Transient Calcium and Dopamine Increase PKA Activity and DARPP-32 Phosphorylation

Reinforcement learning theorizes that strengthening of synaptic connections in medium spiny neurons of the striatum occurs when glutamatergic input (from cortex) and dopaminergic input (from substantia nigra) are received simultaneously. Subsequent to learning, medium spiny neurons with strengthened synapses are more likely to fire in response to cortical input alone. This synaptic plasticity is produced by phosphorylation of AMPA receptors, caused by phosphorylation of various signalling molecules. A key signalling molecule is the phosphoprotein DARPP-32, highly expressed in striatal medium spiny neurons. DARPP-32 is regulated by several neurotransmitters through a complex network of intracellular signalling pathways involving cAMP (increased through dopamine stimulation) and calcium (increased through glutamate stimulation). Since DARPP-32 controls several kinases and phosphatases involved in striatal synaptic plasticity, understanding the interactions between cAMP and calcium, in particular the effect of transient stimuli on DARPP-32 phosphorylation, has major implications for understanding reinforcement learning. We developed a computer model of the biochemical reaction pathways involved in the phosphorylation of DARPP-32 on Thr34 and Thr75. Ordinary differential equations describing the biochemical reactions were implemented in a single compartment model using the software XPPAUT. Reaction rate constants were obtained from the biochemical literature. The first set of simulations using sustained elevations of dopamine and calcium produced phosphorylation levels of DARPP-32 similar to that measured experimentally, thereby validating the model. The second set of simulations, using the validated model, showed that transient dopamine elevations increased the phosphorylation of Thr34 as expected, but transient calcium elevations also increased the phosphorylation of Thr34, contrary to what is believed. When transient calcium and dopamine stimuli were paired, PKA activation and Thr34 phosphorylation increased compared with dopamine alone. This result, which is robust to variation in model parameters, supports reinforcement learning theories in which activity-dependent long-term synaptic plasticity requires paired glutamate and dopamine inputs

Subcellular Location of PKA Controls Striatal Plasticity: Stochastic Simulations in Spiny Dendrites

Dopamine release in the striatum has been implicated in various forms of reward dependent learning. Dopamine leads to production of cAMP and activation of protein kinase A (PKA), which are involved in striatal synaptic plasticity and learning. PKA and its protein targets are not diffusely located throughout the neuron, but are confined to various subcellular compartments by anchoring molecules such as A-Kinase Anchoring Proteins (AKAPs). Experiments have shown that blocking the interaction of PKA with AKAPs disrupts its subcellular location and prevents LTP in the hippocampus and striatum; however, these experiments have not revealed whether the critical function of anchoring is to locate PKA near the cAMP that activates it or near its targets, such as AMPA receptors located in the post-synaptic density. We have developed a large scale stochastic reaction-diffusion model of signaling pathways in a medium spiny projection neuron dendrite with spines, based on published biochemical measurements, to investigate this question and to evaluate whether dopamine signaling exhibits spatial specificity post-synaptically. The model was stimulated with dopamine pulses mimicking those recorded in response to reward. Simulations show that PKA colocalization with adenylate cyclase, either in the spine head or in the dendrite, leads to greater phosphorylation of DARPP-32 Thr34 and AMPA receptor GluA1 Ser845 than when PKA is anchored away from adenylate cyclase. Simulations further demonstrate that though cAMP exhibits a strong spatial gradient, diffusible DARPP-32 facilitates the spread of PKA activity, suggesting that additional inactivation mechanisms are required to produce spatial specificity of PKA activity

