NeuroD1 administration ameliorated neuroinflammation and boosted neurogenesis in a mouse model of subarachnoid hemorrhage.

BACKGROUND: Subarachnoid hemorrhage (SAH) causes significant long-term neurocognitive dysfunction, which is associated with hippocampal neuroinflammation. Growing evidences have shown that astrocytes played a significant role in mediating neuroinflammation. Recently, in vivo reprogramming of astrocytes to neurons by NeuroD1 or PTBP1 administration has generated a lot of interests and controversies. While the debates centered on the source of neurogenesis, no attention has been paid to the changes of the astrocytes-mediated neuroinflammation and its impact on endogenous neurogenesis after NeuroD1 administration. METHODS: 80 adult male C57BL/6 mice were used in this study. SAH was established by pre-chiasmatic injection of 100 µl blood. AAV-NeuroD1-GFP virus was injected to the hippocampus 3 day post-SAH. Neurocognitive function, brain water content, in vivo electrophysiology, Golgi staining, western blot and immunofluorescent staining were assessed at day 14 post-virus injection. RESULTS: NeuroD1 administration markedly attenuated reactive astrocytes-mediated neuroinflammation by reversing neurotoxic A1 astrocytes transformation, decreasing the secretion of neuroinflammatory cytokines, and reducing the activation of harmful microglia. NeuroD1 treatment significantly reversed the brain-blood barrier impairment and promoted the release of neurotrophic factors pleiotrophin (PTN), all of which contributed to the improvement of cellular microenvironment and made it more suitable for neurogenesis. Interestingly, besides neurogenesis in the hippocampus from cells transfected with NeuroD1 at the early phase of SAH, NeuroD1 administration significantly boosted the endogenous neurogenesis at the late phase of SAH, which likely benefited from the improvement of the neuroinflammatory microenvironment. Functionally, NeuroD1 treatment significantly alleviated neurocognitive dysfunction impaired by SAH. CONCLUSIONS: NeuroD1 significantly promoted neurofunctional recovery by attenuating reactive astrocytes-mediated neuroinflammation and boosting neurogenesis decimated by SAH. Specifically, NeuroD1 efficiently converted transfected cells, most likely astrocytes, to neurons at the early phase of SAH, suppressed astrocytes-mediated neuroinflammation and boosted endogenous neurogenesis at the late phase of SAH.

Astrocytic connexin 43 deletion ameliorates SNI-induced neuropathic pain by reducing microglia activation.

Neuropathic pain (NP) is a chronic disease caused by damage to the peripheral or central nervous system. Connexin 43 (Cx43), the primary connexin expressed by astrocytes, has been reported to be significantly increased in NP. However, the roles and mechanisms of Cx43 in the development and maintenance of NP remain largely unknown, while microglia activation has been commonly regarded as a key factor of NP. In the present study, we found that Cx43 deletion significantly ameliorated spared nerve injury (SNI)-induced NP and suppressed SNI induced c-Fos expression in the spinal cord. Notably, Cx43 deletion led to much less SNI-induced microglia activation in the spinal cord. These results suggest that astrocyte Cx43 may play a significant role in regulating microglial activation and NP.

Bexarotene enhances astrocyte phagocytosis via ABCA1-mediated pathways in a mouse model of subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Enhancing phagocytosis can facilitate the removal of inflammatory molecules, limit the toxicity of dead cells and debris, and promote recovery after brain injury. In this study, we aimed to explore the role of bexarotene (Bex), a retinoid X receptor (RXR) agonist, in promoting astrocyte phagocytosis and neurobehavioral recovery after subarachnoid hemorrhage (SAH). METHODS: Mice SAH model was induced by pre-chiasmatic injection of blood. Modified Garcia score, novel object recognition, rotarod test, and Morris water maze were performed to assess neurological function. Immunofluorescence and electron microscopy were used to evaluate astrocyte phagocytosis in vivo. In addition， ABCA1/MEGF10&GULP1, the primary astrocyte phagocytosis pathway, were stimulated by Bex or suppressed by HX531 (a RXR antagonist) to evaluate their impacts on astrocyte phagocytosis and neurological recovery. RESULTS: Astrocytes phagocytosis of blood components were observed in mice after SAH induction, which is further increased by Bex treatment. Bex dramatically attenuated neuroinflammation, reduced brain edema, improved early neurological performance and promoted neurocognitive recovery. Meanwhile, Bex decreased neurotoxic reactive astrocytes and preserved neurogenesis after SAH. Bex increased the expression of astrocyte phagocytosis-related proteins ABCA1, MEGF10, and GULP1. Bex also increased the lysosomal processing of engulfed blood components in astrocytes. Moreover, Bex significantly promoted astrocytes to phagocytize debris in vitro by increasing the expression of ABCA1, MEGF10 and GULP1, while HX531 inhibited astrocyte phagocytosis and decreased these protein levels. CONCLUSIONS: Bex enhanced astrocyte phagocytosis through the ABCA1-mediated pathways, and promoted neurobehavior recovery in mice after SAH induction.

