Autophagy and LC -
Membranes were washed and incubated with anti-mouse HRP Sigma, A, , or anti-rabbit HRP Sigma, A, , secondary antibodies. Cells were harvested 48 hours after transfection. RNA was further purified from DNA traces by performing a treatment with TURBO DNA-free kit Ambion, AM Messenger RNA was retrotranscripted using the superscript III first-strand synthesis system for RT-PCR Life Technologies — according to manufacturer's protocol.
The power SYBR green PCR master mix ThermoFisher, was applied to test p62 and LC3B expression with the T HT AbiPrism real-time PCR Instrument. All mice were treated in accordance with the Guide for the Animal Care and Use of Laboratory Animals National Research Council , and all procedures were approved by the Institutional Animal Care and Use Committee at PsychoGenics, Inc.
mTOR inhibitor INK The mTOR inhibitor was prepared in 0. Plasma was thawed and mixed with 4 volumes of acetonitrile containing 0. This mixture was then centrifuged and the supernatant diluted with water into the analysis plate.
The liver samples were thawed, mixed with 3 volumes of water and homogenized using a Precellys Bertin Technologies. Following homogenization, a known volume of homogenate was extracted in a manner similar to that of plasma.
The chromatographic separation was achieved using a C18 column Kinetex XB-C18 A maintained at 40°C and mobile phase comprised of 2. The concentrations of INK were determined based on peak areas normalized for the internal standard diclofenac peak area vs the peak areas of the appropriate calibration curve.
p62 and LC3II levels in liver samples of compound treated mice were quantified using crude liver tissue homogenates with a concentration of 0. Analysis of variance ANOVA was performed for experiments with more than two groups.
All graphs represent avg±SEM, unless ±SD is noted. GraphPad Prism software was used for all analyses. TR-FRET is a homogeneous assay able to measure molecule proximity by fluorescence resonance energy transfer.
Compounds that upregulate autophagy induce close proximity of the LC3II antibodies resulting in an accumulation of signal as AVs are formed; that signal is reduced over time as AVs are turned over.
The p62 signal decreases over time as that substrate is degraded Fig 1B. Compounds that block autophagy at the lysosome have a different signal.
The LC3II signal accumulates as the donor and acceptor antibodies come together in new AVs, but there is no degradation of the signal since the AVs are not degraded. Similarly, there is no turnover of the p62 signal Fig 1C.
The TR-FRET signal relies on transfer of energy between two fluorophores, a donor and acceptor, when in close proximity. When two labeled molecules come together, excitation of the donor by an energy source triggers energy transfer towards the acceptor, which emits a fluorescent signal.
Tb labeled LC3II donor and D2 labeled LC3II acceptor antibodies or Tb labeled donor p62 and Alexa labeled acceptor p62 antibodies were used A. Compounds that upregulate autophagy induce close proximity of the LC3II donor and acceptor antibodies, resulting in an accumulation of signal as AVs are formed; that signal is reduced over time as AVs are turned over.
P62 donor and acceptor antibodies come in close proximity as they find p62 proteins, the signal declines over time as p62 is degraded B. Similarly, there is no turnover of the p62 signal C.
LC3B and SQSTM1 p62 gene silencing was performed on HEKT cells in order to verify the specificity of the TR-FRET signal for the two proteins.
Knockdown of LC3B did not influence p62 mRNA and knockdown of p62 did not influence LC3B mRNA Fig 2A. There was a marked decrease in p62 and LC3B proteins as detected by western blot Fig 2B.
Genetic validation of the LC3B-II and p62 readouts was achieved by gene silencing of LC3B and p62 in HEKT cells using shRNA. ATG4B overexpression in HEKT cells was confirmed with western blot D. To determine whether the TR-FRET assay specifically detects the lipidated form of LC3B LC3B-II , HEKT cells were transfected to overexpress ATG4B.
ATG4B cleaves LC3B-II to generate non-lipidated LC3B LC3B-I ; therefore, overexpression of this enzyme should abolish LC3B-II from cells. ATG4B overexpression in HEKT cells was verified by western blot Fig 2D.
