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1.
Brain ; 137(Pt 1): 78-91, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24277722

ABSTRACT

The chemokine fractalkine modulates microglial responses in neurodegenerative diseases, including tauopathies, but the mechanistic processes and their relevance in human brain pathologies is not yet known. Here, we show that hippocampal HT22 cells expressing human TAU(P301L) mutant protein produce fractalkine, which in microglia activates AKT, inhibits glycogen synthase kinase-3ß and upregulates the transcription factor NRF2/NFE2L2 and its target genes including heme oxygenase 1. In a mouse model of tauopathy based on stereotaxic delivery in hippocampus of an adeno-associated viral vector for expression of TAU(P301L), we confirmed that tau-injured neurons express fractalkine. NRF2- and fractalkine receptor-knockout mice did not express heme oxygenase 1 in microglia and exhibited increased microgliosis and astrogliosis in response to neuronal TAU(P301L) expression, demonstrating a crucial role of the fractalkine/NRF2/heme oxygenase 1 pathway in attenuation of the pro-inflammatory phenotype. The hippocampus of patients with Alzheimer's disease also exhibits increased expression of fractalkine in TAU-injured neurons that recruit microglia. These events correlated with increased levels of NRF2 and heme oxygenase 1 proteins, suggesting an attempt of the diseased brain to limit microgliosis. Our combined results indicate that fractalkine mobilizes NRF2 to limit over-activation of microglia and identify this new target to control unremitting neuroinflammation in tauopathies.


Subject(s)
Chemokine CX3CL1/pharmacology , Gliosis/drug therapy , Heme Oxygenase-1/metabolism , NF-E2-Related Factor 2/metabolism , Tauopathies/drug therapy , Aged , Aged, 80 and over , Alzheimer Disease/pathology , Animals , Blotting, Western , Brain/pathology , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cytosol/drug effects , Cytosol/metabolism , Female , Fluorescent Antibody Technique , Gliosis/etiology , Gliosis/pathology , Humans , Immunohistochemistry , Inflammation/pathology , Lentivirus/genetics , Male , Mice , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Tauopathies/complications , Tauopathies/pathology
2.
Mol Cell Biol ; 32(17): 3486-99, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22751928

ABSTRACT

The transcription factor NF-E2-related factor 2 (Nrf2) is a master regulator of a genetic program, termed the phase 2 response, that controls redox homeostasis and participates in multiple aspects of physiology and pathology. Nrf2 protein stability is regulated by two E3 ubiquitin ligase adaptors, Keap1 and ß-TrCP, the latter of which was only recently reported. Here, two-dimensional (2D) gel electrophoresis and site-directed mutagenesis allowed us to identify two serines of Nrf2 that are phosphorylated by glycogen synthase kinase 3ß (GSK-3ß) in the sequence DSGISL. Nuclear magnetic resonance studies defined key residues of this phosphosequence involved in docking to the WD40 propeller of ß-TrCP, through electrostatic and hydrophobic interactions. We also identified three arginine residues of ß-TrCP that participate in Nrf2 docking. Intraperitoneal injection of the GSK-3 inhibitor SB216763 led to increased Nrf2 and heme oxygenase-1 levels in liver and hippocampus. Moreover, mice with hippocampal absence of GSK-3ß exhibited increased levels of Nrf2 and phase 2 gene products, reduced glutathione, and decreased levels of carbonylated proteins and malondialdehyde. This study establishes the structural parameters of the interaction of Nrf2 with the GSK-3/ß-TrCP axis and its functional relevance in the regulation of Nrf2 by the signaling pathways that impinge on GSK-3.


