A murine model of Charcot-Marie-Tooth disease 4F reveals a role for the C-terminus of periaxin in the formation and stabilization of Cajal bands

Abstract

Charcot-Marie-Tooth (CMT) disease comprises up to 80 monogenic inherited neuropathies of the peripheral nervous system (PNS) that collectively result in demyelination and axon degeneration. The majority of CMT disease is primarily either dysmyelinating or demyelinating in which mutations affect the ability of Schwann cells to either assemble or stabilize peripheral nerve myelin. CMT4F is a recessive demyelinating form of the disease caused by mutations in the Periaxin ( PRX) gene . Periaxin (Prx) interacts with Dystrophin Related Protein 2 (Drp2) in an adhesion complex with the laminin receptor Dystroglycan (Dag). In mice the Prx/Drp2/Dag complex assembles adhesive domains at the interface between the abaxonal surface of the myelin sheath and the cytoplasmic surface of the Schwann cell plasma membrane. Assembly of these appositions causes the formation of cytoplasmic channels called Cajal bands beneath the surface of the Schwann cell plasma membrane. Loss of either Periaxin or Drp2 disrupts the appositions and causes CMT in both mouse and man. In a mouse model of CMT4F, complete loss of Periaxin first prevents normal Schwann cell elongation resulting in abnormally short internodal distances which can reduce nerve conduction velocity, and subsequently precipitates demyelination. Distinct functional domains responsible for Periaxin homodimerization and interaction with Drp2 to form the Prx/Drp2/Dag complex have been identified at the N-terminus of Periaxin. However, CMT4F can also be caused by a mutation that results in the truncation of Periaxin at the extreme C-terminus with the loss of 391 amino acids. By modelling this in mice, we show that loss of the C-terminus of Periaxin results in a surprising reduction in Drp2. This would be predicted to cause the observed instability of both appositions and myelin, and contribute significantly to the clinical phenotype in CMT4F.

Direct binding of the flexible C-terminal segment of periaxin to β4 integrin suggests a molecular basis for CMT4F

“…In order to identify putative protein ligands for the C-terminal segment of L-PRX, which is missing in the presence of the CMT4F R1070X mutation (24,28), we carried out a yeast 2-hybrid screen of a rat sciatic nerve cDNA library with the C-terminal segment, missing upon the R1070X mutation, as bait. As a result, we obtained three separate clones containing the β4-FNIII-3 domain (Figure 1A).…”

“…Using the mouse model for CMT4F (24), which carries a truncating mutation mimicking human L-PRX with the R1070X mutation, we carried out proteomics experiments to follow the expression level of possible PRX ligand proteins in this model. PRX was immunoprecipitated from mouse nerves after crosslinking, and the bound proteins were analyzed.…”

“…PRX and β4 integrin in the pull-down fractions were identified by Western blot. PRX antibodies have been described (24). Antibodies against β4 integrin were raised in rabbits using a peptide corresponding to amino acids 1756-1772, to which an N-terminal cysteine was attached for coupling to keyhole limpet hemocyanin (CTEPFLIDGLTLGTQRLE), as described (20).…”

“…Proteomics analyses were carried out exactly as described (24), comparing the PRX-bound proteomes of PRX -/- mice carrying a transgene for either wild-type PRX (PrxTg/PRX -/- ) or PRX with a C-terminal truncation mutation (mouse L-PRX truncated at residue 1016; ΔCPrxTg/PRX -/- ) corresponding to human CMT4F R1070X (28,29). Proteins from mouse nerve lysates were crosslinked followed by immunoprecipitation with a PRX antibody, and then subjected to mass spectrometry (MS) analyses.…”

“…Proteins from mouse nerve lysates were crosslinked followed by immunoprecipitation with a PRX antibody, and then subjected to mass spectrometry (MS) analyses. Normalized abundance of β4 integrin was determined by MS using Progenesis (Nonlinear Dynamics) as described (24).…”