Recombinant lectin Gg for brain imaging of glycan structure and formation in the CNS node of Ranvier.

The nodes of Ranvier are unmyelinated gaps in the axon, important for the efficient transmission of action potentials. Despite the identification of several glycoproteins involved in node formation and maintenance, glycans' structure and formation in the node remain unclear. Previously, we developed a recombinant lectin from the Clostridium botulinum neurotoxin complex, specific to the galactose and N-acetylgalactosamine terminal epitopes (Gg). Gg stained Neuro2a cells. Here, we show Gg punctuate staining in mouse brain cryosections. Thus, we hypothesized that Gg could help study glycans in the node of Ranvier. Lectin histochemistry on mouse brain cryosections confirmed that Gg binds specifically to the node of Ranvier in the central nervous system (CNS). Using a combination of lectin blotting, glycosidase treatment on tissue sections, and lectin histochemistry, Gg ligands were identified as α-galactose terminal glycoproteins in the perinodal extracellular matrix. Furthermore, we detected the spatiotemporal distribution of galactosylated glycans in the CNS node of Ranvier in mouse brain tissues at different postnatal times. Finally, we observed impaired clustering of galactosylated glycans in the nodes during demyelination and remyelination in cuprizone-induced demyelination and remyelination mouse model. In conclusion, Gg can serve as a novel brain imaging tool in glycobiology and report glycoprotein formation and alterations in the CNS node of Ranvier. Our findings might serve as a first step to establish the role of glycans in the node of Ranvier.

Fut9 Deficiency Causes Abnormal Neural Development in the Mouse Cerebral Cortex and Retina.

α1,3-Fucosyltransferase 9 (Fut9) is responsible for the synthesis of Lewis X [LeX, Galß1-4(Fucα1-3)GlcNAc] carbohydrate epitope, a marker for pluripotent or multipotent tissue-specific stem cells. Although Fut9-deficient mice show anxiety-related behaviors, structural and cellular abnormalities in the brain remain to be investigated. In this study, using in situ hybridization and immunohistochemical techniques in combination, we clarified the spatiotemporal expression of Fut9, together with LeX, in the brain and retina. We found that Fut9-expressing cells are positive for Ctip2, a marker of neurons residing in layer V/VI, and TLE4, a marker of corticothalamic projection neurons (CThPNs) in layer VI, of the cortex. A birthdating analysis using 5-ethynyl-2'-deoxyuridine at embryonic day (E)11.5, 5-bromo-2'-deoxyuridine at E12.5, and in utero electroporation of a GFP expression plasmid at E14.5 revealed a reduction in the percentage of neurons produced at E11.5 in layer VI/subplate of the cortex and in the ganglion cell layer of the retina in P0 Fut9-/- mice. Furthermore, this reduction in layer VI/subplate neurons persisted into adulthood, leading to a reduction in the number of Ctip2strong/Satb2- excitatory neurons in layer V/VI of the adult Fut9-/- cortex. These results suggest that Fut9 plays significant roles in the differentiation, migration, and maturation of neural precursor cells in the cortex and retina.

Exploring the factors underlying remyelination arrest by studying the post-transcriptional regulatory mechanisms of cystatin F gene.

