Introduction

Oligodendrocytes (OLs) are the cells involved in synthesizing and wrapping myelin around the axons in the CNS. They arise from oligodendrocyte progenitor cells (OPCs) following CNS development [1]. Numerous studies, mostly carried out in vitro, characterized the differentiation and maturation progress through OPC—pro-OL—immature OLs—mature OLs, each of them expresses specific antigens [2–6]. Among them, mature OLs are responsible for myelination, which is necessary to insure efficient information processing. In addition, the impairment of either OL development or inhibition of myelin repair results in demyelinating diseases, such as multiple sclerosis (MS), metachromatic leukodystrophy or even schizophrenia [7]. Therefore, considerable efforts have been devoted to promoting the differentiation and maturation of OLs.

In the last decade, transcriptional regulation of OL differentiation has been intensively studied and a series of OL specific transcription factors (TFs) have been identified. Positive TFs such as Olig1/2, Sox10, NKx2.2, and YY1 interact with negative TFs like ID2/4, Hes5, and Tcf4 to coordinate the expression of myelin related genes [8, 9]. As OLs sit in a special microenvironment containing neurons, ASTs, and microglia, cell–cell interaction should be considered as an important issue related to myelination. For instance, when OPCs migrate towards naked axons, the axon expresses numerous inhibitory cues (such as Jagged1, PSA-NCAM) preventing OL differentiation to ensure enough time for the proper spatial arrangement of OLs and temporal control of myelination.

Recently, it was found that intercellular interactions between competing OLs/OPCs also influence the number and length of myelin internodes as well as the initiation of differentiation [10, 11]. At the same time, a typical feature of glial cells is their high expression level of Cxs, the molecular constituents of gap junction channels and hemichannels [12]. Increasing evidence indicates that Cx-mediated communication among ASTs, among OLs or between ASTs and OLs may be important for the development of OLs, including proliferation, differentiation and myelination. Recently, two reviews have discussed glial cells and Cxs. However, they were focused on separated aspects of the involvement of Cxs in physiological condition and myelin disruption. In fact, one review has considered the role of Cxs in myelinating glial cells [13] while the other on demyelination diseases [14]. Here, we will discuss the involvement of Cxs in oligodendroglial lineage cells and their role in myelination and remyelination (Figs. 1 and 2), as well as the consequence of lack of one or two Cxs on dysmyelination (Fig. 3). Associated mechanisms will also be suggested and discussed. Based on this, we think that the understanding of these issues can be improved and might provide valuable insights into therapeutic strategies for demyelination related diseases.

Fig. 1
figure 1

Developmental schedule of myelination. Blue symbols: OPCs appear in the rat spinal cord as early as embryonic day E15. These precursor cells actively proliferate and migrate throughout the spinal cord until shortly after birth, when the number of OPCs is relatively constant. Beginning around postnatal day P7, a gradual decline in the number of OPCs can be observed, which is followed by the appearing of differentiated OLs [10]. Red symbols: during brain development, Cx47 is first detected in the embryonic stage and its expression increases successively mainly in regions populated by developing oligodendroglia. The expression declines postnatally toward adulthood and immunoreactivity is restricted to a few specific areas, such as corpus callosum, striatum, cerebellum, and spinal cord. Cx32 is first detected on E17.5, Cx29 is detected on P0 and their expressions increase to a higher level in adult brain [45]. Gray symbols: in cultures, homotypic ionic and dye coupling only exhibit in OLs while the heterotypic dye coupling first appears in 3-week co-cultures of ASTs and OPCs and increases to an incidence of 25 % after 6 weeks [48]

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Fig. 2
figure 2

Adult OPCs and connexins participate in de- and remyelination. Blue symbols: Following white matter injuries or in some demyelinating diseases, the inflammation is characterized by activated microglia and reactive ASTs. Adult OPCs in and around the damaged area rapidly undergo changes from an essentially quiescent state to that of proliferative expansion, migration toward regions containing naked axons and subsequent differentiation into OLs (new OLs). Red symbols: Cx47 and Cx32 in mature OLs decrease with myelin degeneration, so do Cx47 and Cx32 gap junctions channels (GJCs). Gray symbols: based on data from the EAE model, we predict that the homotypic GJCs (Cx47 GJC, Cx32 GJC) gradually increase but do not reach a naïve level, while the heterotypic GJCs are not determined yet (ND) [107]. Note: the starting point of this process is not zero (arrows) because the adult OPCs maintain a potential of proliferation, differentiation and myelination even under normal condition. During this progress, data about those oligodendroglial Cxs expression pattern are not available (ND)

