Localization of inositol 1,4,5-trisphosphate receptors in mouse retinal ganglion cells

Inositol 1,4,5-trisphosphate receptors (IP(3)R) are ligand-gated intracellular Ca(2+)channels that mediate release of Ca(2+) from intracellular stores into the cytosol on activation by second messenger IP(3.). Similarly, IP(3)R mediated changes in cytosolic Ca(2+) concentrations control neuronal functions ranging from synaptic transmission to differentiation and apoptosis. IP(3)R-generated cytosolic Ca(2+) transients also control intracellular Ca(2+) release and subsequent retinal ganglion cell (RGC) physiology and pathophysiology. The distribution of IP(3)R isotypes in primary adult mouse RGC cultures was determined to identify molecular substrates of IP(3)R mediated signaling in these neurons. Immunocytochemical labeling of IP(3)Rs in retinal sections and cultured RGCs was carried out using isoform specific antibodies and was detected with fluorescence microscopy. RGCs were identified by the use of morphologic criteria and RGC-specific immunocytochemical markers, neurofilament 68 kDa, Thy 1.1, and Thy 1.2. RGC morphology and immunoreactivity to neurofilament 68 kDa and Thy 1.1 or Thy 1.2 were identified in both RGC primary cultures and tissue cryosections. RGCs showed localization on intracellular membranes with a differential distribution of IP(3)R isoforms 1, 2, and 3. IP(3)R Types 1 and 3 were detected intracellularly throughout the cell whereas Type 2 was expressed predominantly in soma. Expression of all three IP(3)Rs by RGCs indicates that all IP(3)R types potentially play a role in Ca(2+) homeostasis and Ca(2+) signaling in these cells. Differential localization of IP(3) receptor subtypes in combination with biophysical properties of IP(3)R types may be an important molecular mechanism by which RGCs organize their cytosolic Ca(2+) signals.     

Gap junction channels coordinate the propagation of intercellular Ca2+ signals generated by P2Y receptor activation

Astrocytes express gap junction proteins and multiple types of P2Y receptors (P2YRs) that contribute to the propagation of intercellular Ca(2+) waves (ICW). To gain access to the role played by gap junctional communication in ICW propagation generated by P2YR activation, we selectively expressed P2Y(1,2,4)R subtypes and Cx43 in the human 1321N1 astrocytoma cell line, which lacks endogenous P2 receptors. Fluorescence recovery after photobleaching revealed that 1321N1 cells are poorly dye-coupled and do not propagate ICW. Forced expression of Cx43 in 1321N1 cells (which did not show functional hemichannels) increased dye coupling and allowed short-range ICW transmission that was mainly mediated by intercellular diffusion of Ca(2+) generated in the stimulated cells. Astrocytoma clones expressing each of the P2YR subtypes were also able to propagate ICWs that were likely dependent on IP(3) generation. These waves exhibited properties particular to each P2YR subtype. Co-expression of eGFP-hCx43 and P2Y(1)R modified the properties of P2Y(1)R-generated ICW to those characteristics of P2Y(2)R. Increased coupling in P2Y(4)R clones induced by expression of eGFP-hCx43 abolished the ICWs observed in uncoupled P2Y(4)R clones. No changes in the behavior of ICWs generated in P2Y(2)R clones were observed after forced expression of Cx43. These data indicate that in 1321N1 cells gap junctional communication provides intercellular integration of Ca(2+) signals generated by P2YR activation, thus coordinating the propagation of intercellular calcium waves.     

