Yuriko Sakamaki

The HOPS complex mediates autophagosome–lysosome fusion through interaction with syntaxin 17

Autophagosome–lysosome fusion requires the autophagosomal SNARE syntaxin 17. Syntaxin 17 interacts with the HOPS-tethering complex. HOPS is required for syntaxin 17–dependent autophagosome–lysosome fusion, besides its function in endolysosomal fusion.

The ATG conjugation systems are important for degradation of the inner autophagosomal membrane

Open sesame! The autophagosome is a double-membraned intracellular structure involved in the disposal of damaged or defunct organelles. Autophagosome formation requires a number of autophagy-related (ATG) proteins. Among them, the key conjugation systems ATG8 and ATG12 are widely exploited in the detection of autophagy in many organisms. However, their precise function in autophagy remains unknown. Tsuboyama et al. identified an unexpected role of ATG3, an important enzyme in the ATG conjugation systems, in efficient degradation and opening of the inner autophagosomal membrane after fusion with lysosomes (see the Perspective by Levine). Their live-imaging system revealed the entire life of an autophagosome in mammalian cells. Science, this issue p. 1036; see also p. 968 The requirements for autophagosome maturation and efficient autophagy within mammalian cells are dissected. In macroautophagy, cytoplasmic contents are sequestered into the double-membrane autophagosome, which fuses with the lysosome to become the autolysosome. It has been thought that the autophagy-related (ATG) conjugation systems are required for autophagosome formation. Here, we found that autophagosomal soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) syntaxin 17–positive autophagosome-like structures could be generated even in the absence of the ATG conjugation systems, although at a reduced rate. These syntaxin 17–positive structures could further fuse with lysosomes, but degradation of the inner autophagosomal membrane was significantly delayed. Accordingly, autophagic activity in ATG conjugation–deficient cells was strongly suppressed. We suggest that the ATG conjugation systems, which are likely required for the closure (i.e., fission) of the autophagosomal edge, are not absolutely essential for autolysosome formation but are important for efficient degradation of the inner autophagosomal membrane.

Small intestinal stem cell identity is maintained with functional Paneth cells in heterotopically grafted epithelium onto the colon

To develop stem cell therapy for small intestinal (SI) diseases, it is essential to determine whether SI stem cells in culture retain their tissue regeneration capabilities. By using a heterotopic transplantation approach, we show that cultured murine SI epithelial organoids are able to reconstitute self-renewing epithelia in the colon. When stably integrated, the SI-derived grafts show many features unique only to the SI but distinct from the colonic epithelium. Our study provides evidence that cultured adult SI stem cells could be a source for cell therapy of intestinal diseases, maintaining their identity along the gastrointestinal tract through an epithelium-intrinsic mechanism.

Deletion of Autophagy-related 5 (Atg5) and Pik3c3 Genes in the Lens Causes Cataract Independent of Programmed Organelle Degradation

Background: The role of autophagy-dependent quality control in the lens remains unclear. Results: Deletion of Atg5 and Pik3c3/Vps34 in the lens does not affect programmed organelle degradation but causes cataract and a developmental defect, respectively. Conclusion: These genes are important for quality control and development of the lens. Significance: This study provides new insights into biology and age-related pathology of the lens. The lens of the eye is composed of fiber cells, which differentiate from epithelial cells and undergo programmed organelle degradation during terminal differentiation. Although autophagy, a major intracellular degradation system, is constitutively active in these cells, its physiological role has remained unclear. We have previously shown that Atg5-dependent macroautophagy is not necessary for lens organelle degradation, at least during the embryonic period. Here, we generated lens-specific Atg5 knock-out mice and showed that Atg5 is not required for lens organelle degradation at any period of life. However, deletion of Atg5 in the lens results in age-related cataract, which is accompanied by accumulation of polyubiquitinated and oxidized proteins, p62, and insoluble crystallins, suggesting a defect in intracellular quality control. We also produced lens-specific Pik3c3 knock-out mice to elucidate the possible involvement of Atg5-independent alternative autophagy, which is proposed to be dependent on Pik3c3 (also known as Vps34), in lens organelle degradation. Deletion of Pik3c3 in the lens does not affect lens organelle degradation, but it leads to congenital cataract and a defect in lens development after birth likely due to an impairment of the endocytic pathway. Taken together, these results suggest that clearance of lens organelles is independent of macroautophagy. These findings also clarify the physiological role of Atg5 and Pik3c3 in quality control and development of the lens, respectively.

