Hippocampal Pyk2 regulates specific social skills: Implications for schizophrenia.

Pyk2 has been shown previously to be involved in several psychological and cognitive alterations related to stress, Huntington's disease, and Alzheimer's disease. All these disorders are accompanied by different types of impairments in sociability, which has recently been linked to improper mitochondrial function. We hypothesize that Pyk2, which regulates mitochondria, could be associated with the regulation of mitochondrial dynamics and social skills. In the present manuscript, we report that a reduction of Pyk2 levels in mouse pyramidal neurons of the hippocampus decreased social dominance and aggressivity. Furthermore, social interactions induced robust Pyk2-dependent hippocampal changes in several oxidative phosphorylation complexes. We also observed that Pyk2 levels were increased in the CA1 pyramidal neurons of schizophrenic subjects, occurring alongside changes in different direct and indirect regulators of mitochondrial function including DISC1 and Grp75. Accordingly, overexpressing Pyk2 in hippocampal CA1 pyramidal cells mimicked some specific schizophrenia-like social behaviors in mice. In summary, our results indicate that Pyk2 might play a role in regulating specific social skills likely via mitochondrial dynamics and that there might be a link between Pyk2 levels in hippocampal neurons and social disturbances in schizophrenia.

PYK2, a hub of signaling networks in breast cancer progression.

Breast cancer (BC) involves complex signaling networks characterized by extensive cross-communication and feedback loops between and within multiple signaling cascades. Many of these signaling pathways are driven by genetic alterations of oncogene and/or tumor-suppressor genes and are influenced by various environmental cues. We describe unique roles of the non-receptor tyrosine kinase (NRTK) PYK2 in signaling integration and feedback looping in BC. PYK2 functions as a signaling hub in various cascades, and its involvement in positive and negative feedback loops enhances signaling robustness, modulates signaling dynamics, and contributes to BC growth, epithelial-to-mesenchymal transition (EMT), stemness, migration, invasion, and metastasis. We also discuss the potential of PYK2 as a therapeutic target in various BC subtypes.

Operant Training for Highly Palatable Food Alters Translating Messenger RNA in Nucleus Accumbens D2 Neurons and Reveals a Modulatory Role of Ncdn.

BACKGROUND: Highly palatable food triggers behavioral responses including strong motivation. These effects involve the reward system and dopamine neurons, which modulate neurons in the nucleus accumbens (NAc). The molecular mechanisms underlying the long-lasting effects of highly palatable food on feeding behavior are poorly understood. METHODS: We studied the effects of 2-week operant conditioning of mice with standard or isocaloric highly palatable food. We investigated the behavioral responses and dendritic spine modifications in the NAc. We compared the translating messenger RNA in NAc neurons identified by the type of dopamine receptors they express, depending on the kind of food and training. We tested the consequences of invalidation of an abundant downregulated gene, Ncdn. RESULTS: Operant conditioning for highly palatable food increased motivation for food even in well-fed mice. In wild-type mice, free choice between regular and highly palatable food increased weight compared with access to regular food only. Highly palatable food increased spine density in the NAc. In animals trained for highly palatable food, translating messenger RNAs were modified in NAc neurons expressing dopamine D2 receptors, mostly corresponding to striatal projection neurons, but not in neurons expressing D1 receptors. Knockout of Ncdn, an abundant downregulated gene, opposed the conditioning-induced changes in satiety-sensitive feeding behavior and apparent motivation for highly palatable food, suggesting that downregulation may be a compensatory mechanism. CONCLUSIONS: Our results emphasize the importance of messenger RNA alterations in D2 striatal projection neurons in the NAc in the behavioral consequences of highly palatable food conditioning and suggest a modulatory contribution of Ncdn downregulation.

BDNF/TrkB pathway activation in D1 receptor-expressing striatal projection neurons plays a protective role against L-DOPA-induced dyskinesia.

