Distinct temporal expression of the GW182 paralog TNRC6A in neurons regulates dendritic arborization.

Precise development of the dendritic architecture is a critical determinant of mature neuronal circuitry. MicroRNA (miRNA)-mediated regulation of protein synthesis plays a crucial role in dendritic morphogenesis, but the role of miRNA-induced silencing complex (miRISC) protein components in this process is less studied. Here, we show an important role of a key miRISC protein, the GW182 paralog TNRC6A, in the regulation of dendritic growth. We identified a distinct brain region-specific spatiotemporal expression pattern of GW182 during rat postnatal development. We found that the window of peak GW182 expression coincides with the period of extensive dendritic growth, both in the hippocampus and cerebellum. Perturbation of GW182 function during a specific temporal window resulted in reduced dendritic growth of cultured hippocampal neurons. Mechanistically, we show that GW182 modulates dendritic growth by regulating global somatodendritic translation and actin cytoskeletal dynamics of developing neurons. Furthermore, we found that GW182 affects dendritic architecture by regulating the expression of actin modulator LIMK1. Taken together, our data reveal a previously undescribed neurodevelopmental expression pattern of GW182 and its role in dendritic morphogenesis, which involves both translational control and actin cytoskeletal rearrangement. This article has an associated First Person interview with the first author of the paper.

APOE4 Affects Basal and NMDAR-Mediated Protein Synthesis in Neurons by Perturbing Calcium Homeostasis.

Apolipoprotein E (APOE), one of the primary lipoproteins in the brain has three isoforms in humans, APOE2, APOE3, and APOE4. APOE4 is the most well-established risk factor increasing the predisposition for Alzheimer's disease (AD). The presence of the APOE4 allele alone is shown to cause synaptic defects in neurons and recent studies have identified multiple pathways directly influenced by APOE4. However, the mechanisms underlying APOE4-induced synaptic dysfunction remain elusive. Here, we report that the acute exposure of primary cortical neurons or synaptoneurosomes to APOE4 leads to a significant decrease in global protein synthesis. Primary cortical neurons were derived from male and female embryos of Sprague Dawley (SD) rats or C57BL/6J mice. Synaptoneurosomes were prepared from P30 male SD rats. APOE4 treatment also abrogates the NMDA-mediated translation response indicating an alteration of synaptic signaling. Importantly, we demonstrate that both APOE3 and APOE4 generate a distinct translation response which is closely linked to their respective calcium signature. Acute exposure of neurons to APOE3 causes a short burst of calcium through NMDA receptors (NMDARs) leading to an initial decrease in protein synthesis which quickly recovers. Contrarily, APOE4 leads to a sustained increase in calcium levels by activating both NMDARs and L-type voltage-gated calcium channels (L-VGCCs), thereby causing sustained translation inhibition through eukaryotic translation elongation factor 2 (eEF2) phosphorylation, which in turn disrupts the NMDAR response. Thus, we show that APOE4 affects basal and activity-mediated protein synthesis responses in neurons by affecting calcium homeostasis.SIGNIFICANCE STATEMENT Defective protein synthesis has been shown as an early defect in familial Alzheimer's disease (AD). However, this has not been studied in the context of sporadic AD, which constitutes the majority of cases. In our study, we show that Apolipoprotein E4 (APOE4), the predominant risk factor for AD, inhibits global protein synthesis in neurons. APOE4 also affects NMDA activity-mediated protein synthesis response, thus inhibiting synaptic translation. We also show that the defective protein synthesis mediated by APOE4 is closely linked to the perturbation of calcium homeostasis caused by APOE4 in neurons. Thus, we propose the dysregulation of protein synthesis as one of the possible molecular mechanisms to explain APOE4-mediated synaptic and cognitive defects. Hence, the study not only suggests an explanation for the APOE4-mediated predisposition to AD, it also bridges the gap in understanding APOE4-mediated pathology.

Distinct regulation of bioenergetics and translation by group I mGluR and NMDAR.

