Nicotine increases lifespan and rescues olfactory and motor deficits in a Drosophila model of Parkinson's disease

Drosophila melanogaster is an attractive model of familial Parkinson's disease, as flies with loss-of-function mutations of the parkin gene exhibit many pathologies observed in PD patients. Progressive motor deficits found in homozygous parkin mutants seem to result from mitochondrial pathology that causes indirect flight muscle and dopaminergic neuronal degeneration [1,2]. We have found that heterozygous parkin mutants have decreased lifespan, generally progressive motor dysfunction and olfactory deficits compared to control flies, suggesting that mutation of this gene produces a dominant phenotype. Tobacco smokers are dose-dependently less likely to develop PD [3,4]; subsequent in vitro and in vivo studies show that nicotine is protective in models of sporadic PD [6]. Literature addressing the potential protection by nicotine in Parkin loss-of-function models spans limited concentrations and selected time points in the organism's lifespan. We have found that parkin heterozygotes have late-onset climbing and flying deficits as well as decreased viability and olfactory deficits that precede motor defects. While chronic nicotine exposure decreases lifespan and climbing and flying abilities in control flies, it can improve viability and flying capability as well as rescue climbing and olfactory deficits in parkin heterozygotes. Dopaminergic neurons are spared in the parkin heterozygote, perhaps because this phenotype is less severe than in the homozygous parkin mutants. Nicotine pretreatment may be protective in sporadic PD patients and models; however, timely diagnosis remains to be an obstacle. Our results suggest that nicotine also may be protective in familial PD patients, who can be easily identified before motor symptoms occur.


Introduction
Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder, occurring in familial and sporadic forms. The most common symptoms observed in PD patients are resting tremors, rigidity, bradykinesia and unsteady gait. Olfactory deficits have been described more recently; they precede motor symptoms and may be useful for early diagnosis [7][8][9]. Etiology of PD remains poorly understood, but epidemiological evidence reports a lower incidence in tobacco smokers [3,4,[10][11][12]. The protection offered by smoking has been at least partly attributed to what appears to be neuroprotective properties of nicotine, which has been shown to maintain cell number and cellular homeostasis in sporadic models of PD by modulation of nicotinic acetylcholine receptors [6,13], or via nicotinic acetylcholine receptor-independent mechanisms [14][15][16]. Studies addressing the potential protective effects of nicotine in models of autosomal recessive-juvenile parkinsonism (AR-JP) are few and inconclusive [17,18].
Loss-of-function mutations in the PARK2 gene may cause up to half of familial PD cases [19]. The Parkin protein, encoded by the human PARK2 gene, is a putative E3 ubiquitin ligase and appears to modulate fission of mitochondria prior to mitophagy. Disruption of this process may cause cell death as a result of the cell's inability to function properly in the presence of increasingly burdensome, non-functional mitochondria [20]. Both sporadic and familial PD patients present with similar symptoms resulting from selective degeneration of dopaminergic neurons in the substantia nigra pars compacta; thus, understanding the development of pathology in patients with inherited forms of PD will likely provide insight to pathology of the sporadic form. Because PARK2 loss-of-function mutations occur in such a high percentage of familial PD cases, and since homozygous park (the Drosophila melanogaster PARK2 ortholog) loss-of-function mutations in Drosophila melanogaster cause Parkinson-like phenotypes and degeneration of dopaminergic neurons, park mutant Drosophila is an attractive model for studying PD pathology and for understanding mechanisms of disease development [1,2,21]. Most cases of AR-JP are caused by homozygous PARK2 mutations; however, several studies report that AR-JP caused by PARK2 mutations can occur in a dominant inheritance pattern as well [22][23][24][25]. Here we describe a heterozygous Drosophila park mutant that exhibits a phenotype that is similar to that of the homozygous mutant [1]. Unlike mouse parkin mutant models, homozygous park Drosophilamutants have symptoms closely related to those seen in PD patients including decreased life span and late onset, progressive motor deficiencies and neuronal degeneration [1,2,21], which are thought to result from mitochondrial abnormalities [1,2].
Currently, there is no treatment to prevent, cure, or halt the progression of PD. A potentially confounding factor in therapeutic strategies is that between 50% and 80% of dopaminergic neurons in the substantia nigra pars compacta have degenerated when observable motor symptoms appear [26,27]. Early detection and treatment may be critical in halting or decreasing the rate of degeneration in PD. In fact, studies show that tobacco smoking time-and dose-dependently decreases incidence and delays onset of sporadic PD [3,11,12].
To determine whether nicotine pretreatment can protect against the heterozygous park 25 (a loss of function allele) phenotype, we continually exposed heterozygous park 25 and control Drosophila to various concentrations of nicotine in food from day one post-eclosion. Nicotine exposure increases median lifespan in park 25 heterozygotes and decreases it in control flies. It also prevents park 25 heterozygote climbing and olfactory deficits and ameliorates flying deficits. Unlike park 25 homozygotes, park 25 heterozygotes have no dopaminergic neuron loss, as determined by tyrosine hydroxylase (TH) expression, compared to control flies. Overall, this study indicates that this model is even more useful than previously thought for understanding of the etiology and development of PD as well as for exploring the mechanisms by which nicotine is protective in vivo.

