(C) PLOS One This story was originally published by PLOS One and is unaltered. . . . . . . . . . . Single cell transcriptomics reveals dysregulated cellular and molecular networks in a fragile X syndrome model [1] ['Elisa Donnard', 'Program In Bioinformatics', 'Integrative Biology', 'University Of Massachusetts Medical School', 'Worcester', 'Massachusetts', 'United States Of America', 'Huan Shu', 'Program In Molecular Medicine', 'Manuel Garber'] Date: 2022-08 Despite advances in understanding the pathophysiology of Fragile X syndrome (FXS), its molecular basis is still poorly understood. Whole brain tissue expression profiles have proved surprisingly uninformative, therefore we applied single cell RNA sequencing to profile an FMRP deficient mouse model with higher resolution. We found that the absence of FMRP results in highly cell type specific gene expression changes that are strongest among specific neuronal types, where FMRP-bound mRNAs were prominently downregulated. Metabolic pathways including translation and respiration are significantly upregulated across most cell types with the notable exception of excitatory neurons. These effects point to a potential difference in the activity of mTOR pathways, and together with other dysregulated pathways, suggest an excitatory-inhibitory imbalance in the Fmr1-knock out cortex that is exacerbated by astrocytes. Our data demonstrate that FMRP loss affects abundance of key cellular communication genes that potentially affect neuronal synapses and provide a resource for interrogating the biological basis of this disorder. Fragile X syndrome is a leading genetic cause of inherited intellectual disability and autism spectrum disorder. It results from the inactivation of a single gene, FMR1 and hence the loss of its encoded protein FMRP. Despite decades of intensive research, we still lack an overview of the molecular and biological consequences of the disease. Using single cell RNA sequencing, we profiled cells from the brain of healthy mice and of knock-out mice lacking the FMRP protein, a common model for this disease, to identify molecular changes that happen across different cell types. We find neurons are the most impacted cell type, where genes in multiple pathways are similarly impacted. This includes transcripts known to be bound by FMRP, which are collectively decreased only in neurons but not in other cell types. Our results show how the loss of FMRP affects the intricate interactions between different brain cell types, which could provide new perspectives to the development of therapeutic interventions. Funding: This work was partially supported by the National Institutes of Health under award number U54HD082013 (M.G.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2022 Donnard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. We profiled the transcriptome of over 18,000 cells from the cerebral cortex of wild type (WT) and Fmr1-KO mice at postnatal day 5. We detected a heterogeneity in the response of different cell types to the loss of FMRP. In particular, we observed a stronger impact on the expression of mRNAs previously identified as FMRP binding targets in the brain [ 36 ], and we show that this effect is more prominent in neurons compared to other cells. We detect a divergent response of pathways downstream of mTOR signaling across different neuron subtypes, which suggests that excitatory neurons do not display a hyperactivation of this pathway. Taken together with the observed dysregulation of synaptic genes in astrocytes as well as neurons, our results suggest an impact in cell-cell communication that can result in a cortical environment of greater excitability. Despite the widespread gene expression of FMRP and the numerous potential mechanisms of action by which FMRP can impact the transcriptome, it was surprising that only very small changes in the mRNA levels of total or polyribosome associated mRNAs were seen in the Fmr1-KO mouse brain [ 36 ]. We and others have observed widespread, albeit very subtle, changes in mRNA levels in the absence of FMRP [ 22 , 23 , 37 ]. One possible cause behind the lack of detection of strong RNA changes is the cell type heterogeneity of brain tissue, where alterations in specific cell types could be masked in global measurements. Alternatively, the FMRP deficient brain could be inherently lacking strong changes at the transcriptome level, but instead display only mild RNA changes that can be challenging to detect using traditional bulk tissue RNA-seq techniques due to low statistical power. Fortunately, both of these scenarios can be addressed using single-cell RNA-seq. Here we describe our effort to revisit this question using an unbiased approach to survey cell type specific transcriptomes in the Fmr1-KO mouse brain. We took advantage of the power of single cell RNA-Seq to determine which cells are affected by the lack of FMRP at an early postnatal development stage. The cell type specific alteration of the transcriptome is a sensitive reflection of the cellular status, and can serve as a first step towards an overview of the molecular impact of FXS. Most of the above-mentioned studies focus on FMRP’s function in neurons, and rightly so, as neurons have the highest FMRP protein levels in the brain [ 16 ]. Evidence from clinical studies with FXS patients and from Fmr1-KO mouse models of the disease supports the view that neurons are the main affected cell type [ 28 , 29 ]. However, other cell types in the brain also express FMRP and FMRP loss has a clear effect on them. Indeed, astrocytes, oligodendrocyte precursor cells, and microglia express FMRP in a brain region and development-dependent manner [ 30 ]. FMRP-depleted astrocytes are more reactive [ 31 ], and this response alone may account for some of the phenotypes seen in FMRP deficient neurons particularly during development [ 32 – 35 ]. The molecular pathophysiology of FXS and FMRP function has been the subject of numerous studies over the past decades [ 4 , 5 ]. The most extensively studied function of FMRP is its role as a translational repressor. FMRP is critical to hippocampal long-term synaptic and spine morphological plasticity, dependent on protein synthesis. More specifically, the absence of FMRP leads to an exaggerated long-term synaptic depression, induced by the metabotropic glutamate receptor 5 (mGLUR5-LTD) [ 6 ]. However, several ambitious clinical trials that aimed to suppress translation or inhibit mGluR pathways have thus far failed [ 7 ]. The significance of FMRP’s role as a translation repressor at synapses is not without challenges. First, several teams show evidence that FRMP can function as a translation activator [ 8 – 10 ]. Secondly, only a few mRNAs that are bound by FMRP showed a consistent increase in protein levels upon loss of FMRP, and increased levels of proteins are not always pathogenic [ 11 – 15 ]. More importantly, focusing on FMRP’s translational function in dendritic synapses overlooks the fact that the great majority of this protein is located in the cell soma [ 16 ]. Alternatively, FMRP could have important functions independent of its role in translation regulation. Indeed, a wide range of research has associated FMRP to multiple steps of the mRNA life cycle, including pre-mRNA splicing [ 17 ], mRNA editing [ 18 , 19 ], miRNA activity [ 20 , 21 ], and mRNA stability [ 22 , 23 ]. Additionally, FMRP may function outside the RNA-binding scope, by chromatin binding and regulating genome stability [ 24 , 25 ], as well as directly binding to and regulating ion channels [ 26 , 27 ]. Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and autism spectrum disorder (ASD). The disease results from the silencing of a single gene (FMR1, FMRP Translational Regulator 1), which encodes the RNA-binding protein FMRP [ 1 ]. The loss of FMRP leads to a neurodevelopmental disorder with an array of well characterized behaviour and cellular abnormalities, such as impaired cognitive functions, repetitive behaviours, altered synaptic morphology and function [ 2 ]; many of which are reproduced in Fmr1-knock out (Fmr1-KO) mouse models [ 3 ]. Results [END] --- [1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010221 Published and (C) by PLOS One Content appears here under this condition or license: Creative Commons - Attribution BY 4.0. via Magical.Fish Gopher News Feeds: gopher://magical.fish/1/feeds/news/plosone/