Charles K. Abrams

Two Distinct Heterotypic Channels Mediate Gap Junction Coupling between Astrocyte and Oligodendrocyte Connexins

Genetic diseases demonstrate that the normal function of CNS myelin depends on connexin32 (Cx32) and Cx47, gap junction (GJ) proteins expressed by oligodendrocytes. GJs couple oligodendrocytes and astrocytes (O/A channels) as well as astrocytes themselves (A/A channels). Because astrocytes express different connexins (Cx30 and Cx43), O/A channels must be heterotypic, whereas A/A channels may be homotypic or heterotypic. Using electrophysiological and immunocytochemical approaches, we found that Cx47/Cx43 and Cx32/Cx30 efficiently formed functional channels, but other potential heterotypic O/A and A/A pairs did not. These results suggest that Cx30/Cx30 and Cx43/Cx43 channels mediate A/A coupling, and Cx47/Cx43 and Cx32/Cx30 channels mediate O/A coupling. Furthermore, Cx47/Cx43 and Cx32/Cx30 channels have distinct macroscopic and single-channel properties and different dye permeabilities. Finally, Cx47 mutants that cause Pelizaeus–Merzbacher-like disease do not efficiently form functional channels with Cx43, indicating that disrupted Cx47/Cx43 channels cause this disease.

Gap Junctions Couple Astrocytes and Oligodendrocytes

In vertebrates, a family of related proteins called connexins form gap junctions (GJs), which are intercellular channels. In the central nervous system (CNS), GJs couple oligodendrocytes and astrocytes (O/A junctions) and adjacent astrocytes (A/A junctions), but not adjacent oligodendrocytes, forming a “glial syncytium.” Oligodendrocytes and astrocytes each express different connexins. Mutations of these connexin genes demonstrate that the proper functioning of myelin and oligodendrocytes requires the expression of these connexins. The physiological function of O/A and A/A junctions, however, remains to be illuminated.

Hereditary spastic paraplegia is a novel phenotype for GJA12/GJC2 mutations

Recessive mutations in GJA12/GJC2, the gene that encodes the gap junction protein connexin47 (Cx47), cause Pelizaeus-Merzbacher-like disease (PMLD), an early onset dysmyelinating disorder of the CNS, characterized by nystagmus, psychomotor delay, progressive spasticity and cerebellar signs. Here we describe three patients from one family with a novel recessively inherited mutation, 99C>G (predicted to cause an Ile>Met amino acid substitution; I33M) that causes a milder phenotype. All three had a late-onset, slowly progressive, complicated spastic paraplegia, with normal or near-normal psychomotor development, preserved walking capability through adulthood, and no nystagmus. MRI and MR spectroscopy imaging were consistent with a hypomyelinating leukoencephalopathy. The mutant protein forms gap junction plaques at cell borders similar to wild-type (WT) Cx47 in transfected cells, but fails to form functional homotypic channels in scrape-loading and dual whole-cell patch clamp assays. I33M forms overlapping gap junction plaques and functional channels with Cx43, however, I33M/Cx43 channels open only when a large voltage difference is applied to paired cells. These channels probably do not function under physiological conditions, suggesting that Cx47/Cx43 channels between astrocytes and oligodendrocytes are disrupted, similar to the loss-of-function endoplasmic reticulum-retained Cx47 mutants that cause PMLD. Thus, GJA12/GJC2 mutations can result in a milder phenotype than previously appreciated, but whether I33M retains a function of Cx47 not directly related to forming functional gap junction channels is not known.

Gap junctions in inherited human disorders of the central nervous system.

CNS glia and neurons express connexins, the proteins that form gap junctions in vertebrates. We review the connexins expressed by oligodendrocytes and astrocytes, and discuss their proposed physiologic roles. Of the 21 members of the human connexin family, mutations in three are associated with significant central nervous system manifestations. For each, we review the phenotype and discuss possible mechanisms of disease. Mutations in GJB1, the gene for connexin 32 (Cx32) cause the second most common form of Charcot-Marie-Tooth disease (CMT1X). Though the only consistent phenotype in CMT1X patients is a peripheral demyelinating neuropathy, CNS signs and symptoms have been found in some patients. Recessive mutations in GJC2, the gene for Cx47, are one cause of Pelizaeus-Merzbacher-like disease (PMLD), which is characterized by nystagmus within the first 6 months of life, cerebellar ataxia by 4 years, and spasticity by 6 years of age. MRI imaging shows abnormal myelination. A different recessive GJC2 mutation causes a form of hereditary spastic paraparesis, which is a milder phenotype than PMLD. Dominant mutations in GJA1, the gene for Cx43, cause oculodentodigital dysplasia (ODDD), a pleitropic disorder characterized by oculo-facial abnormalities including micropthalmia, microcornia and hypoplastic nares, syndactyly of the fourth to fifth fingers and dental abnormalities. Neurologic manifestations, including spasticity and gait difficulties, are often but not universally seen. Recessive GJA1 mutations cause Hallermann-Streiff syndrome, a disorder showing substantial overlap with ODDD. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and functions.

