The EC (entorhinal cortex) is fundamental for cognitive and mnesic functions. Thus damage to this area appears as a key element in the progression of AD (Alzheimer's disease), resulting in memory deficits arising from neuronal and synaptic alterations as well as glial malfunction. In this paper, we have performed an in-depth analysis of astroglial morphology in the EC by measuring the surface and volume of the GFAP (glial fibrillary acidic protein) profiles in a triple transgenic mouse model of AD [3xTg-AD (triple transgenic mice of AD)]. We found significant reduction in both the surface and volume of GFAP-labelled profiles in 3xTg-AD animals from very early ages (1 month) when compared with non-Tg (non-transgenic) controls (48 and 54%, reduction respectively), which was sustained for up to 12 months (33 and 45% reduction respectively). The appearance of Aβ (amyloid β-peptide) depositions at 12 months of age did not trigger astroglial hypertrophy; nor did it result in the close association of astrocytes with senile plaques. Our results suggest that the AD progressive cognitive deterioration can be associated with an early reduction of astrocytic arborization and shrinkage of the astroglial domain, which may affect synaptic connectivity within the EC and between the EC and other brain regions. In addition, the EC seems to be particularly vulnerable to AD pathology because of the absence of evident astrogliosis in response to Aβ accumulation. Thus we can consider that targeting astroglial atrophy may represent a therapeutic strategy which might slow down the progression of AD.
Alzheimer's disease; astrocyte; dementia; entorhinal cortex; glial fibrillary acidic protein (GFAP); memory; AD, Alzheimer's disease; Aβ, amyloid β-peptide; EC, entorhinal cortex; GFAP, glial fibrillary acidic protein; GFAP-IR, glial fibrillary acidic protein-immunoreactive; HC, hippocampus; LEC, lateral entorhinal cortex; NFT, neurofibril tangle; non-Tg, non-transgenic; Nv, numerical density; PB, phosphate buffer; PHF, paired helical filament; RT, room temperature; SP, senile plaque; 3xTg-AD, triple transgenic mice of AD; TS, Tizma-based saline
While the presence of an inflammatory response in AD (Alzheimer's disease) is well known, the data on inflammation are conflicting, suggesting that inflammation either attenuates pathology, exacerbates it or has no effect. Our goal was to more fully characterize the inflammatory response in APP (amyloid precursor protein) transgenic mice with and without disease progression. In addition, we have examined how anti-Aβ (amyloid β-peptide) immunotherapy alters this inflammatory response. We have used quantitative RT–PCR (reverse transcription–PCR) and protein analysis to measure inflammatory responses ranging from pro-inflammatory to anti-inflammatory and repair factors in transgenic mice that develop amyloid deposits only (APPSw) and amyloid deposits with progression to tau pathology and neuron loss [APPSw/NOS2−/− (nitric oxide synthase 2−/−)]. We also examined tissues from previously published immunotherapy studies. These studies were a passive immunization study in APPSw mice and an active vaccination study in APPSw/NOS2−/− mice. Both studies have already been shown to lower amyloid load and improve cognition. We have found that amyloid deposition is associated with high expression of alternative activation and acquired deactivation genes and low expression of pro-inflammatory genes, whereas disease progression is associated with a mixed phenotype including increased levels of some classical activation factors. Immunotherapy targeting amyloid deposition in both mouse models resulted in decreased alternative inflammatory markers and, in the case of passive immunization, a transient increase in pro-inflammatory markers. Our results suggest that an alternative immune response favours retention of amyloid deposits in the brain, and switching away from this state by immunotherapy permits removal of amyloid.
alternative activation; amyloid deposition; immunotherapy; microglia; neuroinflammation; Aβ, amyloid β-peptide; AD, Alzheimer's disease; AG1, arginase 1; APP, amyloid precursor protein; IL-1β, interleukin 1β; LPS, lipopolysaccharide; MMP, matrix metalloprotease; MR, mannose receptor; Mrc1, mannose receptor C1; NFT, neurofibrillary tangle; NOS2, nitric oxide synthase 2; RT–PCR, reverse transcription–PCR; SPHK, sphingosine kinase; TGFβ, transforming growth factor β; TNFα, tumour necrosis factor α; WT, wild-type
Studies in humans and animal models link maternal infection and imbalanced levels of inflammatory mediators in the foetal brain to the aetiology of neuropsychiatric disorders. In a number of animal models, it was shown that exposure to viral or bacterial agents during a period that corresponds to the second trimester in human gestation triggers brain and behavioural abnormalities in the offspring. However, little is known about the early cellular and molecular events elicited by inflammation in the foetal brain shortly after maternal infection has occurred. In this study, maternal infection was mimicked by two consecutive intraperitoneal injections of 200 μg of LPS (lipopolysaccharide)/kg to timed-pregnant rats at GD15 (gestational day 15) and GD16. Increased thickness of the CP (cortical plate) and hippocampus together with abnormal distribution of immature neuronal markers and decreased expression of markers for neural progenitors were observed in the LPS-exposed foetal forebrains at GD18. Such effects were accompanied by decreased levels of reelin and the radial glial marker GLAST (glial glutamate transporter), and elevated levels of pro-inflammatory cytokines in maternal serum and foetal forebrains. Foetal inflammation elicited by maternal injections of LPS has discrete detrimental effects on brain development. The early biochemical and morphological changes described in this work begin to explain the sequelae of early events that underlie the neurobehavioural deficits reported in humans and animals exposed to prenatal insults.