L-DOPA-induced dyskinesia (LID) is a frequent adverse side effect of L-DOPA treatment in Parkinson's disease (PD). Understanding the mechanisms underlying the development of these motor disorders is needed to reduce or prevent them. We investigated the role of TrkB receptor in LID, in hemiparkinsonian mice treated by chronic L-DOPA administration. Repeated L-DOPA treatment for 10 days specifically increased full-length TrkB receptor mRNA and protein levels in the dopamine-depleted dorsal striatum (DS) compared to the contralateral non-lesioned DS or to the DS of sham-operated animals. Dopamine depletion alone or acute L-DOPA treatment did not significantly increase TrkB protein levels. In addition to increasing TrkB protein levels, chronic L-DOPA treatment activated the TrkB receptor as evidenced by its increased tyrosine phosphorylation. Using specific agonists for the D1 or D2 receptors, we found that TrkB increase is D1 receptor-dependent. To determine the consequences of these effects, the TrkB gene was selectively deleted in striatal neurons expressing the D1 receptor. Mice with TrkB floxed gene were injected with Cre-expressing adeno-associated viruses or crossed with Drd1-Cre transgenic mice. After unilateral lesion of dopamine neurons in these mice, we found an aggravation of axial LID compared to the control groups. In contrast, no change was found when TrkB deletion was induced in the indirect pathway D2 receptor-expressing neurons. Our study suggests that BDNF/TrkB signaling plays a protective role against the development of LID and that agonists specifically activating TrkB could reduce the severity of LID.

Adult-specific Reelin expression alters striatal neuronal organization: implications for neuropsychiatric disorders.

In addition to neuronal migration, brain development, and adult plasticity, the extracellular matrix protein Reelin has been extensively implicated in human psychiatric disorders such as schizophrenia, bipolar disorder, and autism spectrum disorder. Moreover, heterozygous reeler mice exhibit features reminiscent of these disorders, while overexpression of Reelin protects against its manifestation. However, how Reelin influences the structure and circuits of the striatal complex, a key region for the above-mentioned disorders, is far from being understood, especially when altered Reelin expression levels are found at adult stages. In the present study, we took advantage of complementary conditional gain- and loss-of-function mouse models to investigate how Reelin levels may modify adult brain striatal structure and neuronal composition. Using immunohistochemical techniques, we determined that Reelin does not seem to influence the striatal patch and matrix organization (studied by µ-opioid receptor immunohistochemistry) nor the density of medium spiny neurons (MSNs, studied with DARPP-32). We show that overexpression of Reelin leads to increased numbers of striatal parvalbumin- and cholinergic-interneurons, and to a slight increase in tyrosine hydroxylase-positive projections. We conclude that increased Reelin levels might modulate the numbers of striatal interneurons and the density of the nigrostriatal dopaminergic projections, suggesting that these changes may be involved in the protection of Reelin against neuropsychiatric disorders.

Operant training for highly palatable food alters translating mRNA in nucleus accumbens D2 neurons and reveals a modulatory role of Neurochondrin.

BACKGROUND: Highly palatable food triggers behavioral alterations reminiscent of those induced by addictive drugs. These effects involve the reward system and dopamine neurons, which modulate neurons in the nucleus accumbens (NAc). The molecular mechanisms underlying the effects of highly palatable food on feeding behavior are poorly understood. METHODS: We studied the effects of 2-week operant conditioning of mice with standard or isocaloric highly palatable food. We investigated the behavioral effects and dendritic spine modifications in the NAc. We compared the translating mRNA in NAc neurons identified by the type of dopamine receptors they express, depending on the type of food and training. We tested the consequences of invalidation of an abundant downregulated gene, Ncdn (Neurochondrin). RESULTS: Operant conditioning for highly palatable food increases motivation for food even in well-fed mice. In control mice, free access to regular or highly palatable food results in increased weight as compared to regular food only. Highly palatable food increases spine density in the NAc. In animals trained for highly palatable food, translating mRNAs are modified in NAc dopamine D2-receptor-expressing neurons, mostly corresponding to striatal projection neurons, but not in those expressing D1-receptors. Knock-out of Ncdn, an abundant down-regulated gene, opposes the conditioning-induced changes in satiety-sensitive feeding behavior and apparent motivation for highly palatable food, suggesting down-regulation may be a compensatory mechanism. CONCLUSIONS: Our results emphasize the importance of mRNA alterations D2 striatal projection neurons in the NAc in the behavioral consequences of highly palatable food conditioning and suggest a modulatory contribution of Ncdn downregulation.

