Nicholas Ktistakis

Autophagy in major human diseases.
Klionsky DJ, Petroni G, Amaravadi RK, Baehrecke EH, Ballabio A, Boya P, Bravo-San Pedro JM, Cadwell K, Cecconi F, Choi AMK, Choi ME, Chu CT, Codogno P, Colombo MI, Cuervo AM, Deretic V, Dikic I, Elazar Z, Eskelinen EL, Fimia GM, Gewirtz DA, Green DR, Hansen M, Jäättelä M, Johansen T, Juhász G, Karantza V, Kraft C, Kroemer G, Ktistakis NT, Kumar S, Lopez-Otin C, Macleod KF, Madeo F, Martinez J, Meléndez A, Mizushima N, Münz C, Penninger JM, Perera RM, Piacentini M, Reggiori F, Rubinsztein DC, Ryan KM, Sadoshima J, Santambrogio L, Scorrano L, Simon HU, Simon AK, Simonsen A, Stolz A, Tavernarakis N, Tooze SA, Yoshimori T, Yuan J, Yue Z, Zhong Q, Galluzzi L, Pietrocola F

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

The EMBO journal, 1, 1, 30 Aug 2021

Inhibition of the SEC61 translocon by mycolactone induces a protective autophagic response controlled by EIF2S1-dependent translation that does not require ULK1 activity.
Hall BS, Dos Santos SJ, Hsieh LT, Manifava M, Ruf MT, Pluschke G, Ktistakis N, Simmonds RE

The exotoxin, mycolactone, is responsible for the immunosuppression and tissue necrosis that characterizes Buruli ulcer. Mycolactone inhibits SEC61-dependent co-translational translocation of proteins into the endoplasmic reticulum and the resultant cytosolic translation triggers degradation of mislocalized proteins by the ubiquitin-proteasome system. Inhibition of SEC61 by mycolactone also activates multiple EIF2S1/eIF2α kinases in the integrated stress response (ISR). Here we show mycolactone increased canonical markers of selective macroautophagy/autophagy LC3B-II, ubiquitin and SQSTM1/p62 in diverse disease-relevant primary cells and cell lines. Increased formation of puncta positive for the early autophagy markers WIPI2, RB1CC1/FIP200 and ATG16L1 indicates increased initiation of autophagy. The mycolactone response was SEC61A1-dependent and involved a pathway that required RB1CC1 but not ULK. Deletion of reduced cell survival in the presence of mycolactone, suggesting this response protects against the increased cytosolic protein burden caused by the toxin. However, reconstitution of baseline SQSTM1 expression in cells lacking all autophagy receptor proteins could not rescue viability. Translational regulation by EIF2S1 in the ISR plays a key role in the autophagic response to mycolactone. Mycolactone-dependent induction of SQSTM1 was reduced in cells while the p-EIF2S1 antagonist ISRIB reversed the upregulation of SQSTM1 and reduced RB1CC1, WIPI2 and LC3B puncta formation. Increased SQSTM1 staining could be seen in Buruli ulcer patient skin biopsy samples, reinforcing genetic data that suggests autophagy is relevant to disease pathology. Since selective autophagy and the ISR are both implicated in neurodegeneration, cancer and inflammation, the pathway uncovered here may have a broad relevance to human disease. ATF4: activating transcription factor 4; ATG: autophagy related; BAF: bafilomycin A; ATG16L1: autophagy related 16 like 1; BU: Buruli ulcer; CQ: chloroquine; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; CALCOCO2: calcium binding and coiled-coil domain 2; DMSO: dimethyl sulfoxide; EIF2S1: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; GFP: green fluorescent protein; HDMEC: human dermal microvascular endothelial cells; HFFF: human fetal foreskin fibroblasts; ISR: integrated stress response; ISRIB: integrated stress response inhibitor; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; Myco: mycolactone; NBR1: NBR1 autophagy cargo receptor; NFE2L2: nuclear factor, erythroid 2 like 2; OPTN: optineurin; PFA: paraformaldehyde; PtdIns3P: phosphatidylinositol-3-phosphate; RB1CC1: RB1-inducible coiled coil 1; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; ULK: unc-51 like autophagy activating kinase; UPS: ubiquitin-proteasome system; WIPI: WD repeat domain, phosphoinositide interacting; WT: wild type.

Autophagy, 1, 1, 23 Aug 2021

Monitoring selective autophagy of mitochondria using super-resolution microscopy.
Li Z, Ktistakis NT

Selective elimination of damaged mitochondria via macroautophagy (mitophagy) is a conserved cellular process that plays an important role in organismal health. In recent years mitophagy has been studied in parallel to the more general, non-selective autophagy pathway induced in response to amino acid starvation with important similarities and differences noted between the two. The elaborate sequence of membrane rearrangements that give rise to autophagosomes in the non-selective pathway have their counterpart in mitophagy, but with the addition of other factors, such as a ubiquitin mark and mitophagy receptors, which mediate cargo recognition. In some types of mitophagy such as the one induced by ivermectin, the forming autophagosomal structure contains six different elements: the targeted mitochondrial fragment, a section of endoplasmic reticulum that provides a cradle, a ubiquitin layer, the mitophagy receptors and the early and late autophagosomal proteins/membranes. Super-resolution microscopy is ideally suited to investigate the spatial relationships between these elements that converge together but retain some distinctive localization, and we provide here a general protocol that can be used for mammalian cells.

Methods in cell biology, 165, 1, 2021