DARPP-32 Is a Robust Integrator of Dopamine and Glutamate Signals

Integration of neurotransmitter and neuromodulator signals in the striatum plays a central role in the functions and dysfunctions of the basal ganglia. DARPP-32 is a key actor of this integration in the GABAergic medium-size spiny neurons, in particular in response to dopamine and glutamate. When phosphorylated by cAMP-dependent protein kinase (PKA), DARPP-32 inhibits protein phosphatase-1 (PP1), whereas when phosphorylated by cyclin-dependent kinase 5 (CDK5) it inhibits PKA. DARPP-32 is also regulated by casein kinases and by several protein phosphatases. These complex and intricate regulations make simple predictions of DARPP-32 dynamic behaviour virtually impossible. We used detailed quantitative modelling of the regulation of DARPP-32 phosphorylation to improve our understanding of its function. The models included all the combinations of the three best-characterized phosphorylation sites of DARPP-32, their regulation by kinases and phosphatases, and the regulation of those enzymes by cAMP and Ca2+ signals. Dynamic simulations allowed us to observe the temporal relationships between cAMP and Ca2+ signals. We confirmed that the proposed regulation of protein phosphatase-2A (PP2A) by calcium can account for the observed decrease of Threonine 75 phosphorylation upon glutamate receptor activation. DARPP-32 is not simply a switch between PP1-inhibiting and PKA-inhibiting states. Sensitivity analysis showed that CDK5 activity is a major regulator of the response, as previously suggested. Conversely, the strength of the regulation of PP2A by PKA or by calcium had little effect on the PP1-inhibiting function of DARPP-32 in these conditions. The simulations showed that DARPP-32 is not only a robust signal integrator, but that its response also depends on the delay between cAMP and calcium signals affecting the response to the latter. This integration did not depend on the concentration of DARPP-32, while the absolute effect on PP1 varied linearly. In silico mutants showed that Ser137 phosphorylation affects the influence of the delay between dopamine and glutamate, and that constitutive phosphorylation in Ser137 transforms DARPP-32 in a quasi-irreversible switch. This work is a first attempt to better understand the complex interactions between cAMP and Ca2+ regulation of DARPP-32. Progressive inclusion of additional components should lead to a realistic model of signalling networks underlying the function of striatal neurons.Synopsis: Projecting neurons of the striatum are a crucial relay of the basal ganglia, involved in motor, psychomotor, and behavioural functions. Their importance is emphasised by their involvement in various dysfunctions, such as Huntington chorea and schizophrenia, but also drug addiction. The main inputs to those neurons come from cortical glutamatergic terminals. Dopamine modulates this transmission, providing a measure of the internal (hedonic) state. In mammal brain, DARPP-32, a protein phosphatase inhibitor, has been identified as a major target for both dopamine and glutamate signalling. The authors present a detailed quantitative model of the regulation of DARPP-32 phosphorylation and dephosphorylation by both signals. Dynamic simulations show that the function of DARPP-32 depends on the delay between the two signals, and therefore the protein not only measures the intensity, but also the coincidence, between signals. This measurement is insensitive to many parameters, whether kinetic constants or concentrations, making it a robust integrator. This shows that a proper understanding of signal integration in the basal ganglia requires quantitative descriptions of the signalling pathways in addition to the neuronal electrophysiological properties.