Nathan Brady

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Phosphorylation of an autophagy receptor restricts pathogenic cytosolic bacterial growth.

Ischemia and reperfusion (I/R) injury is associated with extensive loss of cardiac myocytes. Bnip3 is a mitochondrial pro-apoptotic Bcl-2 protein which is expressed in the adult myocardium. To investigate if Bnip3 plays a role in I/R injury, we generated a TAT-fusion protein encoding the carboxyl terminal transmembrane deletion mutant of Bnip3 (TAT-Bnip3DeltaTM) which has been shown to act as a dominant negative to block Bnip3-induced cell death. Perfusion with TAT-Bnip3DeltaTM conferred protection against I/R injury, improved cardiac function, and protected mitochondrial integrity. Moreover, Bnip3 induced extensive fragmentation of the mitochondrial network and increased autophagy in HL-1 myocytes. 3D rendering of confocal images revealed fragmented mitochondria inside autophagosomes. Enhancement of autophagy by ATG5 protected against Bnip3-mediated cell death, whereas inhibition of autophagy by ATG5K130R enhanced cell death. These results suggest that Bnip3 contributes to I/R injury which triggers a protective stress response with upregulation of autophagy and removal of damaged mitochondria.

Cardiac myocytes undergo programmed cell death as a result of ischemia/reperfusion (I/R). One feature of I/R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/R injury. We employed simulated I/R of cardiac HL-1 cells as an in vitro model of I/R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation (autophagic flux), as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. We found that I/R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/R and significantly reduced activation of pro-apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-xL were protective against I/R injury, and expression of a Beclin1 Bcl-2/-xL binding domain mutant resulted in decreased autophagic flux and did not protect against I/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell. Cardiac myocytes undergo programmed cell death as a result of ischemia/reperfusion (I/R). One feature of I/R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/R injury. We employed simulated I/R of cardiac HL-1 cells as an in vitro model of I/R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation (autophagic flux), as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. We found that I/R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/R and significantly reduced activation of pro-apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-xL were protective against I/R injury, and expression of a Beclin1 Bcl-2/-xL binding domain mutant resulted in decreased autophagic flux and did not protect against I/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell. Autophagy involves processes for the turnover of long lived macromolecules and organelles via the lysosomal degradative pathway (1Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2988) Google Scholar, 2Cuervo A.M. Mol. Cell. Biochem. 2004; 263: 55-72Crossref PubMed Scopus (395) Google Scholar). Macroautophagy (referred to hereafter as autophagy) is a specific mode of autophagy in which isolation membranes envelop a portion of the cytosol, containing nonspecific cytosolic components, selectively targeted toxic protein aggregates (3Ravikumar B. Vacher C. Berger Z. Davies J.E. Luo S. Oroz L.G. Scaravilli F. Easton D.F. Duden R. O'Kane C.J. Rubinsztein D.C. Nat. Genet. 2004; 36: 585-595Crossref PubMed Scopus (1990) Google Scholar), intracellular pathogens (4Gutierrez M.G. Master S.S. Singh S.B. Taylor G.A. Colombo M.I. Deretic V. Cell. 2004; 119: 753-766Abstract Full Text Full Text PDF PubMed Scopus (1769) Google Scholar), or organelles such as mitochondria (5Xue L. Fletcher G.C. Tolkovsky A.M. Curr. Biol. 2001; 11: 361-365Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 6Priault M. Salin B. Schaeffer J. Vallette F.M. di Rago J.P. Martinou J.C. Cell Death Differ. 2005; 12: 1613-1621Crossref PubMed Scopus (242) Google Scholar). The autophagosomes are then delivered to the lysosome, forming the autophagolysosome, for subsequent degradation of their contents by lysosomal hydrolases (Fig. 10). Interest in autophagy has increased recently, because of the recognition of its involvement in caspase-independent programmed cell death (PCD 2The abbreviations used are: PCD, programmed cell death; AVs, autophagic vacuoles; GFP, green fluorescent protein; KH, Krebs-Henseleit; 3-MA, 3-methyladenine; PI3K, phosphatidylinositol 3-kinase; I/R, ischemia/reperfusion; sI/R, simulated ischemia/reperfusion; RNAi, RNA interference; PI3P, phosphatidylinositol 3-phosphate; mTOR, mammalian target of rapamycin; ER, endoplasmic reticulum. type II) and its regulation by components of the apoptotic death pathway (PCD type I) (7Saeki K. Yuo A. Okuma E. Yazaki Y. Susin S.A. Kroemer G. Takaku F. Cell Death Differ. 2000; 7: 1263-1269Crossref PubMed Scopus (172) Google Scholar, 8Yanagisawa H. Miyashita T. Nakano Y. Yamamoto D. Cell Death Differ. 2003; 10: 798-807Crossref PubMed Scopus (91) Google Scholar, 9Shimizu S. Kanaseki T. Mizushima N. Mizuta T. Arakawa-Kobayashi S. Thompson C.B. Tsujimoto Y. Nat. Cell Biol. 2004; 6: 1221-1228Crossref PubMed Scopus (1192) Google Scholar). Anti-apoptotic Bcl-2 and Bcl-xL have been linked to the autophagic pathway via an interaction with Beclin1, a key mediator of autophagic activity (9Shimizu S. Kanaseki T. Mizushima N. Mizuta T. Arakawa-Kobayashi S. Thompson C.B. Tsujimoto Y. Nat. Cell Biol. 2004; 6: 1221-1228Crossref PubMed Scopus (1192) Google Scholar, 10Liang X.H. Kleeman L.K. Jiang H.H. Gordon G. Goldman J.E. Berry G. Herman B. Levine B. J. Virol. 1998; 72: 8586-8596Crossref PubMed Google Scholar). Autophagy is a vital process in the heart, presumably participating in the removal of dysfunctional cytosolic components and serving as a catabolic energy source during times of starvation. For example, autophagy in cardiac myocytes has been suggested to provide a necessary source of energy between birth and suckling (11Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. Nature. 2004; 432: 1032-1036Crossref PubMed Scopus (2405) Google Scholar), and in a GFP-LC3 transgenic mouse, cardiac myocytes from starved animals displayed high numbers of autophagosomes, some of which contained mitochondria (12Mizushima N. Yamamoto A. Matsui M. Yoshimori T. Ohsumi Y. Mol. Biol. Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1942) Google Scholar). On the other hand, impaired autophagy may play a causative role in cardiac disease. Incomplete autophagic removal of mitochondria may be the source of lipofuscin, a toxic waste product that builds up during the life span (13Brunk U.T. Terman A. Eur. J. Biochem. 2002; 269: 1996-2002Crossref PubMed Scopus (608) Google Scholar), and chronic impairment of the lysosome results in reduced myocardial function (14Saftig P. Tanaka Y. Lullmann-Rauch R. von Figura K. Trends Mol. Med. 2001; 7: 37-39Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Furthermore, disruption of the autophagic pathway may contribute to cardiac cell death under conditions where lysosomal integrity is lost and lysosomal proteases are released into the cytosol (15Decker R.S. Wildenthal K. Am. J. Pathol. 1980; 98: 425-444PubMed Google Scholar). In the study presented here, we investigated the role and regulation of autophagy during ischemia/reperfusion (I/R) injury. Following a bout of ischemia (a reduction of blood flow resulting in oxygen and nutrient starvation), reperfusion must be achieved in order to rescue affected tissue. However, reperfusion can activate pathways that either preserve cell viability (preconditioning) or lead to cell death (I/R injury). Autophagy may be a protective response to I/R injury, as increased prevalence of autophagosomes has been documented in response to sub-lethal ischemia in the perfused heart (15Decker R.S. Wildenthal K. Am. J. Pathol. 1980; 98: 425-444PubMed Google Scholar). Moreover, it was recently reported that increased Beclin1 expression in the heart correlated with the onset of protection in an in vivo model of myocardial stunning (16Yan L. Vatner D.E. Kim S.J. Ge H. Masurekar M. Massover W.H. Yang G. Matsui Y. Sadoshima J. Vatner S.F. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13807-13812Crossref PubMed Scopus (455) Google Scholar). The cardiac HL-1 cell line was subjected to simulated I/R (sI/R) as an in vitro model of I/R injury to the heart. Using three-dimensional high resolution fluorescence imaging, we analyzed the autophagic response to sI/R. Our results indicate that in HL-1 cardiac myocytes subjected to sI/R, autophagic flux is impaired at the level of both induction and degradation, yet remains a vital underlying protective response against sI/R injury. Moreover, increasing autophagic capacity of the cardiac myocyte is protective against sI/R injury. Reagents—3-Methyladenine, wortmannin, rapamycin, pepstatin A methyl ester, E64D, and bafilomycin A1 were purchased from EMD Biosciences. Cell Culture and Transfections—Cells of the atrially derived cardiac cell line HL-1 (17Claycomb W.C. Lanson Jr., N.A. Stallworth B.S. Egeland D.B. Delcarpio J.B. Bahinski A. Izzo Jr., N.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2979-2984Crossref PubMed Scopus (1243) Google Scholar) were plated in gelatin/fibronectin-coated culture vessels and maintained in Claycomb medium (JRH Biosciences) supplemented with 10% fetal bovine serum, 0.1 mm norepinephrine, 2 mm l-glutamine, 100 units/ml penicillin, 100 units/ml streptomycin, and 0.25 μg/ml amphotericin B. Cells were transfected with the indicated vectors using the transfection reagents Effectene (Qiagen) or Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions, achieving at least 40 and 60% transfection efficiency, respectively. RNA Interference—Sequences with 100% homology to regions within the open reading frame of mouse Beclin1 (gi: 27764874) were generated using the BLOCK-iT™ RNAi Designer, a construct that embeds a small hairpin RNA within a micro RNA fold, which is then processed by the endogenous RNAi machinery (18Amarzguioui M. Rossi J.J. Kim D. FEBS Lett. 2005; 579: 5974-5981Crossref PubMed Scopus (170) Google Scholar). The obtained target sequences, 5′-tgaaacttcagacccatctta-3′ (a) and 5′-taatggagctgtgagttcctg-3′ (b), showed no significant homology to other mouse proteins as determined by Blast analysis. The sequence was used to generate oligonucleotide pairs, which were inserted into the pcDNA™6.2-GW/EmGFP-miR, which has co-cistronic expression of EmGFP, allowing for determination of transfection efficiency by fluorescence microscopy. The vectors, pcDNA™6.2-GW/EmGFP-miR-Beclin1 (a) and -Beclin1 (b), were sequence-verified, and cells were co-transfected with both vectors to achieve maximal knockdown. To control for nonspecific RNAi effects, the construct pcDNA™6.2-GW/EmGFP-miR-LacZ (targeting β-galactosidase) was used as a control. Simulated Ischemia/Reperfusion (sI/R)—Cells were plated in 14-mm diameter glass bottom microwell dishes (MatTek), and ischemia was introduced by a buffer exchange to ischemia-mimetic solution (in mm: 125 NaCl, 8 KCl, 1.2 KH2PO4, 1.25 MgSO4, 1.2 CaCl2, 6.25 NaHCO3, 5 sodium lactate, 20 HEPES, pH 6.6) and placing the dishes in hypoxic pouches (GasPak™ EZ, BD Biosciences) equilibrated with 95% N2, 5% CO2. After 2 h of ischemia, reperfusion was initiated by a buffer exchange to normoxic Krebs-Henseleit solution (KH, in mm: 110 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.25 MgSO4, 1.2 CaCl2, 25 NaHCO3, 15 glucose, 20 HEPES, pH 7.4) and incubation at 95% room air, 5% CO2. Controls incubated in normoxic KH solution were run in parallel for each condition for periods of time that corresponded with those of the experimental groups. Under control conditions cell viability was not compromised. Wide Field Fluorescence Microscopy—Cells were observed through a Nikon TE300 fluorescence microscope (Nikon) equipped with a ×10 lens (0.3 N.A., Nikon), a ×40 Plan Fluor, and a ×60 Plan Apo objective (1.4 N.A. and 1.3 N.A. oil immersion lenses; Nikon), a Z-motor (ProScanII, Prior Scientific), a cooled CCD camera (Orca-ER, Hamamatsu), and automated excitation and emission filter wheels controlled by a LAMBDA 10–2 (Sutter Instrument) operated by MetaMorph 6.2r4 (Universal Imaging). Fluorescence was excited through an excitation filter for fluorescein isothiocyanate (HQ480/×40) and Texas Red (D560/×40). Fluorescent light was collected via a polychroic beam splitter (61002bs) and an emission filter for fluorescein isothiocyanate (HQ535/50m) and Texas Red (D630/60m). All filters were from Chroma. Acquired wide field Z-stacks were routinely deconvolved using 10 iterations of a three-dimensional blind deconvolution algorithm (AutoQuant) to maximize spatial resolution. Unless stated otherwise, representative images shown are maximum projections of Z-stacks taken with 0.3- μm increments capturing total cellular volume. Quantification of Cellular Autophagosome Content—LC3 forms I and II are known to be differentially recognized by the LC3 antibodies (19Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1117) Google Scholar). Furthermore, in our hands immunodetection of endogenous LC3 in HL-1 cells was inconclusive (data not shown). Therefore, cellular contents of autophagosomal structures were quantified via fluorescence imaging of GFP-LC3 (20Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5468) Google Scholar) or mCherry-LC3. To generate pmCherry-LC3, mCherry was amplified from the pRSET-mCherry vector (21Shaner N.C. Campbell R.E. Steinbach P.A. Giepmans B.N. Palmer A.E. Tsien R.Y. Nat. Biotechnol. 