Subject(s)
Glycogen Synthase Kinase 3/metabolism , NF-E2-Related Factor 2/chemistry , NF-E2-Related Factor 2/metabolism , beta-Transducin Repeat-Containing Proteins/metabolism , Amino Acid Sequence , Animals , Cells, Cultured , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3 beta , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , NF-E2-Related Factor 2/genetics , Phosphorylation , Protein Structure, Tertiary , Serine/genetics , Serine/metabolism , beta-Transducin Repeat-Containing Proteins/chemistry
3.
Hum Mol Genet ; 21(14): 3173-92, 2012 Jul 15.
Article in English | MEDLINE | ID: mdl-22513881

ABSTRACT

Although α-synuclein (α-SYN) aggregation is a hallmark of sporadic and familial Parkinson's disease (PD), it is not known how it contributes to early events of PD pathogenesis such as oxidative and inflammatory stress. Here, we addressed this question in a new animal model based on stereotaxic delivery of an adeno-associated viral vector (rAAV) for expression of human α-SYN in the ventral midbrain of mice lacking the transcription factor Nrf2 (Nrf2(-/-)). Two months after surgery, Nrf2(-/-) mice exhibited exacerbated degeneration of nigral dopaminergic neurons and increased dystrophic dendrites, reminiscent of Lewy neurites, which correlated with impaired proteasome gene expression and activity. Dopaminergic neuron loss was associated with an increase in neuroinflammation and gliosis that were intensified in Nrf2(-/-) mice. In response to exogenously added α-SYN, Nrf2(-/-) microglia failed to activate the expression of two anti-inflammatory genes, heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate quinone oxidorreductase-1 (NQO1). This impaired Nrf2 response correlated with a shift in the microglial activation profile, towards increased production of proinflammatory markers, IL-6, IL-1ß and iNOS and reduced phagocytic capacity of fluorescent beads, and lower messenger RNA levels for TAM receptors Axl and Mer. Postmortem brain tissue samples from patients in early- to middle-stage progression of PD showed increased HO-1 expression in astrocytes and microglia, suggesting an attempt of the diseased brain to compensate these hallmarks of PD through activation of the Nrf2 pathway. This study demonstrates that α-SYN and Nrf2 deficiency cooperate on protein aggregation, neuroinflammation and neuronal death and provides a bifactorial animal model to study early-stage PD.


Subject(s)
NF-E2-Related Factor 2/deficiency , Neurons/cytology , Parkinson Disease/genetics , Parkinson Disease/immunology , alpha-Synuclein/genetics , Animals , Cell Death , Cells, Cultured , Disease Models, Animal , Gene Expression , Heme Oxygenase-1/genetics , Heme Oxygenase-1/immunology , Humans , Mice , Mice, Knockout , Mice, Transgenic , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/immunology , Neurons/immunology , Parkinson Disease/pathology , Parkinson Disease/physiopathology , alpha-Synuclein/chemistry , alpha-Synuclein/immunology
4.
J Neuroinflammation ; 9: 8, 2012 Jan 16.
Article in English | MEDLINE | ID: mdl-22248049

ABSTRACT

BACKGROUND: Alzheimer's disease (AD) brain shows an ongoing inflammatory condition and non-steroidal anti-inflammatories diminish the risk of suffering the neurologic disease. Cannabinoids are neuroprotective and anti-inflammatory agents with therapeutic potential. METHODS: We have studied the effects of prolonged oral administration of transgenic amyloid precursor protein (APP) mice with two pharmacologically different cannabinoids (WIN 55,212-2 and JWH-133, 0.2 mg/kg/day in the drinking water during 4 months) on inflammatory and cognitive parameters, and on ¹8F-fluoro-deoxyglucose (¹8FDG) uptake by positron emission tomography (PET). RESULTS: Novel object recognition was significantly reduced in 11 month old Tg APP mice and 4 month administration of JWH was able to normalize this cognitive deficit, although WIN was ineffective. Wild type mice cognitive performance was unaltered by cannabinoid administration. Tg APP mice showed decreased ¹8FDG uptake in hippocampus and cortical regions, which was counteracted by oral JWH treatment. Hippocampal GFAP immunoreactivity and cortical protein expression was unaffected by genotype or treatment. In contrast, the density of Iba1 positive microglia was increased in Tg APP mice, and normalized following JWH chronic treatment. Both cannabinoids were effective at reducing the enhancement of COX-2 protein levels and TNF-α mRNA expression found in the AD model. Increased cortical ß-amyloid (Aß) levels were significantly reduced in the mouse model by both cannabinoids. Noteworthy both cannabinoids enhanced Aß transport across choroid plexus cells in vitro. CONCLUSIONS: In summary we have shown that chronically administered cannabinoid showed marked beneficial effects concomitant with inflammation reduction and increased Aß clearance.


