Fibronectin modulation by Aβ amyloid peptide (25–35) in cultured astrocytes of newborn rat cortex (original) (raw)
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Neuroscience, 1998
Aggregated 13/A4-amyloid is known to increase intraneuronal calcium by various mechanisms and to lead eventually to the death of the cultured neuron. This study deals with the role of 13/A4-amyloid and several of its fragments in calcium homeostasis, glial fibrillary acid protein and S10013 staining, morphology and survival of cultured rat hippocampal astrocytes as determined by Fura imaging, indirect immunofluorescence and life/death assays. In contrast to cultured neurons, none of the 12 different 13/A4 fragments tested caused an increase in intra-astrocytic free calcium. However, among the compounds evaluated, the fragments 10 20mer, 25-35mer and the full-length peptides (1-40, 1~42 and l~43mer), at 5 and 10gM, decreased free intra-astrocytic calcium statistically significantly after the cells had been incubated for 48 and 72 h. This occurred both for astrocytes treated with vehicle alone or the reversed sequence of the l~,0mer, i.e. the 40 lmer. However, survival was not altered under the conditions examined, even when there was a change in free intracellular calcium. Concomitant with the decrease in intracellular free calcium, the shape of the astrocytes became more spider-like, normally an indication of activated astrocytes, and markedly more intense anti-S 10013 and anti-glial fibrillary acidic protein staining was seen.
Brain Research, 1998
. A common feature of many neurodegenerative disorders is an abundance of activated glial cells astrocytes and microglia . In Ž . Alzheimer's disease AD , activated astrocytes are in close apposition to and surrounding the amyloid plaques. The mechanisms by which the astrocytes become activated in AD and the consequences of reactive astrocytosis to disease progression are not known. We examined Ž . the possibility that the amyloid-b A b peptide, a major constituent of the amyloid plaque, could act as a stimulus leading to activation. We found that treatment of rat cortical astrocyte cultures with aggregated A b 1-42 peptide induces activation, as assessed by reactive morphological changes and upregulation of selective glial mRNA and proteins, such as the inflammatory cytokine interleukin-1b. A b Ž . Ž . also stimulates inducible nitric oxide synthase iNOS mRNA levels and nitric oxide NO release. A b 1-42, a major form of amyloid associated with neurotoxicity, activated astrocytes in a time-and dose-dependent manner, whereas a scrambled A b 1-42 sequence or A b 17-42 had little or no effect. We also determined that the A b activity can be found in a supernatant fraction containing soluble A b oligomers. Our data suggest that A b plays a role in the reactive astrocytosis of AD and that the inflammatory response induced upon glial activation is a critical component of the neurodegenerative process. q 1998 Elsevier Science B.V.
Effects of Alzheimer's peptide and α1-antichymotrypsin on astrocyte gene expression
Neurobiology of Aging, 2007
We employed gene array technology to investigate the effects of ␣1-antichymotrypsin (ACT), soluble or fibrillar Alzheimer's peptide (A 1-42 ) alone and the combination of ACT/A 1-42 on human astrocytes. Using a 1.2-fold change as significance threshold, 398 astrocyte genes showed altered expression in response to these treatments compared to controls. Of the 276 genes affected by the ACT/soluble A 1-42 combination, 195 (70.6%) were suppressed. The ACT/fibrillar A 1-42 combination affected expression of 64 genes of which 58 (90.5%) were up-regulated. The most prominent gene expression changes in response to the ACT/soluble A 1-42 , were the down-regulation of at least 60 genes involved in transcription, signal transduction, apoptosis and neurogenesis. The ACT/fibril A 1-42 increased the expression of genes involved in transcription regulation and signal transduction. Surprisingly, gene expression of astrocytes exposed to soluble or fibrillar A 1-42 alone was largely unaffected. Thus, the molecular forms generated by the combination of ACT/A 1-42 alter expression of astrocyte genes more profoundly in breadth and magnitude than soluble or fibrillar A 1-42 alone, suggesting that pathogenic effects of A 1-42 may occur as a consequence of its association with other proteins.
