Alternative autophagy dampens UVB-induced NLRP3 inflammasome activation in human keratinocytes.

Sunlight exposure results in an inflammatory reaction of the skin commonly known as sunburn, which increases skin cancer risk. In particular, the ultraviolet B (UVB) component of sunlight induces inflammasome activation in keratinocytes to instigate the cutaneous inflammatory responses. Here, we explore the intracellular machinery that maintains skin homeostasis by suppressing UVB-induced inflammasome activation in human keratinocytes. We found that pharmacological inhibition of autophagy promoted UVB-induced NLRP3 inflammasome activation. Unexpectedly, however, gene silencing of Atg5 or Atg7, which are critical for conventional autophagy, had no effect, whereas gene silencing of Beclin1, which is essential not only for conventional autophagy but also for Atg5/Atg7-independent alternative autophagy, promoted UVB-induced inflammasome activation, indicating an involvement of alternative autophagy. We found that damaged mitochondria were highly accumulated in UVB-irradiated keratinocytes when alternative autophagy was inhibited, and they appear to be recognized by NLRP3. Overall, our findings indicate that alternative autophagy, rather than conventional autophagy, suppresses UVB-induced NLRP3 inflammasome activation through the clearance of damaged mitochondria in human keratinocytes and illustrate a previously unknown involvement of alternative autophagy in inflammation. Alternative autophagy may be a new therapeutic target for sunburn and associated cutaneous disorders.

Beclin1 is essential for the pancreas development.

Beclin1 (Becn1) is a multifunctional protein involved in autophagy regulation, membrane trafficking, and tumor suppression. In this study, we examined the roles of Becn1 in the pancreas development by generating mice with conditional deletion of Becn1 in the pancreas using pancreatic transcriptional factor 1a (Ptf1a)-Cre mice (Becn1f/f; Ptf1aCre/+). Surprisingly, loss of Becn1 in the pancreas resulted in severe pancreatic developmental defects, leading to insufficient exocrine and endocrine pancreatic function. Approximately half of Becn1f/f; Ptf1aCre/+ mice died immediately after birth. However, duodenum and neural tissue development were almost normal, indicating that pancreatic insufficiency was the cause of death. These findings demonstrated a novel role for Becn1 in pancreas morphogenesis, differentiation, and growth, and suggested that loss of this factor leaded to pancreatic agenesis at birth.

Subcellular localization and ER-mediated cytotoxic function of α1A and α1ACT in spinocerebellar ataxia type 6.

Spinocerebellar ataxia type 6 (SCA6) is a polyglutamine (polyQ) disease, which is caused by the elongation of CAG repeats encoding polyQ in the CACNA1A gene. The CACNA1A gene encodes two proteins, namely, α1A (a subunit of the plasma membrane calcium channel), which is translated in its entire length, and α1ACT, which is translated from the second cistron, and both proteins have a polyQ tract. The α1A-polyQ and α1ACT-polyQ proteins with an elongated polyQ stretch have been reported to form aggregates in cells and induce neuronal cell death, but the subcellular localization of these proteins and their cytotoxic properties remain unclear. In this study, we first analyzed SCA6 model mice and found that α1A-polyQlong localized mainly to the Golgi apparatus, whereas a portion of α1ACT-polyQlong localized to the nucleus. Analysis using Neuro2a cells also showed similar subcellular localizations of these proteins, and a proportion of both proteins localized to the endoplasmic reticulum (ER). Cytotoxic studies demonstrated that both proteins induce both the ER stress response and apoptosis, indicating that they are able to induce ER stress-induced apoptosis.

Absence of ULK1 decreases AMPK activity in the kidney, leading to chronic kidney disease progression.

AMP-activated protein kinase (AMPK) inactivation in chronic kidney disease (CKD) leads to energy status deterioration in the kidney, constituting the vicious cycle of CKD exacerbation. Unc-51-like kinase 1 (ULK1) is considered a downstream molecule of AMPK; however, it was recently reported that the activity of AMPK could be regulated by ULK1 conversely. We demonstrated that AMPK and ULK1 activities were decreased in the kidneys of CKD mice. However, whether and how ULK1 is involved in the underlying mechanism of CKD exacerbation remains unknown. In this study, we investigated the ULK1 involvement in CKD, using ULK1 knockout mice. The CKD model of Ulk1-/- mice exhibited significantly exacerbated renal function and worsening renal fibrosis. In the kidneys of the CKD model of Ulk1-/- mice, reduced AMPK and its downstream ß-oxidation could be observed, leading to an energy deficit of increased AMP/ATP ratio. In addition, AMPK signaling in the kidney was reduced in control Ulk1-/- mice with normal renal function compared to control wild-type mice, suggesting that ULK1 deficiency suppressed AMPK activity in the kidney. This study is the first to present ULK1 as a novel therapeutic target for CKD treatment, which regulates AMPK activity in the kidney.

