Cellular mechanisms of connexin32 mutations associated with CNS manifestations

Both oligodendrocytes and myelinating Schwann cells express the gap junction protein connexin32 (Cx32). Mutations in the gene encoding Cx32 (GJB1) cause the X-linked form of Charcot-Marie-Tooth disease (CMTX). Although most CMTX patients do not have clinical central nervous system (CNS) manifestations, subclinical evidence of CNS dysfunction is common. We investigated the cellular effects of a subgroup of GJB1/Cx32 mutations that have been reported to cause clinical CNS dysfunction. We hypothesized that these mutants have dominant-negative effects on other connexins expressed by oligodendrocytes, specifically Cx45. We expressed these and other Cx32 mutants in communication-incompetent as well as Cx45-expressing HeLa cells, and analyzed the transfected cells by immunocytochemistry and immunoblotting. In communication-incompetent cells, the mutants associated with CNS phenotypes failed to reach the cell membrane and were instead retained in the endoplasmic reticulum (A39V, T55I) or Golgi apparatus (M93V, R164Q, R183H), although rare gap junction plaques were found in cells expressing M93V or R183H. In HeLa cells stably expressing Cx45, these Cx32 mutants showed a similar expression pattern, and did not alter the pattern of Cx45 expression. These results indicate that Cx32 mutants that are associated with a CNS phenotype do not interact with Cx45, but may instead have other toxic effects in oligodendrocytes.     

Dominant Cx26 mutants associated with hearing loss have dominant-negative effects on wild type Cx26

Mutations in GJB2, the gene encoding the human gap junction protein connexin26 (Cx26), cause either non-syndromic hearing loss or syndromes affecting both hearing and skin. We have investigated whether dominant Cx26 mutants can interact physically with wild type Cx26. HeLa cells stably expressing wild type Cx26 were transiently transfected to co-express nine individual dominant Cx26 mutants; six associated with non-syndromic hearing loss (W44C, W44S, R143Q, D179N, R184Q, and C202F) and three associated with hearing loss and palmoplantar keratoderma (G59A, R75Q, and R75W). All mutants co-localized and co-immunoprecipitated with wild type Cx26, indicating that they interact physically, likely by forming admixed heteromeric/heterotypic channels. Furthermore, all nine mutants inhibited the transfer of calcein in cells stably expressing Cx26, demonstrating that they each have dominant effects on wild type Cx26. Taken together, these results show that dominant-negative effects of these Cx26 mutants likely contribute to the pathogenesis of hearing loss.     

A new mutation in GJC2 associated with subclinical leukodystrophy

Recessive mutations in GJC2, the gene-encoding connexin 47 (Cx47), cause Pelizaeus–Merzbacher-like disease type 1, a severe dysmyelinating disorder. One recessive mutation (p.Ile33Met) has been associated with a much milder phenotype––hereditary spastic paraplegia type 44. Here, we present evidence that a novel Arg98Leu mutation causes an even milder phenotype––a subclinical leukodystrophy. The Arg98Leu mutant forms gap junction plaques in HeLa cells comparable to wild-type Cx47, but electrical coupling was 20-fold lower in cell pairs expressing Arg98Leu than for cell pairs expressing wild-type Cx47. On the other hand, coupling between Cx47Arg98Leu and Cx43WT expressing cells did not show such reductions. Single channel conductance and normalized steady-state junctional conductance–junctional voltage (G _j–V _j) relations differed only slightly from those for wild-type Cx47. Our data suggest that the minimal phenotype in this patient results from a reduced efficiency of opening of Cx47 channels between oligodendrocyte and oligodendrocyte with preserved coupling between oligodendrocyte and astrocyte, and support a partial loss of function model for the mild Cx47 associated disease phenotypes.     

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.     