Drosophila strains and maintenance
The park 25 mutants [1] were a generous gift from Leo Pallanck at the University of Washington in Seattle. Control flies (w 1118 , the strain from which the park 25 mutants were generated) were obtained from the Bloomington Drosophila Stock Center, Indiana University, Bloomington. All flies were raised on standard cornmealmolasses food at 25 • C. (−)-Nicotine hemisulfate salt (Sigma-Aldrich, Saint Louis, MO) was added to the food to achieve final concentrations of 0, 3, 6, 9 or 12 g/ml in the food after it had cooled below 70 • C (free base concentrations of nicotine are 1.05, 2.11, 3.16 or 4.21 g/ml, respectively). Flies were placed on the nicotinecontaining food on the day of eclosion and transferred every other day to new nicotine-supplemented food.

Mortality assay
For each condition, at least ten vials containing twenty flies were maintained on food with or without nicotine from day one post eclosion. Food was changed every other day, and all deaths were recorded. Data are presented as lethal time 50 (LT50), which represents the median lifespan, or the day at which 50% of the flies were dead. A Gehan-Breslow-Wilcoxon test was performed to measure differences between survival curves.

Climbing assay
The climbing assay was performed as previously described [28]. Approximately 10 flies were knocked to the bottom of the climbing vial and allowed 18 s to climb 5 cm up the vial. The percentage of flies passing a 5 cm mark was recorded, and the averages of approximately 20 separate vials per condition were recorded. Flies were tested on days 5, 10, 15 and 20 post eclosion. Student's t-tests were used to determine the effect of genotype on each day of assay, and a one-way ANOVA was performed in order to determine the effects of nicotine on each day of assay. Dunnett t-tests were performed in order to determine post hoc significance.

Flight assay
The flight assay was adapted from a previously published protocol [29] using a 28 cm 3 Plexiglas flight box. Individual flies were dropped into the box through an entry hole (3 cm in diameter) that was cut into the center of top of the box. A fly was determined to be capable of flight if it kept a steady elevation and flew in a controlled manner after being dropped. Flies that could not perform this task fell directly from the top of the box without direction or were unable to maintain elevation but were able to slow their fall and/or fall with some change in directionality. Flight assays were performed on days 5, 10, 15 and 20 post eclosion. Data represent the average of the percent of flies capable of flight for at least 8 vials containing about 20 flies per condition. Student's t-tests were used to determine the effect of genotype on each day of assay, and a one-way ANOVA was performed in order to determine the effects of nicotine on each day of assay. Dunnett t-tests were performed in order to determine post hoc significance.

Olfactory assay
The olfactory assay has been previously described [30]. Briefly, flies are attracted by the odor of food found in the end of the olfactory trap, but are unable to escape once they have reached the food. Because males are much smaller than females, the olfactory traps are customized for each sex in order to effectively keep them with the food. Therefore, males were separated from females, and 10 flies of each sex were put into each trap. After the allotted time (60 h for males and 86 h for females), the number of flies in the trap was recorded as percentages of flies capable of following olfactory cues. The average of at least 10 traps per condition was measured. Flies were tested on day 5 post eclosion, before climbing deficiencies are present. Student's t-tests were employed to determine differences in genotype or sex for each nicotine concentration. A one-way ANOVA was performed to determine the effect of nicotine. Dunnett t-tests were performed in order to determine post hoc significance.

