iTRAQ-based proteomics analysis of autophagy-mediated immune responses against the vascular fungal pathogen Verticillium dahliae in Arabidopsis

The mechanisms underlying the functional link between autophagy and plant innate immunity remain largely unknown. In this study, we investigated the autophagy-mediated plant defense responses against Verticillium dahliae (V. dahliae) infection by comparative proteomics and cellular analyses. An assessment of the autophagy activity and disease development showed that autophagic processes were tightly related to the tolerance of Arabidopsis plant to Verticillium wilt. An isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics analysis was performed, and we identified a total of 780 differentially accumulated proteins (DAPs) between wild-type and mutant atg10-1 Arabidopsis plants upon V. dahliae infection, of which, 193 ATG8-family-interacting proteins were identified in silico and their associations with autophagy were verified for several selected proteins. Three important aspects of autophagy-mediated defense against V. dahliae infection were revealed: 1) autophagy is required for the activation of upstream defense responses; 2) autophagy-mediated mitochondrial degradation (mitophagy) occurs and is an important player in the defense process; and 3) autophagy promotes the transdifferentiation of perivascular cells and the formation of xylem hyperplasia, which are crucial for protection against this vascular disease. Together, our results provide several novel insights for understanding the functional association between autophagy and plant immune responses.     

Quantitative chemical proteomics profiling of de novo protein synthesis during starvation-mediated autophagy

Autophagy is an intracellular degradation mechanism in response to nutrient starvation. Via autophagy, some nonessential cellular constituents are degraded in a lysosome-dependent manner to generate biomolecules that can be utilized for maintaining the metabolic homeostasis. Although it is known that under starvation the global protein synthesis is significantly reduced mainly due to suppression of MTOR (mechanistic target of rapamycin serine/threonine kinase), emerging evidence demonstrates that de novo protein synthesis is involved in the autophagic process. However, characterizing these de novo proteins has been an issue with current techniques. Here, we developed a novel method to identify newly synthesized proteins during starvation-mediated autophagy by combining bio-orthogonal noncanonical amino acid tagging (BONCAT) and isobaric tags for relative and absolute quantitation (iTRAQ^TM). Using bio-orthogonal metabolic tagging, L-azidohomoalanine (AHA) was incorporated into newly synthesized proteins which were then enriched with avidin beads after a click reaction between alkyne-bearing biotin and AHA's bio-orthogonal azide moiety. The enriched proteins were subjected to iTRAQ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Via the above approach, we identified and quantified a total of 1176 proteins and among them 711 proteins were found to meet our defined criteria as de novo synthesized proteins during starvation-mediated autophagy. The characterized functional profiles of the 711 newly synthesized proteins by bioinformatics analysis suggest their roles in ensuring the prosurvival outcome of autophagy. Finally, we performed validation assays for some selected proteins and found that knockdown of some genes has a significant impact on starvation-induced autophagy. Thus, we think that the BONCAT-iTRAQ approach is effective in the identification of newly synthesized proteins and provides useful insights to the molecular mechanisms and biological functions of autophagy.     

A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications

The lack of a rapid and quantitative autophagy assay has substantially hindered the development and implementation of autophagy-targeting therapies for a variety of human diseases. To address this critical issue, we developed a novel autophagy assay using the newly developed Cyto-ID fluorescence dye. We first verified that the Cyto-ID dye specifically labels autophagic compartments with minimal staining of lysosomes and endosomes. We then developed a new Cyto-ID fluorescence spectrophotometric assay that makes it possible to estimate autophagy flux based on measurements of the Cyto-ID-stained autophagic compartments. By comparing to traditional autophagy approaches, we found that this assay yielded a more sensitive, yet less variable, quantification of the stained autophagic compartments and the estimate of autophagy flux. Furthermore, we tested the potential application of this autophagy assay in high throughput research by integrating it into an RNA interference (RNAi) screen and a small molecule screen. The RNAi screen revealed WNK2 and MAP3K6 as autophagy-modulating genes, both of which inhibited the MTOR pathway. Similarly, the small molecule screen identified sanguinarine and actinomycin D as potent autophagy inducers in leukemic cells. Moreover, we successfully detected autophagy responses to kinase inhibitors and chloroquine in normal or leukemic mice using this assay. Collectively, this new Cyto-ID fluorescence spectrophotometric assay provides a rapid, reliable quantification of autophagic compartments and estimation of autophagy flux with potential applications in developing autophagy-related therapies and as a test to monitor autophagy responses in patients being treated with autophagy-modulating drugs.     