Voltage opens unopposed gap junction hemichannels formed by a connexin 32 mutant associated with X-linked Charcot-Marie-Tooth disease

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is an inherited peripheral neuropathy that arises in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32), which is expressed by Schwann cells. We recently showed that Cx32 containing the CMTX-associated mutation, Ser-85-Cys (S85C), forms functional cell–cell channels in paired Xenopus oocytes. Here, we describe that this mutant connexin also shows increased opening of hemichannels in nonjunctional surface membrane. Open hemichannels may damage the cells through loss of ionic gradients and small metabolites and increased influx of Ca2+, and provide a mechanism by which this and other mutant forms of Cx32 may damage cells in which they are expressed. Evidence for open hemichannels includes: (i) oocytes expressing the Cx32(S85C) mutant show greatly increased conductance at inside positive potentials, significantly larger than in oocytes expressing wild-type Cx32 (Cx32WT); and (ii) the induced currents are similar to those previously described for several other connexin hemichannels, and exhibit slowly developing increases with increasing levels of positivity and reversible reduction when intracellular pH is decreased or extracellular Ca2+ concentration is increased. Although increased currents are seen, oocytes expressing Cx32(S85C) have lower levels of the protein in the surface and in total homogenates than do oocytes expressing Cx32WT; thus, under the conditions examined here, hemichannels in the surface membrane formed of the Cx32(S85C) mutant have a higher open probability than hemichannels formed of Cx32WT. This increase in functional hemichannels may damage Schwann cells and ultimately lead to loss of function in peripheral nerves of patients harboring this mutation.

Cx29 and Cx32, two connexins expressed by myelinating glia, do not interact and are functionally distinct.

In rodents, oligodendrocytes and myelinating Schwann cells express connexin32 (Cx32) and Cx29, which have different localizations in the two cell types. We show here that, in contrast to Cx32, Cx29 does not form gap junction plaques or functional gap junctions in transfected cells. Furthermore, when expressed together, Cx29 and Cx32 are not colocalized and do not coimmunoprecipitate. To determine the structural basis of their divergent behavior, we generated a series of chimeric Cx32‐Cx29 proteins by exchanging their intracellular loops and/or their C‐terminal cytoplasmic tails. Although some chimerae reach the cell membrane, others appear to be largely localized intracellularly; none form gap junction plaques or functional gap junctions. Substituting the C‐terminus or the intracellular loop and the C‐terminus of Cx32 with those of Cx29 does not disrupt their colocalization or coimmunoprecipitation with Cx32. Substituting the C‐terminus of Cx29 with that of Cx32 does not disrupt the coimmunoprecipitation or the colocalization with Cx29, whereas substituting both the intracellular loop and the C‐terminus of Cx32 with those of Cx29 diminishes the coimmunoprecipitation with Cx29. Conversely, the Cx32 chimera that contains the intracellular loop of Cx29 coimmunoprecipitates with Cx29, indicating that the intracellular loop participates in Cx29‐Cx29 interactions. These data indicate that homomeric interactions of Cx29 and especially Cx32 largely require other domains: the N‐terminus, transmembrane domains, and extracellular loops. Substituting the intracellular loop and/or tail of Cx32 with those of Cx29 appears to prevent Cx32 from forming functional gap junctions.

How do mutations in GJB1 cause X-linked Charcot-Marie-Tooth disease?

The X-linked form of Charcot-Marie-Tooth disease (CMT1X) is the second most common form of hereditary motor and sensory neuropathy. The clinical phenotype is characterized by progressive weakness, atrophy, and sensory abnormalities that are most pronounced in the distal extremities. Some patients have CNS manifestations. Affected males have moderate to severe symptoms, whereas heterozygous females are usually less affected. Neurophysiology shows intermediate slowing of conduction and length-dependent axonal loss. Nerve biopsies show more prominent axonal degeneration than de/remyelination. Mutations in GJB1, the gene that encodes the gap junction (GJ) protein connexin32 (Cx32) cause CMT1X; more than 400 different mutations have been described. Many Cx32 mutants fail to form functional GJs, or form GJs with abnormal biophysical properties. Schwann cells and oligodendrocytes express Cx32, and the GJs formed by Cx32 play an important role in the homeostasis of myelinated axons. Animal models of CMT1X demonstrate that loss of Cx32 in myelinating Schwann cells causes a demyelinating neuropathy. Effective therapies remain to be developed. This article is part of a Special Issue entitled Electrical Synapses.