Oligodendrocytes in mouse corpus callosum are coupled via gap junction channels formed by connexin47 and connexin32

According to previously published ultrastructural studies, oligodendrocytes in white matter exhibit gap junctions with astrocytes, but not among each other, while in vitro oligodendrocytes form functional gap junctions. We have studied functional coupling among oligodendrocytes in acute slices of postnatal mouse corpus callosum. By whole‐cell patch clamp we dialyzed oligodendrocytes with biocytin, a gap junction‐permeable tracer. On average 61 cells were positive for biocytin detected by labeling with streptavidin‐Cy3. About 77% of the coupled cells stained positively for the oligodendrocyte marker protein CNPase, 9% for the astrocyte marker GFAP and 14% were negative for both CNPase and GFAP. In the latter population, the majority expressed Olig2 and some NG2, markers for oligodendrocyte precursors. Oligodendrocytes are known to express Cx47, Cx32 and Cx29, astrocytes Cx43 and Cx30. In Cx47‐deficient mice, the number of coupled cells was reduced by 80%. Deletion of Cx32 or Cx29 alone did not significantly reduce the number of coupled cells, but coupling was absent in Cx32/Cx47‐double‐deficient mice. Cx47‐ablation completely abolished coupling of oligodendrocytes to astrocytes. In Cx43‐deficient animals, oligodendrocyte‐astrocyte coupling was still present, but coupling to oligodendrocyte precursors was not observed. In Cx43/Cx30‐double deficient mice, oligodendrocyte‐to‐astrocyte coupling was almost absent. Uncoupled oligodendrocytes showed a higher input resistance. We conclude that oligodendrocytes in white matter form a functional syncytium predominantly among each other dependent on Cx47 and Cx32 expression, while astrocytic connexins expression can promote the size of this network. © 2010 Wiley‐Liss, Inc.     

Unique distributions of the gap junction proteins connexin29, connexin32, and connexin47 in oligodendrocytes

Oligodendrocytes of adult rodents express three different connexins: connexin29 (Cx29), Cx32, and Cx47. In this study, we show that Cx29 is localized to the inner membrane of small myelin sheaths, whereas Cx32 is localized on the outer membrane of large myelin sheaths; Cx29 does not colocalize with Cx32 in gap junction plaques. All oligodendrocytes appear to express Cx47, which is largely restricted to their perikarya. Cx32 and Cx47 are colocalized in many gap junction plaques on oligodendrocyte somata, particularly in gray matter. Cx45 is detected in the cerebral vasculature, but not in oligodendrocytes or myelin sheaths. This diversity of connexins in oligodendrocytes (in different populations of cells and in different subcellular compartments) likely reflects functional differences between these connexins and perhaps the oligodendrocytes themselves.     

Changes in object recognition and anxiety-like behaviour in mice expressing a cx47 mutation that causes pelizaeus-merzbacher-like disease

Pelizaeus-Merzbacher-like disease is characterized by impaired psychomotor development, ataxia, progressive spasticity and mental retardation. It is induced by mutations in the gap junction gene GJC2 that encodes for the gap junction protein connexin 47. Mice bearing a human Cx47M283T missense mutation have been generated as a transgenic mouse model of Pelizaeus-Merzbacher-like disease. Homozygous expression of the mutant connexin 47 gene in oligodendrocytes resulted in a complex and variable neuropathologic phenotype, which was associated with impairments in motor coordination in juvenile, but not adult mice. In the present study, we have investigated anxiety-like behaviour and spatial working memory in juvenile (P23) and adult (3-month-old) Cx47M282T mutant mice. Adult Cx47M282T mice were also evaluated in terms of neuromotor functions and in the novel object recognition test. Juvenile Cx47M282T mutant mice exhibited an increase in anxiety-like behaviour in the open field test, but no changes in spatial working memory performance. No significant changes in anxiety-like behaviour, spatial working memory or neuromotor functions were observed in the adult Cx47M282T mutant mice. However, novel object recognition was significantly impaired in adult Cx47M282T mice. Our results suggest that the expression of the human Cx47M282T mutation in the mouse causes changes in anxiety-like behaviour in juvenile and novel object recognition impairments in adult mice. It appears that the distortion of panglial gap junction coupling in white and grey matter tissue in the Cx47M282T mice is associated with a complex age-dependent behavioural phenotype including changes in psychomotor, emotional and memory functions.     

Cx32 and Cx47 mediate oligodendrocyte:astrocyte and oligodendrocyte:oligodendrocyte gap junction coupling

In addition to the extensive gap junction coupling between astrocytes themselves, oligodendrocytes are thought to be exclusively coupled to astrocytes (O:A coupling) via heterotypic gap junctions composed of Cx47:Cx43 and Cx32:Cx30. We used fluorescent dyes to examine functional coupling in acute slices from the cerebra of mice lacking Cx32 and/or Cx47. In the corpus callosum, unexpectedly, oligodendrocytes appeared to be directly and exclusively coupled to other oligodendrocytes (O:O coupling), and electron microscopy revealed gap junctions between adjacent oligodendrocytes. O:O coupling was more affected in mice lacking Cx32 than in mice lacking Cx47. In the neocortex, oligodendrocytes appeared to be directly and exclusively coupled to astrocytes; Cx47, but not Cx32, was required for O:A coupling.     

