Pikachurin interaction with dystroglycan is diminished by defective O-mannosyl glycosylation in congenital muscular dystrophy models and rescued by LARGE overexpression

Abstract

Congenital muscular dystrophies (CMD) such as muscle-eye-brain disease caused by defective glycosylation of α-dystroglycan (α-DG) exhibit defective photoreceptor synaptic function. Mouse knockouts of dystroglycan and its extracellular matrix binding partner pikachurin recapitulate this phenotype. In this study, pikachurin-α-dystroglycan interactions in several mouse models of CMD were examined by pikachurin overlay experiments. The results show that hypoglycosylation of α-dystroglycan resulted in markedly reduced pikachurin-α-dystroglycan interactions. Expression of pikachurin is abolished at the outer plexiform layer of two mouse models, protein O-mannose N-acetylglucosaminyl transferase 1 (POMGnT1) knockout and Large(myd) mice. Overexpressing LARGE restored this interaction in POMGnT1 knockout cells. These results indicate that pikachurin interactions with α-dystroglycan and its localization at the photoreceptor ribbon synapse require normal glycosylation of α-dystroglycan.

Figure 1

Hypoglycosylation of α-DG led to dramatically reduced laminin binding. Glycoproteins were isolated from the whole brains and cultured neural stem cells by WGA-agarose, separated on SDS-PAGE, and transferred onto PVDF membranes. Pikachurin overlay and Western blot then were carried out. (A) Recombinant pikachurin (detected with anti-c-Myc) was detected in conditioned medium from HEK293 cells transfected with pikachurin expression plasmid. (B) Whole brain lysates. (C) Neural stem cells. Pikachurin binding was readily detected in the wildtype but dramatically reduced or abolished in Dag1^{f/f;Nestin-Cre(+)}, POMGnT1 knockout, and Large^myd mice and in POMT2-deficient neural stem cells. Abbreviations: β-DG, β-dystroglycan; KO, knockout.

Figure 2

LARGE overexpression enhances pikachurin binding in POMGnT1 knockout cells. Wildtype and POMGnT1-deficient neural stem cells were infected with Ad-LARGE. Pikachurin overlay and IIH6C4 immunoblot analysis were then carried out on glycoproteins isolated from cell lysates. Pikachurin binding was dramatically increased by LARGE overexpression in both wildtype and POMGnT1-deficient neural stem cells.

Figure 3

Diminished pikachurin expression in the outer plexiform layer of the mutant mouse retinas. Retinal sections were immunofluorescence stained with antibodies against Bassoon (A, C, E, and G) and pikachurin (B, D, F, and H). (A and B) Wildtype retina. Bassoon and pikachurin immunofluorescence were observed at the ribbon synapses in the outer plexiform layer. (C and D) POMGnT1 knockout retina. While ribbon synapses were immunoreactive to anti-Bassoon, immunoreactivity to anti-pikachurin was lost. (E and F) Large^myd retina. Pikachurin immunoreactivity at the ribbon synapse was lost. (G and H) Dag1^{f/f;Nestin-Cre(+)} retina. While no noticeable changes were observed for the density of Bassoon immunoreactive ribbon synapses, the density of pikachurin-positive puncta was reduced. Each pikachurin-positive puncta showed similar fluorescence intensity to the wildtype. Scale bar in H: 10 μm.

Figure 4

Ribbon synapses devoid of pikachurin were present in Dag1^{f/f;Nestin-Cre(+)} retinas. Retinal sections were immunostained with antibodies against Bassoon (red fluorescence) and pikachurin (green fluorescence). (A) Wildtype retina. Virtually all Basson-positive ribbon synapses were accompanied by immunoreactivity to anti-pikachurin. (B) Dag1^{f/f;Nestin-Cre(+)} retina. Basson-positive ribbon synapses devoid of pikachurin immunoreactivity were frequently observed (arrowheads). Scale bar: 10 μm.