The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human β-amyloid peptide (Aβ). Induced expression of Aβ in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate Aβ toxicity.
Accumulation of the β-amyloid peptide (Aβ) is generally believed to be central to the induction of Alzheimer’s disease, but the relevant mechanism(s) of toxicity are still unclear. Aβ is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human Aβ. Depending on the tissue or timing of Aβ expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of Aβ toxicity. For example, transgenic worms with pan-neuronal Aβ expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an Aβ transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter Aβ toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.
The protocol we have described can be effectively used to assay the effects of compounds on Aβ toxicity. Published examples include the effects of Ginkgo biloba extract constituents (Wu et al. 2006) and reserpine (Arya et al. 2009). Compounds that have been protective against Aβ toxicity in other systems have also been shown to be protective in this model (e.g., memantine, our unpublished results). An important rationale for establishing this model is that the well-developed genetic and molecular tools available in C. elegans can be used to investigate mechanisms of Aβ toxicity. Thus, the effects specific mutations on Aβ toxicity can be assayed (e.g., the effect of reducing insulin-like signaling by introducing a daf-2 loss-of-function mutation, Florez-McClure et al. 2007), and this model can also be used to examine the effects of over-expression transgenes (Fonte et al. 2008). We have recently made extensive use of this model to examine the effects on toxicity of single amino acid substitutions in Aβ (unpublished data).
The transgenic model described here assays the effect of acute Aβ expression on muscle cell function. At the time of paralysis muscle cells and their sarcomere are intact; the paralysis phenotype is not the result of muscle cell death. However, after paralysis ( ~48 hr after initial upshift) there is a breakdown of muscle integrity and eventual death of the worms. We have not investigated this downstream effect of Aβ expression or used it as a toxicity marker.
The inducible Aβ expression model described here is not appropriate for investigating treatments that modulate β-amyloid formation per se. Although transgenic worms with constitutive Aβ expression form readily detectable amyloid deposits (Fay et al. 1998; Link et al. 2001), transgenic worms with inducible Aβ expression rarely have amyloid deposits and the paralysis phenotype appears independent of amyloid deposition (Drake et al. 2003). Our results are consistent with the view that acute toxicity of induced Aβ expression results from the accumulation of soluble oligomeric Aβ, not amyloid deposition.
The authors have nothing to disclose.
We would like to thank current and former members of the Link lab who helped develop the protocols described here, particularly Gin Fonte, who developed the original procedure. This work has been supported by awards from the NIH (AG12423) and the Alzheimer’s Association (Zenith Award) to C.D.L.
Reagents | Quantity/2L | Company/Catalogue # | Comments |
NaCl | 6g | Sigma-Aldrich / S9888 | |
Agar | 40g | Sigma-Aldrich / A7002 | |
Peptone | 5g | Sigma-Aldrich / P0556 | |
ddH2O | 2L | ||
1M CaCl2 | 2mL | 147.02mg/mL in ddH2O | |
Uracil | 2mL | 2mg/mL in ddH2O | |
Cholesterol | 2mL | 5mg/mL in ethanol (do not autoclave) | |
1M MgSO4 | 2mL | 246.5mg/mL in ddH2O | |
1M KPO4 | 50mL | 98mg KH2PO4.mL, 48mg K2HPO4/mL in ddH2O, pH | |
Erlenmeyer Flask | VWR |