Characteristics of Electrical Synapses, C-terminals and Small-conductance Ca2+ activated Potassium Channels in the Sexually Dimorphic Cremaster Motor Nucleus in Spinal Cord of Mouse and Rat.

Sexually dimorphic motoneurons (MNs) located in lower lumbar spinal cord are involved in mating and reproductive behaviours and are known to be coupled by electrical synapses. The cremaster motor nucleus in upper lumbar spinal cord has also been suggested to support physiological processes associated with sexual behaviours in addition to its thermoregulatory and protective role in maintaining testes integrity. Using immunofluorescence approaches, we investigated whether cremaster MNs also exhibit features reflecting their potential for electrical synaptic communication and examined some of their other synaptic characteristics. Both mice and rats displayed punctate immunolabelling of Cx36 associated with cremaster MNs, indicative of gap junction formation. Transgenic mice with enhanced green fluorescent protein (eGFP) reporter for connexin36 expression showed that subpopulations of cremaster MNs in both male and female mice express eGFP, with greater proportions of those in male mice. The eGFP+ MNs within the cremaster nucleus vs. eGFP- MNs inside and outside this nucleus displayed a 5-fold greater density of serotonergic innervation and exhibited a paucity of innervation by C-terminals arising from cholinergic V0c interneurons. All MNs within the cremaster motor nucleus displayed prominent patches of immunolabelling for SK3 (K+) channels around their periphery, suggestive of their identity as slow MNs, many though not all of which were in apposition to C-terminals. The results provide evidence for electrical coupling of a large proportion of cremaster MNs and suggest the existence of two populations of these MNs with possibly differential innervation of their peripheral target muscles serving different functions.

On the Organization of Connexin36 Expression in Electrically Coupled Cholinergic V0c Neurons (Partition Cells) in the Spinal Cord and Their C-terminal Innervation of Motoneurons.

Large cholinergic neurons (V0c neurons; aka, partition cells) in the spinal cord project profusely to motoneurons on which they form C-terminal contacts distinguished by their specialized postsynaptic subsurface cisterns (SSCs). The V0c neurons are known to be rhythmically active during locomotion and release of acetylcholine (ACh) from their terminals is known to modulate the excitability of motoneurons in what appears to be a task-dependent manner. Here, we present evidence that a subpopulation of V0c neurons express the gap junction forming protein connexin36 (Cx36), indicating that they are coupled by electrical synapses. Based on immunofluorescence imaging and the use of Cx36BAC-enhanced green fluorescent protein (eGFP) mice in which C-terminals immunolabelled for their marker vesicular acetylcholine transporter (vAChT) are also labelled for eGFP, we found a heterogeneous distribution of eGFP+ C-terminals on motoneurons at cervical, thoracic and lumber spinal levels. The density of C-terminals on motoneurons varied as did the proportion of those that were eGFP+ vs. eGFP-. We present evidence that fast vs. slow motoneurons have a greater abundance of these terminals and fast motoneurons also have the highest density that were eGFP+. Thus, our results indicate that a subpopulation of V0c neurons projects preferentially to fast motoneurons, suggesting that the capacity for synchronous activity conferred by electrical synapses among networks of coupled V0c neurons enhances their dynamic capabilities for synchronous regulation of motoneuron excitability during high muscle force generation. The eGFP+ vs. eGFP- V0c neurons were more richly innervated by serotonergic terminals, suggesting their greater propensity for regulation by descending serotonergic systems.

Structural and Intermolecular Associations Between Connexin36 and Protein Components of the Adherens Junction-Neuronal Gap Junction Complex.

