Differential accumulation of soluble amyloid β peptides 1-40 and 1-42 in human monocytic and neuroblastoma cell lines (original) (raw)

Differential accumulation of soluble amyloid β peptides 1–40 and 1–42 in human monocytic and neuroblastoma cell lines

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.

Differential accumulation of soluble amyloid beta 1-40 and 1-42 in human monocytic and neuroblastoma cell lines: implications for cerebral amyloidogenesis

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.

Internalization and resistance to degradation of Alzheimer's Aβ1–42 at nanomolar concentrations in THP-1 human monocytic cell line

Neuroscience Letters, 1999

Microglial cell involvement in Alzheimer's disease has been related to amyloid b (Ab) internalization, the release of inflammatory cytokines and the development of neuritic plaques. The human monocyte/macrophage THP-1 cell line has been widely used as a model of human microglial cells. We used THP-1 cells to study the adsorption, internalization and resistance to degradation of Ab1-40 and Ab1-42 isoforms offered at nanomolar concentrations and free of large aggregates, conditions that may mimic a pre-fibrillar stage of Ab in the brain. Under these conditions, Abs did not induce THP-1 activation, as assessed by interleukin-1b expression. Ab1-42 showed a preferential adsorption and intracellular accumulation as compared to Ab1-40, supporting that competent nuclei for Ab1-42 ordered aggregation may be formed inside microglial cells. In light of the possible neurotoxicity of soluble Ab1-42, we propose that amyloid formation within brain phagocytic cells may be a protective mechanism in early stages of the disease.

Intraneuronal Aβ42 Accumulation in Human Brain

American Journal of Pathology, 2000

Alzheimer's disease (AD) is characterized by the deposition of senile plaques (SPs) and neurofibrillary tangles (NFTs) in vulnerable brain regions. SPs are composed of aggregated ␤-amyloid (A␤) 40/42(43) peptides. Evidence implicates a central role for A␤ in the pathophysiology of AD. Mutations in ␤APP and presenilin 1 (PS1) lead to elevated secretion of A␤, especially the more amyloidogenic A␤42. Immunohistochemical studies have also emphasized the importance of A␤42 in initiating plaque pathology. Cell biological studies have demonstrated that A␤ is generated intracellularly. Recently , endogenous A␤42 staining was demonstrated within cultured neurons by confocal immunofluorescence microscopy and within neurons of PS1 mutant transgenic mice. A central question about the role of A␤ in disease concerns whether extracellular A␤ deposition or intracellular A␤ accumulation initiates the disease process. Here we report that human neurons in AD-vulnerable brain regions specifically accumulate ␥-cleaved A␤42 and suggest that this intraneuronal A␤42 immunoreactivity appears to precede both NFT and A␤ plaque deposition. This study suggests that intracellular A␤42 accumulation is an early event in neuronal dysfunction and that preventing intraneuronal A␤42 aggregation may be an important therapeutic direction for the treatment of AD. (Am J Pathol 2000, 156:15-20)

Amyloid-β42 protofibrils are internalized by microglia more extensively than monomers

Brain Research, 2016

One pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid-β peptide (Aβ) in the affected brain. While there are numerous deleterious effects of Aβ accumulation, there is general agreement that a sustained inflammatory response to aggregated Aβ contributes to progressive neurodegeneration in AD and microglial cells play a significant role in this process. Our laboratory and others have shown that small soluble aggregates of Aβ activate a microglia-mediated inflammatory response. One component of the response involves internalization of extracellular Aβ, and this process is likely very sensitive to Aβ structure. In this study we analyzed the proclivity of microglia for internalization of Aβ42 monomers and protofibrils using fluorescentlylabeled Aβ. Both Aβ42 species were labeled directly via amino linkage with an Alexa Fluor 488 tetrafluorophenyl ester (AF488-TFP) and then isolated individually by chromatography. Aβ42 protofibrils retained their size and morphological properties after labeling but monomers had a much higher stoichiometry of labeling compared to protofibrils. Primary murine microglia internalized AF488-Aβ42 protofibrils rapidly and in significant amounts compared to AF488-Aβ42 monomers. Microglial internalization of protofibrils was dependent on time and concentration, and corresponded with tumor necrosis factor α secretion. In competition studies, unlabeled Aβ42 protofibril internalization, detected by immunostaining, did not diminish AF488-protofibril uptake. Internalized AF488-Aβ42 protofibrils were found widely dispersed in the cytosol with some lysosomal accumulation but little degradation. These studies highlight the sensitivity that microglia exhibit to Aβ structure in the internalization process and emphasize their affinity for soluble Aβ protofibrils.

