Caenorhabditis elegans Model for Initial Screening and Mechanistic Evaluation of Potential New Drugs for Aging and Alzheimer’s Disease

Yuan Luo; Yanjue Wu; Marishka Brown; Christopher D. Link

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Buccafusco JJ, editor. Methods of Behavior Analysis in Neuroscience. 2nd edition. Boca Raton (FL): CRC Press/Taylor & Francis; 2009.

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Methods of Behavior Analysis in Neuroscience. 2nd edition.

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Chapter 16Caenorhabditis elegans Model for Initial Screening and Mechanistic Evaluation of Potential New Drugs for Aging and Alzheimer’s Disease

Yuan Luo, Yanjue Wu, Marishka Brown, and Christopher D. Link.

16.1. INTRODUCTION

Alzheimer’s disease (AD) is one of the most devastating CNS disorders of old age, which has led to a serious public health problem. Currently about five million Americans are affected at a proximate cost of up to $100 billion per year. It has been estimated that by the year 2050, the number of AD patients will be over 14 million if no new treatments are developed [1]. The existing two classes of Food and Drug Administration (FDA)-approved drugs for treatment of AD—acetylcholineesterase (AChE) inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists—only provide symptomatic benefit for some mild to moderate AD patients. The means to prevent or reduce the rate of this disorder is a high priority for medical research. Development of new drugs with disease-modifying or preventive properties for this devastating disease continues to be a challenging job. The apparent difficulty is because of the lack of validated therapeutic targets and adequate animal models [2].

The transgenic mouse model of AD has been useful for understanding the mechanisms by which specific mutations might lead to an AD-related behavior phenotype and for testing possible therapeutics [3–6]. Despite the availability of a variety of transgenic mouse models of AD [7,8], drug evaluations with these animals are expensive and time consuming. It is recognized that assays based on inexpensive in vivo models amenable to high-throughput screening (HTS), such as the round worm Caenorhabditis elegans [9] and the fruit fly Drosophila melanogaster [10] provide attractive platforms for streamlining efficient drug discovery and drug target identification [11].

Modeling AD in a simple microscopic organism such as C. elegans is a relatively new approach [12]. Worms genetically engineered to express the human Aβ_1–42 peptide in muscle cells accumulate both immunoreactive deposits of Aβ_1–42 and insoluble β-amyloid, as is observed in senile plaques in AD brains. (This muscle cell expression model may more directly model inclusion body myositis, a severe human myopathy associated with intramuscular accumulation of Aβ.) Accumulation of Aβ_1–42 in this model is associated with a progressive paralysis, providing a simple biological readout of Aβ toxicity [9]. The relatively short life span of the worms (they only live about 20 days) allows us to evaluate the time sequence of events in these animals during their entire lives. Thus, transgenic C. elegans expressing Aβ₄₂ have been used extensively for the mechanistic study of Aβ₋₄₂ toxicity (1) because of its ability to express muscle-specific human Aβ peptide, which forms intracellular Aβ deposits; [13] and (2) its up-regulated stress response genes [14], which are known to be elevated in the AD brain [15,16]. Most importantly, the transgenic strain develops a concomitant progressive paralysis phenotype (CL4176) [17], which can be easily scored and quantified.

To understand the early events in the progression of AD for development of therapeutic strategies, it is fundamentally important to determine the temporal sequence of events leading to neurodegeneration. C. elegans is a suitable tool for mechanistic examination of the transgene products, as well as for pharmacological analysis of time course and the kinetics of drug effects [18]. For example, a relationship between Aβ amino-acid sequence and amyloid aggregation was established using this model. Yatin et al. [19] showed that methionine (Met [35]) is critical for free radical production by Aβ_1–42, and it is also critical for β-sheet formation in the transgenic C. elegans lines [20].

Although the worms may not be universally accepted as having a direct relevance for AD pathology, they are well suited for validation of target Aβ toxicity in vivo [12,21]. The absence of endogenous Aβ production in the worms offers an opportunity to find a direct role of the Aβ involvement in pathological behaviors [22]. In addition, predominantly intracellular expression of Aβ provides another tool to address specific roles of intracellular Aβ in relation to its toxicity. Substantial evidence implicates intracellular Aβ oligomers in early events related to AD [16]. Intracellular Aβ has also been observed in human brain neurons [23] and in triple transgenic AD mouse models, where its accumulation preceded neurofibrillary tangle formation [24]. This evidence supports the notion that Aβ toxicity assayed in the worm model reflects Aβ toxicity in mammalian neurons. A recent study indicates that the transgenic C. elegans model may be generally relevant to the proteotoxicity underlying neurodegenerative diseases [25]. Additionally, the strain has been used to investigate the role of insulin-like signaling and heat-shock factor in Aβ proteotoxicity [26,27], providing excellent examples for the relevance of the C. elegans model to AD.

