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Characterising protein oligomers and their role in neurodegenerative disease in humans

Periodic Reporting for period 4 - Oligomers (Characterising protein oligomers and their role in neurodegenerative disease in humans)

Reporting period: 2020-03-01 to 2021-08-31

Alzheimer’s and Parkinson’s disease are a major health problem for society as we live for longer but we still do not know the molecular cause of these diseases and hence cannot rationally design therapies for treatment. Small soluble protein aggregates are believed to play a key role in the development and spreading of these diseases but there is currently a lack of suitable methods to detect and characterise these aggregates, due to their low concentration and variable size and structure . The objectives of the project are to develop new methods to image and characterise these aggregates and then apply these methods to clinical samples of cerebral spinal fluid and serum from humans and post-mortem human brain samples to determine which aggregates are actually present in patients with Alzheimer’s or Parkinson’s diseases and what changes occur as the disease progresses . Overall we hope to establish the real aggregates that are present in humans and cause these disease and that should be targeted by therapies and our work may also provide new methods for early diagnosis of disease.
Soluble protein aggregates of tau, beta amyloid and a-synuclein with a range of different sizes and structures are formed during the initiation and development of neurodegenerative disease and are toxic to cells by a variety of different mechanisms. However, currently there is a lack of methods to detect and characterise these soluble aggregates, especially in human-derived samples, due to their low concentration and heterogeneity. To address this issue, in this project we have developed a suite of methods to detect and characterise individual protein aggregates in cells, cell media , cell lysate, CSF or serum and post-mortem human brain samples. Our methods, based on single-molecule fluorescence, can measure the number and composition of individual protein aggregates, their size down to 20 nm, their resistance to proteinase K (PK) degradation and association with other molecules, such as APOE, as well their ability to permeabilise lipid membranes and cause inflammation.

We have applied these method to induced pluripotent stem cell models of neurogenerative disease, clinical samples of cerebrospinal fluid and serum and post-mortem human brain. Our main findings are that aggregates of a range of sizes , 20-150 nm, are formed under normal conditions but there is a change in the size distribution and structure and composition of the aggregates during the development of neurodegenerative disease due to celllular stress, without changes in aggregate number. In particular, we find that the formation of long strings (protofilaments) of amyloid-beta occurs during the development of Alzheimer's disease which cause inflammation via a receptor called toll-like receptor 4. This makes toll-like receptor 4 a potential therapeutic tartget. We also found an increased proportion of a-synuclein aggregates in the serum of patients with Parkinson's disease making this a potential biomarker for disease detection. In the brain of Alzheimer's disease patients we detected inflammatory aggregates in all brain regions but found an increased proportion of smaller inflammatory aggregates in the regions where disease was starting to develop. This suggests the same process is occuring to a greater or lesser extent in different regions of the brain with the formation of more inflammatory aggregates in certain brain regions driving disease progression. We also showed that aggregate-induced inflammation caused long-term potentiation deficit in brain slices, a correlate of memory loss, linking aggregate induced inflammation to the cognitive decline observed in the disease process.

Overall, by exploiting our new methods we have found that small changes in aggregate size and shape occur during the development of Alzheimer's and Parkinson's disease leading to increased inflammation which drives further aggregation and spread of disease through the brain. Blocking inflammation may be a potential treatment and monitoring changes in aggregate size and composition has the potential for early disease diagnosis.

We have published 24 paper during the project describing these new methods and their application to disease models and human samples. We have also presented these results at international meetings on neurodegenerative disease.
We have developed a suite of new methods to image and characterise protein oligomers that can be used on other relevant proteins to neurodegenerative disease such as tau and prion protein and follow their aggregation mechanism without the need to label the protein. This should provide new insights into the mechanism of aggregation and in particular so called prion-like replication where an aggregate can grow and fragment to produce many aggregates and hence spread from cell to cell. There are also potential applications in early cancer detection where our methods can be used to detect aggregates of P53. Our assay which measures the ability of aggregates to permeabilise a lipid membrane can test the possible immunotherapies that target protein aggregates by adding antibodies directly to human CSF and seeing if there is a reduction in toxicity. This may be used to determine the antibody concentration needed for effective treatment and if the treatment might work in humans before undertaking costly clinical trials.

Our results show that Toll-like receptor 4 is a potential therapeutic target for Alzheimer's and Parkinson's disease and more tractable than targeting the heterogeneous protein aggregates. We have also developed a potential biomarker for Parkinson's disease which may allow early disease detection and to monitor the efectiveness of potential therapies.
Detecting aggregates in post-mortem brain