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Fig. 3
figure 3

Scheme of connexin-based panglial networking and the associated pathological changes found in Cx-related transgenic mice in CNS. a In the normal CNS, homotypic gap junctions are formed between neighboring OLs (Cx47-Cx47, Cx32-Cx32), neighboring ASTs (Cx43-Cx43, Cx30-Cx30), and heterotypic gap junctions formed between ASTs and OLs (Cx47-Cx43, Cx32-Cx30). b In Cx47/Cx32 dKO mice, only homotypic gap junctions between ASTs are preserved, axons are wrapped by thinner or vacuolated myelin sheaths. Other pathological features are detected such as vacuolated myelin sheath, enlarged periaxonal space, OL cell death and axonal loss. Activated microglia containing myelin debris is occasionally encountered. c In Cx43/Cx30dKO mice, gap junctions between ASTs or between ASTs and OLs are lacking. Widespread pathologies of white matter comprising vacuolated OLs, myelin vacuolation, edematous and hyperplasia ASTs and degenerated axons are observed. Moreover, there are less mature OLs. d Mice lacking Cx43 and Cx32 cannot form heterotypic gap junctions but homotypic coupling is preserved. Pathological features are characterized by thinner myelin sheaths, myelin debris and reduced ASTs with fragmentized plasma membrane. e Mice lacking Cx47 and Cx30 cannot form heterotypic gap junctions but homotypic coupling is preserved. Axons are wrapped by thinner or vacuolated myelin sheaths, the pathologies are also featured by vacuolated OLs, decreased OLs number, enlarged periaxonal collars, myelin debris, severe astrogliosis and microglia activation. These schematic drawings are based on [46, 50, 67, 77]. Note that there is no mention about Cx hemichannels since there is no data related to the Cx channel function in these transgenic animals

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Connexin-mediated glial networking and myelin integrity in the central nervous system

Connexin-mediated glial networks in the central nervous system

Gap junctions are localized at cell contacts and are composed by Cxs that are characterized by four transmembrane domains with cytoplasmic COOH and NH2 terminals. In each cell membrane, six Cxs bind together to form a hemichannel, or connexon, and two facing hemichannels from opposing cell membranes form a gap junction channel. Finally, aggregates of gap junction channels cluster in the cell membrane to form gap junction plaques [15]. Individual connexon can be formed by the same or different subtypes of Cxs (homomeric and heteromeric channels, respectively). Also, a single gap junction channel can be composed of the same or different subtypes of connexons (homotypic and heterotypic channels, respectively) [16]. In vertebrates, gap junction channels are permeable to ions and molecules smaller than 1–1.2 kDa, including cyclic nucleotides, metabolites, vitamins, amino acids, and ions [17]. The permeability of gap junction channels is regulated by the intracellular calcium concentration, transmembrane voltage and/or transjunctional voltage, phosphorylation of Cxs and other intracellular factors [18–20].

In ASTs, two major Cxs are expressed, Cx30 and Cx43, and to a less extent Cx26 [21]. Cx30 is co-expressed with Cx43 in ASTs located in junctional plaques of gray matter but is undetectable in white matter tracts [22]. Cx26 is, however, expressed within some subcortical regions at a very low level. Cx channels can also function as hemichannels formed by Cx43 in ASTs that are closed in physiological conditions to maintain cellular integrity [23]. These hemichannels, however, can open under certain conditions and become permeable to glutamate, ATP or glucose, after being triggered by metabolic inhibition, proinflammatory cytokines and a moderate increase in intracellular Ca2+ concentration [24, 25].

In OLs, three kinds of Cxs including Cx29, Cx32, and Cx47 are expressed and each Cx is uniquely localized in different brain regions and subcellular regions of the cells [26, 27]. For instance, Cx29 tend to be localized to the inner membrane of small myelin sheaths and thus may not form intercellular channels, whereas Cx32 is localized on the outer membrane of large myelin sheaths and between adjacent layers of myelin sheath at the “paranode”. Cx47 is the main Cx in OLs and is largely restricted to cell bodies as well as the initial processes of OLs. Co-localization of Cx32 and Cx47 is found in many gap junction plaques in OL somata and outer membrane of myelin sheath [28, 29].

