Adult human subventricular, subgranular, and subpial zones contain astrocytes with a specialized intermediate filament cytoskeleton

Abstract

Human glial fibrillary acidic protein-delta (GFAP-delta) is a GFAP protein isoform that is encoded by an alternative splice variant of the GFAP-gene. As a result, GFAP-delta protein differs from the predominant splice form, GFAP-alpha, by its C-terminal protein sequence. In this study, we show that GFAP-delta protein is not expressed by all GFAP-expressing astrocytes but specifically by a subpopulation located in the subpial zone of the cerebral cortex, the subgranular zone of the hippocampus, and, most intensely, by a ribbon of astrocytes following the ependymal layer of the cerebral ventricles. Therefore, at least in the sub ventricular zone (SVZ), GFAP-delta specifically marks the population of astrocytes that contain the neural stem cells in the adult human brain. Interestingly, the SVZ astrocytes actively splice GFAP-delta transcripts, in contrast to astrocytes adjacent to this layer. Furthermore, we show that GFAP-delta protein, unlike GFAP-alpha, is not upregulated in astrogliosis. Our data therefore indicate a different functional role for GFAP-delta in astrocyte physiology. Finally, transfection studies showed that GFAP-delta protein expression has a negative effect on GFAP filament formation, and therefore could be important for modulating intermediate filament cytoskeletal properties, possibly facilitating astrocyte motility. Further studies on GFAP-delta and the cells that express it are important for gaining insights into its function during differentiation, migration and during health and disease.

GFAPδ in radial glia and subventricular zone progenitors in the developing human cortex

“…Sections were washed with phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 1.8 mM KH 2 PO 4 and 4 mM Na 2 HPO 4 , pH 7.4) and incubated for 30 min in 10% normal goat serum (Harlan SeraLab, Loughborough, Leicestershire, UK). After incubation with polyclonal rabbit pan-GFAP, which recognizes all GFAP isoforms (DAKO, Glostrup, Denmark; 1:4000), and GFAPd [bleeding 100501; 1:500 (Roelofs et al, 2005)] primary antibodies in PBS overnight at 4°C, we washed the sections in PBS. We then used the ready-for-use Powervision peroxidase system (Immunologic, Duiven, The Netherlands) and 3,3Ј-diaminobenzidine (DAB; Sigma) as a chromogen to visualize the antibodies.…”

“…To obtain recombinant protein samples for western blotting, expression plasmids pcDNA3-GFAP and pcDNA3-GFAPd (Roelofs et al, 2005) were transfected separately into SH-SY5Y neuroblastoma cells, which do not express GFAP endogenously. These cells were cultured in high-glucose DMEM, supplemented with 10% heat-inactivated foetal calf serum, 100 U/ml penicillin and 100 g/ml streptomycin, at 37°C with 5% CO 2 .…”

“…Subsequently, they were run on a 7.5% SDS-PAGE gel and blotted semi-dry on nitrocellulose. Blots were probed overnight with polyclonal rabbit pan-GFAP (DAKO; 1:20 000), GFAPd [bleeding 100501; 1:500 (Roelofs et al, 2005)] or polyclonal goat C-terminal GFAP (GFAP C-term; Santa Cruz Biotechnology; 1:200) antibodies diluted in Supermix (0.05 M Tris, 0.9% NaCl, 0.25% gelatin and 0.5% Triton X-100, pH 7.4) antibodies. The next day, the blots were washed with TBS-T (TBS; 100 mM Tris-HCl pH 7.4, 150 mM NaCl, with 0.2% tween-20) and incubated with secondary antibody anti-rabbit IRDye800 or anti-goat IRDye800 (1:5000; Rockland Immunochemicals, Gilbertsville, USA) in Supermix for one hour at room temperature.…”

“…These neural stem cells express glial fibrillary acidic protein (GFAP) and are located along the length of the lateral ventricles. In the human brain, intense expression of a splice variant of GFAP, termed GFAPd, was found at this specific location (Roelofs et al, 2005), which suggests that GFAPd-expressing cells might be the neural stem cells of the adult human brain.…”

Neurogenesis in the human hippocampus and its relevance to temporal lobe epilepsies

“…Unfortunately, most established markers for human brain stem and progenitor cells are nonspecific and include GFAP, which is expressed in both astrocytes and in stem cells (Doetsch, 2003), whereas the delta isoform has been postulated to identify adult neural stem cells (Roelofs et al, 2005). The intermediate filament nestin is a cytoskeletal protein that has been originally identified in fetal stem cells (Lendahl et al, 1990), but is also expressed in adult stem cells and reactive astrocytes of the hippocampus (Blumcke et al, 2001;Gu et al, 2002).…”