Loss of function mutations of the GJB2 gene detected in patients with DFNB1-associated hearing impairment

Mutations in GJB2, which encodes the gap junction protein connexin 26 (Cx26), are one of the major causes for inherited and sporadic nonsyndromic hearing impairment. This study aimed to functionally characterize more frequent GJB2 mutations identified in patients showing nonsyndromic hearing impairment. Following injection of wild type and mutated cRNA in Xenopus oocytes, Cx26 hemichannel activity was measured by depolarization activated conductance in noncoupled oocytes. All mutants showed a partially or completely defective phenotype, except (V27I)Cx26, a polymorphism tested as positive control. Coexpression of wild type and mutant Cx26 injected at equimolar levels revealed that p.M34T, p.V37I and p.I82M, but not p.G59V, p.L90P, p.R127H and p.R143W exert a dominant inhibitory effect. When coexpressed with Cx30, a connexin partially colocalized with Cx26 in the cochlea, all mutants had a dominant behavior. This study provides data that might be important for the improvement of genetic diagnosis and counseling for patients with hearing impairment.     

Gap junction disorders of myelinating cells

Gap junctions (GJs) are channels that allow the diffusion of ions and small molecules across apposed cell membranes. In peripheral nerves, Schwann cells express the GJ proteins connexin32 (Cx32) and Cx29, which have distinct localizations. Cx32 forms GJs through non-compact myelin areas, whereas Cx29 forms hemichannels in the innermost layers of myelin apposing axonal Shaker-type K+ channels. In the CNS, rodent oligodendrocytes express Cx47, Cx32 and Cx29. Cx47 is expressed by all types of oligodendrocytes both in the white and grey matter and forms GJs on cell bodies and proximal processes, as well as most of the intercellular channels with astrocytes. Cx32 is expressed mostly by white matter oligodendrocytes and is localized in the myelin sheath of large diameter fibers. Cx29, and its human ortholog Cx31.3, appear to be restricted to oligodendrocytes that myelinate small caliber fibers, likely forming hemichannels. The importance of intercellular and intracellular GJs in myelinating cells are demonstrated by human disorders resulting from mutations affecting GJ proteins. The X-linked Charcot Marie Tooth disease (CMT1X) is caused by hundreds of mutations affecting Cx32. Patients with CMT1X present mainly with a progressive peripheral neuropathy, which may be accompanied by CNS myelin dysfunction. Mutations in Cx47 may cause a devastating leukodystrophy called Pelizaeus-Merzbacher-like disease or a milder spastic paraplegia. In addition, CNS demyelination may be caused by defects in genes expressing astrocytic GJ proteins, which are essential for oligodendrocytes. Findings from in vitro and in vivo models of these disorders developed over the last decade indicate that most mutations cause loss of function and an inability of the mutant connexins to form functional GJs. Here we review the clinical, genetic, and neurobiological aspects of GJ disorders affecting the PNS and CNS myelinating cells.     

Mutations in connexin 32: the molecular and biophysical bases for the X-linked form of Charcot–Marie–Tooth disease

The connexins are a family of homologous integral membrane proteins that form channels that provide a low resistance pathway for the transmission of electrical signals and the diffusion of small ions and non-electrolytes between coupled cells. Individuals carrying mutations in the gene encoding connexin 32 (Cx32), a gap junction protein expressed in the paranodal loops and Schmidt–Lantermann incisures of myelinating Schwann cells, develop a peripheral neuropathy — the X-linked form of Charcot–Marie–Tooth disease (CMTX). Over 160 different mutations in Cx32 associated with CMTX have been identified. Some mutations will lead to complete loss of function with no possibility of expression of functional channels. Some mutations in Cx32 lead to the abnormal accumulation of Cx32 proteins in the cytoplasm, particularly in the Golgi apparatus; CMTX may arise due to incorrect trafficking of Cx32 or to interference with trafficking of other proteins. On the other hand, many mutant forms of Cx32 can form functional channels. Some functional mutants have conductance voltage relationships that are disrupted to a degree which would lead to a substantial reduction in the available gap junction mediated communication pathway. Others have essentially normal steady-state g–V relations. In one of these cases (Ser26Leu), the only change introduced by the mutation is a reduction in the pore diameter from 7 Å for the wild-type channel to less than 3 Å for Ser26Leu. This reduction in pore diameter may restrict the passage of important signaling molecules. These findings suggest that in some, if not all cases of CMTX, loss of function of normal Cx32 is sufficient to cause CMTX.     