2004; 22: 1567-1572Crossref PubMed Scopus (3507) Google Scholar) and swapped with enhanced GFP of the vector pEGFP-LC3 (20Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5468) Google Scholar); HL-1 cells were transfected with (mCherry-/)GFP-LC3, and 48 h after transfection, cells were subjected to sI/R as indicated. Cells were fixed with 4% formaldehyde in phosphate-buffered saline, pH 7.4, for 15 min. To quantify the autophagic response in a population of cells, cells were inspected at ×60 magnification and classified as either having predominantly diffuse (mCherry-/)GFP-LC3 fluorescence or as having numerous punctate (mCherry-/)GFP-LC3 structures, representing autophagic vacuoles, AVs. At least 150 cells were scored in each of three or more independent experiments. For quantification of the autophagic response of single cells, Z-stacks of (mCherry-/)GFP-LC3 fluorescence of 7–10 representative cells per condition in three separate experiments were acquired through the ×60 oil immersion lens with 0.3-μm increments through the entire volume of the cell. Z-stacks were thresholded, and total number and volume of the autophagosome per cell were determined (AutoQuant). Determination of LC3-II Degradation—To analyze autophagic flux, (mCherry-/)GFP-LC3-expressing cells were subjected to the indicated experimental conditions with and without a mixture of the cell-permeable lysosomal inhibitors bafilomycin A1 (100 nm, vacuolar H+-ATPase inhibitor) to inhibit autophagosome-lysosome fusion (22Yamamoto A. Tagawa Y. Yoshimori T. Moriyama Y. Masaki R. Tashiro Y. Cell Struct. Funct. 1998; 23: 33-42Crossref PubMed Scopus (1077) Google Scholar), E64D (5 μg/ml, inhibitor of cysteine proteases, including cathepsin B), and pepstatin A methyl ester (5 μg/ml, cathepsin D inhibitor) to inhibit lysosomal protease activity. Fluorescence microscopy of GFP-LC3 was used to determine cellular autophagosomal content as described above. Activity of the Lysosomal Compartment—LysoTracker Red is a cell-permeable acidotropic probe that selectively labels vacuoles with low internal pH and thus can be used to label functional lysosomes (23Bucci C. Thomsen P. Nicoziani P. McCarthy J. van Deurs B. Mol. Biol. Cell. 2000; 11: 467-480Crossref PubMed Scopus (804) Google Scholar). Following sI/R and control experiments, cells were loaded with 50 nm LysoTracker Red for 5 min in KH solution; the medium was then exchanged with dye-free KH solution, and cells were analyzed by fluorescence microscopy. Activity and intracellular distribution of cathepsin B, a predominant lysosomal protease, were assessed using (z-RR)2-MagicRed-Cathepsin B substrate (B-Bridge). MagicRed cathepsin B substrate was added to the cells during the last 30 min of an experiment according to the manufacturer's instructions. Quantification of Cellular Injury—GFP-Bax (24Wolter K.G. Hsu Y.T. Smith C.L. Nechushtan A. Xi X.G. Youle R.J. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1577) Google Scholar) or mCherry-Bax (25Brady N.R. Hamacher-Brady A. Gottlieb R.A. Biochim. Biophys. Acta. 2006; 1757: 667-678Crossref PubMed Scopus (96) Google Scholar) distribution was used as a parameter to quantify irreversible cellular injury. Cells were cotransfected with (mCherry-/)GFP-Bax and the indicated vectors and allowed to express for 48 h. Cells were then subjected to 2 h of ischemia in hypoxic pouches followed by 5 h of reperfusion, and live cells were analyzed by fluorescence microscopy. Cells were classified as cells with either diffuse or punctate mitochondrial (mCherry-/)GFP-Bax fluorescence. Approximately 300 transfected cells per condition were scored at ×60 magnification in each of three independent experiments. Immunoblotting—Cells were harvested by scraping and centrifugation at 550 × g for 5 min at 4 °C and washed once with cold phosphate-buffered saline, pH 7.4. To prepare whole cell lysates, cell pellets were suspended in cold RIPA buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 1 mm EDTA, 1 mm Na3VO4, 1 mm NaF, and 1× complete protease inhibitor mixture (Roche Applied Science)) and left on ice for 20 min. The cell extracts were centrifuged at 20,000 × g for 5 min to remove cellular debris. After addition of sample buffer and reducing agent (Bio-Rad), samples were incubated at 95 °C for 5 min, electrophoresed on SDS-polyacrylamide gels, and transferred to nitrocellulose membranes (Bio-Rad). Immunodetection was performed using antibodies against actin (clone AC-40; Sigma), Bcl-2 (C-2, Santa Cruz Biotechnology), Bcl-xL (H-5, Santa Cruz Biotechnology), Beclin1 (D-18; Santa Cruz Biotechnology), and fluorescent protein (BD Biosciences). Attempts to detect endogenous LC3-I and -II using F-14 and H-50 (Santa Cruz Biotechnology) and A0973 (Biosignatures) antibodies were unsatisfactory because of inconsistencies in immunoreactivity and nonspecificity, perhaps due to the fact that these are mouse antibodies being used against mouse proteins. Blots shown are representative of at least three independent experiments. Statistics—The probability of statistical differences between experimental groups was determined by the Student's t test. Values are expressed as mean ± S.E. of at least three independent experiments unless stated otherwise. sI/R Induces Programmed Cell Death in HL-1 Cardiac Myocytes—The HL-1 cell line is an excellent model for studying many aspects of cardiac cell physiology (26White S.M. Constantin P.E. Claycomb W.C. Am. J. Physiol. 2004; 286: H823-H829Crossref PubMed Scopus (333) Google Scholar). In our hands HL-1 cells reproducibly underwent PCD in response to simulated I/R via pathways resembling in vivo cardiac I/R injury (25Brady N.R. Hamacher-Brady A. Gottlieb R.A. Biochim. Biophys. Acta. 2006; 1757: 667-678Crossref PubMed Scopus (96) Google Scholar). One key feature of sI/R-induced cell death is the participation of the pro-apoptotic Bcl-2 protein Bax in the mitochondrial death pathway. Bax activation, a point-of-noreturn in the PCD pathway, is reflected by a redistribution from the cytosol to punctate clusters at the mitochondria and can be quantified via fluorescent imaging of a GFP-Bax fusion protein (Fig. 1A) (24Wolter K.G. Hsu Y.T. Smith C.L. Nechushtan A. Xi X.G. Youle R.J. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1577) Google Scholar). Using GFP-Bax redistribution as an index to monitor activation of PCD, we found that sI/R induced PCD in a hypoxia/reoxygenation-dependent manner (Fig. 1B). Overexpression of both Bcl-2 and Bcl-xL, known protectors against cardiac I/R injury (27Brocheriou V. Hagege A.A. Oubenaissa A. Lambert M. Mallet V.O. Duriez M. Wassef M. Kahn A. Menasche P. Gilgenkrantz H. J. Gene Med. 2000; 2: 326-333Crossref PubMed Scopus (155) Google Scholar, 28Huang J. Nakamura K. Ito Y. Uzuka T. Morikawa M. Hirai S. Tomihara K. Tanaka T. Masuta Y. Ishii K. Kato K. Hamada H. Circulation. 2005; 112: 76-83Crossref PubMed Scopus (49) Google Scholar, 29Imahashi K. Schneider M.D. Steenbergen C. Murphy E. Circ. Res. 2004; 95: 734-741Crossref PubMed Scopus (168) Google Scholar), significantly reduced sI/R-induced GFP-Bax redistribution, further demonstrating suitability of the model (Fig. 1, C and D). Cellular Autophagosomal Content Is Increased during the Early Phase of sI/R Injury—We used HL-1 cells to explore the role of autophagy during sI/R injury. To determine whether autophagic activity is modulated in response to sI/R, we first characterized changes in cellular autophagosomal content using high resolution three-dimensional imaging of GFP-LC3. During the initiation of autophagy, cytosolic LC3 (LC3-I) is cleaved and lipidated to form LC3-II (19Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1117) Google Scholar, 30Tanida I. Minematsu-Ikeguchi N. Ueno T. Kominami E. Autophagy. 2005; 1: 84-91Crossref PubMed Scopus (940) Google Scholar). LC3-II is then recruited to the autophagosomal membrane (31Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1161) Google Scholar). Thus, punctate GFP-LC3-labeled structures represent autophagosomes, also referred to as autophagic vacuoles (AVs). Importantly, overexpression of (GFP-)LC3 does not affect autophagic activity, and transgenic mice expressing GFP-LC3 display no detectable abnormalities (12Mizushima N. Yamamoto A. Matsui M. Yoshimori T. Ohsumi Y. Mol. Biol. Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1942) Google Scholar, 32Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (714) Google Scholar). We transfected HL-1 cardiac myocytes with GFP-LC3 and compared the abundance of AVs in cells subjected to sI/R to normoxic control cells. Under normoxic conditions in KH solution, GFP-LC3 was diffusely distributed throughout the cell, with very few detectable AVs (Fig. 2A, upper panel). Cells subjected to sI/R, however, displayed increased numbers of AVs (Fig. 2A, bottom panel). In addition, in control cells the few pre-existing AVs were randomly distributed, whereas AVs in cells subjected to sI/R were typically more clustered at the center of the cell. This distinctive distribution contrasts with the autophagic response to starvation in hepatocytes, where no such clustering was observed (33Kochl R. Hu X.W. Chan E.Y. Tooze S.A. Traffic. 2006; 7: 129-145Crossref PubMed Scopus (345) Google Scholar). To quantify the increase in GFP-LC3-labeled AVs, the percentage of cells displaying numerous punctate GFP-LC3 structures was determined. Only a small fraction of cells displayed punctate GFP-LC3 fluorescence when incubated in fully supplemented medium or KH solution (Fig. 2B). In cells subjected to sI/R, however, the number of cells with numerous AVs was significantly increased (Fig. 2B). Quantitative analysis performed on Z-stacks of GFP-LC3 fluorescence revealed that sI/R significantly increased the number of AVs per cell and, likewise, the total autophagosomal volume (Fig. 2C). Changes in Autophagic Activity during Ischemia and Reperfusion—Our results demonstrate that cellular AV content was increased early in the reperfusion period. We subsequently addressed the effect of sI/R on actual autophagic activity. Autophagy involves the delivery of the autophagosomes and their contents to lysosomes that contain the degradative enzymes needed to complete the catabolic processes of autophagy (1Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2988) Google Scholar). Therefore, the increased presence of AVs may reflect enhanced formation of AVs, impaired fusion of AVs with lysosomes to generate autophagolysosomes, or a combination of the two. Moreover, LC3-II may be removed by lysosomal degradation at a rate that exceeds our imaging capabilities, i.e. the transit is so rapid and/or the AVs so small that only a few AVs can be detected at any given time. Accordingly, a low number of GFP-LC3-labeled AVs may be due to either low or high autophagic activity. To characterize autophagic activity, we therefore determined two relevant parameters of autophagosome-lysosome fusion, the index of LC3-II degradation and downstream lysosomal activity. Flux of LC3-II Degradation during sI/R—Using an approach based on the inhibition of downstream lysosomal degradation of AVs and their cargo, we determined whether the increase in cellular AVs during sI/R was indicative of increased or impaired autophagy. Cells were subjected to various experimental conditions and treated with a mixture of lysosomal inhibitors to inhibit autophagolysosome formation (with bafilomycin A1) and lysosomal protease activity (with E64D and pepstatin A). By analyzing the lysosomal inhibitor-mediated increase in GFP-LC3-II (AV) accumulation within a cell population, we were able to obtain a quantitative index of the flux of AV formation and degradation. Bar graphs with offset superimposed bars depict the percentage of cells exhibiting high AV levels in the absence and presence of lysosomal inhibitors, per condition. The difference between the two bars (see values in graphs) is a measure of the percentage of cells demonstrating high autophagic activity, or flux. We found that in KH solution AV content was dramatically increased in the presence of inhibitors (Fig. 3, A and B). Thus, under control conditions in KH solution, which lacks the serum and amino acid component of full medium, autophagy was strongly active. Notably, this response is only revealed through the use of inhibitors; based on GFP-LC3 imaging alone (Fig. 2), low autophagic activity in KH solution would be incorrectly assumed. sI/R augmented the number of cells with increased numbers of AVs (Fig. 3B). Under lysosomal inhibition the number of cells with high AV content was increased only slightly more, indicating that the previously described increase in cellular AV content in sI/R (Fig. 2 and 3B) is a reflection of an accumulation of AVs, presumably due to impairment in the autophagic pathway at a point(s) following AV formation and before AV degradation. As the level of AV accumulation was substantially smaller than the inhibitor-mediated response seen in KH solution for the same period of time, it can be concluded that autophagy is also impaired at the level of AV formation. Most AVs were formed during the reperfusion period, as cells fixed immediately after the ischemic period were essentially devoid of AVs, either with or without lysosomal inhibitors, indicating a complete blockage of autophagy during the ischemic period (Fig. 3C). Interestingly, hypoxia was a necessary component of the insult, as cells incubated in ischemia/mimetic solution alone, under normoxic conditions, exhibited only a minor reduction of autophagic flux, which recovered completely upon reperfusion (Fig. 3B). Lysosomal Activity during sI/R—One possible explanation for the observed accumulation of AVs during sI/R was a nonfunctional lysosomal compartment. To investigate down-stream lysosomal activity, HL-1 cells were incubated in LysoTracker Red, which labels the highly acidic lysosomal vacuoles and thus reports activity of the vacuolar H+-ATPase (v-ATPase). Before and after sI/R, we observed similar patterns of LysoTracker Red fluorescence, indicating that, consistent with its importance in cell survival during I/R (34Karwatowska-Prokopczuk E. Nordberg J.A. Li H.L. Engler R.L. Gottlieb R.A. Circ. Res. 1998; 82: 1139-1144Crossref PubMed Scopus (75) Google Scholar), activity of the v-ATPase is maintained during the reperfusion period (Fig. 4A). Furthermore, we determined the activity and subcellular localization of cathepsin B, a predominant lysosomal protease, using a MagicRed substrate that fluoresces when cleaved by cathepsin B (35Lamparska-Przybysz M. Gajkowska B. Motyl T. J. Physiol. Pharmacol. 2005; 56: 159-179PubMed Google Scholar). We did not detect a decrease in cathepsin B activity following sI/R, as MagicRed fluorescence was still punctate (lysosomal) and displayed an intensity comparable with the normoxic control (Fig. 4B). Moreover, we found that cathepsin B activity was not detected in the cytosol, indicating that cathepsin B is not released from the lysosomes following sI/R. Together, these results indicate a functional lysosomal compartment durin

