Human immunodeficiency virus‐1 (HIV) is a public health issue and a major complication of the disease is NeuroAIDS. In vivo, microglia/macrophages are the main cells infected. However, a low but significant number of HIV‐infected astrocytes has also been detected, but their role in the pathogenesis of NeuroAIDS is not well understood. Our previous data indicate that gap junction channels amplify toxicity from few HIV‐infected into uninfected astrocytes. Now, we demonstrated that HIV infection of astrocytes results in the opening of connexin43 hemichannels (HCs). HIV‐induced opening of connexin43 HCs resulted in dysregulated secretion of dickkopf‐1 protein (DKK1, a soluble wnt pathway inhibitor). Treatment of mixed cultures of neurons and astrocytes with DKK1, in the absence of HIV infection, resulted in the collapse of neuronal processes. HIV infection of mixed cultures of human neurons and astrocytes also resulted in the collapse of neuronal processes through a DKK1‐dependent mechanism. In addition, dysregulated DKK1 expression in astrocytes was observed in human brain tissue sections of individuals with HIV encephalitis as compared to tissue sections from uninfected individuals. Thus, we demonstrated that HIV infection of astrocytes induces dysregulation of DKK1 by a HC‐dependent mechanism that contributes to the brain pathogenesis observed in HIV‐infected individuals.
Monoamine neurotransmitters should be immediately removed from the synaptic cleft to avoid excessive neuronal activity. Recent studies have shown that astrocytes and neurons are involved in monoamine removal. However, the mechanism of monoamine transport by astrocytes is not entirely clear. We aimed to elucidate the transporters responsible for monoamine transport in 1321N1, a human astrocytoma‐derived cell line. First, we confirmed that 1321N1 cells transported dopamine, serotonin, norepinephrine, and histamine in a time‐ and dose‐dependent manner. Kinetics analysis suggested the involvement of low‐affinity monoamine transporters, such as organic cation transporter (OCT) 2 and 3 and plasma membrane monoamine transporter (PMAT). Monoamine transport in 1321N1 cells was not Na+/Cl? dependent but was inhibited by decynium‐22, an inhibitor of low‐affinity monoamine transporters, which supported the importance of low‐affinity transporters. RT‐PCR assays revealed that 1321N1 cells expressed OCT3 and PMAT but no other neurotransmitter transporters. Another human astrocytoma‐derived cell line, U251MG, and primary human astrocytes also exhibited the same gene expression pattern. Gene‐knockdown assays revealed that 1321N1 and primary human astrocytes could transport monoamines predominantly through PMAT and partly through OCT3. These results might indicate that PMAT and OCT3 in human astrocytes are involved in monoamine clearance.
Heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF), a vascular‐derived trophic factor, belongs to the epidermal growth factor (EGF) family of neuroprotective, hypoxia‐inducible proteins released by astrocytes in CNS injuries. It was suggested that HB–EGF can replace fetal calf serum (FCS) in astrocyte cultures. We previously demonstrated that in contrast to standard 2D cell culture systems, Bioactive3D culture system, when used with FCS, minimizes the baseline activation of astrocytes and preserves their complex morphology. Here, we show that HB‐EGF induced EGF receptor (EGFR) activation by Y1068 phosphorylation, Mapk/Erk pathway activation, and led to an increase in cell proliferation, more prominent in Bioactive3D than in 2D cultures. HB‐EGF changed morphology of 2D and Bioactive3D cultured astrocytes toward a radial glia‐like phenotype and induced the expression of intermediate filament and progenitor cell marker protein nestin. Glial fibrillary acidic protein (GFAP) and vimentin protein expression was unaffected. RT‐qPCR analysis demonstrated that HB‐EGF affected the expression of Notch signaling pathway genes, implying a role for the Notch signaling in HB‐EGF‐mediated astrocyte response. HB‐EGF can be used as a FCS replacement for astrocyte expansion and in vitro experimentation both in 2D and Bioactive3D culture systems; however, caution should be exercised since it appears to induce partial de‐differentiation of astrocytes.
Treatments to inhibit or repair neuronal cell damage sustained during focal ischemia/reperfusion injury in stroke are largely unavailable. We demonstrate that dietary supplementation with the antioxidant di‐tert‐butyl‐bisphenol (BP) before injury decreases infarction and vascular complications in experimental stroke in an animal model. We confirm that BP, a synthetic polyphenol with superior radical‐scavenging activity than vitamin E, crosses the blood–brain barrier and accumulates in rat brain. Supplementation with BP did not affect blood pressure or endogenous vitamin E levels in plasma or cerebral tissue. Pre‐treatment with BP significantly lowered lipid, protein and thiol oxidation and decreased infarct size in animals subjected to middle cerebral artery occlusion (2 h) and reperfusion (24 h) injury. This neuroprotective action was accompanied by down‐regulation of hypoxia inducible factor‐1α and glucose transporter‐1 mRNA levels, maintenance of neuronal tissue ATP concentration and inhibition of pro‐apoptotic factors that together enhanced cerebral tissue viability after injury. That pre‐treatment with BP ameliorates oxidative damage and preserves cerebral tissue during focal ischemic insult indicates that oxidative stress plays at least some causal role in promoting tissue damage in experimental stroke. The data strongly suggest that inhibition of oxidative stress through BP scavenging free radicals in vivo contributes significantly to neuroprotection.
