Elsevier

Brain Research

Volume 746, Issues 1–2, 23 January 1997, Pages 275-284
Brain Research

Research report
Preferential adsorption, internalization and resistance to degradation of the major isoform of the Alzheimer's amyloid peptide, Aβ1–42, in differentiated PC12 cells

https://doi.org/10.1016/S0006-8993(96)01262-0Get rights and content

Abstract

A central question in Alzheimer's disease (AD) is the role of amyloid in pathogenesis. Recent discoveries implicating the longer Aβ1–42 form of amyloid in pathogenesis led us to characterize the interaction of Aβ with cells to elucidate differences that might account for these observations. We characterized the adsorption, internalization and degradation of radiolabeled Aβ in NGF-differentiated PC12 cells under conditions that are not acutely toxic. All Aβ peptides examined adsorb to the surface of PC12 cells and are internalized; however the adsorption and internalization of Aβ1–42 is significantly greater than that of Aβ1–40 and Aβ1–28. The adsorption of Aβ1–42 is decreased by treatment of the cells with neuraminidase, but not heparitinase. The fate of the internalized Aβ1–42 is also very different than shorter Aβ peptides: a fraction of the internalized Aβ1–42 accumulates intracellularly and is resistant to degradation for at least 3 days while Aβ1–40 and shorter peptides are eliminated with a half life of about 1 h. Aβ1–42 does not appear to inhibit lysosomal hydrolases, since Aβ1–28 is degraded at the same rate in the presence or absence of Aβ1–42. The intracellular Aβ1–42 is located in a dense organellar compartment and colocalizes with the lysosomal markers Lucifer Yellow and horseradish peroxidase. These data indicate that there are significant differences in the cell surface adsorption, internalization and catabolism of Aβ1–42 compared to Aβ1–40 and Aβ1–28. These differences may be important for the preferential accumulation of the longer Aβ1–42 isoform and its association with AD pathogenesis.

Introduction

The most prevalent form of senile dementia, Alzheimer's disease (AD), is characterized by proteinaceous deposits in the cerebrovasculature (CVA) and in the parenchyma as senile plaques (SP) [48]. The major component of SP and CVA is the 4.5 kDa Aβ peptide 10, 25, that is derived from the transmembrane amyloid precursor protein (APP) 9, 19. Biochemical analysis of the amyloid peptides from AD brain indicate that Aβ1–42 is the principal species associated with SP amyloid 19, 26, 36, while both Aβ1–42 and Aβ1–40 are abundant in CVA deposits 32, 36. Aβ peptides ending in 42 display a higher degree of amino-terminal heterogeneity than peptides ending in 40 and also contain more d-Asp residues, iso-l-Asp peptide bonds and non-enzymatic glycation products 35, 45. These posttranslational modifications are hallmarks of long-lived proteins like lens crystallins and their presence in Aβ peptides ending in residue 42 and absence in Aβ1–40 raises the question of whether the life cycles of these two Aβ isoforms may be quite different.

Recent evidence indicates that the longer Aβ1–42(43) forms may be more closely related to pathogenesis than the shorter Aβ1–40 isoform. The development of monoclonal antibodies that specifically recognize the carboxyl termini of Aβ1–42(43) and Aβ1–40 and their use in immunohistochemical studies led to discovery that Aβ1–42(43) is nearly exclusively deposited in senile plaque in human AD brain, confirming the results obtained from biochemical analysis of AD brain amyloid 18, 43. Mutations associated with inherited, familial forms of AD (FAD) [reviewed in [27]] have been shown to alter the processing of the APP to favor pathways that generate the amyloid peptide and favor the production of the longer Aβ1–42 isoform 41, 44, 15, 53, 54. FAD mutations within the transmembrane domain of APP (at codon 717 of APP; corresponding to position 46 from the beginning of Aβ) may alter the specificity of APP processing enzyme, gamma-secretase, to favor the production of the longer Aβ1–42(43) forms of Aβ. Although the FAD mutation on chromosome 14 does not occur within the APP gene, this mutation also appears to lead to the production of an increased amount of Aβ1–42(43) [53]. Transfected cells expressing APP molecules with mutations in the transmembrane domain produce more of the longer Aβ1–42(43) than cells expressing wild type APP 53, 54, 24.

