Oxidative Stress-Induced Damage to the Developing Hippocampus Is Mediated by GSK3ß.

Neonatal brain injury renders the developing brain vulnerable to oxidative stress, leading to cognitive deficit. However, oxidative stress-induced damage to hippocampal circuits and the mechanisms underlying long-term changes in memory and learning are poorly understood. We used high oxygen tension or hyperoxia (HO) in neonatal mice of both sexes to investigate the role of oxidative stress in hippocampal damage. Perinatal HO induces reactive oxygen species and cell death, together with reduced interneuron maturation, inhibitory postsynaptic currents, and dentate progenitor proliferation. Postinjury interneuron stimulation surprisingly improved inhibitory activity and memory tasks, indicating reversibility. With decreased hippocampal levels of Wnt signaling components and somatostatin, HO aberrantly activated glycogen synthase kinase 3 ß activity. Pharmacological inhibition or ablation of interneuron glycogen synthase kinase 3 ß during HO challenge restored progenitor cell proliferation, interneuron development, inhibitory/excitatory balance, as well as hippocampal-dependent behavior. Biochemical targeting of interneuron function may benefit learning deficits caused by oxidative damage.SIGNIFICANCE STATEMENT Premature infants are especially vulnerable to oxidative stress, as their antioxidant defenses are underdeveloped. Indeed, high oxygen tension is associated with poor neurologic outcomes. Because of its sustained postnatal development and role in learning and memory, the hippocampus is especially vulnerable to oxidative damage in premature infants. However, the role of oxidative stress in the developing hippocampus has yet to be explored. With ever-rising rates of neonatal brain injury and no universally viable approach to maximize functional recovery, a better understanding of the mechanisms underlying neonatal brain injury is needed. Addressing this need, this study uses perinatal hyperoxia to study cognitive deficits, pathophysiology, and molecular mechanisms of oxidative damage in the developing hippocampus.

Glial Factors Regulating White Matter Development and Pathologies of the Cerebellum.

The cerebellum is a brain region that undergoes extremely dynamic growth during perinatal and postnatal development which is regulated by the proper interaction between glial cells and neurons with a complex concert of growth factors, chemokines, cytokines, neurotransmitters and transcriptions factors. The relevance of cerebellar functions for not only motor performance but also for cognition, emotion, memory and attention is increasingly being recognized and acknowledged. Since perturbed circuitry of cerebro-cerebellar trajectories can play a role in many central nervous system pathologies and thereby contribute to neurological symptoms in distinct neurodevelopmental and neurodegenerative diseases, is it the aim with this mini-review to highlight the pathways of glia-glia interplay being involved. The designs of future treatment strategies may hence be targeted to molecular pathways also playing a role in development and disease of the cerebellum.

Transgenic overexpression of Sox17 promotes oligodendrocyte development and attenuates demyelination.

We have previously demonstrated that Sox17 regulates cell cycle exit and differentiation in oligodendrocyte progenitor cells. Here we investigated its function in white matter (WM) development and adult injury with a newly generated transgenic mouse overexpressing Sox17 in the oligodendrocyte lineage under the CNPase promoter. Sox17 overexpression in CNP-Sox17 mice sequentially promoted postnatal oligodendrogenesis, increasing NG2 progenitor cells from postnatal day (P) 15, then O4+ and CC1+ cells at P30 and P120, respectively. Total Olig2+ oligodendrocyte lineage cells first decreased between P8 and P22 through Sox17-mediated increase in apoptotic cell death, and thereafter significantly exceeded WT levels from P30 when cell death had ceased. CNP-Sox17 mice showed increased Gli2 protein levels and Gli2+ cells in WM, indicating that Sox17 promotes the generation of oligodendrocyte lineage cells through Hedgehog signaling. Sox17 overexpression prevented cell loss after lysolecithin-induced demyelination by increasing Olig2+ and CC1+ cells in response to injury. Furthermore, Sox17 overexpression abolished the injury-induced increase in TCF7L2/TCF4+ cells, and protected oligodendrocytes from apoptosis by preventing decreases in Gli2 and Bcl-2 expression that were observed in WT lesions. Our study thus reveals a biphasic effect of Sox17 overexpression on cell survival and oligodendrocyte formation in the developing WM, and that its potentiation of oligodendrocyte survival in the adult confers resistance to injury and myelin loss. This study demonstrates that overexpression of this transcription factor might be a viable protective strategy to mitigate the consequences of demyelination in the adult WM.

