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.

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.

Identification of an endocannabinoid gut-brain vagal mechanism controlling food reward and energy homeostasis.

The regulation of food intake, a sine qua non requirement for survival, thoroughly shapes feeding and energy balance by integrating both homeostatic and hedonic values of food. Unfortunately, the widespread access to palatable food has led to the development of feeding habits that are independent from metabolic needs. Among these, binge eating (BE) is characterized by uncontrolled voracious eating. While reward deficit seems to be a major contributor of BE, the physiological and molecular underpinnings of BE establishment remain elusive. Here, we combined a physiologically relevant BE mouse model with multiscale in vivo approaches to explore the functional connection between the gut-brain axis and the reward and homeostatic brain structures. Our results show that BE elicits compensatory adaptations requiring the gut-to-brain axis which, through the vagus nerve, relies on the permissive actions of peripheral endocannabinoids (eCBs) signaling. Selective inhibition of peripheral CB1 receptors resulted in a vagus-dependent increased hypothalamic activity, modified metabolic efficiency, and dampened activity of mesolimbic dopamine circuit, altogether leading to the suppression of palatable eating. We provide compelling evidence for a yet unappreciated physiological integrative mechanism by which variations of peripheral eCBs control the activity of the vagus nerve, thereby in turn gating the additive responses of both homeostatic and hedonic brain circuits which govern homeostatic and reward-driven feeding. In conclusion, we reveal that vagus-mediated eCBs/CB1R functions represent an interesting and innovative target to modulate energy balance and counteract food-reward disorders.

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.

Conditional BDNF Delivery from Astrocytes Rescues Memory Deficits, Spine Density, and Synaptic Properties in the 5xFAD Mouse Model of Alzheimer Disease.

It has been well documented that neurotrophins, including brain-derived neurotrophic factor (BDNF), are severely affected in Alzheimer's disease (AD), but their administration faces a myriad of technical challenges. Here we took advantage of the early astrogliosis observed in an amyloid mouse model of AD (5xFAD) and used it as an internal sensor to administer BDNF conditionally and locally. We first demonstrate the relevance of BDNF release from astrocytes by evaluating the effects of coculturing WT neurons and BDNF-deficient astrocytes. Next, we crossed 5xFAD mice with pGFAP:BDNF mice (only males were used) to create 5xFAD mice that overexpress BDNF when and where astrogliosis is initiated (5xF:pGB mice). We evaluated the behavioral phenotype of these mice. We first found that BDNF from astrocytes is crucial for dendrite outgrowth and spine number in cultured WT neurons. Double-mutant 5xF:pGB mice displayed improvements in cognitive tasks compared with 5xFAD littermates. In these mice, there was a rescue of BDNF/TrkB downstream signaling activity associated with an improvement of dendritic spine density and morphology. Clusters of synaptic markers, PSD-95 and synaptophysin, were also recovered in 5xF:pGB compared with 5xFAD mice as well as the number of presynaptic vesicles at excitatory synapses. Additionally, experimentally evoked LTP in vivo was increased in 5xF:pGB mice. The beneficial effects of conditional BDNF production and local delivery at the location of active neuropathology highlight the potential to use endogenous biomarkers with early onset, such as astrogliosis, as regulators of neurotrophic therapy in AD.SIGNIFICANCE STATEMENT Recent evidence places astrocytes as pivotal players during synaptic plasticity and memory processes. In the present work, we first provide evidence that astrocytes are essential for neuronal morphology via BDNF release. We then crossed transgenic mice (5xFAD mice) with the transgenic pGFAP-BDNF mice, which express BDNF under the GFAP promoter. The resultant double-mutant mice 5xF:pGB mice displayed a full rescue of hippocampal BDNF loss and related signaling compared with 5xFAD mice and a significant and specific improvement in all the evaluated cognitive tasks. These improvements did not correlate with amelioration of ß amyloid load or hippocampal adult neurogenesis rate but were accompanied by a dramatic recovery of structural and functional synaptic plasticity.

Fluorescence-activated sorting of fixed nuclei: a general method for studying nuclei from specific cell populations that preserves post-translational modifications.

