Assessing gene function in human B cells: CRISPR/Cas9-based gene editing and mRNA-based gene expression in healthy and tumor cells.

Robust methods for manipulation of human B cells, isolated from healthy donors and patients with B cell disorders, has the potential to significantly accelerate B cell research. Our work describes a step-by-step protocol to perform electroporation-based screening of gene function in B cells through the use of Cas9 ribonuclecomplexes and in vitro produced mRNA.

miR-218 Inhibits Mitochondrial Clearance by Targeting PRKN E3 Ubiquitin Ligase.

The selective elimination of dysfunctional mitochondria through mitophagy is crucial for preserving mitochondrial quality and cellular homeostasis. The most described mitophagy pathway is regulated by a positive ubiquitylation feedback loop in which the PINK1 (PTEN induced kinase 1) kinase phosphorylates both ubiquitin and the E3 ubiquitin ligase PRKN (Parkin RBR E3 ubiquitin ligase), also known as PARKIN. This event recruits PRKN to the mitochondria, thus amplifying ubiquitylation signal. Here we report that miR-218 targets PRKN and negatively regulates PINK1/PRKN-mediated mitophagy. Overexpression of miR-218 reduces PRKN mRNA levels, thus also reducing protein content and deregulating the E3 ubiquitin ligase action. In fact, following miR-218 overexpression, mitochondria result less ubiquitylated and the autophagy machinery fails to proceed with correct mitochondrial clearance. Since mitophagy defects are associated with various human diseases, these results qualify miR-218 as a promising therapeutic target for human diseases.

A protective variant of the autophagy receptor CALCOCO2/NDP52 in Multiple Sclerosis (MS).

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, which has been found associated with dysfunctional mitochondria. In order to advance our understanding of the complex molecular mechanisms underlying this disease, we analyzed mitophagy, a process fundamental for the elimination of damaged mitochondria through the autophagic process, in peripheral blood mononuclear cells (PBMCs) of MS patients. Through a genetic analysis carried out on 203 MS patients and 1000 healthy controls, we identified a natural variant of CALCOCO2/NDP52, a well-known autophagic receptor, associated with and protective in MS. Structural modeling of the CALCOCO2 variant and functional studies highlighted an amino acid substitution (G140E) located near the LC3-interacting region (LIR) motif of CALCOCO2, crucial in controlling mitophagy. In addition, we found that among PBMCs, CALCOCO2 is mainly expressed in B cells and, by mediating mitophagy, it reduces pro-inflammatory cytokine production following stimulation of these cells. Here we summarize these recent findings, discuss the putative protective roles of CALCOCO2 in B cells and its novel association with an autoimmune disease such as MS.Abbreviations: LIR: LC3-interacting region; MS: multiple sclerosis; PBMC: peripheral blood mononuclear cells; RR-MS: relapsing-remitting MS; TLR: toll like receptor.

Characterization of a natural variant of human NDP52 and its functional consequences on mitophagy.

The role of mitophagy, a process that allows the removal of damaged mitochondria from cells, remains unknown in multiple sclerosis (MS), a disease that is found associated with dysfunctional mitochondria. Here we have qualitatively and quantitatively studied the main players in PINK1-mediated mitophagy in peripheral blood mononuclear cells (PBMCs) of patients with relapsing-remitting MS. We found the variant c.491G>A (rs550510, p.G140E) of NDP52, one of the major mitophagy receptor genes, associated with a MS cohort. Through the characterization of this variant, we discovered that the residue 140 of human NDP52 is a crucial modulator of NDP52/LC3C binding, promoting the formation of autophagosomes in order to drive efficient mitophagy. In addition, we found that in the PBMC population, NDP52 is mainly expressed in B cells and by ensuring efficient mitophagy, it is able to limit the production of the proinflammatory cytokine TNF-α following cell stimulation. In sum, our results contribute to a better understanding of the role of NDP52 in mitophagy and underline, for the first time, a possible role of NDP52 in MS.

HUWE1 controls MCL1 stability to unleash AMBRA1-induced mitophagy.

