Synaptic Protein Translation Research Articles

Lack of FMRP in the retina: Evidence of a retinal specific transcriptomic profile

Fragile X Syndrome (FXS), the most common inherited form of human intellectual disability, is a monogenic neurodevelopmental disorder caused by a loss-of-function mutation of the FMR1 gene. FMR1 is encoding the Fragile X Messenger Ribonucleo Protein (FMRP) an RNA-binding protein that regulates the translation of synaptic proteins. The absence of FMRP expression has many important consequences on synaptic plasticity and function, leading to the FXS clinical phenotype. Over the last decade, a visual neurosensorial phenotype had been described in the FXS patients as well as in the murine model (Fmr1-/ymice), characterized by retinal deficits associated to retinal perception alterations. However, although the transcriptomic profile in the absence of FMRP has been studied in the cerebral part of the central nervous system (CNS), there are no actual data for the retina which is an extension of the CNS. Herein, we investigate the transcriptomic profile of mRNA from whole retinas of Fmr1-/ymice. Interestingly, we found a specific signature of Fmrp absence on retinal mRNA expression with few common genes compared to other brain studies. Gene Ontology on these retinal specific genes demonstrated an enrichment in retinal development genes as well as in synaptic genes. These alterations could be linked to the reported retinal phenotype of the FXS condition. In conclusion, we describe for the first time, retinal-specific transcriptomic changes in the absence of FMRP.

Mutations in FUS lead to synaptic dysregulation in ALS-iPSC derived neurons

Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurodegenerative disorder characterized by progressive muscular weakness due to the selective loss of motor neurons. Mutations in the gene Fused in Sarcoma (FUS) were identified as one cause of ALS. Here, we report that mutations in FUS lead to upregulation of synaptic proteins, increasing synaptic activity and abnormal release of vesicles at the synaptic cleft. Consequently, FUS-ALS neurons showed greater vulnerability to glutamate excitotoxicity, which raised neuronal swellings (varicose neurites) and led to neuronal death. Fragile X mental retardation protein (FMRP) is an RNA-binding protein known to regulate synaptic protein translation, and its expression is reduced in the FUS-ALS lines. Collectively, our data suggest that a reduction of FMRP levels alters the synaptic protein dynamics, leading to synaptic dysfunction and glutamate excitotoxicity. Here, we present a mechanistic hypothesis linking dysregulation of peripheral translation with synaptic vulnerability in the pathogenesis of FUS-ALS.

Mitochondrial Targeting against Alzheimer's Disease: Lessons from Hibernation.

Alzheimer's disease (AD) is the most common cause of dementia worldwide and yet remains without effective therapy. Amongst the many proposed causes of AD, the mitochondrial cascade hypothesis is gaining attention. Accumulating evidence shows that mitochondrial dysfunction is a driving force behind synaptic dysfunction and cognitive decline in AD patients. However, therapies targeting the mitochondria in AD have proven unsuccessful so far, and out-of-the-box options, such as hibernation-derived mitochondrial mechanisms, may provide valuable new insights. Hibernators uniquely and rapidly alternate between suppression and re-activation of the mitochondria while maintaining a sufficient energy supply and without acquiring ROS damage. Here, we briefly give an overview of mitochondrial dysfunction in AD, how it affects synaptic function, and why mitochondrial targeting in AD has remained unsuccessful so far. We then discuss mitochondria in hibernation and daily torpor in mice, covering current advancements in hibernation-derived mitochondrial targeting strategies. We conclude with new ideas on how hibernation-derived dual mitochondrial targeting of both the ATP and ROS pathways may boost mitochondrial health and induce local synaptic protein translation to increase synaptic function and plasticity. Further exploration of these mechanisms may provide more effective treatment options for AD in the future.

Impaired synaptic incorporation of AMPA receptors in a mouse model of fragile X syndrome.

