α-Synuclein toxicity in yeast and human cells is caused by cell cycle re-entry and autophagy degradation of ribonucleotide reductase 1.

Belém Sampaio‐Marques,Joris Winderickx,Tiago F Outeiro,Paula Ludovico,William C Burhans,Igor Vasilevskiy,Ana Guedes,Susana Gonçalves

doi:10.1111/acel.12922

Abstract

α‐Synuclein (aSyn) toxicity is associated with cell cycle alterations, activation of DNA damage responses (DDR), and deregulation of autophagy. However, the relationships between these phenomena remain largely unknown. Here, we demonstrate that in a yeast model of aSyn toxicity and aging, aSyn expression induces Ras2‐dependent growth signaling, cell cycle re‐entry, DDR activation, autophagy, and autophagic degradation of ribonucleotide reductase 1 (Rnr1), a protein required for the activity of ribonucleotide reductase and dNTP synthesis. These events lead to cell death and aging, which are abrogated by deleting RAS2, inhibiting DDR or autophagy, or overexpressing RNR1. aSyn expression in human H4 neuroglioma cells also induces cell cycle re‐entry and S‐phase arrest, autophagy, and degradation of RRM1, the human homologue of RNR1, and inhibiting autophagic degradation of RRM1 rescues cells from cell death. Our findings represent a model for aSyn toxicity that has important implications for understanding synucleinopathies and other age‐related neurodegenerative diseases.

Highlights

Inappropriate re‐entry into the cell cycle of postmitotic neurons followed by the death of these cells is frequently observed in Parkinson's disease (PD) and other synucleinopathies (Folch et al, 2012; Lee et al, 2003; FIGURE 5 aSyn promotes S‐phase cell cycle arrest, increases autophagy, and decreases RRM1 levels in human cells. (a) Cellular viability and (b) aSyn levels of H4 cells transiently transfected with the vector control or wt aSyn in the absence or presence of bafilomycin A1 (Baf) (10 nM for 24 hr). (c) Representative blot of LC3 processing and p62 for autophagy flux assessment. (d,e) Graphical representation of the LC3II/actin and p62/actin, respectively
Deregulation of cellular proteolytic systems, autophagy, appears to play an important role in synucleinopathies (Sampaio‐Marques & Ludovico, 2015; Xilouri, Brekk, & Stefanis, 2013). How these phenotypes relate to each other and how they might contribute to aSyn toxicity have remained poorly understood. We show both in the budding yeast chronological aging model and in human neuroglioma cells that expression of aSyn induces inappropriate cell cycle re‐entry of quiescent postmitotic cells in the absence of the nutrient signaling in budding yeast or growth factor signaling in H4 neuroglioma cells that normally drive entry of these cells into the cell cycle
Cell cycle re‐entry of postmitotic neurons in synucleinopathies and other neurodegenerative disorders, which is frequently assessed by bromodeoxyuridine incorporation as these cells enter S‐phase, is a generally, but not universally, accepted phenotype, because it can be difficult to distinguish small amounts of DNA replication from DNA repair

Summary

| INTRODUCTION

Α‐Synuclein (aSyn) is a presynaptic neuronal protein that is genetically and neuropathologically linked to a group of age‐related neurodegenerative diseases called synucleinopathies. Mounting evidence suggests that macroautophagy (hereafter autophagy), a self‐degradation pathway, plays a key role in the DDR by controlling the levels of proteins involved in cell cycle checkpoints and DNA synthesis/repair mechanisms. Expression of aSyn in human H4 neuroglioma cells induces the accumulation of cells in S‐phase, autophagy and the degradation of RRM1, the human homologue of Rnr, and cell death, which is blocked by inhibiting autophagy. These findings reveal a novel mechanism for aSyn toxicity in aged postmitotic cells that involves the inappropriate entry of cells into S‐phase followed by DDR and the autophagy‐dependent loss of RNR activity

| RESULTS

| DISCUSSION

| EXPERIMENTAL PROCEDURES

Findings

CONFLICT OF INTEREST