Connexin 32 increases the proliferative response of Schwann cells to neuregulin-1 (Nrg1)

Connexin 32 (Cx32), a gap junction protein, is found within the para-nodal region and Schmidt-Lanterman incisures of myelinating Schwann cells (SCs). In developing and regenerating peripheral nerves, pro-myelinating SCs express Cx32 mRNA and protein in conjunction with the expression of myelin specific genes. Neuregulin-1 (Nrg1), a member of the neuregulin family of growth factors, controls SC proliferation and differentiation depending on the cellular environment and the particular stage of SC maturation. Primary cultures of purified SCs from newborn mouse sciatic nerve were used to characterize both the role of Nrg1 in the expression of Cx32 and, conversely, the role of Cx32 in SC responsiveness to Nrg1. Glial growth factor 2, an isoform of Nrg1, up-regulated Cx32 in both proliferating and non-proliferating SCs. However, SCs from Cx32-KO mice exhibited a significantly smaller mitogenic response to glial growth factor 2. Electrical coupling between Cx32-KO SCs did not differ from that between WT SCs, indicating the presence of other connexins. These results suggest a link between Cx32 expression and Nrg1 regulation of SC proliferation that does not involve Cx32-mediated intercellular communication.

Human oligodendrocytes express Cx31.3: function and interactions with Cx32 mutants

Murine oligodendrocytes express the gap junction (GJ) proteins connexin32 (Cx32), Cx47, and Cx29. CNS phenotypes in patients with X-linked Charcot-Marie-Tooth disease may be caused by dominant effects of Cx32 mutations on other connexins. Here we examined the expression of Cx31.3 (the human ortholog of murine Cx29) in human brain and its relation to the other oligodendrocyte GJ proteins Cx32 and Cx47. Furthermore, we investigated in vitro whether Cx32 mutants with CNS manifestations affect the expression and function of Cx31.3. Cx31.3 was localized mostly in the gray matter along small myelinated fibers similar to Cx29 in rodent brain and was co-expressed with Cx32 in a subset of human oligodendrocytes. In HeLa cells Cx31.3 was localized at the cell membrane and appeared to form hemichannels but no GJs. Cx32 mutants with CNS manifestations were retained intracellularly, but did not alter the cellular localization or function of co-expressed Cx31.3. Thus, Cx31.3 shares many characteristics with its ortholog Cx29. Cx32 mutants with CNS phenotypes do not affect the trafficking or function of Cx31.3, and may have other toxic effects in oligodendrocytes.

Functional Requirement for a Highly Conserved Charged Residue at Position 75 in the Gap Junction Protein Connexin 32

Background: Arg75 in connexins is highly conserved. Results: Disease-causing mutations at this position cause loss of function; for Cx32, loss of a positive charge appears to be critical. Conclusion: Positive charge at position 75 is required for normal Cx32 function. Significance: Better understanding of the effects of mutations of this position in Cx32 may have relevance to pathogenesis of several human diseases. Charcot Marie Tooth disease (CMT) is a group of inherited disorders characterized clinically by exclusively or predominantly peripheral nerve dysfunction. CMT1X, the most common form of X-linked CMT is caused by mutations in connexin 32 (Cx32). In this work, we used dual whole cell patch clamp recording to examine the functional effects of mutations at the Arg75 position. This residue is highly conserved among members of the connexin family, and disease-causing mutations have been identified at this (or the corresponding) position in Cx26, Cx43, and Cx46. Thus, a better understanding of the effects of mutations of this position in Cx32 may have relevance to pathogenesis of a number of different human diseases. All three mutants associated with CMT1X (R75P, R75Q, and R75W) showed very low levels of coupling similar to those of the cells transfected with vector alone. Heterotypic pairing with Cx32 WT showed that the absence of coupling for these mutants in the homotypic configuration could be explained by shifts in their hemichannel Gj-Vj relations. Examination of the expression levels and gating characteristics of seven additional mutants (R75A, R75D, R75E, R75H, R75K, R75L, and R75V) at this position suggest that the positive charge at position 75 in Cx32 is required for normal channel function but not for gap junction assembly. Our studies also suggest that disease treatment strategies for CMT1X, which correct trafficking abnormalities in Cx32, may be ineffective for the group of mutations also conferring changes in gating properties of Cx32 channels.