The effects of a dominant connexin32 mutant in myelinating Schwann cells

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

Connexin29 expression,immunocytochemistry and freeze-fracture replica immunogold labelling (FRIL) in sciatic nerve

The recently discovered connexin29 (Cx29) was reported to be present in the central and peripheral nervous systems (CNS and PNS), and its mRNA was found in particular abundance in peripheral nerve. The expression and localization of Cx29 protein in sciatic nerve were investigated using an antibody against Cx29. The antibody recognized Cx29 in HeLa cells transfected with Cx29 cDNA, while nontransfected HeLa cells were devoid of Cx29. Immunoblotting of sciatic nerve homogenate revealed monomeric and possibly higher molecular weight forms of Cx29. These were distinguished from connexin32 (Cx32), which also is expressed in peripheral nerve. Double immunofluorescence labelling for Cx29 and Cx32 revealed only partial colocalization of the two connexins, with codistribution at intermittent, conical-shaped striations along nerve fibers. By freeze-fracture replica immunogold labelling (FRIL), Cx32 was found in gap junctions in the outermost layers of myelin, whereas Cx29-immunogold labelling was found only in the innermost layer of myelin in close association with hexagonally arranged intramembrane particle (IMP) 'rosettes' and gap junction-like clusters of IMPs. Although both Cx32 and Cx29 were detected in myelin of normal mice, only Cx29 was present in Schwann cell membranes in Cx32 knockout mice. The results confirm that Cx29 is a second connexin expressed in Schwann cells of sciatic nerve. In addition, Cx29 is present in distinctive IMP arrays in the inner most layer of myelin, adjacent to internodal axonal plasma membranes, where this connexin may have previously unrecognized functions.     

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

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

Expression of IP3 receptor isoforms at the nodes of Ranvier in rat sciatic nerve

Inositol 1,4,5-trisphosphate receptors (IP3R) are modulated by the second messenger IP3, which induces intracellular calcium release. Using immunohistochemical techniques, we show that the three isoforms are expressed in sciatic nerve. IP3R1 and IP3R2 are mainly present in the nucleus of Schwann cells. IP3R1 is also expressed in Schmidt-Lanterman incisures. IP3R3 is primarily localized at very high levels in nonmyelinating Schwann cells. Interestingly, the three isoforms are expressed at the nodes of Ranvier. IP3R1 is clustered at the node of Ranvier, in a distribution that is similar to the Nav1.6 sodium channels in the sciatic nerve. IP3R3 is present in the paranodal regions of the nodes. IP3R2 is concentrated in the vicinity of the node, and the outer Schwann cell cytoplasm similar to the Kv1.5 potassium channel.     

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.     

Cellular mechanisms of connexin32 mutations associated with CNS manifestations

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

Gap junctional communication and connexin expression in cultured olfactory ensheathing cells

The olfactory ensheathing cell (OEC) is a unique glial cell able to support neurite outgrowth in the CNS throughout life. The OEC has been described as having both Schwann cell-like and astrocyte-like characteristics. The purpose of this study was to compare gap junctional communication and connexin (Cx) expression in cultured olfactory ensheathing cells with both astrocytes and Schwann cells to establish which of these two cells types they most closely resemble. We examined the Cx mRNA profile of OECs, astrocytes, and Schwann cells using primers to Cx26, Cx32, Cx37, Cx43, Cx46, and Cx50. All connexins tested except Cx50 were expressed by all three cell types when initially cultured. However, we observed differences in the levels of expression of Cx32 and Cx26 between astrocytes, Schwann cells, and OECs that became pronounced with time. All three cell types show limited and variable gap junctional communication in culture as assessed by the transfer of microinjected Lucifer yellow. OECs had limited coupling compared with Schwann cells and astrocytes, although the extent of the dye spread through OECs was more comparable to that seen with Schwann cells than astrocytes. Thus, OECs display a profile of Cx expression that more closely resembles the Cx expression of Schwann cells rather than astrocytes.     