Remyelination plays an important role in determining the fate of demyelinating disorders. However, it is arrested during chronic disease states. Cystatin F, a papain-like lysosomal cysteine proteinase inhibitor, is a crucial regulator of demyelination and remyelination. Using hemizygous proteolipid protein transgenic 4e (PLP4e/- ) mice, an animal model of chronic demyelination, we found that cystatin F mRNA expression was induced at 2.5 months of age and up-regulated in the early phase of demyelination, but significantly decreased in the chronic phase. We next investigated cystatin F regulatory factors as potential mechanisms of remyelination arrest in chronic demyelinating disorders. We used the CysF-STOP-tetO::Iba-mtTA mouse model, in which cystatin F gene expression is driven by the tetracycline operator. Interestingly, we found that forced cystatin F mRNA over-expression was eventually decreased. Our findings show that cystatin F expression is modulated post-transcriptionally. We next identified embryonic lethal, abnormal vision, drosophila like RNA-binding protein 1 (ELAVL-1), and miR29a as cystatin F mRNA stabilizing and destabilizing factors, respectively. These roles were confirmed in vitro in NIH3T3 cells. Using postmortem plaque samples from human multiple sclerosis patients, we also confirmed that ELAVL-1 expression was highly correlated with the previously reported expression pattern of cystatin F. These data indicate the important roles of ELAVL-1 and miR29a in regulating cystatin F expression. Furthermore, they provide new insights into potential therapeutic targets for demyelinating disorders.

Life-Long Neural Stem Cells Are Fate-Specified at an Early Developmental Stage.

The origin and life-long fate of quiescent neural stem cells (NSCs) in the adult mammalian brain remain largely unknown. A few neural precursor cells in the embryonic brain elongate their cell cycle time and subsequently become quiescent postnatally, suggesting the possibility that life-long NSCs are selected at an early embryonic stage. Here, we utilized a GFP-expressing lentivirus to investigate the fate of progeny from individual lentivirus-infected NSCs by identifying the lentiviral integration site. Our data suggest that NSCs become specified to two or more lineages prior to embryonic day 13.5 in mice: one NSC lineage produces cells only for the cortex and another provides neurons to the olfactory bulb. The majority of neurosphere-forming NSCs in the adult brain are relatively dormant and generate very few cells, if any, in the olfactory bulb or cortex, and this NSC population could serve as a reservoir that is occasionally reactivated later in life.

Region- and Cell Type-Specific Facilitation of Synaptic Function at Destination Synapses Induced by Oligodendrocyte Depolarization.

The axonal conduction of action potentials affects the absolute time it takes to transmit nerve impulses as well as temporal summation at destination synapses. At the physiological level, oligodendrocyte depolarization facilitates axonal conduction along myelinated fibers in the hippocampus; however, the functional significance of this facilitation is largely unknown. In this study, we examined the physiology of the facilitation of axonal conduction by investigating the changes in synaptic responses at destination synapses using male and female mice in which channelrhodopsin-2 expression was restricted to oligodendrocytes. The subiculum, one of the projection areas of the examined axons at the alveus of the hippocampus, is divided into three regions (proximal, mid, and distal) and contains two types of principal neurons: regular firing and bursting pyramidal cells. We found a significant increase in excitatory synaptic responses following optogenetic oligodendrocyte depolarization in bursting neurons at two of the three regions, but not in regular firing neurons at any region. The long-term potentiation (LTP) induced by theta burst stimulation at the synapses showing a significant increase was also enhanced after oligodendrocyte depolarization. Conversely, the reduction of oligodendrocyte depolarization during theta burst stimulation, which was achieved by photostimulation of archaerhodopsin-T expressed selectively on oligodendrocytes, reduced the magnitude of LTP. These results show that oligodendrocyte depolarization contributes to the fine control of synaptic activity between the axons they myelinate and targets subicular cells in a region- and cell type-specific manner, and suggest that oligodendrocyte depolarization during conditioning of stimuli is involved in the induction of LTP.SIGNIFICANCE STATEMENT All activity in the nervous system depends on the propagation of action potentials. Changes in the axonal conduction of action potentials influence the timing of synaptic transmission and information processing in neural circuits. At the physiological level, oligodendrocyte depolarization facilitates axonal conduction along myelinated fibers. In this study, we investigated the functional significance of the facilitation of axonal conduction induced by physiological oligodendrocyte depolarization. Using optogenetics and electrophysiological recordings, we demonstrated that oligodendrocyte depolarization in mice expressing channelrhodopsin-2 on oligodendrocytes increased excitatory synaptic responses and enhanced the induction of long-term potentiation at destination synapses in a region- and cell type-specific manner. This facilitation may have a hitherto unappreciated influence on the transfer of information between regions in the nervous system.

