Probing the extracellular space for modulators of protein aggregation in neurodegeneration

BBSRC strategic theme: Bioscience for an integrated understanding of health

Protein misfolding and aggregation in vivo is associated with a wide range of human disorders including Alzheimer's disease, Parkinson's disease and systemic amyloidosis. The pathological hallmark of these diseases is amyloid fibrils that are formed when soluble proteins undergo multiple diverse and ever-changing conformations over time resulting in proteinaceous aggregates.

As the world's population is ageing, the impact of "amyloid diseases" is an increasingly prevalent health concern; therefore, it is critical to understand the mechanisms by which disease-associated peptides and proteins behave aberrantly.

Although the mechanisms of protein misfolding and aggregation are well studied in vitro using purified proteins and controlled conditions, this process occurs in a complex cellular environment that will highly influence protein misfolding and disease progression. Amyloid-B peptide (AB) deposits in the brain are a hallmark of Alzheimer's disease (AD), however, a large number of AD patients also develop cerebral amyloid angiopathy (CAA) with deposits of AB in the walls of cerebral arteries and capillaries.

As well, studies have shown that in cognitively healthy subjects, high baseline plasma AB1-42 levels may be associated with increased future risk of AD; therefore, looking at factors within blood homeostasis that impacts on the behaviour of AB is important for understanding the disease in a cellular context.

Platelets are the main cellular component of haemostasis. Upon stimulation, platelets expose phosphatidylserine (PS) and release PS-exposing extracellular vesicles (EVs) that circulate through the blood. The circulating level of these PS-exposing EVs is increased in many inflammatory and cardiovascular diseases.

Intriguingly, artificial lipid membrane bilayers containing PS have been reported to affect the oligomerisation and fibril growth of AB. Our first research question therefore focuses on the EVs themselves: to what extent do these physiologically and pathologically relevant circulating EVs modulate the aggregation of disease-relevant proteins? Activated platelets also release a wide array of biomolecules.

To date, several extracellular chaperones (ECs) such as clusterin and alpha-2-macroglobulin, have been isolated from blood plasma, are released by activated platelets, and found to potently inhibit AB aggregation. Interestingly, clusterin has been shown to decrease neuroinflammatory effects of AB aggregates. Our second research question therefore focuses on the factors released by platelets: do activated platelets release biomolecules that may act as enhancers or inhibitors of AB aggregation?

We will isolate EVs from stimulated platelets and erythrocytes and characterise them by flow cytometry and biophysical techniques (such as dynamic light scattering). The impact of the isolated extracellular factors on protein aggregation will be assessed using fibril forming kinetics assays to gain (with the Kumita lab) and identification of EV components will be probed using immunoblotting and proteomics-based methods.

Isolation and detailed biochemical studies of the inhibitory effects of putative modulators will be done, using cell-based assays to understand their impact on neuroinflammation caused by AB aggregates.