prenatal inflammation; lipopolysaccharide (LPS); brain development; cytokine; maternal infection; neurodevelopmental disorder; Arc, activity-regulated cytoskeletal-associated protein; CNS, central nervous system; CP, cortical plate; DAPI, 4′,6-diamidino-2-phenylindole; GD, gestational day; GFAP, glial fibrillary acidic protein; GLAST, glial glutamate transporter; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H&E, haematoxylin and eosin; IEG, immediate early gene; IL, interleukin; IZ, intermediate zone; LPS, lipopolysaccharide; MgZ, marginal zone; PFA, paraformaldehyde; poly(I:C), polyriboinosinic-polyribocytidilic acid; qRT–PCR, quantitative real-time PCR; SVZ/VZ, subventricular zone/ventricular zone; TNFα, tumour necrosis factor α
YY1 (Yin and Yang 1) is a multifunctional, ubiquitously expressed, zinc finger protein that can act as a transcriptional activator, repressor, or initiator element binding protein. Previous studies have shown that YY1 modulates the activity of reporter genes driven by the myelin PLP (proteolipid protein) (PLP1/Plp1) promoter. However, it is known that Plp1 intron 1 DNA contains regulatory elements that are required for the dramatic increase in gene activity, coincident with the active myelination period of CNS (central nervous system) development. The intron in mouse contains multiple prospective YY1 target sites including one within a positive regulatory module called the ASE (anti-silencer/enhancer) element. Results presented here demonstrate that YY1 has a negative effect on the activity of a Plp1-lacZ fusion gene [PLP(+)Z] in an immature oligodendroglial cell line (Oli-neu) that is mediated through sequences present in Plp1 intron 1 DNA. Yet YY1 does not bind to its alleged site in the ASE (even though the protein is capable of recognizing a target site in the promoter), indicating that the down-regulation of PLP(+)Z activity by YY1 in Oli-neu cells does not occur through a direct interaction of YY1 with the ASE sequence. Previous studies with Yy1 conditional knockout mice have demonstrated that YY1 is essential for the differentiation of oligodendrocyte progenitors. Nevertheless, the current study suggests that YY1 functions as a repressor (not an activator) of Plp1 gene expression in immature oligodendrocytes. Perhaps YY1 functions to keep the levels of PLP in check in immature cells before vast quantities of the protein are needed in mature myelinating oligodendrocytes.
gene expression; gene regulation; myelin proteolipid protein gene; Oli-neu cells; repression; YY1; ASE, anti-silencer/enhancer; β-gal, β-galactosidase; CMV, cytomegalovirus; CNS, central nervous system; DMEM/F-12, Dulbecco's modified Eagle medium/Ham's F-12 nutrient mixture; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; F-YY1, FLAG-tagged YY1; PLP, proteolipid protein; RLU, relative light units; RSVL, Rous sarcoma virus-luciferase; TBST, Tris-buffered saline with Tween-20; YY1, Yin and Yang 1
The neuronal RNA-binding protein HuD plays a critical role in the post-transcriptional regulation of short-lived mRNAs during the initial establishment and remodelling of neural connections. We have generated transgenic mice overexpressing this protein (HuD-Tg) in adult DGCs (dentate granule cells) and shown that their mossy fibres contain high levels of GAP-43 (growth-associated protein 43) and exhibit distinct morphological and electrophysiological properties. To investigate the basis for these changes and identify other molecular targets of HuD, DGCs from HuD-Tg and control mice were collected by LCM (laser capture microscopy) and RNAs analysed using DNA microarrays. Results show that 216 known mRNAs transcripts and 63 ESTs (expressed sequence tags) are significantly up-regulated in DGCs from these transgenic mice. Analyses of the 3′-UTRs (3′-untranslated regions) of these transcripts revealed an increased number of HuD-binding sites and the presence of several known instability-conferring sequences. Among these, the mRNA for TTR (transthyretin) shows the highest level of up-regulation, as confirmed by qRT–PCR (quantitative reverse transcription–PCR) and ISH (in situ hybridization). GO (gene ontology) analyses of up-regulated transcripts revealed a large over-representation of genes associated with neural development and axogenesis. In correlation with these gene expression changes, we found an increased length of the infrapyramidal mossy fibre bundle in HuD-Tg mice. These results support the notion that HuD stabilizes a number of developmentally regulated mRNAs in DGCs, resulting in increased axonal elongation.
axonal outgrowth; dentate granule cell; gene profiling; HuD; post-transcriptional mechanisms; RNA-binding protein; αCaMKII, α-Ca2+/calmodulin-dependent protein kinase II; ARE, AU-rich element; ARED, ARE database; CP, choroid plexus; Dcx, doublecortin; DGC, dentate granule cell; DIG, digoxigenin; EST, expressed sequence tag; FISH, fluorescence in situ hybridization; GAP-43, growth-associated protein 43; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GO, gene ontology; GRE, GU-rich element; IPA, ingenuity pathway analysis; IPB, infrapyramidal bundle; ISH, in situ hybridization; KO, knockout; LCM, laser capture microscopy; Mtap1b, microtubule-associated protein 1b; NIH, National Institutes of Health; PFA, paraformaldehyde; PPF, paired-pulse facilitation; qRT–PCR, quantitative reverse transcription–PCR; RNA-IP, RNA-immunoprecipitation; SMA, spinal muscular atrophy; SPB, suprapyramidal bundle; T3, 3,3′,5-tri-iodothyronine; T4, thyroxine; TTR, transthyretin; 3′-UTR, 3′-untranslated region; WT, wild-type
Infantile Krabbe disease results in the accumulation of lipid-raft-associated galactosylsphingosine (psychosine), demyelination, neurodegeneration and premature death. Recently, axonopathy has been depicted as a contributing factor in the progression of neurodegeneration in the Twitcher mouse, a bona fide mouse model of Krabbe disease. Analysis of the temporal-expression profile of MBP (myelin basic protein) isoforms showed unexpected increases of the 14, 17 and 18.5 kDa isoforms in the sciatic nerve of 1-week-old Twitcher mice, suggesting an abnormal regulation of the myelination process during early postnatal life in this mutant. Our studies showed an elevated activation of the pro-apoptotic protease caspase 3 in sciatic nerves of 15- and 30-day-old Twitcher mice, in parallel with increasing demyelination. Interestingly, while active caspase 3 was clearly contained in peripheral axons at all ages, we found no evidence of caspase accumulation in the soma of corresponding mutant spinal cord motor neurons. Furthermore, active caspase 3 was found not only in unmyelinated axons, but also in myelinated axons of the mutant sciatic nerve. These results suggest that axonal caspase activation occurs before demyelination and following a dying-back pattern. Finally, we showed that psychosine was sufficient to activate caspase 3 in motor neuronal cells in vitro in the absence of myelinating glia. Taken together, these findings indicate that degenerating mechanisms actively and specifically mediate axonal dysfunction in Krabbe disease and support the idea that psychosine is a pathogenic sphingolipid sufficient to cause axonal defects independently of demyelination.
