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A quantitative ELISA assay for the fragile x mental retardation 1 protein.

ABSTRACT: Non-coding (CGG-repeat) expansions in the fragile X mental retardation 1 (FMR1) gene result in a spectrum of disorders involving altered neurodevelopment (fragile X syndrome), neurodegeneration (late-onset fragile X-associated tremor/ataxia syndrome), or primary ovarian insufficiency. While reliable and quantitative assays for the number of CGG repeats and FMR1 mRNA levels are now available, there has been no scalable, quantitative assay for the FMR1 protein (FMRP) in non-transformed cells. Using a combination of avian and murine antibodies to FMRP, we developed a sensitive and highly specific sandwich enzyme-linked immunosorbent assay (ELISA) for FMRP in peripheral blood lymphocytes. This ELISA method is capable of quantifying FMRP levels throughout the biologically relevant range of protein concentrations and is specific for the intact FMRP protein. Moreover, the ELISA is well-suited for replicate protein determinations across serial dilutions in non-transformed cells and is readily scalable for large sample numbers. The FMRP ELISA is potentially a powerful tool in expanding our understanding of the relationship between FMRP levels and the various FMR1-associated clinical phenotypes.

SUBMITTER: Iwahashi C

PROVIDER: S-EPMC2710703 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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A quantitative ELISA assay for the fragile x mental retardation 1 protein.

Iwahashi Christine C Tassone Flora F Hagerman Randi J RJ Yasui Dag D Parrott George G Nguyen Danh D Mayeur Greg G Hagerman Paul J PJ

The Journal of molecular diagnostics : JMD 20090521 4

Non-coding (CGG-repeat) expansions in the fragile X mental retardation 1 (FMR1) gene result in a spectrum of disorders involving altered neurodevelopment (fragile X syndrome), neurodegeneration (late-onset fragile X-associated tremor/ataxia syndrome), or primary ovarian insufficiency. While reliable and quantitative assays for the number of CGG repeats and FMR1 mRNA levels are now available, there has been no scalable, quantitative assay for the FMR1 protein (FMRP) in non-transformed cells. Usin ...[more]

PMID: 19460937

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Project description:Fragile X syndrome (FXS), the most common inherited determinant of intellectual disability and autism spectrum disorders, is caused by loss of the fragile X mental retardation 1 (FMR1) gene product (FMRP), an mRNA-binding translational repressor. A number of conserved FMRP targets have been identified in the well-characterized Drosophila FXS disease model, but FMRP is highly pleiotropic in function and the full spectrum of FMRP targets has yet to be revealed. In this study, screens for upregulated neural proteins in Drosophila fmr1 (dfmr1) null mutants reveal strong elevation of two synaptic heparan sulfate proteoglycans (HSPGs): GPI-anchored glypican Dally-like protein (Dlp) and transmembrane Syndecan (Sdc). Our recent work has shown that Dlp and Sdc act as co-receptors regulating extracellular ligands upstream of intracellular signal transduction in multiple trans-synaptic pathways that drive synaptogenesis. Consistently, dfmr1 null synapses exhibit altered WNT signaling, with changes in both Wingless (Wg) ligand abundance and downstream Frizzled-2 (Fz2) receptor C-terminal nuclear import. Similarly, a parallel anterograde signaling ligand, Jelly belly (Jeb), and downstream ERK phosphorylation (dpERK) are depressed at dfmr1 null synapses. In contrast, the retrograde BMP ligand Glass bottom boat (Gbb) and downstream signaling via phosphorylation of the transcription factor MAD (pMAD) seem not to be affected. To determine whether HSPG upregulation is causative for synaptogenic defects, HSPGs were genetically reduced to control levels in the dfmr1 null background. HSPG correction restored both (1) Wg and Jeb trans-synaptic signaling, and (2) synaptic architecture and transmission strength back to wild-type levels. Taken together, these data suggest that FMRP negatively regulates HSPG co-receptors controlling trans-synaptic signaling during synaptogenesis, and that loss of this regulation causes synaptic structure and function defects characterizing the FXS disease state.

Dataset Information

A quantitative ELISA assay for the fragile x mental retardation 1 protein.

Publications

A quantitative ELISA assay for the fragile x mental retardation 1 protein.

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