Integrating the response to stressed mitochondria.

Chakrabarty et al.1 demonstrate that phospho-EIF2α (pEIF2α), the translation initiation factor that mediates the integrated stress response (ISR), is necessary and sufficient for the autophagic degradation of mitochondria following the addition of mitochondrial stressors.

STING induces LUBAC-mediated synthesis of linear ubiquitin chains to stimulate innate immune signaling.

STING activation by cyclic dinucleotides in mammals induces interferon- and NFκB -related gene expression, and the lipidation of LC3B at Golgi membranes. While mechanisms of the interferon response are well understood, the mechanisms of NFκB activation mediated by STING remain unclear. We report that STING activation induces K63- and M1-linked/linear ubiquitin chain formation at LC3B-associated Golgi membranes. Loss of the LUBAC E3 ubiquitin ligase prevents formation of linear, but not K63-linked ubiquitin chains or STING activation and inhibits STING-induced NFκB and IRF3-mediated signaling in monocytic THP1 cells. The proton channel activity of STING is also important for both K63 and linear ubiquitin chain formation, and NFκB- and interferon-related gene expression. Thus, LUBAC synthesis of linear ubiquitin chains regulates STING-mediated innate immune signaling.

Nix interacts with WIPI2 to induce mitophagy.

Nix is a membrane-anchored outer mitochondrial protein that induces mitophagy. While Nix has an LC3-interacting (LIR) motif that binds to ATG8 proteins, it also contains a minimal essential region (MER) that induces mitophagy through an unknown mechanism. We used chemically induced dimerization (CID) to probe the mechanism of Nix-mediated mitophagy and found that both the LIR and MER are required for robust mitophagy. We find that the Nix MER interacts with the autophagy effector WIPI2 and recruits WIPI2 to mitochondria. The Nix LIR motif is also required for robust mitophagy and converts a homogeneous WIPI2 distribution on the surface of the mitochondria into puncta, even in the absence of ATG8s. Together, this work reveals unanticipated mechanisms in Nix-induced mitophagy and the elusive role of the MER, while also describing an interesting example of autophagy induction that acts downstream of the canonical initiation complexes.

Loss of STING in parkin mutant flies suppresses muscle defects and mitochondria damage.

The early pathogenesis and underlying molecular causes of motor neuron degeneration in Parkinson's Disease (PD) remains unresolved. In the model organism Drosophila melanogaster, loss of the early-onset PD gene parkin (the ortholog of human PRKN) results in impaired climbing ability, damage to the indirect flight muscles, and mitochondrial fragmentation with swelling. These stressed mitochondria have been proposed to activate innate immune pathways through release of damage associated molecular patterns (DAMPs). Parkin-mediated mitophagy is hypothesized to suppress mitochondrial damage and subsequent activation of the cGAS/STING innate immunity pathway, but the relevance of this interaction in the fly remains unresolved. Using a combination of genetics, immunoassays, and RNA sequencing, we investigated a potential role for STING in the onset of parkin-null phenotypes. Our findings demonstrate that loss of Drosophila STING in flies rescues the thorax muscle defects and the climbing ability of parkin-/- mutants. Loss of STING also suppresses the disrupted mitochondrial morphology in parkin-/- flight muscles, suggesting unexpected feedback of STING on mitochondria integrity or activation of a compensatory mitochondrial pathway. In the animals lacking both parkin and sting, PINK1 is activated and cell death pathways are suppressed. These findings support a unique, non-canonical role for Drosophila STING in the cellular and organismal response to mitochondria stress.

TNIP1 inhibits selective autophagy via bipartite interaction with LC3/GABARAP and TAX1BP1.

Mitophagy is a form of selective autophagy that disposes of superfluous and potentially damage-inducing organelles in a tightly controlled manner. While the machinery involved in mitophagy induction is well known, the regulation of the components is less clear. Here, we demonstrate that TNIP1 knockout in HeLa cells accelerates mitophagy rates and that ectopic TNIP1 negatively regulates the rate of mitophagy. These functions of TNIP1 depend on an evolutionarily conserved LIR motif as well as an AHD3 domain, which are required for binding to the LC3/GABARAP family of proteins and the autophagy receptor TAX1BP1, respectively. We further show that phosphorylation appears to regulate its association with the ULK1 complex member FIP200, allowing TNIP1 to compete with autophagy receptors, which provides a molecular rationale for its inhibitory function during mitophagy. Taken together, our findings describe TNIP1 as a negative regulator of mitophagy that acts at the early steps of autophagosome biogenesis.

