ABSTRACT
Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons, resulting in progressive locomotor dysfunction. Identification of genes required for the maintenance of these neurons should help to identify potential therapeutic targets. However, little is known regarding the factors that render dopaminergic neurons selectively vulnerable to PD. Here, we show that Drosophila melanogaster scarlet mutants exhibit an age-dependent progressive loss of dopaminergic neurons, along with subsequent locomotor defects and a shortened lifespan. Knockdown of Scarlet specifically within dopaminergic neurons is sufficient to produce this neurodegeneration, demonstrating a unique role for Scarlet beyond its well-characterized role in eye pigmentation. Both genetic and pharmacological manipulation of the kynurenine pathway rescued loss of dopaminergic neurons by promoting synthesis of the free radical scavenger kynurenic acid (KYNA) and limiting the production of the free radical generator 3-hydroxykynurenine (3-HK). Finally, we show that expression of wild-type Scarlet is neuroprotective in a model of PD, suggesting that manipulating kynurenine metabolism may be a potential therapeutic option in treating PD.
This article has an associated First Person interview with the first author of the paper.
INTRODUCTION
Most neurodegenerative diseases are characterized by the loss of selectively vulnerable populations of neurons in the central nervous system. The neurons rendered most vulnerable in Parkinson's disease (PD) are dopaminergic neurons in the substantia nigra. However, the factors that render these neurons particularly vulnerable in this disease are poorly understood. To better understand the genes responsible for this selective vulnerability, we conducted a screen to identify mutants that display a progressive loss of dopaminergic neurons.
Drosophila models of PD have proven to be useful for uncovering the cellular and molecular mechanisms responsible for the loss of dopaminergic neurons (Feany and Bender, 2000). These include 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 organized into eight well-defined clusters (Mao and Davis, 2009), allowing for easy identification and quantification of neuronal loss (Barone and Bohmann, 2013). Moreover, the progressive loss of dopaminergic neurons results in defects in locomotor function, analogous to what is seen in PD patients (Babcock et al., 2015; Feany and Bender, 2000).
Previous studies have demonstrated that Drosophila eye color mutants display a variety of phenotypes that are independent of eye pigmentation. For example, both deep-orange and carnation mutants are defective in late endosomal biogenesis, lysosomal delivery, SNARE-mediated trafficking to lysosomes or programmed autophagy (Akbar et al., 2009; Lindmo et al., 2006; Sevrioukov et al., 1999; Sriram et al., 2003). It has also been shown that white and brown mutants enhance the severity of neurodegeneration in a tauopathy model (Ambegaokar and Jackson, 2010). White mutants also show courtship behavioral changes with higher sexual arousal in males (Krstic et al., 2013). The cardinal mutant is associated with neurodegeneration leading to age-dependent memory loss and synaptic pathology (Savvateeva et al., 2000). Finally, the mutations cinnabar and vermilion are protective in a model of Huntington's disease (Campesan et al., 2011).
Here, we demonstrate the neurodegeneration of dopaminergic neurons in scarlet mutants. Scarlet encodes an ABC transporter (Ewart and Howells, 1998) responsible for the transport of metabolites such as 3-hydroxykynurenine (3-HK) across the membrane of pigment granules, leading to the development of brown eye pigments (Mackenzie et al., 2000). Scarlet mutants, which are defective in this transport, exhibit a bright red eye color (Mackenzie et al., 2000). We show that scarlet mutants display locomotor defects as well as a shortened lifespan. We also show that manipulation of the kynurenine pathway can rescue this neurodegeneration. Finally, we describe a neuroprotective role of the Scarlet protein that is mediated via inhibiting α-Synuclein-mediated toxicity in a PD model.
