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1.  The ABCA1 cholesterol transporter associates with one of two distinct dystrophin-based scaffolds in Schwann cells 
Glia  2008;56(6):611-618.
Cytoskeletal scaffolding complexes help organize specialized membrane domains with unique functions on the surface of cells. In this study, we define the scaffolding potential of the Schwann cell dystrophin glycoprotein complex (DGC) by establishing the presence of four syntrophin isoforms, (α1, β1, β2, and γ2), and one dystrobrevin isoform, (α–dystrobrevin-1), in the abaxonal membrane. Furthermore, we demonstrate the existence of two separate DGCs in Schwann cells that divide the abaxonal membrane into spatially distinct domains, the DRP2/periaxin rich plaques and the Cajal bands that contain Dp116, utrophin, α-dystrobrevin-1 and four syntrophin isoforms. Finally, we show that the two different DGCs can scaffold unique accessory molecules in distinct areas of the Schwann cell membrane. Specifically, the cholesterol transporter ABCA1, associates with the Dp116/syntrophin complex in Cajal bands and is excluded from the DRP2/periaxin rich plaques.
PMCID: PMC4335170  PMID: 18286648
2.  Remodelling of motor nerve terminals in demyelinating axons of periaxin null mutant mice 
Glia  2008;56(4):471-479.
Myelin formation around axons increases nerve conduction velocity and regulates phenotypic characteristics of the myelinated axon. In the peripheral nervous system, demyelinating forms of hereditary Charcot-Marie-Tooth (CMT) diseases, due to Schwann-cell intrinsic molecular defects, leads to reduced nerve conduction velocity and changes in the axonal phenotype. Several mouse models of CMT diseases have been generated, allowing the study of consequences of demyelination in peripheral nerve fibres. Nevertheless, the effect of demyelination at the level of the neuromuscular synapse has been largely overlooked. Here we show that in the periaxin knock-out mice, a model of CMT condition, neuromuscular junctions develop profound morphological changes in pre-terminal region of motoraxons. These changes include extensive preterminal branches which originate in demyelinated regions of the nerve fibre and axonal swellings associated with residually-myelinated regions of the fibre. Using intracellular recording from muscle fibres we detected asynchronous failure of action potential transmission at high but not low stimulation frequencies, a phenomenon consistent with branch point failure. Taken together, our morphological and electrophysiological findings suggest that preterminal branching due to segmental demyelination near the neuromuscular synapse in periaxin KO mice may underlie phenotypic disabilities present in this mouse model of CMT disease. These results opens a new avenue of research in order to understand the cellular changes responsible for clinical disabilities in demyelinating conditions.
PMCID: PMC4335188  PMID: 18205176
3.  Age-Related Changes in Astrocytic and Ependymal Cells of the Subventricular Zone 
Glia  2014;62(5):790-803.
Neurogenesis persists in the adult subventricular zone (SVZ) of the mammalian brain. During aging, the SVZ neurogenic capacity undergoes a progressive decline, which is attributed to a decrease in the population of neural stem cells (NSCs). However, the behavior of the NSCs that remain in the aged brain is not fully understood. Here we performed a comparative ultrastructural study of the SVZ niche of 2-month-old and 24-month-old male C57BL/6 mice, focusing on the NSC population. Using thymidine-labeling, we showed that residual NSCs in the aged SVZ divide less frequently than those in young mice. We also provided evidence that ependymal cells are not newly generated during senescence, as others studies suggest. Remarkably, both astrocytes and ependymal cells accumulated a high number of intermediate filaments and dense bodies during aging, resembling reactive cells. A better understanding of the changes occurring in the neurogenic niche during aging will allow us to develop new strategies for fighting neurological disorders linked to senescence.
PMCID: PMC4322944  PMID: 24677590
subventricular zone; neural stem cells; astrocytes; ependymal cells; aging; ultrastructure
4.  Transgenic Analysis of GFAP Promoter Elements 
Glia  2013;61(9):1488-1499.
Transcriptional regulation of the glial fibrillary acidic protein gene (GFAP) is of interest because of its astrocyte specificity and its upregulation in response to CNS injuries. We have used a transgenic approach instead of cell transfection to identify promoter elements of the human GFAP gene, since previous observations show that GFAP transcription is regulated differently in transfected cultured cells from in the mouse. We previously showed that block mutation of enhancer regions spanning from bp −1488 to −1434 (the C1.1 segment) and −1443 to −1399 (C1.2) resulted in altered patterns of expression and loss of astrocyte specificity, respectively. This analysis has now been extended upstream to bp −1612 to −1489 (the B region), which previously has been shown especially important for expression. Block mutation of each of four contiguous sequences, which together span the B region, each decreased the level of transgene activity by at least 50%, indicating that multiple sites contribute to the transcriptional activity in a cooperative manner. Several of the block mutations also altered the brain region pattern of expression, astrocyte specificity and/or the developmental time course. Transgenes were then analyzed in which mutations were limited to specific transcription factor binding sites in each of the 4 B block segments as well as in C1.1 and C1.2. Whereas mutation of the conserved consensus AP-1 site unexpectedly had little effect on transgene expression; NFI, SP1, STAT3, and NF-κB were identified as having important roles in regulating the strength of GFAP promoter activity and/or its astrocyte specificity.
