Disruption of the KH1 domain of Fmr1 leads to transcriptional alterations and attentional deficits in rats

Abstract

(to CEMG). Carla E. M. Golden is a Seaver Graduate Fellow and Michael S.Breen is a Seaver Postdoctoral Fellow. We thank Eilam Doron, who contributed to this work. Abstract:Fragile X Syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene. FXS is a leading monogenic cause of autism spectrum disorder (ASD) and inherited intellectual disability (ID). In most cases, the mutation is an expansion of a microsatellite (CGG triplet), which leads to suppressed expression of the fragile X mental retardation protein (FMRP), an RNA-binding protein involved in multiple aspects of mRNA metabolism. Interestingly, we found that the previously published Fmr1 knockout rat model of FXS expresses a transcript with an in-frame deletion of a K-homology (KH) domain, KH1. KH domains are RNA-binding domains of FMR1 and several of the few, known point mutations associated with FXS are found within them. We observed that this deletion leads to medial prefrontal cortex (mPFC)-dependent attention deficits, similar to those observed in FXS, and to alterations in transcriptional profiles within the mPFC, which mapped to two weighted gene coexpression network analysis modules. We demonstrated that these modules are conserved inND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/338988 doi: bioRxiv preprint first posted online Jun. 9, 2018; 2 human frontal cortex, are enriched for known FMRP targets and for genes involved in neuronal and synaptic processes, and that one is enriched for genes that are implicated in ASD, ID, and schizophrenia. Hub genes in these conserved modules represent potential targets for FXS.These findings provide support for a prefrontal deficit in FXS, indicate that attentional testing might be a reliable cross-species tool for investigating the pathophysiology of FXS and a potential readout for pharmacotherapy testing, and identify dysregulated gene expression modules in a relevant brain region. Significance Statement:The significance of the current study lies in two key domains. First, this study demonstrates that deletion of the Fmrp-KH1 domain is sufficient to cause major mPFCdependent attention deficits in both males and females, like those observed in both individuals with FXS and in knockout mouse models for FXS. Second, the study shows that deletion of the KH1 domain leads to alterations in the transcriptional profiles within the medial prefrontal cortex (mPFC), which are of potential translational value for subjects with FXS. These findings indicate that attentional testing might be a reliable cross-species tool for investigating the pathophysiology of FXS and a potential readout for pharmacotherapy testing and also highlight hub genes for follow up.

Reduced axonal caliber and white matter changes in a rat model of Fragile X syndrome with a deletion of a K Homology domain of Fmr1

“…The decrease in diffusion that we observed could be related to the integrity of the dopaminergic neurons in Fmr1-Δ exon 8 -/rats. All of these anatomical perturbations could be related to the deficits we see in behavior and transcription of Fmr1-Δ exon 8 rats [11]. This is worth further exploration.…”

“…The Fmr1-Δ exon 8 rat model was generated using zinc finger nucleases (ZFNs) in the outbred Sprague-Dawley background. The design and cloning of the ZFN, as well as the embryonic microinjection and screening for positive founder rats were performed by Horizon Labs (Boyertown, PA USA) as previously described [14]. The best performing ZFN pair targeting the CATGAACAGTTTATCgtacgaGAAGATCTGATGGGT sequence, located between 18686bp-18721bp in the Fmr1 gene (NCBI reference sequence NC_005120.4), was used for embryo microinjection.…”

“…The remaining images (fractional anisotropy (FA), mean diffusion (MD), axial diffusion (AD), radial diffusion (RD)) were resampled to the T2 template with their respective concatenated transforms to allow both voxel-wise and ROI analyses. 11 We used DTI to evaluate white matter specifically, as it is affected in individuals with FXS, with AD, RD, MD, and total FA as measures of possible changes. FA is enhanced when parallel diffusivity is facilitated and/or perpendicular diffusivity is restricted, RD is increased following myelin damage, and AD is reduced after axonal damage [18].…”

“…A Kolmogorov-Smirnov test was used to test for the distance between the empirical distribution functions of axon caliber. Data points that were outside 1.5 times the interquartile range for each rat were removed and a linear mixed model where the image variable 14 was nested in the rat ID which was nested in the cohort number in order to determine the effect of genotype was computed using custom scripts written in the R statistical programming environment (R Development Core Team, 2006).…”

“…We recently identified a deficit in sustained attention in both sexes of a rat model of FXS that has a deletion of an mRNA-binding domain, K-homology 1 (KH1), the Fmr1-Δ exon 8 rat [11].…”