Motor learning requires myelination to reduce asynchrony and spontaneity in neural activity.

Myelination increases the conduction velocity in long-range axons and is prerequisite for many brain functions. Impaired myelin regulation or impairment of myelin itself is frequently associated with deficits in learning and cognition in neurological and psychiatric disorders. However, it has not been revealed what perturbation of neural activity induced by myelin impairment causes learning deficits. Here, we measured neural activity in the motor cortex during motor learning in transgenic mice with a subtle impairment of their myelin. This deficit in myelin impaired motor learning, and was accompanied by a decrease in the amplitude of movement-related activity and an increase in the frequency of spontaneous activity. Thalamocortical axons showed variability in axonal conduction with a large spread in the timing of postsynaptic cortical responses. Repetitive pairing of forelimb movements with optogenetic stimulation of thalamocortical axon terminals restored motor learning. Thus, myelin regulation helps to maintain the synchrony of cortical spike-time arrivals through long-range axons, facilitating the propagation of the information required for learning. Our results revealed the pathological neuronal circuit activity with impaired myelin and suggest the possibility that pairing of noninvasive brain stimulation with relevant behaviors may ameliorate cognitive and behavioral abnormalities in diseases with impaired myelination.

A Potential Serum N-glycan Biomarker for Hepatitis C Virus-Related Early-Stage Hepatocellular Carcinoma with Liver Cirrhosis.

Detection of early-stage hepatocellular carcinoma (HCC) is beneficial for prolonging patient survival. However, the serum markers currently used show limited ability to identify early-stage HCC. In this study, we explored human serum N-glycans as sensitive markers to diagnose HCC in patients with cirrhosis. Using a simplified fluorescence-labeled N-glycan preparation method, we examined non-sialylated and sialylated N-glycan profiles from 71 healthy controls and 111 patients with hepatitis and/or liver cirrhosis (LC) with or without HCC. We found that the level of serum N-glycan A2G1(6)FB, a biantennary N-glycan containing core fucose and bisecting GlcNAc residues, was significantly higher in hepatitis C virus (HCV)-infected cirrhotic patients with HCC than in those without HCC. In addition, A2G1(6)FB was detectable in HCV-infected patients with early-stage HCC and could be a more accurate marker than alpha-fetoprotein (AFP) or protein induced by vitamin K absence or antagonists-II (PIVKA-II). Moreover, there was no apparent correlation between the levels of A2G1(6)FB and those of AFP or PIVKA-II. Thus, simultaneous use of A2G1(6)FB and traditional biomarkers could improve the accuracy of HCC diagnosis in HCV-infected patients with LC, suggesting that A2G1(6)FB may be a reliable biomarker for early-stage HCC patients.

Role of Purinergic Receptor P2Y1 in Spatiotemporal Ca2+ Dynamics in Astrocytes.

