Enhanced mGluR5 function is causally associated with the pathophysiology of Fragile X Syndrome (FXS), a leading inherited cause of intellectual disability and autism. Here we provide evidence that altered mGluR5-Homer scaffolds contribute to mGluR5 dysfunction and phenotypes in the FXS mouse model, Fmr1 KO. In Fmr1 KO mice mGluR5 is less associated with long Homer isoforms, but more associated with the short Homer1a. Genetic deletion of Homer1a restores mGluR5- long Homer scaffolds and corrects multiple phenotypes in Fmr1 KO mice including altered mGluR5 signaling, neocortical circuit dysfunction, and behavior. Acute, peptide-mediated disruption of mGluR5-Homer scaffolds in wildtype mice mimics many Fmr1 KO phenotypes. In contrast, Homer1a deletion does not rescue altered mGluR-dependent long-term synaptic depression or translational control of FMRP target mRNAs. Our findings reveal novel functions for mGluR5-Homer interactions in the brain and delineate distinct mechanisms of mGluR5 dysfunction in a mouse model of cognitive dysfunction and autism.
Summary The activity-dependent transcription factor, Myocyte Enhancer Factor-2 (MEF2), induces excitatory synapse elimination in mouse neurons which requires Fragile X Mental Retardation Protein (FMRP), an RNA binding protein implicated in human cognitive dysfunction and autism. We report here that protocadherin-10 (Pcdh10), an autism-spectrum disorders gene, is necessary for this process. MEF2 and FMRP cooperatively regulate the expression of Pcdh10. Upon MEF2 activation, PSD-95 is ubiquitinated by the ubiquitin E3 ligase, murine double minute-2 (Mdm2) and then binds to Pcdh10, which links it to the proteasome for degradation. Blockade of the Pcdh10 -proteasome interaction inhibits MEF2-induced PSD-95 degradation and synapse elimination. In FMRP-lacking neurons, elevated protein levels of eukaryotic translation elongation factor 1-alpha (EF1α), an Mdm2 interacting protein and FMRP target mRNA, sequester Mdm2 and prevent MEF2-induced PSD-95 ubiquitination and synapse elimination. Together our findings reveal novel roles for multiple autism-linked genes in activity-dependent synapse elimination.
SUMMARY A novel experience induces Arc/Arg3.1 gene as well as plasticity of CA1 neural networks. To understand how these are linked, we briefly exposed GFP reporter mice of Arc transcription to a novel environment. Excitatory synaptic function of CA1 neurons with recent in vivo Arc-induction (ArcGFP+) was similar to neighboring non-induced neurons (ArcGFP–). However, in response to group 1 mGluR activation, ArcGFP+ neurons preferentially displayed long-term synaptic depression (mGluR-LTD) and robust increases in dendritic Arc protein. mGluR-LTD in ArcGFP+ neurons required rapid protein synthesis and Arc suggesting that dendritic translation of Arc underlies the priming of mGluR-LTD. In support of this idea, novelty exposure increased Arc mRNA in CA1 dendrites and promoted mGluR-induced translation of Arc in hippocampal synaptoneurosomes. Repeated experience suppressed synaptic transmission onto ArcGFP+ neurons and occluded mGluR-LTD ex vivo. mGluR-LTD priming in neurons with similar Arc activation history may contribute to encoding a novel environment.
RIP140 (receptor-interacting protein 140) is a transcriptional co-repressor that regulates diverse genes such as those responsive to hormones and involved in metabolic processes. The expression of RIP140 is regulated by multiple hormonal activities in adipose tissue and cancer cell lines. However, it is unclear whether and how RIP140 is regulated post-transcriptionally. Using 5'RACE (rapid amplification of 5' cDNA ends), we have identified a novel 5' splice variant of RIP140 mRNA in mouse brain and P19 cells. A target sequence for miRNA (microRNA) mir-346 was found in the 5'UTR (5'-untranslated region) of RIP140 mRNA; this miRNA is also expressed endogenously in mouse brain and P19 cells. Gain- and loss-of-function studies demonstrated that mir-346 elevates RIP140 protein levels by facilitating association of its mRNA with the polysome fraction. Furthermore, the activity of mir346 does not require Ago-2 (Argonaute 2). The expression of mir-346 enhances the gene repressive activity of RIP140. This is the first report demonstrating post-transcriptional regulation of RIP140 mRNA, involving the enhancing effect of a specific miRNA that targets RIP140's 5'UTR.