apoptosis; caspase 3; dying-back pathology; Krabbe disease; leukodystrophies; myelin; Twitcher mouse; APC, adenomatous polyposis coli; CCT, central conduction time; CNS, central nervous system; CMAP, compound motor action potential; cMEP, cortical motor evoked potential; DAB, diaminobenzidine; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; MCV, motor conduction velocity; NCAM, neural cell adhesion molecule; NF-H, neurofilament heavy chain; PFA, paraformaldehyde; WT, wild-type
MS (multiple sclerosis) is a chronic autoimmune and neurodegenerative pathology of the CNS (central nervous system) affecting approx. 2.5 million people worldwide. Current and emerging DMDs (disease-modifying drugs) predominantly target the immune system. These therapeutic agents slow progression and reduce severity at early stages of MS, but show little activity on the neurodegenerative component of the disease. As the latter determines permanent disability, there is a critical need to pursue alternative modalities. VIP (vasoactive intestinal peptide) and PACAP (pituitary adenylate cyclase-activating peptide) have potent anti-inflammatory and neuroprotective actions, and have shown significant activity in animal inflammatory disease models including the EAE (experimental autoimmune encephalomyelitis) MS model. Thus, their receptors have become candidate targets for inflammatory diseases. Here, we will discuss the immunomodulatory and neuroprotective actions of VIP and PACAP and their signalling pathways, and then extensively review the structure–activity relationship data and biophysical interaction studies of these peptides with their cognate receptors.
autoimmunity; drug-design; experimental autoimmune encephalomyelitis (EAE); neuroprotection; PACAP; VIP; AC, adenylate cyclase; AD, Alzheimer's disease; ADNF, activity-dependent neurotrophic factor; ADNP, activity-dependent neuroprotective protein; APC, antigen-presenting cells; BM, bone marrow; Bz-Phe, benzophenone-Phe; CCR, CC chemokine receptor, ; CIA, collagen-induced arthritis; CNS, central nervous system; DC, dendritic cell; DMD, disease-modifying drug; DSS, dextran sodium sulfate; EAE, experimental autoimmune encephalomyelitis; FDA, Food and Drug Administration; GPCR, G-protein-coupled receptor; IFN, interferon; IL-6, interleukin 6; JNK, c-Jun N-terminal kinase; KO, knockout; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein 1; MIP-2, macrophage inflammatory protein 2; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; MSC, mesenchymal stem cell; NAP, neutrophil-activating protein; NMDA, N-methyl-d-aspartate; PACAP, pituitary adenylate cyclase-activating peptide; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PMCAO, permanent middle cerebral artery occlusion; PPMS, primary progressive form of MS; RAMP, receptor-activity-modifying protein; RANTES, regulated upon activation, normal T-cell expressed and secreted; SAPK, stress-activated protein kinase; SCG, superior cervical ganglion; SPMS, secondary progressive form of MS; S-SCAM, synaptic scaffolding molecule; STAT, signal transducer and activator of transcription; TGFβ, transforming growth factor β; TM, transmembrane; TNFα, tumour necrosis factor α; Treg, regulatory T; VIP, vasoactive intestinal peptide; WT, wild-type
Most malignant brain tumours contain various numbers of cells with characteristics of activated or dysmorphic macrophages/microglia. These cells are generally considered part of the tumour stroma and are often described as TAM (tumour-associated macrophages). These types of cells are thought to either enhance or inhibit brain tumour progression. Recent evidence indicates that neoplastic cells with macrophage characteristics are found in numerous metastatic cancers of non-CNS (central nervous system) origin. Evidence is presented here suggesting that subpopulations of cells within human gliomas, specifically GBM (glioblastoma multiforme), are neoplastic macrophages/microglia. These cells are thought to arise following mitochondrial damage in fusion hybrids between neoplastic stem cells and macrophages/microglia.
fusion; glioblastoma multiforme; glioma; macrophage; microglia; phagocytosis; AIF1, allograft inflammatory factor 1; CNS, central nervous system; GFAP, glial fibrillary acidic protein; GBM, glioblastoma multiforme; IL, interleukin; MDSC, myeloid-derived suppressor cell; MNGC, multinucleated giant cell; PXA, pleomorphic xanthoastrocytoma; ROS, reactive oxygen species; RTG, retrograde; TAM, tumour-associated macrophage; VM, vasculogenic mimicry
GBM (glioblastoma multiforme) is a highly aggressive brain tumour with very poor prognosis despite multi-modalities of treatment. Furthermore, recent failure of targeted therapy for these tumours highlights the need of appropriate rodent models for preclinical studies. In this review, we highlight the most commonly used rodent models (U251, U86, GL261, C6, 9L and CNS-1) with a focus on the pathological and genetic similarities to the human disease. We end with a comprehensive review of the CNS-1 rodent model.
U251; U87; GL261; C6; 9L; CNS-1; Akt, protein kinase B; BBB, blood–brain barrier; BEHAB, brain-enriched hyaluronan-binding; CNS, central nervous system; CXCR4, CXC chemokine receptor 4; EGFR, endothelial growth factor receptor; GBM, glioblastoma multiforme; GFAP, glial fibrillary acidic protein; H/E, haematoxylin/eosin; HIF-1α, hypoxia-inducible factor-1α; IGF-1, insulin-like growth factor 1; IL-1α, interleukin-1α; MMP, matrix metalloproteinase; MNU, methylnitrosourea; MRI, magnetic resonance imaging; NCAM, neural cell adhesion molecule; PI3K, phosphoinositide 3-kinase; TNFα, tumour necrosis factor α; VEGF, vascular endothelial growth factor; PTEN, phosphatase and tensin homologue deleted on chromosome 10
Hypoxic preconditioning reprogrammes the brain's response to subsequent H/I (hypoxia–ischaemia) injury by enhancing neuroprotective mechanisms. Given that astrocytes normally support neuronal survival and function, the purpose of the present study was to test the hypothesis that a hypoxic preconditioning stimulus would activate an adaptive astrocytic response. We analysed several functional parameters 24 h after exposing rat pups to 3 h of systemic hypoxia (8% O2). Hypoxia increased neocortical astrocyte maturation as evidenced by the loss of GFAP (glial fibrillary acidic protein)-positive cells with radial morphologies and the acquisition of multipolar GFAP-positive cells. Interestingly, many of these astrocytes had nuclear S100B. Accompanying their differentiation, there was increased expression of GFAP, GS (glutamine synthetase), EAAT-1 (excitatory amino acid transporter-1; also known as GLAST), MCT-1 (monocarboxylate transporter-1) and ceruloplasmin. A subsequent H/I insult did not result in any further astrocyte activation. Some responses were cell autonomous, as levels of GS and MCT-1 increased subsequent to hypoxia in cultured forebrain astrocytes. In contrast, the expression of GFAP, GLAST and ceruloplasmin remained unaltered. Additional experiments utilized astrocytes exposed to exogenous dbcAMP (dibutyryl-cAMP), which mimicked several aspects of the preconditioning response, to determine whether activated astrocytes could protect neurons from subsequent excitotoxic injury. dbcAMP treatment increased GS and glutamate transporter expression and function, and as hypothesized, protected neurons from glutamate excitotoxicity. Taken altogether, these results indicate that a preconditioning stimulus causes the precocious differentiation of astrocytes and increases the acquisition of multiple astrocytic functions that will contribute to the neuroprotection conferred by a sublethal preconditioning stress.
