Molecular mechanisms of neuronal mRNA localization in an animal model of Fragile X syndrome

Abstract

Messenger RNA (mRNA) localization to neuronal dendrites and activity-induced nascent protein synthesis from the locally available pool of synaptic mRNAs are required for long-term structural and functional remodeling that underlies learning and memory. Dysregulation of mRNAs at synapses is a common cause of neurodevelopmental disorders such as Fragile X syndrome (FXS), the most prevalent single gene cause of autism and learning disability. FXS is primarily caused by the transcriptional silencing of Fragile X messenger ribonucleoprotein (FMRP), a ubiquitous RNA-binding protein (RBP) that can bind to and regulate approximately 4% of the brain transcriptome, the majority of which encode proteins for synaptic function. The breadth of FMRP's mRNA targets makes it an excellent model system for identifying the fundamental molecular rules that govern mRNA localization across hundreds of microns in distant neuronal processes. Using FMRP, and a dozen of its mRNA targets, we systematically mapped the spatial organization of localized mRNAs across multiple hippocampal cell types in vivo. Combining high-resolution microscopy, multiplex single molecule fluorescent in situ hybridization (smFISH) and computational analyses, we discovered that FMRP target mRNAs in the hippocampal neuropil exist in heterogeneous copy number states and are predominantly co-packaged into multi-RNA containing mRNP (ribonucleoprotein particles) granules for constitutive localization. Notably, this co-packaging pattern is best explained by stochastic interaction-driven assembly which in turn is determined by the local abundance of each mRNA species. This contrasts with earlier models proposing that neuronal mRNAs predominantly travel as single molecules, each subject to precise and independent regulation. Our abundance-driven model of mRNA co-localization is further supported by in vitro studies of mRNA biophysical properties and is consistent with the broader principle of energy minimization, suggesting that neurons achieve efficient mRNA localization under basal conditions through population-level strategies, rather than individually regulated, assembly mechanisms. To determine whether FMRP loss affects its target mRNAs uniformly or selectively, both under basal conditions and following neuronal activity, we visualized a subset of these mRNAs in hippocampal CA1 and CA2 neurons, which are critical for encoding spatial and social memory with pathological phenotypes observed in FXS. We compared wildtype and Fmr1 KO mice either housed in their home cage or subjected to 60 minutes of enriched environment exploration, a well-established paradigm for inducing synaptic activity in vivo. Under basal conditions, loss of FMRP did not alter the steady-state abundance of its mRNA targets in either cell type, suggesting that FMRP is largely dispensable for constitutive mRNA localization -likely because other RBPs are able to compensate for its absence under resting conditions. Interestingly, localization of specific mRNA target (Dlg4) of FMRP was significantly downregulated following experience-driven neuronal activity in Fmr1 KO mice, in both CA1 and CA2 neuropil. As Dlg4 mRNA serves as the template for a critical structural scaffolding protein of the post-synapse, PSD-95, we further quantified and observed an increased spine density phenotype in the distal dendrites of both cell types in Fmr1 KO mice. Lastly, we failed to detect a cell-specific role of FMRP in localized mRNA regulation in contrast to conclusions drawn by recent studies suggesting that activity-dependent localization mechanisms of FMRP targets are likely similar across these hippocampal circuits. Taken together, these findings support a two-layered model of neuronal mRNA localization. Under basal conditions, constitutive mRNA localization is governed by stochastic, abundance-driven co-packaging of multi-mRNA molecules and is largely FMRP-independent, as complementary/ redundant RBPs can compensate for its loss. However, under states of activity, additional regulatory mechanisms/ localization pathways are recruited that critically depend on FMRP. The selective failure to upregulate or maintain activity-driven localization of a functionally critical subset of synaptic mRNAs, such as Dlg4, in the absence of FMRP likely contributes to the inability to remodel synapses, the presumed cause of clinically relevant learning disabilities observed in FXS. In summary, these findings extend previous observations on localized mRNA regulation in neurons by moving beyond a parts list view of mRNA associations toward a working model of mRNA co-regulation governed by population localization principles that seems energy efficient. Novel observations on FMRP-dependent activity-driven mRNA localization in intact hippocampal circuits, that are similar across cell types and not cell-autonomous, offer a potential mechanistic basis for understanding how disruption of activity-dependent mRNA localization in parallel with translational dysregulation contributes to the synaptic pathophysiology, and learning and memory deficits of FXS.