RESULTS
Progressive loss of dopaminergic neurons in scarlet mutants
To identify novel genes associated with degeneration of dopaminergic neurons, we screened through a collection of mutants previously identified as having progressive vacuolar pathology in the brain (Palladino et al., 2002). We specifically examined degeneration of dopaminergic neurons located in the protocerebral posterior lateral 1 (PPL1) cluster within the central brain. In parkin mutants there is significant neurodegeneration within the PPL1 cluster that is not observed in other DA neuron clusters (Whitworth et al., 2005). In wild-type (WT) flies there are between 12±0.3 PPL1 neurons (mean±s.e.m.) per cluster (Fig. 1A). We identified several uncharacterized mutants, referred to as M1–M4 that displayed progressive loss of dopaminergic neurons. When raised at 29°C, these mutants lost nearly one third of their PPL1 neurons by day 21 (Fig. 1B). Because all of these mutants had a scarlet phenotype, we tested whether the scarlet mutation itself is responsible for this neurodegeneration. Interestingly, we found that st1 flies also had a loss of dopaminergic neurons (Fig. 1B), demonstrating that a scarlet mutation itself promotes degeneration of dopaminergic neurons. Lines M1, M2, M3 and M4 also failed to compliment the st1 allele, suggesting that this loss of dopaminergic neurons is due to the scarlet mutation (Fig. S1A).
Progressive loss of dopaminergic neurons in scarlet mutants. (A) Diagram illustrating the location of dopaminergic neurons in the posterior region of the brain. Protocerebral posterior lateral 1 (PPL1) clusters are outlined. (B) Comparing the number of PPL1 dopaminergic neurons in WT flies to those in scarlet mutants as well as other mutants generated in a scarlet background. (C) Number of dopaminergic neurons in PPL1 clusters in both WT and scarlet mutant 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 mutants at 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 heterozygous with 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<0.0001; NS, not significant (Student's t-test with Bonferroni correction for multiple comparisons, and log rank test). For DA neuron quantification, sample sizes were of n=10. Scale bars: 200 µm (A), 20 µm (D, for D–I).
Progressive loss of dopaminergic neurons in scarlet mutants. (A) Diagram illustrating the location of dopaminergic neurons in the posterior region of the brain. Protocerebral posterior lateral 1 (PPL1) clusters are outlined. (B) Comparing the number of PPL1 dopaminergic neurons in WT flies to those in scarlet mutants as well as other mutants generated in a scarlet background. (C) Number of dopaminergic neurons in PPL1 clusters in both WT and scarlet mutant 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 mutants at 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 heterozygous with 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<0.0001; NS, not significant (Student's t-test with Bonferroni correction for multiple comparisons, and log rank test). For DA neuron quantification, sample sizes were of n=10. Scale bars: 200 µm (A), 20 µm (D, for D–I).
To test whether the decreased number of neurons is due to neurodegeneration rather than improper development, we also analyzed dopaminergic neurons in these mutants at an earlier age. On day 3, scarlet flies showed no significant difference in the number of dopaminergic neurons compared to WT controls (Fig. 1C–G). By day 21, however, this number had decreased to an average of 8.5 neurons per cluster. This result suggests that there is an age-dependent progressive loss of dopaminergic neurons in scarlet mutants. To distinguish between a loss of dopaminergic neurons versus lower levels of tyrosine hydroxylase, we drove expression of a nuclear red fluorescent protein (UAS-RedStinger) in dopaminergic neurons using the TH-Gal4 driver. We used these transgenes to determine the number of PPL1 neurons in a WT and scarlet mutant background. We found a progressive loss of RedStinger+ neurons over time in scarlet mutants compared to controls (Fig. S1B–F). These results suggest that there is a progressive loss of dopaminergic neurons in scarlet mutants. Dopaminergic neuron loss appears to be a temperature-sensitive phenotype of st1 mutants, as flies raised at 25° do not lose dopaminergic neurons, even by day 42 (Fig. S1G).
To verify that st1 is responsible for the loss of dopaminergic neurons, we also examined st1 heterozygous with a deficiency that uncovers this locus (Ryder et al., 2007), and found that these flies also exhibited loss of dopaminergic neurons (Fig. 1H–J). These flies had difficulty living to day 21 and displayed an earlier onset of neurodegeneration by day 18. These results demonstrate that loss of scarlet function is sufficient to promote degeneration of dopaminergic neurons.
Scarlet flies display a shortened lifespan and locomotor dysfunction
To further characterize the phenotype of scarlet mutants, we also performed lifespan analysis. We found that WT flies have a median lifespan of ∼40 days when maintained at 29°. By contrast, scarlet flies have a lifespan that is 30% shorter, with a median lifespan of 27 days (Fig. 2A). Scarlet mutants over the deficiency have an even shorter median lifespan of 17 days (Fig. S2B). Because loss of dopaminergic neurons is often associated with locomotor defects (Feany and Bender, 2000), we examined whether scarlet flies also display motor impairment in a climbing assay (Barone and Bohmann, 2013). WT flies are very active at day 4 and only slightly less so by day 18. In contrast, scarlet flies performed fairly well on day 4, but showed a substantial decrease in climbing ability by day 11 with an even further decrease by day 18 (Fig. 2B). The climbing defect of scarlet over deficiency flies was even more severe, emerging as soon as day 4 (Fig. 2B). These results suggest that the loss of dopaminergic neurons in scarlet mutants is associated with locomotor defects and a shortened lifespan.
