Steven W. Levison

Both Oligodendrocytes and Astrocytes Develop from Progenitors in the Subventricular Zone of Postnatal .Rat Forebrain

The developmental fates of subventricular zone (SVZ) cells of the postnatal rat forebrain were determined by retroviral-mediated gene transfer and immunolabeling for glial antigens. A beta-galactosidase-containing retrovirus injected stereotactically into the SVZ infected small, immature cells. By 28 days post-injection labeled cells had appeared in both gray and white matter of the ipsilateral hemisphere. White matter contained labeled oligodendrocytes, but few astrocytes, while neocortex and striatum contained both glial types, often appearing in tightly knit clusters. An analysis after simultaneously injecting alkaline phosphatase- and beta-galactosidase-containing retroviruses showed that cells in each cortical cluster were related. Most clusters contained a single cell type, but approximately 15% contained both astrocytes and oligodendrocytes. These observations strongly suggest that a single SVZ cell can differentiate into both glial types.

Diabetic Retinopathy: More Than Meets the Eye

Retinal microvascular dysfunction in diabetes is a major component of diabetic retinopathy. This review highlights recent observations regarding the cellular anatomy that contributes to the blood-retinal barrier and its breakdown, the alterations of macroglial, neuronal, and microglial cells in diabetes, and how these changes lead to loss of vision. In addition, the effects of systemic pathophysiologic influences, including metabolic control, blood pressure, and fluid volume on the formation of diabetic macular edema are discussed. Finally, an overview of inflammatory mechanisms and responses in the retina in diabetes is provided. Together, these new observations provide a broader clinical and research perspective on diabetic retinal vascular dysfunction than previously considered, and provide new avenues for improved treatments to prevent loss of vision.

Pro‐regenerative properties of cytokine‐activated astrocytes

The prevailing view of the astrocytic response to injury is that reactive astrocytes impede the regenerative process by forming scar tissue. As the levels of many cytokines dramatically increase following CNS insult and as this increase in cytokine expression precedes the production of the glial scar, a long‐standing view has been that cytokines diminish neuronal survival and regeneration by stimulating the formation of astrogliotic scar tissue. However, there is a wealth of data indicating that cytokines ‘activate’ astrocytes, and that cytokine‐stimulated astrocytes can promote the recovery of CNS function. Supporting evidence demonstrates that cytokine‐activated astrocytes produce energy substrates and trophic factors for neurons and oligodendrocytes, act as free radical and excess glutamate scavengers, actively restore the blood–brain barrier, promote neovascularization, restore CNS ionic homeostasis, promote remyelination and also stimulate neurogenesis from neural stem cells. Accordingly, a re‐assessment of cytokine‐activated astrocytes is necessary. Here, we review studies that promote the thesis that cytokines elicit potent neuroprotective and regenerative responses from astrocytes.

Interleukin-1: A master regulator of neuroinflammation

Interleukins 1α and 1β (IL‐1) are very potent signaling molecules that are expressed normally at low levels, but are induced rapidly in response to local or peripheral insults. IL‐1 coordinates systemic host defense responses to pathogens and to injury and not surprisingly it has similar effects within the central nervous system (CNS). Numerous reports have correlated the presence of IL‐1 in the injured or diseased brain, and its effects on neurons and nonneuronal cells in the CNS, but it is only recently that the importance of IL‐1 signaling has been recognized. This article reviews studies that demonstrate that IL‐1 is at or near the top of the hierarchical cytokine signaling cascade in the CNS that results in the activation of endogenous microglia and vascular endothelial cells to recruit peripheral leukocytes (i.e., neuroinflammation). The IL‐1 system thus provides an attractive target for therapeutic intervention to ameliorate the destructive consequences of neuroinflammation.

The role of inflammation in perinatal brain injury.

