Immunofluorescence mapping of dystrophin in the rat brain: astrocytes contain the splice variant Dp71f, but this is confined to subpopulations

Abstract

Dystrophins are membrane-associated actin-binding proteins, recognized at first in muscular dystrophies. In the brain the full-length Dp427 has been detected, as well as Dp140 and Dp71 of the shorter variants. Dp71 seems to be their major representative in the brain, and it occurs as splice variants, Dp71f and Dp71d. Dystrophins have been demonstrated mainly in neurons. In tissue cultures, the glial data, mainly in situ, are still insufficient. The present mapping study reveals the astroglial localization of the splice variant Dp71f, using a monoclonal antibody (5F3, developed by D. Mornet) specific for its additional 31 last amino acids. In parallel, another monoclonal antibody was used (Dys2, Novocastra) that detects the Dp71d, Dp427, as well as Dp140 and other short variants. Rats were overdosed with ether and perfused transcardially with 4% phosphate-buffered paraformaldehyde solution. Floating Vibratome sections were processed for immunohistochemical labeling with fluorescent secondary antibodies. In some animals the reactive glia were investigated following stab wounds in ketamine-xylazine anesthesia. Only the 5F3 antibody labeled astrocytes, however, not in general but in special localizations, mainly along the glia limitans of the pial surface, below the ependyma and in the reactive glia. Perivascular astrocytes were consistently labeled only where the vessels entered the brain, and in some circumventricular organs. The 5F3 antibody also labeled the ependyma and the residual subventricular zone. In contrast to the astrocytes, neurons were labeled throughout the brain. Dys2 antibody (to Dp71d and longer isoforms) labeled neurons in a distribution similar to that of 5F3, but rarely labeled astroglia and only in perivascular rings. Dp71f positivity seems to occur in those astrocyte populations that proved to be immunopositive to glial fibrillary acidic protein (GFAP) and produced laminin in former studies.

Identification of Dp71 Isoforms Expressed in PC12 Cells: Subcellular Localization and Colocalization with β-Dystroglycan and α1-Syntrophin

“…Differential distributions of Dp71d and Dp71f isoforms have been reported in different cell types and tissues (Aleman et al 2001;Marquez et al 2003;Szabó et al 2004;Fuentes-Mera et al 2006;Daoud et al 2010). In this work, we found that Dp71a, Dp71ab, and Dp71e isoforms are mainly localized in the cell periphery and cytoplasm of undifferentiated PC12 Tet ON cells (Fig.…”

“…The three Dp71 isoform groups have unique C-terminal ends (Fig. 1), and to date, reported data have suggested that the C-terminus of Dp71 is involved in its specific functions (Aleman et al 2001;Szabó et al 2004;Aragon et al 2011). Dp71 has been reported to have roles in cell division and membrane stabilization.…”

Dystrophin Dp71: The Smallest but Multifunctional Product of the Duchenne Muscular Dystrophy Gene

“…Dp71 mRNA and protein have been shown to be expressed in different brain structures and cell types, including perivascular astrocytes [46,91], MGC in retina [92], neurons in hippocampus and olfactory bulb [34], cultured hippocampal neurons and forebrain astrocytes [55], and post-synaptic densities in vivo [41]. Interestingly, an early study showed that activation of glutamate receptors by kainate alters mRNA expression of dystrophin and Dp71 in rat hippocampus, suggesting the involvement of the DMD gene in neuronal plasticity [93].…”

Expression of α-dystrobrevin in blood-tissue barriers: sub-cellular localisation and molecular characterisation in normal and dystrophic mice

“…This abnormality has been attributed to a dramatic reduction of aquaporin-4 in astroglial end-feet. DAPC serves as an anchoring site for aquaporin and other membrane channel proteins (Amiry- Moghaddam et al 2004) and astrocytes are rich in DAPs (Khurana et al 1992;Jancsik and Hajós 1999;Szabó et al 2004;Haenggi et al 2004). Thus, the loss of aquaporin is probably the result of the lack of its anchoring site in the glial end-feet membrane (Vajda et al 2002).…”