The mechanisms and roles of selective autophagy in mammals.

Autophagy is a process that targets various intracellular elements for degradation. Autophagy can be non-selective - associated with the indiscriminate engulfment of cytosolic components - occurring in response to nutrient starvation and is commonly referred to as bulk autophagy. By contrast, selective autophagy degrades specific targets, such as damaged organelles (mitophagy, lysophagy, ER-phagy, ribophagy), aggregated proteins (aggrephagy) or invading bacteria (xenophagy), thereby being importantly involved in cellular quality control. Hence, not surprisingly, aberrant selective autophagy has been associated with various human pathologies, prominently including neurodegeneration and infection. In recent years, considerable progress has been made in understanding mechanisms governing selective cargo engulfment in mammals, including the identification of ubiquitin-dependent selective autophagy receptors such as p62, NBR1, OPTN and NDP52, which can bind cargo and ubiquitin simultaneously to initiate pathways leading to autophagy initiation and membrane recruitment. This progress opens the prospects for enhancing selective autophagy pathways to boost cellular quality control capabilities and alleviate pathology.

VPS13D promotes peroxisome biogenesis.

The VPS13 gene family consists of VPS13A-D in mammals. Although all four genes have been linked to human diseases, their cellular functions are poorly understood, particularly those of VPS13D. We generated and characterized knockouts of each VPS13 gene in HeLa cells. Among the individual knockouts, only VPS13D-KO cells exhibit abnormal mitochondrial morphology. Additionally, VPS13D loss leads to either partial or complete peroxisome loss in several transformed cell lines and in fibroblasts derived from a VPS13D mutation-carrying patient with recessive spinocerebellar ataxia. Our data show that VPS13D regulates peroxisome biogenesis.

Image-based pooled whole-genome CRISPRi screening for subcellular phenotypes.

Genome-wide CRISPR screens have transformed our ability to systematically interrogate human gene function, but are currently limited to a subset of cellular phenotypes. We report a novel pooled screening approach for a wider range of cellular and subtle subcellular phenotypes. Machine learning and convolutional neural network models are trained on the subcellular phenotype to be queried. Genome-wide screening then utilizes cells stably expressing dCas9-KRAB (CRISPRi), photoactivatable fluorescent protein (PA-mCherry), and a lentiviral guide RNA (gRNA) pool. Cells are screened by using microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated and isolated via flow cytometry, and the gRNAs are identified by sequencing. A proof-of-concept screen accurately identified PINK1 as essential for Parkin recruitment to mitochondria. A genome-wide screen identified factors mediating TFEB relocation from the nucleus to the cytosol upon prolonged starvation. Twenty-one of the 64 hits called by the neural network model were independently validated, revealing new effectors of TFEB subcellular localization. This approach, AI-photoswitchable screening (AI-PS), offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale.

Ubiquitin signaling in neurodegenerative diseases: an autophagy and proteasome perspective.

Ubiquitin signaling is a sequence of events driving the fate of a protein based on the type of ubiquitin modifications attached. In the case of neurodegenerative diseases, ubiquitin signaling is mainly associated with degradation signals to process aberrant proteins, which form aggregates often fatal for the brain cells. This signaling is often perturbed by the aggregates themselves and leads to the accumulation of toxic aggregates and inclusion bodies that are deleterious due to a toxic gain of function. Decrease in quality control pathways is often seen with age and is a critical onset for the development of neurodegeneration. Many aggregates are now thought to propagate in a prion-like manner, where mutated proteins acting like seeds are transitioning from cell to cell, converting normal proteins to toxic aggregates. Modulation of ubiquitin signaling, by stimulating ubiquitin ligase activation, is a potential therapeutic strategy to treat patients with neurodegeneration diseases.