Neurological Dysfunction And Axonal Degeneration In Charcot-Marie-Tooth Disease Type 1A

More than 130 different mutations in the gap junction integral plasma membrane protein connexin32 (Cx32) have been linked to the human peripheral neuropathy X-linked Charcot-Marie-Tooth disease (CMTX). How these various mutants are processed by the cell and the mechanism(s) by which they cause CMTX are unknown. To address these issues, we have studied the intracellular transport, assembly, and degradation of three CMTX-linked Cx32 mutants stably expressed in PC12 cells. Each mutant had a distinct fate: E208K Cx32 appeared to be retained in the endoplasmic reticulum (ER), whereas both the E186K and R142W mutants were transported to perinuclear compartments from which they trafficked either to lysosomes (R142W Cx32) or back to the ER (E186K Cx32). Despite these differences, each mutant was soluble in nonionic detergent but unable to assemble into homomeric connexons. Degradation of both mutant and wild-type connexins was rapid (t(1/2) < 3 h) and took place at least in part in the ER by a process sensitive to proteasome inhibitors. The mutants studied are therefore unlikely to cause disease by accumulating in degradation-resistant aggregates but instead are efficiently cleared from the cell by quality control processes that prevent abnormal connexin molecules from traversing the secretory pathway.     

Functional alterations in gap junction channels formed by mutant forms of connexin 32: evidence for loss of function as a pathogenic mechanism in the X-linked form of Charcot-Marie-Tooth disease

CMTX, the X-linked form of Charcot-Marie-Tooth disease, is an inherited peripheral neuropathy arising in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32). In this communication, we describe the expression levels and biophysical parameters of seven mutant forms of Cx32 associated with CMTX, when expressed in paired Xenopus oocytes. Paired oocytes expressing the R15Q and H94Q mutants show junctional conductances not statistically different from that determined for Cx32WT, though both show a trend toward reduced levels. The S85C and G12S mutants induce reduced levels of junctional conductance. Three other mutants (R15W, H94Y and V139M) induce no conductance above baseline when expressed in paired oocytes. Analysis of the conductance voltage relations for these mutants shows that the reduced levels of conductance are entirely (H94Y and V139M) or partly (S85C and R15W) explicable by a reduced open probability of the mutant hemichannels. The R15Q and H94Q mutations also show alterations in the conductance voltage relations that would be expected to minimally (H94Q) or moderately (R15Q) reduce the available gap junction communication pathway. The reduction in G12S induced conductance cannot be explained by alterations in hemichannel open probability and are more likely due to reduced junction formation. These results demonstrate that many CMTX mutations lead to loss of function of Cx32. For these mutations, the loss of function model is likely to explain the pathogenesis of CMTX.     

