Mitogen-activated protein kinase phosphatase-2 deletion impairs synaptic plasticity and hippocampal-dependent memory

Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer’s disease. Thus there is great interest in understanding the signalling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2-/- mice), we show that long-term potentiation (LTP) is impaired in MKP-2-/- mice compared to MKP-2+/+ controls whereas neuronal excitability, evoked synaptic transmission and paired-pulse facilitation remain unaltered. Furthermore, spontaneous excitatory postsynaptic currents (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2-/- mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures which may account for the increase in spontaneous EPSC frequency. In addition no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2-/- mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signalling

Mitogen-activated protein kinase phosphatase-2 deletion modifies ventral tegmental area function and connectivity and alters reward processing

Mitogen-activated protein kinases (MAPKs) regulate normal brain functioning, and their dysfunction is implicated in a number of brain disorders. Thus, there is great interest in understanding the signalling systems that control MAPK functioning. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in foetal development, the immune system, cancer and synaptic plasticity and memory. In the present study, we performed an unbiased investigation using MKP-2-/- mice to assess whether MKP-2 plays a global role in modulating brain function. Local cerebral glucose utilization is significantly increased in the ventral tegmental area (VTA) of MKP-2-/- mice, with connectivity analysis revealing alterations in VTA functional connectivity, including a significant reduction in connectivity to the nucleus accumbens and hippocampus. In addition, spontaneous excitatory postsynaptic current frequency, but not amplitude, onto putative dopamine neurons in the VTA is increased in MKP-2-/- mice, which indicates that increased excitatory drive may account for the increased VTA glucose utilization. Consistent with modified VTA function and connectivity, in behavioural tests MKP-2-/- mice exhibited increased sucrose preference and impaired amphetamine-induced hyperlocomotion. Overall, these data reveal that MKP-2 plays a role in modulating VTA function and that its dysfunction may contribute to brain disorders in which altered reward processing is present

Modulation of SF1 neuron activity coordinately regulates both feeding behaviour and associated emotional states

Feeding requires the integration of homeostatic drives with emotional states relevant to food procurement in potentially hostile environments. The ventromedial hypothalamus (VMH) regulates feeding and anxiety, but how these are controlled in a concerted manner remains unclear. Using pharmacogenetic, optogenetic, and calcium imaging approaches with a battery of behavioral assays, we demonstrate that VMH steroidogenic factor 1 (SF1) neurons constitute a nutritionally sensitive switch, modulating the competing motivations of feeding and avoidance of potentially dangerous environments. Acute alteration of SF1 neuronal activity alters food intake via changes in appetite and feeding-related behaviors, including locomotion, exploration, anxiety, and valence. In turn, intrinsic SF1 neuron activity is low during feeding and increases with both feeding termination and stress. Our findings identify SF1 neurons as a key part of the neurocircuitry that controls both feeding and related affective states, giving potential insights into the relationship between disordered eating and stress-associated psychological disorders in humans

RNA polymerase II primes Polycomb-repressed developmental genes throughout terminal neuronal differentiation

Polycomb repression in mouse embryonic stem cells (ESCs) is tightly associated with promoter co-occupancy of RNA polymerase II (RNAPII) which is thought to prime genes for activation during early development. However, it is unknown whether RNAPII poising is a general feature of Polycomb repression, or is lost during differentiation. Here, we map the genome-wide occupancy of RNAPII and Polycomb from pluripotent ESCs to non-dividing functional dopaminergic neurons. We find that poised RNAPII complexes are ubiquitously present at Polycomb-repressed genes at all stages of neuronal differentiation. We observe both loss and acquisition of RNAPII and Polycomb at specific groups of genes reflecting their silencing or activation. Strikingly, RNAPII remains poised at transcription factor genes which are silenced in neurons through Polycomb repression, and have major roles in specifying other, non-neuronal lineages. We conclude that RNAPII poising is intrinsically associated with Polycomb repression throughout differentiation. Our work suggests that the tight interplay between RNAPII poising and Polycomb repression not only instructs promoter state transitions, but also may enable promoter plasticity in differentiated cells

The Mechanistic Classification of Addictive Drugs

The authors review recent research on the molecular mechanisms of addiction and propose a new classification for addictive drugs

Protein restriction during pregnancy alters Cdkn1c silencing, dopamine circuitry and offspring behaviour without changing expression of key neuronal marker genes

