A new Drosophila model to study the interaction between genetic and environmental factors in Parkinson׳s disease

brain research 1583 (2014) 277–286

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Research Report

A new Drosophila model to study the interaction between genetic and environmental factors in Parkinson's disease Scott J. Varga, Cheng Qi, Eric Podolsky, Daewoo Leen Neuroscience Program, Department of Biological Sciences, Ohio University, Athens, OH 45701, USA

art i cle i nfo

ab st rac t

Article history:

The fruit fly Drosophila melanogaster has long been used as a model organism for human

Accepted 9 August 2014

diseases, including Parkinson's disease (PD). Its short lifespan, simple maintenance, and

Available online 15 August 2014

the widespread availability of genetic tools allow researchers to study disease mechanisms

Keywords:

as well as potential drug therapies. Many different PD models have already been

Drosophila larvae α-Synuclein

developed, including ones utilizing mutated α-Syn and chronic exposure to rotenone. However, few animal models have been used to study interaction between the PD causing

Rotenone

factors. In this study, we developed a new model of PD for use in the larval stage in order to

Dopaminergic neurons

study interaction between genetic and environmental factors. First, the 3rd instar larvae

Locomotion

(90–94 hours after egg laying) expressing a mutated form of human α-Syn (A53T) in dopaminergic (DA) neurons were video-taped and quantified for locomotion (e.g. crawling pattern and speed) using ImageJ software. A53T mutant larvae showed locomotion deficits and also loss of DA neurons in age-dependent manner. Similarly, larvae chronically exposed to rotenone (10 μM in food) showed age-dependent decline in locomotion accompanied by loss of DA neurons. We further show that combining the two models, by exposing A53T mutant larvae to rotenone, causes a much more severe PD phenotype (i.e. locomotor deficit). Our finding shows interaction between genetic and environmental factors underlying development of PD symptoms. This model can be used to further study mechanisms underlying the interaction between genes and different environmental PD factors, as well as to explore potential therapies for PD treatment. & 2014 Elsevier B.V. All rights reserved.

1.

Introduction

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder, after Alzheimer's disease. It is marked by a progressive loss of dopaminergic (DA) neurons and a

subsequent development of locomotor deficits. The cause of most cases of PD is currently unknown. However, a small proportion (
5%) can be linked to specific genetic mutations (de Lau and Breteler, 2006). Mutations in genes such as α-Synuclein, parkin, and LRRK2 have been used to model PD

n Correspondence to: 213 Life Science Building, Department of Biological Sciences, Ohio University, Athens, OH 45701, USA. Fax: þ1 740 593 0300. E-mail address: [email protected] (D. Lee).

http://dx.doi.org/10.1016/j.brainres.2014.08.021 0006-8993/& 2014 Elsevier B.V. All rights reserved.

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in several organisms and have provided key insights into disease mechanisms (Dawson et al., 2010). In addition to genetic models, several other model systems have been developed to take advantage of epidemiological data suggesting a link between PD development and exposure to pesticides such as rotenone and paraquat (Cannon and Greenamyre, 2010; Tanner et al., 2011). All these findings strongly suggest that a combination of genetic and environmental factors contributes to the development of PD. However, it is not well understood how genetic and environmental PD factors interact to manifest the disease. PD develops in such a slow fashion that we cannot easily use human subjects to study the mechanisms underlying its pathogenesis. An alternative animal model has been used, suitable for genetic manipulations. During the last several decades, many PD models have been developed either using genetic means or challenge by PD toxins (Shimohama et al., 2003; Lim and Ng, 2009; Botella et al., 2009; Goldman, 2014). Studies with the fruit fly Drosophila melanogaster over the last decade have contributed to pivotal advances in our understanding of PD due to the powerful genetic tools and resources available in flies (Bilen and Bonini, 2005; Whitworth, 2011). The reason that Drosophila continues to contribute important insights of relevance to PD is that gene sequence and function are highly conserved between flies and humans (Rubin et al., 2000). The Drosophila genome encodes orthologs of all the genes that have thus far been implicated in PD, with the only exception being the α-Syn gene (Whitworth et al., 2006). However, transgenic flies overexpressing human α-Syn have also shown age-dependent loss of DA neurons (Feany and Bender, 2000; Auluck et al., 2002; Botella et al., 2009) and locomotor deficit (Pendleton et al., 2002), which are two pathological hallmarks of PD. In addition, toxin-induced fly models are also established (Coulom and Birman, 2004; Lawal et al., 2010). However, few Drosophila models have been used to study interaction between the PD causing factors. In this study, we chose α-Syn and rotenone as a genetic factor and an environmental factor, respectively. Drosophila