Long-lasting brain alterations that underlie learning and memory are triggered by synaptic activity. How activity can exert long-lasting effects on neurons is a major question in neuroscience. Signalling pathways from cytoplasm to nucleus and the resulting changes in transcription and epigenetic modifications are particularly relevant in this context. However, a major difficulty in their study comes from the cellular heterogeneity of brain tissue. A promising approach is to directly purify identified nuclei. Using mouse striatum we have developed a rapid and efficient method for isolating cell type-specific nuclei from fixed adult brain (fluorescence-activated sorting of fixed nuclei; FAST-FIN). Animals are quickly perfused with a formaldehyde fixative that stops enzymatic reactions and maintains the tissue in the state it was at the time of death, including nuclear localisation of soluble proteins such as GFP and differences in nuclear size between cell types. Tissue is subsequently dissociated with a Dounce homogeniser and nuclei prepared by centrifugation in an iodixanol density gradient. The purified fixed nuclei can then be immunostained with specific antibodies and analysed or sorted by flow cytometry. Simple criteria allow distinction of neurons and non-neuronal cells. Immunolabelling and transgenic mice that express fluorescent proteins can be used to identify specific cell populations, and the nuclei from these populations can be efficiently isolated, even rare cell types such as parvalbumin-expressing interneurons. FAST-FIN allows the preservation and study of dynamic and labile post-translational protein modifications. It should be applicable to other tissues and species, and allow study of DNA and its modifications.

Nucleus accumbens D1- and D2-expressing neurons control the balance between feeding and activity-mediated energy expenditure.

Accumulating evidence points to dysregulations of the Nucleus Accumbens (NAc) in eating disorders (ED), however its precise contribution to ED symptomatic dimensions remains unclear. Using chemogenetic manipulations in male mice, we found that activity of dopamine D1 receptor-expressing neurons of the NAc core subregion facilitated effort for a food reward as well as voluntary exercise, but decreased food intake, while D2-expressing neurons have opposite effects. These effects are congruent with D2-neurons being more active than D1-neurons during feeding while it is the opposite during running. Chronic manipulations of each subpopulations had limited effects on energy balance. However, repeated activation of D1-neurons combined with inhibition of D2-neurons biased behavior toward activity-related energy expenditure, whilst the opposite manipulations favored energy intake. Strikingly, concomitant activation of D1-neurons and inhibition of D2-neurons precipitated weight loss in anorexia models. These results suggest that dysregulations of NAc dopaminoceptive neurons might be at the core of EDs.

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.

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.

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.

The Addiction-Susceptibility TaqIA/Ankk1 Controls Reward and Metabolism Through D2 Receptor-Expressing Neurons.

BACKGROUND: A large body of evidence highlights the importance of genetic variants in the development of psychiatric and metabolic conditions. Among these, the TaqIA polymorphism is one of the most commonly studied in psychiatry. TaqIA is located in the gene that codes for the ankyrin repeat and kinase domain containing 1 kinase (Ankk1) near the dopamine D2 receptor (D2R) gene. Homozygous expression of the A1 allele correlates with a 30% to 40% reduction of striatal D2R, a typical feature of addiction, overeating, and other psychiatric pathologies. The mechanisms by which the variant influences dopamine signaling and behavior are unknown. METHODS: Here, we used transgenic and viral-mediated strategies to reveal the role of Ankk1 in the regulation of activity and functions of the striatum. RESULTS: We found that Ankk1 is preferentially enriched in striatal D2R-expressing neurons and that Ankk1 loss of function in the dorsal and ventral striatum leads to alteration in learning, impulsivity, and flexibility resembling endophenotypes described in A1 carriers. We also observed an unsuspected role of Ankk1 in striatal D2R-expressing neurons of the ventral striatum in the regulation of energy homeostasis and documented differential nutrient partitioning in humans with or without the A1 allele. CONCLUSIONS: Overall, our data demonstrate that the Ankk1 gene is necessary for the integrity of striatal functions and reveal a new role for Ankk1 in the regulation of body metabolism.

Hypothalamic astrocytes control systemic glucose metabolism and energy balance.

The hypothalamus is key in the control of energy balance. However, strategies targeting hypothalamic neurons have failed to provide viable options to treat most metabolic diseases. Conversely, the role of astrocytes in systemic metabolic control has remained largely unexplored. Here, we show that obesity promotes anatomically restricted remodeling of hypothalamic astrocyte activity. In the paraventricular nucleus (PVN) of the hypothalamus, chemogenetic manipulation of astrocytes results in bidirectional control of neighboring neuron activity, autonomic outflow, glucose metabolism, and energy balance. This process recruits a mechanism involving the astrocytic control of ambient glutamate levels, which becomes defective in obesity. Positive or negative chemogenetic manipulation of PVN astrocyte Ca2+ signals, respectively, worsens or improves metabolic status of diet-induced obese mice. Collectively, these findings highlight a yet unappreciated role for astrocytes in the direct control of systemic metabolism and suggest potential targets for anti-obesity strategy.

The non-receptor tyrosine kinase Pyk2 modulates acute locomotor effects of cocaine in D1 receptor-expressing neurons of the nucleus accumbens.

