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Two papers in Cell uncover reciprocal crosstalk of local translation and mitochondria in neurons. Rangaraju et al. (2019) observe tethered compartments of stable mitochondria in dendrites that provide a local energy supply for mRNA translation at synapses. Cioni et al. (2019) report a novel association of axonal RNA granules with Rab7a-late endosomes that provides a platform for local translation supporting mitochondria., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Local protein synthesis occurs in axons and dendrites of neurons, enabling fast and spatially restricted responses to a dynamically changing extracellular environment. Prior to local translation, mRNA that is to be translated is packed into ribonucleoprotein particles (RNPs) where RNA binding proteins ensure mRNA silencing and provide a link to molecular motors. ZBP1 is a component of RNP transport particles and is known for its role in the local translation of β-actin mRNA. Its binding to mRNA is regulated by tyrosine 396 phosphorylation, and this particular modification was shown to be vital for axonal growth and dendritic branching. Recently, additional phosphorylation of ZBP1 at serine 181 (Ser181) was described in non-neuronal cells. In the present study, we found that ZBP1 is also phosphorylated at Ser181 in neurons in a mammalian/mechanistic target of rapamycin complex 2-, Src kinase-, and mRNA binding-dependent manner. Furthermore, Ser181 ZBP1 phosphorylation was essential for the proper dendritic branching of hippocampal neurons that were cultured in vitro and for the proper ZBP1 dendritic distribution and motility.

Fragile X syndrome (FXS) is caused by the loss of the fragile X mental retardation protein (FMRP), an RNA binding protein that regulates translation of numerous target mRNAs, some of which are dendritically localized. Our previous biochemical studies using synaptoneurosomes demonstrate a role for FMRP and miR-125a in regulating the translation of PSD-95 mRNA. However, the local translation of PSD-95 mRNA within dendrites and spines, as well as the roles of FMRP or miR-125a, have not been directly studied. Herein, local synthesis of a Venus-PSD-95 fusion protein was directly visualized in dendrites and spines using single-molecule imaging of a diffusion-restricted Venus-PSD-95 reporter under control of the PSD-95 3'UTR. The basal translation rates of Venus-PSD-95 mRNA was increased in cultured hippocampal neurons from Fmr1 KO mice compared with WT neurons, which correlated with a transient elevation of endogenous PSD-95 within dendrites. Following mGluR stimulation with (S)-3,5-dihydroxyphenylglycine, the rate of Venus-PSD-95 mRNA translation increased rapidly in dendrites of WT hippocampal neurons, but not in those of Fmr1 KO neurons or when the binding site of miR125a, previously shown to bind PSD-95 3'UTR, was mutated. This study provides direct support for the hypothesis that local translation within dendrites and spines is dysregulated in FXS. Impairments in the regulated local synthesis of PSD-95, a critical regulator of synaptic structure and function, may affect the spatiotemporal control of PSD-95 levels and affect dendritic spine development and synaptic plasticity in FXS., (Copyright © 2015 the authors 0270-6474/15/357116-15$15.00/0.)

Long-term synaptic plasticity involves changes in the expression and membrane insertion of cell-surface proteins. Interestingly, the mRNAs encoding many cell-surface proteins are localized to dendrites, but whether dendritic protein synthesis is required for activity-induced surface expression of specific proteins is unknown. Herein, we used microfluidic devices to demonstrate that dendritic protein synthesis is necessary for activity-induced insertion of GluN2A-containing NMDA receptors in rat hippocampal neurons. Furthermore, visualization of activity-induced local translation of GluN2A mRNA and membrane insertion of GluN2A protein in dendrites was directly observed and shown to depend on a 3' untranslated region cytoplasmic polyadenylation element and its associated translation complex. These findings uncover a novel mechanism for cytoplasmic polyadenylation element-mediated posttranscriptional regulation of GluN2A mRNA to control NMDA receptor surface expression during synaptic plasticity.

The small GTPase RhoA has critical functions in regulating actin dynamics affecting cellular morphogenesis through the RhoA/Rho kinase (ROCK) signaling cascade. RhoA signaling controls stress fiber and focal adhesion formation and cell motility in fibroblasts. RhoA signaling is involved in several aspects of neuronal development, including neuronal migration, growth cone collapse, dendrite branching, and spine growth. Altered RhoA signaling is implicated in cancer and neurodegenerative disease and is linked to inherited intellectual disabilities. Although much is known about factors regulating RhoA activity and/or degradation, little is known about molecular mechanisms regulating RhoA expression and the subsequent effects on RhoA signaling. We hypothesized that posttranscriptional control of RhoA expression may provide a mechanism to regulate RhoA signaling and downstream effects on cell morphology. Here we uncover a cellular function for the mRNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) Q1 in the control of dendritic development and focal adhesion formation that involves the negative regulation of RhoA synthesis and signaling. We show that hnRNP-Q1 represses RhoA translation and knockdown of hnRNP-Q1 induced phenotypes associated with elevated RhoA protein levels and RhoA/ROCK signaling. These morphological changes were rescued by ROCK inhibition and/or RhoA knockdown. These findings further suggest that negative modulation of RhoA mRNA translation can provide control over downstream signaling and cellular morphogenesis.