Loss of TAX1BP1-Directed Autophagy Results in Protein Aggregate Accumulation in the Brain.

Protein aggregates disrupt cellular homeostasis, causing toxicity linked to neurodegeneration. Selective autophagic elimination of aggregates is critical to protein quality control, but how aggregates are selectively targeted for degradation is unclear. We compared the requirements for autophagy receptor proteins: OPTN, NBR1, p62, NDP52, and TAX1BP1 in clearance of proteotoxic aggregates. Endogenous TAX1BP1 is recruited to and required for the clearance of stress-induced aggregates, whereas ectopic expression of TAX1BP1 increases clearance through autophagy, promoting viability of human induced pluripotent stem cell-derived neurons. In contrast, TAX1BP1 depletion sensitizes cells to several forms of aggregate-induced proteotoxicity. Furthermore, TAX1BP1 is more specifically expressed in the brain compared to other autophagy receptor proteins. In vivo, loss of TAX1BP1 results in accumulation of high molecular weight ubiquitin conjugates and premature lipofuscin accumulation in brains of young TAX1BP1 knockout mice. TAX1BP1 mediates clearance of a broad range of cytotoxic proteins indicating therapeutic potential in neurodegenerative diseases.

STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain.

Following the detection of cytosolic double-stranded DNA from viral or bacterial infection in mammalian cells, cyclic dinucleotide activation of STING induces interferon ß expression to initiate innate immune defenses. STING activation also induces LC3B lipidation, a classical but equivocal marker of autophagy, that promotes a cell-autonomous antiviral response that arose before evolution of the interferon pathway. We report that STING activation induces LC3B lipidation onto single-membrane perinuclear vesicles mediated by ATG16L1 via its WD40 domain, bypassing the requirement of canonical upstream autophagy machinery. This process is blocked by bafilomycin A1 that binds and inhibits the vacuolar ATPase (V-ATPase) and by SopF, a bacterial effector that catalytically modifies the V-ATPase to inhibit LC3B lipidation via ATG16L1. These results indicate that activation of the cGAS-STING pathway induces V-ATPase-dependent LC3B lipidation that may mediate cell-autonomous host defense, an unanticipated mechanism that is distinct from LC3B lipidation onto double-membrane autophagosomes.

Mitochondrial Quality Control and Restraining Innate Immunity.

Maintaining mitochondrial health is essential for the survival and function of eukaryotic organisms. Misfunctioning mitochondria activate stress-responsive pathways to restore mitochondrial network homeostasis, remove damaged or toxic proteins, and eliminate damaged organelles via selective autophagy of mitochondria, a process termed mitophagy. Failure of these quality control pathways is implicated in the pathogenesis of Parkinson's disease and other neurodegenerative diseases. Impairment of mitochondrial quality control has been demonstrated to activate innate immune pathways, including inflammasome-mediated signaling and the antiviral cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING)-regulated interferon response. Immune system malfunction is a common hallmark in many neurodegenerative diseases; however, whether inflammation suppresses or exacerbates disease pathology is still unclear. The goal of this review is to provide a historical overview of the field, describe mechanisms of mitochondrial quality control, and highlight recent advances on the emerging role of mitochondria in innate immunity and inflammation.

Mitochondrial damage-associated inflammation highlights biomarkers in PRKN/PINK1 parkinsonism.