Protocols,Toolboxes and Resource papers

In the August 2009 issue of Autophagy, I indicated that we were launching a new category of article, Protocols. At that time, I noted that we would ultimately be placing these articles on a new site online. Well, that time has finally arrived (see www.landesbioscience.com/journals/autophagy/protocols/ for links to these papers). Therefore, it seems appropriate for me to briefly distinguish among three types of community-oriented papers, Protocol, Toolbox and Resource.     

Autophagy Regulates Ageing in C. elegans

The role of autophagy in ageing regulation has been suggested based on studies in C. elegans, in which knockdown of the expression of bec-1 (ortholog of the yeast and mammalian autophagy genes ATG6/VPS30 and beclin 1, respectively) shortens the lifespan of the daf-2(e1370) mutant C. elegans. However, Beclin1/ATG6 is also known to be involved in other cellular functions in addition to autophagy. In the current study, we knocked down two other autophagy genes, atg-7 and atg-12, in C. elegans using RNAi. We showed that RNAi shortened the lifespan of both wild type and daf-2 mutant C. elegans, providing strong support for a role of autophagy in ageing regulation.     

An Optimization of a Bioassay for Toluene Analogs using Bioluminescence Reporter Strain KG1206

A genetically engineered strain of P. putida mt-2 KG1206 used in this study contains the intact TOL plasmid and a plasmid with the P _m-lux gene, and bioluminescence was produced by direct (m-toluate and benzoate) and indirect inducers (toluene analogs). Much less bioluminescence was produced by benzoate and o-xylene among the tested inducers. This bioluminescence producing strain was used for the quantification of m-toluate in soil, and a quantification protocol for pollutant was developed for standardization. Values determined by bioluminescence were in the range of 75 (min.) ～158 (max.) % of their true concentration as determined by HPLC analysis. Statistical analysis indicates that this bioluminescence strain is useful for quantifying specific pollutant in environmental system. However, more investigation is required for mixture pollutants in the environment.     

Detection and Quantification of Dehalococcoides-Related Bacteria in a Chlorinated Ethene-Contaminated Aquifer Undergoing Natural Attenuation

Detection and quantification of bacteria related to Dehalococcoides is essential for the development of effective remediation strategies for tetrachloroethene (PCE)-contaminated sites. In this study, the authors applied three methods for quantifying Dehalococcoides-like bacteria in a PCE-contaminated aquifer undergoing natural attenuation in Grenchen, Switzerland: a catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) protocol, a competitive nested polymerase chain reaction (PCR) approach, and a direct PCR end point quantification with external standards. For the investigated aquifer, multiple lines of evidence indicated that reductive dechlorination (and likely dehalorespiration) was an active process. Both PCR-based quantification methods indicated that low numbers of mostly sediment-bound Dehalococcoides were present in the contaminated zone of the Grenchen aquifer. Estimates based on the quantitative PCR methods ranged from 2.1 × 10⁷ to 1.5 × 10⁸ sediment-bound Dehalococcoides 16S rRNA gene copies per liter of aquifer volume. In contrast, the liquid phase only contained between 8 and 80 copies per liter aquifer volume. CARD-FISH was not sensitive enough for the quantification of Dehalococcoides cell numbers in this aquifer. Cloning and sequencing of the PCR products revealed the presence of sequences closely related to Dehalococcoides isolates such as D. ethenogenes and Dehalococcoides sp. BAV1. An apparently abundant group (termed “Grenchen Cluster”) of sequences more distantly related to Dehalococcoides was also identified, so far without cultured representatives.     