Neonatal hyperoxia exposure disrupts axon-oligodendrocyte integrity in the subcortical white matter.

The pathological mechanisms underlying neurological deficits observed in individuals born prematurely are not completely understood. A common form of injury in the preterm population is periventricular white matter injury (PWMI), a pathology associated with impaired brain development. To mitigate or eliminate PWMI, there is an urgent need to understand the pathological mechanism(s) involved on a neurobiological, structural, and functional level. Recent clinical data suggest that a percentage of premature infants experience relative hyperoxia. Using a hyperoxic model of premature brain injury, we have previously demonstrated that neonatal hyperoxia exposure in the mouse disrupts development of the white matter (WM) by delaying the maturation of the oligodendroglial lineage. In the present study, we address the question of how hyperoxia-induced alterations in WM development affect overall WM integrity and axonal function. We show that neonatal hyperoxia causes ultrastructural changes, including: myelination abnormalities (i.e., reduced myelin thickness and abnormal extramyelin loops) and axonopathy (i.e., altered neurofilament phosphorylation, paranodal defects, and changes in node of Ranvier number and structure). This disruption of axon-oligodendrocyte integrity results in the lasting impairment of conduction properties in the adult WM. Understanding the pathology of premature PWMI injury will allow for the development of interventional strategies to preserve WM integrity and function.

Oligodendroglial alterations and the role of microglia in white matter injury: relevance to schizophrenia.

Schizophrenia is a chronic and debilitating mental illness characterized by a broad range of abnormal behaviors, including delusions and hallucinations, impaired cognitive function, as well as mood disturbances and social withdrawal. Due to the heterogeneous nature of the disease, the causes of schizophrenia are very complex; its etiology is believed to involve multiple brain regions and the connections between them, and includes alterations in both gray and white matter regions. The onset of symptoms varies with age and severity, and there is some debate over a degenerative or developmental etiology. Longitudinal magnetic resonance imaging studies have detected progressive gray matter loss in the first years of disease, suggesting neurodegeneration; but there is also increasing recognition of a temporal association between clinical complications at birth and disease onset that supports a neurodevelopmental origin. Presently, neuronal abnormalities in schizophrenia are better understood than alterations in myelin-producing cells of the brain, the oligodendrocytes, which are the predominant constituents of white matter structures. Proper white matter development and its structural integrity critically impacts brain connectivity, which affects sensorimotor coordination and cognitive ability. Evidence of defective white matter growth and compromised white matter integrity has been found in individuals at high risk of psychosis, and decreased numbers of mature oligodendrocytes are detected in schizophrenia patients. Inflammatory markers, including proinflammatory cytokines and chemokines, are also associated with psychosis. A relationship between risk of psychosis, white matter defects and prenatal inflammation is being established. Animal models of perinatal brain injury are successful in producing white matter damage in the brain, typified by hypomyelination and/or dysmyelination, impaired motor coordination and prepulse inhibition of the acoustic startle reflex, recapitulating structural and functional characteristics observed in schizophrenia. In addition, elevated expression of inflammation-related genes in brain tissue and increased production of cytokines by blood cells from patients with schizophrenia indicate immunological dysfunction and abnormal inflammatory responses, which are also important underlying features in experimental models. Microglia, resident immune defenders of the central nervous system, play important roles in the development and protection of neural cells, but can contribute to injury under pathological conditions. This article discusses oligodendroglial changes in schizophrenia and focuses on microglial activity in the context of the disease, in neonatal brain injury and in various experimental models of white matter damage. These include disorders associated with premature birth, and animal models of perinatal bacterial and viral infection, oxygen deprivation (hypoxia) and excess (hyperoxia), and elevated systemic proinflammatory cytokine levels. We briefly review the effects of treatment with antipsychotic and anti-inflammatory agents in models of perinatal brain injury, and comment on the therapeutic potential of these strategies. By understanding the neurobiological basis of oligodendroglial abnormalities in schizophrenia, it is hoped that patients will benefit from the availability of targeted and more efficacious treatment options.

