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.

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.

Modeling of Retina and Optic Nerve Ischemia-Reperfusion Injury through Hypoxia-Reoxygenation in Human Induced Pluripotent Stem Cell-Derived Retinal Ganglion Cells.

Retinal ganglion cells (RGCs) are specialized projection neurons that constitute part of the retina, and the death of RGCs causes various eye diseases, but the mechanism of RGC death is still unclear. Here, we induced cell death in human induced pluripotent stem cell (hiPSC)-derived RGC-rich retinal tissues using hypoxia-reoxygenation in vitro. Flow cytometry, immunochemistry, and Western blotting showed the apoptosis and necrosis of RGCs under hypoxia-reoxygenation, and they were rescued by an apoptosis inhibitor but not by a necrosis inhibitor. This revealed that the cell death induced in our model was mainly due to apoptosis. To our knowledge, this is the first model to reproduce ischemia-reperfusion in hiPSC-derived RGCs. Thus, the efficacy of apoptosis inhibitors and neuroprotective agents can be evaluated using this model, bringing us closer to clinical applications.

An Overview of Golgi Membrane-Associated Degradation (GOMED) and Its Detection Methods.

Autophagy is a cellular mechanism that utilizes lysosomes to degrade its own components and is performed using Atg5 and other molecules originating from the endoplasmic reticulum membrane. On the other hand, we identified an alternative type of autophagy, namely, Golgi membrane-associated degradation (GOMED), which also utilizes lysosomes to degrade its own components, but does not use Atg5 originating from the Golgi membranes. The GOMED pathway involves Ulk1, Wipi3, Rab9, and other molecules, and plays crucial roles in a wide range of biological phenomena, such as the regulation of insulin secretion and neuronal maintenance. We here describe the overview of GOMED, methods to detect autophagy and GOMED, and to distinguish GOMED from autophagy.

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.

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.

Involvement of casein kinase 1 epsilon/delta (Csnk1e/d) in the pathogenesis of familial Parkinson's disease caused by CHCHD2.

Parkinson's disease (PD) is a common neurodegenerative disorder that results from the loss of dopaminergic neurons. Mutations in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) gene cause a familial form of PD with α-Synuclein aggregation, and we here identified the pathogenesis of the T61I mutation, the most common disease-causing mutation of CHCHD2. In Neuro2a cells, CHCHD2 is in mitochondria, whereas the T61I mutant (CHCHD2T61I ) is mislocalized in the cytosol. CHCHD2T61l then recruits casein kinase 1 epsilon/delta (Csnk1e/d), which phosphorylates neurofilament and α-Synuclein, forming cytosolic aggresomes. In vivo, both Chchd2T61I knock-in and transgenic mice display neurodegenerative phenotypes and aggresomes containing Chchd2T61I , Csnk1e/d, phospho-α-Synuclein, and phospho-neurofilament in their dopaminergic neurons. Similar aggresomes were observed in a postmortem PD patient brain and dopaminergic neurons generated from patient-derived iPS cells. Importantly, a Csnk1e/d inhibitor substantially suppressed the phosphorylation of neurofilament and α-Synuclein. The Csnk1e/d inhibitor also suppressed the cellular damage in CHCHD2T61I -expressing Neuro2a cells and dopaminergic neurons generated from patient-derived iPS cells and improved the neurodegenerative phenotypes of Chchd2T61I mutant mice. These results indicate that Csnk1e/d is involved in the pathogenesis of PD caused by the CHCHD2T61I mutation.

Biogenesis of fibrils requires C-mannosylation of PMEL.

Premelanosome protein (PMEL), a melanocyte-specific glycoprotein, has an essential role in melanosome maturation, assembling amyloid fibrils for melanin deposition. PMEL undergoes several post-translational modifications, including N- and O-glycosylations, which are associated with proper melanosome development. C-mannosylation is a rare type of protein glycosylation at a tryptophan residue that might regulate the secretion and localization of proteins. PMEL has one putative C-mannosylation site in its core amyloid fragment (CAF); however, there is no report focusing on C-mannosylation of PMEL. To investigate this, we expressed recombinant PMEL in SK-MEL-28 human melanoma cells and purified the protein. Mass spectrometry analyses demonstrated that human PMEL is C-mannosylated at multiple tryptophan residues in its CAF and N-terminal fragment (NTF). In addition to the W153 or W156 residue (CAF), which lies in the consensus sequence for C-mannosylation, the W104 residue (NTF) was C-mannosylated without the consensus sequence. To determine the effects of the modifications, we deleted the PMEL gene by using CRISPR/Cas9 technology and re-expressed wild-type or C-mannosylation-defective mutants of PMEL, in which the C-mannosylated tryptophan was replaced with a phenylalanine residue (WF mutation), in SK-MEL-28 cells. Importantly, fibril-containing melanosomes were significantly decreased in W104F mutant PMEL-re-expressing cells compared with wild-type PMEL, observed using transmission electron microscopy. Furthermore, western blot and immunofluorescence analysis suggested that the W104F mutation may cause mild endoplasmic reticulumretention, possibly associated with early misfolding, and lysosomal misaggregation, thus reducing functional fibril formation. Our results demonstrate that C-mannosylation of PMEL is required for proper melanosome development by regulating PMEL-derived fibril formation.

