ULTRASENSITIVE MEASUREMENT OF MUTANT HUNTINGTIN IN HUMAN CSF AND RODENT SAMPLES

Huntington disease (HD) is a neurodegenerative disorder caused by a genetic defect in Huntingtin gene (<i>HTT</i>) that leads to the expression of an expanded poly-glutamine protein. HD has a broad impact on a person's functional abilities due to progressive motor dysfunction, cognitive decline, and psychiatric disturbance, probably caused by both neuronal dysfunction and neuronal cell death. New strategies for drug discovery and new therapeutic approaches are now leading to slow the progression of HD; one of these promising strategies consists in reducing the mutant protein level through its direct reduction (gene silencing) or increasing its clearance (compound treatment). In this framework mutant HTT protein can be a biomarker for treatment efficacy and disease progression, raising the necessity to develop highly sensitive immunoassays to quantify its level in biological fluids. The Single Molecule Counting (SMC?) technology, powered by Singulex?, enabled the ultrasensitive measurement of mutant HTT at levels previously undetectable in human Cerebrospinal fluid (CSF). The immunoassay was developed using the 2B7 and the MW1 antibodies that recognize the N-terminal region and the poly-Q trait of the protein respectively and was successfully applied to human and murine derived fluids and murine tissues. Here is presented a summary of results obtained from the assay validation process, including the evaluation of calibration curve performance, specificity, matrix effect (spike recovery, parallelism and dilution linearity), selectivity, accuracy, precision and stabilit

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

<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

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

The temperature dependence of the 2B7-MW1 TR-FRET signal is inversely proportional to polyQ length.

<p>A.-B. Fluorescence signals obtained from dilution curves of N548 proteins bearing Q16, Q19, Q25, Q33, Q39 and Q55, tested by TR-FRET (1 ng/µl of 2B7-Tb and 10 ng/µl of MW1-d2) at RT (A) and 4°C (B). C. Ratio between the maximum value of the TR-FRET signal obtained at 4°C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves presented in A and B. D. Ratio obtained and expressed as in C after performing TR-FRET with 2B7-Tb (1 ng/µl) and MW1-d2 (10 ng/µl) antibody combination (as before) or MW1-Tb (1 ng/µl) and 2B7-Alexa647 (10 ng/µl) antibody combination on N548 proteins bearing Q16 and Q55 repeats. In C and D data are represented as mean ± S.D. of three independent experiments; significance was calculated using the one-way ANOVA test and Bonferroni's multiple comparison post-test (** p<0.01 and * p<0.05).</p

Temperature- and polyQ- dependent conformational changes detected on HTT full length protein (Q18 and Q83) using the 2B7-MW1 TR-FRET assay in a biological sample (lysates from transfected cells).

<p>A–B. TR-FRET values obtained at RT (A) or 4°C (B) using 2B7-Tb (1 n/µl) - MW1-d2 (10 ng/µl) on lysates, confirming the temperature- and polyQ-dependent conformational effect. C. Absolute ratios between the maximum value of the TR-FRET signal obtained at 4°C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves in A and B. D. Absolute ratios between the maximum value of the TR-FRET signal obtained at 4°C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves achieved using a 1∶1 ratio of 2B7-Tb and MW1-d2 (1 n/µl for both antibodies). E-F. TR-FRET values obtained at RT (E) or 4°C (F) using 2B7-Tb and 4C9-Alexa647 on the same lysates, confirming the requirement for polyQ detection to observe conformational changes. In C, D and G data are represented as mean ± S.D. of three independent experiments; significance was calculated using the one-way ANOVA test and Bonferroni's multiple comparison post-test (** = p<0.01; *** = p<0.005).</p

Temperature-dependent alpha-helical variations in thioredoxin-tagged exon 1 HTT proteins are not due to variations in tag helicity, and are also observed in untagged, larger N548 HTT purified recombinant proteins.

<p>A. MRE values at 222 nm (at which alpha-helical structure has a signature peak) for Q25 and Q46 THRX-HTT exon 1 at each temperature tested. Red asterisks denote measurements taken at 4°C and RT, the temperatures used in the TR-FRET experiments. Black asterisks denote <i>p</i>-values as determined by the Student's <i>t</i>-test. *<i>p</i><0.05. **<i>p</i><0.01. ns, not significant. All data are represented as mean ± S.D. of three independent experiments. B. CD spectra for the THRX fusion partner alone at indicated temperatures. C. MRE spectra for the Q25 N548 HTT protein at −10°C and 37°C, showing that the temperature effect can still be seen in this longer fragment. D. MRE values at 222 nm at each indicated temperature relative to MRE at 37°C for N548 HTT of increasing polyQ length (Q19, Q22, Q25, Q42, Q52, Q55).</p