Cellular changes underlying hyperoxia-induced delay of white matter development.

Impaired neurological development in premature infants frequently arises from periventricular white matter injury (PWMI), a condition associated with myelination abnormalities. Recently, exposure to hyperoxia was reported to disrupt myelin formation in neonatal rats. To identify the causes of hyperoxia-induced PWMI, we characterized cellular changes in the white matter (WM) using neonatal wild-type 2-3-cyclic nucleotide 3-phosphodiesterase-enhanced green fluorescent protein (EGFP) and glial fibrillary acidic protein (GFAP)-EGFP transgenic mice exposed to 48 h of 80% oxygen from postnatal day 6 (P6) to P8. Myelin basic protein expression and CC1(+) oligodendroglia decreased after hyperoxia at P8, but returned to control levels during recovery between P12 and P15. At P8, hyperoxia caused apoptosis of NG2(+)O4(-) progenitor cells and reduced NG2(+) cell proliferation. This was followed by restoration of the NG2(+) cell population and increased oligodendrogenesis in the WM after recovery. Despite apparent cellular recovery, diffusion tensor imaging revealed WM deficiencies at P30 and P60. Hyperoxia did not affect survival or proliferation of astrocytes in vivo, but modified GFAP and glutamate-aspartate transporter expression. The rate of [(3)H]-d-aspartic acid uptake in WM tissue was also decreased at P8 and P12. Furthermore, cultured astrocytes exposed to hyperoxia showed a reduced capacity to protect oligodendrocyte progenitor cells against the toxic effects of exogenous glutamate. This effect was prevented by 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide treatment. Our analysis reveals a role for altered glutamate homeostasis in hyperoxia-induced WM damage. Understanding the cellular dynamics and underlying mechanisms involved in hyperoxia-induced PWMI will allow for future targeted therapeutic intervention.

SRY-box containing gene 17 regulates the Wnt/ß-catenin signaling pathway in oligodendrocyte progenitor cells.

The SRY-box (Sox) transcription factors regulate oligodendrocyte differentiation, but their signaling targets are largely unknown. We have identified a major signal transduction pathway regulated by Sox containing gene 17 (Sox17) in the oligodendrocyte lineage. Microarray analysis in oligodendrocyte progenitor cells (OPCs) after Sox17 attenuation revealed upregulated genes associated with cell cycle control and activation of the Wingless and integration site (Wnt)/ß-catenin pathway. Sox17 knockdown also increases the levels of cyclin D1, Axin2, and activated ß-catenin. In OPCs, the expression pattern of Sox17, cyclin D1, and secreted Frizzled-related protein-1 in the presence of platelet-derived growth factor (PDGF) was coordinately accelerated by addition of thyroid hormone, indicating differentiation-induced regulation of Sox17 targets. In developing white matter, decreased total ß-catenin, activated ß-catenin, and cyclin D1 levels coincided with the peak of Sox17 expression, and immunoprecipitates showed a developmentally regulated interaction among Sox17, T-cell transcription factor 4, and ß-catenin proteins. In OPCs, PDGF stimulated phosphorylation of glycogen synthase 3ß and the Wnt coreceptor LRP6, and enhanced ß-catenin-dependent gene expression. Sox17 overexpression inhibited PDGF-induced TOPFLASH and cyclin D1 promoter activity, and decreased endogenous cyclin D1, activated ß-catenin, as well as total ß-catenin levels. Recombinant Sox17 prevented Wnt3a from repressing myelin protein expression, and inhibition of Sox17-mediated proteasomal degradation of ß-catenin blocked myelin protein induction. These results indicate that Sox17 suppresses cyclin D1 expression and cell proliferation by directly antagonizing ß-catenin, whose activity in OPCs is stimulated not only by Wnt3a, but also by PDGF. Our identification of downstream targets of Sox17 thus defines signaling pathways and molecular mechanisms in OPCs that are regulated by Sox17 during cell cycle exit and the onset of differentiation in oligodendrocyte development.

