TGF? signaling in the brain increases with aging and signals to astrocytes and innate immune cells in the weeks after stroke.
ABSTRACT: TGF? is both neuroprotective and a key immune system modulator and is likely to be an important target for future stroke therapy. The precise function of increased TGF-?1 after stroke is unknown and its pleiotropic nature means that it may convey a neuroprotective signal, orchestrate glial scarring or function as an important immune system regulator. We therefore investigated the time course and cell-specificity of TGF? signaling after stroke, and whether its signaling pattern is altered by gender and aging.We performed distal middle cerebral artery occlusion strokes on 5 and 18 month old TGF? reporter mice to get a readout of TGF? responses after stroke in real time. To determine which cell type is the source of increased TGF? production after stroke, brain sections were stained with an anti-TGF? antibody, colocalized with markers for reactive astrocytes, neurons, and activated microglia. To determine which cells are responding to TGF? after stroke, brain sections were double-labelled with anti-pSmad2, a marker of TGF? signaling, and markers of neurons, oligodendrocytes, endothelial cells, astrocytes and microglia.TGF? signaling increased 2 fold after stroke, beginning on day 1 and peaking on day 7. This pattern of increase was preserved in old animals and absolute TGF? signaling in the brain increased with age. Activated microglia and macrophages were the predominant source of increased TGF? after stroke and astrocytes and activated microglia and macrophages demonstrated dramatic upregulation of TGF? signaling after stroke. TGF? signaling in neurons and oligodendrocytes did not undergo marked changes.We found that TGF? signaling increases with age and that astrocytes and activated microglia and macrophages are the main cell types that undergo increased TGF? signaling in response to post-stroke increases in TGF?. Therefore increased TGF? after stroke likely regulates glial scar formation and the immune response to stroke.
Project description:Astrocytes limit inflammation after CNS injury, at least partially by physically containing it within an astrocytic scar at the injury border. We report here that astrocytic transforming growth factor-beta (TGF?) signaling is a second, distinct mechanism that astrocytes utilize to limit neuroinflammation. TGF?s are anti-inflammatory and neuroprotective cytokines that are upregulated subacutely after stroke, during a clinically accessible time window. We have previously demonstrated that TGF?s signal to astrocytes, neurons and microglia in the stroke border days after stroke. To investigate whether TGF? affects astrocyte immunoregulatory functions, we engineered "Ast-Tbr2DN" mice where TGF? signaling is inhibited specifically in astrocytes. Despite having a similar infarct size to wildtype controls, Ast-Tbr2DN mice exhibited significantly more neuroinflammation during the subacute period after distal middle cerebral occlusion (dMCAO) stroke. The peri-infarct cortex of Ast-Tbr2DN mice contained over 60% more activated CD11b(+) monocytic cells and twice as much immunostaining for the activated microglia and macrophage marker CD68 than controls. Astrocytic scarring was not altered in Ast-Tbr2DN mice. However, Ast-Tbr2DN mice were unable to upregulate TGF-?1 and its activator thrombospondin-1 2 days after dMCAO. As a result, the normal upregulation of peri-infarct TGF? signaling was blunted in Ast-Tbr2DN mice. In this setting of lower TGF? signaling and excessive neuroinflammation, we observed worse motor outcomes and late infarct expansion after photothrombotic motor cortex stroke. Taken together, these data demonstrate that TGF? signaling is a molecular mechanism by which astrocytes limit neuroinflammation, activate TGF? in the peri-infarct cortex and preserve brain function during the subacute period after stroke.
Project description:Ischemic stroke, which accounts for 75-80% of all strokes, is the predominant cause of morbidity and mortality worldwide. The post-stroke immune response has recently emerged as a new breakthrough target in the treatment strategy for ischemic stroke. Glial cells, including microglia, astrocytes, and oligodendrocytes, are the primary components of the peri-infarct environment in the central nervous system (CNS) and have been implicated in post-stroke immune regulation. However, increasing evidence suggests that glial cells exert beneficial and detrimental effects during ischemic stroke. Microglia, which survey CNS homeostasis and regulate innate immune responses, are rapidly activated after ischemic stroke. Activated microglia release inflammatory cytokines that induce neuronal tissue injury. By contrast, anti-inflammatory cytokines and neurotrophic factors secreted by alternatively activated microglia are beneficial for recovery after ischemic stroke. Astrocyte activation and reactive gliosis in ischemic stroke contribute to limiting brain injury and re-establishing CNS homeostasis. However, glial scarring hinders neuronal reconnection and extension. Neuroinflammation affects the demyelination and remyelination of oligodendrocytes. Myelin-associated antigens released from oligodendrocytes activate peripheral T cells, thereby resulting in the autoimmune response. Oligodendrocyte precursor cells, which can differentiate into oligodendrocytes, follow an ischemic stroke and may result in functional recovery. Herein, we discuss the mechanisms of post-stroke immune regulation mediated by glial cells and the interaction between glial cells and neurons. In addition, we describe the potential roles of various glial cells at different stages of ischemic stroke and discuss future intervention targets.