A Kinetic Model of Dopamine- and Calcium-Dependent Striatal Synaptic Plasticity

Corticostriatal synapse plasticity of medium spiny neurons is regulated by glutamate input from the cortex and dopamine input from the substantia nigra. While cortical stimulation alone results in long-term depression (LTD), the combination with dopamine switches LTD to long-term potentiation (LTP), which is known as dopamine-dependent plasticity. LTP is also induced by cortical stimulation in magnesium-free solution, which leads to massive calcium influx through NMDA-type receptors and is regarded as calcium-dependent plasticity. Signaling cascades in the corticostriatal spines are currently under investigation. However, because of the existence of multiple excitatory and inhibitory pathways with loops, the mechanisms regulating the two types of plasticity remain poorly understood. A signaling pathway model of spines that express D1-type dopamine receptors was constructed to analyze the dynamic mechanisms of dopamine- and calcium-dependent plasticity. The model incorporated all major signaling molecules, including dopamine- and cyclic AMP-regulated phosphoprotein with a molecular weight of 32 kDa (DARPP32), as well as AMPA receptor trafficking in the post-synaptic membrane. Simulations with dopamine and calcium inputs reproduced dopamine- and calcium-dependent plasticity. Further in silico experiments revealed that the positive feedback loop consisted of protein kinase A (PKA), protein phosphatase 2A (PP2A), and the phosphorylation site at threonine 75 of DARPP-32 (Thr75) served as the major switch for inducing LTD and LTP. Calcium input modulated this loop through the PP2B (phosphatase 2B)-CK1 (casein kinase 1)-Cdk5 (cyclin-dependent kinase 5)-Thr75 pathway and PP2A, whereas calcium and dopamine input activated the loop via PKA activation by cyclic AMP (cAMP). The positive feedback loop displayed robust bi-stable responses following changes in the reaction parameters. Increased basal dopamine levels disrupted this dopamine-dependent plasticity. The present model elucidated the mechanisms involved in bidirectional regulation of corticostriatal synapses and will allow for further exploration into causes and therapies for dysfunctions such as drug addiction

Transfer Functions for Protein Signal Transduction: Application to a Model of Striatal Neural Plasticity

We present a novel formulation for biochemical reaction networks in the context of signal transduction. The model consists of input-output transfer functions, which are derived from differential equations, using stable equilibria. We select a set of 'source' species, which receive input signals. Signals are transmitted to all other species in the system (the 'target' species) with a specific delay and transmission strength. The delay is computed as the maximal reaction time until a stable equilibrium for the target species is reached, in the context of all other reactions in the system. The transmission strength is the concentration change of the target species. The computed input-output transfer functions can be stored in a matrix, fitted with parameters, and recalled to build discrete dynamical models. By separating reaction time and concentration we can greatly simplify the model, circumventing typical problems of complex dynamical systems. The transfer function transformation can be applied to mass-action kinetic models of signal transduction. The paper shows that this approach yields significant insight, while remaining an executable dynamical model for signal transduction. In particular we can deconstruct the complex system into local transfer functions between individual species. As an example, we examine modularity and signal integration using a published model of striatal neural plasticity. The modules that emerge correspond to a known biological distinction between calcium-dependent and cAMP-dependent pathways. We also found that overall interconnectedness depends on the magnitude of input, with high connectivity at low input and less connectivity at moderate to high input. This general result, which directly follows from the properties of individual transfer functions, contradicts notions of ubiquitous complexity by showing input-dependent signal transmission inactivation.Comment: 13 pages, 5 tables, 15 figure

Casein Kinase 1 Enables Nucleus Accumbens Amphetamine-Induced Locomotion by Regulating AMPA Receptor Phosphorylation

The closely related δ and ε isoforms of the serine/threonine protein kinase casein kinase 1 (Csnk1) have been implicated in the generation of psychostimulant-induced behaviors. In this study, we show that Csnk1δ/ε produces its effects on behavior by acting on the Darpp-32-PP1 signaling pathway to regulate AMPA receptor phosphorylation in the nucleus accumbens (NAcc). Inhibiting Csnk1δ/ε in the NAcc with the selective inhibitor PF-670462 blocks amphetamine induced locomotion and its ability to increase phosphorylation of Darpp-32 at S137 and T34, decrease PP1 activity and increase phosphorylation of the AMPA receptor subunit at S845. Consistent with these findings, preventing GluR1 phosphorylation with the alanine mutant GluR1(S845A) reduces glutamate-evoked currents in cultured medium spiny neurons and blocks the locomotor activity produced by NAcc amphetamine. Thus, Csnk1 enables the locomotor and likely the incentive motivational effects of amphetamine by regulating Darrp-32-PP1-GlurR1(S845) signaling in the NAcc. As such, Csnk1 may be a critical target for intervention in the treatment of drug use disorders