The Ratio of Monomeric to Aggregated Forms of Aβ40 and Aβ42 Is an Important Determinant of Amyloid-β Aggregation, Fibrillogenesis, and Toxicity

Journal of Biological Chemistry, 2008

Aggregation and fibril formation of amyloid-␤ (A␤) peptides A␤40 and A␤42 are central events in the pathogenesis of Alzheimer disease. Previous studies have established the ratio of A␤40 to A␤42 as an important factor in determining the fibrillogenesis, toxicity, and pathological distribution of A␤. To better understand the molecular basis underlying the pathologic consequences associated with alterations in the ratio of A␤40 to A␤42, we probed the concentration-and ratio-dependent interactions between well defined states of the two peptides at different stages of aggregation along the amyloid formation pathway. We report that monomeric A␤40 alters the kinetic stability, solubility, and morphological properties of A␤42 aggregates and prevents their conversion into mature fibrils. A␤40, at approximately equimolar ratios (A␤40/A␤42 ϳ 0.5-1), inhibits (>50%) fibril formation by monomeric A␤42, whereas inhibition of protofibrillar A␤42 fibrillogenesis is achieved at lower, substoichiometric ratios (A␤40/A␤42 ϳ 0.1). The inhibitory effect of A␤40 on A␤42 fibrillogenesis is reversed by the introduction of excess A␤42 monomer. Additionally, monomeric A␤42 and A␤40 are constantly recycled and compete for binding to the ends of protofibrillar and fibrillar A␤ aggregates. Whereas the fibrillogenesis of both monomeric species can be seeded by fibrils composed of either peptide, A␤42 protofibrils selectively seed the fibrillogenesis of monomeric A␤42 but not monomeric A␤40. Finally, we also show that the amyloidogenic propensities of different individual and mixed A␤ species correlates with their relative neuronal toxicities. These findings, which highlight specific points in the amyloid peptide equilibrium that are highly sensitive to the ratio of A␤40 to A␤42, carry important implications for the pathogenesis and current therapeutic strategies of Alzheimer disease.

A Dynamic Relationship between Intracellular and Extracellular Pools of Aβ

The American Journal of Pathology, 2006

The accumulation of the amyloid-␤ peptide (A␤) in the brain is considered to have a primary role in Alzheimer's disease (AD). In addition to the extracellular accumulation of A␤ in the parenchyma and cerebrovasculature, emerging evidence indicates that intraneuronal A␤ also plays a pathophysiological role in AD. It is unclear, however, if the intracellular and extracellular pools of A␤ are unrelated or connected. In these studies, we sought to establish a relationship between these two pools of A␤. We identified an inverse relationship between intracellular and extracellular A␤ in the 3xTg-AD transgenic model of AD. Using an immunotherapy approach, we further found that extracellular A␤ was cleared before intracellular A␤. After the antibody dissipated, however, the reappearance of extracellular plaques was preceded by the accumulation of intraneuronal A␤. Taken together, these results provide strong experimental evidence that intraneuronal A␤ may serve as a source for some of the extracellular amyloid deposits. (Am J Pathol 2006, 168:184 -194; DOI: 10.2353/ajpath.2006.050593)

Fibrillar beta-amyloid (Aβ) (1–42) elevates extracellular Aβ in cultured hippocampal neurons of adult rats

Brain Research, 2007

Alzheimer's disease (AD) is a chronic disorder with progressive neurodegeneration associated with aging and is characterized by fibrillar beta-amyloid (Aβ) deposits in the brain. Although the increased production of Aβ seems to play a noticeable role in AD pathogenesis and its progression, all the mechanisms which are involved in this extracellular Aβ elevation are not known completely. In the present study, we used adult hippocampal neuronal culture as an in vitro model which is favorable for adult neurodegenerative diseases' studies. We introduced a toxic concentration for fibrillar Aβ1-42 in adult neurons which was much lower from the toxic concentration in embryonic neurons. To determine the effect of fibrillar Aβ1-42 which is the most toxic part of amyloid plaques, on extracellular Aβ1-40, as the main part of βAPP proteolysis products, we treated the neurons with fibrillar Aβ1-42 at nontoxic concentrations of 2 × 10 − 6 , 2×10 − 5 and 2 × 10 − 4 μM and measured extracellular Aβ1-40. Our findings show that even very low levels of fibrillar Aβ1-42 can contribute to subsequent extracellular Aβ elevation in a dose dependent manner. These results suggest that even low levels of fibrillar Aβ may have deleterious actions if it remains in extracellular space for a period of time.