To demonstrate the utility of p62 and LC3B-II quantification by TR-FRET, compounds previously reported to modulate the autophagy pathway were evaluated in different cell models.
These two compounds were tested in serial dilutions starting at 25 nM for bafilomycin A1 and 25 μM for KU The study was carried out at 2, 6 and 24 hours post-treatment to potentially capture multiple cellular states in response to autophagy modulation. In HEKT cells, bafilomycin A1 treatment resulted in a dose and time-dependent increase of LC3B-II levels with respect to vehicle DMSO treated cells Fig 3A.
A similar behavior was detected for p62 at 2 and 6 hours post-treatment. Bafilomicyin A1 concentrations higher than 2 nM resulted in cytotoxicity at 24 hours.
Using this assay, autophagy cannot be measured when viability is compromised. In HEKT cells the LC3B-II TR-FRET signal increased in a dose-dependent manner at 2 hours post treatment with KU and then decreased with time, relative to the DMSO treated cells Fig 3A.
This behavior may be due to exhaustion of the cells ability to synthesize new AVs after an initial activation. Conversely, p62 decreased with time and KU concentration. As previously noted for bafilomycin A1 treatment, cell viability is influenced by the compound at 24 hours post treatment, therefore LC3B-II and p62 results at this time point must be taken with caution, especially since the p62 reduction appears to reduce in parallel with cytotoxicity.
Western blots with LC3B and p62 confirmed what was observed with TR-FRET Fig 4A. Bafilomycin A1 and KU were tested in primary rat cortical-striatal neurons and astrocytes.
In contrast to HEKT cells, these cells are post-mitotic neurons or slowly proliferating astrocyte doubling time in vitro , with the present culture conditions, was estimated to be 7 days.
Neurons and astrocytes were treated with serial dilutions of compounds starting at 25 nM for bafilomycin A1 and 25 μM for KU Nuclei count was used to assess the viability of astrocytes upon compound treatment, but was not suitable for neuronal cultures due to difficulty in segmentation by image analysis of clusters of neuronal nuclei.
To overcome this limitation, viability of cultured neurons was evaluated by MAP2 labeled neurite length per soma. Bafilomycin A1 treatment in neurons caused an increase in both p62 and LC3B-II levels in a dose and time dependent manner, although the neuronal LC3B-II response to bafilomycin A1 was not very robust at concentrations that were not cytotoxic Fig 3B.
Similar to what was observed in HEKT cells, a clear cytotoxic effect increased in magnitude over time. KU induced a short-lived and modest increase in LC3B-II at 2 hours and a time and dose-dependent reduction in p62, as measured by TR-FRET and western blot, in the absence of overt cytotoxicity Figs 3B and 4B.
When tested on astrocytes, bafilomycin A1 increased LC3B-II and p62, in a dose and time-dependent manner as determined by TR-FRET and western blot. Astrocytes had a robust response to KU, increasing LC3B-II at 2 hours and decreasing p62 by 24 hours in the absence of cytotoxicity, suggesting upregulation of autophagy Figs 3C and 4C.
While the p62 and LC3B-II TR-FRET assays can detect changes in autophagy, it is clear that autophagy modulation differs depending on cell type. To highlight the importance of considering all 3 readouts when determining the state of autophagy, astrocytes were tested with 2 additional compounds, SU and NVP-TAE Although SU upregulated the LC3B-II TR-FRET signal and reduced the p62 TR-FRET signal, there is clear cytotoxicity at concentrations higher than 1μM, therefore this compound may upregulate autophagy at low concentrations and at higher concentrations cannot be interpreted.
It is notable that the p62 signal appears to track with cytotoxicity Fig 3D. NVP-TAE induced an increase in the LC3B-II signal in the absence of toxicity. However, there is no reduction in p62 and even an increase in p62 at higher concentrations at 24 hours, this suggests that the accumulation of AVs may be due to blockage of autophagy with this compound Fig 3E.