Neuroscience Letters, 1993
The GFAP mRNA levels were compared to the density of the senile plaques (SP) in postmortem brain samples of 8 cases, either non-demented or affected by senile dementia of the Alzheimer type. In the frontal neocortex, the GFAP mRNA level is not affected, even if SP are present. In the temporal neocortex, a positive correlation between GFAP mRNA level and SP density was highly significant. This shows that in this area, astrocytes are altered at transcriptional or post-transcriptional levels, or both. The different responses of this astrogliosis marker in each area may be related to the loss of specific neurotransmitter system.
Proteomic analysis of glial fibrillary acidic protein in Alzheimer's disease and aging brain
Neurobiology of …, 2005
Chronic inflammation is known to play an important role in the heterogeneous pathogenesis of Alzheimer's disease (AD). Activated astrocytes expressing glial fibrillary acidic protein (GFAP) are closely associated with AD pathology, such as tangles, neuritic plaques and amyloid depositions. Altogether, 46 soluble isoforms of GFAP were separated and most of them quantified by two-dimensional immunoblotting in frontal cortices of AD patients and age-matched controls. A 60% increase in the amount of more acidic isoforms of GFAP was observed in AD and these isoforms were both phosphorylated and N-glycosylated, while more basic isoforms were O-glycosylated and exhibited no quantitative differences between post-mortem AD and control brains. These data highlight the importance of exploring isoform-specific levels of proteins in pathophysiological conditions since modifications of proteins determine their activity state, localization, turnover and interaction with other molecules. Mechanisms, structures and functional consequences of modification of GFAP isoforms remain to be clarified. D
Proceedings of the National Academy of Sciences, 1998
The major pathological features of Alzheimer's disease (AD) include amyloid plaques composed primarily of the -amyloid (A) peptide, degenerating neurons and neurofibrillary tangles, and the presence of numerous activated astrocytes and microglia. Although extensive genetic data implicate A in the neurodegenerative cascade of AD, the molecular mechanisms underlying its effects on neurons and glia and the relationship between glial activation and neuronal death are not well defined. A has been shown to induce glial activation, and a growing body of evidence suggests that activated glia contribute to neurotoxicity through generation of inf lammatory cytokines and neurotoxic free radicals, such as nitric oxide (NO), potent sources of oxidative stress known to occur in AD. It is therefore crucial to identify specific A-induced molecular pathways mediating these responses in activated glia. We report that A stimulates the activation of the transcription factor NFB in rat astrocytes, that NFB activation occurs selectively from p65 transactivation domain 2, and that A-induced NO synthase expression and NO production occur through an NFB-dependent mechanism. This demonstration of how A couples an intracellular signal transduction pathway involving NFB to a potentially neurotoxic response provides a key mechanistic link between A and the generation of oxidative damage. Our results also suggest possible molecular targets upon which to focus future drug discovery efforts for AD. MATERIALS AND METHODS Cell Culture and Amyloid  1-42 Peptide (A42) Preparation. Cultured rat cortical astrocytes were prepared and tertiary cultures made as described (22). Cells were maintained in ␣MEM supplemented with 10% fetal bovine serum (FBS) (HyClone) and antibiotics [100 units/ml penicillin͞100 g/ml streptomycin (GIBCO͞BRL)]. Twenty-four hours before stimulation, astrocyte medium was removed, cells were washed once with prewarmed PBS, and then serum-free ␣MEM containing N2 media supplement (GIBCO͞BRL) was added to the cultures. A peptides [A 1-42 or scrambled 1-42 sequence (A42scr) KVKGLIDGAHIGDLVYEFMD-SNSAIFREGVGAGHVHVAQVEF] were either purchased The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
1998