larvae expressing mutant human α-Syn showed agedependent decline in locomotion and also a significant loss of DA neurons in the brain. Similarly, larvae chronically exposed to rotenone (10 μM) showed significant reduction in locomotion accompanied by loss of DA neurons in the brain. We further showed that combining these two models, by exposing α-Syn mutant larvae to rotenone, causes a much more severe locomotor deficit. Our results show that Drosophila larvae can be an excellent model to easily quantify PD symptoms at the levels of DA neurons and its resultant behavior and thus to further study mechanisms underlying the interaction between genes and different environmental PD factors.

2.

Results

2.1. Mutant human alpha-Synuclein causes locomotor deficits and age-dependent degeneration of dopaminergic neurons in Drosophila larvae A small presynaptic protein, alpha-Synuclein (α-Syn) has been extensively studied as it gives rises to a dominant heritable form of Parkinson's disease (Polymeropoulos et al., 1997; Moore et al., 2005; Whitworth, 2011). Therefore, in this study, we chose Drosophila larvae expressing human α-Syn (wild type or a mutant form A53T) to examine DA neuronal degeneration and locomotor deficits. Expression of human α-Syn in DA neurons was driven by using a DA specific driver, TH-Gal4 (Friggi-Grelin et al., 2003). Crawling speed of the third instar larvae (90–94 h after egg laying) was quantified by videotaping locomotion for 30 sec (see Experimental procedure). The video clip was analyzed using ImageJ in order to calculate the path length. Thus we were able to quantify locomotion score (speed) as the distance traveled per minute (mm/min). Fig. 1 shows that A53T-expressing larvae have significantly slower locomotion (58.5þ/2.2 mm/min) compared to wild type Canton-S (CS) fly lines. In addition, human α-Syn wild type-expressing larvae showed reduced locomotion speed (81.9þ/3.4 mm/min)

Fig. 1 – Larvae expressing a mutant human α-Syn (A53T) show abnormal locomotor behaviors and decreased speed. Sample crawling paths (from 4 different larvae) are shown for wild type Canton-S (CS) as control (A) and TH-A53T ( ¼TH-Gal4xUAS-αSyn (A53T)) larvae (B). Scale bar¼1 cm. (C) A graph showing the average crawling speed as a means of quantifying larval locomotion (mm/min) for Canton S wild type (CS), w1118 (white eye wild type), TH-Gal4, TH-α-Syn (wild type human α-Syn which is different from WT fly line) and TH-A53T larvae. TH-α-Syn and TH-A53T larvae showed significantly lower crawling speed compare to other control lines. Student t-test, n ¼ po0.05, nn ¼ po0.01. Number (n) of repeated experiments: CS (29), w1118 (15), TH-Gal4 (10), TH-α-Syn (10), TH-A53T (16), respectively.

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although it was faster than A53T-expressing larvae. In contrast, the locomotion speed of wild type fly, Canton-S (94.4þ/  2.1 mm/min) was similar in another wild type fly, w1118 (93.7þ/ 3.7 mm/min) and a transgenic line, TH-Gal4 (91.8þ/  6.1 mm/min), respectively, demonstrating that slower locomotion in α-Syn (wild type or A53T) larvae is not due to nonspecific genetic background effects. Next, we wanted to examine whether DA neurons in the mutant α-Syn larval brain are degenerated. Brains of the third instar larvae were dissected and immunostained with an antibody for the DA synthesis enzyme tyrosine hydroxylase (TH). In normal larval brains, there are three major clusters of DA neurons (Blanco et al., 2011): dorsomedial (DM), dorsolateral 1 (DL1), and dorsolateral 2 (DL2). In a wild type Canton-S (CS) larval brain, it was easy to identify three clusters (DM, DL2, DL2) of anti-TH(þ) neurons in each hemisphere (circles