Homeostatic p62 levels and inclusion body formation in CHCHD2 knockout mice.

Inactivation of constitutive autophagy results in the formation of cytoplasmic inclusions in neurones, but the relationship between impaired autophagy and Lewy bodies (LBs) remains unknown. α-Synuclein and p62, components of LBs, are the defining characteristic of Parkinson's disease (PD). Until now, we have analyzed mice models and demonstrated p62 aggregates derived from an autophagic defect might serve as 'seeds' and can potentially be a cause of LB formation. P62 may be the key molecule for aggregate formation. To understand the mechanisms of LBs, we analyzed p62 homeostasis and inclusion formation using PD model mice. In PARK22-linked PD, intrinsically disordered mutant CHCHD2 initiates Lewy pathology. To determine the function of CHCHD2 for inclusions formation, we generated Chchd2-knockout (KO) mice and characterized the age-related pathological and motor phenotypes. Chchd2 KO mice exhibited p62 inclusion formation and dopaminergic neuronal loss in an age-dependent manner. These changes were associated with a reduction in mitochondria complex activity and abrogation of inner mitochondria structure. In particular, the OPA1 proteins, which regulate fusion of mitochondrial inner membranes, were immature in the mitochondria of CHCHD2-deficient mice. CHCHD2 regulates mitochondrial morphology and p62 homeostasis by controlling the level of OPA1. Our findings highlight the unexpected role of the homeostatic level of p62, which is regulated by a non-autophagic system, in controlling intracellular inclusion body formation, and indicate that the pathologic processes associated with the mitochondrial proteolytic system are crucial for loss of DA neurones.

Identification of small compounds that inhibit multiple myeloma proliferation by targeting c-Maf transcriptional activity.

Multiple myeloma displays the clonal B cell expansion and the overproduction of monoclonal immunoglobulins. Genetic translocations at 14q32, particularly with partners like 16q23, lead to the dysregulation of oncogene expression, including the significant enhancement of c-Maf. This aberrant expression of c-Maf has prompted research into strategies for targeting this transcription factor as a potential therapeutic avenue for multiple myeloma treatment. In this study, we introduce a screening pipeline to test small compounds for their ability to inhibit c-Maf. Using a luciferase indicator driven by the Ccl8 gene promoter, we identified two small compounds that inhibit transcriptional activity of c-Maf. These molecules impede the proliferation of c-Maf-expressing myeloma cells, and repress the expression of c-Maf target genes such as ITGB7 and CCR1. Importantly, these molecules target c-Maf-expressing multiple myeloma cells, but not c-Maf-negative myeloma cells, showing potential for tailoring therapeutic intervention. In conclusion, our screening pipeline is effective to explore leads for a novel c-Maf inhibitor for multiple myeloma therapy.

Nickel particles are present in Crohn's disease tissue and exacerbate intestinal inflammation in IBD susceptible mice.

Crohn's disease is an inflammatory disease of the gut caused by a complex interplay among genetic, microbial, and environmental factors. The intestinal tract is constantly exposed to metals and other trace elements ingested as food. Synchrotron radiation-induced X-ray fluorescence spectroscopy and X-ray absorption fine structure analysis revealed the deposition of nickel particles within Crohn's disease tissue specimens. After nickel particle stimulation, THP-1 cells showed filopodia formation and autophagic vacuoles containing lipid bodies. Nickel particles precipitated colitis in mice bearing mutations of the IBD susceptibility protein A20/TNFAIP3. Nickel particles also exacerbated dextran sulfate sodium-induced colitis in mice harboring myeloid cell-specific Atg5 deficiency. These findings illustrate that nickel particle ingestion may worsen Crohn's disease by perturbing autophagic processes in the intestine, providing new insights into environmental factors in Crohn's disease pathogenesis.

Autophagy involvement in oncogenesis.

Various clinical and experimental findings have revealed the causal relationship between autophagy failure and oncogenesis, and several mechanisms have been suggested to explain this relationship. We recently proposed two additional mechanisms: centrosome number dysregulation and the failure of autophagic cell death. Here, we detail the mechanical relationship between autophagy failure and oncogenesis.