A role for diacylglycerol in antibacterial autophagy

Antibacterial autophagy is understood to be a key cellular immune response to invading microbes. However, the mechanism(s) by which bacteria are selected as targets of autophagy remain unclear. We recently identified diacylglycerol as a novel signaling molecule that targets bacteria to the autophagy pathway, and show that it acts via protein kinase C activation. We also found that Pkc1 is required for autophagy in yeast, indicating that this kinase plays a conserved role in autophagy regulation.Key words: bacteria, Salmonella, innate immunity, adaptor, lipid second messenger, diacylglycerol, ubiquitin, NDP52, p62, SQSTM1The mechanism by which bacteria and other subcellular targets are identified and degraded by the autophagy pathway is an area of intense research. Ubiquitin has been recently found to act as an essential signal required for the autophagy of bacteria and proteins. We have previously observed ubiquitin on autophagy-targeted Salmonella enterica serovar Typhimurium (S. Typhimurium) but were surprised to see that only 50% of these bacteria were positive for ubiquitin. This indicated the possibility that an alternate signal was required for efficient autophagic targeting of the nonubiquitinated population of these bacteria.We initially performed a screen quantifying the colocalization of different lipid second messengers (diacylglycerol (DAG), PtdIns(3)P, PtdIns(4,5)P₂, PtdIns(3,4) P₂, and PtdIns(3,4,5)P₃) with autophagytargeted (i.e., LC3⁺) S. Typhimurium. We observed that DAG preferentially localizes with LC3⁺ bacteria. A kinetic analysis revealed that maximal DAG colocalization with bacteria (45 min post-infection) precedes maximal autophagy of the bacteria (60 min post-infection). Using pharmacological agents, siRNA and dominant negative constructs we were able to determine that DAG localization to the bacteria requires the action of phospholipase D (PLD; phosphatidylcholine to phosphatidic acid conversion) and phosphatidic acid phosphatase (PAP; phosphatidic acid to DAG conversion). We observed that inhibition of these pathways significantly reduces DAG localization to bacteria as well as concomitant autophagy of the bacteria, indicating a role for this lipid second messenger in the regulation of this process.Having determined that DAG is necessary for autophagy of bacteria we subsequently wanted to identify the effector through which it was signaling. Conventional and novel isoforms of the protein kinase C (PKC) family contain DAG-binding C1 domains. Accordingly, we targeted PKC isoforms using pharmacological agents, siRNA and knockout cell lines and were able to determine that DAG is signaling through the δ isoform of PKC. Inhibition of this serine/threonine kinase results in significant inhibition of antibacterial autophagy. Furthermore, bacterial replication in PKCδ knockout mouse embryonic fibroblasts is significantly higher compared to control fibroblasts, consistent with previous observations demonstrating that autophagy impairs intracellular replication of S. Typhimurium (Birmingham et al. 2006).We addressed the possibility that DAG and ubiquitin are functioning in a cooperative manner to target Salmonella for degradation by autophagy. We simultaneously inhibited both pathways using siRNA or pharmacological agents and observed additive inhibitory effects on autophagy of the bacteria. While this is indicative of two independent pathways, we cannot discount the possibility that there is still cooperation between the two pathways, especially as we did observe a small population of bacteria that were positive for both DAG and ubiquitin (Fig. 1). There are also a number of technical limitations in the methods we used, such as detection levels of the probes and antibodies that warrant caution in concluding that the two pathways are completely independent. Nonetheless, our studies clearly demonstrate a role for both DAG (Shahnazari et al. 2010) and ubiquitin (Zheng et al. 2009) in autophagy of S. Typhimurium. Future studies are required to further examine how these signals contribute to regulation of antibacterial autophagy.Open in a separate windowFigure 1Autophagic targeting of Salmonella Typhimurium. Invading S. Typhimurium can be targeted to the autophagy pathway by two independent signaling mechanisms. The first requires ubiquitin and the autophagy adaptors p62 and NDP52. The second requires DAG generation and PKCδ function. DAG generation on the SCV may occur through interaction of the SCV with DAG-positive endocytic vesicles (pathway 1) or through direct DAG production on the SCV (pathway 2). SCV, Salmonella-containing vacuole; PA, phosphatidic acid; DAG, diacylglycerol; PAP, phosphatidic acid phosphatase; PKCδ, protein kinase C delta; Ub, ubiquitin.Having characterized this pathway in antibacterial autophagy we were interested in determining whether these components were required for general autophagy. We therefore tested whether DAG localizes with rapamycin-induced autophagosomes. We observed DAG on these compartments and also found a requirement for PAP and PKCδ in this process. Other PKC isoforms are involved in alternate types of autophagy including ER stress-induced autophagy (Sakaki et al. 2008) as well as hypoxia-induced autophagy (Chen et al. 2009). As a result, we were interested in determining whether PKC function in autophagy was evolutionarily conserved. We therefore tested a role for the yeast ortholog, Pkc1, in this process and observed that it is required for starvation-induced autophagy in Saccharomyces cerevisiae.Having identified and characterized a novel signal and effector for antibacterial autophagy, further work still remains to be done in order to obtain a complete picture of this process. This includes additional study of the mechanism by which DAG is generated and the subcellular localization of PLD and PAP during this process. It is possible that DAG⁺ endocytic vesicles fuse with the Salmonella-containing vacuole (SCV) coating this compartment with DAG (pathway 1, see Fig. 1). It is also possible that both PLD and PAP function directly on the SCV, converting phosphatidylcholine to DAG via the phosphatidic acid intermediate (pathway 2, Fig. 1).More work also needs to be done to dissect DAG and ubiquitin signaling contributions to this pathway. Questions to be answered include the identification of the ubiquitinated protein(s) on the SCV, which may be host or bacterial proteins. Additionally, while we know that DAG is present on the SCV we do not yet know the signal that induces its generation. One intriguing possibility is that DAG generation occurs in response to bacterial-induced damage to the SCV during invasion. To date, PKC has been implicated in at least three different types of autophagy, and the possibility exists that other PKC isoforms (DAG responsive or not) are also involved in this process.     