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shown in Fig. 2A). Fig. 2D shows mean number of anti-TH(þ) neurons in each cluster of wild type Canton-S (CS) larval brains. Total number of DA neurons in a wild type (CS) brain was 39.8þ/0.4, which is very similar to that of previously reported (Blanco et al., 2011). In A53T-expressing larvae, a decreased number of DA neurons were observed in each cluster (Fig. 2B). The quantitative analysis showed this reduction was grossly in all three major clusters (Fig. 2D). Total number of DA neurons in A53T was 31.9þ/ 1.1, significantly lower than wild type CS (Fig. 2E). In addition, wild type α-Syn showed an intermediate level of DA degeneration (34.4þ/ 2.0, n¼ 7), consistent with the locomotion defects (Fig. 1C). Since A53T causes more severe degeneration, we decided to use A53T in the following experiments. Taken together, our results demonstrated that Drosophila larvae expressing human α-Syn (wild type or A53T) are an excellent new animal

Fig. 2 – Whole-brain immunostaining with a dopaminergic marker, anti-TH antibody and anti-α-Syn antibody. Sample images of immunostained wild type Canton-S (CS) (A) and A53T ( ¼ TH-A53T) brains (B). Three major cell groups (DM, DL1, and DL2) are visible in both brains (circled in CS brain). (C) A53T brain stained with anti-α-Syn antibody (green) showed expression of α-Syn in the third instar larval brain. (Inset) Examples of α-Syn-positive neurons (indicated by a dotted square in C) co-stained with anti-TH (yellow). Outer white lines indicate the boundary of the brain. White dotted lines are borders between brain and ventral nerve cord (lower part). Scale bar¼ 50 μm. (D) Number of DA neurons counted from the three different regions (DM, DL1, DL2) for WT and A53T larvae (L, left hemisphere; R, right). (E) Total number of TH-positive neurons in the brain of CS and A53T larvae. Student t-test, n ¼po0.05, nn ¼po0.01 difference from WT. Number (n) of brains: CS (16), A53T (14).

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Fig. 3 – Whole-brain immunostaining of the first instar larval brains with anti-TH antibody. (A) Image of wild type Canton-S (CS) 1st instar larval brain showing anti-TH positive neurons in three different regions (DM, DL1 & DL2) like those in the third instar brain. (B) Image of TH-A53T 1st instar larval brain showing anti-TH positive neurons. White lines are borders between brain and ventral nerve cord. Scale bar¼ 30 μm. (C) Number of TH-positive neurons in the brain for WT control (n ¼15) and TH-A53T larvae (n¼ 15).

model for PD research as we observed two cardinal hallmarks of PD symptoms in α-Syn larvae – locomotor deficits and DA neurodegeneration. However, to be a good animal model for the neurodegeneration study, it is important to demonstrate age-dependent degeneration in the nervous system. Therefore, we stained brains of the first instar larvae with anti-TH antibody. As shown in Fig. 3A, the first instar brain from wild type CantonS (CS) fly larvae possessed DA neurons (37.3þ/ 0.4), very comparable to that from the third instar brains (see above). Total number of DA neurons in the first instar brain of A53T was 36.5þ/ 0.5, not significantly different from that from wild type (CS) larvae (Fig. 3C). All the data confirm that a mutant human α-Syn (A53T) causes age-dependent degeneration of dopaminergic system in the brain.

2.2. Rotenone exposure causes concentration-dependent locomotor deficits and breakdown of dopaminergic neurons In addition to genetic PD causing factors such as α-Syn, environmental factors can cause the selective loss of DA neurons as studied with neurotoxin-based PD models (Bové et al., 2005; Cannon and Greenamyre, 2010). The insecticide rotenone has become a popular toxin to study mechanisms underlying PD pathology induced by environmental factors. Therefore, we wanted to develop a rotenone model of PD using Drosophila larvae. First, it was necessary to determine an ideal concentration for modeling PD using the larvae. Wild type Canton-S (CS) larvae were grown in the presence of various concentrations (0.1–100 μM) of rotenone to determine an appropriate, non-lethal dose. Table 1 lists the percent of larvae surviving to the third instar stage when grown in various concentrations of rotenone. Chronic exposure to rotenone is mildly toxic starting at approximately 1 μM. At 30 μM, the majority of larvae (56.4%) are killed before reaching the third instar and no larva survived to the late third instar at 100 μM. We also examined effects of rotenone exposure on the locomotor abilities of surviving larvae (Fig. 4). At 1 μM or higher, rotenone causes a significant reduction in