Calcium Input Frequency, Duration and Amplitude Differentially Modulate the Relative Activation of Calcineurin and CaMKII

NMDA receptor dependent long-term potentiation (LTP) and long-term depression (LTD) are two prominent forms of synaptic plasticity, both of which are triggered by post-synaptic calcium elevation. To understand how calcium selectively stimulates two opposing processes, we developed a detailed computational model and performed simulations with different calcium input frequencies, amplitudes, and durations. We show that with a total amount of calcium ions kept constant, high frequencies of calcium pulses stimulate calmodulin more efficiently. Calcium input activates both calcineurin and Ca2+/calmodulin-dependent protein kinase II (CaMKII) at all frequencies, but increased frequencies shift the relative activation from calcineurin to CaMKII. Irrespective of amplitude and duration of the inputs, the total amount of calcium ions injected adjusts the sensitivity of the system to calcium input frequencies. At a given frequency, the quantity of CaMKII activated is proportional to the total amount of calcium. Thus, an input of a small amount of calcium at high frequencies can induce the same activation of CaMKII as a larger amount, at lower frequencies. Finally, the extent of activation of CaMKII signals with high calcium frequency is further controlled by other factors, including the availability of calmodulin, and by the potency of phosphatase inhibitors

Dopamine and cAMP Regulated Phosphoprotein, 32 kDA: A Novel Therapeutic in Traumatic Brain Injury

Traumatic brain injury (TBI) represents a significant cause of death and disability in industrialized countries. Of particular importance to patients is the chronic effect that TBI has on cognitive function. Therapeutic strategies have been difficult to evaluate because of the complexity of injuries and variety of patient presentations within a TBI population. Experimental therapies based upon cortical and hippocampal neuroprotection have not translated clinically. However, pharmacotherapies targeting dopamine (DA) have consistently shown benefits in attention, behavioral outcome, executive function, and memory. Striatal damage causes deficits in executive function, learning, and memory. Dopamine and cAMP regulated phosphoprotein 32 (DARPP-32), expressed within striatal medium spiny neurons, is known to regulate several substrates of cognition. We found that controlled cortical impact injury in rats produces a chronic decrease in DARPP-32 threonine-34 phosphorylation and increase in protein phosphatase-1 activity. There is no effect of injury on threonine-75 phosphorylation or DARPP-32 protein. Amantadine has known benefits on post-TBI cognitive deficits and when given daily for two weeks reversed the DARPP-32 and protein phosphatase-1 changes. Amantadine also decreased the phosphorylation of threonine-75 consistent with activity as a partial N-methyl-D-aspartic acid receptor antagonist and partial dopamine agonist. FK-506, also known as tacrolimus, is a calcineurin inhibitor that has been shown to decrease cell death in the hippocampus following a fluid percussion experimental TBI. Calcineurin is also an important regulator of DARPP-32 phosphorylation in the striatum. We evaluated the effect of FK-506 on the hippocampus and DARPP-32 in the striatum to better detail its effects after a TBI. An acute administration of FK-506 following controlled cortical impact reversed the effects of TBI on DARPP-32 phosphorylation seen chronically. We then evaluated the effect of a combined drug therapy on cognitive deficits post TBI. An acute treatment with FK-506 post TBI followed by chronic Amantadine therapy demonstrated an improvement in both motor behavior and Morris water maze deficits seen following TBI. Neither drug produced benefit when given alone. These data demonstrate that DARPP-32 represents a promising new therapeutic target for TBI induced cognitive deficits

Regulation of Nociceptive Plasticity Threshold and DARPP-32 Phosphorylation in Spinal Dorsal Horn Neurons by Convergent Dopamine and Glutamate Inputs

