Abstract Endosomal transport and positioning are involved in establishing neuronal compartment architecture, dynamics and function, contributing to neuronal intracellular logistics. Furthermore, endo-lysosomal dysfunction has been identified as a common mechanism in neurodegenerative diseases. Here, we analyzed endolysosomal transport following the external application of α-synuclein (α-syn) fibrillar polymorphs, ß-amyloid (Aß) fibrils and oligomers on primary cultures of mouse cortical neurons. We used a simple readout to measure this transport: the spontaneous endocytosis of fluorescent nanodiamonds — a perfectly stable nano-emitter — in cultured neurons. We then performed a high-throughput automatic extraction and quantification of the directed motions of these nanodiamonds. α-syn fibrillar polymorphs, Aß fibrils and oligomers halved the proportion of nanodiamonds transported along microtubules, but only slightly decreased their interactions with cortical neurons. This large decrease in endosomal transport would be expected to have a huge impact on neuronal homeostasis. We then assessed lysosomal dynamics with Lysotracker. The exposure of neurons to Aß oligomers led to an increase in the number of lysosomes, a decrease in the fraction of moving lysosomes and an increase in their size, reminiscent of findings for the APP transgenic model of Alzheimer’s disease. We then analyzed the effect of α-syn fibrillar polymorphs, Aß fibrils and oligomers on endosomal and lysosomal transport and quantified the directed transport of these assemblies within cortical neurons. We report different impacts on endosomal and lysosomal transport parameters and differences in trajectory length for cargoes loaded with pathogenic protein assemblies. Our results suggest that the internalization and transport of intraneuronal pathogenic protein aggregates are potential targets for novel neuroprotective treatment strategies. Significance Statement Neurodegenerative diseases (NDs) are characterized by the deposition of protein aggregates with broad-range neuronal toxicity. Defects of endolysosomal trafficking are increasingly being seen as key pathological features of NDs, probably contributing to synaptic dysfunction and ultimate neuronal death. We used fast fluorescence videomicroscopy to investigate endosomal and lysosomal dynamics in the branches of mouse cortical neurons in primary cultures following the application of α-syn fibrillar polymorphs (fibrils and ribbons) and Aß assemblies (oligomers and fibrils). We provide new insight into the differential effects of these pathogenic protein assemblies on endosomal and lysosomal transport, and reveal differences in the transport characteristics of the compartments loaded with these protein assemblies relative to endosomes.

Impact of α-synuclein fibrillar strains and ß-amyloid assemblies on the endolysosomal logistics of mouse cortical neurons