Neuronal activity is responsible for the high energy consumption in the brain. However, the cellular mechanisms draining ATP upon the arrival of a stimulus are yet to be explored systematically at the post-synapse. Here, we provide evidence that a significant fraction of ATP is consumed upon glutamate stimulation to energize mGluR-induced protein synthesis. We find that both mGluR and NMDAR alter protein synthesis and ATP consumption with distinct kinetics at the synaptic-dendritic compartments. While mGluR activation leads to a rapid and sustained reduction in neuronal ATP levels, NMDAR activation has no immediate impact on the same. ATP consumption correlates inversely with the kinetics of protein synthesis for both receptors. We observe a persistent elevation in protein synthesis within 5 minutes of mGluR activation and a robust inhibition of the same within 2 minutes of NMDAR activation, assessed by the phosphorylation status of eEF2 and metabolic labeling. However, a delayed protein synthesis-dependent ATP expenditure ensues after 15 minutes of NMDAR stimulation. We identify a central role for AMPK in the correlation between protein synthesis and ATP consumption. AMPK is dephosphorylated and inhibited upon mGluR activation, while it is phosphorylated upon NMDAR activation. Perturbing AMPK activity disrupts receptor-specific modulations of eEF2 phosphorylation and protein synthesis. Our observations, therefore, demonstrate that the regulation of the AMPK-eEF2 signaling axis by glutamate receptors alters neuronal protein synthesis and bioenergetics.

Effect of early maternal separation stress on attention, spatial learning and social interaction behaviour.

Early life stress is known to influence affective and cognitive functions in later life but comprehensive explanation for the impact of early life stress on attentional functions, behavioural control and social behaviour is inadequate. The early life stress was induced by exposing rat pups to 6 h of maternal separation and isolation (MS) stress from postnatal days 4-14 i.e. during SHRP period. The long-term impact of MS in these rats was evaluated by assessing anxiety, sociability, social preference, spatial learning and memory along with a detailed evaluation of attentional functions during young adulthood period. Adult male MS rats showed increased anxiety-like behaviour, impaired flexibility in social interactions, and increased reward-seeking behaviour. MS rats also showed faster spatial learning in the partially baited radial arm maze and exhibited moderately enhanced sustained attention in the 5-choice serial reaction time task (5CSRTT). These results suggest that early MS has both positive and negative consequences in adulthood. Increased cognitive ability in MS rats, as evidenced by the improved sustained attention and spatial learning and memory, is usually advantageous and indicates positive influences of early stressors that might lead to the development of resilience and enhanced compensatory mechanisms later in adulthood. MS stress has compromised flexibility in social behaviour that promotes solitary lifestyle and social isolation. Heightened reward-seeking behaviour, as shown by the MS rats, could be a predisposing factor for substance abuse and addiction. Thus, our study highlights the crucial and differential impact of early life challenges on behaviour during adulthood and suggests that the positive aspects could be an asset that may be utilized to suppress the negative effects of early life stress in adulthood.

Reversible inhibition of PSD-95 mRNA translation by miR-125a, FMRP phosphorylation, and mGluR signaling.

The molecular mechanism for how RISC and microRNAs selectively and reversibly regulate mRNA translation in response to receptor signaling is unknown but could provide a means for temporal and spatial control of translation. Here we show that miR-125a targeting PSD-95 mRNA allows reversible inhibition of translation and regulation by gp1 mGluR signaling. Inhibition of miR-125a increased PSD-95 levels in dendrites and altered dendritic spine morphology. Bidirectional control of PSD-95 expression depends on miR-125a and FMRP phosphorylation status. miR-125a levels at synapses and its association with AGO2 are reduced in Fmr1 KO. FMRP phosphorylation promotes the formation of an AGO2-miR-125a inhibitory complex on PSD-95 mRNA, whereas mGluR signaling of translation requires FMRP dephosphorylation and release of AGO2 from the mRNA. These findings reveal a mechanism whereby FMRP phosphorylation provides a reversible switch for AGO2 and microRNA to selectively regulate mRNA translation at synapses in response to receptor activation.

A 3' untranslated region variant in FMR1 eliminates neuronal activity-dependent translation of FMRP by disrupting binding of the RNA-binding protein HuR.