Project description:Transforming growth factor ? (TGF-?) has been reported to play important roles in neurogenesis and angiogenesis in the injured brain. The present study characterizes a novel role for TGF? in oligodendrocyte lineage cell survival and white matter integrity after ischemic stroke. Three days after transient (60?min) middle cerebral artery occlusion (tMCAO), TGF? expression was significantly increased in microglia/macrophages and neurons. Expression of the receptor of TGF?-epidermal growth factor receptor (EGFR)-was increased in glial cells after ischemia, including in oligodendrocyte lineage cells. TGF? knockout enlarged brain infarct volumes and exacerbated cell death in oligodendrocyte precursor cells (OPCs) and oligodendrocytes three days after tMCAO. TGF?-deficient mice displayed long-term exacerbation of sensorimotor deficits after tMCAO, and these functional impairments were accompanied by loss of white matter integrity and impaired oligodendrocyte replacement. In vitro studies confirmed that 5 or 10?ng/mL TGF? directly protected OPCs and oligodendrocytes against oxygen and glucose deprivation (OGD)-induced cell death, but exerted no effects on OPC differentiation. Further studies identified STAT3 as a key transcription factor mediating the effects of TGF? on OPCs and oligodendrocytes. In conclusion, TGF? provides potent oligodendrocyte protection against cerebral ischemia, thereby maintaining white matter integrity and improving neurological recovery after stroke.
Project description:We explored the hypothesis that injured neurons release lipocalin-2 as a help me signal.In vivo lipocalin-2 responses were assessed in rat focal cerebral ischemia and human stroke brain samples using a combination of ELISA and immunostaining. In vitro, microglia and astrocytes were exposed to lipocalin-2, and various markers and assays of glial activation were quantified. Functional relevance of neuron-to-glia lipocalin-2 signaling was examined by transferring conditioned media from lipocalin-2-activated microglia and astrocytes onto neurons to see whether activated glia could protect neurons against oxygen-glucose deprivation and promote neuroplasticity.In human stroke samples and rat cerebral ischemia, neuronal expression of lipocalin-2 was significantly increased. In primary cell cultures, exposing microglia and astrocytes to lipocalin-2 resulted in glial activation. In microglia, lipocalin-2 converted resting ramified shapes into a long-rod morphology with reduced branching, increased interleukin-10 release, and enhanced phagocytosis. In astrocytes, lipocalin-2 upregulated glial fibrillary acid protein, brain-derived neurotropic factor, and thrombospondin-1. Conditioned media from lipocalin-2-treated astrocytes upregulated synaptotagmin, and conditioned media from lipocalin-2-treated microglia upregulated synaptophysin and post-synaptic density 95 (PSD95) and protected neurons against oxygen-glucose deprivation.These findings provide proof of concept that lipocalin-2 is released by injured neurons as a help me distress signal that activates microglia and astrocytes into potentially prorecovery phenotypes.
Project description:Microglia are myeloid cells of the CNS that participate both in normal CNS function and in disease. We investigated the molecular signature of microglia and identified 239 genes and 8 microRNAs that were uniquely or highly expressed in microglia versus myeloid and other immune cells. Of the 239 genes, 106 were enriched in microglia as compared with astrocytes, oligodendrocytes and neurons. This microglia signature was not observed in microglial lines or in monocytes recruited to the CNS, and was also observed in human microglia. We found that TGF-? was required for the in vitro development of microglia that express the microglial molecular signature characteristic of adult microglia and that microglia were absent in the CNS of TGF-?1-deficient mice. Our results identify a unique microglial signature that is dependent on TGF-? signaling and provide insights into microglial biology and the possibility of targeting microglia for the treatment of CNS disease.
Project description:Reactive astrocytes are strongly induced by central nervous system (CNS) injury and disease, but their role is poorly understood. Here we show that a subtype of reactive astrocytes, which we termed A1, is induced by classically activated neuroinflammatory microglia. We show that activated microglia induce A1 astrocytes by secreting Il-1?, TNF and C1q, and that these cytokines together are necessary and sufficient to induce A1 astrocytes. A1 astrocytes lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis, and induce the death of neurons and oligodendrocytes. Death of axotomized CNS neurons in vivo is prevented when the formation of A1 astrocytes is blocked. Finally, we show that A1 astrocytes are abundant in various human neurodegenerative diseases including Alzheimer's, Huntington's and Parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis. Taken together these findings help to explain why CNS neurons die after axotomy, strongly suggest that A1 astrocytes contribute to the death of neurons and oligodendrocytes in neurodegenerative disorders, and provide opportunities for the development of new treatments for these diseases.
