Convergent dopamine and ALK4 signaling to PCBP1 controls FosB alternative splicing and cocaine behavioral sensitization.

ΔfosB is an alternatively spliced product of the FosB gene that is essential for dopamine-induced reward pathways and that acts as a master switch for addiction. However, the molecular mechanisms of its generation and regulation by dopamine signaling are unknown. Here, we report that dopamine D1 receptor signaling synergizes with the activin/ALK4/Smad3 pathway to potentiate the generation of ΔFosB mRNA in medium spiny neurons (MSNs) of the nucleus accumbens (NAc) via activation of the RNA-binding protein PCBP1, a regulator of mRNA splicing. Concurrent activation of PCBP1 and Smad3 by D1 and ALK4 signaling induced their interaction, nuclear translocation, and binding to sequences in exon-4 and intron-4 of FosB mRNA. Ablation of either ALK4 or PCBP1 in MSNs impaired ΔFosB mRNA induction and nuclear translocation of ΔFosB protein in response to repeated co-stimulation of D1 and ALK4 receptors. Finally, ALK4 is required in NAc MSNs of adult mice for behavioral sensitization to cocaine. These findings uncover an unexpected mechanism for ΔFosB generation and drug-induced sensitization through convergent dopamine and ALK4 signaling.

Adult medial habenula neurons require GDNF receptor GFRα1 for synaptic stability and function.

The medial habenula (mHb) is an understudied small brain nucleus linking forebrain and midbrain structures controlling anxiety and fear behaviors. The mechanisms that maintain the structural and functional integrity of mHb neurons and their synapses remain unknown. Using spatiotemporally controlled Cre-mediated recombination in adult mice, we found that the glial cell-derived neurotrophic factor receptor alpha 1 (GFRα1) is required in adult mHb neurons for synaptic stability and function. mHb neurons express some of the highest levels of GFRα1 in the mouse brain, and acute ablation of GFRα1 results in loss of septohabenular and habenulointerpeduncular glutamatergic synapses, with the remaining synapses displaying reduced numbers of presynaptic vesicles. Chemo- and optogenetic studies in mice lacking GFRα1 revealed impaired circuit connectivity, reduced AMPA receptor postsynaptic currents, and abnormally low rectification index (R.I.) of AMPARs, suggesting reduced Ca2+ permeability. Further biochemical and proximity ligation assay (PLA) studies defined the presence of GluA1/GluA2 (Ca2+ impermeable) as well as GluA1/GluA4 (Ca2+ permeable) AMPAR complexes in mHb neurons, as well as clear differences in the levels and association of AMPAR subunits with mHb neurons lacking GFRα1. Finally, acute loss of GFRα1 in adult mHb neurons reduced anxiety-like behavior and potentiated context-based fear responses, phenocopying the effects of lesions to septal projections to the mHb. These results uncover an unexpected function for GFRα1 in the maintenance and function of adult glutamatergic synapses and reveal a potential new mechanism for regulating synaptic plasticity in the septohabenulointerpeduncular pathway and attuning of anxiety and fear behaviors.

Lack of Cdk5 activity is involved on Dopamine Transporter expression and function: Evidences from an animal model of Attention-Deficit Hyperactivity Disorder.

Attention deficit/Hyperactivity disorder (ADHD) is one of the most diagnosed psychiatric disorders nowadays. The core symptoms of the condition include hyperactivity, impulsiveness and inattention. The main pharmacological treatment consists of psychostimulant drugs affecting Dopamine Transporter (DAT) function. We have previously shown that genetically modified mice lacking p35 protein (p35KO), which have reduced Cdk5 activity, present key hallmarks resembling those described in animal models useful for studying ADHD. The p35KO mouse displays spontaneous hyperactivity and shows a calming effect of methylphenidate or amphetamine treatment. Interestingly, dopaminergic neurotransmission is altered in these mice as they have an increased Dopamine (DA) content together with a low DA turnover. This led us to hypothesize that the lack of Cdk5 activity affects DAT expression and/or function in this animal model. In this study, we performed biochemical assays, cell-based approaches, quantitative fluorescence analysis and functional studies that allowed us to demonstrate that p35KO mice exhibit decreased DA uptake and reduced cell surface DAT expression levels in the striatum (STR). These findings are supported by in vitro observations in which the inhibition of Cdk5 activity in N2a cells induced a significant increase in constitutive DAT endocytosis with a concomitant increase in DAT localization to recycling endosomes. Taken together, these data provide evidences regarding the role of Cdk5/p35 in DAT expression and function, thus contributing to the knowledge of DA neurotransmission physiology and also providing therapeutic options for the treatment of DA pathologies such as ADHD.

