A synthetic amyloid lawn system for high-throughput analysis of amyloid toxicity and drug screening
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.
Section snippets
Materials
Human amyloid-β (Aβ) 42 was obtained from rPeptide Co. (Athens, GA). Bovine serum albumin (BSA), 3-aminopropyltriethoxysilane (APTS), N,N′-disuccinimidyl carbonate (DSC), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), dimethyl sulfoxide (Me2SO), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), and Hoechst 33258 were purchased from Sigma–Aldrich (St. Louis, MO). Micro-cover glasses were obtained from VWR Scientific (West Chester, PA). Culture media and supplements were obtained
Results
The concept of cell culture over amyloid lawn is illustratively described in Fig. 1A. We cultured cells over Aβ lawns, which consisted of three different forms of Aβ aggregate states like – monomeric, oligomeric, and fibrillar. The representative AFM images of Aβ lawn surfaces in Fig. 1A show oligomeric or fibrillar aggregates, respectively. Cells were well adhered onto the surface of these Aβ lawns as observed under inverted optical microscope. The number of adhered cells was similar to the
Discussion
Over the last two decades, accumulating evidences had implicated abnormal protein aggregation as a mechanistic feature of many amyloid diseases, including Alzheimer's disease [5], [35]. Although significant progress has been made in understanding the mechanism of amyloid fibril formation [36], there still remains uncertainty with regard to Aβ-mediated neuronal cell death [37]. Studies on the neurotoxic properties of Aβ aggregates had proven it difficult to interpret because of significant
Conclusion
We have established an in vitro system for evaluating the cellular toxicity of amyloid by introducing a new concept of amyloid lawn. According to our results, cell viability was differentially affected when grown on the top of the synthetic amyloid lawn, depending on the precursor peptide (i.e. Aβ and insulin) and the aggregate size (i.e. monomeric, oligomeric, and fibrillar). The results suggest that amyloid aggregates can cause cell death through contact, not by their uptake inside cell,
Acknowledgement
This research was supported by grants from the KAIST Institute for the BioCentury, the BioGreen 21 Program (20070301034038), and the Korea Research Foundation (KRF-2006-D00078). The authors thank the National NanoFab Center (NNFC) in Korea for technical supports in the characterization.
References (40)
The molecular pathology of Alzheimer's disease
Neuron
(1991)- et al.
Amyloid β-protein fibrillogenesis. Structure and biological activity of protofibrillar intermediates
J Biol Chem
(1999) - et al.
Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability
J Biol Chem
(2002) - et al.
Amyloid beta peptide induces necrosis rather than apoptosis
Brain Res
(1994) Amyloid β-protein induces necrotic cell death mediated by ICE cascade in PC12 cells
Exp Cell Res
(1997)- et al.
Dramatic aggregation of Alzheimer Aβ by Cu(II) is induced by conditions representing physiological acidosis
J Biol Chem
(1998) - et al.
Surface-catalyzed amyloid fibril formation
J Biol Chem
(2002) - et al.
Pathways and intermediates of amyloid fibril formation
J Mol Biol
(2007) - et al.
Genotoxic effects in M5 cells and Chinese hamster V79 cells after exposure to 7Li-beam (LET = 60 keV/microm) and correlation of their survival dynamics to nuclear damages and cell death
Mutat Res
(2007) - et al.
β-Amyloid peptide in vitro toxicity: lot-to-lot variability
Neurobiol Aging
(1992)