Interrogating the role of evolutionarily conserved dysferlin exon 40a in muscle membrane repair, lipidomics and muscular dystrophy

Frances Lemckert, PhD

Children’s Medical Research Institute

Dr. Lemckert is a senior research officer at The Children's Hospital at Westmead & The Children's Medical Research Institute, Kid's Neuroscience Centre.

Research Projects

Objective: 
To dissect the role of exon 40a in membrane repair and muscle disease, and also determine a beneficial threshold of dysferlin protein that prevents development of dystrophy.

Dysferlin plays a role in resealing acute membrane injury in skeletal muscle.  Calcium influx through the membrane breach activates calpain-mediated cleavage of dysferlin in the amino acid residues coded by the alternatively spliced exon 40a, yielding a C-terminal “mini-dysferlinC72”.  We believe mini-dysferlinC72 is the specialised repair module of dysferlin that recruits to sites of membrane injury and carries out a vesicle fusion role for emergency membrane repair.

To address this hypothesis we generated CRISPR/Cas9 gene-edited mice that do not express dysferlin protein isoforms carrying the residues encoded by exon 40a.  We have three mouse lines, each with a different genomic deletion event in and around exon 40a, and each expressing different levels of total dysferlin protein (~90%, 50% and 10% of normal).  In this project we are investigating dysferlin recruitment and repair capacity in skeletal muscle from our exon 40a-KO mouse lines compared to WT mice and BLAJ dysferlin-deficient mice.  We will characterise any muscular dystrophy present in these mice and perform lipidomics and proteomics analysis of 40a-KO muscle to look for perturbation of normal, as dysferlin-deficient mice display an altered lipidome well before muscle disease is detected.

With three mouse lines available to study, we hope to be able to specifically dissect the role of exon 40a in membrane repair and muscle disease, and also determine a beneficial threshold of dysferlin protein that prevents development of dystrophy. These finding may help inform future gene therapy approaches in the dysferlinopathies.