Abstract
Cells are extremely complex systems able to actively modify their metabolism and behavior in response to environmental conditions and stimuli such as pathogenic agents or drugs. The comprehension of these responses is central to understand the molecular bases of human pathologies, including amyloid misfolding diseases. Conventional bulk biological assays are limited by intrinsic cellular heterogeneity in gene, protein and metabolite expression, and can investigate only indirectly cellular reactions in non-physiological conditions. Here we employ a label-free nanomotion sensor to study single neuroblastoma cells exposed to extracellular monomeric and amyloid α-synuclein species in real-time and in physiological conditions. Combining this technique with fluorescence microscopy, we demonstrate multispecies cooperative cytotoxic effect of amyloids and aggregate-induced loss of cellular membrane integrity. Notably, the method can study cellular reactions and cytotoxicity an order of magnitude faster, and using 100-fold smaller volume of reagents when compared to conventional bulk analyses. This rapidity and sensitivity will allow testing novel pharmacological approaches to stop or delay a wide range of human diseases.
Highlights
The complexity of the brain architecture and its cellular heterogeneity are central limitations to evaluate the specific responses, biological functions and causes of cytotoxicity in living organisms when subjected to external stimuli such as the exposition to pathogenic agents or drugs
We considered at the following α-syn preparations: (a) freshly filtrated monomers, (b) a solution of mature fibrillar aggregates and (c) a stable mixture of oligomeric and mature fibrillar species, which we called crude mixture.[20,21,22]
Most of the available evidences indicate that the aggregation of α-syn is essential for the induction of the pathological effects associated with Parkinson’s disease (PD).[28,29,30]
Summary
The complexity of the brain architecture and its cellular heterogeneity are central limitations to evaluate the specific responses, biological functions and causes of cytotoxicity in living organisms when subjected to external stimuli such as the exposition to pathogenic agents or drugs. By only considering the average cellular reactions from a large population of cells,[2,3] they might conceal important but subtle individual cellular responses.[4] these bulk assays can assess mid- and long-term cellular responses generally arising long after the exposure to chemical or physical stimuli. For this reason, these methods could not account for a phenotypic modification in the studied cells during the assessment and they could mask the dynamics of early events that are important in the initiation and long-term propagation of cellular responses to this exposure
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