DARPP-32 is a robust integrator of dopamine and glutamate signals

Abstract

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 Ca(2+) signals. Dynamic simulations allowed us to observe the temporal relationships between cAMP and Ca(2+) 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 Ca(2+) regulation of DARPP-32. Progressive inclusion of additional components should lead to a realistic model of signalling networks underlying the function of striatal neurons.

Figure 1. Biological Model of DARPP-32 Regulation

The various endogenous external signals affecting DARPP-32 through cAMP and calcium are represented, as well as external drugs. (A) nigro-striatal medium-sized spiny GABAergic neuron; (B) nigro-pallidal medium-size spiny GABAergic neuron. Arrow-ending lines represent stimulation, bar-ending lines represent inhibition, circle-ending lines represent enzymatic reactions. Dashed lines represent reactions only present in model B. Source is [9].

Figure 2. Biochemical Model of DARPP-32 Regulation

Graphical representation of the models implemented in this study. Arrow-ending lines represent transition, either phosphorylations or binding. Note that the bindings are reversible. Circle-ending lines represent enzymatic reactions. The effects of kinases and phosphatases on DARPP-32 have been represented only once for clarity, but each couple of enzymes effectively acts on every pair of arrows of the same colour. The different thicknesses of the red-arrowed lines represent the catalytic rates for the various DARPP-32 species. Dashed lines represent reactions only present in model B. Colour code of the molecular species is the same as for Figure 1.

Figure 3. Effect of a Pulse of cAMP on DARPP-32 Phosphorylation

Time-course of DARPP-32 isoforms after a pulse of cAMP. Brown line represents the number of cAMP molecules. Orange line represents the total number of PKA catalytic subunits not bound to regulatory subunits. DARPP-32 species are represented in black (unphosphorylated), red (D34*), blue (D75*), and green (D137*).

Figure 4. Effects of a Train of Ca²⁺ Spikes on DARPP-32 Phosphorylation

Time-course of DARPP-32 isoforms triggered by a train of Ca²⁺ spikes. Bordeaux line represents the number of calcium ions in the dendritic spine. Colour code of DARPP-32 isoforms is the same as for Figure 3. In the absence of a cAMP signal, the phosphorylation on Thr34 remains null. (A) model A; (B) model B.

Figure 5. Effect of One Pulse of cAMP Followed by a Train of Ca²⁺ Spikes on DARPP-32 Phosphorylation

Time-course of DARPP-32 isoforms triggered by a pulse of cAMP followed by a train of Ca²⁺ spikes. Colour code of DARPP-32 isoforms is the same as for Figure 3. Relax and Thr34min show the two readouts used in sensitivity analysis. (A) model A; (B) model B.

Figure 6. Effect of the Delay between cAMP and Calcium Stimuli

(A) Time-course of D34* in model B, triggered by a pulse of cAMP, followed, after a variable delay, by a train of Ca²⁺ spikes. (B) Relaxation time of DARPP-32 response to calcium in function of the delay between cAMP pulse and Ca²⁺ spikes. Green diamonds represent the response of “wild-type” DARPP-32, while red triangles represent the response of a mutant without Ser137 phosphorylation.

Figure 7. Cross-Sensitivity to the Inhibition of PKA by DARPP-32 and the Activity of CDK5 or the Stimulation of PP2A by PKA

Values corresponding to model A are blue, while values corresponding to model B are magenta. (A) Cross-sensitivity to the inhibition of PKA by DARPP-32 and the activity of CDK5. Note the inverse relationship between CDK5 activity and Thr34min for strong inhibition of PKA (low kcat) while the relationship is reversed at weak inhibition. (B) Cross-sensitivity to the inhibition of PKA by DARPP-32 and the stimulation of PP2A by PKA.

Figure 8. Sensitivity to the Auto-Phosphorylation Activity of CK1

(A) Sensitivity of Thr34min to the autophosphorylation activity of CK1. The maximal values of the x-axis correspond to a very fast auto-inhibition of CK1, with effects identical to a Ser137Ala mutation, see red curve on Figure 10A. (B) Sensitivity of the relaxation time after calcium signal (the “sharpness” of the response) to the autophosphorylation activity of CK1.

Figure 9. Dependency of the Signal Integration on the Concentration of DARPP-32

Same simulation paradigm as the one depicted in Figure 5, but with different concentrations of DARPP-32, all the other parameters being conserved. Only D34* of model B is plotted. While the x-axis remains the same for all time-courses, the y-axis is scaled to superpose all the traces. The vertical scaling is roughly linear, that is, a 2-fold increase between successive values of DARPP-32. (A) Calcium spikes started 50 s after the pulse of cAMP. (B) Calcium spikes started 200 s after the pulse of cAMP.

Figure 10. In Silico Site-Directed Mutagenesis of DARPP-32

Same simulation paradigm as the one depicted in Figure 5, but describing the predicted behaviour of mutants by model B. Wild-type DARPP-32 species are represented in green, Ser137Ala in red, and constitutive Ser137P in blue. (A) D34*; (B) D75*.