TGFbeta1 stimulates the over-production of white matter astrocytes from precursors of the "brain marrow" in a rodent model of neonatal encephalopathy.
ABSTRACT: In children born prematurely and those surviving cerebral ischemia there are white matter abnormalities that correlate with neurological dysfunction. Since this injury occurs in the immature brain, when the majority of subventricular zone (SVZ) cells generate white matter oligodendrocytes, we sought to study the effect this injury has on gliogenesis from the SVZ. We hypothesized that there is aberrant glial cell generation from the SVZ after neonatal hypoxia ischemia (H/I) that contributes to an increased astrogliogenesis with concomitant oligodendroglial insufficiency. Mechanistically we hypothesized that an increase in specific locally produced cytokines during recovery from injury were modifying the differentiation of glial progenitors towards astrocytes at the expense of the more developmentally-appropriate oligodendrocytes.For these studies we used the Vannucci H/I rat model where P6 rats are subjected to unilateral common carotid ligation followed by 75 min of systemic hypoxia. Retroviral lineage tracing studies combined with morphological and immunohistochemical analyses revealed the preferential generation of SVZ-derived white matter astrocytes instead of oligodendrocytes post hypoxia/ischemia. Microarray and QRT-PCR analyses of the damaged SVZ showed increased expression of several cytokines and receptors that are known to promote astrocyte differentiation, such as EGF, LIF and TGFbeta1 signaling components. Using gliospheres to model the neonatal SVZ, we evaluated the effects of these cytokines on signal transduction pathways regulating astrocyte generation, proliferation and differentiation. These studies demonstrated that combinations of EGF, LIF and TGFbeta1 reconstituted the increased astrogliogenesis. TGFbeta1-induced Smad 2/3 phosphorylation and the combination of EGF, LIF and TGFbeta1 synergistically increased STAT3 phosphorylation over single or double cytokine combinations. Pharmacologically inhibiting ALK5 signaling in vitro antagonized the TGFbeta1-induced increase in astrocyte generation and antagonizing ALK5 signaling in vivo similarly inhibited astrogliogenesis within the SVZ during recovery from H/I.Altogether, these data indicate that there is aberrant specification of glial precursors within the neonatal SVZ during recovery from neonatal H/I that is a consequence of altered cytokine signaling. Our studies further suggest that antagonizing the ALK5 receptor will restore the normal pattern of cell differentiation after injury to the immature brain.
Project description:Neural stem/progenitor cells (NSPCs) originating from the subventricular zone (SVZ) contribute to brain repair during CNS disease. The microenvironment within the SVZ stem cell niche controls NSPC fate. However, extracellular factors within the niche that trigger astrogliogenesis over neurogenesis during CNS disease are unclear. Here, we show that blood-derived fibrinogen is enriched in the SVZ niche following distant cortical brain injury in mice. Fibrinogen inhibited neuronal differentiation in SVZ and hippocampal NSPCs while promoting astrogenesis via activation of the BMP receptor signaling pathway. Genetic and pharmacologic depletion of fibrinogen reduced astrocyte formation within the SVZ after cortical injury, reducing the contribution of SVZ-derived reactive astrocytes to lesion scar formation. We propose that fibrinogen is a regulator of NSPC-derived astrogenesis from the SVZ niche via BMP receptor signaling pathway following injury.
