J Biol Chem. 2015 Nov 20;290(47):28343-52. doi: 10.1074/jbc.M115.676072.

Amyloid Oligomers and Mature Fibrils Prepared from an Innocuous Protein Cause Diverging Cellular Death Mechanisms.
 

Harte NP1, Klyubin I2, McCarthy EK3, Min S1, Garrahy SA4, Xie Y1, Davey GP5, Boland JJ3, Rowan MJ2, Mok KH6.

  • 1From the Trinity Biomedical Sciences Institute (TBSI), School of Biochemistry and Immunology, Trinity College Dublin, the University of Dublin, Dublin 2, Ireland.
  • 2the Department of Pharmacology and Therapeutics, Trinity College Institute of Neuroscience (TCIN), Trinity College Dublin, the University of Dublin, Dublin 2, Ireland.
  • 3the School of Chemistry and Centre for Research on Adaptive Nanostructures and Nanodevices (CRANN), Trinity College Dublin, the University of Dublin, Dublin 2, Ireland, and.
  • 4TCIN, Trinity College Dublin, the University of Dublin, Dublin 2, Ireland.
  • 5From the Trinity Biomedical Sciences Institute (TBSI), School of Biochemistry and Immunology, Trinity College Dublin, the University of Dublin, Dublin 2, Ireland, TCIN, Trinity College Dublin, the University of Dublin, Dublin 2, Ireland.
  • 6From the Trinity Biomedical Sciences Institute (TBSI), School of Biochemistry and Immunology, Trinity College Dublin, the University of Dublin, Dublin 2, Ireland, Centre for Research on Adaptive Nanostructures and Nanodevices (CRANN), Trinity College Dublin, the University of Dublin, Dublin 2, Ireland, and mok1@tcd.ie.

 

Summery:

  • Background: Although oligomers are considered more important, mature fibrils also show evidence as cytotoxic agents in neurodegenerative diseases.
  • Results: Oligomers and fibrils both kill PC12 cells albeit mechanistically differently. In vivo, only oligomers inhibit hippocampal long term potentiation.
  • Conclusion: Protein aggregates, even those irrelevant to disease, are capable of inducing different toxic actions in neuronal cells.
  • Significance: Understanding these toxic mechanisms is vital in improving amyloidosis therapy

 

Abstract

Despite significant advances, the molecular identity of the cytotoxic species populated during in vivo amyloid formation crucial for the understanding of neurodegenerative disorders is yet to be revealed. In this study lysozyme prefibrillar oligomers and fibrils in both mature and sonicated states have been isolated through an optimized ultrafiltration/ultracentrifugation method and characterized with various optical spectroscopic techniques, atomic force microscopy, and transmission electron microscopy. We examined their level and mode of toxicity on rat pheochromocytoma (PC12) cells in both differentiated and undifferentiated states. We find that oligomers and fibrils display cytotoxic capabilities toward cultured cells in vitro, with oligomers producing elevated levels of cellular injury toward undifferentiated PC12 cells (PC12(undiff)). Furthermore, dual flow cytometry staining experiments demonstrate that the oligomers and mature fibrils induce divergent cellular death pathways (apoptosis and secondary necrosis, respectively) in these PC12 cells. We have also shown that oligomers but not sonicated mature fibrils inhibit hippocampal long term potentiation, a form of synaptic plasticity implicated in learning and memory, in vivo. We conclude that our in vitro and in vivo findings confer a level of resistance toward amyloid fibrils, and that the PC 12-based comparative cytotoxicity assay can provide insights into toxicity differences between differently aggregated protein species.

KEYWORDS: amyloid; apoptosis; cell death; fibril; long term potentiation; neurodegenerative disease; oligomer; protein aggregation; protein misfolding

PMID: 26221033

 

Supplement:

  • The aggregation of a non-disease-related protein in brain tissue caused toxic effects similar to those with key roles in devastating neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s and Creutzfeld Jakob Diseases.
  • This suggests that presence alone is as important as the specific form or identity of a protein in causing toxic effects

In this paper, we have been able to show that the mere presence of protein aggregates may be as important as the form and identity of these proteins in inducing cell death in brain tissue.

 

For over a decade, much research has focused on determining which form of protein aggregates is the most important in mediating cell degeneration (‘small oligomers’ or ‘mature amyloid fibrils’) in these debilitating diseases.

 

Our team of interdisciplinary scientists encompassing the biomedical, neuro, and nanosciences, all of whom are based at Trinity College Dublin, adopted a “neutral territory” approach by comparing the effects of these two forms of a non-disease-related protein. We made comparisons in cells and animal models, and via experiments that studied electrical ‘brainwave’ responses in these cells.

 

We were able to show that both forms killed cells and impacted on potential memory processes in a similar way to specific proteins such as beta amyloid (Alzheimer’s), alpha-synuclein (Parkinson’s), huntingtin (Huntington’s) and prions (Creutzfeld Jakob) – despite the protein aggregates coming from a non-harmful hen egg protein called lysozyme.

 

It was really surprising that both forms of the protein aggregates operated in a similar way to the neurodegenerative-disease-related proteins. Their toxic effects, which included cell death, came about via different mechanisms, but the big news here is that the mere presence of these protein aggregates seems to endow them with the properties that are so damaging to brain cells – it is a surprise that a non-harmful protein like lysozyme can have such an effect. In addition, we have shown that rather than one or the other, both the oligomeric and mature fibril forms are clearly toxic, providing an answer to a topic that had been debated for many years.

 

Scientists all across the world, including ourselves, are tirelessly working to develop drug candidates that will prevent such protein aggregation from occurring. It is hoped that preventing or reversing this process will turn the tide in the fight against these debilitating neurodegenerative diseases.

 

Contact:

K. H. Mok, Ph.D.

Associate Professor of Biochemistry

Protein Folding and Biomolecular NMR Spectroscopy Laboratory

Director, TBSI NMR Facility

 

School of Biochemistry & Immunology, Room 504

Trinity Biomedical Sciences Institute (TBSI)

Trinity College Dublin

Dublin 2

Ireland

 

Telephone:  +353 1 896 3190 (Office)

FAX:              +353 1 677 2400

e-mail:          mok1@tcd.ie

Web pages:

http://www.tcd.ie/Biochemistry/research/k_h_mok.php

http://www.crann.tcd.ie/Research/Investigators/School-of-Biochemistry-and-Immunology/Dr-Kenneth-Mok.aspx

KM FIG1

 

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