Fragile X syndrome is a common cause of intellectual disability and autism spectrum disorder. The gene underlying the disorder, fragile X mental retardation 1 (FMR1), is silenced in most cases by a CGG-repeat expansion mutation in the 5' untranslated region (UTR). Recently, we identified a variant located in the 3'UTR of FMR1 enriched among developmentally delayed males with normal repeat lengths. A patient-derived cell line revealed reduced levels of endogenous fragile X mental retardation protein (FMRP), and a reporter containing a patient 3'UTR caused a decrease in expression. A control reporter expressed in cultured mouse cortical neurons showed an expected increase following synaptic stimulation that was absent when expressing the patient reporter, suggesting an impaired response to neuronal activity. Mobility-shift assays using a control RNA detected an RNA-protein interaction that is lost with the patient RNA, and HuR was subsequently identified as an associated protein. Cross-linking immunoprecipitation experiments identified the locus as an in vivo target of HuR, supporting our in vitro findings. These data suggest that the disrupted interaction of HuR impairs activity-dependent translation of FMRP, which may hinder synaptic plasticity in a clinically significant fashion.

Distinct calcium sources regulate temporal profiles of NMDAR and mGluR-mediated protein synthesis.

Calcium signaling is integral for neuronal activity and synaptic plasticity. We demonstrate that the calcium response generated by different sources modulates neuronal activity-mediated protein synthesis, another process essential for synaptic plasticity. Stimulation of NMDARs generates a protein synthesis response involving three phases-increased translation inhibition, followed by a decrease in translation inhibition, and increased translation activation. We show that these phases are linked to NMDAR-mediated calcium response. Calcium influx through NMDARs elicits increased translation inhibition, which is necessary for the successive phases. Calcium through L-VGCCs acts as a switch from translation inhibition to the activation phase. NMDAR-mediated translation activation requires the contribution of L-VGCCs, RyRs, and SOCE. Furthermore, we show that IP3-mediated calcium release and SOCE are essential for mGluR-mediated translation up-regulation. Finally, we signify the relevance of our findings in the context of Alzheimer's disease. Using neurons derived from human fAD iPSCs and transgenic AD mice, we demonstrate the dysregulation of NMDAR-mediated calcium and translation response. Our study highlights the complex interplay between calcium signaling and protein synthesis, and its implications in neurodegeneration.

Dephosphorylation-induced ubiquitination and degradation of FMRP in dendrites: a role in immediate early mGluR-stimulated translation.

Fragile X syndrome is caused by the loss of fragile X mental retardation protein (FMRP), which represses and reversibly regulates the translation of a subset of mRNAs in dendrites. Protein synthesis can be rapidly stimulated by mGluR-induced and protein phosphatase 2a (PP2A)-mediated dephosphorylation of FMRP, which is coupled to the dissociation of FMRP and target mRNAs from miRNA-induced silencing complexes. Here, we report the rapid ubiquitination and ubiquitin proteasome system (UPS)-mediated degradation of FMRP in dendrites upon DHPG (3,5-dihydroxyphenylglycine) stimulation in cultured rat neurons. Using inhibitors to PP2A and FMRP phosphomutants, degradation of FMRP was observed to depend on its prior dephosphorylation. Translational induction of an FMRP target, postsynaptic density-95 mRNA, required both PP2A and UPS. Thus, control of FMRP levels at the synapse by dephosphorylation-induced and UPS-mediated degradation provides a mode to regulate protein synthesis.

NMDAR mediated dynamic changes in m6A inversely correlates with neuronal translation.

Epitranscriptome modifications are crucial in translation regulation and essential for maintaining cellular homeostasis. N6 methyladenosine (m6A) is one of the most abundant and well-conserved epitranscriptome modifications, which is known to play a pivotal role in diverse aspects of neuronal functions. However, the role of m6A modifications with respect to activity-mediated translation regulation and synaptic plasticity has not been studied. Here, we investigated the role of m6A modification in response to NMDAR stimulation. We have consistently observed that 5 min NMDAR stimulation causes an increase in eEF2 phosphorylation. Correspondingly, NMDAR stimulation caused a significant increase in the m6A signal at 5 min time point, correlating with the global translation inhibition. The NMDAR induced increase in the m6A signal is accompanied by the redistribution of the m6A marked RNAs from translating to the non-translating pool of ribosomes. The increased m6A levels are well correlated with the reduced FTO levels observed on NMDAR stimulation. Additionally, we show that inhibition of FTO prevents NMDAR mediated changes in m6A levels. Overall, our results establish RNA-based molecular readout which corelates with the NMDAR-dependent translation regulation which helps in understanding changes in protein synthesis.