Dopamine can influence NMDA receptor function and regulate glutamate-triggered long-term changes in synaptic strength in several regions of the CNS. In spinal cord, regulation of the threshold of synaptic plasticity may determine the proneness to undergo sensitization and hyperresponsiveness to noxious input. In the current study, we increased endogenous dopamine levels in the dorsal horn by using re-uptake inhibitor GBR 12935. During the so-induced hyperdopaminergic transmission, conditioning low-frequency (1 Hz) stimulation (LFS) to the sciatic nerve induced long-termpotentiation (LTP) of C-fiber-evoked potentials in dorsal horn neurons. The magnitude of LTP was attenuated by blockade of either dopamine D1-like receptors (D1LRs) by with SCH 23390 or NMDA receptor subunit NR2B with antagonist Ro25-6981. Conditioning LFS during GBR 12935 administration increased phosphorylation of dopamine-and cAMP-regulated phosphoprotein of Mr 32kDa (DARPP-32) at threonine 34 residue in synaptosomal (P3) fraction of dorsal horn homogenates, as assessed by Western blot analysis, which was partially prevented by NR2B blockade prior to conditioning stimulation. Conditioning LFS also was followed by higher co-localization of phosphorylated form of NR2B at tyrosine 1472 and pDARPP-32(Thr34)-with postsynaptic marker PSD-95 in transverse L5 dorsal horn sections. Such increase could be significantly attenuated by D1LR blockade with SCH 23390. The current results support that coincidental endogenous recruitment of D1LRs and NR2B in dorsal horn synapses plays a role in regulating afferent-induced nociceptive plasticity. Parallel increases in DARPP-32 phosphorylationupon LTP induction suggests a role for this phosphoprotein as intracellular detector of convergent D1L- and NMDA receptor activation.This work was supported by the Basque Government (Eusko Jaurlaritza), Grant IT782-13 to JJA, URL: http://www.euskadi.eus and by the Ministerio de Economia y Compettividad(Spanish Government), Grant SAF2013-44533-P to JJA, URL: www.mineco.gob.es

DARPP-32, Jack of All Trades… Master of Which?

DARPP-32 (PPP1R1B) was discovered as a substrate of cAMP-dependent protein kinase (PKA) enriched in dopamine-innervated brain areas. It is one of three related, PKA-regulated inhibitors of protein phosphatase-1 (PP1). These inhibitors seem to have appeared in early vertebrate ancestors, possibly Gnathostomes. DARPP-32 has additional important biochemical properties including inhibition of PKA when phosphorylated by Cdk5 and regulation by casein kinases 1 and 2. It is highly enriched in specific neuronal populations, especially striatal medium-size spiny neurons. As PP1 inhibitor DARPP-32 amplifies and/or mediates many actions of PKA at the plasma membrane and in the cytoplasm, with a broad spectrum of potential targets and functions. DARPP-32 also undergoes a continuous and tightly regulated cytonuclear shuttling. This trafficking is controlled by phosphorylation of Ser-97, which is necessary for nuclear export. When phosphorylated on Thr-34 and dephosphorylated on Ser-97, DARPP-32 can inhibit PP1 in the nucleus and modulate signaling pathways involved in the regulation of chromatin response. Recent work with multiple transgenic and knockout mutant mice has allowed the dissection of DARPP-32 function in striato-nigral and striato-pallidal neurons. It is implicated in the action of therapeutic and abused psychoactive drugs, in prefrontal cortex function, and in sexual behavior. However, the contribution of DARPP-32 in human behavior remains poorly understood. Post-mortem studies in humans suggest possible alterations of DARPP-32 levels in schizophrenia and bipolar disorder. Genetic studies have revealed a polymorphism with possible association with psychological and psychopathological traits. In addition, a short isoform of DARPP-32, t-DARPP, plays a role in cancer, indicating additional signaling properties. Thus, DARPP-32 is a non-essential but tightly regulated signaling hub molecule which may improve the general performance of the neuronal circuits in which it is expressed