Intimate structural and functional relationships between gap junctions and adherens junctions have been demonstrated in peripheral tissues, but have not been thoroughly examined in the central nervous system, where adherens junctions are often found in close proximity to neuronal gap junctions. Here, we used immunofluorescence approaches to document the localization of various protein components of adherens junctions in relation to those that we have previously reported to occur at electrical synapses formed by neuronal gap junctions composed of connexin36 (Cx36). The adherens junction constituents N-cadherin and nectin-1 were frequently found to localize near or overlap with Cx36-containing gap junctions in several brain regions examined. This was also true of the adherens junction-associated proteins α-catenin and ß-catenin, as well as the proteins zonula occludens-1 and AF6 (aka, afadin) that were reported constituents of both adherens junctions and gap junctions. The deployment of the protein constituents of these junctions was especially striking at somatic contacts between primary afferent neurons in the mesencephalic trigeminal nucleus (MesV), where the structural components of adherens junctions appeared to be maintained in connexin36 null mice. These results support emerging views concerning the multi-molecular composition of electrical synapses and raise possibilities for various structural and functional protein-protein interactions at what now can be considered the adherens junction-neuronal gap junction complex. Further, the results point to intracellular signaling pathways that could potentially contribute to the assembly, maintenance and turnover of this complex, as well as to the dynamic nature of neuronal communication at electrical synapses.

Connexin36 Expression in Primary Afferent Neurons in Relation to the Axon Reflex and Modality Coding of Somatic Sensation.

Electrical coupling mediated by connexin36-containing gap junctions that form electrical synapses is known to be prevalent in the central nervous system, but such coupling was long ago reported also to occur between cutaneous sensory fibers. Here, we provide evidence supporting the capability of primary afferent fibers to engage in electrical coupling. In transgenic mice with enhanced green fluorescent protein (eGFP) serving as a reporter for connexin36 expression, immunofluorescence labeling of eGFP was found in subpopulations of neurons in lumbar dorsal root and trigeminal sensory ganglia, and in fibers within peripheral nerves and tissues. Immunolabeling of connexin36 was robust in the sciatic nerve, weaker in sensory ganglia than in peripheral nerve, and absent in these tissues from Cx36 null mice. Connexin36 mRNA was detected in ganglia from wild-type mice, but not in those from Cx36 null mice. Labeling of eGFP was localized within a subpopulation of ganglion cells containing substance P and calcitonin gene-releasing peptide, and in peripheral fibers containing these peptides. Expression of eGFP was also found in various proportions of sensory ganglion neurons containing transient receptor potential (TRP) channels, including TRPV1 and TRPM8. Ganglion cells labeled for isolectin B4 and tyrosine hydroxylase displayed very little co-localization with eGFP. Our results suggest that previously observed electrical coupling between peripheral sensory fibers occurs via electrical synapses formed by Cx36-containing gap junctions, and that some degree of selectivity in the extent of electrical coupling may occur between fibers belonging to subpopulations of sensory neurons identified according to their sensory modality responsiveness.

Immunofluorescence reveals unusual patterns of labelling for connexin43 localized to calbindin-D28K-positive interstitial cells in the pineal gland.

Gap junctions between cells in the pineal gland have been described ultrastructurally, but their connexin constituents have not been fully characterized. We used immunofluorescence in combination with markers of pineal cells to document the cellular localization of connexin43 (Cx43). Immunofluorescence labelling of Cx43 with several different antibodies was widely distributed throughout the pineal, whereas another connexin examined, connexin26, was not found in pineal but only in surrounding leptomeninges. Labelling apparently associated with plasma membranes was visualized either as fine Cx43-puncta (1-2 µm) or as unusually large pools of Cx43 ranging up to 4-7 µm in diameter or length. These puncta and pools were highly concentrated in perivascular spaces, where they were associated with numerous cells devoid of labelling for markers of pinealocytes (e.g. tryptophan hydroxylase and serotonin), and where they were minimally associated with blood vessels and lacked association with resident macrophages. Astrocytes labelled for glial fibrillary acidic protein were largely restricted to the anterior pole of the pineal gland, where they displayed only fine and sparse Cx43-puncta along their processes. Labelling for Cx43 was localized largely though not exclusively to the somata and long processes of a subpopulation of perivascular interstitial cells that were immunopositive for calbindin-D28K. These cells were often located among dense bundles or termination areas of sympathetic fibres labelled for tyrosine hydroxylase or serotonin. The results indicate that interstitial cells form abundant gap junctions composed of Cx43, and suggest that gap junction-mediated intracellular communication by these cells supports the activities of pinealocytes.