The striatum is critical for cocaine-induced locomotor responses. Although the role of D1 receptor-expressing neurons is established, underlying molecular pathways are not fully understood. We studied the role of Pyk2, a non-receptor, calcium-dependent protein-tyrosine kinase. The locomotor coordination and basal activity of Pyk2 knock-out mice were not altered and major striatal protein markers were normal. Cocaine injection increased Pyk2 tyrosine phosphorylation in mouse striatum. Pyk2-deficient mice displayed decreased locomotor response to acute cocaine injection. In contrast, locomotor sensitization and conditioned place preference were normal. Cocaine-activated ERK phosphorylation, a signaling pathway essential for these late responses, was unaltered. Conditional deletion of Pyk2 in the nucleus accumbens or in D1 neurons reproduced decreased locomotor response to cocaine, whereas deletion of Pyk2 in the dorsal striatum or in A2A receptor-expressing neurons did not. In mice lacking Pyk2 in D1-neurons locomotor response to D1 agonist SKF-81297, but not to an anticholinergic drug, was blunted. Our results identify Pyk2 as a regulator of acute locomotor responses to psychostimulants. They highlight the role of tyrosine phosphorylation pathways in striatal neurons and suggest that changes in Pyk2 expression or activation may alter specific responses to drugs of abuse, or possibly other behavioral responses linked to dopamine action.

Circulating Triglycerides Gate Dopamine-Associated Behaviors through DRD2-Expressing Neurons.

Energy-dense food alters dopaminergic (DA) transmission in the mesocorticolimbic (MCL) system and can promote reward dysfunctions, compulsive feeding, and weight gain. Yet the mechanisms by which nutrients influence the MCL circuitry remain elusive. Here, we show that nutritional triglycerides (TGs), a conserved post-prandial metabolic signature among mammals, can be metabolized within the MCL system and modulate DA-associated behaviors by gating the activity of dopamine receptor subtype 2 (DRD2)-expressing neurons through a mechanism that involves the action of the lipoprotein lipase (LPL). Further, we show that in humans, post-prandial TG excursions modulate brain responses to food cues in individuals carrying a genetic risk for reduced DRD2 signaling. Collectively, these findings unveil a novel mechanism by which dietary TGs directly alter signaling in the reward circuit to regulate behavior, thereby providing a new mechanistic basis by which energy-rich diets may lead to (mal)adaptations in DA signaling that underlie reward deficit and compulsive behavior.

Pyk2 in the amygdala modulates chronic stress sequelae via PSD-95-related micro-structural changes.

Major depressive disorder (MDD) is a common disorder with a variety of symptoms including mood alterations, anhedonia, sleep and appetite disorders, and cognitive disturbances. Stressful life events are among the strongest risk factors for developing MDD. At the cellular level, chronic stress results in the modification of dendritic spine morphology and density. Here, we study the role of Pyk2 in the development of depressive-like symptoms induced by a model of chronic unpredictable mild stress (CUMS). Pyk2 is a non-receptor calcium-dependent protein-tyrosine kinase highly expressed in the forebrain principal neurons and involved in spine structure and density regulation. We show that Pyk2 knockout mice are less affected to anxiety-like and anhedonia-like phenotypes induced by the CUMS paradigm. Using region-specific knockout, we demonstrate that this phenotype is fully recapitulated by selective Pyk2 inactivation in the amygdala. We also show that in the absence of Pyk2 the spine alterations, PSD-95 clustering, and NMDA receptors changes induced by the CUMS paradigm are prevented. Our results reveal a possible role for Pyk2 in the response to stress and in synaptic markers expression and spine density regulation in the amygdala. We suggest that Pyk2 contributes to stress-induced responses through micro-structural changes and that its deficit may contribute to the resilience to chronic stress.

MiR-21 is an Ngf-modulated microRNA that supports Ngf signaling and regulates neuronal degeneration in PC12 cells.

The neurotrophins Ngf, Bdnf, NT-3, NT4-5 have key roles in development, survival, and plasticity of neuronal cells. Their action involves broad gene expression changes at the level of transcription and translation. MicroRNAs (miRs)-small RNA molecules that control gene expression post-transcriptionally-are increasingly implicated in regulating development and plasticity of neural cells. Using PC12 cells as a model system, we show that Ngf modulates changes in expression of a variety of microRNAs, including miRs known to be modulated by neurotrophins-such as the miR-212/132 cluster-and several others, such as miR-21, miR-29c, miR-30c, miR-93, miR-103, miR-207, miR-691, and miR-709. Pathway analysis indicates that Ngf-modulated miRs may regulate many protein components of signaling pathways involved in neuronal development and disease. In particular, we show that miR-21 enhances neurotrophin signaling and controls neuronal differentiation induced by Ngf. Notably, in a situation mimicking neurodegeneration-differentiated neurons deprived of Ngf-this microRNA is able to preserve the neurite network and to support viability of the neurons. These findings uncover a broad role of microRNAs in regulating neurotrophin signaling and suggest that aberrant expression of one or more Ngf-modulated miRs may be involved in neurodegenerative diseases.