Cognitive and Emotional Symptoms Induced by Chronic Stress Are Regulated by EGR1 in a Subpopulation of Hippocampal Pyramidal Neurons.

Chronic stress is a core risk factor for developing a myriad of neurological disorders, including major depression. The chronicity of such stress can lead to adaptive responses or, on the contrary, to psychological maladaptation. The hippocampus is one of the most affected brain regions displaying functional changes in chronic stress. Egr1, a transcription factor involved in synaptic plasticity, is a key molecule regulating hippocampal function, but its role in stress-induced sequels has been poorly addressed. Emotional and cognitive symptoms were induced in mice by using the chronic unpredictable mild stress (CUMS) protocol. We used inducible double-mutant Egr1-CreERT2 x R26RCE mice to map the formation of Egr1-dependent activated cells. Results show that short- (2 days) or long-term (28 days) stress protocols in mice induce activation or deactivation, respectively, of hippocampal CA1 neural ensembles in an Egr1-activity-dependent fashion, together with an associated dendritic spine pathology. In-depth characterization of these neural ensembles revealed a deep-to-superficial switch in terms of Egr1-dependent activation of CA1 pyramidal neurons. To specifically manipulate deep and superficial pyramidal neurons of the hippocampus, we then used Chrna7-Cre (to express Cre in deep neurons) and Calb1-Cre mice (to express Cre in superficial neurons). We found that specific manipulation of superficial but not deep pyramidal neurons of the CA1 resulted in the amelioration of depressive-like behaviors and the restoration of cognitive impairments induced by chronic stress. In summary, Egr1 might be a core molecule driving the activation/deactivation of hippocampal neuronal subpopulations underlying stress-induced alterations involving emotional and cognitive sequels.

DNA methylation and hydroxymethylation characterize the identity of D1 and D2 striatal projection neurons.

Neuronal DNA modifications differ from those in other cells, including methylation outside CpG context and abundant 5-hydroxymethylation whose relevance for neuronal identities are unclear. Striatal projection neurons expressing D1 or D2 dopamine receptors allow addressing this question, as they share many characteristics but differ in their gene expression profiles, connections, and functional roles. We compare translating mRNAs and DNA modifications in these two populations. DNA methylation differences occur predominantly in large genomic clusters including differentially expressed genes, potentially important for D1 and D2 neurons. Decreased gene body methylation is associated with higher gene expression. Hydroxymethylation differences are more scattered and affect transcription factor binding sites, which can influence gene expression. We also find a strong genome-wide hydroxymethylation asymmetry between the two DNA strands, particularly pronounced at expressed genes and retrotransposons. These results identify novel properties of neuronal DNA modifications and unveil epigenetic characteristics of striatal projection neurons heterogeneity.

Plea for a Simple But Radical Change in Scientific Publication: To Improve Openness, Reliability, and Reproducibility, Let's Deposit and Validate Our Results before Writing Articles.