Oncogenic KRas reprograms pancreatic ductal adenocarcinoma (PDAC) cells to states which are highly resistant to apoptosis. Thus, a major preclinical goal is to identify effective strategies for killing PDAC cells. Artesunate (ART) is an anti-malarial that specifically induces programmed cell death in different cancer cell types, in a manner initiated by reactive oxygen species (ROS)-generation. In this study we demonstrate that ART specifically induced ROS- and lysosomal iron-dependent cell death in PDAC cell lines. Highest cytotoxicity was obtained in PDAC cell lines with constitutively-active KRas, and ART did not affect non-neoplastic human pancreatic ductal epithelial (HPDE) cells. We determined that ART did not induce apoptosis or necroptosis. Instead, ART induced ferroptosis, a recently described mode of ROS- and iron-dependent programmed necrosis which can be activated in Ras-transformed cells. Co-treatment with the ferroptosis inhibitor ferrostatin-1 blocked ART-induced lipid peroxidation and cell death, and increased long-term cell survival and proliferation. Importantly, analysis of PDAC patient mRNA expression indicates a dependency on antioxidant homeostasis and increased sensitivity to free intracellular iron, both of which correlate with Ras-driven sensitivity to ferroptosis. Overall, our findings suggest that ART activation of ferroptosis is an effective, novel pathway for killing PDAC cells.

BH3-only proteins integrate apoptosis and autophagy pathways, yet regulation and functional consequences of pathway cross-talk are not fully resolved. The BH3-only protein Bnip3 is an autophagy receptor that signals autophagic degradation of mitochondria (mitophagy) via interaction of its LC3-interacting region (LIR) with Atg8 proteins. Here we report that phosphorylation of serine residues 17 and 24 flanking the Bnip3 LIR promotes binding to specific Atg8 members LC3B and GATE-16. Using quantitative multispectral image-based flow cytometry, we demonstrate that enhancing Bnip3-Atg8 interactions via phosphorylation-mimicked LIR mutations increased mitochondrial sequestration, lysosomal delivery, and degradation. Importantly, mitochondria were targeted by mitophagy prior to cytochrome c release, resulting in reduced cellular cytochrome c release capacity. Intriguingly, pro-survival Bcl-x(L) positively regulated Bnip3 binding to LC3B, sequestration, and mitochondrial autophagy, further supporting an anti-apoptotic role for Bnip3-induced mitophagy. The ensemble of these results demonstrates that the phosphorylation state of the Bnip3 LIR signals either the induction of apoptosis or pro-survival mitophagy.

Mitochondria are an essential source of ATP for cellular function, but when damaged, mitochondria generate a plethora of stress signals, which lead to cellular dysfunction and eventually programmed cell death. Thus, a major component of maintaining cellular homeostasis is the recognition and removal of dysfunctional mitochondria through autophagy-mediated degradation, i.e., mitophagy. Mitophagy further constitutes a developmental program, and undergoes a high degree of crosstalk with apoptosis. Reduced mitochondrial quality control is linked to disease pathogenesis, suggesting the importance of process elucidation as a clinical target. Recent work has revealed multiple mitophagy programs that operate independently or undergo crosstalk, and require modulated autophagy receptor activities at outer membranes of mitochondria. Here, we review these mitophagy programs, focusing on pathway mechanisms which recognize and target mitochondria for sequestration by autophagosomes, as well as mechanisms controlling pathway activities. Furthermore, we provide an introduction to the currently available methods for detecting mitophagy.

Abstract The mitophagy receptor Nix interacts with LC3/GABARAP proteins, targeting mitochondria into autophagosomes for degradation. Here we present evidence for phosphorylation-driven regulation of the Nix:LC3B interaction. Isothermal titration calorimetry and NMR indicate a ~100 fold enhanced affinity of the serine 34/35-phosphorylated Nix LC3-interacting region (LIR) to LC3B and formation of a very rigid complex compared to the non-phosphorylated sequence. Moreover, the crystal structure of LC3B in complex with the Nix LIR peptide containing glutamic acids as phosphomimetic residues and NMR experiments revealed that LIR phosphorylation stabilizes the Nix:LC3B complex via formation of two additional hydrogen bonds between phosphorylated serines of Nix LIR and Arg11, Lys49 and Lys51 in LC3B. Substitution of Lys51 to Ala in LC3B abrogates binding of a phosphomimetic Nix mutant. Functionally, serine 34/35 phosphorylation enhances autophagosome recruitment to mitochondria in HeLa cells. Together, this study provides cellular, biochemical and biophysical evidence that phosphorylation of the LIR domain of Nix enhances mitophagy receptor engagement.

The antimalarial agent artesunate (ART) activates programmed cell death (PCD) in cancer cells in a manner dependent on the presence of iron and the generation of reactive oxygen species. In malaria parasites, ART cytotoxicity originates from interactions with heme-derived iron within the food vacuole. The analogous digestive compartment of mammalian cells, the lysosome, similarly contains high levels of redox-active iron and in response to specific stimuli can initiate mitochondrial apoptosis. We thus investigated the role of lysosomes in ART-induced PCD and determined that in MCF-7 breast cancer cells ART activates lysosome-dependent mitochondrial outer membrane permeabilization. ART impacted endolysosomal and autophagosomal compartments, inhibiting autophagosome turnover and causing perinuclear clustering of autophagosomes, early and late endosomes, and lysosomes. Lysosomal iron chelation blocked all measured parameters of ART-induced PCD, whereas lysosomal iron loading enhanced death, thus identifying lysosomal iron as the lethal source of reactive oxygen species upstream of mitochondrial outer membrane permeabilization. Moreover, lysosomal inhibitors chloroquine and bafilomycin A1 reduced ART-activated PCD, evidencing a requirement for lysosomal function during PCD signaling. ART killing did not involve activation of the BH3-only protein, Bid, yet ART enhanced TNF-mediated Bid cleavage. We additionally demonstrated the lysosomal PCD pathway in T47D and MDA-MB-231 breast cancer cells. Importantly, non-tumorigenic MCF-10A cells resisted ART-induced PCD. Together, our data suggest that ART triggers PCD via engagement of distinct, interconnected PCD pathways, with hierarchical signaling from lysosomes to mitochondria, suggesting a potential clinical use of ART for targeting lysosomes in cancer treatment.

Tumor cells activate autophagy in response to chemotherapy-induced DNA damage as a survival program to cope with metabolic stress. Here, we provide in vitro and in vivo evidence that histone deacetylase (HDAC)10 promotes autophagy-mediated survival in neuroblastoma cells. We show that both knockdown and inhibition of HDAC10 effectively disrupted autophagy associated with sensitization to cytotoxic drug treatment in a panel of highly malignant V-MYC myelocytomatosis viral-related oncogene, neuroblastoma derived-amplified neuroblastoma cell lines, in contrast to nontransformed cells. HDAC10 depletion in neuroblastoma cells interrupted autophagic flux and induced accumulation of autophagosomes, lysosomes, and a prominent substrate of the autophagic degradation pathway, p62/sequestosome 1. Enforced HDAC10 expression protected neuroblastoma cells against doxorubicin treatment through interaction with heat shock protein 70 family proteins, causing their deacetylation. Conversely, heat shock protein 70/heat shock cognate 70 was acetylated in HDAC10-depleted cells. HDAC10 expression levels in high-risk neuroblastomas correlated with autophagy in gene-set analysis and predicted treatment success in patients with advanced stage 4 neuroblastomas. Our results demonstrate that HDAC10 protects cancer cells from cytotoxic agents by mediating autophagy and identify this HDAC isozyme as a druggable regulator of advanced-stage tumor cell survival. Moreover, these results propose a promising way to considerably improve treatment response in the neuroblastoma patient subgroup with the poorest outcome.

The most common cause of the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal dementia is a hexanucleotide repeat expansion in C9orf72. Here we report a study of the C9orf72 protein by examining the consequences of loss of C9orf72 functions. Deletion of one or both alleles of the C9orf72 gene in mice causes age-dependent lethality phenotypes. We demonstrate that C9orf72 regulates nutrient sensing as the loss of C9orf72 decreases phosphorylation of the mTOR substrate S6K1. The transcription factor EB (TFEB), a master regulator of lysosomal and autophagy genes, which is negatively regulated by mTOR, is substantially up-regulated in C9orf72 loss-of-function animal and cellular models. Consistent with reduced mTOR activity and increased TFEB levels, loss of C9orf72 enhances autophagic flux, suggesting that C9orf72 is a negative regulator of autophagy. We identified a protein complex consisting of C9orf72 and SMCR8, both of which are homologous to DENN-like proteins. The depletion of C9orf72 or SMCR8 leads to significant down-regulation of each other's protein level. Loss of SMCR8 alters mTOR signaling and autophagy. These results demonstrate that the C9orf72-SMCR8 protein complex functions in the regulation of metabolism and provide evidence that loss of C9orf72 function may contribute to the pathogenesis of relevant diseases.

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.

