Potential role of senescent macrophages in radiation-induced pulmonary fibrosis

Radiation-induced pulmonary fibrosis (RIPF) is a late toxicity of therapeutic radiation in clinic with poor prognosis and limited therapeutic options. Previous results have shown that senescent cells, such as fibroblast and type II airway epithelial cell, are strongly implicated in pathology of RIPF. However, the role of senescent macrophages in the development RIPF is still unknown. In this study, we report that ionizing radiation (IR) increase cellular senescence with higher expression of senescence-associated β-galactosidase (SA-β-Gal) and senescence-specific genes (p16, p21, Bcl-2, and Bcl-xl) in irradiated bone marrow-derived monocytes/macrophages (BMMs). Besides, there’s a significant increase in the expression of pro-fibrogenic factors (TGF-β1 and Arg-1), senescence-associated secretory phenotype (SASP) proinflammatory factors (Il-1α, Il-6, and Tnf-α), SASP chemokines (Ccl2, Cxcl10, and Ccl17), and SASP matrix metalloproteinases (Mmp2, Mmp9 and Mmp12) in BMMs exposed to 10 Gy IR. In addition, the percentages of SA-β-Gal⁺ senescent macrophages are significantly increased in the macrophages of murine irradiated lung tissue. Moreover, robustly elevated expression of p16, SASP chemokines (Ccl2, Cxcl10, and Ccl17) and SASP matrix metalloproteinases (Mmp2, Mmp9, and Mmp12) is observed in the macrophages of irradiated lung, which might stimulate a fibrotic phenotype in pulmonary fibroblasts. In summary, irradiation can induce macrophage senescence, and increase the secretion of SASP in senescent macrophages. Our findings provide important evidence that senescent macrophages might be the target for prevention and treatment of RIPF.Subject terms: Senescence, Chemokines     

Arginase‐2, a miR‐1299 target,enhances pigmentation in melasma by reducing melanosome degradation via senescence‐induced autophagy inhibition

Expression profiles revealed miR‐1299 downregulation concomitant with arginase‐2 (ARG2) upregulation in hyperpigmented skin of melasma patients. Opposite regulation of tyrosinase and PMEL17 by miR‐1299 and inverse relationship between miR‐1299 and ARG2 expression denoted a role of miR‐1299 in pigmentation with ARG2 as a miR‐1299 target. ARG2 overexpression or knock‐down in keratinocytes, the main source of ARG2 in epidermis, positively regulated tyrosinase and PMEL17 protein levels, but not mRNA levels or melanosome transfer. ARG2 overexpression in keratinocytes reduced autophagy equivalent to 3‐MA, an autophagy inhibitor which also increased tyrosinase and PMEL17 protein levels, whereas ARG2 knock‐down induced opposite results. Autophagy inducer rapamycin reduced ARG2‐increased tyrosinase and PMEL17 protein levels. Also, autophagy was reduced in late passage‐induced senescent keratinocytes showing ARG2 upregulation. ARG2, but not 3‐MA, stimulated keratinocyte senescence. These results suggest that ARG2 reduces autophagy in keratinocytes by stimulating cellular senescence, resulting in skin pigmentation by reducing degradation of transferred melanosomes.     

Autophagy induction can regulate skin pigmentation by causing melanosome degradation in keratinocytes and melanocytes

Autophagy regulates cellular turnover by disassembling unnecessary or dysfunctional constituents. Recent studies demonstrated that autophagy and its regulators play a wide variety of roles in melanocyte biology. Activation of autophagy is known to induce melanogenesis and regulate melanosome biogenesis in melanocytes. Also, autophagy induction was reported to regulate physiologic skin color via melanosome degradation, although the downstream effectors are not yet clarified. To determine the role of autophagy as a melanosome degradation machinery, we administered several autophagy inducers in human keratinocytes and melanocytes. Our results showed that the synthetic autophagy inducer PTPD‐12 stimulated autophagic flux in human melanocytes and in keratinocytes containing transferred melanosomes. Increased autophagic flux led to melanosome degradation without affecting the expression of MITF. Furthermore, the color of cell pellets of both melanocytes and keratinocytes was visibly lightened. Inhibition of autophagic flux by chloroquine resulted in marked attenuation of PTPD‐12‐induced melanosome degradation, whereas the expression of melanogenesis pathway genes and proteins remained unaffected. Taken together, our results suggest that the modulation of autophagy can contribute to the regulation of melanocyte biology and skin pigmentation.     

