Protein aggregation is a complex process resulting in the formation of heterogeneous mixtures of aggregate populations that are closely linked to neurodegenerative conditions, such as Alzheimer’s disease. Here, we find that soluble aggregates formed at different stages of the aggregation process of amyloid beta (Aβ42) induce the disruption of lipid bilayers and an inflammatory response to different extents. Further, by using gradient ultracentrifugation assay, we show that the smaller aggregates are those most potent at inducing membrane permeability and most effectively inhibited by antibodies binding to the C-terminal region of Aβ42. By contrast, we find that the larger soluble aggregates are those most effective at causing an inflammatory response in microglia cells and more effectively inhibited by antibodies targeting the N-terminal region of Aβ42. These findings suggest that different toxic mechanisms driven by different soluble aggregated species of Aβ42 may contribute to the onset and progression of Alzheimer’s disease.

Soluble α-synuclein aggregates varying in size, structure, and morphology have been closely linked to neuronal death in Parkinson’s disease. However, the heterogeneity of different co-existing aggregate species makes it hard to isolate and study their individual toxic properties. Here, we show a reliable non-perturbative method to separate a heterogeneous mixture of protein aggregates by size. We find that aggregates of wild-type α-synuclein smaller than 200 nm in length, formed during an in vitro aggregation reaction, cause inflammation and permeabilization of single-liposome membranes and that larger aggregates are less toxic. Studying soluble aggregates extracted from post-mortem human brains also reveals that these aggregates are similar in size and structure to the smaller aggregates formed in aggregation reactions in the test tube. Furthermore, we find that the soluble aggregates present in Parkinson’s disease brains are smaller, largely less than 100 nm, and more inflammatory compared to the larger aggregates present in control brains. This study suggests that the small non-fibrillar α-synuclein aggregates are the critical species driving neuroinflammation and disease progression.

Aggregation of α-synuclein plays a key role in the development of Parkinson’s disease. Soluble aggregates are present not only within human brain but also the CSF and blood. Characterizing the aggregates present in these biofluids may provide insights into disease mechanisms and also have potential for aiding diagnosis. We used two optical single-molecule imaging methods called aptamer DNA-PAINT and single-aggregate confocal fluorescence, together with high-resolution atomic force microscopy for specific detection and characterization of individual aggregates with intermolecular β-sheet structure, present in the CSF and serum of 15 early stage Parkinson’s disease patients compared to 10 healthy age-matched controls. We found aggregates ranging in size from 20 nm to 200 nm, in both CSF and serum. There was a difference in aggregate size distribution between Parkinson’s disease and control groups with a significantly increased number of larger aggregates (longer than 150 nm) in the serum of patients with Parkinson’s disease. To determine the chemical composition of the aggregates, we performed aptamer DNA-PAINT on serum following α-synuclein and amyloid-β immunodepletion in an independent cohort of 11 patients with early stage Parkinson’s disease and 10 control subjects. β-Sheet aggregates in the serum of Parkinson’s disease patients were found to consist of, on average, 50% α-synuclein and 50% amyloid-β in contrast to 30% α-synuclein and 70% amyloid-β in control serum [the differences in the proportion of these aggregates were statistically significant between diseased and control groups (P = 1.7 × 10−5 for each species)]. The ratio of the number of β-sheet α-synuclein aggregates to β-sheet amyloid-β aggregates in serum extracted using our super-resolution method discriminated Parkinson’s disease cases from controls with an accuracy of 98.2% (AUC = 98.2%, P = 4.3 × 10−5). Our data suggest that studying the protein aggregates present in serum can provide information about the disruption of protein homeostasis occurring in Parkinson’s disease and warrants further investigation as a potential biomarker of disease.

Protein aggregation likely plays a key role in the initiation and spreading of Alzheimer’s disease pathology through the brain. Soluble aggregates of amyloid beta are believed to play a key role in this process. However, the aggregates present in humans are still poorly characterised due to a lack of suitable methods required for characterising the low concentration of heterogeneous aggregates present. We have used a variety of biophysical methods to characterise the aggregates present in human Alzheimer’s disease brains at Braak stage III. We find soluble amyloid beta-containing aggregates in all regions of the brain up to 200 nm in length, capable of causing an inflammatory response. Rather than aggregates spreading through the brain as disease progresses, it appears that aggregation occurs all over the brain and that different brain regions are at earlier or later stages of the same process, with the later stages causing increased inflammation.