An Ultra-sensitive Assay to Measure Oligomer Induced Toxicity in Human Cerebrospinal Fluid

Project Information

Neurotoxic

Grant agreement ID: 701013

DOI

10.3030/701013

Project closed

EC signature date 24 February 2016

Start date 1 August 2016

End date 31 July 2018

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 195 454,80

EU contribution

€ 195 454,80

Coordinated by

THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE
United Kingdom

Periodic Reporting for period 1 - Neurotoxic (An Ultra-sensitive Assay to Measure Oligomer Induced Toxicity in Human Cerebrospinal Fluid)

Reporting period: 2016-08-01 to 2018-07-31

Summary of the context and overall objectives of the project

There are an estimated 50 million people worldwide living with dementia and the total estimated worldwide cost of dementia is $1 trillion. Alzheimer’s disease is most common cause of dementia which accounts for an estimated approximately 60% to 80% percent of cases. The conversion of peptides or proteins from their soluble native states into insoluble amyloid deposits has been described as a hallmark of many neurodegenerative disorders including Alzheimer’s disease (AD). Misfolded oligomeric intermediates, rather than matured fibrils, formed during the aggregation process have been identified as the primary pathogenic agents. It has been shown that targeting these oligomeric species with antibodies can reduce pathology in both mouse models and the human brain. This highly heterogeneous conformers of protein aggregation are transient species which only constitute a very small fraction as compared to the amyloid and the non-aggregated native form of the specific protein or peptide. Therefore, despite the importance of oligomers as the key cause of pathogenesis of many neurodegenerative diseases, it has been extremely challenging to characterize experimentally the functions and structures of these small soluble aggregates both within aggregation mixture at physiological condition (in the range of pM concentration) as well as in human bio-markers such as Cerebrospinal fluid. Moreover, due to the complex mechanism of their formation and their heterogeneous and dynamical nature, traditional bulk methods of structural biology cannot easily be applied. Therefore, it is important to develop methods that can detect and quantify the toxic form of aggregate present in the aggregation mixture as well as in complex biofluids. Such a method would then allow to study molecules that bind these toxic forms and delineate effective therapeutic strategies for protein aggregation disorders.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Development of ultrasensitive assay to measure protein aggregate induced Ca2+ influx:

I have developed a high-throughput biophysical assay which identifies oligomers as the most toxic form of protein aggregates in the complex aggregation mixtures of Aβ42 peptide. For that fabricated nanosized single vesicles as an optochemical probe consists of a native-like model system. Small encapsulation of these vesicles (10-18 L) makes the effective concentrations of encapsulated molecules very high which enables to study protein aggregate induced toxicity study in picomolar protein concentration.
Therefore, thousands of surface immobilize single vesicles have been tethered to the surface of glass coverslides using biotin-neutravidin linkage. Tethering of single vesicles have been shown to maintain their ion permeability, spherical morphology and diffusivity of membranes. In this single vesicle assay to quantify protein aggregate induced toxicity, the fellow has imaged the membrane permeabilization by visualizing Calcium influx into hundreds of these individual vesicles per field of view (2500 µm2) in a high throughput manner using highly sensitive Total internal reflection fluorescence microscopy (TIRFM) approach.

This work has been published in Angewandte Chemie 2017, 56, 7750

Measurement of oligomer induced Ca2+ influx for oligomers - aβ, αS, tau and PrP

Using this assay can identify the most toxic species formed during a protein aggregation reaction. Monomeric Aβ42 was added to the vesicles and no notable membrane disruption was detected, suggesting that the monomeric protein has no toxic effect. Then, monomeric amyloid beta with 42 residues was incubated at 37 °C and an aggregation mixture has been taken at the end of the lag-phase to test its potential toxic effect. At that time of the aggregation the presence of both small protein aggregates and monomeric protein has been shown. After addition and incubation of this aggregation mixture, localized fluorescence increase was detected due to aggregate induced membrane disruption and calcium influx. This suggest membrane disruption suggesting that the aggregated protein gained a toxic function compared to the monomeric protein. The fellow also has tested an aggregation reaction that reached the plateau phase for which most of the monomeric protein has been reported to be converted into fibrils and detected a decreased toxic effect compared with the aggregation reaction at the end of the lag phase. Similarly, we have used this to measure a-Synuclein, tau and PrP oligomer induced membrane permeation.

Part of this work has been published in ACS Chemical biology 2018, 13, 636 and Angewandte Chemie 2018, 57, 4886; ACS NANO 2018 (10.1021/acsnano.8b03575) and Nano Letters 2018 (10.1021/acs.nanolett.8b02916)

Screening of inhibitor to block the Ca2+ influx:

Using protein aggregate specific antibody we are able block protein aggregate induced membrane permeation. I test the effect of different antibodies/nanobodies which are effective at reducing the Calcium influx by inhibiting the membrane permeating effect as a function of antibody concentration. This assay serves as a powerful screening platform for antibodies and nanobodies and also contribute significantly to the development of novel diagnostic tools.

Part of this work has been published in ACS Chemical biology 2018, 13, 636 and Cell Reports 218, 23, 3492.


Translation of this assay to measure toxicity induced by protein aggregate present in human CSF

I have translated this study to the protein aggregate present in CSF of individual affected with Alzheimer’s disease and shown the presence amyloid beta aggregate. I have used various antibodies and nanobodies and compare their inhibition potency using membrane permeation assay. This work is extremely interesting for screening therapeutic agent with target toxic protein aggregates associated with Alzheimer’s diseases.
This work has been published in Cell Reports 20

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The accumulation of senile plaques of amyloid-beta proteins in brain tissue of affected individuals is one of the hallmarks of Alzheimer's disease. However, the extent of plaque deposition in brain poorly correlate with the extent of dementia; for example, many middle aged and elderly people have extensive plaque deposition without any signs of AD such as memory loss and difficulties with thinking or language. In recent years, many researchers demonstrate that small, soluble clusters of amyloid-beta, known as oligomers, rather than large conspicuous amyloid plaques, are the primary disease causing species. These oligomers can kill nerve cell and thereby suppress learning and memory. Oligomers are highly heterogeneous in size and structure, are low in abundance human brain. Thus, the actual identity of these species participating in initiation and progression of Alzheimer's disease remains elusive. Therefore, measuring the toxicity of protein aggregates is of vital importance because these species appear to constitute the main toxic agents in many neurodegenerative diseases. Moreover, the structural investigation of these individual aggregates is of vital importance because these species appear to constitute the main toxic agents in many neurodegenerative diseases. Beside, measuring effectivity of antibodies using this highly sensitive assay by measuring their inhibition ability allows to develop a screening platform .for therapeutic agent for Alzheimer’s disease.

Ultrasensitive assay to measure protein aggregate induced Calcium influx