There are several advantages of C. elegans over the mouse model for initial drug screening and target characterization. First, there are highly conserved biochemical pathways between worms and humans. Second, established transgenic mutant linking of human Aβ expression with pathological behavioral phenotypes are easy to score. The worms have a relatively low cost of cultivation because of their small size, rapid life cycle, and short life span [28], which allow screening of thousands of animals over multiple generations on microtiter plates. The simple structure of its nervous system, consisting of only 302 neurons in an adult nematode, makes it valuable for screening drugs against age-associated neurodegeneration and the ease of genetic manipulations, which is evident in the availability of mutants and application of RNA interference (RNAi) knockdown. Several examples illustrate the power of C. elegans in screening for new drugs [29], including many known human drugs [30,31]. Some lead molecules originating from worm-based screening assays are in advanced stages of drug discovery [11].

Using the C. elegans model in the past years, we have uncovered effects of natural compounds on extension of the worms’ life span; [32] on a stress response protein, the small heat-shock protein hsp-16.2; [33] on age-related behavioral declines; [34] on muscle degeneration; [35] and on Aβ-expression-induced pathological behaviors [22]. Most of those experiments would be difficult and might be impossible to perform in mice. In this chapter, we describe methods we have employed for compound screening and pharmacological evaluations of potential AD drugs using the C. elegans model.

16.2. METHODS

16.2.1. Animal Subjects

Since Sydney Brenner introduced the soil nematode C. elegans into laboratory practices in the 1960s, it has continuously proven its merit as a model organism in scientific research. C. elegans’ diet consists primarily of bacteria (Escherichia coli), and under optimal laboratory conditions the nematode has a 3-day growth-reproduction cycle. C. elegans has two sexes: hermaphrodite and male. An adult hermaphrodite grows to ~1 mm in length and 80 μm in diameter and produces close to 300 progeny by self-fertilization. Besides the ability to grow a homogenous population, the worm is small enough that it can be handled in large numbers and it is easily cultivated in the laboratory. It is also amendable to genetic manipulations, which is evidenced by the large mutant library. The fully sequenced worm genome revealed 60%–80% of the genes shared with humans (available at the Wormbase: http://www.wormbase.org). There are many biochemical pathways that have been evolutionarily conserved between the nematode and humans that make it an ideal model for discovering novel drug targets.

16.2.2. Equipment

Temperature-controlled incubator (Sheldon Manufacturing, Cornelius, Oregon, USA)
Automated timer
Platinum wire loop or inoculating loop
NGM-Nematode Growth Media (for 500 mL)
1. Before autoclaving, add:
  1. 9.5 g agar
  2. 1.25 g peptone
  3. 1.5 g NaCl
  4. 487.5 mL distilled H₂O
  5. 0.5 mL cholesterol (5 mg/mL in ethanol)
  6. Autoclave mixture for 30 min
2. After autoclaving, add:
  1. 0.5 mL CaCl₂ (1 M)
  2. 0.5 mL MgSO₄ (1 M)
  3. 12.5 mL KH₂PO₄ (1 M) (pH 6.0)
Microscope: Nikon SMZ1000 stereoscopic zoom microscope
Petri dishes (60 × 15 mm)
OP50 food source (E. coli) (to make 500 mL)
1. 5.0 g tryptone
2. 2.5 g NaCl
3. 500 mL distilled H₂O
Note: Add tryptone and NaCl to a 500 mL glass bottle. Pour in 500 mL of distilled H₂O and add a stirring rod. Stir on hot plate without heat until the tryptone is dissolved. Place the culture media and two empty covered 500 mL Erlenmeyer flasks in the autoclave for 30 min. Leave for several hours to cool at room temperature. After the culture media has reached room temperature, place 5 mL into two 15 mL presterilized centrifuge tubes. Using aseptic technique, sterilize an inoculating loop and carefully place colonies of E. coli into each tube. Let incubate at 37°C for 18–24 hr. Place 250 mL of the culture media into each sterilized flask. Add the 5 mL of liquid OP50 to each flask. Incubate at 37°C for 18–24 hr. Pour OP50 into 50 mL presterilized tubes and keep at 4°C until needed.
M9 buffer (to make 1 L)
1. 3 g KH₂PO₄
2. 6 g Na₂HPO₄
3. 5 g NaCl
4. 1 mL MgSO₄ (1 M)
5. 1000 mL distilled H₂O
6. Autoclave mixture

