Probes for FMR1

Short trinucleotide expansions at the FMR1 locus are associated with the late-onset condition fragile X-associated tremor/ataxia syndrome (FXTAS), which shows very different clinical and pathological features from fragile X syndrome (associated with longer expansions), with no clear molecular explanation for these marked differences. One prevailing theory posits that the shorter, premutation expansion uniquely causes extreme neurotoxic increases in FMR1 mRNA (i.e., four to eightfold increases), but evidence to support this hypothesis is largely derived from analysis of peripheral blood. We applied single-nucleus RNA sequencing to postmortem frontal cortex and cerebellum from 7 individuals with premutation and matched controls (n = 6) to assess cell type-specific molecular neuropathology. We found only modest upregulation (~1.3-fold) of FMR1 in some glial populations associated with premutation expansions. In premutation cases, we also identified decreased astrocyte proportions in the cortex. Differential expression and gene ontology analysis demonstrated altered neuroregulatory roles of glia. Using network analyses, we identified cell type-specific and region-specific patterns of FMR1 protein target gene dysregulation unique to premutation cases, with notable network dysregulation in the cortical oligodendrocyte lineage. We used pseudotime trajectory analysis to determine how oligodendrocyte development was altered and identified differences in early gene expression in oligodendrocyte trajectories in premutation cases specifically, implicating early cortical glial developmental perturbations. These findings challenge dogma regarding extremely elevated FMR1 increases in FXTAS and implicate glial dysregulation as a critical facet of premutation pathophysiology, representing potential unique therapeutic targets directly derived from the human condition.

Fragile X syndrome (FXS), resulting from a mutation in the Fmr1 gene, is the most common monogenic cause of autism and inherited intellectual disability. Fmr1 encodes the Fragile X Messenger Ribonucleoprotein (FMRP), and its absence leads to cognitive, emotional, and social deficits compatible with the nucleus accumbens (NAc) dysfunction. This structure is pivotal in social behavior control, consisting mainly of spiny projection neurons (SPNs), distinguished by dopamine D1 or D2 receptor expression, connectivity, and associated behavioral functions. This study aims to examine how FMRP absence differentially affects SPN cellular properties, which is crucial for categorizing FXS cellular endophenotypes.We utilized a novel Fmr1-/y::Drd1a-tdTomato mouse model, which allows in-situ identification of SPN subtypes in FXS mice. Using RNA-sequencing, RNAScope and ex-vivo patch-clamp in adult male mice NAc, we comprehensively compared the intrinsic passive and active properties of SPN subtypes.Fmr1 transcripts and their gene product, FMRP, were found in both SPNs subtypes, indicating potential cell-specific functions for Fmr1. The study found that the distinguishing membrane properties and action potential kinetics typically separating D1- from D2-SPNs in wild-type mice were either reversed or abolished in Fmr1-/y::Drd1a-tdTomato mice. Interestingly, multivariate analysis highlighted the compound effects of Fmr1 ablation by disclosing how the phenotypic traits distinguishing each cell type in wild-type mice were altered in FXS.Our results suggest that the absence of FMRP disrupts the standard dichotomy characterizing NAc D1- and D2-SPNs, resulting in a homogenous phenotype. This shift in cellular properties could potentially underpin select aspects of the pathology observed in FXS. Therefore, understanding the nuanced effects of FMRP absence on SPN subtypes can offer valuable insights into the pathophysiology of FXS, opening avenues for potential therapeutic strategies.