Table 1 – Survival of wild type Canton-S (CS) fly larvae exposed to various concentrations of rotenone. Concentration (μM)

% Survival

nn

0 0.1 0.3 1 3 10 30 100

91.9 92.9 89.9 83.0 81.4 72.5 43.6 0

170 579 159 112 585 222 204 50

n

Number (n) of larvae used for each concentration.

speed of locomotion. For example, locomotive speed was reduced by
44% at 10 μM rotenone. Based on these results, 10 μM rotenone was selected as an appropriate dose to model PD in larvae, given that the majority of larvae survive (72.5%) to the third instar stage but still show a significant decrease in locomotion (Fig. 4A). They also exhibit defective crawling patterns (Fig. 4B) compared to wild type (CS) fly line (Fig. 1A). In our study, larvae were exposed to rotenone for the larval lifespan. However, several animal studies with rotenone including adult flies (Betarbet et al., 2000; Coulom and Birman, 2004; Cannon et al., 2009) applied for a short-term exposure (e.g. days) or after development. In order to confirm whether a short-term treatment of rotenone is enough to cause locomotion deficits, we exposed wild type (WT) larvae to rotenone for only 24 or 48 h prior to the locomotion test instead of the entire lifespan. 10 μM rotenone for 24 h did not induce any locomotion deficits. In contrast, 48 h exposure to 10 μM rotenone caused significant deficits in locomotion (80.5þ/ 5./min; Table 2). 100 μM exposure for both 24 and 48 h resulted in locomotion deficits. This data strongly supports the notion that rotenone-induced locomotion defects are due to neurodegeneration, not due to developmental deficits. Our data also show that rotenone causes locomotion defects in a dose- and exposure time-dependent manner. Therefore, in the subsequent experiments, we wanted to

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Fig. 4 – Crawling speed and patterns of wild type larvae treated with rotenone. (A) Crawling speed quantified at varying concentrations of rotenone (0.1–100 μM). Rotenone concentrations at 1 μM or higher significantly slowed wild type larval crawling. Wild type Canton-S (CS) control is shown by an open circle in the top left. (B) Sample paths of larvae treated with 10 μM rotenone. Scale bar¼ 1 cm. Number (n) of repeated experiments: control (8), 0.1 μM (6), 0.3 μM (10), 1 μM (13), 3 μM (13), 10 μM (11), 30 μM (5), 50 μM (8). Table 2 – Effects of the short-term exposed rotenone on Drosophila larval locomotion. a

Treatments

No rotenone 10 μM rotenone 100 μM rotenone

Exposure 24 h (nb)

48 h (nb)

97.9þ/ 5.1 mm/min (12) 100.7þ/7.3 mm/min (11) 80.9þ/ 3.4 mm/min (12)n

95.7þ/4.4 mm/min (18) 80.4þ/5.8 mm/min (12)n 74.5þ/3.7 mm/min (18)nn

a

Wild type Canton-S (CS) larvae were used for this study. Number (n) of larvae examined. n Student t-test, po0.05. nn Student t-test, po0.01. b

Fig. 5 – Whole-brain immunostaining with anti-TH antibody. (A) Image of wild type Canton-S (CS) larval brain treated with 10 μM rotenone. Scale bar¼ 50 μm. (B) Number of TH-positive neurons in different brain regions for CS control (n ¼14) and rotenone-treated larvae (n ¼ 10). (C) Number of DA neurons counted from the three different brain regions for CS and rotenonetreated larvae. Student t-test, n ¼po0.05, nn ¼po0.01 difference from CS.