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 ß-cell line and Atg7-deficient ß-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 ceramide analogue N-(1-hydroxy-3-morpholino-1-phenylpropan-2-yl)decanamide induces large lipid droplet accumulation and highlights the effect of LAMP-2 deficiency on lipid droplet degradation.

Excess accumulation of intracellular lipids leads to various diseases. Lipid droplets (LDs) are ubiquitous cellular organelles for lipid storage. LDs are hydrolyzed via cytosolic lipases (lipolysis) and also degraded in lysosomes through autophagy; namely, lipophagy. A recent study has shown the size-dependent selection of LDs by the two major catabolic pathways (lipolysis and lipophagy), and thus experimental systems that can manipulate the size of LDs are now needed. The ceramide analogue N-(1-hydroxy-3-morpholino-1-phenylpropan-2-yl)decanamide (PDMP) affects the structures and functions of lysosomes/late endosomes and the endoplasmic reticulum (ER), and alters cholesterol homeostasis. We previously reported that PDMP induces autophagy via the inhibition of mTORC1. In the present study, we found that PDMP induced the accumulation of LDs, especially that of large LDs, in mouse fibroblast (L cells). Surprisingly, the LD accumulation was relieved by PDMP in L cells deficient in lysosome-associated membrane protein-2 (LAMP-2), which is reportedly important for lipophagy. An electron microscopy analysis demonstrated that the LAMP-2 deficiency caused enlarged autophagosomes/autolysosomes in L cells, which may promote the sequestration and degradation of the PDMP-dependent large LDs. Accordingly, PDMP will be useful to explore the mechanism of LD degradation, by inducing large LDs.

Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response.

The innate immune system senses RNA viruses by pattern recognition receptors (PRRs) and protects the host from virus infection. PRRs mediate the production of immune modulatory factors and direct the elimination of RNA viruses. Here, we show a unique PRR that mediates antiviral response. Tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP ribose) polymerase (TIPARP), a Cysteine3 Histidine (CCCH)-type zinc finger-containing protein, binds to Sindbis virus (SINV) RNA via its zinc finger domain and recruits an exosome to induce viral RNA degradation. TIPARP typically localizes in the nucleus, but it accumulates in the cytoplasm after SINV infection, allowing targeting of cytoplasmic SINV RNA. Redistribution of TIPARP is induced by reactive oxygen species (ROS)-dependent oxidization of the nuclear pore that affects cytoplasmic-nuclear transport. BCL2-associated X protein (BAX) and BCL2 antagonist/killer 1 (BAK1), B-cell leukemia/lymphoma 2 (BCL2) family members, mediate mitochondrial damage to generate ROS after SINV infection. Thus, TIPARP is a viral RNA-sensing PRR that mediates antiviral responses triggered by BAX- and BAK1-dependent mitochondrial damage.

Mitochondrial E3 Ubiquitin Ligase Parkin: Relationships with Other Causal Proteins in Familial Parkinson's Disease and Its Substrate-Involved Mouse Experimental Models.

Parkinson's disease (PD) is a common neurodegenerative disorder. Recent identification of genes linked to familial forms of PD has revealed that post-translational modifications, such as phosphorylation and ubiquitination of proteins, are key factors in disease pathogenesis. In PD, E3 ubiquitin ligase Parkin and the serine/threonine-protein kinase PTEN-induced kinase 1 (PINK1) mediate the mitophagy pathway for mitochondrial quality control via phosphorylation and ubiquitination of their substrates. In this review, we first focus on well-characterized PINK1 phosphorylation motifs. Second, we describe our findings concerning relationships between Parkin and HtrA2/Omi, a protein involved in familial PD. Third, we describe our findings regarding inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS), a member of PINK1 and Parkin substrates, involved in neurodegeneration during PD. IPAS is a dual-function protein involved in transcriptional repression of hypoxic responses and the pro-apoptotic activities.

Prediction of intracellular targets of a small compound by analyzing peptides presented on MHC class I.

In chemical biology, the elucidation of chemical target is crucial for successful drug development. Because MHC class I molecules present peptides from intracellular damaged proteins, it might be possible to identify targets of a chemical by analyzing peptide sequences on MHC class I. Therefore, we treated cells with the autophagy-inducing chemical TMD-457 and identified the peptides presented on MHC class I. Many of the peptides were derived from molecules involved in ER trafficking and ER stress, which were confirmed by morphological and biochemical analyses. Therefore, our results demonstrate that analyzing MHC class I peptides is useful for the detection of chemical targets.

[Nomenclature on Cell Death, apoptosis and autophagic cell death.]

