UC San Diego

Alcohol use disorder (AUD) has major societal impact and few utilized treatments. New molecular targets with therapeutic potential and better understanding of the neurobiology of AUD are needed. Chronic ethanol exposure and consumption can lead to neurobiological changes in the brain’s reward and motivation systems that are viewed as that perpetuate harmful drinking. The development of compulsive drinking putatively involves a shift in striatal control from the NAc to the dorsomedial and then dorsolateral striatum as well as an imbalance between the dopamine receptor D1-direct and D2-indirect medium spiny neuron (MSN) pathways, whereby overactive dMSNs increase ethanol seeking and drinking behavior and underactive iMSNs putatively reduce inhibitory self-regulation. PDE10A is an enzyme of the phosphodiesterase enzyme family, a class of enzymes that regulate cellular responses by hydrolyzing cAMP/cGMP. PDE10A appears to encode the main PDE isoforms that control cAMP and cGMP levels of MSNs in the striatum, and the majority of PDE10A in the mouse striatum is the membrane-enriched PDE10A2 isoform. Recent studies suggest that inhibition of PDE10A family isoforms with different selective inhibitors can reduce alcohol selfadministration in models of excessive or dependent intake, which has raised interest in PDE10A inhibitors as potential treatments for AUD. AMG 579 is a small molecule potent, selective PDE10A inhibitor that recently showed promise for its drug-like physiochemical properties (e.g., lipophilic efficiency) and ability to dose-dependently reduce ethanol consumption in rats. However, it is unknown how AMG 579 alters activation of striatal D1-direct and D2-indirect MSN pathways at doses that are considered effective to reduce drinking as well as whether there is regional variation or sex differences in these effects. There also is uncertainty as to the specific role of the PDE10A2 isoform in the anti-drinking or molecular actions of PDE10A inhibitors like AMG 579. I decided to test the hypothesis that Pde10a2-knockout mice would show increased expression of Drd1, a direct pathway MSN marker, and the immediate-early genes (IEG) Egr1 and Fos as compared to wild-type (WT) mice reflecting decreased regulation in cAMP due to Pde10a2-knockout (KO). I also hypothesized that treatment with the PDE10A inhibitor AMG 579 would increase IEG expression, similar to what was observed previously with the PDE10A inhibitors MR1916 and TAK-063. Further, I hypothesized that treatment effects seen on IEG expression would be in wild-type mice only, which would indicate that the membrane-bound PDE10A2 isoform was involved in the ability of the PDE10A inhibitor to alter MSN activation, and thus absent in Pde10a2-knockout mice. These results were expected to be more pronounced in the dorsal striatum than the NAc, because PDE10A mRNA and protein are expressed at greatest levels in the caudate-putamen, and in male animals more their female counterparts, due to previously reported sex differences in synaptic striatal PDE10A levels and activation effects of the PDE10A inhibitor papaverine in vitro. MSN marker expression was measured using two methods: qPCR to measure overall Drd1, Drd2, Penk, Fos, and Egr1 expression and a method of fluorescent in-situ hybridization known as RNAscope to measure Drd1, Drd2 and Egr1 expression within Drd1- and Drd2- positive cell types. qPCR results show that both treatment with AMG 579 and Pde10a2- knockout decreased expression of Drd1, Drd2, Fos, and Egr1. Pde10a2-knockout seemed to reduce Penk expression as well. There were trends that suggested that there was a stronger reduction in Drd2 expression in AMG 579 KO mice in males than females but not in expression in Pde10a, Drd1, or either of the IEGs. RNAscope results showed that Egr1 expression within Drd1- and Drd2-expressing cells types as well as undefined cell types decreased as a result of treatment with AMG 579. The DMS and, to a lesser extent, the DLS, showed greater significance in Treatment effect than the NAc. Treatment with AMG 579 seemed to have a sexually dimorphic effect in the DMS that varied by gene target or cell type. Overall, the results show that AMG 579 acts differently than other PDE10A inhibitors in that it reduces, instead of increases, MSN marker and IEG expression; further study is needed to determine which method is better suited to modulate problematic drinking behavior