Immunohistochemistry
Brains were dissected in PBS from control (N = 10) and park 25 heterozygote flies (N = 11) on day 20 and fixed with 4% formaldehyde in PBS for 30 min. Brains were washed in 0.1% Triton X-100 in PBS and blocked for 2 h in wash solution with 10% normal goat serum. Brains were then probed with anti-tyrosine hydroxylase antibody AB152 (1:100, Millipore, Billerica, MA) overnight at 4 • C in blocking solution, followed by washing and incubation in an Alexa 488-conjugated secondary antibody (1:400 in blocking solution). PBS rinsed brains were mounted on microscope slides using Vectashield (Vectorlabs, Burlingame, CA) and visualized on a Zeiss Axio Imager.Z1 using a Colibri Illumination System, a 40× oil objective and Zeiss filter set 38 HE (Carl Zeiss Imaging, Germany). Tyrosine hydroxylase-expressing neurons in protocerebral posterior lateral clusters 1 and 2 (PPL1 and PPL2, respectively) and protocerebral posterior medial clusters 2 and 3 (PPM2 and PPM3, respectively) were counted manually. Student's t-tests were used to explore potential differences in TH-stained cell numbers for each brain region between park 25 heterozygotes and control flies.  25 ("park 25 het") mutation causes increased mortality. (B) Heterozygous park 25 flies raised on 9 g/ml nicotine have increased LT50. (C) Control flies (w 1118 ) raised on 9 or 12 g/ml of nicotine in food have decreased LT50. Gehan-Breslow-Wilcoxon survival curve analysis shows significant differences between curves compared to untreated. Error bars represent the standard error of the mean (SEM). *P < 0.05; **P < 0.01; ****P < 0.0001.

Heterozygous park 25 Drosophila have increased mortality rates
Our study confirms that, like park 25 homozygotes, park 25 heterozygotes have decreased median lifespan compared to control (w 1118 ) Drosophila (Fig. 1A). For each condition, at least 200 flies were maintained on normal food beginning on day 1 post eclosion. Food was changed every other day, and all deaths were recorded. Nicotine had no effect on food consumption based on individual fly weight data (not shown). A Gehan-Breslow-Wilcoxon test was performed to measure differences between survival curves. The LT 50 (median life span) for control flies was 64 days, while the LT 50 for park 25 heterozygotes was 26 days (P < 0.0001).

Nicotine decreases mortality rates in heterozygous park 25 mutants
In order to determine whether nicotine exposure can be protective against increased mortality in this model of PD, at least 200 heterozygous park 25 flies were maintained on 0, 9 or 12 g/ml of nicotine in food beginning on day 1 post eclosion and deaths were recorded every other day when food was changed (Fig. 1B). A Gehan-Breslow-Wilcoxon test was performed to measure differences between survival curves. Nicotine increased LT 50 by 5 days or 15%. As previously noted, the LT 50 for park 25 heterozygotes not exposed to nicotine was 26 days. Continuous exposure to 9 g/ml increased park 25 heterozygote LT 50 values to 31 days (P < 0.05), while 12 g/ml had no effect on median lifespan (LT 50 = 27, P = 0.92).

Nicotine increases mortality rates in control flies
To determine whether the life-prolonging effects of nicotine observed in the park 25 flies could be measured in normal flies, control (w 1118 ) populations were maintained on food containing nicotine from day 1 post eclosion and deaths were recorded every other day ( Fig. 1C). At least 200 flies were maintained on food with or without nicotine for each condition, and a Gehan-Breslow-Wilcoxon test was performed to measure differences between survival curves. As reported above, the LT 50 of control flies raised on 0 g/ml nicotine in food was 64 days. When control flies were exposed to 9 g/ml nicotine, LT 50 decreased to 55 days (P < 0.05). Control flies exposed to 12 g/ml of nicotine had an LT 50 of 50 days (P < 0.01 when compared to 0 g/ml nicotine). Taken together, our results suggest that the beneficial effects of nicotine on mortality are limited to park 25 heterozygotes and that nicotine is dose-dependently toxic to control flies.

