Binding and uptake of Aβ1-42 by primary human astrocytes in vitro (original) (raw)
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Glia, 2000
Intracerebral accumulation of amyloid-b (Ab) leading to Ab plaque formation, is the main hallmark of Alzheimer's disease and might be caused by defective Ab-clearance. We previously found primary human astrocytes and microglia able to bind and ingest Ab1-42 in vitro, which appeared to be limited by Ab1-42 fibril formation. We now confirm that astrocytic Ab-uptake depends on size and/or composition of Ab-aggregates as astrocytes preferably take up oligomeric Ab over fibrillar Ab. Upon exposure to either fluorescence-labelled Ab1-42 oligomers (Ab oligo ) or fibrils (Ab fib ), a larger (3.7 times more) proportion of astrocytes ingested oligomers compared to fibrils, as determined by flow cytometry. Ab-internalization was verified using confocal microscopy and live-cell imaging. Neither uptake of Ab oligo nor Ab fib , triggered proinflammatory activation of the astrocytes, as judged by quantification of interleukin-6 and monocyte-chemoattractant protein-1 release. Amyloid-associated proteins, including a1antichymotrypsin (ACT), serum amyloid P component (SAP), C1q and apolipoproteins E (ApoE) and J (ApoJ) were earlier found to influence Ab-aggregation. Here, astrocytic uptake of Ab fib increased when added to the cells in combination with SAP and C1q (SAP/C1q), but was unchanged in the presence of ApoE, ApoJ and ACT. Interestingly, ApoJ and ApoE dramatically reduced the number of Ab oligo -positive astrocytes, whereas SAP/C1q slightly reduced Ab oligo uptake. Thus, amyloid-associated proteins, especially ApoJ and ApoE, can alter Ab-uptake in vitro and hence may influence Ab clearance and plaque formation in vivo.
The Amyloid-42 Proxy, Amyloid-25-35 , Induces Normal Human Cerebral Astrocytes to Produce Amyloid-42
Astrocytes in amyloid-(A) 42-accumulating human brains afflicted with Alzheimer's disease (AD) upregulate vascular endothelial growth factor (VEGF)-A synthesis and also become loaded with A 42. We have already shown that A 25-35 (surrogate of A 42)-induced VEGF-A production in 'normoxic' cultures of early passage normal human cerebral astrocytes (NAHAs) is mediated by the stabilization of VEGF gene-stimulating hypoxia-inducible factor (HIF)-1 and nuclear translocation of HIF-1 • HIF-1 complexes. We have now found that treating these NAHAs with A 25-35 also stimulates them to make A 42 (appearing in immunoblots as several bands with M r 's from 8 kDa upwards), whose levels peak at 48 h (2.8-fold versus 0 h, p < 0.001) and then start falling slowly. This rise of A 42 peptide production coincides with a transiently increased flow of HIF-1 (therefore HIF-1 • HIF-1 complexes; at 24 h, 1.5-fold versus 0 h, p < 0.001) into the nucleus and transient surges first of-secretase (BACE-1/-S) mRNA expression (1.2-fold versus 0 h, p = 0.013) and activity peaking at 24-h (1.4-fold versus 0 h, p = 0.001), and then of-secretase (-S) activity cresting at 48 h (1.6-fold versus 0 h, p < 0.001) that cleave the A 42 peptides from amyloid-protein precursor. Since the genes encoding components of these two secretases have the same HIF-1 • HIF-1-responsive elements in their promoters as the VEGF gene, these observations suggest that the A 42 released from neurons in the AD brain can recruit associated astrocytes via HIF-1 • HIF-1 signaling into the pool of A 42-producing cells. In other words, A 42 begets A 42 in NAHAs.
Alzheimer's disease (AD) is characterized by the massive deposition in the brain of the 40-42-residue amyloid β protein (Aβ). While Aβ1-40 predominates in the vascular system, Aβ1-42 is the major component of the senile plaques in the neuropil. The concentration of both Aβ species required to form amyloid fibrils in vitro is micromolar, yet soluble Aβs found in normal and AD brains are in the low nanomolar range. It has been recently proposed that the levels of Aβ sufficient to trigger amyloidogenesis may be reached intracellularly. To study the internalization and intracellular accumulation of the major isoforms of Aβ, we used THP-1 and IMR-32 neuroblastoma cells as models of human monocytic and/or macrophagic and neuronal lineages, respectively. We tested whether these cells were able to internalize and accumulate 125 I-Aβ1-40 and 125 I-Aβ1-42 differentially when offered at nanomolar concentrations and free of large aggregates, conditions that mimic a prefibrillar stage of Aβ in AD brain. Our results showed that THP-1 monocytic cells internalized at least 10 times more 125 I-Aβs than IMR-32 neuroblastoma cells, either isolated or in a coculture system. Moreover, 125 I-Aβ1-42 presented a higher adsorption, internalization, and accumulation of undigested peptide inside cells, as opposed to 125 I-Aβ1-40. These results support that Aβ1-42, the major pathogenic form in AD, may reach supersaturation and generate competent nuclei for amyloid fibril formation intracellularly. In light of the recently reported strong neurotoxicity of soluble, nonfibrillar Aβ1-42, we propose that intracellular amyloidogenesis in microglia is a protective mechanism that may delay neurodegeneration at early stages of the disease.