Because OLs and ASTs express non-overlapping sets of Cxs, the intercellular channels formed between them are heterotypic (i.e., asymmetric) with regard to their Cx content. Thus, homotypic gap junction channels are formed between neighboring ASTs and between neighboring OLs, while heterotypic gap junction channels are formed between ASTs and OLs [30]. Several models for such heterotypic interactions have been proposed. One recent in vitro study states that OL Cx47 can form heterotypic channels with AST Cx43 or Cx30 but not Cx26, whereas OL Cx32 can form channels with AST Cx30 or Cx26 but not Cx43. Thus, four types of heterotypic intercellular channels, Cx47-Cx43, Cx47-Cx30, Cx32-Cx30, and Cx32-Cx26 could contribute to OL-AST communication in vivo [30]. In addition, ASTs and OLs can establish “reflexive” or “autologous” gap junctions within the same cell [31, 32].

Connexin expression patterns are correlated with myelination

Similar to neurons and ASTs, OLs arise from pluripotent neuroepithelial precursor cells of the CNS. In the mouse spinal cord, ~85 % of OPCs are generated from the motor neuron progenitor domain in the ventral ventricular zones, starting at about embryonic day E12.5. Around E15, the secondary wave of the precursors’ generation appears in more dorsal regions by trans-differentiation of radial glia (RG) [33, 34]. Moreover, there are also ventral and dorsal sources in the forebrain. In the telencephalon, the most ventral-precursors in the medial ganglionic eminence are produced around E12.5, generation of the lateral ganglionic eminence derived precursors starts a few days later, and generation of the cortex-derived precursors occurs mainly after birth [35].

Once specified, OPCs exhibit multidirectional migration from their origins to occupy all areas of the CNS. During migration, OPCs maintain their proliferative property and only exit the cell cycle after reaching their destination with a suitable cell number. Then, these cells undergo differentiation through immature OLs to mature myelinating OLs. Normally mature OLs (appear after P8 in mice) start to express myelin proteins and extends multiple exploratory processes around axons after receiving a set of promoting signals around P11 in mice [36]. Although most of the OPCs differentiate into mature OLs during CNS development, some of them remain in an undifferentiated state after completion of myelination, which are named as adult OPCs. These cells have the potential to remyelinate axons after demyelination [37]. As an endogenous source of progenitor cells, adult OPCs represent 2 to 9 % of the CNS cell population [38]. Current evidence indicates that these adult OPCs express the same markers (PDGFRα and NG2) and show similar morphology characters as the perinatal progenitors [39].

During brain development, Cx43 emerges early in development, around 12 days of gestation in rat RG development processes. RG is a class of undifferentiated cerebral cortical progenitor cells that give rise to excitatory neurons and ASTs [40]. Cortical neurogenesis in the mouse normally occurs between E11 and E17, after which RGs generate mainly ASTs [41, 42]. Thus, astroglial Cx43 is expressed early on E18 and increases throughout brain development. Moreover, astroglial Cx30 is expressed in juvenile rodents between the second and third postnatal weeks [22, 43, 44]. On the other hand, Cx47 is detected in the embryonic mouse brain at E10.5 and its expression increases successively mainly in regions populated by developing oligodendroglial cells. The expression of Cx47 declines postnatally toward adulthood and its immunoreactivity is restricted to a few specific areas, such as corpus callosum (CC), striatum, cerebellum and spinal cord. Cx32 is first detected on E17.5 while Cx29 is detected on P0 and their expression increases to a higher level in adult brain [45]. In mouse spinal cord, temporal profile in the level of Cx47 mRNA is similar to that of other myelin related genes such as Plp, which encodes an abundant intrinsic membrane protein of compact myelin [46]. The levels of Cx47, Cx32, and Plp mRNA are much lower in Plp mutant rats that results in a failure of OL maturation and myelin formation [47]. Together, these data support the idea that oligodendroglial Cxs and perhaps, astroglial Cxs, are part of the program correlated with myelin gene expression (Fig. 1). To address this issue, the functional gap junctions formed between ASTs and oligodendroglia lineage cells in vivo need to be determined.

So far, there are only in vitro studies showing the time specific development pattern of homotypic gap junctions between the OLs and the heterotypic gap junctions between OLs and ASTs. OPCs identified with A2B5 antibodies were characterized by the lack of ionic and dye coupling, whereas immature OLs, identified with galactosylceramide (GalC) antibodies, exhibit both types of intercellular communication. In co-cultures of ASTs and OLs, Lucifer yellow is exchanged in both directions between the two glial cell types. This heterotypic dye coupling first appears after 3 weeks in culture and increases to an incidence of 25 % after 6 weeks [48] (Fig. 1). Such a dynamic progress suggests that glial gap junctions are highly correlated with myelination.