examine whether DA neurons in the larval brain exposed to rotenone are degenerated. Application of 10 μM rotenone to wild type (CS) larvae causes a significant decrease in the number (34.9þ/0.9) of DA neurons in the whole larval brain (Fig. 5). Interestingly, DA neurons in DM and DL2 (left) clusters

were significantly degenerated by rotenone while DA neurons in DL1 and DL2 (right) clusters were less damaged. It is not clear at this time whether this is due to differences between environmental vs genetic PD causing factors, or due to uneven penetration of rotenone itself. Nonetheless, our

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results clearly show that chronic exposure to rotenone recapitulates two cardinal hallmarks of PD symptoms in Drosophila larvae and thus provides a new model for studying the mechanisms of DA neurodegeneration by environmental factors.

2.3. Expression of mutant α-Synuclein and exposure to rotenone do not cause olfactory defects Along with the more visible motor symptoms, there are many non-motor PD symptoms (Chaudhuri and Schapira, 2009). One such early non-motor symptom is olfactory deficit. Indeed, one study showed that majority of individuals diagnosed with PD had some sort of olfactory deficit (Fritsch et al., 2012). Therefore, we wanted to examine whether α-Syn or rotenone models of PD developed in this study also showed defects in olfaction. To determine whether the larval PD models show deficits in the olfactory abilities, naïve olfaction test (Honjo and Furukubo-Tokunaga, 2005) was performed using third-instar larvae expressing mutant human α-Syn (A53T). Expression of A53T did not show any difference in olfaction as its olfactory score (0.34þ/ 0.01) compared to that of wild type CS (0.35þ/ 0.02; Fig. 6). Wild type (CS) larvae exposed to rotenone also did not show any significant decrease in olfactory ability (0.33þ/ 0.02). Since the Drosophila larval model of PD did not show alterations in olfaction, these data strongly support the notion that locomotor deficits observed in our two PD larval models are not the results of global degeneration of brain function.

2.4.

Interaction between rotenone and α-Synuclein

Once we established that mutant human α-Syn (A53T) and chronic exposure to rotenone can cause PD-like symptoms in Drosophila larvae, we wanted to determine whether there is an interaction between these genetic and environmental models. In this experiment, α-Syn (A53T) mutant larvae were exposed to 10 μM rotenone and their locomotion score was tested throughout the larval lifespan (1st–3rd instar). As shown in Fig. 7, A53T expression or rotenone exposure alone did not show any

Fig. 6 – Olfactory response is not altered in TH-α-Syn (A53T) and wild type (CS)-treated with rotenone (10 μM). In this experiment, we used traditional naïve olfactory test described in Experimental procedures. Number (n) of experiments repeated: wild type CS (8), A53T (7), CSþRot (7). 10 μM rotenone was used.

Fig. 7 – Stage-dependent changes in locomotion speeds. For this study, three different stages of larvae were used: 1st (34–40 h old), 2nd (59–63 h old), 3rd instar (90–94 h old). Locomotion speed was increased as the larvae (CS, CSþRot, A53T) developed. However, TH-α-Syn A53T ( ¼A53T) larvae treated with rotenone (10 μM) did not show an increase of locomotion speed as they developed. Also locomotion speed was significantly lower throughout the larval stage compared to CS, CSþRot or A53T. Rotenone was present in the food throughout the entire larval development. ANOVA, n ¼po0.05, nn ¼po0.01, nn ¼po0.001 (difference from CS). significant deficits in locomotion for the first instar larvae. However, locomotion in the second instar larvae became significantly slower for both A53T and rotenone-treated, compared to wild type (CS). This difference was even greater in the third instar. Our results demonstrate that deficit in locomotion caused by A53 or rotenone is age-dependent, as expected in PD patients. After confirming an age-dependent locomotor deficit in A53T or rotenone-treated larvae, we examined using a similar approach to determine if exposure of A53T mutant larvae to rotenone (A53TþRot) caused a further, significant locomotor deficit, which worsens with age. A53TþRot larvae also exhibited defective crawling patterns compared to wild type (data not shown). In the subsequent experiments, we examined whether A53TþRot larvae show degeneration of DA neurons in the brain. Application of 10 μM rotenone to A53T larvae caused a significant decrease in the number (32.1þ/ 1.5) of DA neurons in the whole larval brain (Fig. 8). In contrast, there were no changes in the brain size and larval development by A53T and rotenone. The brain size (386.5þ/20.5 μM, n ¼13) of wild type Canton-S (CS) was similar to that (371.2þ/ 23.7, n¼ 13) of A53TþRot larvae. In addition, there was no difference in the body size between CS (5.61þ/0.14 mm, n ¼12) and A53TþRot third instar larvae (5.73þ/ 0.20, n¼12). All the data show that combining the two models, by exposing A53T mutant larvae to rotenone, causes a much more severe PD phenotype (i.e. locomotor deficit), strongly indicating potential interaction between genetic and environmental factors underlying the development of PD symptoms.

