In vivo induction of membrane damage by β-amyloid peptide oligomers

Abstract

Exposure to the β-amyloid peptide (Aβ) is toxic to neurons and other cell types, but the mechanism(s) involved are still unresolved. Synthetic Aβ oligomers can induce ion-permeable pores in synthetic membranes, but whether this ability to damage membranes plays a role in the ability of Aβ oligomers to induce tau hyperphosphorylation, or other disease-relevant pathological changes, is unclear. To examine the cellular responses to Aβ exposure independent of possible receptor interactions, we have developed an in vivo C. elegans model that allows us to visualize these cellular responses in living animals. We find that feeding C. elegans E. coli expressing human Aβ induces a membrane repair response similar to that induced by exposure to the CRY5B, a known pore-forming toxin produced by B. thuringensis. This repair response does not occur when C. elegans is exposed to an Aβ Gly37Leu variant, which we have previously shown to be incapable of inducing tau phosphorylation in hippocampal neurons. The repair response is also blocked by loss of calpain function, and is altered by loss-of-function mutations in the C. elegans orthologs of BIN1 and PICALM, well-established risk genes for late onset Alzheimer's disease. To investigate the role of membrane repair on tau phosphorylation directly, we exposed hippocampal neurons to streptolysin O (SLO), a pore-forming toxin that induces a well-characterized membrane repair response. We find that SLO induces tau hyperphosphorylation, which is blocked by calpain inhibition. Finally, we use a novel biarsenical dye-tagging approach to show that the Gly37Leu substitution interferes with Aβ multimerization and thus the formation of potentially pore-forming oligomers. We propose that Aβ-induced tau hyperphosphorylation may be a downstream consequence of induction of a membrane repair process.

Keywords: Alzheimer’s disease; Caenorhabditis elegans; Pore-forming toxin; Tau; β-amyloid.

Fig. 1

Membrane repair model. This model is based on the mechanism for repair of plasma membrane (PM) pores created in mammalian cells by exposure to the bacterial pore-forming toxin SLO [32]. ASM is acidic spingomyelinase that is stored in lysosomes, which fuse to the PM in response to an influx of Ca2+ through the pore created by SLO (Step 1), thereby releasing ASM at the cell surface (Step 2). The localized release of ASM cleaves off the phosphoryl head group of sphingomyelinase in the vicinity of the pore to generate ceramide in that area (Step 3). Consequently, the PM around the pore undergoes inward curvature and endocytosis such that the pore is removed from the PM (Step 4)

Fig. 2

Induction of intestinal endosomes in C. elegans fed E. coli that express human Aβ peptide. a Live images of C. elegans reporter strain KWN117 (vha-6::mCherry) fed E. coli expressing either vector control, CRY5B toxin, wild type Aβ_1–42, or Aβ_1–42 Gly³⁷Leu. Note that both CRY5B and wild type Aβ_1–42 induce intestinal endosomes (arrows) containing the vha-6::mCherry reporter usually associated with the lumenal membrane of the intestine. This induction does not occur in worms fed E. coli expressing the non-pore-forming Aβ_1–42 Gly³⁷Leu variant. b Quantification of endosome induction. c Super resolution image of intestinal endosomes induced in strain KWN117 by wild type Aβ_1–42. The treated worm was subsequently fixed, permeabilized, and probed using anti-Aβ antibody 6E10. Note that the Aβ staining co-localizes with the membrane-associated vha-6::mCherry reporter (arrow), not the endosomal lumen. *p < 0.05 and **p < 0.01 when compared with the vehicle control

Fig. 3

Induction of intestinal endosomes is blocked by a mutation in the acid sphingomyelinase gene asm-1. a Introduction of the asm-1(tm5267) deletion allele into reporter strain KWN117 blocks the ability of E. coli expressing wild type Aβ_1–42 to induce intestinal endosomes. b The asm-1(tm5267) mutation also blocks endosome induction by the CRY5B toxin. *p < 0.05 and ***p < 0.001 when compared with the vehicle control

