Generation of dystrophin short product-specific tag-insertion mouse: distinct Dp71 glycoprotein complexes at inhibitory postsynapse and glia limitans

Abstract

Duchenne muscular dystrophy (DMD), the most severe form of dystrophinopathies, is a fatal X-linked recessive neuromuscular disorder characterized by progressive muscle degeneration and various extents of intellectual disabilities. Physiological and pathological roles of the responsible gene, dystrophin, in the brain remain elusive due to the presence of multiple dystrophin products, mainly full-length dystrophin, Dp427, and the short product, Dp71. In this study, we generated a Dp71-speci c hemagglutinin (HA) peptide tag-insertion mice to enable speci c detection of intrinsic Dp71 expression by anti-HA tag antibodies. Immunohistochemical detections in the transgenic mice demonstrated Dp71 expression not only at the blood-brain barrier, where astrocytic endfeet surround the microvessels, but also at the inhibitory postsynapse of hippocampal dentate granule neurons. Interestingly, hippocampal cornu ammonis (CA)1 pyramidal neurons were negative for Dp71 although Dp427 detected by anti-dystrophin antibody was clearly present at the inhibitory postsynapse, suggesting cell-type dependent dystrophin expressions. Precise examination using the primary hippocampal culture validated exclusive localization of Dp71 at the inhibitory postsynaptic compartment but not at the excitatory synapse in neurons. We further performed interactome analysis and found that Dp71 formed distinct molecular complexes, i.e. synapse-associated Dp71 interacted with dystroglycan (Dg) and dystrobrevinb (Dtnb) whereas glia-associated Dp71 did with Dg and dystrobrevina (Dtna). Thus, our data indicates that Dp71 and its binding partners are relevant to the inhibitory postsynaptic function of hippocampal granule neurons and the novel Dp71-transgenic mouse provides a valuable tool to understand precise physiological expressions and functions of Dp71 and its interaction proteins in vivo and in vitro.

Dp71 Point Mutations Induce Protein Aggregation, Loss of Nuclear Lamina Integrity and Impaired Braf35 and Ibraf Function in Neuronal Cells

“…Because the C272Y and E299del mutations are mapped on the β-dystrolgycan binding domain of Dp71 [ 39 , 40 , 41 ], we analyzed the impact of expressing these Dp71 mutants on the subcellular localization of β-DG. The distribution of β-DG was partitioned between the nucleus and the cytoplasm in SH-SY5Y/N1E-115 cells expressing GFP alone or GFP-Dp71; however, a polarized cytoplasmic distribution of β-DG with some aggregates was commonly observed in SH-SY5Y/N1E-115 cells expressing both Dp71 mutants ( Figure 4 ).…”

Using digital pathology to analyze the murine cerebrovasculature

“…DP71 localizes to astrocyte endfeet. 34,35 Because endfeet directly contact blood vessels, DP71 can be used as a protein marker for visualization. 36 Anti-DP71 (Figure 3(e)) showed moderate vessel staining, but also had a moderately high background, including staining of cell bodies.…”