Norihiko Furuya

Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene

Malignant cells often display defects in autophagy, an evolutionarily conserved pathway for degrading long-lived proteins and cytoplasmic organelles. However, as yet, there is no genetic evidence for a role of autophagy genes in tumor suppression. The beclin 1 autophagy gene is monoallelically deleted in 40-75% of cases of human sporadic breast, ovarian, and prostate cancer. Therefore, we used a targeted mutant mouse model to test the hypothesis that monoallelic deletion of beclin 1 promotes tumorigenesis. Here we show that heterozygous disruption of beclin 1 increases the frequency of spontaneous malignancies and accelerates the development of hepatitis B virus-induced premalignant lesions. Molecular analyses of tumors in beclin 1 heterozygous mice show that the remaining wild-type allele is neither mutated nor silenced. Furthermore, beclin 1 heterozygous disruption results in increased cellular proliferation and reduced autophagy in vivo. These findings demonstrate that beclin 1 is a haplo-insufficient tumor-suppressor gene and provide genetic evidence that autophagy is a novel mechanism of cell-growth control and tumor suppression. Thus, mutation of beclin 1 or other autophagy genes may contribute to the pathogenesis of human cancers.

The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function.

Atg6/Beclin 1 is an evolutionarily conserved protein family that has been shown to function in vacuolar protein sorting (VPS) in yeast; in autophagy in yeast, Drosophila, Dictyostelium, C.elegans, and mammals; and in tumor suppression in mice. Atg6/Beclin 1 is thought to function as a VPS and autophagy protein as part of a complex with Class III PI3 kinase, Vps34. However, nothing is known about which domains of Atg6/Beclin 1 are required for its functional activity and binding to Vps34. We hypothesized that the most highly conserved region of human Beclin 1 spanning from amino acids 244-337 is essential for Vps34 binding, autophagy, and tumor suppressor function. To investigate this hypothesis, we evaluated the effects of wild-type and mutant beclin 1 gene transfer in autophagy-deficient MCF7 human breast carcinoma cells. We found that, unlike wildtype Beclin 1, a Beclin 1 mutant lacking aa 244-337 (Beclin 1deltaGECD), is unable to enhance starvation-induced autophagy in low Beclin 1-expressing MCF7 human breast carcinoma cells. In contrast to wild-type Beclin 1, mutant Beclin 1£GECD is unable to immunoprecipitate Vps34, has no Beclin 1-associated Vps34 kinase activity, and lacks tumor suppressor function in an MCF7 scid mouse xenograft tumor model. The maturation of cathepsin D, which requires intact Vps34-dependent VPS function, is comparable in autophagy-deficient low-Beclin 1 expressing MCF7 cells, autophagydeficient MCF7 cells transfected with Beclin 1deltaGECD, and autophagy-competent MCF7 cells transfected with wild-type Beclin 1. These findings identify an evolutionarily conserved domain of Beclin 1 that is essential for Vps34 interaction, autophagy function, and tumor suppressor function. Furthermore, they suggest a connection between Beclin 1-associated Class III PI-3 kinase-dependent autophagy, but not VPS, function and the mechanism of Beclin 1 tumor suppressor action in human breast cancer cells.

Amino Acids and Insulin Control Autophagic Proteolysis through Different Signaling Pathways in Relation to mTOR in Isolated Rat Hepatocytes

