroprotective in scarlet mutants. Interestingly,the cn1;st1 double mutant did not rescue the locomotor defects orshortened lifespan seen in st1mutants alone (Fig. S2A,B). However,these results are likely due to the fact that cn1 mutants alone havelocomotor impairments and a shortened lifespan despite the fact thatthey maintain all of the PPL1 dopaminergic neurons (Fig. S2A,B).We also tested whether pharmacological manipulation of the

kynurenine pathway could affect dopaminergic neuron loss inscarletmutants. Specifically, we tested whether increasing levels ofthe free radical scavenger KYNA would also rescue loss ofdopaminergic neurons by raising WT and scarlet flies on standardmedium or medium supplemented with KYNA (5 mg/ml) for21 days at 29°. We found that KYNA-supplemented food had noimpact on the number of dopaminergic neurons in WT flies.However, the KYNA-supplemented food significantly rescued thedopaminergic neuron loss in scarlet mutants (Fig. 3G–K). Theseresults show that both genetic and pharmacological manipulation ofthe kynurenine pathway through inhibiting 3-HK or promotingKYNA has neuroprotective effects in scarlet mutants.

Scarlet mutants show elevated levels of reactiveoxygen speciesRecent evidence has shown that defects in the kynurenine metabolicpathway are linked to elevated oxidative stress, primarily with theaccumulation of reactive oxygen species (ROS) (Ferreira et al.,2018). Because dopaminergic neurons are particularly vulnerable tooxidative stress (Szabo et al., 2011), we hypothesized that increasedROS production could explain the defects seen in scarlet mutantflies. To test this, we used a 2′,7′-dichlorofluorescein (H2DCF) dye,which is used to measure ROS production (Owusu-Ansah et al.,2008). We measured the fluorescence intensity of H2DCF in st1

mutant brains along with WT controls at 21 days of age. We foundsignificantly higher levels of ROS production in st1 flies relative tocontrols. Interestingly, we also found that cn1;st1 double mutants

had lower levels of ROS production compared to st1 mutants alone(Fig. 4A–C,E,F), suggesting that limiting 3-HK accumulationreduces oxidative stress in st1 mutants. Additionally, we found thatdopaminergic neuron-specific expression of Scarlet prevented ROSaccumulation in scarlet mutant brains (Fig. 4A–D), suggesting thatthe protective role of Scarlet involves limiting oxidative stress.

Scarlet is neuroprotective in a PD modelBecause neurodegeneration occurs in scarlet mutants, we alsoexamined whether expression of normal Scarlet protein exerts aneuroprotective function. Neurodegeneration of dopaminergicneurons is common in Drosophila models of PD. One of thesemodels involves expressing α-Synuclein in dopaminergic neuronsusing a TH-Gal4 driver, which causes a substantial loss of PPL1neurons (Babcock et al., 2015; Feany and Bender, 2000). To testwhether expression of Scarlet can rescue α-Synuclein-mediatedtoxicity in dopaminergic neurons, we generated transgenic fliesexpressing UAS-Scarlet::venus. Similar to what was observed inprevious studies (Feany andBender, 2000), we found that expressionof WT human α-Synuclein in dopaminergic neurons resulted inneurodegeneration within 21 days. We also found that expression ofA30P and A53T mutant isoforms of α-Synuclein, which areassociated with PD (Feany and Bender, 2000), also produced aloss of PPL1 neurons (Fig. 5A–I). However, when Scarlet isexpressed together with α-Synuclein, the loss of dopaminergicneurons is prevented. We found that Scarlet expression was able torescue the degeneration caused by all three versions of α-Synuclein.To test whether this rescue is due to Scarlet as opposed to adiminished Gal4 activity caused by additional UAS constructs, wealso examined dopaminergic neuron loss when expressing α-Synuclein together with UAS-mCherry. We found that expression ofScarlet in addition to UAS-mCherry did not alleviate α-Synuclein-dependent loss of dopaminergic neurons (Fig. 5A–I). These resultsdemonstrate that increased levels of Scarlet in dopaminergic neuronscan exert a neuroprotective function in a Drosophila model of PD.