Control of brown adipose tissue adaptation to nutrient stress by the activin receptor ALK7.

Adaptation to nutrient availability is crucial for survival. Upon nutritional stress, such as during prolonged fasting or cold exposure, organisms need to balance the feeding of tissues and the maintenance of body temperature. The mechanisms that regulate the adaptation of brown adipose tissue (BAT), a key organ for non-shivering thermogenesis, to variations in nutritional state are not known. Here we report that specific deletion of the activin receptor ALK7 in BAT resulted in fasting-induced hypothermia due to exaggerated catabolic activity in brown adipocytes. After overnight fasting, BAT lacking ALK7 showed increased expression of genes responsive to nutrient stress, including the upstream regulator KLF15, aminoacid catabolizing enzymes, notably proline dehydrogenase (POX), and adipose triglyceride lipase (ATGL), as well as markedly reduced lipid droplet size. In agreement with this, ligand stimulation of ALK7 suppressed POX and KLF15 expression in both mouse and human brown adipocytes. Treatment of mutant mice with the glucocorticoid receptor antagonist RU486 restored KLF15 and POX expression levels in mutant BAT, suggesting that loss of BAT ALK7 results in excessive activation of glucocorticoid signaling upon fasting. These results reveal a novel signaling pathway downstream of ALK7 which regulates the adaptation of BAT to nutrient availability by limiting nutrient stress-induced overactivation of catabolic responses in brown adipocytes.

ALK4 coordinates extracellular and intrinsic signals to regulate development of cortical somatostatin interneurons.

Although the role of transcription factors in fate specification of cortical interneurons is well established, how these interact with extracellular signals to regulate interneuron development is poorly understood. Here we show that the activin receptor ALK4 is a key regulator of the specification of somatostatin interneurons. Mice lacking ALK4 in GABAergic neurons of the medial ganglionic eminence (MGE) showed marked deficits in distinct subpopulations of somatostatin interneurons from early postnatal stages of cortical development. Specific losses were observed among distinct subtypes of somatostatin+/Reelin+ double-positive cells, including Hpse+ layer IV cells targeting parvalbumin+ interneurons, leading to quantitative alterations in the inhibitory circuitry of this layer. Activin-mediated ALK4 signaling in MGE cells induced interaction of Smad2 with SATB1, a transcription factor critical for somatostatin interneuron development, and promoted SATB1 nuclear translocation and repositioning within the somatostatin gene promoter. These results indicate that intrinsic transcriptional programs interact with extracellular signals present in the environment of MGE cells to regulate cortical interneuron specification.

Correction: MAG induces apoptosis in cerebellar granule neurons through p75NTR demarcating granule layer/white matter boundary.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

MAG induces apoptosis in cerebellar granule neurons through p75NTR demarcating granule layer/white matter boundary.

MAG (Myelin-associated glycoprotein) is a type I transmembrane glycoprotein expressed by Schwann cells and oligodendrocytes, that has been implicated in the control of axonal growth in many neuronal populations including cerebellar granule neurons (CGNs). However, it is unclear whether MAG has other functions in central nervous system, in particular, in cerebellar development and patterning. We find that MAG expression in the cerebellum is compartmentalised resulting in increased MAG protein levels in the cerebellar white matter. MAG induces apoptosis in developing CGNs through p75NTR signalling. Deletion of p75NTR in vivo reduced the number of apoptotic neurons in cerebellar white matter during development leading to reduction in the size of white matter in the adulthood. Furthermore, we show that MAG impairs CGNs neurite outgrowth as consequence of MAG-induced apoptosis in CGNs. Mechanistically, we find that MAG/NgR1-induced cell death is dependent of p75NTR-mediated activation of JNK/cell death signalling pathway. Together, these findings identify the mechanisms by which MAG induces CGNs apoptotic activity, a crucial event that facilitates cerebellar layer refinement during development.

Cdk5 Is Essential for Amphetamine to Increase Dendritic Spine Density in Hippocampal Pyramidal Neurons.