Fine processes of astrocytes enwrap synapses and are well positioned to sense neuronal information via synaptic transmission. In rodents, astrocyte processes sense synaptic transmission via Gq-protein coupled receptors (GqPCR), including the P2Y1 receptor (P2Y1R), to generate Ca2+ signals. Astrocytes display numerous spontaneous microdomain Ca2+ signals; however, it is not clear whether such signals are due to local synaptic transmission and/or in what timeframe astrocytes sense local synaptic transmission. To ask whether GqPCRs mediate microdomain Ca2+ signals, we engineered mice (both sexes) to specifically overexpress P2Y1Rs in astrocytes, and we visualized Ca2+ signals via a genetically encoded Ca2+ indicator, GCaMP6f, in astrocytes from adult mice. Astrocytes overexpressing P2Y1Rs showed significantly larger Ca2+ signals in response to exogenously applied ligand and to repetitive electrical stimulation of axons compared with controls. However, we found no evidence of increased microdomain Ca2+ signals. Instead, Ca2+ waves appeared and propagated to occupy areas that were up to 80-fold larger than microdomain Ca2+ signals. These Ca2+ waves accounted for only 2% of total Ca2+ events, but they were 1.9-fold larger and 2.9-fold longer in duration than microdomain Ca2+ signals at processes. Ca2+ waves did not require action potentials for their generation and occurred in a probenecid-sensitive manner, indicating that the endogenous ligand for P2Y1R is elevated independently of synaptic transmission. Our data suggest that spontaneous microdomain Ca2+ signals occur independently of P2Y1R activation and that astrocytes may not encode neuronal information in response to synaptic transmission at a point source of neurotransmitter release.SIGNIFICANCE STATEMENT Astrocytes are thought to enwrap synapses with their processes to receive neuronal information via Gq-protein coupled receptors (GqPCRs). Astrocyte processes display numerous microdomain Ca2+ signals that occur spontaneously. To determine whether GqPCRs play a role in microdomain Ca2+ signals and the timeframe in which astrocytes sense neuronal information, we engineered mice whose astrocytes specifically overexpress the P2Y1 receptor, a major GqPCR in astrocytes. We found that overexpression of P2Y1 receptors in astrocytes did not increase microdomain Ca2+ signals in astrocyte processes but caused Ca2+ wavelike signals. Our data indicate that spontaneous microdomain Ca2+ signals do not require activation of P2Y1 receptors.

Mechanical regulation of oligodendrocyte morphology and maturation by the mechanosensor p130Cas.

Oligodendrocytes (OLs) are myelinating cells of the central nervous system. Recent studies have shown that mechanical factors influence various cell properties. Mechanical stimulation can be transduced into intracellular biochemical signals through mechanosensors, such as integrin, p130Cas, talin and vinculin. However, the molecular mechanisms underlying the mechanical regulation of OLs by mechanosensors remain largely unknown. We found that morphology of OL was affected by knockdown of the mechanosensors p130Cas or talin1. Stretching of OL precursor cells induced the phosphorylation of p130Cas and talin-associated assembly of vinculin. Shear stress decreased the number of OL processes, whereas these effects were mechanically suppressed by dominant-negative (DN) p130Cas, but not by DN-talin1. To investigate the roles of p130Cas in post-natal OLs in vivo, we constructed a novel p130Cas knock-in mouse and found overexpression of p130Cas in vivo affected the number of mature OLs in the cortex. These results indicate that the mechanosensor p130Cas controls both OL morphogenesis and maturation.

Cathepsin C modulates myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease characterized by immune-mediated inflammation, which attacks the myelin sheath. MS pursues a relapsing and remitting course with varying intervals between symptoms. The main clinical pathological features include inflammation, myelin sheath destruction and plaque formation in the central nervous system (CNS). We previously reported that cystatin F (CysF) expression is induced in demyelinating lesions that are accompanied by active remyelination (referred to as shadow plaques) but is down-regulated in chronic demyelinated lesions (plaques) in the spinal cord of MS patients and in several murine models of demyelinating disease. CysF is a cathepsin protease inhibitor whose major target is cathepsin C (CatC), which is co-expressed in demyelinating regions in Plp4e/- mice, a model of chronic demyelination. Here, we report the time course of CatC and CysF expression and describe the symptoms in a mouse experimental autoimmune encephalomyelitis (EAE) model using CatC knockdown (KD) and CatC over-expression (OE) mice. In myelin oligodendrocyte glycoprotein (MOG)-EAE, CatC positive cells were found to infiltrate the CNS at an early stage prior to any clinical signs, in comparison to WT mice. CysF expression was not observed at this early stage, but appeared later within shadow plaques. CatC expression was found in chronic demyelinated lesions but was not associated with CysF expression, and CatCKD EAE mouse showed delayed demyelination. Whereas, CatCOE in microglia significantly increased severity of demyelination in the MOG-EAE model. Thus, these results demonstrate that CatC plays a major role in MOG-EAE.