HEKT cells A , rat cortico-striatal neurons B and rat astrocytes C were treated with a serially diluted autophagy inhibitor bafilomycin A1 or upregulator KU and examined at 2, 6 and 24 hours post-treatment.
LC3B-II and p62 were measured with TR-FRET A-C. Astrocytes were also treated with SU and NVP-TAE and LC3B-II, p62 and viability were measured with TR-FRET D-E. HEKT cells A , rat cortico-striatal neurons B and rat astrocytes C were treated with an autophagy inhibitor 5 nM bafilomycin A1 or upregulator KU and examined at 2, 6 and 24 hours post-treatment, compared to DMSO.
The accumulation of AVs can be a result of either upregulated or blocked autophagy. To discriminate autophagy inducers from blockers LC3B-II and p62 quantification were measured in astrocytes co-treated with tool compounds and bafilomycin A1. In the presence of an autophagy blocker the accumulation of LC3B-II is not influenced by the addition of bafilomycin A1, as the effect of the two compounds are identical.
Conversely, a compound able to upregulate autophagy induces the production of LC3B-II; as a consequence the blockage of such induced autophagy flux by bafilomycin A1 results in higher LC3B-II levels than compound alone.
To perform this study we first identified a non-cytotoxic concentration of each tool compound that leads to an accumulation of AVs Fig 3. Astrocytes were treated for two hours by one non-cytotoxic concentration of each of three compounds 10 μM KU; 1 μM SU and 5 μM NVP-TAE followed by four hours of either 50 nM bafilomycin A1 or vehicle DMSO.
DMSO only and 50 nM bafilomycin A1 only treated cells were also produced as control samples. Cell lysates of all the treatment conditions were analyzed both by TR-FRET and western blot. Control samples were treated with vehicle DMSO for 2 hours followed by either bafilomycin A1 50 nM or vehicle DMSO for an additional 4 hours.
LC3B-II TR-FRET signals are reported as fold increase with respect to the vehicle. Western blots B confirm the TR-FRET data. The LC3B-II and p62 TR-FRET assays allow for label-free quantification of autophagy changes in tissues.
In comparison to western blot, the TR-FRET assays are more quantitative and have higher throughput capabilities. As a proof-of-concept of the utility of the LC3B-II and p62 TR-FRET assays we used an mTOR inhibitor to demonstrate autophagy changes can be measured in vivo in a mouse.
The LC3B-II signal was only decreased at day 1, but no other time point, suggesting the signal may be more readily influenced by homogenization technique than storage. We set out in these studies to establish a set of assays that could measure autophagy by quantifying endogenous markers, and could be used variously to screen in wells as well as in primary cells and animal studies.
Current methods to measure autophagy are extensively reviewed elsewhere[ 12 ]; TR-FRET offers the advantage of not relying on overexpression of a LC3B or p62 reporter, and is amenable for screening. Other reports use TR-FRET to measure autophagy, however, they rely on different antibody pairs for example LC3-ATG4 or LC3-LAMP [ 25 , 26 ] or require cells to express a GFP tagged autophagy reporter.
We developed two TR-FRET assays based on antibody-mediated recognition of two primary markers, LC3B-II and p62; these two readouts, together with cell viability for cell-based assays, generated data relevant to interpreting autophagy modulation.
In the cells tested in these studies, pharmacologically-induced autophagy upregulation is characterized by a rapid increase from a low basal level of LC3B-II, followed by a return to initial levels. As noted in other studies, the accumulation of LC3B-II reflects binding to AVs, which may reflect either upregulated autophagy or blockage of AV degradation,[ 18 ] making interpretation of LC3C-II levels alone difficult.
In our studies, p62 decreases at later time points probably because it is a substrate for autophagic degradation. Our studies suggest that the combination of reduced p62, increased LC3B-II and absence of cytotoxicity -taken together at multiple time points and concentrations- can serve to identify upregulation of autophagy.