Heterozygous park 25 Drosophila have delayed-onset, decreased climbing ability
In order to determine whether having one copy of the park 25 allele was sufficient to decrease climbing behavior, untreated heterozygous park 25 Drosophila were compared to untreated control (w 1118 ) flies in the climbing assay on days 5, 10, 15 and 20 ( Fig. 2A). For each condition, vials containing approximately 10 flies were tested, and the percentage of flies per vial that were able to climb 5 cm was recorded. Data represent the averages of about 20 vials per condition. Student's t-tests were used in order to determine the effects of genotype on each day of assay. One-way ANOVA indicates that climbing capability in control flies remained steady up to day 20 (P = 0.11), while climbing in park 25 heterozygotes was consistently less than that of control after day 5 (55.4 ± 5.6% vs. 60.8 ± 6.6%, P = 0.35 for day 5; 4.7 ± 1.5% vs. 70.9 ± 3.5%, P < 0.0001 on day 10; 7.90 ± 1.9% vs. 73.8 ± 3.0%, P < 0.0001 on day 15; and 25.9 ± 3.4% vs. 70.8 ± 3.8% on day 20, P < 0.0001).

Nicotine protects against climbing deficits in heterozygous park 25 flies
To determine whether nicotine could protect against deficits in climbing ability caused by the park 25 mutation, untreated park 25 heterozygotes were compared to park 25 heterozygotes treated with nicotine (Fig. 2B). For each condition, vials containing approximately 10 flies were tested, and the percentage of flies able to climb was recorded. Data represent the averages of about 20 vials per condition. A one-way ANOVA was performed in order to determine the effects of nicotine treatment on each day of assay. Two-sided Dunnett t-tests were performed in order to determine post hoc significance. Nicotine-induced increases in climbing capacity were Control flies (w 1118 ) have increased climbing on day 5 with both concentrations of nicotine; however, by day 20, nicotine exposure has no effect or decreases climbing behavior. Statistical analyses were performed using Student's t-tests or one-way ANOVA followed by a Dunnett post hoc test. Error bars represent SEM. ***P < 0.001; ****P < 0.0001. observed on all days tested. On day 5, flies raised on 12 g/ml nicotine had increased climbing ability compared to untreated flies (from 55.4 ± 5.6% to 84.2 ± 2.3%, P < 0.0001). By day 10, a significant increase in climbing was observed in park 25 heterozygotes treated with either 9 or 12 g/ml nicotine (from 4.7 ± 1.5% to 31.0 ± 4.3%, P < 0.0001 for 9 g/ml; to 45.2 ± 3.9%, P < 0.0001 for 12 g/ml). On day 15, nicotine exposure increased climbing in park 25 heterozygotes at both concentrations (from 7.9 ± 1.9% to 49.6 ± 5.4%, P < 0.0001 and 39.4 ± 6.4%, P < 0.0001, respectively). Finally, on day 20, only exposure to 9 g/ml of nicotine increased climbing ability from 25.9 ± 3.4% to 50.8 ± 3.2% (P < 0.0001). Student's t-tests show that there is no difference in climbing ability between park 25 heterozygotes raised on 9 g/ml nicotine and untreated control flies on days 15 and 20 (P = 0.25 and 0.15, respectively), indicating that 9 g/ml nicotine rescued climbing ability in heterozygous park 25 flies by day 15 ( Fig. 2A and B). After day 5, exposure to 9 g/ml nicotine consistently increased climbing ability, while the protective effect of continuous exposure to 12 g/ml nicotine decreased from day 10 to 20.
3.6. Exposure to increasing concentrations of nicotine causes a progressive decline in climbing ability in control Drosophila Control (w 1118 ) flies were treated with the same concentrations of nicotine, and their ability to climb was evaluated in order to determine whether nicotine-mediated improvement in climbing observed in the park 25 heterozygotes was specific for deficits caused by the park mutation (Fig. 2C). For each condition, vials containing approximately 10 flies were tested, and the percentage of flies able to climb was recorded. Data represent the averages of about 20 vials per condition. A one-way ANOVA was performed in order to determine the effects of nicotine treatment on each assay day. Two sided Dunnett t-tests were performed in order to determine post hoc significance. On day 5, flies treated with 9 or 12 g/ml nicotine had increased climbing capability compared to untreated flies (from 60.8 ± 6.6% to 88.8 ± 1.9%, P < 0.0001 for 9 g/ml; to 83.5 ± 2.7%, P < 0.0001 for 12 g/ml). By day 10, a significant increase in climbing was observed in control flies treated with 12 g/ml (from 70.9 ± 3.5% to 92.0 ± 1.9%, P < 0.001). On day 20, climbing behavior decreased in flies treated with 12 g/ml (70.7 ± 3.8% to 45.4 ± 5.5%, P < 0.001). Although both nicotine concentrations improved climbing capabilities on day 5, neither was able to sustain this effect, and 12 g/ml nicotine inhibits climbing in control flies treated for 20 days.

