Role of 4E-BP2 in neuronal translation

Translational control is a powerful means to alter gene expression and regulates synaptic plasticity, learning and memory. 4E-BP2 (Eif4ebp2, Eukaryotic Initiation Factor 4E-Binding Protein 2) is the predominantly expressed 4E-BP in the mammalian brain and represses cap-dependent translation initiation, by binding to eIF4E (Eif4e, eukaryotic Initiation Factor 4E). As a master regulator of protein synthesis in the mammalian brain, 4E-BP2 has been implicated in learning, memory and Autism Spectrum Disorder (ASD). Upon phosphorylation by mTOR (mammalian Target Of Rapamycin, mTOR) which occurs in most tissues, 4E-BP2 cannot bind to eIF4E, failing to repress translation initiation. However, in early postnatal brain development, 4E-BP2 undergoes brain-specific post-translational deamidation on asparagines N99 and N102, which are converted to aspartic acid. Asparagine deamidation is not catalysed by enzymes but can occur spontaneously and is induced by alkaline pH. Deamidated 4E-BP2 was shown to regulate the kinetics of excitatory synaptic transmission in early postnatal brain development, suggesting that it may be important for synaptic function during that crucial developmental period. N99/N102 deamidation decreases the affinity of 4E-BP2 for eIF4E and increases its binding to the mTORC1 protein Raptor. The significance of enhanced Raptor binding to deamidated 4E-BP2 is yet unclear because 4E-BP2 phosphorylation is very low in adult brain. Moreover, the role of deamidated 4E-BP2 and the downstream effects of deamidated 4E-BP2 translational control in the mammalian brain are not known but of cardinal importance given the pervasive role of 4E-BP2 in regulating brain function. In this thesis, we describe a previously unidentified mechanism during early postnatal brain development, whereby the constitutively deamidated form of the cardinal brain translation initiation repressor 4E-BP2 is more susceptible to ubiquitin proteasomal degradation (as compared to unmodified, WT protein) because it binds with higher affinity to a complex, comprising the mTORC1 protein Raptor and the ubiquitin E3 ligase CUL4B. Deamidated 4E-BP2 (2D) stability is regulated by mTORC1 and AMPAR activity but not NMDARs. We also showed that 4E-BP2 deamidation is neuron-specific and occurs in human brain. We explored whether deamidated and WT 4E-BP2 have a similar subcellular distribution in neurons, indicating that there is very low co-localization of them in both soma and dendrites. We studied WT and 2D structures, with Synchrotron radiation circular dichroism (SCRD), Small angle X-ray scattering (SAXS) and Nuclear magnetic resonance (NMR) spectroscopy of full-length recombinant 4E-BP2 (WT or 2D) expressed in E. Coli and purified, and we identified that they share a similar structure, with only minor differences in a few residues. Moreover, using unbiased translatome mapping, we discovered that overexpression of deamidated 4E-BP2 represses the translation of a distinct pool of mRNAs linked to cerebral development, mitochondria and chiefly NF- κB activity. Collectively, these data describe a previously unidentified brain-specific translational control mechanism that could be crucial for postnatal brain development in neurodevelopmental disorders such as ASD

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

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"

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

MAPK (mitogen-activated protein kinase) interacting protein kinases 1 and 2 (Mnk1/2) regulate a plethora of functions, presumably via phosphorylation of their best characterised substrate, eukaryotic translation initiation factor 4E (eIF4E) on Ser209. Here, we show that whereas deletion of Mnk1/2 (Mnk DKO) impairs synaptic plasticity and memory in mice, ablation of phosho-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 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 since Syngap1 immunoprecipitated with Mnk1 and showed reduced phosphorylation (S788) in Mnk DKO mice. Knock-down of Syngap1 reversed memory deficits in Mnk DKO 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