Shortened lifespan and locomotor dysfunction in scarlet mutants. (A) Lifespan analysis of both control (WT) and scarlet mutant flies maintained at 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 control flies 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 representative images for control and RNAi knockdown of Scarlet in dopaminergic neurons using TH-Gal4 (TH>stIR) and in glial cells using Repo-Gal4 (Repo>stIR) along with 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 represent the mean values for each genotype. *P<0.01; ***P<0.0001; N.S., not significant (Student's t-test with Bonferroni correction for multiple comparisons). Sample sizes for lifespan were 300 flies per genotype. Sample sizes for climbing were 100 flies per genotype. Scale bar: 20 µm (E, for E–J).
Shortened lifespan and locomotor dysfunction in scarlet mutants. (A) Lifespan analysis of both control (WT) and scarlet mutant flies maintained at 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 control flies 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 representative images for control and RNAi knockdown of Scarlet in dopaminergic neurons using TH-Gal4 (TH>stIR) and in glial cells using Repo-Gal4 (Repo>stIR) along with 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 represent the mean values for each genotype. *P<0.01; ***P<0.0001; N.S., not significant (Student's t-test with Bonferroni correction for multiple comparisons). Sample sizes for lifespan were 300 flies per genotype. Sample sizes for climbing were 100 flies per genotype. Scale bar: 20 µm (E, for E–J).
Other eye color mutants do not show loss of dopaminergic neurons
Recent studies have demonstrated that several eye color mutants are involved in processes unrelated to eye pigmentation (Akbar et 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 specific to 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, demonstrating that degeneration of dopaminergic neurons is specific to mutation of scarlet (Fig. 2C).
To test whether loss of dopaminergic neurons in scarlet mutants is cell autonomous, we knocked down expression of scarlet within specific cell types through RNAi (Dietzl et al., 2007). We used TH-Gal4 (Friggi-Grelin et al., 2003) to drive expression specifically within dopaminergic neurons, and we also used Repo-Gal4 (Sepp et al., 2001) to specifically target glial cells. When scarlet was knocked down in glial cells, we observed no change in the number of dopaminergic neurons (Fig. 2D,F–H). However, when scarlet was knocked down in dopaminergic neurons, we observed a loss of neurons in the PPL1 cluster (Fig. 2D,E,G,I). These results suggest that scarlet is working cell autonomously in the degeneration of dopaminergic neurons. To determine whether expression of Scarlet specifically in dopaminergic neurons can rescue the degeneration seen in scarlet mutants, we used TH-Gal4 to drive expression of UAS-Scarlet::venus in a scarlet mutant background as well as in controls. We found that expression of the Scarlet transgene in dopaminergic neurons was sufficient to rescue neuronal loss in scarlet mutants (Fig. 2D,J). We also found that dopaminergic neuron expression of Scarlet rescues the progressive climbing defect in scarlet mutants (Fig. 2B), suggesting that dopaminergic neuron expression of Scarlet is necessary and sufficient for these conditions. Interestingly, expression of the Scarlet transgene in dopaminergic neurons did not have a significant impact on lifespan (Fig. S2B), suggesting that the role of Scarlet in longevity requires more than expression in dopaminergic neurons.