Heterozygous park 25 mutants have delayed-onset, progressively decreasing flight ability
In order to determine whether having one copy of the park 25 allele was sufficient to decrease flight capability, untreated heterozygous park 25 Drosophila were compared to untreated control (w 1118 ) flies in the flight assay on days 5, 10, 15 and 20 (Fig. 3A). For each condition, at least 8 vials of about 20 flies were tested, and the percentage of flies per vial that were able to fly was recorded. Onetailed Student's t-tests were performed in order to determine the effects of the mutation. One way ANOVA reveals that flight capability in control flies remained unchanged up to day 20 (P = 0.06), while park 25 heterozygotes had a progressively decreasing trend in flight ability. There was no effect of the park mutation on day 5 (83.6 ± 3.5% vs. 88.7 ± 1.5% for control, P = 0.38). Heterozygous park mutation progressively caused more severe flight deficits on day 10 (68.5 ± 2.9% vs. 87.4 ± 2.5%, P < 0.01), day 15 (64.1 ± 4.2% vs. 92.8 ± 1.9%, P < 0.0001), and day 20 (32.5 ± 4.0% vs. 92.2 ± 2.7%, P < 0.0001).

Nicotine improves flight deficits in heterozygous park 25 flies
Nicotine was continuously administered to park 25 heterozygotes to determine whether it could protect against the degradation in flight ability caused by the heterozygous park 25 mutation (Fig. 3B). Flight capability was recorded on days 5, 10, 15 and 20. For each condition, at least 7 vials of about 20 flies were tested, and the percentage of flies per vial that were able to fly was recorded. A one-way ANOVA was performed in order to determine the effects of nicotine treatment on each day of assay, and Dunnett t-tests were performed in order to determine post hoc significance. On day 5, when there was no effect of the park mutation (Fig. 3A), nicotine did not affect flight (P = 0.83 for flies treated with 9 g/ml,  25 heterozygotes. (C) Prolonged exposure to nicotine decreases flight behavior in control (w 1118 ) flies. Statistical analyses were performed using Student's t-tests or one-way ANOVA followed by a Dunnett post hoc test. Error bars represent SEM, which is very small for some of the data and thus not visible. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. P = 0.36 for those treated with 12 g/ml). By day 15, 12 g/ml nicotine increased flight abilities by about 13% (from 64.1 ± 4.2% to 76.9 ± 3.3%, P < 0.05). On day 20, when the park 25 mutant flight deficiencies are the most severe (32.5 ± 4.0% of untreated flies were capable of flight), nicotine was able to dose-dependently increase flight at both concentrations (51.3 ± 6.1%, P < 0.05 for 9 g/ml and 53.5 ± 6.5%, P < 0.01 for 12 g/ml). As flight in park 25 heterozygotes decreased progressively, nicotine was able to provide more protection against the effects of the mutation.

Nicotine decreases flight ability in control flies
Nicotine was administered to control flies (w 1118 ) to determine its effects on flight ability. For each condition, at least 7 vials of about 20 flies were tested, and the percentage of flies per vial that were able to fly was recorded. A one-way ANOVA was performed in order to determine the effects of nicotine treatment on each day of Statistical analyses were done using Student's t-tests or one-way ANOVA followed by a Dunnett post hoc test. Error bars represent SEM. *P < 0.05; **P < 0.01; ****P < 0.0001 for effect of sex and genotype. + P < 0.05; ++ P < 0.01 for the effect of nicotine compared to untreated flies. assay. Dunnett t-tests were performed in order to determine post hoc significance. Flies were maintained on food containing nicotine from day 1 post eclosion, and flight capability was recorded on days 5, 10, 15 and 20 (Fig. 3C). Nicotine had no effect on flight on day 5 (P = 0.65 and 0.77, for 9 and 12 g/ml respectively) or on day 10 (P = 1.0 and 0.68 for 9 and 12 g/ml, respectively). By day 15, flies raised on 9 g/ml were less capable of flight (79.3 ± 3.1% compared to 92.8 ± 1.9%, P < 0.001). By day 20, 12 g/ml nicotine exposure decreased flight capabilities in control flies (77.6 ± 3.4% compared to 92.2 ± 2.7%, P < 0.05).

