Phenotypes of X-linked Charcot-Marie-Tooth disease and altered trafficking of mutant Connexin 32 (GJB1)

Abstract

To clarify the pathomechanism in three patients with X-linked Charcot-Marie-Tooth disease (CMTX) and unique clinical features, we studied three connexin (Cx) 32 (GJB1) mutants with respect to cellular localization in cultured cells. Wild-type Cx32 and three Cx32 mutants (Val63Ile and Glu186Lys, obtained from CMTX patients with hearing impairment; and Arg22Gln, obtained from a CMTX patient with a fair number of onion-bulb formations) were transfected to rat pheochromocytoma cells (PC12). We investigated the expression of Cx32 protein in each clone by immunoblotting and immunohistochemical staining. While Cx32 protein with the Arg22Gln mutation was detectable immunohistochemically only in the cytoplasm, Cx32 protein with the Val63Ile or Glu186Lys mutation was detected in both the plasma membrane and the cytoplasm. Cx32 protein with the wild-type sequence was detected mostly in the plasma membrane, with plaques indicating the existence of active gap junction formation. These three Cx32 mutations associated with CMTX patients with unique clinical and pathological findings caused altered trafficking of the Cx32 protein. These altered expressions indicated loss of active gap junction formation with different expression abnormalities in these CMTX patients.

Lysosomal and Proteasomal Degradation Play Distinct Roles in the Life Cycle of Cx43 in Gap Junctional Intercellular Communication-deficient and -competent Breast Tumor Cells

“…While considerable information exists on the degradation of connexins in GJIC-competent cell models, where connexins are efficiently assembled into gap junctions, less is known about the degradation of connexins in GJIC-deficient cells, where wild-type or mutant connexins aberrantly accumulate within cells (23,24,29,30). An abnormal GJIC phenotype is predominantly observed in malignant cells that may continue to express connexins, yet little is known about the mechanisms involved in aberrant connexin processing and assembly in tumor cells (23).…”

“…In addition, we previously reported that misfolded DsRed-tagged Cx43 also accumulated within lysosomes in HBL-100 cells, whereas wildtype Cx43 was readily assembled into gap junctions (24). Likewise, Cx32 mutants linked to Charcot-Marie-Tooth disease displayed altered trafficking propensities resulting in a variety of phenotypes where the mutants have an intracellular organelle localization pattern (29,30). These latter cell models suggest that connexins with trafficking defects are unlikely to be properly assembled into gap junctions, reflecting a quality control mechanism that exists to degrade misfolded or mutant connexins.…”

Properties of human connexin 31, which is implicated in hereditary dermatological disease and deafness

“…Investigations from a number of laboratories suggest that most CMTXassociated mutations lead to a loss of Cx32 function (13). Data suggest this loss of function arises either through defects in trafficking of the mutant protein (14)(15)(16)(17)(18) or through alteration of properties of the junctions it forms (19)(20)(21)(22)(23)(24)(25). Similarly, studies of human Cx31 (hCx31) suggest that mutations in this connexin may also lead to alterations in trafficking or channel function (26)(27)(28), and some mutations increase cell death when expressed in HeLa, NIH 3T3, or NEB1 cells (26,29).…”

The Role of Gap Junctions in Charcot-Marie-Tooth Disease

“…When expressed in mammalian cells with more stringent protein trafficking requirements, Cx32 mutants are often retained intracellularly, even if they form rare GJ-like plaques (Omori et al, 1996;Yoshimura et al, 1998;Yum et al, 2002). They are localized predominately in the ER or Golgi (Deschênes et al, 1997;Oh et al, 1997;Martin et al, 2000;Matsuyama et al, 2001;Kleopa et al, 2002Yum et al, 2002) and are degraded via endosomal and proteasomal pathways (VanSlyke et al, 2000;Kleopa et al, 2002). Several mutants, the majority of which occur in the C-terminal domain, were mainly localized on the cell membrane and show no significant difference from WT protein , but they may be less stable or have abnormal biophysical properties (Rabadan-Diehl et al, 1994;Castro et al, 1999;Abrams et al, 2000).…”