Connexin-mediated nutritional support to oligodendrocytes

In white matter tracts, axons are almost completely encapsulated by myelin, and reside in a milieu that is virtually defined by ensheathing OLs. Previously, the traditional view was that axons can only receive energy from ASTs at the nodes of Ranvier, but recently a new function of OLs in energy supporting to neurons has been proposed. Myelinating glial cells no longer carry the role as a passive insulator, they appear to be actively involved in myelin independent sensing axonal energy needs and maintaining long-term axonal integrity [49]. The OL-AST gap junction provides OLs a physical connection to the blood–brain-barrier (BBB) and to the nutritive support by brain capillaries. Under working conditions, this “panglial” networking likely represents a primary source for axonal energy metabolism. Indeed, ASTs provide main metabolites to support myelin-forming OLs from early development stage, which is reflected in the lethal phenotype of Cx47/Cx30 deficient mice [50]. Moreover, the entrance of glucose through GLUT1 and its trafficking through gap junctions [51] could be used for glycolysis in OLs. Pyruvate is then metabolized in mitochondria for ATP generation (tricarboxylic acid cycle, TCA). With the onset of myelination (“developmental switch”), glucose also serves the synthesis of fatty acid (FAS) and myelin lipids from acetyl-CoA [52]. In postmyelination OLs, glycolysis can yield sufficient ATP to support OL survival. Glycolysis products are used by myelinated axons when energy levels are low. In the latter condition, lactate may first be transferred through the multiple myelin sheaths by reflexive gap junction formed by Cx32. Then it can directly be transferred via monocarboxylic acid transporters (MCT1, MCT2), which reside in myelin internodes and the axonal compartment, thus lactate can be rapidly used by axons [49].

Glial connexins contribute to the homeostasis of the microenvironment important for oligodendrocyte function

It has been demonstrated that the strong negative resting potential and relatively high permeability to K+ are a typical feature of ASTs. These properties could facilitate the diffusion of the absorbed K+ into available sinks, such as local capillaries after extensive neuronal activity [53]. Several mechanisms have been proposed to account for K+ uptake and release, including Na+/K+ ATPases, ion co-transporters and K+ channels, especially the inwardly rectifying K+ channel Kir4.1 [54], an important contributor to K+ homeostasis in the CNS [55]. In addition, inclusion of OLs into the network may improve the spatial buffering capacity. In support of this idea, there is a striking similarity in the phenotypes found in the Cx47/Cx32 dKO mice, Cx43/Cx30 dKO mice as well as the Kir4.1 knockout mice [56]. Kir4.1 is expressed in OLs and is highly enriched at the perivascular endfeet of ASTs [57]. Genetic and physiological evidence indicates that OL gap junctions contribute to spatial buffering of potassium released during neuronal activity. In Cx47/Cx32 dKOs, vacuolation correlates with neuronal stimulation [58]. The expression of Cx29 hemichannel and Na+/K+ ATPase isoform in OL periaxonal cytoplasm might facilitates import of K+ into the periaxonal cytoplasm. Then, K+ may be redistributed through “reflexive” gap junctions between the layers of myelin at paranodes [29]. This pathway could dramatically shorten the distance for diffusion from the inner adaxonal to the outer abaxonal cytoplasm, as has been shown in the case of myelinating Schwann cells (SCs) [17]. After this, gap junctions between ASTs and OLs could allow K+ access from OLs to ASTs cytosol. Finally, as proposed in the original spatial buffer hypothesis, extensive gap junctional coupling in ASTs provides a pathway for dispersal of K+ to distal locations, by a passive mechanism. The latter includes K+ release from capillaries and pia mater via Kir4.1. Thus, glial gap junction channels might play an important role in brain homeostasis by redistributing K+ following axonal activity [58, 59], which is certainly essential for myelin formation and maintenance.

Connexin-mediated glial networking functions in myelination

What connexin knockout mouse models tell us about myelination

Although the function of glial networking in myelination has not been intensively investigated, there are Cx-KO models and non-genetic models indicating the essential role of Cxs-mediated gap junctional communication, and perhaps hemichannels activity in OL differentiation and myelination.