Cathepsin C promotes microglia M1 polarization and aggravates neuroinflammation via activation of Ca2+-dependent PKC/p38MAPK/NF-κB pathway.

BACKGROUND: Microglia-derived lysosomal cathepsins are important inflammatory mediators to trigger signaling pathways in inflammation-related cascades. Our previous study showed that the expression of cathepsin C (CatC) in the brain is induced predominantly in activated microglia in neuroinflammation. Moreover, CatC can induce chemokine production in brain inflammatory processes. In vitro studies further confirmed that CatC is secreted extracellularly from LPS-treated microglia. However, the mechanisms of CatC affecting neuroinflammatory responses are not known yet. METHODS: CatC over-expression (CatCOE) and knock-down (CatCKD) mice were treated with intraperitoneal and intracerebroventricular LPS injection. Morris water maze (MWM) test was used to assess the ability of learning and memory. Cytokine expression in vivo was detected by in situ hybridization, quantitative PCR, and ELISA. In vitro, microglia M1 polarization was determined by quantitative PCR. Intracellular Ca2+ concentration was determined by flow cytometry, and the expression of NR2B, PKC, p38, IkBα, and p65 was determined by western blotting. RESULTS: The LPS-treated CatCOE mice exhibited significantly increased escape latency compared with similarly treated wild-type or CatCKD mice. The highest levels of TNF-α, IL-1ß, and other M1 markers (IL-6, CD86, CD16, and CD32) were found in the brain or serum of LPS-treated CatCOE mice, and the lowest levels were detected in CatCKD mice. Similar results were found in LPS-treated microglia derived from CatC differentially expressing mice or in CatC-treated microglia from wild-type mice. Furthermore, the expression of NR2B mRNA, phosphorylation of NR2B, Ca2+ concentration, phosphorylation of PKC, p38, IκBα, and p65 were all increased in CatC-treated microglia, while addition of E-64 and MK-801 reversed the phosphorylation of above molecules. CONCLUSION: The data suggest that CatC promotes microglia M1 polarization and aggravates neuroinflammation via activation of Ca2+-dependent PKC/p38MAPK/NF-κB pathway. CatC may be one of key molecular targets for alleviating and controlling neuroinflammation in neurological diseases.

Differential expression of N-linked oligosaccharides in methotrexate-resistant primary central nervous system lymphoma cells.

BACKGROUND: Oligosaccharides of glycoprotein, particularly negatively-charged sialylated N-glycans, on the surface of lymphomas play important roles in cell-cell interactions and bind immunoglobulin-like lectins, causing inflammatory responses and bioregulation. However, their characterizations have largely been unknown in central nervous system (CNS) lymphoma. METHODS: Here, we investigated expression patterns of N-linked oligosaccharides of glycoproteins in cells derived from CNS lymphomas and clinical specimens. RESULTS: We first generated methotrexate (MTX)-resistant cells derived from HKBML and TK as CNS lymphoma, and RAJI as non-CNS lymphoma and determined N-linked oligosaccharide structures in these cells and other non-CNS lymphoma-derived cells including A4/FUK, OYB, and HBL1. Major components of the total oligosaccharides were high-mannose type N-glycans, whose level increased in MTX-resistant HKBML and TK but decreased in MTX-resistant RAJI. We also detected sialylated biantennary galactosylated N-glycans with α1,6-fucosylation, A2G2F, and A2G2FB from HKBML, TK, and RAJI. Sialylated A4G4F was specifically isolated from RAJI. However, the ratios of these sialylated N-glycans slightly decreased against MTX-resistant compared to non-resistant cells. Interestingly, almost all complex-type oligosaccharides were α2,6-sialylated. DISCUSSION: This is the first study for the expression profile of N-oligosaccharides on MTX-resistant primary CNS lymphoma-derived cells HKBML and TK, and tumor tissues resected from patients with CNS lymphoma, CONCLUSION: These results propose a possibility that the differential expression of high-mannose types and sialylated A2G2F, A2G2FB, and A4G4F on the surface of CNS lymphomas may provide a hint for targets for diagnoses and treatments of the oligosaccharide type-specific lymphomas.