Manipulation of the kynurenine pathway reveals a neuroprotective function of kynurenic acid in scarlet mutant flies
Previous studies have shown that Drosophila eye color mutations can modify the outcomes in various models of neurodegenerative diseases. For example, white and brown mutations exacerbate neurodegeneration seen in a Tau model (Ambegaokar and Jackson, 2010). More recently, mutation of eye color proteins that act as enzymes in kynurenine metabolism have been shown to be protective in a model of Huntington's disease (Campesan et al., 2011). Manipulation of the kynurenine metabolic pathway can result in the accumulation of metabolites such as kynurenic acid (KYNA), a free radical scavenger (Lugo-Huitron et al., 2011), as well as 3-hydroxykynurenine (3-HK), a free radical generator (Wang et al., 2012b) (Fig. 3A). The kynurenine pathway is well conserved between humans and invertebrates, and the oxidative stress 3-HK imposes, specifically on mitochondria, is involved in numerous neurodegenerative diseases (Sas et al., 2007; Tan et al., 2012). For example, PD patients show increased levels of 3-HK and lower levels of KYNA (Ogawa et al., 1992). In mammals, 3-HK is converted into quinolinic acid (QUIN). In PD, QUIN upregulates NMDA-R activity in DA neurons, which causes mitochondrial overactivation leading to oxidative stress and ultimately apoptosis. KYNA is thought to be protective in PD by acting to downregulate NMDA-R activity, and therefore preventing DA neuron apoptosis (Tan et al., 2012). Increasing the levels of KYNA while simultaneously reducing the levels of QUIN in a rat brain has been shown to be neuroprotective in a PD model (Miranda et al., 1997). In Drosophila, cinnabar encodes the homolog of kynurenine 3-monooxygenase (KMO), the enzyme needed to convert kynurenine into 3-HK. The abnormal eye color in scarlet mutants is due in large part to the inability to take up 3-HK into pigment granules, where it can be processed into xanthommatin, thus resulting in a buildup of 3-HK (Mackenzie et al., 2000). Consequently, we examined whether 3-HK accumulation could be responsible for the neurodegeneration of dopaminergic neurons in scarlet.
Altering kynurenine metabolism prevents dopaminergic neuron loss in scarlet mutants. (A) Diagram of the kynurenine pathway illustrating the relationship between kynurenine and its metabolites. Eye color mutants involved in this pathway are highlighted in red. The proposed role for Scarlet is highlighted in blue. (B–F) Number of dopaminergic neurons in PPL1 clusters in control (WT) flies, and cinnabar, scarlet, and cinnabar; scarlet double-mutant flies. Flies were maintained for 21 days at 29°C. (G–K) Number of dopaminergic neurons in PPL1 clusters in control flies, control flies raised on KYNA-supplemented medium, scarlet flies and scarlet flies raised on KYNA-supplemented medium. Flies were maintained for 21 days at 29°C. Dopaminergic neurons are stained with anti-tyrosine antibody. Black bars in B and G represent the mean values for each condition. ***P<0.0001; N.S., not significant (Student's t-test with Bonferroni correction for multiple comparisons). For DA neuron quantification, sample sizes were of n=10. Scale bars: 20 µm (C,H for C–F and H–K).
Altering kynurenine metabolism prevents dopaminergic neuron loss in scarlet mutants. (A) Diagram of the kynurenine pathway illustrating the relationship between kynurenine and its metabolites. Eye color mutants involved in this pathway are highlighted in red. The proposed role for Scarlet is highlighted in blue. (B–F) Number of dopaminergic neurons in PPL1 clusters in control (WT) flies, and cinnabar, scarlet, and cinnabar; scarlet double-mutant flies. Flies were maintained for 21 days at 29°C. (G–K) Number of dopaminergic neurons in PPL1 clusters in control flies, control flies raised on KYNA-supplemented medium, scarlet flies and scarlet flies raised on KYNA-supplemented medium. Flies were maintained for 21 days at 29°C. Dopaminergic neurons are stained with anti-tyrosine antibody. Black bars in B and G represent the mean values for each condition. ***P<0.0001; N.S., not significant (Student's t-test with Bonferroni correction for multiple comparisons). For DA neuron quantification, sample sizes were of n=10. Scale bars: 20 µm (C,H for C–F and H–K).