Altered gene expression in Schwann cells of connexin32 knockout animals

The discovery that the dominant X-linked form of Charcot-Marie-Tooth disease (CMTX), a genetic disease of the peripheral nervous system (PNS), is associated with mutations in connexin32 (Cx32) has brought attention to the importance of connexins in glial cell biology. To gain further insight into the consequences of Cx32 deficiency, we have undertaken a detailed characterization of the gene expression profile of Schwann cells isolated from the sciatic nerve of wild-type and Cx32-null mice. Schwann cells exhibit two distinct phenotypes, myelinating and nonmyelinating, which are defined by their different morphology with respect to axons and by their unique profile of gene expression. Our findings show that, regardless of the mouse genotype, cultured Schwann cells express similar levels of messages for a number of connexins and for genes characteristic of both the myelinating and the nonmyelinating phenotypes. Furthermore, we have identified Cx36, a member of the gamma subclass of connexins, which are preferentially expressed in neuronal cells of mouse brain and retina, as an additional connexin present in Schwann cells. Mice lacking Cx32, however, exhibited a marked up-regulation of glial fibrillary acidic protein (GFAP), a cytoskeletal protein usually synthesized only by nonmyelinating Schwann cells. This observation was extended to the PNS in vivo and did not reflect a general perturbation of the expression of other nonmyelinating Schwann cell genes. These findings demonstrate that the absence of Cx32 results in a distinct pattern of gene dysregulation in Schwann cells and that Schwann cell homeostasis is critically dependent on the correct expression of Cx32 and not just any connexin. Identifying the relationship between increased GFAP expression and the absence of Cx32 could lead to the definition of specific roles for Cx32 in the control of myelin homeostasis and in the development of CMTX.     

CSF‐1‐activated macrophages are target‐directed and essential mediators of schwann cell dedifferentiation and dysfunction in Cx32‐deficient mice

We investigated connexin 32 (Cx32)‐deficient mice, a model for the X‐linked form of Charcot‐Marie‐Tooth neuropathy (CMT1X), regarding the impact of low‐grade inflammation on Schwann cell phenotype. Whereas we previously identified macrophages as amplifiers of the neuropathy, we now explicitly focus on the impact of the phagocytes on Schwann cell dedifferentiation, a so far not‐yet addressed disease‐related mechanism for CMT1X. Using mice heterozygously deficient for Cx32 and displaying both Cx32‐positive and ‐negative Schwann cells in one and the same nerve, we could demonstrate that macrophage clusters rather than single macrophages precisely associate with mutant but not with Cx32‐positive Schwann cells. Similarly, in an advanced stage of Schwann cell perturbation, macrophage clusters were strongly associated with NCAM‐ and L1‐positive, dedifferentiated Schwann cells. To clarify the role of macrophages regarding Schwann cell dedifferentiation, we generated Cx32‐deficient mice additionally deficient for the macrophage‐directed cytokine colony‐stimulating factor (CSF)?1. In the absence of CSF‐1, Cx32‐deficient Schwann cells not only showed the expected amelioration in myelin preservation but also failed to upregulate the Schwann cell dedifferentiation markers NCAM and L1. Another novel and unexpected finding in the double mutants was the retained activation of ERK signaling, a pathway which is detrimental for Schwann cell homeostasis in myelin mutant models. Our findings demonstrate that increased ERK signaling can be compatible with the maintenance of Schwann cell differentiation and homeostasis in vivo and identifies CSF‐1‐activated macrophages as crucial mediators of detrimental Schwann cell dedifferentiation in Cx32‐deficient mice. GLIA 2015;63:977–986     

Functional analysis of connexin-32 mutants associated with X-linked dominant Charcot-Marie-Tooth disease

To investigate the pathogenic role of connexin-32 (Cx32) mutation in X-linked dominant Charcot-Marie-Tooth disease (CMTX), dual whole-cell voltage-clamp recordings and tracer coupling were performed to investigate functional properties of wild-type and 22 CMTX mutant Cx32 proteins expressed in N2A cells. Ten mutant Cx32 proteins either formed defective junctional channels (Y65C, V95M, R107W, L156R, R164W and G199R) or failed to form gap junctions (G12S, S182T, E208K and Y211stop). Except (G12S) and (E208K) mutants, other mutant Cx32 proteins were localized in the cell membrane despite their impaired ability to form functional gap junctions. Twelve CMTX mutations (V13L, R15Q, R22Q, I30N, V35M, V63I, R75Q, Q80R, W133R, P158A, P172S and N205S) did not affect the ability of Cx32 to form homotypic gap junctions in N2A cells. Our results indicate that 10 of 22 CMTX Cx32 mutations studied in the present investigation could lead to the assembly of defective Cx32 gap junctions, which in turn may result in peripheral neuropathy. However, further studies are required to elucidate the exact mechanism by which CMTX mutant Cx32 proteins, which retain the ability to form homotypic junctional channels, damage Schwann cells and cause demyelinating neuropathy.     

