Kanako Itoh

Astrocytes Promote Oligodendrogenesis after White Matter Damage via Brain-Derived Neurotrophic Factor

Oligodendrocyte precursor cells (OPCs) in the adult brain contribute to white matter homeostasis. After white matter damage, OPCs compensate for oligodendrocyte loss by differentiating into mature oligodendrocytes. However, the underlying mechanisms remain to be fully defined. Here, we test the hypothesis that, during endogenous recovery from white matter ischemic injury, astrocytes support the maturation of OPCs by secreting brain-derived neurotrophic factor (BDNF). For in vitro experiments, cultured primary OPCs and astrocytes were prepared from postnatal day 2 rat cortex. When OPCs were subjected to chemical hypoxic stress by exposing them to sublethal CoCl2 for 7 d, in vitro OPC differentiation into oligodendrocytes was significantly suppressed. Conditioned medium from astrocytes (astro-medium) restored the process of OPC maturation even under the stressed conditions. When astro-medium was filtered with TrkB-Fc to remove BDNF, the BDNF-deficient astro-medium no longer supported OPC maturation. For in vivo experiments, we analyzed a transgenic mouse line (GFAPcre/BDNFwt/fl) in which BDNF expression is downregulated specifically in GFAP+ astrocytes. Both wild-type (GFAPwt/BDNFwt/fl mice) and transgenic mice were subjected to prolonged cerebral hypoperfusion by bilateral common carotid artery stenosis. As expected, compared with wild-type mice, the transgenic mice exhibited a lower number of newly generated oligodendrocytes and larger white matter damage. Together, these findings demonstrate that, during endogenous recovery from white matter damage, astrocytes may promote oligodendrogenesis by secreting BDNF. SIGNIFICANCE STATEMENT The repair of white matter after brain injury and neurodegeneration remains a tremendous hurdle for a wide spectrum of CNS disorders. One potentially important opportunity may reside in the response of residual oligodendrocyte precursor cells (OPCs). OPCs may serve as a back-up for generating mature oligodendrocytes in damaged white matter. However, the underlying mechanisms are still mostly unknown. Here, we use a combination of cell biology and an animal model to report a new pathway in which astrocyte-derived BDNF supports oligodendrogenesis and regeneration after white matter damage. These findings provide new mechanistic insight into white matter physiology and pathophysiology, which would be broadly and clinically applicable to CNS disease.

Mechanisms of cell-cell interaction in oligodendrogenesis and remyelination after stroke.

White matter damage is a clinically important aspect of several central nervous system diseases, including stroke. Cerebral white matter primarily consists of axonal bundles ensheathed with myelin secreted by mature oligodendrocytes, which play an important role in neurotransmission between different areas of gray matter. During the acute phase of stroke, damage to oligodendrocytes leads to white matter dysfunction through the loss of myelin. On the contrary, during the chronic phase, white matter components promote an environment, which is favorable for neural repair, vascular remodeling, and remyelination. For effective remyelination to take place, oligodendrocyte precursor cells (OPCs) play critical roles by proliferating and differentiating into mature oligodendrocytes, which help to decrease the burden of axonal injury. Notably, other types of cells contribute to these OPC responses under the ischemic conditions. This mini-review summarizes the non-cell autonomous mechanisms in oligodendrogenesis and remyelination after white matter damage, focusing on how OPCs receive support from their neighboring cells. This article is part of a Special Issue entitled SI: Cell Interactions In Stroke.

Astrocyte-Derived Pentraxin 3 Supports Blood–Brain Barrier Integrity Under Acute Phase of Stroke

Background and Purpose— Pentraxin 3 (PTX3) is released on inflammatory responses in many organs. However, roles of PTX3 in brain are still mostly unknown. Here we asked whether and how PTX3 contributes to blood–brain barrier dysfunction during the acute phase of ischemic stroke. Methods— In vivo, spontaneously hypertensive rats were subjected to focal cerebral ischemia by transient middle cerebral artery occlusion. At day 3, brains were analyzed to evaluate the cellular origin of PTX3 expression. Correlations with blood–brain barrier breakdown were assessed by IgG staining. In vitro, rat primary astrocytes and rat brain endothelial RBE.4 cells were cultured to study the role of astrocyte-derived PTX3 on vascular endothelial growth factor–mediated endothelial permeability. Results— During the acute phase of stroke, reactive astrocytes in the peri-infarct area expressed PTX3. There was negative correlation between gradients of IgG leakage and PTX3-positive astrocytes. Cell culture experiments showed that astrocyte-conditioned media increased levels of tight junction proteins and reduced endothelial permeability under normal conditions. Removing PTX3 from astrocyte-conditioned media by immunoprecipitation increased endothelial permeability. PTX3 strongly bound vascular endothelial growth factor in vitro and was able to decrease vascular endothelial growth factor–induced endothelial permeability. Conclusions— Astrocytes in peri-infarct areas upregulate PTX3, which may support blood–brain barrier integrity by regulating vascular endothelial growth factor–related mechanisms. This response in astrocytes may comprise a compensatory mechanism for maintaining blood–brain barrier function after ischemic stroke.