Function of FMRP Domains in Regulating Distinct Roles of Neuronal Protein Synthesis.

The Fragile-X Mental Retardation Protein (FMRP) is an RNA binding protein that regulates translation of mRNAs essential for synaptic development and plasticity. FMRP interacts with a specific set of mRNAs, aids in their microtubule-dependent transport and regulates their translation through its association with ribosomes. However, the biochemical role of FMRP's domains in forming neuronal granules and associating with microtubules and ribosomes is currently undefined. We report that the C-terminus domain of FMRP is sufficient to bind to ribosomes akin to the full-length protein. Furthermore, the C-terminus domain alone is essential and responsible for FMRP-mediated neuronal translation repression. However, dendritic distribution of FMRP and its microtubule association is favored by the synergistic combination of FMRP domains rather than individual domains. Interestingly, we show that the phosphorylation of hFMRP at Serine-500 is important in modulating the dynamics of translation by controlling ribosome association. This is a fundamental mechanism governing the size and number of FMRP puncta that contain actively translating ribosomes. Finally through the use of pathogenic mutations, we emphasize the hierarchical contribution of FMRP's domains in translation regulation.

Astrocytic reactivity triggered by defective autophagy and metabolic failure causes neurotoxicity in frontotemporal dementia type 3.

Frontotemporal dementia type 3 (FTD3), caused by a point mutation in the charged multivesicular body protein 2B (CHMP2B), affects mitochondrial ultrastructure and the endolysosomal pathway in neurons. To dissect the astrocyte-specific impact of mutant CHMP2B expression, we generated astrocytes from human induced pluripotent stem cells (hiPSCs) and confirmed our findings in CHMP2B mutant mice. Our data provide mechanistic insights into how defective autophagy causes perturbed mitochondrial dynamics with impaired glycolysis, increased reactive oxygen species, and elongated mitochondrial morphology, indicating increased mitochondrial fusion in FTD3 astrocytes. This shift in astrocyte homeostasis triggers a reactive astrocyte phenotype and increased release of toxic cytokines, which accumulate in nuclear factor kappa b (NF-κB) pathway activation with increased production of CHF, LCN2, and C3 causing neurodegeneration.

The Role of Dynamic miRISC During Neuronal Development.

Activity-dependent protein synthesis plays an important role during neuronal development by fine-tuning the formation and function of neuronal circuits. Recent studies have shown that miRNAs are integral to this regulation because of their ability to control protein synthesis in a rapid, specific and potentially reversible manner. miRNA mediated regulation is a multistep process that involves inhibition of translation before degradation of targeted mRNA, which provides the possibility to store and reverse the inhibition at multiple stages. This flexibility is primarily thought to be derived from the composition of miRNA induced silencing complex (miRISC). AGO2 is likely the only obligatory component of miRISC, while multiple RBPs are shown to be associated with this core miRISC to form diverse miRISC complexes. The formation of these heterogeneous miRISC complexes is intricately regulated by various extracellular signals and cell-specific contexts. In this review, we discuss the composition of miRISC and its functions during neuronal development. Neurodevelopment is guided by both internal programs and external cues. Neuronal activity and external signals play an important role in the formation and refining of the neuronal network. miRISC composition and diversity have a critical role at distinct stages of neurodevelopment. Even though there is a good amount of literature available on the role of miRNAs mediated regulation of neuronal development, surprisingly the role of miRISC composition and its functional dynamics in neuronal development is not much discussed. In this article, we review the available literature on the heterogeneity of the neuronal miRISC composition and how this may influence translation regulation in the context of neuronal development.

Emerging Role of microRNAs in Dementia.