We hypothesized that if the neurodegeneration in scarlet mutants is due to accumulation of 3-HK, then inhibiting 3-HK synthesis should rescue this phenotype. To test this hypothesis, we generated cinnabar, scarlet (cn;st) double mutants and analyzed them for loss of dopaminergic neurons. We found that there was no loss of dopaminergic neurons in cinnabar mutants alone. Moreover, loss of dopaminergic neurons in scarlet mutants (Fig. 3B–F) was rescued in cn;st double mutants, suggesting that preventing 3-HK accumulation is neuroprotective in scarlet mutants. Interestingly, the cn1;st1 double mutant did not rescue the locomotor defects or shortened lifespan seen in st1 mutants alone (Fig. S2A,B). However, these results are likely due to the fact that cn1 mutants alone have locomotor impairments and a shortened lifespan despite the fact that they 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 in scarlet mutants. Specifically, we tested whether increasing levels of the free radical scavenger KYNA would also rescue loss of dopaminergic neurons by raising WT and scarlet flies on standard medium or medium supplemented with KYNA (5 mg/ml) for 21 days at 29°. We found that KYNA-supplemented food had no impact on the number of dopaminergic neurons in WT flies. However, the KYNA-supplemented food significantly rescued the dopaminergic neuron loss in scarlet mutants (Fig. 3G–K). These results show that both genetic and pharmacological manipulation of the kynurenine pathway through inhibiting 3-HK or promoting KYNA has neuroprotective effects in scarlet mutants.
Scarlet mutants show elevated levels of reactive oxygen species
Recent evidence has shown that defects in the kynurenine metabolic pathway are linked to elevated oxidative stress, primarily with the accumulation of reactive oxygen species (ROS) (Ferreira et al., 2018). Because dopaminergic neurons are particularly vulnerable to oxidative stress (Szabo et al., 2011), we hypothesized that increased ROS production could explain the defects seen in scarlet mutant flies. 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 found significantly higher levels of ROS production in st1 flies relative to controls. 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 accumulation reduces oxidative stress in st1 mutants. Additionally, we found that dopaminergic neuron-specific expression of Scarlet prevented ROS accumulation in scarlet mutant brains (Fig. 4A–D), suggesting that the protective role of Scarlet involves limiting oxidative stress.
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 for 21 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<0.0001 (Student's t-test with Bonferroni correction for multiple comparisons).
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 for 21 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<0.0001 (Student's t-test with Bonferroni correction for multiple comparisons).
Scarlet is neuroprotective in a PD model
Because neurodegeneration occurs in scarlet mutants, we also examined whether expression of normal Scarlet protein exerts a neuroprotective function. Neurodegeneration of dopaminergic neurons is common in Drosophila models of PD. One of these models involves expressing α-Synuclein in dopaminergic neurons using a TH-Gal4 driver, which causes a substantial loss of PPL1 neurons (Babcock et al., 2015; Feany and Bender, 2000). To test whether expression of Scarlet can rescue α-Synuclein-mediated toxicity in dopaminergic neurons, we generated transgenic flies expressing UAS-Scarlet::venus. Similar to what was observed in previous studies (Feany and Bender, 2000), we found that expression of WT human α-Synuclein in dopaminergic neurons resulted in neurodegeneration within 21 days. We also found that expression of A30P and A53T mutant isoforms of α-Synuclein, which are associated with PD (Feany and Bender, 2000), also produced a loss of PPL1 neurons (Fig. 5A–I). However, when Scarlet is expressed together with α-Synuclein, the loss of dopaminergic neurons is prevented. We found that Scarlet expression was able to rescue the degeneration caused by all three versions of α-Synuclein. To test whether this rescue is due to Scarlet as opposed to a diminished Gal4 activity caused by additional UAS constructs, we also examined dopaminergic neuron loss when expressing α-Synuclein together with UAS-mCherry. We found that expression of Scarlet in addition to UAS-mCherry did not alleviate α-Synuclein-dependent loss of dopaminergic neurons (Fig. 5A–I). These results demonstrate that increased levels of Scarlet in dopaminergic neurons can exert a neuroprotective function in a Drosophila model of PD.
Scarlet overexpression is neuroprotective in a model of PD. (A) Number of dopaminergic neurons in PPL1 clusters in control (TH/+) 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 for 21 days at 29°C. (B–I) Representative images of PPL1 clusters of dopaminergic neurons for each genotype. Dopaminergic neurons 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 mean values for each condition. Scale bar: 20 µm (B, for B–I). *P<0.05; ***P<0.0001 (Student's t-test with Bonferroni correction for multiple comparisons). Error bars represent s.e.m. For DA neuron quantification, sample sizes were of n=10. For climbing assay, n=100 flies of each genotype were assessed.