Psychostimulant drugs of abuse increase dendritic spine density in reward centers of the brain. However, little is known about their effects in the hippocampus, where activity-dependent changes in the density of dendritic spine are associated with learning and memory. Recent reports suggest that Cdk5 plays an important role in drug addiction, but its role in psychostimulant's effects on dendritic spines in hippocampus remain unknown. We used in vivo and in vitro approaches to demonstrate that amphetamine increases dendritic spine density in pyramidal neurons of the hippocampus. Primary cultures and organotypic slice cultures were used for cellular, molecular, pharmacological and biochemical analyses of the role of Cdk5/p25 in amphetamine-induced dendritic spine formation. Amphetamine (two-injection protocol) increased dendritic spine density in hippocampal neurons of thy1-green fluorescent protein (GFP) mice, as well as in hippocampal cultured neurons and organotypic slice cultures. Either genetic or pharmacological inhibition of Cdk5 activity prevented the amphetamine-induced increase in dendritic spine density. Amphetamine also increased spine density in neurons overexpressing the strong Cdk5 activator p25. Finally, inhibition of calpain, the protease necessary for the conversion of p35 to p25, prevented amphetamine's effect on dendritic spine density. We demonstrate, for the first time, that amphetamine increases the density of dendritic spine in hippocampal pyramidal neurons in vivo and in vitro. Moreover, we show that the Cdk5/p25 signaling and calpain activity are both necessary for the effect of amphetamine on dendritic spine density. The identification of molecular mechanisms underlying psychostimulant effects provides novel and promising therapeutic approaches for the treatment of drug addiction.

Mice lacking p35 display hyperactivity and paradoxical response to psychostimulants.

Cyclin-dependent kinase 5/p35 kinase complex plays a critical role in dopaminergic neurotransmission. Dysregulation of dopamine (DA) signaling is associated with neurological and neuropsychiatric disorders. As cyclin-dependent kinase 5 (Cdk5) requires association with p35 for its proper activation, we hypothesized that dysregulation of Cdk5 activity might have an effect on striatal-mediated behavior. We used a mutant mouse, deficient in p35 protein (p35 KO), which displayed reduced Cdk5 activity. Throughout behavioral and biochemical characterization of naïve and psychostimulant-treated mice, we demonstrated that only juvenile p35 KO mice displayed spontaneous hyperactivity, responded with a paradoxical hypolocomotor effect to psychostimulant drugs and exhibited deficit on proper behavioral inhibition. Strong immunolabeling for tyrosine-hydroxylase and high striatal DA synthesis and contents with a low DA turnover, which were reverted by psychostimulants, were also found in mutant mice. Our results demonstrate that p35 deficiency is critically involved in the expression of a hyperactive behavioral phenotype with hyper-functioning of the dopaminergic system, emphasizing the importance of proper Cdk5 kinase activity for normal motor and emotional features. Thus, p35 KO mice may be another useful animal model for understanding cellular and molecular events underlying attention deficit hyperactivity disorder-like disorders.

Transient enhanced expression of Cdk5 activator p25 after acute and chronic d-amphetamine administration.

The cellular and molecular mechanisms of sensitization in the addictive process are still unclear. Recently, chronic treatment with cocaine has been shown to upregulate the expression of cyclin-dependent kinase 5 (cdk5) and its specific activator, p35, in the striatum, as a downstream target gene of DeltaFosB, and has been implicated in compensatory adaptive changes associated with psychostimulants. Cdk5 is a serine/threonine kinase and its activation is achieved through association with a regulatory subunit, either p35 or p39. P35 is cleaved by the protease calpain, which results in the generation of a truncated product termed p25, which contains all elements necessary for cdk5 activation. The cdk5/p35 complex plays an essential role in neuronal development and survival. It has also been involved in neuronal trafficking and transport and in dopaminergic transmission, indicating its role either in presynaptic and postsynaptic signaling. In this study we report that the cdk5/p35 complex participates in acute and chronic d-amphetamine (AMPH)-evoked behavioral events, and we show a surprisingly transient enhanced expression of p25 and a lasting increased expression of p35 in dorsal striatal synaptosomes after acute and chronic AMPH administration. Pak1, a substrate for cdk5, is also enriched in the synaptosomal fraction of acute AMPH-treated rats. Our data suggest that the transient upregulation of p25 may regulate the activity of cdk5 in phosphorylating particular substrates, such as Pak1, implicated in the compensatory adaptive morphophysiologic changes associated with the process of behavioral sensitization to psychostimulants.