3.

Discussion

In this study, we show that Parkinson's disease (PD) can be effectively modeled in Drosophila larvae to study both genetic

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Fig. 8 – Whole-brain immunostaining with anti-TH antibody. (A) Image of A53T larval brain treated with 10 μM rotenone (A53TþRot). Scale bar¼ 50 μm. (B) Number of DA neurons counted from the three different regions (DM, DL1, DL2) for wild type (CS) and A53TþRot larvae. (C) Total number of TH-positive neurons in the brain of CS and A53TþRot larvae. Student t-test, n ¼ po0.05, nn ¼ po0.01, difference from CS. Number (n) of brains: CS (9), A53TþRot (10).

and environmental factors involved in disease development. As an accurate PD model system, these larvae replicate two major hallmarks of PD symptoms. The locomotion assay provides an efficient test for PD symptoms, and immunofluorescence imaging can provide a look at the cellular degeneration underlying PD. Drosophila larvae expressing mutated α-Syn (A53T) demonstrated age-dependent locomotion deficits and DA degeneration in the brain. This matches with the previous reports (Feany and Bender, 2000; Auluck et al. 2002; Trinh et al., 2008) in which they used adult flies expressing the mutated human α-Syn. Similarly, larvae chronically exposed to rotenone show an almost identical pattern of age-dependent decline in locomotor ability accompanied by loss of DA neurons. The models of PD used in this study, the A53T mutation and chronic rotenone exposure, are already established in adult Drosophila and other animals. Thus, one might question why a larval model is beneficial for PD research. In fact, larvae possess several advantages for studying PD. They contain the full spectrum of genetic tools that make Drosophila such a useful model organism. Like adult flies, larvae demonstrate age-dependent PD symptoms. However, the much shorter life cycle of larvae (
4 days) allows for more rapid screening of potential PD drugs and therapies. They are also simpler to work with, and their brains contain clearly defined groups of DA neurons. The locomotion assay available for larvae is quantitative for the speed and also allows further examination of crawling patterns whereas adult locomotion assay provides a simple binary score – pass the line or fail (ShaltielKaryo et al., 2012). Compared to their adult Drosophila model (250–500 μM rotenone; Coulom and Birman, 2004), our larval model is much more sensitive to rotenone exposure (10 μM). As seen in Figs. 1 and 4, larvae affected by A53T and rotenone show different patterns, respectively, which will be an interesting subject to further explore. In addition, due to the semitransparent cuticle, larval PD model can be used for an increasingly popular optogenetic technique (e.g. channelrhodopsin 2; Störtkuhl KF, 2011) to manipulate DA neuronal

