Akitsugu Yamamoto

Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice

Autophagy is an intracellular bulk degradation process through which a portion of the cytoplasm is delivered to lysosomes to be degraded. Although the primary role of autophagy in many organisms is in adaptation to starvation, autophagy is also thought to be important for normal turnover of cytoplasmic contents, particularly in quiescent cells such as neurons. Autophagy may have a protective role against the development of a number of neurodegenerative diseases. Here we report that loss of autophagy causes neurodegeneration even in the absence of any disease-associated mutant proteins. Mice deficient for Atg5 (autophagy-related 5) specifically in neural cells develop progressive deficits in motor function that are accompanied by the accumulation of cytoplasmic inclusion bodies in neurons. In Atg5-/- cells, diffuse, abnormal intracellular proteins accumulate, and then form aggregates and inclusions. These results suggest that the continuous clearance of diffuse cytosolic proteins through basal autophagy is important for preventing the accumulation of abnormal proteins, which can disrupt neural function and ultimately lead to neurodegeneration.

The role of autophagy during the early neonatal starvation period

At birth the trans-placental nutrient supply is suddenly interrupted, and neonates face severe starvation until supply can be restored through milk nutrients. Here, we show that neonates adapt to this adverse circumstance by inducing autophagy. Autophagy is the primary means for the degradation of cytoplasmic constituents within lysosomes. The level of autophagy in mice remains low during embryogenesis; however, autophagy is immediately upregulated in various tissues after birth and is maintained at high levels for 3–12 h before returning to basal levels within 1–2 days. Mice deficient for Atg5, which is essential for autophagosome formation, appear almost normal at birth but die within 1 day of delivery. The survival time of starved Atg5-deficient neonates (∼ 12 h) is much shorter than that of wild-type mice (∼ 21 h) but can be prolonged by forced milk feeding. Atg5-deficient neonates exhibit reduced amino acid concentrations in plasma and tissues, and display signs of energy depletion. These results suggest that the production of amino acids by autophagic degradation of ‘self’ proteins, which allows for the maintenance of energy homeostasis, is important for survival during neonatal starvation.

LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation.

Rat LC3, a homologue of yeast Atg8 (Aut7/Apg8), localizes to autophagosomal membranes after post-translational modifications. The C-terminal fragment of LC3 is cleaved immediately following synthesis to yield a cytosolic form called LC3-I. A subpopulation of LC3-I is further converted to an autophagosome-associating form, LC3-II. Because yeast Atg8 is conjugated with phosphatidylethanolamine (PE) by a ubiquitin-like system, it has been hypothesized that LC3 is modified in a similar manner. Here, we show that [14C]-ethanolamine was preferentially incorporated into LC3-II, suggesting that LC3-II is a PE-conjugated form. LC3-II can be a substrate of mammalian Atg4B, a homologue of yeast Atg8-PE deconjugase, supporting the idea that LC3-II is LC3-PE. Moreover, two other mammalian homologues of yeast Atg8, γ-aminobutyric-acid-type-A-receptor-associated protein (GABARAP) and Golgi-associated ATPase enhancer of 16 kDa (GATE16) also generate form II, which are recovered in membrane fractions. Generation of the form II correlates with autophagosome association of GABARAP and GATE16. These results suggest that all mammalian Atg8 homologues receive a common modification to associate with autophagosomal membrane as the form II.

A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation

Autophagy is a bulk degradation process in eukaryotic cells and has fundamental roles in cellular homeostasis.The origin and source of autophagosomal membranes are long-standing questions in the field. Using electron microscopy, we show that, in mammalian culture cells, the endoplasmic reticulum (ER) associates with early autophagic structures called isolation membranes (IMs). Overexpression of an Atg4B mutant, which causes defects in autophagosome formation, induces the accumulation of ER–IM complexes. Electron tomography revealed that the ER–IM complex appears as a subdomain of the ER that formed a cradle encircling the IM, and showed that both ER and isolation membranes are interconnected.

Subcellular Localization of Toll-Like Receptor 3 in Human Dendritic Cells

Toll-like receptor (TLR)3 recognizes dsRNA and transduces signals to activate NF-κB and IFN-β promoter. Type I IFNs (IFN-α/β) function as key cytokines in anti-viral host defense. Human fibroblasts express TLR3 on the cell surface, and anti-TLR3 mAb inhibits dsRNA-induced IFN-β secretion by fibroblasts, suggesting that TLR3 acts on the cell surface to sense viral infection. In this study, we examined the expression and localization of human TLR3 in various DC subsets using anti-TLR3 mAb. In monocyte-derived immature dendritic cells (iDCs), TLR3 predominantly resided inside the cells but not on the cell surface. iDCs produced IL-12p70 and IFN-α and -β in response to poly(I:C). Similar response was observed in iDCs treated with rotavirus-derived dsRNA. These responses could not be blocked by pretreatment of the cells with anti-TLR3 mAb. In CD11c+ blood DCs, cytoplasmic retention of TLR3 was also observed as in monocyte-derived iDCs, again endorsing a different TLR3 distribution profile from fibroblasts. In precursor DC2, however, TLR3 could not be detected inside or outside the cells. Of note, there was a putative centrosomal protein that shared an epitope with TLR3 in myeloid DCs and precursor DC2, but not peripheral blood monocytes. Immunoelectron microscopic analysis revealed that TLR3, when stably expressed in the murine B cell line Ba/F3, was specifically accumulated in multivesicular bodies, a subcellular compartment situated in endocytic trafficking pathways. Thus, regulation and localization of TLR3 are different in each cell type, which may reflect participation of cell type-specific multiple pathways in antiviral IFN induction via TLR3.

Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure

Heart failure is a leading cause of morbidity and mortality in industrialized countries. Although infection with microorganisms is not involved in the development of heart failure in most cases, inflammation has been implicated in the pathogenesis of heart failure. However, the mechanisms responsible for initiating and integrating inflammatory responses within the heart remain poorly defined. Mitochondria are evolutionary endosymbionts derived from bacteria and contain DNA similar to bacterial DNA. Mitochondria damaged by external haemodynamic stress are degraded by the autophagy/lysosome system in cardiomyocytes. Here we show that mitochondrial DNA that escapes from autophagy cell-autonomously leads to Toll-like receptor (TLR) 9-mediated inflammatory responses in cardiomyocytes and is capable of inducing myocarditis and dilated cardiomyopathy. Cardiac-specific deletion of lysosomal deoxyribonuclease (DNase) II showed no cardiac phenotypes under baseline conditions, but increased mortality and caused severe myocarditis and dilated cardiomyopathy 10 days after treatment with pressure overload. Early in the pathogenesis, DNase II-deficient hearts showed infiltration of inflammatory cells and increased messenger RNA expression of inflammatory cytokines, with accumulation of mitochondrial DNA deposits in autolysosomes in the myocardium. Administration of inhibitory oligodeoxynucleotides against TLR9, which is known to be activated by bacterial DNA, or ablation of Tlr9 attenuated the development of cardiomyopathy in DNase II-deficient mice. Furthermore, Tlr9 ablation improved pressure overload-induced cardiac dysfunction and inflammation even in mice with wild-type Dnase2a alleles. These data provide new perspectives on the mechanism of genesis of chronic inflammation in failing hearts.

Pivotal Role of the Cell Death Factor BNIP3 in Ceramide-Induced Autophagic Cell Death in Malignant Glioma Cells

The sphingolipid ceramide has been recognized as an important second messenger implicated in regulating diverse signaling pathways especially for apoptosis. Very little is known, however, about the molecular mechanisms underlying nonapoptotic cell death induced by ceramide. In the present study, we first demonstrate that ceramide induces nonapoptotic cell death in malignant glioma cells. The cell death was accompanied by several specific features characteristic of autophagy: presence of numerous autophagic vacuoles in the cytoplasm, development of the acidic vesicular organelles, autophagosome membrane association of microtubule-associated protein light chain 3 (LC3), and a marked increase in expression levels of two forms of LC3 protein (LC3-I and LC3-II). We additionally demonstrate that ceramide decreases mitochondrial membrane potential and activates the transcription of death-inducing mitochondrial protein, BNIP3, resulting in increased expression levels of its mRNA and protein in malignant glioma cells. Moreover, tumor cells transfected with BNIP3 gene undergo autophagy in the absence of ceramide. These results suggest that ceramide induces autophagic cell death in malignant glioma cells via activation of BNIP3. This study adds a new concept to characterize the pathways by which ceramide acts to induce nonapoptotic autophagic cell death in malignant gliomas.

Autophagy Is Essential for Preimplantation Development of Mouse Embryos

After fertilization, maternal proteins in oocytes are degraded and new proteins encoded by the zygotic genome are synthesized. We found that autophagy, a process for the degradation of cytoplasmic constituents in the lysosome, plays a critical role during this period. Autophagy was triggered by fertilization and up-regulated in early mouse embryos. Autophagy-defective oocytes derived from oocyte-specific Atg5 (autophagy-related 5) knockout mice failed to develop beyond the four- and eight-cell stages if they were fertilized by Atg5-null sperm, but could develop if they were fertilized by wild-type sperm. Protein synthesis rates were reduced in the autophagy-null embryos. Thus, autophagic degradation within early embryos is essential for preimplantation development in mammals.

An Atg4B Mutant Hampers the Lipidation of LC3 Paralogues and Causes Defects in Autophagosome Closure

In the process of autophagy, a ubiquitin-like molecule, LC3/Atg8, is conjugated to phosphatidylethanolamine (PE) and associates with forming autophagosomes. In mammalian cells, the existence of multiple Atg8 homologues (referred to as LC3 paralogues) has hampered genetic analysis of the lipidation of LC3 paralogues. Here, we show that overexpression of an inactive mutant of Atg4B, a protease that processes pro-LC3 paralogues, inhibits autophagic degradation and lipidation of LC3 paralogues. Inhibition was caused by sequestration of free LC3 paralogues in stable complexes with the Atg4B mutant. In mutant overexpressing cells, Atg5- and ULK1-positive intermediate autophagic structures accumulated. The length of these membrane structures was comparable to that in control cells; however, a significant number were not closed. These results show that the lipidation of LC3 paralogues is involved in the completion of autophagosome formation in mammalian cells. This study also provides a powerful tool for a wide variety of studies of autophagy in the future.

Identification of a vesicular nucleotide transporter

ATP is a major chemical transmitter in purinergic signal transmission. Before secretion, ATP is stored in secretory vesicles found in purinergic cells. Although the presence of active transport mechanisms for ATP has been postulated for a long time, the proteins responsible for its vesicular accumulation remains unknown. The transporter encoded by the human and mouse SLC17A9 gene, a novel member of an anion transporter family, was predominantly expressed in the brain and adrenal gland. The mouse and bovine counterparts were associated with adrenal chromaffin granules. Proteoliposomes containing purified transporter actively took up ATP, ADP, and GTP by using membrane potential as the driving force. The uptake properties of the reconstituted transporter were similar to that of the ATP uptake by synaptic vesicles and chromaffin granules. Suppression of endogenous SLC17A9 expression in PC12 cells decreased exocytosis of ATP. These findings strongly suggest that SLC17A9 protein is a vesicular nucleotide transporter and should lead to the elucidation of the molecular mechanism of ATP secretion in purinergic signal transmission.