The impact of autophagy on the development of senescence in primary tubular epithelial cells

Autophagy and senescence are 2 distinct pathways that are importantly involved in acute kidney injury and renal repair. Recent data indicate that the 2 processes might be interrelated. To investigate the potential link between autophagy and senescence in the kidney we isolated primary tubular epithelial cells (PTEC) from wild-type mice and monitored the occurrence of cellular senescence during autophagy activation and inhibition. We found that the process of cell isolation and transfer into culture was associated with a strong basal autophagic activation in PTEC. Specific inhibition of autophagy by silencing autophagy-related 5 (Atg5) counteracted the occurrence of senescence hallmarks under baseline conditions. Reduced senescent features were also observed in Atg5 silenced PTEC after γ-irradiation and during H-Ras induced oncogenic senescence, but the response was less uniform in these stress models. Senescence inhibition was paralleled by better preservation of a mature epithelial phenotype in PTEC. Interestingly, treatment with rapamycin, which acts as an activator of autophagy, also counteracted the occurrence of senescence features in PTEC. While we interpret the anti-senescent effect of rapamycin as an autophagy-independent effect of mTOR-inhibition, the more specific approach of Atg5 silencing indicates that overactivated autophagy can have pro-senescent effects in PTEC. These results highlight the complex interaction between cell culture dependent stress mechanisms, autophagy and senescence.     

Golgi membrane‐associated degradation pathway in yeast and mammals

Autophagy is a cellular process that degrades subcellular constituents, and is conserved from yeast to mammals. Although autophagy is believed to be essential for living cells, cells lacking Atg5 or Atg7 are healthy, suggesting that a non‐canonical degradation pathway exists to compensate for the lack of autophagy. In this study, we show that the budding yeast Saccharomyces cerevisiae, which lacks Atg5, undergoes bulk protein degradation using Golgi‐mediated structures to compensate for autophagy when treated with amphotericin B1, a polyene antifungal drug. We named this mechanism Golgi membrane‐associated degradation (GOMED) pathway. This process is driven by the disruption of PI(4)P‐dependent anterograde trafficking from the Golgi, and it also exists in Atg5‐deficient mammalian cells. Biologically, when an Atg5‐deficient β‐cell line and Atg7‐deficient β‐cells were cultured in glucose‐deprived medium, a disruption in the secretion of insulin granules from the Golgi occurred, and GOMED was induced to digest these (pro)insulin granules. In conclusion, GOMED is activated by the disruption of PI(4)P‐dependent anterograde trafficking in autophagy‐deficient yeast and mammalian cells.     

The role of autophagy in neonatal tissues: just a response to amino acid starvation?

Autophagy is activated soon after birth in neonatal tissues and is essential for survival because mice deficient in Atg5 or Atg7 autophagy genes die within 1 day after birth. Amino acid starvation has been considered as a major deleterious effect of autophagy deficiency, since the concentration of amino acids in plasma was decreased by 20% in the two knockout models, whereas blood glucose and fatty acid levels were apparently not affected. However, autophagy may have other important functions in neonatal physiology, including glycogen degradation, programmed cell remodeling and response to oxidative stress.     