Nicotine prevents decreases in olfaction in heterozygous park 25 mutants
On day 5, olfactory assays were performed on male (Fig. 4A) and female (Fig. 4B) heterozygous park 25 and control flies in order to determine whether mutation in the park gene causes olfactory deficits that precede motor deficits in this PD model as it does in humans. Ten flies, male or female, treated or untreated, were assayed in the olfactory trap as detailed in the materials and methods section. Data represent means for at least 10 traps per condition. One-tailed Student's t-tests were performed to determine the effect of the mutation for flies raised on 0, 3, 6, 9 or 12 g/ml nicotine. On day 5, when no climbing or flying deficits were observed in the park 25 heterozygotes, both male and female heterozygous park 25 Drosophila had olfactory deficits (2.7 ± 2.1% and 14.3 ± 7.8%) compared to control (w 1118 ) males and females (45.0 ± 9.2%, P < 0.01 and 95.5 ± 1.7%, P < 0.0001, respectively; Fig. 4A and B). Nicotine treatment prevented olfactory deficiencies in male and female heterozygous park 25 Drosophila in a generally dose-dependent manner ( Fig. 4A and B). Performance in the olfactory assay for female flies raised on 6 g/ml nicotine was not different from that of untreated control flies (49.0 ± 11.4% compared to 84.0 ± 8.8% for control, P = 0.08). Additionally, males and females raised on 12 g/ml nicotine had no olfactory deficits compared to control flies (28.0 ± 8.4% compared to 40.0 ± 9.7%, P = 0.32 for males; 62.0 ± 12.5% compared to 86.1 ± 5.4%, P = 0.10 for females). One-way ANOVA analyses followed by Dunnett t-tests were performed in order to determine the effect of nicotine. Compared to untreated heterozygous park 25 Drosophila, both males and female mutant flies treated with nicotine had increased olfactory function when fed nicotine concentrations of 9 g/ml (from 2.7 ± 2.1% to 22.1 ± 7.5%, P < 0.05 for males; from 14.3 ± 7.8% to 66.0 ± 9.7%, P < 0.01 for females) and 12 g/ml (to 28.0 ± 8.1%, P < 0.01 for males; to 62.0 ± 12.5%, P < 0.01 for females; Fig. 4A and B). There was no effect of nicotine treatment on control flies (P = 0.56 for males, P = 0.50 for females).

Female Drosophila have increased olfactory function compared to males
Because females are larger than males, flies were separated by sex for the olfactory assay because the entry orifice of the olfactory trap is just large enough to allow passage of the respective sex and thus prevent them from leaving. Since the olfactory assay requires motor function to follow olfactory cues, and because motor deficits occur in park 25 heterozygotes after day 5, this assay was only performed on day 5 post eclosion. The necessary separation by sex allowed for the observation of an interesting difference in olfaction between sexes. Unpaired student's t-tests were performed to determine differences in olfaction between sexes at each nicotine concentration. As seen in Fig. 4A and B, control (w 1118 ) females performed better than control males when untreated as well as when raised on all concentrations of nicotine (the difference between means are: 50.5% at 0 g/ml, P < 0.0001; 52.8% at 3 g/ml, P < 0.0001; 26.1% at 6 g/ml, P < 0.05; 42.0% at 9 g/ml, P < 0.001; 46.1% at 12 g/ml; P < 0.001).

park 25 heterozygote pathology does not result from loss of tyrosine hydroxylase-expressing cells
At least 10 brains were dissected from control and park 25 heterozygous flies on day 20 post-eclosion. TH-expressing cells in the protocerebral posterior lateral regions 1 and 2 (PPL1 and PPL2, respectively) and protocerebral posterior medial regions 2 and 3 (PPM2 and PPM3, respectively) were counted. There are no differences in TH-labeled cell number for park 25 heterozygotes vs. control in any brain region when analyzed with unpaired Student's t-test (Fig. 5). For PPL1, control flies (w 1118 ) and park 25 heterozygotes had an average of 12.9 and 13.0 cells, respectively (P = 0.85). Control flies had an average of 9.5 TH-labeled cells in PPL2, while park 25 heterozygotes had a mean of 9.2 (P = 0.30). The mean number of TH-labeled cells in PPM2 was 10.3 and 10.7 for control flies and park 25 heterozygotes, respectively (P = 0.31). The mean number of TH-labeled cells in PPM3 was 11.6 and 11.9 for control flies and park 25 heterozygotes, respectively (P = 0.40).

