Interaction between NADPH‐oxidase and Rho‐kinase in angiotensin II‐induced microglial activation

Previous studies have shown that the brain renin–angiotensin system may play a major role, via angiotensin type 1 (AT1) receptors, in the regulation of neuroinflammation, oxidative stress and progression of dopaminergic degeneration. Angiotensin‐induced activation of the microglial nicotinamide adenine dinucleotide phosphate (NADPH)‐oxidase complex and microglial Rho‐kinase are particularly important in this respect. However, it is not known whether crosstalk between Rho‐kinase and NADPH‐oxidase leads to microglial activation. In the present study, we found that, in the substantia nigra of rats, NADPH‐oxidase activation was involved in angiotensin‐induced Rho‐kinase activation, which, in turn, was involved in angiotensin‐induced NADPH‐oxidase activation. In N9 microglial cell line and primary microglial cultures, a crosstalk signaling between NADPH‐oxidase and Rho‐kinase occurred in a positive feedback fashion during angiotensin‐induced microglial activation. Angiotensin‐induced NADPH‐oxidase activation and superoxide generation led to NF‐кB translocation and Rho‐kinase activation. Rho‐kinase activation was involved in regulation of NADPH‐oxidase activation via p38 mitogen‐activated protein kinase. Moreover, Rho‐kinase activation, via NF‐кB, upregulated AT1 receptor expression in microglial cells through a feed‐forward mechanism. NADPH‐oxidase and Rho‐kinase pathways are known to be responsible for major components of the microglial response, such as changes involving microglial motility and phagocytosis, generation of superoxide, and release of inflammatory cytokines. The present results show that both pathways are linked by a common mechanism that may constitute a basic means of coordinating the microglial response. GLIA 2015;63:466–482     

Toll‐like receptor 4 is required for α‐synuclein dependent activation of microglia and astroglia

Alpha‐synucleinopathies (ASP) are neurodegenerative disorders, characterized by accumulation of misfolded α‐synuclein, selective neuronal loss, and extensive gliosis. It is accepted that microgliosis and astrogliosis contribute to the disease progression in ASP. Toll‐like receptors (TLRs) are expressed on cells of the innate immune system, including glia, and TLR4 dysregulation may play a role in ASP pathogenesis. In this study we aimed to define the involvement of TLR4 in microglial and astroglial activation induced by different forms of α‐synuclein (full length soluble, fibrillized, and C‐terminally truncated). Purified primary wild type (TLR4^+/+) and TLR4 deficient (TLR4^?/?) murine microglial and astroglial cell cultures were treated with recombinant α‐synuclein and phagocytic activity, NFκB nuclear translocation, cytokine release, and reactive oxygen species (ROS) production were measured. We show that TLR4 mediates α‐synuclein‐induced microglial phagocytic activity, pro‐inflammatory cytokine release, and ROS production. TLR4^?/? astroglia present a suppressed pro‐inflammatory response and decreased ROS production triggered by α‐synuclein treatment. However, the uptake of α‐synuclein by primary astroglia is not dependent on TLR4 expression. Our results indicate the C‐terminally truncated form as the most potent inductor of TLR4‐dependent glial activation. The current findings suggest that TLR4 plays a modulatory role on glial pro‐inflammatory responses and ROS production triggered by α‐synuclein. In contrast to microglia, the uptake of alpha‐synuclein by astroglia is not dependent on TLR4. Our data provide novel insights into the mechanisms of α‐synuclein‐induced microglial and astroglial activation which may have an impact on understanding the pathogenesis of ASP. © 2012 Wiley Periodicals, Inc.     