Limited reproducibility and validity are major sources of concern in biology and other fields of science. Their origins have been extensively described and include material variability, incomplete materials and methods report, results selection, defective experimental design, lack of power, inappropriate statistics, overinterpretation, and reluctance to publish negative results. Promoting complete and accurate communication of positive and negative results is a major objective. Multiple steps in this direction are taken, but they are not sufficient and the general construction of articles has not been questioned. I propose here a simple change with a potentially strong positive impact. First, when they complete a substantial coherent set of experiments, scientists deposit their positive or negative results in a database ["deposited results," (DRs)], including detailed materials, methods, raw data, analysis, and processed results. The DRs are technically reviewed and validated as "validated DRs" (vDRs) or rejected until satisfactory. vDR databases are open (after an embargo period if requested by the authors) and can later be updated by them or others with replications or replication failures, providing a comprehensive active log of scientific data. Articles, in this proposal, are then built as they currently are, except they only include vDRs as strong and open building blocks. I argue that this approach would increase the transparency, reproducibility, and reliability of scientific publications and have additional advantages including accurate author credit, better material for evaluation, exhaustive scientific archiving, and increased openness of life science material.

PYK2 senses calcium through a disordered dimerization and calmodulin-binding element.

Multidomain kinases use many ways to integrate and process diverse stimuli. Here, we investigated the mechanism by which the protein tyrosine kinase 2-beta (PYK2) functions as a sensor and effector of cellular calcium influx. We show that the linker between the PYK2 kinase and FAT domains (KFL) encompasses an unusual calmodulin (CaM) binding element. PYK2 KFL is disordered and engages CaM through an ensemble of transient binding events. Calcium increases the association by promoting structural changes in CaM that expose auxiliary interaction opportunities. KFL also forms fuzzy dimers, and dimerization is enhanced by CaM binding. As a monomer, however, KFL associates with the PYK2 FERM-kinase fragment. Thus, we identify a mechanism whereby calcium influx can promote PYK2 self-association, and hence kinase-activating trans-autophosphorylation. Collectively, our findings describe a flexible protein module that expands the paradigms for CaM binding and self-association, and their use for controlling kinase activity.

Hippocampal Egr1-Dependent Neuronal Ensembles Negatively Regulate Motor Learning.

Motor skills learning is classically associated with brain regions including cerebral and cerebellar cortices and basal ganglia nuclei. Less is known about the role of the hippocampus in the acquisition and storage of motor skills. Here, we show that mice receiving a long-term training in the accelerating rotarod display marked hippocampal transcriptional changes and reduced pyramidal neurons activity in the CA1 region when compared with naive mice. Then, we use mice in which neural ensembles are permanently labeled in an Egr1 activity-dependent fashion. Using these mice, we identify a subpopulation of Egr1-expressing pyramidal neurons in CA1 activated in short-term (STT) and long-term (LTT) trained mice in the rotarod task. When Egr1 is downregulated in the CA1 or these neuronal ensembles are depleted, motor learning is improved whereas their chemogenetic stimulation impairs motor learning performance. Thus, Egr1 organizes specific CA1 neuronal ensembles during the accelerating rotarod task that limit motor learning. These evidences highlight the role of the hippocampus in the control of this type of learning and we provide a possible underlying mechanism.SIGNIFICANCE STATEMENT It is a major topic in neurosciences the deciphering of the specific circuits underlying memory systems during the encoding of new information. However, the potential role of the hippocampus in the control of motor learning and the underlying mechanisms has been poorly addressed. In the present work we show how the hippocampus responds to motor learning and how the Egr1 molecule is one of the major responsible for such phenomenon controlling the rate of motor coordination performances.

Pyk2 Regulates MAMs and Mitochondrial Dynamics in Hippocampal Neurons.

Pyk2 is a non-receptor tyrosine kinase enriched in hippocampal neurons, which can be activated by calcium-dependent mechanisms. In neurons, Pyk2 is mostly localised in the cytosol and dendritic shafts but can translocate to spines and/or to the nucleus. Here, we explore the function of a new localisation of Pyk2 in mitochondria-associated membranes (MAMs), a subdomain of ER-mitochondria surface that acts as a signalling hub in calcium regulation. To test the role of Pyk2 in MAMs' calcium transport, we used full Pyk2 knockout mice (Pyk2-/-) for in vivo and in vitro studies. Here we report that Pyk2-/- hippocampal neurons present increased ER-mitochondrial contacts along with defective calcium homeostasis. We also show how the absence of Pyk2 modulates mitochondrial dynamics and morphology. Taken all together, our results point out that Pyk2 could be highly relevant in the modulation of ER-mitochondria calcium efflux, affecting in turn mitochondrial function.