PMCID: PMC4319660  PMID: 23832770
transgenic mice; transcription; GFAP; AP-1; NFI; SP1; STAT3; GATA; NF-κB
5.  Regional, Developmental, and Cell Cycle-Dependent Differences in μ, and δ, and κ-Opioid Receptor Expression among Cultured Mouse Astrocytes 
Glia  1998;22(3):249-259.
The diversity of opioid receptor expression was examined in astrocytes in low-density and non-dividing (confluent) cultures from the cerebral cortex, hippocampus, cerebellum, and striatum of 1-day-old mice. μ, δ, and κ Opioid receptor expression was assessed in individual cells immunocytochemically, by using flow cytometry, and functionally by examining agonist-induced changes in intracellular calcium ([Ca2+]i). Significant spatial and temporal differences were evident in the pattern of expression of μ, δ, and κ receptors among astrocytes. In low-density cultures, greater proportions of astrocytes expressed μ-opioid receptor immunoreactivity in the cerebral cortex and hippocampus (26-34%) than in the cerebellum or striatum (7-12%). At confluence, a greater percentage of astrocytes in cerebellar (26%) and striatal (30%) cultures expressed μ-immunoreactivity. Fewer astrocytes possessed δ-immunoreactivity in low-density striatal cultures (8%) compared to other regions (16-22%). The proportion of δ receptor-expressing astrocytes declined in the cerebellum but increased in the hippocampus. κ-Opioid receptors were uniformly expressed by 27-34% of astrocytes from all regions, except in cortical cultures where the proportion of κ expressing cells was 38% at low-density and decreased to 22% at confluence. Selective μ (PLO 17; H-Tyr-Pro-Phe (N-Me) -D-Pro-NH2, δ ([D-Pen2, D-Pen5] enkephalin) or κ (U50,488H; trans-(±)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl] benzeneacetamide methanesulfonate) opioid receptor agonists increased [Ca2+]i in subpopulations of astrocytes indicating the presence of functional receptors. Lastly, opioid receptor immunofluorescence varied during the cell division cycle. A greater proportion of astrocytes in the G2/M phase of the cell cycle were μ or δ receptor immunofluorescence than at G0/G1. When astrocytes were reversibly arrested in G1, significantly fewer cells expressed δ receptor immunofluorescence; however, upon reentry into the cell cycle immunofluorescent cells reappeared. In conclusion, opioid phenotype varies considerably among individual cultured astrocytes, and this diversity was determined by regional and developmental (age and cell cycle dependent) differences in the brain. These in vitro findings suggest astroglia contribute to regional and developmental idiosyncrasies in opioid function within the brain.
PMCID: PMC4319791  PMID: 9482211
Mu opioid receptors; Delta opioid receptors; Kappa opioid receptors; Intracellular calcium; cell proliferation; Drug Abuse
6.  [No title available] 
PMCID: PMC4096126  PMID: 24339157
7.  Arylsulfatase B modulates neurite outgrowth via astrocyte chondroitin-4-sulfate: dysregulation by ethanol 
Glia  2013;62(2):259-271.
In utero ethanol exposure causes Fetal Alcohol Spectrum Disorders, associated with reduced brain plasticity; the mechanisms of these effects are not well understood, particularly with respect to glial involvement.
Astrocytes release factors that modulate neurite outgrowth. We explored the hypothesis that ethanol inhibits neurite outgrowth by increasing the release of inhibitory chondroitin sulfate proteoglycans (CSPGs) from astrocytes.
Astrocyte treatment with ethanol inhibited the activity of arylsulfatase B (ARSB), the enzyme that removes sulfate groups from chondroitin-4-sulfate (C4S) and triggers the degradation of C4S, increased total sulfated glycosaminoglycans (GAGs), C4S, and neurocan core-protein content and inhibited neurite outgrowth in neurons co-cultured with ethanol-treated astrocytes in vitro, effects reversed by treatment with recombinant ARSB.
Ethanol also inhibited ARSB activity and increased sulfate GAG and neurocan levels in the developing hippocampus after in vivo ethanol exposure.
ARSB silencing increased the levels of sulfated GAGs, C4S, and neurocan in astrocytes and inhibited neurite outgrowth in co-cultured neurons, indicating that ARSB activity directly regulates C4S and affects neurocan expression.