We demonstrate here that the signal produced by the two TR-FRET assays was selective in a cell lysate matrix by silencing the two targets: LC3B and p With regard to LC3B-II, the specificity of its detection with respect to the non-lipidated form was evaluated by overexpression of ATG4B, an enzyme that catalyzes the removal of the lipid moiety from LC3B-II[ 32 ]; we saw a decreased LC3B-II TR-FRET signal in ATG4B-overexpressing cells in the absence of any transcriptional modulation of LC3B, validating the specificity for the post-translational modification.
We further validated the suite of assays by treatment with KU mTOR inhibitor and bafilomycin A1 AVs-lysosome fusion inhibitor , which are known to induce and block autophagy, respectively.
LC3B-II and p62 levels in each cell type were generally in agreement with the above described kinetic change of LC3B-II and p However, under the culturing conditions used, astrocytes produced the most robust response to autophagy stimuli. Furthermore, in the same concentration range a clear influence of cell viability of the two markers is detectable in HEKT cells, especially at 24 hours post treatment.
Assay results for unknown compounds should be interpreted carefully when cytotoxicity is evident. We also compared measurement of LC3B-II using TR-FRET versus western blotting. One common method to discriminate between autophagy inducers and blockers is to block autophagy by bafilomycin A1 a few hours after treatment with the compound under investigation and evaluate the amount of LC3B-II by western blot.
As demonstrated with KU, an autophagy inducer results in an increase in LC3B-II, while co-treatment with bafilomycin A1 augments the signal, since the additional AVs that were made are not degraded by the lysosome. In contrast, NVP-TAE, an autophagy blocker results in an increase in LC3B-II, but the signal is not altered by bafilomycin A1; additional blockage had no influence on the LC3B-II signal since there was no upregulation of AV generation.
In both cases the TR-FRET and western blot gave similar results. The TR-FRET method is highly quantitative with its direct measurement of fluorescent emission, in comparison to western blots which are more qualitative and require a secondary antibody for visualization.
It is noteworthy that the combination of the LC3B-II and p62 TR-FRET signals, plus cytotoxicity, predicts whether a compound is an autophagy stimulator or inhibitor, similar to bafilomycin A1 co-treatment. HEK cells were lysed in PBS buffers with different detergent Tween20, Triton-X , ionic strength NaCl and glycerol concentrations.
Bulk amounts of brain homogenates A or HEKT cell lysates treated with or without bafilomycin A1 B were distributed into aliquots. Data is presented as the difference between bafilomycin A1 treated and untreated cells. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.
Article Authors Metrics Comments Media Coverage Reader Comments Figures. Abstract Autophagy is a cellular mechanism that can generate energy for cells or clear misfolded or aggregated proteins, and upregulating this process has been proposed as a therapeutic approach for neurodegenerative diseases.
Introduction Macroautophagy is a cellular process that leads to the inclusion of cytoplasmic contents by double-membraned vesicles called autophagic vacuoles AV; also called autophagosomes and fusion to lysosomes for degradation, and is the prevalent process in a more general pathway; here we will refer to this process as autophagy throughout.
Compound treatments Compound treatments were performed in well clear plates Greiner, and well black plates to evaluate viability Greiner, Time-resolved fluorescence resonance energy transfer TR-FRET assays TR-FRET assays were performed transferring a proper amount of cell lysate to a low volume well plate Greiner, and adding the antibody pairs diluted in lysis buffer.
Cell viability evaluation by immunofluorescence Immunofluorescence in 96 well black plates Greiner, was used to evaluate viability after cell treatment with each compound. Mouse studies All mice were treated in accordance with the Guide for the Animal Care and Use of Laboratory Animals National Research Council , and all procedures were approved by the Institutional Animal Care and Use Committee at PsychoGenics, Inc.
Pharmacodynamic analysis p62 and LC3II levels in liver samples of compound treated mice were quantified using crude liver tissue homogenates with a concentration of 0.
Results TR-FRET TR-FRET is a homogeneous assay able to measure molecule proximity by fluorescence resonance energy transfer. Download: PPT. Technical development and evaluation of p62 and LC3B-II TR-FRET assay specificity LC3B and SQSTM1 p62 gene silencing was performed on HEKT cells in order to verify the specificity of the TR-FRET signal for the two proteins.
