Limb Girdle Muscular Dystrophies type 2I (LGMD2I), a recessive autosomal muscular dystrophy, is caused by mutations in the Fukutin Related Protein (FKRP) gene. It has been proposed that FKRP, a ribitol-5-phosphate transferase, is a participant in α-dystroglycan (αDG) glycosylation, which is important to ensure the cell/matrix anchor of muscle fibers. A LGMD2I knock-in mouse model was generated to express the most frequent mutation (L276I) encountered in patients. The expression of FKRP was not altered neither at transcriptional nor at translational levels, but its function was impacted since abnormal glycosylation of αDG was observed. Skeletal muscles were functionally impaired from 2 months of age and a moderate dystrophic pattern was evident starting from 6 months of age. Gene transfer with a rAAV2/9 vector expressing Fkrp restored biochemical defects, corrected the histological abnormalities and improved the resistance to eccentric stress in the mouse model. However, injection of high doses of the vector induced a decrease of αDG glycosylation and laminin binding, even in WT animals. Finally, intravenous injection of the rAAV-Fkrp vector into a dystroglycanopathy mouse model due to Fukutin (Fktn) knock-out indicated a dose-dependent toxicity. These data suggest requirement for a control of FKRP expression in muscles.

To identify new molecular chaperones of cannabinoid type-1 receptors (CB1R), we used a proteomic approach in HEK293T cells, transfected with two spicing variants of this receptor, CB1R and CB1AR. These two isoforms differ only in the composition of the N-terminus domain. Surprisingly, the only protein found to interact differently with CB1R and CB1AR was transactive response DNA-Binding Protein 43 (TDP-43), which is known to be involved in the pathology of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The specific interactions between CB1R and TDP-43 were also confirmed by co-immunoprecipitation not only in transfected HEK293 cells, as well at the endogenous level in rat crude spinal cord. TDP-43 overexpression decreased CB1R plasma membrane levels by stimulating receptor internalization, but it did not change the total receptor levels. Further, TDP-43 enhanced CB1R ubiquitination to similar levels, as induced by the full CB1R agonist, CP 55,940. Also, overexpression of TDP-43 resulted in reduced effects of CB1R stimulation on cAMP, P-ERK1/2, and P-CREB responses. Present results revealed TDP-43 as an unexpected molecular chaperone of CB1R and these findings may have significance in the regulation of CB1R intracellular trafficking and possibly in the treatment of ALS and FTLD.