Translational profiling of mouse dopaminoceptive neurons reveals region-specific gene expression, exon usage, and striatal prostaglandin E2 modulatory effects.

Forebrain dopamine-sensitive (dopaminoceptive) neurons play a key role in movement, action selection, motivation, and working memory. Their activity is altered in Parkinson's disease, addiction, schizophrenia, and other conditions, and drugs that stimulate or antagonize dopamine receptors have major therapeutic applications. Yet, similarities and differences between the various neuronal populations sensitive to dopamine have not been systematically explored. To characterize them, we compared translating mRNAs in the dorsal striatum and nucleus accumbens neurons expressing D1 or D2 dopamine receptor and prefrontal cortex neurons expressing D1 receptor. We identified genome-wide cortico-striatal, striatal D1/D2 and dorso/ventral differences in the translating mRNA and isoform landscapes, which characterize dopaminoceptive neuronal populations. Expression patterns and network analyses identified novel transcription factors with presumptive roles in these differences. Prostaglandin E2 (PGE2) was a candidate upstream regulator in the dorsal striatum. We pharmacologically explored this hypothesis and showed that misoprostol, a PGE2 receptor agonist, decreased the excitability of D2 striatal projection neurons in slices, and diminished their activity in vivo during novel environment exploration. We found that misoprostol also modulates mouse behavior including by facilitating reversal learning. Our study provides powerful resources for characterizing dopamine target neurons, new information about striatal gene expression patterns and regulation. It also reveals the unforeseen role of PGE2 in the striatum as a potential neuromodulator and an attractive therapeutic target.

Mouse Modeling Dissecting Macrophage-Breast Cancer Communication Uncovered Roles of PYK2 in Macrophage Recruitment and Breast Tumorigenesis.

Macrophage infiltration in mammary tumors is associated with enhanced tumor progression, metastasis, and poor clinical outcome, and considered as target for therapeutic intervention. By using different genetic mouse models, the authors show that ablation of the tyrosine kinase PYK2, either in breast cancer cells, only in the tumor microenvironment, or in both, markedly reduces the number of infiltrating tumor macrophages and concomitantly inhibits tumor angiogenesis and tumor growth. Strikingly, PYK2 ablation only in macrophages is sufficient to induce similar effects. These phenotypic changes are associated with reduced monocyte recruitment and a substantial decrease in tumor-associated macrophages (TAMs). Mechanistically, the authors show that PYK2 mediates mutual communication between breast cancer cells and macrophages through critical effects on key receptor signaling. Specifically, PYK2 ablation inhibits Notch1 signaling and consequently reduces CCL2 secretion by breast cancer cells, and concurrently reduces the levels of CCR2, CXCR4, IL-4Rα, and Stat6 activation in macrophages. These bidirectional effects modulate monocyte recruitment, macrophage polarization, and tumor angiogenesis. The expression of PYK2 is correlated with infiltrated macrophages in breast cancer patients, and its effects on macrophage infiltration and pro-tumorigenic phenotype suggest that PYK2 targeting can be utilized as an effective strategy to modulate TAMs and possibly sensitize breast cancer to immunotherapy.

Cell-type- and region-specific modulation of cocaine seeking by micro-RNA-1 in striatal projection neurons.