There is increasing evidence for a role of inflammation in Parkinson's disease. Recent research in murine models suggests that parkin and PINK1 deficiency leads to impaired mitophagy, which causes the release of mitochondrial DNA (mtDNA), thereby triggering inflammation. Specifically, the CGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) pathway mitigates activation of the innate immune system, quantifiable as increased interleukin-6 (IL6) levels. However, the role of IL6 and circulating cell-free mtDNA in unaffected and affected individuals harbouring mutations in PRKN/PINK1 and idiopathic Parkinson's disease patients remain elusive. We investigated IL6, C-reactive protein, and circulating cell-free mtDNA in serum of 245 participants in two cohorts from tertiary movement disorder centres. We performed a hypothesis-driven rank-based statistical approach adjusting for multiple testing. We detected (i) elevated IL6 levels in patients with biallelic PRKN/PINK1 mutations compared to healthy control subjects in a German cohort, supporting the concept of a role for inflammation in PRKN/PINK1-linked Parkinson's disease. In addition, the comparison of patients with biallelic and heterozygous mutations in PRKN/PINK1 suggests a gene dosage effect. The differences in IL6 levels were validated in a second independent Italian cohort; (ii) a correlation between IL6 levels and disease duration in carriers of PRKN/PINK1 mutations, while no such association was observed for idiopathic Parkinson's disease patients. These results highlight the potential of IL6 as progression marker in Parkinson's disease due to PRKN/PINK1 mutations; (iii) increased circulating cell-free mtDNA serum levels in both patients with biallelic or with heterozygous PRKN/PINK1 mutations compared to idiopathic Parkinson's disease, which is in line with previous findings in murine models. By contrast, circulating cell-free mtDNA concentrations in unaffected heterozygous carriers of PRKN/PINK1 mutations were comparable to control levels; and (iv) that circulating cell-free mtDNA levels have good predictive potential to discriminate between idiopathic Parkinson's disease and Parkinson's disease linked to heterozygous PRKN/PINK1 mutations, providing functional evidence for a role of heterozygous mutations in PRKN or PINK1 as Parkinson's disease risk factor. Taken together, our study further implicates inflammation due to impaired mitophagy and subsequent mtDNA release in the pathogenesis of PRKN/PINK1-linked Parkinson's disease. In individuals carrying mutations in PRKN/PINK1, IL6 and circulating cell-free mtDNA levels may serve as markers of Parkinson's disease state and progression, respectively. Finally, our study suggests that targeting the immune system with anti-inflammatory medication holds the potential to influence the disease course of Parkinson's disease, at least in this subset of patients.

Two different axes CALCOCO2-RB1CC1 and OPTN-ATG9A initiate PRKN-mediated mitophagy.

PINK1 and PRKN, proteins mutated in Parkinson disease, selectively amplify ubiquitin signals on damaged mitochondria for elimination via mitophagy. Because all five macroautophagy/autophagy receptors in mammals possess domains binding to ubiquitin and Atg8-family proteins, they were thought to recruit Atg8-family protein labeled phagophores from a cytosolic pool. However, our recent findings show that, in addition to Atg8-family protein binding, two of the receptors CALCOCO2 and OPTN interact with RB1CC1 and ATG9A, respectively, indicating that two different axes, CALCOCO2-RB1CC1 and OPTN-ATG9A, can initiate de novo biogenesis of autophagic membranes on ubiquitin-coated damaged mitochondria. These results explain the critical roles of the autophagy receptors CALCOCO2 and OPTN in mitochondrial degradation, and their abilities to simultaneously bind multiple autophagy core proteins propose a new function, i.e. a scaffold to build multivalent interactions for the orchestrated assembly of autophagy proteins near the ubiquitinated cargo. ABBREVIATIONS: ATG: autophagy-related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CRABP2: cellular retinoic acid binding protein 2; LIR: MAP1LC3/LC3-interacting region; MAP1LC3: microtubule associated protein 1 light chain 3; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SNIPER: specific and nongenetic IAP-dependent protein eraser; SQSTM1/p62: sequestosome 1; ULK: unc-51 like autophagy activating kinase.

ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer.

The autophagy-initiating human ULK complex consists of the kinase ULK1/2, FIP200, ATG13, and ATG101. Hydrogen-deuterium exchange mass spectrometry was used to map their mutual interactions. The N-terminal 640 residues (NTD) of FIP200 interact with the C-terminal IDR of ATG13. Mutations in these regions abolish their interaction. Negative stain EM and multiangle light scattering showed that FIP200 is a dimer, while a single molecule each of the other subunits is present. The FIP200NTD is flexible in the absence of ATG13, but in its presence adopts the shape of the letter C ∼20 nm across. The ULK1 EAT domain interacts loosely with the NTD dimer, while the ATG13:ATG101 HORMA dimer does not contact the NTD. Cryo-EM of the NTD dimer revealed a structural similarity to the scaffold domain of TBK1, suggesting an evolutionary similarity between the autophagy-initiating TBK1 kinase and the ULK1 kinase complex.