α-Synuclein expression in Drosophila dopaminergic neurons isalso known to cause progressive locomotor defects (Feany andBender, 2000). To test whether expression of Scarlet indopaminergic neurons is protective against these defects, weperformed a climbing assay on flies expressing the three forms of

Fig. 4. Scarlet mutants show elevated levels of reactive oxygen species (ROS). (A) Fluorescent intensities (arbitrary units; a.u.) of control, scarlet mutant,cinnabar mutant, cn1;st1 double mutants and scarlet mutants expressing the Scarlet transgene (TH>St::venus) in dopaminergic neurons. Flies were raised for21 days at 29°C. Error bars represent s.e.m. (B–F) Representative fluorescence images of brains stained with the 2′,7′-dichlorofluorescein (H2DCF) dye.Measurements of the fluorescent intensities were taken of the protocerebral area of the brain, represented by the white outline in B. Scale bar: 20 µm (B, for B–F).Sample sizes were of n=10. ***P

α-Synuclein, with and without UAS-Scarlet expression. We foundthat, at day 4, flies expressing the α-Synuclein isoforms performedsimilarly to WT controls. However, progressive climbing defectsemerged with age for all isoforms of α-Synuclein. These climbingdefects were rescued in all cases with co-expression ofUAS-Scarlet,but not with UAS-mCherry (Fig. 5J). These results demonstrate thatScarlet has a functionally protective role in these models of PD.Interestingly, we find that expression of all forms of α-Synucleinhad no impact on lifespan despite the loss of dopaminergic neurons(Fig. S2C). This is in agreement with our above result showing thatrescue of dopaminergic neurons in scarlet mutants does not restorelifespan, but rather involves more-complex mechanisms (Fig. S2B).

DISCUSSIONWe identified scarlet as a target gene whose function is required toprevent age-dependent loss of dopaminergic neurons inDrosophila.

We found that loss of scarlet activity causes a progressive loss ofdopaminergic neurons, induces locomotor defects, shortens lifespanand functions cell autonomously within dopaminergic neurons.Additionally, we found that this neurodegeneration can be modifiedby genetically and pharmacologically manipulating levels ofmetabolites within the kynurenine pathway, and that Scarlet has aneuroprotective role in a model of PD. Future studies aimed atidentifying genes that interact with scarlet, either directly orindirectly, should further aid in understanding why dopaminergicneurons are particularly vulnerable to degeneration. Identifyingadditional genes that are required to maintain dopaminergic neuronswill help further research into therapeutic and preventativetreatments for PD patients.

Because scarlet mutants are defective in transport of 3-HKacross the pigment granule membrane within pigment cells, apossible mechanism underlying the loss of dopaminergic neurons

Fig. 5. Scarlet overexpression is neuroprotective in a model of PD. (A) Number of dopaminergic neurons in PPL1 clusters in control (TH/+) flies, fliesexpressing α-Synuclein(WT), α-Synuclein(A53T), α-Synuclein(A30P), and the three isoforms co-expressing either Scarlet protein or mCherry using TH-Gal4.Flies were maintained for 21 days at 29°C. (B–I) Representative images of PPL1 clusters of dopaminergic neurons for each genotype. Dopaminergicneurons are stained with anti-tyrosine antibody. (J) Climbing index of control flies, flies expressing α-Synuclein(WT), α-Synuclein(A53T), α-Synuclein(A30P),and the three isoforms co-expressing either Scarlet protein or mCherry using TH-Gal4. Flies were maintained at 29°. Black bars in A represent the meanvalues for each condition. Scale bar: 20 µm (B, for B–I). *P

is increasing accumulation of 3-HK relative to KYNA, leading tomore free radical generators and oxidative stress. Interestingly, theinability of 3-HK to be transported across the pigment granules isalso seen in white mutants. However, neither white nor brownmutants exhibit loss of dopaminergic neurons, suggesting that therole of scarlet in neurodegeneration does not require formation ofa complex with white as it does within pigment cells. Thisobservation may indicate that scarlet functions independentlywithin dopaminergic neurons, or perhaps in association with othercurrently unidentified proteins.Previous studies have also demonstrated that expression of