16.2.3. Procedure 1: Aβ-Induced Paralysis Behavior Assay

Unlike human amyloid precursor protein (APP) gene, C. elegans homologue gene apl-1 cannot produce neurotoxic peptide Aβ_1–42 [36]. However, Aβ toxicity can be investigated by engineering C. elegans to express human Aβ_1–42 in either neurons [12] or body wall muscle [9]. In developing an inducible transgenic C. elegans AD model, the transgene plasmid was introduced into C. elegans by gonad microinjection with visible marker gene rol-6. Abnormally long 3′ untranslated region (UTR) in the transgene plasmid makes transgene expression under the control of messenger RNA (mRNA) surveillance system. At 16°C, the mRNA surveillance system in the transgenic strain monitors the expression of abnormally long 3′ UTR sequence in the Aβ plasmid and degrades these transcripts. After temperature upshifts to 23°C, the mRNA surveillance system in C. elegans becomes inactive, which allows the stabilized expression of the Aβ plasmid [14]. After Aβ expression in the muscle cells for 24 hr, transgenic worms gradually become paralyzed [9] (Figure 16.1A).

FIGURE 16.1

(a) Images of Aβ-induced paralysis in the transgenic CL4176 strain without transgene expression (no Aβ), untreated with EGb 761 (Ctrl, left panel), and in the transgenic CL4176 strain (muscle Aβ strain) fed with (EGb, right) or (more...)

Transgenic C. elegans CL4176 (smg-1^ts [myo-3/Aβ_1–42 long 3′-UTR]) and control strain CL1175 (smg-1^ts) are propagated at 16°C on solid nematode growth medium (NGM) plates seeded with 100 μL OP50. To prepare age-synchronized animals, transgenic C. elegans are transferred to fresh NGM plates upon reaching reproductive maturity at L4 stage and allowed to lay eggs for 4–6 hr on a food lawn containing compounds or vehicle. Young larvae from the synchronized eggs are cultured at 16°C in a temperature-controlled incubator (Sheldon Manufacturing, Model 2005, Cornelius, Oregon, USA). Aβ transgene expression in muscle cells is induced by upshifting the temperature from 16°C to 23°C at the 36th hour after egg laying and lasts until the end of the paralysis assay. Usually after temperature upshifts for 24 hr, the number of paralyzed worms is scored under the microscope (Nikon SMZ1000, Japan) at 1-hr intervals until the last worm becomes paralyzed. To identify the paralysis, each worm will be gently touched with a platinum loop (VWR, Bridgeport, New Jersey, USA). The worm is considered paralyzed if it moves its head only or does not move after touching. Paralysis time course is plotted thereafter. To quantify the paralysis, PT₅₀ is used to measure the rate of the paralysis. It is defined as a mean time duration at which 50% worms are paralyzed [37].

Note: It is important to keep the upshift temperature consistent (23°C). Even a difference by 1°C would significantly affect the onset and the duration of paralysis.

