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.     

Disruption of oligodendrocyte gap junctions in experimental autoimmune encephalomyelitis

Gap junctions (GJs) are vital for oligodendrocyte survival and myelination. In order to examine how different stages of inflammatory demyelination affect oligodendrocyte GJs, we studied the expression of oligodendrocytic connexin32 (Cx32) and Cx47 and astrocytic Cx43 in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis (MS) induced by recombinant myelin oligodendrocyte glycoprotein. EAE was characterized by remissions and relapses with demyelination and axonal loss. Formation of GJ plaques was quantified in relation to the lesions and in normal appearing white matter (NAWM). During acute EAE at 14 days postimmunization (dpi) both Cx47 and Cx32 GJs were severely reduced within and around lesions but also in the NAWM. Cx47 was localized intracellularly in oligodendrocytes while protein levels remained unchanged, and this redistribution coincided with the loss of Cx43 GJs in astrocytes. Cx47 and Cx32 expression increased during remyelination at 28 dpi but decreased again at 50 dpi in the relapsing phase. Oligodendrocyte GJs remained reduced even in NAWM, despite increased formation of Cx43 GJs toward lesions indicating astrogliosis. EAE induced in Cx32 knockout mice resulted in an exacerbated clinical course with more demyelination and axonal loss compared with wild-type EAE mice of the same backcross, despite similar degree of inflammation, and an overall milder loss of Cx47 and Cx43 GJs. Thus, EAE causes persistent impairment of both intra- and intercellular oligodendrocyte GJs even in the NAWM, which may be an important mechanism of MS progression. Furthermore, GJ deficient myelinated fibers appear more vulnerable to CNS inflammatory demyelination.     

A de novo p.Arg756Cys mutation in ATP1A3 causes a distinct phenotype with prolonged weakness and encephalopathy triggered by fever

Patients with a mutation at Arg756 in ATP1A3 have been known to exhibit a distinct phenotype, characterized by prolonged weakness and encephalopathy, triggered by febrile illness. With only eight reports published to date, more evidence is required to correlate clinical features with a mutation at Arg756. Here we report an additional case with an Arg756Cys mutation in ATP1A3. A four-year-old boy showed mild developmental delay with recurrent paroxysmal episodes of weakness and encephalopathy from nine months of age. Motor deficits, which included bilateral hypotonia, ataxia, dysmetria, limb incoordination, dysarthria, choreoathetosis, and dystonia, were observed from one year and three months. Whole-exome sequencing detected a heterozygous de novo variant at c.2266C > T (p.Arg756Cys) in ATP1A3. The episodic course and clinical features of this case were consistent with previously reported cases with mutations at Arg756. Furthermore, his phenotype of marked ataxia was more similar to that of an Arg756Cys patient with relapsing encephalopathy and cerebellar ataxia syndrome, than to those with Arg756His and Arg756Leu mutations. This report therefore provides evidence of genotype-phenotype correlations in ATP1A3-related disorders as well as in patients with mutations at Arg756 in ATP1A3.     

Modulation of junctional conductance between rat carotid body glomus cells by hypoxia, cAMP and acidity

Short-term cultures of glomus cells (up to seven days), were employed to study intercellular electrical communications. Bidirectional electric coupling was established under current clamping after impaling two adjacent glomus cells with microelectrodes, and alternate stimulation and recording. Their resting potential (V_m) and input resistance (R_o) were thus measured. Both coupled cells were then voltage clamped at a level between their V_ms. Current pulses applied to either cell elicited a transjunctional voltage (V_j) and current (I_j), used to calculate the junctional conductance (G_j). G_j was 1.52±0.29 nS (mean±S.E.; n=147). V_j linearly influenced G_j, suggesting ohmic junctions. G_j was not affected by V_m in 50% of the cases. However, there was V_m-dependence in the others, but voltage changes had to be large (>±40 mV from the V_m). Therefore, physiologically or pharmacologically induced glomus cell depolarization or hyperpolarization may not significantly affect intercellular coupling unless there are large variations in V_m. Hypoxia (induced by Na₂S₂O₄ 1 mM or 100% N₂) decreased G_j in 60–80% of the pairs while producing tighter coupling in the rest. Similar effects were obtained when the medium was acidified with lactic acid 1–10 mM. Cobalt chloride (3 mM) prevented, diminished or reversed the changes in G_j observed during low PO₂, suggesting that [Ca²⁺]_i changes are important in hypoxic uncoupling. However, non-specific cationic effects of Co²⁺ have not been ruled out. Applications of the membrane-permeant dB-cAMP 1 mM tightened coupling in almost all cell pairs. This is important because endogenous cAMP increases during hypoxia. Our results suggest that multiple factors modulate junctional conductance between glomus cells. Changes in G_j by ‘natural' stimuli and/or cAMP may play an important role in chemoreception, especially in titrating the release of transmitters toward the carotid nerve terminals.     

