Analysis of ATG4C function <i>in vivo</i>

Despite the great advances in macroautophagy/autophagy research in the last years, the in vivo role of the different members of the four mammalian orthologs of yeast Atg4 protease (ATG4A-D) remain unclear. To gain further insights into the functional relevance of Atg4 orthologs, we have generated mutant mice deficient in Atg4c. These mice are viable and fertile, and do not display any obvious abnormalities, indicating that they are able to develop the autophagic response required during the early neonatal period. However, they show tissue-specific autophagy alterations, including reduced autophagic flux in diaphragm and show decreased breathing and locomotor activity after fasting. In addition, atg4c-/- mice show reduced number of circulating T and B lymphocytes, which is associated with accumulation of apoptotic cells in the spleen and an increased susceptibility to develop chemically-induced fibrosarcomas. Moreover, through the analysis of cells and mice simultaneously deficient for ATG4C and ATG4D proteases we also reveal a role for ATG4C in mATG8 proteins delipidation. ATG4 (autophagy related 4 cysteine peptidase); ATG4A (autophagy related 4A cysteine peptidase); ATG4B (autophagy related 4B cysteine peptidase); ATG4C (autophagy related 4C cysteine peptidase); ATG4D (autophagy related 4D cysteine peptidase); Atg8 (autophagy related 8); GABARAP (GABA type A receptor-associated protein); GABARAPL1(GABA type A receptor-associated protein like 1); GABARAPL2 (GABA type A receptor-associated protein like 2); MAP1LC3A/LC3A (microtubule associated protein 1 light chain 3 alpha); MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta); mATG8 (mammalian Atg8); PE (phosphatidylethanolamine); PS (phosphatydylserine); SQSTM1/p62 (sequestosome 1).</p

MetR specifically regulates induction of autophagy.

<p>MET⁺, Δ<i>met15</i> and Δ<i>met2</i> strains from chronological aging experiments were analyzed for vacuolar ALP activity (with a fluorescent plate reader) (A) (n = 6), and GFP-Atg8p processing (by Western-blot analysis) (B). (C) GFP-Atg8p localization was determined by using fluorescent microscopy (white arrows indicate vacuolar localization or autophagosome formation) and statistical analysis thereof (330–600 cells of each GFP-Atg8p expressing strain were evaluated from two independent samples) (D). (E) <i>MET2</i> deletion strain (Δ<i>met2</i>) was grown to stationary phase in SCD (supplemented with all aa) and shifted to SCD media with given methionine concentrations. Autophagy was measured by means of ALP activity with a fluorescent plate reader (Tecan, Genios Pro) (n = 6). (F) ALP assays of chronological aging of MET⁺ strain, in SCD media supplemented with all aa except for methionine which was added at given concentrations (n = 2). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004347#pgen.1004347.s003" target="_blank">Figure S3</a>.</p

Model of MetR-mediated longevity.

<p>MetR specifically enhances autophagy, either by interfering upstream of TOR-pathway or presumably by impinging on (metabolic) pathways that potentially target autophagy directly, downstream of the TOR-pathway. MetR-specific vacuolar acidification is dependent on autophagy and elongates CLS. High levels of methionine inhibit autophagy induction during early phases of chronological aging, enhancing ROS and diminishing acidic vacuoles in a cell population, which leads to cell death.</p

Methionine determines yeast chronological lifespan.

<p>(A) Chronological aging of methionine prototroph (MET⁺), semi-auxotroph (Δ<i>met15</i>) and auxotroph (Δ<i>met2</i>) isogenic yeast strains in SCD media supplemented with all amino acids (aa). Cell survival was estimated as colony formation of 500 cells plated at given time points, normalized to cell survival on day one (n = 4). (B) Chronological aging of Δ<i>met15</i> strain, in SCD media supplemented with all aa except for methionine which was added at given concentrations. Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 4). (C) <i>MET2</i> deletion strain (Δ<i>met2</i>) was grown to stationary phase in SCD (supplemented with all aa) and shifted to SCD media with different methionine concentrations. Cell survival of 500 cells plated at given time points, normalized to cell survival before the shift (n = 4). (D) Day 8 from experiment shown in C (n = 4). (E) Chronological aging of EUROSCARF BY4741 (also used above as Δ<i>met15</i> reference strain) and mating type α wild type strain BY4742, as well as a methionine semi-auxotrophic variant thereof (BY4742 Δ<i>met15</i>), in SCD media supplemented with all aa. Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 3). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004347#pgen.1004347.s001" target="_blank">Figure S1</a>.</p

MetR enhancement of vacuolar acidification is autophagy-dependent and necessary for longevity.

<p>Fluorescent microscopy of acidic vacuoles during chronological aging of MET+, Δ<i>met2</i>, and Δ<i>met2</i>/Δ<i>atg5</i> strains, by means of quinacrine accumulation and statistical analysis thereof. (>1000 cells of each strain from 3 to 5 independent samples at each time point were evaluated. Only cells with acidic vacuoles without an additionally stained cytoplasm were counted as positive, resulting in cell counts that represent cells which have a clearly intact pH-homeostasis. Positively counted cells are indicated by white arrowheads) (A and B). (C) Statistical analysis of fluorescent microscopy of acidic vacuoles by means of quinacrine accumulation. Strains Δ<i>met2</i> and Δ<i>met2</i>/Δ<i>atg5</i> were grown to stationary phase under excess of methionine and shifted to media with the indicated amounts of methionine (>500 cells from each strain from 2 independent samples) and assayed for quinacrine accumulation after ∼20 hours (D) Chronological aging of the MET⁺ strain overexpressing Vma1p or Vph2p. Cell death was measured via propidium iodide staining of cells that have lost integrity and subsequent flow cytometry analysis (BD LSRFortessa) (n = 6 to 8). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004347#pgen.1004347.s006" target="_blank">Figure S6</a>.</p

Autophagy is crucial for MetR-mediated longevity.

<p>(A) Chronological aging of <i>MET2</i> deletion strains carrying an additional gene deletion (Δ<i>atg5</i>) and MET⁺ strain in SCD media supplemented with all aa. Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 6). (B) Chronological aging of MET⁺ strain treated with indicated amounts of rapamycin (Rap). Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 3; p***). Autophagy was measured by means of ALP activity with a fluorescent plate reader (Tecan, Genios Pro) and normalized to untreated controls at indicated time points (also compare to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004347#pgen-1004347-g004" target="_blank">Figure 4E</a>) (C) (n = 4). (D) Chronological aging of MET⁺ strain deleted for <i>TOR1</i>. Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 6; p***). (E) Chronological aging of the MET⁺ strain deleted for <i>TOR1</i> and <i>ATG5</i> or <i>ATG5</i> alone. Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 4–6). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004347#pgen.1004347.s004" target="_blank">Figure S4</a>.</p

MetR is epistatic to other longevity treatments involving <i>TOR1</i> inhibition.

<p>(A and B) Chronological aging of <i>MET2</i> and <i>MET15</i> deletion strains deleted for <i>TOR1</i>. Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 6). Chronological aging of <i>MET2</i> (C) and <i>MET15</i> (D) deletion strains treated with indicated amounts of rapamycin (Rap). Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 3). Autophagy was measured by means of ALP activity with a fluorescent plate reader (Tecan, Genios Pro) and normalized to untreated controls at indicated time points (also compare to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004347#pgen-1004347-g003" target="_blank">Figure 3C</a>) (E) (n = 4). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004347#pgen.1004347.s005" target="_blank">Figure S5</a>.</p