GAP junctions: multifaceted regulators of neuronal differentiation

ABSTRACT

Gap junctions are intercellular membrane channels consisting of connexin proteins, which contribute to direct cytoplasmic exchange of small molecules, substrates and metabolites between adjacent cells. These channels play important roles in neuronal differentiation, maintenance, survival and function. Gap junctions regulate differentiation of neurons from embryonic, neural and induced pluripotent stem cells. In addition, they control transdifferentiation of neurons from mesenchymal stem cells. The expression and levels of several connexins correlate with cell cycle changes and different stages of neurogenesis. Connexins such as Cx36, Cx45, and Cx26, play a crucial role in neuronal function. Several connexin knockout mice display lethal or severely impaired phenotypes. Aberrations in connexin expression is frequently associated with various neurodegenerative disorders. Gap junctions also act as promising therapeutic targets for neuronal regenerative medicine, because of their role in neural stem cell integration, injury and remyelination.

1. Introduction: what are gap junctions?

Gap junctions (GJ) are intercellular membrane channels which facilitate direct cytoplasmic exchange of small molecules, substrates and metabolites between adjacent cells.^1–3 The basic structural unit of the GJ is the connexon, consisting of 2 hemichannels (HCs) forming a cylinder like structure with a hydrophilic channel (Figure 1,⁴). These connexons span the plasma membranes of closely aligned cells, forming intercellular channels that help with the exchange of small molecules (less than 1200 Da). The GJ proteins, called connexin (Cx) (Figure 1), are named according to their predicted molecular weights.^5,6 These proteins are encoded by a multi-gene family consisting of at least 20 members in mammals.^3,7 Connexin protein subunits are tetra-spanning membrane proteins with three conserved extracellular cysteine residues. These conserved residues are crucial for docking.⁸ The connexin subunits differ mostly in their cytoplasmic loop and carboxy-terminal region. The S–S structure in the protein represents conserved disulfide bonds in the extracellular domains of connexins.⁸ There are several types of connexins and these connexins display distinguishing distribution phenotypes in various tissues, with many tissues and cells expressing multiple connexins. In the central nervous system there are eleven subtypes of connexins.³ The specific subset and the varied expression of connexins depend on cell type as well as the developmental stage^3,9 (Table 1). Most electrophysiological studies of connexins have concentrated on studying the conductance of homotypic GJ channels, consisting of identical connexin proteins (Figure 1). GJ channels in a given cell type can also develop by the union of HCs each comprising homologous, but different connexin proteins. GJ channels connect the cytoplasm of contacting cells and coordinate electric and metabolic activity, whereas HCs assist in the communication between intra- and extracellular compartments thereby providing a diffusional scheme for ions and small molecules.³⁹ The main function of connexin HCs is attributed to the formation of GJ channels, however, recent research also suggests the presence of functional connexin HCs in non-junctional membranes. These channels facilitate uptake of small molecules as well as release of autocrine/paracrine signaling molecules to the extracellular compartments. In the central nervous system, GJ channels and HCs play a critical role in mediating ischemic tolerance⁴⁰ and they establish adhesive interactions.⁴¹ HCs release physiological molecules that are relevant for intercellular signaling under normal brain conditions. However, HCs also play a significant role in inducing homeostatic imbalance that is frequently observed in diverse brain disease. Each HC represents an assembly of six connexin protein subunits, and thus a GJ constitutes a total of 12 connexins.⁸ A single cell can express more than one connexin type that initiates expression of nonidentical connexins resulting in a heteromeric HC or connexon.⁵ Three different types of GJs have been reported depending on the composition: homomeric/homotypic, heteromeric and heterotypic⁸ (Figure 1). Homomeric or heteromeric HCs are composed of identical or different types of connexin isoforms, respectively. Homotypic or heterotypic GJs comprise two identical or two different types of HCs, respectively. Pannexins are another form of membrane channels.⁴² Of the three family members only Panx1 and Panx2 are expressed in the central nervous system (CNS).⁴³ Panx1 has paracrine functions as an ATP release channel along with the release of “find-me” signals for apoptotic cell clearance. Panx1 was also found to be connected to calcium signaling, and tumor growth suppression of gliomas. Panx2 along with gap junctions are known to control differentiation in neurons.⁴⁴ Gap junction plaques are relatively large structures on the plasma membrane, composed of hundreds or up to thousands of single individual channels, where gap junction channels are concentrated in specialized plaques of plasma membrane when cells are in close apposition.⁴⁵ These structures form as free HCs that are exposed to the plaque area of the plasma membrane, where each cell adds newly synthetized HCs (Figure 1d, [46]).⁴⁵ These structures are important for exchanging information between neighboring cells, thereby supporting synchronized and concerted responses⁴⁵ (Figure 2).

Figure 1.

