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.

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.

Sideroflexin 3 is an α-synuclein-dependent mitochondrial protein that regulates synaptic morphology.

α-Synuclein plays a central role in Parkinson's disease, where it contributes to the vulnerability of synapses to degeneration. However, the downstream mechanisms through which α-synuclein controls synaptic stability and degeneration are not fully understood. Here, comparative proteomics on synapses isolated from α-synuclein-/- mouse brain identified mitochondrial proteins as primary targets of α-synuclein, revealing 37 mitochondrial proteins not previously linked to α-synuclein or neurodegeneration pathways. Of these, sideroflexin 3 (SFXN3) was found to be a mitochondrial protein localized to the inner mitochondrial membrane. Loss of SFXN3 did not disturb mitochondrial electron transport chain function in mouse synapses, suggesting that its function in mitochondria is likely to be independent of canonical bioenergetic pathways. In contrast, experimental manipulation of SFXN3 levels disrupted synaptic morphology at the Drosophila neuromuscular junction. These results provide novel insights into α-synuclein-dependent pathways, highlighting an important influence on mitochondrial proteins at the synapse, including SFXN3. We also identify SFXN3 as a new mitochondrial protein capable of regulating synaptic morphology in vivo.

eIF4E phosphorylation recruits ß-catenin to mRNA cap and promotes Wnt pathway translation in dentate gyrus LTP maintenance.

The mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), is crucial for translation and regulated by Ser209 phosphorylation. However, the biochemical and physiological role of eIF4E phosphorylation in translational control of long-term synaptic plasticity is unknown. We demonstrate that phospho-ablated Eif4eS209A Knockin mice are profoundly impaired in dentate gyrus LTP maintenance in vivo, whereas basal perforant path-evoked transmission and LTP induction are intact. mRNA cap-pulldown assays show that phosphorylation is required for synaptic activity-induced removal of translational repressors from eIF4E, allowing initiation complex formation. Using ribosome profiling, we identified selective, phospho-eIF4E-dependent translation of the Wnt signaling pathway in LTP. Surprisingly, the canonical Wnt effector, ß-catenin, was massively recruited to the eIF4E cap complex following LTP induction in wild-type, but not Eif4eS209A, mice. These results demonstrate a critical role for activity-evoked eIF4E phosphorylation in dentate gyrus LTP maintenance, remodeling of the mRNA cap-binding complex, and specific translation of the Wnt pathway.

A seeding-based neuronal model of tau aggregation for use in drug discovery.

Intracellular accumulation of tau protein is a hallmark of Alzheimer's Disease and Progressive Supranuclear Palsy, as well as other neurodegenerative disorders collectively known as tauopathies. Despite our increasing understanding of the mechanisms leading to the initiation and progression of tau pathology, the field still lacks appropriate disease models to facilitate drug discovery. Here, we established a novel and modulatable seeding-based neuronal model of full-length 4R tau accumulation using humanized mouse cortical neurons and seeds from P301S human tau transgenic animals. The model shows specific and consistent formation of intraneuronal insoluble full-length 4R tau inclusions, which are positive for known markers of tau pathology (AT8, PHF-1, MC-1), and creates seeding competent tau. The formation of new inclusions can be prevented by treatment with tau siRNA, providing a robust internal control for use in qualifying the assessment of potential therapeutic candidates aimed at reducing the intracellular pool of tau. In addition, the experimental set up and data analysis techniques used provide consistent results in larger-scale designs that required multiple rounds of independent experiments, making this is a versatile and valuable cellular model for fundamental and early pre-clinical research of tau-targeted therapies.

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.

The Role of the Eukaryotic Translation Initiation Factor 4E (eIF4E) in Neuropsychiatric Disorders.

Protein synthesis in eukaryotic cells is a complex, multi-step and tightly regulated process. Translation initiation, the rate limiting step in protein synthesis, is dependent on the activity of eukaryotic translation Initiation Factor 4E (eIF4E). eIF4E is the cap-binding protein which, in synergy with proteins such as the helicase eIF4A and the scaffolding protein eIF4G, binds to mRNA, allowing the recruitment of ribosomes and translation initiation. The function of eIF4E is tightly regulated in cells under normal physiological conditions and can be controlled by post-translational modifications, such as phosphorylation, and by the binding of inhibitory proteins, including eIF4E binding proteins (4E-BPs) and CYFIP1. Recent studies have highlighted the importance of eIF4E in normal or aberrant function of the nervous system. In this mini-review, we will highlight the role of eIF4E function and regulation in the pathophysiology of neurodevelopmental and neuropsychiatric disorders.

Translational profiling of dorsal root ganglia and spinal cord in a mouse model of neuropathic pain.

Acute pain serves as a protective mechanism, guiding the organism away from actual or potential tissue injury. In contrast, chronic pain is a debilitating condition without any obvious physiological function. The transition to, and the maintenance of chronic pain require new gene expression to support biochemical and structural changes within the pain pathway. The regulation of gene expression at the level of mRNA translation has emerged as an important step in the control of protein expression in the cell. Recent studies show that signaling pathways upstream of mRNA translation, such as mTORC1 and ERK, are upregulated in chronic pain conditions, and their inhibition effectively alleviates pain in several animal models. Despite this progress, mRNAs whose translation is altered in chronic pain conditions remain largely unknown. Here, we performed genome-wide translational profiling of dorsal root ganglion (DRG) and spinal cord dorsal horn tissues in a mouse model of neuropathic pain, spared nerve injury (SNI), using the ribosome profiling technique. We identified distinct subsets of mRNAs that are differentially translated in response to nerve injury in both tissues. We discovered key converging upstream regulators and pathways linked to mRNA translational control and neuropathic pain. Our data are crucial for the understanding of mechanisms by which mRNA translation promotes persistent hypersensitivity after nerve injury.

Molecular neuropathology of the synapse in sheep with CLN5 Batten disease.

AIMS: Synapses represent a major pathological target across a broad range of neurodegenerative conditions. Recent studies addressing molecular mechanisms regulating synaptic vulnerability and degeneration have relied heavily on invertebrate and mouse models. Whether similar molecular neuropathological changes underpin synaptic breakdown in large animal models and in human patients with neurodegenerative disease remains unclear. We therefore investigated whether molecular regulators of synaptic pathophysiology, previously identified in Drosophila and mouse models, are similarly present and modified in the brain of sheep with CLN5 Batten disease. METHODS: Gross neuropathological analysis of CLN5 Batten disease sheep and controls was used alongside postmortem MRI imaging to identify affected brain regions. Synaptosome preparations were then generated and quantitative fluorescent Western blotting used to determine and compare levels of synaptic proteins. RESULTS: The cortex was particularly affected by regional neurodegeneration and synaptic loss in CLN5 sheep, whilst the cerebellum was relatively spared. Quantitative assessment of the protein content of synaptosome preparations revealed significant changes in levels of seven out of eight synaptic neurodegeneration proteins investigated in the motor cortex, but not cerebellum, of CLN5 sheep (α-synuclein, CSP-α, neurofascin, ROCK2, calretinin, SIRT2, and UBR4). CONCLUSIONS: Synaptic pathology is a robust correlate of region-specific neurodegeneration in the brain of CLN5 sheep, driven by molecular pathways similar to those reported in Drosophila and rodent models. Thus, large animal models, such as sheep, represent ideal translational systems to develop and test therapeutics aimed at delaying or halting synaptic pathology for a range of human neurodegenerative conditions.