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-specific hemagglutinin (HA) peptide tag-insertion mice to enable specific 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 dystrobrevinβ (Dtnb), whereas glia-associated Dp71 did with Dg and dystrobrevinα (Dtna). Thus, our data indicate 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.
Highlights
Dystrophin is the causative gene for X-linked recessive Duchenne/Becker muscular dystrophy (DMD/BMD) and consists of 79 exons over 2.2Mb of genomic DNA [1,2,3,4,5]
Dp71d and Dp71f proteins ectopically expressed in HEK293T cells were used as the positive controls, and immunoprecipitates by anti-HA beads from the HEK293T cell extracts and mouse cerebrum tissue extracts were blotted by anti-HA, -Pan dystrophin, -Dp71d, or -Dp71f antibodies (Fig. 1e)
Because it is known that Dp427 is expressed at the inhibitory postsynapses within CA1 pyramidal neurons, we examined whether HADp71 is present at the inhibitory postsynapses of the CA1 area and revealed that gephyrin-positive synaptic puncta were negative for HA-Dp71 signal while HA-Dp71 was strongly detected at a perivascular glia limitans (Fig. 4a)
Summary
Dystrophin is the causative gene for X-linked recessive Duchenne/Becker muscular dystrophy (DMD/BMD) and consists of 79 exons over 2.2Mb of genomic DNA [1,2,3,4,5]. Fatal clinical symptom of DMD is progressive muscle degeneration, non-muscular manifestations have been described, including cognitive impairments, epilepsy and retinal electrophysiology [6,7,8,9,10,11,12]. Dp427 and the C-terminal short product, Dp71, which is transcribed from intron and encoded by exons to 79, are expressed before and after birth [14,15,16,17,18,19,20,21,22] and its defect is suspected to associate with cognitive impairments in DMD [6, 9]. Multiple Dp71 splicing isoforms, such as Dp71d, Dp71f and Dp40 shortest isoform, are potentially expressed from the intron 62 promoter [9, 28, 29]
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