cell death; glutamate; glutamine synthetase; excitotoxicity; stroke; transporter; CNS, central nervous system; CP, ceruloplasmin; dbcAMP, dibutyryl-cAMP; DMEM, Dulbecco's modified Eagle's medium; EAAT-1, excitatory amino acid transporter-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; GS, glutamine synthetase; HBSS, Hepes-buffered salt solution; H/I, hypoxia–ischaemia; IL-1, interleukin-1; LDH, lactate dehydrogenase; MCT-1, monocarboxylate transporter-1; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; P6, postnatal day 6; TBS, Tris-buffered saline
OLs (oligodendrocytes) are the myelinating cells of the CNS (central nervous system), wrapping axons in conductive sheathes to ensure effective transmission of neural signals. The regulation of OL development, from precursor to mature myelinating cell, is controlled by a variety of inhibitory and inductive signalling factors. The dorsal spinal cord contains signals that inhibit OL development, possibly to prevent premature and ectopic precursor differentiation. The Wnt and BMP (bone morphogenic protein) signalling pathways have been identified as dorsal spinal cord signals with overlapping temporal activity, and both have similar inhibitory effects on OL differentiation. Both these pathways feature prominently in many developmental processes and demyelinating events after injury, and they are known to interact in complex inductive, inhibitive and synergistic manners in many developing systems. The interaction between BMP and Wnt signalling in OL development, however, has not been extensively explored. In the present study, we examine the relationship between the canonical Wnt and BMP pathways. We use pharmacological and genetic paradigms to show that both Wnt3a and BMP4 will inhibit OL differentiation in vitro. We also show that when the canonical BMP signalling pathway is blocked, neither Wnt3a nor BMP4 have inhibitory effects on OL differentiation. In contrast, abrogating the Wnt signalling pathway does not alter the actions of BMP4 treatment. Our results indicate that the BMP signalling pathway is necessary for the canonical Wnt signalling pathway to exert its effects on OL development, but not vice versa, suggesting that Wnt signals upstream of BMP.
bone morphogenic protein (BMP); development; glia; myelin; oligodendrocyte (OL); Wnt; β-Cat-null, β-catenin loss of function mutant mice; Bmpr1 DKO, Bmpr1 double knockout; BMP, bone morphogenic protein; CNP, 2′,3′ cyclic nucleotide 3′-phosphodiesterase; CNS, central nervous system; DAPI, 4′,6-diamidino-2-phenylindole; Dkk-1, Dikkopf-1; DKO, double knockout; DM, differentiation medium; GalC, galactocerebroside; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; HDAC, histone deacetylase; HRP, horseradish peroxidase; ID, inhibitor of DNA-binding protein; IHC, immunohistochemistry; KO, knockout; LEF, lymphoid enhancer factor; MBP, myelin basic protein; OL, oligodendrocyte; OPC, OL precursor cell; PLP, proteolipid protein; QPCR, quantitative real-time PCR; Shh, sonic hedgehog; TCF, T-cell factor
HD (Huntington's disease) is characterized by dysfunction and death of striatal MSNs (medium-sized spiny neurons). Excitotoxicity, transcriptional dysregulation and mitochondrial abnormalities are among the mechanisms that are proposed to play roles in HD pathogenesis. To determine the extent of cell-autonomous effects of mhtt (mutant huntingtin) protein on vulnerability to excitotoxic insult in MSNs in vivo, we measured the number of degenerating neurons in response to intrastriatal injection of QA (quinolinic acid) in presymptomatic and symptomatic transgenic (D9-N171-98Q, also known as DE5) mice that express mhtt in MSNs but not in cortex. After QA, the number of degenerating neurons in presymptomatic DE5 mice was not significantly different from the number in WT (wild-type) controls, suggesting the early, increased vulnerability to excitotoxicity demonstrated in other HD mouse models has a largely non-cell-autonomous component. Conversely, symptomatic DE5 mice showed significantly fewer degenerating neurons relative to WT, implying the resistance to excitotoxicity observed at later ages has a primarily cell-autonomous origin. Interestingly, mitochondrial complex II respiration was enhanced in striatum of symptomatic mice, whereas it was reduced in presymptomatic mice, both relative to their age-matched controls. Consistent with the QA data, MSNs from symptomatic mice showed decreased NMDA (N-methyl-d-aspartate) currents compared with age-matched controls, suggesting that in addition to aging, cell-autonomous mechanisms mitigate susceptibility to excitotoxicity in the symptomatic stage. Also, symptomatic DE5 mice did not display some of the electrophysiological alterations present in other HD models, suggesting that blocking the expression of mhtt in cortical neurons may restore corticostriatal function in HD.
