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Characterising protein oligomers and their role in neurodegenerative disease in humans

Periodic Reporting for period 3 - Oligomers (Characterising protein oligomers and their role in neurodegenerative disease in humans)

Reporting period: 2018-09-01 to 2020-02-29

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 from humans 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.
We have continued to make good progress against all the objectives of the project . We have developed a range of assays to detect and characterise the small protein aggregates, oligomer, that are thought to play a key role in the development of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Specifically we have developed a liposome based assay to sensitively detect oligiomers that can permeabilise a lipid membrane, by detecting the resulting entry of calcium ions using fluorescence. We can also directly image the aggregates in cerebral spinal fluid by adding a dye, thioflavin T, and detect aggregates that are fibrillar.

We have now completed and published our study in Cell reports that uses these assays to compare the number and toxicity of the oligomers in control and cerebral spinal fluid from patients with Alzheimer's disease. We find that both samples contain similar number of oligomers and can have similar levels of toxicity as measured using our assay. The toxicity is caused by oligomers containing beta amlyoid. We found that both a nanobody to beta amyloid and Bapineuzumab, a humanised monoclonal antibody, can efficiently reduce calcium ion influx due to beta amyloid oligomers in buffer but are far less effective in CSF, particularly Bapipeuzumab. Our results suggest that the other species present in CSF reduces the effectiveness of oligomer binding. This method may have potential to test the effectiveness of therapies before costly clinical trials.

We have developed a method to characterize protein aggregates at the nanometer scale without the need for a conjugated fluorophore. The technique utilizes short complementary strands of DNA; a“docking” strand is extended from an aptamer, which selectively binds aggregates, whilst its complementary “imaging” strand is labelled with an organic fluorophore. The repeated transient binding of the imaging strand to the docking strand allows the imaging of the aggregate with 20 nm resolution Using this method we have demonstrated that this technique is able to detect and superresolve a range of aggregated species, including those formed by α-synuclein and amyloid-β. Additionally, this method enables endogenous protein aggregates to be characterized; we found that neuronal cells derived from patients with Parkinson’s disease contain a larger number of protein aggregates than those from healthy controls.

We have developed rationally designed single chain domain antibodies that scan the length of beta amyloid to image oligomers and map their toxic regions. We have also synthesised a high affinity version of a dimer of thioflavin T with a biotin attached and show that this can bind alpha synuclein oligomers. We have then used this to pull-down the oligomers in AD, PD and control CSF for analysis by mass spectrometry. However our sensitivity is not sufficient to detect the aggregates present in human CSF by about a factor of 10. We are therefore exploring a number of different ways of improving the assay to get the required sensitivity.

Overall our results to date using these methods suggest that there is change in the structure or size of a small number of oligomers that is important in driving the development of Alzheimer's disease. We will use a combination of our new methods to identify these oligomers.
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.