Journal of Cellular Physiology, 2003
The modulation of CD44, VCAM-1 and CD71 expression was analysed by flow cytometry in the 1321N1 astrocytoma cell line in the presence of interleukin-1b (IL1b), tumour necrosis factor-a (TNFa) and 1-40 or 25-35 b-amyloid (Ab) fragments. The percentage of 1321N1 astrocytoma cell line expressing these markers increased significantly after treatment with TNFa or IL1b. The presence of Ab 1-40 fragment, alone or in combination with IL1b, induced an increase in the percentage of cells expressing CD44, but not VCAM-1. However, the concomitant presence of Ab 1-40 fragment and of IL1b or TNFa caused an increase in the percentage of CD71 positive cells. In contrast, the shorter Ab 25-35 fragment was always inactive. These results indicates that Ab 1-40 fragment, in association with cytokines, can activate this astrocyte-derived cell line and add further elements in favour of the hypothesis that b-amyloid can act as immunological mediator.
Cell and Tissue Research, 1999
Alzheimer's disease (AD) is characterized by the massive deposition in the brain of the 40-42-residue amyloid β protein (Aβ). While Aβ1-40 predominates in the vascular system, Aβ1-42 is the major component of the senile plaques in the neuropil. The concentration of both Aβ species required to form amyloid fibrils in vitro is micromolar, yet soluble Aβs found in normal and AD brains are in the low nanomolar range. It has been recently proposed that the levels of Aβ sufficient to trigger amyloidogenesis may be reached intracellularly. To study the internalization and intracellular accumulation of the major isoforms of Aβ, we used THP-1 and IMR-32 neuroblastoma cells as models of human monocytic and/or macrophagic and neuronal lineages, respectively. We tested whether these cells were able to internalize and accumulate 125 I-Aβ1-40 and 125 I-Aβ1-42 differentially when offered at nanomolar concentrations and free of large aggregates, conditions that mimic a prefibrillar stage of Aβ in AD brain. Our results showed that THP-1 monocytic cells internalized at least 10 times more 125 I-Aβs than IMR-32 neuroblastoma cells, either isolated or in a coculture system. Moreover, 125 I-Aβ1-42 presented a higher adsorption, internalization, and accumulation of undigested peptide inside cells, as opposed to 125 I-Aβ1-40. These results support that Aβ1-42, the major pathogenic form in AD, may reach supersaturation and generate competent nuclei for amyloid fibril formation intracellularly. In light of the recently reported strong neurotoxicity of soluble, nonfibrillar Aβ1-42, we propose that intracellular amyloidogenesis in microglia is a protective mechanism that may delay neurodegeneration at early stages of the disease.
Alzheimer's disease (AD) is characterized by the massive deposition in the brain of the 40-42-residue amyloid β protein (Aβ). While Aβ1-40 predominates in the vascular system, Aβ1-42 is the major component of the senile plaques in the neuropil. The concentration of both Aβ species required to form amyloid fibrils in vitro is micromolar, yet soluble Aβs found in normal and AD brains are in the low nanomolar range. It has been recently proposed that the levels of Aβ sufficient to trigger amyloidogenesis may be reached intracellularly. To study the internalization and intracellular accumulation of the major isoforms of Aβ, we used THP-1 and IMR-32 neuroblastoma cells as models of human monocytic and/or macrophagic and neuronal lineages, respectively. We tested whether these cells were able to internalize and accumulate 125 I-Aβ1-40 and 125 I-Aβ1-42 differentially when offered at nanomolar concentrations and free of large aggregates, conditions that mimic a prefibrillar stage of Aβ in AD brain. Our results showed that THP-1 monocytic cells internalized at least 10 times more 125 I-Aβs than IMR-32 neuroblastoma cells, either isolated or in a coculture system. Moreover, 125 I-Aβ1-42 presented a higher adsorption, internalization, and accumulation of undigested peptide inside cells, as opposed to 125 I-Aβ1-40. These results support that Aβ1-42, the major pathogenic form in AD, may reach supersaturation and generate competent nuclei for amyloid fibril formation intracellularly. In light of the recently reported strong neurotoxicity of soluble, nonfibrillar Aβ1-42, we propose that intracellular amyloidogenesis in microglia is a protective mechanism that may delay neurodegeneration at early stages of the disease.
Neurochemistry International, 2007
Recent studies indicate that astrocytes may be the primary target of secreted amyloid-beta 1-42 peptides, with the neurotoxicity representing a secondary response to astrocytic stress. Our purpose was to clarify the astrocytic stress response induced by amyloid-beta peptides in human and rat astrocytes. Human amyloid-beta 1-42 peptides and fibrils induced the appearance of cytoplasmic vacuoles in normal human astrocytes (NHA) and CCFsttg1 astrocytoma cells. Vacuoles appeared 9-12h after the amyloid-beta exposure and remained present for several days. Rat primary neonatal astrocytes showed similar but less prominent vacuolar response. Human amyloid-beta peptides 1-16, 1-28, 10-20, 17-21 and 25-35 did not cause vacuole formation. Electron microscopic observations revealed large endocytic vacuoles containing fibrillar amyloid material. Stress marker analysis did not show any increase in protein levels of HSP70, HSP90, GRP78 and GRP94. However, the protein level of clusterin/apoJ, a secreted chaperone, was strongly increased both in NHA and CCFsttg1 astrocytes. Endocytic response associated with the accumulation of clusterin/apoJ protein suggests that clusterin/apoJ has a role in the clearance of amyloid-beta peptides.
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.