Nestin+/CD31+ cells in the hypoxic perivascular niche regulate glioblastoma chemoresistance by upregulating JAG1 and DLL4.

BACKGROUND: Failure of glioblastoma (GBM) therapy is often ascribed to different types of glioblastoma stem-like cell (GSLC) niche; in particular, a hypoxic perivascular niche (HPVN) is involved in GBM progression. However, the cells responsible for HPVNs remain unclear. METHODS: Immunostaining was performed to determine the cells involved in HPVNs. A hypoxic chamber and 3-dimensional (3D) microfluidic chips were designed to simulate a HPVN based on the pathological features of GBM. The phenotype of GSLCs was evaluated by fluorescence scanning in real time and proliferation and apoptotic assays. The expression of JAG1, DLL4, and Hes1 was determined by immunostaining, ELISA, Western blotting, and quantitative PCR. Their clinical prognostic significance in GBM HPVNs and total tumor tissues were verified by clinical data and The Cancer Genome Atlas databases. RESULTS: Nestin+/CD31+ cells and pericytes constitute the major part of microvessels in the HPVN, and the high ratio of nestin+/CD31+ cells rather than pericytes are responsible for the poor prognosis of GBM. A more real HPVN was simulated by a hypoxic coculture system in vitro, which consisted of 3D microfluidic chips and a hypoxic chamber. Nestin+/CD31+ cells in the HPVN were derived from GSLC transdifferentiation and promoted GSLC chemoresistance by providing more JAG1 and DLL4 to induce downstream Hes1 overexpression. Poor GBM prognosis correlated with Hes1 expression of tumor cells in the GBM HPVN, and not with total Hes1 expression in GBM tissues. CONCLUSIONS: These results highlight the critical role of nestin+/CD31+ cells in HPVNs that acts in GBM chemoresistance and reveal the distinctive prognostic value of these molecular markers in HPVNs.

Dual pathways mediate ß-amyloid stimulated glutathione release from astrocytes.

Oxidative stress plays an important role in the progression of Alzheimer's disease (AD) and other neurodegenerative conditions. Glutathione (GSH), the major antioxidant in the central nervous system, is primarily synthesized and released by astrocytes. We determined if ß-amyloid (Aß42), crucially involved in Alzheimer's disease, affected GSH release. Monomeric Aß (mAß) stimulated GSH release from cultured cortical astrocytes more effectively than oligomeric Aß (oAß) or fibrillary Aß (fAß). Monomeric Aß increased the expression of the transporter ABCC1 (also referred to as MRP1) that is the main pathway for GSH release. GSH release from astrocytes, with or without mAß stimulation, was reduced by pharmacological inhibition of ABCC1. Astrocytes robustly express connexin proteins, especially connexin43 (Cx43), and mAß also stimulated Cx43 hemichannel-mediated glutamate and GSH release. Aß-stimulation facilitated hemichannel opening in the presence of normal extracellular calcium by reducing astrocyte cholesterol level. Aß treatment did not alter the intracellular concentration of reduced or oxidized glutathione. Using a mouse model of AD with early onset Aß deposition (5xFAD), we found that cortical ABCC1 was significantly increased in temporal register with the surge of Aß levels in these mice. ABCC1 levels remained elevated from 1.5 to 3.5 months of age in 5xFAD mice, before plunging to subcontrol levels when amyloid plaques appeared. Similarly, in cultured astrocytes, prolonged incubation with aggregated Aß, but not mAß, reduced induction of ABCC1 expression. These results support the hypothesis that in the early stage of AD pathogenesis, less aggregated Aß increases GSH release from astrocytes (via ABCC1 transporters and Cx43 hemichannels) providing temporary protection from oxidative stress which promotes AD development.

Cell-density-dependent manifestation of partial characteristics for neuronal precursors in a newly established human gliosarcoma cell line.

