Glial Gap Junction Pathology in the Spinal Cord of the 5xFAD Mouse Model of Early-Onset Alzheimer’s Disease (original) (raw)
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Amyloid-β regulates gap junction protein connexin 43 trafficking in cultured primary astrocytes
Journal of Biological Chemistry, 2020
Altered expression and function of astroglial gap junction protein Connexin 43 (Cx43) has increasingly been associated to neurotoxicity in Alzheimer disease (AD). While earlier studies have examined the effect of increased amyloid-β (Aβ) on Cx43 expression and function leading to neuronal damage, underlying mechanisms by which Aβ modulates Cx43 in astrocytes remain elusive. Here, using mouse primary astrocyte cultures, we have examined the cellular processes by which Aβ can alter Cx43 gap junctions. We show that Aβ 25-35 impairs functional gap junction coupling yet increases hemichannel activity. Interestingly, Aβ 25-35 increased the intracellular pool of Cx43 with a parallel decrease in gap junction assembly at the surface. Intracellular Cx43 was found to be partly retained in the endoplasmic reticulumassociated cell compartments. However, forward trafficking of the newly synthesized Cx43 that already reached the Golgi was not affected in Aβ 25-35 exposed astrocytes. Supporting this, treatment with 4-phenylbutyrate, a well-known chemical chaperone that improves trafficking of several transmembrane proteins, restored Aβinduced impaired gap junction coupling between astrocytes. We further show that interruption of Cx43 endocytosis in Aβ 25-35 exposed astrocytes resulted in their retention at the cell surface in the form of functional gap junctions indicating that Aβ 25-35 causes rapid internalization of Cx43 gap junctions. Additionally, in silico molecular docking suggests that Aβ can bind favorably to Cx43. Our study thus provides novel insights into the cellular mechanisms by which Aβ modulates Cx43 function in astrocytes, the basic understanding of which is vital for the development of alternative therapeutic strategy targeting connexin channels in AD.
Astroglial Connexins in Neurodegenerative Diseases
Frontiers in Molecular Neuroscience, 2021
Astrocytes play a crucial role in the maintenance of the normal functions of the Central Nervous System (CNS). During the pathogenesis of neurodegenerative diseases, astrocytes undergo morphological and functional remodeling, a process called reactive astrogliosis, in response to the insults to the CNS. One of the key aspects of the reactive astrocytes is the change in the expression and function of connexins. Connexins are channel proteins that highly expressed in astrocytes, forming gap junction channels and hemichannels, allowing diffusional trafficking of small molecules. Alterations of astrocytic connexin expression and function found in neurodegenerative diseases have been shown to affect the disease progression by changing neuronal function and survival. In this review, we will summarize the role of astroglial connexins in neurodegenerative diseases including Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Also, we will discu...
The cellular localization, relation to other glial connexins (Cx30, Cx32, and Cx43), and developmental expression of Cx29 were investigated in the mouse central nervous system (CNS) with an anti-Cx29 antibody. Cx29 was enriched in subcellular fractions of myelin, and immunofluorescence for Cx29 was localized to oligodendrocytes and myelinated fibers throughout the brain and spinal cord. Oligodendrocyte somata displayed minute Cx29-immunopositive puncta around their periphery and intracellularly. In developing brain, Cx29 levels increased during the first few postnatal weeks and were highest in the adult brain. Immunofluorescence labeling for Cx29 in oligodendrocyte somata was intense at young ages and was dramatically shifted in localization primarily to myelinated fibers in mature CNS. Labeling for Cx32 also was localized to oligodendrocyte somata and myelin and absent in Cx32 knockout mice. Cx29 and Cx32 were minimally colocalized on oligodendrocytes somata and partly colocalized along myelinated fibers. At gap junctions on oligodendrocyte somata, Cx43/Cx32 and Cx30/Cx32 were strongly associated, but there was minimal association of Cx29 and Cx43. Cx32 was very sparsely associated with astrocytic connexins along myelinated fibers. With Cx26, Cx30, and Cx43 expressed in astrocytes and Cx29, Cx32, and Cx47 expressed in oligodendrocytes, the number of connexins localized to gap junctions of glial cells is increased to six. The results suggested that Cx29 in mature CNS contributes minimally to gap junctional intercellular communication in oligodendrocyte cell bodies but rather is targeted to myelin, where it, with Cx32, may contribute to connexin-mediated communication between adjacent layers of uncompacted myelin. © 2003 Wiley-Liss, Inc.
The connexin family of proteins (Cx) that form intercellular gap junctions in vertebrates is well represented in the mammalian central nervous system. Among these, Cx30 and Cx43 are present in gap junctions of astrocytes. Cx32 is expressed by oligodendrocytes and is present in heterologous gap junctions between oligodendrocytes and astrocytes as well as at autologous gap junctions between successive myelin layers. Cx36 mRNA has been identified in neurons, and Cx36 protein has been localized at ultrastructurally defined interneuronal gap junctions. Cx26 is also expressed in the CNS, primarily in the leptomeningeal linings, but is also reported in astrocytes and in neurons of developing brain and spinal cord. To establish further the regional, cellular, and subcellular localization of Cx26 in neural tissue, we investigated this connexin in adult mouse brain and in rat brain and spinal cord using biochemical and immunocytochemical methods. Northern blotting, western blotting, and immunofluorescence studies indicated widespread and heterogeneous Cx26 expression in numerous subcortical areas of both species. By confocal microscopy, Cx26 was colocalized with both Cx30 and Cx43 in leptomeninges as well as along blood vessels in cortical and subcortical structures. It was also localized at the surface of oligodendrocyte cell bodies, where it was coassociated with Cx32. Freeze-fracture replica immunogold labeling (FRIL) demonstrated Cx26 in most gap junctions between cells of the pia mater by postnatal day 4. By postnatal day 18 and thereafter, Cx26 was present at gap junctions between astrocytes and in the astrocyte side of most gap junctions between astrocytes and oligodendrocytes. In perinatal spinal cord and in five regions of adult brain and spinal cord examined by FRIL, no evidence was obtained for the presence of Cx26 in neuronal gap junctions. In addition to its established localization in leptomeningeal gap junctions, these results identify Cx26 as a third connexin (together with Cx30 and Cx43) within astrocytic gap junctions and suggest a further level of complexity to the heterotypic connexin channel combinations formed at these junctions. © 2001 Wiley-Liss, Inc.
We identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructurally-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocyte gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytes, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gapjunctions contained Cx36, whereas Cx26 was present in leptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with "permissive" connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.
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. © 2003 Wiley-Liss, Inc.
Journal of Neuroscience, 2009
Astrocytes are coupled via gap junctions (GJs) comprising connexin 43 (Cx43) (Gja1) and Cx30 (Gjb6), which facilitate intercellular exchange of ions. Astrocyte connexins also form heterotypic GJs with oligodendrocytic somata and lamellae. Loss of oligodendrocyte gap junctions results in oligodendrocyte and myelin pathology. However, whether loss of astrocyte GJs affects oligodendrocytes and myelin is not known. To address this question, mice with astrocyte-targeted deletion of Cx43 and global loss of Cx30 [double knock-out (dKO)] were studied using Western blotting, immunohistochemistry, electron microscopy, and functional assays. Commencing around postnatal day 23 and persisting into old age, we found widespread pathology of white matter tracts comprising vacuolated oligodendrocytes and intramyelinic edema. In contrast, gray matter pathology was restricted to the CA1 region of the hippocampus, and consisted of edematous astrocytes. No differences were observed in synaptic density or total NeuN ϩ cells in the hippocampus, or olig2 ϩ cells in the corpus callosum. However, in dKO mice, fewer CC1-positive mature oligodendrocytes were detected, and Western blotting indicated reduced myelin basic protein. Pathology was not noted in mice expressing a single allele of either Cx43 or Cx30. When compared with single connexin knock-outs, dKO mice were impaired in sensorimotor (rotarod, balance beam assays) and spatial memory tasks (object recognition assays). We conclude that loss of astrocytic GJs can result in white matter pathology that has functional consequences.
Connexin-43 in relation to gap junctions between astrocytes and between other cell types in rat spinal cord was investigated immunohistochemically. In gray matter, connexin-43 was distributed thoughout all laminae, but was more concentrated in the substantia gelatinosa and around the central canal. Ultrastructurally, immunostaining was present in the cytoplasm of, and at gap junctions between, fine astrocytic processes, most of which ensheathed neuronal elements. In white matter, connexin-43 was localized to somata of fibrous astrocytes, their glial fibrillary acidic protein-positive processes running parallel to myelinated axons, and at gap junctions between these processes. Labelling was also evident in thick radially-directed astrocytic processes displaying pockets of staining near immunopositive gap junctions. Near the cord surface, staining was present in cell bodies of subpial astrocytes and at gap junctions between their tangential processes which formed most of the glia limitans. Radially-directed processes of subpial astrocytes formed symmetrically- and asymmetrically-labelled gap junctions with each other and extended fine branches into surrounding white matter where they made contact and often formed gap junctions with oligodendrocytic processes at the outer surface of myelinated fibres. Immunopositive astrocyte processes also made heterologous gap junctions with unstained oligodendrocyte cell bodies. Ependymal cells lining the central canal exhibited apical cytoplasmic labelling, as well as symmetrically-labelled gap junctions at their apices. Ependymal cells also formed asymmetrically-labelled gap junctions at which the junctional membranes of unlabelled cells, presumed to be tanycytes, were unstained. The results indicate the expression of connexins in addition to connexin-43 at asymmetrically-labelled gap junctions between some astrocytic processes, between astrocytes and oligodendrocytes and between some ependymal cells. The presence of gap junctions between astrocyte and oligodendrocyte processes at the outer surface of myelin suggests incorporation of the latter into the extensive gap junctionally-coupled astrocytic syncytium.
Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS
This review article summarizes early and recent literature on the structure, distribution and composition of gap junctions between astrocytes and oligodendrocytes, and the differential expression of glial connexins in adult and developing mammalian CNS. In addition to an overview of the topic, discussion is focused on the organization of homologous gap junctional interactions between astrocytes and between oligodendrocytes as well as on heterologous junctional coupling between astrocytes and oligodendrocytes. The homotypic and heterotypic nature of these gap junctions is related to the connexins known to be produced by glial cells in the intact brain and spinal cord. Emphasis is placed on the ultrastructural level of analysis required to attribute gap junction and connexin deployment to particular cell types and subcellular locations. Our aim is to provide a firm basis for consideration of anticipated rapid advances in understanding of structural relationships of gap junctions and connexins within the glial gap junctional syncytium. Conclusions to date suggest that the glial syncytium is more complex than previously appreciated and that glial pathways of junctional communication may not only be determined by the presence of gap junctions, but also by the connexin composition and conductance regulation of junctional channels. (C) 2000 Elsevier Science B.V.