Connexins, connexons and gap junction channels. A, Connexins have four transmembrane domains, which are connected by two extracellular loops and one intracellular loop. The N- and C-termini are both located in the cytosol. B, Connexins form hexamers called connexons. Connexins can combine with either the same or different connexin isoforms, forming homomeric or heteromeric connexons, respectively. C, Connexons form gap junction channels by interacting with either identical homomeric or heteromeric connexons in adjacent cells, forming homotypic channels, or with different homomeric or heteromeric connexons, forming heterotypic channels. (From [4]) D, Connexin proteins synthesized in the endoplasmic reticulum (ER) oligomerize to form connexon complexes. The connexons are transported to the cell surface and inserted into the plasma membrane where they form hemichannels. These hemichannels can dock with hemichannels of an opposing cell and cluster to form a gap junction plaque, characterized by a 2–4 nm gap between the two cell membranes. Gap junction plaques are removed from the cell surface through endoexocytosis, which results in the formation of an annular gap junction. The annular gap junction is then degraded through lysosomal proteolysis (From [46]).

Table 1.

Connexin subtype and cellular expression in the CNS, adapted from [3]

Connexin subtype	Cell type (expressed stage)	References
26	neuron	10–12
	astrocyte	13
29	oligodendrocyte	14
30	astrocyte (matured)	15,16
32	neuron (matured)	17
	oligodendrocyte	18–23
36	neuron	24–26
	oligodendrocyte	27
	microglia	27
37	neuron	28
40	neuron (developing)	28
	astrocyte	13
43	neuron (mainly developing)	10,17,29,30
	astrocyte	1,31,32
	microglia (activated)	33,34
45	neuron	35,36
	astrocyte	13
46	astrocyte	13
47	neuron	37
	astrocyte	9
	oligodendrocyte	38

Figure 2.

Schematic representation of GJ intercellular communication and HC-mediated cell-to-extracellular environment communication. Cxns, composed of 4 transmembrane domains and an intracellular carboxy-tail, are organized into homomeric or heteromeric HCs. GJ plaques are structures of hundreds up to thousands of single channels, which mediate exchanges of small molecules, substrates and metabolites. Those structures show free HCs exposed to the plaque border, where each cell adds newly synthetized HCs. These structures are crucial players of the GJIC and HC-mediated cell-to-extracellular environment communication and lead to the information exchanges between neighboring cells favoring synchronized and concerted responses. Cx, connexin; HC, hemichannel; GJ, gap junction; GJIC, gap junctional intercellular communication. [From 45].

2. What is neuronal differentiation?

Neuronal differentiation is a complex cellular process that integrates several signaling pathways and extrinsic factors to fuel distinct electrophysiological, morphological, and transcriptional changes, leading to the development of neurons⁴⁷ (Figure 3, [48]). This process depends on the molecular signals received from several hormones including neurotransmitters, accessory neurotrophic factors (such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4)), and chemokines that are translated into downstream intracellular responses, largely by G-protein-coupled receptors. Components of these processes that regulate neurite outgrowth have significant implications in neuronal survival after injury.⁴⁷ Neuronal differentiation in vivo occurs during development. Neuronal replacement therapies usually utilize the in vitro differentiated neurons from embryonic or induced pluripotent stem cells, or the direct reprogramming of differentiated adult cells though the expression of transcription factors, signaling molecules or treatment with small molecules^47,49 (Figure 3, [48]).

Figure 3.

Pathway from pluripotent stem cells to neural cell populations. Fertilization and subsequent cellular divisions create the embryonic blastocyst, where pluripotent ESCs are derived (from the inner cell mass; ICM). Additionally, pluripotent and multipotent-like cells can be created via transduction of various factors into differentiated tissue, such as fibroblasts. In vitro analyses of pluripotent and multipotent neural stem cells are integral for understanding aspects of neural differentiation. The in vivo niche of stem cells contains a considerable diversity of biomolecules whose roles still need be deciphered. Exposure of ESCs to various growth factors in serum-free media such as fibroblast growth factor 2 (FGF2) and epithelial growth factor (EGF) allows selection of cell lines possessing a neural fate. Neural stem cells can also be acquired from adult tissue and expanded in vitro. (From [48]).

