桜井 一

The Golgi stress response is an important cytoprotective system that enhances Golgi function in response to cellular demand, while cells damaged by prolonged Golgi stress undergo cell death. OSW-1, a natural compound with anticancer activity, potently inhibits OSBP that transports cholesterol and phosphatidylinositol-4-phosphate (PI4P) at contact sites between the endoplasmic reticulum and the Golgi apparatus. Previously, we reported that OSW-1 induces the Golgi stress response, resulting in Golgi stress-induced transcription and cell death. However, the underlying molecular mechanism has been unknown. To reveal the mechanism of a novel pathway of the Golgi stress response regulating transcriptional induction and cell death (the PI4P pathway), we performed a genome-wide knockout screen and found that transcriptional induction as well as cell death induced by OSW-1 was repressed by the loss of regulators of PI4P synthesis, such as PITPNB and PI4KB. Our data indicate that OSW-1 induces Golgi stress-dependent transcriptional induction and cell death through dysregulation of the PI4P metabolism in the Golgi.

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.

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.

Abstract The Golgi stress response is an important cytoprotective system that enhances Golgi function in response to cellular demand, while cells damaged by prolonged Golgi stress undergo cell death to ensure the survival of organisms. OSW-1, a natural compound with anticancer activity, acts as a potent inhibitor of OSBP that transports cholesterol and phosphatidylinositol-4-phosphate (PI4P) at contact sites between the endoplasmic reticulum and the Golgi apparatus. Previously, we reported that OSW-1 induces the Golgi stress response, resulting in Golgi stress-induced transcription and cell death. However, the underlying molecular mechanism has been unknown. To reveal the mechanism of a novel pathway of the Golgi stress response regulating transcriptional induction and cell death (the cholesterol pathway), we performed a genome-wide knockout screen and found that transcriptional induction as well as cell death induced by OSW-1 was repressed in HeLa cells deficient in factors involved in the PI4P metabolism, such as PITPNB and PI4KB genes. Our data indicate that OSW-1 induces Golgi stress-dependent transcriptional induction and cell death through dysregulation of the PI4P metabolism in the Golgi apparatus.

Abstract The Golgi stress response is a homeostatic mechanism that augments Golgi function when Golgi function becomes insufficient (Golgi stress). Glycosylation of the core proteins of proteoglycans is one of the important functions of the Golgi. If the production of core proteins is increased and the amount of glycosylation enzymes for proteoglycans becomes insufficient (PG-type Golgi stress), the proteoglycan pathway of the Golgi stress response is activated, resulting in the transcriptional induction of glycosylation enzymes, including NDST2, HS6ST1 and GLCE. The transcriptional induction of these glycosylation enzymes is regulated by the enhancer element, PGSE-A; however, transcription factors that induce transcription from PGSE-A have not yet been identified. We herein proposed KLF2 and KLF4 as candidate transcription factors for transcriptional induction from PGSE-A, and revealed that their expression was up-regulated in response to PG-type Golgi stress. These results suggest that KLF2 and KLF4 are important regulators of the proteoglycan pathways of the mammalian Golgi stress response.

Abstract 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.

Autophagy is a cellular process that degrades intracellular components, including misfolded proteins and damaged organelles. Many neurodegenerative diseases are considered to progress via the accumulation of misfolded proteins and damaged organelles; therefore, autophagy functions in regulating disease severity. There are at least two types of autophagy (canonical autophagy and alternative autophagy), and canonical autophagy has been applied to therapeutic strategies against various types of neurodegenerative diseases. In contrast, the role of alternative autophagy has not yet been clarified, but it is speculated to be involved in the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease.

<title>Abstract</title> 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<italic>-</italic>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.

We have developed two types of fluorescent probes, DALGreen and DAPGreen, for monitoring autophagy, that exhibit fluorescence upon being incorporated into autophagosomes. DALGreen enhances its fluorescence at acidic pH, which is favorable for monitoring late-phase autophagy, whereas DAPGreen remains fluorescent with almost constant brightness during the autophagic process. With these probes that stain autophagosomes as they are being formed, the real-time change of autophagic phenomena of live cells may be traced, which is an advantage over conventional approaches with small molecules that stain mature autophagosomes. The use of both dyes allows monitoring of the membrane dynamics of autophagy in any type of cell without the need for genetic engineering, and therefore, will be useful as a tool to study autophagic phenomena.