Some insight into the mechanisms for the production of Aβ has been obtained from studies of the intracellular trafficking and proteolytic processing of APP (reviewed in 37, 16). These studies have demonstrated that soluble 4 kDa Aβ and truncated 3 kDa peptides lacking the amino-terminal 16 residues of Aβ are secreted into the cell culture medium, cerebrospinal fluid and serum of both normal and AD cells 14, 38. The fact that relatively little if any 13, 37soluble Aβ is detected in association with cells suggests that if gamma type cleavage occurs within endosomes, it is rapidly externalized or that the gamma secretase resides on the plasma membrane. Recent studies have demonstrated that transfected cells expressing the Swedish mutant APP give rise to an intracellular pool of Aβ that is produced by a pathway that is distinct from the one that produces soluble Aβ[24]. Although the role of intracellular Aβ in amyloid deposition remains to be established, recent studies from our laboratory indicate that intracellular Aβ1–42 aggregates establish an autocatalytic, solid-phase pathway, that results in the accumulation of insoluble amyloidogenic fragments of APP that may ultimately give rise to insoluble Aβ[50].

Aβ has been shown to elicit both trophic and toxic responses in neurons in vitro. Soluble, non-aggregated Aβ increases the survival of cultured hippocampal neurons and augments neurite elongation and branching 46, 47, 52, while aggregated or fibrillar Aβ induces neurodegeneration in cultured neurons 51, 52, 30, 31. The longer Aβ1–42 peptide is equally as toxic as the shorter Aβ1–39 isoform [30], indicating that the differential association of Aβ1–42 with pathogenesis in vivo is not reflected in the cell culture model of acute toxicity in vitro. The explanation for this apparent discrepancy is not yet clear, however the cell culture models for neuronal toxicity have been optimized to demonstrate toxicity requiring extended incubation times to preaggregate Aβ1–39 [30]. Although the toxic effects of Aβ on neurons have been the subject of many investigations, little is known about the interaction of Aβ with cultured neurons. In a previous study of the interaction of Aβ with human fibroblasts, we found that Aβ1–42 accumulates intracellularly and is resistant to degradation for at least 3 days, while Aβ1–39 does not accumulate [20]. In this study we have examined the adsorption, uptake and degradation of freshly solubilized Aβ peptides in differentiated, PC12 cells to elucidate any differences in the interaction of the pathological Aβ1–42 isoform of Aβ with neuron-like cells. The rationale for this is that these conditions may more closely reflect the situation in normal brain prior to the accumulation of insoluble amyloid deposits. Here we report that Aβ1–42 preferentially adsorbs to the surface of differentiated PC12 cells and is internalized at a higher rate than Aβ1–40. The intracellular Aβ1–42 is largely resistant to degradation and accumulates in late endosomes or secondary lysosomes, while Aβ1–40 and Aβ1–28 are rapidly degraded and do not accumulate. Both of these findings may serve to explain why the longer Aβ1–42 isoform is more closely associated with AD pathogenesis and preferentially accumulates in AD.

Section snippets

Peptide preparation and iodination

Aβ1–42 (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA), Aβ1–40 and Aβ1–28 (corresponding to the first 40 or 28 amino acids of this sequence) were synthesized, purified, lyophilized and characterized by electrospray mass spectroscopy as previously described [4]. Both Aβ1–42 and Aβ1–40 peptides contain approximately 10% of a population of heterogeneous failure sequences, which is typical for synthetic peptides of this size, even though Aβ1–40 appears to be homogeneous by inspection of peak shape on

Cell surface adsorption of Aβ

We initially characterized the adsorption of Aβ1–42, Aβ1–40 and Aβ1–28 to the surface of differentiated PC12 cells at 4°C, where no internalization occurs (Fig. 1A). Similar results were obtained when phenylarsine oxide was used to inhibit endocytosis (data not shown). The amount of Aβ associated with the cell surface increases nearly linearly with Aβ concentration in the medium up to the limit of the solubility of the peptide. The fact that the amount of cell-associated peptide does not

Discussion

We examined the interaction of different Aβ isoforms with cultured neuron-like PC12 cells with the goal of identifying interactions that could account for the preferential accumulation of the longer Aβ1–42 isoform in AD brain tissue and its close association with pathogenesis. We have identified two prominent differences in the manner in which Aβ1–42 and Aβ1–40 interacts with PC12 cells: The longer Aβ1–42 isoform preferentially adsorbs to the cell surface and as a consequence is preferentially

Acknowledgements

We express our gratitude to B. Soreghan, Dr. A. Yang, and Dr. D. Knauer for helpful discussions and comments on the manuscript. We also would like to thank Dr. C. Pike for help with initial studies in rat hippocampal neurons. This work was supported in part by grants from the NIH, GM07311 (D.B.), NS31230 and AG00538, and the American Health Assistance Foundation (C.G.).

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