Under construction: building the macromolecular superstructure and signaling components of an electrical synapse.

A great deal is now known about the protein components of tight junctions and adherens junctions, as well as how these are assembled. Less is known about the molecular framework of gap junctions, but these also have membrane specializations and are subject to regulation of their assembly and turnover. Thus, it is reasonable to consider that these three types of junctions may share macromolecular commonalities. Indeed, the tight junction scaffolding protein zonula occluden-1 (ZO-1) is also present at adherens and gap junctions, including neuronal gap junctions. On the basis of these earlier observations, we more recently found that two additional proteins, AF6 and MUPP1, known to be associated with ZO-1 at tight and adherens junctions, are also components of neuronal gap junctions in rodent brain and directly interact with connexin36 (Cx36) that forms these junctions. Here, we show by immunofluorescence labeling that the cytoskeletal-associated protein cingulin, commonly found at tight junctions, is also localized at neuronal gap junctions throughout the central nervous system. In consideration of known functions related to ZO-1, AF6, MUPP1, and cingulin, our results provide a context in which to examine functional relationships between these proteins at Cx36-containing electrical synapses in brain--specifically, how they may contribute to regulation of transmission at these synapses, and how they may govern gap junction channel assembly and/or disassembly.

The effector and scaffolding proteins AF6 and MUPP1 interact with connexin36 and localize at gap junctions that form electrical synapses in rodent brain.

Electrical synapses formed by neuronal gap junctions composed of connexin36 (Cx36) occur in most major structures in the mammalian central nervous system. These synapses link ensembles of neurons and influence their network properties. Little is known about the macromolecular constituents of neuronal gap junctions or how transmission through electrical synapses is regulated at the level of channel conductance or gap junction assembly/disassembly. Such knowledge is a prerequisite to understanding the roles of gap junctions in neuronal circuitry. Gap junctions share similarities with tight and adhesion junctions in that all three reside at close plasma membrane appositions, and therefore may associate with similar structural and regulatory proteins. Previously, we reported that the tight junction-associated protein zonula occludens-1 (ZO-1) interacts with Cx36 and is localized at gap junctions. Here, we demonstrate that two proteins known to be associated with tight and adherens junctions, namely AF6 and MUPP1, are components of neuronal gap junctions in rodent brain. By immunofluorescence, AF6 and MUPP1 were co-localized with Cx36 in many brain areas. Co-immunoprecipitation and pull-down approaches revealed an association of Cx36 with AF6 and MUPP1, which required the C-terminus PDZ domain interaction motif of Cx36 for interaction with the single PDZ domain of AF6 and with the 10th PDZ domain of MUPP1. As AF6 is a target of the cAMP/Epac/Rap1 signalling pathway and MUPP1 is a scaffolding protein that interacts with CaMKII, the present results suggest that AF6 may be a target for cAMP/Epac/Rap1 signalling at electrical synapses, and that MUPP1 may contribute to anchoring CaMKII at these synapses.

Ablation of connexin30 in transgenic mice alters expression patterns of connexin26 and connexin32 in glial cells and leptomeninges.

