Elliot L. Hertzberg

Gap junctions: New tools, new answers, new questions

The big news is that gap junctions of different kinds are formed by a number of homologous proteins termed connexins, which are encoded by a gene family. Specific connexins are expressed in more than one tissue, and a single cell type may express more than one connexin

LM and EM immunolocalization of the gap junctional protein connexin 43 in rat brain

A site-specific antibody against the principal gap junctional protein in heart (connexin 43) was used to determine immunohistochemically the cellular localization of this protein in rat brain. Structures labelled with the antibody included gap junctional membranes between glial, ependymal, pial and arachnoid cells as well as cytoplasmic membranes and intracellular organelles in close proximity to junctions between these various cell types. No labelling was detected within cell bodies of oligodendrocytes and neurons and no labelled neuronal gap junctions were found. The results suggest that connexin 43 is one of the major gap junctional proteins utilized for junctional coupling between astrocytes and between cells lining the surfaces of the brain.

Elevated connexin43 immunoreactivity at sites of amyloid plaques in alzheimer's disease

The distribution of the astrocytic gap junctional protein, connexin43 (Cx43) was compared immunohistochemically with that of amyloid plaques in Alzheimers Disease (AD) brain. By light microscopy, cortical areas containing numerous beta/A4 amyloid plaques exhibited increased immunostaining density for Cx43 and some plaques corresponded exactly to sites of intensified Cx43 immunoreactivity. By electron microscopy, Cx43 was localized to astrocytic gap junctions in AD brain. Increased Cx43 expression in AD may represent an attempt to maintain tissue homeostasis by augmented intercellular communication via gap junction formation between astrocytic processes that invest senile plaques, or alternatively, an aberrant induction of astrocytic Cx43 expression which may further compromise homeostasis and exacerbate pathological conditions in the microenvironment of amyloid plaques.

Oligodendrocytes express gap junction proteins connexin32 and connexin45

Oligodendrocytes, the myelin‐forming glia of brain, are connected by gap junctions in situ and in culture. Cultured oligodendrocytes from adult bovine and porcine brains were studied using immunocytochemical, molecular, and electrophysiological techniques in order to characterize the gap junction types. The expression of connexin32 was substantiated by the detection of low, but significant, signals using connexin‐specific probes in Northern and Western blot analyses. Connexin43, which comprises gap junctions in astrocytes, was not detectable in pure oligodendrocytic cultures; mRNAs of connexin40 and connexin37 and connexin26 were also not detected. By means of two specific antibodies directed to the recently cloned connexin45 and by RT‐PCR we were able to identify this connexin as a second oligodendrocytic gap junction protein. Whole cell voltage clamp recording provided evidence for electrical coupling between pairs of cultured oligodendrocytes (mean junctional conductance 3.9 nS, n = 38 pairs) and intracellular Lucifer Yellow injection indicated that oligodendrocytes were usually only weakly dye coupled, with spread generally being restricted to nearest neighbors. Unitary conductances ranged from >20 to <150 pS with modes of distribution at about 100 to 120pS and 40 to 20 pS, respectively.

CLIP-115, a Novel Brain-Specific Cytoplasmic Linker Protein, Mediates the Localization of Dendritic Lamellar Bodies

Intracellular localization of organelles may depend in part on specific cytoplasmic linker proteins (CLIPs) that link membranous organelles to microtubules. Here, we characterize rat cDNAs encoding a novel, brain-specific CLIP of 115 kDa. This protein contains two N-terminal microtubule-binding domains and a long coiled-coil region; it binds to microtubules and is homologous to CLIP-170, a protein mediating the binding of endosomes to microtubules. CLIP-115 is enriched in the dendritic lamellar body (DLB), a recently discovered organelle predominantly present in bulbous dendritic appendages of neurons linked by dendrodendritic gap junctions. Local microtubule depolymerization leads to a temporary reduction of DLBs. These results suggest that CLIP-115 operates in the control of brain-specific organelle translocations.

