Objective: To test whether PIKfyve inhibition results in a lysosomal-driven reduction of alpha-synuclein aggregates in a mutant neuroblastoma cell line overexpressing an aggregation-prone form of alpha-synuclein.

Background: Presence of alpha-synuclein insoluble aggregates is a typical hallmark of different neurodegenerative disorders characterised by the presence of Lewy bodies, particularly Parkinson’s disease (PD). Clearing such aggregates, or preventing their formation, is a crucial test for any therapy aiming to reverse or halt the progression of PD. 

Boosting lysosomal activity is a promising route to clear synuclein aggregates since, physiologically, lysosomes are the cellular organelle responsible for removing proteins and peptides. Lysosomal biogenesis is finely regulated by transcription factor EB (TFEB), which is generally detected in the cell cytoplasm in its inactive form. Upon signal transduction, TFEB is translocated into the nuclei, where it activates a series of pathways responsible for lysosomal biogenesis. A well-known mechanism for TFEB translocation into the nuclei is PIKfyve inhibition, which prevents mTORC1-dependant phosphorylation of TFEB.

Method: In order to test whether PIKfyve inhibition results in a lysosomal-driven reduction of alpha-synuclein aggregates, we exploited a mutant neuroblastoma cell line overexpressing an aggregation-prone form of synuclein (3K-synuclein) and treated it with PIKfyve inhibitor YM201636. Our preliminary findings revealed a significant reduction (P<0.001) of a-synuclein aggregates as early as 24h post-treatment compared to the untreated group. Following this result, we investigated whether the reduction would still be present in a neuron-like cell model. We used BDNF and retinoic acid to differentiate the 3K-synuclein-expressing cells into neuron-like cells and treated them with YM201636.

Results: PIKfyve inhibition proved to be able to significantly (p<0.01) drop a-synuclein aggregates in differentiated cells. Ultimately, to investigate whether YM201636 effects were lysosomal driven, we co-treated cells with bafilomycin to disrupt proper lysosomal function. Impairment of lysosomes prevented the YM201636 reduction of a-synuclein in both models tested.

Conclusion: These results suggest that PIKfyve inhibition could be exploited as a target to reduce toxic a-synuclein aggregates species.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/lysosomal-driven-reduction-of-alpha-synuclein-aggregates-in-a-neuron-like-model-of-parkinsons-disease/