C. elegans expressing D76N β2-microglobulin: a model for in vivo screening of drug candidates targeting amyloidosis

Abstract

The availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease and the exploration of new therapeutic avenues. The natural human variant of β₂-microglobulin (D76N β₂-m) is associated with a fatal familial form of systemic amyloidosis. Hitherto, no animal model has been available for studying in vivo the pathogenicity of this protein. We have established a transgenic C. elegans line, expressing the human D76N β₂-m variant. Using the INVertebrate Automated Phenotyping Platform (INVAPP) and the algorithm Paragon, we were able to detect growth and motility impairment in D76N β₂-m expressing worms. We also demonstrated the specificity of the β₂-m variant in determining the pathological phenotype by rescuing the wild type phenotype when β₂-m expression was inhibited by RNA interference (RNAi). Using this model, we have confirmed the efficacy of doxycycline, an inhibitor of the aggregation of amyloidogenic proteins, in rescuing the phenotype. In future, this C. elegans model, in conjunction with the INVAPP/Paragon system, offers the prospect of high-throughput chemical screening in the search for new drug candidates.

Figure 1

D76N β₂-m C. elegans strain genotype characterization and β₂-microglobulin expression. (a) Scheme of C. elegans manipulation highlighting the stage in which the β₂-m expression is activated by temperature switch. Eggs were maintained at 16 °C and L1 larvae were up-shifted to 23–25 °C in order to induce the expression of the protein. (b) PCR genotyping of adult transgenic nematodes. The expected size of PCR products (about 800 bp) was observed after DNA electrophoresis on 1.5% agarose gel (lane 1: EZ Load Precision, BioRad, lane 2,4: pcr products of DNA extracted from smg-1 (cc546) ancestral strain, lane 3,5: pcr products of DNA extracted from D76N β₂-m worms. (c) Representative western blot of β₂-m expression. Equal amounts of protein (10 μg) were loaded for each sample from smg-1 (-ctrl) or D76N β₂-m expressing nematodes and immunoblotted with polyclonal anti-human β₂-m antibody (Dako) and anti-GAPDH antibody used as loading control (M = Molecular weight standard: Precision Plus Western C, BioRad). Uncropped scans of immunoblots are shown in Supplementary Fig. S1.

Figure 2

Self-assembly of β₂-m D76N in transgenic C. elegans strain. (a) Immunoblot analysis of soluble (SF) and insoluble fraction (IF) of worm lysates grown at 25 °C resolved via 8–18% SDS PAGE and detected with anti-β₂-m antibody (DAKO). The insoluble fraction was washed twice in PBS buffer before loading onto gel. (M = Molecular weight standard: Precision Plus Western C, BioRad). (b–d) Size-excluded soluble proteins from β₂-m expressing nematodes and recombinant D76N β₂-m. Shown is the absorbance at 280 nm (Abs 280) of eluted material against elution volume. Fractions (1 ml) were collected. (c–e) Immunoblot analysis of size-excluded fractions (6–20) of β₂-m expressing worms’ lysates and recombinant β₂-m resolved via 8–18% SDS PAGE and detected with anti-β₂-m antibody (DAKO). Uncropped scans of immunoblots are shown in Supplementary Fig. S2a.

Figure 3

Movement index analysis using the INVAPP/Paragon system. 3 L4 worms/well were placed onto NGM 6-well plates, fed with OP50 E. coli, and maintained at 25 °C for six days (a). Worms were imaged using INVAPP Paragon software (b). Data are mean of movement index parameter ± SEM; which was obtained after processing the acquired images (c). Three independent experiments were carried out and the results were plotted using GraphPad Prism (v6), **p < 0.01 vs the control (smg-1) according to t-test.