PINK1/Parkin Influences Cell Cycle by Sequestering TBK1 at Damaged Mitochondria, Inhibiting Mitosis.

PINK1 and Parkin are established mediators of mitophagy, the selective removal of damaged mitochondria by autophagy. PINK1 and Parkin have been proposed to act as tumor suppressors, as loss-of-function mutations are correlated with enhanced tumorigenesis. However, it is unclear how PINK1 and Parkin act in coordination during mitophagy to influence the cell cycle. Here we show that PINK1 and Parkin genetically interact with proteins involved in cell cycle regulation, and loss of PINK1 and Parkin accelerates cell growth. PINK1- and Parkin-mediated activation of TBK1 at the mitochondria during mitophagy leads to a block in mitosis due to the sequestration of TBK1 from its physiological role at centrosomes during mitosis. Our study supports a diverse role for the far-reaching, regulatory effects of mitochondrial quality control in cellular homeostasis and demonstrates that the PINK1/Parkin pathway genetically interacts with the cell cycle, providing a framework for understanding the molecular basis linking PINK1 and Parkin to mitosis.

Mitochondria-Striking a balance between host and endosymbiont.

Mitochondria are organelles with their own genome that arose from α-proteobacteria living within single-celled Archaea more than a billion years ago. This step of endosymbiosis offered tremendous opportunities for energy production and metabolism and allowed the evolution of fungi, plants, and animals. However, less appreciated are the downsides of this endosymbiosis. Coordinating gene expression between the mitochondrial genomes and the nuclear genome is imprecise and can lead to proteotoxic stress. The clonal reproduction of mitochondrial DNA requires workarounds to avoid mutational meltdown. In metazoans that developed innate immune pathways to thwart bacterial and viral infections, mitochondrial components can cross-react with pathogen sensors and invoke inflammation. Here, I focus on the numerous and elegant quality control processes that compensate for or mitigate these challenges of endosymbiosis.

Neurolastin, a dynamin family GTPase, translocates to mitochondria upon neuronal stress and alters mitochondrial morphology in vivo.

Neurolastin is a dynamin family GTPase that also contains a RING domain and exhibits both GTPase and E3 ligase activities. It is specifically expressed in the brain and is important for synaptic transmission, as neurolastin knockout animals have fewer dendritic spines and exhibit a reduction in functional synapses. Our initial study of neurolastin revealed that it is membrane-associated and partially co-localizes with endosomes. Using various biochemical and cell-culture approaches, we now show that neurolastin also localizes to mitochondria in HeLa cells, cultured neurons, and brain tissue. We found that the mitochondrial localization of neurolastin depends upon an N-terminal mitochondrial targeting sequence and that neurolastin is imported into the mitochondrial intermembrane space. Although neurolastin was only partially mitochondrially localized at steady state, it displayed increased translocation to mitochondria in response to neuronal stress and mitochondrial fragmentation. Interestingly, inactivation or deletion of neurolastin's RING domain also increased its mitochondrial localization. Using EM, we observed that neurolastin knockout animals have smaller but more numerous mitochondria in cerebellar Purkinje neurons, indicating that neurolastin regulates mitochondrial morphology. We conclude that the brain-specific dynamin GTPase neurolastin exhibits stress-responsive localization to mitochondria and is required for proper mitochondrial morphology.

Spatiotemporal Control of ULK1 Activation by NDP52 and TBK1 during Selective Autophagy.

Selective autophagy recycles damaged organelles and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization (CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis. We demonstrate that ectopic placement of NDP52 on mitochondria or peroxisomes is sufficient to initiate selective autophagy by focally localizing and activating the ULK1 complex. The capability of NDP52 to induce mitophagy is dependent on its interaction with the FIP200/ULK1 complex, which is facilitated by TBK1. Ectopically tethering ULK1 to cargo bypasses the requirement for autophagy receptors and TBK1. Focal activation of ULK1 occurs independently of AMPK and mTOR. Our findings provide a parsimonious model of selective autophagy, which highlights the coordination of ULK1 complex localization by autophagy receptors and TBK1 as principal drivers of targeted autophagosome biogenesis.