Skeletal muscle IP3R1 receptors amplify physiological and pathological synaptic calcium signals

Ca(2+) release from internal stores is critical for mediating both normal and pathological intracellular Ca(2+) signaling. Recent studies suggest that the inositol 1,4,5-triphosphate (IP(3)) receptor mediates Ca(2+) release from internal stores upon cholinergic activation of the neuromuscular junction (NMJ) in both physiological and pathological conditions. Here, we report that the type I IP(3) receptor (IP(3)R(1))-mediated Ca(2+) release plays a crucial role in synaptic gene expression, development, and neuromuscular transmission, as well as mediating degeneration during excessive cholinergic activation. We found that IP(3)R(1)-mediated Ca(2+) release plays a key role in early development of the NMJ, homeostatic regulation of neuromuscular transmission, and synaptic gene expression. Reducing IP(3)R(1)-mediated Ca(2+) release via siRNA knockdown or IP(3)R blockers in C2C12 cells decreased calpain activity and prevented agonist-induced acetylcholine receptor (AChR) cluster dispersal. In fully developed NMJ in adult muscle, IP(3)R(1) knockdown or blockade effectively increased synaptic strength at presynaptic and postsynaptic sites by increasing both quantal release and expression of AChR subunits and other NMJ-specific genes in a pattern resembling muscle denervation. Moreover, in two mouse models of cholinergic overactivity and NMJ Ca(2+) overload, anti-cholinesterase toxicity and the slow-channel myasthenic syndrome (SCS), IP(3)R(1) knockdown eliminated NMJ Ca(2+) overload, pathological activation of calpain and caspase proteases, and markers of DNA damage at subsynaptic nuclei, and improved both neuromuscular transmission and clinical measures of motor function. Thus, blockade or genetic silencing of muscle IP(3)R(1) may be an effective and well tolerated therapeutic strategy in SCS and other conditions of excitotoxicity or Ca(2+) overload.     

Selective defects in channel permeability associated with Cx32 mutations causing X-linked Charcot-Marie-Tooth disease

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is caused by mutations in connexin32 (Cx32), a gap junction protein expressed by Schwann cells where it forms reflexive channels that allow the passage of ions and signaling molecules across the myelin sheath. Although most mutations result in loss of function, several studies have reported that some retain the ability to form homotypic intercellular channels. To gain insight into the molecular defect of three functional CMTX variants, S26L, Delta111-116 and R220stop, we have used several fluorescent tracers of different size and ionic charge to compare their permeation properties to those of wild-type Cx32. Although all mutations allowed the passage of the dye with the smallest molecular mass, they exhibited a clear reduction in the permeability of either one or all of the probes with respect to wild-type channels, as assessed by the percentage of injections showing dye coupling. These data reveal that a lower size cutoff distinguishes these functional CMTX variants from wild-type channels and suggest that this defect may be of pathophysiological relevance.     

Analysis of connexin expression during mouse Schwann cell development identifies connexin29 as a novel marker for the transition of neural crest to precursor cells