Gliosarcoma cell line K308 was established from a primary tumor specimen removed from a 51-year-old male Han Chinese patient. Besides the typical characteristics of gliosarcoma cells, K308 cells express abundant glutaminase and can release large amount of glutamate. K308 exhibited cell-density-dependent expression of neuronal precursor markers, particularly nestin. At low density, the majority of K308 cells were nestin negative (approximately 70%) and nestin levels remained homogenous within each single-cell-derived colony when K308 proliferated. After reaching confluence, however, the majority of K308 cells turned nestin positive. These confluent K308 cells were also Sox2 positive and could form tumor spheres even in serum-containing media.

Activation, permeability, and inhibition of astrocytic and neuronal large pore (hemi)channels.

Astrocytes and neurons express several large pore (hemi)channels that may open in response to various stimuli, allowing fluorescent dyes, ions, and cytoplasmic molecules such as ATP and glutamate to permeate. Several of these large pore (hemi)channels have similar characteristics with regard to activation, permeability, and inhibitor sensitivity. Consequently, their behaviors and roles in astrocytic and neuronal (patho)physiology remain undefined. We took advantage of the Xenopus laevis expression system to determine the individual characteristics of several large pore channels in isolation. Expression of connexins Cx26, Cx30, Cx36, or Cx43, the pannexins Px1 or Px2, or the purinergic receptor P2X7 yielded functional (hemi)channels with isoform-specific characteristics. Connexin hemichannels had distinct sensitivity to alterations of extracellular Ca(2+) and their permeability to dyes and small atomic ions (conductance) were not proportional. Px1 and Px2 exhibited conductance at positive membrane potentials, but only Px1 displayed detectable fluorescent dye uptake. P2X7, in the absence of Px1, was permeable to fluorescent dyes in an agonist-dependent manner. The large pore channels displayed overlapping sensitivity to the inhibitors Brilliant Blue, gadolinium, and carbenoxolone. These results demonstrated isoform-specific characteristics among the large pore membrane channels; an open (hemi)channel is not a nonselective channel. With these isoform-specific properties in mind, we characterized the divalent cation-sensitive permeation pathway in primary cultured astrocytes. We observed no activation of membrane conductance or Cx43-mediated dye uptake in astrocytes nor in Cx43-expressing C6 cells. Our data underscore that although Cx43-mediated transport is observed in overexpressing cell systems, such transport may not be detectable in native cells under comparable experimental conditions.

Glutamate/glutamine metabolism coupling between astrocytes and glioma cells: neuroprotection and inhibition of glioma growth.

Glioma glutamate release has been shown to promote the growth of glioma cells and induce neuronal injuries from epilepsy to neuronal death. However, potential counteractions from normal astrocytes against glioma glutamate release have not been fully evaluated. In this study, we investigated the glutamate/glutamine cycling between glioma cells and astrocytes and their impact on neuronal function. Co-cultures of glioma cells with astrocytes (CGA) in direct contact were established under different mix ratio of astrocyte/glioma. Culture medium conditioned in these CGAs were sampled for HPLC measurement, for neuronal ratiometric calcium imaging, and for neuronal survival assay. We found: (1) High levels of glutaminase expression in glioma cells, but not in astrocytes, glutaminase enables glioma cells to release large amount of glutamate in the presence of glutamine. (2) Glutamate levels in CGAs were directly determined by the astrocyte/glioma ratios, indicating a balance between glioma glutamate release and astrocyte glutamate uptake. (3) Culture media from CGAs of higher glioma/astrocyte ratios induced stronger neuronal Ca(2+) response and more severe neuronal death. (4) Co-culturing with astrocytes significantly reduced the growth rate of glioma cells. These results indicate that normal astrocytes in the brain play pivotal roles in glioma growth inhibition and in reducing neuronal injuries from glioma glutamate release. However, as tumor growth, the protective role of astrocytes gradually succumb to glioma cells.

Novel hypoglycemic injury mechanism: N-methyl-D-aspartate receptor-mediated white matter damage.