Connexin abnormality in OLs induces dysmyelination

With the development and application of multiple Cx-transgenic mice, increasing data has been found concerning the role of Cxs in myelination and remyelination. Indeed, it was reported that mice lacking either Cx47 or Cx32 show mild myelin vacuolation [60, 61]. However, animals lacking both Cxs die by postnatal week 6 and exhibit profound abnormalities in central myelin characterized by thin or absent myelin sheaths, vacuolation, enlarged periaxonal spaces, OL death, and axon loss (Fig. 3). These data provided the first evidence showing that ASTs-OLs gap junctional communication is crucial for myelination in CNS [46]. The interesting point is that demyelination in this dKO mice show regional variation.

Charcot Marie Tooth disease (CMT1X), caused by mutations in the gene encoding Cx32 in Schwann cells, is a peripheral neuropathy characterized by axonal alterations, with an impact on myelin thickness and loose myelin. However, this pathology also exhibits some CNS manifestations including cerebellar ataxia or encephalopathy under stress conditions [62]. Different kinds of mutation show different function abnormalities, but it is clear that altered synthesis of protein and loss of gap junction function is the fundamental mechanism in most CMT1X mutants.

Pelizaeus-Merzbacher disease (PMLD) caused by mutations in Cx47, is characterized by severe CNS myelin deficiency. Patients with PMLD often exhibit nystagmus, impaired motor development, ataxia, progressive spasticity and sometimes a mild peripheral neuropathy. It has been found that Cx47 ablation can completely abolish coupling of OLs to ASTs while the number of coupled OLs was reduced by 80 % [63], suggesting the important role of Cx-mediated gap junctions between OLs and ASTs in myelin formation.

Connexin abnormality in ASTs induces dysmyelination

As regarding astroglial Cxs, mutations in the human GJA1 gene encoding Cx43 have been identified in a rare, mostly autosomal dominant syndrome called oculodentodigital dysplasia (ODDD). About 30 % of the ODDD patients show neurological malfunctioning, which may be the result of dysmyelination. Since MRI imaging studies have brought up diffuse bilateral abnormalities in the subcortical cerebral white matter, possibly indicating a slow progressive leukodystrophy [64]. In animal models, mutations in Cx43 or Cx30 alone do not show signs of myelin abnormality [65, 66]. However, double knockout of Cx43/Cx30 mice have been found to show glial “edema” and myelin vacuolation in white matter, with the hippocampus CA1 region (a non-myelinated region) as the only area where obvious pathology is found in gray matter (Fig. 3). In these dKO mice, fewer CC1+ mature OLs and reduced MBP expression were detected, and no difference was observed considering Olig2+ cells in the CC, raising the possibility that OL differentiation is influenced by AST gap junctional communication [67]. In addition, conditional deletion of the astroglial Cx43 leads to the loss of OPCs coupled with OLs which may be a result of affected OPC population [63]. This is of great interest regarding the role of astroglial Cx43 in myelination as the interaction between OLs and OPCs has been shown important for maturation of OLs [68].

However, a special attention should be paid to Cx43, as this Cx has been shown to regulate a large number of genes, many of which are not gap junction-dependent [69]. In addition, results from studies in cultured ASTs suggest a role of Cx43-based reflexive gap junctions in cytoskeleton reorganization and process formation [31]. Cx43 also interacts with AQP4 (a water-selective channel in AST membrane) to influence the cytoskeleton and change the morphology of ASTs in culture [70]. Besides ASTs, Cx43 is also expressed in either undifferentiated or differentiated neurospheres, and phosphorylation of Cx43 is strongly increased in adherent neurospheres suggesting the post-translational regulation of Cx43 during differentiation of neural stem cells (NSCs) [71]. Indeed, phosphorylation of Cx43 has been implicated in several aspects of gap junction channel function and could be tightly linked to the program of cell differentiation [72–74]. Moreover, Cx43 knockout can induce downregulation of P2Y1 receptor and following calcium signals alteration and thus impact the proliferation and migration of neuronal progenitors. This can be regarded as a non-channel function of Cx43 to regulate proliferation and differentiation of nearby cells [75]. Finally, a new study provides strong evidence that astroglial Cx43 expression is necessary for phosphorylation and stability of oligodendroglial Cx47 proteins [76]. These facts reveal the complexity of Cx43 function in ASTs that should be considered when dealing with Cx43 mutant mice.