Autophagy, a major bulk proteolytic pathway, contributes to intracellular protein turnover, together with protein synthesis. Both are subject to dynamic control by amino acids and insulin. The mechanisms of signaling and cross-talk of their physiological anabolic effects remain elusive. Recent studies established that amino acids and insulin induce p70 S6 kinase (p70S6k) phosphorylation by mTOR, involved in translational control of protein synthesis. Here, the signaling mechanisms of amino acids and insulin in macroautophagy in relation to mTOR were investigated. In isolated rat hepatocytes, both regulatory amino acids (RegAA) and insulin coordinately activated p70S6k phosphorylation, which was completely blocked by rapamycin, an mTOR inhibitor. However, rapamycin blocked proteolytic suppression by insulin, but did not block inhibition by RegAA. These contrasting results suggest that insulin controls autophagy through the mTOR pathway, but amino acids do not. Furthermore, micropermeabilization with Saccharomyces aureus α-toxin completely deprived hepatocytes of proteolytic responsiveness to RegAA and insulin, but still maintained p70S6k phosphorylation by RegAA. In contrast, Leu8-MAP, a non-transportable leucine analogue, did not mimic the effect of leucine on p70S6k phosphorylation, but maintained the activity on proteolysis. Finally, BCH, a System L-specific amino acid, did not affect proteolytic suppression or mTOR activation by leucine. All the results indicate that mTOR is not common to the signaling mechanisms of amino acids and insulin in autophagy, and that the amino acid signaling starts extracellularly with their “receptor(s),” probably other than transporters, and is mediated through a novel route distinct from the mTOR pathway employed by insulin.

The MAP1-LC3 conjugation system is involved in lipid droplet formation.

Lipid droplets (LDs) are ubiquitous in eukaryotic cells, while excess free fatty acids and glucose in plasma are converted to triacylglycerol (TAG) and stored as LDs. However, the mechanism for the generation and growth of LDs in cells is largely unknown. We show here that the LC3 lipidation system essential for macroautophagy is involved in LD formation. LD formation accompanied by accumulation of TAG induced by starvation was largely suppressed in the hepatocytes that cannot execute autophagy. Under starvation conditions, LDs in addition to autophagosomes were abundantly formed in the cytoplasm of these tissue cells. Moreover, LC3 was localized on the surface of LDs and LC3-II (lipidation form) was fractionated to a perilipin (LD marker)-positive lipid fraction from the starved liver. Taken together, these results indicate that the LC3 conjugation system is critically involved in lipid metabolism via LD formation.

Liver autophagy contributes to the maintenance of blood glucose and amino acid levels

Both anabolism and catabolism of the amino acids released by starvation-induced autophagy are essential for cell survival, but their actual metabolic contributions in adult animals are poorly understood. Herein, we report that, in mice, liver autophagy makes a significant contribution to the maintenance of blood glucose by converting amino acids to glucose via gluconeogenesis. Under a synchronous fasting-initiation regimen, autophagy was induced concomitantly with a fall in plasma insulin in the presence of stable glucagon levels, resulting in a robust amino acid release. In liver-specific autophagy (Atg7)-deficient mice, no amino acid release occurred and blood glucose levels continued to decrease in contrast to those of wild-type mice. Administration of serine (30 mg/animal) exerted a comparable effect, raising the blood glucose levels in both control wild-type and mutant mice under starvation. Thus, the absence of the amino acids that were released by autophagic proteolysis is a major reason for a decrease in blood glucose. Autophagic amino acid release in control wild-type livers was significantly suppressed by the prior administration of glucose, which elicited a prompt increase in plasma insulin levels. This indicates that insulin plays a dominant role over glucagon in controlling liver autophagy. These results are the first to show that liver-specific autophagy plays a role in blood glucose regulation.

CHCHD2 mutations in autosomal dominant late-onset Parkinson's disease: a genome-wide linkage and sequencing study