Mitochondrial Dynamics in Physiology and Pathology of Myelinated Axons.

Mitochondria play essential roles in neurons and abnormal functions of mitochondria have been implicated in neurological disorders including myelin diseases. Since mitochondrial functions are regulated and maintained by their dynamic behavior involving localization, transport, and fusion/fission, modulation of mitochondrial dynamics would be involved in physiology and pathology of myelinated axons. In fact, the integration of multimodal imaging in vivo and in vitro revealed that mitochondrial localization and transport are differentially regulated in nodal and internodal regions in response to the changes of metabolic demand in myelinated axons. In addition, the mitochondrial behavior in axons is modulated as adaptive responses to demyelination irrespective of the cause of myelin loss, and the behavioral modulation is partly through interactions with cytoskeletons and closely associated with the pathophysiology of demyelinating diseases. Furthermore, the behavior and functions of axonal mitochondria are modulated in congenital myelin disorders involving impaired interactions between axons and myelin-forming cells, and, together with the inflammatory environment, implicated in axonal degeneration and disease phenotypes. Further studies on the regulatory mechanisms of the mitochondrial dynamics in myelinated axons would provide deeper insights into axo-glial interactions mediated through myelin ensheathment, and effective manipulations of the dynamics may lead to novel therapeutic strategies protecting axonal and neuronal functions and survival in primary diseases of myelin.

Interactions between mitochondria and endoplasmic reticulum in demyelinated axons.

Demyelination leads to axonal changes that involve the functions and dynamics of axonal mitochondria supporting metabolism and survival of axons. However, the changes in the physical interactions between mitochondria and endoplasmic reticulum, called mitochondria-associated membranes, are poorly understood in demyelinated axons. In this study, we investigated the three-dimensional ultrastructural changes in membrane juxtapositions between mitochondria and endoplasmic reticulum in axons of a chronic progressive demyelination mouse model caused by extra copies of proteolipid protein (PLP4e). In the optic nerve of PLP4e mice, most axons were ensheathed by myelin by age 1 month, but were demyelinated by age 5 months. At age 1 month, mitochondria in PLP4e mice were slightly larger than those in wild-type mice, while the size and frequency of juxtaposition were similar. At age 5 months, the sizes of mitochondria and size of juxtaposition in PLP4e mice were prominently larger than those in wild-type mice. In degenerating axons under demyelination, the enlargement of mitochondria was diminished, while the density and frequency of juxtaposition were similar to those of non-degenerating axons. These results suggest that interactions between mitochondria and ER are enhanced in chronically demyelinated axons and maintained during axonal degeneration in hereditary myelin diseases.

Branched Sialylated N-glycans Are Accumulated in Brain Synaptosomes and Interact with Siglec-H.