Double knock out of glial connexins in a mixed manner induces dysmyelination

To detect functions of the complex glial networking, mice mutant for either OL Cxs or AST Cxs have been developed: i.e., dKO of Cx47/Cx43, dKO of Cx43/Cx32 and dKO of Cx47/Cx30. Surprisingly, while the phenotype of the Cx47/Cx43 dKO mice was mild as predicted, the Cx43/Cx32dKO mice exhibited myelin vacuolation accompanied by little effect on OLs but marked increase in cell death of ASTs. It is speculated that Cx32 reflexive gap junction loss caused myelin vacuolation. Cx43/Cx32 together may mediate signaling events promoting AST survival but the nature of the signals and the details of the mechanism underlying AST pathology remain to be determined [77] (Fig. 3).

In the Cx47/30dKO mouse model, homotypic coupling between OLs and between ASTs still exists due to the preservation of oligodendroglial Cx32 and astroglial Cx43, respectively, while heterotypic gap junctions between OLs and ASTs are lacking. With the development of white matter, myelin pathology such as severe vacuolation in all white matter tracts of the CNS is observed and about 40 % of the mutant animals die within postnatal 42 to 90 days, accompanied by severe motor impairments. Furthermore, Cx47/Cx30 double deficient mice exhibit a decrease of the number of OLs together with a severe astrogliosis and microglial activation in white matter tracts (Fig. 3). Obvious myelin lesions and OL death start only in later development stage, suggesting that although necessary for proper myelin development, the formation of panglial networks seems more important with regard to myelin maintenance and brain function. As regarding the mechanism, the author presumes that osmotic dysregulation or local hypoxic conditions may be the cause of the observed phenomenon since panglial networking has been considered to be important for ion homeostasis, especially concerning K+ siphoning upon neuronal activity [50, 78].

Connexin-related demyelination in non-genetic models

In several non-genetic animal models, a relationship between Cx-mediated glial communication and demyelination can also be found. A rapid correction of hyponatremia can usually cause demyelination but the underlying mechanism remains a mystery. Recently, it has been found that rapid osmotic changes can trigger apoptosis of ASTs followed by a downregulation of Cx43 and Cx47, secondary inflammation, microglial activation and finally demyelination. Thus, it is predictable that Cx-mediated communication between glial cells is important for myelin maintenance [79].

In an inflammation-induced demyelination model, the injection of lipopolysaccharide (LPS) into the white matter of spinal cord initiates microglia activation followed by a functional disturbance of ASTs, mainly reflected by retraction of astrocytic foot processes with loss of AQP-4 and reduced expression of Cx30 and Cx43 [80]. Moreover, injection of LPS into the CC of rats results in a downregulation of Cx43 and GFAP following by OL cell apoptosis and demyelination [81]. This time course suggests that demyelination may be a secondary consequence of AST dysfunction, probably due to damages in Cxs-mediated glial networks.

Recently, the involvement of Cxs in MS pathology has been studied. Compared to the brains in the control group, both Cx32 and Cx47 gap junction plaques and protein levels are reduced in and around MS lesions, while Cx43 is increased as part of astrogliosis. In the normal appearing white matter (NAWM), Cx32 is significantly reduced along myelinated fibers whereas Cx47 shows increased expression mainly in OPCs. On the other hand, Cx43 shows a modestly increase while gap junction plaques are unchanged. These findings indicate that OL gap junctions are affected not only in chronic MS lesions but also in NAWM [82].

Connexin-mediated calcium waves may be important for transcriptional regulation of the oligodendroglial differentiation

The development of OLs lineage cells and myelination of individual axon is a highly regulated process controlled by a number of mechanisms, including intracellular and extracellular factors. In this process, the inner and outer environment changes with time and sonic hedgehog (Shh), Bone morphogenetic protein (BMP), Notch as well as Wnt signaling pathways are either activated or inhibited followed by spatio-temporal expression of OL lineage specific transcription factors (TFs), which in turn regulate proliferation, migration and the terminal maturation of OLs [83–86]. In the last decade, transcriptional regulation of OLs differentiation has been intensively studied and a series of OL specific TFs has been identified. Indeed, positive TFs such as Olig1/2, Sox10, NKx2.2, YY1 interact with the negative TFs like ID2/4, Hes5, Tcf4 to coordinate the expression of myelin related genes [8, 9]. Among them, valuable insight has been provided concerning the role of basic helix-loop-helix TFs Olig1 and Olig2. Indeed, Olig2 is essential for oligodendroglial lineage specification while Olig1 plays a crucial role in the maturation of OLs [87–89]. Our recent work shows that stage-specific deletion of Olig2 conveys opposing functions on differentiation and maturation of OLs, and that this stage-specific role of Olig2 seems to depend on the function of Olig1 [90]. Interestingly, the function of Olig1 is highly regulated by its phosphorylated state as phosphorylated Olig1 translocates into cytoplasm to promote membrane expansion and maturation of OLs [91].