activities without any light penetration concerns like adult flies. In early 2000, an intriguing PD model has been developed using adult transgenic flies that express a mutant human αSyn protein (Feany and Bender, 2000; Auluck et al., 2002). These adult transgenic flies exhibit typical anatomical and behavioral symptoms of PD, including an age-dependent loss of DA neurons (Feany and Bender, 2000; Auluck et al., 2002; Yang et al., 2003) and locomotor deficits (Feany and Bender, 2000; Pendleton et al., 2002). However, some studies (Pesah et al., 2005) reported no degeneration of DA neurons in addition to no locomotor deficits by expression of mutant human α-Syn, suggesting the necessity of developing new PD models. Furthermore, several rodent PD models also failed to recapitulate PD symptoms such as selective DA cell loss (Beal, 2010). For example, α-Syn knockout mice do not reliably recapitulate the PD symptoms such as loss of DA neurons (Masliah et al., 2000). It was also shown that parkin-null mice do not manifest any DA neuronal loss or behavioral abnormality (Dawson et al., 2010). Considering all these, our larval model expressing α-Syn (A53T) showing reduced locomotion and also in the number of DA neurons in the brain, strongly supports the usefulness of this genetic PD model in Drosophila larvae. In addition, a cellular PD model using Drosophila neuronal culture has been well established for mutant α-Syn and a PD toxin, MPPþ (Park and Lee, 2006; Wiemerslage et al., 2013). Therefore, this larval PD model will be an excellent complementary system to study genetic and environmental PD causing factors by adding intact brain morphology and behavioral characteristics to the cell culture model, which is suitable for biochemical, sub-cellular structure (e.g. synapse) and electrophysiological analyses (Lee and O’Dowd, 1999; Rohrbough et al., 2003). The fact that the larvae show locomotor deficits is particularly interesting because in some Drosophila models, including A53T and park25, locomotor deficits used as a PD symptomatic marker are age-dependent (Bilen and Bonini, 2005). These flies begin with almost normal locomotion as

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young adults but as they age their scores fail to increase at the same rate as control flies. If they show deficits in the larval stage but not as young adults, there must be certain mechanisms by which the flies rescue these deficits during the metamorphosis from larvae to adult. A study that could be very informative would trace the fate of affected larval DA neurons into the adulthood. DA neurons have previously been traced from the larval to the adult stage in normal flies (Budnik and White, 1988; Truman, 1990). It shows that many of the larval neurons are maintained in adults. This carryover means that in PD models, young adults possibly have some DA neurons affected by PD causing factors during the larval stage. Understanding how these flies avoid PD symptoms could lead to a way to regenerate or repair the neurons lost in PD. This could lead to an important breakthrough in curing PD, as opposed to simply lessening the symptoms. In this study, we further show that combining the two models, by exposing A53T mutant larvae to rotenone, causes a much more severe PD phenotype. We believe the interaction between α-Syn (A53T) and rotenone is not due to simple additive effect. First, rotenone with higher concentrations (i.e. 30–50 μM in Fig. 4) further reduced the locomotion by only 7–19% while A53TþRot caused 40% reduction in locomotion compared to Rot control (Fig. 7), strongly indicating that the effect of rotenone is potentiated by A53T mutation. Second, the number of DA neurons in A53TþRot did not show a proportional loss of DA neurons and was not statistically different to that of A53T or rotenone alone although more severe locomotion defects were observed in A53TþRot larvae. Our immunostaining assay with anti-TH antibody is limited to address the relative health or synaptic connectivity of surviving neurons. Therefore, electrophysiological recordings could be taken to measure their neural activity to determine whether these neurons act normally. In addition, it would be beneficial to measure the overall level of DA in the brain of these model larvae as previously shown in adult flies (Powell et al., 2005). This would determine whether more severe locomotion defects in A53TþRot larvae is due to the reduced DA neuronal activity and thus reduced synaptic DA release, not by reduced TH expression. So far, little has been done to study the interactions between environmental and genetic PD factors. One such example was reported by Meulener et al. (2005) showing that DJ-1 mutant flies are more sensitive to a herbicide paraquat although DJ-1 mutant flies did not show locomotor deficits and DA neuronal loss. Another example was to show that expression of a PD gene hLRRK2 increases the sensitivity to rotenone (100 μM), measured with 50% survival and DA neurodegeneration (Venderova et al., 2009). Therefore, our new larval model can be used to further probe interactions between genetic and environmental factors. It will be a good system to examine the multiple hit hypotheses (more than one risk factors) for DA neuronal loss in PD proposed by Carvey et al. (2006) as the hypotheses have not been thoroughly tested due to the lack of an appropriate model system. In this study, we have presented a new model to examine PD symptoms and behaviors in Drosophila larvae. This model can be used in future studies to examine in more detail of the interactions between genes and different environmental factors, as well as exploring potential therapies for PD treatment.

4.

Experimental procedures

4.1.

