The Drosophila JIL-1 kinase is known to phosphorylate histone H3 at Ser10 (H3S10) during interphase. This modification is associated with transcriptional activation, but its function is not well understood. Here we present evidence suggesting that JIl-1-mediated H3S10 phosphorylation is dependent on chromatin remodeling by the brahma complex and is required during early transcription elongation to release RNA polymerase II (Pol II) from promoter-proximal pausing. JIL-1 localizes to transcriptionally active regions and is required for activation of the E75A ecdysone-responsive and hsp70 heat-shock genes. The heat-shock transcription factor, the promoter-paused form of Pol II (Pol IIo ser5 ), and the pausing factor DSIF (DRB sensitivity-inducing factor) are still present at the hsp70 loci in JIL-1-null mutants, whereas levels of the elongating form of Pol II (Pol IIo ser2 ) and the P-TEFb kinase are dramatically reduced. These observations suggest that phosphorylation of H3S10 takes place after transcription initiation but prior to recruitment of P-TEFb and productive elongation. Western analyses of global levels of both forms of Pol II further suggest that JIL-1 plays a general role in early elongation of a broad range of genes. Taken together, the results introduce H3S10 phosphorylation by JIL-1 as a hallmark of early transcription elongation in Drosophila.

Dopamine D2 receptor antagonism is a unifying property of all antipsychotic drugs in clinical use for schizophrenia. While often effective at ameliorating psychosis, these drugs are largely ineffective at treating negative and cognitive symptoms. Increasing attention is being focused on the complex genetics of the illness and the signaling pathways implicated in its pathophysiology. We review targeted approaches for pharmacotherapy involving the glutamatergic, GABAergic and cholinergic pathways. We also describe a number of the major genetic findings that identify signaling pathways representing potential targets for novel pharmacological intervention. These include genes in the 22q11 locus, DISC1, neuregulin/ERB4, and components of the Akt/GSK-3 pathway.

Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMAT's role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H+ antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects.

The dopamine transporter (DAT) is the primary molecular target responsible for the rewarding properties of the psychostimulants amphetamine (AMPH) and cocaine. AMPH increases extracellular dopamine (DA) by promoting its nonexocytotic release via DAT-mediated efflux. Previous studies in heterologous cells have shown that phosphorylation of the amino terminus of DAT is required for AMPH-induced DA efflux but not for DA uptake. However, the identity of many of the modulatory proteins and the molecular mechanisms that coordinate efflux and the ensuing behavioral effects remain poorly defined. Here we establish a robust assay for AMPH-induced hyperlocomotion in Drosophila melanogaster larvae. Using a variety of genetic and pharmacological approaches we demonstrate that this behavioral response is dependent on DA and on DAT and its phosphorylation. We also show that methylphenidate (MPH), which competitively inhibits DA uptake but does not induce DAT-mediated DA efflux, also leads to DAT-dependent hyperlocomotion, but this response is independent of DAT phosphorylation. Moreover, we demonstrate that the membrane raft protein Flotillin1 is required for AMPH-induced but not MPH-induced hyperlocomotion. These results are the first evidence of a role for a raft protein in an AMPH-mediated behavior. Thus, using our assay we are able to translate molecular and cellular findings to a behavioral level and to differentiate in vivo the distinct mechanisms of two psychostimulants.