Human embryonic stem cell (hESC) research has significantly advanced human neural differentiation research. Using the right culture conditions, neural progenitor (NP) cells are generated from pluripotent hESCs.^50–58 There is potential to generate other lineage cells (such as embryoid bodies) as contaminants in this process.⁵² Under suitable culture conditions, when the hESCs differentiate into NP cells, the NP cells continue to express SOX2 and begin expressing other neuroepithelial markers, such as Nestin, SOX1, SOX3, PSA-NCAM, and MUSASHI-1.⁵² The NP cells next form “neural rosettes” another morphological marker of hESC differentiation into neural cells.^52,59 Several signaling molecules, supplements/additives and conditioned media have been used by researchers to help the process of differentiation of hESCs into NP cells.^{51,52,57,60–62} Stromal-derived inducing activity (SDIA)-mediated differentiation is one method that can promote efficient neural tissue generation, however, this co-culture technique can introduce unknown stromal factors of non-human origin in culture, which may cause problems in comprehending neural differentiation,⁵²). Retinoic acid (RA) is another factor that is crucial for many aspects of neural development and activity, varying from axon regeneration in the adult, to neuronal differentiation and patterning of the neural plate and neural tube in the early embryo.^52,63 Retinoic acid is usually used for neuronal differentiation media at concentrations between 1–10 μM.^{51,52,61,62,64–68} The BMP-antagonist noggin is also used in the neuronal differentiation of hESC cultures grown adherently on laminin or matrigel^52,64,69,70 or in suspension.^52,71 FGF is another factor used as a supplement for neuronal differentiation. FGF molecules might play two different roles during neuronal development in the embryo: they are capable of inducing a “pro” neural state at an early stage, while acting as an antagonist to BMP signaling, which helps in establishing the neural phenotype.^52,53

3. The role of gap junctions in neurons

In the CNS, various cell types are coupled by GJs, which play an important role in maintaining normal function.³ Neuronal GJs are critical for electrical coupling and also contribute to the functional recovery after cell injury.³ Recent studies indicate the critical importance of the temporal pattern of connexin expression and gap junctional coupling during neuronal differentiation.³ The connexins that are usually found in neurons are Cx36, Cx30.2, Cx31.1, Cx40, Cx45, Cx50 and Cx57.^24,72–79 The important role that connexins play in the functioning of neurons is listed in Table 2. GJs control cell signaling, differentiation, and growth¹⁰⁰ by supporting intercellular communication at plaques on the cell-to-cell interface and also by mediating GJ-independent signaling.^45,101,102 The free HCs present throughout the plasma membrane are also made up of connexin, allowing complex chemical trafficking between cytoplasm and the extracellular environment.^45,103,104 GJs are crucial for electrotonic coupling between neurons. Electrotonic coupling has been shown in many areas of the CNS and is important for the regulation of neuronal synchronization.^3,105–108 In the developmental stages, a high degree of intercellular coupling between neurons has been observed.^3,109–112 These developmental areas consist of mitotically active epithelial cells lining the ventricles that express Cx43 and Cx26,^3,10 and this coupling involves both neuronal precursors and radial glia.^3,11 Thus, gap junctional intercellular communication (GJIC) creates cortical domains in the developing neocortex that give rise to the adult architecture^{3,109,111,113} (Figures 4, 114). Cx26^12,25 and Cx36^24,26 are expressed in the developing brain, consistent with the transient gap junctional coupling reported in the neocortex. Cx47 is also expressed in the CNS.³⁷ Cx37 is expressed mostly in motor neurons and Cx40 is evident in developing neurons of the spinal cord.²⁸ In the adult CNS, neurons in the cortex and hippocampus, express mainly Cx36²⁵ and Cx45³⁶ (Table 1). These neuronal GPs are crucial in the formation of electrical synapses.^89,90 Cx45 is also highly expressed in the developing brain and while in the adult brain its expression is localized to the olfactory nerves, cerebral cortex, hippocampus (part of allocortex) and thalamus.^36,115 Cx45 are the most highly expressed Cxs in neurons and Cx32 is found in oligodendrocytes. Cx45 is also expressed in glial cells. In the retina, Cx26 is expressed in horizontal cells¹¹⁶ and Cx36 in AII amacrine cells,¹¹⁷ in spite of the fact that Cx26 is not present in the neurons of adult cortex and hippocampus.¹¹⁸ HCs of horizontal cells regulate the activity of the Ca2+ channels and subsequent glutamate release.¹¹⁶ Cx43 knockout mice display abnormal migration of the neural crest.^96,119 The expression of Cx32 in mature neurons is an area of controversy.^3,25,120 Cx32 knockout mice show demyelination in the peripheral nervous system^3,83,84 along with neuronal hyperexcitability as well as myelin defects in the neocortex,⁸⁵ and amplified apoptosis.¹²¹ Cx36 knockout mice show selective impairment of hippocampal gamma oscillations.⁸⁸ Knockout of other neuronal candidates, Cx26, Cx45, is lethal.³⁵ In vitro studies have helped elucidate gap junctional coupling in neuronal differentiation. During the neuronal differentiation of NT2 human embryonal carcinoma cells in response to retinoic acid (RA) the expression of Cx43 and the level of gap junctional coupling progressively disappear.^3,122 Blocking of GJs disrupts retinoic acid-induced neuronal differentiation of both human NT2¹²³ and mouse P19 cells.¹⁰ In addition, blocking of HCs also affects the differentiation of NT2/D1 progenitor cells, signifying HCs also contribute to neuronal differentiation.¹²⁴ Programmed cell death is critical for development of the CNS as it regulates neuronal circuit formation and establishes the final number of neurons.⁷² A role for GJs in cell death/survival during development, glutamate-mediated excitotoxicity and neuronal injury has been demonstrated (Figure 5⁷² Several connexins appear to be important for this process, however, the role of Cx36 in the death or survival of neurons is still controversial.⁷²

Table 2.