Minocycline protects oligodendroglial precursor cells against injury caused by oxygen-glucose deprivation.

Ischemic brain injury is widely modeled in vitro with paradigms of oxygen-glucose deprivation (OGD), which leads to cell death. The prevention and attenuation of brain injury by the tetracycline antibiotic minocycline has been attributed largely to suppression of microglial activation, but its benefits in oligodendrocyte cells have not been well characterized. Using primary cultures of rat oligodendroglial precursor cells (OPC) exposed to OGD, we investigated the direct effects of minocycline on the survival, proliferation, and maturation of oligodendroglial lineage cells. OGD for 2 hr caused a decrease in the total number of OPC and the amount of proliferating progenitors by 50%, which was attenuated by inclusion of minocycline. The reduced numbers of immature oligodendroglial cells at 72 hr and of mature oligodendrocytes at 120 hr after OGD were partially restored by minocycline. In OPC, OGD caused an increase of reactive oxygen species (ROS) and production of TUNEL-positive cell numbers, which was abolished by minocycline. Minocycline preferentially increased the expression of superoxide dismutase under OGD but not in control OPC. Minocycline also prevented the OGD-induced downregulation of the transcription factors Sox10 and Olig2 and of myelin-specific genes 2'3' cyclic nucleotide phosphodiesterase (CNP) and myelin basic protein (MBP) in response to OGD. These studies demonstrate direct protective actions of minocycline on oligodendroglial-lineage cells, suggesting potential benefit in white matter injury involving OGD.

Mechanisms of regulation of oligodendrocyte development by p38 mitogen-activated protein kinase.

Many extracellular and intrinsic factors regulate oligodendrocyte development, but their signaling pathways remain poorly understood. Although the p38 mitogen-activated protein kinase (MAPK)-dependent pathway is implicated in oligodendrocyte progenitor cell (OPC) lineage progression, its molecular targets involved in myelinogenesis are mostly unidentified. We have analyzed mechanisms by which p38MAPK regulates oligodendrocyte development and demonstrate that p38MAPK inhibition prevents OPC lineage progression and inhibits MBP (myelin basic protein) promoter activity and Sox10 function. In white-matter tissue, differential levels of MAPK phosphorylation are observed in oligodendrocyte lineage cells. Phosphorylated p38MAPK was found in CC1- and CNP-expressing differentiated oligodendrocytes of the adult brain and was temporally associated with a decline in the levels of phosphorylated extracellular signal-regulated kinase (ERK) in cells of this lineage. PDGF stimulates the phosphorylation of ERK, p38MAPK, and c-Jun N-terminal kinase (JNK), and p38MAPK inhibition was associated with increased ERK, JNK, and c-Jun phosphorylation. In the presence of PDGF, simultaneous inhibition of p38MAPK and either MAPK kinase (MEK) or JNK significantly alleviates the repression of myelin gene expression and lineage progression induced by p38MAPK inhibition alone. Dominant-negative c-Jun reverses the inhibition of myelin promoter activity by active MEK1 or dominant-negative p38MAPKalpha mutants, and phosphorylated c-Jun was detected at the MBP promoter after p38MAPK inhibition, indicating c-Jun as a negative mediator of p38MAPK action. Our findings indicate that p38MAPK activity in the brain supports myelin gene expression through distinct mechanisms via positive and negative regulatory targets. We show that oligodendrocyte differentiation involves p38-mediated Sox10 regulation and cross talk with parallel ERK and JNK pathways to repress c-Jun activity.