Expression of connexin26 (Cx26), Cx30 and Cx43 in astrocytes and expression of Cx29, Cx32 and Cx47 in oligodendrocytes of adult rodent brain has been well documented, as has the interdependence of connexin expression patterns of macroglial cells in Cx32- and Cx47-knockout mice. To investigate this interdependence further, we examined immunofluorescence labelling of glial connexins in transgenic Cx30 null mice. Ablation of astrocytic Cx30, confirmed by the absence of immunolabelling for this connexin in all brain regions, resulted in the loss of its coupling partner Cx32 on the oligodendrocyte side of astrocyte-oligodendrocyte (A/O) gap junctions, but had no effect on the localization of astrocytic Cx43 and oligodendrocytic Cx47 at these junctions or on the distribution of Cx32 along myelinated fibres. Surprisingly, gene deletion of Cx30 led to the near total elimination of immunofluorescence labelling for Cx26 in all leptomeningeal tissues covering brain surfaces as well as in astrocytes of brain parenchyma. Moreover northern blot analysis revealed downregulation of Cx26 mRNA in Cx30-knockout brains. Our results support earlier observations on the interdependency of Cx30/Cx32 targeting to A/O gap junctions and further suggest that Cx26 mRNA expression is affected by Cx30 gene expression. In addition, Cx30 protein may be required for co-stabilization of gap junctions or for co-trafficking in cells.

Connexin26 expression in brain parenchymal cells demonstrated by targeted connexin ablation in transgenic mice.

Astrocytes are known to express the gap junction forming proteins connexin30 (Cx30) and connexin43 (Cx43), but it has remained controversial whether these cells also express connexin26 (Cx26). To investigate this issue further, we examined immunofluorescence labelling of glial connexins in wild-type vs. transgenic mice with targeted deletion of Cx26 in neuronal and glial cells (Cx26fl/fl:Nestin-Cre mice). The Cx26 antibodies utilized specifically recognized Cx26 and lacked cross reaction with highly homologous Cx30, as demonstrated by immunoblotting and immunofluorescence in Cx26-transfected and Cx30-transfected C6 glioma cells. Punctate immunolabelling of Cx26 with these antibodies was observed in leptomeninges and subcortical brain regions. This labelling was absent in subcortical areas of Cx26fl/fl:Nestin-Cre mice, but persisted in leptomeningeal tissues of these mice, thereby distinguishing localization of Cx26 between parenchymal and non-parenchymal tissue. In subcortical brain parenchyma, Cx26-positive puncta were often co-localized with astrocytic Cx43, and some were localized along astrocyte cell bodies and processes immunolabelled for glial fibrillary acidic protein. Cx26-positive puncta were also co-localized with punctate labelling of Cx47 around oligodendrocyte somata. Comparisons of Cx26 labelling in rodent species revealed a lower density of Cx26-positive puncta and a more restricted distribution in subcortical regions of mouse compared with rat brain, perhaps partly explaining reported difficulties in detection of Cx26 in mouse brain parenchyma using antibodies or Cx26 gene reporters. These results support our earlier observations of Cx26 expression in astrocytes and its ultrastructural localization in individual gap junction plaques formed between astrocytes as well as in heterotypic gap junctions between astrocytes and oligodendrocytes.

Spatial relationships of connexin36, connexin57 and zonula occludens-1 in the outer plexiform layer of mouse retina.

Horizontal cells form gap junctions with each other in mammalian retina, and lacZ reporter analyses have recently indicated that these cells express the Cx57 gene, which codes for the corresponding gap junctional protein. Using anti-connexin57 antibodies, we detected connexin57 protein in immunoblots of mouse retina, and found punctate immunolabeling of this connexin co-distributed with calbindin-positive horizontal cells in the retinal outer plexiform layer. Double immunofluorescence labeling was conducted to determine the spatial relationships of connexin36, connexin57, the gap junction-associated protein zonula occludens-1 and the photoreceptor ribbon synapse-associated protein bassoon in the outer plexiform layer. Connexin36 was substantially co-localized with zonula occludens-1 in the outer plexiform layer, and both of these proteins were frequently located in close spatial proximity to bassoon-positive ribbon synapses. Connexin57 was often found adjacent to, but not overlapping with, connexin36-positive and zonula occludens-1-positive puncta, and was also located adjacent to bassoon-positive ribbon synapses at rod spherules, and intermingled with such synapses at cone pedicles. These results suggest zonula occludens-1 interaction with connexin36 but not with Cx57 in the outer plexiform layer, and an absence of connexin57/connexin36 heterotypic gap junctional coupling in mouse retina. Further, an arrangement of synaptic contacts within rod spherules is suggested whereby gap junctions between horizontal cell terminals containing connexin57 occur in very close proximity to ribbon synapses formed by rod photoreceptors, as well as in close proximity to Cx36-containing gap junctions between rods and cones.