The persistent and experience-dependent nature of drug addiction may result in part from epigenetic alterations, including non-coding micro-RNAs (miRNAs), which are both critical for neuronal function and modulated by cocaine in the striatum. Two major striatal cell populations, the striato-nigral and striato-pallidal projection neurons, express, respectively, the D1 (D1-SPNs) and D2 (D2-SPNs) dopamine receptor, and display distinct but complementary functions in drug-evoked responses. However, a cell-type-specific role for miRNAs action has yet to be clarified. Here, we evaluated the expression of a subset of miRNAs proposed to modulate cocaine effects in the nucleus accumbens (NAc) and dorsal striatum (DS) upon sustained cocaine exposure in mice and showed that these selected miRNAs were preferentially upregulated in the NAc. We focused on miR-1 considering the important role of some of its predicted mRNA targets, Fosb and Npas4, in the effects of cocaine. We validated these targets in vitro and in vivo. We explored the potential of miR-1 to regulate cocaine-induced behavior by overexpressing it in specific striatal cell populations. In DS D1-SPNs miR-1 overexpression downregulated Fosb and Npas4 and reduced cocaine-induced CPP reinstatement, but increased cue-induced cocaine seeking. In DS D2-SPNs miR-1 overexpression reduced the motivation to self-administer cocaine. Our results indicate a role of miR1 and its target genes, Fosb and Npas4, in these behaviors and highlight a precise cell-type- and region-specific modulatory role of miR-1, illustrating the importance of cell-specific investigations.

The Non-receptor Tyrosine Kinase Pyk2 in Brain Function and Neurological and Psychiatric Diseases.

Pyk2 is a non-receptor tyrosine kinase highly enriched in forebrain neurons. Pyk2 is closely related to focal adhesion kinase (FAK), which plays an important role in sensing cell contacts with extracellular matrix and other extracellular signals controlling adhesion and survival. Pyk2 shares some of FAK's characteristics including recruitment of Src-family kinases after autophosphorylation, scaffolding by interacting with multiple partners, and activation of downstream signaling pathways. Pyk2, however, has the unique property to respond to increases in intracellular free Ca2+, which triggers its autophosphorylation following stimulation of various receptors including glutamate NMDA receptors. Pyk2 is dephosphorylated by the striatal-enriched phosphatase (STEP) that is highly expressed in the same neuronal populations. Pyk2 localization in neurons is dynamic, and altered following stimulation, with post-synaptic and nuclear enrichment. As a signaling protein Pyk2 is involved in multiple pathways resulting in sometimes opposing functions depending on experimental models. Thus Pyk2 has a dual role on neurites and dendritic spines. With Src family kinases Pyk2 participates in postsynaptic regulations including of NMDA receptors and is necessary for specific types of synaptic plasticity and spatial memory tasks. The diverse functions of Pyk2 are also illustrated by its role in pathology. Pyk2 is activated following epileptic seizures or ischemia-reperfusion and may contribute to the consequences of these insults whereas Pyk2 deficit may contribute to the hippocampal phenotype of Huntington's disease. Pyk2 gene, PTK2B, is associated with the risk for late-onset Alzheimer's disease. Studies of underlying mechanisms indicate a complex contribution with involvement in amyloid toxicity and tauopathy, combined with possible functional deficits in neurons and contribution in microglia. A role of Pyk2 has also been proposed in stress-induced depression and cocaine addiction. Pyk2 is also important for the mobility of astrocytes and glioblastoma cells. The implication of Pyk2 in various pathological conditions supports its potential interest for therapeutic interventions. This is possible through molecules inhibiting its activity or increasing it through inhibition of STEP or other means, depending on a precise evaluation of the balance between positive and negative consequences of Pyk2 actions.

Pyk2 in dorsal hippocampus plays a selective role in spatial memory and synaptic plasticity.