Analysis of a lung defect in autophagy-deficient mouse strains

Yeast Atg1 initiates autophagy in response to nutrient limitation. The Ulk gene family encompasses the mammalian orthologs of yeast ATG1. We created mice deficient for both Ulk1 and Ulk2 and found that the mice die within 24 h of birth. When found alive, pups exhibited signs of respiratory distress. Histological sections of lungs of the Ulk1/2 DKO pups showed reduced airspaces with thickened septae. A similar defect was seen in Atg5-deficient pups as both Ulk1/2 DKO and Atg5 KO lungs show numerous glycogen-laden alveolar type II cells by electron microscopy, PAS staining, and increased levels of glycogen in lung homogenates. No abnormalities were noted in expression of genes encoding surfactant proteins but the ability to incorporate exogenous choline into phosphatidylcholine, the major phospholipid component of surfactant, was increased in comparison to controls. Despite this, there was a trend for total phospholipid levels in lung tissue to be lower in Ulk1/2 DKO and Atg5 KO compared with controls. Autophagy was abundant in lung epithelial cells from wild-type mice, but lacking in Atg5 KO and Ulk1/2 DKO mice at P1. Analysis of the autophagy signaling pathway showed the existence of a negative feedback loop between the ULK1 and 2 and MTORC1 and 2, in lung tissue. In the absence of autophagy, alveolar epithelial cells are unable to mobilize internal glycogen stores independently of surfactant maturation. Together, the data suggested that autophagy plays a vital role in lung structural maturation in support of perinatal adaptation to air breathing.     

Analysis of a lung defect in autophagy-deficient mouse strains

Yeast Atg1 initiates autophagy in response to nutrient limitation. The Ulk gene family encompasses the mammalian orthologs of yeast ATG1. We created mice deficient for both Ulk1 and Ulk2 and found that the mice die within 24 h of birth. When found alive, pups exhibited signs of respiratory distress. Histological sections of lungs of the Ulk1/2 DKO pups showed reduced airspaces with thickened septae. A similar defect was seen in Atg5-deficient pups as both Ulk1/2 DKO and Atg5 KO lungs show numerous glycogen-laden alveolar type II cells by electron microscopy, PAS staining, and increased levels of glycogen in lung homogenates. No abnormalities were noted in expression of genes encoding surfactant proteins but the ability to incorporate exogenous choline into phosphatidylcholine, the major phospholipid component of surfactant, was increased in comparison to controls. Despite this, there was a trend for total phospholipid levels in lung tissue to be lower in Ulk1/2 DKO and Atg5 KO compared with controls. Autophagy was abundant in lung epithelial cells from wild-type mice, but lacking in Atg5 KO and Ulk1/2 DKO mice at P1. Analysis of the autophagy signaling pathway showed the existence of a negative feedback loop between the ULK1 and 2 and MTORC1 and 2, in lung tissue. In the absence of autophagy, alveolar epithelial cells are unable to mobilize internal glycogen stores independently of surfactant maturation. Together, the data suggested that autophagy plays a vital role in lung structural maturation in support of perinatal adaptation to air breathing.     

Autophagy gene ATG7 regulates ultraviolet radiation-induced inflammation and skin tumorigenesis

Macroautophagy (hereafter autophagy) is a cellular “self-eating” process that is implicated in many human cancers, where it can act to either promote or suppress tumorigenesis. However, the role of autophagy in regulation of inflammation during tumorigenesis remains unclear. Here we show that autophagy is induced in the epidermis by ultraviolet (UV) irradiation and autophagy gene Atg7 promoted UV-induced inflammation and skin tumorigenesis. Atg7 regulated UV-induced cytokine expression and secretion, and promoted Ptgs2/Cox-2 expression through both a CREB1/CREB-dependent cell autonomous mechanism and an IL1B/IL1β-dependent non-cell autonomous mechanism. Adding PGE₂ increased UV-induced skin inflammation and tumorigenesis, reversing the epidermal phenotype in mice with Atg7 deletion in keratinocytes. Similar to ATG7 knockdown in human keratinocytes, ATG5 knockdown inhibited UVB-induced expression of PTGS2 and cytokines. Furthermore, ATG7 loss increased the activation of the AMPK pathway and the phosphorylation of CRTC1, and led to endoplasmic reticulum (ER) accumulation and reduction of ER stress. Inducing ER stress and inhibiting calcium influx into the ER by thapsigargin reverses the inflammation and tumorigenesis phenotype in mice with epidermal Atg7 deletion. Taken together, these findings demonstrate that deleting autophagy gene Atg7 leads to a suppression of carcinogen-induced protumorigenic inflammatory microenvironment and tumorigenesis of the epithelium.     