Discussion
We have shown that heterozygous park 25 flies have decreased median life spans as well as motor and olfactory deficits, suggesting that heterozygous Parkin loss-of-function mutations can produce a mutant phenotype. Results from climbing and flying assays indicate that park 25 heterozygotes have delayed onset of motor deficits and that flight deficits are progressive. Increased mortality and climbing and flying deficiencies have been reported in homozygous park 25 mutants; however, olfaction has not been explored in these flies. Our results indicate that heterozygous park 25 mutants have olfactory deficits that precede motor symptoms. Our evidence also suggests that chronic nicotine exposure from day one post eclosion increases lifespan, improves flight and prevents climbing and olfactory deficiencies in park 25 heterozygotes. These improvements are not observed in control flies, suggesting that nicotine is selectively protective against the Parkin loss-of-function phenotype in our model.
Homozygous park mutants have phenotypes related to those seen in PD patients including decreased life span, late onset progressive motor deficiencies and neuronal degeneration [1,2,21], which are thought to result from mitochondrial abnormalities [1,2]. Most cases of AR-JP are caused by homozygous PARK2 mutations; however, several studies report that AR-JP caused by PARK2 mutation can occur in a dominant inheritance pattern [22][23][24][25]. In Drosophila, it appears that Parkin loss-of-function causes a dose-dependent phenotype. A previous study corroborates our observations that park 25 heterozygotes have significantly shorter life spans than control flies [1]. Climbing and flying indices for homozygous park 25 mutants have been shown to be decreased compared to control, but since methods of measuring climbing and flight differ in this study, comparing abilities between homozygous and heterozygous park 25 mutants is difficult. Notably, climbing and flight deficits in homozygous park 25 mutants appear before day 5 [1], while our results indicate that decreases in climbing and flight abilities in heterozygous park 25 mutants occur after day 5, substantiating the possibility that being homozygous for park mutations causes a more severe phenotype in Drosophila. Evidence suggests that the pathological phenotype in homozygous park 25 flies results from decreased mitochondrial function, which leads to a loss of dopaminergic cells in the fly brain [2,21]. We did not detect a loss of dopaminergic cells in the PPL1, PPL2, PPM2 or PPM3 neuronal clusters of 20 day heterozygous park 25 mutants. Perhaps this is because park 25 heterozygote pathology is less severe, and as such, the milder mitochondrial pathology may affect the function but not the viability of dopaminergic cells. Alternatively, the decrease in PPL1 dopaminergic cells in park 25 homozygotes [21], while statistically and likely physiologically significant, is only a matter of a few cells. The potential loss of one or two of these cells in park 25 heterozygotes, which have a milder pathology, would be difficult to detect.
Although there appears to be no difference in olfaction between male and female Drosophila when sex pheromones are used as chemoattractants [30], we have shown that control male flies perform much worse than females in the olfactory assay when standard food is used as a chemoattractant on day 5 post eclosion. park 25 heterozygotes perform worse than controls of the same sex. This is an important phenotype in this model because a potentially confounding factor in therapeutic strategies for PD is that between 50% and 80% of dopaminergic neurons in the substantia nigra pars compacta have degenerated when observable motor symptoms appear [26,27]. Early detection and treatment may be critical in halting or decreasing the rate of degeneration in PD. Because olfactory assays were performed before climbing deficits were observed, we can assume that decreased entry of park 25 heterozygotes into the olfactory trap was due to decreased olfaction and not to decreased climbing activity. Thus, as in PD patients, park 25 heterozygotes have olfactory deficits that precede disturbances in motor behavior. Therefore, it may be possible to utilize this model to identify beneficial therapies following olfaction loss but prior to motor deficits.
