Autophagy is controlled by AMPK and mTOR, both of which associate with ULK1 and control the production of phosphatidylinositol 3-phosphate (PtdIns3P), a prerequisite for autophagosome formation. Here we report that WIPI3 and WIPI4 scaffold the signal control of autophagy upstream of PtdIns3P production and have a role in the PtdIns3P effector function of WIPI1-WIPI2 at nascent autophagosomes. In response to LKB1-mediated AMPK stimulation, WIPI4-ATG2 is released from a WIPI4-ATG2/AMPK-ULK1 complex and translocates to nascent autophagosomes, controlling their size, to which WIPI3, in complex with FIP200, also contributes. Upstream, WIPI3 associates with AMPK-activated TSC complex at lysosomes, regulating mTOR. Our WIPI interactome analysis reveals the scaffold functions of WIPI proteins interconnecting autophagy signal control and autophagosome formation. Our functional kinase screen uncovers a novel regulatory link between LKB1-mediated AMPK stimulation that produces a direct signal via WIPI4, and we show that the AMPK-related kinases NUAK2 and BRSK2 regulate autophagy through WIPI4.

Background/Aims As autophagy is linked to several pathological conditions, like cancer and neurodegenerative diseases, it is crucial to understand its regulatory signaling network. In this study, we investigated the role of the serum- and glucocorticoid-induced protein kinase 1 (SGK1) in the control of autophagy. Methods To measure autophagic activity in vivo, we quantified the abundance of the autophagy conjugates LC3-PE (phosphatidylethanolamine) and ATG12-ATG5 in tissue extracts of SGK1 wild-type (Sgk1 +/+) and knockout (Sgk1 −/−) mice that were either fed or starved for 24 h prior sacrifice. In vitro, we targeted SGK1 by RNAi using GFP-WIPI1 expressing U-2 OS cells to quantify the numbers of cells displaying newly formed autophagosomes. In parallel, these cells were also assessed with regard to LC3 and ULK1 by quantitative Western blotting. Results The abundance of both LC3-PE (LC3-II) and ATG12-ATG5 was significantly increased in red muscle tissues of SGK1 knockout mice. This was found in particular in fed conditions, suggesting that SGK1 may keep basal autophagy under control in red muscle in vivo. Under starved conditions, significant differences were observed in SGK1-deficient white muscle tissue and, under fed conditions, also in the liver. In vitro, we found that SGK1 silencing provoked a significant increase of cells displaying WIPI1-positive autophagosomes and autophagosomal LC3 (LC3-II). Moreover, autophagic flux assessments revealed that autophagic degradation significantly increased in the absence of SGK1, strongly suggesting that SGK1 inhibits both autophagosome formation and autophagic degradation in vitro. In addition, more ULK1 protein lacking the inhibitory, TORC1-specific phosphorylation at serine 758 was detected in the absence of SGK1. Conclusions Combined, our data strongly support the idea that SGK1 inhibits the process of autophagy. Mechanistically, our data suggest that SGK1 should act upstream of ULK1 in regulating autophagy, and we hypothesize that SGK1 contributes to the regulation of ULK1 gene expression.

Antimitotic drugs are extensively used in the clinics to treat different types of cancer. They can retain cells in a prolonged mitotic arrest imposing two major fates, mitotic slippage, or mitotic cell death. While the former is molecularly well characterized, the mechanisms that control mitotic cell death remain poorly understood. Here, we performed quantitative proteomics of HeLa cells under mitotic arrest induced with paclitaxel, a microtubule-stabilizer drug, to identify regulators of such cell fate decision. We identified alterations in several apoptosis-related proteins, among which the mitochondrial fission protein Drp1 presented increased levels. We found that Drp1 depletion during prolonged mitotic arrest led to strong mitochondrial depolarization and faster mitotic cell death as well as enhanced mitophagy, a mechanism to remove damaged mitochondria. Our findings support a new role of Drp1 in orchestrating the cellular stress responses during mitosis, where mitochondrial function and distribution into the daughter cells need to be coordinated with cell fate. This novel function of Drp1 in the cell cycle becomes best visible under conditions of prolonged mitotic arrest.

The aim of this study was to characterize the ophthalmic and genetic features of Bardet Biedl (BBS) syndrome in a cohort of patients from a German specialized ophthalmic care center. Sixty-one patients, aged 5–56 years, underwent a detailed ophthalmic examination including visual acuity and color vision testing, electroretinography (ERG), visually evoked potential recording (VEP), fundus examination, and spectral domain optical coherence tomography (SD-OCT). Adaptive optics flood illumination ophthalmoscopy was performed in five patients. All patients had received diagnostic genetic testing and were selected upon the presence of apparent biallelic variants in known BBS-associated genes. All patients had retinal dystrophy with morphologic changes of the retina. Visual acuity decreased from ~0.2 (decimal) at age 5 to blindness 0 at 50 years. Visual field examination could be performed in only half of the patients and showed a concentric constriction with remaining islands of function in the periphery. ERG recordings were mostly extinguished whereas VEP recordings were reduced in about half of the patients. The cohort of patients showed 51 different likely biallelic mutations—of which 11 are novel—in 12 different BBS-associated genes. The most common associated genes were BBS10 (32.8%) and BBS1 (24.6%), and by far the most commonly observed variants were BBS10 c.271dup;p.C91Lfs*5 (21 alleles) and BBS1 c.1169T>G;p.M390R (18 alleles). The phenotype associated with the different BBS-associated genes and genotypes in our cohort is heterogeneous, with diverse features without genotype–phenotype correlation. The results confirm and expand our knowledge of this rare disease.

The aim of this study was to investigate coenzyme Q10 (CoQ10) biosynthesis pathway defects in inherited retinal dystrophy. Individuals affected by inherited retinal dystrophy (IRD) underwent exome or genome sequencing for molecular diagnosis of their condition. Following negative IRD gene panel analysis, patients carrying biallelic variants in CoQ10 biosynthesis pathway genes were identified. Clinical data were collected from the medical records. Haplotypes harbouring the same missense variant were characterised from family genome sequencing (GS) data and direct Sanger sequencing. Candidate splice variants were characterised using Oxford Nanopore Technologies single molecule sequencing. The CoQ10 status of the human plasma was determined in some of the study patients. 13 individuals from 12 unrelated families harboured candidate pathogenic genotypes in the genes: PDSS1, COQ2, COQ4 and COQ5. The PDSS1 variant c.589 A > G was identified in three affected individuals from three unrelated families on a possible ancestral haplotype. Three variants (PDSS1 c.468-25 A > G, PDSS1 c.722-2 A > G, COQ5 c.682-7 T > G) were shown to lead to cryptic splicing. 6 affected individuals were diagnosed with non-syndromic retinitis pigmentosa and 7 had additional clinical findings. This study provides evidence of CoQ10 biosynthesis pathway gene defects leading to non-syndromic retinitis pigmentosa in some cases. Intronic variants outside of the canonical splice-sites represent an important cause of disease. RT-PCR nanopore sequencing is effective in characterising these splice defects.