The observed abnormalities in connexin knockout mice, adapted from [3]

Knockout mouse	Abnormalities	References
Cx26	embryonal lethal	80
(Cx26fl/fl, Otog-cre)	hearing impairment	81
Cx30	severe hearing impairment	82
Cx32	demyelination in PNS	83,84
	hyperexcitability in CNS	85
	enhanced neuronal injury in ischemia	86
Cx36	visual deficit	87
	synchronization defect in hippocampus	88
	disturbed synchronous inhibitory activity	89
	disrupted gamma frequency oscillations in cortex Vision; electrical coupling between mouse rods and cones	90 91
Cx43	neonatal lethal	92,93
	subtle neural changes in embryo	94
	neural crest migration defect	95,96
	disturbance of neural migration in embryonic neocortex Memory consolidation	17 92
(Cx43+/−)	enhanced neuronal injury in ischemia	97,98
(Cx43fl/fl,GFAP-cre)	increased spreading depression in hippocampus	97
	enhanced neuronal injury in ischemia
Cx45	embryonic lethal	35
Cx47	vacuolation in nerve fibers	38
(Cx47/32 double)	severe demyelination in CNS	38,99

Figure 4.

The role of gap junction coupling and hemichannels in radial glial cell proliferation. Radial glial coupling and hemichannel activity are regulated during the course of neurogenesis and within each cell cycle. Radial glial cell gap junction coupling is greatest during mid-neurogenesis and decreases in late neurogenesis. During the cell cycle, cells uncouple from clusters during M phase and recouple during S phase in mid-neurogenesis or late S or G2 phase in late neurogenesis. During late neurogenesis, hemichannels on S phase radial glia initiate Ca2+ waves by releasing ATP which binds to P2Y1 receptors on adjacent cells inducing an IP3-mediated release of Ca2+ from intracellular stores. Additionally, during each cell cycle, the levels of Cx26 and Cx43 fluctuate such that Cx26 and Cx43 levels are at their peak during M phase and S phase, respectively. Pharmacologically blocking coupling or Ca2+ waves inhibits entry into S phase of the cell cycle. [From 114].

Figure 5.

Glutamate-dependent excitotoxicity during neuronal injury. (a) Traditional model of the mechanisms for glutamate-dependent excitotoxicity. (b) Alternative model of the mechanisms of glutamate-dependent excitotoxicity. (c) This figure illustrates key points of the alternative model. In all figures:, Neuronal death caused directly by overactivation of N-methyl-D-aspatate receptors (NMDARs); (Baharvand), Existing neuronal gap junctions (GJs) contribute substantially to neuronal death caused by overactivation of NMDARs; (Baharvand), New neuronal gap junctions are induced by activation of group II metabotropic glutamate receptors (mGluRs) and also contribute to glutamate-dependent neuronal death; (Akopian), Pharmacological or genetic blockade of neuronal gap junction channels (GJCs) reduces glutamate-dependent neuronal death. In a,b: the sign (+) indicates the increase in receptor activity or expression of Cx36. In b: a possibility is illustrated that some Ca2+-dependent molecules (or Ca2+ itself) serve as gap junction-permeable death signals (b(Baharvand)); Ca2+ overload also directly induces neuronal death (b), however, this is not the main driver of neuronal death, rather the key determinant for expansion of cell death is neuronal GJCs (b)⁶⁴ [From 72].

4. The role of GAP junctions in neuropathological conditions

GJs regulate not only development, but also the maintenance of the physiological functions of multicellular organisms.^24,35,45,125 As a result, disturbance in the functioning of GJs, HCs, and Cxs, disrupts healthy cellular communication, leading to pathological conditions with different degrees of severity, including brain cancer and degenerative diseases. ^{126, 45, 127}Modifications in Cxs can lead to severe complications of tissue functioning and even lethal phenotypes.^45,128,129 Accordingly, the expression of Cxs in tissues and organs occurs throughout life starting from the embryo stage to adult stages and is stringently regulated,⁴⁵ and often a cause of neurodegenerative disorders.