Project description:Astrocyte-derived ciliary neurotrophic factor (CNTF) promotes adult subventricular zone (SVZ) neurogenesis. We found that focal adhesion kinase (FAK) and JNK, but not ERK or P38, repress CNTF in vitro. Here, we defined the FAK-JNK pathway and its regulation of CNTF in mice, and the related leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), which promote stem cell renewal at the expense of neurogenesis. Intrastriatal injection of FAK inhibitor, FAK14, in adult male C57BL/6 mice reduced pJNK and increased CNTF expression in the SVZ-containing periventricular region. Injection of a JNK inhibitor increased CNTF without affecting LIF and IL-6, and increased SVZ proliferation and neuroblast formation. The JNK inhibitor had no effect in CNTF-/- mice, suggesting that JNK inhibits SVZ neurogenesis by repressing CNTF. Inducible deletion of FAK in astrocytes increased SVZ CNTF and neurogenesis, but not LIF and IL-6. Intrastriatal injection of inhibitors suggested that P38 reduces LIF and IL-6 expression, whereas ERK induces CNTF and LIF. Intrastriatal FAK inhibition increased LIF, possibly through ERK, and IL-6 through another pathway that does not involve P38. Systemic injection of FAK14 also inhibited JNK while increasing CNTF, but did not affect P38 and ERK activation, or LIF and IL-6 expression. Importantly, systemic FAK14 increased SVZ neurogenesis in wild-type C57BL/6 and CNTF+/+ mice, but not in CNTF-/- littermates, indicating that it acts by upregulating CNTF. These data show a surprising differential regulation of related cytokines and identify the FAK-JNK-CNTF pathway as a specific target in astrocytes to promote neurogenesis and possibly neuroprotection in neurological disorders.
Project description:Inflammation promotes epidermal wound healing but is considered detrimental to recovery from central nervous system injury. Sick infants have increased levels of cytokines in their cerebrospinal fluid that correlate with poor neurological outcome. In this study, we investigated the role of neuroinflammation and more specifically interleukin 6 (IL-6) in the amplification of subventricular zone (SVZ) and subgranular zone (SGZ) neural precursors after neonatal brain injury.Neonatal hypoxia/ischemia (H/I) was induced in P6 rat pups, and IL-6 was quantified with or without indomethacin administration. Neural precursor responses were evaluated by neurosphere assays as well as by stereological analyses. Studies were performed to determine how IL-6 and leukemia-inhibiting factor (LIF) affect SVZ cell expansion, proliferation, and self-renewal.Consistent with earlier studies, medially situated SVZ cells expanded after H/I. Contrary to our expectations, indomethacin significantly decreased both the initial reactive increase in these precursors and their ability to self-renew. By contrast, indomethacin increased proliferation in the SGZ and lateral SVZ. Indomethacin diminished the accumulation of microglia/macrophages and IL-6 production after H/I. In vitro IL-6 enhanced neurosphere growth, self-renewal, and tripotentiality and was more effective than LIF in promoting self-renewal. Enhanced precursor self-renewal also was obtained using prostaglandin E2, which is downstream of cyclooxygenase 2 and a target of indomethacin.These data implicate neuroinflammation and in particular IL-6 as a positive effector of primitive neural precursor expansion after neonatal brain injury. These findings have important clinical implications, as indomethacin and other anti-inflammatory agents are administered to premature infants for a variety of reasons.
Project description:Focal brain ischemia in adult rats rapidly and robustly induces neurogenesis in the subventricular zone (SVZ) but there are few and inconsistent reports in mice, presenting a hurdle to genetically investigate the endogenous neurogenic regulators such as ciliary neurotrophic factor (CNTF). Here, we first provide a platform for further studies by showing that middle cerebral artery occlusion in adult male C57BL/6 mice robustly enhances neurogenesis in the SVZ only under very specific conditions, i.e., 14days after a 30min occlusion. CNTF expression paralleled changes in the number of proliferated, BrdU-positive, SVZ cells. Stroke-induced proliferation was absent in CNTF-/- mice, suggesting that it is mediated by CNTF. MCAO-increased CNTF appears to act on C cell proliferation and by inducing FGF2 expression but not via EGF expression or Notch1 signaling of neural stem cells in the SVZ. CNTF is unique, as expression of other gp130 ligands, IL-6 and LIF, did not predict SVZ proliferation or showed no or only small compensatory increases in CNTF-/- mice. Expression of tumor necrosis factor-?, which can inhibit neurogenesis, and the presence of leukocytes in the SVZ were inversely correlated with neurogenesis, but pro-inflammatory cytokines did not affect CNTF expression in cultured astrocytes. These results suggest that slowly up-regulated CNTF in the SVZ mediates stroke-induced neurogenesis and is counteracted by inflammation. Further pharmacological stimulation of endogenous CNTF might be a good therapeutic strategy for cell replacement after stroke as CNTF regulates normal patterns of neurogenesis and is expressed almost exclusively in the nervous system.
