C. elegans expressing human β2-microglobulin: a novel model for studying the relationship between the molecular assembly and the toxic phenotype

Abstract

Availability of living organisms to mimic key step of amyloidogenesis of human protein has become an indispensable tool for our translation approach aiming at filling the deep gap existing between the biophysical and biochemical data obtained in vitro and the pathological features observed in patients. Human β(2)-microglobulin (β(2)-m) causes systemic amyloidosis in haemodialysed patients. The structure, misfolding propensity, kinetics of fibrillogenesis and cytotoxicity of this protein, in vitro, have been studied more extensively than for any other globular protein. However, no suitable animal model for β(2)-m amyloidosis has been so far reported. We have now established and characterized three new transgenic C. elegans strains expressing wild type human β(2)-m and two highly amyloidogenic isoforms: P32G variant and the truncated form ΔN6 lacking of the 6 N-terminal residues. The expression of human β(2)-m affects the larval growth of C. elegans and the severity of the damage correlates with the intrinsic propensity to self-aggregate that has been reported in previous in vitro studies. We have no evidence of the formation of amyloid deposits in the body-wall muscles of worms. However, we discovered a strict correlation between the pathological phenotype and the presence of oligomeric species recognized by the A11 antibody. The strains expressing human β(2)-m exhibit a locomotory defect quantified with the body bends assay. Here we show that tetracyclines can correct this abnormality confirming that these compounds are able to protect a living organism from the proteotoxicity of human β(2)-m.

Figure 1. Genotype of C. elegans transgenic strains.

(A) PCR genotyping of adult transgenic worms transfected with the empty vector (vector) or vectors for expression of wild type β₂-m (WT), P32G or 7–99 truncated form (ΔN6). The expected size of PCR products (about 360 bp) was observed. (B) Human β₂-m mRNA expression in different transgenic strains was normalized to worm cell division cycle 42 (cdc-42, GTP binding protein) as endogenous reference. Data are expressed as mean ± SD of three independent experiments.

Figure 2. Human β₂-m protein expression.

(A) Representative dot blot of β₂-m (polyclonal anti-human β₂-m antibody) in transgenic worms and (B) quantification of β₂-m immunoreactive bands. Data are mean values of density of immunoreactive bands/µg of protein ± SE of three independent experiments (N = 6). (C) Representative western blot of β₂-m in control worms (vector), wild type β₂-m expressing worms (WT), and in nematodes expressing P32G (P32G) or ΔN6 β₂-m isoform (ΔN6). Day 1 adult worms were collected, processed as described in Methods section, and equal amounts of proteins (40 µg) were loaded on each lane and immunoblotted with polyclonal anti-human β₂-m antibody (Dako). (D) Representative dot blot developed by antibody recognizing oligomers (A11) in transgenic worms and (E) quantification of A11-immunoreactive bands. Data are expressed as mean of density of A11 immunoreactive bands/µg of protein ± SE of three independent experiments (N = 9); *p<0.01 vs WT, according to one-way ANOVA.

Figure 3. Localization of β₂-m in transgenic C. elegans strains.

Overlay of bright field and immunofluorescence images of head, vulva and tail of transgenic C. elegans strains. All animals depicted are 2 days adult worms. A specific β₂-m related signal (red, using a polyclonal anti human β₂-m antibody) was observed at the vulva muscles and anal sphincter muscle in the tail (red arrows) whereas no signal was observed in the head muscles. Scale bar, 50 µm.

Figure 4. Behavioural phenotypes of transgenic C. elegans strains.

(A) Larval growth of control worms (Vector), wild type β₂-m expressing worms (WT) and nematodes expressing P32G or 7–99 truncated form of β₂-m (ΔN6). One hundred synchronized eggs were placed into fresh NMG plates seeded with OP50 as food, and the number of L1/L2, L2/L3 and L4/adult worms were scored after 24, 48 and 72 hours, respectively. Data are expressed as percentage of total worms in the plate at each time point and are given as mean of three independent experiments (N = 300). (B) Correlation between oligomers of β₂-m and reduction in growth rate of transgenic C. elegans strains. Percentage of adult worms of each transgenic strain, scored 72 after egg synchronization, was correlated to the the amount of A11-positive oligomeric assemblies detected by dot blotting. Data of both graphic axes represent mean of three independent experiments. (C) Kaplan-Meier survival curves of transgenic hermaphrodite adult nematodes. Animals were placed in plates seeded with OP50 starting from L4, cultured at 20°C and transferred to fresh plates for each consecutive other days. Survival rate was scored every day and expressed as percent of survival. Plots are representative of three independent experiments (N = 30). (D) Body bends in liquid of transgenic worms. At least three independent assays were performed (N = 100 animals for each group). Data are given as mean of number of body bends/min ± SE, *p<0.05 and **p<0.01 vs. the vector, °°p<0.01 vs. WT, according to one-way ANOVA. (E) Superoxide anions production in control worms (Vector), wild type β₂-m expressing worms (WT) and in nematodes expressing P32G or 7–99 truncated form of β₂-m (ΔN6). Age-synchronized worms were collected in PBS containing 1.6 ml of 1% Tween 20 and colorimetric NBT assay was carried out as described in Materials and Methods. Results show the fold increase in superoxide production calculated as NBT absorbance/mg of proteins (% NBT) compared to Vector; *p<0.05 vs. vehicle and ° p<0.05 vs. WT, according to one-way ANOVA. Error bars indicate SD.

Figure 5. Effect of tetracycline on β₂-m induced locomotory defect in transgenic C. elegans strains.

Egg-synchronized control worms (vector), wild type β₂-m expressing worms (WT), P32G-mutated β₂-m and ΔN6-truncated β₂-m expressing nematodes (ΔN6) were placed at 20°C into fresh NMG plates seeded with tetracycline-resistant E. coli. At their L3/L4 larval stage, animals were fed with 50–100 µM tetracycline hydrochloride or 100 µM doxycycline (100 µl/plate). Body bends in liquid were scored after 24 hours. At least three independent assays were performed. Data are mean of number of body bends/min ± SD; **p<0.01 vs. the Vector, °°p<0.01 vs. the respective untreated group, according to one-way ANOVA (N = 60 animals for each group).