Distinct functions of Ulk1 and Ulk2 in the regulation of lipid metabolism in adipocytes

ULK1 (unc-51 like kinase 1) is a serine/threonine protein kinase that plays a key role in regulating the induction of autophagy. Recent studies using autophagy-defective mouse models, such as atg5- or atg7-deficient mice, revealed an important function of autophagy in adipocyte differentiation. Suppression of adipogenesis in autophagy-defective conditions has made it difficult to study the roles of autophagy in metabolism of differentiated adipocytes. In this study, we established autophagy defective-differentiated 3T3-L1 adipocytes, and investigated the roles of Ulk1 and its close homolog Ulk2 in lipid and glucose metabolism using the established adipocytes. Through knockdown approaches, we determined that Ulk1 and Ulk2 are important for basal and MTORC1 inhibition-induced autophagy, basal lipolysis, and mitochondrial respiration. However, unlike other autophagy genes (Atg5, Atg13, Rb1cc1/Fip200, and Becn1) Ulk1 was dispensable for adipogenesis without affecting the expression of CCAAT/enhancer binding protein α (CEBPA) and peroxisome proliferation-activated receptor gamma (PPARG). Ulk1 knockdown reduced fatty acid oxidation and enhanced fatty acid uptake, the metabolic changes that could contribute to adipogenesis, whereas Ulk2 knockdown had opposing effects. We also found that the expression levels of insulin receptor (INSR), insulin receptor substrate 1 (IRS1), and glucose transporter 4 (SLC2A4/GLUT4) were increased in Ulk1-silenced adipocytes, which was accompanied by upregulation of insulin-stimulated glucose uptake. These results suggest that ULK1, albeit its important autophagic role, regulates lipid metabolism and glucose uptake in adipocytes distinctly from other autophagy proteins.     