<h2>Summary</h2> It is unclear why some SARS-CoV-2 patients readily resolve infection while others develop severe disease. By interrogating metabolic programs of immune cells in severe and recovered coronavirus disease 2019 (COVID-19) patients compared with other viral infections, we identify a unique population of T cells. These T cells express increased Voltage-Dependent Anion Channel 1 (VDAC1), accompanied by gene programs and functional characteristics linked to mitochondrial dysfunction and apoptosis. The percentage of these cells increases in elderly patients and correlates with lymphopenia. Importantly, T cell apoptosis is inhibited <i>in vitro</i> by targeting the oligomerization of VDAC1 or blocking caspase activity. We also observe an expansion of myeloid-derived suppressor cells with unique metabolic phenotypes specific to COVID-19, and their presence distinguishes severe from mild disease. Overall, the identification of these metabolic phenotypes provides insight into the dysfunctional immune response in acutely ill COVID-19 patients and provides a means to predict and track disease severity and/or design metabolic therapeutic regimens.

Abstract Cancer cells that transit from primary tumours into the circulatory system are known as circulating tumour cells (CTCs). These cancer cells have unique phenotypic and genotypic characteristics which allow them to survive within the circulation, subsequently extravasate and metastasise. CTCs have emerged as a useful diagnostic tool using “liquid biopsies” to report on the metastatic potential of cancers. However, CTCs by their nature interact with components of the blood circulatory system on a constant basis, influencing both their physical and morphological characteristics as well as metastatic capabilities. These properties and the associated molecular profile may provide critical diagnostic and prognostic capabilities in the clinic. Platelets interact with CTCs within minutes of their dissemination and are crucial in the formation of the initial metastatic niche. Platelets and coagulation proteins also alter the fate of a CTC by influencing EMT, promoting pro-survival signalling and aiding in evading immune cell destruction. CTCs have the capacity to directly hijack immune cells and utilise them to aid in CTC metastatic seeding processes. The disruption of CTC clusters may also offer a strategy for the treatment of advance staged cancers. Therapeutic disruption of these heterotypical interactions as well as direct CTC targeting hold great promise, especially with the advent of new immunotherapies and personalised medicines. Understanding the molecular role that platelets, immune cells and the coagulation cascade play in CTC biology will allow us to identify and characterise the most clinically relevant CTCs from patients. This will subsequently advance the clinical utility of CTCs in cancer diagnosis/prognosis.

Once considered simply as the main source of ATP, mitochondria are now implicated in the control of many additional aspects of cell physiology, such as calcium signaling, and pathology, as in injury incurred on ischemia and subsequent reperfusion (I/R). Mitochondrial respiration is ordinarily accompanied by low-level ROS production, but they can respond to elevated ROS concentrations by increasing their own ROS production, a phenomenon termed ROS-induced ROS release (RIRR). Two modes of RIRR have been described. In the first mode of RIRR, enhanced ROS leads to mitochondrial depolarization via activation of the MPTP, yielding a short-lived burst of ROS originating from the mitochondrial electron transport chain (ETC). The second mode of RIRR is MPTP independent but is regulated by the mitochondrial benzodiazepine receptor (mBzR). Increased ROS in the mitochondrion triggers opening of the inner mitochondrial membrane anion channel (IMAC), resulting in a brief increase in ETC-derived ROS. Both modes of RIRR have been shown to transmit localized mitochondrial perturbations throughout the cardiac cell in the form of oscillations or waves but are kinetically distinct and may involve different ROS that serve as second messengers. In this review, we discuss the mechanisms of these different modes of RIRR.

Viruses have played an important role in human evolution and have evolved diverse strategies to co-exist with their hosts. As obligate intracellular pathogens, viruses exploit and manipulate different host cell processes, including cellular trafficking, metabolism and immunity-related functions, for their own survival. In this article, we review evidence for how autophagy, a highly conserved cellular degradative pathway, serves either as an antiviral defense mechanism or, alternatively, as a pro-viral process during virus infection. Furthermore, we highlight recent reports concerning the role of selective autophagy in virus infection and how viruses manipulate autophagy to evade lysosomal capture and degradation.

There is compelling evidence to support the idea that autophagy has a protective function in neurons and its disruption results in neurodegenerative disorders. Neuronal damage is well-documented in the brains of HIV-infected individuals, and evidence of inflammation, oxidative stress, damage to synaptic and dendritic structures, and neuronal loss are present in the brains of those with HIV-associated dementia. We investigated the role of autophagy in microglia-induced neurotoxicity in primary rodent neurons, primate and human models. We demonstrate here that products of simian immunodeficiency virus (SIV)-infected microglia inhibit neuronal autophagy, resulting in decreased neuronal survival. Quantitative analysis of autophagy vacuole numbers in rat primary neurons revealed a striking loss from the processes. Assessment of multiple biochemical markers of autophagic activity confirmed the inhibition of autophagy in neurons. Importantly, autophagy could be induced in neurons through rapamycin treatment, and such treatment conferred significant protection to neurons. Two major mediators of HIV-induced neurotoxicity, tumor necrosis factor-alpha and glutamate, had similar effects on reducing autophagy in neurons. The mRNA level of p62 was increased in the brain in SIV encephalitis and as well as in brains from individuals with HIV dementia, and abnormal neuronal p62 dot structures immunoreactivity was present and had a similar pattern with abnormal ubiquitinylated proteins. Taken together, these results identify that induction of deficits in autophagy is a significant mechanism for neurodegenerative processes that arise from glial, as opposed to neuronal, sources, and that the maintenance of autophagy may have a pivotal role in neuroprotection in the setting of HIV infection.

Autophagy mediates lysosomal degradation of cytosolic components. Recent work has associated autophagic dysfunction with pathologies, including cancer and cardiovascular disease. To date, the identification of clinically-applicable drugs that modulate autophagy has been hampered by the lack of standardized assays capable of precisely reporting autophagic activity.We developed and implemented a high-content, flow-cytometry-based screening approach for rapid, precise, and quantitative measurements of pharmaceutical control over autophagy. Our assay allowed for time-resolved individual measurements of autolysosome formation and degradation, and endolysosomal activities under both basal and activated autophagy conditions. As proof of concept, we analyzed conventional autophagy regulators, including cardioprotective compounds aminoimidazole carboxamide ribonucleotide (AICAR), rapamycin, and resveratrol, and revealed striking conditional dependencies of rapamycin and autophagy inhibitor 3-methyladenine (3-MA). To identify novel autophagy modulators with translational potential, we screened the Prestwick Chemical Library of 1,120 US Food and Drug Administration (FDA)-approved compounds for impact on autolysosome formation. In all, 38 compounds were identified as potential activators, and 36 as potential inhibitors of autophagy. Notably, amongst the autophagy enhancers were cardiac glycosides, from which we selected digoxin, strophanthidin, and digoxigenin for validation by standard biochemical and imaging techniques. We report the induction of autophagic flux by these cardiac glycosides, and the concentrations allowing for specific enhancement of autophagic activities without impact on endolysosomal activities.Our systematic analysis of autophagic and endolysosomal activities outperformed conventional autophagy assays and highlights the complexity of drug influence on autophagy. We demonstrate conditional dependencies of established regulators. Moreover, we identified new autophagy regulators and characterized cardiac glycosides as novel potent inducers of autophagic flux.

Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV), several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or them TOR inhibitor Torin1.We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. In addition, endolysosomal trafficking was suppressed, while lysosomal activities were increased.We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable overexpression of p62 significantly suppressed DENV replication, suggesting a novel role for p62 as a viral restriction factor. Overall, our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an antiviral role, which is countered by DENV.Autophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the replication cycle of many viruses. However, thus far the dynamics of autophagy in case of Dengue virus (DENV) infections has not been systematically quantified. Therefore, we used high-content, imaging-based flow cytometry to quantify autophagic flux and endolysosomal trafficking in response to DENV infection. We report that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Further, lysosomal activity was increased, but endolysosomal trafficking was suppressed confirming the block of autophagic flux. Importantly, we provide evidence that p62, an autophagy receptor, restrict DENV replication and was specifically depleted in DENV-infected cells via increased proteasomal degradation. These results suggest that during DENV infection autophagy shifts from a proviral to an antiviral cellular process, which is counteracted by the virus.

Abstract How the network around ROS protects against oxidative stress and Parkinson’s disease (PD), and how processes at the minutes timescale cause disease and aging after decades, remains enigmatic. Challenging whether the ROS network is as complex as it seems, we built a fairly comprehensive version thereof which we disentangled into a hierarchy of only five simpler subnetworks each delivering one type of robustness. The comprehensive dynamic model described in vitro data sets from two independent laboratories. Notwithstanding its five-fold robustness, it exhibited a relatively sudden breakdown, after some 80 years of virtually steady performance: it predicted aging. PD-related conditions such as lack of DJ-1 protein or increased α-synuclein accelerated the collapse, while antioxidants or caffeine retarded it. Introducing a new concept (aging-time-control coefficient), we found that as many as 25 out of 57 molecular processes controlled aging. We identified new targets for “life-extending interventions”: mitochondrial synthesis, KEAP1 degradation, and p62 metabolism.

<h2>Summary</h2> Diagnosis and assessment of patients with prostate cancer is dependent on accurate interpretation and grading of histopathology. However, morphology does not necessarily reflect the complex biological changes occurring in prostate cancer disease progression, and current biomarkers have demonstrated limited clinical utility in patient assessment. This study aimed to develop biomarkers that accurately define prostate cancer biology by distinguishing specific pathological features that enable reliable interpretation of pathology for accurate Gleason grading of patients. Online gene expression databases were interrogated and a pathogenic pathway for prostate cancer was identified. The protein expression of key genes in the pathway, including adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (Appl1), Sortilin and Syndecan-1, was examined by immunohistochemistry (IHC) in a pilot study of 29 patients with prostate cancer, using monoclonal antibodies designed against unique epitopes. Appl1, Sortilin, and Syndecan-1 expression was first assessed in a tissue microarray cohort of 112 patient samples, demonstrating that the monoclonal antibodies clearly illustrate gland morphologies. To determine the impact of a novel IHC-assisted interpretation (the utility of Appl1, Sortilin, and Syndecan-1 labelling as a panel) of Gleason grading, versus standard haematoxylin and eosin (H&E) Gleason grade assignment, a radical prostatectomy sample cohort comprising 114 patients was assessed. In comparison to H&E, the utility of the biomarker panel reduced subjectivity in interpretation of prostate cancer tissue morphology and improved the reliability of pathology assessment, resulting in Gleason grade redistribution for 41% of patient samples. Importantly, for equivocal IHC-assisted labelling and H&E staining results, the cancer morphology interpretation could be more accurately applied upon re-review of the H&E tissue sections. This study addresses a key issue in the field of prostate cancer pathology by presenting a novel combination of three biomarkers and has the potential to transform clinical pathology practice by standardising the interpretation of the tissue morphology.

Macroautophagy is a vital process in the cardiac myocyte: it plays a protective role in the response to ischemic injury, and chronic perturbation is causative in heart disease. Recent findings evidence a link between the apoptotic and autophagic pathways through the interaction of the antiapoptotic proteins Bcl-2 and Bcl-XL with Beclin 1. However, the nature of the interaction, either in promoting or blocking autophagy, remains unclear. Here, using a highly sensitive, macroautophagy-specific flux assay allowing for the distinction between enhanced autophagosome production and suppressed autophagosome degradation, we investigated the control of Beclin 1 and Bcl-2 on nutrient deprivation-activated macroautophagy. We found that in HL-1 cardiac myocytes the relationship between Beclin 1 and Bcl-2 is subtle: Beclin 1 mutant lacking the Bcl-2-binding domain significantly reduced autophagic activity, indicating that Beclin 1-mediated autophagy required an interaction with Bcl-2. Overexpression of Bcl-2 had no effect on the autophagic response to nutrient deprivation; however, targeting Bcl-2 to the sarco/endoplasmic reticulum (S/ER) significantly suppressed autophagy. The suppressive effect of S/ER-targeted Bcl-2 was in part due to the depletion of S/ER calcium stores. Intracellular scavenging of calcium by BAPTA-AM significantly blocked autophagy, and thapsigargin, an inhibitor of sarco/endoplasmic reticulum calcium ATPase, reduced autophagic activity by approximately 50%. In cells expressing Bcl-2-ER, thapsigargin maximally reduced autophagic flux. Thus, our results demonstrate that Bcl-2 negatively regulated the autophagic response at the level of S/ER calcium content rather than via direct interaction with Beclin 1. Moreover, we identify calcium homeostasis as an essential component of the autophagic response to nutrient deprivation.