Subject(s)
Amyloid beta-Peptides/metabolism , Cannabinoids/administration & dosage , Cognition Disorders/prevention & control , Encephalitis/prevention & control , Administration, Oral , Alzheimer Disease/complications , Alzheimer Disease/genetics , Amyloid beta-Protein Precursor/genetics , Analysis of Variance , Animals , Benzoxazines/administration & dosage , Choroid Plexus/metabolism , Choroid Plexus/pathology , Cognition Disorders/diagnostic imaging , Cognition Disorders/etiology , Corpus Striatum/drug effects , Corpus Striatum/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Disease Models, Animal , Encephalitis/diagnostic imaging , Encephalitis/etiology , Enzyme-Linked Immunosorbent Assay , Fluorodeoxyglucose F18/metabolism , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Mice , Mice, Transgenic , Microglia/drug effects , Microglia/pathology , Morpholines/administration & dosage , Naphthalenes/administration & dosage , Positron-Emission Tomography , RNA, Messenger/metabolism , Receptor, Cannabinoid, CB2/metabolism , Time Factors
5.
Antioxid Redox Signal ; 14(12): 2347-60, 2011 Jun 15.
Article in English | MEDLINE | ID: mdl-21254817

ABSTRACT

Current therapies for motor symptoms of Parkinson's disease (PD) are based on dopamine replacement. However, the disease progression remains unaffected, because of continuous dopaminergic neuron loss. Since oxidative stress is actively involved in neuronal death in PD, pharmacological targeting of the antioxidant machinery may have therapeutic value. Here, we analyzed the relevance of the antioxidant phase II response mediated by the transcription factor NF-E2-related factor 2 (Nrf2) on brain protection against the parkinsonian toxin methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Intraperitoneal administration of the potent Nrf2 activator sulforaphane (SFN) increased Nrf2 protein levels in the basal ganglia and led to upregulation of phase II antioxidant enzymes heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase (NQO1). In wild-type mice, but not in Nrf2-knockout mice, SFN protected against MPTP-induced death of nigral dopaminergic neurons. The neuroprotective effects were accompanied by a decrease in astrogliosis, microgliosis, and release of pro-inflammatory cytokines. These results provide strong pharmacokinetic and biochemical evidence for activation of Nrf2 and phase II genes in the brain and also offer a neuroprotective strategy that may have clinical relevance for PD therapy.


Subject(s)
Basal Ganglia/physiology , NF-E2-Related Factor 2/metabolism , Parkinsonian Disorders/drug therapy , Thiocyanates/therapeutic use , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Animals , Anticarcinogenic Agents/therapeutic use , Antioxidants/metabolism , Basal Ganglia/drug effects , Disease Progression , Dopamine/metabolism , Dopamine Agents/pharmacology , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Isothiocyanates , Mice , Mice, Knockout , NAD(P)H Dehydrogenase (Quinone)/genetics , NAD(P)H Dehydrogenase (Quinone)/metabolism , NF-E2-Related Factor 2/genetics , Neuroprotective Agents/pharmacology , Neurotoxins/pharmacology , Oxidative Stress , Parkinsonian Disorders/genetics , Parkinsonian Disorders/physiopathology , Sulfoxides
6.
PLoS One ; 5(7): e11838, 2010 Jul 28.
Article in English | MEDLINE | ID: mdl-20676377