Pyk2 is a Ca2+-activated non-receptor tyrosine kinase enriched in the forebrain, especially in pyramidal neurons of the hippocampus. Previous reports suggested its role in hippocampal synaptic plasticity and spatial memory but with contradictory findings possibly due to experimental conditions. Here we address this issue and show that novel object location, a simple test of spatial memory induced by a single training session, is altered in Pyk2 KO mice and that re-expression of Pyk2 in the dorsal hippocampus corrects this deficit. Bilateral targeted deletion of Pyk2 in dorsal hippocampus CA1 region also alters novel object location. Long term potentiation (LTP) in CA1 is impaired in Pyk2 KO mice using a high frequency stimulation induction protocol but not with a theta burst protocol, explaining differences between previous reports. The same selective LTP alteration is observed in mice with Pyk2 deletion in dorsal hippocampus CA1 region. Thus, our results establish the role of Pyk2 in specific aspects of spatial memory and synaptic plasticity and show the dependence of the phenotype on the type of experiments used to reveal it. In combination with other studies, we provide evidence for a selective role of non-receptor tyrosine kinases in specific aspects of hippocampal neurons synaptic plasticity.

Dopamine D1 receptor-expressing neurons activity is essential for locomotor and sensitizing effects of a single injection of cocaine.

Dopamine D1 receptors play an important role in the effects of cocaine. Here, we investigated the role of neurons which express these receptors (D1-neurons) in the acute locomotor effects of cocaine and the locomotor sensitization observed after a second injection of this drug, using the previously established two-injection protocol of sensitization. We inhibited D1-neurons using double transgenic mice conditionally expressing the inhibitory Gi-coupled designer receptor exclusively activated by designer drugs (Gi-DREADD) in D1-neurons. Chemogenetic inhibition of D1-neurons by a low dose of clozapine (0.1 mg/kg) decreased the cocaine-induced expression of Fos in striatal neurons. It diminished the basal locomotor activity and acute hyper-locomotion induced by cocaine (20 mg/kg). Clozapine 0.1 mg/kg had no effect by itself and did not alter cocaine effects in wild-type mice. Inhibition of D1-neurons during the first cocaine administration prevented the sensitization of the locomotor response in response to a second cocaine administration 10 days later. On Day 11, inhibition of D1-neurons by clozapine stimulation of Gi-DREADD blocked cocaine-induced locomotion including in sensitized mice, whereas on Day 12, in the absence of clozapine and D1-neurons inhibition, all mice displayed a sensitized response to cocaine. These results show that chemogenetic inhibition of D1-neurons decreases spontaneous and cocaine-induced locomotor activity. It prevents sensitization induction and blocks sensitized locomotion in a two-injection protocol of sensitization but does not reverse established sensitization. Our study further supports the central role of D1-neurons in mediating the acute locomotor effects of cocaine and its sensitization.

DARPP-32 40 years later.

DARPP-32 (dopamine- and cAMP-regulated phosphoprotein with an apparent Mr of 32,000), now also known as phosphoprotein phosphatase 1 regulatory subunit 1B (PPP1R1B), is a potent inhibitor of protein phosphatase 1 (PP1, also known as PPP1) when phosphorylated at Thr34 by cAMP-dependent protein kinase (PKA). DARPP-32 exhibits a remarkable regional distribution in brain, roughly similar to that of dopamine innervation. Its discovery was a culmination of the long-standing effort of Paul Greengard to understand the mechanisms through which neurotransmitters such as dopamine exert their effects on target neurons. DARPP-32 is particularly enriched in striatal projection neurons where it is regulated by numerous signals through which it integrates and amplifies responses to many stimuli. Molecular studies of DARPP-32 have revealed that its regulation and function are more complex than anticipated. It is phosphorylated on multiple sites by several protein kinases that modulate DARPP-32 properties. Primarily, when phosphorylated at Thr34 DARPP-32 is a potent inhibitor of PP1, whereas when phosphorylated at Thr75 by Cdk5 it inhibits PKA. Phosphorylation at serine residues by CK1 and CK2 modulates its intracellular localization and its sensitivity to kinases or phosphatases. Modeling studies provide evidence that the signaling pathways including DARPP-32 are endowed of strong robustness and bistable properties favoring switch-like responses. Thus DARPP-32 combined with a set of other distinct signaling molecules enriched in striatal projection neurons plays a key role in the characteristic properties and physiological function of these neurons.