OBJECTIVE: Hypoglycemia is a common adverse event and can injure central nervous system (CNS) white matter (WM). We determined whether glutamate receptors were involved in hypoglycemic WM injury. METHODS: Mouse optic nerves (MON), CNS WM tracts, were maintained at 37°C with oxygenated artificial cerebrospinal fluid (ACSF) containing 10mM glucose. Aglycemia was produced by switching to 0 glucose ACSF. Supramaximal compound action potentials (CAPs) were elicited using suction electrodes, and axon function was quantified as the area under the CAP. Amino acid release was measured using high-performance liquid chromatography. Extracellular lactate concentration ([lactate(-)]o) was measured using an enzyme electrode. RESULTS: About 50% of MON axons were injured after 60 minutes of aglycemia (90% after 90 minutes); injury extent was not affected by animal age. Blockade of N-methyl-D-aspartate (NMDA)-type glutamate receptors improved recovery after 90 minutes of aglycemia by 250%. Aglycemic injury was increased by reducing [Mg(2+)]o or increasing [glycine]o , and decreased by lowering pHo , expected results for NMDA receptor-mediated injury. pHo increased during aglycemia due to a drop in [lactate(-)]o. Aglycemic injury was dramatically reduced in the absence of [Ca(2+)]o. Extracellular aspartate, a selective NMDA receptor agonist, increased during aglycemia ([glutamate]o fell). INTERPRETATION: Aglycemia injured WM by a unique excitotoxic mechanism involving NMDA receptors (located primarily on oligodendrocytes). During WM aglycemia, the selective NMDA agonist aspartate is released, probably from astrocytes. Injury is mediated by Ca(2+) influx through aspartate-activated NMDA receptors made permeable by an accompanying alkaline shift in pHo caused by a fall in [lactate(-)]o. These insights have important clinical implications.

Pharmacological "cross-inhibition" of connexin hemichannels and swelling activated anion channels.

The study of ion channels has relied heavily on the use of pharmacological blocking agents. However, many of these agents have multiple effects, which may compromise interpretation of results when the affected mechanisms/pathways mediate similar functions. Volume regulated anion channels (VRAC) and connexin hemichannels can both mediate the release of glutamate and taurine, although these channels have distinct activation stimuli and hemichannels, but not VRAC, are permeable to Lucifer Yellow (LY). It has been reported that some anion channel blockers may inhibit connexin hemichannels. We further examined the effects of classic gap junction/hemichannel blockers and anion channel blockers on these channels. The typical VRAC blockers, NPPB, IAA-94, and tamoxifen blocked low divalent cation-induced glutamate and taurine release and LY loading, presumed due to hemichannel opening. The blocking action of these compounds on hemichannels was concentration dependent and fell within the same range where the drugs classically block VRACs. Conversely, carbenoxolone (CBX), the most widely used gap junction/hemichannel blocker, was an effective blocker of VRAC-mediated glutamate and taurine release, and blocked these channels at similar concentrations at which it blocked hemichannels. The CBX effect on VRACs was verified using astrocytes from connexin 43 knock out (Cx43 KO) animals. In these cells, the hypotonic induced amino acid flux was retained whereas the low divalent cation solution-induced flux was lost. These results extend our knowledge about "cross-inhibition" of VRACs and gap junctions/hemichannels by certain pharmacological agents. Given the overlap in function of these two types of channels, great care must be exerted in using pharmacological blockers to identify one channel from the other.

Functional connexin "hemichannels": a critical appraisal.

"Hemichannels" are defined as the halves of gap junction channels (also termed connexons) that are contributed by one cell; "hemichannels" are considered to be functional if they are open in nonjunctional membranes in the absence of pairing with partners from adjacent cells. Several recent reviews have summarized the blossoming literature regarding functional "hemichannels", in some cases encyclopedically. However, most of these previous reviews have been written with the assumption that all data reporting "hemichannel" involvement really have studied phenomena in which connexons actually form the permeability or conductance pathway. In this review, we have taken a slightly different approach. We review the concept of "hemichannels", summarize properties that might be expected of half gap junctions and evaluate the extent to which the properties of presumptive "hemichannels" match expectations. Then we consider functions attributed to hemichannels, provide an overview of other channel types that might fulfill similar roles and provide sets of criteria that might be applied to verify involvement of connexin hemichannels in cell and tissue function. One firm conclusion is reached. The study of hemichannels is technically challenging and fraught with opportunities for misinterpretation, so that future studies must apply rigorous standards for detection of hemichannel expression and function. At the same time there are reasons to expect surprises, including the possibility that some time honored techniques for studying gap junctions may prove unsuitable for detecting hemichannels. We advise hemichannel researchers to proceed with caution and an open mind.

Sensitization to brain antigens after stroke is augmented by lipopolysaccharide.