突变缝隙连接蛋白32在X连锁Charcot-Marie-Tooth病1型患者的外周血管内皮细胞异常表达

目的 观察突变缝隙连接蛋白32( Cx32)在X连锁的Charcot-Marie-Tooth病1型(CMTX1)患者血管内皮细胞的表达规律。方法 对3例经Cx32基因检查证实的CMTX1患者（突变位点分别为c.379A＞T、c.533A＞G和c.590C＞T点突变）进行腓肠神经活体组织检查,同时取非CMTXI患者的神经作为对照。以第Ⅷ因子、缝隙连接蛋白40和Cx32单克隆抗体为一抗,对患者及对照者标本中的神经滋养血管进行免疫荧光标记。同时构建携带上述3个点突变的以增强型绿色荧光蛋白(enhanced green fluorescence protein,EGFP)为报告基因的重组质粒pEGFP-N1-CX32质粒,转染至HeLa细胞,并进行Cx32和内质网标志蛋白葡萄糖调节蛋白78免疫荧光染色,以观察Cx32蛋白在HeLa细胞的分布规律。结果 Cx32蛋白呈一定间隔规律性点状表达于对照组血管内皮细胞间的缝隙连接部位,与Cx40蛋白共表达;3例Cx32基因突变患者的血管内皮细胞膜的Cx32蛋白表达显著减少。细胞转染显示c.379A＞T突变的Cx32蛋白主要以团块状蓄积在HeLa细胞的胞质内,c.533A＞G突变的Cx32蛋白仅有少量表达于核周,c.590C＞T突变的Cx32蛋白在胞膜上和胞质内均有点状分布,较野生型显著减少。3种突变的Cx32蛋白在HeLa细胞内的分布和内质网标记不重叠。结论 不同Cx32基因突变均可导致CMTX1的血管内皮细胞膜Cx32蛋白表达下降,该蛋白主要聚集在细胞的内质网外。     

The effects of a dominant connexin32 mutant in myelinating Schwann cells

Mutations in GJB1, the gene encoding the gap junction protein connexin32 (Cx32), cause X-linked Charcot-Marie-Tooth disease, an inherited demyelinating peripheral neuropathy. We generated transgenic mice that express the R142W mutation in myelinating Schwann cells. The R142W mutant protein was aberrantly localized to the Golgi, indicating that it does not traffic properly, but the molecular organization of the myelin sheath, including the localization of Cx29, another connexin expressed by myelinating Schwann cells, was not disrupted. In a wild type background, this mutation dramatically decreased the level of wild type mouse Cx32 in immunoblots of sciatic nerve and caused demyelination. The expression of wild type human Cx32 with the same transgenic construct had different effects-increased amounts of Cx32, normal localization of Cx32 at nodes and incisures, and split myelin sheaths. Thus, the R142W mutant protein has dominant effects that are distinct from overexpression.     

Salvage Therapy With Thalidomide In Patients With Advanced Relapsed/Refractory Multiple Myeloma

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(S35C) 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(S35C) 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.     

MUTATIONS IN CONNEXIN 32: THE MOLECULAR AND BIOPHYSICAL BASES FOR THE X-LINKED FORM OF CHARCOT-MARIE-TOOTH DISEASE

The connexins are a family of homologous integral membrane proteins that form channels that provide a low resistance pathway for the transmission of electrical signals and the diffusion of small ions and non-electrolytes between coupled cells. Individuals carrying mutations in the gene encoding connexin 32 (Cx32), a gap junction protein expressed in the paranodal loops and Schmidt-Lantermann incisures of myelinating Schwann cells, develop a peripheral neuropathy—the X-linked form of Charcot-Marie-Tooth disease (CMTX). Over 160 different mutations in Cx32 associated with CMTX have been identified. Some mutations will lead to complete loss of function with no possibility of expression of functional channels. Some mutations in Cx32 lead to the abnormal accumulation of Cx32 proteins in the cytoplasm, particularly in the Golgi apparatus; CMTX may arise due to incorrect trafficking of Cx32 or to interference with trafficking of other proteins. On the other hand, many mutant forms of Cx32 can form functional channels. Some functional mutants have conductance voltage relationships that are disrupted to a degree which would lead to a substantial reduction in the available gap junction mediated communication pathway. Others have essentially normal steady-state g-V relations. In one of these cases (Ser26Leu), the only change introduced by the mutation is a reduction in the pore diameter from 7 Angstrom for the wild-type channel to less than 3 Angstrom for Ser26Leu. This reduction in pore diameter may restrict the passage of important signaling molecules. These findings suggest that in some, if not all cases of CMTX, loss of function of normal Cx32 is sufficient to cause CMTX.     