Project description:In traumatic brain injury, absent in melanoma 2 (AIM2) has been demonstrated to be involved in pyroptotic neuronal cell death. Although the pathophysiological mechanism of spinal cord injury is similar to that of brain injury, the expression and cellular localization of AIM2 after spinal cord injury is still not very clear. In the present study, we used a rat model of T9 spinal cord contusive injury, produced using the weight drop method. The rats were randomly divided into 1-hour, 6-hour, 1-day, 3-day and 6-day (post-injury time points) groups. Sham-operated rats only received laminectomy at T9 without contusive injury. Western blot assay revealed that the expression levels of AIM2 were not significantly different among the 1-hour, 6-hour and 1-day groups. The expression levels of AIM2 were markedly higher in the 1-hour, 6-hour and 1-day groups compared with the sham, 3-day and 7-day groups. Double immunofluorescence staining demonstrated that AIM2 was expressed by NeuN+ (neurons), GFAP+ (astrocytes), CNPase+ (oligodendrocytes) and CD11b+ (microglia) cells in the sham-operated spinal cord. In rats with spinal cord injury, AIM2 was also found in CD45+ (leukocytes) and CD68+ (activated microglia/macrophages) cells in the spinal cord at all time points. These findings indicate that AIM2 is mainly expressed in neurons, astrocytes, microglia and oligodendrocytes in the normal spinal cord, and that after spinal cord injury, its expression increases because of the infiltration of leukocytes and the activation of astrocytes and microglia/macrophages.
Project description:Interleukin-17 (IL-17) secreted by T helper 17 (Th17) cells is essential in the development of experimental autoimmune encephalomyelitis (EAE). However, it remains unclear how IL-17-mediated signaling in different cellular compartments participates in the central nervous system (CNS) inflammatory process. We examined CNS inflammation in mice with specific deletion of Act1, a critical component required for IL-17 signaling, in endothelial cells, macrophages and microglia, and neuroectoderm (neurons, astrocytes, and oligodendrocytes). In Act1-deficient mice, Th17 cells showed normal infiltration into the CNS but failed to recruit lymphocytes, neutrophils, and macrophages. Act1 deficiency in endothelial cells or in macrophages and microglia did not substantially impact the development of EAE. However, targeted Act1 deficiency in neuroectoderm-derived CNS-resident cells resulted in markedly reduced severity in EAE. Specifically, Act1-deficient astrocytes showed impaired IL-17-mediated inflammatory gene induction. Thus, astroctyes are critical in IL-17-Act1-mediated leukocyte recruitment during autoimmune-induced inflammation of the CNS.
Project description:Erythropoietin (EPO) promotes oligodendrogenesis and attenuates white matter injury in neonatal rats. However, it is unknown whether this effect extends to adult mice and whether EPO regulate microglia polarization after ischemic stroke. Male adult C57BL/6 mice (25-30g) were subjected to 45 min of middle cerebral artery occlusion (MCAO). EPO (5000 IU/kg) or saline was injected intraperitoneally every other day after reperfusion. Neurological function was evaluated using the rotarod test at 1, 3, 7 and 14 days after MCAO. Brain tissue loss volume was determined by hematoxylin-eosin staining. Immunofluorescence staining and Western blot were also used to assess the severity of white matter injury and phenotypic changes in microglia/macrophages. Bromodeoxyuridine (BrdU) was injected intraperitoneally daily for 1 week to analyze the number of newly proliferating glia cells (oligodendrocytes, microglia, and astrocytes). We found that EPO significantly reduced Brain tissue loss volume, ameliorated white matter injury, and improved neurobehavioral outcomes at 14 days after MCAO (P<0.05). In addition, EPO also increased the number of newly generated oligodendrocytes and attenuated the rapid hypertrophy and hyperplasia of microglia and astrocytes after ischemic stroke (P<0.05). Furthermore, EPO reduced M1 microglia and increased M2 microglia (P<0.05). Taken together, our results suggest that EPO treatment improves white matter integrity after cerebral ischemia, which could be attributed to EPO attenuating gliosis and facilitating the microglial polarization toward the beneficial M2 phenotype to promote oligodendrogenesis.
Project description:Pathological findings in neonatal brain injury associated with preterm birth include focal and/or diffuse white matter injury (WMI). Despite the heterogeneous nature of this condition, reactive astrogliosis and microgliosis are frequently observed. Thus, molecular mechanisms by which glia activation contribute to WMI were investigated.Postmortem brains of neonatal brain injury were investigated to identify molecular features of reactive astrocytes. The contribution of astrogliosis to WMI was further tested in a mouse model in genetically engineered mice.Activated STAT3 signaling in reactive astrocytes was found to be a common feature in postmortem brains of neonatal brain injury. In a mouse model of neonatal WMI, conditional deletion of STAT3 in astrocytes resulted in exacerbated WMI, which was associated with delayed maturation of oligodendrocytes. Mechanistically, the delay occurred in association with overexpression of transforming growth factor (TGF)?-1 in microglia, which in healthy controls decreased with myelin maturation in an age-dependent manner. TGF?-1 directly and dose-dependently inhibited the maturation of purified oligodendrocyte progenitors, and pharmacological inhibition of TGF?-1 signaling in vivo reversed the delay in myelin development. Factors secreted from STAT3-deficient astrocytes promoted elevated TGF?-1 production in cultured microglia compared to wild-type astrocytes.These results suggest that myelin development is regulated by a mechanism involving crosstalk between microglia and oligodendrocyte progenitors. Reactive astrocytes may modify this signaling in a STAT3-dependent manner, preventing the pathological expression of TGF?-1 in microglia and the impairment of oligodendrocyte maturation.