Reactive oxygen species (ROS) can trigger a transient burst of mitochondrial ROS production via ROS activation of the mitochondrial permeability transition pore (MPTP), a phenomenon termed ROS-induced ROS release (RIRR). The goal of this study was to investigate if the generation of ROS in a discrete region of a cardiomyocyte could serve to propagate RIRR-mediated mitochondrial depolarizations throughout a cell. Our experiments revealed that localized RIRR activated either RIRR-mediated fluctuations in mitochondrial membrane potential (time period: 3-10 min) or a traveling wave of depolarization of the cell's mitochondria (velocity: approximately 5 microm/min). Both phenomena appeared to be mediated by the mitochondrial permeability transition pore and eventually encompassed the majority of the mitochondrial population of both isolated rat and rabbit cardiomyocytes. Furthermore, depolarization was often reversible; the waves of depolarization were then followed by a rapid (approximately 40 microm/min) repolarization wave of the mitochondria. We show that the RIRR can function to communicate the mitochondrial permeability transition from one mitochondrion to another in the isolated adult cardiomyocyte.

Bnip3 is a member of the 'BH3-only' Bcl-2 subfamily which has been implicated in apoptotic,(1) necrotic(2) and autophagic cell death.(3,4) We recently reported that Bnip3 is a key mediator of mitochondrial dysfunction and cell death in the ex vivo heart following ischemia/reperfusion (I/R).(5) Moreover, we found that Bnip3 was involved in upregulation of autophagy in I/R and that Bnip3-mediated mitochondrial dysfunction correlated with upregulation of autophagy. Using a model of simulated I/R and overexpression of Bnip3 in HL-1 cardiac myocytes, we determined that Bnip3-mediated upregulation of autophagic activity constituted a protective response against Bnip3 death signaling. Here we present additional evidence that enhanced autophagic activity functions as a cytoprotective pathway to oppose ischemia/reperfusion-related apoptosis.

The objective of this study was to evaluate mitochondrial alterations in a cell-based model of myocardial ischemia/reperfusion (I/R) injury. Using GFP-biosensors and fluorescence deconvolution microscopy, we investigated mitochondrial morphology in relation to Bax and Bid activation in the HL-1 cardiac cell line. Mitochondria underwent extensive fragmentation during ischemia. Bax translocation from cytosol to mitochondria was initiated during ischemia and proceeded during reperfusion. However, Bax translocation was not sufficient to induce cell death or mitochondrial dysfunction. Bid processing was caspase-8 dependent, and Bid translocation to mitochondria occurred after Bax translocation and clustering, and minutes before cell death. Clustering of Bax into distinct regions on mitochondria could be prevented by CsA, an inhibitor of the mitochondrial permeability transition pore, and also by SB203580, an inhibitor of p38 MAPK. Surprisingly, mitochondrial fragmentation which occurred during ischemia and before Bax translocation could be reversed by the addition of the p38 inhibitor SB203580 at reperfusion. Taken together, these results implicate p38 MAPK in the mitochondrial remodeling response to I/R that facilitates Bax recruitment to mitochondria.

Exposure to high glucose (HG) stimulates reactive oxygen species (ROS) production by NADPH oxidase in cardiomyocytes, but the underlying mechanism remains elusive. In this study, we have dissected the link between glucose transport and metabolism and NADPH oxidase activation under hyperglycaemic conditions.Primary cultures of adult rat cardiomyocytes were exposed to HG concentration (HG, 21 mM) and compared with the normal glucose level (LG, 5 mM). HG exposure activated Rac1GTP and induced p47phox translocation to the plasma membrane, resulting in NADPH oxidase (NOX2) activation, increased ROS production, insulin resistance, and eventually cell death. Comparison of the level of O-linked N-acetylglucosamine (O-GlcNAc) residues in LG- and HG-treated cells did not reveal any significant difference. Inhibition of the pentose phosphate pathway (PPP) by 6-aminonicotinamide counteracted ROS production in response to HG but did not prevent Rac-1 upregulation and p47phox translocation leading to NOX2 activation. Modulation of glucose uptake barely affected oxidative stress and toxicity induced by HG. More interestingly, non-metabolizable glucose analogues (i.e. 3-O-methyl-D-glucopyranoside and α-methyl-D-glucopyranoside) reproduced the toxic effect of HG. Inhibition of the sodium/glucose cotransporter SGLT1 by phlorizin counteracted HG-induced NOX2 activation and ROS production.Increased glucose metabolism by itself does not trigger NADPH oxidase activation, although PPP is required to provide NOX2 with NADPH and to produce ROS. NOX2 activation results from glucose transport through SGLT1, suggesting that an extracellular metabolic signal transduces into an intracellular ionic signal.

Intrinsic apoptosis involves BH3-only protein activation of Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP). Consequently, cytochrome c is released from the mitochondria to activate caspases, and Smac (second mitochondria-derived activator of caspases) to inhibit XIAP-mediated caspase suppression. Dysfunctional mitochondria can be targeted for lysosomal degradation via autophagy (mitophagy), or directly through mitochondria-derived vesicle transport. However, the extent of autophagy and lysosomal interactions with apoptotic mitochondria remains largely unknown. We describe here a novel pathway of endolysosomal processing of mitochondria, activated in response to canonical BH3-only proteins and mitochondrial depolarization. We report that expression of canonical BH3-only proteins, tBid, BimEL, Bik, Bad, and mitophagy receptor mutants of atypical BH3-only proteins, Bnip3 and Bnip3L/Nix, leads to prominent relocalization of endolysosomes into inner mitochondrial compartments, in a manner independent of mitophagy. As an upstream regulator, we identified the XIAP E3 ligase. In response to mitochondrial depolarization, XIAP actuates Bax-mediated MOMP, even in the absence of BH3-only protein signaling. Subsequently, in an E3 ligase-dependent manner, XIAP rapidly localizes inside all the mitochondria, and XIAP-mediated mitochondrial ubiquitylation catalyses interactions of Rab membrane targeting components Rabex-5 and Rep-1 (RFP-tagged Rab escort protein-1), and Rab5- and Rab7-positive endolysosomes, at and within mitochondrial membrane compartments. While XIAP-mediated MOMP permits delayed cytochrome c release, within the mitochondria XIAP selectively signals lysosome- and proteasome-associated degradation of its inhibitor Smac. These findings suggest a general mechanism to lower the mitochondrial apoptotic potential via intramitochondrial degradation of Smac.

Mitochondrial outer membrane permeabilization (MOMP) is a core event in apoptosis signaling. However, the underlying mechanism of BAX and BAK pore formation remains incompletely understood. We demonstrate that mitochondria are globally and dynamically targeted by endolysosomes (ELs) during MOMP. In response to pro-apoptotic BH3-only protein signaling and pharmacological MOMP induction, ELs increasingly form transient contacts with mitochondria. Subsequently, ELs rapidly accumulate within the entire mitochondrial compartment. This switch-like accumulation period temporally coincides with mitochondrial BAX clustering and cytochrome c release. Remarkably, interactions of ELs with mitochondria control BAX recruitment and pore formation. Knockdown of Rab5A, Rab5C, or USP15 interferes with EL targeting of mitochondria and functionally uncouples BAX clustering from cytochrome c release, while knockdown of the Rab5 exchange factor Rabex-5 impairs both BAX clustering and cytochrome c release. Together, these data reveal that EL-mitochondrial inter-organelle communication is an integral regulatory component of functional MOMP execution during cellular apoptosis signaling.

Gleason scoring is used within a five-tier risk stratification system to guide therapeutic decisions for patients with prostate cancer. This study aimed to compare the predictive performance of routine H&E or biomarker-assisted ISUP (International Society of Urological Pathology) grade grouping for assessing the risk of biochemical recurrence (BCR) and clinical recurrence (CR) in patients with prostate cancer. This retrospective study was an assessment of 114 men with prostate cancer who provided radical prostatectomy samples to the Australian Prostate Cancer Bioresource between 2006 and 2014. The prediction of CR was the primary outcome (median time to CR 79.8 months), and BCR was assessed as a secondary outcome (median time to BCR 41.7 months). The associations of (1) H&E ISUP grade groups and (2) modified ISUP grade groups informed by the Appl1, Sortilin and Syndecan-1 immunohistochemistry (IHC) labelling were modelled with BCR and CR using Cox proportional hazard approaches. IHC-assisted grading was more predictive than H&E for BCR (C-statistic 0.63 vs. 0.59) and CR (C-statistic 0.71 vs. 0.66). On adjusted analysis, IHC-assisted ISUP grading was independently associated with both outcome measures. IHC-assisted ISUP grading using the biomarker panel was an independent predictor of individual BCR and CR. Prospective studies are needed to further validate this biomarker technology and to define BCR and CR associations in real-world cohorts.

The search for cancer cell-specific targets suffers from a lack of integrative approaches that take into account the relative contributions of several mechanisms or pathways involved in cell death. A systematic experimental and computational comparison of murine glioma cells with astrocytes, their nontransformed counterparts, identified differences in the sphingolipid (SL) rheostat linked to an increased lysosomal instability in glioma cells. In vitro and in silico analyses indicate that sphingosine metabolized in lysosomes was preferentially recycled into ceramide, the prodeath member of the rheostat, in astrocytes. In glioma cells, it preferentially was used for production of the prosurvival sphingosine-1-phosphate (S1P). A combination of tumor necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS), and interferon gamma (IFN-gamma) strongly decreased S1P production that resulted in abnormal lysosome enlargement and cell death associated with mitochondrial dysfunction of glioma cells only. Lack of intracellular S1P in glioma cells was concomitant with protein and lipid accumulation in enlarged lysosomes, indicating a blockade in lysosome recycling, and hence a role for S1P in membrane trafficking. A pharmacological sphingosine kinase inhibitor efficiently replaced the TNF-alpha, LPS, and IFN-gamma combination and killed murine and human glioma cells without affecting astrocytes. Our study provides evidence for a novel mechanism of lysosomal death dependent upon the SL rheostat that can be specifically triggered in glioma cells. It further strengthens the potential of cancer therapies based on specific ceramide pathway alterations.

Mitochondria exist as a network of interconnected organelles undergoing constant fission and fusion. Current approaches to study mitochondrial morphology are limited by low data sampling coupled with manual identification and classification of complex morphological phenotypes. Here we propose an integrated mechanistic and data-driven modeling approach to analyze heterogeneous, quantified datasets and infer relations between mitochondrial morphology and apoptotic events. We initially performed high-content, multi-parametric measurements of mitochondrial morphological, apoptotic, and energetic states by high-resolution imaging of human breast carcinoma MCF-7 cells. Subsequently, decision tree-based analysis was used to automatically classify networked, fragmented, and swollen mitochondrial subpopulations, at the single-cell level and within cell populations. Our results revealed subtle but significant differences in morphology class distributions in response to various apoptotic stimuli. Furthermore, key mitochondrial functional parameters including mitochondrial membrane potential and Bax activation, were measured under matched conditions. Data-driven fuzzy logic modeling was used to explore the non-linear relationships between mitochondrial morphology and apoptotic signaling, combining morphological and functional data as a single model. Modeling results are in accordance with previous studies, where Bax regulates mitochondrial fragmentation, and mitochondrial morphology influences mitochondrial membrane potential. In summary, we established and validated a platform for mitochondrial morphological and functional analysis that can be readily extended with additional datasets. We further discuss the benefits of a flexible systematic approach for elucidating specific and general relationships between mitochondrial morphology and apoptosis.

Apoptosis is essential to prevent oncogenic transformation by triggering self-destruction of harmful cells, including those unable to differentiate. However, the mechanisms linking impaired cell differentiation and apoptosis during development and disease are not well understood. Here we report that the Drosophila transcription factor Cut coordinately controls differentiation and repression of apoptosis via direct regulation of the pro-apoptotic gene reaper. We also demonstrate that this regulatory circuit acts in diverse cell lineages to remove uncommitted precursor cells in status nascendi and thereby interferes with their potential to develop into cancer cells. Consistent with the role of Cut homologues in controlling cell death in vertebrates, we find repression of apoptosis regulators by Cux1 in human cancer cells. Finally, we present evidence that suggests that other lineage-restricted specification factors employ a similar mechanism to put the brakes on the oncogenic process.