Sox17 Promotes Oligodendrocyte Regeneration by Dual Modulation of Hedgehog and Wnt Signaling.

Signaling pathways that promote oligodendrocyte development improve oligodendrocyte regeneration and myelin recovery from demyelinating pathologies. Sox factors critically control myelin gene expression and oligodendroglial fate, but little is known about signaling events underlying Sox-mediated oligodendroglial regeneration. In this study of the SoxF member Sox17, we demonstrate that Sox17-induced oligodendrocyte regeneration in adult myelin lesions occurs by suppressing lesion-induced Wnt/beta-catenin signaling which is inhibitory to oligodendrocyte regeneration and by increasing Sonic Hedgehog/Smoothened/Gli2 activity. Hedgehog signaling through Smoothened critically supports adult oligodendroglial viability and is an upstream regulator of beta-catenin. Gli2 ablation in adult oligodendrocyte progenitor cells indicates that Gli2 regulates beta-catenin differentially in wild-type and Sox17-overexpressing white matter. Myelin lesions in Sox17-deficient mice show beta-catenin hyperactivation, regenerative failure, and loss of oligodendrogenesis, despite exogenous Hedgehog stimulation. These studies indicate the benefit of Sox17 signaling targets to enhance oligodendrocyte regeneration after demyelination injury by modulating both Hedgehog and Wnt/beta-catenin signaling.

The Yin and Yang of Sox proteins: Activation and repression in development and disease.

The general view of development consists of the acquisition of committed/differentiated phenotypes following a period of self-renewal and progenitor expansion. Lineage specification and progression are phenomena of antagonistic events, silencing tissue-specific gene expression in precursors to allow self-renewal and multipotentiality, and subsequently suppressing proliferation and embryonic gene expression to promote the restricted expression of tissue-specific genes during maturation. The high mobility group-containing Sox family of transcription factors constitutes one of the earliest classes of genes to be expressed during embryonic development. These proteins not only are indispensable for progenitor cell specification but also are critical for terminal differentiation of multiple cell types in a wide variety of lineages. Sox transcription factors are now known to induce or repress progenitor cell characteristics and cell proliferation or to activate the expression of tissue-specific genes. Sox proteins fulfill their diverse functions in developmental regulation by distinct molecular mechanisms. Not surprisingly, in addition to DNA binding and bending, Sox transcription factors also interact with different protein partners to function as coactivators or corepressors of downstream target genes. Here we seek to provide an overview of the current knowledge of Sox gene functional mechanisms, in an effort to understand their roles in both development and pathology.

Sox17 Regulates a Program of Oligodendrocyte Progenitor Cell Expansion and Differentiation during Development and Repair.

Sox17, a SoxF family member transiently upregulated during postnatal oligodendrocyte (OL) development, promotes OL cell differentiation, but its function in white matter development and pathology in vivo is unknown. Our analysis of oligodendroglial- and OL-progenitor-cell-targeted ablation in vivo using a floxed Sox17 mouse establishes a dependence of postnatal oligodendrogenesis on Sox17 and reveals Notch signaling as a mediator of Sox17 function. Following Sox17 ablation, reduced numbers of Olig2-expressing cells and mature OLs led to developmental hypomyelination and motor dysfunction. After demyelination, Sox17 deficiency inhibited OL regeneration. OL decline was unexpectedly preceded by transiently increased differentiation and a reduction of OL progenitor cells. Evidence of a dual role for Sox17 in progenitor cell expansion by Notch and differentiation involving TCF7L2 expression were found. A program of progenitor expansion and differentiation promoted by Sox17 through Notch thus contributes to OL production and determines the outcome of white matter repair.