In summary, this study reports two major findings: ARSB modulates sulfated GAG and neurocan levels in astrocytes and astrocyte-mediated neurite outgrowth in co-cultured neurons; and ethanol inhibits the activity of ARSB, increases sulfated GAG, C4S, and neurocan levels, and thereby inhibits astrocyte-mediated neurite outgrowth.
An unscheduled increase in CSPGs in the developing brain may lead to altered brain connectivity and to premature decrease in neuronal plasticity and therefore represents a novel mechanism by which ethanol can exert its neurodevelopmental effects.
PMCID: PMC4272875  PMID: 24311516
Astrocytes; arylsulfatase B; fetal alcohol; chondroitin sulfate; neurocan
8.  Molecular scaffolds underpinning macroglial polarization: an analysis of retinal Müller cells and brain astrocytes in mouse 
Glia  2012;60(12):2018-2026.
Key roles of macroglia are inextricably coupled to specialized membrane domains. The perivascular endfoot membrane has drawn particular attention, as this domain contains a unique complement of aquaporin-4 (AQP4) and other channel proteins that distinguishes it from perisynaptic membranes. Recent studies indicate that the polarization of macroglia is lost in a number of diseases, including temporal lobe epilepsy and Alzheimer’s disease. A better understanding is required of the molecular underpinning of astroglial polarization, particularly when it comes to the significance of the dystrophin associated protein complex (DAPC). Here we employ immunofluorescence and immunogold cytochemistry to analyze the molecular scaffolding in perivascular endfeet in macroglia of retina and three regions of brain (cortex, dentate gyrus and cerebellum), using AQP4 as a marker. Compared with brain astrocytes, Müller cells (a class of retinal macroglia) exhibit lower densities of the scaffold proteins dystrophin and α-syntrophin (a DAPC protein), but higher levels of AQP4. In agreement, depletion of dystrophin or α-syntrophin – while causing a dramatic loss of AQP4 from endfoot membranes of brain astrocytes – had only modest or insignificant effect, respectively, on the AQP4 pool in endfoot membranes of Müller cells. Also, while polarization of brain macroglia was less affected by dystrophin depletion than by targeted deletion of α-syntrophin, the reverse was true for retinal macroglia. These data indicate that the molecular scaffolding in perivascular endfeet is more complex than previously assumed and that macroglia are heterogeneous with respect to the mechanisms that dictate their polarization.
PMCID: PMC4306326  PMID: 22987438
AQP4; aquaporin; dystrophin; endfeet; glia; mdx3cv; syntrophin
9.  Opioid System Diversity in Developing Neurons, Astroglia and Oligodendroglia in the Subventricular Zone and Striatum: Impact on Gliogenesis In Vivo 
Glia  2001;36(1):78-88.
Accumulating evidence, obtained largely in vitro, indicates that opioids regulate the genesis of neurons and glia and their precursors in the nervous system. Despite this evidence, few studies have assessed opioid receptor expression in identified cells within germinal zones or examined opioid effects on gliogenesis in vivo. To address this question, the role of opioids was explored in the subventricular zone (SVZ) and/or striatum of 2-5 day-old and/or adult ICR mice. The results showed that subpopulations of neurons, astrocytes, and oligodendrocytes in the SVZ and striatum differentially express μ, δ, and/or κ receptor immunoreactivity in a cell-type-specific and developmentally regulated manner. In addition, DNA synthesis was assessed by examining 5-bromo-2′-deoxyuridine (BrdU) incorporation into glial and non-glial precursors. In GFAP+ cells, morphine (a preferential μ agonist) significantly decreased BrdU-labeled astroglia compared to controls or mice co-treated with naltrexone plus morphine. Alternatively, in S100β+ cells, morphine did not significantly decrease BrdU incorporation; however, significant differences were noted between morphine and morphine plus naltrexone-treated mice. The majority of cells were non-GFAP+/non-S100β+. When BrdU incorporation was assessed within the total population (glia and non-glia), morphine had no net effect but naltrexone alone markedly increased BrdU incorporation. This suggests DNA synthesis in non-GFAP+/non-S100β+ cells is tonically suppressed by endogenous opioids. Assuming S100β and GFAP, respectively, distinguish among younger and older astroglia, this implies that astroglial replication becomes increasingly sensitive to morphine during maturation, and suggests that opioids differentially regulate the development of distinct subpopulations of glia and glial precursors.
PMCID: PMC4303466  PMID: 11571786
astrocytes; oligodendrocytes; cell division; subventricular zone; striatum; μ opioid receptors; δ opioid receptors; κ opioid receptors; central nervous system development; opiate drug abuse; glial fibrillary acidic protein; S100β; drug abuse
Glia  2009;57(16):1754-1764.