Programmed cell death, which is required for the development and homeostasis of metazoans, includes mechanisms such as apoptosis, autophagic cell death, and necrotic (or type III) death. Members of the Bcl2 family regulate apoptosis, among which Bax and Bak act as a mitochondrial gateway. Although embryonic fibroblasts from Bax/Bak double-knockout (DKO) mice are resistant to apoptosis, we previously demonstrated that these cells die through an autophagy-dependent mechanism in response to various types of cellular stressors. To determine the physiological role of autophagy-dependent cell death, we generated Atg5/Bax/Bak triple-knockout (TKO) mice, in which autophagy is greatly suppressed compared with DKO mice. Embryonic fibroblasts and thymocytes from TKO mice underwent autophagy much less frequently, and their viability was much higher than DKO cells in the presence of certain cellular stressors, providing genetic evidence that DKO cells undergo Atg5-dependent death. Compared with wild-type embryos, the loss of interdigital webs was significantly delayed in DKO embryos and was even further delayed in TKO embryos. Brain malformation is a distinct feature observed in DKO embryos on the 129 genetic background, but not in those on a B6 background, whereas such malformations appeared in TKO embryos even on a B6 background. Taken together, our data suggest that Atg5-dependent cell death contributes to the embryonic development of DKO mice, implying that autophagy compensates for the deficiency in apoptosis.

Programmed cell death, which is required for the development and homeostasis of metazoans, includes mechanisms such as apoptosis, autophagic cell death, and necrotic (or type III) death. Members of the Bcl2 family regulate apoptosis, among which Bax and Bak act as a mitochondrial gateway. Although embryonic fibroblasts from Bax/Bak double-knockout (DKO) mice are resistant to apoptosis, we previously demonstrated that these cells die through an autophagy-dependent mechanism in response to various types of cellular stressors. To determine the physiological role of autophagy-dependent cell death, we generated Atg5/Bax/Bak triple-knockout (TKO) mice, in which autophagy is greatly suppressed compared with DKO mice. Embryonic fibroblasts and thymocytes from TKO mice underwent autophagy much less frequently, and their viability was much higher than DKO cells in the presence of certain cellular stressors, providing genetic evidence that DKO cells undergo Atg5-dependent death. Compared with wild-type embryos, the loss of interdigital webs was significantly delayed in DKO embryos and was even further delayed in TKO embryos. Brain malformation is a distinct feature observed in DKO embryos on the 129 genetic background, but not in those on a B6 background, whereas such malformations appeared in TKO embryos even on a B6 background. Taken together, our data suggest that Atg5-dependent cell death contributes to the embryonic development of DKO mice, implying that autophagy compensates for the deficiency in apoptosis.

RAB5 GTPases act as molecular switches that regulate various endosomal functions in animal cells, including homotypic fusion of early endosomes, endosomal motility, endosomal signaling, and subcompartmentalization of the endosomal membrane. RAB5 proteins fulfill these diverse functions through interactions with downstream effector molecules. Two canonical RAB5 members, ARA7 and RAB HOMOLOG1 (RHA1), are encoded in the Arabidopsis thaliana genome. ARA7 and RHA1 play crucial roles in endocytic and vacuolar trafficking pathways. Plant RAB5 GTPases function via interactions with effector molecules, whose identities and functions are currently unclear. In this study, we searched for canonical RAB5 effector molecules of Arabidopsis and identified a candidate, which we called ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN (EREX). The intimate genetic interaction between EREX and RAB5 members, the results from subcellular colocalization experiments, and the direct interaction observed in an in vitro pull-down assay strongly suggest that EREX is a genuine effector of canonical RAB5s in Arabidopsis. We further found that close homologs of EREX play partially redundant functions with EREX in the transport of seed storage proteins. Our results indicate that canonical plant RAB5s acquired distinct effector molecules from those of non-plant systems to fulfill their functions.