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.

The function of local protein synthesis in synaptic plasticity and its dysregulation in fragile X syndrome (FXS) is well studied, however the contribution of regulated mRNA transport to this function remains unclear. We report a function for the fragile X mental retardation protein (FMRP) in the rapid, activity-regulated transport of mRNAs important for synaptogenesis and plasticity. mRNAs were deficient in glutamatergic signaling-induced dendritic localization in neurons from Fmr1 KO mice, and single mRNA particle dynamics in live neurons revealed diminished kinesis. Motor-dependent translocation of FMRP and cognate mRNAs involved the C terminus of FMRP and kinesin light chain, and KO brain showed reduced kinesin-associated mRNAs. Acute suppression of FMRP and target mRNA transport in WT neurons resulted in altered filopodia-spine morphology that mimicked the FXS phenotype. These findings highlight a mechanism for stimulus-induced dendritic mRNA transport and link its impairment in a mouse model of FXS to altered developmental morphologic plasticity.

Activity-dependent regulation of synaptic AMPA receptor (AMPAR) number is critical to NMDA receptor (NMDAR)-dependent synaptic plasticity. Using quantitative high-resolution in situ hybridization, we show that mRNAs encoding the AMPA-type glutamate receptor subunits (GluRs) 1 and 2 are localized to dendrites of hippocampal neurons and are regulated by paradigms that alter synaptic efficacy. A substantial fraction of synaptic sites contain AMPAR mRNA, consistent with strategic positioning and availability for "on-site" protein synthesis. NMDAR activation depletes dendritic levels of AMPAR mRNAs. The decrease in mRNA occurs via rise in intracellular Ca2+, activation of extracellular signal-regulated kinase/mitogen-activated protein kinase signaling, and transcriptional arrest at the level of the nucleus. The decrease in mRNA is accompanied by a long-lasting reduction in synaptic AMPAR number, consistent with reduced synaptic efficacy. In contrast, group I metabotropic GluR signaling promotes microtubule-based trafficking of existing AMPAR mRNAs from the soma to dendrites. Bidirectional regulation of dendritic mRNA abundance represents a potentially powerful means to effect long-lasting changes in synaptic strength.

Fragile X syndrome is caused by the absence of the mRNA-binding protein Fragile X mental retardation protein (FMRP), which may play a role in activity-regulated localization and translation of mRNA in dendrites and at synapses. We investigated whether neuronal activity and glutamatergic signals regulate trafficking of FMRP and its encoding Fmr1 mRNA into dendrites or at synapses. Using high-resolution fluorescence and digital imaging microscopy in cultured hippocampal neurons, FMRP and Fmr1 mRNA were localized in granules throughout dendrites and within spines. KCl depolarization rapidly increased FMRP and Fmr1 mRNA levels in dendrites. Metabotropic glutamate receptor (mGluR) activation, in particular mGluR5 activation, was necessary for localization of FMRP into dendrites. Blockade of either PKC or internal calcium prevented mGluR-dependent localization of both FMRP and Fmr1 mRNA in dendrites. The activity-dependent localization of FMRP was not dependent on protein synthesis. Fluorescence recovery after photobleaching analysis of live neurons transfected with enhanced green fluorescent protein-FMRP revealed increased granule trafficking in response to KCl depolarization. In contrast to its dendritic localization, mGluR activation diminished FMRP, but not Fmr1 mRNA, localization at synapses. These results demonstrate regulation of FMRP and Fmr1 mRNA trafficking in dendrites and synapses in response to specific glutamatergic signals.

The dendritic transport and local translation of mRNA may be an essential mechanism to regulate synaptic growth and plasticity. We investigated the molecular mechanism and function of beta-actin mRNA localization in dendrites of cultured hippocampal neurons. Previous studies have shown that beta-actin mRNA localization to the leading edge of fibroblasts or the growth cones of developing neurites involved a specific interaction between a zipcode sequence in the 3' untranslated region and the mRNA-binding protein zipcode-binding protein-1 (ZBP1). Here, we show that ZBP1 is required for the localization of beta-actin mRNA to dendrites. Knock-down of ZBP1 using morpholino antisense oligonucleotides reduced dendritic levels of ZBP1 and beta-actin mRNA and impaired growth of dendritic filopodia in response to BDNF treatment. Transfection of an enhanced green fluorescent protein (EGFP)-beta-actin construct, which contained the zipcode, increased the density of dendritic filopodia and filopodial synapses. Transfection of an EGFP construct, also with the zipcode, resulted in recruitment of endogenous ZBP1 and beta-actin mRNA into dendrites and similarly increased the density of dendritic filopodia. However, the beta-actin zipcode did not affect filopodial length or the density of mature spines. These results reveal a novel function for an mRNA localization element and its binding protein in the regulation of dendritic morphology and synaptic growth via dendritic filopodia.