The gene encoding leucine‐rich repeat kinase 2 (LRRK2) comprises a major risk factor for Parkinson's disease. Recently, it has emerged that LRRK2 plays important roles in the immune system. LRRK2 is induced by interferon‐γ (IFN‐γ) in monocytes, but the signaling pathway is not known. Here, we show that IFN‐γ‐mediated induction of LRRK2 was suppressed by pharmacological inhibition and RNA interference of the extracellular signal‐regulated kinase 5 (ERK5). This was confirmed by LRRK2 immunostaining, which also revealed that the morphological responses to IFN‐γ were suppressed by ERK5 inhibitor treatment. Both human acute monocytic leukemia THP‐1 cells and human peripheral blood monocytes stimulated the ERK5‐LRRK2 pathway after differentiation into macrophages. Thus, LRRK2 is induced via a novel, ERK5‐dependent IFN‐γ signal transduction pathway, pointing to new functions of ERK5 and LRRK2 in human macrophages.
Parkinson's disease is the second most common neurodegenerative disease and its pathogenesis is closely associated with oxidative stress. Deposition of aggregated α‐synuclein (α‐Syn) occurs in familial and sporadic forms of Parkinson's disease. Here, we studied the effect of oligomeric α‐Syn on one of the major markers of oxidative stress, lipid peroxidation, in primary co‐cultures of neurons and astrocytes. We found that oligomeric but not monomeric α‐Syn significantly increases the rate of production of reactive oxygen species, subsequently inducing lipid peroxidation in both neurons and astrocytes. Pre‐incubation of cells with isotope‐reinforced polyunsaturated fatty acids (D‐PUFAs) completely prevented the effect of oligomeric α‐Syn on lipid peroxidation. Inhibition of lipid peroxidation with D‐PUFAs further protected cells from cell death induced by oligomeric α‐Syn. Thus, lipid peroxidation induced by misfolding of α‐Syn may play an important role in the cellular mechanism of neuronal cell loss in Parkinson's disease.
Synaptic dysfunction and neuronal death are responsible for cognitive and behavioral deficits in Alzheimer's disease (AD). It is well known that such neurological abnormalities are preceded by long‐term exposure of amyloid β‐peptide (Aβ) and/or hyperphosphorylated tau prior. In addition to the neurological deficit, astrocytes as a major glial cell type in the brain, significantly participate in the neuropathogenic mechanisms underlying synaptic modulation. Although astrocytes play a significant key role in modulating synaptic transmission, little is known on whether astrocyte dysfunction caused by such long‐term Aβ exposure affects synapse formation and function. Here, we show that synapse formation and synaptic transmission are attenuated in hippocampal‐naïve neurons co‐cultured with astrocytes that have previously experienced chronic Aβ1‐40 exposure. In this abnormal astrocytic condition, hippocampal neurons exhibit decrements of evoked excitatory post‐synaptic currents (EPSCs) and miniature EPSC frequency. Furthermore, size of readily releasable synaptic pools and number of excitatory synapses were also significantly decreased. Contrary to these negative effects, release probability at individual synapses was significantly increased in the same astrocytic condition. Taken together, our data indicate that lower synaptic transmission caused by astrocytes previously, and chronically, exposed to Aβ1–40 is attributable to a small number of synapses with higher release probability.
Inflammation within the CNS is a major component of many neurodegenerative diseases. A characteristic feature is the generation of microglia‐derived factors that play an essential role in the immune response. IL‐1β is a pro‐inflammatory cytokine released by activated microglia, able to exacerbate injury at elevated levels. In the presence of caspase‐1, pro‐IL‐1β is cleaved to the mature cytokine following NOD‐like receptor pyrin domain containing 3 (NLRP3) inflammasome activation. Growing evidence suggests that ceramide plays a critical role in NLRP3 inflammasome assembly, however, the relationship between ceramide and inflammasome activation in microglia remains unknown. Here, we investigated potential mechanistic links between ceramide as a modulator of NLRP3 inflammasome assembly and the resulting secretion of IL‐1β using small bioactive enzyme stimulators and inhibitors of ceramide signaling in wild‐type and apoptosis‐associated speck‐like protein containing a CARD knockout (ASC?/?) primary microglia. To induce the expression of inflammasome components, microglia were primed prior to experiments. Treatment with sodium palmitate (PA) induced de novo ceramide synthesis via modulation of its synthesizing protein serine palmitoyl transferase resulting in increased IL‐1β secretion in microglia. Exposure of microglia to the serine palmitoyl transferase‐inhibitor l ‐cycloserine significantly prevented PA‐induced IL‐1β secretion. Application of the ceramide analogue C2 and the sphingosine‐1‐phosphate‐receptor agonist Fingolimod (FTY720) up‐regulated levels of IL‐1β and cleaved caspase‐1 in wild‐type microglia, whereas ASC?/? microglia were unaffected. HPA‐12 inhibition of ceramide transport did not affect inflammasome activation. Taken together, our findings reveal a critical role for ceramide as a positive modulator of NLRP3 inflammasome assembly and the resulting release of IL‐1β.