ABSTRACT

BACKGROUND: The transcription factor Nrf2 (NF-E2-related factor 2) and its target gene products, including heme oxygenase-1 (HO-1), elicit an antioxidant response that may have therapeutic value for Parkinson's disease (PD). However, HO-1 protein levels are increased in dopaminergic neurons of Parkinson's disease (PD) patients, suggesting its participation in free-iron deposition, oxidative stress and neurotoxicity. Before targeting Nrf2 for PD therapy it is imperative to determine if HO-1 is neurotoxic or neuroprotective in the basal ganglia. METHODOLOGY: We addressed this question by comparing neuronal damage and gliosis in Nrf2- or HO-1-knockout mice submitted to intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for five consecutive days. Nrf2-knockout mice showed exacerbated gliosis and dopaminergic nigrostriatal degeneration, as determined by immunohistochemical staining of tyrosine hydroxylase in striatum (STR) and substantia nigra (SN) and by HPLC determination of striatal dopamine and 3,4- dihydroxyphenylacetic acid (DOPAC). On the other hand, the severity of gliosis and dopaminergic degeneration in HO-1-null mice was neither increased nor reduced. Regarding free-iron deposition, both Nrf2- and HO-1-deficient mice exhibited similar number of deposits as determined by Perl's staining, therefore indicating that these proteins do not contribute significantly to iron accumulation or clearance in MPTP-induced Parkinsonism. CONCLUSIONS: These results suggest that HO-1 does not protect or enhance the sensitivity to neuronal death in Parkinson's disease and that pharmacological or genetic intervention on Nrf2 may provide a neuroprotective benefit as add on therapy with current symptomatic protocols.


Subject(s)
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Heme Oxygenase-1/metabolism , NF-E2-Related Factor 2/physiology , Animals , Chromatography, High Pressure Liquid , Dopamine/metabolism , Fluorescent Antibody Technique , Gliosis/chemically induced , Gliosis/genetics , Heme Oxygenase-1/genetics , Immunoblotting , Immunohistochemistry , Iron/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-E2-Related Factor 2/genetics , Neurotoxins/pharmacology , Parkinson Disease/genetics , Parkinson Disease/metabolism , Polymerase Chain Reaction
7.
Glia ; 58(5): 588-98, 2010 Apr.
Article in English | MEDLINE | ID: mdl-19908287

ABSTRACT

Neural injury leads to inflammation and activation of microglia that in turn may participate in progression of neurodegeneration. The mechanisms involved in changing microglial activity from beneficial to chronic detrimental neuroinflammation are not known but reactive oxygen species (ROS) may be involved. We have addressed this question in Nrf2-knockout mice, with hypersensitivity to oxidative stress, submitted to daily inoculation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 4 weeks. Basal ganglia of these mice exhibited a more severe dopaminergic dysfunction than wild type littermates in response to MPTP. The amount of CD11b-positive/CD45-highly-stained cells, indicative of peripheral macrophage infiltration, did not increase significantly in response to MPTP. However, Nrf2-deficient mice exhibited more astrogliosis and microgliosis as determined by an increase in messenger RNA and protein levels for GFAP and F4/80, respectively. Inflammation markers characteristic of classical microglial activation, COX-2, iNOS, IL-6, and TNF-alpha were also increased and, at the same time, anti-inflammatory markers attributable to alternative microglial activation, such as FIZZ-1, YM-1, Arginase-1, and IL-4 were decreased. These results were confirmed in microglial cultures stimulated with apoptotic conditioned medium from MPP(+)-treated dopaminergic cells, further demonstrating a role of Nrf2 in tuning balance between classical and alternative microglial activation. This study demonstrates a crucial role of Nrf2 in modulation of microglial dynamics and identifies Nrf2 as molecular target to control microglial function in Parkinson's disease (PD) progression.


Subject(s)
Brain/metabolism , Inflammation/etiology , MPTP Poisoning/complications , MPTP Poisoning/pathology , Microglia/physiology , NF-E2-Related Factor 2/metabolism , Analysis of Variance , Animals , Antigens, Differentiation/metabolism , Brain/pathology , CD11b Antigen/metabolism , Calcium-Binding Proteins/metabolism , Cell Line, Transformed , Culture Media, Conditioned/pharmacology , Cyclooxygenase 2/metabolism , Cytokines/metabolism , Disease Models, Animal , Dopamine/metabolism , Dopamine Plasma Membrane Transport Proteins/metabolism , Flow Cytometry/methods , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Glial Fibrillary Acidic Protein/metabolism , Gliosis/etiology , Inflammation/metabolism , Leukocyte Common Antigens/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microfilament Proteins , NF-E2-Related Factor 2/deficiency , Neurons/chemistry , Neurons/drug effects , Neurons/metabolism , Nitric Oxide Synthase Type II/metabolism , Reactive Oxygen Species/metabolism , Tyrosine 3-Monooxygenase/metabolism
8.
Curr Opin Neurol ; 22(3): 308-14, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19359988