Development of small fluorescent probes for the analysis of autophagy kinetics.

Autophagy is a dynamic process that degrades subcellular constituents, and its activity is measured by autophagic flux. The tandem proteins RFP-GFP-LC3 and GFP-LC3-RFP-LC3ΔG, which enable the visualization of autophagic vacuoles of different stages by differences in their fluorescent color, are useful tools to monitor autophagic flux, but they require plasmid transfection. In this study, we hence aimed to develop a new method to monitor autophagic flux using small cell-permeable fluorescent probes. We previously developed two green-fluorescent probes, DALGreen and DAPGreen, which detect autolysosomes and multistep autophagic vacuoles, respectively. We here developed a red-fluorescent autophagic probe, named DAPRed, which recognizes various autophagic vacuoles. By the combinatorial use of these green- and red-fluorescent probes, we were able to readily detect autophagic flux. Furthermore, these probes were useful not only for the visualization of canonical autophagy but also for alternative autophagy. DAPRed was also applicable for the detection of autophagy in living organisms.

Lactiplantibacillus plantarum OLL2712 Induces Autophagy via MYD88 and Strengthens Tight Junction Integrity to Promote the Barrier Function in Intestinal Epithelial Cells.

Autophagy is an important system conserved in eukaryotes that maintains homeostasis by degrading abnormal proteins. Autophagy incompetence in intestinal epithelial cells causes the abnormal function of intestinal stem cells and other cells and damages intestinal barrier function. The disruption of the intestinal barrier causes chronic inflammation throughout the body, followed by impaired glucose and lipid metabolism. Lactiplantibacillus plantarum OLL2712 (OLL2712) is a lactic acid bacterium that induces interleukin-10 production from immune cells, alleviates chronic inflammation, and improves glucose and lipid metabolism. In this study, we hypothesized that OLL2712 exerts anti-inflammatory effects by inducing autophagy and ameliorating intestinal barrier dysfunction, and we investigated its autophagy-inducing activities and functions. Caco-2 cells stimulated with OLL2712 for 24 h showed an increased number of autolysosomes per cell, compared with unstimulated cells. Therefore, the permeability of fluorescein isothiocyanate dextran 4000 (FD-4) was suppressed by inducing autophagy. In contrast, mucin secretion in HT-29-MTX-E12 cells was also increased by OLL2712 but not via autophagy induction. Finally, the signaling pathway involved in autophagy induction by OLL2712 was found to be mediated by myeloid differentiation factor 88 (MYD88). In conclusion, our findings suggest that OLL2712 induces autophagy in intestinal epithelial cells via MYD88, and that mucosal barrier function is strengthened by inducing autophagy.

Identification of a novel type of focal adhesion remodelling via FAK/FRNK replacement, and its contribution to cancer progression.

Numerous studies have investigated the various cellular responses against genotoxic stress, including those mediated by focal adhesions. We here identified a novel type of focal adhesion remodelling that occurs under genotoxic stress conditions, which involves the replacement of active focal adhesion kinase (FAK) with FAK-related non-kinase (FRNK). FRNK stabilized focal adhesions, leading to strong cell-matrix adhesion, and FRNK-depleted cells were easily detached from extracellular matrix upon genotoxic stress. This remodelling occurred in a wide variety of cells. In vivo, the stomachs of Frnk-knockout mice were severely damaged by genotoxic stress, highlighting the protective role of FRNK against genotoxic stress. FRNK was also found to play a vital role in cancer progression, because FRNK depletion significantly inhibited cancer dissemination and progression in a mouse cancer model. Furthermore, in human cancers, FRNK was predominantly expressed in metastatic tissues and not in primary tissues. We hence conclude that this novel type of focal adhesion remodelling reinforces cell adhesion and acts against genotoxic stress, which results in the protection of normal tissues, but in turn facilitates cancer progression.

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.

Uncovering the Localization and Function of a Novel Read-Through Transcript 'TOMM40-APOE'.