Downregulation of miR‐7116‐5p in microglia by MPP+ sensitizes TNF‐α production to induce dopaminergic neuron damage

Activation of microglia resulting in exacerbated inflammation expression plays an important role in degeneration of dopaminergic (DA) neurons in the pathogenesis of Parkinson's disease (PD). However, how this enhanced inflammation is induced in microglia remains largely unclear. Here, in the mouse PD model induced by 1‐methyl‐4‐phenyl‐1,2,3,6‐tetra hydropyridine (MPTP), we found that miR‐7116‐5p in microglia has a crucial role in this inflammation. 1‐methyl‐4‐phenylpyridinium (MPP⁺) is uptaken by microglia through organic cation transporter 3 (OCT3) to downregulate miR‐7116‐5p, an miRNA found to target tumor necrosis factor alpha (TNF‐α). Production of TNF‐α in microglia is specifically potentiated by MPP⁺ via downregulation of miR‐7116‐5p to elicit subsequent inflammatory responses. Furthermore, enhancement of miR‐7116‐5p expression in microglia in mice inhibits the production of TNF‐α and the activation of glia, and further prevents loss of DA neurons. Together, our studies suggest that MPP⁺ suppresses miR‐7116‐5p level in microglia and potentiates TNF‐α production and inflammatory responses to contribute to DA neuron damage.     

Antipsychotic‐induced DRD2 upregulation and its prevention by α‐lipoic acid in SH‐SY5Y Neuroblastoma cells

Most antipsychotic (AP) drugs are dopamine (DA) D2 receptor (DRD2) antagonists and remain the main pharmacological treatment of schizophrenia. Long‐term AP use can give rise to tardive dyskinesia. It has been reported that chronic treatment with APs induces DRD2 upregulation and oxidative stress, which have been associated with tardive dyskinesia. We showed previously that H₂O₂‐induced oxidative stress increased DRD2 expression in human SH‐SY5Y neuroblastoma cells. We report here the effects of AP drugs on DRD2 expression levels in the same cell line and the effects of the inhibition of oxidative phenomena by (±)‐α‐lipoic acid treatment. Haloperidol, a first‐generation AP, induced an increase in DRD2 protein and mRNA levels, whereas amisulpride, a second‐generation AP, had no significant effect. (±)‐α‐Lipoic acid pretreatment reversed the haloperidol‐induced DRD2 upregulation in mRNA and protein levels. Furthermore, haloperidol induced a larger increase of oxidative stress biomarkers (protein carbonylation, lipid peroxidation, and superoxide anion production) than amisulpride. (±)‐α‐Lipoic acid also attenuated AP‐induced oxidative stress. Inhibition of catecholamine synthesis by α‐methyl‐DL ‐tyrosine (AMPT) increased DRD2 expression and prevented further increase by APs. Our results suggest that haloperidol‐induced DRD2 upregulation is linked to oxidative stress and provide potential mechanisms by which (±)‐α‐lipoic acid can be considered as a therapeutic agent to prevent and treat side effects related to the use of first‐generation APs. Synapse, 2011. © 2010 Wiley‐Liss, Inc.     

The unlikely partnership between LRRK2 and α‐synuclein in Parkinson's disease

Our understanding of the mechanisms underlying Parkinson's disease, the once archetypical nongenetic neurogenerative disorder, has dramatically increased with the identification of α‐synuclein and LRRK2 pathogenic mutations. While α‐synuclein protein composes the aggregates that can spread through much of the brain in disease, LRRK2 encodes a multidomain dual‐enzyme distinct from any other protein linked to neurodegeneration. In this review, we discuss emergent datasets from multiple model systems that suggest these unlikely partners do interact in important ways in disease, both within cells that express both LRRK2 and α‐synuclein as well as through more indirect pathways that might involve neuroinflammation. Although the link between LRRK2 and disease can be understood in part through LRRK2 kinase activity (phosphotransferase activity), α‐synuclein toxicity is multilayered and plausibly interacts with LRRK2 kinase activity in several ways. We discuss common protein interactors like 14‐3‐3s that may regulate α‐synuclein and LRRK2 in disease. Finally, we examine cellular pathways and outcomes common to both mutant α‐synuclein expression and LRRK2 activity and points of intersection. Understanding the interplay between these two unlikely partners in disease may provide new therapeutic avenues for PD.     