Induction of Apoptosis and Autophagy Using Ectopic DSCR1 Expression in Breast Cancer Cells

Down syndrome critical region 1 gene (DSCR1) is an anti-angiogenesis gene that inhibits the growth of tumor cells. In this study, the role of autophagy and apoptosis in DSCR1-induced cytotoxicity were investigated in MDA-MB-468 breast cancer cells. Lentivirus vector harboring DSCR1 (LV-DSCR1⁺) was constructed in HEK 293 cells and the optimal dosage of lentivirus vector for infection was determined by the MTT assay. After infection of cells using LV-DSCR1⁺, acridine orange and ethidium bromide staining was performed to investigation of apoptosis and autophagy. Expression of DSCR1 and marker genes for angiogenesis (VEGF), apoptosis (Bax and Bcl2) and autophagy (LC3 and Beclin) were determined by Real time PCR. The cellular morphological changes related to apoptosis and autophagy was happened after 48 hours of viral infection. Fragmented bright orange nucleuses and vacuoles were observed due to the cell apoptosis and autophagy after acridine orange and ethidium bromide staining. Upregulation of Bax, Lc3, DSCR1 and Beclin1 and downregulation of Bcl2 and VEGF was detected due to treatment with LV-DSCR1⁺. These results demonstrated that LV-DSCR1⁺ can induce apoptosis and autophagy, therefore suggesting that it may serves as an efficient tool to breast cancer treatment.     

Inhibition of autophagy as a therapeutic strategy of iron-induced brain injury after hemorrhage

Premenopausal women have better survival than men after intracerebral hemorrhage, which is associated with iron overproduction and autophagy induction. To examine the participation of neuronal autophagy and estrogen receptor α (ERα) in the E₂–mediated protection, PC12 neurons treated with Atg7 (autophagy-related protein 7) siRNA, rapamycin (an autophagy inducer), or Erα siRNA were applied. To study whether autophagy involves in β-estradiol 3-benzoate (E₂)-mediated neuroprotection against iron-induced striatal injury, castration and E₂ capsule implantation were performed at 2 weeks and 24 h, respectively, before ferrous citrate (FC) infusion into the caudate nucleus (CN) of Sprague Dawley male and female rats. Furthermore, the role of neuronal autophagy in the sex difference of FC-induced CN injury was confirmed by using conditional knockout Atg7 in dopamine receptor 2 (DRD2)-containing neurons in mice. The results showed that the suppression of FC-induced autophagy by E₂ was abolished by Erα siRNA preincubation. Atg7 silencing simulates and rapamycin diminishes E₂-mediated neuroprotection against FC-induced neurotoxicity. In vivo, FC induced a lower degree of autophagy, autophagic cell death, injury severity, histological lesion and behavioral deficit in female rats than in males. E₂ implantation decreased the levels of both FC-induced autophagy and injury in ovariectomized rats. Moreover, the sex difference of FC-induced CN injury was diminished in Atg7 knockout mice. Thus, suppression of autophagy by E₂ via ERα contributes to less severity of iron-induced brain injury in females than in male. This finding opens up the prospect for a therapeutic strategy targeting autophagic inhibition for patients suffering from intracerebral iron overload.     

High‐density lipoprotein inhibits mechanical stress‐induced cardiomyocyte autophagy and cardiac hypertrophy through angiotensin II type 1 receptor‐mediated PI3K/Akt pathway

Mechanical stress triggers cardiac hypertrophy and autophagy through an angiotensin II (Ang II) type 1 (AT1) receptor‐dependent mechanism. Low level of high density lipoprotein (HDL) is an independent risk factor for cardiac hypertrophy. This study was designed to evaluate the effect of HDL on mechanical stress‐induced cardiac hypertrophy and autophagy. A 48‐hr mechanical stretch and a 4‐week transverse aortic constriction were employed to induce cardiomyocyte hypertrophy in vitro and in vivo, respectively, prior to the assessment of myocardial autophagy using LC3b‐II and beclin‐1. Our results indicated that HDL significantly reduced mechanical stretch‐induced rise in autophagy as demonstrated by LC3b‐II and beclin‐1. In addition, mechanical stress up‐regulated AT1 receptor expression in both cultured cardiomyocytes and in mouse hearts, whereas HDL significantly suppressed the AT1 receptor. Furthermore, the role of Akt phosphorylation in HDL‐mediated action was assessed using MK‐2206, a selective inhibitor for Akt phosphorylation. Our data further revealed that MK‐2206 mitigated HDL‐induced beneficial responses on cardiac remodelling and autophagy. Taken together, our data revealed that HDL inhibited mechanical stress‐induced cardiac hypertrophy and autophagy through downregulation of AT1 receptor, and HDL ameliorated cardiac hypertrophy and autophagy via Akt‐dependent mechanism.     

