Folliculin Regulates Ampk-Dependent Autophagy and Metabolic Stress Survival

<div><p>Dysregulation of AMPK signaling has been implicated in many human diseases, which emphasizes the importance of characterizing AMPK regulators. The tumor suppressor <i>FLCN</i>, responsible for the Birt-Hogg Dubé renal neoplasia syndrome (BHD), is an AMPK-binding partner but the genetic and functional links between FLCN and AMPK have not been established. Strikingly, the majority of naturally occurring <i>FLCN</i> mutations predisposing to BHD are predicted to produce truncated proteins unable to bind AMPK, pointing to the critical role of this interaction in the tumor suppression mechanism. Here, we demonstrate that FLCN is an evolutionarily conserved negative regulator of AMPK. Using <i>Caenorhabditis elegans</i> and mammalian cells, we show that loss of FLCN results in constitutive activation of AMPK which induces autophagy, inhibits apoptosis, improves cellular bioenergetics, and confers resistance to energy-depleting stresses including oxidative stress, heat, anoxia, and serum deprivation. We further show that AMPK activation conferred by FLCN loss is independent of the cellular energy state suggesting that FLCN controls the AMPK energy sensing ability. Together, our data suggest that FLCN is an evolutionarily conserved regulator of AMPK signaling that may act as a tumor suppressor by negatively regulating AMPK function.</p></div

Loss of FLCN stimulates cellular energy production and resistance to energy stress.

<p>(A) Relative ATP levels measured in the indicated worm strains treated with or without PQ. (B) Percent survival of wild-type and <i>flcn-1(ok975)</i> nematodes upon heat stress (35°C). (C) Recovery rate of wild-type and <i>flcn-1(ok975)</i> strains after 26 hours anoxic injury. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004273#pgen.1004273.s013" target="_blank">Tables S4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004273#pgen.1004273.s014" target="_blank">S5</a>. Data represent the mean ± SEM, n≥3.</p

Loss of <i>flcn-1</i> confers an <i>aak-2</i>-dependent resistance to oxidative stress.

<p>(A, B, C, D) Percent survival of indicated worm strains treated with 4 mM or 100 mM PQ. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004273#pgen.1004273.s011" target="_blank">Tables S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004273#pgen.1004273.s012" target="_blank">S3</a>. (E) Western blot analysis of pAAK-2 (Thr234) protein levels in indicated worm strains. Levels were normalized to Tubulin. (F) Fold change in pAAK-2 levels in <i>flcn-1(ok975);par-4(it57)</i> and <i>par-4(it57)</i> animals. Data represent the means ± SEM, n≥3.</p

The FLCN-dependent regulation of AMPK, autophagy, and metabolic stress survival is evolutionarily conserved.

<p>(A) Percent survival of wild-type, <i>Flcn</i>^−/− and FLCN-rescued MEFs (resc.) upon serum starvation (-FBS). (B) Western blot analysis of pAMPK (Thr172) and AMPK protein levels in indicated MEFs lines. (C) Percent survival of the indicated MEF cell lines upon serum starvation. Data represent the means ± SEM, n≥3. (D and E) Representative immunofluorescence pictures (D) and quantification (E) of LC3 positive GFP puncta (arrows) in wild-type or <i>Flcn</i>^−/− MEFs under basal or 24 hours serum starvation conditions (-FBS). When indicated, cells were pretreated with chloroquine (CQ) 12 hours prior to serum starvation, N>200 cells for every trial. (F) Percent survival of indicated cell lines upon serum starvation, treated with or without 10 µM CQ. Data represent the mean ± SEM, n≥3. (G) Relative ATP levels measured in the indicated MEFs lines, pre-treated with or without 10 µM CQ prior to serum starvation. (H) Graphical model that summarizes findings of this study.</p

Loss of <i>flcn-1</i> confers resistance to oxidative stress in <i>C. elegans</i>.

<p>(A) Amino acid alignment of the human and <i>C. elegans</i> Folliculin sequences (accession numbers: human AF517523, <i>C. elegans</i> HE963850). Identical (black) and similar (grey) amino acids are highlighted. (B) Genomic structure of <i>flcn-1</i>. The <i>ok975</i> mutation and the genomic region targeted by RNAi are indicated. (C) Western blot analysis of FLCN-1 protein levels in wild-type and <i>flcn-1(ok975)</i> worm protein lysates. (D) Lifespan of wild-type and <i>flcn-1(ok975)</i> nematodes at 20°C (also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004273#pgen.1004273.s010" target="_blank">Table S1</a>). (E-H) Percent survival of indicated worm strains treated with 4 mM or 100 mM PQ (also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004273#pgen.1004273.s011" target="_blank">Tables S2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004273#pgen.1004273.s012" target="_blank">S3</a>). (I) Percent survival of indicated worm strains treated with 7.5 mM H₂O₂.</p

Loss of <i>flcn-1</i> activates autophagy resulting in oxidative stress resistance in <i>C. elegans</i>.

<p>(A and B) Representative electron micrographs from longitudinal sections of the hypodermis in indicated nematodes strains. Arrows represent autophagic vacuoles (A) and autophagosomal membranes (B). Scale bars: 0.2 µm (A and B). (C) Quantification of the autophagic events observed in defined surface area of 4.25 um² of electron micrographs taken from at least 5 animals. Red lines represent the mean of autophagosome numbers per area indicated strains and treatment condition. (D) Number of GFP::LGG-1 positive autophagosome puncta in the seam cells of the indicated worm strains. (E) Western blot analysis of the GFP::LGG-1 cleavage profile (LGG-1-II, GFP) in worm protein extracts. (F, G, H, I). Percent survival of indicated strains upon 4 mM (F and H) or 100 mM (G and I) PQ. Data represent the means ± SEM, n≥3. Also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004273#pgen.1004273.s011" target="_blank">Tables S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004273#pgen.1004273.s012" target="_blank">S3</a>.</p