A synthetic amyloid lawn system for high-throughput analysis of amyloid toxicity and drug screening

Abstract

Amyloid-β (Aβ) is the major constituent of senile plaques in the brains of Alzheimer's disease patients. In order to develop an efficient in vitro system for studying the interaction of cells with Aβ aggregates, we have prepared a synthetic amyloid lawn by immobilizing Aβ peptides over a functionalized glass surface and subsequently incubating the template in a fresh Aβ solution. On the top of different types of amyloid lawns (e.g. monomeric, oligomeric, and fibrillar), we cultivated PC12 cells, creating physical contacts between the cells and the lawns. Results indicated that cell viability was differentially affected when grown atop different Aβ lawns while cells were well adhered onto the surface of these Aβ lawns. The mode of cell death by Aβ lawn was confirmed to be apoptotic rather than necrotic, showing that cells undergo suicide by just contact with Aβ lawn. While conventional ‘solution-based’ methods for testing amyloid toxicity suffer from problems such as lot-to-lot variations, continued fibrillation, and heterogeneous population of aggregates, our ‘surface-based’ lawn system is suitable for high-throughput analysis of amyloid toxicity, which may enable high-throughput screening of potential drug candidates for treating amyloid diseases with the goal of reducing the cell death on the lawn.

Introduction

Alzheimer's disease (AD) is characterized by the deposition of insoluble amyloid aggregates as amyloid plaques in the neuropil as well as the accumulation of neuro-fibrillary tangles in the cell bodies of neurons [1]. The majority of amyloid aggregates are composed of the amyloid-β (Aβ) peptides, a 39–43 amino acid residue peptide produced by cleavage from a larger amyloid precursor protein, APP [2], [3]. Aβ is present in unaffected individuals and has a normal physiological role; however, in AD patients the Aβ forms ordered aggregates that deposit extracellularly as amyloid or senile plaques in the neuropil, and as vascular deposits [4], [5]. Numerous studies indicated a central role for Aβ in AD pathogenesis; however, none of these studies definitively specify the form or the site of action of Aβ neurotoxicity. Strong circumstantial evidences supported the notion that prefibrillar aggregates of Aβ might be important for neurotoxicity [6], [7], [8], [9], but some studies also explicated that fibrillar Aβ, rather than amorphous aggregates of Aβ, were causing neuronal cell death in vitro [3], [10], [11], [12]. The mode of cell death induced by Aβ is also very controversial. Some groups confirmed the necrotic mode of cell death induced by Aβ [13], [14], whereas several studies indicated that apoptosis was induced in the presence of this peptide [15], [16]. Thus, it will be difficult to explain the topography of neurodegeneration until both the form of Aβ causing neurotoxicity and the mode of cell death induced by Aβ is clarified.

Several studies on the effect of Aβ on neuronal function had been explored so far involving the addition of exogenous Aβ peptides to neuronal preparations, with the concomitant uncertainties regarding peptide aggregation state and their access to sub-cellular compartments [17], [18]. Many reports demonstrated the toxicity assay of different Aβ aggregates prepared by initially dissolving fresh Aβ peptides into a buffered solution [19], [20], [21], but the solution phase should become to contain Aβ aggregates of different sizes/conformations resulting in a heterogeneous mixture, and the solution-based method cannot avoid a continuous fibrillation of Aβ during incubation. Furthermore, in vivo formation of amyloid aggregates had been known to be highly affected by the presence of a solid surface, and the mechanism of amyloid deposition over surface was observed to be significantly different and more physiologically relevant compared to that in solution phase [22], [23]. Recently, we have successfully developed an ‘amyloid lawn’ system by immobilizing Aβ peptide onto a functionalized solid surface, giving rise to different forms of aggregates such as monomers, oligomers, or fully grown fibrils on the surface [24], [25]. The purpose of this study was to apply the amyloid lawn system for observing the behavior of cells grown over these surfaces in vitro. For the analysis of amyloid lawn and cell viability, multiple analytical tools were utilized including ex situ atomic force microscopy (AFM), scanning electron microscopy (SEM), fluorescence microscopy, and confocal laser scanning microscopy. Our ‘surface-based’ amyloid lawn system is suitable for high-throughput analysis of amyloid toxicity since it can be easily transformed into a microarray format on solid substrate, which may lead to high-throughput screening of potential drug candidates for treating amyloid diseases with the goal of reducing the cell death on the lawn.