Immunorecognition, ultrastructure and phosphorylation status of astrocytic gap junctions and connexin43 in rat brain after cerebral focal ischaemia

Gap junctions between astrocytes support a functional syncytium that is thought to play an important role in neural homeostasis. In order to investigate regulation of this syncytium and of connexin43 (Cx43), a principal astrocytic gap junction protein, we determined the sequelae of gap junction and Cx43 disposition in a rat cerebral focal ischaemia model with various ischaemia/reperfusion times using sequence‐specific anti‐Cx43 antibodies (designated 13‐8300, 18A, 16A and 71‐0700) that exhibit differential recognition of Cx43, perhaps reflecting functional aspects of gap junctions. Antibody 13‐8300 specifically detects only an unphosphorylated form of Cx43 in both Western blots and tissue sections. In hypothalamus after brief (15 min) ischaemic injury, Cx43 at intact gap junctions undergoes dephosphorylation, accompanied by reduced epitope recognition by antibodies 16A and 71‐0700. Tissue examined 24 h after reperfusion showed that these effects were reversible. Astrocytic gap junction internalization occurring 1 h after ischaemia was accompanied by decreased immunodetection with 13‐8300. At this time, gap junctions were absent in the ischaemic core, coinciding with a loss of Cx43 recognition with 18A and 13‐8300, but elevated labelling of internalized Cx43 with 16A and 71‐0700. Unphosphorylated Cx43 persisted at intact gap junctions confined to a thin corridor at the ischaemic penumbra which contained presumptive apoptotic cell profiles. Similar results were obtained in ischaemic striatum and cerebral cortex, though with a delayed time course that depended on the severity of the ischaemic insult. These results demonstrate that astrocytic Cx43 epitope masking, dephosphorylation and cellular redistribution occur after ischaemic brain injury, proceed as a temporally and spatially ordered sequence of events and culminate in differential patterns of Cx43 modification and sequestration at the lesion centre and periphery. These observations suggest an attempt by astrocytes in the vicinity of injury to remodel the junctional syncytium according to altered tissue homeostatic requirements.

Effects of cGMP-dependent phosphorylation on rat and human connexin43 gap junction channels

The effects of 8-bromoguanosine 3′:5′-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (gj) in rat Cx43-transfected cells by 24.0±3.7% (mean±SEM, n=5), whereas gj was not affected in human Cx43-transfected cells by the same treatment. The relaxation of gj in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (γj) of about 20, 40–45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the γj distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [32P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [32P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased gj, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.

Ischemia-induced cellular redistribution of the astrocytic gap junctional protein connexin43 in rat brain

The distribution and levels of the astrocytic gap junction protein, connexin43 (Cx43) was analyzed in various regions of brain as a function of time after neuronal loss and consequent reactive gliosis induced by bilateral carotid occlusion in rats. In the striatum 2 days after induction of ischemia, immunostaining intensity for Cx43 increased in animals exhibiting mild to moderate striatal damage, whereas areas of reduced staining surrounded by elevated levels of Cx43 immunoreactivity were observed in animals with severe ischemic damage. Immunolabelling of glial cell bodies was evident in ischemic, but not normal, striatum. Similar, though less dramatic, changes were seen at 7 days post-ischemia. Compared with the fine punctate pattern of Cx43 staining seen in normal striatum, ischemic striatal areas contained large aggregates of punctate profiles. In the hippocampus, increased immunostaining was seen at 2 and 7 days post-ischemia and, unlike normal hippocampus, neurons in the CA3 pyramidal cell layer were surrounded by a network of Cx43-immunoreactive puncta at the latter survival time. Immuno-EM analysis of ischemic tissue revealed numerous immunolabelled gap junctions among astrocytic processes in the vicinity of degenerating neurons and elevated levels of intracellular Cx43 immunoreactivity in astrocytic processes and cell bodies. No differences in protein levels or phosphorylation states of Cx43 were detected in either hippocampus or striatum by Western blot analyses of ischemic and control tissue. These results suggest that astrocytes respond to an ischemic insult by reorganizing their gap junctions, that the qualitative nature of their response is dependent on the severity of neuronal damage or loss, and that a pool of Cx43 normally undetectable by immunohistochemistry may contribute to the ischemia-induced elevations of immunolabelling for this protein.