Scarlet overexpression is neuroprotective in a model of PD. (A) Number of dopaminergic neurons in PPL1 clusters in control (TH/+) 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 for 21 days at 29°C. (B–I) Representative images of PPL1 clusters of dopaminergic neurons for each genotype. Dopaminergic neurons 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 mean values for each condition. Scale bar: 20 µm (B, for B–I). *P<0.05; ***P<0.0001 (Student's t-test with Bonferroni correction for multiple comparisons). Error bars represent s.e.m. For DA neuron quantification, sample sizes were of n=10. For climbing assay, n=100 flies of each genotype were assessed.
α-Synuclein expression in Drosophila dopaminergic neurons is also known to cause progressive locomotor defects (Feany and Bender, 2000). To test whether expression of Scarlet in dopaminergic neurons is protective against these defects, we performed a climbing assay on flies expressing the three forms of α-Synuclein, with and without UAS-Scarlet expression. We found that, at day 4, flies expressing the α-Synuclein isoforms performed similarly to WT controls. However, progressive climbing defects emerged with age for all isoforms of α-Synuclein. These climbing defects were rescued in all cases with co-expression of UAS-Scarlet, but not with UAS-mCherry (Fig. 5J). These results demonstrate that Scarlet has a functionally protective role in these models of PD. Interestingly, we find that expression of all forms of α-Synuclein had no impact on lifespan despite the loss of dopaminergic neurons (Fig. S2C). This is in agreement with our above result showing that rescue of dopaminergic neurons in scarlet mutants does not restore lifespan, but rather involves more-complex mechanisms (Fig. S2B).
DISCUSSION
We identified scarlet as a target gene whose function is required to prevent age-dependent loss of dopaminergic neurons in Drosophila. We found that loss of scarlet activity causes a progressive loss of dopaminergic neurons, induces locomotor defects, shortens lifespan and functions cell autonomously within dopaminergic neurons. Additionally, we found that this neurodegeneration can be modified by genetically and pharmacologically manipulating levels of metabolites within the kynurenine pathway, and that Scarlet has a neuroprotective role in a model of PD. Future studies aimed at identifying genes that interact with scarlet, either directly or indirectly, should further aid in understanding why dopaminergic neurons are particularly vulnerable to degeneration. Identifying additional genes that are required to maintain dopaminergic neurons will help further research into therapeutic and preventative treatments for PD patients.
Because scarlet mutants are defective in transport of 3-HK across the pigment granule membrane within pigment cells, a possible mechanism underlying the loss of dopaminergic neurons is increasing accumulation of 3-HK relative to KYNA, leading to more free radical generators and oxidative stress. Interestingly, the inability of 3-HK to be transported across the pigment granules is also seen in white mutants. However, neither white nor brown mutants exhibit loss of dopaminergic neurons, suggesting that the role of scarlet in neurodegeneration does not require formation of a complex with white as it does within pigment cells. This observation may indicate that scarlet functions independently within dopaminergic neurons, or perhaps in association with other currently 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-mediated toxicity and accumulation of ROS. Thus, the protective role of Scarlet 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, alternative effects can arise from the use of these markers. Deep-orange, carnation, white, brown, cardinal, vermilion and cinnabar mutations have all previously been shown to exhibit a number of phenotypes that are not associated with eye pigmentation. In this paper, we have established that scarlet mutations directly cause the loss of dopaminergic neurons. Together, these results suggest that caution should be used when interpreting data using fly stocks bearing chromosomes marked with these eye color mutations. The identification of a neuroprotective role for Scarlet should help in characterizing the selective vulnerability of dopaminergic neurons in PD. ABC transporters are a large family of proteins that carry out diverse biological functions across several species (Dean et al., 2001). These roles include an involvement in neurodegenerative diseases; for example, ABCB1 shows a decreased function in PD patients, resulting in dysfunction of blood--brain barrier transport (Bartels et al., 2008). Although this is not a direct homolog of scarlet, it does highlight the importance of ABC transporters in mediating neurodegeneration. Thus, investigating mechanisms uncovered here should be helpful for uncovering potential therapeutic targets to prevent the loss of these neurons.
MATERIALS AND METHODS
Fly stocks and husbandry
Oregon-R was used as the WT control for all experiments. The following stocks 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) was provided by the Vienna Drosophila Resource Center. M1, M2, M3, and M4 were previously described (Palladino et al., 2002). UAS-Scarlet::Venus was generated in the Babcock laboratory and is described below.