Fly strains

Flies were grown in standard cornmeal/agar media with 0.4% propionic acid on a 12-hour light/dark cycle at 25 1C. Fly strains used in this study were: wild-type (Canton-S or CS), w1118 (from Bloomington Stock Center), TH-Gal4 (a kind gift from Dr. J. Hirsh, University of Virginia), UAS-α-Syn (wild type) & UAS-α-Syn (A53T); TH-GAL4 (A53T, a gift from Dr. L. Pallanck, University of Washington). Expression of wild type α-Syn was reported in Trinh et al. (2008) while α-Syn (A53) expression was examined in this study using anti-α-Syn antibody (Fig. 2C). Rotenone-treated larvae were grown in standard food supplemented with varying concentrations of rotenone dissolved in DMSO. For the generation of a survival curve, flies were placed in an egg collection bottle with a food dish containing rotenone. The flies were allowed to lay eggs for a hour. Each plate had roughly 50 eggs on its surface. The plate was then incubated at 25 1C using a 12-hour light/dark cycle. In the late third instar stage (90–94 h after egg-laying) the larvae were separated from the food, and then used for locomotion and survival assays.

4.2.

Locomotion assay

Individual larvae were separated from the food using a 15% glucose solution and rinsed with distilled water. They were then placed on the surface of a plate of 2.5% agar mixed with 1 mL India ink (to have a black background). The larvae were allowed to acclimate for 1 min and a video was then recorded for 30 s at approximately 10 frames per second using a Moticam3 digital camera (Motic) and Motic Images Plus 2.0 software. The video was analyzed using the MTrack2 plug-in (from http://valelab.ucsf.edu/
nico/IJplugins/MTrack2.html) for ImageJ (from http://rsb.info.nih.gov/ij/). The path length was recorded; scores were quantified as the distance traveled per minute.

4.3.

Larval whole-brain immunostaining

Larval brains were isolated from late third instar larvae and fixed for 30 min in a solution containing 4% paraformaldehyde in 1  phosphate buffered saline (PBS). After 1 h in blocking solution (2.5% normal goat serum and 0.15% TritonX 100 in 1x PBS), brains were stained overnight at 4 1C with a mouse monoclonal antibody to tyrosine hydroxylase (TH, 1:750)(Immunostar) or α-Syn (LB509, Zymed). They were then incubated for 2 h in goat anti-mouse IgG labeled with Alexa Fluor 546 (diluted 1:750 in 1xPBS). Brains were mounted on 50  22 mm glass coverslips and images were taken on a Zeiss LSM510 confocal microscope. The number of TH-positive cells in the 1st and 3rd instar brains was counted manually, as previously described in Blanco et al. (2011). The cells were categorized into three major groups based on their position in the brain: dorsomedial (DM), dorsolateral 1 (DL1), and dorsolateral 2 (DL2).

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4.4.

Larval olfaction assay

First, a test plate was prepared by adding 2.5% agar into a 100  15 mm Petri dish (BD Falcon, BD Biosciences). A 1 cm2 piece of Whatman filter paper was placed on either side of the plate. Next, third-instar larvae (90–94 h after egg-laying) were isolated from their food mixture using a 15% glucose solution. The larvae were filtered using a 500 μm sieve (U.S.A. Standard Test Sieve, Newark Wire Cloth Co.) and rinsed three times with distilled water. They were then moved to the surface of the testing plate, and 2.5 μL of pentyl acetate was placed on the filter paper on one side – the “odor side”. The other side had 2.5 μL of distilled water. The larvae were allowed to crawl freely for 5 min, and the number within 3 cm of each side was counted. A response index (RI) was quantified as described in Honjo and Furukubo-Tokunaga (2005). The RI is given by
# Larvae on odor side – # larvae on opposite ½no odor side Total # larvae

Acknowledgments This work was partially supported by the International Collaboration Grant from Korea Institute of Science and Technology (Brain Science Institute), Seoul, Republic of Korea. We thank Charles Bunce for help with larval brain immunostaining and Dr. R.A. Colvin for valuable inputs on previous versions of this manuscript. SJV was a recipient of Ohio University Provost's Undergraduate Research Fund.

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