2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a ubiquitous environmental pollutant that could induce significant toxic effects in the human nervous system. However, the underlying molecular mechanism has not been entirely elucidated. Reactive astrogliosis has implicated in various neurological diseases via the production of a variety of pro‐inflammatory mediators. Herein, we investigated the potential role of TCDD in facilitating astrocyte activation and the underlying molecular mechanisms. We showed that TCDD induced rapid astrocyte activation following TCDD exposure, which was accompanied by significantly elevated expression of Src‐Suppressed‐C Kinase Substrate (SSeCKS), a protein involved in protein kinase C (PKC)‐mediated Nuclear Factor kappa B signaling, suggesting a possible involvement of PKC‐induced SSeCKS activation in TCDD‐triggered reactive astroglia. In keeping with the finding, we found that the level of phosphorylated Nuclear Factor kappa B p65 was remarkably increased after TCDD treatment. Furthermore, interference of SSeCKS attenuated TCDD‐induced inducible nitric oxide synthase, glial fibrillary acidic protein, phospho‐p65 expression, and tumor necrosis factor‐α secretion in astrocytes. In addition, pre‐treatment with PKC inhibitor also attenuated TCDD‐induced astrocyte activation, as well as SSeCKS expression. Interestingly, we found that TCDD treatment could lead to SSeCKS perinuclear localization, which could be abolished after treatment with PKC inhibitor. Finally, we showed that inhibition of PKC activity or SSeCKS expression would impair TCDD‐triggered tumor necrosis factor‐α secretion. Our results suggested that TCDD exposure could lead to astrocyte activation through PKC/SSeCKS‐dependent mechanisms, highlighting that astrocytes might be important target of TCDD‐induced neurotoxicity.
Reduced expression of a ~150 kDa protein was unexpectedly observed while investigating Norrin protein in a transgenic murine model in which Müller cells can be selectively and inducibly disrupted. Isolation of this unknown protein via ion exchange and hydrophobic interaction chromatography followed by Tandem mass spectrometry identified it as Inter‐photoreceptor retinoid‐binding protein (IRBP). Significantly reduced IRBP mRNA expression was observed at the early and late stages after Müller cell disruption. IRBP protein expression was also consistently reduced to 5.7% of the control level as early as 1 week after Müller cell disruption. This down‐regulation of IRBP was accompanied by focal hyperfluorescent dots and cytotoxic N‐retinylidene‐N‐retinylethanolamine (A2E) accumulation. In vitro treatment of cone photoreceptor cell lines with conditioned medium collected from stressed Müller cells suggested that Müller cells regulated photoreceptors expression of IRBP via secreted factor(s). In vivo studies suggested that one of these secreted factors was tumour necrosis factor alpha (TNFα). These findings suggest that dysregulation of IRBP expression caused by Müller cell dysfunction may be an important early event in photoreceptor degeneration in some retinal diseases.
Stroke is a devastating clinical condition for which an effective neuroprotective treatment is currently unavailable. S‐allyl cysteine (SAC), the most abundant organosulfur compound in aged garlic extract, has been reported to possess neuroprotective effects against stroke. However, the mechanisms underlying its beneficial effects remain poorly defined. The present study tests the hypothesis that SAC attenuates ischemic neuronal injury by activating the nuclear factor erythroid‐2‐related factor 2 (Nrf2)‐dependent antioxidant response in both in vitro and in vivo models. Our findings demonstrate that SAC treatment resulted in an increase in Nrf2 protein levels and subsequent activation of antioxidant response element pathway genes in primary cultured neurons and mice. Exposure of primary neurons to SAC provided protection against oxygen and glucose deprivation‐induced oxidative insults. In wild‐type (Nrf2+/+) mice, systemic administration of SAC attenuated middle cerebral artery occlusion‐induced ischemic damage, a protective effect not observed in Nrf2 knockout (Nrf2?/?) mice. Taken together, these findings provide the first evidence that activation of the Nrf2 antioxidant response by SAC is strongly associated with its neuroprotective effects against experimental stroke and suggest that targeting the Nrf2 pathway may provide therapeutic benefit for the treatment of stroke.