Proper N-glycosylation of proteins is important for normal brain development and nervous system function. Identification of the localization, carrier proteins and interacting partners of N-glycans is essential for understanding the roles of glycoproteins. The present study examined the N-glycan A2G'2F (Galß1-3GlcNAcß1-2Manα1-6[Galß1-3GlcNAcß1-2Manα1-3]Manß1-4GlcNAcß1-4[Fucα1-6]GlcNAc-). A2G'2F has a branched sialic acid structural feature, and branched sialylated A2G'2F is a major N-glycan in the mouse brain. Its expression in the mouse brain increases during development, suggesting that branched sialylated N-glycans play essential roles during brain development. However, the carrier proteins, interacting partners and localization of branched sialylated N-glycans remain unknown. We previously improved our method for analyzing N-glycans from trace samples, and here we succeeded in detecting A2G'2F in small fragments excised from the two-dimensional electrophoresis gels of subcellular fractionated mouse brain proteins. A2G'2F was accumulated in mouse brain synaptosomes. We identified calreticulin as one of the candidate A2G'2F carriers and found calreticulin expression in both the endoplasmic reticulum and synaptosomal fractions. Calreticulin was observed in dendritic spines of cultured cortical neurons. Synthesized branched sialylated glycan clusters interacted with sialic acid-binding immunoglobulin-like lectin H (Siglec-H), which is known to be a microglia-specific molecule. Taken together, these results suggest that branched sialylated A2G'2F in synaptosomes plays a role in the interaction of dendritic spines with microglia.Key words: N-glycan, subcellular fractionation, calreticulin, dendritic spine, Siglec-H.

Length of myelin internodes of individual oligodendrocytes is controlled by microenvironment influenced by normal and input-deprived axonal activities in sensory deprived mouse models.

Oligodendrocytes myelinate neuronal axons to increase conduction velocity in the vertebrate central nervous system (CNS). Recent studies revealed that myelin formed on highly active axons is more stable compared to activity-silenced axons, and length of the myelin sheath is longer in active axons as well in the zebrafish larva. However, it is unclear whether oligodendrocytes preferentially myelinate active axons compared to sensory input-deprived axons in the adult mammalian CNS. It is also unknown if a single oligodendrocyte forms both longer myelin sheaths on active axons and shorter sheaths on input-deprived axons after long-term sensory deprivation. To address these questions, we applied simultaneous labeling of both neuronal axons and oligodendrocytes to mouse models of long-term monocular eyelid suturing and unilateral whisker removal. We found that individual oligodendrocytes evenly myelinated normal and input-deprived axons in the adult mouse CNS, and myelin sheath length on normal axons and input-deprived axons formed by a single oligodendrocyte were comparable. Importantly, the average length of the myelin sheath formed by individual oligodendrocytes did change depending on relative abundance of normal against sensory-input deprived axons, indicating an abundance of deprived axons near an oligodendrocyte impacts on myelination program by a single oligodendrocyte.

Aberrant astrocyte Ca2+ signals "AxCa signals" exacerbate pathological alterations in an Alexander disease model.

Alexander disease (AxD) is a rare neurodegenerative disorder caused by gain of function mutations in the glial fibrillary acidic protein (GFAP) gene. Accumulation of GFAP proteins and formation of Rosenthal fibers (RFs) in astrocytes are hallmarks of AxD. However, malfunction of astrocytes in the AxD brain is poorly understood. Here, we show aberrant Ca2+ responses in astrocytes as playing a causative role in AxD. Transcriptome analysis of astrocytes from a model of AxD showed age-dependent upregulation of GFAP, several markers for neurotoxic reactive astrocytes, and downregulation of Ca2+ homeostasis molecules. In situ AxD model astrocytes produced aberrant extra-large Ca2+ signals "AxCa signals", which increased with age, correlated with GFAP upregulation, and were dependent on stored Ca2+ . Inhibition of AxCa signals by deletion of inositol 1,4,5-trisphosphate type 2 receptors (IP3R2) ameliorated AxD pathogenesis. Taken together, AxCa signals in the model astrocytes would contribute to AxD pathogenesis.

Ectopic positioning of Bergmann glia and impaired cerebellar wiring in Mlc1-over-expressing mice.