We tracked the consequences of in utero protein restriction in mice throughout their development and life course using a luciferase-based allelic reporter of imprinted Cdkn1c. Exposure to gestational low-protein diet (LPD) results in the inappropriate expression of paternally inherited Cdkn1c in the brains of embryonic and juvenile mice. These animals were characterised by a developmental delay in motor skills, and by behavioural alterations indicative of reduced anxiety. Exposure to LPD in utero resulted in significantly more tyrosine hydroxylase positive (dopaminergic) neurons in the midbrain of adult offspring as compared to age-matched, control-diet equivalents. Positron emission tomography (PET) imaging revealed an increase in striatal dopamine synthesis capacity in LPD-exposed offspring, where elevated levels of dopamine correlated with an enhanced sensitivity to cocaine. These data highlight a profound sensitivity of the developing epigenome to gestational protein restriction. Our data also suggest that loss of Cdkn1c imprinting and p57KIP2 upregulation alters the cellular composition of the developing midbrain, compromises dopamine circuitry, and thereby provokes behavioural abnormalities in early postnatal life. Molecular analyses showed that despite this phenotype, exposure to LPD solely during pregnancy did not significantly change the expression of key neuronal- or dopamine-associated marker genes in adult offspring

Electrophysiological Properties of Embryonic Stem Cell-Derived Neurons

In vitro generation of functional neurons from embryonic stem (ES) cells and induced pluripotent stem cells offers exciting opportunities for dissecting gene function, disease modelling, and therapeutic drug screening. To realize the potential of stem cells in these biomedical applications, a complete understanding of the cell models of interest is required. While rapid advances have been made in developing the technologies for directed induction of defined neuronal subtypes, most published works focus on the molecular characterization of the derived neural cultures. To characterize the functional properties of these neural cultures, we utilized an ES cell model that gave rise to neurons expressing the green fluorescent protein (GFP) and conducted targeted whole-cell electrophysiological recordings from ES cell-derived neurons. Current-clamp recordings revealed that most neurons could fire single overshooting action potentials; in some cases multiple action potentials could be evoked by depolarization, or occurred spontaneously. Voltage-clamp recordings revealed that neurons exhibited neuronal-like currents, including an outward current typical of a delayed rectifier potassium conductance and a fast-activating, fast-inactivating inward current, typical of a sodium conductance. Taken together, these results indicate that ES cell-derived GFP+ neurons in culture display functional neuronal properties even at early stages of differentiation

Tumour compartment transcriptomics demonstrates the activation of inflammatory and odontogenic programmes in human adamantinomatous craniopharyngioma and identifies the MAPK/ERK pathway as a novel therapeutic target

Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. β-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP. KEYWORDS: Craniopharyngioma; IL1-β; Inflammasome; MAPK/ERK pathway; Odontogenesis; Paracrine signalling; Trametini

Antenatal glucocorticoid treatment induces adaptations in adult midbrain dopamine neurons, which underpin sexually dimorphic behavioral resilience

We demonstrated previously that antenatal glucocorticoid treatment (AGT, gestational days 16-19) altered the size and organization of the adult rat midbrain dopaminergic (DA) populations. Here we investigated the consequences of these AGT-induced cytoarchitectural disturbances on indices of DA function in adult rats. We show that in adulthood, enrichment of striatal DA fiber density paralleled AGT-induced increases in the numbers of midbrain DA neurons, which retained normal basal electrophysiological properties. This was co-incident with changes in (i) striatal D2-type receptor levels (increased, both sexes); (ii) D1-type receptor levels (males decreased; females increased); (iii) DA transporter levels (males increased; females decreased) in striatal regions; and (iv) amphetamine-induced mesolimbic DA release (males increased; females decreased). However, despite these profound, sexually dimorphic changes in markers of DA neurotransmission, in-utero glucocorticoid overexposure had a modest or no effect on a range of conditioned and unconditioned appetitive behaviors known to depend on mesolimbic DA activity. These findings provide empirical evidence for enduring AGT-induced adaptive mechanisms within the midbrain DA circuitry, which preserve some, but not all, functions, thereby casting further light on the vulnerability of these systems to environmental perturbations. Furthermore, they demonstrate these effects are achieved by different, often opponent, adaptive mechanisms in males and females, with translational implications for sex biases commonly found in midbrain DA-associated disorders