Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (<i>GFAP</i>) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

<div><p>The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. <i>Gfap</i> 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 <i>Gfapδ</i> mRNA and Gfapδ protein. RT-qPCR analysis showed that <i>Gfapδ</i> mRNA and <i>Gfapα</i> mRNA expression is coordinately increased in the post-natal period. Immunohistochemical staining of developing mouse brain samples showed that Gfapδ 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 Gfapδ into the Gfap intermediate filament network and overlap in Gfapδ and Gfapα subcellular localization. Subcellular mRNA localization studies identified different localization patterns of <i>Gfapδ</i> and <i>Gfapα</i> mRNA in mouse primary astrocytes. A larger fraction of <i>Gfapα</i> mRNA showed mRNA localization to astrocyte protrusions compared to <i>Gfapδ</i> mRNA. The differential mRNA localization patterns were dependent on the different 3′-exon sequences included in <i>Gfapδ</i> and <i>Gfapα</i> mRNA. The presented results show that alternative <i>Gfap</i> 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.</p></div

<i>Gfapα</i> and <i>Gfapδ</i> have isoform specific subcellular mRNA localization.

<p>(A) RT-qPCR analysis of the relative localization of <i>Gfapα, Gfapκ</i> and <i>Gfapδ</i> mRNA from the astrocyte protrusion fraction (PF) and the cell body fraction (CF). The localization ratio is visualized relative to the localization ratio for <i>Arpc3</i> mRNA given the value 1. <i>actb</i> mRNA localization was also examined. Results are presented as mean +/− SD and represent three independent RT-qPCR experiments from material representing 2 independent Boyden chamber inserts. P-values were calculated by a Student’s unpaired two tailed t-test. (B) Western blotting analysis of Gfapδ, pan-Gfap, Actb and histone H3 protein from astrocyte protrusion fraction (PF) and cell body fraction (CF). (*) indicates an uncharacterized band of approximately 60 kD enriched in protrusions. (C) <i>Gfap</i> minigene based mRNA localization analyses. Mouse primary astrocytes and NIH3T3 cells were transfected with a <i>Gfap</i> minigene inserted in the pTAG4 vector. Following a Boyden chamber analysis purified RNA samples from protrusions and cell bodies were analyzed by RT-qPCR with primer combinations specific for <i>Gfapα</i> and <i>Gfapδ</i> mRNA expressed from the pTAG4 minigene and mRNA localization ratios determined by division. Results are presented as mean +/− SD and represent three independent RT-qPCR experiments from 2 independent Boyden chamber inserts. P-values were calculated by a Student’s unpaired two tailed t-test. (D–E) FISH analyses showing representative examples of <i>Gfapα</i> and <i>Gfapδ</i> mRNA localization in mouse primary astrocytes. The cells were probed with a mixture of 8 <i>Gfapδ</i> mRNA Cy3.5 labeled probes and 8 Gfap<i>α</i> mRNA Cy3 labeled probes (left panels). FISH images were analyzed for spot detection (central panels) as described in the materials and methods section. The nuclei were counterstained with DAPI. A merged image is shown with <i>Gfapα</i> labeled red, <i>Gfapδ</i> labeled green and DAPI labeled blue. Arrowheads show astrocyte protrusions and for all panels scale bars represent 20 µm.</p

Immunofluorescence analysis of Gfapδ.

<p>(A) Co-localization analysis of Gfapα and Gfapδ. Mouse primary astrocytes were stained with primary Gfapδ antibody and Gfapα antibody. The nuclei were counterstained with DAPI. Merged image is included with Gfapα labeled green, Gfapδ labeled red and DAPI labeled blue. (B) Gfapδ and Vimentin have partial co-localization. Mouse primary astrocytes were stained with primary Gfapδ antibody and Vimentin antibody. The nuclei were counterstained with DAPI. Merged image is included with Vimentin labeled red, Gfapδ labeled green and DAPI labeled blue. Scale bar 10 µm.</p

mRNA for the alternative spliced <i>Gfap</i> isoform <i>Gfapδ</i> is expressed in the postnatal mouse brain.

<p>(A) Schematic drawing of the mouse <i>Gfap</i> locus. pA indicate positions of poly-adenylation signals in exons E7a and E9. (*) indicate positions of translational stop codons in exons E7a and E9. Coding regions are shown in grey. (B–C) Expression analysis of <i>Gfapδ</i> (B) and <i>Gfapα</i> (C) mRNA in postnatal mouse brain samples. RNA was isolated from cortices at the indicated time points. cDNA was used in RT-qPCR experiments with primer combinations specific for <i>Gfapδ</i> and <i>Gfapα</i> cDNA. Results are normalized to the <i>b2m</i> mRNA expression level which is determined as reference gene by GeNorm analysis. Results are presented as mean +/− SD. P-values for the expression levels compared to the P12 expression level were calculated by a Student’s unpaired two tailed t-test. The results represent three independent RT-qPCR experiments from one brain sample cohort. Another independent brain sample cohort gave similar results. (D) The post-natal expression ratio between <i>Gfapα</i> and <i>Gfapδ</i> mRNA is constant. The expression ratio at different time points using the values from panels B and C was calculated and presented as mean +/− SD.</p

Immunohistochemical staining of Gfapδ in the mouse brain.

<p>(A–B) Immunohistochemical staining of mouse P70 brain for Gfapδ (1), pan-Gfap (2), Vimentin (3) and Nestin (4). The sections were counterstained with haematoxylin solution. Upper section panels (A) illustrate lateral ventricle and lower section panels (B) the roof of 3rd ventricle. Ep, Ependymal lining; V, 3rd ventricle; CC, corpus callosum. (C–D) Immunohistochemical staining of mouse P10 brain. Upper section panels illustrate hippocampus (C) and lower section panels brain stem (D). Experimental settings were similar to panels in A–B. Ep, Ependymal lining; V, 4rd ventricle; H, Dentate Hilus; DG, Dentate granule cells; M; Dentate molecular layer; LV, lateral ventricle; DC, Diencephalon. (E) Immunohistochemical staining of mouse P3 brain. Panels illustrate brain stem. Experimental settings were similar to panels in A–B. Ep, Ependymal lining; V, 4rd ventricle. In all Gfapδ panels arrowheads indicate representative Gfapδ antibody stained cells and the corresponding regions also indicated by arrowheads in the pan-Gfap, Vimentin and Nestin stained sections. For all panels scale bar 20 µm.</p