POSTER NO: 453

A Molecular Basis for the Central Nervous System Defects Exhibited in Duchenne Muscular Dystrophy

¹D.J. Stevens, ¹E. Goh, ²P.N. Ray
¹University of Toronto, Toronto, Canada, ²Hospital for Sick Children, Toronto, Canada

Individuals with a disruption in the gene responsible for producing the protein dystrophin are afflicted with Duchenne muscular dystrophy (DMD). This disease is characterised by progressive skeletal muscle degeneration and irregularities of the central nervous system (CNS), including cognitive deficiencies and abnormal retinal electrophysiology. With the goal of determining the molecular basis of these abnormalities we have focused our investigations on the retina, a small-defined portion of the CNS that demonstrates a clear and quantifiable electrophysiology defect in DMD patients.

Immunohistochemical studies have demonstrated that dystrophin and dystrophin associated proteins are localised to subcellular regions of the brain and retina that play a vital role in regulating extracellular ion concentrations. The nature of the CNS deficiencies of DMD patients together with this subcellular localisation suggests that the dystrophin complex is associated with ion channels, and that in the absence of dystrophin these channels are disrupted. As members of the dystrophin complex, the PDZ domain containing syntrophin proteins are ideal candidates for mediating this interaction; PDZ domain containing proteins are often involved in anchoring integral membrane proteins by binding a c-terminal S/T-X-V-COOH consensus motif. In order to identify candidate syntrophin interacting channels we used literature and Swissprot database searches to generate a list of proteins that are present in the retina at the same regions as the dystrophin complex and contain an S/T-X-V-COOH motif. Using this strategy we identified a potassium channel, Kir4.1, that if mislocalised might result in the abnormal retinal electrophysiology observed in DMD patients.

To determine if the function of the Kir4.1 potassium channel is affected by the absence of dystrophin we have used dystrophin null mice (MDX-3CV) as a model of Duchenne muscular dystrophy. We have demonstrated that the loss of dystrophin results in the Kir4.1 potassium channels being present in the retina in a very dispersed manner, in contrast to their normally defined aggregated localisation. To demonstrate that proper localisation is mediated by syntrophin proteins we co-expressed Kir4.1 and alpha1-syntrophin in COS-7 cells and investigated their pattern of expression with immunoflourescence. Expression of Kir4.1 alone resulted in the potassium channel being distributed in a dispersed manner throughout the cell membrane, while co-transfection with alpha1-syntrophin resulted in both proteins concentrating into distinct aggregates. Using site directed mutagenesis we demonstrated that missense mutations within the c-terminus of Kir4.1 were capable of abrogating this effect. Co-immunoprecipitation experiments demonstrate that syntrophin and Kir4.1 physically interact within these aggregates, and that this interaction is abolished by mutations within the c- terminus of Kir4.1.

Our results demonstrate that dystrophin is required for the correct localisation of potassium channels within the retina, suggesting for the first time a molecular basis for the abnormal retinal electrophysiology observed in DMD patients. By extension these studies suggest that the cognitive deficiencies observed in these patients may be a result of an imbalance in ion homeostasis. Our future investigations will be directed towards confirming a direct connection between the disruption of the molecular associations we have demonstrated in vitro with the CNS defects of DMD patients.