Taki Nishimura

De novo mutations in the autophagy gene WDR45 cause static encephalopathy of childhood with neurodegeneration in adulthood.

Static encephalopathy of childhood with neurodegeneration in adulthood (SENDA) is a recently established subtype of neurodegeneration with brain iron accumulation (NBIA). By exome sequencing, we found de novo heterozygous mutations in WDR45 at Xp11.23 in two individuals with SENDA, and three additional WDR45 mutations were identified in three other subjects by Sanger sequencing. Using lymphoblastoid cell lines (LCLs) derived from the subjects, aberrant splicing was confirmed in two, and protein expression was observed to be severely impaired in all five. WDR45 encodes WD-repeat domain 45 (WDR45). WDR45 (also known as WIPI4) is one of the four mammalian homologs of yeast Atg18, which has an important role in autophagy. Lower autophagic activity and accumulation of aberrant early autophagic structures were demonstrated in the LCLs of the affected subjects. These findings provide direct evidence that an autophagy defect is indeed associated with a neurodegenerative disorder in humans.

The HOPS complex mediates autophagosome–lysosome fusion through interaction with syntaxin 17

Autophagosome–lysosome fusion requires the autophagosomal SNARE syntaxin 17. Syntaxin 17 interacts with the HOPS-tethering complex. HOPS is required for syntaxin 17–dependent autophagosome–lysosome fusion, besides its function in endolysosomal fusion.

Mammalian Atg2 proteins are essential for autophagosome formation and important for regulation of size and distribution of lipid droplets

Autophagy is an intracellular degradation process that is mediated by autophagosomes. Mammalian Atg2 proteins Atg2A and Atg2B are identified and characterized as essential for autophagy. They are also present on lipid droplets and are involved in regulation of lipid droplet volume and distribution.

FIP200 regulates targeting of Atg16L1 to the isolation membrane.

Autophagosome formation is a dynamic process that is strictly controlled by autophagy‐related (Atg) proteins. However, how these Atg proteins are recruited to the autophagosome formation site or autophagic membranes remains poorly understood. Here, we found that FIP200, which is involved in proximal events, directly interacts with Atg16L1, one of the downstream Atg factors, in an Atg14‐ and phosphatidylinositol 3‐kinase‐independent manner. Atg16L1 deletion mutants, which lack the FIP200‐interacting domain, are defective in proper membrane targeting. Thus, FIP200 regulates not only early events but also late events of autophagosome formation through direct interaction with Atg16L1.

LC3, a microtubule-associated protein1A/B light chain3, is involved in cytoplasmic lipid droplet formation.

The cytoplasmic lipid droplet (LD) is one of organelles that has a neutral lipid core with a single phospholipid layer. LDs are believed to be generated between the two leaflets of the endoplasmic reticulum (ER) membrane and to play various roles, such as high effective energy storage. However, it remains largely unknown how LDs are generated and grow in the cytoplasm. We have previously shown that the Atg conjugation system that is essential for autophagosome formation is involved in LD formation in hepatocytes and cardiac myocytes. We show here that LC3 itself is involved in LD formation by using RNA interference (RNAi). All cultured cell lines examined, in which the expression of LC3 was suppressed by RNAi, showed reduced LD formation. Triacylglycerol, a major component of LDs, was synthesized and degraded in LC3 mRNA-knockdown cells as well as in control cells. Interestingly, potential of the bulk protein degradation in the knockdown-cells was also evident in the control cells. These findings indicate that LC3 is involved in the LD formation regardless of the bulk degradation, and that LC3 has two pivotal roles in cellular homeostasis mediated by autophagy and lipid metabolism.

Inhibition of cholesterol biosynthesis by 25-hydroxycholesterol is independent of OSBP.

25‐hydroxycholesterol (25‐HC) is a potent suppressor of cholesterol synthesis gene transcription in cultured cells. A high affinity binding protein for 25‐HC, oxysterol‐binding protein (OSBP), has been identified from tissue cytosol. OSBP translocates from the cytosol to the Golgi apparatus membranes after addition of 25‐HC to cell cultures and is thought to mediate 25‐HC action on cholesterol metabolism through association to the Golgi apparatus. However, direct evidence to prove this hypothesis was lacking. In this study, we knocked down expression of OSBP by using duplex siRNAs specific for OSBP to examine the relationship between OSBP and 25‐HC‐induced inhibition of cholesterol synthesis gene transcription. We found that decreasing OSBP expression by ∼90% did not affect 25‐HC‐induced inhibition of transcription of 3‐hydoxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase and squalene epoxidase to any extent. Exogenous lysophosphatidylcholine (LPC), which is known to cause the efflux of cellular cholesterol into the medium and to increase cholesterol synthesis, was found to rescue the 25‐HC‐induced down‐regulation of sterol regulated genes, while LPC did not affect 25‐HC‐induced association of OSBP with the Golgi apparatus. These results suggest that inhibition of cholesterol biosynthesis genes by 25‐HC is OSBP‐independent.