BACKGROUND Identification of causative genes in mendelian forms of Parkinsons disease is valuable for understanding the cause of the disease. We did genetic studies in a Japanese family with autosomal dominant Parkinsons disease to identify novel causative genes. METHODS We did a genome-wide linkage analysis on eight affected and five unaffected individuals from a family with autosomal dominant Parkinsons disease (family A). Subsequently, we did exome sequencing on three patients and whole-genome sequencing on one patient in family A. Variants were validated by Sanger sequencing in samples from patients with autosomal dominant Parkinsons disease, patients with sporadic Parkinsons disease, and controls. Participants were identified from the DNA bank of the Comprehensive Genetic Study on Parkinsons Disease and Related Disorders (Juntendo University School of Medicine, Tokyo, Japan) and were classified according to clinical information obtained by neurologists. Splicing abnormalities of CHCHD2 mutants were analysed in SH-SY5Y cells. We used the Fishers exact test to calculate the significance of allele frequencies between patients with sporadic Parkinsons disease and unaffected controls, and we calculated odds ratios and 95% CIs of minor alleles. FINDINGS We identified a missense mutation (CHCHD2, 182C>T, Thr61Ile) in family A by next-generation sequencing. We obtained samples from a further 340 index patients with autosomal dominant Parkinsons disease, 517 patients with sporadic Parkinsons disease, and 559 controls. Three CHCHD2 mutations in four of 341 index cases from independent families with autosomal dominant Parkinsons disease were detected by CHCHD2 mutation screening: 182C>T (Thr61Ile), 434G>A (Arg145Gln), and 300+5G>A. Two single nucleotide variants (-9T>G and 5C>T) in CHCHD2 were confirmed to have different frequencies between sporadic Parkinsons disease and controls, with odds ratios of 2·51 (95% CI 1·48-4·24; p=0·0004) and 4·69 (1·59-13·83, p=0·0025), respectively. One single nucleotide polymorphism (rs816411) was found in CHCHD2 from a previously reported genome-wide association study; however, there was no significant difference in its frequency between patients with Parkinsons disease and controls in a previously reported genome-wide association study (odds ratio 1·17, 95% CI 0·96-1·19; p=0·22). In SH-SY5Y cells, the 300+5G>A mutation but not the other two mutations caused exon 2 skipping. INTERPRETATION CHCHD2 mutations are associated with, and might be a cause of, autosomal dominant Parkinsons disease. Further genetic studies in other populations are needed to confirm the pathogenicity of CHCHD2 mutations in autosomal dominant Parkinsons disease and susceptibility for sporadic Parkinsons disease, and further functional studies are needed to understand how mutant CHCHD2 might play a part in the pathophysiology of Parkinsons disease. FUNDING Japan Society for the Promotion of Science; Japanese Ministry of Education, Culture, Sports, Science and Technology; Japanese Ministry of Health, Labour and Welfare; Takeda Scientific Foundation; Cell Science Research Foundation; and Nakajima Foundation.

Ileal bile acid-binding protein, functionally associated with the farnesoid X receptor or the ileal bile acid transporter, regulates bile acid activity in the small intestine

Bile acids secreted in the small intestine are reabsorbed in the ileum where they activate the nuclear farnesoid X receptor (FXR), which in turn stimulates expression of the ileal bile acid-binding protein (I-BABP). We first hypothesized that I-BABP may negatively regulate the FXR activity by competing for the ligands, bile acids. Reporter assays using stable HEK293 cell lines expressing I-BABP revealed that I-BABP enhances rather than attenuates FXR activity. In these cells I-BABP localizes predominantly in the cytosol and partially in the nucleus, a distribution that does not shift in response to FXR expression. In vitro binding assays reveal that recombinant I-BABP is able to bind 35S-labeled FXR and that chenodeoxycholic acid (CDCA) stimulates this interaction modestly. When FLAG-tagged FXR was expressed in stable cells, the FXR·I-BABP complex in the nuclear extracts was more efficiently immunoprecipitable with anti-FLAG antibodies in the presence of CDCA. These results indicate that I-BABP stimulates FXR activity through a mutual interaction augmented by bile acids. When stable cells were transfected with an expression plasmid of the ileal bile acid transporter 14(IBAT) essential for the reabsorption of conjugated bile acids, the C-labeled conjugated bile acid, glycocholic acid, was more efficiently imported via IBAT in the presence than absence of I-BABP, whereas no change was observed in 14C-labeled CDCA uptake, which is independent of IBAT. Immunofluorescent staining analysis revealed that these two proteins co-localize in the vicinity of the plasma membrane in stable cells. Taken together, the current data provide the first evidence that I-BABP is functionally associated with FXR and IBAT in the nucleus and on the membrane, respectively, stimulating FXR transcriptional activity and the conjugated bile acid uptake mediated by IBAT in the ileum.