Efficient apoptosis requires Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), which releases death-promoting proteins cytochrome c and Smac to the cytosol, which activate apoptosis and inhibit X-linked inhibitor of apoptosis protein (XIAP) suppression of executioner caspases, respectively. We recently identified that in response to Bcl-2 homology domain 3 (BH3)-only proteins and mitochondrial depolarization, XIAP can permeabilize and enter mitochondria. Consequently, XIAP E3 ligase activity recruits endolysosomes into mitochondria, resulting in Smac degradation. Here, we explored mitochondrial XIAP action within the intrinsic apoptosis signaling pathway. Mechanistically, we demonstrate that mitochondrial XIAP entry requires Bax or Bak and is antagonized by pro-survival Bcl-2 proteins. Moreover, intramitochondrial Smac degradation by XIAP occurs independently of Drp1-regulated cytochrome c release. Importantly, mitochondrial XIAP actions are activated cell-intrinsically by typical apoptosis inducers TNF and staurosporine, and XIAP overexpression reduces the lag time between the administration of an apoptotic stimuli and the onset of mitochondrial permeabilization. To elucidate the role of mitochondrial XIAP action during apoptosis, we integrated our findings within a mathematical model of intrinsic apoptosis signaling. Simulations suggest that moderate increases of XIAP, combined with mitochondrial XIAP preconditioning, would reduce MOMP signaling. To test this scenario, we pre-activated XIAP at mitochondria via mitochondrial depolarization or by artificially targeting XIAP to the intermembrane space. Both approaches resulted in suppression of TNF-mediated caspase activation. Taken together, we propose that XIAP enters mitochondria through a novel mode of mitochondrial permeabilization and through Smac degradation can compete with canonical MOMP to act as an anti-apoptotic tuning mechanism, reducing the mitochondrial contribution to the cellular apoptosis capacity. Efficient apoptosis requires Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), which releases death-promoting proteins cytochrome c and Smac to the cytosol, which activate apoptosis and inhibit X-linked inhibitor of apoptosis protein (XIAP) suppression of executioner caspases, respectively. We recently identified that in response to Bcl-2 homology domain 3 (BH3)-only proteins and mitochondrial depolarization, XIAP can permeabilize and enter mitochondria. Consequently, XIAP E3 ligase activity recruits endolysosomes into mitochondria, resulting in Smac degradation. Here, we explored mitochondrial XIAP action within the intrinsic apoptosis signaling pathway. Mechanistically, we demonstrate that mitochondrial XIAP entry requires Bax or Bak and is antagonized by pro-survival Bcl-2 proteins. Moreover, intramitochondrial Smac degradation by XIAP occurs independently of Drp1-regulated cytochrome c release. Importantly, mitochondrial XIAP actions are activated cell-intrinsically by typical apoptosis inducers TNF and staurosporine, and XIAP overexpression reduces the lag time between the administration of an apoptotic stimuli and the onset of mitochondrial permeabilization. To elucidate the role of mitochondrial XIAP action during apoptosis, we integrated our findings within a mathematical model of intrinsic apoptosis signaling. Simulations suggest that moderate increases of XIAP, combined with mitochondrial XIAP preconditioning, would reduce MOMP signaling. To test this scenario, we pre-activated XIAP at mitochondria via mitochondrial depolarization or by artificially targeting XIAP to the intermembrane space. Both approaches resulted in suppression of TNF-mediated caspase activation. Taken together, we propose that XIAP enters mitochondria through a novel mode of mitochondrial permeabilization and through Smac degradation can compete with canonical MOMP to act as an anti-apoptotic tuning mechanism, reducing the mitochondrial contribution to the cellular apoptosis capacity.

Mitochondria are semi-autonomous organelles that supply energy for cellular biochemistry through oxidative phosphorylation. Within a cell, hundreds of mobile mitochondria undergo fusion and fission events to form a dynamic network. These morphological and mobility dynamics are essential for maintaining mitochondrial functional homeostasis, and alterations both impact and reflect cellular stress states. Mitochondrial homeostasis is further dependent on production (biogenesis) and the removal of damaged mitochondria by selective autophagy (mitophagy). While mitochondrial function, dynamics, biogenesis and mitophagy are highly-integrated processes, it is not fully understood how systemic control in the cell is established to maintain homeostasis, or respond to bioenergetic demands. Here we used agent-based modeling (ABM) to integrate molecular and imaging knowledge sets, and simulate population dynamics of mitochondria and their response to environmental energy demand. Using high-dimensional parameter searches we integrated experimentally-measured rates of mitochondrial biogenesis and mitophagy, and using sensitivity analysis we identified parameter influences on population homeostasis. By studying the dynamics of cellular subpopulations with distinct mitochondrial masses, our approach uncovered system properties of mitochondrial populations: (1) mitochondrial fusion and fission activities rapidly establish mitochondrial sub-population homeostasis, and total cellular levels of mitochondria alter fusion and fission activities and subpopulation distributions; (2) restricting the directionality of mitochondrial mobility does not alter morphology subpopulation distributions, but increases network transmission dynamics; and (3) maintaining mitochondrial mass homeostasis and responding to bioenergetic stress requires the integration of mitochondrial dynamics with the cellular bioenergetic state. Finally, (4) our model suggests sources of, and stress conditions amplifying, cell-to-cell variability of mitochondrial morphology and energetic stress states. Overall, our modeling approach integrates biochemical and imaging knowledge, and presents a novel open-modeling approach to investigate how spatial and temporal mitochondrial dynamics contribute to functional homeostasis, and how subcellular organelle heterogeneity contributes to the emergence of cell heterogeneity.

Mitochondria are key regulators of apoptosis. In response to stress, BH3-only proteins activate pro-apoptotic Bcl2 family proteins Bax and Bak, which induce mitochondrial outer membrane permeabilization (MOMP). While the large-scale mitochondrial release of pro-apoptotic proteins activates caspase-dependent cell death, a limited release results in sub-lethal caspase activation which promotes tumorigenesis. Mitochondrial autophagy (mitophagy) targets dysfunctional mitochondria for degradation by lysosomes, and undergoes extensive crosstalk with apoptosis signaling, but its influence on apoptosis remains undetermined. The BH3-only protein Bnip3 integrates apoptosis and mitophagy signaling at different signaling domains. Bnip3 inhibits pro-survival Bcl2 members via its BH3 domain and activates mitophagy through its LC3 Interacting Region (LIR), which is responsible for binding to autophagosomes. Previously, we have shown that Bnip3-activated mitophagy prior to apoptosis induction can reduce mitochondrial activation of caspases, suggesting that a reduction to mitochondrial levels may be pro-survival. An outstanding question is whether organelle dynamics and/or recently discovered subcellular variations of protein levels responsible for both MOMP sensitivity and crosstalk between apoptosis and mitophagy can influence the cellular apoptosis decision event. To that end, here we undertook a systems biology analysis of mitophagy-apoptosis crosstalk at the level of cellular mitochondrial populations.Based on experimental findings, we developed a multi-scale, hybrid model with an individually adaptive mitochondrial population, whose actions are determined by protein levels, embedded in an agent-based model (ABM) for simulating subcellular dynamics and local feedback via reactive oxygen species signaling. Our model, supported by experimental evidence, identified an emergent regulatory structure within canonical apoptosis signaling. We show that the extent of mitophagy is determined by levels and spatial localization of autophagy capacity, and subcellular mitochondrial protein heterogeneities. Our model identifies mechanisms and conditions that alter the mitophagy decision within mitochondrial subpopulations to an extent sufficient to shape cellular outcome to apoptotic stimuli.Overall, our modeling approach provides means to suggest new experiments and implement findings at multiple scales in order to understand how network topologies and subcellular heterogeneities can influence signaling events at individual organelle level, and hence, determine the emergence of heterogeneity in cellular decisions due the actions of the collective intra-cellular population.

A genetic mutation in the C9orf72 gene causes the most common forms of neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The C9orf72 protein, predicted to be a DENN-family protein, is reduced in ALS and FTD, but its functions remain poorly understood. Using a 3110043O21Rik/C9orf72 knockout mouse model, as well as cellular analysis, we have found that loss of C9orf72 causes alterations in the signaling states of central autophagy regulators. In particular, C9orf72 depletion leads to reduced activity of MTOR, a negative regulator of macroautophagy/autophagy, and concomitantly increased TFEB levels and nuclear translocation. Consistent with these alterations, cells exhibit enlarged lysosomal compartments and enhanced autophagic flux. Loss of the C9orf72 interaction partner SMCR8 results in similar phenotypes. Our findings suggest that C9orf72 functions as a potent negative regulator of autophagy, with a central role in coupling the cellular metabolic state with autophagy regulation. We thus propose C9orf72 as a fundamental component of autophagy signaling with implications in basic cell physiology and pathophysiology, including neurodegeneration.

Abstract By interrogating metabolic programs in the peripheral blood mononuclear cells (PBMC) of acutely infected COVID-19 patients, we identified novel and distinct immune cell subsets Our studies identified a non-clonal population of T cells expressing high H3K27me3 and voltage-dependent anion channel (VDAC) with mitochondrial dysfunction and increased susceptibility to cell death. Characterized by dysmorphic mitochondria and increased cytoplasmic cytochrome c , apoptosis of these cells was inhibited by preventing VDAC aggregation or blocking caspase activation. Further, we observed a marked increase in Hexokinase II + polymorphonuclear-myeloid derived suppressor cells (PMN-MDSC). While PMN-MDSC were also found in the PBMC of patients with other viral infections, the Hexokinase II + PMN-MDSC were found exclusively in the acute COVID-19 patients with moderate or severe disease. Finally, we identified a population of monocytic MDSC (M-MDSC) expressing high carnitine palmitoyltransferase I (CPT1a) and VDAC, which were present in the PBMC of the acute COVID-19 patients, but not recovered COVID-19 patients and whose presence correlated with severity of disease. Overall, these unique populations of immune cells provide insight into the pathogenesis of SARS-CoV-2 infection and provide a means to predict and track disease severity as well as an opportunity to design and evaluate novel therapeutic regimens. One Sentence Summary Metabolic programs define unique immune cells among COVID-19 patients with severe diease.

Neuroblastoma is the most common extracranial solid tumor in childhood. Despite intense multimodal therapy and many improvements through basic scientific and clinical research, the successful response of advanced-stage patients to chemotherapy remains poor. Autophagy is a cytoprotective mechanism that may help advanced cancer cells survive stressful conditions such as chemotherapy. Here we review our recent findings describing HDAC10 as a promoter of autophagy-mediated survival in neuroblastoma cells and identifying this HDAC isozyme as a druggable regulator of advanced-stage tumor cell survival. These results propose a new and promising way to considerably improve treatment response in the neuroblastoma patient subgroup with the poorest outcome.

The majority of melanomas have been shown to harbor somatic mutations in the RAS-RAF-MEK-MAPK and PI3K-AKT pathways, which play a major role in regulation of proliferation and survival. The prevalence of these mutations makes these kinase signal transduction pathways an attractive target for cancer therapy. However, tumors have generally shown adaptive resistance to treatment. This adaptation is achieved in melanoma through its ability to undergo neovascularization, migration and rearrangement of signaling pathways. To understand the dynamic, nonlinear behavior of signaling pathways in cancer, several computational modeling approaches have been suggested. Most of those models require that the pathway topology remains constant over the entire observation period. However, changes in topology might underlie adaptive behavior to drug treatment. To study signaling rearrangements, here we present a new approach based on Fuzzy Logic (FL) that predicts changes in network architecture over time. This adaptive modeling approach was used to investigate pathway dynamics in a newly acquired experimental dataset describing total and phosphorylated protein signaling over four days in A375 melanoma cell line exposed to different kinase inhibitors. First, a generalized strategy was established to implement a parameter-reduced FL model encoding non-linear activity of a signaling network in response to perturbation. Next, a literature-based topology was generated and parameters of the FL model were derived from the full experimental dataset. Subsequently, the temporal evolution of model performance was evaluated by leaving time-defined data points out of training. Emerging discrepancies between model predictions and experimental data at specific time points allowed the characterization of potential network rearrangement. We demonstrate that this adaptive FL modeling approach helps to enhance our mechanistic understanding of the molecular plasticity of melanoma.