α-Synuclein in dopaminergic neurons results in oxidative stress(Trinh et al., 2008). Here, we demonstrate that dopaminergic neuron-specific expression of Scarlet prevents both α-Synuclein-mediatedtoxicity and accumulation of ROS. Thus, the protective role ofScarlet is possibly due to its role in combating oxidative stress.Drosophila eye color is commonly used as a phenotypic marker

for chromosomes due to the ease of scoring. However, alternativeeffects can arise from the use of these markers. Deep-orange,carnation, white, brown, cardinal, vermilion and cinnabarmutations have all previously been shown to exhibit a number ofphenotypes that are not associated with eye pigmentation. In thispaper, we have established that scarlet mutations directly cause theloss of dopaminergic neurons. Together, these results suggest thatcaution should be used when interpreting data using fly stocksbearing chromosomes marked with these eye color mutations. Theidentification of a neuroprotective role for Scarlet should help incharacterizing the selective vulnerability of dopaminergic neuronsin PD. ABC transporters are a large family of proteins that carry outdiverse biological functions across several species (Dean et al.,2001). These roles include an involvement in neurodegenerativediseases; for example, ABCB1 shows a decreased function in PDpatients, resulting in dysfunction of blood–brain barrier transport(Bartels et al., 2008). Although this is not a direct homolog ofscarlet, it does highlight the importance of ABC transporters inmediating neurodegeneration. Thus, investigating mechanismsuncovered here should be helpful for uncovering potentialtherapeutic targets to prevent the loss of these neurons.

MATERIALS AND METHODSFly stocks and husbandryOregon-R was used as the WT control for all experiments. The followingstocks were obtained from the Bloomington Drosophila Stock Center:Oregon-R, st1, bw1 (Srb, 1990), w1118 (Hazelrigg et al., 1984), v1 (Shapard,1960), ry506 (Gelbart et al., 1974), cn1 (Warren et al., 1996), TH-Gal4,Repo-Gal4, UAS-mCherry, UAS- α-Synuclein.WT, UAS-α-Synuclein.A53T, UAS-αSynuclein.A30P,UAS-RedStinger,Df(3L)ED4606.UAS-stIR (109793) wasprovided by the Vienna Drosophila Resource Center.M1,M2,M3, andM4were previously described (Palladino et al., 2002). UAS-Scarlet::Venus wasgenerated in the Babcock laboratory and is described below.

Creation of transgenic fliesGeneration of UAS-venus-st was achieved by cloning scarlet cDNA intopBID-UASC-VG (Addgene #35206 deposited by Brian McCabe; Wanget al., 2012a) for placement of a Venus tag at the N-terminal. Microinjectionof constructs was performed by BestGene Inc (Chino Hills, CA), with theconstruct inserted on the second chromosome at VK00002.

ImmunohistochemistryBrains were dissected and stained as previously described (Babcock et al.,2015). Briefly, brains were dissected in PBS and fixed in 4% formaldehydein PBS for 20 min at room temperature. Samples were then washed fourtimes in PBS with 0.3% Triton X-100 (PBS-T) and placed in blockingbuffer (PBS with 0.2% Triton X-100 and 0.1% normal goat serum) for 1 h at

4°C. Samples were then incubated with primary antibody for 48 h at 4°C.Samples were then washed four times in PBS-T and incubated in secondaryantibody for 2 h at room temperature. Finally, samples were washed fourtimes in PBS-T, mounted with Vectashield (Vector Laboratories), and storedat −20°. The primary antibody used was rabbit anti-tyrosine hydroxylase(1:100) (AB152, Millipore). The secondary antibody used was Alexa Fluor488 goat anti-rabbit-IgG (1:200) (Invitrogen).

Image analysisImages were captured on a Zeiss LSM 880 confocal microscope. Sequential1.5-µm optical slices were taken using a 20× objective. Brightness andcontrast were adjusted using ImageJ software (NIH) and Adobe PhotoshopCS5 (Adobe). Dopaminergic neurons were counted as previously described(Barone and Bohmann, 2013; Whitworth et al., 2005). In brief, mountedbrains were observed under a Nikon Eclipse Ni-U fluorescent microscopeand the number of PPL1 neurons in each cluster was counted. Aminimum of10 brains were used for each condition and all experiments were performedin triplicate. Analysis was performed by a researcher that was blind withrespect to the genotype.

