Quantifying autophagy using novel LC3B and p62 TR-FRET assays

<div><p>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. Here we describe a novel set of LC3B-II and p62 time-resolved fluorescence resonance energy transfer (TR-FRET) assays that can detect changes in autophagy in the absence of exogenous labels. Lipidated LC3 is a marker of autophagosomes, while p62 is a substrate of autophagy. These assays can be employed in high-throughput screens to identify novel autophagy upregulators, and can measure autophagy changes in cultured cells or tissues after genetic or pharmacological interventions. We also demonstrate that different cells exhibit varying autophagic responses to pharmacological interventions. Overall, it is clear that a battery of readouts is required to make conclusions about changes in autophagy.</p></div

Evaluation of LC3B-II and p62 TR-FRET specificity.

<p>Genetic validation of the LC3B-II and p62 readouts was achieved by gene silencing of LC3B and p62 in HEK293T cells using shRNA. <i>LC3B</i> and <i>p62</i> mRNA were reduced after shRNA of each gene, compared to shRNA scramble, as verified by qRT-PCR (N = 3; avg±SD; Student’s t-test (unpaired; two-tailed); *p<0.001), expression levels were calculated using the 2^-ΔΔCT method and expressed relative to scramble control (A). A corresponding reduction of protein levels was observed by western blot (B) and TR-FRET (N = 4; avg±SD; Student’s t-test (unpaired; two-tailed); *p<0.005; expression relative to scramble control; C). ATG4B overexpression in HEK293T cells was confirmed with western blot (D). ATG4B overexpression did not alter LC3B mRNA levels as seen by qRT-PCR (N = 3, avg±SD; Student’s t-test (unpaired, two-tailed) p>0.05, expression levels were calculated using the 2^-ΔΔCT method and expressed relative to scramble control (E) but clearly reduced LC3B-II detection, as measured by TR-FRET (expressed relative to empty vector; N = 3, unpaired t-test, *p<0.001 (F) and western blot (D). LC3B-II quantification by TR-FRET (fluorescence ratio of 665/615 nm) in HEK293 cells showed detection with as few as 2000 cells/well (N = 2; avg±SD; G). Different concentrations of purified p62 were measured and the p62 TR-FRET assay is sensitive enough to detect 1ng/ml purified recombinant p62 protein (signal expressed as fluorescence ratio of 665/615 nm; N = 2; avg±SEM; H).</p

LC3B-II and p62 quantification in response to tool compounds treatment.

<p>HEK293T cells (A), rat cortico-striatal neurons (B) and rat astrocytes (C) were treated with a serially diluted autophagy inhibitor (bafilomycin A1) or upregulator (KU0063794) and examined at 2, 6 and 24 hours post-treatment. LC3B-II and p62 were measured with TR-FRET (A-C). The response to compound treatment is reported as percentage average of three replicates with respect to vehicle (DMSO) treated samples (100%). Cell viability was evaluated by H33342 stained nuclei count for HEK293T (A) and astrocytes (C-E) and by neurite length/soma (morphometric readout) on MAP2 stained neurons (B). Astrocytes were also treated with SU11652 and NVP-TAE684 and LC3B-II, p62 and viability were measured with TR-FRET (D-E). Each data point is the mean±SEM (N = 3).</p

LC3B-II and p62 protein responses after tool compound.

<p>HEK293T cells (A), rat cortico-striatal neurons (B) and rat astrocytes (C) were treated with an autophagy inhibitor (5 nM bafilomycin A1) or upregulator (KU0063794) and examined at 2, 6 and 24 hours post-treatment, compared to DMSO. Western blot analysis is presented for LC3B-I/II and p62 levels, with a GAPDH loading control.</p

<i>In vivo</i> mTOR inhibition resulted in a measurable stimulation of autophagy.

<p>6 month mice were treated one time with a mTOR inhibitor and sacrificed 0.5 or 2 hours afterwards. mTOR inhibition increased LC3B-II (A) and reduced p62 (B) levels in the mouse liver, as measured by TR-FRET and expressed as the fluorescence ration (665/615 nm) ΔF; N = 5; one-way ANOVA, p<0.01; Tukey’s multiple comparison test, *p<0.05, **p≤0.01.</p

Co-treatment with bafilomycin A1 to distinguish autophagy enhancers versus blockers.

<p>Rat primary astrocytes were treated with 10 μM KU0063794, 1 μM SU11652 or 5 μM NVP-TAE684 (concentration selected from information in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194423#pone.0194423.g002" target="_blank">Fig 2</a>) followed by either 50 nM bafilomycin A1 (+Baf) or vehicle (DMSO, -Baf) for an additional 4 hours. 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. Co-treatment of KU0063794 and bafilomycin A1 increased LC3B-II, compared to KU0063794 or bafilomycin A1 alone (N = 2, one-way ANOVA, p<0.01; Tukey’s multiple comparison test, *p<0.05); co-treatment of SU11652 and bafilomycin A1 increased LC3B-II, compared to SU11652 or bafilomycin A1 alone (N = 2, one-way ANOVA, p<0.01; Tukey’s multiple comparison test, *p<0.05); co-treatment of NVP-TAE684 and bafilomycin A1 did not alter the LC3B-II signal (N = 2, one-way ANOVA, p>0.05; (A). Western blots (B) confirm the TR-FRET data.</p