Single molecule imaging of protein aggregation in Dementia: Methods, insights and prospects

The formation of protein aggregates in the brain is a central aspect of the pathology of many neurodegenerative diseases. This self-assembly of specific proteins into filamentous aggregates, or fibrils, is a fundamental biophysical process that can easily be reproduced in the test tube. However, it has been difficult to obtain a clear picture of how the biophysical insights thus obtained can be applied to the complex, multi-factorial diseases and what this means for therapeutic strategies. While new, disease-modifying therapies are now emerging, for the most devastating disorders, such as Alzheimer's and Parkinson's disease, they still fall well short of offering a cure, and few drug design approaches fully exploit the wealth of mechanistic insights that has been obtained in biophysical studies. Here, I attempt to provide a new perspective on the role of protein aggregation in disease, by phrasing the problem in terms of a system that, under constant energy consumption, attempts to maintain a healthy, aggregate-free state against the thermodynamic driving forces that inexorably push it toward pathological aggregation.

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SynPull: a novel method for studying neurodegeneration-related aggregates in synaptosomes using super-resolution microscopy

Kedia,

Fertan,

et al. 2024

Preprint

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Synaptic dysfunction is one of the primary hallmarks of both Alzheimer’s and Parkinson’s disease, leading to cognitive and behavioural decline. While alpha-synuclein, beta-amyloid, and tau are involved in the physiological functioning of synapses, their pathological aggregation has been linked to synaptic dysfunction. However, the methodology for studying the small (sub-diffraction limit) and soluble aggregates -often called oligomers, formed by these proteins is limited. Here we describe SynPull, a novel method combining single-molecule pulldown, super-resolution microscopy, and advanced computational analyses, in order to reliably study the quantity and morphology of the oligomeric alpha-synuclein, beta-amyloid, and AT8-positive tau aggregates in synaptosomes harvested from post-mortem human brain samples and mouse models. Using SynPull, we show that AT8-positive tau is the predominant aggregate type in AD, with significantly more aggregates compared to the control samples, yet the aggregate size does not differ between disease and control samples. Meanwhile, the relatively smaller amount of alpha-synuclein and beta-amyloid aggregates found in the synapses are larger than the extra-synaptic ones. Collectively, these results show the utility of SynPull to study pathological aggregates in dementia, which can help further understand the disease mechanisms causing synaptic dysfunction.Abstract FigureGraphical abstract.Human post-mortem orbitofrontal cortex samples from subjects with neuropathological diagnosis of Alzheimer’s and Parkinson’s disease, as well as age-matched controls cut into ∼300 mg sections, and MI2, APPNL-G-F, P301S, and C57Bl/6J mouse brains were first homogenised in synaptosome buffer using a Dounce homogeniser and then filtered and centrifuged to separate nuclei and organelles from the synaptic fragments. Then, the isolated synaptosomes were incubated on the SiMPull surface with anti-neurexin antibody overnight, followed by fixation and permeabilisation. Imaging antibodies against beta-amyloid, alpha-synuclein, and AT8-positive tau were added to the samples anddSTORM imaging was performed to super-resolve the aggregates.

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Single molecule imaging of protein aggregation in Dementia: Methods, insights and prospects

Cited by 3 publications

References 105 publications

Advances in the study of organelle interactions and their role in neurodegenerative diseases enabled by super-resolution microscopy

Advances in the study of organelle interactions and their role in neurodegenerative diseases enabled by super-resolution microscopy

The thermodynamics of neurodegenerative disease

SynPull: a novel method for studying neurodegeneration-related aggregates in synaptosomes using super-resolution microscopy

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