ABSTRACT

PURPOSE OF REVIEW: This review discusses some of the emerging concepts on how modulation of redox homeostasis in microglia is crucial to restore its inactive state and modulate inflammation in neurologic diseases. RECENT FINDINGS: Reactive oxygen species generated by microglia help to eliminate pathogens in the extracellular milieu but also act on microglia itself, altering the intracellular redox balance and functioning as second messengers in induction of proinflammatory genes. Recent findings indicate that restoration of redox balance may be determinant in driving microglia back to the resting state. Thus, deficiency of the transcription factor NF-E2-related factor-2 (Nrf2), guardian of redox homeostasis, results in exacerbated inflammatory response to neurotoxins whereas inducers of Nrf2 and its target heme oxygenase-1 downmodulate inflammation. SUMMARY: New available information indicates that downregulation of microglia is a matter closely correlated with control of oxidative stress in this cell type and points to Nrf2 as a new therapeutic target for modulation of inflammation in neurodegenerative diseases.


Subject(s)
Inflammation/immunology , Microglia/metabolism , Neurodegenerative Diseases , Animals , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Homeostasis , Humans , Inflammation/genetics , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Neurodegenerative Diseases/immunology , Neurodegenerative Diseases/pathology , Oxidation-Reduction , Reactive Oxygen Species/metabolism , Signal Transduction/physiology
9.
J Immunol ; 181(1): 680-9, 2008 Jul 01.
Article in English | MEDLINE | ID: mdl-18566435

ABSTRACT

Because chronic neuroinflammation is a hallmark of neurodegenerative diseases and compromises neuron viability, it is imperative to discover pharmacologic targets to modulate the activation of immune brain cells, the microglia. In this study, we identify the transcription factor Nrf2, guardian of redox homeostasis, as such target in a model of LPS-induced inflammation in mouse hippocampus. Nrf2 knockout mice were hypersensitive to the neuroinflammation induced by LPS, as determined by an increase in F4/80 mRNA and protein, indicative of an increase in microglial cells, and in the inflammation markers inducible NO synthase, IL-6, and TNF-alpha, compared with the hippocampi of wild-type littermates. The aliphatic isothiocyanate sulforaphane elicited an Nrf2-mediated antioxidant response in the BV2 microglial cell line, determined by flow cytometry of cells incubated with the redox sensitive probe dihydrodichlorofluorescein diacetate, and by the Nrf2-dependent induction of the phase II antioxidant enzyme heme oxygenase-1. Animals treated with sulforaphane displayed a 2-3-fold increase in heme oxygenase-1, a reduced abundance of microglial cells in the hippocampus and an attenuated production of inflammation markers (inducible NO synthase, IL-6, and TNF-alpha) in response to LPS. Considering that release of reactive oxygen species is a property of activated microglia, we propose a model in which late induction of Nrf2 intervenes in the down-regulation of microglia. This study opens the possibility of targeting Nrf2 in brain as a means to modulate neuroinflammation.


Subject(s)
Encephalitis/metabolism , Encephalitis/pathology , NF-E2-Related Factor 2/metabolism , Animals , Cell Line , Encephalitis/chemically induced , Encephalitis/genetics , Gene Expression Regulation, Enzymologic/drug effects , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Hippocampus/drug effects , Hippocampus/metabolism , Isothiocyanates , Lipopolysaccharides/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microglia/drug effects , Microglia/metabolism , NF-E2-Related Factor 2/deficiency , NF-E2-Related Factor 2/genetics , Sulfoxides , Thiocyanates/pharmacology
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