Ablation of Cx47 in transgenic mice leads to the loss of MUPP1, ZONAB and multiple connexins at oligodendrocyte-astrocyte gap junctions

Oligodendrocytes in CNS are linked to astrocytes by heterotypic gap junctions composed of Cx32 and Cx47 in oligodendrocytes and Cx30 and Cx43 in astrocytes. These gap junctions also harbour regulatory proteins, including ZO-1 and ZONAB. Here, we investigated the localization of multi-PDZ domain protein 1 (MUPP1) at these gap junctions and examined accessory proteins and connexins associated with oligodendrocytes in Cx47-knockout mice. In every CNS region tested, punctate immunolabelling for MUPP1 was found on all oligodendrocyte somata in wild-type mice. These MUPP1-positive puncta were colocalized with punctate labelling for oligodendrocytic Cx32 or Cx47, and with astrocytic Cx30 or Cx43 at oligodendrocyte-astrocyte (O/A) gap junctions, but were not found at astrocyte-astrocyte gap junctions. In Cx47-knockout mice, immunolabelling of MUPP1 and ZONAB was absent on oligodendrocytes, whereas some ZO-1-positive puncta remained. In Cx32-knockout mice, MUPP1 and ZONAB persisted at O/A gap junctions. The absence of Cx47 in Cx47-knockout mice was accompanied by a total loss of punctate labelling for Cx30, Cx32 and Cx43 on oligodendrocyte somata, and by a dramatic increase in immunolabelling for Cx32 along myelinated fibers. These results demonstrate MUPP1 at O/A gap junctions and Cx47-dependent targeting of connexins to the plasma membranes of oligodendrocyte somata. Further, it appears that deficits in myelination reported in Cx47-knockout mice may arise not only from a loss of Cx47 but also from the accompanied loss of gap junctions and their regulatory proteins at oligodendrocyte somata, and that loss of Cx47 may be partly compensated for by elevated levels of Cx32 along myelinated fibers.     

Intercellular communication in spinal cord astrocytes: fine tuning between gap junctions and P2 nucleotide receptors in calcium wave propagation.

Electrophysiological properties of gap junction channels and mechanisms involved in the propagation of intercellular calcium waves were studied in cultured spinal cord astrocytes from sibling wild-type (WT) and connexin43 (Cx43) knock-out (KO) mice. Comparison of the strength of coupling between pairs of WT and Cx43 KO spinal cord astrocytes indicates that two-thirds of total coupling is attributable to channels formed by Cx43, with other connexins contributing the remaining one-third of junctional conductance. Although such a difference in junctional conductance was expected to result in the reduced diffusion of signaling molecules through the Cx43 KO spinal cord syncytium, intercellular calcium waves were found to propagate with the same velocity and amplitude and to the same number of cells as between WT astrocytes. Measurements of calcium wave propagation in the presence of purinoceptor blockers indicate that calcium waves in Cx43 KO spinal cord astrocytes are mediated primarily by extracellular diffusion of ATP; measurements of responses to purinoceptor agonists revealed that the functional P2Y receptor subtype is shifted in the Cx43 KO astrocytes, with a markedly potentiated response to ATP and UTP. Thus, the reduction in gap junctional communication in Cx43 KO astrocytes leads to an increase in autocrine communication, which is a consequence of a functional switch in the P2Y nucleotide receptor subtype. Intercellular communication via calcium waves therefore is sustained in Cx43 null mice by a finely tuned interaction between gap junction-dependent and independent mechanisms.     