β2 Adrenergic receptor activation induces microglial NADPH oxidase activation and dopaminergic neurotoxicity through an ERK‐dependent/protein kinase A‐independent pathway

Activation of the β2 adrenergic receptor (β2AR) on immune cells has been reported to possess anti‐inflammatory properties, however, the pro‐inflammatory properties of β2AR activation remain unclear. In this study, using rat primary mesencephalic neuron‐glia cultures, we report that salmeterol, a long‐acting β2AR agonist, selectively induces dopaminergic (DA) neurotoxicity through its ability to activate microglia. Salmeterol selectively increased the production of reactive oxygen species (ROS) by NADPH oxidase (PHOX), the major superoxide‐producing enzyme in microglia. A key role of PHOX in mediating salmeterol‐induced neurotoxicity was demonstrated by the inhibition of DA neurotoxicity in cultures pretreated with diphenylene‐iodonium (DPI), an inhibitor of PHOX activity. Mechanistic studies revealed the activation of microglia by salmeterol results in the selective phosphorylation of ERK, a signaling pathway required for the translocation of the PHOX cytosolic subunit p47^phox to the cell membrane. Furthermore, we found ERK inhibition, but not protein kinase A (PKA) inhibition, significantly abolished salmeterol‐induced superoxide production, p47^phox translocation, and its ability to mediate neurotoxicity. Together, these findings indicate that β2AR activation induces microglial PHOX activation and DA neurotoxicity through an ERK‐dependent/PKA‐independent pathway. © 2009 Wiley‐Liss, Inc.     

Nigral burden of α‐synuclein correlates with striatal dopamine deficit

Aggregated α‐synuclein is the hallmark of Parkinson's disease (PD), diffuse Lewy body disease (DLBD), and multiple system atrophy (MSA). Physiologically, α‐synuclein ensures normal functions of dopamine transporter (DAT) and tyrosine hydoxylase. In α‐synucleinopathies, it accumulates in neuronal cytoplasm and neurites through several stages. It is unclear whether the accumulation of pathological α‐synuclein in the substantia nigra in PD correlates with the dopaminergic deficit in the striatal target. We evaluated the impact of the nigral burden of pathological α‐synuclein immunoreactivity in 27 α‐synucleinopathy brains by morphometric immunohistochemistry. DAT immunoreactivity in the striatum inversely correlates with the total α‐synuclein burden in the substantia nigra but not with cytoplasmic inclusion counts only. This result has implications for imaging, clinicopathological correlative studies, and staging of the disease process. © 2008 Movement Disorder Society     

Stat3 mediates LIF‐induced protection of astrocytes against toxic ROS by upregulating the UPC2 mRNA pool

Reactive oxygen species (ROS) have been implicated in various types of CNS damage, including stroke. We used a cultured astrocyte model to explore mechanisms of survival of CNS cells following ROS damage. We found that pretreatment with leukemia inhibitory factor (LIF) preserves astrocytes exposed to toxic levels of t‐BHP by inhibiting an increase in intracellular ROS following t‐BHP treatment. Astrocytes lacking functional Stat3 did not benefit from the pro‐survival or antioxidant effects of LIF. Inhibition of mitochondrial uncoupling protein 2 (UCP2) using a chemical inhibitor or siRNA abrogates the prosurvival effects of LIF, indicating a critical role for UCP2 in modulation of mitochondrial ROS production in survival following ROS exposure. LIF treatment of astrocytes results in increased UCP2 mRNA that is accompanied by an increase in Stat3 binding to the UCP2 promoter region. Although treatment with LIF alone did not increase UCP2 protein, a combination of LIF treatment and ROS stress led to increased UCP2 protein levels. We conclude that LIF protects astrocytes from ROS‐induced death by increasing UCP2 mRNA, allowing cells to respond to ROS stress by rapidly producing UCP2 protein that ultimately decreases endogenous mitochondrial ROS production. GLIA 2014;62:159–170     

Cannabinoids prevent the amyloid β‐induced activation of astroglial hemichannels: A neuroprotective mechanism