Inflammation is increasingly recognized as being a critical contributor to both normal development and injury outcome in the immature brain. The focus of this Review is to highlight important differences in innate and adaptive immunity in immature versus adult brain, which support the notion that the consequences of inflammation will be entirely different depending on context and stage of CNS development. Perinatal brain injury can result from neonatal encephalopathy and perinatal arterial ischaemic stroke, usually at term, but also in preterm infants. Inflammation occurs before, during and after brain injury at term, and modulates vulnerability to and development of brain injury. Preterm birth, on the other hand, is often a result of exposure to inflammation at a very early developmental phase, which affects the brain not only during fetal life, but also over a protracted period of postnatal life in a neonatal intensive care setting, influencing critical phases of myelination and cortical plasticity. Neuroinflammation during the perinatal period can increase the risk of neurological and neuropsychiatric disease throughout childhood and adulthood, and is, therefore, of concern to the broader group of physicians who care for these individuals.

Cycling cells in the adult rat neocortex preferentially generate oligodendroglia

Gliogenesis in the mammalian central nervous system does not cease abruptly like neurogenesis. Instead, glia accumulate over a time period that extends into adulthood. To determine whether new glial cells in the adult cortex arise from resident progenitors and to determine the glial types to which these progenitors give rise to, cells in the perinatal subventricular zone (SVZ) were labeled with replication‐deficient retroviral vectors, and clonal clusters of glia in the neocortex were examined from 1 week to 8 months of age. The average clonal cluster size increased during the first month of life. Interestingly, clusters containing oligodendrocyte lineage cells preferentially expanded with age, on average doubling every 3 months. Unexpectedly, the number of cells in astrocyte clusters decreased over time. In heterogeneous clusters, the numbers of oligodendroglia increased, whereas the number of astrocytes did not. Moreover, clonal clusters containing mature glia also contained less mature cells, indicating that clonally related progenitors do not differentiate synchronously in vivo. Thus, progenitors from the SVZ continue to cycle, resulting in an accumulation of oligodendroglia in the neocortex. These slowly cycling cells likely express the NG2 proteoglycan because a subset of the clonal clusters contained NG2+ cells and these NG2+ cells accumulated with time. J. Neurosci. Res. 57:435–446, 1999.

Activation of the Mammalian Target of Rapamycin (mTOR) Is Essential for Oligodendrocyte Differentiation

Although both extrinsic and intrinsic factors have been identified that orchestrate the differentiation and maturation of oligodendrocytes, less is known about the intracellular signaling pathways that control the overall commitment to differentiate. Here, we provide evidence that activation of the mammalian target of rapamycin (mTOR) is essential for oligodendrocyte differentiation. Specifically, mTOR regulates oligodendrocyte differentiation at the late progenitor to immature oligodendrocyte transition as assessed by the expression of stage specific antigens and myelin proteins including MBP and PLP. Furthermore, phosphorylation of mTOR on Ser 2448 correlates with myelination in the subcortical white matter of the developing brain. We demonstrate that mTOR exerts its effects on oligodendrocyte differentiation through two distinct signaling complexes, mTORC1 and mTORC2, defined by the presence of the adaptor proteins raptor and rictor, respectively. Disrupting mTOR complex formation via siRNA mediated knockdown of raptor or rictor significantly reduced myelin protein expression in vitro. However, mTORC2 alone controlled myelin gene expression at the mRNA level, whereas mTORC1 influenced MBP expression via an alternative mechanism. In addition, investigation of mTORC1 and mTORC2 targets revealed differential phosphorylation during oligodendrocyte differentiation. In OPC-DRG cocultures, inhibiting mTOR potently abrogated oligodendrocyte differentiation and reduced numbers of myelin segments. These data support the hypothesis that mTOR regulates commitment to oligodendrocyte differentiation before myelination.

