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U.S.A."],"published-print":{"date-parts":[[2022,10,11]]},"abstract":"<jats:p>\n                    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 \u03b1-synuclein, tau, and amyloid-\u03b2 aggregates, achieving \u223c4 nm precision. Applying AT630 to\n                    <jats:italic>\n                      App\n                      <jats:sup>NL-G-F<\/jats:sup>\n                    <\/jats:italic>\n                    mouse brain tissues or aggregates extracted from a Parkinson\u2019s disease donor, we demonstrate excellent agreement with antibodies specific for amyloid-\u03b2 or \u03b1-synuclein, respectively, confirming the specificity of AT630. Subsequently, we use AT630 to reveal a linear relationship between \u03b1-synuclein aggregate size and cellular toxicity and discovered that aggregates smaller than 450 \u00b1 60 nm (aggregate\n                    <jats:sub>450nm<\/jats:sub>\n                    ) readily penetrated the plasma membrane. We determine aggregate\n                    <jats:sub>450nm<\/jats:sub>\n                    concentrations in six Parkinson\u2019s 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 \u00b1 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.\n                  <\/jats:p>","DOI":"10.1073\/pnas.2205591119","type":"journal-article","created":{"date-parts":[[2022,10,7]],"date-time":"2022-10-07T14:05:15Z","timestamp":1665151515000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":47,"title":["Quantitative super-resolution imaging of pathological aggregates reveals distinct toxicity profiles in different synucleinopathies"],"prefix":"10.1073","volume":"119","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1206-6133","authenticated-orcid":false,"given":"Michael J.","family":"Morten","sequence":"first","affiliation":[{"name":"Department of Brain Sciences, Imperial College London, London W12 0NN, United Kingdom"},{"name":"UK Dementia Research Institute at Imperial College London, London W12 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