Neurodegenerative diseases are one of the major causes of disability and death in patients. Extensive research has been done to develop appropriate therapeutic strategies⁴⁵ to control these diseases. The involvement of Cxs in the initiation and progression of various neurodegenerative diseases makes these proteins excellent therapeutic targets.^{45,126,130,131} Many researchers have shown that homeostatic imbalances observed during neurodegeneration are regulated by GJ-independent increased membrane permeability related to HC activity in the CNS.^45,132–134 GJs can also cause increased secondary harmful effects through cytotoxicity and inflammatory responses, resulting in secondary cell death and neuronal loss.^45,135,136

Gap junctional communication between activated microglia at the site of a pathophysiological condition might enhance the release of cytokines, leading to inflammatory responses that cause neurodegenerative effects. This is how several degenerative disorders, such as glaucoma¹³⁵), traumatic brain injury,^137,138 stroke,^139,140 Alzheimer’s disease^{141, 142} and amyotrophic lateral sclerosis (ALS)-related motor neuron loss¹⁴³ are initiated and progress. These neurodegenerative conditions are associated with reactive astrogliosis, mononuclear phagocyte activation, neuronal injury, and neuronal death. These varied responses are regulated by connexins such as Cx36, Cx43, Cx30, Cx32, Cx29, and Cx47.^126,127 Depending on the nature of the injury and the cell types affected either upregulation or downregulation of a specific connexon could signal either “pro-death” or “pro-survival” effects in neurons.^126,127,135 For example, Cx43, one of the highly expressed Cxs in the CNS regulates different mechanisms in the CNS ranging from the regulation of the blood brain barrier (BBB) to the modulation of integrative brain functions (i.e., learning, memory, and behavior).¹⁴⁴ Aberrant Cx43 expression can increase GJ-mediated coupling, and cause increased HC activity during in vivo astrocyte-mediated toxicity.¹⁴³ Blocking GJs or HCs provide neuroprotection to motor neurons cultured with SOD1G93A astrocytes (astrocytes carrying the superoxide dismutase 1 (SOD1G93A) mutation induce wild-type motor neuron degeneration in vivo), suggesting a prodegenerative role of Cx43 in amyotrophic lateral sclerosis or ALS.¹⁴³ Similar effects are seen in hypoxia, ischemia, Alzheimer’s disease, and glaucoma. ^{130,137,145,146}. GJ-mediated neuronal death is usually caused by overactivation of N-Methyl-D-aspartate receptors.⁷² Activation of group II metabotropic glutamate receptors cause formation of new GJs, which also assist in glutamate-dependent neuronal death.^72,147 In contrast, pharmacological^72,148 or genetic^147,148 blocking of neuronal GJs decrease this glutamate-dependent neuronal death. Ca2+-dependent molecules (or Ca2+ itself) can also serve as GJ-permeable cell death signals,^72,149 and in such cases Ca2+ overload triggers neuronal death.⁷² Astrocyte GJs are commonly associated with neuronal disorders, such as brain ischemia, Alzheimer’s disease and epilepsy. GJs may also contribute to the degree of malignancy and metastasis of brain tumors.³ Moreover, as hypersynchronous neuronal activity is a characteristic feature of convulsions, increased gap junctional communication also contributes to the initiation and maintenance of seizures.^150–152 The importance of connexins, GJs and HCs in CNS neuropathology deserves further study especially the function of GJs in the neural cell network, involving neurons, astrocytes, microglia and oligodendrocytes.³

5. The role of GAP junctions in embryonic cortical development and differentiation

Multiple putative roles of neuronal GJ communication during development have been proposed. These functions are based on observations from studies investigating developmental changes, GJ communication and Cx36 expression, as well as utilization of knockdown, knock-in and mutational approaches. In total these studies support the concept that neuronal GJs can regulate synaptogenesis,^72,76 neuronal differentiation,^72,153 migration^72,154 and neural circuit formation as well as maturation.^155,156 GJs can regulate these processes through exchange of Ca2+, metabolites and second messengers (such as glutamate, glutamine, D-serine, adenosine triphosphate (ATP) and lactate) between cells, which provide clues for the coordination of metabolic and transcriptional activities in developing neurons.¹²⁵ Furthermore, GJs also assist in generation of the highly synchronized excitatory electrical activity. This network-driven activity is a hallmark of the developing brain¹⁵⁷ and it depends on collaboration between GJs and chemical neurotransmitter receptors.^72,155,158

The development of the cerebral cortex from the neuroepithelium into a complex brain architecture supporting advanced cognition is highly dependent on the formation of a network of intercellular signaling.¹¹⁴ GJs are abundantly expressed during embryonic development and they support cell–cell contact and communication.¹¹⁴ Even though GJs aid in neural progenitor coupling, they also act as HCs facilitating the spread of calcium waves across progenitor cell populations, which serve as adhesive molecules helping in neuronal migration. In total, GJs have complex roles during the cortical development stages and utilize diverse strategies for effective intercellular communication¹¹⁴ (Figure 6).

Figure 6.

The adult subventricular zone (SVZ) and subgrandular zone (SGZ) support neurogenesis. Adult neural progenitor cells (NPCs) are defined by their capacity to proliferate and replenish neuronal and glial cell numbers. Antigenic markers used to distinguish between lineages are listed. [From 159].

