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 neurons. Both plaques and diffusible, non-plaque Aβ oligomers, are thought to constitute the underlying pathogenic forms of Aβ.

Laboratory study of neuronal amylopathies is complicated by the fact that these conditions progress with time. Therefore, it is important to develop genetically tractable animal models-complementary to mice-with short life span. These models can be used to elucidate specific aspects of amylopathies-typically cellular and molecular-and by virtue of their simplicity, help to capture the essence of the problem. The worm Caenorhabditis elegans falls is this category. It has a short life span, ~20 days and in addition basic cellular processes including regulation of gene expression, protein trafficking, neuronal connectivity, synaptogenesis, cell signaling, and death are similar to mammalian ⁷. Unique features of the worm include powerful genetics and lack of a vessel system, which enables to study neuronal damage independently of vascular damage. On the other hand, the lack of a brain limits the use of C. elegans to studying many aspects of neurodegeneration. In addition, the reproduction and identification of anatomical distributions of lesions cannot be performed in this organism. Other limitations include the difficulty to assess both differences in gene expression profiles and impairment of complex behavior and memory function. Here we describe methods to generate C. elegans models of amylopathies.

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 empty plasmid DNA to a final concentration of 150 ng/μl. Centrifuge the injection mixture to remove any contaminant. This step is crucial because contaminants reduce transformation efficiency. Transfer the top 5 μl (debris and other contaminants collect in the bottom) to a fresh tube to be used in injection.
   4. Load injection mix by capillarity. The bottom of the pipette is immersed in the injection mix. Typically 0.5 μl are sufficient to inject 100 worms.
   5. Load injection pipette. Insert the pipette onto the holder of the micromanipulator and break it open by rubbing its tip against the edge of a cover slip mounted on a glass slide or alternatively against debris on the agarose pad. This is a crucial step as too large tips damage the worm and too small tips are easily clogged. The quality of the broken tip can be judged by the shape and most importantly by the flow rate. The flow rate can be assessed by the size of the bubbles flowing from the open tip immersed in a drop of halocarbon 700 oil in response to an injection pressure.
   6. Transfer worms to injection pad. Place a drop of 700 halocarbon oil on an injection pad and transfer several (1 or 2 for beginners) worms to the oil. Use a worm pick to push the worms down onto the pad until they adhere to the agarose. Worms should be oriented in rows with ventral sides facing the same direction. Avoid touching worm's head. If after several attempts the worms fail to adhere to the pad, replace with a fresh pad or increase agarose thickness and/or concentration.
   7. Insert the pipette into the worm. By moving the stage, position the worm under the pipette. Position the pipette in the central core of the gonad because its cytoplasm is shared by many germ cell nuclei. This increases the likelihood to deliver injected DNA to many progeny. The pipette should lie almost parallel to the worm (~15°-25° angle). Push vertically the tip of the pipette down the worm's body until the skin is depressed. Then gently tap on the manipulator to induce tip's insertion. For best results inject both gonads.
   8. Inject the DNA solution. Apply pressure to the pipette until the gonad swells. Stop the flow and pull the worm off the pipette. Test the needle for flow and then move to the next worm and repeat injection steps.
   9. Recover the worms: Add a drop (~10 μl) of recovery buffer on the worms and incubate until the worms begin swimming. Add an equal volume of M9, wait until worms resume swimming and repeat several times until the solution is mostly M9. Individually transfer worms to seeded plates.
2. Integration.
   1. Place 40 healthy, well-fed, L4 worms into a fresh plate
   2. Irradiate with γ-ray with 4,000 rads for 40 min. Transfer irradiated worms (P0) in fresh, OP50 seeded plates (4 worms/plate). Worms can alternatively be irradiated with a dose of 300 J/m² UVs.
   3. Transfer 10-20 F1 transformants from each plate to individual plates, label the plates with the corresponding P0 origin.
   4. Single out 2-4 F2 transformants for each F1 to separate plates. Check the F3 progeny for 100% transmission of the transformation marker. Typically, 1-3% of progeny from an irradiated worm will have an integration event.
   5. Make stocks of three or more independent transformant lines.