Recent advancements in genome analysis technology have revealed the presence of read-through transcripts in which transcription continues by skipping the polyA signal. We here identified and characterized a new read-through transcript, TOMM40-APOE. With cDNA amplification from THP-1 cells, the TOMM40-APOE3 product was successfully generated. We also generated TOMM40-APOE4, another isoform, by introducing point mutations. Notably, while APOE3 and APOE4 exhibited extracellular secretion, both TOMM40-APOE3 and TOMM40-APOE4 were localized exclusively to the mitochondria. But functionally, they did not affect mitochondrial membrane potential. Cell death induction studies illustrated increased cell death with TOMM40-APOE3 and TOMM40-APOE4, and we did not find any difference in cellular function between the two isoforms. These findings indicated that the new mitochondrial protein TOMM40-APOE has cell toxic ability.

FLIP-based autophagy-detecting technique reveals closed autophagic compartments.

Autophagy results in the degradation of cytosolic components via two major membrane deformations. First, the isolation membrane sequesters components from the cytosol and forms autophagosomes, by which open structures become closed compartments. Second, the outer membrane of the autophagosomes fuses with lysosomes to degrade the inner membrane and its contents. The efficiency of the latter degradation process, namely autophagic flux, can be easily evaluated using lysosomal inhibitors, whereas the dynamics of the former process is difficult to analyze because of the challenges in identifying closed compartments of autophagy (autophagosomes and autolysosomes). To resolve this problem, we here developed a method to detect closed autophagic compartments by applying the FLIP technique, and named it FLIP-based Autophagy Detection (FLAD). This technique visualizes closed autophagic compartments and enables differentiation of open autophagic structures and closed autophagic compartments in live cells. In addition, FLAD analysis detects not only starvation-induced canonical autophagy but also genotoxic stress-induced alternative autophagy. By the combinational use of FLAD and LC3, we were able to distinguish the structures of canonical autophagy from those of alternative autophagy in a single cell.

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.

Molecular mechanisms and biological roles of GOMED.

We previously discovered an autophagy-like proteolysis mechanism that uses the Golgi membrane, namely, Golgi membrane-associated degradation (GOMED). Morphologically, GOMED resembles canonical autophagy, but the two mechanisms have different cellular functions, as they degrade different substrates and use different membrane sources. Furthermore, although the molecules involved partially overlap, the core molecules are completely different. GOMED preferentially degrades Golgi-trafficking proteins, including insulin granules in pancreatic ß-cells and ceruloplasmin in neurons, and is involved in a wide variety of physiological events.

Oxidized Phospholipids and Neutrophil Elastase Coordinately Play Critical Roles in NET Formation.

Neutrophil extracellular traps (NETs) are web-like structures consisting of decondensed chromatin DNA and contents of granules, such as myeloperoxidase (MPO) and neutrophil elastase (NE). NETs are usually released from neutrophils undergoing NETosis, a neutrophil-specific cell death mode characterized by the collapse and disappearance of cell membranes and nuclear envelopes. It is well known that production of reactive oxygen species (ROS) triggers NETosis and NET formation. However, details of intracellular signaling downstream of ROS production during NETosis and NET formation remains uncertain. Here, we demonstrated that the peroxidation of phospholipids plays a critical role in NETosis and NET formation induced by phorbol 12-myristate13-acetate (PMA) or immune complex in vitro and by lipopolysaccharide (LPS) in vivo. This phospholipid peroxidation is mediated by the enzymatic activity of MPO. On the other hand, NE, which was previously reported to be released from granules to cytosol by MPO during NET formation, is not required for either the peroxidation of phospholipids or the execution of NETosis, but contributes to chromatin decondensation and nuclear swelling independently of MPO-mediated oxidized phospholipids. Analysis of isolated nuclei clearly demonstrated that oxidized phospholipids and NE differently yet synergistically execute chromatin decondensation and nuclear swelling, and the subsequent release of nuclear contents. These findings indicate the dual roles of MPO in NETosis and NET formation, and provide new insight into the molecular mechanism of these phenomena.

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.

Wipi3 is essential for alternative autophagy and its loss causes neurodegeneration.

Alternative autophagy is an Atg5/Atg7-independent type of autophagy that contributes to various physiological events. We here identify Wipi3 as a molecule essential for alternative autophagy, but which plays minor roles in canonical autophagy. Wipi3 binds to Golgi membranes and is required for the generation of isolation membranes. We establish neuron-specific Wipi3-deficient mice, which show behavioral defects, mainly as a result of cerebellar neuronal loss. The accumulation of iron and ceruloplasmin is also found in the neuronal cells. These abnormalities are suppressed by the expression of Dram1, which is another crucial molecule for alternative autophagy. Although Atg7-deficient mice show similar phenotypes to Wipi3-deficient mice, electron microscopic analysis shows that they have completely different subcellular morphologies, including the morphology of organelles. Furthermore, most Atg7/Wipi3 double-deficient mice are embryonic lethal, indicating that Wipi3 functions to maintain neuronal cells via mechanisms different from those of canonical autophagy.

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.