MicroRNAs are small non-coding RNAs regulating mRNA translation. They play a crucial role in regulating homeostasis in neurons, especially in regulating local and stimulation dependent protein synthesis. Since activity-mediated protein synthesis in neurons is critical for memory and cognition, microRNAs have become key players in modulating these processes. Dementia is a broad term used for symptoms involving decline of memory and cognition. Several studies have implicated the dysregulation of microRNAs in many brain diseases like neurodegenerative diseases, neurodevelopmental disorders, brain injuries and dementia. In this review, we give an overview of microRNA-mediated regulation of proteins and cellular processes affected in dementia pathology, hence illustrating the importance of microRNAs in normal functioning. We also focus on a relatively less explored area in dementia pathology-the importance of activity-mediated protein synthesis at the synapse and the role of microRNAs in modulating this. Overall, this review will be helpful in looking at the significance of microRNAs in dementia from the perspective of defective regulation of protein synthesis and synaptic dysfunction.

Generation of a FMR1 homozygous knockout human embryonic stem cell line (WAe009-A-16) by CRISPR/Cas9 editing.

Mutations in FMR1 gene is the cause of Fragile X Syndrome (FXS) leading inherited cause of intellectual disability and autism spectrum disorders. FMR1 gene encodes Fragile X Mental Retardation Protein (FMRP) which is a RNA binding protein and play important role in synaptic plasticity and translational regulation in neurons. We have generated a homozygous FMR1 knockout (FMR1-KO) hESC line using CRISPR/Cas9 based genome editing. It created a homozygous 280 nucleotide deletion at exon1, removing the start codon. This FMR1-KO cell line maintains stem cell like morphology, pluripotency, normal karyotype and ability to in-vitro differentiation.

BDNF Induced Translation of Limk1 in Developing Neurons Regulates Dendrite Growth by Fine-Tuning Cofilin1 Activity.

Dendritic growth and branching are highly regulated processes and are essential for establishing proper neuronal connectivity. There is a critical phase of early dendrite development when these are heavily regulated by external cues such as trophic factors. Brain-derived neurotrophic factor (BDNF) is a major trophic factor known to enhance dendrite growth in cortical neurons, but the molecular underpinnings of this response are not completely understood. We have identified that BDNF induced translational regulation is an important mechanism governing dendrite development in cultured rat cortical neurons. We show that BDNF treatment for 1 h in young neurons leads to translational up-regulation of an important actin regulatory protein LIM domain kinase 1 (Limk1), increasing its level locally in the dendrites. Limk1 is a member of serine/threonine (Ser/Thr) family kinases downstream of the Rho-GTPase pathway. BDNF induced increase in Limk1 levels leads to increased phosphorylation of its target protein cofilin1. We observed that these changes are maintained for long durations of up to 48 h and are mediating increase in number of primary dendrites and total dendrite length. Thus, we show that BDNF induced protein synthesis leads to fine-tuning of the actin cytoskeletal reassembly and thereby mediate dendrite development.

NMDAR mediated translation at the synapse is regulated by MOV10 and FMRP.

Protein synthesis is crucial for maintaining synaptic plasticity and synaptic signalling. Here we have attempted to understand the role of RNA binding proteins, Fragile X Mental Retardation Protein (FMRP) and Moloney Leukemia Virus 10 (MOV10) protein in N-Methyl-D-Aspartate Receptor (NMDAR) mediated translation regulation. We show that FMRP is required for translation downstream of NMDAR stimulation and MOV10 is the key specificity factor in this process. In rat cortical synaptoneurosomes, MOV10 in association with FMRP and Argonaute 2 (AGO2) forms the inhibitory complex on a subset of NMDAR responsive mRNAs. On NMDAR stimulation, MOV10 dissociates from AGO2 and promotes the translation of its target mRNAs. FMRP is required to form MOV10-AGO2 inhibitory complex and to promote translation of MOV10 associated mRNAs. Phosphorylation of FMRP appears to be the potential switch for NMDAR mediated translation and in the absence of FMRP, the distinct translation response to NMDAR stimulation is lost. Thus, FMRP and MOV10 have an important regulatory role in NMDAR mediated translation at the synapse.

Differential Regulation of Syngap1 Translation by FMRP Modulates eEF2 Mediated Response on NMDAR Activity.

