A large number of environmental toxins are neurotoxic, and may increase the risk of PD (Chou et al., 2008
; Costello et al., 2009
; Di Monte et al., 2002
). The cytotoxic effects of DA may also play a role in the survival of DA neurons and the pathophysiology of PD (Caudle et al., 2007
; Graham, 1978
; Kuhn et al., 1999
; Stokes et al., 1999
; Ritz et al., 2009
). However, despite much speculation on this topic, the actual interplay between pesticides and genes such as DAT and VMAT that regulate cellular DA remains poorly understood, and the notion that VMAT might alter the neurotoxic effects of pesticides in an animal model had not been investigated. We have used the genetic model organism Drosophila melanogaster
and DVMAT to directly test this hypothesis.
Dopamine homeostasis is controlled by VMAT in both mammals (Fon et al., 1997
; Liu et al., 1992
; Mooslehner et al., 2001
; Takahashi et al., 1997
; Wang et al., 1997
) and the fly (Chang et al., 2006
; Greer et al., 2005
). We have previously shown that overexpression of DVMAT protein in flies can decrease DA cell death caused by the overexpression of a human mutant form of parkin
(Sang et al., 2007
). In addition, RNAi directed against dVMAT
modestly exacerbated the parkin
overexpression phenotype (Sang et al., 2007
). We have recently characterized a null dVMAT
loss of function mutation, which provides an even more potent method than RNAi for reducing dVMAT
expression (Simon et al., 2009
). Here we have explored the influence of dVMAT
on the neurotoxic effects of pesticides, using both the loss of function dVMAT
mutant and DVMAT overexpression.
We find that dVMAT
mutants contain fewer DA neurons than wildtype at eclosion (the onset of the adult phase) in the absence of toxin exposure. Interestingly, this appeared to be the case from the earliest adult time point, more consistent with a developmental rather than a degenerative effect. In contrast, VMAT2 knockdown mice retaining ~20% of wild type VMAT2 levels show a decrease in DA neurons with age, but wild type numbers during development (Caudle et al., 2008
). To our knowledge, similar stereological measurements have not been performed in VMAT2 knockouts, which die soon after birth (Fon et al., 1997
; Takahashi et al., 1997
; Wang et al., 1997
). Our data suggest the possibility that a complete loss of VMAT2 activity in mice might decrease the number of DA in mice as it does in flies.
mutants show a greater cell loss than wild type flies when treated with either one of two pesticides, rotenone and paraquat, both of which have been used previously in animal models of DA cell death (Betarbet et al., 2000
; Chaudhuri et al., 2007
; Coulom and Birman, 2004
; McCormack et al., 2002
). Conversely, overexpression of DVMAT blocks DA cell death caused by rotenone. Inhibition of a mammalian VMAT with reserpine has been shown to increase the cytotoxicity of rotenone in cultured cells (Sai et al., 2008
). However, our data are the first to show that VMAT expression can influence the toxic effects of pesticides in vivo
, and together with previous studies (Park et al., 2007
; Sang et al., 2007
), strongly support the idea that VMAT, and by extension the sequestration of cellular DA, can play a neuroprotective role for DA neurons in an intact organism.
The preservation of DA neurons in DVMAT-overexpressing flies treated with rotenone appears to represent a gene x environment interaction since the observed effects are not additive: DVMAT-overexpression alone does not increase the number of DA neurons, and its effects on DA cell number are only observed when rotenone is administered. In contrast, it is not yet clear whether the interaction of the dVMAT loss of function allele is additive or synergistic. Since the baseline number of DA cells in the mutant is lower, the percentage of cells that are lost in mutant flies is consistently greater than wild type. In contrast, if we measure the absolute number of cells that are lost, then the mutant’s cell loss in response to pesticide exposure is generally similar to wild type (however, see ). Additional experiments will be needed to determine whether the mutants are truly more sensitive than the wild type to pesticides, or alternatively, whether DVMAT expression might compromise the health of DA neurons via two independent mechanisms.