Locomotor behaviorAdult flies were collected shortly after eclosion and separated into 10cohorts consisting of 10 flies (100 total) for each genotype. Flies weremaintained at 29°C and transferred to fresh food every 3 days. For theclimbing assay, each cohort was transferred to an empty glass cylinder(diameter, 2.5 cm; height, 20 cm), and allowed to acclimatize for 5 min. Foreach trial, flies were tapped down to the bottom of the vial, and thepercentage of flies able to cross an 8-cm mark successfully within 10 s wasrecorded as the climbing index. Five trials were performed for each cohort,with a 1-min recovery period between each trial.

LifespanAdult flies were collected shortly after eclosion and separated into 30cohorts consisting of 10 flies (300 total) for each genotype. Flies weremaintained at 29°C and transferred to fresh food every 2 days, at which timethe percentage of surviving flies was recorded.

KYNA feeding and drug treatmentStandard fly medium was prepared and 5 mg/ml KYNA (Sigma) was addedto the liquid medium as previously described (Campesan et al., 2011). Flieswere collected shortly after eclosion and placed on KYNA-supplementedfood or standard food as a control. Flies were maintained at 29°C. Flies weretransferred to fresh food every 3 days.

ROS quantificationThe dissection and staining protocol was followed for the 2′,7′-dichlorofluorescein (H2DCF) dye (Fisher) was as previously described(Owusu-Ansah et al., 2008). The H2DCF dye was reconstituted inanhydrous dimethyl sulfoxide (DMSO) (Fisher) immediately beforedissection to a 10 mM stock solution. 1 µl of the stock solution wasdissolved in 1 ml of 1× PBS for a final concentration of 10 µM, thenvortexed for 20 s. Brains were dissected and incubated in the dye for 10 minon a shaker in the dark at room temperature, then the samples went throughthree 5-min washes with 1× PBS under the same conditions. Brains weremounted in Vectashield and immediately imaged on a confocal microscope(Zeiss) at 20× magnification. Imaging analysis was conducted byquantifying the fluorescence intensity of the protocerebral area usingImageJ software (NIH).

Statistical analysisSignificance values for lifespan survival curves was analyzed using the log-rank test. Comparisons for both climbing behavior and dopaminergicneuron loss were analyzed using Student’s t-test, with Bonferronicorrections for multiple comparisons when applicable. Statistical analysiswas performed using SPSS software (IBM Corporation) and GraphPadPrism (Graphpad Software, Inc.).

Any reagents will be made available upon request.

7

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs216697. doi:10.1242/jcs.216697

Journal

ofCe

llScience

AcknowledgementsThe authors would like to thank members of the Babcock and Ganetzky laboratoriesfor helpful suggestions.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: D.T.B.; Methodology: P.C.C., K.W., D.T.B.; Validation: D.T.B.;Formal analysis: P.C.C., D.T.B.; Investigation: P.C.C., K.W., D.T.B.; Resources:B.G., D.T.B.; Writing - original draft: P.C.C., D.T.B.; Writing - review & editing: P.C.C.,B.G., D.T.B.; Visualization: P.C.C., K.W., D.T.B.; Supervision: B.G., D.T.B.; Fundingacquisition: B.G., D.T.B.

FundingThis research was supported by the National Institutes of Health (R01 NS15390to B.G.) and Lehigh University start-up funds (D.T.B.). Deposited in PMC for releaseafter 12 months.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.216697.supplemental

ReferencesAkbar, M. A., Ray, S. and Krämer, H. (2009). The SM protein Car/Vps33Aregulates SNARE-mediated trafficking to lysosomes and lysosome-relatedorganelles. Mol. Biol. Cell 20, 1705-1714.

Ambegaokar, S. S. and Jackson, G. R. (2010). Interaction between eye pigmentgenes and tau-induced neurodegeneration in Drosophila melanogaster.Genetics186, 435-442.

Auluck, P. K., Chan, H. Y., Trojanowski, J. Q., Lee, V. M. andBonini, N. M. (2002).Chaperone suppression of alpha-synuclein toxicity in a Drosophila model forParkinson’s disease. Science 295, 865-868.

Babcock, D. T., Shen, W. and Ganetzky, B. (2015). A neuroprotective function ofNSF1 sustains autophagy and lysosomal trafficking in Drosophila. Genetics199, 511-522.

