ATG4D is the main ATG8 delipidating enzyme in mammalian cells and protects against cerebellar neurodegeneration

Isaac Tamargo-Gómez,Carlos López-Otín,Helena Codina-Martínez,Patrice Codogno,Etienne Morel,Guillermo Mariño,Gemma G Martínez-García,Nicolas Dupont,María F Suárez,Antonio Fueyo,Sylvie Souquere,Gerard Pierron,Gabriel Bretones,Xurde M Caravia,Roberto Cabo,Verónica Rey,Cristina Tomás-Zapico,Álvaro F Fernández

doi:10.1038/s41418-021-00776-1

Abstract

Despite the great advances in autophagy research in the last years, the specific functions of the four mammalian Atg4 proteases (ATG4A-D) remain unclear. In yeast, Atg4 mediates both Atg8 proteolytic activation, and its delipidation. However, it is not clear how these two roles are distributed along the members of the ATG4 family of proteases. We show that these two functions are preferentially carried out by distinct ATG4 proteases, being ATG4D the main delipidating enzyme. In mammalian cells, ATG4D loss results in accumulation of membrane-bound forms of mATG8s, increased cellular autophagosome number and reduced autophagosome average size. In mice, ATG4D loss leads to cerebellar neurodegeneration and impaired motor coordination caused by alterations in trafficking/clustering of GABAA receptors. We also show that human gene variants of ATG4D associated with neurodegeneration are not able to fully restore ATG4D deficiency, highlighting the neuroprotective role of ATG4D in mammals.

Highlights

Autophagy is a catabolic process, which has remained well conserved through evolution in all eukaryotic cells
SNAP-LC3B/Membrane Impermeable SNAP-Surface® Ligand (MIL)+, LAMP-1+ double-positive structures will represent autolysosomes showing lipidated SNAP-LC3B-II at their cytosolic membrane leaflet (Fig. 4H). This assay revealed a significant increase of these SNAP-LC3B/MIL+, LAMP-1+ double-positive structures in Atg4d−/− cells in different experimental conditions (Fig. 4I), in line with our previous results pointing to a delipidation defect in the absence of ATG4D (Fig. 4I). These results show that the accumulation of the membrane-bound forms of LC3s and GABARAPs in the absence of ATG4D stems from a major role for ATG4D in their delipidation
We have shown that ATG4D is the main delipidating enzyme for mammalian mATG8s, which accumulate in their membrane-bound form in the absence of this protease

Summary

Introduction

Autophagy is a catabolic process, which has remained well conserved through evolution in all eukaryotic cells. One of the essential components for the elongation and expansion of nascent autophagosomes is the Atg4/Atg ubiquitin-like conjugation system, in which cytoplasmic protein Atg must be cleaved after a Gly residue by the cysteine proteinase Atg4 [3]. After this priming, Atg is conjugated with membrane-bound phosphatidylethanolamine (PE), allowing the correct formation, closure, and maturation of autophagosomes. There are four human orthologues of Atg protease in mammalian cells (ATG4A-D) [4] and six Atg orthologues

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Results

Conclusion