It is conceivable that DVMAT provides neuroprotection against rotenone by sequestering it into synaptic vesicles, thereby preventing its access to mitochondria. However, rotenone is structurally quite distinct from biogenic amines or other known VMAT substrates, such as the toxin MPP+
or amphetamines. Rather, the simplest interpretation of our results is that cytosolic DA can interact with rotenone to increase its cytotoxic potential, and that VMAT functions as a neuroprotectant by decreasing cytosolic DA concentrations. Rotenone inhibits complex I of the mitochondrial respiratory chain (Betarbet et al., 2000
; Chance et al., 1963
; Panov et al., 2005
), but neurotoxicity may also be influenced by its ability to disrupt microtubules (Feng, 2006
), and at least one study has questioned the importance of complex I for the neurotoxic effects of rotenone in DA cells (Choi et al., 2008
). Although it remains unclear which of these mechanisms is correct, one common outcome may be an increase in oxidative stress on the cell, or if both complex I and microtubules are impaired, a more specific increase in the cell body (Feng, 2006
; Ren et al., 2009
). Cytosolic DA would be expected to potentiate this effect (Guillot and Miller, 2009
; Hastings et al., 1996
; Michel and Hefti, 1990
; Mosharov et al., 2009
), and the convergence of these two weak oxidative stressors might explain the relative specificity of rotenone to kill DA neurons.
The developmental loss of DA neurons due to loss of VMAT activity has not been reported previously, and as noted above may not be due to same mechanism by which DVMAT influences the animal’s response to pesticides. It is possible that an unknown exogenous toxin interacts with DA during development. Alternatively, the ability of DA cells to protect themselves against oxidative stress (or other effects of DA) during development may be relatively weak, such that additional toxins are not needed. Finally, it is conceivable that DA cell loss in the mutant is a non-cell autonomous effect caused indirectly by the loss of serotonergic or octopaminergic signaling, a hypothesis that can be tested using genetic rescue in specific aminergic cell types. Regardless of the precise mechanism, our findings raise the possibility that dVMAT mutant flies might be used to further study how the health of DA cells early in life can influence their function at later time points. Importantly, for PD patients, it is not known how the premorbid function of DA neurons may influence the pathogenesis of PD later in life.
The death of DA neurons in DVMAT overexpression flies exposed to paraquat demonstrates the limitations of VMAT’s neuroprotective effects, and may also reflect differences between the neurotoxic mechanisms of paraquat versus rotenone. If DA functions primarily as an oxidant, it would suggest that the neuroprotective effects of DVMAT might be overwhelmed by toxins with a greater oxidative potential than rotenone. Paraquat is thought to cause a massive increase in oxidative stress (Bus and Gibson, 1984
; Przedborski and Ischiropoulos, 2005
) and it is likely that the cellular target(s) of paraquat and rotenone are different (Ramachandiran et al., 2007
). The high oxidative potential of paraquat may supersede whatever contribution cytosolic DA might have on the overall oxidative stress experienced by a DA cell. Thus, at least in the overexpression experiments we show here, reducing the weak oxidative stress caused by dopamine may be inconsequential in face of the more potent effects of paraquat.
However, it is also possible that the rapid administration of paraquat was the most important factor in its inability to interact with DVMAT overexpression. Ideally, rotenone and paraquat would be administered similarly, and both assayed for relatively long-term (days to weeks) rather than acute (hours to days) neurodegenerative effects. In our hands, low dose (2 mM) paraquat exposure for four weeks did not cause a detectable loss of DA neurons (Supplemental Fig. 4
). We therefore used acute exposure to high dose paraquat as previously reported (Chaudhuri et al., 2007
) with the resultant loss of DA cells occurring over the course of hours. Thus, it is possible that over-expression of DVMAT failed to provide neuroprotection against paraquat because cell death occurred too rapidly, rather than because of a higher oxidative potential than rotenone. Further experiments will be needed to determine how the rate of cell death, versus the intrinsic activities of paraquat and rotenone affect the ability of DVMAT to provide neuroprotection.