After stroke, the blood-brain barrier is transiently disrupted, allowing leukocytes to enter the brain and brain antigens to enter the peripheral circulation. This encounter of normally sequestered brain antigens by the systemic immune system could therefore present an opportunity for an autoimmune response to brain to occur after stroke. In this study, we assessed the immune response to myelin basic protein (MBP) in animals subjected to middle cerebral artery occlusion (MCAO). Some animals received an intraperitoneal injection of lipopolysaccharide (LPS; 1 mg/kg) at reperfusion to stimulate a systemic inflammatory response. At 1 month after MCAO, animals exposed to LPS were more likely to be sensitized to MBP (66.7% versus 22.2%; P=0.007) and had more profound and persistent neurologic deficits than non-LPS-treated animals. Exposure to LPS was associated with increased expression of the costimulatory molecule B7.1 early after stroke onset (P=0.009) and increased brain atrophy 1 month after MCAO (P=0.03). These data suggest that animals subjected to a systemic inflammatory insult at the time of stroke are predisposed to develop an autoimmune response to brain, and that this response is associated with worse outcome. These data may partially explain why patients who become infected after stroke experience increased morbidity.

Functional hemichannels in astrocytes: a novel mechanism of glutamate release.

Little is known about the expression and possible functions of unopposed gap junction hemichannels in the brain. Emerging evidence suggests that gap junction hemichannels can act as stand-alone functional channels in astrocytes. With immunocytochemistry, dye uptake, and HPLC measurements, we show that astrocytes in vitro express functional hemichannels that can mediate robust efflux of glutamate and aspartate. Functional hemichannels were confirmed by passage of extracellular lucifer yellow (LY) into astrocytes in nominal divalent cation-free solution (DCFS) and the ability to block this passage with gap junction blocking agents. Glutamate/aspartate release (or LY loading) in DCFS was blocked by multivalent cations (Ca2+, Ba2+, Sr2+, Mg2+, and La3+) and by gap junction blocking agents (carbenoxolone, octanol, heptanol, flufenamic acid, and 18alpha-glycyrrhetinic acid) with affinities close to those reported for blockade of gap junction intercellular communication. Glutamate efflux via hemichannels was also accompanied by greatly reduced glutamate uptake. Glutamate release in DCFS, however, was not significantly mediated by reversal of the glutamate transporter: release did not saturate and was not blocked by glutamate transporter blockers. Control experiments in DCFS precluded glutamate release by volume-sensitive anion channels, P2X7 purinergic receptor pores, or general purinergic receptor activation. Blocking intracellular Ca2+ mobilization by BAPTA-AM or thapsigargin did not inhibit glutamate release in DCFS. Divalent cation removal also induced glutamate release from intact CNS white matter (acutely isolated optic nerve) that was blocked by carbenoxolone, suggesting the existence of functional hemichannels in situ. Our results indicated that astrocyte hemichannels could influence CNS levels of extracellular glutamate with implications for normal and pathological brain function.

Modulation of glial glutamate transport through cell interactions with the extracellular matrix.

Glial glutamate transport plays a pivotal role in maintaining glutamate homeostasis in the central nervous system. Expression of glutamate transporters is highly regulated during brain development, and a number of pathological conditions are associated with deficits in expression and/or function of glutamate transports. While several soluble factors have been shown to regulate the expression of glutamate transporter, the contribution of cell-cell interaction and cell-environmental interaction in the regulation of glutamate transport is unknown. Extracellular matrix (ECM) molecules are essential components in cell-cell and cell-environmental interactions, and the ECM has been shown to play critical role in normal development and during brain pathogenesis. We, therefore, investigated the possibility that ECM molecules may regulate astrocytic glutamate transport. Therefore, we cultured rat cortical astrocytes with different ECMs and determined expression levels of the two astrocytic glutamate transporters GLT-1 and GLAST by Western Blot and determined transporter activity through measurements of 3H-D-aspartate uptake. Astrocytes grown on poly-ornithine or poly-D/L-lysine showed approximately two-fold higher GLT-1 expression than sister cells grown on plastic dishes without ECM. Naturally occurring ECM's, including laminin and collagen, showed a dose-dependent regulation of GLT-1 protein expression. These effects were specific for GLT-1 as GLAST expression was unaffected by different ECMs. Surprisingly, however, none of the examined ECMs altered the apparent glutamate uptake activity. In probing blots side-by-side for expression of Na(+)/K(+)-ATPase, we found that ECMs affected expression of Na(+)/K(+)-ATPase and GLT-1 in a reciprocal fashion. Poly-ornithine, for example, enhanced GLT-1 expression, but reduced expression of Na(+)/K(+)-ATPase. Na(+) transport may, thus, be a limiting factor for glutamate uptake.