Multipotential and lineage restricted precursors coexist in the mammalian perinatal subventricular zone

Developmental studies have shown that both neurons and glia arise from the subventricular zone (SVZ) but there have been no clonal analyses to determine whether a single progenitor can produce both. Therefore, we used replication deficient retroviral vectors to analyze the clonal progeny of single rat SVZ cells that were maintained in culture media permissive or non‐permissive for neuronal differentiation. When maintained in medium supplemented with 5% fetal bovine serum, all surviving progenitors generated glial cell clones. Within these glial clones we often observed both type 1 astrocytes and O‐2A lineage cells. When SVZ cells were maintained in medium permissive for neurogenesis approximately 50% of the total clones contained at least one antigenically defined neuron. Of those clones that contained neurons, 60% contained neurons and glia. The other 50% of the total clones were either comprised of only astrocytes, astrocytes and oligodendrocytes, or were unidentifiable. Since the culture environment permitted multilineage clone formation, yet many homogeneous neuronal or astrocytic clones were obtained, some progenitors must become developmentally restricted while they are in the germinal zone. Therefore, we conclude that the perinatal SVZ is a mosaic of multipotential, bipotential, and lineage restricted precursors, and that the lack of postnatal neocortical neurogenesis is not due to the absence of potential neuroblasts. J. Neurosci. Res. 48:83–94, 1997.

Hypoxia/Ischemia Depletes the Rat Perinatal Subventricular Zone of Oligodendrocyte Progenitors and Neural Stem Cells

Cerebral hypoxia/ischemia of the newborn has a frequency of 4/1,000 births and remains a major cause of cerebral palsy, epilepsy, and mental retardation. Despite progress in understanding the pathogenesis of hypoxic-ischemic injury, the data are incomplete regarding the mechanisms leading to permanent brain injury. Here we tested the hypothesis that cerebral hypoxia/ischemia damages stem/progenitor cells in the subventricular zone (SVZ), resulting in a permanent depletion of oligodendrocytes. We used a widely accepted rat model and examined animals at recovery intervals ranging from 4 h to 3 weeks. Within hours after the hypoxic-ischemic insult 20% of the total cells were deleted from the SVZ. The residual damaged cells appeared necrotic. During 48 h of recovery deaths accumulated; however, these later deaths were predominantly apoptotic. Many apoptotic SVZ cells stained with a marker for immature oligodendrocytes. At 3 weeks survival, the SVZ was smaller and markedly less cellular, and it contained less than 1/4 the normal complement of neural stem cells. The corresponding subcortical white matter was dysmyelinated, relatively devoid of oligodendrocytes and enriched in astrocytes. We conclude that neural stem cells and oligodendrocyte progenitors in the SVZ are vulnerable to hypoxia/ischemia. Consequently, the developmental production of oligodendrocytes is compromised and regeneration of damaged white matter oligodendrocytes does not occur resulting in failed regeneration of CNS myelin in periventricular loci. The resulting dysgenesis of the brain that occurs subsequent to perinatal hypoxic/ischemic injury may contribute to the cognitive and motor dysfunction that results from asphyxia of the newborn.

Neural Stem/Progenitor Cells Participate in the Regenerative Response to Perinatal Hypoxia/Ischemia

Perinatal hypoxia/ischemia (H/I) is the leading cause of neurologic injury resulting from birth complications. Recent advances in critical care have dramatically improved the survival rate of infants suffering this insult, but ∼50% of survivors will develop neurologic sequelae such as cerebral palsy, epilepsy or cognitive deficits. Here we demonstrate that tripotential neural stem/progenitor cells (NSPs) participate in the regenerative response to perinatal H/I as their numbers increase 100% by 3 d and that they alter their intrinsic properties to divide using expansive symmetrical cell divisions. We further show that production of new striatal neurons follows the expansion of NSPs. Increased proliferation within the NSP niche occurs at 2 d after perinatal H/I, and the proliferating cells express nestin. Of those stem-cell related genes that change, the membrane receptors Notch1, gp-130, and the epidermal growth factor receptor, as well as the downstream transcription factor Hes5, which stimulate NSP proliferation and regulate stem cellness are induced before NSP expansion. The mechanisms for the reactive expansion of the NSPs reported here reveal potential therapeutic targets that could be exploited to amplify this response, thus enabling endogenous precursors to restore a normal pattern of brain development after perinatal H/I.