During the different stages of embryonic development, embryonic cells proliferate as well as develop specific features and differentiate into different cell types, which ultimately become organized into different tissues and organs.¹⁰ Development of the nervous system involves an intricate series of events involving coordinated neuronal and astroglial differentiation.¹⁰ This coordination of growth and differentiation is regulated, at least partly, by exchange of small ions and molecules through the intercellular GJ channels.¹²³ Studies utilizing NTera2/clone D1 (NT2/D1) cells, which are CNS precursors, show that these cells differentiate into NT2-N neurons in the CNS after treatment with retinoic acid (RA) and antiproliferative agents.¹²³ In another study, the effect of GJ blockers, 18 alpha-glycyrrhetinic acid (GRA) and carbenoxolone (CBX) on neuronal differentiation was compared to the effect induced by oleanolic acid (OLA) and glycyrrhizic acid (GZA), which are inactive GRA analogs.¹²³ They observed that both control- (inactive blockers) and GJ blocker-treated samples expressed decreased Cx43, after 4 weeks of RA treatment. While control cells treated with RA displayed decreased cytokeratin, vimentin and nestin expression, supporting the characteristics of NT2-N neuronal differentiation,¹²³ cells treated with GJ blockers did not show any significant decrease. Moreover, the average number of MAP2 (a neuronal differentiation marker)-positive NT2-N differentiated neurons in the group treated with the GJ blockers was less than 7% of that of the control group.¹²³ These results support the importance of GJ intercellular communications in this differentiation process.

The occurrence of gap-junctional coupling is also significantly correlated with specific developmental events.¹⁶⁰ HC activation represents a vital step in the establishment of Ca2+ waves, which help in coordinating cell cycle events during early prenatal neurogenesis, whereas both HCs and/or GJs regulate precursor cell division and migration during late prenatal neurogenesis.¹⁶⁰ A novel group of proteins conspicuously expressed in the brain, called pannexins also form HCs. This suggests that there are proteins other than connexins that mediate these intricate processes.¹⁶⁰

Radial glial coupling and HC activity are also important during neurogenesis. Radial glial cell GJ coupling is observed to be highest during mid-neurogenesis, which later decreases during late neurogenesis.^114,161 The HCs present on S phase radial glia initiate waves of Ca2+ signaling by releasing ATP, which then binds to P2Y1 receptors on the adjacent cells causing an IP3-mediated release of Ca2+ during late neurogenesis stages.^114,162 Moreover, expression of the GJ proteins Cx26 and Cx43 levels are observed to be at their peak during M phase and S phase, respectively.^114,163 The importance of coupling is implied by pharmacological studies where blocking coupling or Ca2+ waves was observed to inhibit entry of the cells into the S phase of the cell cycle.^114,161,162 After radial glia divide asymmetrically, the newly formed neurons need to migrate to the appropriate lamina of the cortical plate.¹¹⁴ Radial glial cells also assist in this process of guiding the new neurons to their destination apart from being the stem cells of the developing cortex. The newly formed neurons migrate in very close association with radial glial fibers^{114,159,164,165} (Figure 7) and GJs possibly facilitate communication between the radial fibers and the migrating neurons.^{17,114,166–168}

Figure 7.

Connexon-Connexon mediated adhesion domains. In addition to forming functional intercellular channels, docking of compatible connexons between radial glia (yellow) and NPCs (gold) directs migration of NPCs Docking and undocking enables the “rolling” of NPCs along their radial glial guides to their final location before terminal differentiation. Adhesion can be channel-independent without requiring exchange of small molecules or functional channel openings. [From 159].

Additionally, studies involving conditionally immortalized mouse hippocampal multipotent progenitor cells (MK31 cells) demonstrate strong coupling by GJs expressing Cx43 during early neuronal ontogeny.¹⁶⁹ However, the coupling strength and expression of Cx43 declined after the progenitor cells went through neuronal differentiation post interleukin 7 (IL-7) treatment alone or after combined treatment with basic fibroblast growth factor, IL-7, and transforming growth factor α¹⁶⁹ In addition, the GJs express Cx40 and Cx33 during the intermediate stage of neuronal differentiation.^169,170,171 These studies point toward a loss of coupling during differentiation of neural progenitor cells involving downregulation of Cx43 coupled with enhanced expression of Cx33 and Cx40,¹⁶⁹ suggesting complex dynamics of Cx expression during neuronal differentiation. Fluctuation in the expression of other connexins such as Cx26, which initially increase and then decrease by the third postnatal week, while that of Cx32 increases, has also been reported).^114,172 The expression of these GJs is also specific to defined cell types: Cx43 and Cx26 are expressed in neurons and radial glia during development,^114,166 but their expression is largely restricted to astrocytes in the adult, whereas Cx26 and Cx45 are the most highly expressed Cxs in neurons and Cx32 is found in oligodendrocytes. Cx45 is also expressed in glial cells. Also, knockdown of Cx43 decreases the proportion of cells that express mature neuronal markers postnatally such as NeuN and MAP2].^166,172 Cx32, Cx43 and Cx31 has also been shown to regulate neurite outgrowth.^{24,114,169,173} GJ-mediated intercellular communication is also crucial for the growth and survival of mouse neural progenitor cells (NPCs).¹⁷⁴ In the presence of basic fibroblast growth factor (bFGF), NPCs express the gap junction protein Cx43 and are Lucifer Yellow fluorescent dye-coupled,¹⁷⁴ however, in the absence of bFGF, expression of Cx43 and dye coupling is reduced, and the cells stop proliferating and instead differentiate into neurons.¹⁷⁴ A role of Panx1 GJ proteins has been proposed in the regulation of neural stem cell survival, neuronal maturation and synaptic plasticity, along with normal cognitive functioning.⁴² Gap junctional communication in this manner helps maintain NPCs in a self-renewing state.¹⁷⁴