Connexin47, connexin29 and connexin32 co-expression in oligodendrocytes and Cx47 association with zonula occludens-1 (ZO-1) in mouse brain.

Gap junctions between glial cells in mammalian CNS are known to contain several connexins (Cx), including Cx26, Cx30 and Cx43 at astrocyte-to-astrocyte junctions, and Cx29 and Cx32 on the oligodendrocyte side of astrocyte-to-oligodendrocyte junctions. Recent reports indicating that oligodendrocytes also express Cx47 prompted the present studies of Cx47 localization and relationships to other glial connexins in mouse CNS. In view of the increasing number of connexins reported to interact directly with the scaffolding protein zonula occludens-1 (ZO-1), we investigated ZO-1 expression and Cx47/ZO-1 interaction capabilities in brain, spinal cord and Cx47-transfected HeLa cells. From counts of over 9000 oligodendrocytes labeled by immunofluorescence in various brain regions, virtually all of these cells were found to express Cx29, Cx32 and Cx47. Oligodendrocyte somata displayed robust Cx47-immunopositive puncta that were co-localized with punctate labeling for Cx32 and Cx43. By freeze-fracture replica immunogold labeling, Cx47 was abundant on the oligodendrocyte-side of oligodendrocyte/astrocyte gap junctions. By immunofluorescence, labeling for Cx47 along myelinated fibers was sparse in most brain regions, whereas Cx29 and Cx32 were previously found to be concentrated along these fibers. By immunogold labeling, Cx47 was found in numerous small gap junctions linking myelin to astrocytes, but not within deeper layers of myelin. Brain subcellular fractionation revealed a lack of Cx47 enrichment in myelin fractions, which nevertheless contained an enrichment of Cx32 and Cx29. Oligodendrocytes were immunopositive for ZO-1, and displayed almost total Cx47/ZO-1 co-localization. ZO-1 was found to co-immunoprecipitate with Cx47, and pull-down assays indicated binding of Cx47 to the second PDZ domain of ZO-1. Our results indicate widespread expression of Cx47 by oligodendrocytes, but with a distribution pattern in relative levels inverse to the abundance of Cx29 in myelin and paucity of Cx29 in oligodendrocyte somata. Further, our findings suggest a scaffolding and/or regulatory role of ZO-1 at the oligodendrocyte side of astrocyte-to-oligodendrocyte gap junctions.

Coupling of astrocyte connexins Cx26, Cx30, Cx43 to oligodendrocyte Cx29, Cx32, Cx47: Implications from normal and connexin32 knockout mice.

