Loss-of-function GJA12/Connexin47 mutations cause Pelizaeus–Merzbacher-like disease
Introduction
Gap junction communication (GJC) facilitates the diffusion of ions and small molecules (less than 1000 Da) between two apposed cells, thereby enabling electrical coupling, metabolic cooperation, and spatial buffering of ions (Bruzzone et al., 1996). The molecular basis of GJC is the gap junction plaque, an aggregation of tens to thousands of individual channels. Two hemichannels (or connexons) on apposing cell membranes form the channel. Hemichannels are composed of six connexins – a family of highly conserved integral membrane proteins that are usually named according to their predicted molecular mass (Willecke et al., 2002). Individual hemichannels can be composed of one (homomeric) or more than one (heteromeric) type of connexin. Similarly, channels can be composed of hemichannels containing the same (homotypic) or different (heterotypic) connexins (Kumar and Gilula, 1996).
Anatomical and functional studies of the rodent CNS have demonstrated that astrocytes and oligodendrocytes are coupled by gap junctions (Massa and Mugnaini, 1982, Ransom and Kettenmann, 1990, Robinson et al., 1993, Rash et al., 1997), and that each cell type expresses different connexins (Dermietzel et al., 1989, Yamamoto et al., 1990, Scherer et al., 1995, Dermietzel et al., 1997, Ochalski et al., 1997, Nagy et al., 1999, Altevogt et al., 2002, Menichella et al., 2003, Odermatt et al., 2003, Kleopa et al., 2004). Astrocyte/astrocyte (A/A) coupling is likely mediated by Cx43/Cx43 and Cx30/Cx30 homotypic channels, and astrocyte/oligodendrocyte (A/O) coupling is most likely mediated by Cx43/Cx47 and Cx30/Cx32 heterotypic channels, with an uncertain contribution of Cx26/Cx32 channels (Rash et al., 2001, Nagy et al., 2003, Altevogt and Paul, 2004, Li et al., 2004). Cx32/Cx32 homotypic channels appear to form gap junctions between layers of CNS myelin sheaths (Kamasawa et al., 2005), as previously described in PNS myelin sheaths (Bergoffen et al., 1993, Scherer et al., 1995, Balice-Gordon et al., 1998, Meier et al., 2004). A/O heterotypic channels appear to be spatially restricted, with Cx43/Cx47 outnumbering Cx30/Cx32 channels at oligodendrocyte somata (Kleopa et al., 2004, Kamasawa et al., 2005). Oligodendrocytes also express Cx29, which does not appear to form gap junction plaques (Altevogt et al., 2002, Kleopa et al., 2004). Mutations in GJB1, the gene encoding Cx32, cause the X-linked form of Charcot–Marie–Tooth disease (CMT1X), an inherited demyelinating neuropathy (Bergoffen et al., 1993, Scherer and Kleopa, 2005). Clinical manifestations of CNS dysfunction in CMT1X are rare, associated with a few of the more than 300 different GJB1 mutations (Taylor et al., 2003), indicating that Cx30/Cx32 A/O coupling is not critical in humans.
Although CNS myelination in Gja12/cx47-null mice is minimally affected (Menichella et al., 2003, Odermatt et al., 2003), recessive GJA12/Cx47 mutations cause a devastating dysmyelinating disease in humans, called Pelizaeus–Merzbacher-like disease (PMLD; Uhlenberg et al., 2004, Bugiani et al., 2006). PMD itself is an X-linked dysmyelinating disorder caused by mutations in Proteolipid Protein 1 (PLP1), which encodes the main intrinsic membrane protein of CNS myelin (Garbern et al., 1999). PMLD patients are clinically similar to those with PMD, with nystagmus, spasticity, and ataxia, as well as widespread changes in CNS white matter by MRI imaging (Hudson et al., 2004), but do not have PLP1 mutations. The clinical phenotype and MRI findings in patients with recessive GJA12/Cx47 mutations (Uhlenberg et al., 2004, Bugiani et al., 2006) provide provocative evidence that Cx47-mediated GJC is essential for the proper functioning of oligodendrocytes. We show here that Cx47 is expressed by oligodendrocytes in the primate brain and that recessive missense mutations in GJA12/Cx47 cause loss-of-function. Thus, the Cx47 mutants associated with PMLD likely disrupt the A/O coupling that is mediated by Cx43/Cx47 heterotypic channels.
Section snippets
The sequence of human Cx47
In their report describing the GJA12/Cx47 mutations that cause PMLD, Uhlenberg et al. (2004) used the amino acid sequence derived from a human Cx47 cDNA clone (NM_020435) that included part of the 5’untranslated region (UTR). Because this 5′UTR contains a potential additional start codon (ATG) that is 9 nucleotides upstream of the ATG conventionally considered to be the connexin start codon, Uhlenberg et al. (2004) added 3 amino acids in their description of the mutations. This alternative ATG
PMLD mutations appear to cause simple loss-of-function
The PMLD patients described to date (Uhlenberg et al., 2004, Bugiani et al., 2006) have similar phenotypes – nystagmus noted in early infancy, and impaired motor development noted by 15 months. Different genotypes – homozygous P128frameshift, M283T, G233S, or L278frameshift mutations, compound heterozygotes for P87S/P327frameshift or Y269D/R237stop mutations – cause the same phenotype, including alleles that would be predicted to disrupt the protein (P128frameshift, R237stop, L278frameshift,
Mutant human Cx47 expression constructs
Using primers developed from a human Cx47 DNA sequence (GenBank accession number AF014643), we amplified the putative open reading frame of Cx47 by RT–PCR (SuperScript II, Invitrogen, Carlsbad, CA) from polyA RNA isolated from human brain, corpus callosum, or spinal cord (Clontech, Mountain View, CA), adding EcoRV and BamHI restriction sites at the 5′ and 3′ ends, respectively. The 5′ primer eliminated an unlikely alternative AUG that has been electronically translated in some sequences as the
Acknowledgments
We thank Jonathan Lee, Julia Beamesderfer, and Kate Hawk for technical assistance, Dr. Mike Koval for advice, Drs. Klaus Willecke and Bruce Nicholson for the HeLa cells, Dr. David Paul for the Cx47 antibody, Dr. Doug Rosene for the rhesus monkey tissue, the Neuropathology Core of the University of Pennsylvania Alzheimer Disease Core Center for human brain tissue, Dr. Jim Garbern for the ASPA antibody, Dr. Virginia Lee for the GFAP antibody, and Dr. Alex Gow for the CHOP antibody. Postmortem
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