Amyloid-like aggregating proteins cause lysosomal defects in neurons via gain-of-function toxicity.

Irene Riera-Tur,Dennis Feigenbutz,Tillman Schäfer,Daniel Hornburg,Matthias Mann,Patrick Auer,Lorena Fernández-Mosquera,Miguel Da Silva Padilha,Irina Dudanova,Nuno Raimundo,Wolfgang Baumeister,Rüdiger Klein,Felix Meissner,Archana Mishra,Rubén Fernández-Busnadiego

doi:10.26508/lsa.202101185

Abstract

The autophagy-lysosomal pathway is impaired in many neurodegenerative diseases characterized by protein aggregation, but the link between aggregation and lysosomal dysfunction remains poorly understood. Here, we combine cryo-electron tomography, proteomics, and cell biology studies to investigate the effects of protein aggregates in primary neurons. We use artificial amyloid-like β-sheet proteins (β proteins) to focus on the gain-of-function aspect of aggregation. These proteins form fibrillar aggregates and cause neurotoxicity. We show that late stages of autophagy are impaired by the aggregates, resulting in lysosomal alterations reminiscent of lysosomal storage disorders. Mechanistically, β proteins interact with and sequester AP-3 μ1, a subunit of the AP-3 adaptor complex involved in protein trafficking to lysosomal organelles. This leads to destabilization of the AP-3 complex, missorting of AP-3 cargo, and lysosomal defects. Restoring AP-3μ1 expression ameliorates neurotoxicity caused by β proteins. Altogether, our results highlight the link between protein aggregation, lysosomal impairments, and neurotoxicity.

Highlights

The autophagy-lysosomal system is a major cellular degradation pathway for long-lived proteins, macromolecular complexes, and damaged organelles (Settembre et al, 2013; Finkbeiner, 2020)
We focused on AP-3μ1 as the most highly enriched β protein interactor
We found a high degree of colocalization between co-transfected AP-3μ1 and β protein aggregates (Fig 7A and B)

Summary

Introduction

The autophagy-lysosomal system is a major cellular degradation pathway for long-lived proteins, macromolecular complexes, and damaged organelles (Settembre et al, 2013; Finkbeiner, 2020). Analysis of the total proteome of transduced neurons (which includes the proteins interacting with the aggregates) did not reveal significant changes in the levels of any of the β protein interactors (Fig S7A–D and Tables S3 and S4). This indicates that their presence in the interactome was not merely a result of their increased amounts in the cells, nor was their sequestration markedly compensated by increased expression. When mocha cells were loaded with LysoTracker Red, we observed reduced numbers of lysosomes per cell (Fig 8E and F), in line with our findings in LysoTracker-labeled β protein neurons (Fig 4C) These results confirm that impairment of the AP-3 complex leads to lysosomal defects. Our data strongly suggest that lysosomal defects mediate, at least in part, the toxicity of β protein aggregates in neurons

Discussion

Findings

Materials and Methods