Dynamic involvement of ATG5 in cellular stress responses

Autophagy maintains cell and tissue homeostasis through catabolic degradation. To better delineate the in vivo function for autophagy in adaptive responses to tissue injury, we examined the impact of compromised autophagy in mouse submandibular glands (SMGs) subjected to main excretory duct ligation. Blocking outflow from exocrine glands causes glandular atrophy by increased ductal pressure. Atg5^f/−;Aqp5-Cre mice with salivary acinar-specific knockout (KO) of autophagy essential gene Atg5 were generated. While duct ligation induced autophagy and the expression of inflammatory mediators, SMGs in Atg5^f/−;Aqp5-Cre mice, before ligation, already expressed higher levels of proinflammatory cytokine and Cdkn1a/p21 messages. Extended ligation period resulted in the caspase-3 activation and acinar cell death, which was delayed by Atg5 knockout. Moreover, expression of a set of senescence-associated secretory phenotype (SASP) factors was elevated in the post-ligated glands. Dysregulation of cell-cycle inhibitor CDKN1A/p21 and activation of senescence-associated β-galactosidase were detected in the stressed SMG duct cells. These senescence markers peaked at day 3 after ligation and partially resolved by day 7 in post-ligated SMGs of wild-type (WT) mice, but not in KO mice. The role of autophagy-related 5 (ATG5)-dependent autophagy in regulating the tempo, duration and magnitude of cellular stress responses in vivo was corroborated by in vitro studies using MEFs lacking ATG5 or autophagy-related 7 (ATG7) and autophagy inhibitors. Collectively, our results highlight the role of ATG5 in the dynamic regulation of ligation-induced cellular senescence and apoptosis, and suggest the involvement of autophagy resolution in salivary repair.Autophagy is a catabolic process that has an essential role in cellular adaptation to multiple types of stress by recycling of superfluous cellular material, safeguarding quality control in organelles, removing protein aggregates, and eliminating intracellular pathogens.¹ Conceptually, autophagy serves a pro-survival mechanism by providing sources of energy and biosynthetic building blocks during starvation, removing dysfunctional organelles and large aggregates toxic to cells to avoid unwarranted cell death. However, upon sustained stress conditions, cell death eventually takes place either by excessive autophagy or by the induction of apoptosis and/or necrosis pathways.² The ATG5, autophagy-related 5, has a pivotal role in autophagosome formation. Mouse neonates systemic deficient for ATG5 die within a day of birth,³ whereas mice depleted of Atg5 in selected tissues have abnormalities ranging from neurodegeneration⁴ and age-related cardiomyopathy⁵ to liver tumors.⁶Autophagy and senescence are two distinct, however functionally intertwined, cellular responses to stress.⁷ Cellular senescence is a state of stable growth arrest that is induced by telomere shortening, DNA-damage, oncogenes or other stresses. In general, senescence is a heterogeneous phenotype, which is characterized by a senescent-associated secretory phenotype (SASP), expression of senescence-associated β-galactosidase (SA-β-gal) and other senescent markers, and increased cell size.⁸ In culture system, inhibiting or enhancing autophagy leads to the opposite effect on premature senescence.^{9, 10, 11, 12} While premature senescence can be induced by a plethora of cell-extrinsic and cell-intrinsic stressors,¹³ little is known about the possible role of autophagy in modulating injury-induced cellular senescence in vivo. Rodent salivary duct ligation has been used as an experimental model system to study salivary gland atrophy, which often occurs in patients with Sjögren''s syndrome or receiving head and neck radiation therapy. Although autophagy induction has been implicated in the repair of rapamycin-treated, post-ligated salivary glands,^14,15 the roles played by autophagy in regulating the injury responses in submandibular glands (SMGs) have not been explored.To explore how autophagy contributes to salivary (patho)physiology, we established a transgenic mouse model deficient for ATG5 in the salivary acinar cells. Previously, we have identified a role for basal autophagy in salivary homeostatic mechanisms that restrict acinar cell size and the number of secretory granules.¹⁶ Here, we report that ligation of the major SMG excretory duct triggers the glandular atrophy and the induction of autophagy. By comparing the acute and subacute stress responses from autophagy-impaired and -competent SMGs with duct obstruction, we established the intrinsic roles of ATG5-dependent autophagy in modulating salivary inflammatory responses, stress-induced senescence and cell death, which all occur sequentially in response to tissue injury. Our results provide in vivo evidence that stress-induced autophagic response is indispensable for resolving premature senescence in duct cells of the ligated glands, whereas ATG5 deficiency leads to delayed acinar cell death.     

Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila

Autophagy is involved with the turnover of intracellular components and the management of stress responses. Genetic studies in mice have shown that suppression of neuronal autophagy can lead to the accumulation of protein aggregates and neurodegeneration. However, no study has shown that increasing autophagic gene expression can be beneficial to an aging nervous system. Here we demonstrate that expression of several autophagy genes is reduced in Drosophila neural tissues as a normal part of aging. The age-dependent suppression of autophagy occurs concomitantly with the accumulation of insoluble ubiquitinated proteins (IUP), a marker of neuronal aging and degeneration. Mutations in the Atg8a gene (autophagy-related 8a) result in reduced lifespan, IUP accumulation and increased sensitivity to oxidative stress. In contrast, enhanced Atg8a expression in older fly brains extends the average adult lifespan by 56% and promotes resistance to oxidative stress and the accumulation of ubiquitinated and oxidized proteins. These data indicate that genetic or age-dependent suppression of autophagy is closely associated with the buildup of cellular damage in neurons and a reduced lifespan, while maintaining the expression of a rate-limiting autophagy gene prevents the age-dependent accumulation of damage in neurons and promotes longevity.     

Autophagy in the intestinal epithelium is not involved in the pathogenesis of intestinal tumors

Autophagy has been demonstrated to be associated with the pathogenesis of cancer, but no consensus has been reached about its precise role. Therefore, we investigated whether autophagy in the intestinal epithelium is involved in the pathogenesis of intestinal tumors. To evaluate the relationship between autophagy and intestinal tumors, GFP-LC3-APC(min/+) mice were generated by mating GFP-LC3 transgenic mice with APC(min/+) mice. Autophagy was weakly induced in the intestinal polyp regions of the mice in comparison to their non-polyp regions. Under starved conditions, autophagy was not induced in the polyp regions, whereas it was observed in the non-polyp regions. Then, to examine whether a lack of autophagy in the intestinal epithelium enhances the induction of intestinal tumor, Atg7flox/flox:vil-cre-APC(min/+) mice, in which Atg7 had been conditionally deleted in the intestinal epithelium, were generated by mating Atg7flox/flox:vil-cre mice with APC(min/+) mice. However, there was no significant difference in the number of intestinal polyps between the Atg7flox/flox:vil-cre-APC(min/+) and the corresponding control Atg7flox/flox-APC(min/+) mice. These results indicate that autophagy in the intestinal epithelium is not involved in the pathogenesis of intestinal tumors, and future research should focus on regulating autophagy as a form of cancer therapy.     