Fragile X syndrome (FXS) is the most common monogenetic cause of inherited intellectual disability and autism in humans. One of the well-characterized molecular phenotypes of Fmr1 KO mice, a model of FXS, is increased translation of synaptic proteins. Although this upregulation stabilizes in adulthood, abnormalities during the critical period of plasticity have long-term effects on circuit formation and synaptic properties. Using high-resolution quantitative proteomics of synaptoneurosomes isolated from the adult, developed brains of Fmr1 KO mice, we show a differential abundance of proteins regulating the postsynaptic receptor activity of glutamatergic synapses. We investigated the AMPA receptor composition and shuttling in adult Fmr1 KO and WT mice using a variety of complementary experimental strategies such as surface protein crosslinking, immunostaining of surface receptors, and electrophysiology. We discovered that the activity-dependent synaptic delivery of AMPARs is impaired in adult Fmr1 KO mice. Furthermore, we show that Fmr1 KO synaptic AMPARs contain more GluA2 subunits that can be interpreted as a switch in the synaptic AMPAR subtype toward an increased number of Ca2+-impermeable receptors in adult Fmr1 KO synapses.

Utilities of Isolated Nerve Terminals in Ex Vivo Analyses of Protein Translation in (Patho)physiological Brain States: Focus on Alzheimer's Disease.

Synapses are the cellular substrates of higher-order brain functions, and their dysfunction is an early and primary pathogenic mechanism across several neurological disorders. In particular, Alzheimer's disease (AD) is categorized by prodromal structural and functional synaptic deficits, prior to the advent of classical behavioral and pathological features. Recent research has shown that the development, maintenance, and plasticity of synapses depend on localized protein translation. Synaptosomes and synaptoneurosomes are biochemically isolated synaptic terminal preparations which have long been used to examine a variety of synaptic processes ex vivo in both healthy and pathological conditions. These ex vivo preparations preserve the mRNA species and the protein translational machinery. Hence, they are excellent in organello tools for the study of alterations in mRNA levels and protein translation in neuropathologies. Evaluation of synapse-specific basal and activity-driven de novo protein translation activity can be conveniently performed in synaptosomal/synaptoneurosomal preparations from both rodent and human brain tissue samples. This review gives a quick overview of the methods for isolating synaptosomes and synaptoneurosomes before discussing the studies that have utilized these preparations to study localized synapse-specific protein translation in (patho)physiological situations, with an emphasis on AD. While the review is not an exhaustive accumulation of all the studies evaluating synaptic protein translation using the synaptosomal model, the aim is to assemble the most relevant studies that have done so. The hope is to provide a suitable research platform to aid neuroscientists to utilize the synaptosomal/synaptoneurosomal models to evaluate the molecular mechanisms of synaptic dysfunction within the specific confines of mRNA localization and protein translation research.

Sex-specific differences in cognitive functions and synaptic dysfunction in an Alzheimer's disease mouse model.

Women have higher burden of Alzheimer's disease (AD) compared to men. Although neuroprotective effect of estrogen is well established, the underlying pathogenic mechanisms explaining the higher burden of AD in women remains unknown. Therefore, we attempted to investigate the sex-specific differences through the progression of AD in familial AD mouse model. Synaptic dysfunction is considered to occur prior to the appearance of the pathophysiological and behavioural symptoms of AD. Akt1-mTOR signaling has a critical role at the synapse in AD. Here, we investigated behavioural and synaptic deficits in female AD mice of different age and compared it male mice of similar age in order to study the progression of AD in mouse models. Recall memory test was assessed by contextual fear conditioning (cFC). We isolated synaptosomes prepared from brain cortices of WT and APP/PS1 mice at different ages. Synaptosomes were resolved on SDS-PAGE and immunoblotted against respective antibodies. WT and APP/PS1 female mice underwent an ovariectomy and sham surgery. After ovariectomy and behavioural assessment we prepared synaptoneurosomes from hippocampus and performed 35 [S] methionine incorporation assay. APP/PS1 female mice protected from cFC recall deficits and activity dependent synaptic protein translation until 8 months of age. However, these mice started showing cFC recall deficits and activity dependent synaptic protein translation after 8 months of age unlike APP/PS1 male mice wherein these deficits were observed very early (2-4 months). Ovariectomized APP/PS1 female mice induced cFC recall deficits and activity dependent protein translation at 4 months of age which is similar to young APP/PS1 male mice. Synaptosomal pAkt1, pGSK3β, pmTOR, p70S6K, p4E-BP1 levels were significantly increased at 4 months but when they entered into menopause (at 8 months of age) these protein levels were significantly decreased in APP/PS1 female mice. We conclude that the appearance of recall deficits and loss of synaptosomal Akt1-mTOR signaling occurred in females only as they aged and attained menopause. Our findings indicate that the neuroprotective effects of estrogen may play an important role in protecting from b-amyloid induced toxicity at the synapse.