Neurotrophin-3 (NT-3) regulates oligodendrocyte (OLG) differentiation by mechanisms that remain poorly understood. Exposure of OLGs to NT-3 induces a significant increase in the levels of myelin basic protein (MBP). However, we found that this stimulation occurs in the absence of measurable effects on MBP gene promoter activation or mRNA expression, suggesting that NT-3 up-regulates MBP protein expression by a posttranscriptional mechanism. Furthermore, NT-3 also causes an increase in the levels of myelin associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG), raising the possibility of a more general effect on myelin protein synthesis. Surprisingly, 35S-methionine incorporation into total OLG proteins demonstrated a 50% increase in labeling following only a brief, 15 minute treatment with NT-3. Such a remarkably fast response is unlikely due to transcriptional activation, reinforcing the possibility that NT-3 may play a crucial role in regulating protein expression by a posttranscriptional mechanism. In support of this idea, we found that NT-3 stimulates the phosphorylation of essential regulators of the initiation machinery, eukaryotic initiation factor 4E (eIF4E) and its inhibitory binding partner 4E binding protein 1 (4EBP1), two crucial players in controlling cap-dependent protein synthesis. This stimulation involves the activation of pathways mediated by ERK1/2 and PI3K/mTOR, implicating these two kinase systems as modulators of protein synthesis in developing OLGs. Altogether, these observations show for the first time that NT-3 has the capacity of targeting the translational machinery and suggest a potential stimulatory effect of this neurotrophin on myelination by direct action on protein translation in the OLGs.
PMCID: PMC4300950  PMID: 19455580
neurotrophin-3; oligodendrocytes; myelination; translational regulation
11.  Synergistic increases in intracellular Ca2+, and the release of MCP-1, RANTES, and IL-6 by astrocytes treated with opiates and HIV-1 Tat‡ 
Glia  2005;50(2):91-106.
Recent evidence suggests that injection drug users who abuse heroin are at increased risk for CNS complications from human immunodeficiency virus (HIV) infection. Opiate drugs may intrinsically alter the pathogenesis of HIV by directly modulating immune function and by directly modifying the CNS response to HIV. Despite this, the mechanisms by which opiates increase the neuropathogenesis of HIV are uncertain. Herein we describe the effect of morphine and the HIV-1 protein toxin Tat1-72 on astroglial function in cultures derived from ICR mice. Astroglia maintain the blood brain barrier and influence inflammatory signaling in the CNS. Astrocytes can express μ opioid receptors, and are likely targets for abused opiates, which preferentially activate μ-opioid receptors. While Tat alone disrupts astrocyte function, when combined with morphine, Tat causes synergistic increases in [Ca2+]i.. Moreover, astrocyte cultures treated with morphine and Tat showed exaggerated increases in chemokine release including monocyte chemoattractant protein-1 (MCP-1) and regulated on activation, normal T cell expressed and secreted (RANTES), as well as interleukin-6 (IL-6). Morphine-Tat interactions were prevented by the μ-opioid receptor antagonist β-funaltrexamine, or by immunoneutralizing Tat1-72 or substituting a non-toxic, deletion mutant (TatΔ31-61). Our findings suggest that opiates may increase the vulnerability of the CNS to viral entry (via recruitment of monocytes/macrophages) and ensuing HIV encephalitis by synergistically increasing MCP-1 and RANTES release by astrocytes. The results further suggest that astrocytes are key intermediaries in opiate-HIV interactions and disruptions in astroglial function and inflammatory signaling may contribute to an accelerated neuropathogenesis in HIV infected individuals who abuse opiates.
PMCID: PMC4301446  PMID: 15630704
AIDS; chemokines; μ-opioid receptors; drug abuse; neuroimmunology; cytokine arrays
12.  Developmental and Post-Injury Cortical Gliogenesis: A Genetic Fate-Mapping Study with Nestin-CreER Mice 
Glia  2009;57(10):1115-1129.
The primary sources of cortical gliogenesis, either during development or after adult brain injury, remain uncertain. We previously generated Nestin-CreER mice to fate-map the progeny of radial glial cells (RG), a source of astrocytes and oligodendrocytes in the nervous system. Here, we show that Nestin-CreER mice label another population of glial progenitors, namely the perinatal subventricular zone (SVZ) glioblasts, if they are crossed with stop-floxed EGFP mice and receive tamoxifen in late embryogenesis (E16-E18). Quantification showed E18 tamoxifen-induction labeled more perinatal SVZ glioblasts than RG and transitional RG combined in the newborn brain (54% vs. 22%). Time-lapse microscopy showed SVZ-glioblasts underwent complex metamorphosis and often-reciprocal transformation into transitional RG. Surprisingly, the E10-dosed RG progenitors produced astrocytes, but no oligodendrocytes, whereas E18-induction fate-mapped both astrocytes and NG2+ oligodendrocyte precursors in the postnatal brain. These results suggest that cortical oligodendrocytes mostly derive from perinatal SVZ glioblast progenitors. Further, by combining genetic fate-mapping and BrdU-labeling, we showed that cortical astrocytes cease proliferation soon after birth (
PMCID: PMC4286201  PMID: 19115384
Subventricular zone (SVZ)-glioblasts; Radial Glia (RG); Gliogenesis; Gliosis; Oligodendrocyte; Astrocyte
Glia  2013;61(12):2009-2022.