Barone, M. C. and Bohmann, D. (2013). Assessing neurodegenerativephenotypes in Drosophila dopaminergic neurons by climbing assays and wholebrain immunostaining. J. Vis. Exp. 74, e50339.

Bartels, A. L., Willemsen, A. T. M., Kortekaas, R., de Jong, B. M., de Vries, R.,de Klerk, O., van Oostrom, J. C. H., Portman, A. and Leenders, K. L. (2008).Decreased blood-brain barrier P-glycoprotein function in the progression ofParkinson’s disease, PSPandMSA. J. Neural. Transm. (Vienna) 115, 1001-1009.

Campesan, S., Green, E. W., Breda, C., Sathyasaikumar, K. V.,Muchowski, P. J., Schwarcz, R., Kyriacou, C. P. and Giorgini, F. (2011). Thekynurenine pathway modulates neurodegeneration in a Drosophila model ofHuntington’s disease. Curr. Biol. 21, 961-966.

Clark, I. E., Dodson, M.W., Jiang, C., Cao, J. H., Huh, J. R., Seol, J. H., Yoo, S. J.,Hay, B. A. and Guo, M. (2006). Drosophila pink1 is required for mitochondrialfunction and interacts genetically with parkin. Nature 441, 1162-1166.

Dean, M., Rzhetsky, A. and Allikmets, R. (2001). The human ATP-bindingcassette (ABC) transporter superfamily. Genome Res. 11, 1156-1166.

Dietzl, G., Chen, D., Schnorrer, F., Su, K.-C., Barinova, Y., Fellner, M.,Gasser, B., Kinsey, K., Oppel, S., Scheiblauer, S. et al. (2007). A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.Nature 448, 151-156.

Ewart, G. D. and Howells, A. J. (1998). ABC transporters involved in transport ofeye pigment precursors in Drosophila melanogaster. Methods Enzymol.292, 213-224.

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

Ferreira, F. S., Biasibetti-Brendler, H., Pierozan, P., Schmitz, F., Berto, C. G.,Prezzi, C. A., Manfredini, V. and Wyse, A. T. S. (2018). Kynurenic acid restoresNrf2 levels and prevents quinolinic acid-induced toxicity in rat striatal slices.Mol. Neurobiol. doi:10.1007/s12035-018-1003-2

Friggi-Grelin, F., Coulom, H., Meller, M., Gomez, D., Hirsh, J. and Birman, S.(2003). Targeted gene expression in Drosophila dopaminergic cells usingregulatory sequences from tyrosine hydroxylase. J. Neurobiol. 54, 618-627.

Gelbart, W. M., McCarron, M., Pandey, J. and Chovnick, A. (1974). Genetic limitsof the xanthine dehydrogenase structural element within the rosy locus inDrosophila melanogaster. Genetics 78, 869-886.

Hazelrigg, T., Levis, R. and Rubin, G. M. (1984). Transformation of white locusDNA in drosophila: dosage compensation, zeste interaction, and position effects.Cell 36, 469-481.

Krstic, D., Boll, W. and Noll, M. (2013). Influence of the White locus on thecourtship behavior of Drosophila males. PLoS One 8, e77904.

Lindmo, K., Simonsen, A., Brech, A., Finley, K., Rusten, T. E. and Stenmark, H.(2006). A dual function for Deep orange in programmed autophagy in theDrosophila melanogaster fat body. Exp. Cell Res. 312, 2018-2027.

Liu, Z., Wang, X., Yu, Y., Li, X., Wang, T., Jiang, H., Ren, Q., Jiao, Y., Sawa, A.,Moran, T. et al. (2008). A Drosophilamodel for LRRK2-linked parkinsonism.Proc.Natl. Acad. Sci. USA 105, 2693-2698.

Lugo-Huitrón, R., Blanco-Ayala, T., Ugalde-Mun ̃iz, P., Carrillo-Mora, P.,Pedraza-Chaverrı,́ J., Silva-Adaya, D., Maldonado, P. D., Torres, I.,Pinzón, E., Ortiz-Islas, E. et al. (2011). On the antioxidant properties ofkynurenic acid: free radical scavenging activity and inhibition of oxidative stress.Neurotoxicol. Teratol. 33, 538-547.