Local Protein Translation and RNA Processing of Synaptic Proteins in Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a heritable neurodevelopmental condition associated with impairments in social interaction, communication and repetitive behaviors. While the underlying disease mechanisms remain to be fully elucidated, dysfunction of neuronal plasticity and local translation control have emerged as key points of interest. Translation of mRNAs for critical synaptic proteins are negatively regulated by Fragile X mental retardation protein (FMRP), which is lost in the most common single-gene disorder associated with ASD. Numerous studies have shown that mRNA transport, RNA metabolism, and translation of synaptic proteins are important for neuronal health, synaptic plasticity, and learning and memory. Accordingly, dysfunction of these mechanisms may contribute to the abnormal brain function observed in individuals with autism spectrum disorder (ASD). In this review, we summarize recent studies about local translation and mRNA processing of synaptic proteins and discuss how perturbations of these processes may be related to the pathophysiology of ASD.

MTORC and PKCε in Regulation of Alcohol Use Disorder.

Alcohol use disorder (AUD) is characterized by compulsive binge alcohol intake, leading to various health and social harms. Protein Kinase C epsilon (PKCε), a specific family of PKC isoenzyme, regulates binge alcohol intake, and potentiates alcohol-related cues. Alcohol via upstream kinases like the mammalian target to rapamycin complex 1 (mTORC1) or 2 (mTORC2), may affect the activities of PKCε or vice versa in AUD. mTORC2 phosphorylates PKCε at hydrophobic and turn motif, and was recently reported to be associated with alcohol-seeking behavior, suggesting the potential role of mTORC2-PKCε interactions in the pathophysiology of AUD. mTORC1 regulates translation of synaptic proteins involved in alcohol-induced plasticity. Hence, in this article, we aimed to review the molecular composition of mTORC1 and mTORC2, drugs targeting PKCε, mTORC1, and mTORC2 in AUD, upstream regulation of mTORC1 and mTORC2 in AUD and downstream cellular mechanisms of mTORCs in the pathogenesis of AUD.

Brain Translation: A Feather Tips the Scale.

In this issue of Molecular Cell, Gonatopoulos-Pournatzis etal. (2020) report a neuron-specific microexon in eIF4G translation initiation factors that dampens synaptic protein translation. Autism-associated disruption of this exon results in increased protein production, likely through reduced coalescence with cytoplasmic ribonucleoprotein granule components, including FMRP.

Application of polysome profiling analysis in the study of synaptic protein translation

Application of polysome profiling analysis in the study of synaptic protein translation

Developmental lead (Pb)-induced deficits in hippocampal protein translation at the synapses are ameliorated by ascorbate supplementation.