2. Behavioral Assays

1. Age-synchronization.
   1. Grow worms in standard 10 cm NGM plates + OP50 E. coli until a large population of gravid adults is reached (3-5 days).
   2. Collect the worms in 50 ml Falcon tubes by suspending them in 1 ml M9 buffer.
   3. Add 5 ml M9 buffer and centrifuge at 450 x g for 3 min. Discard supernatant. Repeat 3-4x.
   4. At the end of the last centrifugation, remove the supernatant and add 10 volumes of basic hypochlorite solution (0.5 M NaOH, 1% hypochlorite freshly mixed) to the pelleted worms. Incubate at RT for ~10 min. The lysis reaction can be monitored by placing a drop of the lysis reaction on a coverslip and examining the worms under a microscope. When roughly 80% of worms are broken the reaction should be stopped.
   5. Stop the lysis reaction by adding the same volume of sterile egg buffer.
   6. Collect the eggs (and carcasses) by centrifugation at 450 x g for 5 min.
   7. Wash the eggs with sterile egg buffer 2-3x.
   8. Incubate the eggs O/N in M9 buffer and seed them on standard NGM plates.
2. Chemotaxis assay.
   1. Prepare several pieces of agar roughly 0.5x0.5x0.5 cm. We deposit the agar in a 10-cm plate and we cut the agar chunks from there.
   2. Soak the agar chunk in a solution containing the desired attractant (at saturating, near-saturating concentrations) for 2 hr. Typical attractants are lysine (0.5 M), biotin (0.2 M).
   3. Deposit an agar chunk in a 10 cm test plate in which the location of a test spot and a control spot have been marked (Figure 1A). Allow equilibration and formation of a gradient O/N (Figure 1B). Prepare 5 plates for a single experiment.
   4. Prior the experiment add 10 μl of 20 mM NaN₃ (anesthetic) to each spot.
   5. Place 20 age-synchronized worms in the center of the plate. Place the plate in the incubator at 20 °C.
   6. After 1 hr, count the animals on the test/control spots and calculate the chemotaxis index, (C.I.) as follow: where N, N_test and N_Cnt., indicate the total number of animals, the number of animals in the test spot and the number of animals in the control spot.

3. Primary Embryonic Cell Culture

1. Lyse worms as described in age-synchronization.
2. Stop the lysis reaction by adding the same volume of sterile egg buffer and centrifuge at 450 x g for 5 min. Gently discard supernatant being careful to not lose pelleted eggs. Repeat 2-3x or until supernatant is clear.
3. Resuspend pelleted eggs (and carcasses) in 2 ml sterile egg buffer and add 2 ml of sterile 60% sucrose in egg buffer. Mix this solution until eggs are completely resuspended (as they tend to form clumps under centrifugation) by hand or by vortexing.
4. Centrifuge at 450 x g for 15 min.
5. Carefully transfer supernatant (containing the eggs) to a sterile tube. Discard pellet which contains carcasses and other by-products of lysis.
6. Remove residual sucrose by resuspending the eggs in egg buffer and centrifuging at 450 x g for 5 min. Gently collect and discard the supernatant. Repeat 3x.
7. Under a laminar hood resuspend pelleted eggs in sterile egg buffer containing 1 U/ml chitinase at RT to digest the eggshells. After 30 min start to monitor the reaction (under an inverted cell culture microscope). Each batch of chitinase has a slightly different activity. Typically digestion is completed in 1 hr.
8. When roughly 70-80% eggshells are digested by chitinase add CM-15 (L-15 cell culture medium containing 10% fetal bovine serum, 50 U/ml penicillin, and 50 μg/ml streptomycin). Dissociate cells using a syringe with a 27 gauge. Filter the cell suspension with a 5.0 μm filter to remove intact embryos, clumps of cells and larvae.
9. Pellet the dissociated cell suspension by centrifugation at 450 x g for 15 min. Remove the supernatant and resuspend the pellet in CM-15 cell culture medium.
10. Plate dissociated cells on glass cover slips previously coated with peanut lectin (0.1 mg/ml) dissolved in water. Note: cells must adhere to the substrate in order to differentiate.
11. Cells can be maintained at RT (16-20 °C) in air for more than 2 weeks.

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