Mackenzie, S. M., Howells, A. J., Cox, G. B. and Ewart, G. D. (2000). Sub-cellularlocalisation of the white/scarlet ABC transporter to pigment granule membraneswithin the compound eye of Drosophila melanogaster. Genetica 108, 239-252.

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.

Miranda, A. F., Boegman, R. J., Beninger, R. J. and Jhamandas, K. (1997).Protection against quinolinic acid-mediated excitotoxicity in nigrostriataldopaminergic neurons by endogenous kynurenic acid. Neuroscience 78,967-975.

Ogawa, T., Matson, W. R., Beal, M. F., Myers, R. H., Bird, E. D., Milbury, P. andSaso, S. (1992). Kynurenine pathway abnormalities in Parkinson’s disease.Neurology 42, 1702-1706.

Owusu-Ansah, E., Yavari, A., Mandal, S. and Banerjee, U. (2008). Distinctmitochondrial retrograde signals control the G1-S cell cycle checkpoint. Nat.Genet. 40, 356-361.

Palladino, M. J., Hadley, T. J. and Ganetzky, B. (2002). Temperature-sensitiveparalytic mutants are enriched for those causing neurodegeneration inDrosophila. Genetics 161, 1197-1208.

Park, J., Lee, S. B., Lee, S., Kim, Y., Song, S., Kim, S., Bae, E., Kim, J.,Shong, M., Kim, J.-M. et al. (2006). Mitochondrial dysfunction in DrosophilaPINK1 mutants is complemented by parkin. Nature 441, 1157-1161.

Ryder, E., Ashburner, M., Bautista-Llacer, R., Drummond, J., Webster, J.,Johnson, G., Morley, T., Chan, Y. S., Blows, F., Coulson, D. et al. (2007). TheDrosDel deletion collection: a Drosophila genomewide chromosomal deficiencyresource. Genetics 177, 615-629.

Sas, K., Robotka, H., Toldi, J. and Vécsei, L. (2007). Mitochondria, metabolicdisturbances, oxidative stress and the kynurenine system, with focus onneurodegenerative disorders. J. Neurol. Sci. 257, 221-239.

Savvateeva, E., Popov, A., Kamyshev, N., Bragina, J., Heisenberg, M.,Senitz, D., Kornhuber, J. and Riederer, P. (2000). Age-dependent memory loss,synaptic pathology and altered brain plasticity in the Drosophila mutant cardinalaccumulating 3-hydroxykynurenine. J. Neural. Transm. (Vienna) 107, 581-601.

Sepp, K. J., Schulte, J. and Auld, V. J. (2001). Peripheral glia direct axon guidanceacross the CNS/PNS transition zone. Dev. Biol. 238, 47-63.

Sevrioukov, E. A., He, J.-P., Moghrabi, N., Sunio, A. and Krämer, H. (1999).A role for the deep orange and carnation eye color genes in lysosomal delivery inDrosophila. Mol. Cell 4, 479-486.

Shapard, P. B. (1960). A physiological study of the vermilion eye color mutants ofDrosophila melanogaster. Genetics 45, 359-376.

Srb, A. M. (1990). G. W. Beadle. Annu. Rev. Genet. 24, 1-4.Sriram, V., Krishnan, K. S. and Mayor, S. (2003). deep-orange and carnation

define distinct stages in late endosomal biogenesis in Drosophila melanogaster.J. Cell Biol. 161, 593-607.

Szabó, N., Kincses, Z. T., Toldi, J. and Vecsei, L. (2011). Altered tryptophanmetabolism in Parkinson’s disease: a possible novel therapeutic approach.J. Neurol. Sci. 310, 256-260.

Tan, L., Yu, J.-T. and Tan, L. (2012). The kynurenine pathway in neurodegenerativediseases: mechanistic and therapeutic considerations. J. Neurol. Sci. 323, 1-8.

Trinh, K., Moore, K., Wes, P. D., Muchowski, P. J., Dey, J., Andrews, L. andPallanck, L. J. (2008). Induction of the phase II detoxification pathwaysuppresses neuron loss in Drosophila models of Parkinson’s disease.J. Neurosci. 28, 465-472.

Wang, J.-W., Beck, E. S. and McCabe, B. D. (2012a). A modular toolset forrecombination transgenesis and neurogenetic analysis of Drosophila. PLoS One7, e42102.