complex II; excitotoxicity; Huntington's disease (HD); mitochondria; N-methyl-d-aspartate (NMDA); striatum; ACSF, artificial cerebrospinal fluid; EPSC, excitatory postsynaptic current; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; FJC, Fluoro-Jade C; GABAA, γ-aminobutyric acid type A; HD, Huntington's disease; htt, huntingtin; IP, intraperitoneally; IPSC, inhibitory postsynaptic current; mEPSC, miniature EPSC; mhtt, mutant htt; MSN, medium-sized spiny neuron; NMDA, N-methyl-d-aspartate; NMDAR, NMDA receptor; PGC-1α, peroxisome-proliferator-activated receptor γ co-activator-1α; QA, quinolinic acid; sEPSC, spontaneous EPSC; sIPSC, spontaneous IPSC; TMPD, N,N,N′,N′-tetramethylphenylenediamine; TTX, tetrodotoxin; WT, wild-type
Neurons born in the postnatal SVZ (subventricular zone) must migrate a great distance before becoming mature interneurons of the OB (olfactory bulb). During migration immature OB neurons maintain an immature morphology until they reach their destination. While the morphological development of these cells must be tightly regulated, the cellular pathways responsible are still largely unknown. Our results show that the non-canonical Wnt pathway induced by Wnt5a is important for the morphological development of OB interneurons both in vitro and in vivo. Additionally, we demonstrate that non-canonical Wnt signalling works in opposition to canonical Wnt signalling in neural precursors from the SVZ in vitro. This represents a novel role for Wnt5a in the development of OB interneurons and suggests that canonical and non-canonical Wnt pathways dynamically oppose each other in the regulation of dendrite maturation.
dendrites; dkk1; non-canonical; sfrp1; subventricular zone (SVZ); wnt signalling; BMP, bone morphogenic protein; CB, calbindin; CSF, cerebrospinal fluid; DAPI, 4′,6-diamidino-2-phenylindole; DKK-1, Dickkopf-related protein 1; JNK, c-Jun N-terminal kinase; LEF, lymphoid enhancer factor; LRP5/6, LDL (low-density lipoprotein)-receptor-related protein 5/6; NPC, neural progenitor cell; OB, olfactory bulb; OSN, olfactory sensory axon; PFA, paraformaldehyde; RMS, rostral migratory stream; sFRP-1, secreted frizzled-related protein 1; SVZ, subventricular zone; TDBTN, total dendritic branch tip number; TH, tyrosine hydroxylase; VGAT, vesicular γ-aminobutyric acid transporter
Dysbindin (also known as dysbindin-1 or dystrobrevin-binding protein 1) was identified 10 years ago as a ubiquitously expressed protein of unknown function. In the following years, the protein and its encoding gene, DTNBP1, have become the focus of intensive research owing to genetic and histopathological evidence suggesting a potential role in the pathogenesis of schizophrenia. In this review, we discuss published results demonstrating that dysbindin function is required for normal physiology of the mammalian central nervous system. In tissues other than brain and in non-neuronal cell types, the protein has been characterized as a stable component of a multi-subunit complex, named BLOC-1 (biogenesis of lysosome-related organelles complex-1), which has been implicated in intracellular protein trafficking and the biogenesis of specialized organelles of the endosomal–lysosomal system. In the brain, however, dysbindin has been proposed to associate into multiple complexes with alternative binding partners, and to play a surprisingly wide variety of functions including transcriptional regulation, neurite and dendritic spine formation, synaptic vesicle biogenesis and exocytosis, and trafficking of glutamate and dopamine receptors. This puzzling array of molecular and functional properties ascribed to the dysbindin protein from brain underscores the need of further research aimed at ascertaining its biological significance in health and disease.
biogenesis of lysosome-related organelles complex-1 (BLOC-1); DTNBP1; dysbindin; dystrobrevin-binding protein; schizophrenia; AP-3, adaptor protein-3; BLOC, biogenesis of lysosome-related organelles complex; coIP, co-immunoprecipitation; HEK-293 cells, human embryonic kidney cells; HPS, Hermansky–Pudlak syndrome; JNK, c-Jun N-terminal kinase; MS/MS, tandem mass spectrometry; RNAi, RNA interference; shRNA, short-hairpin RNA; siRNA, small-interfering RNA; WASH, Wiskott–Aldrich syndrome protein and SCAR homologue; VAMP-7, vesicle-associated membrane protein 7; WAVE, WASP (Wiskott–Aldrich syndrome protein) verprolin homologous; Y2H, yeast two-hybrid
We have previously shown that overexpression of the Glud1 (glutamate dehydrogenase 1) gene in neurons of C57BL/6 mice results in increased depolarization-induced glutamate release that eventually leads to selective neuronal injury and cell loss by 12 months of age. However, it is known that isogenic lines of Tg (transgenic) mice produced through back-crossing with one strain may differ in their phenotypic characteristics from those produced using another inbred mouse strain. Therefore, we decided to introduce the Glud1 transgene into the Balb/c strain that has endogenously lower levels of GLUD1 (glutamate dehydrogenase 1) enzyme activity in the brain as compared with C57BL/6. Using an enzyme-based MEA (microelectrode array) that is selective for measuring glutamate in vivo, we measured depolarization-induced glutamate release. Within a discrete layer of the striatum, glutamate release was significantly increased in Balb/c Tg mice compared with wt (wild-type) littermates. Furthermore, Balb/c mice released approx. 50–60% of the amount of glutamate compared with C57BL/6 mice. This is similar to the lower levels of endogenous GLUD1 protein in Balb/c compared with C57BL/6 mice. The development of these Glud1-overexpressing mice may allow for the exploration of key molecular events produced by chronic exposure of neurons to moderate, transient increases in glutamate release, a process hypothesized to occur in neurodegenerative disorders.
amperometry; biosensor; electrochemistry; excitotoxicity; neurodegeneration; neurotransmission; CNS, central nervous system; DLAR, Division of Animal Laboratory Resources; DV, dorsoventral; GLUD1, glutamate dehydrogenase 1; MEA, microelectrode array; m-POD, micro-peroxidase; 1-MPMS, 1-methoxy-5-methylphenazinium methyl-sulfate; Pt, platinum; Tg, transgenic; wt, wild-type
NSCs (neural stem cells) are undifferentiated neural cells endowed with a high potential for proliferation and a capacity for self-renewal with retention of multipotency to differentiate into neurons and glial cells. It has been recently reported that GD3, a b-series ganglioside, is a marker molecule for identifying and isolating mouse NSCs. However, the expression of gangliosides in human NSCs is largely unknown. In the present study, we analysed the expression of gangliosides, GD2 and GD3, in human NSCs that were isolated from human brains at gestational week 17 in the form of neurospheres, which are floating clonal aggregates formed by NSCs in vitro. Employing immunocytochemistry, we found that human NSCs were strongly reactive to anti-GD2 antibody and relatively weakly reactive to anti-GD3 antibody. Treatment of these cells with an organic solvent such as 100% methanol, which selectively removes glycolipids from plasma membrane, abolished the immunoreactivity with those antibodies, indicating that the reactivity was due to GD2 and GD3, but not to GD2-/GD3-like glycoproteins or proteoglycans. The immunoreactivity of human NSCs to antibody against SSEA-1 (stage-specific embryonic antigen-1), a well-known carbohydrate antigen of NSCs, was not decreased by the treatment with 100% methanol, indicating that SSEA-1 is mainly carried by glycoproteins and/or proteoglycans in human NSCs. Our study suggests that GD2 and GD3 can be marker gangliosides for identifying human NSCs.