Cytokines and myelination in the central nervous system.

Myelin abnormalities that reflect damage to developing and mature brains are often found in neurological diseases with evidence of inflammatory infiltration and microglial activation. Many cytokines are virtually undetectable in the uninflamed central nervous system (CNS), so that their rapid induction and sustained elevation in immune and glial cells contributes to dysregulation of the inflammatory response and neural cell homeostasis. This results in aberrant neural cell development, cytotoxicity, and loss of the primary myelin-producing cells of the CNS, the oligodendrocytes. This article provides an overview of cytokine and chemokine activity in the CNS with relevance to clinical conditions of neonatal and adult demyelinating disease, brain trauma, and mental disorders with observed white matter defects. Experimental models that mimic human disease have been developed in order to study pathogenic and therapeutic mechanisms, but have shown mixed success in clinical application. However, genetically altered animals, and models of CNS inflammation and demyelination, have offered great insight into the complexities of neuroimmune interactions that impact oligodendrocyte function. The intracellular signaling pathways of selected cytokines have also been highlighted to illustrate current knowledge of receptor-mediated events. By learning to interpret the actions of cytokines and by improving methods to target appropriate predictors of disease risk selectively, a more comprehensive understanding of altered immunoregulation will aid in the development of advanced treatment options for patients with inflammatory white matter disorders.

Age-Dependent Cellular and Behavioral Deficits Induced by Molecularly Targeted Drugs Are Reversible.

Newly developed targeted anticancer drugs inhibit signaling pathways commonly altered in adult and pediatric cancers. However, as these pathways are also essential for normal brain development, concerns have emerged of neurologic sequelae resulting specifically from their application in pediatric cancers. The neural substrates and age dependency of these drug-induced effects in vivo are unknown, and their long-term behavioral consequences have not been characterized. This study defines the age-dependent cellular and behavioral effects of these drugs on normally developing brains and determines their reversibility with post-drug intervention. Mice at different postnatal ages received short courses of molecularly targeted drugs in regimens analagous to clinical treatment. Analysis of rapidly developing brain structures important for sensorimotor and cognitive function showed that, while adult administration was without effect, earlier neonatal administration of targeted therapies attenuated white matter oligodendroglia and hippocampal neuronal development more profoundly than later administration, leading to long-lasting behavioral deficits. This functional impairment was reversed by rehabilitation with physical and cognitive enrichment. Our findings demonstrate age-dependent, reversible effects of these drugs on brain development, which are important considerations as treatment options expand for pediatric cancers.Significance: Targeted therapeutics elicit age-dependent long-term consequences on the developing brain that can be ameliorated with environmental enrichment. Cancer Res; 78(8); 2081-95. ©2018 AACR.

Identification of Sox17 as a transcription factor that regulates oligodendrocyte development.

Microarray analysis of oligodendrocyte lineage cells purified by fluorescence-activated cell sorting (FACS) from 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP)-enhanced green fluorescent protein (EGFP) transgenic mice revealed Sox17 (SRY-box containing gene 17) gene expression to be coordinately regulated with that of four myelin genes during postnatal development. In CNP-EGFP-positive (CNP-EGFP+) cells, Sox17 mRNA and protein levels transiently increased between postnatal days 2 and 15, with white matter O4+ preoligodendrocytes expressing greater Sox17 levels than Nkx2.2+ (NK2 transcription factor related, locus 2) NG2+, or GalC+ (galactocerebroside) cells. In spinal cord, Sox17 protein expression was undetectable in the primary motor neuron domain between embryonic days 12.5 and 15.5 but was evident in Nkx2.2+ and CC1+ cells. In cultured oligodendrocyte progenitor cells (OPCs), Sox17 levels were maximal in O4+ cells and peaked during the phenotypic conversion from bipolar to multipolar. Parallel increases in Sox17 and p27 occurred before MBP protein expression, and Sox17 upregulation was prevented by conditions inhibiting differentiation. Sox17 downregulation with small interfering RNAs increased OPC proliferation and decreased lineage progression after mitogen withdrawal, whereas Sox17 overexpression in the presence of mitogen had opposite effects. Sox17 overexpression enhanced myelin gene expression in OPCs and directly stimulated MBP gene promoter activity. These findings support important roles for Sox17 in controlling both oligodendrocyte progenitor cell cycle exit and differentiation.