Project description:The extent of stroke damage in patients affects the range of subsequent pathophysiological responses that influence recovery. Here we investigate the effect of lesion size on development of new blood vessels as well as inflammation and scar formation and cellular responses within the subventricular zone (SVZ) following transient focal ischemia in rats (n?=?34). Endothelin-1-induced stroke resulted in neurological deficits detected between 1 and 7 days (P<0.001), but significant recovery was observed beyond this time. MCID image analysis revealed varying degrees of damage in the ipsilateral cortex and striatum with infarct volumes ranging from 0.76-77 mm3 after 14 days, where larger infarct volumes correlated with greater functional deficits up to 7 days (r?=?0.53, P<0.05). Point counting of blood vessels within consistent sample regions revealed that increased vessel numbers correlated significantly with larger infarct volumes 14 days post-stroke in the core cortical infarct (r?=?0.81, P<0.0001), core striatal infarct (r?=?0.91, P<0.005) and surrounding border zones (r?=?0.66, P<0.005; and r?=?0.73, P<0.05). Cell proliferation within the SVZ also increased with infarct size (P<0.01) with a greater number of Nestin/GFAP positive cells observed extending towards the border zone in rats with larger infarcts. Lesion size correlated with both increased microglia and astrocyte activation, with severely diffuse astrocyte transition, the formation of the glial scar being more pronounced in rats with larger infarcts. Thus stroke severity affects cell proliferation within the SVZ in response to injury, which may ultimately make a further contribution to glial scar formation, an important factor to consider when developing treatment strategies that promote neurogenesis.
Project description:Adult neural stem cells (NSCs) are located in the subventricular zone (SVZ), a specialized brain niche located on the walls of the lateral ventricle. Under physiological conditions, NSCs generate a large number of young neurons and some oligodendrocytes, however the mechanisms controlling cell proliferation and migration are unclear. In vitro, epidermal growth factor (EGF) signaling has been shown to be an important mediator of cell proliferation and migration in the adult brain; however, the primary SVZ progenitors that respond to EGF are not well known. In this study, we isolated SVZ type-B astrocytes and cultured them under different EGF concentrations. We found a dose-dependent effect of EGF on proliferation rates and migration of SVZ type-B astrocytes. We found that GFAP+ type-B astrocytes gave rise to highly migratory and proliferating cells that expressed Olig2 and NG2. After EGF withdrawal, a significant number of EGF-stimulated cells differentiated into S100beta+/O4+ oligodendrocytes. This study provides new insights about the production of oligodendrocytes derived from the astrocyte NSCs residing in the adult SVZ. To be able to manipulate the endogenous adult progenitors, it is crucial to identify and isolate the responding primary precursors and determine the extracellular signals that regulate their cell division, migration, and fate.
Project description:Astrocytes play important roles in brain development and injury response. Transcription factors STAT3 and Smad1, activated by leukemia inhibitory factor (LIF) and bone morphogenetic protein 2 (BMP2), respectively, form a complex with the coactivator p300 to synergistically induce astrocytes from neuroepithelial cells (NECs) (K. Nakashima, M. Yanagisawa, H. Arakawa, N. Kimura, T. Hisatsune, M. Kawabata, K. Miyazono, and T. Taga, Science 284:479-482, 1999). However, the mechanisms that govern astrogliogenesis during the determination of the fate of neural stem cells remain elusive. Here we found that LIF induces expression of BMP2 via STAT3 activation and leads to the consequent activation of Smad1 to efficiently promote astrogliogenic differentiation of NECs. The BMP antagonist Noggin abrogated LIF-induced Smad1 activation and astrogliogenesis by inhibiting BMPs produced by NECs. NECs deficient in suppressor of cytokine signaling 3 (SOCS3), a negative regulator of STAT3, readily differentiated into astrocytes upon activation by LIF not only due to sustained activation of STAT3 but also because of the consequent activation of Smad1. Our study suggests a novel LIF-triggered positive regulatory loop that enhances astrogliogenesis.