Wang, X.-D., Notarangelo, F. M., Wang, J.-Z. and Schwarcz, R. (2012b).Kynurenic acid and 3-hydroxykynurenine production from D-kynurenine in mice.Brain Res. 1455, 1-9.

Warren, W. D., Palmer, S. and Howells, A. J. (1996). Molecular characterization ofthe cinnabar region of Drosophila melanogaster: identification of the cinnabartranscription unit. Genetica 98, 249-262.

Whitworth, A. J., Theodore, D. A., Greene, J. C., Benes, H., Wes, P. D. andPallanck, L. J. (2005). Increased glutathione S-transferase activity rescuesdopaminergic neuron loss in a Drosophila model of Parkinson’s disease. Proc.Natl. Acad. Sci. USA 102, 8024-8029.

8

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs216697. doi:10.1242/jcs.216697

Journal

ofCe

llScience

http://jcs.biologists.org/lookup/doi/10.1242/jcs.216697.supplementalhttp://jcs.biologists.org/lookup/doi/10.1242/jcs.216697.supplementalhttp://dx.doi.org/10.1091/mbc.e08-03-0282http://dx.doi.org/10.1091/mbc.e08-03-0282http://dx.doi.org/10.1091/mbc.e08-03-0282http://dx.doi.org/10.1534/genetics.110.119545http://dx.doi.org/10.1534/genetics.110.119545http://dx.doi.org/10.1534/genetics.110.119545http://dx.doi.org/10.1126/science.1067389http://dx.doi.org/10.1126/science.1067389http://dx.doi.org/10.1126/science.1067389http://dx.doi.org/10.1534/genetics.114.172403http://dx.doi.org/10.1534/genetics.114.172403http://dx.doi.org/10.1534/genetics.114.172403http://dx.doi.org/10.3791/50339http://dx.doi.org/10.3791/50339http://dx.doi.org/10.3791/50339http://dx.doi.org/10.1007/s00702-008-0030-yhttp://dx.doi.org/10.1007/s00702-008-0030-yhttp://dx.doi.org/10.1007/s00702-008-0030-yhttp://dx.doi.org/10.1007/s00702-008-0030-yhttp://dx.doi.org/10.1016/j.cub.2011.04.028http://dx.doi.org/10.1016/j.cub.2011.04.028http://dx.doi.org/10.1016/j.cub.2011.04.028http://dx.doi.org/10.1016/j.cub.2011.04.028http://dx.doi.org/10.1038/nature04779http://dx.doi.org/10.1038/nature04779http://dx.doi.org/10.1038/nature04779http://dx.doi.org/10.1101/gr.GR-1649Rhttp://dx.doi.org/10.1101/gr.GR-1649Rhttp://dx.doi.org/10.1038/nature05954http://dx.doi.org/10.1038/nature05954http://dx.doi.org/10.1038/nature05954http://dx.doi.org/10.1038/nature05954http://dx.doi.org/10.1016/S0076-6879(98)92017-1http://dx.doi.org/10.1016/S0076-6879(98)92017-1http://dx.doi.org/10.1016/S0076-6879(98)92017-1http://dx.doi.org/10.1038/35006074http://dx.doi.org/10.1038/35006074http://dx.doi.org/10.1007/s12035-018-1003-2http://dx.doi.org/10.1007/s12035-018-1003-2http://dx.doi.org/10.1007/s12035-018-1003-2http://dx.doi.org/10.1007/s12035-018-1003-2http://dx.doi.org/10.1002/neu.10185http://dx.doi.org/10.1002/neu.10185http://dx.doi.org/10.1002/neu.10185http://dx.doi.org/10.1016/0092-8674(84)90240-Xhttp://dx.doi.org/10.1016/0092-8674(84)90240-Xhttp://dx.doi.org/10.1016/0092-8674(84)90240-Xhttp://dx.doi.org/10.1371/journal.pone.0077904http://dx.doi.org/10.1371/journal.pone.0077904http://dx.doi.org/10.1016/j.yexcr.2006.03.002http://dx.doi.org/10.1016/j.yexcr.2006.03.002