Receptor-mediated mitophagy is a crucial process involved in mitochondria quality control. AMBRA1 is a mitophagy receptor for the selective removal of damaged mitochondria in mammalian cells. A critical unresolved issue is how AMBRA1-mediated mitophagy is controlled in response to cellular stress. Here, we investigated the role of BCL2-family proteins on AMBRA1-dependent mitophagy and showed that MCL1 delays AMBRA1-dependent mitophagy. Indeed, MCL1 overexpression is sufficient to inhibit recruitment to mitochondria of the E3 Ubiquitin ligase HUWE1, a crucial dynamic partner of AMBRA1, upon AMBRA1-mediated mitophagy induction. In addition, we found that during mitophagy induced by AMBRA1, MCL1 levels decreased but were sustained by inhibition of the GSK-3ß kinase, which delayed AMBRA1-mediated mitophagy. Also, we showed that MCL1 was phosphorylated by GSK-3ß at a conserved GSK-3 phosphorylation site (S159) during AMBRA1-mediated mitophagy and that this event was accompanied by HUWE1-dependent MCL1 degradation. Altogether, our results demonstrate that MCL1 stability is regulated by the kinase GSK-3ß and the E3 ubiquitin ligase HUWE1 in regulating AMBRA1-mediated mitophagy. Our work thus defines MCL1 as an upstream stress-sensitive protein, functional in AMBRA1-mediated mitophagy.

Correction to: HUWE1 controls MCL1 stability to unleash AMBRA1-induced mitophagy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction: JNK1 and ERK1/2 modulate lymphocyte homeostasis via BIM and DRP1 upon AICD induction.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

JNK1 and ERK1/2 modulate lymphocyte homeostasis via BIM and DRP1 upon AICD induction.

The Activation-Induced Cell Death (AICD) is a stimulation-dependent form of apoptosis used by the organism to shutdown T-cell response once the source of inflammation has been eliminated, while allowing the generation of immune memory. AICD is thought to progress through the activation of the extrinsic Fas/FasL pathway of cell death, leading to cytochrome-C release through caspase-8 and Bid activation. We recently described that, early upon AICD induction, mitochondria undergo structural alterations, which are required to promote cytochrome-C release and execute cell death. Here, we found that such alterations do not depend on the Fas/FasL pathway, which is instead only lately activated to amplify the cell death cascade. Instead, such alterations are primarily dependent on the MAPK proteins JNK1 and ERK1/2, which, in turn, regulate the activity of the pro-fission protein Drp1 and the pro-apoptotic factor Bim. The latter regulates cristae disassembly and cooperate with Drp1 to mediate the Mitochondrial Outer Membrane Permeabilization (MOMP), leading to cytochrome-C release. Interestingly, we found that Bim is also downregulated in T-cell Acute Lymphoblastic Leukemia (T-ALL) cells, this alteration favouring their escape from AICD-mediated control.

Mitophagy could fight Parkinson's disease through antioxidant action.

During aging, the process of mitophagy, a system that allows the removal of dysfunctional mitochondria through lysosomal degradation, starts to malfunction. Because of this defect, damaged mitochondria are not removed correctly, and their decomposing components accumulate inside the cells. Dysfunctional mitochondria that are not removed by mitophagy produce high amounts of reactive oxygen species (ROS) and, thus, cause oxidative stress. Oxidative stress, in turn, is very harmful for the cells, neuronal cells, in particular. Consequently, the process of mitophagy plays a crucial role in mitochondria-related disease. Mitochondrial dysfunctions and oxidative stress are well-established factors contributing to Parkinson's disease (PD), one of the most common neurodegenerative disorders. In this review, we report various known antioxidants for PD treatments and describe the stimulation of mitophagy process as a novel and exciting method for reducing oxidative stress in PD patients. We describe the different mechanisms responsible for mitochondria removal through the mitophagy process. In addition, we review the functional connection between mitophagy induction and reduction of oxidative stress in several in vitro models of PD and also agents (drugs and natural compounds) already known to be antioxidants and to be able to activate mitophagy. Finally, we propose that there is an urgent need to test the use of mitophagy-inducing antioxidants in order to fight PD.

Reversible induction of mitophagy by an optogenetic bimodular system.

Autophagy-mediated degradation of mitochondria (mitophagy) is a key process in cellular quality control. Although mitophagy impairment is involved in several patho-physiological conditions, valuable methods to induce mitophagy with low toxicity in vivo are still lacking. Herein, we describe a new optogenetic tool to stimulate mitophagy, based on light-dependent recruitment of pro-autophagy protein AMBRA1 to mitochondrial surface. Upon illumination, AMBRA1-RFP-sspB is efficiently relocated from the cytosol to mitochondria, where it reversibly mediates mito-aggresome formation and reduction of mitochondrial mass. Finally, as a proof of concept of the biomedical relevance of this method, we induced mitophagy in an in vitro model of neurotoxicity, fully preventing cell death, as well as in human T lymphocytes and in zebrafish in vivo. Given the unique features of this tool, we think it may turn out to be very useful for a wide range of both therapeutic and research applications.