Reactive astrogliosis, characterized by cellular hypertrophy and various alterations in gene expression and proliferative phenotypes, is considered to contribute to brain injuries and diseases as diverse as trauma, neurodegeneration, and ischemia. KCa3.1 (intermediate‐conductance calcium‐activated potassium channel), a potassium channel protein, has been reported to be up‐regulated in reactive astrocytes after spinal cord injury in vivo. However, little is known regarding the exact role of KCa3.1 in reactive astrogliosis. To elucidate the role of KCa3.1 in regulating reactive astrogliosis, we investigated the effects of either blocking or knockout of KCa3.1 channels on the production of astrogliosis and astrocytic proliferation in response to transforming growth factor (TGF)‐β in primary cultures of mouse astrocytes. We found that TGF‐β increased KCa3.1 protein expression in astrocytes, with a concomitant marked increase in the expression of reactive astrogliosis, including glial fibrillary acidic protein and chondroitin sulfate proteoglycans. These changes were significantly attenuated by the KCa3.1 inhibitor 1‐((2‐chlorophenyl) (diphenyl)methyl)‐1H‐pyrazole (TRAM‐34). Similarly, the increase in glial fibrillary acidic protein and chondroitin sulfate proteoglycans in response to TGF‐β was attenuated in KCa3.1?/? astrocytes. TRAM‐34 also suppressed astrocytic proliferation. In addition, the TGF‐β‐induced phosphorylation of Smad2 and Smad3 proteins was reduced with either inhibition of KCa3.1 with TRAM‐34 or in KCa3.1?/? astrocytes. These findings highlight a novel role for the KCa3.1 channel in reactive astrogliosis phenotypic modulation and provide a potential target for therapeutic intervention for brain injuries.
Recent evidence suggests that the predominant astrocyte glutamate transporter, GLT‐1/ Excitatory Amino Acid Transporter 2 (EAAT2) is associated with mitochondria. We used primary cultures of mouse astrocytes to assess co‐localization of GLT‐1 with mitochondria, and tested whether the interaction was dependent on neurons, actin polymerization or the kinesin adaptor, TRAK2. Mouse primary astrocytes were transfected with constructs expressing V5‐tagged GLT‐1, pDsRed1‐Mito with and without dominant negative TRAK2. Astrocytes were visualized using confocal microscopy and co‐localization was quantified using Volocity software. Image analysis of confocal z‐stacks revealed no co‐localization between mitochondria and GLT‐1 in pure astrocyte cultures. Co‐culture of astrocytes with primary mouse cortical neurons revealed more mitochondria in processes and a positive correlation between mitochondria and GLT‐1. This co‐localization was not further enhanced after neuronal depolarization induced by 1 h treatment with 15 mM K+. In pure astrocytes, a rho kinase inhibitor, Y27632 caused the distribution of mitochondria to astrocyte processes without enhancing GLT‐1/mitochondrial co‐localization, however, in co‐cultures, Y27632 abolished mitochondrial:GLT‐1 co‐localization. Disrupting potential mitochondrial: kinesin interactions using dominant negative TRAK2 did not alter GLT‐1 distribution or GLT‐1: mitochondrial co‐localization. We conclude that the association between GLT‐1 and mitochondria is modest, is driven by synaptic activity and dependent on polymerized actin filaments.
Acrolein, an α,β‐unsaturated aldehyde and a reactive product of lipid peroxidation, has been suggested as a key factor in neural post‐traumatic secondary injury in spinal cord injury (SCI), mainly based on in vitro and ex vivo evidence. Here, we demonstrate an increase of acrolein up to 300%; the elevation lasted at least 2 weeks in a rat SCI model. More importantly, hydralazine, a known acrolein scavenger can provide neuroprotection when applied systemically. Besides effectively reducing acrolein, hydralazine treatment also resulted in significant amelioration of tissue damage, motor deficits, and neuropathic pain. This effect was further supported by demonstrating the ability of hydralazine to reach spinal cord tissue at a therapeutic level following intraperitoneal application. This suggests that hydralazine is an effective neuroprotective agent not only in vitro, but in a live animal model of SCI as well. Finally, the role of acrolein in SCI was further validated by the fact that acrolein injection into the spinal cord caused significant SCI‐like tissue damage and motor deficits. Taken together, available evidence strongly suggests a critical causal role of acrolein in the pathogenesis of spinal cord trauma. Since acrolein has been linked to a variety of illness and conditions, we believe that acrolein‐scavenging measures have the potential to be expanded significantly ensuring a broad impact on human health.
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