The major pathological features of Alzheimer's disease (AD) include amyloid plaques composed primarily of the -amyloid (A) peptide, degenerating neurons and neurofibrillary tangles, and the presence of numerous activated astrocytes and microglia. Although extensive genetic data implicate A in the neurodegenerative cascade of AD, the molecular mechanisms underlying its effects on neurons and glia and the relationship between glial activation and neuronal death are not well defined. A has been shown to induce glial activation, and a growing body of evidence suggests that activated glia contribute to neurotoxicity through generation of inf lammatory cytokines and neurotoxic free radicals, such as nitric oxide (NO), potent sources of oxidative stress known to occur in AD. It is therefore crucial to identify specific A-induced molecular pathways mediating these responses in activated glia. We report that A stimulates the activation of the transcription factor NFB in rat astrocytes, that NFB activation occurs selectively from p65 transactivation domain 2, and that A-induced NO synthase expression and NO production occur through an NFB-dependent mechanism. This demonstration of how A couples an intracellular signal transduction pathway involving NFB to a potentially neurotoxic response provides a key mechanistic link between A and the generation of oxidative damage. Our results also suggest possible molecular targets upon which to focus future drug discovery efforts for AD. MATERIALS AND METHODS Cell Culture and Amyloid  1-42 Peptide (A42) Preparation. Cultured rat cortical astrocytes were prepared and tertiary cultures made as described (22). Cells were maintained in ␣MEM supplemented with 10% fetal bovine serum (FBS) (HyClone) and antibiotics [100 units/ml penicillin͞100 g/ml streptomycin (GIBCO͞BRL)]. Twenty-four hours before stimulation, astrocyte medium was removed, cells were washed once with prewarmed PBS, and then serum-free ␣MEM containing N2 media supplement (GIBCO͞BRL) was added to the cultures. A peptides [A 1-42 or scrambled 1-42 sequence (A42scr) KVKGLIDGAHIGDLVYEFMD-SNSAIFREGVGAGHVHVAQVEF] were either purchased The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
β-Amyloid of Alzheimer's Disease Induces Reactive Gliosis That Inhibits Axonal Outgrowth
Experimental Neurology, 1993
Pathological lesions in the brains of patients with Alzheimer's disease (AD) are characterized by dense deposits of the protein ,B-amyloid. The link between the deposition of ,B-amyloid in senile plaques and AD-associated pathology is, at present, controversial since there have been conflicting reports on whether the 39-43 amino acid ,B-amyloid sequence is toxic or trophic to neurons. In this report, we show that ,8-amyloid peptide when presented as an insoluble substrate which mimics its conformation in vivo can induce cortical glial cells in vitro and in vivo to locally deposit chondroitin sulfate containingproteoglycan. In vitro the proteoglycan-containing matrix deposited by glia on ,8-amyloid blocks the usual ability of the peptide to allow cortical neurons to adhere and grow. Chondroitin sulfate-containing proteoglycan was also found in senile plaques of human AD tissue. We suggest that an additional effect of ,8-amyloid in the brain, which compounds the direct effects of ,8amyloid on neurons, is mediated by the stimulation of astroglia to become reactive. Once in the reactive state, glial cells deposit large amounts of growth-inhibitory molecules within the neuropil which could impair neuronal process survival and regeneration leading to neurite retraction and/or dystrophy around senile plaques in AD.
Amyloid-β fibril formation is not necessarily required for microglial activation by the peptides
Neurochemistry International, 2005
There is increasing evidence that microglial activation has pathogenic influence on Alzheimer's disease. According to in vitro studies, microglia activated by amyloid-b (Ab) peptides have been reported to damage or kill neurons by the release of neurotoxic molecules such as tumor necrosis factor-a (TNF-a), interleukin-1b, nitric oxide or reactive oxygen species. Although the relationship between the aggregational state of Ab peptides and their neurotoxic activities has been well investigated, little is known about the relationship between the aggregational state of Ab peptides and their ability to induce microglial activation. In the present study, we thus performed both structural and biochemical studies to clarify the relationship between the aggregational state of Ab peptides and their ability to activate microglia. Our results have shown that, in the presence of interferon-g, the Ab25-35(M 35 Nle) peptide had almost the same potency of activating microglia and producing TNF-a as the Ab25-35 peptide on both protein and mRNA levels, in spite of the fact that former peptide represented much less amyloid fibril formation than the latter in a thioflavine-T fluorometric assay. These results suggest that Ab fibril formation is not necessarily required for microglial activation by the peptides.