The mechanisms involved in Alzheimer's disease are not completely understood and how astrocytes and their gliotransmission contribute to this neurodegenerative disease remains to be fully elucidated. Previous studies have shown that amyloid‐β peptide (Aβ) induces neuronal death by a mechanism that involves the excitotoxic release of ATP and glutamate associated to astroglial hemichannel opening. We have demonstrated that synthetic and endogenous cannabinoids (CBs) reduce the opening of astrocyte Cx43 hemichannels evoked by activated microglia or inflammatory mediators. Nevertheless, whether CBs could prevent the astroglial hemichannel‐dependent death of neurons evoked by Aβ is unknown. Astrocytes as well as acute hippocampal slices were treated with the active fragment of Aβ alone or in combination with the following CBs: WIN, 2‐AG, or methanandamide (Meth). Hemichannel activity was monitored by single channel recordings and by time‐lapse ethidium uptake while neuronal death was assessed by Fluoro‐Jade C staining. We report that CBs fully prevented the hemichannel activity and inflammatory profile evoked by Aβ in astrocytes. Moreover, CBs fully abolished the Aβ‐induced release of excitotoxic glutamate and ATP associated to astrocyte Cx43 hemichannel activity, as well as neuronal damage in hippocampal slices exposed to Aβ. Consequently, this work opens novel avenues for alternative treatments that target astrocytes to maintain neuronal function and survival during AD. GLIA 2016 GLIA 2017;65:122–137     

Regulatory role of cytosolic phospholipase A2α in NADPH oxidase activity and in inducible nitric oxide synthase induction by aggregated Aβ1–42 in microglia

In Alzheimer's disease, extracellular deposits of amyloid β_1–42 (Aβ_1–42) may induce activation of microglial cells by releasing proinflammatory factors that contribute to the neurodegeneration process. Since the activation of cytosolic phospholipase A₂α (cPLA₂α) has been reported in inflammatory conditions, its role in primary rat microglial cell activated by aggregated Aβ_1–42 was elucidated. The results of the present study show that activation of microglia by 5 μM aggregated Aβ_1–42 (as evident by the amoeboid morphology and increased CD68 immunofluorescence reactivity) caused an immediate activation of cPLA₂α, measured by its phosphorylated form and its specific activity, followed by a gradual elevation of its expression and activity during 24 h. Inhibition of cPLA₂α expression and activity by the presence of 1 μM specific antisense resulted in a significant decrease in NADPH oxidase activity that releases superoxides, PGE₂ formation, iNOS expression, and NO production, indicating a major role for cPLA₂α in the regulation of these inflammatory processes. NADPH oxidase activity, which is under cPLA₂α regulation, was found to upregulate cPLA₂α and COX‐2 protein expression through the redox‐sensitive NFκB activation as evident by its phosphorylation on Ser‐536, resulting in increased PGE₂ formation. The secreted PGE₂ induced the synthesis of iNOS and the production of NO through the PKA‐CREB pathway. Taken together, our results suggest that the response of cPLA₂α to aggregated Aβ_1–42 is probably a key player in the oxidative stress present in AD, regulating potent oxidative agents: the production of superoxides by NADPH oxidase and NO formation by iNOS. © 2009 Wiley‐Liss, Inc.     

Interleukin‐1β protects astrocytes against oxidant‐induced injury via an NF‐κB‐Dependent upregulation of glutathione synthesis

Astrocytes produce and export the antioxidant glutathione (GSH). Previously, we found that interleukin‐1β (IL‐1β) enhanced the expression of astrocyte system x_c^?, the transporter that delivers the rate‐limiting substrate for GSH synthesis—cyst(e)ine. Herein, we demonstrate directly that IL‐1β mediates a time‐dependent increase in extracellular GSH levels in cortical astrocyte cultures, suggesting both enhanced synthesis and export. This increased GSH production was blocked by inhibition of nuclear factor‐κB (NF‐κB) activity but not by inhibition of p38 MAPK. To determine whether this increase could provide protection against oxidative stress, the oxidants tert‐butyl hydroperoxide (tBOOH) and ferrous sulfate (FeSO₄) were employed. IL‐1β treatment prevented the increase in reactive oxygen species produced in astrocytes following tBOOH exposure. Additionally, the toxicity induced by tBOOH or FeSO₄ exposure was significantly attenuated following treatment with IL‐1β, an effect reversed by concomitant exposure to l ‐buthionine‐S,R‐sulfoximine (BSO), which prevented the IL‐1β‐mediated rise in GSH production. IL‐1β failed to increase GSH or to provide protection against t‐BOOH toxicity in astrocyte cultures derived from IL‐1R1 null mutant mice. Overall, our data indicate that under certain conditions IL‐1β may be an important stimulus for increasing astrocyte GSH production, and potentially, total antioxidant capacity in brain, via an NF‐κB‐dependent process. GLIA 2015;63:1568–1580     