Pharmacological approaches to intervention in hypomyelinating and demyelinating white matter pathology.

White matter disease afflicts both developing and mature central nervous systems. Both cell intrinsic and extrinsic dysregulation result in profound changes in cell survival, axonal metabolism and functional performance. Experimental models of developmental white matter (WM) injury and demyelination have not only delineated mechanisms of signaling and inflammation, but have also paved the way for the discovery of pharmacological approaches to intervention. These reagents have been shown to enhance protection of the mature oligodendrocyte cell, accelerate progenitor cell recruitment and/or differentiation, or attenuate pathological stimuli arising from the inflammatory response to injury. Here we highlight reports of studies in the CNS in which compounds, namely peptides, hormones, and small molecule agonists/antagonists, have been used in experimental animal models of demyelination and neonatal brain injury that affect aspects of excitotoxicity, oligodendrocyte development and survival, and progenitor cell function, and which have been demonstrated to attenuate damage and improve WM protection in experimental models of injury. The molecular targets of these agents include growth factor and neurotransmitter receptors, morphogens and their signaling components, nuclear receptors, as well as the processes of iron transport and actin binding. By surveying the current evidence in non-immune targets of both the immature and mature WM, we aim to better understand pharmacological approaches modulating endogenous oligodendroglia that show potential for success in the contexts of developmental and adult WM pathology. This article is part of the Special Issue entitled 'Oligodendrocytes in Health and Disease'.

Sirt1 regulates glial progenitor proliferation and regeneration in white matter after neonatal brain injury.

Regenerative processes in brain pathologies require the production of distinct neural cell populations from endogenous progenitor cells. We have previously demonstrated that oligodendrocyte progenitor cell (OPC) proliferation is crucial for oligodendrocyte (OL) regeneration in a mouse model of neonatal hypoxia (HX) that reproduces diffuse white matter injury (DWMI) of premature infants. Here we identify the histone deacetylase Sirt1 as a Cdk2 regulator in OPC proliferation and response to HX. HX enhances Sirt1 and Sirt1/Cdk2 complex formation through HIF1α activation. Sirt1 deacetylates retinoblastoma (Rb) in the Rb/E2F1 complex, leading to dissociation of E2F1 and enhanced OPC proliferation. Sirt1 knockdown in culture and its targeted ablation in vivo suppresses basal and HX-induced OPC proliferation. Inhibition of Sirt1 also promotes OPC differentiation after HX. Our results indicate that Sirt1 is an essential regulator of OPC proliferation and OL regeneration after neonatal brain injury. Therefore, enhancing Sirt1 activity may promote OL recovery after DWMI.

A novel secreted splice variant of vascular endothelial cell growth inhibitor.