Autophagy genes are required for normal lipid levels in C. elegans

Autophagy is a cellular catabolic process in which various cytosolic components are degraded. For example, autophagy can mediate lipolysis of neutral lipid droplets. In contrast, we here report that autophagy is required to facilitate normal levels of neutral lipids in C. elegans. Specifically, by using multiple methods to detect lipid droplets including CARS microscopy, we observed that mutants in the gene bec-1 (VPS30/ATG6/BECN1), a key regulator of autophagy, failed to store substantial neutral lipids in their intestines during development. Moreover, loss of bec-1 resulted in a decline in lipid levels in daf-2 [insulin/IGF-1 receptor (IIR) ortholog] mutants and in germline-less glp-1/Notch animals, both previously recognized to accumulate neutral lipids and have increased autophagy levels. Similarly, inhibition of additional autophagy genes, including unc-51/ULK1/ATG1 and lgg-1/ATG8/MAP1LC3A/LC3 during development, led to a reduction in lipid content. Importantly, the decrease in fat accumulation observed in animals with reduced autophagy did not appear to be due to a change in food uptake or defecation. Taken together, these observations suggest a broader role for autophagy in lipid remodeling in C. elegans.     

Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.Download video file.^{(23M, mov)}     

Fluorescently labeled liposomes for monitoring cholera toxin binding to epithelial cells

Vibrio cholerae, the causative agent for cholera, expresses a toxin required for virulence consisting of two subunits: the pentameric cholera toxin B (CTB) and cholera toxin A (CTA). CTB is frequently used as an indicator of the presence of pathogenic V. cholerae and binds to the G_M1 ganglioside on the surface of epithelial cells. To study V. cholerae virulence (CTB expression) in the presence of human epithelia, we devised an inexpensive, simple, and rapid method for quantifying CTB bound on epithelial surfaces in microtiter plates. G_M1 ganglioside was incorporated into the lipid bilayer of liposomes both encapsulating the fluorescent dye sulforhodamine B (SRB) and with SRB tagged to lipids in the bilayer (BEGs). In addition, G_M1-embedded liposomes encapsulating SRB only (EGs) and with SRB in their bilayers only (BGs) were synthesized. The three types of liposomes were compared with respect to their efficacy for both visualizing and quantifying CTB attached to the surface of Caco-2 cells. The BEGs were the most effective overall, providing both visualization under a fluorescence microscope and quantification after lysis in a microtiter plate reader. A limit of detection corresponding to 0.28 μg/ml applied CTB was attained for the on-cell assay using the microtiter plate reader approach, whereas as low as 2 μg/ml applied CTB could be observed under the fluorescence microscope.     

Quantification of In Vitro and In Vivo Cryptosporidium parvum Infection by Using Real-Time PCR

Established methods for quantifying experimental Cryptosporidium infection are highly variable and subjective. We describe a new technique using quantitative real-time PCR (qPCR) that can be used to measure in vitro and in vivo laboratory infections with Cryptosporidium. We show for the first time that qPCR permits absolute quantification of the parasite while simultaneously controlling for the amount of host tissue and correlates significantly with established methods of quantification in in vitro and in vivo laboratory models of infection.     

Gas chromatographic–mass spectrometric procedures for determination of the catechol-O-methyltransferase (COMT) activity and for detection of unstable catecholic metabolites in human and rat liver preparations after COMT catalyzed in statu nascendi derivatization using S-adenosylmethionine

