Miles Trupp et al. (2002) West Coast Worm Meeting "A Genetic Selection for Amphetamine Resistance"

Miles Trupp, Steven L McIntire

[West Coast Worm Meeting, 2002]

We are using amphetamine (AMPH) as a pharmacological agent to study monoaminergic function in C. elegans. Previous studies in vertebrates have shown that AMPH can bind and inhibit the plasma membrane reuptake transporters for dopamine, serotonin and norepinephrine, resulting in a prolonged increase in amine transmitters in the synaptic cleft. Also, AMPH has been shown to block the uptake of dopamine by the C. elegans plasma membrane transporter (ceDAT) in vitro (Jayanthi, L.D. et al., Mol Pharm 54:601-9). In worms, AMPH inhibits both pharyngeal pumping and egg-laying, which correlates well with the behavioral effects of exogenous dopamine, but not serotonin. Chronic exposure to 5mM AMPH from early larval stages results in a dramatic slowing of the rate of development. We have used EMS mutagenesis and a genetic selection for resistance to chronic AMPH in order to identify mutants which may be altered in monoaminergic function. From an initial screen of 4500 genomes, 12 mutants were isolated which show faster growth on 5mM AMPH relative to N2. Isolated mutants demonstrate varying degrees of resistance to developmental delay, with some mutants growing at nearly wild-type rates on 5mM AMPH. Also, some of the mutants isolated in the chronic AMPH screen show resistance to the acute effects of AMPH on both egg-laying and pharyngeal pumping. We have also begun to analyze the responsiveness of these mutants to exogenous dopamine and serotonin and are attempting to genetically map those mutations which result in altered post-synaptic function.

Miles Trupp et al. (2001) International C. elegans Meeting "Steps Toward Solving the Drug Problem in Worms"

Miles Trupp, Steven L McIntire, Lucinda Carnell

[International C. elegans Meeting, 2001]

Amphetamine (AMPH) and its methylated derivatives are among the most widely abused illegal drugs. These compounds are known to inhibit the reuptake of monoamines by various plasma membrane transporters; and the rewarding and addictive properties are thought to be mediated by an increase in synaptic monoamines. We are using a genetic screen in C. elegans to further characterize the monoaminergic signalling pathways in worms and possibly to identify additional sites of action of psychostimulants that may retain a conserved function in vertebrates. Toward this end, we have begun to characterize the behavioral modifications seen with chronic and acute treatments of AMPH. Based upon the inhibition of reuptake of multiple monoamine transmitters, it is expected that AMPH would have myriad effects upon C. elegans behavior; and indeed AMPH does have inhibitory effects upon locomotion, pharyngeal pumping, egg laying and the overall rate of development. Within a single behavior, AMPH exhibits distinct concentration dependent effects. For instance, egg-laying of drug naïve wild-type worms on AMPH containing plates is stimulated at lower concentrations during the initial 15-30 minutes but inhibited immediately at higher concentrations. Locomotion is also suppressed with increasing dosage to the point of paralysis, while pre-treatment with a lower dose results in uncoordinated movement. Pharyngeal pumping is also dramatically suppressed by acute AMPH, and perhaps consequently, chronic treatment with AMPH from embryonic or L1 stages slows the rate of development to adulthood. The inhibitory effects seen in locomotion, egg-laying and pumping upon acute exposure to amphetamine are reversible, and the length of time to recovery is dose dependent. Importantly, cat-1 (vesicular monoamine transporter) and cat-2 (tyrosine hydroxylase) mutants are partially resistant to some of these AMPH effects. We have initiated genetic selections for resistance to AMPH, focusing on the selection of mutants that can escape the AMPH induced slowing of the rate of development. Screening of 4500 EMS mutagenized haploid genomes yielded 12 mutants that exhibit varying degrees of resistance to AMPH. Classes of mutants include those that exhibit hyperforaging behavior as well as mutants that exhibit increased egg-laying at normally inhibitory concentrations of AMPH. Genetic mapping and molecular characterization of these loci may further our understanding of the mechanism of action of psychostimulants.

Miles Trupp et al. (2005) International Worm Meeting "Amphetamine Suppression of Feeding and Growth in C. elegansRequires the Membrane Protein Encoded by egl-28."

Steven L McIntire, Miles Trupp

[International Worm Meeting, 2005]

Amphetamine and its derivatives are a class of psychostimulants with wide-ranging therapeutic uses which are limited by the potential for abuse and toxicity. While it is well established that synaptic increases in amine neurotransmitters are responsible for rewarding and addictive properties of amphetamine, other effects, including the paradoxical calming of ADHD patients, appetite suppression and neuronal toxicity are poorly understood. To identify additional targets of amphetamine, we have used a genetic selection to identify amphetamine resistant mutants based upon the suppression of growth by chronic amphetamine. One isolated mutant, egl-28(eg814),exhibits pronounced resistance to amphetamine inhibition of growth and is also resistant to acute and chronic inhibition of pharyngeal pumping by amphetamine. SNP mapping, cosmid rescue and RNAi studies indicate that egl-28encodes a 12 transmembrane spanning domain protein related to the nose-resistant to fluoxetine (prozac) mutants nrf-6and ndg-4.Blast searches of genome databases identify a large family of C. elegansputative transport proteins that has likely expanded from a single ancestral gene which is conserved in vertebrate genomes. egl-28is expressed in neurons, intestine and head muscle of C. elegans.Tissue specific expression and phenotypic analysis indicate that egl-28functions in neurons and muscle to regulate normal behavior as well as sensitivity to amphetamine. Similar to nrf-6and ndg-4mutants, egl-28animals exhibit strong resistance to fluoxetine-induced nose contraction. egl-28animals are also resistant to fluoxetine induced egg-laying, but respond normally to exogenous serotonin. Since three members of this family exhibit overlapping expression patterns and function, this suggests that the vertebrate orthologue may also mediate certain actions of fluoxetine and amphetamine. Further studies including biochemical assays using amphetamine and fluoxetine will be carried out to characterize the function of this family of proteins in worms and vertebrates.

Miles Trupp et al. (2004) West Coast Worm Meeting "A Putative Transporter Related to NRF-6 and NDG-4 is Required for Amphetamine Sensitivity in C. elegans."

Miles Trupp, Steven L McIntire

[West Coast Worm Meeting, 2004]

Amphetamine is a strong psychostimulant that is widely abused. Amphetamine also has wide ranging therapeutic uses that are limited by its neurotoxic and addictive properties. Some of the neurobehavioral effects of amphetamine are mediated by inhibition of synaptic transporters responsible for reuptake of the brain neurotransmitters dopamine and serotonin. However, other behavioral and neurotoxic effects of amphetamine have not clearly been linked to altered monoamine reuptake. To identify additional targets of amphetamine, we have used a genetic selection to isolate amphetamine-resistant mutants. One of the mutants, eg814 , exhibits strong resistance to amphetamine inhibition of growth as well as resistance to behavioral effects of the drug. SNP mapping, cosmid rescue and RNAi studies indicate that eg814 encodes a multi-pass transmembrane protein related to a class of putative transporters defined by the C. elegans fluoxetine (Prozac) resistant mutants nrf-6 and ndg-4 . Members of this family are found in other invertebrate and vertebrate genomes. eg814 is expressed in neurons, intestine and head muscle, and tissue specific expression and behavioral analysis indicate that eg814 functions in neurons. Members of the nrf-6 family of putative transporters may be physiological targets of amphetamine in C. elegans and other systems. Further studies including transport assays will be carried out to characterize the function of this family of proteins in worms and vertebrates.