Mlc1 is a causative gene for megalencephalic leukoencephalopathy with subcortical cysts, and is expressed in astrocytes. Mlc1-over-expressing mice represent an animal model of early-onset leukoencephalopathy, which manifests as astrocytic swelling followed by myelin membrane splitting in the white matter. It has been previously reported that Mlc1 is highly expressed in Bergmann glia, while the cerebellar phenotypes of Mlc1-over-expressing mouse have not been characterized. Here, we examined the cerebellum of Mlc1-over-expressing mouse and found that the distribution of Bergmann glia (BG) was normally compacted along the Purkinje cell (PC) layer until postnatal day 10 (P10), while most BG were dispersed throughout the molecular layer by P28. Ectopic BG were poorly wrapped around somatodendritic elements of PCs and exhibited reduced expression of the glutamate transporter glutamate-aspartate transporter. Extraordinarily slow and small climbing fiber (CF)-mediated excitatory post-synaptic currents, which are known to be elicited under accelerated glutamate spillover, emerged at P20-P28 when BG ectopia was severe, but not at P9-P12 when ectopia was mild. Furthermore, maturation of CF wiring, which translocates the site of innervation from somata to proximal dendrites, was also impaired. Manipulations that restricted the Mlc1-over-expressing period successfully generated mice with and without BG ectopia, depending on the over-expressing period. Together, these findings suggest that there is a critical time window for mechanisms that promote the positioning of BG in the PC layer. Once normal positioning of BG is affected, the differentiation of BG is impaired, leading to insufficient glial wrapping, exacerbated glutamate spillover, and aberrant synaptic wiring in PCs. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14199.

Dysregulation of schizophrenia-related aquaporin 3 through disruption of paranode influences neuronal viability.

Myelinated axons segregate the axonal membrane into four defined regions: the node of Ranvier, paranode, juxtaparanode, and internode. The paranodal junction consists of specific component proteins, such as neurofascin155 (NF155) on the glial side, and Caspr and Contactin on the axonal side. Although paranodal junctions are thought to play crucial roles in rapid saltatory conduction and nodal assembly, the role of their interaction with neurons is not fully understood. In a previous study, conditional NF155 knockout in oligodendrocytes led to disorganization of the paranodal junctions. To examine if disruption of paranodal junctions affects neuronal gene expression, we prepared total RNA from the retina of NF155 conditional knockout, and performed expression analysis. We found that the expression level of 433 genes changed in response to paranodal junction ablation. Interestingly, expression of aquaporin 3 (AQP3) was significantly reduced in NF155 conditional knockout mice, but not in cerebroside sulfotransferase knockout (CST-KO) mice, whose paranodes are not originally formed during development. Copy number variations have an important role in the etiology of schizophrenia (SCZ). We observed rare duplications of AQP3 in SCZ patients, suggesting a correlation between abnormal AQP3 expression and SCZ. To determine if AQP3 over-expression in NF155 conditional knockout mice influences neuronal function, we performed adeno-associated virus (AAV)-mediated over-expression of AQP3 in the motor cortex of mice and found a significant increase in caspase 3-dependent neuronal apoptosis in AQP3-transduced cells. This study may provide new insights into therapeutic approaches for SCZ by regulating AQP3 expression, which is associated with paranodal disruption.

Oligodendrocyte-Neuron Interactions: Impact on Myelination and Brain Function.

In the past, glial cells were considered to be 'glue' cells whose primary role was thought to be merely filling gaps in neural circuits. However, a growing number of reports have indicated the role of glial cells in higher brain function through their interaction with neurons. Myelin was originally thought to be just a sheath structure surrounding neuronal axons, but recently it has been shown that myelin exerts effects on the conduction velocity of neuronal axons even after myelin formation. Therefore, the investigation of glial cell properties and the neuron-glial interactions is important for understanding higher brain function. Moreover, since there are many neurological disorders caused by glial abnormalities, further understanding of glial cell-related diseases and the development of effective therapeutic strategies are warranted. In this review, we focused on oligodendrocyte-neuron interactions, with particular attention on (1) axonal signals underlying oligodendrocyte differentiation and myelination, (2) neuronal activity-dependent myelination and (3) the effects of myelination on higher brain function.