Enhanced generation of intracellular Aβ42 amyloid peptide by mutation of presenilins PS1 and PS2

The accumulation of amyloid β‐peptide (Aβ) in the brain is a critical pathological process in Alzheimer's disease (AD). Recent studies have implicated intracellular Aβ in neurodegeneration in AD. To investigate the generation of intracellular Aβ, we established human neuroblastoma SH‐SY5Y cells stably expressing wild‐type amyloid precursor protein (APP), Swedish mutant APP, APP plus presenilin 1 (PS1) and presenilin 2 (PS2; wild‐type or familial AD‐associated mutant), and quantified intracellular Aβ40 and Aβ42 in formic acid extracts by sensitive Western blotting. Levels of both intracellular Aβ40 and Aβ42 were 2–3‐fold higher in cells expressing Swedish APP, compared with those expressing wild‐type APP. Intracellular Aβ42/Aβ40 ratios were approximately 0.5 in these cells. These ratios were increased markedly in cells expressing mutant PS1 or PS2 compared with those expressing their wild‐type counterparts, consistent with the observed changes in secreted Aβ42/Aβ40 ratios. High total levels of intracellular Aβ were observed in cells expressing mutant PS2 because of a marked elevation of Aβ42. Immunofluorescence staining additionally revealed more intense Aβ42 immunoreactivity in mutant PS2‐expressing cells than in wild‐type cells, which was partially colocalized with immunoreactivity for the trans‐Golgi network and endosomes. The data collectively indicate that PS mutations promote the accumulation of intracellular Aβ42, which appears to be localized in multiple subcellular compartments.     

Connexin43 and connexin47 alterations after neural precursor cells transplantation in experimental autoimmune encephalomyelitis

Exogenous transplanted neural precursor cells (NPCs) exhibit miscellaneous immune‐modulatory effects in models of autoimmune demyelination. However, the regional interactions of NPCs with the host brain tissue in remissive inflammatory events have not been adequately studied. In this study we used the chronic MOG‐induced Experimental Autoimmune Encephalomyelitis (EAE) model in C57BL/six mice. Based on previous data, we focused on neuropathology at Day 50 post‐induction (D50) and studied the expression of connexin43 (Cx43) and Cx47, two of the main glial gap junction (GJ) proteins, in relation to the intraventricular transplantation of GFP⁺NPCs and their integration with the host tissue. By D50, NPCs had migrated intraparenchymally and were found in the corpus callosum at the level of the lateral ventricles and hippocampus. The majority of GFP⁺ cells differentiated with simple or ramified processes expressing mainly markers of mature GLIA (GFAP and NogoA) and significantly less of precursor glial cells. GFP⁺NPCs expressed connexins and formed GJs around the hippocampus more than lateral ventricles. The presence of NPCs did not alter the increase in Cx43 GJ plaques at D50 EAE, but prevented the reduction of oligodendrocytic Cx47, increased the number of oligodendrocytes, local Cx47 levels and Cx47 GJ plaques per cell. These findings suggest that transplanted NPCs may have multiple effects in demyelinating pathology, including differentiation and direct integration into the panglial syncytium, as well as amelioration of oligodendrocyte GJ loss, increasing the supply of potent myelinating cells to the demyelinated tissue. GLIA 2015;63:1772–1783     

Dynamic expression of Cx47 in mouse brain development and in the cuprizone model of myelin plasticity

The study shows the dynamic expression of connexin47 (Cx47) in oligodendrocytes and myelin of mice, either in myelinogenesis occurring in early development or in an experimental model of new‐myelinogenesis of adult mice. Cx47 first appeared in the embryonic mouse brain at E10.5 successively the expression increased, principally in regions populated by developing oligodendrocytes. The expression declined postnatally toward adulthood and immunoreactivity was restricted to a few specific areas, such as the corpus callosum, the striatum, the cerebellum, and the spinal cord. Since the expression of Cx47 in developing oligodendrocytes preceded those of Cx32 and Cx29, a role of Cx47 in myelinogenesis was postulated. This hypothesis was tested in a model of re‐myelination, which principally involved the corpus callosum, occurring in adult mice by treatment with cuprizone. Cx47 was upregulated during demyelination and recovered during the remyelination phase. During demyelination, Cx47 was first over‐expressed in the corpus callosum and later, when the myelin virtually disappeared in the injured areas, Cx47 was expressed in astrocytes located inside and closely around the demyelinated areas. The remyelination of injured areas occurred after stopping the administration of cuprizone and continued to complete recovery. In this period the expression of Cx47 shifted from astrocytes to newly‐formed myelin. Thus, Cx47 exhibits in this model a transient and de novo expression in astrocytes with a topographic segregation in the injured areas, only when oligodendrocytes and the myelin were most severely affected. Taken as a whole the evidence suggests that Cx47 play a key role in myelination. © 2010 Wiley‐Liss, Inc.