BackgroundLead (Pb) is a persistent environmental neurotoxin and its exposure even in minute quantities has been known to induce neuronal defects. The immature brain is singularly sensitive to Pb neurotoxicity, and its exposure during development has permanent detrimental effects on the brain developmental trajectory and neuronal signaling and plasticity, culminating into compromises in the cognitive and behavioral attributes which persists even later in adulthood. Several molecular pathways have been implicated in the Pb-mediated disruption of neuronal signaling, including elevated oxidative stress, alterations in neurotransmitter biology, and mitochondrial dysfunction. Nevertheless, the neuronal targets and biochemical pathways underlying these Pb-mediated alterations in synaptic development and function have not been completely deduced. In this respect, recent studies have shown that synaptic signaling and its maintenance and plasticity are critically dependent on localized de novo protein translation at the synaptic terminals.Materials and methodsThe present study hence aimed to assess the alterations in the synapse-specific translation induced by developmental Pb exposure. To this end, in vitro protein translation rate was analyzed in the hippocampal synaptoneurosomal fractions of rat pups pre- and postnatally exposed to Pb using a puromycin incorporation assay. Moreover, we evaluated the therapeutic effects of ascorbic acid supplementation against Pb-induced deficits in synapse-localized protein translation.ResultsWe observed a significant loss in the rates of de novo protein translation in synaptoneurosomes of Pb-exposed pups compared to age-matched control pups. Interestingly, ascorbate supplementation lead to an appreciable recovery in Pb-induced translational deficits. Moreover, the deficit in activity-dependent synaptic protein translation was found to correlate significantly with the increase in the blood Pb levels.ConclusionDysregulation of synapse-localized de novo protein translation is a potentially critical determinant of Pb-induced synaptic dysfunction and the consequent deficits in behavioral, social, and psychological attributes of the organisms. In addition, our study establishes ascorbate supplementation as a key ameliorative agent against Pb-induced neurotoxicity.

Mammalian target of rapamycin complex 1 and its downstream effector collapsin response mediator protein-2 drive reinstatement of alcohol reward seeking.

Alcohol use disorder is a chronic relapsing disease. Maintaining abstinence represents a major challenge for alcohol-dependent patients. Yet the molecular underpinnings of alcohol relapse remain poorly understood. In the present study, we investigated the potential role of the mammalian target of rapamycin complex 1 (mTORC1) in relapse to alcohol-seeking behavior by using the reinstatement of a previously extinguished alcohol conditioned place preference (CPP) response as a surrogate relapse paradigm. We found that mTORC1 is activated in the nucleus accumbens shell following alcohol priming-induced reinstatement of alcohol place preference. We further report that the selective mTORC1 inhibitor, rapamycin, abolishes reinstatement of alcohol place preference. Activation of mTORC1 initiates the translation of synaptic proteins, and we observed that reinstatement of alcohol CPP is associated with increased protein levels of one of mTORC1's downstream targets, collapsin response mediator protein-2 (CRMP2), in the nucleus accumbens. Importantly, the level of mTORC1 activation and CRMP2 expression positively correlate with the CPP score during reinstatement. Finally, we found that systemic administration of the CRMP2 inhibitor, lacosamide, attenuates alcohol priming-induced reinstatement of CPP. Together, our results reveal that mTORC1 and its downstream target, CRMP2, contribute to mechanisms underlying reinstatement of alcohol reward seeking. Our results could have important implications for the treatment of relapse to alcohol use and position the Food and Drug Administration approved drugs, rapamycin and lacosamide, for the treatment of alcohol use disorder.

Neonatal maternal deprivation impairs localized de novo activity-induced protein translation at the synapse in the rat hippocampus.

Neonatal neuropsychiatric stress induces alterations in neurodevelopment that can lead to irreversible damage to neuronal physiology, and social, behavioral, and cognitive skills. In addition, this culminates to an elevated vulnerability to stress and anxiety later in life. Developmental deficits in hippocampal synaptic function and plasticity are among the primary contributors of detrimental alterations in brain function induced by early-life stress. However, the underlying molecular mechanisms are not completely understood. Localized protein translation, occurring at the synapse and triggered by neuronal activity, is critical for synapse function, maintenance, and plasticity. We used a rodent model of chronic maternal deprivation to characterize the effects of early-life neuropsychiatric stress on localized de novo protein translation at synaptic connections between neurons. Synaptoneurosomal preparations isolated biochemically from the hippocampi of rat pups that were subjected to maternal deprivation were deficient in depolarization-induced activity-dependent protein translation when compared with littermate controls. Conversely, basal unstimulated protein translation was not affected. Moreover, deficits in activity-driven synaptic protein translation were significantly correlated with a reduction in phosphorylated cell survival protein kinase protein B or Akt (p473 Ser and p308 Thr), but not phosphorylated extracellular signal-regulated kinase.