16.2.4. Procedure 2: Chemotaxis Behavior Assay

C. elegans can detect attractants or repellents with chemosensory neurons. The chemotaxis response is mediated by activation of several sensory neurons and interneurons to stimulate the motor neurons [38]. The chemotaxis assay was used to identify a neuronal behavioral phenotype in the transgenic C. elegans strain CL2355 in which Aβ is expressed in neurons [21]. Before chemotaxis assay, synchronized transgenic C. elegans CL2355 (smg-1^ts [snb-1/Aβ_1–42 long 3′-UTR]) and its control strain CL2122 (mtl-2/green fluorescent protein) are cultured in 16°C for 36 hr and then in 23°C for another 36 hr. The worms are then collected and washed with M9 buffer three times and transferred to 100 × 15 mm plates (VWR, Bridgeport, New Jersey, USA) containing 1.9% agar, 1 mM CaCL₂, 1 mM MgSO₄, and 25 mM phosphate buffer, pH 6.0. Twenty worms are placed to the center of the plate. After all animals are transferred to the plate, 1 μL of odorant (0.1% benzaldehyde in 100% ethanol) along with 1 μL of 1 M sodium azide is added to the original position. On the opposite side of the attractant, 1 μL drop of sodium azide and 1 μL of control odorant (100% ethanol) are added. Assay plates are incubated at 23°C for 1 hr (Figure 16.1B). The chemotaxis behavior is scored and expressed as chemotaxis index (CI). CI is defined as (number of worms at the attractant location - number of worms at the control location)/total number of worms on the plate.

Note: The chemotaxis behavior is affected by age. Therefore, it is critical to compare CI in a given age, e.g., at L4 stage.

16.2.5. Procedure 3: Serotonin Sensitivity Assay and Egg-Laying Assay

C. elegans has the ability to take up exogenous serotonin (5-HT). 5-HT sensitivity assay is used to determine whether 5-HT mediated neurotransmission is affected by Aβ depositions in the neurons. 5-HT is a key neurotransmitter that modulates several behaviors of C. elegans, including egg laying, locomotion, and olfactory learning [39]. This 5-HT sensitivity assay has previously been used to identify 5-HT hypersensitive mutants, which revealed the relationship of the genes involved in 5-HT signaling [40,41].

Synchronized transgenic worms (CL2355) and control strain (CL2122) are collected at 36 hr after temperature upshift. 5-HT (serotonin, creatinine sulfate salt; Sigma) is dissolved in M9 buffer to 1 mM. Twenty worms in each group are washed with M9 three times and transferred into 200 μL serotonin solution in a 96-well assay plate (VWR, Bridgeport, New Jersey, USA). The number of worms is scored after 5 min as active or paralyzed (immobile for 5 sec) (Figure 16.2). Data are expressed as percent not paralyzed.

FIGURE 16.2

Summary of experiments for paralysis, survival, and biochemical assays in C. elegans

Egg laying, a 5-HT controlled behavior in C. elegans, has been used as a simple genetic system to identify and characterize the action of drugs on neurotransmitter pathways that modulate a particular behavior [42]. Egg laying requires the harmonious orchestration of several different neurons, neurotransmitters, and muscles. Neurotransmitters activate the vulval muscles and the fertilized eggs are ejected out of the uterus by contractions of these muscles. The rate of egg laying is controlled by the availability of food. If the animals are starved, then there is a cessation of egg laying and development continues to occur inside the adult worm.

To perform the egg-laying assay, age-synchronized, well-fed L4 young larvae are transferred to fresh plates seeded with OP50 and allowed to develop ~20 hr at 20°C. The resultant young adults will be used in the assay. To test the response to drugs, individual young adults (that are fed our drug) will be transferred to a 96-well plate containing 100 μL of the following drug concentrations: 5 mg/mL solution of 5-HT, serotonin creatinine sulfate complex (Sigma); 0.75 mg/mL imipramine; 0.5 mg/mL fluoxetine. All drugs are dissolved in M9 buffer. The number of eggs released at room temperature will be scored after 60 min. The egg-laying assay protocol and concentrations are well established [40].

16.2.6. Procedure 4: Pharyngeal Pumping Assay and Life Span Assay

The neuromuscular pharynx allows the worm to ingest the bacteria. Rhythmic contraction and relaxation of this organ transports food from the mouth to the intestines. This rate of pharyngeal pumping varies between individual worms and the environment that the worms are in. If there is an abundant food source, the rate of pharyngeal pumping can exceed 250 pumps/min⁻¹. However, when there is a paucity of resources, i.e., food source, then the rate of pharyngeal pumping declines. The pumping rates also progressively decline as the animals age. Recent research has demonstrated that pharyngeal pumping rates can be pharmacologically modulated [43]. Administration of Epigallocatechin gallate (EGCG), a component of green tea, delayed the age-related functional decline of pharyngeal pumping rates [34] (Figure 16.2). Pharyngeal pumping spans have been positively correlated with the life span of the animal.