P2X7‐dependent,but differentially regulated release of IL‐6, CCL2, and TNF‐α in cultured mouse microglia

ATP is an important regulator of microglia and its effects on microglial cytokine release are currently discussed as important contributors in a variety of brain diseases. We here analyzed the effects of ATP on the production of six inflammatory mediators (IL‐6, IL‐10, CCL2, IFN‐γ, TNF‐α, and IL‐12p70) in cultured mouse primary microglia. Stimulation of P2X7 receptor by ATP (1 mM) or BzATP (500 µM) evoked the mRNA expression and release of proinflammatory cytokines IL‐6, TNF‐α, and the chemokine CCL2 in WT cells but not in P2X7^?/? cells. The effects of ATP and BzATP were inhibited by the nonselective P2 receptor antagonists PPADs and suramin. Various selective P2X7 receptor antagonists blocked the P2X7‐dependent release of IL‐6 and CCL2, but, surprisingly, had no effect on BzATP‐induced release of TNF‐α in microglia. Calcium measurements confirmed that P2X7 is the main purine receptor activated by BzATP in microglia and showed that all P2X7 antagonists were functional. It is also presented that pannexin‐1 hemichannel function and potential P2X4/P2X7 heterodimers are not involved in P2X7‐dependent release of IL‐6, CCL2, and TNF‐α in microglia. How P2X7‐specific antagonists only affect P2X7‐dependent IL‐6 and CCL2 release, but not TNF‐α release is at the moment unclear, but indicates that the P2X7‐dependent release of cytokines in microglia is differentially regulated. GLIA 2014;62:592–607     

γ‐Aminobutyric acid‐mediated regulation of the activity‐dependent olfactory bulb dopaminergic phenotype

γ‐Aminobutyric acid (GABA) regulates the proliferation and migration of olfactory bulb (OB) interneuron progenitors derived from the subventricular zone (SVZ), but the role of GABA in the differentiation of these progenitors has been largely unexplored. This study examines the role of GABA in the differentiation of OB dopaminergic interneurons using neonatal forebrain organotypic slice cultures prepared from transgenic mice expressing green fluorescent protein (GFP) under the control of the tyrosine hydroxylase (Th) gene promoter (ThGFP). KCl‐mediated depolarization of the slices induced ThGFP expression. The addition of GABA to the depolarized slices further increased GFP fluorescence by inducing ThGFP expression in an additional set of periglomerular cells. These findings show that GABA promoted differentiation of SVZ‐derived OB dopaminergic interneurons and suggest that GABA indirectly regulated Th expression and OB dopaminergic neuron differentiation through an acceleration of the maturation rate for the dopaminergic progenitors. Additional studies revealed that the effect of GABA on ThGFP expression required activation of L‐ and P/Q‐type Ca²⁺ channels as well as GABA_A and GABA_B receptors. These voltage‐gated Ca²⁺ channels and GABA receptors have previously been shown to be required for the coexpressed GABAergic phenotype in the OB interneurons. Together, these findings suggest that Th expression and the differentiation of OB dopaminergic interneurons are coupled to the coexpressed GABAergic phenotype and demonstrate a novel role for GABA in neurogenesis. © 2009 Wiley‐Liss, Inc.     

Spinal injection of TNF‐α‐activated astrocytes produces persistent pain symptom mechanical allodynia by releasing monocyte chemoattractant protein‐1

