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Updated: Mar 18, 2026
Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons
Published on: January 7, 2019
Aberrant trafficking of a Leu89Pro connexin32 mutant associated with X-linked dominant Charcot-Marie-Tooth disease
Yuwei Da1, Wei Wang1, Zhongfeng Liu2
1a Department of Neurology , Xuan Wu Hospital, Capital Medical University , Beijing , China.
Objective:
To determine the functional abnormalities of the Leu89Pro mutation in connexin32 (CX32), which we have previously reported is present within an X-linked dominant Charcot-Marie-Tooth disease family. In this family, male patients were moderately to severely affected.
Methods:
We performed immunofluorescence to investigate whether the Leu89Pro CX32 protein was transported to the cell membrane in HeLa and Schwann cells. First, we constructed the eukaryotic express plasmids expressing CX32 (wild-type or Leu89Pro) and enhanced green fluorescent protein by the gene recombination technology. Then the recombinant plasmids were transiently transfected into communication-incompetent HeLa cells and human Schwann cells by the lipofectamine method. Later, we double-labeled cells for both CX32 and markers of the ER (calnexin) or the Golgi (58-kDa protein) at 24 h or 48 h. The images were collected using a Leica TCS SP5 II confocal microscope.
Results:
The mutant CX32 protein was localized in the endoplasmic reticulum and failed to reach the cell membrane to form gap junctions.
Conclusion:
Our results indicated that the Leu89Pro substitution in the second transmembrane domain of CX32 disrupts the trafficking of the protein, inhibiting the assembly of CX32 gap junctions, which in turn may result in peripheral neuropathy. This functional abnormality may explain the moderate to severe phenotype seen in Leu89Pro patients, and as such represents a promising therapeutic target in the treatment of this subset of CMTX patients.
Insights
The Leu89Pro mutation in connexin32 (CX32) prevents protein transport to the cell membrane, inhibiting gap junction formation. This defect may cause Charcot-Marie-Tooth disease (CMTX) and represents a therapeutic target.
Area of Science:
- Neuroscience
- Cell Biology
- Genetics
Background:
- Connexin32 (CX32) mutations are linked to X-linked dominant Charcot-Marie-Tooth disease (CMTX).
- The Leu89Pro mutation in CX32 has been identified in a CMTX family with affected males.
Purpose of the Study:
- To investigate the functional consequences of the Leu89Pro mutation in CX32.
- To determine if the Leu89Pro mutation affects CX32 protein trafficking and gap junction formation.
Main Methods:
- Constructed eukaryotic expression plasmids for wild-type and Leu89Pro CX32.
- Transfected HeLa and human Schwann cells with CX32 plasmids.
- Used immunofluorescence and confocal microscopy to assess protein localization, including co-labeling for ER and Golgi markers.
Main Results:
- The Leu89Pro CX32 mutant protein was retained in the endoplasmic reticulum.
- The mutant protein failed to traffic to the cell membrane.
- Gap junction assembly was inhibited by the Leu89Pro mutation.
Conclusions:
- The Leu89Pro substitution disrupts CX32 protein trafficking, preventing gap junction formation.
- This disruption may underlie the peripheral neuropathy observed in CMTX patients with this mutation.
- The identified functional abnormality offers a potential therapeutic target for this CMTX subset.
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