A procedure is presented for determination of the catechol-O-methyltransferase (COMT) activity in liver cytosolic preparations using 3,4-dihydroxyphenethylamine as substrate and by quantifying the product 3-methoxy-4-hydroxyphenethylamine (3-MHP). For quantification of 3-MHP in liver cytosolic preparations a gas chromatographic–mass spectrometric procedure after liquid–liquid extraction and acetylation was established and validated. The intra- and inter-day accuracy and precision were better than 15% and 20%, respectively. Extraction efficiency and selectivity were also sufficient. For in statu nascendi derivatization of unstable catecholic metabolites in liver microsome preparations, cytosolic preparations with COMT activities of at least 1 nmol product/min/mg protein were used after addition of S-adenosylmethionine. Such catecholic metabolites, which are claimed to be responsible for toxic effects in vivo, e.g., neurotoxicity or carcinogenesis, must not be overlooked in in vitro metabolism studies. Using this trick, gas chromatography–mass spectrometry (GC–MS) was suitable for the determination of catecholic metabolites in human and rat liver preparations after the same sample preparation as for 3-MHP quantification. The applicability was exemplified for the antidepressant paroxetine.     

Impaired autophagy flux is associated with neuronal cell death after traumatic brain injury

Dysregulation of autophagy contributes to neuronal cell death in several neurodegenerative and lysosomal storage diseases. Markers of autophagy are also increased after traumatic brain injury (TBI), but its mechanisms and function are not known. Following controlled cortical impact (CCI) brain injury in GFP-Lc3 (green fluorescent protein-LC3) transgenic mice, we observed accumulation of autophagosomes in ipsilateral cortex and hippocampus between 1 and 7 d. This accumulation was not due to increased initiation of autophagy but rather to a decrease in clearance of autophagosomes, as reflected by accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1). This was confirmed by ex vivo studies, which demonstrated impaired autophagic flux in brain slices from injured as compared to control animals. Increased SQSTM1 peaked at d 1–3 but resolved by d 7, suggesting that the defect in autophagy flux is temporary. The early impairment of autophagy is at least in part caused by lysosomal dysfunction, as evidenced by lower protein levels and enzymatic activity of CTSD (cathepsin D). Furthermore, immediately after injury both autophagosomes and SQSTM1 accumulated predominantly in neurons. This was accompanied by appearance of SQSTM1 and ubiquitin-positive puncta in the affected cells, suggesting that, similar to the situation observed in neurodegenerative diseases, impaired autophagy may contribute to neuronal injury. Consistently, GFP-LC3 and SQSTM1 colocalized with markers of both caspase-dependent and caspase-independent cell death in neuronal cells proximal to the injury site. Taken together, our data indicated for the first time that autophagic clearance is impaired early after TBI due to lysosomal dysfunction, and correlates with neuronal cell death.     

Stimulation of autophagy suppresses the intracellular survival of Burkholderia pseudomallei in mammalian cell lines

Burkholderia pseudomallei is the causative agent of melioidosis, a tropical infection of humans and other animals. The bacterium is an intracellular pathogen that can escape from endosomes into the host cytoplasm, where it replicates and infects adjacent cells. We investigated the role played by autophagy in the intracellular survival of B. pseudomallei in phagocytic and non-phagocytic cell lines. Autophagy was induced in response to B. pseudomallei invasion of murine macrophage (RAW 264.7) cells and a proportion of the bacteria co-localized with the autophagy effector protein LC3, a marker for autophagosome formation. Pharmacological stimulation of autophagy in RAW 264.7 and murine embryonic fibroblast (MEF) cell lines resulted in increased co-localization of B. pseudomallei with LC3 while basal levels of co-localization could be abrogated using inhibitors of the autophagic pathway. Furthermore, induction of autophagy decreased the intracellular survival of B. pseudomallei in these cell lines, but bacterial survival was not affected in MEF cell lines deficient in autophagy. Treatment of infected macrophages with chloramphenicol increased the proportion of bacteria within autophagosomes indicating that autophagic evasion is an active process relying on bacterial protein synthesis. Consistent with this hypothesis, we identified a B. pseudomallei type III secreted protein, BopA, which plays a role in mediating bacterial evasion of autophagy. We conclude that the autophagic pathway is a component of the innate defense system against invading B. pseudomallei, but which the bacteria can actively evade. However, when autophagy is pharmacologically induced using rapamycin, bacteria are actively sequestered in autophagosomes, ultimately decreasing their survival.