ganglioside; glycosphingolipid (GSL); neural stem cell (NSC); neurosphere; stage-specific embryonic antigen-1 (SSEA-1); bFGF, basic fibroblast growth factor; GSL, glycosphingolipid; LAMP-1, lysosome-associated membrane protein-1; NSC, neural stem cell; SSEA-1, stage-specific embryonic antigen-1. Abbreviations for gangliosides follow Svennerholm's nomenclature system (Svennerholm, 1963)
DMF (dimethyl fumarate) exerts anti-inflammatory and pro-metabolic effects in a variety of cell types, and a formulation (BG-12) is being evaluated for monotherapy in multiple sclerosis patients. DMF modifies glutathione (GSH) levels that can induce expression of the anti-inflammatory protein HO-1 (haem oxygenase-1). In primary astrocytes and C6 glioma cells, BG-12 dose-dependently suppressed nitrite production induced by either LI [LPS (lipopolysaccharide) at 1 μg/ml plus IFNγ (interferon γ) at 20 units/ml] or a mixture of pro-inflammatory cytokines, with greater efficacy in C6 cells. BG-12 reduced NOS2 (nitric oxide synthase 2) mRNA levels and activation of a NOS2 promoter, reduced nuclear levels of NF-κB (nuclear factor κB) p65 subunit and attenuated loss of IκBα (inhibitory κBα) in both cell types, although with greater effects in astrocytes. In astrocytes, LI decreased mRNA levels for GSHr (GSH reductase) and GCL (c-glutamylcysteine synthetase), and slightly suppressed GSHs (GSH synthetase) mRNAs. Co-treatment with BG-12 prevented those decreased and increased levels above control values. In contrast, LI reduced GSHp (GSH peroxidase) and GCL in C6 cells, and BG-12 had no effect on those levels. BG-12 increased nuclear levels of Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2), an inducer of GSH-related enzymes, in astrocytes but not C6 cells. In astrocytes, GSH was decreased by BG-12 at 2 h and increased at 24 h. Prior depletion of GSH using buthionine-sulfoximine increased the ability of BG-12 to reduce nitrites. In astrocytes, BG-12 increased HO-1 mRNA levels and effects on nitrite levels were blocked by an HO-1 inhibitor. These results demonstrate that BG-12 suppresses inflammatory activation in astrocytes and C6 glioma cells, but with distinct mechanisms, different dependence on GSH and different effects on transcription factor activation.
nitric oxide synthase 2 (NOS2); inflammation; glial cells; glutathione; multiple sclerosis; BSO, buthionine-sulfoximine; DMEM, Dulbecco's modified Eagle's medium; DMF, dimethyl fumarate; EAEEMSA, experimental autoimmune encephalomyelitiselectrophoretic mobility-shift assay; FAE, fumaric acid ester; FCS, fetal calf serum; GSHp, GSH peroxidase; GSHr, GSH reductase; GSHs, GSH synthetase; HO-1, haem oxygenase-1; HSP32, heat-shock protein 32; IFNγ, interferon γ; IκBα, inhibitory κBα; IL, interleukin; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; LI, LPS at 1 μg/ml plus IFNγ at 20 units/ml; MSNF-κB, multiple sclerosisi; nuclear factor κB; Nrf2, nuclear factor-erythroid 2 p45 subunit-related factor 2; NOS2, nitric oxide synthase 2; qPCR, quantitative PCR; TNFα, tumour necrosis factor α; ZnPP, zinc protoporphyrin IX
Neuronal death pathways following hypoxia–ischaemia are sexually dimorphic, but the underlying mechanisms are unclear. We examined cell death mechanisms during OGD (oxygen-glucose deprivation) followed by Reox (reoxygenation) in segregated male (XY) and female (XX) mouse primary CGNs (cerebellar granule neurons) that are WT (wild-type) or Parp-1 [poly(ADP-ribose) polymerase 1] KO (knockout). Exposure of CGNs to OGD (1.5 h)/Reox (7 h) caused cell death in XY and XX neurons, but cell death during Reox was greater in XX neurons. ATP levels were significantly lower after OGD/Reox in WT-XX neurons than in XY neurons; this difference was eliminated in Parp-1 KO-XX neurons. AIF (apoptosis-inducing factor) was released from mitochondria and translocated to the nucleus by 1 h exclusively in WT-XY neurons. In contrast, there was a release of Cyt C (cytochrome C) from mitochondria in WT-XX and Parp-1 KO neurons of both sexes; delayed activation of caspase 3 was observed in the same three groups. Thus deletion of Parp-1 shunted cell death towards caspase 3-dependent apoptosis. Delayed activation of caspase 8 was also observed in all groups after OGD/Reox, but was much greater in XX neurons, and caspase 8 translocated to the nucleus in XX neurons only. Caspase 8 activation may contribute to increased XX neuronal death during Reox, via caspase 3 activation. Thus, OGD/Reox induces death of XY neurons via a PARP-1-AIF-dependent mechanism, but blockade of PARP-1-AIF pathway shifts neuronal death towards a caspase-dependent mechanism. In XX neurons, OGD/Reox caused prolonged depletion of ATP and delayed activation of caspase 8 and caspase 3, culminating in greater cell death during Reox.
