Quantitative super-resolution imaging of pathological aggregates reveals distinct toxicity profiles in different synucleinopathies

Protein aggregation is a hallmark of major neurodegenerative disorders. Increasing data suggest that smaller aggregates cause higher toxic response than filamentous aggregates (fibrils). However, the size of small aggregates has challenged their detection within biologically relevant environments. Here, we report approaches to quantitatively super-resolve aggregates in live cells and ex vivo brain tissues. We show that Amytracker 630 (AT630), a commercial aggregate-activated fluorophore, has outstanding photophysical properties that enable super-resolution imaging of a-synuclein, tau, and amyloid-beta aggregates, achieving similar to 4 nm precision. Applying AT630 to App(NL-G-F) mouse brain tissues or aggregates extracted from a Parkinson's disease donor, we demonstrate excellent agreement with antibodies specific for amyloid-beta or a-synuclein, respectively, confirming the specificity of AT630. Subsequently, we use AT630 to reveal a linear relationship between a-synuclein aggregate size and cellular toxicity and discovered that aggregates smaller than 450 +/- 60 nm (aggregate(450nm)) readily penetrated the plasma membrane. We determine aggregate450nm concentrations in six Parkinson's disease and dementia with Lewy bodies donor samples and show that aggregates in different synucleinopathies demonstrate distinct potency in toxicity. We further show that cell-penetrating aggregates are surrounded by proteasomes, which assemble into foci to gradually process aggregates. Our results suggest that the plasma membrane effectively filters out fibrils but is vulnerable to penetration by aggregates of 450 +/- 60 nm. Together, our findings present an exciting strategy to determine specificity of aggregate toxicity within heterogeneous samples. Our approach to quantitatively measure these toxic aggregates in biological environments opens possibilities to molecular examinations of disease mechanisms under physiological conditions.

Automated summary

Protein aggregation is a characteristic feature of major neurodegenerative disorders. This study presents an approach to quantitatively super-resolve aggregates in live cells and brain tissues, and reveals a linear relationship between aggregate size and cellular toxicity for neurodegenerative proteins. The results suggest that smaller aggregates can penetrate the plasma membrane and have higher toxicity. This finding provides a potential strategy to determine the specificity of aggregate toxicity and opens up possibilities for studying disease mechanisms in physiological conditions.