Autophagosome formation is initiated at phosphatidylinositol synthase‐enriched ER subdomains

The autophagosome, a double‐membrane structure mediating degradation of cytoplasmic materials by macroautophagy, is formed in close proximity to the endoplasmic reticulum (ER). However, how the ER membrane is involved in autophagy initiation and to which membrane structures the autophagy‐initiation complex is localized have not been fully characterized. Here, we were able to biochemically analyze autophagic intermediate membranes and show that the autophagy‐initiation complex containing ULK and FIP200 first associates with the ER membrane. To further characterize the ER subdomain, we screened phospholipid biosynthetic enzymes and found that the autophagy‐initiation complex localizes to phosphatidylinositol synthase (PIS)‐enriched ER subdomains. Then, the initiation complex translocates to the ATG9A‐positive autophagosome precursors in a PI3P‐dependent manner. Depletion of phosphatidylinositol (PI) by targeting bacterial PI‐specific phospholipase C to the PIS domain impairs recruitment of downstream autophagy factors and autophagosome formation. These findings suggest that the autophagy‐initiation complex, the PIS‐enriched ER subdomain, and ATG9A vesicles together initiate autophagosome formation.

Oligo-astheno-teratozoospermia in mice lacking ORP4, a sterol-binding protein in the OSBP-related protein family.

Oligo‐astheno‐teratozoospermia (OAT), a condition that includes low sperm number, low sperm motility and abnormal sperm morphology, is the commonest cause of male infertility. Because genetic analysis is frequently impeded by the infertility phenotype, the genetic basis of many of OAT conditions has been hard to verify. Here, we show that deficiency of ORP4, a sterol‐binding protein in the oxysterol‐binding protein (OSBP)‐related protein family, causes male infertility due to severe OAT in mice. In ORP4‐deficient mice, spermatogonia proliferation and subsequent meiosis occurred normally, but the morphology of elongating and elongated spermatids was severely distorted, with round‐shaped head, curled back head or symplast. Spermatozoa derived from ORP4‐deficient mice had little or no motility and no fertilizing ability in vitro. In ORP4‐deficient testis, postmeiotic spermatids underwent extensive apoptosis, leading to a severely reduced number of spermatozoa. At the ultrastructural level, nascent acrosomes appeared to normally develop in round spermatids, but acrosomes were detached from the nucleus in elongating spermatids. These results suggest that ORP4 is essential for the postmeiotic differentiation of germ cells.

Functional analysis of GS28, an intra‐Golgi SNARE, in Caenorhabditis elegans

Intra‐Golgi retrograde transport is assumed to maintain Golgi function by recycling Golgi‐resident proteins to younger cisternae in the progression of entire Golgi stack from cis to trans. GS28 (Golgi SNARE of 28 kDa, also known as GOS28) is a Golgi‐localized SNARE protein and has been implicated in intra‐Golgi retrograde transport. However, the in vivo functions of GS28, and consequently, the roles of the intra‐Golgi retrograde transport in animal development are largely unknown. In this study, we generated deletion mutants of Caenorhabditis elegans GS28 and performed a synthetic lethal RNAi screen using GS28 mutants. We found that another Golgi‐localized SNARE, Ykt6, functions cooperatively with GS28 in embryonic development. During post‐embryonic development, GS28 mutants exhibited reduced seam cell numbers and a missing ray phenotype under Ykt6 knockdown conditions, suggesting that cell proliferation and/or differentiation of stem cell‐like seam cells are impaired in GS28‐ and Ykt6‐depleted worms. We also demonstrated that GS28 and Ykt6 act redundantly for the proper expression of Golgi‐resident proteins in adult intestinal cells. This study reveals the in vivo importance of the Golgi‐localized SNAREs GS28 and Ykt6.

Oxysterol-binding protein (OSBP) is required for the perinuclear localization of intra-Golgi v-SNAREs

OSBP regulates the Golgi cholesterol level. This study demonstrates that OSBP and cholesterol are essential for localization of Golgi v-SNAREs. Knockdown of ArfGAP1 restores v-SNARE localization in OSBP-depleted cells, suggesting that OSBP-regulated cholesterol ensures proper COP-I vesicle transport.