Based on these observations, we hypothesize that these TFs can also be regulated by calcium signals. Indeed, Ca2+ regulates the expression of downstream genes and it is well known that gap junction channels or/and hemichannels contribute to the propagation of intercellular Ca2+ waves in glial cells [92]. On the other hand, it has been shown that the spreading of Ca2+ waves through gap junctions between ASTs and OLs is bi-directional [93], suggesting that both cell types can influence each other their respective Ca2+ signaling pathways. It is most likely that AST can regulate OL differentiation by calcium information through gap junctions. Thus, insufficient calcium signals due to Cx-KO in those genetic models may be one reason for abnormal OL differentiation and hypomyelination. Furthermore, other small molecules transferred by AST-OL gap junctions may also participate in gene expression regulation. For instance, some linear molecules, like polypeptides or siRNA, might “wiggle through” gap junctions formed by Cx43 and result in abnormal gene expression or impairment of cellular functions [94].

Connexins-mediated glial networking functions in demyelination and remyelination

Comparison of myelination and remyelination

Myelin is essential for speeding up the signaling transduction as well as for nutrition support to axons. However, it is easily damaged by immune attack, inflammation, and other brain insults. What happens after myelin injury is therefore an important question that needs to be addressed. It is now accepted that both developmental myelination and remyelination contain the same key stages of OPC expansion within or toward regions containing naked axons by proliferation, migration, and subsequent differentiation into myelin-forming OLs. Indeed, following white matter injury, adult OPCs near the damaged area rapidly transit from the quiescent state to the proliferative state which is associated with upregulation of myelin related genes [95]. Genetic fate-mapping studies show that adult OPCs have the capacity to myelinate continuously throughout normal adult life [96]. However, in demyelination diseases such as MS, remyelination is only observed in the early stage of the acute demyelination lesion. This process becomes incomplete and eventually fails even though adult OPCs are present in the demyelinated areas [97]. Additionally, as adult OPCs divide more slowly than their developmental counterparts [98], applying mitogens to compensate this potential shortage does not enhance remyelination [99]. These results indicate that frustrated differentiation of OLs is likely the barrier impeding myelin repair rather than the recruitment of OPCs [100].

Increasing evidences support the notion that remyelination is not the simple recapitulation of myelin formation. These two processes differ in many aspects. Firstly, in striking contrast to developmental myelination, CNS remyelination can also be mediated by SCs, the myelin-forming cells in the peripheral nervous system. Transgenic fate-mapping studies revealed that the majority of these myelin-forming cells are derived from OPCs except a few from SCs [101]. One possibility is that the extrinsic cues within demyelinated lesion lead PDGFRα/NG2-expressing cells (CNS-resident glial progenitor cells) to differentiate into SCs, and this case occurs in areas where astrogliosis is absent [101]. Secondly, remyelination is a progress associated with inflammation circumstance in which microglia and ASTs are activated by myelin debris with a large amount of bioactivity factors released. Among them, mitogens can promote adult OPC proliferation while chemotactic factor induce migration of these cells to the impaired regions [102]. On the other hand, activated microglia can also remove myelin debris and thus promote OLs differentiation. In this immune response environment, adult OPCs proliferate to a certain amount and then exit the cell cycle, differentiate into mature OLs for myelin repair [103]. Finally, the most obvious difference between myelination and remyelination is the relationship between axon diameter and length and thickness of myelin sheaths. In developmental myelination, larger diameter axons are invested with proportionally thicker myelin and longer internodes. In remyelination, however, the length and thickness of myelin sheaths remain roughly constant regardless of the axon diameter. Furthermore, both Notch and Jagged are presented around lesions where remyelination are observed, suggesting that they do not block myelination as they do in development, but rather they may regulate the kinetics of myelin repair [104].

Taking all these points into account, it is difficult to totally understand remyelination with the present knowledge regarding development myelination. However, most regulators appear to be well conserved in these two progresses (see Figs. 1 and 2). Thus providing a favorable environment for adult OPC differentiation and maintaining the adult OPC pool is meaningful to promote remyelination.