Schwann cell (SC) migration is an important step preceding myelination and remyelination in the peripheral nervous system, and can be promoted by peptide factors like neuregulins. Here we present evidence that a lipid factor, lysophosphatidic acid (LPA), influences both SC migration and peripheral myelination through its cognate G protein-coupled receptor (GPCR) known as LPA1. Ultrastructural analyses of peripheral nerves in mouse null-mutants for LPA1 showed delayed SC-to-axon segregation, polyaxonal myelination by single SCs, and thinner myelin sheaths. In primary cultures, LPA promoted SC migration through LPA1, while analysis of conditioned media from purified dorsal root ganglia neurons using HPLC/MS supported the production of LPA by these neurons. The heterotrimeric G-alpha protein, Gαi, and the small GTPase, Rac1, were identified as important downstream signaling components of LPA1. These results identify receptor mediated LPA signaling between neurons and SCs that promote SC migration and contribute to the normal development of peripheral nerves through effects on SC-axon segregation and myelination.
PMCID: PMC3941654  PMID: 24115248
LPA; Schwann cell; Rac1; Gi; myelination; Gi; Rac1
Glia  2014;62(4):580-591.
The oligodendrocyte (OL), the myelinating cell of the central nervous system, undergoes dramatic changes in the organization of its cytoskeleton as it differentiates from a precursor (oligodendrocyte precursor cells) to a myelin-forming cell. These changes include an increase in its branching cell processes, a phenomenon necessary for OL to myelinate multiple axon segments. We have previously shown that levels and activity of non-muscle myosin II (NMII), a regulator of cytoskeletal contractility, decrease as a function of differentiation and that inhibition of NMII increases branching and myelination of OL in coculture with neurons. We have also found that mixed glial cell cultures derived from NMIIB knockout mice display an increase in mature myelin basic protein-expressing OL compared with wild-type cultures. We have now extended our studies to investigate the role of NMIIB ablation on myelin repair following focal demyelination by lysolecithin. To this end, we generated an oligodendrocyte-specific inducible knockout model using a Plp-driven promoter in combination with a temporally activated CRE-ER fusion protein. Our data indicate that conditional ablation of NMII in adult mouse brain, expedites lesion resolution and remyelination by Plp+ oligodendrocyte-lineage cells when compared with that observed in control brains. Taken together, these data validate the function of NMII as that of a negative regulator of OL myelination in vivo and provide a novel target for promoting myelin repair in conditions such as multiple sclerosis.
PMCID: PMC4135430  PMID: 24470341
oligodendrocytes; remyelination; cytoskeleton; myosin II
Glia  2014;62(6):964-970.
Multiple system atrophy (MSA) is a progressive neurodegenerative disease presenting clinically with parkinsonian, cerebellar, and autonomic features. α-Synuclein (αsyn), encoded by the gene SNCA, is the main constituent of glial cytoplasmic inclusion (GCI) found in oligodendrocytes in MSA, but the methods of its accumulation have not been established. The aim of this study is to investigate alterations in regional and cellular SNCA mRNA expression in MSA as a possible substrate for GCI formation. Quantitative reverse transcription polymerase chain reaction (qPCR) was performed on postmortem brain samples from 15 MSA, 5 IPD, and 5 control cases to investigate regional expression in the frontal and occipital regions, dorsal putamen, pontine base, and cerebellum. For cellular expression analysis, neurons and oligodendrocytes were isolated by laser-capture microdissection from five MSA and five control cases. SNCA mRNA expression was not significantly different between the MSA, IPD and control cases in all regions (multilevel model, P = 0.14). After adjusting for group effect, the highest expression was found in the occipital cortex while the lowest was in the putamen (multilevel model, P < 0.0001). At the cellular level, MSA oligodendrocytes expressed more SNCA than control oligodendrocytes and expression in MSA neurons was slightly lower than that in controls, however, these results did not reach statistical significance. We have demonstrated regional variations in SNCA expression, which is higher in cortical than subcortical regions. This study is the first to demonstrate SNCA mRNA expression by oligodendrocytes in human postmortem tissue using qPCR and, although not statistically significant, could suggest that this may be increased in MSA compared to controls.
