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Alternative mRNA splicing from the glial fibrillary acidic protein (GFAP) gene generates isoforms with distinct subcellular mRNA localization patterns in astrocytes.

ABSTRACT: The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfap? is the most predominant isoform. The Gfap? isoform is expressed in proliferating neurogenic astrocytes of the developing human brain and in the adult human and mouse brain. Here we provide a characterization of mouse Gfap? mRNA and Gfap? protein. RT-qPCR analysis showed that Gfap? mRNA and Gfap? mRNA expression is coordinately increased in the post-natal period. Immunohistochemical staining of developing mouse brain samples showed that Gfap? is expressed in the sub-ventricular zones in accordance with the described localization in the developing and adult human brain. Immunofluorescence analysis verified incorporation of Gfap? into the Gfap intermediate filament network and overlap in Gfap? and Gfap? subcellular localization. Subcellular mRNA localization studies identified different localization patterns of Gfap? and Gfap? mRNA in mouse primary astrocytes. A larger fraction of Gfap? mRNA showed mRNA localization to astrocyte protrusions compared to Gfap? mRNA. The differential mRNA localization patterns were dependent on the different 3'-exon sequences included in Gfap? and Gfap? mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential to participate in subcellular region-specific intermediate filament dynamics during brain development, maintenance and in disease.

SUBMITTER: Thomsen R

PROVIDER: S-EPMC3753360 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Alternative mRNA splicing from the glial fibrillary acidic protein (GFAP) gene generates isoforms with distinct subcellular mRNA localization patterns in astrocytes.

Thomsen Rune R Daugaard Tina F TF Holm Ida E IE Nielsen Anders Lade AL

PloS one 20130826 8

The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapα is the most predominant isoform. The Gfapδ isoform is expressed in proliferating neurogenic astrocytes of the developing human brain and in the adult human and mouse brain. Here we provide a characterization of mouse Gfapδ mRNA and Gfapδ protein. RT-qPCR analysis showed that Gfapδ ...[more]

PMID: 23991052

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Project description:BackgroundGlial fibrillary acidic protein (GFAP) has emerged as a promising fluid biomarker for several neurological indications including traumatic brain injury (TBI), a leading cause of death and disability worldwide. In humans, serum or plasma GFAP levels can predict brain abnormalities including hemorrhage on computed tomography (CT) scans and magnetic resonance imaging (MRI). However, assays to quantify plasma or serum GFAP in preclinical models are not yet available.MethodsWe developed and validated a novel sensitive GFAP immunoassay assay for mouse plasma on the Meso Scale Discovery immunoassay platform and validated assay performance for robustness, precision, limits of quantification, dilutional linearity, parallelism, recovery, stability, selectivity, and pre-analytical factors. To provide proof-of-concept data for this assay as a translational research tool for TBI and Alzheimer's disease (AD), plasma GFAP was measured in mice exposed to TBI using the Closed Head Impact Model of Engineered Rotational Acceleration (CHIMERA) model and in APP/PS1 mice with normal or reduced levels of plasma high-density lipoprotein (HDL).ResultsWe performed a partial validation of our novel assay and found its performance by the parameters studied was similar to assays used to quantify human GFAP in clinical neurotrauma blood specimens and to assays used to measure murine GFAP in tissues. Specifically, we demonstrated an intra-assay CV of 5.0%, an inter-assay CV of 7.2%, a lower limit of detection (LLOD) of 9.0 pg/mL, a lower limit of quantification (LLOQ) of 24.8 pg/mL, an upper limit of quantification (ULOQ) of at least 16,533.9 pg/mL, dilution linearity of calibrators from 20 to 200,000 pg/mL with 90-123% recovery, dilution linearity of plasma specimens up to 32-fold with 96-112% recovery, spike recovery of 67-100%, and excellent analyte stability in specimens exposed to up to 7 freeze-thaw cycles, 168 h at 4 °C, 24 h at room temperature (RT), or 30 days at - 20 °C. We also observed elevated plasma GFAP in mice 6 h after TBI and in aged APP/PS1 mice with plasma HDL deficiency. This assay also detects GFAP in serum.ConclusionsThis novel assay is a valuable translational tool that may help to provide insights into the mechanistic pathophysiology of TBI and AD.

Dataset Information

Alternative mRNA splicing from the glial fibrillary acidic protein (GFAP) gene generates isoforms with distinct subcellular mRNA localization patterns in astrocytes.

Publications

Alternative mRNA splicing from the glial fibrillary acidic protein (GFAP) gene generates isoforms with distinct subcellular mRNA localization patterns in astrocytes.

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