AMBRA1-Mediated Mitophagy Counteracts Oxidative Stress and Apoptosis Induced by Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells.

Therapeutic strategies are needed to protect dopaminergic neurons in Parkinson's disease (PD) patients. Oxidative stress caused by dopamine may play an important role in PD pathogenesis. Selective autophagy of mitochondria (mitophagy), mainly regulated by PINK1 and PARKIN, plays an important role in the maintenance of cell homeostasis. Mutations in those genes cause accumulation of damaged mitochondria, leading to nigral degeneration and early-onset PD. AMBRA1ActA is a fusion protein specifically expressed at the mitochondria, and whose expression has been shown to induce a powerful mitophagy in mammalian cells. Most importantly, the pro-autophagy factor AMBRA1 is sufficient to restore mitophagy in fibroblasts of PD patients carrying PINK1 and PARKIN mutations. In this study, we investigated the potential neuroprotective effect of AMBRA1-induced mitophagy against 6-hydroxydopamine (6-OHDA)- and rotenone-induced cell death in human neuroblastoma SH-SY5Y cells. We demonstrated that AMBRA1ActA overexpression was sufficient to induce mitochondrial clearance in SH-SY5Y cells. We found that apoptosis induced by 6-OHDA and rotenone was reversed by AMBRA1-induced mitophagy. Finally, transfection of SH-SY5Y cells with a vector encoding AMBRA1ActA significantly reduced 6-OHDA and rotenone-induced generation of reactive oxygen species (ROS). Altogether, our results indicate that AMBRA1ActA is able to induce mitophagy in SH-SY5Y cells in order to suppress oxidative stress and apoptosis induced by both 6-OHDA and rotenone. These results strongly suggest that AMBRA1 may have promising neuroprotective properties with an important role in limiting ROS-induced dopaminergic cell death, and the utmost potential to prevent PD or other neurodegenerative diseases associated with mitochondrial oxidative stress.

HUWE1 E3 ligase promotes PINK1/PARKIN-independent mitophagy by regulating AMBRA1 activation via IKKα.

The selective removal of undesired or damaged mitochondria by autophagy, known as mitophagy, is crucial for cellular homoeostasis, and prevents tumour diffusion, neurodegeneration and ageing. The pro-autophagic molecule AMBRA1 (autophagy/beclin-1 regulator-1) has been defined as a novel regulator of mitophagy in both PINK1/PARKIN-dependent and -independent systems. Here, we identified the E3 ubiquitin ligase HUWE1 as a key inducing factor in AMBRA1-mediated mitophagy, a process that takes place independently of the main mitophagy receptors. Furthermore, we show that mitophagy function of AMBRA1 is post-translationally controlled, upon HUWE1 activity, by a positive phosphorylation on its serine 1014. This modification is mediated by the IKKα kinase and induces structural changes in AMBRA1, thus promoting its interaction with LC3/GABARAP (mATG8) proteins and its mitophagic activity. Altogether, these results demonstrate that AMBRA1 regulates mitophagy through a novel pathway, in which HUWE1 and IKKα are key factors, shedding new lights on the regulation of mitochondrial quality control and homoeostasis in mammalian cells.

Prosurvival AMBRA1 turns into a proapoptotic BH3-like protein during mitochondrial apoptosis.

Autophagy and apoptosis are 2 stress-response mechanisms that are closely interconnected. However, the molecular interplays between these 2 pathways remain to be clarified. Here we report that the crucial proautophagic factor AMBRA1 can act as a positive mediator of mitochondrial apoptosis. Indeed, we show that, in a proapoptotic positive feedback loop, the C-terminal part of AMBRA1, generated by CASP/CASPASE cleavage upon apoptosis induction, inhibits the antiapoptotic factor BCL2 by a direct binding through its BH3-like domain. The mitochondrial AMBRA1-BCL2 complex is thus at the crossroad between autophagy and cell death and may represent a novel target in development of therapeutic approaches in clinical diseases.