NMDAR‐dependent Argonaute 2 phosphorylation regulates miRNA activity and dendritic spine plasticity

MicroRNAs (miRNAs) repress translation of target mRNAs by associating with Argonaute (Ago) proteins to form the RNA‐induced silencing complex (RISC), underpinning a powerful mechanism for fine‐tuning protein expression. Specific miRNAs are required for NMDA receptor (NMDAR)‐dependent synaptic plasticity by modulating the translation of proteins involved in dendritic spine morphogenesis or synaptic transmission. However, it is unknown how NMDAR stimulation stimulates RISC activity to rapidly repress translation of synaptic proteins. We show that NMDAR stimulation transiently increases Akt‐dependent phosphorylation of Ago2 at S387, which causes an increase in binding to GW182 and a rapid increase in translational repression of LIMK1 via miR‐134. Furthermore, NMDAR‐dependent down‐regulation of endogenous LIMK1 translation in dendrites and dendritic spine shrinkage requires phospho‐regulation of Ago2 at S387. AMPAR trafficking and hippocampal LTD do not involve S387 phosphorylation, defining this mechanism as a specific pathway for structural plasticity. This work defines a novel mechanism for the rapid transduction of NMDAR stimulation into miRNA‐mediated translational repression to control dendritic spine morphology.

Targeting PERK signaling with the small molecule GSK2606414 prevents neurodegeneration in a model of Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder, leading to the progressive decline of motor control due to the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Accumulating evidence suggest that altered proteostasis is a salient feature of PD, highlighting perturbations to the endoplasmic reticulum (ER), the main compartment involved in protein folding and secretion. PERK is a central ER stress sensor that enforces adaptive programs to recover homeostasis through a block of protein translation and the induction of the transcription factor ATF4. In addition, chronic PERK signaling results in apoptosis induction and neuronal dysfunction due to the repression in the translation of synaptic proteins. Here we confirmed the activation of PERK signaling in postmortem brain tissue derived from PD patients and three different rodent models of the disease. Pharmacological targeting of PERK by the oral administration of GSK2606414 demonstrated efficient inhibition of the pathway in the SNpc after experimental ER stress stimulation. GSK2606414 protected nigral-dopaminergic neurons against a PD-inducing neurotoxin, improving motor performance. The neuroprotective effects of PERK inhibition were accompanied by an increase in dopamine levels and the expression of synaptic proteins. However, GSK2606414 treated animals developed secondary effects possibly related to pancreatic toxicity. This study suggests that strategies to attenuate ER stress levels may be effective to reduce neurodegeneration in PD.

788. Local Translation of Synaptic Proteins Supports Plasticity Specifically at Distal Hippocampal Synapses

788. Local Translation of Synaptic Proteins Supports Plasticity Specifically at Distal Hippocampal Synapses

PATHOLOGICAL TAU IMPAIRS RIBOSOMAL FUNCTION AND DECREASES PROTEIN SYNTHESIS

Alzheimer’s disease (AD) is one of 20 crippling neurodegenerative disorders characterized by the aberrant intracellular deposition of the microtubule-associated protein tau. One of the first symptoms of tauopathic patients is progressive memory loss and cognitive decline. Since the pathogenic mechanism by which tau induces neurotoxicity and leads to memory impairment is unknown, therapeutic strategies are limited. We performed subcellular fractionation of human AD and control brains coupled with tandem mass spectrometry peptide identification to identify tau-protein interactions. We performed cell free in vitro translation assays to measure translation of green fluorescent protein (GFP) in the presence of oligomeric proteins. Finally, we performed cell culture assays in immortalized lines and primary neuronal cultures to measure changes in protein translation as a result of pathological tau. We show that pathological tau associates differentially with endoplasmic reticulum (ER) proteins in AD compared to control brains. We also show that pathological tau associates with ribosomes in AD brains, and that this association leads to a decrease in overall translation as well as translation of vital synaptic proteins. Our data suggest that pathological tau impairs protein production. Since protein biosynthesis is necessary for memory formation, our work establishes a direct link between tau aberrations and memory impairment. We also show that there is a decrease in translation of synaptic proteins, which could be a reason for neuronal dysfunction observed in diseased brains. These data support the exploration of the tau-ribosome complex for therapeutic target identification, and it opens a new window of treatment strategies for tauopathies.

Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons

Fragile X syndrome (FXS) is caused by the absence of the Fragile X Mental Retardation Protein (FMRP) in neurons. In the mouse, the lack of FMRP is associated with an excessive translation of hundreds of neuronal proteins, notably including postsynaptic proteins. This local protein synthesis deregulation is proposed to underlie the observed defects of glutamatergic synapse maturation and function and to affect preferentially the hundreds of mRNA species that were reported to bind to FMRP. How FMRP impacts synaptic protein translation and which mRNAs are most important for the pathology remain unclear. Here we show by cross-linking immunoprecipitation in cortical neurons that FMRP is mostly associated with one unique mRNA: diacylglycerol kinase kappa (Dgkκ), a master regulator that controls the switch between diacylglycerol and phosphatidic acid signaling pathways. The absence of FMRP in neurons abolishes group 1 metabotropic glutamate receptor-dependent DGK activity combined with a loss of Dgkκ expression. The reduction of Dgkκ in neurons is sufficient to cause dendritic spine abnormalities, synaptic plasticity alterations, and behavior disorders similar to those observed in the FXS mouse model. Overexpression of Dgkκ in neurons is able to rescue the dendritic spine defects of the Fragile X Mental Retardation 1 gene KO neurons. Together, these data suggest that Dgkκ deregulation contributes to FXS pathology and support a model where FMRP, by controlling the translation of Dgkκ, indirectly controls synaptic proteins translation and membrane properties by impacting lipid signaling in dendritic spine.

Rapamycin reduces motivated responding for cocaine and alters GluA1 expression in the ventral but not dorsal striatum

The mechanistic target of rapamycin complex 1 (mTORC1) regulates synaptic protein synthesis and therefore synaptic function and plasticity. A role for mTORC1 has recently been demonstrated for addiction-related behaviors. For example, central or intra-accumbal injections of the mTORC1 inhibitor rapamycin attenuates several indices of cocaine-seeking including progressive ratio (PR) responding and reinstatement. These behavioral effects are associated with decreased mTORC1 activity and synaptic protein translation in the nucleus accumbens (NAC) and point to a possible therapeutic role for rapamycin in the treatment of addiction. Currently, it is unclear whether similar behavioral and biochemical effects can be achieved by administering rapamycin systemically, which represents a more clinically-appropriate route of administration. Here, we assessed the effects of repeated, systemic administration of rapamycin (10mg/kg, i.p.) on PR responding for cocaine. We also assessed whether systemic rapamycin was associated with changes in measures of mTORC1 activity and GluA1 expression in the ventral and dorsal striatum. We report that systemic rapamycin treatment reduced PR breakpoints to levels comparable to intra-NAC rapamycin. Systemic rapamycin treatment also reduced phosphorylated p70S6K and GluA1 AMPARs within the NAC but not dorsal striatum. Thus, systemic administration of rapamycin is as effective at reducing drug seeking behavior and measures of mTORC1 activity compared to direct accumbal application and may therefore represent a possible therapeutic option in the treatment of addiction. Possible caveats of this treatment approach are discussed.

Bidirectional Synaptic Structural Plasticity after Chronic Cocaine Administration Occurs through Rap1 Small GTPase Signaling

Dendritic spines are the sites of most excitatory synapses in the CNS, and opposing alterations in the synaptic structure of medium spiny neurons (MSNs) of the nucleus accumbens (NAc), a primary brain reward region, are seen at early versus late time points after cocaine administration. Here we investigate the time-dependent molecular and biochemical processes that regulate this bidirectional synaptic structural plasticity of NAc MSNs and associated changes in cocaine reward in response to chronic cocaine exposure. Our findings reveal key roles for the bidirectional synaptic expression of the Rap1b small GTPase and an associated local synaptic protein translation network in this process. The transcriptional mechanisms and pathway-specific inputs to NAc that regulate Rap1b expression are also characterized. Collectively, these findings provide a precise mechanism by which nuclear to synaptic interactions induce "metaplasticity" in NAc MSNs, and we reveal the specific effects of this plasticity on reward behavior in a brain circuit-specific manner.