In particular, it will be important to determine how DVMAT may influence the oxidative stress in DA neurons caused by each agent, and the time course of these effects. At least one recent study testing the effects of expressing PD-related gene in DA cells used immunolabeling to measure oxidative changes in these cells (Imai et al., 2008
). Ingestion of pesticides is likely to cause a general increase in oxidative stress throughout the brain, rather than specifically in DA neurons. Under these conditions, the detection of oxidative changes in DA neurons may be difficult, and require the use of other biochemical methods.
Regardless of the precise mechanisms by which rotenone and parquat kill DA cells, the difference we observe between the effects of dVMAT
on paraquat versus rotenone underscore the important idea that its potential for neuroprotection must be considered within the context of both the specific type of toxin and the manner of exposure. This idea may be relevant to future gene-environment interaction studies in mammalian systems, and for epidemiological data on the potential effects of polymorphisms in the human VMAT2 gene (Glatt et al., 2006
). If we can extrapolate from our data generated in flies, it would suggest that polymorphisms that increase the expression of VMAT2 might confer neuroprotection only in people exposed to specific pesticides, or alternatively only to relatively low-dose, chronic exposures rather than higher-dose, acute exposures. To test this hypothesis, epidemiological studies of PD may be used to correlate the exposure to specific pesticides and expression of particular VMAT2 haplotypes. One potentially important comparison could be the high-dose exposure of farm workers who periodically handle pesticides directly, versus others chronically exposed to lower levels of pesticides.
We have explored the possibility of using two other pesticides (ziram and benomyl) to model DA cell death in the fly. Both may increase the risk of PD in exposed populations (Ritz personal communication) and show cytotoxicity in vitro
(Chou et al., 2008
; Wang et al., 2006b
). However, feeding high-dose ziram has no effect on fly survival. We therefore believe that it is unlikely to be useful for more specific studies of neurotoxicity in the fly, at least using our current exposure paradigm. Benomyl shows a weak effect at high dose suggesting that it is capable of at least some toxic effects in flies, but does not affect climbing ability, limiting the likelihood that it could be used as a model for neurotoxicity. It is possible that limited absorption or other pharmacokinetic parameters can reduce the toxicity of these agents. Alternatively, their toxicity may be revealed in Drosophila
only with additional genetic risk factors. Here we did not pursue either toxin for more specific studies on DA cell death.
To facilitate our neurotoxicity studies, we have optimized the rotenone exposure paradigm, starting with methods published by others using a relatively short exposure (Coulom and Birman, 2004
). We have modified the paradigm such that flies are exposed to rotenone for ten days, then cultured on normal food for six weeks. Using this protocol, we observe an increase in DA cell death relative to a more acute (ten day) exposure paradigm. The more robust loss of DA cells has helped to demonstrate the neuroprotective effects of DVMAT, and may be useful for other studies of neurodegeneration in the fly.
In addition to directly counting DA neurons, we have used survival and behavioral tests following exposure of flies to pesticides. DVMAT overexpression may slightly increase survival in flies exposed to paraquat, but does not increase the survival of flies exposed to rotenone as compared to the UAS-DVMAT
transgene alone. These data indicate that DVMAT and its expression in dopaminergic cells are limited in their ability to mitigate effects of pesticides in the organism as a whole. In contrast, other genes such as DJ1 and pink may have cytoprotective effects in a variety of cell types, and their absence can markedly decrease survival in animals exposed to pesticides (Clark et al., 2006
; Meulener et al., 2005
). Studies in Drosophila
of these, more widely expressed genes are likely to enhance our understanding of basic cellular mechanisms of neurodegeneration relevant to PD. In contrast, future mechanistic studies of DVMAT will help determine how DA contributes to the remarkable sensitivity of dopaminergic cells to environmental as well as genetic insults. In addition, if VMAT is indeed able to protect DA cells against some pesticides, we speculate that genetic or drug screens in Drosophila
may be useful to develop strategies to increase VMAT activity and thus reduce toxin-induced DA cell death.