SYNGAP1, a Synaptic Ras-GTPase activating protein, regulates synapse maturation during a critical developmental window. Heterozygous mutation in SYNGAP1 (SYNGAP1 -/+) has been shown to cause Intellectual Disability (ID) in children. Recent studies have provided evidence for altered neuronal protein synthesis in a mouse model of Syngap1 -/+. However, the molecular mechanism behind the same is unclear. Here, we report the reduced expression of a known translation regulator, FMRP, during a specific developmental period in Syngap1 -/+ mice. Our results demonstrate that FMRP interacts with and regulates the translation of Syngap1 mRNA. We further show reduced Fmr1 translation leads to decreased FMRP level during development in Syngap1 -/+ which results in an increase in Syngap1 translation. These developmental changes are reflected in the altered response of eEF2 phosphorylation downstream of NMDA Receptor (NMDAR)-mediated signaling. In this study, we propose a cross-talk between FMRP and SYNGAP1 mediated signaling which can also explain the compensatory effect of impaired signaling observed in Syngap1 -/+ mice.

Dysregulated metabotropic glutamate receptor-dependent translation of AMPA receptor and postsynaptic density-95 mRNAs at synapses in a mouse model of fragile X syndrome.

Fragile X syndrome, a common form of inherited mental retardation, is caused by the loss of fragile X mental retardation protein (FMRP), an mRNA binding protein that is hypothesized to regulate local mRNA translation in dendrites downstream of gp1 metabotropic glutamate receptors (mGluRs). However, specific FMRP-associated mRNAs that localize to dendrites in vivo and show altered mGluR-dependent translation at synapses of Fmr1 knock-out mice are unknown so far. Using fluorescence in situ hybridization, we discovered that GluR1/2 and postsynaptic density-95 (PSD-95) mRNAs are localized to dendrites of cortical and hippocampal neurons in vivo. Quantitative analyses of their dendritic mRNA levels in cultured neurons and synaptoneurosomes did not detect differences between wild-type and Fmr1 knock-out (KO) mice. In contrast, PSD-95, GluR1/2, and calcium/calmodulin-dependent kinase IIalpha (CaMKIIalpha) mRNA levels in actively translating polyribosomes were dysregulated in synaptoneurosomes from Fmr1 knock-out mice in response to mGluR activation. [35S]methionine incorporation into newly synthesized proteins similarly revealed impaired stimulus-induced protein synthesis of CaMKIIalpha and PSD-95 in synaptoneurosomes from Fmr1 KO mice. Quantitative analysis of mRNA levels in FMRP-specific immunoprecipitations from synaptoneurosomes demonstrated the association of FMRP with CaMKIIalpha, PSD-95, and GluR1/2 mRNAs. These findings suggest a novel mechanism whereby FMRP regulates the local synthesis AMPA receptor (AMPAR) subunits, PSD-95, and CaMKIIalpha downstream of mGluR-activation. Dysregulation of local translation of AMPAR and associated factors at synapses may impair control of the molecular composition of the postsynaptic density and consequently alter synaptic transmission, causing impairments of neuronal plasticity observed in Fmr1 knock-out mice and fragile X syndrome.

FMRP Interacts with C/D Box snoRNA in the Nucleus and Regulates Ribosomal RNA Methylation.

FMRP is an RNA-binding protein that is known to localize in the cytoplasm and in the nucleus. Here, we have identified an interaction of FMRP with a specific set of C/D box snoRNAs in the nucleus. C/D box snoRNAs guide 2'O methylations of ribosomal RNA (rRNA) on defined sites, and this modification regulates rRNA folding and assembly of ribosomes. 2'O methylation of rRNA is partial on several sites in human embryonic stem cells, which results in ribosomes with differential methylation patterns. FMRP-snoRNA interaction affects rRNA methylation on several of these sites, and in the absence of FMRP, differential methylation pattern of rRNA is significantly altered. We found that FMRP recognizes ribosomes carrying specific methylation patterns on rRNA and the recognition of methylation pattern by FMRP may potentially determine the translation status of its target mRNAs. Thus, FMRP integrates its function in the nucleus and in the cytoplasm.