Evidence for the co-localization of another connexin with connexin-43 at astrocytic gap junctions in rat brain

Gap junctions between astrocytes as well as between astrocytes and oligodendrocytes in rat brain were immunohistochemically labelled with a monoclonal and an affinity-purified polyclonal antibody generated against connexin-26. By light microscopy, the immunolabelling patterns obtained were, with a few exceptions, remarkably similar to previously described distribution patterns of the gap junctional protein connexin-43, which is expressed by astrocytes and is localized at astrocytic gap junctions. By electron microscopy, immunoreactivity with these two anti-connexin-26 antibodies was restricted to astrocytes; inter-astrocytic gap junctional membranes were symmetrically labelled, heterologous oligo-astrocytic junctional membranes were asymmetrically labelled only on the astrocyte side and oligo-oligodendrocyte junctions were unlabelled. Two additional anti-connexin-26 antibodies that were found to produce punctate labelling in leptomeninges and liver failed to do so in brain parenchyma, consistent with reports indicating the absence of authentic connexin-26 in this tissue. Antibodies that labelled astrocytic gap junctions exhibited no cross-reaction with connexin-43 or connexin-32, as demonstrated by western blotting, but recognized liver connexin-26 as well as several brain proteins, including an approximately 32000 mol. wt protein that did not correspond to connexin-32 and a 26000 mol. wt protein that co-migrated with liver connexin-26. These results suggest that connexin-26, or more likely a protein having sequence homology with connexin-26, is targeted to astrocytic gap junctions and raise the possibility of the existence of connexins that may be co-expressed with connexin-43 in most, but perhaps not all, astrocytes.

Connexin43 and astrocytic gap junctions in the rat spinal cord after acute compression injury

To examine the possible role of interastrocytic gap junctions in the maintenance of tissue homeostasis after spinal cord damage, we initiated studies of the astrocytic gap junctional protein connexin43 (Cx43) in relation to temporal and spatial parameters of neuronal loss, reactive gliosis, and white matter survival in a rat model of traumatic spinal cord injury (SCI). Cx43 immunolocalization in normal and compression‐injured spinal cord was compared by using two different sequence‐specific anti‐Cx43 antibodies that have previously exhibited different immunorecognition properties at lesion sites in brain. At 1‐ and 3‐day survival times, gray matter areas with mild to moderate neuronal depletion exhibited a loss of immunolabeling with one of the two antibodies. At the lesion epicenter, these areas consisted of a zone that separated normal staining distal to the lesion from intensified labeling seen with both antibodies immediately adjacent to the lesion. Loss of immunoreactivity with only one of the two antibodies suggested masking of the corresponding Cx43 epitope. By 7 days post‐SCI, Cx43 labeling was absent with both antibodies in all regions extending up to 1 mm from the lesion site. Reactive astrocytes displaying glial fibrillary acidic protein (GFAP) appeared by 1 day and were prominent by 3 days post‐SCI. Their distribution in white and gray matter corresponded closely to that of Cx43 staining at 1 day, but less so at 3 days when GFAP‐positive profiles were present at sites where Cx43 labeling was absent. By 7 days post‐SCI, Cx43 again co‐localized with GFAP‐positive cells in the surviving subpial rim, and with astrocytic processes on radially oriented vascular profiles investing the central borders of the lesion. The results indicate that alterations in Cx43 cellular localization and Cx43 molecular modifications reflected by epitope masking, which were previously correlated with gap junction remodeling following excitotoxin‐induced lesions in brain, are not responses limited to exogenously applied excitotoxins; they also occur in damaged spinal cord and are evoked by endogenous mechanisms after traumatic SCI. The GFAP/Cx43 co‐localization results suggest that during their transformation to a reactive state, spinal cord astrocytes undergo a transitional phase marked by altered Cx43 localization or expression. J. Comp. Neurol. 382:199‐214, 1997.