apoptosis; caspase 3; caspase 8; hypoxia–ischaemia; neuronal death; sexual dimorphism; AIF, apoptosis-inducing factor; AM: acetoxymethyl ester, ; CGN, cerebellar granule neuron; Cyt C, cytochrome c; DAPI, 4′,6-diamidino-2-phenylindole; DIV 9, 9 days in vitro; HBSS, Hanks' balanced salt solution; HI, hypoxia–ischaemia; HRP, horseradish peroxidase; KO, knockout; LDH, lactate dehydrogenase; MB, mitochondrial buffer; OGD, oxygen-glucose deprivation; PI, propidium iodide; pNA, p-nitroaniline; Parp-1/PARP-1, poly(ADP-ribose) polymerase-1; Reox, reoxygenation; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling; VDAC, voltage-dependent anion channel; WT, wild-type
The ability of an AEF (axolemma-enriched fraction) to influence the proliferation, survival and differentiation of OPC (oligodendrocyte progenitor cells) was evaluated. Following addition of AEF to cultured OPC, the AEF associated with the outer surface of OPC so that subsequent metabolic events were likely mediated by direct AEF-OPC contact. Addition of AEF to the cultured OPC resulted in a dose- and time-dependent increase in proliferation that was partially dependent on Akt (protein kinase B) and MAPK (mitogen-activated protein kinase) activation. The major mitogen in an AEF-SE (soluble 2.0 M NaCl extract of the AEF) was identified as aFGF (acidic fibroblast growth factor) and accounted for 50% of the mitogenicity. The remaining 50% of the mitogenicity had properties consistent with bFGF (basic fibroblast growth factor) but was not unequivocally identified. Under conditions that limit the survival of OPC in culture, AEF treatment prolonged the survival of the OPC. Antigenic and morphological examination of the AEF-treated OPC indicated that the AEF treatment helped the OPC survive in a more immature state. The potential downstream metabolic pathways potentially activated in OPC by AEF and the consequences of these activated pathways are discussed. The results of these studies are consistent with the view that direct contact of axons with OPC stimulates their proliferation and survival while preventing their differentiation.
acidic fibroblast growth factor (aFGF); axolemma-enriched fraction (AEF); axonal–oligodendrocyte signalling; CNS trophic factors; multiple sclerosis; oligodendrocyte differentiation; oligodendrocyte progenitor cell; AEF, axolemma-enriched fraction; AEF-SE, soluble 2.0 M NaCl extract of the AEF; aFGF, acidic fibroblast growth factor; Akt, protein kinase B; BCA, bicinchoninic acid; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; CNPase, 2′,3′-cyclic nucleotide 3′-phosphodiesterase; CNS, central nervous system; CREB, cAMP-response-element-binding protein; DAPI, 4′,6-diamidino-2-phenylindole; F-12, Ham's F12 nutrient medium; DMEM–F12, Dulbecco's modified Eagle medium nutrient mixture F-12; DSHB, Developmental Studies Hybridoma Bank; ERK, extracellular-signal-regulated kinase; FCS, fetal calf serum; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; GalC, galactosylcerebroside; GFAP, glial fibrillary acidic protein; LIF, leukaemia inhibitory factor; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NRG, neuregulin; OPC, oligodendrocyte progenitor cell; RIPA buffer, radio immunoprecipitation assay buffer
It has been 10 years since the seminal work of Dwight Bergles and collaborators demonstrated that NG2 (nerve/glial antigen 2)-expressing oligodendrocyte progenitor cells (NG2 cells) receive functional glutamatergic synapses from neurons (Bergles et al., 2000), contradicting the old dogma that only neurons possess the complex and specialized molecular machinery necessary to receive synapses. While this surprising discovery may have been initially shunned as a novelty item of undefined functional significance, the study of neuron-to-NG2 cell neurotransmission has since become a very active and exciting field of research. Many laboratories have now confirmed and extended the initial discovery, showing for example that NG2 cells can also receive inhibitory GABAergic synapses (Lin and Bergles, 2004) or that neuron-to-NG2 cell synaptic transmission is a rather ubiquitous phenomenon that has been observed in all brain areas explored so far, including white matter tracts (Kukley et al., 2007; Ziskin et al., 2007; Etxeberria et al., 2010). Thus, while still being in its infancy, this field of research has already brought many surprising and interesting discoveries, and has become part of a continuously growing effort in neuroscience to re-evaluate the long underestimated role of glial cells in brain function (Barres, 2008). However, this area of research is now reaching an important milestone and its long-term significance will be defined by its ability to uncover the still elusive function of NG2 cells and their synapses in the brain, rather than by its sensational but transient successes at upsetting the old order established by neuronal physiology. To participate in the effort to facilitate such a transition, here we propose a critical review of the latest findings in the field of NG2 cell physiology – discussing how they inform us on the possible function(s) of NG2 cells in the brain – and we present some personal views on new directions the field could benefit from in order to achieve lasting significance.
α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR); nerve/glial antigen 2 (NG2) cells; neuron; oligodendrocyte progenitor cell (OPC); postsynaptic density (PSD); AMPAR, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor; CNP-GFP, C-type natriuretic peptide-green fluorescent protein; CNS, central nervous system; EGFP, enhanced green fluorescent protein; EPSC, excitatory postsynaptic current; GABA, γ-aminobutyric acid; GABAAR, GABA type A receptor; LTP, long-term potentiation; MBP, maltose-binding protein; NG2, nerve/glial antigen 2; NMDAR, N-methyl-d-aspartate receptor; OL, oligodendrocyte lineage; OPC, oligodendrocyte progenitor cell; PDGFRα, platelet-derived growth factor receptor α; PSD, postsynaptic density; SCP, Schwann cell progenitor
Axonally specific microtubule-associated protein tau is an important component of neurofibrillary tangles found in AD (Alzheimer's disease) and other tauopathy diseases such as CTE (chronic traumatic encephalopathy). Such tau aggregate is found to be hyperphosphorylated and often proteolytically fragmented. Similarly, tau is degraded following TBI (traumatic brain injury). In the present study, we examined the dual vulnerability of tau to calpain and caspase-3 under neurotoxic and neurodegenerative conditions. We first identified three novel calpain cleavage sites in rat tau (four-repeat isoform) as Ser130↓Lys131, Gly157↓Ala158 and Arg380↓Glu381. Fragment-specific antibodies to target the major calpain-mediated TauBDP-35K (35 kDa tau-breakdown product) and the caspase-mediated TauBDP-45K respectively were developed. In rat cerebrocortical cultures treated with excitotoxin [NMDA (N-methyl-d-aspartate)], tau is significantly degraded into multiple fragments, including a dominant signal of calpain-mediated TauBDP-35K with minimal caspase-mediated TauBDP-45K. Following apoptosis-inducing EDTA treatment, tau was truncated only to TauBDP-48K/45K-exclusively by caspase. Cultures treated with another apoptosis inducer STS (staurosporine), dual fragmentation by calpain (TauBDP-35K) and caspase-3 (TauBDP-45K) was observed. Tau was also fragmented in injured rat cortex following TBI in vivo to BDPs of 45–42 kDa (minor), 35 kDa and 15 kDa, followed by TauBDP-25K. Calpain-mediated TauBDP-35K-specific antibody confirmed robust signals in the injured cortex, while caspase-mediated TauBDP-45K-specific antibody only detected faint signals. Furthermore, intravenous administration of a calpain-specific inhibitor SNJ-1945 strongly suppressed the TauBDP-35K formation. Taken together, these results suggest that tau protein is dually vulnerable to calpain and caspase-3 proteolysis under different neurotoxic and injury conditions.