Beneficial Effects of Estrogen in a Mouse Model of Cerebrovascular Insufficiency

Background The M5 muscarinic acetylcholine receptor is known to play a crucial role in mediating acetylcholine dependent dilation of cerebral blood vessels. Previously, we reported that male M5 muscarinic acetylcholine knockout mice (M5R −/− mice) suffer from a constitutive constriction of cerebral arteries, reduced cerebral blood flow, dendritic atrophy, and short-term memory loss, without necrosis and/or inflammation in the brain. Methodology/Principal Findings We employed the Magnetic Resonance Angiography to study the area of the basilar artery in male and female M5R −/− mice. Here we show that female M5R −/− mice did not show the reduction in vascular area observed in male M5R −/− mice. However, ovariectomized female M5R −/− mice displayed phenotypic changes similar to male M5R −/− mice, strongly suggesting that estrogen plays a key role in the observed gender differences. We found that 17β-estradiol (E2) induced nitric oxide release and ERK activation in a conditional immortalized mouse brain cerebrovascular endothelial cell line. Agonists of ERα, ERβ, and GPR30 promoted ERK activation in this cell line. Moreover, in vivo magnetic resonance imaging studies showed that the cross section of the basilar artery was restored to normal in male M5R −/− mice treated with E2. Treatment with E2 also improved the performance of male M5R −/− mice in a cognitive test and reduced the atrophy of neural dendrites in the cerebral cortex and hippocampus. M5R −/− mice also showed astrocyte swelling in cortex and hippocampus using the three-dimensional reconstruction of electron microscope images. This phenotype was reversed by E2 treatment, similar to the observed deficits in dendrite morphology and the number of synapses. Conclusions/Significance Our findings indicate that M5R −/− mice represent an excellent novel model system to study the beneficial effects of estrogen on cerebrovascular function and cognition. E2 may offer new therapeutic perspectives for the treatment of cerebrovascular insufficiency related memory dysfunction.

Myosin light chain kinase expression induced via tumor necrosis factor receptor 2 signaling in the epithelial cells regulates the development of colitis-associated carcinogenesis.

It has been suggested that prolonged inflammatory bowel diseases (IBD) may lead to colitis-associated carcinogenesis (CAC). We previously observed that the NF-κB activation in colonic epithelial cells is associated with increased tumor necrosis factor receptor 2 (TNFR2) expression in CAC development. However, the mechanism by which epithelial NF-κB activation leading to CAC is still unclear. Myosin light chain kinase (MLCK) has been reported to be responsible for the epithelial permeability associated with TNF signaling. Therefore we focused on the role of MLCK expression via TNFR2 signaling on CAC development. Pro-tumorigenic cytokines such as IL-1β, IL-6 and MIP-2 production as well as INF-γ and TNF production at the lamina propria were increased in the setting of colitis, and further in tumor tissues in associations with up-regulated TNFR2 and MLCK expressions in the epithelial cells of a CAC model. The up-regulated MLCK expression was observed in TNF-stimulated colonic epithelial cells in a dose-dependent fashion in association with up-regulation of TNFR2. Silencing TNFR2, but not TNFR1, resulted in restoration of epithelial tight junction (TJ) associated with decreased MLCK expression. Antibody-mediated blockade of TNF signaling also resulted in restoration of TJ in association with suppressed MLCK expression, and interestingly, similar results were observed with suppressing TNFR2 and MLCK expressions by inhibiting MLCK in the epithelial cells. Silencing of MLCK also resulted in suppressed TNFR2, but not TNFR1, expression, suggesting that the restored TJ leads to reduced TNFR2 signaling. Such suppression of MLCK as well as blockade of TNFR2 signaling resulted in restored TJ, decreased pro-tumorigenic cytokines and reduced CAC development. These results suggest that MLCK may be a potential target for the prevention of IBD-associated tumor development.

Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17

Macroautophagy is an evolutionarily conserved catabolic mechanism that delivers intracellular constituents to lysosomes using autophagosomes. To achieve degradation, lysosomes must fuse with closed autophagosomes. We previously reported that the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) protein syntaxin (STX) 17 translocates to autophagosomes to mediate fusion with lysosomes. In this study, we report an additional mechanism. We found that autophagosome–lysosome fusion is retained to some extent even in STX17 knockout (KO) HeLa cells. By screening other human SNAREs, we identified YKT6 as a novel autophagosomal SNARE protein. Depletion of YKT6 inhibited autophagosome–lysosome fusion partially in wild-type and completely in STX17 KO cells, suggesting that YKT6 and STX17 are independently required for fusion. YKT6 formed a SNARE complex with SNAP29 and lysosomal STX7, both of which are required for autophagosomal fusion. Recruitment of YKT6 to autophagosomes depends on its N-terminal longin domain but not on the C-terminal palmitoylation and farnesylation that are essential for its Golgi localization. These findings suggest that two independent SNARE complexes mediate autophagosome–lysosome fusion.

Porphyromonas gingivalis, a periodontal pathogen, enhances myocardial vulnerability, thereby promoting post-infarct cardiac rupture

There is a strong association between periodontal disease (PD) and atherosclerosis. However, it remains unknown whether PD is also involved in myocardial damage. We hypothesized that infection with periodontal pathogens could cause an adverse outcome after myocardial infarction (MI). C57BL/6J mice were inoculated with Porphyromonas gingivalis (P.g.), a major periodontal pathogen, or injected with phosphate-buffered saline (PBS) into a subcutaneously-implanted steelcoil chamber before and after coronary artery ligation. A significant increase in mortality, due to cardiac rupture, was observed in the P.g.-inoculated MI mice. Ultrastructural examinations revealed that P.g. invaded the ischemic myocardium of the P.g.-inoculated MI mice. The expression of p18 Bax, an active form of pro-apoptotic Bax protein, markedly increased in the P.g.-inoculated MI hearts. In vitro experiments demonstrated that gingipain, a protease uniquely secreted from P.g., cleaved wild type Bax at Arg34, as evidenced by the observation that the cleavage of Bax by gingipain was completely abolished by the Arg34Ala mutation in Bax. Treatment with immunoglobulin Y against gingipain significantly decreased the mortality of the P.g.-inoculated MI mice caused by cardiac rupture. Furthermore, inoculation of P.g. also resulted in an increase of MMP-9 activity in the post-MI myocardium by enhancing oxidative stress, possibly through impairing the selective autophagy-mediated clearance of damaged mitochondria. In conclusion, infection with P.g. during MI plays a detrimental role in the healing process of the infarcted myocardium by invasion of P.g. into the myocardium, thereby promoting apoptosis and the MMP-9 activity of the myocardium, which, in turn, causes cardiac rupture.

Accumulation of undegraded autophagosomes by expression of dominant-negative STX17 (syntaxin 17) mutants

ABSTRACT Macroautophagy/autophagy, which is one of the main degradation systems in the cell, is mediated by a specialized organelle, the autophagosome. Purification of autophagosomes before fusion with lysosomes is important for both mechanistic and physiological studies of the autophagosome. Here, we report a simple method to accumulate undigested autophagosomes. Overexpression of the autophagosomal Qa-SNARE STX17 (syntaxin 17) lacking the N-terminal domain (NTD) or N-terminally tagged GFP-STX17 causes accumulation of autophagosomes. A HeLa cell line, which expresses GFP-STX17ΔNTD or full-length GFP-STX17 under the control of the tetracycline-responsive promoter, accumulates a large number of undigested autophagosomes devoid of lysosomal markers or early autophagy factors upon treatment with doxycycline. Using this inducible cell line, nascent autophagosomes can be easily purified by OptiPrep density-gradient centrifugation and immunoprecipitation. This novel method should be useful for further characterization of nascent autophagosomes.

Differential requirement for ATG2A domains for localization to autophagic membranes and lipid droplets

ATG2 is one of the autophagy‐related (ATG) proteins essential for autophagosome formation and localizes to isolation membranes and lipid droplets in mammalian cells. Here, we investigated the requirement of regions in ATG2A for its organellar localization and function. The N‐terminal amino acids 1–198 and the C‐terminal amino acids 1830–1938 are required for the localization to isolation membranes and lipid droplets, respectively. The C‐terminal region is not required for the localization to isolation membranes and for autophagy. We also identified an amphipathic helix in ATG2A that is required for both its localization to organelles and autophagosome formation. These data suggest that the dual localization of ATG2A is regulated by different regions.