Figure 4

D76N β₂-m expression is prevented by RNA-interference (RNAi). Equal amounts of proteins (40 µg) were loaded on each lane and immunoblotted with polyclonal anti-human β₂-m antibody (a) and with anti-GAPDH (b). Lanes: M = Precision Plus Western C (Bio Rad) molecular weight standard. smg-1 incubated at 25 °C and collected at day 1 of adulthood. D76N β₂-m expressing nematodes incubated at 25 °C and collected at day 1 of adulthood and fed with HT115 bacteria transformed, as reported in the Methods section, with PAV2 plasmid for RNA-interfering (black box) or with control bacteria. Uncropped scans of immunoblots are shown in Supplementary Fig. S3. (c) Percentage of β₂-m expression is given as D76N β₂-m/GAPDH ratio of the WB band density of the RNAi strain relative to the control at their first day of adulthood. Density of the bands was determined by Image Studio Lite (LI-COR Biosciences). Three independent WB experiments were carried out and the results were plotted using GraphPad Prism (v6), p = 0.0052 vs the control level of expression (black) according to one sample t-test. (d) Index movement analysis. Three L4 worms were placed into NGM plates, fed with HT115/L4440 or HT115 bacteria transformed with PAV2 for RNA interference. Plates were maintained at 25 °C for 5 days. At this stage, the plates with adult worms and progenies were analysed by INVAPP Paragon software. Three independent experiments were carried out and the results were plotted using GraphPad Prism (v6). Data are mean of movement index parameter ± SEM; **p < 0.01 vs ctrl worms and °p < 0.05 vs the non-silenced D76N β₂-m expressing according to one-way Anova.

Figure 5

Characterization of the phenotype of D76N β₂-m strain by classic behavioral assays. (a) Egg-synchronized control worms (smg-1) and D76N β₂-m expressing worms were placed at 23 °C into fresh NMG plates seeded with OP50 E. coli. At day 1, 5 and 6 of adulthood body bends were scored in liquid. At least three independent assays were performed. Data are mean of number of body bends/min ± SEM; **p < 0.01 and *p < 0.05 vs the control strain (smg-1), according to one-way ANOVA (N = 40 animals for each group). (b) Kaplan–Meier survival curves of control nematodes and D76N β₂-m strain. Data are expressed as mean of three independent experiments (N = 40 animals for each group, χ² = 5.52, p = 0.019 according to Peto-Peto-Prentice test). (c) Total eggs deposition for smg-1 and D76N β₂m strains maintained at 23 °C. At least three independent assays were performed. Error bars represent the SEM, *p < 0.05 vs. the control strain (smg-1) according to t-test. (d) Percentage of not-hatched eggs of smg-1 and D76N β₂-m strains analyzed 24 h after deposition. At least three independent assays were performed. Error bars represent the SEM, ***p < 0.001 vs. the control strain according to t-test.

Figure 6

(a) Movement index analysis in presence and absence of doxycycline. Three L4 worms/well were placed onto NGM 6-well plates, fed with OP50 E. coli, and incubated at 25 °C for six days in presence of 0 or 100 μM of doxycycline from the L4 larval stage. Then the plates with adult worms and progenies were analyzed by INVAPP Paragon software. Three independent experiments were carried out and the results were plotted using GraphPad Prism (v6), ***p < 0.001 vs the untreated control (D76N β₂-m expressing nematodes) and °p < 0.5 vs. the ctrl worms according to one-way Anova. (b) Egg-synchronized control worms (smg-1) and D76N β₂-m expressing worms were placed at 23 °C into fresh NMG plates seeded with OP50 E. coli. At day 5 of adulthood body bends were scored in liquid. At least three independent assays were performed. Data are mean of number of body bends/min ± SEM; ***p < 0.001 vs the untreated control (D76N β₂-m expressing nematodes) and ****p < 0.0001 vs the ctrl worms, according to one-way ANOVA (N = 40 animals for each group). (c) Percentage of β₂-m in eluted fractions from gel filtration of D76N β₂-m expressing worms treated with 0 or 100 μM of doxycycline, is given as ratio of D76N β₂-m quantity of eluted fraction/soluble fraction starting material quantified from WB bands density related to the monomeric molecular weight (mean ± SEM, n = 2). Uncropped scans of immunoblots are shown in Supplementary Fig. S6. Density of the bands was determined by Image Studio Lite (LI-COR Biosciences).