Electrical coupling between cultured glomus cells of the rat carotid body: observations with current and voltage clamping

Electrically coupled pairs of cultured rat glomus cells were used. In one group of experiments, both cells were current-clamped. Delivery of positive or negative pulses to Cell 1 elicited appreciable voltage noise in this cell and large action potentials (probably Ca²⁺ spikes) in about 10% of them. Both passive and active electrical events spread to Cell 2, presumably through the gap junctions between them. The coupling coefficient (K_c) was larger for the spikes than for non-regenerative voltage noise. In another group of experiments, Cell 1 was current-clamped and Cell 2 was voltage-clamped at Cell 1 E_M. Pulses of either polarity, delivered to Cell 1, produced current flow through the intercellular junction and allowed direct measurement of junctional currents (I_j) and total conductances (G_j). I_j had a mean value of about 12.5 pA and G_j of 391 pS. Unitary (presumably single channel) conductance (g_j) was about 78 pS.     

Astrocyte‐restricted disruption of connexin‐43 impairs neuronal plasticity in mouse barrel cortex

There is intensive gap‐junctional coupling between glial processes, but their significance in sensory functions remains unknown. Connexin‐43 (Cx43), a major component of astrocytic gap‐junction channels, is abundantly expressed in astrocytes. To investigate the role of Cx43‐mediated gap junctions between astrocytes in sensory functions, we generated Cx43 knockout (KO) mice with a mouse line carrying loxP sites flanking exon 2 of the Cx43 gene and the transgenic line expressing Cre recombinase under control of the glial fibrillary acidic protein promoter, which exhibited a significant loss of Cx43 in astrocytes in the barrel cortex. Although Cx43 expression between the astrocytes measured by immunohistochemistry was virtually abolished in Cx43 KO mice, they had normal architecture in the barrel cortex but the intensity of cytochrome oxide histochemistry decreased significantly. In vivo electrophysiological analysis revealed that the long‐term potentiation of the vibrissal evoked responses in the barrel cortex evoked by high‐frequency rhythmic vibrissal stimuli (100 Hz, 1 s) was abolished in Cx43 KO mice. Current source density analysis also revealed that astrocytic Cx43 was important to the flow of excitation within the laminar connections in barrel cortex. Behavioral tests showed that the ability of Cx43 KO mice to sense the environment with their whiskers decreased. Even so, the jump‐stand experiment showed that they could still discriminate rough from smooth surfaces. Our findings suggest that Cx43‐mediated gap‐junctional coupling between astrocytes is important in the neuron–glia interactions required for whisker‐related sensory functions and plasticity.     

pH Dependence of transmission at electronic synapses of the crayfish septate axon

Gap junctions between segments of the crayfish septate axon mediate electronic transmission of impulses propagating along the length of the nerve cord. We simultaneously measured intracellular pH (pH_i) and gap junctional conductance (g_j) while axons were exposed to saline equilibrated with CO₂, weak acids, and the weak base ammonium chloride. Normal pH_i is about 7.1. When pH_i is elevated, g_j is unaffected. When pH_i is reduced, g_j declines with an apparent pK of about 6.7 and a Hill coefficient of about 2.7. We also measured effects of pH_i on non-junctional conductance (g_nj) and on the coupling coefficient, k. Over the pH_i range 6.2–8, g_nj increases approximately linearly with pH_i. Since k is a function of g_j and g_nj, it reached a maximum at about pH_i 7.1, decreasing at higher and lower pH_i. The pH_i dependence of g_j in crayfish septate axon is less steep and has a lower apparent pK than the g_j-pH_i relation in two vertebrate embryos previously examined. This finding illustrates a difference in gating among analoguos and possibly homologous membrane channels.     

Electrical coupling among Bergmann glial cells and its modulation by glutamate receptor activation

We studied the characteristics of electrical coupling between Bergmann glial cells in mouse cerebellar slices using Lucifer Yellow injection, patch-clamping cell pairs, and ultrastructural inspection. While early postnatal cells (days 5–7) were not coupled, coupling was abundant at postnatal days 20–24. Coupled cells were arranged perpendicular to the parallel fibers in a parasagittal section, forming a string, rather than a cluster of cells. Electron microscopy revealed that gap junctions were abundant in the distal parts of the processes. Gap junctions between cell bodies and processes were very rare, and no gap junctions were found between cell bodies of adjacent Bergmann glial cells. The junctional conductance was voltage and time independent and could be markedly reduced by halothane. Alkalization of cells (by applying NH₄⁺ increased the junctional conductance to 150%, while acidification of the cell interior (by removing NH₄⁺) led to a decrease to 70%. Activation of AMPA receptors induced a blockade of the junctional conductance to 30% of the control. This link is most likely mediated by the influx of Ca²⁺ via the receptor since this effect was not observed in Ca²⁺-free medium, suggesting that Ca²⁺ entry via the kainate receptor pore led to the closure of gap junctions. These studies indicate that electrical coupling between Bergmann glial cells is not only developmentally regulated but also controlled by physiological stimuli. © 1996 Wiley-Liss, Inc.     