PMCID: PMC4238782  PMID: 24590631
α-synuclein; multiple system atrophy; oligodendrocytes; glial cytoplasmic inclusions; laser-capture microdissection
Glia  2014;62(7):1066-1074.
Thymic stromal lymphopoietin (TSLP) is an epithelial cytokine expressed at barrier surfaces of the skin, gut, nose, lung, and the maternal/fetal interphase. At these sites, it is important for the generation and maintenance of non-inflammatory, tissue-resident dendritic cell responses. We show here that TSLP is also expressed in the central nervous system (CNS) where it is produced by choroid plexus epithelial cells and astrocytes in the spinal cord. Under conditions of low-grade myelin degeneration, the numbers of TSLP-expressing astrocytes increase, and microglia express transcripts for the functional TSLP receptor dimer indicating that these cells are targets for TSLP in the myelin-degenerative CNS.
PMCID: PMC4237118  PMID: 24668732
thymic stromal lymphopoietin; astrocytes; microglia; myelin degeneration
Glia  2014;62(5):804-817.
Microglia are resident antigen-presenting cells in the central nervous system (CNS) that either suppress or promote disease depending on their activation phenotype and the microenvironment. Multiple sclerosis (MS) is a chronic inflammatory disease causing demyelination and nerve loss in the CNS, and experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that is widely used to investigate pathogenic mechanisms and therapeutic effects. We isolated and cultured microglia from adult mouse brains and exposed them to specific combinations of stimulatory molecules and cytokines, the combination of IL-4, IL-10, and TGF-β yielding the optimal regime for induction of an immunosuppressive phenotype (M2). M2 microglia were characterized by decreased expression or production of CD86, PD-L1, nitric oxide, and IL-6, increased expression of PD-L2, and having a potent capacity to retain their phenotype on secondary proinflammatory stimulation. M2 microglia induced regulatory T cells, suppressed T-cell proliferation, and downmodulated M1-associated receptor expression in M1 macrophages. Myelin oligodendrocyte glycoprotein (MOG)-induced EAE was induced in DBA/1 mice and at different time points (0, 5, 12, or 15 days postimmunization) 3 × 105 M2 microglia were transferred intranasally. A single transfer of M2 microglia attenuated the severity of established EAE, which was particularly obvious when the cells were injected at 15 days postimmunization. M2 microglia-treated mice had reduced inflammatory responses and less demyelination in the CNS. Our findings demonstrate that adult M2 microglia therapy represents a novel intervention that alleviated established EAE and that this therapeutic principle may have relevance for treatment of MS patients.
PMCID: PMC4237117  PMID: 24677019
EAE; microglia; macrophages; phenotype; cell therapy
Glia  2013;61(11):10.1002/glia.22556.
Following transient forebrain ischemia, astrocytes play a key role in determining whether or not neurons in the hippocampal CA1 sector go on to die in a delayed fashion. MicroRNAs (miRNAs) are a novel class of RNAs that control gene expression at the post-transcriptional level and the miR-29 family is highly expressed in astrocytes. In this study we assessed levels of miR-29 in hippocampus following forebrain ischemia and found that after transient forebrain ischemia and short periods of reperfusion, miR-29a significantly increased in the resistant dentate gyrus, but decreased in the vulnerable CA1 region of the hippocampus. We demonstrate that miR-29a targets BH3-only pro-apoptotic BCL2 family member PUMA by luciferase reporter assay and by Western blot. Comparing primary neuron and astrocyte cultures, and postnatal brain, we verified the strongly astrocytic expression of miR-29a. We further found that miR-29a mimic protects and miR-29a inhibitor aggravates cell injury and mitochondrial function after ischemia-like stresses in vitro. Lastly, by overexpressing and reducing miR-29a we demonstrate the protective effect of miR-29a on CA1 delayed neuronal death after forebrain ischemia. Our data suggest that by targeting a pro-apoptotic BCL2 family member, increasing levels of miR-29a might emerge as a strategy for protection against ischemia-reperfusion injury.
PMCID: PMC3810393  PMID: 24038396
microRNA; PUMA; astrocyte
Glia  2013;62(1):17-25.
