A Caenorhabditis elegans Model System for Amylopathy Study

Summary

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human Aβ₄₂ in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

Abstract

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (Aβ) in tissues with time. Aβ is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy ^1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study Aβ-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human Aβ₄₂ into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing Aβ₄₂ is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

Introduction

Amyloid beta (Aβ) is a peptide of 36-43 amino acids that is formed after sequential cleavage of the amyloid precursor protein (APP) by β and γ secretases ¹. The γ secretase processes the C-terminal end of the Aβ peptide and is responsible for its variable lengths ⁵. The most common forms of Aβ are Aβ₄₀ and Aβ₄₂, the latter being more commonly associated to pathologic conditions such as AD ⁵. At high concentrations Aβ form β-sheets that aggregate to form amyloid fibrils ⁶. Fibrils deposits are the main component of senile plaques surrounding ne....

Protocol

1. Construction of Transgenic Worms

1. Transformation.
   1. Prepare injection pads. Place a drop of hot, 2% agarose dissolved in water, onto a glass coverslip. Quickly place a second coverslip on the drop and lightly tap it. After the agarose is solidified, slide coverslips apart, and bake the coverslip-pad in a vacuum oven at 80 °C O/N.
   2. Pull pipettes. We use a Sutter P-97 puller to pull 1/0.5 mm O.D./I.D. borosilicate capillaries with filament. Pipettes are forged with closed tip which is broken open at a later stage.
   3. Prepare injection mix. Make the injection mixture containing the DNA of interest (20 ng/μl per construct) plus em....

Results

With our protocols we study the effects of human Aβ₄₂ oligomer on neuronal function ⁸. A fragment encoding human Aβ₄₂ and the artificial signal peptide coding sequence of Fire vector pPD50.52 was amplified from construct PCL12 ⁹ using primers that introduced a Sma 1 restriction endonuclease site at the ends. The fragment was then inserted into a construct containing a 2,481-bp flp-6 promoter sequence in the pPD95.75 Fire vector between the unique Sma 1 site ¹⁰

Discussion

Here we describe a combined approach, to study cellular and molecular aspects of amylopathies using C. elegans. The advantages of this approach include: 1) low cost. C.elegans is maintained in normal Petri dish seeded with bacteria, at room temperature. 2) Powerful genetics. Transgenic animals can be obtained in few months and a wide array of promoter sequences is available to drive expression of the desired gene in specific neurons. 3) Simple, well-characterized, nervous system. C. elegans pos.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1. NGM
Sodium Chloride	Sigma-Aldrich	S5886	3 g
Bacteriological agar	AMRESCO	J637	17 g
Bacto-peptone	AMRESCO	J636	2.5 g
Distilled Water			Bring to 975 ml
Sterilized by autoclaving, then add the following items and mix well
Magnesium sulfate	Sigma-Aldrich	M2643	1 ml of 1 M stock
Calcium Chloride	Sigma-Aldrich	C5670	1 ml of 1 M stock
Cholesterol	Sigma-Aldrich	C3045	1 ml of 5 mg/ml stock( in ethanol)
Potassium phosphate buffer			25 ml of 1M stock
2. Potassium phosphate buffer
Potassium phosphate monobasic	Sigma-Aldrich	P5655	108.3 g
Potassium phosphate dibasic	Sigma-Aldrich	P3786	35.6 g
Distilled Water			Bring to 1 L
Sterilized by autoclaving
3. M9 buffer
Potassium phosphate monobasic	Sigma-Aldrich	P5655	3 g
Sodium phosphate dibasic	Sigma-Aldrich	S5136	6 g
Sodium Chloride	Sigma-Aldrich	S5886	5 g
Magnesium sulfate	Sigma-Aldrich	M2643	1 ml of 1 M stock
Distilled Water			Bring to 1 L
Sterilized by autoclaving
4. Egg buffer (pH 7.3, 340 mOsm)
Sodium Chloride	Sigma-Aldrich	S5886	118 mM
Potassium Chloride	Sigma-Aldrich	P5405	48 mM
Calcium Chloride	Sigma-Aldrich	C5670	2 mM
Magnesium Chloride	Sigma-Aldrich	M4880	2 mM
HEPES	Fisher Scientific	BP310	25 mM
Distilled Water			Bring to 1 L
Sterilized by autoclaving
5. CM-15
L-15 culture medium	Gibco	11415	450 ml
Fetal Bovine Serum	Gibco	10437-028	50 ml
Penicillin	Gibco	15140	50 units/ml
Streptomycin	Gibco	15140	50 g/ml
Adjust to 340 mOsm with sucrose then sterile filter into autoclaved bottles and store at 4 °C
6. Other Reagents
Halocarbon 700 oil	Halocarbon Products	9002-83-9
5 μm Acrodisc Syringe Filter	PALL Co.	4199
Chitinase	Sigma-Aldrich	C6137-5UN
Lectin (peanut)	Sigma-Aldrich	L0881-10MG
Sodium hydroxide	Fisher Scientific	S320
Lysine	Sigma-Aldrich	L5501
Biotin	Sigma-Aldrich	B4639
Sodium hypochlorite solution	Sigma-Aldrich	425044
Sodium azide	Sigma-Aldrich	71289
Sucrose	Sigma-Aldrich	S0389