Do glial connexins participate in demyelination and remyelination found in animal models?

As collective data suggest an important role of Cxs in myelin formation during the progression normal development, it is reasonable to consider the significant position of Cxs in myelin regeneration or remyelination process. Although adult OLs are cell type responsible for remyelination, ASTs also play a crucial role in establishing a communication networking and creating a favorable environment for remyelination [105]. In a chronic guinea pig model of experimental allergic encephalomyelitis (EAE), Cx43 expression shows a dynamic pattern: absent in demyelinated lesions but considerably increased in remyelinating regions, indicating that astroglial Cx43 expression, and possibly intercellular communication (OL-AST, AST-AST) are required for effective myelin repair [106]. In another EAE model, during acute pathology phase, both Cx47 and Cx32 were severely reduced within and around lesions. Cx47, however, was localized intracellularly in OLs while protein levels remained unchanged. This redistribution coincided with the loss of Cx43 gap junctions in ASTs. The expression of Cx47 and Cx32 increased with remyelination but decreased following relapsing. The author thus suggested that the impairment of both intra- and intercellular OL gap junctions cased by EAE may be an important mechanism for MS progression [107].

There is also a dynamic expression pattern of Cx47 in a cuprizone-induced demyelination mouse model. Normally Cx47 is slightly expressed in myelin and absent in ASTs. Nevertheless, under cuprizone-induced demyelination, Cx47 is absent from OLs after a short term upregulation in myelin sheaths, and then transiently expressed in ASTs. Following remyelinating progress, Cx47 is re-expressed in newly formed myelin [45]. These evidences suggest that Cx47 may play an important role in remyelination. Moreover, in the recruitment phase of remyelination, adult OPCs rapidly migrate to lesions, which need the cooperation of calcium and cytoskeletons in the cytoplasm. As Cx-mediate GJ channels and hemichannels are important for the propagation of intercellular calcium wave among glial cells (AST-AST and AST-OL), one can hypothesize that Cx-mediate glial networking is important for myelin repair. In addition, it has been reported that the levels of Cx expression and gap junction coupling are highly sensitive and controlled by external signals in astroglia [108]. Thus, the panglial Cx-mediated networking is expected to be also highly regulated and it is likely that in demyelinating diseases gap junction abnormality also play an important role in the pathogenic progress. Recently, we observed that in transgenic mouse lacking Cx43 in ASTs, obtained by interbreeding Cx43-flox mice with mice carrying hGFAP-cre, the proliferation of adult OPCs is inhibited (unpublished data). Based on this, we propose that astroglial Cx43 might participate in the regulation of the adult OPCs pool and thus influence remyelination process.

Conclusions and perspectives

In this review, we summarized the structure of Cxs, their physical properties and functions of hemichannels and gap junction channels in CNS glia. OLs and ASTs express non-overlapping kinds of Cxs, which emerge in a special time and location, thus gap junctions formed between them are time-space specific. To emphasize the essential role of Cx-mediated networking in myelinating and remyelinating progress, we reviewed the data about the Cx associated diseases in human, dysmyelination in transgenic mice and Cx expression pattern in some non-genetic model for different subsets of Cxs. The mechanisms by which these glial networks regulate the myelination and remyelination progress are most concerned. As regarding the extracellular aspects, Cx-mediated glial networking is important for maintaining CNS homeostasis and the nutritional support for OLs. In intracellular aspects, gliotransmitters released from hemichannels and cell signaling transported through gap junctions can regulate migration, proliferation and gene expression of the oligodendroglial lineage cells. But what we must pay attention is that the exact pathways about how these glial networks participate in the regulation of OPCs/adult OPCs development remains unknown. In addition, the dynamic formation of gap junctions between oligodendroglial lineage cells and ASTs in vivo during myelination and remyelination needs to be determined in order to better clarify the functional consequences of these networks.

All we discussed here on Cxs is to get a better understanding of the role of Cxs in the genesis, development, myelination and remyelination behavior of OLs. Clarification of these mechanisms can help a lot to treat demyelination diseases such as MS, PMLD, CMTX1, and so on. For instance, figuring out the pathway involved in Cx-mediated regulation of OL differentiation may provide clues for novel strategy to rescue hereditary dysmyelinating diseases and to improve remyelination by manipulating Cx expression or enhancing gap junction channel/hemichannel function.