Fragile X syndrome is a neurodevelopmental disorder caused by the absence of the mRNA-binding protein fragile X messenger ribonucleoprotein (FMRP). Because FMRP is a highly pleiotropic protein controlling the expression of hundreds of genes, viral vector-mediated gene replacement therapy is viewed as a potential viable treatment to correct the fundamental underlying molecular pathology inherent in the disorder. Here, we studied the safety profile and therapeutic effects of a clinically relevant dose of a self-complementary adeno-associated viral (AAV) vector containing a major human brain isoform of FMRP after intrathecal injection into wild-type and fragile X-KO mice. Analysis of the cellular transduction in the brain indicated primarily neuronal transduction with relatively sparse glial expression, similar to endogenous FMRP expression in untreated wild-type mice. AAV vector-treated KO mice showed recovery from epileptic seizures, normalization of fear conditioning, reversal of slow-wave deficits as measured via electroencephalographic recordings, and restoration of abnormal circadian motor activity and sleep. Further assessment of vector efficacy by tracking and analyzing individual responses demonstrated correlations between the level and distribution of brain transduction and drug response. These preclinical findings further demonstrate the validity of AAV vector-mediated gene therapy for treating the most common genetic cause of cognitive impairment and autism in children.

Identifying causative variants in cis-regulatory elements (CRE) in neurodevelopmental disorders has proven challenging. We have used in vivo functional analyses to categorize rigorously filtered CRE variants in a clinical cohort that is plausibly enriched for causative CRE mutations: 48 unrelated males with a family history consistent with X-linked intellectual disability (XLID) in whom no detectable cause could be identified in the coding regions of the X chromosome (chrX). Targeted sequencing of all chrX CRE identified six rare variants in five affected individuals that altered conserved bases in CRE targeting known XLID genes and segregated appropriately in families. Two of these variants, FMR1CRE and TENM1CRE, showed consistent site- and stage-specific differences of enhancer function in the developing zebrafish brain using dual-color fluorescent reporter assay. Mouse models were created for both variants. In male mice Fmr1CRE induced alterations in neurodevelopmental Fmr1 expression, olfactory behavior and neurophysiological indicators of FMRP function. The absence of another likely causative variant on whole genome sequencing further supported FMR1CRE as the likely basis of the XLID in this family. Tenm1CRE mice showed no phenotypic anomalies. Following the release of gnomAD 2.1, reanalysis showed that TENM1CRE exceeded the maximum plausible population frequency of a XLID causative allele. Assigning causative status to any ultra-rare CRE variant remains problematic and requires disease-relevant in vivo functional data from multiple sources. The sequential and bespoke nature of such analyses renders them time-consuming and challenging to scale for routine clinical use.

Mature protoplasmic astroglia in the mammalian central nervous system (CNS) uniquely possess a large number of fine processes that have been considered primary sites to mediate astroglia to neuron synaptic signaling. However, localized mechanisms for regulating interactions between astroglial processes and synapses, especially for regulating the expression of functional surface proteins at these fine processes, are largely unknown. Previously, we showed that the loss of the RNA binding protein FMRP in astroglia disrupts astroglial mGluR5 signaling and reduces expression of the major astroglial glutamate transporter GLT1 and glutamate uptake in the cortex of Fmr1 conditional deletion mice. In the current study, by examining ribosome localization using electron microscopy and identifying mRNAs enriched at cortical astroglial processes using SNS/TRAP and RNA-Seq in wild type and FMRP-deficient male mice, our results reveal interesting localization-dependent functional clusters of mRNAs at astroglial processes. We further showed that the lack of FMRP preferentially alters the subcellular localization and expression of process-localized mRNAs. Taken together, we defined the role of FMRP in altering mRNA localization and expression at astroglial processes at the postnatal development (P30-40) and provided new candidate mRNAs that are potentially regulated by FMRP in cortical astroglia.SIGNIFICANCE STATEMENTLocalized mechanisms for regulating interactions between astroglial processes and synapses, especially for regulating the expression of functional surface proteins at these fine processes, are largely unknown. Previously, we showed that the loss of the RNA binding protein FMRP in astroglia disrupts expression of several astroglial surface proteins such as mGluR5 and major astroglial glutamate transporter GLT1 in the cortex of FMRP-deficient mice. Our current study examined ribosome localization using electron microscopy and identified mRNAs enriched at cortical astroglial processes in wild type and FMRP-deficient mice. These results reveal interesting localization-dependent functional clusters of mRNAs at astroglial processes and demonstrate that the lack of FMRP preferentially alters the subcellular localization and expression of process-localized mRNAs.