To measure pharyngeal pumping, individual worms are placed on NGM agar plates at room temperature containing a lawn of OP50. Assessment of pumping behavior was done by observing the number of times the terminal bulb of the pharynx contracted over a 1-min interval. Worms that display no pharyngeal contractions in the intervening time period are classified as not pumping. Animals that display 1–24 contractions are classified as slow pumping, and 25 or more pharyngeal contractions are classified as fast pumping. Pharyngeal pumping is recorded every day until the death of the worms. Adult day 1 animals are divided into a control group and a treated group. Each NGM plate contains one worm and there are 12 total plates for each group. The number of pharyngeal pumps is counted for each group and an average is obtained.

For life span assay, adult hermaphrodites are allowed to lay eggs for 2–4 hr on a lawn of OP50. The age-synchronized young larvae (L1) are transferred to plates that contain either the vehicle and/or other compounds and are maintained at 20°C. The worms are then transferred to fresh plates on the fourth day after hatching. Once the worms reach adulthood, they should be transferred daily for six consecutive days until the cessation of egg laying to avoid overlapping generations. After these six consecutive days, the worms can then be transferred every other day. Worms are counted around the same time every day and scored as dead if they do not respond to a touch stimulus by a platinum wire. This is repeated until all animals have died (Figure 16.3A).

FIGURE 16.3

(a) Time course of paralysis assays in CL4176 fed with different drugs. Synchronized eggs of CL4176 C. elegans were maintained at 16°C, on the 35 × 10 mm culture plates (~ 35 eggs/plate) containing vehicle (Ctrl), EGb 761 (100 μg/mL), (more...)

16.3. TYPICAL APPLICATIONS

16.3.1. Target Identification and Validation

An example of using the worms for target identification was described by Ashrafi et al. using a genome-wide RNAi library to screen for genes that regulate fat storage in order to identify potential targets for the treatment of obesity [44,45]. This approach has recently been reviewed in depth [18].

The Aβ-expressing worm bearing pathological behavior [9] serves as an excellent example of target validation for Aβ, which has become well accepted as a disease-modifying target for AD. The amyloid cascade hypothesis predicts that increased production, aggregation, and accumulation of Aβ lead to senile plaques, neurotoxicity, and the clinical symptoms of AD. Accordingly, many attempts have been made to target Aβ aggregates as a disease-modifying therapeutic strategy in AD [6,46]. Increasing experimental evidence suggests that specific oligomeric forms of Aβ constitute the toxic species [47,48]. Thus, specific inhibition of the toxic species of Aβ is of importance for therapeutic development of new drugs for AD [43], which was validated in the transgenic C. elegans. [37]

16.3.2. Mechanism of Action Using Mutant Worms and RNAi Feeding

Using mutant worms can help us determine the mechanism of action of a particular drug or compound. The insulin/IGF-1 (insulin growth factor-1) pathway has been well studied in the nematode. Mutations in this pathway have been shown to prolong the life span of the worms or accelerate aging. Amazingly enough, certain natural compounds have also been shown to have life-span-extending properties that are related to caloric restriction, the process of reducing food intake, which has been shown to increase the life spans of several species. One study demonstrated that resveratrol, a component found in the skin of grapes, was able to extend the life span of the worms by activating SIR2-like proteins (sirtuins), a family of NAD+ dependent deacetylases that are evolutionarily conserved from bacteria to humans [49]. Mutant worms that lacked this particular gene, SIR2.1, failed to have their life spans extended in response to resveratrol. In fact, the adult life span of the sir 2.1–null mutants was not significantly different from that of the wild-type worms. Another study demonstrated the effect of blueberry polyphenols on C. elegans life span and used mutants to show that this life span extension required the CaMKII pathway [50]. Treatment of several mutants with blueberry phenol, including sir 2.1, the daf-16 mutant, which promotes an increased life span and stress resistance, and skn-1, which promotes resistance to oxidative stress, all had an increase in longevity. From these results it was determined that these gene products were not required for the beneficial effects of the blueberry polyphenols.

Using mutant animals is also a very effective way to determine the site of drug action of compounds at pre- or post-synaptic components. For example, the tph-1 mutant is deficient in presynaptic serotonin biosynthesis. The mod-5 gene encodes the only known presynaptic 5-HT transporter in the worm (SERT). The mod-1, ser-1 and, ser-4 mutants all encode postsynaptic 5-HT receptors in C. elegans. If a drug acts presynaptically, then the outcome of the 5-HT-controlled egg-laying behavior would be unchanged in the mod-1, ser-1 and, ser-4 mutants fed with that drug. If the drug acts postsynaptically, then the egg-laying results would differ in these worms compared to the wild-type worms.