Cells form stress granules (SGs) in response to environmental stresses, which constitute cytoplasmic domains where mRNAs are stored and translation is halted. Although several components are found in SGs, it is poorly understood as to how SGs are formed and dissolved. We identified growth factor receptor‐bound protein 7 (Grb7), an RNA‐binding, translational regulator, as an integral component of SGs, which directly interacts with Hu antigen R (HuR) and is required for cells to form SGs. When stress is terminated, Grb7 is hyperphosphorylated by focal adhesion kinase (FAK), loses its ability to directly interact with HuR and is dissociated from SG components, thereby disrupting SGs in recovering cells. Consistently, dominant‐negative hypophospho mutants of FAK and Grb7 significantly attenuate SG disassembly during recovery. FAK activation followed by its phosphorylating Grb7 constitutes a cell‐autonomous signalling pathway that regulates the disassembly of SGs and translational stimulation during recovery. This is the first reported pathway actively regulating the dynamics of SGs.
We previously reported a novel biological activity of Netrin‐1 in translational stimulation of kappa opioid receptor (KOR). We now identify Grb7 as a new RNA‐binding protein that serves as the molecular adaptor for transmitting Netrin‐1 signals, through focal adhesion kinase (FAK), to the translation machinery. Grb7 binds specifically to the first stem loop of kor mRNA 5′‐UTR through a new RNA‐binding domain located in its amino terminus. Upon binding to its capped, target mRNA, Grb7 blocks the recruitment of eIF4E, rendering mRNA untranslatable. The RNA‐binding and translation‐repressive activity is reduced by FAK‐mediated hyperphosphorylation on two tyrosine residues of its carboxyl terminus. This study reports an adaptor protein Grb7 that transmits the stimulating signals of Netrin‐1 to the translational machinery to rapidly regulate mRNA translation.
Cells form stress granule (SG), in response to unfavorable environments, to avoid apoptosis, but it is unclear whether and how SG formation and cellular apoptosis are coordinately regulated. In this study we detected the small GTPase, Ras homolog gene family member A (RhoA), and its downstream kinase, Rho-associated, coiled-coil containing protein kinase 1 (ROCK1), in SG, and found that their stress-induced activities were important for SG formation and subsequent global translational repression. Importantly, only activated RhoA and ROCK1 were sequestered into SG. Sequestration of activated ROCK1 into SG prevented ROCK1 from interacting with JNK-interacting protein 3 (JIP-3) and its activation of c-Jun N-terminal kinase (JNK), a pathway triggering apoptosis, thereby protecting cells from apoptosis. This study identifies a specific signaling pathway, mediated by RhoA and ROCK1, which determines cell fate by promoting SG formation or initiating apoptosis during stress.
SUMMARY Experience refines synaptic connectivity through neural activity-dependent regulation of transcription factors. Although activity-dependent regulation of transcription factors has been well described, it is unknown if synaptic activity and local, dendritic regulation of the induced transcripts are necessary for mammalian synaptic plasticity in response to transcription factor activation. Neuronal depolarization activates the Myocyte Enhancer Factor 2 (MEF2) family of transcription factors which suppresses excitatory synapse number. We report that activation of metabotropic glutamate receptor 5 (mGluR5) on the dendrites, but not cell soma, of hippocampal CA1 neurons is required for MEF2-induced functional and structural synapse elimination. We present evidence that mGluR5 is necessary for synapse elimination to stimulate dendritic translation of the MEF2-target gene Arc/Arg3.1. Arc is required for MEF2-induced synapse elimination, where it plays an acute, cell autonomous and postsynaptic role. This work reveals a role for dendritic activity in local translation of specific transcripts in synapse refinement.
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