Reference compound profiling on HEKT and primary cultures of rat neurons and astrocytes To demonstrate the utility of p62 and LC3B-II quantification by TR-FRET, compounds previously reported to modulate the autophagy pathway were evaluated in different cell models.
Fig 3. LC3B-II and p62 quantification in response to tool compounds treatment. Confirmation of autophagy inducers vs blockers The accumulation of AVs can be a result of either upregulated or blocked autophagy.
Fig 5. Co-treatment with bafilomycin A1 to distinguish autophagy enhancers versus blockers. Quantification of autophagy in tissues The LC3B-II and p62 TR-FRET assays allow for label-free quantification of autophagy changes in tissues.
Fig 6. In vivo mTOR inhibition resulted in a measurable stimulation of autophagy. Conclusions We set out in these studies to establish a set of assays that could measure autophagy by quantifying endogenous markers, and could be used variously to screen in wells as well as in primary cells and animal studies.
Supporting information. S1 Fig. LC3B-II TR-FRET lysis buffer optimization. s PDF. S2 Fig. Pharmodynamics of mTOR inhibitor. S3 Fig. Evaluation of the storage of LC3B-II and p62 protein in cell lysates and mouse brain homogenates.
Acknowledgments We thank Psychogenics for conducting the mouse work and Charles River for the PK analysis. References 1. Feng Y, He D, Yao Z, Klionsky DJ. Using the double tag LC3B and the GFP-Rab7 degradation assays, Hundeshagen et al. Cardiac glycosides are used in treatment of heart failure and arrhythmia.
It is known that increases in intracellular calcium induce autophagy, so it is perhaps not surprising that cardiac glycosides activate autophagy. Cardiac glycosides have been suggested for cancer therapy, and their stimulatory effect on autophagy may be important in this context.
There is one further proviso about screening strategy. Hundeshagen et al. The importance of the strategy chosen for the primary screen is emphasized by results from another group [ 10 ], who screened a collection of 3, chemicals, including the chemical library Hundeshagen et al.
The difference was that Balgi et al. In this screen perhexiline, niclosamide, amiodarone and rottlerin were identified as autophagy modulators [ 10 ]. Considering these different results, and the caveats and shortcomings that different assays have, it seems necessary to use a combination of different assays to perform exhaustive screens for small molecule autophagic modulators.
That said, the work of Hundeshagen and colleagues [ 5 ] is clearly a step forward in quantitative cell population based monitoring of distinct steps of the autophagy pathway in the screening for autophagy modulators. Further development and refinement of autophagy screening protocols from this and other groups are to be expected.
Levine B, Kroemer G: Autophagy in the pathogenesis of disease. Article PubMed Central CAS PubMed Google Scholar. Mizushima N, Levine B, Cuervo AM, Klionsky DJ: Autophagy fights disease through cellular self-digestion. Johansen T, Lamark T: Selective autophagy mediated by autophagic adapter proteins.
Mathew R, White E: Autophagy in tumorigenesis and energy metabolism: friend by day, foe by night. Curr Opin Genet Dev. Hundeshagen P, Hamacher-Brady A, Eils R, Brady NR: Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy.
BMC Biology. Mizushima N, Yoshimori T, Levine B: Methods in mammalian autophagy research. Shvets E, Fass E, Elazar Z: Utilizing flow cytometry to monitor autophagy in living mammalian cells. Article CAS PubMed Google Scholar. Kimura S, Noda T, Yoshimori T: Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3.
J Biol Chem. Balgi AD, Fonseca BD, Donohue E, Tsang TC, Lajoie P, Proud CG, Nabi IR, Roberge M: Screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mTORC1 signaling.
PLoS One. Article PubMed Central PubMed Google Scholar. Download references. This work was supported by grants from the FRIBIO program of the Norwegian Research Council, the Norwegian Cancer Society, the Aakre Foundation and the Blix Foundation to T.
Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø, , Tromsø, Norway. You can also search for this author in PubMed Google Scholar.
Correspondence to Terje Johansen. This article is published under license to BioMed Central Ltd. Reprints and permissions. Hansen, T. Following autophagy step by step.
BMC Biol 9 , 39 Download citation. Received : 27 May Accepted : 02 June Published : 02 June Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative.
Skip to main content. Search all BMC articles Search. Download PDF. Abstract Autophagy is an evolutionarily conserved lysosomal degradation route for soluble components of the cytosol and organelles. Autophagy and disease Research during the last decade has made it increasingly clear that autophagy plays important roles in most of the major human diseases as well as in infection and immunity [ 1 , 2 ], with increasing evidence for selective autophagy of protein aggregates, organelles and pathogens [ 3 ].
Figure 1. Full size image. Monitoring autophagic flux It is important to distinguish effects on lysosomal activity from effects on autophagosome formation.
Figure 2. The significance of cardiac glycosides, and some provisos A general problem when attempting to measure autophagic flux is the interconnection between the endocytic and autophagosomal pathways see Figure 1 , which makes it difficult to ensure that the observed effects are on autophagic flux and not the endolysosomal pathway.
References Levine B, Kroemer G: Autophagy in the pathogenesis of disease. Article PubMed Central CAS PubMed Google Scholar Mizushima N, Levine B, Cuervo AM, Klionsky DJ: Autophagy fights disease through cellular self-digestion.
Article PubMed Central CAS PubMed Google Scholar Johansen T, Lamark T: Selective autophagy mediated by autophagic adapter proteins. Article PubMed Central CAS PubMed Google Scholar Mathew R, White E: Autophagy in tumorigenesis and energy metabolism: friend by day, foe by night.
Article PubMed Central CAS PubMed Google Scholar Hundeshagen P, Hamacher-Brady A, Eils R, Brady NR: Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy. Article PubMed Central CAS PubMed Google Scholar Mizushima N, Yoshimori T, Levine B: Methods in mammalian autophagy research.
Article PubMed Central CAS PubMed Google Scholar Shvets E, Fass E, Elazar Z: Utilizing flow cytometry to monitor autophagy in living mammalian cells. Article CAS PubMed Google Scholar Kimura S, Noda T, Yoshimori T: Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3.
Autophagy and LC is the annd widely used marker of autophagosomes. LC3 was originally Hypertension and potassium-rich foods as a microtubule associated protein in abd brain. MAP1LC3B is a member of the highly conserved ATG8 Autphagy family. ATG8 proteins are present uAtophagy all known eukaryotic organisms. The animal ATG8 family comprises three subfamilies: i microtubule-associated protein 1 light chain 3 MAP1LC3 ; ii Golgi-associated ATPase enhancer of 16 kDa GATE ; and iii γ-amino-butyric acid receptor-associate protein GABARAP. MAP1LC3B is one of the four genes in the MAP1LC3 subfamily others include MAP1LC3AMAP1LC3Cand MAP1LC3B2. Newly synthesized LC3's C-terminus is hydrolyzed by a cysteine protease called ATG4B exposing Gly, termed LC3-I. Thank Autophagg for visiting nature. Autophagy and LC are using a browser version with limited Autophgy for CSS. To obtain the Top Fat Burner experience, Hypertension and potassium-rich foods recommend you use a more up to date browser or Autlphagy Autophagy and LC compatibility mode in Boost insulin sensitivity naturally Explorer. In anf meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Autophagy is an evolutionarily conserved process across eukaryotes that degrades cargoes like aggregate-prone proteins, pathogens, damaged organelles and macromolecules via delivery to lysosomes. The process involves the formation of double-membraned autophagosomes that engulf the cargoes destined for degradation, sometimes with the help of autophagy receptors like p62, which are themselves autophagy substrates. LC3-II, a standard marker for autophagosomes, is generated by the conjugation of cytosolic LC3-I to phosphatidylethanolamine PE on the surface of nascent autophagosomes.
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