Creation of transgenic flies
Generation of UAS-venus-st was achieved by cloning scarlet cDNA into pBID-UASC-VG (Addgene #35206 deposited by Brian McCabe; Wang et al., 2012a) for placement of a Venus tag at the N-terminal. Microinjection of constructs was performed by BestGene Inc (Chino Hills, CA), with the construct inserted on the second chromosome at VK00002.
Immunohistochemistry
Brains were dissected and stained as previously described (Babcock et al., 2015). Briefly, brains were dissected in PBS and fixed in 4% formaldehyde in PBS for 20 min at room temperature. Samples were then washed four times in PBS with 0.3% Triton X-100 (PBS-T) and placed in blocking buffer (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 secondary antibody for 2 h at room temperature. Finally, samples were washed four times in PBS-T, mounted with Vectashield (Vector Laboratories), and stored at −20°. The primary antibody used was rabbit anti-tyrosine hydroxylase (1:100) (AB152, Millipore). The secondary antibody used was Alexa Fluor 488 goat anti-rabbit-IgG (1:200) (Invitrogen).
Image analysis
Images were captured on a Zeiss LSM 880 confocal microscope. Sequential 1.5-µm optical slices were taken using a 20× objective. Brightness and contrast were adjusted using ImageJ software (NIH) and Adobe Photoshop CS5 (Adobe). Dopaminergic neurons were counted as previously described (Barone and Bohmann, 2013; Whitworth et al., 2005). In brief, mounted brains were observed under a Nikon Eclipse Ni-U fluorescent microscope and the number of PPL1 neurons in each cluster was counted. A minimum of 10 brains were used for each condition and all experiments were performed in triplicate. Analysis was performed by a researcher that was blind with respect to the genotype.
Locomotor behavior
Adult flies were collected shortly after eclosion and separated into 10 cohorts consisting of 10 flies (100 total) for each genotype. Flies were maintained at 29°C and transferred to fresh food every 3 days. For the climbing assay, each cohort was transferred to an empty glass cylinder (diameter, 2.5 cm; height, 20 cm), and allowed to acclimatize for 5 min. For each trial, flies were tapped down to the bottom of the vial, and the percentage of flies able to cross an 8-cm mark successfully within 10 s was recorded as the climbing index. Five trials were performed for each cohort, with a 1-min recovery period between each trial.
Lifespan
Adult flies were collected shortly after eclosion and separated into 30 cohorts consisting of 10 flies (300 total) for each genotype. Flies were maintained at 29°C and transferred to fresh food every 2 days, at which time the percentage of surviving flies was recorded.
KYNA feeding and drug treatment
Standard fly medium was prepared and 5 mg/ml KYNA (Sigma) was added to the liquid medium as previously described (Campesan et al., 2011). Flies were collected shortly after eclosion and placed on KYNA-supplemented food or standard food as a control. Flies were maintained at 29°C. Flies were transferred to fresh food every 3 days.
ROS quantification
The 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 in anhydrous dimethyl sulfoxide (DMSO) (Fisher) immediately before dissection to a 10 mM stock solution. 1 µl of the stock solution was dissolved in 1 ml of 1× PBS for a final concentration of 10 µM, then vortexed for 20 s. Brains were dissected and incubated in the dye for 10 min on a shaker in the dark at room temperature, then the samples went through three 5-min washes with 1× PBS under the same conditions. Brains were mounted in Vectashield and immediately imaged on a confocal microscope (Zeiss) at 20× magnification. Imaging analysis was conducted by quantifying the fluorescence intensity of the protocerebral area using ImageJ software (NIH).
Statistical analysis
Significance values for lifespan survival curves was analyzed using the log-rank test. Comparisons for both climbing behavior and dopaminergic neuron loss were analyzed using Student's t-test, with Bonferroni corrections for multiple comparisons when applicable. Statistical analysis was performed using SPSS software (IBM Corporation) and GraphPad Prism (Graphpad Software, Inc.).
Any reagents will be made available upon request.
Acknowledgements
The authors would like to thank members of the Babcock and Ganetzky laboratories for helpful suggestions.
Footnotes
Author contributions
Conceptualization: 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.; Funding acquisition: B.G., D.T.B.
Funding
This research was supported by the National Institutes of Health (R01 NS15390 to B.G.) and Lehigh University start-up funds (D.T.B.). Deposited in PMC for release after 12 months.
References
Competing interests
The authors declare no competing or financial interests.