Apoptotic cell death due to mitochondrial abnormalities is thought to cause inferior flight muscle fiber and dopaminergic neuron degeneration in homozygous park 25 flies [1,2,21]. Additionally, dopaminergic neurons in homozygous park 25 Drosophila seem to be selectively sensitive to disruptions in mitochondrial fission caused by Parkin loss-of-function [2,21]. Studies done in cultured mammalian cells and in Drosophila suggest that Parkin functions downstream of phosphotensin-induced kinase 1, or PINK1 [31,32] to mediate mitochondrial fission events that precede mitophagy when the mitochondrial membrane potential dissipates [33][34][35]. The loss of a cell's ability to dispose of malfunctioning, burdensome mitochondria may trigger the initiation of apoptosis in AR-JP caused by Parkin or PINK1 loss-of-function. Although it is likely that the deficits we have observed in park 25 heterozygotes result from the same mechanism of pathology, we have not yet explored this possibility. The role of Parkin as a mediator of mitochondrial fission appears to be conserved in humans, as fibroblasts harvested from homozygous mutant PARK2 patients have elongated mitochondria [36]. Although Parkin and PINK1 are expressed throughout the human brain, dopaminergic cells may be particularly sensitive to mitochondrial malfunction [37,38]. Drosophila dopaminergic neurons and inferior flight muscles also may be particularly sensitive to the loss of Parkin function.
The protection from PD that seems to be offered by tobacco smoking has been at least partly attributed to neuroprotective properties of nicotine, which has been shown to maintain cell number and cellular homeostasis in sporadic models of PD by modulation of nicotinic acetylcholine receptors [6,13] or via nicotinic acetylcholine receptor-independent mechanisms [14][15][16]. A previous study has demonstrated that 0.1 g/ml of nicotine in food could not prevent dopaminergic cell loss in park 25 homozygotes [18]. Our mortality, climbing, flying and olfaction assay results suggest that therapeutic nicotine concentrations are higher. However, one possibility that we have not pursued is that nicotine treatment may be more effective in the less severe heterozygous park 25 phenotype. Results from our climbing and olfactory assays suggest that while pretreatment with both 9 and 12 g/ml nicotine can improve performance, 9 g/ml may be the optimal dose, since the protective effect of nicotine diminishes with 12 g/ml exposure. In this study, the beneficial effects of nicotine are limited to park 25 heterozygotes; in fact, 9 and 12 g/ml nicotine treatment reduced median lifespan in control flies. There was no effect observed with 9 g/ml nicotine on climbing behavior in control flies after day 5, and 12 g/ml nicotine reduced climbing and flying in this population by day 20. In general, the beneficial effects of 12 g/ml nicotine decreased over time, perhaps indicating a long-term change in receptor availability and/or sensitivity. Drosophila have ten putative nicotinic acetylcholine receptor (DnAChR) subunit encoding genes, three of which share up to 45% protein sequence identity with the human ␣7 nAChR subunit [39]. Like mammalian nAChR, DnAChR modulate fast neurotransmission by binding acetylcholine. Nicotine, a powerful insecticide, has been shown to displace binding of alpha7-containing nAChR selective antagonist ␣-bungarotoxin in Drosophila head homogenates [40]. Nicotine also elicits an electrophysiological response in cultured Drosophila cholinergic neurons [41]. We must also consider the possibility that nicotine may provide protection in this model by affecting mitochondrial function in a nAChR-independent fashion. Therefore, future studies will address whether nicotine pretreatment protects against this mutant phenotype via modulation of DnAChR or via DnAChRindependent mechanisms.
Literature addressing whether cognitive and motor symptoms in PD patients improve following various nicotine administration regimens is inconclusive [42][43][44][45]. Since advanced neurodegeneration has occurred by the time motor symptoms are observable, this type of intervention may be ineffective when implemented after diagnosis. Tobacco smokers who benefit most from the putatively protective properties of nicotine have been exposed to it for decades prior to PD diagnosis. For this reason, we chose to administer nicotine continually from day one, post eclosion. Identification of AR-JP patients requires a simple genetic test; thus, nicotine treatment could begin well before symptoms appear. Early diagnosis of sporadic PD remains an obstacle to early intervention. Although insufficient for diagnosis, olfactory deficits are common in PD patients, and as with our model, they typically precede motor symptoms. We believe identification of olfactory deficits in sporadic PD patients may aid in early diagnosis and allow for earlier treatment and potential benefit via nicotine therapy.