p38 MAP kinase mediates transforming-growth factor-β1-induced upregulation of matrix metalloproteinase-9 but not -2 in human brain pericytes

Pericytes are vascular mural cells embedded within the basal lamina of blood micro-vessels. Within the neurovascular unit, pericytes play important roles in regulating neurovascular homeostasis by secreting soluble factors, such as matrix metalloproteinases (MMPs). However, little is known about the regulatory signaling pathways in brain pericytes. Here we show that transforming growth factor-β1 (TGF-β1) induces MMP-9 upregulation in pericytes via p38 mitogen-activated protein (MAP) kinase signaling. Cultured human brain vascular pericytes were used in this study. When the brain pericytes were treated with purified human TGF-β1 (0.1-10ng/mL for 24h), the levels of MMP-2 and MMP-9 in culture media were significantly increased in a concentration dependent manner as measured by gelatin zymography. WST assay confirmed that TGF-β1 did not affect cell survival of the brain pericytes. A TGF-β-receptor inhibitor SB431542 (0.5-5μM) decreased the TGF-β1-induced upregulation of MMP-2 and MMP-9. To assess the underlying intracellular mechanisms, we focused on p38 MAP kinase signaling, which is one of the major downstream kinases for TGF-β1. A well-validated p38 MAP kinase inhibitor SB203580 (0.5-5μM) cancelled the effect of TGF-β1 in upregulation of MMP-9 but not MMP-2. Western blotting confirmed that TGF-β1 treatment increased the level of p38 MAP kinase phosphorylation in pericytes, and again, the TGF-β-receptor inhibitor SB431542 (0.5-5μM) blocked the TGF-β1-induced phosphorylation of p38 MAP kinase. Both TGF-β1 and MMP-9 are major neurovascular mediators, and therefore, our current finding may suggest a novel mechanism for how pericytes regulate neurovascular homeostasis.

Magnesium sulfate protects oligodendrocyte lineage cells in a rat cell-culture model of hypoxic-ischemic injury.

Hypoxic-ischemic (HI) brain injury in newborns results in serious damage. Magnesium sulfate has been clinically used as a cyto-protective agent against HI brain injury in newborns in some countries, including Japan. However, it is not clear how magnesium exerts this effect and how it acts on the individual types of cells within the newborn brain. In this study, we exposed cultured rat oligodendrocyte precursor cells to magnesium sulfate during the period when they differentiate into oligodendrocytes, and showed that magnesium-exposed oligodendrocytes exhibited more resistance to HI injury. Our data may support the use of magnesium sulfate in the clinical setting.

Effects of Aging on Neural Stem/Progenitor Cells and Oligodendrocyte Precursor Cells After Focal Cerebral Ischemia in Spontaneously Hypertensive Rats.

Aging and vascular comorbidities such as hypertension comprise critical cofactors that influence how the brain responds to stroke. Ischemic stress induces neurogenesis and oligodendrogenesis in younger brains. However, it remains unclear whether these compensatory mechanisms can be maintained even under pathologically hypertensive and aged states. To clarify the age-related remodeling capacity after stroke under hypertensive conditions, we assessed infarct volume, behavioral outcomes, and surrogate markers of neurogenesis and oligodendrogenesis in acute and subacute phases after transient focal cerebral ischemia in 3- and 12-month-old spontaneously hypertensive rats (SHRs). Hematoxylin and eosin staining showed that 3- and 12-month-old SHRs exhibited similar infarction volumes at both 3 and 14 days after focal cerebral ischemia. However, recovery of behavioral deficits (neurological score assessment and adhesive removal test) was significantly less in 12-month-old SHRs compared to 3-month-old SHRs. Concomitantly, numbers of nestin+ neural stem/progenitor cells (NSPCs) near the infarct border area or subventricular zone in 12-month-old SHRs were lower than 3-month-old SHRs at day 3. Similarly, numbers of PDGFR-α+ oligodendrocyte precursor cells (OPCs) in the corpus callosum were lower in 12-month-old SHRs at day 3. Lower levels of NSPC and OPC numbers were accompanied by lower expression levels of phosphorylated CREB. By day 14 postischemia, NSPC and OPC numbers in 12-month-old SHRs recovered to similar levels as in 3-month-old SHRs, but the numbers of proliferating NSPCs (Ki-67+nestin+ cells) and proliferating OPCs (Ki-67+PDGFR-α+ cells) remained lower in the older brains even at day 14. Taken together, these findings suggest that aging may also decrease poststroke compensatory responses for neurogenesis and oligodendrogenesis even under hypertensive conditions.

Neuregulin1-β decreases interleukin-1β-induced RhoA activation, myosin light chain phosphorylation, and endothelial hyperpermeability.