6. The role of GAP junctions in transdifferentiation toward neurons

The term transdifferentiation, which is also known as lineage reprogramming, was first used by Selman and Kafatos in 1974.^175,176 During this complex phenomena, one type of mature somatic cell transforms into another type of mature somatic cell bypassing an intermediate pluripotent state.^175,176 This process is induced mainly by the exogenous expression of lineage-specific transcription factors (TFs) and by chemical mediators.¹⁷⁶ Recently, in vitro transdifferentiation of patient-derived mesenchymal stem/stromal cells (MSCs) into neurons has been reported and this process could potentially be important for the treatment of neurodegenerative diseases.¹⁷⁷ During this process, MSC markers such as CD73, CD90, CD105, and CD166 were downregulated and the neuronal marker, Tuj1, was upregulated.¹⁷⁷ The MSCs transformed from a fibroblastoid morphology into a neuronal morphology with round cell bodies and neurite-like processes. In addition, they were functionally active as depolarization evoked action potentials in the transdifferentiated cells. MSCs express Cx43 and Cx45 as well as trace levels of Cx26, Cx37- and Cx40, which allowed transfer of microinjected Lucifer yellow. However, the differentiation process elevated the levels of Cx26 and decreased Cx43, while reducing the dye transfer. Treatment with a GJ coupling inhibitor, carbenoxolone (CBX), changed connexin expression modestly and had little effect on neuronal differentiation.¹⁷⁷ These studies highlight the complexity of GJ functioning in neuronal transdifferentiation from MSCs. Human embryonic fibroblasts and mouse embryonic fibroblasts can also be converted into neuronal cells using chemical modifiers, along with forced expression-specific transcriptional factors.^178,179 However, the role of GJs in this transdifferentiation process from fibroblasts has not at this time been reported.

7. The role of GAP junctions in neural stem cell graft integration

Neural stem cell (NSC)-based regeneration is a promising treatment strategy for neuropathological conditions NSCs secrete soluble factors and can differentiate into neurons, astrocytes and oligodendrocytes.¹⁷⁶ NSCs can be obtained by either direct isolation from primary tissues, differentiation from pluripotent stem cells or by transdifferentiation from somatic cells.¹⁷⁶ NSC transplantation-based therapies for the treatment of numerous neural defects and injuries have been widely investigated in animal models and clinical trials.¹⁷⁶

Spinal cord injury (SCI) triggers severe damage to the neuronal elements of the spinal cord including separating of axon tracts and cell death. The primary mechanical damage is followed by multiple secondary damaging cascades that lead to loss of tissue and functioning.¹⁸⁰ Connexins play a prominent role in the secondary phase of SCI by regulating cell death signaling through widespread glial networks.¹⁸⁰ These gap junction-mediated networks are supported by connexin intercellular channels and the formation of pore-like hemichannels formed by both connexins and pannexins. Several studies have been conducted on the distribution and functional role of connexins in the spinal cord and these studies show that connexin-formed gap junctions undergo cell-to-cell coupling between neurons, adjacent astrocytes, and astrocytes/oligodendrocytes.¹⁸⁰ Cx36 is expressed in neuron GJs, Cx30 and Cx43 are expressed in astrocyte/astrocyte GJs, Cx29, Cx32, are expressed in oligodendrocyte/astrocyte GJs and Cx47 is associated with the major heterotypic channels composed of Cx43-Cx47 and Cx30-Cx32¹⁸⁰ (Figure 8a). Following SCI, Cx43 expression is elevated in astrocytes resulting in aberrant release of ATP from the perilesional region. The ATP release initiates destructive inflammatory responses including recruitment of microglia and macrophages to the damaged area, resulting in increased secondary expansion of the primary lesion¹⁸⁰ (Figure 8b). As described earlier, the coupling of neurons by GJs (i.e., electrical synapses) and the expression of the neuronal GJ protein, Cx36, is transiently increased during early postnatal development,⁷² which decreases and then remains low in the adult, confined to specific subsets of neurons. However, following neuronal injury [such as ischemia, traumatic brain injury (TBI), and epilepsy], the coupling and expression of Cx36 increases.⁷²

Figure 8.