Loss-of-function GJA12/Connexin47 mutations cause Pelizaeus-Merzbacher-like disease

Recessive mutations in GJA12/Cx47, the gene encoding the gap junction protein connexin47 (Cx47), cause Pelizaeus–Merzbacher-like disease (PMLD), which is characterized by severe CNS dysmyelination. Three missense PMLD mutations, P87S, Y269D and M283T, were expressed in communication-incompetent HeLa cells, and in each case the mutant proteins appeared to at least partially accumulate in the ER. Cells expressing each mutant did not pass Lucifer Yellow or neurobiotin in scrape loading assays, in contrast to robust transfer in cells expressing wild type Cx47. Dual whole-cell patch clamping of transfected Neuro2A cells demonstrated that none of the mutants formed functional channels, in contrast to wild type Cx47. Immunostaining sections of primate brains demonstrated that oligodendrocytes express Cx47, which is primarily localized to their cell bodies. Thus, the Cx47 mutants associated with PMLD likely disrupt the gap junction coupling between astrocytes and oligodendrocytes.     

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.     

MEGF10 related myopathies: A new case with adult onset disease with prominent respiratory failure and review of reported phenotypes

Recessive mutations in MEGF10 (multiple epidermal growth factor 10) have been reported in a severe early onset disorder named Early Myopathy, Areflexia, Respiratory Distress and Dysphagia, and a milder form with cores in the muscle biopsy; and a possible genotype–phenotype correlation determining the clinical presentation has been suggested. We undertook exome sequencing in a 66 year old male with a 20 year history of progressive proximal and distal weakness of upper and lower limbs, facial weakness and dysphagia, who developed respiratory failure requiring ventilation while still ambulant in his 50s. Muscle biopsy demonstrated myopathic changes with aggregation of myofibrillar proteins. Mutations in MEGF10 were identified: a novel essential splice site (c.1426+1G>T) and a novel missense variant (c.352T>C, p.(Cys118Arg)). We performed a detailed review of all reported MEGF10 cases (n = 20), and confirmed the presence of a genotype–phenotype correlation, namely that with ≥1 null mutation onset of respiratory dysfunction occurs in the first year of life, whereas with 2 missense mutations, respiratory dysfunction occurs at 10 years old or much later, as in the patient reported here. Our findings expand the phenotype of MEGF10 mutations to include onset in the 5th decade, and discuss the spectrum of MEGF10 related disease.     

Genetic background determines phenotypic severity of the Plp rumpshaker mutation

The rumpshaker mutation of the proteolipid protein (Plp) gene causes dysmyelination in man and mouse. We show that the phenotype in the mouse depends critically on the genetic background in which the mutation is expressed. On the C3H background there is normal longevity whereas changing to a C57BL/6 strain results in seizures and death at around postnatal day 30. The more severe phenotype is associated with less myelin and reduced levels of major myelin proteins. There are also more apoptotic cells, including oligodendrocytes, increased numbers of proliferating cells, increased numbers of NG2+ oligodendrocyte progenitors and increased microglia compared to the milder phenotype. The number of mature oligodendrocytes is similar to wild-type in both strains of mutant, however, suggesting that increased oligodendrocyte death is matched by increased generation from progenitors. The dichotomy of phenotype probably reflects the influence of modifying loci. The localization of these putative modifying genes and their mode of action remain to be determined.     