Accumulating evidence suggests that spinal astrocytes play an important role in the genesis of persistent pain, by increasing the activity of spinal cord nociceptive neurons, i.e., central sensitization. However, direct evidence of whether activation of astrocytes is sufficient to induce chronic pain symptoms is lacking. We investigated whether and how spinal injection of activated astrocytes could produce mechanical allodynia, a cardinal feature of chronic pain, in naïve mice. Spinal (intrathecal) injection of astrocytes, which were prepared from cerebral cortexes of neonatal mice and briefly stimulated by tumor necrosis factor‐alpha (TNF‐α), induced a substantial decrease in paw withdrawal thresholds, indicating the development of mechanical allodynia. This allodynia was prevented when the astrocyte cultures were pretreated with a peptide inhibitor of c‐Jun N‐terminal kinase (JNK), D‐JNKI‐1. Of note a short exposure of astrocytes to TNF‐α for 15 min dramatically increased the expression and release of the chemokine monocyte chemoattractant protein‐1 (MCP‐1), even 3 h after TNF‐α withdrawal, in a JNK‐dependent manner. In parallel, intrathecal administration of TNF‐α induced MCP‐1 expression in spinal cord astrocytes. In particular, mechanical allodynia induced by TNF‐α‐activated astrocytes was reversed by a MCP‐1 neutralizing antibody. Finally, pretreatment of astrocytes with MCP‐1 siRNA attenuated astrocytes‐induced mechanical allodynia. Taken together, our results suggest that activated astrocytes are sufficient to produce persistent pain symptom in naïve mice by releasing MCP‐1. © 2010 Wiley‐Liss, Inc.     

The comparison of mouse full metallothionein‐1 versus α and β domains and metallothionein‐1‐to‐3 mutation following traumatic brain injury reveals different biological motifs

Traumatic injury to the brain is one of the leading causes of injury‐related death or disability, but current therapies are limited. Previously it has been shown that the antioxidant proteins metallothioneins (MTs) are potent neuroprotective factors in animal models of brain injury. The exogenous administration of MTs causes effects consistent with the roles proposed from studies in knock‐out mice. We herewith report the results comparing full mouse MT‐1 with the independent α and β domains, alone or together, in a cryoinjury model. The lesion of the cortex caused the mice to perform worse in the horizontal ladder beam and the rota‐rod tests; all the proteins showed a modest effect in the former test, while only full MT‐1 improved the performance of animals in the rota‐rod, and the α domain showed a rather detrimental effect. Gene expression analysis by RNA protection assay demonstrated that all proteins may alter the expression of host‐response genes such as GFAP, Mac1 and ICAM, in some cases being the β domain more effective than the α domain or even the full MT‐1. A MT‐1‐to‐MT‐3 mutation blunted some but not all the effects caused by the normal MT‐1, and in some cases increased its potency. Thus, splitting the two MT‐1 domains do not seem to eliminate all MT functions but certainly modifies them, and different motifs seem to be present in the protein underlying such functions. © 2010 Wiley‐Liss, Inc.     

17β‐Oestradiol Anti‐Inflammatory Effects in Primary Astrocytes Require Oestrogen Receptor β‐Mediated Neuroglobin Up‐Regulation

Neuroglobin (Ngb), so named after its initial discovery in brain neurones, has received great attention as a result of its neuroprotective effects both in vitro and in vivo. Recently, we demonstrated that, in neurones, Ngb is a 17β‐oestradiol (E₂) inducible protein that is pivotal for hormone‐induced anti‐apoptotic effects against H₂O₂ toxicity. The involvement of Ngb in other brain cell populations, as well as in other neuroprotective effects of E₂, is completely unknown at present. We demonstrate Ngb immunoreactivity in reactive astrocytes located in the proximity of a penetrating cortical injury in vivo and the involvement of Ngb in the E₂‐mediated anti‐inflammatory effect in primary cortical astrocytes. Upon binding to oestrogen receptor (ER)β, E₂ enhances Ngb levels in a dose‐dependent manner. Although with a lesser degree than E₂, the pro‐inflammatory stimulation with lipopolysaccharide (LPS) also induces the increase of Ngb protein levels via nuclear factor‐(NF)κB signal(s). Moreover, a negative cross‐talk between ER subtypes and NFκB signal(s) has been demonstrated. In particular, ERα‐activated signals prevent the NFκB‐mediated Ngb increase, whereas LPS impairs the ERβ‐induced up‐regulation of Ngb. Therefore, the co‐expression of both ERα and ERβ is pivotal for mediating E₂‐induced Ngb expression in the presence of NFκB‐activated signals. Interestingly, Ngb silencing prevents the effect of E₂ on the expression of inflammatory markers (i.e. interleukin 6 and interferon γ‐inducible protein 10). Ngb can be regarded as a key mediator of the different protective effects of E₂ in the brain, including protection against oxidative stress and the control of inflammation, both of which are at the root of several neurodegenerative diseases.