Autophagy is a vesicle-mediated pathway for lysosomal degradation, essential under basal and stressed conditions. Various cellular components, including specific proteins, protein aggregates, organelles and intracellular pathogens, are targets for autophagic degradation. Thereby, autophagy controls numerous vital physiological and pathophysiological functions, including cell signaling, differentiation, turnover of cellular components and pathogen defense. Moreover, autophagy enables the cell to recycle cellular components to metabolic substrates, thereby permitting prolonged survival under low nutrient conditions. Due to the multi-faceted roles for autophagy in maintaining cellular and organismal homeostasis and responding to diverse stresses, malfunction of autophagy contributes to both chronic and acute pathologies.We applied a systems biology approach to improve the understanding of this complex cellular process of autophagy. All autophagy pathway vesicle activities, i.e. creation, movement, fusion and degradation, are highly dynamic, temporally and spatially, and under various forms of regulation. We therefore developed an agent-based model (ABM) to represent individual components of the autophagy pathway, subcellular vesicle dynamics and metabolic feedback with the cellular environment, thereby providing a framework to investigate spatio-temporal aspects of autophagy regulation and dynamic behavior. The rules defining our ABM were derived from literature and from high-resolution images of autophagy markers under basal and activated conditions. Key model parameters were fit with an iterative method using a genetic algorithm and a predefined fitness function. From this approach, we found that accurate prediction of spatio-temporal behavior required increasing model complexity by implementing functional integration of autophagy with the cellular nutrient state. The resulting model is able to reproduce short-term autophagic flux measurements (up to 3 hours) under basal and activated autophagy conditions, and to measure the degree of cell-to-cell variability. Moreover, we experimentally confirmed two model predictions, namely (i) peri-nuclear concentration of autophagosomes and (ii) inhibitory lysosomal feedback on mTOR signaling.Agent-based modeling represents a novel approach to investigate autophagy dynamics, function and dysfunction with high biological realism. Our model accurately recapitulates short-term behavior and cell-to-cell variability under basal and activated conditions of autophagy. Further, this approach also allows investigation of long-term behaviors emerging from biologically-relevant alterations to vesicle trafficking and metabolic state.

Monoclonal antibodies (mAbs) have emerged as a promising tool for cancer therapy. Differing approaches utilize mAbs to either deliver a drug to the tumor cells or to modulate the host's immune system to mediate tumor kill. The rate by which a therapeutic antibody is being internalized by tumor cells is a decisive feature for choosing the appropriate treatment strategy. We herein present a novel method to effectively quantitate antibody uptake of tumor cells by using image-based flow cytometry, which combines image analysis with high throughput of sample numbers and sample size. The use of this method is established by determining uptake rate of an anti-EpCAM antibody (HEA125), from single cell measurements of plasma membrane versus internalized antibody, in conjunction with inhibitors of endocytosis. The method is then applied to two mAbs (L1-9.3, L1-OV52.24) targeting the neural cell adhesion molecule L1 (L1CAM) at two different epitopes. Based on median cell population responses, we find that mAb L1-OV52.24 is rapidly internalized by the ovarian carcinoma cell line SKOV3ip while L1 mAb 9.3 is mainly retained at the cell surface. These findings suggest the L1 mAb OV52.24 as a candidate to be further developed for drug-delivery to cancer cells, while L1-9.3 may be optimized to tag the tumor cells and stimulate immunogenic cancer cell killing. Furthermore, when analyzing cell-to-cell variability, we observed L1 mAb OV52.24 rapidly transition into a subpopulation with high-internalization capacity. In summary, this novel high-content method for measuring antibody internalization rate provides a high level of accuracy and sensitivity for cell population measurements and reveals further biologically relevant information when taking into account cellular heterogeneity.

Chronic lymphocytic leukemia (CLL) cells fail to enter apoptosis in vivo as opposed to their non-malignant B-lymphocyte counterparts. The ability of CLL cells to escape apoptosis is highly dependent on their microenvironment. Compared to non-malignant B cells, CLL cells are more responsive to complex stimuli that can be reproduced in vitro by the addition of cytokines. To understand the molecular mechanism of the environment-dependent anti-apoptotic signaling circuitry of CLL cells, we quantified the effect of the SDF-1, BAFF, APRIL, anti-IgM, interleukin-4 (IL4) and secreted CD40L (sCD40L) on the survival of in vitro cultured CLL cells and found IL4 and sCD40L to be most efficient in rescuing CLL cells from apoptosis. In quantitative dose–response experiments using cell survival as readout, the binding affinity of IL4 to its receptor was similar between malignant and non-malignant cells. However, the downstream signaling in terms of the amount of STAT6 and its degree of phosphorylation was highly stimulated in CLL cells. In contrast, the response to sCD40L showed a loss of cooperative binding in CLL cells but displayed a largely increased ligand binding affinity. Although a high-throughput microscopy analysis did not reveal a significant difference in the spatial CD40 receptor organization, the downstream signaling showed an enhanced activation of the NF-kB pathway in the malignant cells. Thus, we propose that the anti-apoptotic phenotype of CLL involves a sensitized response for IL4 dependent STAT6 phosphorylation, and an activation of NF-kB signaling due to an increased affinity of sCD40L to its receptor.

Epidermal growth factor (EGF) receptor (EGFR) overexpression is a hallmark of many cancers. EGFR endocytosis is a critical step in signal attenuation, raising the question of how receptor expression levels affect the internalization process. Here we combined quantitative experimental and mathematical modeling approaches to investigate the role of the EGFR expression level on the rate of receptor internalization. Using tetramethylrhodamine-labeled EGF, we established assays for quantifying EGF-triggered EGFR internalization by both high resolution confocal microscopy and flow cytometry. We determined that the flow cytometry approach was more sensitive for examining large populations of cells. Mathematical modeling was used to investigate the relationship between EGF internalization kinetics, EGFR expression, and internalization machinery. We predicted that the standard parameter used to assess internalization kinetics, the temporal evolution r(t) of the ratio of internalized versus surface-located ligand.receptor complexes, does not describe a straight line, as proposed previously. Instead, a convex or concave curve occurs depending on whether initial receptor numbers or internalization adaptors are limiting the uptake reaction, respectively. To test model predictions, we measured EGF-EGFR binding and internalization in cells expressing different levels of green fluorescent protein-EGFR. As expected, surface binding of rhodamine-labeled EGF increased with green fluorescent protein-EGFR expression level. Unexpectedly, internalization of ligand. receptor complexes increased linearly with increasing receptor expression level, suggesting that receptors and not internalization adaptors were limiting the uptake in our experimental model. Finally, determining the ratio of internalized versus surface-located ligand.receptor complexes for this cell line confirmed that it follows a convex curve, supporting our model predictions.

TFEB (transcription factor EB) regulates metabolic homeostasis through its activation of lysosomal biogenesis following its nuclear translocation. TFEB activity is inhibited by mTOR phosphorylation, which signals its cytoplasmic retention. To date, the temporal relationship between alterations to mTOR activity states and changes in TFEB subcellular localization and concentration has not been sufficiently addressed. mTOR was activated by renewed addition of fully-supplemented medium, or inhibited by Torin1 or nutrient deprivation. Single-cell TFEB protein levels and subcellular localization in HeLa and MCF7 cells were measured over a time course of 15 hours by multispectral imaging cytometry. To extract single-cell level information on heterogeneous TFEB activity phenotypes, we developed a framework for identification of TFEB activity subpopulations. Through unsupervised clustering, cells were classified according to their TFEB nuclear concentration, which corresponded with downstream lysosomal responses. Bulk population results revealed that mTOR negatively regulates TFEB protein levels, concomitantly to the regulation of TFEB localization. Subpopulation analysis revealed maximal sensitivity of HeLa cells to mTOR activity stimulation, leading to inactivation of 100 % of the cell population within 0.5 hours, which contrasted with a lower sensitivity in MCF7 cells. Conversely, mTOR inhibition increased the fully active subpopulation only fractionally, and full activation of 100 % of the population required co-inhibition of mTOR and the proteasome. Importantly, mTOR inhibition activated TFEB for a limited duration of 1.5 hours, and thereafter the cell population was progressively re-inactivated, with distinct kinetics for Torin1 and nutrient deprivation treatments. TFEB protein levels and subcellular localization are under control of a short-term rheostat, which is highly responsive to negative regulation by mTOR, but under conditions of mTOR inhibition, restricts TFEB activation in a manner dependent on the proteasome. We further identify a long-term, mTOR-independent homeostatic control negatively regulating TFEB upon prolonged mTOR inhibition. These findings are of relevance for developing strategies to target TFEB activity in disease treatment. Moreover, our quantitative approach to decipher phenotype heterogeneity in imaging datasets is of general interest, as shifts between subpopulations provide a quantitative description of single cell behaviour, indicating novel regulatory behaviors and revealing differences between cell types.

Tumour metastasis accounts for over 90% of cancer related deaths. The platelet is a key blood component, which facilitates efficient metastasis. This study aimed to understand the molecular mechanisms involved in tumour-platelet cell interactions. The interaction between cancer cells and platelets was examined in 15 epithelial cell lines, representing 7 cancer types. Gene expression analysis of EMT-associated and cancer stemness genes was performed by RT-PCR. Whole transcriptome analysis (WTA) was performed using Affymetrix 2.0ST arrays on a platelet co-cultured ovarian model. Platelet adhesion and activation occurred across all tumour types. WTA identified increases in cellular movement, migration, invasion, adhesion, development, differentiation and inflammation genes and decreases in processes associated with cell death and survival following platelet interaction. Increased invasive capacity was also observed in a subset of cell lines. A cross-comparison with a platelet co-cultured mouse model identified 5 common altered genes; PAI-1, PLEK2, CD73, TNC, and SDPR. Platelet cancer cell interactions are a key factor in driving the pro-metastatic phenotype and appear to be mediated by 5 key genes which have established roles in metastasis. Targeting these metastasis mediators could improve cancer patient outcomes.

&lt;div class="section abstract"&gt;&lt;div class="htmlview paragraph"&gt;The application of cooperative adaptive cruise control (CACC) to heavy-duty trucks known as truck platooning has shown fuel economy improvements over test track ideal driving conditions. However, there are limited test data available to assess the performance of CACC under real-world driving conditions. As part of the Cummins-led U.S. Department of Energy Funding Opportunity Announcement award project, truck platooning with CACC has been tested under real-world driving conditions and the results are presented in this paper. First, real-world driving conditions are characterized with the National Renewable Energy Laboratory’s Fleet DNA database to define the test factors. The key test factors impacting long-haul truck fuel economy were identified as terrain and highway traffic with and without advanced driver-assistance systems (ADAS). Track and on-highway testing guided by SAE J1321 procedures were conducted to assess truck platooning operation under the characterized real-world driving conditions. On-highway testing is done on a route in Indiana representing operation of long-haul Class 8 trucks in the United States. The road includes low-, medium-, and high-grade segments. The highway test results of a two-truck platooning configuration indicate considerable fuel-saving reduction comparing to the test track data collected under ideal driving conditions. The test data indicate that platooning could lead to increases in fuel consumption during traffic or high-grade portions of the route, causing reduction of the overall fuel saving on the road comparing to test track results. However, integration of ADAS features on the lead truck during on-road tests leads to significant improvement of fuel saving for both trucks in CACC operation.&lt;/div&gt;&lt;/div&gt;

Abstract Damaged mitochondria can be subject to lysosomal degradation via mitophagy. However, whole-organelle degradation exhibits relatively slow kinetics and thus its impact may be limited in response to acute, fast-acting cellular stress. We previously reported that in Parkin-deficient cells endolysosomes directly target mitochondria when subjected to bioenergetic stress. Here, using high-resolution live cell imaging we reveal a striking level of dynamic targeting of Rab5+ early endosomes to stressed mitochondria, culminating in a switch-like accumulation in the entire mitochondrial population, independently of canonical autophagy. This process of rapid, largescale Rab5+ vesicle trafficking to mitochondria coincides with, and is mediated by, XIAP E3 ligase activated mitochondrial ubiquitylation and results in ultrastructural changes to, and degradation of, intra-mitochondrial components. Mitochondria-targeting vesicles include early endosomal subpopulations marked by Rab5 effector APPL1 and ubiquitin-binding endocytic adaptors OPTN, TAX1BP1 and Tollip, and Rab7-positive late endosomes/lysosomes. In Parkin expressing cells, XIAP- and Parkin-dependent mitochondrial targeting and resulting processing modes are competitively regulated. Together, our data suggest that XIAP-mediated targeting of endolysosomes to mitochondria functions as a stress-responsive, sub-organelle level mitochondrial processing mode that is distinct from, and competitive to, Parkin-mediated mitophagy.

Extracellular vesicles (EVs) are produced by all domains of life including Bacteria, Archaea and Eukarya. EVs are critical for cellular physiology and contain varied cargo: virulence factors, cell wall remodeling enzymes, extracellular matrix components and even nucleic acids and metabolites. While various protocols for isolating EVs have been established for mammalian cells, the field is actively developing tools to study EVs in other organisms. In this protocol we describe our methods to perform density gradient purification of EVs in bacterial cells, allowing for separation of EV subpopulations, followed by protection assays for EV cargo characterization. Furthermore, we devised a protocol which incorporates a fluorescent conjugate of fatty acids into EVs, the first to allow live-cell EV tracking to observe release of EVs, including during infection of mammalian cells by pathogenic bacteria. These protocols are powerful tools for EV researchers as they enable the observation of EV release and the study of the mechanisms of their formation and release.