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     

TNFα Inhibits Schwann Cell Proliferation, Connexin46 Expression, and Gap Junctional Communication

Schwann cell responses to nerve injury are stimulated, in part, by inflammatory cytokines. This study compares changes in the phenotype of cultured Schwann cells after exposure to the cytokine tumor necrosis factor (TNF)-α or the mitogen neu differentiation factor (NDF)-β. TNFα inhibited proliferation in a dose-dependent manner without altering Schwann cell survival. TNFα also reduced both gap junctional conductance and Lucifer yellow dye coupling between Schwann cells. Moreover, both P₀and glial fibrillary acidic protein (GFAP) immunoreactivity were reduced. By contrast, NDFβ initially had little effect on cell division although it reduced junctional coupling within 8 h. However, by 48 h, NDFβ stimulated proliferation with a concomitant increase in coupling. Dividing Schwann cells (BrdU⁺) were preferentially dye coupled compared to nondividing cells, indicating an association between proliferation and coupling. Moreover, cultured Schwann cells expressed connexin46 mRNA and protein, and changes in the levels of the protein correlated with the degree of proliferation and coupling. The data thus provide evidence for cytokine-induced modulation of Schwann cell antigenic phenotype, proliferation, and gap junction properties. These observations suggest that enhanced gap junctional communication among Schwann cells after nerve injury could help to coordinate cellular responses to the injury, and that TNFα may be a signal which terminates proliferation as well as junctional communication.     

Aberrant trafficking of a Leu89Pro connexin32 mutant associated with X-linked dominant Charcot–Marie–Tooth disease

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.     

Panglial gap junctional communication is essential for maintenance of myelin in the CNS

In this study, we have investigated the contribution of oligodendrocytic connexin47 (Cx47) and astrocytic Cx30 to panglial gap junctional networks as well as myelin maintenance and function by deletion of both connexin coding DNAs in mice. Biocytin injections revealed complete disruption of oligodendrocyte-to-astrocyte coupling in the white matter of 10- to 15-d-old Cx30/Cx47 double-deficient mice, while oligodendrocyte-to-oligodendrocyte coupling was maintained. There were no quantitative differences regarding cellular networks in acute brain slices obtained from Cx30/Cx47 double-null mice and control littermates, probably caused by the upregulation of oligodendrocytic Cx32 in Cx30/Cx47 double-deficient mice. We observed early onset myelin pathology, and ～40% of Cx30/Cx47 double-deficient animals died within 42 to 90 d after birth, accompanied by severe motor impairments. Histological and ultrastructural analyses revealed severe vacuolization and myelination defects in all white matter tracts of the CNS. Furthermore, Cx30/Cx47 double-deficient mice exhibited a decreased number of oligodendrocytes, severe astrogliosis, and microglial activation in white matter tracts. Although less affected concerning motor impairment, surviving double-knock-out (KO) mice showed behavioral alterations in the open field and in the rotarod task. Vacuole formation and thinner myelin sheaths were evident also with adult surviving double-KO mice. Since interastrocytic coupling due to Cx43 expression and interoligodendrocytic coupling because of Cx32 expression are still maintained, Cx30/Cx47 double-deficient mice demonstrate the functional role of both connexins for interastrocytic, interoligodendrocytic, and panglial coupling, and show that both connexins are required for maintenance of myelin.     

Genotypic and phenotypic spectrum of CADASIL in Japan: the experience at a referral center in Kumamoto University from 1997 to 2014

This study elucidates the genotypic and phenotypic spectrum and histopathological findings related to cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) in Japan. For this single-center retrospective observational study, we enrolled 215 patients who were clinically suspected of having CADASIL and were examined at Kumamoto University from 1997 to 2014, and we diagnosed CADASIL in 70 patients. We found 19 different NOTCH3 mutations in the patients, with the NOTCH3 Arg133Cys mutation being found most frequently. We also found the Arg75Pro mutation, a cysteine-sparing NOTCH3 mutation. CADASIL patients with this Arg75Pro mutation were frequently found throughout Japan, and fewer patients with the Arg75Pro mutation showed MRI hyperintensity in the anterior temporal pole compared with patients with other NOTCH3 mutations. Significantly more CADASIL patients with the NOTCH3 Arg133Cys mutation had hyperintensity in the external capsule compared with CADASIL patients with the other mutations not including the NOTCH3 Arg75Pro mutation. We also showed postmortem pathological findings of the first Japanese CADASIL case with the NOTCH3 Arg133Cys mutation, and histopathological findings of fresh frozen skin biopsy specimens of CADASIL patients. In conclusions, the spectrum of NOTCH3 mutations in Japanese CADASIL patients may be partially explained by founder effects. Genotype–phenotype correlations may exist in CADASIL, which should be considered so as to make an accurate diagnosis of CADASIL in each population. Fresh frozen skin biopsy specimens may aid detection of Notch3 deposits on vascular walls for an improved diagnosis of CADASIL.     

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.