Connexins are transmembrane proteins forming gap junction channels for direct intercellular and, for example in myelinating glia cells, intracellular communication. In mature myelin-forming Schwann cells, expression of multiple connexins, i.e. connexin (Cx) 43, Cx29, Cx32, and Cx46 (after nerve injury) has been detected. However, little is known about connexin protein expression during Schwann cell development. Here we use histochemical methods on wildtype and Cx29lacZ transgenic mice to investigate the developmental expression of connexins in the Schwann cell lineage. Our data demonstrate that in the mouse Cx43, Cx29, and Cx32 protein expression is activated in a developmental sequence that is clearly correlated with major developmental steps in the lineage. Only Cx43 was expressed from neural crest cells onwards. Cx29 protein expression was absent from neural crest cells but appeared as neural crest cells generated precursors (embryonic day 12) both in vivo and in vitro. This identifies Cx29 as a novel marker for cells of the defined Schwann cell lineage. The only exception to this were dorsal roots, where the expression of Cx29 was delayed four days relative to ventral roots and spinal nerves. Expression of Cx32 commenced postnatally, coinciding with the onset of myelination. Thus, the coordinated expression of connexin proteins in cells of the embryonic and postnatal Schwann cell lineage might point to a potential role in peripheral nerve development and maturation.     

Astrocytes in adult rat brain express type 2 inositol 1,4,5-trisphosphate receptors

Astrocytes respond to neuronal activity by propagating Ca(2+) waves elicited through the inositol 1,4,5-trisphosphate pathway. We have previously shown that wave propagation is supported by specialized Ca(2+) release sites, where a number of proteins, including inositol 1,4,5-trisphosphate receptors (IP(3)R), occur together in patches. The specific IP(3)R isoform expressed by astrocytes in situ in rat brain is unknown. In the present report, we use isoform-specific antibodies to localize immunohistochemically the IP(3)R subtype expressed in astrocytes in rat brain sections. Astrocytes were identified using antibodies against the astrocyte-specific markers, S-100 beta, or GFAP. Dual indirect immunohistochemistry showed that astrocytes in all regions of adult rat brain express only IP(3)R2. High-resolution analysis showed that hippocampal astrocytes are endowed with a highly branched network of processes that bear fine hair-like extensions containing punctate patches of IP(3)R2 staining in intimate contact with synapses. Such an organization is reminiscent of signaling microdomains found in cultured glial cells. Similarly, Bergmann glial cell processes in the cerebellum also contained fine hair-like processes containing IP(3)R2 staining. The IP(3)R2-containing fine terminal branches of astrocyte processes in both brain regions were found juxtaposed to presynaptic terminals containing synaptophysin as well as PSD 95-containing postsynaptic densities. Corpus callosum astrocytes had an elongated morphology with IP(3)R2 studded processes extending along fiber tracts. Our data suggest that PLC-mediated Ca(2+) signaling in astrocytes in rat brain occurs predominantly through IP(3)R2 ion channels. Furthermore, the anatomical arrangement of the terminal astrocytic branches containing IP(3)R2 ensheathing synapses is ideal for supporting glial monitoring of neuronal activity.     

Connexin channels in Schwann cells and the development of the X-linked form of Charcot-Marie-Tooth disease

Charcot-Marie-Tooth disease comprises a group of genetically heterogenous disorders of the peripheral nervous system. The X-linked form of Charcot-Marie-Tooth (CMTX) is associated with mutations in the gene encoding the gap junction protein connexin32 (Cx32), which is expressed in Schwann cells. Immunocytochemical evidence suggests that Cx32 is localized to the incisures of Schmidt–Lanterman and the paranodes of myelinating Schwann cells, where it appears to form reflexive gap junctions. It is currently thought that this cytoplasmic continuity provides a much shorter diffusion pathway for the transport of ions, metabolites and second messenger molecules through intracellular channels between the adaxonal and peri-nuclear regions of Schwann cells, across the myelin sheath. This review summarizes our current understanding of the role of connexins in Schwann cells and focuses on the lessons for channel function and disease pathophysiology derived from the functional analysis of Cx32 mutations. One of the most intriguing aspects emerging from this work is that several mutations retain functional competence, although the mutated channels exhibit altered gating properties. This suggests that partial and/or selective disruption of the radial communication pathway formed by Cx32 is sufficient to cause a functional deficit and lead to the development of CMTX. The next challenge will be to define, at the molecular level, the sequence of events involved in the disease process. The presence of a group of functional mutations should help understand the cellular basis of CMTX, by allowing the identification of the specific molecules that need to be exchanged through Cx32 channels, but are excluded from the mutated ones.