Apoptosis had been considered as a sole form of cell death in living body. However, non-apoptotic forms of cell death have recently attracted more attention, and about 20 forms of non-apoptotic cell death have been identified. In this review, I introduce apoptosis and these non-apoptotic cell death, particularly autophagic cell death.

Identification of PPM1D as an essential Ulk1 phosphatase for genotoxic stress-induced autophagy.

Autophagy is an evolutionary conserved process that degrades subcellular constituents. Unlike starvation-induced autophagy, the molecular mechanism of genotoxic stress-induced autophagy has not yet been fully elucidated. In this study, we analyze the molecular mechanism of genotoxic stress-induced autophagy and identify an essential role of dephosphorylation of the Unc51-like kinase 1 (Ulk1) at Ser637, which is catalyzed by the protein phosphatase 1D magnesium-dependent delta isoform (PPM1D). We show that after exposure to genotoxic stress, PPM1D interacts with and dephosphorylates Ulk1 at Ser637 in a p53-dependent manner. The PPM1D-dependent Ulk1 dephosphorylation triggers Ulk1 puncta formation and induces autophagy. This happens not only in mouse embryonic fibroblasts but also in primary thymocytes, where the genetic ablation of PPM1D reduces the dephosphorylation of Ulk1 at Ser637, inhibits autophagy, and accelerates apoptosis induced by X-ray irradiation. This acceleration of apoptosis is caused mainly by the inability of the autophagic machinery to degrade the proapoptotic molecule Noxa. These findings indicate that the PPM1D-Ulk1 axis plays a pivotal role in genotoxic stress-induced autophagy.

TRF2 Protein Interacts with Core Histones to Stabilize Chromosome Ends.

Mammalian chromosome ends are protected by a specialized nucleoprotein complex called telomeres. Both shelterin, a telomere-specific multi-protein complex, and higher order telomeric chromatin structures combine to stabilize the chromosome ends. Here, we showed that TRF2, a component of shelterin, binds to core histones to protect chromosome ends from inappropriate DNA damage response and loss of telomeric DNA. The N-terminal Gly/Arg-rich domain (GAR domain) of TRF2 directly binds to the globular domain of core histones. The conserved arginine residues in the GAR domain of TRF2 are required for this interaction. A TRF2 mutant with these arginine residues substituted by alanine lost the ability to protect telomeres and induced rapid telomere shortening caused by the cleavage of a loop structure of the telomeric chromatin. These findings showed a previously unnoticed interaction between the shelterin complex and nucleosomal histones to stabilize the chromosome ends.

Molecular mechanisms and physiological roles of Atg5/Atg7-independent alternative autophagy.

ATG5 and ATG7 are considered to be essential molecules for the induction of autophagy. However, we found that cells lacking ATG5 or ATG7 can still form autophagosomes/autolysosomes and perform autophagic protein degradation when subjected to certain types of stress. Although the lipidation of LC3 is accepted as a good indicator of autophagy, this did not occur during ATG5/ATG7-independent alternative autophagy. Unlike conventional autophagy, autophagosomes appeared to be generated in a Rab9-dependent manner by the fusion of the phagophores with vesicles derived from the trans-Golgi and late endosomes. Therefore, mammalian autophagy can occur via at least two different pathways; the ATG5/ATG7-dependent conventional pathway and an ATG5/ATG7-independent alternative pathway.

Autophagy mediates the mitotic senescence transition.

As a stress response, senescence is a dynamic process involving multiple effector mechanisms whose combination determines the phenotypic quality. Here we identify autophagy as a new effector mechanism of senescence. Autophagy is activated during senescence and its activation is correlated with negative feedback in the PI3K-mammalian target of rapamycin (mTOR) pathway. A subset of autophagy-related genes are up-regulated during senescence: Overexpression of one of those genes, ULK3, induces autophagy and senescence. Furthermore, inhibition of autophagy delays the senescence phenotype, including senescence-associated secretion. Our data suggest that autophagy, and its consequent protein turnover, mediate the acquisition of the senescence phenotype.

Alternative autophagy plays a central role in mitochondria elimination from reticulocytes.

Mitophagy is a mitochondrial quality control mechanism where damaged and surplus mitochondria are degraded by macroautophagy. Mitophagy is associated with various physiological and pathological events such as mitochondrial clearance during the terminal differentiation of reticulocytes. There are two different mammalian autophagy pathways: the Atg5-dependent conventional pathway and Atg5-independent alternative pathway; the latter is involved in reticulocyte's mitophagy.