Autophagy and ammonia

Autophagy plays an important role in the cellular response to a variety of metabolic stress conditions thus contributing to the maintenance of intracellular homeostasis. Studies in yeast have defined the genetic components involved in the initiation of autophagy as well as the progression through the autophagic cascade. The yeast kinase Atg1 initiates autophagy in response to nutrient limitation in a TOR-dependent manner. The ulk family of genes encodes the mammalian orthologue of yeast Atg1. Our recent work using mouse embryonic fibroblast (MEF) cell lines deficient for both ulk1 and ulk2, has revealed that autophagy induction is more complex in mammals than in yeast. Furthermore, these data confirm the surprising finding that a by-product of amino acid metabolism, ammonia, is a strong inducer of autophagy, as first shown by the Abraham laboratory.     

Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice

Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. This mechanism is involved in the preservation of nutrients under starvation condition as well as the normal turnover of cytoplasmic component. Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies. Here, we generated conditional knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 was essential for ATG conjugation systems and autophagosome formation, amino acid supply in neonates, and starvation-induced bulk degradation of proteins and organelles in mice. Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates. Our results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.     

Autophagy in the intestinal epithelium reduces endotoxin-induced inflammatory responses by inhibiting NF-κB activation

Autophagy is a lysosomal degradation pathway that is essential for survival, differentiation, development and homeostasis. There is growing evidence that impaired autophagy leads to the pathogenesis of diverse diseases. However, the role of autophagy in intestinal epithelium is not clearly understood, although previous studies have pointed out the possibility for the relationships of autophagy with bowel inflammation. In this study, we investigated the involvement of autophagy in intestinal epithelium with inflammatory responses. We generated the mice with a conditional deletion of Atg7, which is one of the autophagy regulated gene, in intestinal epithelium. In Atg7-deficient small intestinal epithelium, LPS-induced production of TNF-α and IL-1β mRNA was enhanced in comparison to the control small intestinal tissues. In addition, the degree of LPS-induced activation of NF-κB was promoted in Atg7-deficient intestinal epithelium. These results demonstrate that autophagy can attenuate endotoxin-induced inflammatory responses in intestinal epithelium resulting in the maintenance of intestinal homeostasis.     

Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder—murine Pompe disease

Autophagy, an intracellular system for delivering portions of cytoplasm and damaged organelles to lysosomes for degradation/recycling, plays a role in many physiological processes and is disturbed in many diseases. We recently provided evidence for the role of autophagy in Pompe disease, a lysosomal storage disorder in which acid alpha-glucosidase, the enzyme involved in the breakdown of glycogen, is deficient or absent. Clinically the disease manifests as a cardiac and skeletal muscle myopathy. The current enzyme replacement therapy (ERT) clears lysosomal glycogen effectively from the heart but less so from skeletal muscle. In our Pompe model, the poor muscle response to therapy is associated with the presence of pools of autophagic debris. To clear the fibers of the autophagic debris, we have generated a Pompe model in which an autophagy gene, Atg7, is inactivated in muscle. Suppression of autophagy alone reduced the glycogen level by 50–60%. Following ERT, muscle glycogen was reduced to normal levels, an outcome not observed in Pompe mice with genetically intact autophagy. The suppression of autophagy, which has proven successful in the Pompe model, is a novel therapeutic approach that may be useful in other diseases with disturbed autophagy.Key words: Pompe disease, lysosomal glycogen storage, myopathy, Atg7, enzyme replacement therapy     

Di-retinoid-pyridinium-ethanolamine (A2E) Accumulation and the Maintenance of the Visual Cycle Are Independent of Atg7-mediated Autophagy in the Retinal Pigmented Epithelium