Neuregulin‐1 (NRG1) is an endogenous growth factor with multiple functions in the embryonic and postnatal brain. The NRG1 gene is large and complex, transcribing more than twenty transmembrane proteins and generating a large number of isoforms in tissue and cell type‐specific patterns. Within the brain, NRG1 functions have been studied most extensively in neurons and glia, as well as in the peripheral vasculature. Recently, NRG1 signaling has been found to be important in the function of brain microvascular endothelial cells, decreasing IL‐1β‐induced increases in endothelial permeability. In the current experiments, we have investigated the pathways through which the NRG1‐β isoform acts on IL‐1β‐induced endothelial permeability. Our data show that NRG1‐β increases barrier function, measured by transendothelial electrical resistance, and decreases IL‐1β‐induced hyperpermeability, measured by dextran‐40 extravasation through a monolayer of brain microvascular endothelial cells plated on transwells. An investigation of key signaling proteins suggests that the effect of NRG1‐β on endothelial permeability is mediated through RhoA activation and myosin light chain phosphorylation, events which affect filamentous actin morphology. In addition, AG825, an inhibitor of the erbB2‐associated tyrosine kinase, reduces the effect of NRG1‐β on IL‐1β‐induced RhoA activation and myosin light chain phosphorylation. These data add to the evidence that NRG1‐β signaling affects changes in the brain microvasculature in the setting of neuroinflammation.

A‐Kinase Anchor Protein 12 Is Required for Oligodendrocyte Differentiation in Adult White Matter

Oligodendrocyte precursor cells (OPCs) give rise to oligodendrocytes in cerebral white matter. However, the underlying mechanisms that regulate this process remain to be fully defined, especially in adult brains. Recently, it has been suggested that signaling via A‐kinase anchor protein 12 (AKAP12), a scaffolding protein that associates with intracellular molecules such as protein kinase A, may be involved in Schwann cell homeostasis and peripheral myelination. Here, we asked whether AKAP12 also regulates the mechanisms of myelination in the CNS. AKAP12 knockout mice were compared against wild‐type (WT) mice in a series of neurochemical and behavioral assays. Compared with WTs, 2‐months old AKAP12 knockout mice exhibited loss of myelin in white matter of the corpus callosum, along with perturbations in working memory as measured by a standard Y‐maze test. Unexpectedly, very few OPCs expressed AKAP12 in the corpus callosum region. Instead, pericytes appeared to be one of the major AKAP12‐expressing cells. In a cell culture model system, conditioned culture media from normal pericytes promoted in‐vitro OPC maturation. However, conditioned media from AKAP12‐deficient pericytes did not support the OPC function. These findings suggest that AKAP12 signaling in pericytes may be required for OPC‐to‐oligodendrocyte renewal to maintain the white matter homeostasis in adult brain. Stem Cells 2018;36:751–760

A Novel Three-Dimensional Culture System for Oligodendrocyte Precursor Cells

Oligodendrocytes are generated from oligodendrocyte precursor cells (OPCs). Mechanisms of OPC differentiation have been extensively examined with two-dimensional cell culture systems. However, these cellular events may be more accurately represented using a three-dimensional (3D) model. In this study, we report the development of a novel 3D OPC culture system using gels composed of a mixture of collagen and hyaluronan, wherein cultured rat primary OPCs can proliferate and differentiate into oligodendrocytes. Our data show that the gel concentration and cell-seeding density are critical factors for the numbers of OPCs and oligodendrocytes in our 3D culture system. In addition, Notch signaling, which supports cell-to-cell communication, may also be important for OPC function in our system because a Notch inhibitor DAPT suppressed OPC proliferation and differentiation. Taken together, cultured rat OPCs can grow in collagen-/hyaluronan-based gels, and our novel 3D OPC culture system may offer a useful platform for examining the mechanisms of OPC function in vitro.

Crosstalk Between Cerebral Endothelium and Oligodendrocyte After Stroke

It is now well-accepted that the cerebrovascular system does not merely provide inert pipes for blood delivery to the brain. Cerebral endothelial cells may compose an embedded bunker of trophic factors that contribute to brain homeostasis and function. Recent findings suggest that soluble factors from cerebral endothelial cells nourish neighboring cells, such as neurons and astrocytes. Although data are strongest in supporting mechanisms of endothelium–neuron and/or endothelium–astrocyte trophic coupling, it is likely that similar interactions also exist between cerebral endothelial cells and oligodendrocyte lineage cells. In this chapter, we summarize current advances in the field of endothelium–oligodendrocyte trophic coupling. These endothelium–oligodendrocyte interactions comprise the oligovascular niche and sustain ongoing angiogenesis/oligodendrogenesis. Importantly, these cell–cell interactions are not static—the trophic coupling is disturbed during the acute phase after brain injury, with the potential to recover in the chronic phase to promote brain remodeling and repairing. Oligodendrocyte lineage cells play critical roles in white matter function. Therefore, a deeper understanding of the mechanisms of endothelium–oligodendrocyte trophic coupling may lead to new therapeutic approaches for diseases related to white matter pathology, such as stroke or vascular dementia.