In neurons, specific mRNAs are transported in a translationally repressed manner along dendrites or axons by transport ribonucleic-protein complexes called RNA granules. ZBP1 is one RNA binding protein present in transport RNPs, where it transports and represses the translation of cotransported mRNAs, including b-actin mRNA. The release of β-actin mRNA from ZBP1 and its subsequent translation depends on the phosphorylation of ZBP1 by Src kinase, but little is known about how this process is regulated. Here we demonstrate that the ribosomal-associated protein RACK1, another substrate of Src, binds the β-actin mRNA/ZBP1 complex on ribosomes and contributes to the release of β-actin mRNA from ZBP1 and to its translation. We identify the Src binding and phosphorylation site Y246 on RACK1 as the critical site for the binding to the bactin mRNA/ZBP1 complex. Based on these results we propose RACK1 as a ribosomal scaffold protein for specific mRNA-RBP complexes to tightly regulate the translation of specific mRNAs. [ABSTRACT FROM AUTHOR]

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 IIα (CaMKIIα) 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 CaMKIIα 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 CaMKIIα, 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 CaMKIIα 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. [ABSTRACT FROM AUTHOR]

The dendritic transport and local translation of mRNA may be an essential mechanism to regulate synaptic growth and plasticity. We investigated the molecular mechanism and function of β-actin mRNA localization in dendrites of cultured hippocampal neurons. Previous studies have shown that β-actin mRNA localization to the leading edge of fibroblasts or the growth cones of developing neurites involved a specific interaction between a zipcode sequence in the 3′ untranslated region and the mRNA-binding protein zipcode-binding protein-1 (ZBP1). Here, we show that ZBP1 is required for the localization of β-actin mRNA to dendrites. Knock-down of ZBP1 using morpholino antisense oligonucleotides reduced dendritic levels of ZBP1 and β-actin mRNA and impaired growth of dendritic filopodia in response to BDNF treatment. Transfection of an enhanced green fluorescent protein (EGFP)-β-actin construct, which contained the zipcode, increased the density of dendritic filopodia and filopodial synapses. Transfection of an EGFP construct, also with the zipcode, resulted in recruitment of endogenous ZBP1 and β-actin mRNA into dendrites and similarly increased the density of dendritic filopodia. However, the β-actin zipcode did not affect filopodial length or the density of mature spines. These results reveal a novel function for an mRNA localization element and its binding protein in the regulation of dendritic morphology and synaptic growth via dendritic filopodia. [ABSTRACT FROM AUTHOR]

Presents information on a study which used fluorescence microscopy and digital imaging techniques applied to both fixed and live cultured hippocampal neurons to visualize the localization of the messenger RNA binding protein, zipcode binding protein 1 (ZBP1) and its dynamic movements. Visualization of ZBP1 granules in dendrites, actin-rich spines and subsynaptic loci with the use of high-resolution fluorescence microscopy; Localization of ZBP1 in dendrites and spines with the use of high-resolution fluorescence imaging; Regulation of messenger RNA localization by neuronal activity and glutamatergic signals.

The RNA binding protein CPEB (cytoplasmic polyadenylation element binding) regulates cytoplasmic polyadenylation and translation in germ cells and the brain. In neurons, CPEB is detected at postsynaptic sites, as well as in the cell body. The related CPEB3 protein also regulates translation in neurons, albeit probably not through polyadenylation; it, as well as CPEB4, is present in dendrites and the cell body. Here, we show that treatment of neurons with ionotropic glutamate receptor agonists causes CPEB4 to accumulate in the nucleus. All CPEB proteins are nucleus-cytoplasm shuttling proteins that are retained in the nucleus in response to calcium-mediated signaling and alpha-calcium/calmodulin-dependent kinase protein II (CaMKII) activity. CPEB2, -3, and -4 have conserved nuclear export signals that are not present in CPEB. CPEB4 is necessary for cell survival and becomes nuclear in response to focal ischemia in vivo and when cultured neurons are deprived of oxygen and glucose. Further analysis indicates that nuclear accumulation of CPEB4 is controlled by the depletion of calcium from the ER, specifically, through the inositol-1,4,5-triphosphate (IP3) receptor, indicating a communication between these organelles in redistributing proteins between subcellular compartments. [ABSTRACT FROM AUTHOR]

Summary: 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. [Copyright &y& Elsevier]