http://dx.doi.org/10.1016/j.yexcr.2006.03.002http://dx.doi.org/10.1073/pnas.0708452105http://dx.doi.org/10.1073/pnas.0708452105http://dx.doi.org/10.1073/pnas.0708452105http://dx.doi.org/10.1016/j.ntt.2011.07.002http://dx.doi.org/10.1016/j.ntt.2011.07.002http://dx.doi.org/10.1016/j.ntt.2011.07.002http://dx.doi.org/10.1016/j.ntt.2011.07.002http://dx.doi.org/10.1016/j.ntt.2011.07.002http://dx.doi.org/10.1023/A:1004115718597http://dx.doi.org/10.1023/A:1004115718597http://dx.doi.org/10.1023/A:1004115718597http://dx.doi.org/10.3389/neuro.04.005.2009http://dx.doi.org/10.3389/neuro.04.005.2009http://dx.doi.org/10.3389/neuro.04.005.2009http://dx.doi.org/10.1016/S0306-4522(96)00655-0http://dx.doi.org/10.1016/S0306-4522(96)00655-0http://dx.doi.org/10.1016/S0306-4522(96)00655-0http://dx.doi.org/10.1016/S0306-4522(96)00655-0http://dx.doi.org/10.1212/WNL.42.9.1702http://dx.doi.org/10.1212/WNL.42.9.1702http://dx.doi.org/10.1212/WNL.42.9.1702http://dx.doi.org/10.1038/ng.2007.50http://dx.doi.org/10.1038/ng.2007.50http://dx.doi.org/10.1038/ng.2007.50http://dx.doi.org/10.1038/nature04788http://dx.doi.org/10.1038/nature04788http://dx.doi.org/10.1038/nature04788http://dx.doi.org/10.1534/genetics.107.076216http://dx.doi.org/10.1534/genetics.107.076216http://dx.doi.org/10.1534/genetics.107.076216http://dx.doi.org/10.1534/genetics.107.076216http://dx.doi.org/10.1016/j.jns.2007.01.033http://dx.doi.org/10.1016/j.jns.2007.01.033http://dx.doi.org/10.1016/j.jns.2007.01.033http://dx.doi.org/10.1007/s007020070080http://dx.doi.org/10.1007/s007020070080http://dx.doi.org/10.1007/s007020070080http://dx.doi.org/10.1007/s007020070080http://dx.doi.org/10.1006/dbio.2001.0411http://dx.doi.org/10.1006/dbio.2001.0411http://dx.doi.org/10.1016/S1097-2765(00)80199-9http://dx.doi.org/10.1016/S1097-2765(00)80199-9http://dx.doi.org/10.1016/S1097-2765(00)80199-9http://dx.doi.org/10.1146/annurev.ge.24.120190.000245http://dx.doi.org/10.1083/jcb.200210166http://dx.doi.org/10.1083/jcb.200210166http://dx.doi.org/10.1083/jcb.200210166http://dx.doi.org/10.1016/j.jns.2011.07.021http://dx.doi.org/10.1016/j.jns.2011.07.021http://dx.doi.org/10.1016/j.jns.2011.07.021http://dx.doi.org/10.1016/j.jns.2012.08.005http://dx.doi.org/10.1016/j.jns.2012.08.005http://dx.doi.org/10.1523/JNEUROSCI.4778-07.2008http://dx.doi.org/10.1523/JNEUROSCI.4778-07.2008http://dx.doi.org/10.1523/JNEUROSCI.4778-07.2008http://dx.doi.org/10.1523/JNEUROSCI.4778-07.2008http://dx.doi.org/10.1371/journal.pone.0042102http://dx.doi.org/10.1371/journal.pone.0042102http://dx.doi.org/10.1371/journal.pone.0042102http://dx.doi.org/10.1016/j.brainres.2012.03.026http://dx.doi.org/10.1016/j.brainres.2012.03.026http://dx.doi.org/10.1016/j.brainres.2012.03.026http://dx.doi.org/10.1007/BF00057589http://dx.doi.org/10.1007/BF00057589http://dx.doi.org/10.1007/BF00057589http://dx.doi.org/10.1073/pnas.0501078102http://dx.doi.org/10.1073/pnas.0501078102http://dx.doi.org/10.1073/pnas.0501078102http://dx.doi.org/10.1073/pnas.0501078102