Abstract Medulloblastoma comprises the most common malignant brain tumor in childhood. Recently, integrated genomic approaches revealed four major biological disease variants: WNT (wingless), SHH (sonic hedgehog), group 3, and group 4. Treatment failure mainly occurs in children harboring metastatic tumors, which typically carry an isochromosome 17 or gain of 17q, a common hallmark of intermediate and high-risk non-WNT/non-SHH medulloblastoma. Thus, novel therapeutic options for these patients are urgently warranted. Through mRNA expression profiling of 64 primary tumor samples, we identified potassium inwardly-rectifying channel J2 (KCNJ2) as one of the most upregulated genes on chromosome 17q in tumors with 17q gain. Notably, recent reports have linked deregulation of voltage-dependent ion channels to the development of other types of cancer. We first validated our microarray findings on KCNJ2 transcript levels using quantitative real-time PCR. High KCNJ2 transcript levels were significantly associated with non-WNT/non-SHH grouping, anaplastic histology, metastatic dissemination, and poor clinical outcome. KCNJ2 protein expression was analyzed by immunohistochemistry in a large cohort of patients (n=199), and high protein expression levels were found to be strongly correlated with 17q gain, metastatic dissemination, and inferior overall and progression-free survival (p&amp;lt;0.0001). To functionally validate the potential role of KCNJ2 in medulloblastoma biology, we performed knockdown experiments by small interfering RNA-mediated silencing in two well characterized medulloblastoma cell lines. Knockdown of KCNJ2 resulted in a reduced proliferation rate and induction of apoptosis. Furthermore, treatment of the medulloblastoma cell lines with Amiodarone and SR 59230A, two inhibitors of this class of Kir channels, phenocopied these promising anti-proliferative and pro-apoptotic effects in a time- and dose-dependent manner. Whole cell patch clamp results revealed a remarkable current reduction upon inhibitor treatment with SR 59230A. In summary, we could delineate KCNJ2 immunopositivity as an independent biomarker for medulloblastoma with dismal prognosis. Thus, pharmacological inhibition of this candidate gene may constitute a new therapeutic option for patients with high-risk medulloblastomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1424. doi:1538-7445.AM2012-1424

ABSTRACT Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secreted extracellular vesicles (EVs) was not pursued due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteriaare implicated in virulence, toxin release and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that is the etiological agent of listeriosis. Here we report that L. monocytogenes produces EVs with diameter ranging from 20-200 nm, containing the pore-forming toxin listeriolysin O(LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Using simultaneous m etabolite, p rotein, and l ipid e xtraction (MPLEx) multi-omics we characterized protein, lipid and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Cell-free EV preparations were toxic to the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC can restrain LLO activity. Using immunogold electron microscopy we detect LLO localization at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes that colocalize with LLO during infection. Our findings demonstrate that L. monocytogenes utilize EVs for toxin release and implicate these structures in mammalian cytotoxicity.

Ultrafast pump-probe spectroscopy shows that the photo-induced monoclinic-to-rutile phase transformation in vanadium dioxide thin films occurs in 40±0.5 ps independent of nanograin morphology, because substrate-induced strain leads to common families of grain orientations.

We use ultrafast pump-probe spectroscopy to demonstrate that the dynamics of the photo-induced structural (monoclinic to rutile) phase transformation in vanadium dioxide is independent of thin-film morphology and substrate-induced strain, and occurs in 40±0.5 ps.

Autophagy is a vesicle-mediated pathway for lysosomal degradation, essential under basal and stressed conditions. Various cellular components, including specific proteins, protein aggregates, organelles and intracellular pathogens, are targets for autophagic degradation. Thereby, autophagy controls numerous vital physiological and pathophysiological functions, including cell signaling, differentiation, turnover of cellular components and pathogen defense. Moreover, autophagy enables the cell to recycle cellular components to metabolic substrates, thereby permitting prolonged survival under low nutrient conditions. Due to the multi-faceted roles for autophagy in maintaining cellular and organismal homeostasis and responding to diverse stresses, malfunction of autophagy contributes to both chronic and acute pathologies. We applied a systems biology approach to improve the understanding of this complex cellular process of autophagy. All autophagy pathway vesicle activities, i.e. creation, movement, fusion and degradation, are highly dynamic, temporally and spatially, and under various forms of regulation. We therefore developed an agent-based model (ABM) to represent individual components of the autophagy pathway, subcellular vesicle dynamics and metabolic feedback with the cellular environment, thereby providing a framework to investigate spatio-temporal aspects of autophagy regulation and dynamic behavior. The rules defining our ABM were derived from literature and from high-resolution images of autophagy markers under basal and activated conditions. Key model parameters were fit with an iterative method using a genetic algorithm and a predefined fitness function. From this approach, we found that accurate prediction of spatio-temporal behavior required increasing model complexity by implementing functional integration of autophagy with the cellular nutrient state. The resulting model is able to reproduce short-term autophagic flux measurements (up to 3 hours) under basal and activated autophagy conditions, and to measure the degree of cell-to-cell variability. Moreover, we experimentally confirmed two model predictions, namely (i) peri-nuclear concentration of autophagosomes and (ii) inhibitory lysosomal feedback on mTOR signaling. Agent-based modeling represents a novel approach to investigate autophagy dynamics, function and dysfunction with high biological realism. Our model accurately recapitulates short-term behavior and cell-to-cell variability under basal and activated conditions of autophagy. Further, this approach also allows investigation of long-term behaviors emerging from biologically-relevant alterations to vesicle trafficking and metabolic state.

Abstract Medulloblastoma comprises the most common malignant brain tumor in children. Non-WNT/SHH tumors define the most refractory medulloblastoma subgroups. Interestingly, 17q gain, the most common genetic aberration in medulloblastoma, comprises a cytogenetic hallmark of these molecular high-risk tumors detected in group 3 (62%), and group 4 (73%). The majority of recurrent tumors harbor 17q gain in the corresponding primary. Virtually all of these tumors develop resistance to current treatment protocols at relapse. The lack of a common molecular target hampers the development of urgently needed novel treatment strategies. Through mRNA expression profiling of 64 primary tumor samples, we identified potassium inwardly-rectifying channel J2 (KCNJ2) as one of the most upregulated genes on chromosome 17q in tumors with 17q gain. High KCNJ2 transcript levels were significantly associated with non-WNT/non-SHH grouping, anaplastic histology, metastatic dissemination, and poor clinical outcome. KCNJ2 protein expression was analyzed by immunohistochemistry in a large cohort of patients (n=199), and high protein expression levels were found to be strongly correlated with 17q gain, metastatic dissemination, and inferior prognosis (p&amp;lt;0.0001). To functionally validate the potential role of KCNJ2 in medulloblastoma biology, we performed knockdown experiments by small interfering RNA-mediated silencing in two well-characterized medulloblastoma cell lines. Transient knockdown of KCNJ2 resulted in a reduced proliferation rate and induction of apoptosis. Furthermore, treatment of the medulloblastoma cell lines and medulloblastoma stem cells with amiodarone and gambogic acid, two inhibitors of this class of Kir channels, phenocopied these effects in a time- and dose-dependent manner. Whole cell patch clamp results revealed a nearly complete current blockade upon inhibitor treatment. Subsequently, we showed that pharmacological inhibition of KCNJ2 and knockdown KCNJ2 significantly reduced tumor growth and resulted in prolonged survival in an orthotopic medulloblastoma mouse model. In summary, our data suggest that pharmacological inhibition of KCNJ2 may constitute a new therapeutic option for patients with high-risk medulloblastomas. Citation Format: Francesca Valdora, Florian Freier, Livia Garzia, Vijay Ramaswamy, Claudia Seyler, Thomas Hielscher, Nathan Brady, Paul A. Northcott, Marcel Kool, David TW Jones, Hendrik Witt, Gian Paolo Tonini, Wolfram Scheurlen, Hugo A. Katus, Andreas E. Kulozik, Edgar Zitron, Andrey Korshunov, Peter Lichter, Michael D. Taylor, Stefan M. Pfister, Marc Remke. KCNJ2 constitutes a marker and therapeutic target of high-risk medulloblastomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5050. doi:10.1158/1538-7445.AM2013-5050

Conclusions and perspectives The pathophysiology of neurodegenerative diseases is attributed to the death of specific neurons, for example the dopaminergic neurons in Parkinson’s Disease (PD). Mitochondrial dysfunction and excessive Reactive Oxygen Species (ROS) generation may play an important role in the development of PD. ROS is produced mostly due to the incomplete reduction of oxygen during oxidative phosphorylation. This process takes place in almost all cells as a side effect of respiratory ATP synthesis. In dopaminergic neurons, the problem of excessive ROS generation is aggravated because of the additional generation of ROS during ROS-induced dopamine degradation. The later is a chain reaction: ROS causes the degradation of dopamine, which in turn produces more ROS. Introduction Detailed network diagram

Abstract FATE1 (fetal and adult testis expressed 1), also known as the cancer-testis antigen BJ-HCC-2, is expressed in testis and tumor tissues. FATE1 was recently described as a major survival factor in tumor cells of various origins, by mediating the degradation of the pro-apoptotic BH3-only protein Bik (Maxfield et al., in Nat Commun 2015 Nov 16;6:8840) and through ER-mitochondrial uncoupling (Doghman-Bouguerra et al., in EMBO Rep 2016 Sep;17(9):1264-80). Interestingly, FATE1 shares high sequence homology with the mitochondrial Drp1 receptor MFF (mitochondrial fission factor). We thus investigated a possible role of FATE1 impact on mitochondrial morphology following the stimulation of apoptosis in cancer cells. We found that, similar to MFF, FATE1 is localized to outer mitochondrial membranes and, unlike MFF, additionally localized to the endoplasmic reticulum. Importantly, in contrast to MFF, FATE1 overexpression does not recruit Drp1 to mitochondria, and instead promotes hyperfusion of mitochondrial networks. Co-immunoprecipitation experiments and reconstitution of Mfn2 or Mfn1 in double knockout mouse embryonic fibroblasts indicate a role for the mitochondrial fusion protein Mfn2, but not Mfn1. As FATE1 overexpressing cancer cells were more resistant to mitochondrial fragmentation in response to TNF and valinomycin treatments, we propose FATE1 as a novel regulator of mitochondrial morphology changes occurring during apoptosis, with possible implications in FATE1-mediated resistance of cancer cells to chemotherapy. Citation Format: Anne Hamacher-Brady, Verena Lang, Nathan R. Brady. FATE1 promotes mitochondrial hyperfusion and supports maintenance of mitochondrial networks following apoptosis stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3324. doi:10.1158/1538-7445.AM2017-3324

Abstract The eminently complex regulatory network protecting the cell against oxidative stress, surfaces in several disease maps, including that of Parkinson’s disease (PD). How this molecular networking achieves its various functionalities and how processes operating at the seconds-minutes time scale cause a disease at a time scale of multiple decennia is enigmatic. By computational analysis, we here disentangle the reactive oxygen species (ROS) regulatory network into a hierarchy of subnetworks that each correspond to a different functionality. The detailed dynamic model of ROS management obtained integrates these functionalities and fits in vitro data sets from two different laboratories. The model shows effective ROS-management for a century, followed by a sudden system’s collapse due to the loss of p62 protein. PD related conditions such as lack of DJ-1 protein or increased α-synuclein accelerated the system’s collapse. Various in-silico interventions (e.g. addition of antioxidants or caffeine) slowed down the collapse of the system in silico , suggesting the model may help discover new medicinal and nutritional therapies.

Apoptotic and autophagic pathways are linked through the interaction of anti-apoptotic Bcl-2 proteins with the autophagy protein Beclin1. However, the nature of the interaction, either in promoting...

Microscopic detection of fluorescent dyes is a powerful tool to monitor dynamics of intracellular parameters, in the living cell. In contrast to green fluorescent proteins (GFPs), which require expertise in molecular biology, the ease at which fluorescent dyes can be used makes them appealing for a larger audience of biologists. In this overview we will highlight certain methodologies and considerations when imaging a selection of cellpermeable fluorescent dyes and endogenous fluorescent molecules. We will do this with an emphasis on pH and mitochondrial energetics in cardiomyocytes.