Autophagy is an evolutionarily conserved catabolic mechanism that relieves cellular stress by removing/recycling damaged organelles and debris through the action of lysosomes. Compromised autophagy has been implicated in many neurodegenerative diseases, including retinal degeneration. Here we examined retinal phenotypes resulting from RPE-specific deletion of the autophagy regulatory gene Atg7 by generating Atg7^flox/flox;VMD2-rtTA-cre+ mice to determine whether autophagy is essential for RPE functions including retinoid recycling. Atg7-deficient RPE displayed abnormal morphology with increased RPE thickness, cellular debris and vacuole formation indicating that autophagy is important in maintaining RPE homeostasis. In contrast, 11-cis-retinal content, ERGs and retinal histology were normal in mice with Atg7-deficient RPE in both fasted and fed states. Because A2E accumulation in the RPE is associated with pathogenesis of both Stargardt disease and age-related macular degeneration (AMD) in humans, deletion of Abca4 was introduced into Atg7^flox/flox;VMD2-rtTA-cre+ mice to investigate the role of autophagy during A2E accumulation. Comparable A2E concentrations were detected in the eyes of 6-month-old mice with and without Atg7 from both Abca4^−/− and Abca4^+/+ backgrounds. To identify other autophagy-related molecules involved in A2E accumulation, we performed gene expression array analysis on A2E-treated human RPE cells and found up-regulation of four autophagy related genes; DRAM1, NPC1, CASP3, and EIF2AK3/PERK. These observations indicate that Atg7-mediated autophagy is dispensable for retinoid recycling and A2E deposition; however, autophagy plays a role in coping with stress caused by A2E accumulation.     

Purification and characterization of a ubiquitin-like system for autophagosome formation

Autophagy refers to the bulk degradation of cellular proteins and organelles through an autophagosome and plays a pivotal role in the development, cellular differentiation, aging, and elimination of aberrant structures. A failure of autophagy has been implicated in a growing list of mammalian disease states, including cancer and cardiomyopathy. Two ubiquitin-like systems are highly involved in autophagy, especially in the formation of autophagosomes. Here, we purified and characterized Atg7 (an E1-like enzyme), and Atg3 and Atg10 (E2-like enzymes) in order to gain an insight into the role played by ubiquitin-like systems in the formation of autophagosomes. Interestingly, we observed that Atg7 forms a homodimer to construct an active conformation, unlike other E1-like enzymes. Although Atg3 was detected as a monomer under physiological conditions, Atg10 existed in an oligomeric form, indicating that the mechanism by which Atg10 functions may differ from that of Atg3.     

Neuronal autophagy: Going the distance to the axon

Autophagy, a regulated cellular degradation process responsible for the turnover of long-lived proteins and organelles, has been increasingly implicated in neurological disorders. Although autophagy is mostly viewed as a stress-induced process, recent studies have indicated that it is constitutively active in central nervous system (CNS) neurons and is protective against neurodegeneration. Neurons are highly specialized, post-mitotic cells that are typically composed of a soma (cell body), a dendritic tree and an axon. The detailed process of autophagy in such a highly differentiated cell type remains to be characterized. To elucidate the physiological role of neuronal autophagy, we generated mutant mice containing a neural cell type-specific deletion of Atg7, an essential gene for autophagy. Establishment of these mutant mice allowed us to examine cell-autonomous events in cerebellar Purkinje cells deficient in autophagy. Our data reveal the indispensability of autophagy in the maintenance of axonal homeostasis and the prevention of axonal dystrophy and degeneration. Furthermore, our study implicates dysfunction of axonal autophagy as a potential mechanism underlying axonopathy, which is linked to neurodegeneration associated with numerous human neurological disorders. Finally, our study has raised a possibility that “constitutive autophagy” in neurons involves processes that are not typical of autophagy in other cell types, but rather is highly adapted to local physiological function in the axon, which is projected in a distance from one neuron to another for transducing neural signals.

Addendum to: Komatsu M, Wang QJ, Holstein GR, Friedrich Jr. VL, Iwata J, Kominami E, Chait BT, Tanaka K, Yue Z. Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration. Proc Natl Acad Sci USA 2007; 104:14489-94.