Mnk1/2 kinases regulate memory and autism-related behaviours via Syngap1.

MAPK interacting protein kinases 1 and 2 (Mnk1/2) regulate a plethora of functions, presumably via phosphorylation of their best characterized substrate, eukaryotic translation initiation factor 4E (eIF4E) on Ser209. Here, we show that, whereas deletion of Mnk1/2 (Mnk double knockout) impairs synaptic plasticity and memory in mice, ablation of phospho-eIF4E (Ser209) does not affect these processes, suggesting that Mnk1/2 possess additional downstream effectors in the brain. Translational profiling revealed only a small overlap between the Mnk1/2- and phospho-eIF4E(Ser209)-regulated translatome. We identified the synaptic Ras GTPase activating protein 1 (Syngap1), encoded by a syndromic autism gene, as a downstream target of Mnk1 because Syngap1 immunoprecipitated with Mnk1 and showed reduced phosphorylation (S788) in Mnk double knockout mice. Knockdown of Syngap1 reversed memory deficits in Mnk double knockout mice and pharmacological inhibition of Mnks rescued autism-related phenotypes in Syngap1+/- mice. Thus, Syngap1 is a downstream effector of Mnk1, and the Mnks-Syngap1 axis regulates memory formation and autism-related behaviours.

Autistic-like behavior and cerebellar dysfunction in Bmal1 mutant mice ameliorated by mTORC1 inhibition.

Although circadian and sleep disorders are frequently associated with autism spectrum disorders (ASD), it remains elusive whether clock gene disruption can lead to autistic-like phenotypes in animals. The essential clock gene Bmal1 has been associated with human sociability and its missense mutations are identified in ASD. Here we report that global Bmal1 deletion led to significant social impairments, excessive stereotyped and repetitive behaviors, as well as motor learning disabilities in mice, all of which resemble core behavioral deficits in ASD. Furthermore, aberrant cell density and immature morphology of dendritic spines were identified in the cerebellar Purkinje cells (PCs) of Bmal1 knockout (KO) mice. Electrophysiological recordings uncovered enhanced excitatory and inhibitory synaptic transmission and reduced firing rates in the PCs of Bmal1 KO mice. Differential expression of ASD- and ataxia-associated genes (Ntng2, Mfrp, Nr4a2, Thbs1, Atxn1, and Atxn3) and dysregulated pathways of translational control, including hyperactivated mammalian target of rapamycin complex 1 (mTORC1) signaling, were identified in the cerebellum of Bmal1 KO mice. Interestingly, the antidiabetic drug metformin reversed mTORC1 hyperactivation and alleviated major behavioral and PC deficits in Bmal1 KO mice. Importantly, conditional Bmal1 deletion only in cerebellar PCs was sufficient to recapitulate autistic-like behavioral and cellular changes akin to those identified in Bmal1 KO mice. Together, these results unveil a previously unidentified role for Bmal1 disruption in cerebellar dysfunction and autistic-like behaviors. Our findings provide experimental evidence supporting a putative role for dysregulation of circadian clock gene expression in the pathogenesis of ASD.

Uncovering memory-related gene expression in contextual fear conditioning using ribosome profiling.

Contextual fear conditioning (CFC) in rodents is the most widely used behavioural paradigm in neuroscience research to elucidate the neurobiological mechanisms underlying learning and memory. It is based on the pairing of an aversive unconditioned stimulus (US; e.g. mild footshock) with a neutral conditioned stimulus (CS; e.g. context of the test chamber) in order to acquire associative long-term memory (LTM), which persists for days and even months. Using genome-wide analysis, several studies have generated lists of genes modulated in response to CFC in an attempt to identify the "memory genes", which orchestrate memory formation. Yet, most studies use naïve animals as a baseline for assessing gene-expression changes, while only few studies have examined the effect of the US alone, without pairing to context, using genome-wide analysis of gene-expression. Herein, using the ribosome profiling methodology, we show that in male mice an immediate shock, which does not lead to LTM formation, elicits pervasive translational and transcriptional changes in the expression of Immediate Early Genes (IEGs) in dorsal hippocampus (such as Fos and Arc), a fact which has been disregarded by the majority of CFC studies. By removing the effect of the immediate shock, we identify and validate a new set of genes, which are translationally and transcriptionally responsive to the association of context-to-footshock in CFC, and thus constitute salient "memory genes".

Monitoring translation in synaptic fractions using a ribosome profiling strategy.

