Golgi-retained Cx32 mutants interfere with gene addition therapy for CMT1X

Abstract

Numerous GJB1 gene mutations cause the X-linked form of Charcot-Marie-Tooth disease (CMT1X). GJB1 encodes connexin32 (Cx32), which forms trans-myelin gap junctions in Schwann cells. Most GJB1 mutations result in loss-of-function mechanisms, supporting the concept of gene replacement therapy. However, interactions between delivered wild type and endogenously expressed mutant Cx32 may potentially occur in the setting of gene replacement therapy. In order to screen for possible interactions of several representative CMT1X mutants with wild type Cx32 that may interfere with the functional gap junction formation, we established an in vitro screening method co-expressing in HeLa cells wild type Cx32 and one of eight different Cx32 mutants including A39P, A39V, T55I, R75W, M93V, L143P, N175D and R183S. Some of the Golgi-retained mutants hindered gap junction plaque assembly by Cx32 on the cell membrane, while co-immunoprecipitation analysis revealed a partial interaction of wild type protein with Golgi-retained mutants. Dye transfer studies confirmed that Golgi-retained R75W, M93V and N175D but not endoplasmic reticulum-retained T55I had a negative effect on wild type Cx32 function. Finally, in vivo intraneural delivery of the gene encoding the wild type Cx32 in mice bearing either the T55I or R75W mutation on Cx32 knockout background showed that virally delivered protein was correctly localized in mice expressing the endoplasmic reticulum-retained T55I whereas it did not traffic normally in mice expressing the Golgi-retained R75W. Thus, certain Golgi-retained Cx32 mutants may interfere with exogenously delivered Cx32. Screening for mutant-wild type Cx32 interactions should be considered prior to planning gene addition therapy for CMT1X.

Charcot-Marie-Tooth: From Molecules to Therapy

“…In those particular mutations, the virally delivered gene therapy does not traffic normally to deliver therapy optimally. Thus, screening for mutant-wild type interaction will be an important caveat to gene replacement therapy strategies [84]. In the proper context, gene replacement therapy might lead to a viable treatment option.…”

Novel GJA1/Cx43 Variant Associated With Oculo-Dento-Digital Dysplasia Syndrome: Clinical Phenotype and Cellular Mechanisms

“…Cx26 variants associated with hearing loss showed intracellular retention in the ER or in the early endosomes, and their loss of function appears to be due to premature degradation (Xiao et al, 2011;Beach et al, 2020). Several Cx32 mutants causing neuropathy showed complete absence (Sargiannidou et al, 2015) or abnormal intracellular retention in the ER and/or Golgi (Kleopa et al, 2002(Kleopa et al, , 2006, often with dominant effects on co-expressed WT Cx32 with demonstrated direct protein-protein interaction, likely during hexamer formation in the Golgi (Kyriakoudi et al, 2017). Other Cx32 mutants reached the cell membrane but showed shifted voltage gating and abnormally increased opening of the channels (Abrams et al, 2002).…”

The role of oligodendrocyte gap junctions in neuroinflammation

“…Virally delivered wild-type (WT) Cx32 could be detected in the non-compact myelin of T55I KO mice, but only rarely in N175D KO or R75W KO mice, suggesting dominant-negative effects of the R75W and N175D mutants but not of the T55I mutant on co-expressed WT Cx32 [77]. Further, in vitro Cx32 WT-mutant co-expression studies demonstrated directly that certain Golgi-retained CMT1X mutants may interfere with expression and intracellular trafficking of WT Cx32 [78], and could thus potentially interfere with gene addition therapy for CMT1X.…”