Development of a Quantitative FMRP Assay for Mouse Tissue Applications

Abstract

Fragile X syndrome results from the absence of the FMR1 gene product—Fragile X Mental Retardation Protein (FMRP). Fragile X animal research has lacked a reliable method to quantify FMRP. We report the development of an array of FMRP-specific monoclonal antibodies and their application for quantitative assessment of FMRP (qFMRPm) in mouse tissue. To characterize the assay, we determined the normal variability of FMRP expression in four brain structures of six different mouse strains at seven weeks of age. There was a hierarchy of FMRP expression: neocortex > hippocampus > cerebellum > brainstem. The expression of FMRP was highest and least variable in the neocortex, whereas it was most variable in the hippocampus. Male C57Bl/6J and FVB mice were selected to determine FMRP developmental differences in the brain at 3, 7, 10, and 14 weeks of age. We examined the four structures and found a developmental decline in FMRP expression with age, except for the brainstem where it remained stable. qFMRPm assay of blood had highest values in 3 week old animals and dropped by 2.5-fold with age. Sex differences were not significant. The results establish qFMRPm as a valuable tool due to its ease of methodology, cost effectiveness, and accuracy.

Efficient delivery ofFMR1across the blood brain barrier using AAVphp construct in adultFMR1KO mice demonstrates the feasibility of gene therapy for fragile X syndrome

“…Neuron-specific targeted delivery of the Fmr1 isoform 7 was selected as most age appropriate for the time of the therapy administration. The level of FMRP detected in the WT+Empty treatment group was comparable with previously observed per structure profile of FMRP expression in FVB mouse strain [23]. Efficient delivery was observed in all tested CNS tissues in treated KO mice (KO+Fmr1).…”

“…Luminex-based method was used for the quantification of FMRP as previously described [23]. In short, qFMRPm assays were prepared in 96-well format using monoclonal antibody mAb 5C2 coupled to xMAP Microplex microspheres (Luminex Corporation, Austin TX).…”

Editorial for the Fragile X Syndrome Genetics Special Issue: May 2023

“…Defining the functions of FMRP in human tissues has limitations, especially in preclinical studies. Adaev et al [ 10 ] develop an FMRP assay to be used in mouse models of FXS to address these limitations. The study characterized an array of FMRP-specific antibodies for quantitative assessments in mouse tissues using an Enzyme-Linked Immunosorbent Assay (ELISA).…”