Oligodendrocytes in vivo form heterologous gap junctions with astrocytes. These oligodendrocyte/astrocyte (A/O) gap junctions contain multiple connexins (Cx), including Cx26, Cx30, and Cx43 on the astrocyte side, and Cx32, Cx29, and Cx47 on the oligodendrocyte side. We investigated connexin associations at A/O gap junctions on oligodendrocytes in normal and Cx32 knockout (KO) mice. Immunoblotting and immunolabeling by several different antibodies indicated the presence of Cx32 in liver and brain of normal mice, but the absence of Cx32 in liver and brain of Cx32 KO mice, confirming the specificity and efficacy of the antibodies, as well as allowing the demonstration of Cx32 expression by oligodendrocytes. Oligodendrocytes throughout brain were decorated with numerous Cx30-positive puncta, which also were immunolabeled for both Cx32 and Cx43. In Cx32 KO mice, astrocytic Cx30 association with oligodendrocyte somata was nearly absent, Cx26 was partially reduced, and Cx43 was present in abundance. In normal and Cx32 KO mice, oligodendrocyte Cx29 was sparsely distributed, whereas Cx47-positive puncta were densely localized on oligodendrocyte somata. These results demonstrate that astrocyte Cx30 and oligodendrocyte Cx47 are widely present at A/O gap junctions. Immunolabeling patterns for these six connexins in Cx32 KO brain have implications for deciphering the organization of heterotypic connexin coupling partners at A/O junctions. The persistence and abundance of Cx43 and Cx47 at these junctions after Cx32 deletion, together with the paucity of Cx29 normally present at these junctions, suggests Cx43/Cx47 coupling at A/O junctions. Reductions in Cx30 and Cx26 after Cx32 deletion suggest that these astrocytic connexins likely form junctions with Cx32 and that their incorporation into A/O gap junctions is dependent on the presence of oligodendrocytic Cx32.

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.

Identification of sequence, protein isoforms, and distribution of the hyaluronan-binding protein RHAMM in adult and developing rat brain.

The protein RHAMM (for "receptor for hyaluronan-mediated motility"; CD168) is a member of the hyaladherin family of hyaluronan-binding proteins. RHAMM has a role in cell signaling, migration, and adhesion via interactions with hyaluronan, microtubules, actin, calmodulin, and components of the extracellular regulated kinase (erk) signaling pathway. Based on previous findings of potentially similar roles in neural cells in culture, we investigated the molecular characteristics, protein expression profile, and distribution of RHAMM in rat brain. Reverse transcriptase-polymerase chain reaction (RT-PCR) using RNA isolated from adult rat brain yielded a single RHAMM sequence of 2.1 kilobases encoding a protein of 82.4 kDa. RHAMM is subject to alternate splicing in other systems, but no RT-PCR evidence was found for splice variants in brain, although our analysis does not rule out this possibility. The amino acid sequence displayed homology with human and murine RHAMM (74% and 80%, respectively) but contained only one copy of a 21-amino-acid sequence that is repeated five times in the murine homologue. By using anti-RHAMM antibodies, several RHAMM isoforms were identified in brain. Immunohistochemically, RHAMM was found in the vast majority of neurons and in many oligodendrocytes throughout brain, with heterogeneous levels among cell populations, and was confined to the somata and initial processes of these cells. RHAMM was detected in neurons of cerebral cortex and most subcortical and brainstem structures at postnatal day 1 and exhibited an adult distribution pattern by postnatal day 5. High levels were detected in oligodendrocytes by postnatal day 10. The widespread expression of RHAMM in adult and developing brain implies a role for this protein and its ligand hyaluronan in key events of cell signaling and cytoskeletal regulation in the CNS.

Subcellular distribution, calmodulin interaction, and mitochondrial association of the hyaluronan-binding protein RHAMM in rat brain.

The CNS contains high levels of the glycosaminoglycan hyaluronan, and neural cells express a variety of proteins that are members of the hyaladherin family of hyaluronan-binding proteins. We have previously shown that the hyaladherin RHAMM (receptor for hyaluronan-mediated motility; CD168) is expressed by neural cells in culture; plays a role in astrocyte motility, neurite migration, and axonal growth; and is widely distributed in neurons and oligodendrocytes of developing and adult rat CNS. Here we demonstrate differential localization of various forms of RHAMM in subcellular fractions of adult rat brain. Western blotting indicated the presence of 66, 75, and 85-90 kDa molecular weight RHAMM forms in whole-brain homogenates. Subfractionation revealed enrichment of the 66 and 85-90 kDa forms in soluble fractions, whereas the 75 kDa form was enriched in mitochondrial fractions. This latter form was retained in osmotically shocked mitochondria, but was liberated by alkali carbonate, suggesting a nonintrinsic mitochondrial membrane association. By double immunohistochemical labeling for RHAMM and the mitochondrial marker cytochrome oxidase, RHAMM was localized to isolated mitochondria in vitro and to neuronal mitochondria in vivo. Hyaluronan-sepharose chromatography and cetylpiridinium chloride precipitation confirmed the hyaluronan-binding capacity of RHAMM forms. By calmodulin-affinity chromatography, endogenously expressed brain RHAMM was demonstrated to bind calmodulin in a Ca2+-dependent manner. These results, together with reports of RHAMM association with actin and microtubules in other systems, suggest a role of RHAMM in calmodulin-mediated cell signaling to cytoskeletal elements and/or mitochondria in the CNS and invoke novel functions of its interactions with hyaluronan.