The arcuate nucleus (ARC) of the hypothalamus plays a key role in sensing metabolic feedback and regulating energy homeostasis. Recent studies revealed activation of microglia in mice with high-fat diet (HFD)-induced obesity (DIO), suggesting a potential pathophysiological role for inflammatory processes within the hypothalamus. To further investigate the metabolic causes and molecular underpinnings of such glial activation, we analyzed the microglial activity in wild-type (WT), monogenic obese ob/ob (leptin deficient), db/db (leptin-receptor mutation), and Type-4 melanocortin receptor knockout (MC4R KO) mice on either a HFD or on standardized chow (SC) diet. Following HFD exposure, we observed a significant increase in the total number of ARC microglia, immunoreactivity of ionized calcium binding adaptor molecule 1 (iba1-ir), cluster of differentiation 68 (CD68-ir), and ramification of microglial processes. The ob/ob mice had significantly less iba1-ir and ramifications. Leptin replacement rescued these phenomena. The db/db mice had similar iba1-ir comparable with WT mice but had significantly lower CD68-ir and more ramifications than WT mice. After 2 weeks of HFD, ob/ob mice showed an increase of iba1-ir, and db/db mice showed increase of CD68-ir. Obese MC4R KO mice fed a SC diet had comparable iba1-ir and CD68-ir with WT mice but had significantly more ramifications than WT mice. Intriguingly, treatment of DIO mice with glucagon-like peptide-1 receptor agonists reduced microglial activation independent of body weight. Our results show that diet type, adipokines, and gut signals, but not body weight, affect the presence and activity levels of hypothalamic microglia in obesity.
PMCID: PMC4213950  PMID: 24166765
leptin; obesity; high calorie diet
Glia  2011;60(4):515-525.
Emerging evidence suggests that p53, a tumor suppressor protein primarily involved in cancer biology, coordinates a wide range of novel functions in the CNS including the mediation of pathways underlying neurodegenerative disease pathogenesis. Moreover, an evolving concept in cell and molecular neuroscience is that glial cells are far more fundamental to disease progression than previously thought, which may occur via a noncell-autonomous mechanism that is heavily dependent on p53 activities. As a crucial hub connecting many intracellular control pathways, including cell-cycle control and apoptosis, p53 is ideally placed to coordinate the cellular response to a range of stresses. Although neurodegenerative diseases each display a distinct and diverse molecular pathology, apoptosis is a widespread hallmark feature and the multimodal capacity of the p53 system to orchestrate apoptosis and glial cell behavior highlights p53 as a potential unifying target for therapeutic intervention in neurodegeneration.
PMCID: PMC4195591  PMID: 22105777
p53; glia; neurodegeneration
Glia  2013;62(2):317-337.
Multiple system atrophy (MSA) is a neurodegenerative disease characterized by the pathological accumulation of alpha-synuclein (α-syn) within oligodendroglial cells. This accumulation is accompanied by neuroinflammation with astrogliosis and microgliosis, that leads to neuronal death and subsequent parkinsonism and dysautonomia. Antidepressants have been explored as neuroprotective agents as they normalize neurotrophic factor levels, increase neurogenesis and reduce neurodegeneration, but their anti-inflammatory properties have not been fully characterized. We analyzed the anti-inflammatory profiles of three different antidepressants (fluoxetine, olanzapine and amitriptyline) in the MBP1-hα-syn transgenic (tg) mouse model of MSA. We observed that antidepressant treatment decreased the number of α-syn-positive cells in the basal ganglia of 11-month old tg animals. This reduction was accompanied with a similar decrease in the colocalization of α-syn with astrocyte markers in this brain structure. Consistent with these results, antidepressants reduced astrogliosis in the hippocampus and basal ganglia of the MBP1-hα-syn tg mice, and modulated the expression levels of key cytokines that were dysregulated in the tg mouse model, such as IL-1β. In vitro experiments in the astroglial cell line C6 confirmed that antidepressants inhibited NF-κB translocation to the nucleus and reduced IL-1β protein levels. We conclude that the anti-inflammatory properties of antidepressants in the MBP1-hα-syn tg mouse model of MSA might be related to their ability to inhibit α-syn propagation from oligodendrocytes to astroglia and to regulate transcription factors involved in cytokine expression. Our results suggest that antidepressants might be of interest as anti-inflammatory and α-syn-reducing agents for MSA and other α-synucleinopathies.
PMCID: PMC4183229  PMID: 24310907
astroglia; cytokine; chemokine; anti-inflammation; neurodegenerative disease
Glia  2013;61(10):1595-1606.