Structure and distribution of the main connexin proteins in the spinal cord before and after spinal cord injury (SCI). (a) Gap junction channels are produced by the conjoining of two hemichannels, with one hemichannel provided by each cell. Compared to the closed state (∼1.8 nm), opened channels (∼2.5 nm) allow the transfer of substances less than 1 kDa between the cytoplasm of two cells, including nutrients, metabolites, K+, ATP, cAMP, and Ca2 + . (b) Each hemichannel is composed of six individual connexin proteins, which contain four transmembrane domains. The cytoplasmic loop (CL), amino (n), and carboxy (c) terminals are located on the cytoplasmic side, with multiple regulatory phosphorylation sites located on the C termini. There are two extracellular loops (E1 and E2) which contain three highly conserved cysteine bonds that play a role in the docking of hemichannels from opposing cells via intramolecular cysteine/cysteine bonds. (C) In the spinal cord, there is a large distribution of connexins. Neuron/neuron gap junctions contain Cx36, astrocyte/astrocyte junctions contain Cx30 and Cx43 and oligodendrocyte/astrocyte gap junctions contain Cx29, Cx32, and Cx47 with the major heterotypic channels composed of Cx43Cx47 and Cx30Cx32. (d) Following SCI, Cx43 expression is increased in astrocytes. This causes an uncontrolled release of the small molecule ATP from the perilesional region. ATP release leads to the activation of a destructive inflammatory response including recruitment of microglia and macrophages to the site of injury, which causes an increased secondary expansion of the lesion. [From 180].

In some studies, astrocytic Cx43 HC is negatively involved in the remyelination process by favoring local inflammation, and thus inhibiting Cx43 HC could be an interesting treatment strategy for injury.¹⁸¹ The process of integration of exogenous NSCs and its progeny into recipient brain is a promising strategy for repair of injury that will benefit from experiments providing more mechanistic insights.¹⁸² GJ-mediated communications help transplanted NSCs to regulate host network activity, including synchronized calcium fluctuations.¹⁸² In addition, the NSCs also have cytoprotective effects on the host neurons, helping them survive death-inducing conditions and thereby reduce symptoms of secondary injury, e.g., reactive astrogliosis. GJs (containing Cx43) that are formed between NSCs and host cells at risk are correlated with rescue of neurons. Both in vitro and in vivo cytoprotective effects of NSCs were eliminated when GJ formation or function was suppressed by pharmacologic and/or RNA-inhibition strategies. This highlights the seminal role of gap-junctional coupling in the regulation of modulatory, homeostatic, and protective actions of NSCs on host systems as well as establishing functional networks for regenerative repair.¹⁸²

Coupling among ependymal cells is down-regulated during post-natal development and increased after spinal cord injury, which correlates with up-regulation of Cx26 and rescued proliferation.¹⁸³ Blocking connexin resulted in decreased injury-induced proliferation of ependymal cells.¹⁸³ Thus, connexins are important for the functioning of ependymal cells in response to injury, representing another strategy of stem cell-based repair (Figure 9).¹⁸⁴

Figure 9.

Schematic representation of how blocking and upregulating different connexins could aid in the repair, differentiation and regeneration of neurons.

8. Conclusion

GJs play essential roles in the formation and functioning of neurons. They mediate cell-to-extracellular environment communication and regulate the information exchanges between neighboring cells that favor synchronized and concerted responses. They regulate not only differentiation from embryonic precursors, but can also regulate transdifferentiation, neuropathological conditions and even neuronal death. Because of these properties they are important in the treatment of spinal cord injury or traumatic brain injury.¹⁷⁹

9. Future perspectives

Humans are unfortunately limited in their ability to regenerate nerve cells, posing significant issues for the treatment of injury and diseases of the nervous system. Neuronal differentiation techniques using embryonic, pluripotent and multipotent stem cells and even transdifferentiation from somatic cells holds great promise for treatment of various neurodegenerative diseases and recovery from injury. Different research groups are working on clinical trials that involve NSC transplantation in patients with ischemic injury and ALS, with preclinical studies being implemented for future cell-based treatments of brain cancer, ischemic spastic paraplegia, chronic spinal cord injury, and chronic stroke. Since GJs play seminal roles in neuronal differentiation, combining existing strategies with appropriate GJ-based therapies utilizing patient-specific stem cells offer significant potential in regenerative medicine by providing patients with genetically identical matches for treatment of diseased and damaged tissue.

Funding Statement

Support from the Human and Molecular Genetics Development Fund, the Virginia Commonwealth University Institute of Molecular Medicine (VIMM), and the VCU Massey Cancer Center is appreciated.

Disclosure statement

No potential conflict of interest was reported by the author(s).