Oligomeric Amyloid-β Toxicity Can Be Inhibited by Blocking Its

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Oligomeric Amyloid-# Toxicity Can be Inhibited by Blocking its Cellular Binding in Cortical Neuronal Cultures with Addition of the Triphenylmethane Brilliant Blue G Dye. Metta K Jana, Roberto Cappai, and Giuseppe D Ciccotosto ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00108 • Publication Date (Web): 03 Jun 2016 Downloaded from http://pubs.acs.org on June 4, 2016

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ACS Chemical Neuroscience

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Oligomeric Amyloid-β Toxicity Can be Inhibited by Blocking its Cellular Binding in Cortical Neuronal Cultures with Addition of the Triphenylmethane Brilliant Blue G Dye.

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Metta K. Jana, Roberto Cappai, and Giuseppe D. Ciccotosto*

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Department of Pathology, The University of Melbourne, VIC 3010, Australia

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ABSTRACT: Accumulation of soluble amyloid beta (Aβ) oligomers in the brain has been suggested to cause neurodegeneration associated with Alzheimer’s disease (AD). Our previous findings showed that the binding of Aβ trimer and tetramer to neurons significantly correlates with Aβ-induced neuronal cell death. We propose that blocking neuronal binding of these neurotoxic Aβ oligomers as a therapeutic strategy for preventing this disease. To test this, a non-toxic triphenylmethane brilliant blue G (BBG) dye, which has been reported to modulate Aβ aggregation and neurotoxicity, was investigated using mouse primary cortical neuronal cultures treated with photo-induced cross-linked toxic Aβ40 oligomers, as well as soluble Aβ40 and Aβ42 peptides. We found that BBG-induced decrease in Aβ binding resulted in significant decrease in its neurotoxicity. These findings support our hypothesis that disrupting cellular Aβ binding event is a promising therapeutic strategy for combating AD.

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KEY WORDS: Alzheimer’s disease (AD), amyloid beta (Aβ), oligomer, tetramer, brilliant blue G, PICUP, toxicity.

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 INTRODUCTION

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Alzheimer’s disease (AD) is the most common form of dementia affecting older people with declining brain cognitive functions. The presence of extracellular amyloid plaques in AD brains are the major pathological feature identifying this disease while the main constituent of these amyloid plaques, amyloid-beta (Aβ) peptide, is regarded as the cause of AD pathogenesis.1, 2 In the aging brain, Aβ peptide will undergo rapid and dynamic aggregation from monomeric state to form soluble oligomers, protofibrils then insoluble fibrils which are deposited as plaques in the brain.3 While the visual presence of post-mortem plaques in the brain is used to define AD, the detection of soluble Aβ oligomers from whole brain extracts correlates with the severity of AD.4, 5 While AD research field agrees that the toxic Aβ species is not the monomer or fibrillar form rather it is one or more of oligomeric species.6-10 We have recently identified that Aβ trimer and tetramer are the key toxic oligomers species that kill brain neurons,11 an event that is mediated by Aβ binding to neurons.12-14 These Aβ trimer and tetramer species have been isolated from human AD brains15, 16 but whether they are the true toxic species in AD remains unresolved.

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A triphenylmethane dye, brilliant blue G (BBG), is commercially known as coomassie blue and is routinely used for protein staining because of its specific binding to proteins.17 BBG is an analogue of a common food dye,18 and has been shown to be non-toxic 19 and blood brain barrier permeable.20 BBG dye has been shown to have neuroprotective properties in AD transgenic mice models 18, 21-23 and in other neurodegenerative disease mouse models including motor neuron disease,24 Huntington’s disease25 and Parkinson’s disease.26 BBG binds to Aβ40 monomers and modulates its aggregation to form non-toxic aggregates,23, 27 but whether it binds to and modulates the behaviour of toxic Aβ oligomers has not been reported. We previously showed that the binding levels of Aβ trimer and tetramer oligomers to neurons correlated with neurotoxicity while the preparation and isolation of Aβ40 oligomeric trimers and tetramers, using the photo-induced cross-linked (PICUP) technique, were shown to be 50-fold more neurotoxic than the soluble monomeric preparation.11 Therefore, based on the very toxic nature of these oligomeric peptides, we investigated the cell binding and toxic behaviour of BBG bound oligomers and soluble Aβ42 and Aβ40. We identified that when BBG was bound to the toxic Aβ oligomeric species, this diminished their ability to bind to neurons resulting in less cell death. Therefore, since BBG can directly target toxic Aβ oligomeric species, this may explain its neuroprotective properties in AD mouse models.18, 22, 23

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 RESULTS AND DISCUSSION

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BBG binds to purified Aβ40 1-4mer. We previously reported that purified cross-linked Aβ40 trimer (3mer) and tetramer (4mer) are the toxic Aβ oligomeric species while Aβ40 dimer (2mer) and monomer (1mer) were non-toxic to mouse cortical cultures.28 For the preparation of Aβ40 1mer-4mer, we used the reported PICUP method where gels were stained with BBG dye to identify the oligomeric bands then excising and purifying Aβ from 2 ACS Paragon Plus Environment

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gels (presence of BBG) 29 and a modified protocol we developed where the gels were not stained with BBG (absence of BBG)28 prior to purifying the Aβ from the gels. The purified Aβ samples in the presence of BBG were coloured blue suggesting that BBG dye is tightly bound to the peptide. Therefore, we initially investigated whether BBG binding to Aβ altered its structural properties using the region specific antibody, W02 which directly binds to amino acid residues 2 to 8 of Aβ30. We found that W02 antibody detected similar levels of Aβ when bound to BBG compared to without BBG sample suggesting BBG is most likely not bind to the epitope region for W02. In contrast, BBG was reported to decrease 4G8 (residues 17-24 of Aβ peptide)31 and 6E10 (residues 1-17 of Aβ peptide)31 anti-Aβ antibody reactivity32 further indicating BBG direct interaction with Aβ. Taken together, our results suggest that BBG is most likely binding the mid portion of Aβ peptide and not the amino terminal end where the W02 epitope is located and therefore will not interfere with our Aβ binding assay.

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To confirm that BBG was bound to the toxic oligomers, we performed mass spectrometry analysis as this technology would identify small changes in the molecular weight of the purified oligomers. While purified Aβ40 species (without BBG) were detected showing molecular weights in agreement with their theoretical masses (Fig. 1B), none of the purified BBG bound to Aβ40 species were detectable using ESI-MS instrument (Fig. 1C). This implies that BBG binding to Aβ can alter its electric charge status (i.e.: masking positive or negatively charged amino acids) and becomes undetectable by ESI-MS. BBG dye preferentially binds to amino acids - arginine > tyrosine, tryptophan, histidine > phenylalanine > lysine and alanine.33 Since the positively charged amino acids- histidine (13 and 14) and lysine (16) and the phenylalanine residues, positioned at 19 and 20 form part of the antibody epitopes for 6E10 and 4G8 respectively,32 taken together, these results suggest that BBG binds within the early to mid-portion Aβ peptide sequence and can bind to toxic Aβ oligomers.

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The toxicity of Aβ oligomers was diminished when bound to BBG. We next tested the purified Aβ40 1mer-4mer with and without BBG bound to it on our primary cortical neurons for 96 hours at two concentrations, 1µM and 2µM, (Fig. 2A and 2B respectively). At 1µM, the toxic Aβ40 4mer oligomer significantly killed neurons but when BBG was bound to it, the toxic nature of this oligomer was significantly inhibited with a 28% increase in cell viability (p