Extracellular adenosine 3′,5′-cyclic monophosphate (3′,5′-cAMP) is an endogenous source of localized adenosine production in many organs. Recent studies suggest that extracellular 2′,3′-cAMP (positional isomer of 3′,5′-cAMP) is also a source of adenosine, particularly in the brain in vivo post-injury. Moreover, in vitro studies show that both microglia and astrocytes can convert extracellular 2′,3′-cAMP to adenosine. Here we examined the ability of primary mouse oligodendrocytes and neurons to metabolize extracellular 2′,3′-cAMP and their respective adenosine monophosphates (2′-AMP and 3′-AMP). Cells were also isolated from mice deficient in 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase). Oligodendrocytes metabolized 2′,3′-cAMP to 2′-AMP with 10-fold greater efficiency than did neurons (and also more than previously examined microglia and astrocytes); whereas, the production of 3′-AMP was minimal in both oligodendrocytes and neurons. The production of 2′-AMP from 2′,3′-cAMP was reduced by 65% in CNPase -/- versus CNPase +/+ oligodendrocytes. Oligodendrocytes also converted 2′-AMP to adenosine, and this was also attenuated in CNPase -/- oligodendrocytes. Inhibition of classic 3′,5′-cAMP-3′-phosphodiesterases with 3-isobutyl-1-methylxanthine did not block metabolism of 2′,3′-cAMP to 2′-AMP and inhibition of classic ecto-5′-nucleotidase (CD73) with α,β-methylene-adenosine-5′-diphosphate did not attenuate the conversion of 2′-AMP to adenosine. These studies demonstrate that oligodendrocytes express the extracellular 2′,3′-cAMP-adenosine pathway (2′,3′-cAMP → 2′-AMP → adenosine). This pathway is more robustly expressed in oligodendrocytes than in all other CNS cell types because CNPase is the predominant enzyme that metabolizes 2′,3′-cAMP to 2-AMP in CNS cells. By reducing levels of 2′,3′-cAMP (a mitochondrial toxin) and increasing levels of adenosine (a neuroprotectant), oligodendrocytes may protect axons from injury.
PMCID: PMC3998092  PMID: 23922219
Glia  2013;61(10):1571-1581.
Studies of the structural organization and functions of the cell body of a neuron (soma) and its surrounding satellite glial cells (SGCs) in sensory ganglia have led to the realization that SGCs actively participate in the information processing of sensory signals from afferent terminals to the spinal cord. SGCs use a variety ways to communicate with each other and with their enwrapped soma. Changes in this communication under injurious conditions often lead to abnormal pain conditions. “What are the mechanisms underlying the neuronal soma and SGC communication in sensory ganglia” and “how do tissue or nerve injuries affect the communication?” are the main questions addressed in this review.
PMCID: PMC3758405  PMID: 23918214
purinergic receptor; pannexin; cytokine; gap junction; pain
Glia  2013;61(7):1029-1040.
Upon peripheral nerve injury, specific molecular events, including increases in the expression of selected neurotrophic factors, are initiated to prepare the tissue for regeneration. However, the mechanisms underlying these events and the nature of the cells involved are poorly understood. We used the injury-induced upregulation of glial cell-derived neurotrophic factor (GDNF) expression as a tool to gain insights into these processes. We found that both myelinating and non-myelinating Schwann cells are responsible for the dramatic increase in GDNF expression after injury. We also demonstrate that the GDNF upregulation is mediated by a signaling cascade involving activation of Schwann cell purinergic receptors, followed by protein kinase C signaling which activates protein kinase D (PKD), which leads to increased GDNF transcription. Given the potent effects of GDNF on survival and repair of injured peripheral neurons, we propose that targeting these pathways may yield therapeutic tools to treat peripheral nerve injury and neuropathies.
PMCID: PMC4165612  PMID: 23553603
GDNF; purinergic receptor; nerve injury; protein kinase D; apyrase; Schwann cell
Glia  2008;56(7):791-800.
Adult progenitor cell proliferation in the subgranular zone (SGZ) of the dentate gyrus is a dynamic process that is modulated by an array of physiological process, including locomotor activity and novel environmental stimuli. In addition, pathophysiological events, such as ischemia and status epilepticus (SE), have been shown to stimulate neurogenesis. Currently, limited information is available regarding the extracellular stimuli, receptors, and downstream intracellular effectors that couple excitotoxic stimulation to progenitor cell proliferation. Here we show that pilocarpine-induced SE triggers a set of signaling events that impinge upon the p42/44 mitogen-activated protein kinase (MAPK) pathway to drive progenitor cell proliferation in the SGZ at 2-days post-SE. Increased proliferation was dependent on insulin-like growth factor-1 (IGF-1), which was localized to activated microglia near the SGZ. Using a combination of techniques, we show that IGF-1 is a CREB-regulated gene and that SE triggered CRE-dependent transcription in microglia at 2-days post-SE. Together, these data identify a potential signaling program that couples SE to progenitor cell proliferation. SE triggers CREB-dependent transcription in reactive microglia. As a CREB-target gene, IGF-1 expression is upregulated, and by 2-days post-SE, IGF-1 triggers MAPK pathway activation in progenitor cells and, in turn, an increase in progenitor cell proliferation.
PMCID: PMC4152854  PMID: 18338791
ERK; MAPK; IGF-1; progenitor cells; seizure; hippocampus; subgranular zone; pilocarpine

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