Scorpion Venom Heat-Resistant Peptide Protects Transgenic Caenorhabditis elegans from β-Amyloid Toxicity

Abstract

Scorpion venom heat-resistant peptide (SVHRP) is a component purified from Buthus martensii Karsch scorpion venom. Our previous studies found SVHRP could enhance neurogenesis and inhibit microglia-mediated neuroinflammation in vivo. Here, we use the transgenic CL4176, CL2006, and CL2355 strains of Caenorhabditis elegans which express the human Aβ1-42 to investigate the effects and the possible mechanisms of SVHRP mediated protection against Aβ toxicity in vivo. The results showed that SVHRP-fed worms displayed remarkably decreased paralysis, less abundant toxic Aβ oligomers, reduced Aβ plaque deposition with respect to untreated animals. SVHRP also suppressed neuronal Aβ expression-induced defects in chemotaxis behavior and attenuated levels of ROS in the transgenic C. elegans. Taken together, these results suggest SVHRP could protect against Aβ-induced toxicity in C. elegans. Further studies need to be conducted in murine models and humans to analyze the effectiveness of the peptide.

Keywords: C. elegans; alzheimer’s disease; amyloid beta-peptide; behavior; scorpion venom heat-resistant peptide.

FIGURE 1

Scorpion venom heat-resistant peptide (SVHRP) delayed β-amyloid induced paralysis in transgenic Caenorhabditis elegans strain CL4176. (A) Diagram illustrating the paralysis assays showing the time at which the temperature was raised in CL4176 and CL802 worms, when the paralysis assay was scored and SVHRP protective effect at different treatment regimens. (B) Time course of Aβ-induced paralysis in the transgenic CL4176 strain treated with a vehicle (Ctrl) or different concentrations of SVHRP (2-80 μg/ml). Data are shown as percentages ± SD of worms not paralyzed (n = 100, three independent assays). (C) The paralysis assays were quantified for mean time duration at which 50% worms were paralyzed (PT₅₀) from the transgenic worms fed with or without SVHRP. Error bars indicate SD (n = 100, three independent assays, ^∗p < 0.05, ^∗∗p < 0.01 vs. control group).

FIGURE 2

Scorpion venom heat-resistant peptide reduced β-amyloid oligomers in C. elegans. (A) Representative western blot of Aβ species in CL2006, CL4176, and CL802 worms fed vehicle or SVHRP. CL2006 maintained at 20°C fed with a vehicle (Ctrl), SVHRP (40 μg/ml) for 96 h. CL4176 and CL802 worms fed with a vehicle (Ctrl), SVHRP (40 μg/ml) were maintained for 36 h at 16°C, then the temperature was raised from 16 to 23°C. 25 h later, the worms were collected and equal amounts of protein were loaded in each lane and immunoblotted with anti-Aβ antibody (6E10) or tubulin. Arrows indicate the Aβ oligomers (20 kDa). Quantification of Aβ oligomers (the band at 20 kDa) in CL2006 (B) and CL4176 (C) worms fed either vehicle or SVHRP using ImageJ software. Data are expressed as mean density of the indicated band based from three independent experiments. Error bars represent SD. ^∗p < 0.05.

FIGURE 3

Aβ deposits in transgenic C. elegans CL2006 fed with or without SVHRP. Representative images of C. elegans with thioflavin S staining in the wild type (a), or in the transgenic strain CL2006 fed with (c) or without SVHRP (b). The number of deposits (arrows) was scored in the worm head (d). Data were obtained from three experiments with 24 worms in each group (n = 72). The quantity is expressed as mean number of β-amyloid deposits in the head region /anterior area of the worm. Error bars indicate SE. ^∗p < 0.05.

FIGURE 4

Effect of SVHRP on reactive oxygen species (ROS) production in transgenic C. elegans strains. Age-synchronized CL4176 or CL802 worms, fed vehicle or different concentrations of SVHRP (2, 20, 40 μg/ml) were collected 36 h after the temperature rise followed by the DCF (2, 7-dichlorofluorescein diacetate) assay for ROS described in Materials and Methods. (A) DCF fluorescent images of nematodes fed vehicle or different concentrations of SVHRP. Scale bars: 100 μm. (B) The DCF fluorescence intensity was detected by a microplate reader at 485 nm excitation and 530 nm emission. Results are expressed as percentage of fluorescence (%DCF) relative to vehicle-treated controls, which is set as 100%. Data were obtained from three experiments with 50 worms in each experiment. Error bars indicate SE, ^∗p < 0.05, ^∗∗p < 0.01 vs. control group).

FIGURE 5

Assays for chemotaxis behavior in neuronal Aβ-expressing strain CL2355. (A) Schematic diagram of the chemotaxis assay. Plates were divided into quadrants two test (A&D) and two controls (B&C). Sodium azide was also included with the attractant and control odorant to paralyze worms. Worms were placed at the center of the plate and after 60 min worms were counted on each quadrant. Schematic examples of neutral and attractant are indicated. (B) The Chemotactic Index (CI) in the neuronal strain CL2355 and the transgenic control strain CL2122 fed with vehicle or different concentration of SVHRP (2, 20, 40 μg/ml). Data were obtained from three experiments with 60 worms in each group. Error bars indicate SD (^∗∗p < 0.01 vs. CL2355 control group).