Fig. 4

Effects of amph-1, unc-11 and clp-4 mutations on Aβ-induced intestinal endosomes. a Strains containing the vha-6::mCherry reporter and deletion mutations in amph-1, unc-11, or clp-4 were fed E. coli expressing wild type Aβ_1–42. Note that the amph-1 and unc-11 mutations increase, while the clp-4 mutation decreases, the accumulation of intestinal endosomes. b Treatment with the calpain inhibitor PD150606 replicates the ability of the clp-4 mutation to block Aβ-induced increases in intestinal endosomes. c RNAi treatments indicate the clp-4 deletion mutation is epistatic to the effects of amph-1 and unc-11 knockdown. *p < 0.05, **p < 0.01 and ***p < 0.001 when compared with the vehicle control

Fig. 5

clp-4 deletion blocks endosome induction by CRY5B and sensitizes worms to CRY5B toxicity. a Induction of endosomes induced by CRY5B is blocked by the clp-4(ok2808) deletion mutation. *p < 0.05 when compared with the vehicle control. ###p < 0.001 when compared with the wt background. b Survival curves of C. elegans strains exposed to E. coli expressing CRY5B. Note that all three mutations that alter endosome accumulation also sensitize worms to CRY5B toxicity. *p < 0.05, **p < 0.01 compared to wild type control. NS = not significant

Fig. 6

Treatment of hippocampal neurons with streptolysin O (SLO) increases tau phosphorylation. a Treatment of hippocampal neurons with SLO increases tau immunoreactivity at the AT8 and PHF1 phospho-epitopes, assayed by quantification of immunofluoresence images and normalized to total tau. b Treatment of hippocampal neurons with SLO increases tau immunoreactivity at the AT8 and PHF1 phospho-epitopes, assayed by quantification of immunoblots. *p < 0.05, **p < 0.01 and ***p < 0.001 when compared with the vehicle control

Fig. 7

Treatment of hippocampal neurons with exogenous bacterial sphingomyelinase increases tau phosphorylation. a Cultured hippocampal neurons were treated with 2.5 mU/ml B. cereus sphingomyelinase, and tau immunoreactivity at the AT8 and PHF1 phospho-epitopes was quantified by immunofluorescence (normalized to total tau). b Cultured hippocampal neurons were treated with 2.5 mU B. cereus sphingomyelinase, and tau immunoreactivity at the AT8 and PHF1 phospho-epitopes was quantified by immunoblot (normalized to total tau). *p < 0.05, **p < 0.01 and ***p < 0.001 when compared with the vehicle control

Fig. 8

Biarsenical dye staining of dicysteine-tagged synthetic wild type and Gly³⁷Leu variant Aβ_1–42 in cultured hippocampal neurons. a Schematic model of how membrane-associated Aβ dimers in a parallel α-helical arrangement could bind the biarsenical FlAsH reagent. b Super-resolution image of cultured hippocampal neurons exposed to synthetic wild type Aβ_1–42, treated with FlAsH reagent, fixed, permeabilized, and probed with anti-Aβ antibody 6E10. Note minimal association of FlAsH signal with Aβ immunoreactivity, expected because this synthetic peptide does not contain dicysteines. c Same experiment as described for panel “B”, except treatment with synthetic dicysteine-tagged wild type Aβ. Note multiple foci of co-localized FlAsH and Aβ staining (arrows). d Same experiment as described for panel “B”, except treatment with synthetic dicysteine-tagged Gly³⁷Leu Aβ. Note this substitution prevents the formation of co-staining foci, supporting the hypothesis that the Gly³⁷Leu substitution inhibits the assembly of (potentially pore-forming) multimers. (The increased neuronal process-associated FlAsH signal in the Gly³⁷Leu Aβ -treated cultures may reflect increased non-specific uptake of the FlAsH dye, because neurons treated with the non-toxic Gly³⁷Leu Aβ are healthier than neurons treated with wild type Aβ peptides.) e Quantification of co-labeling foci from multiple image fields acquired in the experiments described in b-d