Sequence, protein expression and extracellular-regulated kinase association of the hyaladherin RHAMM (receptor for hyaluronan mediated motility) in PC12 cells.

Differentiation of PC12 cells by nerve growth factor (NGF) or fibroblast growth factor-2 (FGF2) is dependent on signaling mediated by extracellular regulated kinase (ERK). We investigated the involvement of receptor for hyaluronan mediated motility (RHAMM) in this signaling pathway. A single RHAMM 3.2 kb transcript was detected in PC12 RNA. Reverse transcriptase-polymerase chain reaction generated a 2141 bp cDNA that had identical sequence to rat brain RHAMM and showed no evidence of alternate splicing. Several RHAMM species were identified by Western blotting. Immunohistochemistry showed RHAMM localization to the cytoskeleton, neurites and growth cones. Following stimulation of PC12 cells with NGF or FGF2 RHAMM was co-immunoprecipitated by phosphorylation-specific anti-ERK antibodies, indicating a role for RHAMM in ERK signaling in PC12 cells.

Enrichment of neuronal and glial connexins in the postsynaptic density subcellular fraction of rat brain.

The similar dense, protein-rich and detergent-resistant characteristics of postsynaptic densities (PSDs) and gap junctions led us to investigate the distribution of gap junctions and their constituent connexins in CNS subcellular fractions containing PSDs. Western blot analysis showed these fractions to be enriched in both neuronal and glial connexins, namely, connexin26, connexin30, connexin36 and connexin43. Connexins were retained in these fractions after treatment with n-lauroyl sarcosine to remove loosely associated proteins. Confocal double immunofluorescence confirmed the presence of connexins in PSD fractions and showed a near total co-localization of glial connexin30 and connexin43, demonstrating preservation of inter-connexin relationships that have been observed in vivo. In contrast, none of the connexins were co-localized with the PSD structural protein PSD-95, indicating their lack of direct association with PSDs. These results show that PSD preparations contain significant levels of connexin proteins, which appear to remain assembled as gap junctions. Thus, protocols used to isolate PSDs may serve as a basis for development of methods to isolate CNS gap junctions, which would aid biochemical identification of regulatory and structural proteins associated with these structures.

The hyaluronan receptor RHAMM in noradrenergic fibers contributes to axon growth capacity of locus coeruleus neurons in an intraocular transplant model.