cell death; neurodegeneration; protease; tau protein; tauopathy; traumatic brain injury (TBI); AD, Alzheimer's disease; CCI, controlled cortical impact; CSF, colony-stimulating factor; CTE, chronic traumatic encephalopathy; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; NMDA, N-methyl-d-aspartate; STS, staurosporine; TAI, traumatic axonal injury; TauBDP-35K, 35 kDa tau-breakdown product; TBI, traumatic brain injury; TBST, TBS and 0.05% Tween-2
Excessive production of Aβ (amyloid β-peptide) has been shown to play an important role in the pathogenesis of AD (Alzheimer's disease). Although not yet well understood, aggregation of Aβ is known to cause toxicity to neurons. Our recent study demonstrated the ability for oligomeric Aβ to stimulate the production of ROS (reactive oxygen species) in neurons through an NMDA (N-methyl-d-aspartate)-dependent pathway. However, whether prolonged exposure of neurons to aggregated Aβ is associated with impairment of NMDA receptor function has not been extensively investigated. In the present study, we show that prolonged exposure of primary cortical neurons to Aβ oligomers caused mitochondrial dysfunction, an attenuation of NMDA receptor-mediated Ca2+ influx and inhibition of NMDA-induced AA (arachidonic acid) release. Mitochondrial dysfunction and the decrease in NMDA receptor activity due to oligomeric Aβ are associated with an increase in ROS production. Gp91ds-tat, a specific peptide inhibitor of NADPH oxidase, and Mn(III)-tetrakis(4-benzoic acid)-porphyrin chloride, an ROS scavenger, effectively abrogated Aβ-induced ROS production. Furthermore, Aβ-induced mitochondrial dysfunction, impairment of NMDA Ca2+ influx and ROS production were prevented by pre-treatment of neurons with EGCG [(−)-epigallocatechin-3-gallate], a major polyphenolic component of green tea. Taken together, these results support a role for NADPH oxidase-mediated ROS production in the cytotoxic effects of Aβ, and demonstrate the therapeutic potential of EGCG and other dietary polyphenols in delaying onset or retarding the progression of AD.
arachidonic acid (AA) release; Ca2+ influx; (−)-epigallocatechin-3-gallate (EGCG); NADPH oxidase; N-methyl-d-aspartate (NMDA) receptor; oligomeric Aβ; reactive oxygen species (ROS); AA, arachidonic acid; Aβ, amyloid β-peptide; ACSF, artificial cerebrospinal fluid; AD, Alzheimer's disease; BCS, bovine calf serum; cPLA2, cytosolic phospholipase A2; DHE, dihydroethidium; DMEM, Dulbecco's modified Eagle's medium; EGCG, (−)-epigallocatechin-3-gallate; ERK1/2, extracellular-signal-regulated kinase 1/2; LDH, lactate dehydrogenase; MAPK, mitogen-activated protein kinase; MnTBAP, Mn(III)-tetrakis(4-benzoic acid)-porphyrin chloride; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NMDA, N-methyl-d-aspartate; ROS, reactive oxygen species; TBS-T, Tris-buffered saline, pH 7.4, with 0.5% Tween 20
Chronic infection with the intracellular protozoan parasite Toxoplasma gondii leads to tissue remodelling in the brain and a continuous requirement for peripheral leucocyte migration within the CNS (central nervous system). In the present study, we investigate the role of MMPs (matrix metalloproteinases) and their inhibitors in T-cell migration into the infected brain. Increased expression of two key molecules, MMP-8 and MMP-10, along with their inhibitor, TIMP-1 (tissue inhibitor of metalloproteinases-1), was observed in the CNS following infection. Analysis of infiltrating lymphocytes demonstrated MMP-8 and -10 production by CD4+ and CD8+ T-cells. In addition, infiltrating T-cells and CNS resident astrocytes increased their expression of TIMP-1 following infection. TIMP-1-deficient mice had a decrease in perivascular accumulation of lymphocyte populations, yet an increase in the proportion of CD4+ T-cells that had trafficked into the CNS. This was accompanied by a reduction in parasite burden in the brain. Taken together, these findings demonstrate a role for MMPs and TIMP-1 in the trafficking of lymphocytes into the CNS during chronic infection in the brain.
astrocyte; cell migration; central nervous system (CNS); T-cell; tissue inhibitor of metalloproteinases-1 (TIMP-1); Toxoplasma gondii; CNS, central nervous system; DAPI, 4′,6-diamidino-2-phenylindole; EAE, experimentally induced autoimmune encephalomyelitis; ECM, extracellular matrix; GFAP, glial fibrillary acidic protein; HFF, human foreskin fibroblast; IFN-γ, interferon-γ; IL, interleukin; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; mfi, mean fluorescent intensity; MMP, matrix metalloproteinase; PECAM-1, platelet/endothelial cell adhesion molecule-1; PFA, paraformaldehyde; RT–PCR, reverse transcription–PCR; sTAg, soluble Toxoplasma antigen; TIMP-1, tissue inhibitor of metalloproteinases-1; TNF, tumour necrosis factor; WT, wild-type