Characterization of Oligomer Formation of Amyloid-β Peptide Using a Split-luciferase Complementation Assay

Abstract

Amyloid-␤ peptide (A␤) is the amyloid component of senile plaques in Alzheimer disease (AD) brains. Recently a soluble oliomeric form of A␤ in A␤ precursor protein transgenic mouse brains and AD brains was identified as a potential causative molecule for memory impairment, suggesting that soluble A␤ oligomers cause neurodegeneration in AD. Further characterization of this species has been hampered, however, because the concentrations are quite small and it is difficult to monitor A␤ oligomers specifically. Here we developed a novel method for monitoring A␤ oligomers using a split-luciferase complementation assay. In this assay, the Nand C-terminal fragments of Gaussia luciferase (Gluc) are fused separately to A␤. We found that conditioned media from both N-and C-terminal fragments of Gluc-tagged A␤1-42 doubly transfected HEK293 cells showed strong luminescence. We used gel filtration analyses to analyze the size of oligomers formed by the luciferase complementation assay, and found that it matched closely with oligomers formed by endogenous A␤ in Tg2576 neurons. Large oligomers (24 -36-mers), 8-mers, trimers, and dimers predominate. In both systems, A␤ formed oligomers intracellularly, which then appear to be secreted as oligomers. We then evaluated several factors that might impact oligomer formation. The level of oligomerization of A␤1-40 was similar to that of A␤1-42. Homodimers formed more readily than heterodimers. The level of oligomerization of murine A␤1-42 was similar to that of human A␤1-42. As expected, the familial AD-linked Arctic mutation (E22G) significantly enhanced oligomer formation. These data suggest that Gluc-tagged A␤ enables the analysis of A␤ oligomers. Alzheimer disease (AD)2 is common progressive neurodegenerative disorder causing dementia. In AD patient's brains, loss of neocortical synapses correlates with cognitive impairment (1). A␤ is the amyloid component of senile plaques in AD brains (2, 3) and is derived from the A␤ precursor protein (APP) through sequential proteolytic cleavage by ␤-secretase and ␥-secretase (4). Based on several genetic and biochemical studies, the amyloid hypothesis is now widely accepted as the pathogenesis of AD (4). It has recently been suggested that picomolar concentrations of dimeric, or oligomeric, rather than monomeric or fibrillar forms of A␤, are the most neurotoxic, but characterization of these forms of soluble A␤ is technically difficult.In previous in vitro A␤ fibrillization studies using synthetic A␤, a variety of soluble prefibrillar species, including A␤ protofibrils (5, 6), A␤-derived diffusible ligands (7), and amylospheroids (8) were reported as neuronal toxic molecules. Recently, soluble A␤ oligomers, A␤*56 (9) and A␤ dimers (10) were isolated from APP transgenic mouse brain and AD brain and these A␤ oligomers caused synaptic dysfunction and memory impairment. Moreover A␤ oligomers were observed to associate with senile plaques and correlate with synaptic loss in AD brains (11). These data suggest that prefibrillar A␤ oligomers may be a ...

A Food and Drug Administration-approved Asthma Therapeutic Agent Impacts Amyloid β in the Brain in a Transgenic Model of Alzheimer Disease

“…A␤ oligomerization and oligomer dissociation assays were performed in vitro using an A␤ split-luciferase complementation assay, as described previously (32). To evaluate the effect of cromolyn sodium on the formation of A␤ oligomers, a HEK293 cell line designed to stably overexpress the N-and C-terminal fragments of Gaussia luciferase conjugated to A␤ 42 was incubated without or with cromolyn sodium at 10 nM, 10 M, or 1 mM for 12 h at 37°C.…”

“…The oligomer dissociation assay was performed by incubating PBS or cromolyn sodium (10 nM, 10 M, or 1 mM) with conditioned media from naïve HEK293 cells overexpressing each half of Gaussia luciferase fused with A␤ 42 for 12 h at 37°C. The luciferase activity was measured as described previously (32).…”

Quantitative analysis of the time course of Aβ oligomerization and subsequent growth steps using tetramethylrhodamine-labeled Aβ

“…More recently, mass spectrometry coupled with ion mobility spectrometry (IM-MS) experiments have provided information regarding the size and shape of the lowmolecular-weight oligomeric forms of Aβ and other amyloidoic proteins (6, 7). A variety of biophysical methods have been used to study the oligomers extensively (2,(7)(8)(9)(10)(11)(12)(13)(14).Despite such studies, the ability to measure the rates of formation of the oligomers remains difficult. Recently, Lee et al (15) reported a method suitable for monitoring the full time course of aggregation showing distinct phases corresponding to oligomerization and fibrillization using Cys-Cys-Aβ and FlAsH dye binding to tetracysteine motifs arising from self-association of Aβ.…”

“…More recently, mass spectrometry coupled with ion mobility spectrometry (IM-MS) experiments have provided information regarding the size and shape of the lowmolecular-weight oligomeric forms of Aβ and other amyloidoic proteins (6, 7). A variety of biophysical methods have been used to study the oligomers extensively (2,(7)(8)(9)(10)(11)(12)(13)(14).…”

Investigation of the effects of carbon-based nanomaterials on A53T alpha-synuclein aggregation using a whole-cell recombinant biosensor

“…32 In this study, we adapted a protein-fragment complementation assay approach to observe the initial A53T αS-A53T αS interactions in living cells utilizing fragments of firefly luciferase that can reconstitute as active luciferase upon αS aggregation. [33][34][35] Here, we generated and characterized a whole-cell bioluminescent sensor which reports A53T αS oligomerization in cell by a fast, sensitive and semiquantitative assay. We demonstrate that this cell-based biosensor in addition to tracking A53T mutant αS oligomerization in cells (Figure 1) can be exploited to assess the effects of NPs on αS oligomerization inside the cells.…”