Perilipin-Mediated Lipid Droplet Formation in Adipocytes Promotes Sterol Regulatory Element-Binding Protein-1 Processing and Triacylglyceride Accumulation

Sterol regulatory element-binding protein-1 (SREBP-1) has been thought to be a critical factor that assists adipogenesis. During adipogenesis SREBP-1 stimulates lipogenic gene expression, and peroxisome proliferator-activated receptor γ (PPARγ) enhances perilipin (plin) gene expression, resulting in generating lipid droplets (LDs) to store triacylglycerol (TAG) in adipocytes. Plin coats adipocyte LDs and protects them from lipolysis. Here we show in white adipose tissue (WAT) of plin−/− mice that nuclear active SREBP-1 and its target gene expression, but not nuclear SREBP-2, significantly decreased on attenuated LD formation. When plin−/− mouse embryonic fibroblasts (MEFs) differentiated into adipocytes, attenuated LDs were formed and nuclear SREBP-1 decreased, but enforced plin expression restored them to their original state. Since LDs are largely derived from the endoplasmic reticulum (ER), alterations in the ER cholesterol content were investigated during adipogenesis of 3T3-L1 cells. The ER cholesterol greatly reduced in differentiated adipocytes. The ER cholesterol level in plin−/− WAT was significantly higher than that of wild-type mice, suggesting that increased LD formation caused a change in ER environment along with a decrease in cholesterol. When GFP-SREBP-1 fusion proteins were exogenously expressed in 3T3-L1 cells, a mutant protein lacking the S1P cleavage site was poorly processed during adipogenesis, providing evidence of the increased canonical pathway for SREBP processing in which SREBP-1 is activated by two cleavage enzymes in the Golgi. Therefore, LD biogenesis may create the ER microenvironment favorable for SREBP-1 activation. We describe the novel interplay between LD formation and SREBP-1 activation through a positive feedback loop.

PARK2/Parkin-mediated mitochondrial clearance contributes to proteasome activation during slow-twitch muscle atrophy via NFE2L1 nuclear translocation.

Skeletal muscle atrophy is thought to result from hyperactivation of intracellular protein degradation pathways, including autophagy and the ubiquitin–proteasome system. However, the precise contributions of these pathways to muscle atrophy are unclear. Here, we show that an autophagy deficiency in denervated slow-twitch soleus muscles delayed skeletal muscle atrophy, reduced mitochondrial activity, and induced oxidative stress and accumulation of PARK2/Parkin, which participates in mitochondrial quality control (PARK2-mediated mitophagy), in mitochondria. Soleus muscles from denervated Park2 knockout mice also showed resistance to denervation, reduced mitochondrial activities, and increased oxidative stress. In both autophagy-deficient and Park2-deficient soleus muscles, denervation caused the accumulation of polyubiquitinated proteins. Denervation induced proteasomal activation via NFE2L1 nuclear translocation in control mice, whereas it had little effect in autophagy-deficient and Park2-deficient mice. These results suggest that PARK2-mediated mitophagy plays an essential role in the activation of proteasomes during denervation atrophy in slow-twitch muscles.

Perlecan inhibits autophagy to maintain muscle homeostasis in mouse soleus muscle.

The autophagy-lysosome system is essential for muscle protein synthesis and degradation equilibrium, and its dysfunction has been linked to various muscle disorders. It has been reported that a diverse collection of extracellular matrix constituents, including decorin, collagen VI, laminin α2, endorepellin, and endostatin, can modulate autophagic signaling pathways. However, the association between autophagy and perlecan in muscle homeostasis remains unclear. The mechanical unloading of perlecan-deficient soleus muscles resulted in significantly decreased wet weights and cross-section fiber area compared with those of control mice. We found that perlecan deficiency in slow-twitch soleus muscles enhanced autophagic activity. This was accompanied by a decrease in autophagic substrates, such as p62, and an increase in LC3II levels. Furthermore, perlecan deficiency caused a reduction in the phosphorylation levels of p70S6k and Akt and increased the phosphorylation of AMPKα. Our findings suggested that perlecan inhibits the autophagic process through the activation of the mTORC1 pathway. This autophagic response may be a novel target for enhancing the efficacy of skeletal muscle atrophy treatment.