Vascular endothelial cell growth inhibitor (VEGI), a member of the tumor necrosis factor (TNF) family, is an endothelial cell-specific inhibitor of angiogenesis. Overexpression by cancer cells of a secretable VEGI fusion protein resulted in abrogation of xenograft tumor progression, but overexpression of full-length VEGI was completely without effect. This finding indicates that secretion is essential for VEGI action. Here we report the identification of two new VEGI isoforms consisting of 251 and 192 amino acid residues. Both isoforms show endothelial cell-specific expression and share a C-terminal 151-residue segment with the previously described VEGI, which comprises 174 residues. The isoforms are generated from a 17 kb human gene by alternative splicing. Their expression is regulated in parallel by inflammatory cytokines TNF-alpha and interferon-gamma. VEGI-251, the most abundant isoform, contains a putative secretion signal. VEGI protein is detected in conditioned media of endothelial cells and VEGI-251-transfected mammalian cells. Overexpression of VEGI-251 in endothelial cells causes dose-dependent cell death. VEGI-251-transfected cancer cells form xenograft tumors of reduced growth rate and microvessel density compared with tumors of empty vector transfectants. These findings support the view that endothelial cell-secreted VEGI may function as an autocrine inhibitor of angiogenesis and a naturally existing modulator of vascular homeostasis.

Finding degrees of separation: experimental approaches for astroglial and oligodendroglial cell isolation and genetic targeting.

The study of CNS glial cell function requires experimental methods to detect, purify, and manipulate each cell population with fidelity and specificity. With the identification and cloning of cell- and stage-specific markers, glial cell analysis techniques have grown beyond physical methods of tissue dissociation and cell culture, and become highly specific with immunoselection of cell cultures in vitro and genetic targeting in vivo. The unique plasticity of glial cells offers the potential for cell replacement therapies in neurological disease that utilize neural cells derived from transplanted neural stem and progenitor cells. In this mini-review, we outline general physical and genetic approaches for macroglial cell generation. We summarize cell culture methods to obtain astrocytes and oligodendrocytes and their precursors, from developing and adult tissue, as well as approaches to obtain human neural progenitor cells through the establishment of stem cells. We discuss popular targeting rodent strains designed for cell-specific detection, selection and manipulation of neuroglial cell progenitors and their committed progeny. Based on shared markers between astrocytes and stem cells, we discuss genetically modified mouse strains with overlapping expression, and highlight SOX-expressing strains available for targeting of stem and progenitor cell populations. We also include recently established mouse strains for detection, and tag-assisted RNA and miRNA analysis. This discussion aims to provide a brief overview of the rapidly expanding collection of experimental approaches and genetic resources for the isolation and targeting of macroglial cells, their sources, progeny and gene products to facilitate our understanding of their properties and potential application in pathology.

Modulation of the Wnt/beta-catenin pathway in human oligodendroglioma cells by Sox17 regulates proliferation and differentiation.

Oligodendrogliomas originate from oligodendrocyte progenitor cells (OPCs), whose development is regulated by the Sonic hedgehog and Wnt/beta-catenin pathways. We investigated the contribution of these pathways in the proliferation and differentiation of human oligodendroglioma cells (HOG). Inhibition of Hedgehog signaling with cyclopamine decreased cell survival and increased phosphorylated beta-catenin without altering myelin protein levels. Conversely, treatment of HOG with the Wnt antagonist secreted frizzled related protein (SFRP1), led to increased myelin protein levels and reduced cell proliferation, suggesting cell cycle arrest and differentiation. Unlike normal primary human OPCs, beta-catenin in HOG cells is not associated with endogenous Sox17 protein despite high levels of both proteins. Retroviral overexpression of recombinant Sox17 increased HOG cell cycle exit and apoptosis, and raised myelin protein levels and the percentage of O4(+) cells, indicating increased differentiation. Recombinant Sox17 also increased beta-catenin-TCF4-Sox17 complex formation and decreased total cellular levels of beta-catenin. These changes were associated with increased SFRP1, and reduced expression of Wnt-1 and Frizzled-1, -3 and -7 RNA, indicating that Sox17 induced a Hedgehog target, and regulated Wnt signaling at multiple levels. Our studies indicate that Wnt signaling regulates HOG cell cycle arrest and differentiation, and that recombinant Sox17 mediates modulation of the Wnt pathway through changes in beta-catenin, SFRP1 and Wnt/Frizzled expression. Our results thus identify Sox17 as a potential molecular target to include in HOG therapeutic strategies.