BACKGROUND: The aim of this study was to develop a method to study genome-wide local translation in biochemically isolated synaptic fractions (synaptoneurosomes). This methodology is of particular interest for neurons, due to the cardinal role of local translational control in neuronal sub-compartments, such as dendrites, for plasticity, learning, memory, and for disorders of the nervous system. NEW METHOD: We combined established methods for purifying synaptoneurosomes with translational profiling (ribosome profiling), a method that employs unbiased next generation sequencing to simultaneously assess transcription and translation in a single sample. RESULTS: The two existing methods are compatible to use in combination and yield high quality sequencing data, which are specific to synaptic compartments. This new protocol provides an easy to implement workflow, which combines biochemical isolation of synaptoneurosomes of varying levels of purity (crude or Percoll gradient purified) with the use of a commercial kit to generate sequencing libraries. COMPARISON WITH EXISTING METHODS: Compared to previous studies of the synaptic translatome, our method shows less contamination with non-neuronal cell types or non-synaptic compartments, increasing the specificity of the data obtained. CONCLUSIONS: Combining the isolation of functional synaptic units with ribosome profiling offers a powerful tool to study local translation in synaptic compartments both in health and disease.

Raptor-Mediated Proteasomal Degradation of Deamidated 4E-BP2 Regulates Postnatal Neuronal Translation and NF-κB Activity.

The translation initiation repressor 4E-BP2 is deamidated in the brain on asparagines N99/N102 during early postnatal brain development. This post-translational modification enhances 4E-BP2 association with Raptor, a central component of mTORC1 and alters the kinetics of excitatory synaptic transmission. We show that 4E-BP2 deamidation is neuron specific, occurs in the human brain, and changes 4E-BP2 subcellular localization, but not its disordered structure state. We demonstrate that deamidated 4E-BP2 is ubiquitinated more and degrades faster than the unmodified protein. We find that enhanced deamidated 4E-BP2 degradation is dependent on Raptor binding, concomitant with increased association with a Raptor-CUL4B E3 ubiquitin ligase complex. Deamidated 4E-BP2 stability is promoted by inhibiting mTORC1 or glutamate receptors. We further demonstrate that deamidated 4E-BP2 regulates the translation of a distinct pool of mRNAs linked to cerebral development, mitochondria, and NF-κB activity, and thus may be crucial for postnatal brain development in neurodevelopmental disorders, such as ASD.

Loss of eIF4E Phosphorylation Engenders Depression-like Behaviors via Selective mRNA Translation.

The MAPK/ERK (mitogen-activated protein kinases/extracellular signal-regulated kinase) pathway is a cardinal regulator of synaptic plasticity, learning, and memory in the hippocampus. One of major endpoints of this signaling cascade is the 5' mRNA cap binding protein eIF4E (eukaryotic Initiation Factor 4E), which is phosphorylated on Ser 209 by MNK (MAPK-interacting protein kinases) and controls mRNA translation. The precise role of phospho-eIF4E in the brain is yet to be determined. Herein, we demonstrate that ablation of eIF4E phosphorylation in male mice (4Eki mice) does not impair long-term spatial or contextual fear memory, or the late phase of LTP. Using unbiased translational profiling in mouse brain, we show that phospho-eIF4E differentially regulates the translation of a subset of mRNAs linked to inflammation, the extracellular matrix, pituitary hormones, and the serotonin pathway. Consequently, 4Eki male mice display exaggerated inflammatory responses and reduced levels of serotonin, concomitant with depression and anxiety-like behaviors. Remarkably, eIF4E phosphorylation is required for the chronic antidepressant action of the selective serotonin reuptake inhibitor fluoxetine. Finally, we propose a novel phospho-eIF4E-dependent translational control mechanism in the brain, via the GAIT complex (gamma IFN activated inhibitor of translation). In summary, our work proposes a novel translational control mechanism involved in the regulation of inflammation and depression, which could be exploited to design novel therapeutics.SIGNIFICANCE STATEMENT We demonstrate that downstream of the MAPK (mitogen-activated protein kinase) pathway, eukaryotic Initiation Factor 4E (eIF4E) Ser209 phosphorylation is not required for classical forms of hippocampal LTP and memory. We reveal a novel role for eIF4E phosphorylation in inflammatory responses and depression-like behaviors. eIF4E phosphorylation is required for the chronic action of antidepressants, such as fluoxetine in mice. These phenotypes are accompanied by selective translation of extracellular matrix, pituitary hormones, and serotonin pathway genes, in eIF4E phospho-mutant mice. We also describe a previously unidentified translational control mechanism in the brain, whereby eIF4E phosphorylation is required for inhibiting the translation of gamma IFN activated inhibitor of translation element-containing mRNAs. These findings can be used to design novel therapeutics for depression.