The hyaluronan receptor for hyaluronic acid-mediated motility (RHAMM) plays a role in cell migration and motility in many systems. Recent observations on the involvement of RHAMM in neurite motility in vitro suggest that it might also be important in axon outgrowth in situ. This was addressed directly by investigating both RHAMM expression in the rat CNS and the ability of anti-RHAMM reagents to interfere with tissue growth and axon outgrowth in intraocular brainstem transplants. By western blotting, anti-RHAMM antibody detected a RHAMM isoform of 75,000 mol. wt in both whole brain homogenate and synaptosome preparations, and a 65,000 mol. wt isoform in synaptosomes. Immunofluorescence of adult brain sections revealed RHAMM-like immunoreactivity in varicose fibers that were also positive for the noradrenergic marker dopamine-beta-hydroxylase. Not all noradrenergic fibers contained RHAMM, nor was RHAMM detected in other monoaminergic fiber types. Lesions of noradrenergic fiber systems with beta-halobenzylamine-N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) eliminated RHAMM-positive fibers, but noradrenergic axons that sprouted extensively after this treatment were strongly RHAMM-positive. To assess RHAMM's role in fiber outgrowth, fetal brainstem tissue containing noradrenergic neurons was grafted into the anterior chamber of the eye. Treatment of grafts with anti-RHAMM antibody caused significant inhibition of tissue growth and axon outgrowth, as did a peptide corresponding to a hyaluronan binding domain of RHAMM. These agents had no such effects on transplants containing serotonergic and dopaminergic neurons. These results suggest that RHAMM, an extracellular matrix receptor previously shown to contribute to migratory and contact behavior of cells, may also be important in the growth and/or regenerative capacity of central noradrenergic fibers originating from the locus coeruleus.

Increased connexin-43 and gap junctional communication correlate with altered phenotypic characteristics of cells overexpressing the receptor for hyaluronic acid-mediated motility.

Gap junctional intercellular communication (GJIC) and connexin expression are often altered during cell migration, growth, and transformation, each of which is accompanied by cytoskeletal reorganization. Recently, transfection of fibroblast cells with various isoforms of the receptor for hyaluronic acid-mediated motility (RHAMM) was shown to have profound and differential effects on motility, growth, and cell contact behavior as well as on elements of the actin-containing cytoskeleton. These cells thus provide an ideal system in which to investigate parameters implicated in regulation of GJIC as well as expression of connexin-43 (Cx43) in fibroblasts. We used 10T1/2 fibroblast cell lines transfected with RHAMM isoforms or a dominant negative mutated form of RHAMM that blocks the function of endogenous RHAMM. Increased RHAMM expression in the various cell lines was correlated with increased Cx43 and GJIC. These changes were accompanied by a loss of contact inhibition and decreased focal adhesions in all, and elevated motility of most but not all, cell lines tested. RHAMM-induced transformation also resulted in elevated GJIC and Cx43 levels. Reversion to normal growth, motility, and focal adhesion density following transfection of H-ras-transformed fibroblasts with the mutant form of RHAMM was associated with decreases in both Cx43 expression and GJIC. Transfection of 10T1/2 fibroblasts with RHAMM II (exons 5-14) produced altered contact behavior and increased both Cx43 and GJIC but had no effect on motility. All cells expressing high levels of RHAMM, regardless of the isoform, exhibited a lower density of focal adhesions, which corresponds to a reduced organizational state of the cytoskeleton. These results indicate that regulation of GJIC most strongly correlates with altered focal adhesion and cytoskeleton organization that can lead to various secondary responses, including motility, growth, and transformation, and suggest that RHAMM regulates GJIC and Cx43 expression possibly through its actions on focal adhesions and the associated cytoskeleton.

Propagation of intercellular calcium waves in PC12 cells overexpressing a carboxy-terminal fragment of amyloid precursor protein.

We have recently found that PC12 cells overexpressing a C-terminal 97 amino acid fragment containing the beta/A4 amyloid peptide of amyloid precursor protein (APP) exhibit an induced production of the gap junctional protein connexin43 (Cx43) and an induction of gap junctional communication as assessed by dye-transfer. In studies of two beta/A4-transfected PC12 clones, we show here that these cells, unlike normal PC12 cells or those transfected with vector alone, have the capacity for intercellular transmission of calcium waves as determined by imaging of calcium with fura-2. Intercellular wave propagation occurred in the absence of extracellular calcium and was blocked by known inhibitors of gap junctional communication (octanol and 12-O-tetradecanoyl-phorbol-13-acetate), suggesting mediation by gap junctions. The results indicate a disruptive influence of the C-terminal region of APP or of beta/A4 amyloid peptide on intercellular signaling via gap junctions, which may be relevant to the normal functions of APP or to pathology in Alzheimer's disease.