Project description:Establishment and maintenance of CNS glial cell identity ensures proper brain development and function, yet the epigenetic mechanisms underlying glial fate control remain poorly understood. Here, we show that the histone deacetylase Hdac3 controls oligodendrocyte-specification gene Olig2 expression and functions as a molecular switch for oligodendrocyte and astrocyte lineage determination. Hdac3 ablation leads to a significant increase of astrocytes with a concomitant loss of oligodendrocytes. Lineage tracing indicates that the ectopic astrocytes originate from oligodendrocyte progenitors. Genome-wide occupancy analysis reveals that Hdac3 interacts with p300 to activate oligodendroglial lineage-specific genes, while suppressing astroglial differentiation genes including NFIA. Furthermore, we find that Hdac3 modulates the acetylation state of Stat3 and competes with Stat3 for p300 binding to antagonize astrogliogenesis. Thus, our data suggest that Hdac3 cooperates with p300 to prime and maintain oligodendrocyte identity while inhibiting NFIA and Stat3-mediated astrogliogenesis, and thereby regulates phenotypic commitment at the point of oligodendrocyte-astrocytic fate decision.
Project description:New neurons and oligodendrocytes are continuously produced in the subventricular zone (SVZ) of adult mammalian brains. Under normal conditions, the SVZ primary precursors (type B1 cells) generate type C cells, most of which differentiate into neurons, with a small subpopulation giving rise to oligodendrocytes. Epidermal growth factor (EGF) signaling induces dramatic proliferation and migration of SVZ progenitors, a process that could have therapeutic applications. However, the fate of cells derived from adult neural stem cells after EGF stimulation remains unknown. Here, we specifically labeled SVZ B1 cells and followed their progeny after a 7-day intraventricular infusion of EGF. Cells derived from SVZ B1 cells invaded the parenchyma around the SVZ into the striatum, septum, corpus callosum, and fimbria-fornix. Most of these B1-derived cells gave rise to cells in the oligodendrocyte lineage, including local NG2+ progenitors, and pre-myelinating and myelinating oligodendrocytes. SVZ B1 cells also gave rise to a population of highly-branched S100beta+/glial fibrillary acidic protein (GFAP)+ cells in the striatum and septum, but no neuronal differentiation was observed. Interestingly, when demyelination was induced in the corpus callosum by a local injection of lysolecithin, an increased number of cells derived from SVZ B1 cells and stimulated to migrate and proliferate by EGF infusion differentiated into oligodendrocytes at the lesion site. This work indicates that EGF infusion can greatly expand the number of progenitors derived from the SVZ primary progenitors which migrate and differentiate into oligodendroglial cells. This expanded population could be used for the repair of white matter lesions.
Project description:OBJECTIVES:To testify that endothelial cells (ECs) induce astrocyte maturation by leukaemia inhibitory factor (LIF) secretion. MATERIALS AND METHODS:In vivo experiments, mice bearing floxed alleles of YAP were crossed with mice expressing a Cre recombinase driven by the endothelial Tek promoter (Tek-Cre) to finally obtain the following three genotypes: YAPf/f , Tek-Cre; YAPf/w , Tek-Cre; and YAPf/f . Retinal vascularization and astrocyte network were evaluated by whole-mount fluorescence and Western blotting. In vitro, experiments were performed in an astrocyte and human microvascular endothelial cell (HMEC-1) coculture model to analyse the mechanisms underlying the effect of endothelial YAP on astrocytes. RESULTS:In vivo, YAPf/f ;Tek-Cre mice showed delayed angiogenesis, sparse vessels and decreased glial fibrillary acidic protein (GFAP)+ astrocytes but aberrant growth of endothelial networks and immature astrocytes (platelet-derived growth factor A, PDGFRA+ astrocytes) overgrowth. In vitro, Yap deletion attenuated the LIF release that delayed the maturation of retinal astrocyte which was consistent with the results of HMEC-1-astrocyte coculture. The effect of YAP overexpression on LIF-LIFR axis in HMEC-1 interferes the GFAP expression of astrocyte. In contrast, LIF protein rescues the astrocytic GFAP expression when EC YAP was inhibited by siRNAs. CONCLUSIONS:We show that EC yes-associated protein (YAP) is not only a critical coactivator of Hippo signalling in retinal vessel development but also plays an essential role in retinal astrocyte maturation by regulating LIF production.