Addressing whether a particular receptor is necessary for Aβ-induced pathological behavior can be done in either of two ways: (1) down-regulate the receptor expression using RNAi followed by testing pathological behavior; or, (2) develop a new double transgenic strain by crossing a transgenic strain with a receptor mutant strain, to be used in a defined behavior assay. The RNAi technique can be employed to knock down certain genes in a Tg C. elegans strain. This newly constructed strain will allow an examination of the relationship between the receptors and Aβ expression by observing their pathological behavioral phenotype [27]. RNAi can be performed in C. elegans by feeding the worms with double-stranded RNA (dsRNA)-containing bacteria. Knock down rate of the target gene will be confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). If RNAi cannot be confirmed by PCR, a green fluorescent protein (GFP) reporter will be used to confirm reduced gene expression. Because of the inherent limitations of RNAi (weak effects for neuronal genes, possibility of off-target effects), alternative “loss of function” experiments can be validated with genetic mutants by using standard genetic crosses.

16.3.3. Rapid Toxicity Assessment for Pharmaceutical Compounds

When interpreting the pharmacological experiments with C. elegans, it is important to ensure that the compounds are not toxic to the organism. Lethality can be determined as a simple signal for toxicity after treatment with specific compounds or their combinations. Toxicity assessment of pharmaceutical compounds using C. elegans is valid for predicting mammalian toxicity [51]. To determine lethality (LD50) of compounds to C. elegans, survival assays will be conducted [31] in a multi-well microplate in solutions containing concentrations that are 10- to 20-fold of the effective concentration (1 μM). If no lethality is observed, egg-laying behavior assays will be performed, since egg laying was much more (30–50 times) sensitive than lethality in the characterization of toxicity in C. elegans. [51,52] In addition, stress response using GFP-hsp16 [32,33] can also be used as a more subtle indicator of toxicity. This approach was successfully employed for fast ranking toxicity of receptor tyrosine kinase inhibitors [53].

16.4. ANALYSIS AND INTERPRETATION

Although there are many potential advantages to using C. elegans for drug screening (particularly for high-throughput studies), there are also scientific and technical limitations inherent in using C. elegans for this purpose. In the case of the AD model worms described here, it is reasonable to question whether Aβ toxicity in worms is directly (or even indirectly) relevant to Aβ toxicity in people. Although the best-supported mechanisms of Aβ toxicity (e.g., membrane disruption, oxidative damage, etc.) would be expected to be operational in invertebrates, it is probably true that the actual relevance of this model will only be known retrospectively, when the mechanisms of AD pathology are fully understood. Given this scientific limitation, it is clear that drug screening in this model is best employed either for in vivo characterization of modes of action of compounds already suggested to have activities in mammalian models, or for broad initial screening of compounds destined to be further validated in mammalian models. A practical disadvantage of this latter approach, at least from the perspective of pharmaceutical development, is that the multiple levels of validation required for compounds found to be active in a worm model may involve time and expense investments that outweigh the advantages of this model system.

The technical limitations of using C. elegans as a target organism for drug development stem primarily from the potential difficulty of getting compounds into worms and/or knowing what tissue concentration results from a given compound dose. C. elegans is surrounded by a fairly impermeant cuticle, which is composed primarily of collagen fibers surrounded by a lipid-rich epicuticle. Although there has been no comprehensive study of which compounds can or cannot diffuse across the cuticle, anecdotal evidence suggests that many compounds that readily enter tissue culture cells do not similarly diffuse into C. elegans. It is also true that compounds that do not directly diffuse into worms can enter the animals by oral uptake during feeding. While this may be an effective way for drugs to get into worms, it precludes establishing any general relationships between compound exposure and resultant tissue concentrations.

Pharyngeal pumping in C. elegans requires the presence of particulate food, which is typically E. coli. Levels of worm food can therefore become a variable affecting compound uptake. In addition, the use of live E. coli as a food source (the typical approach for C. elegans cultivation) can obviously influence drug exposure if the bacteria degrade, modifies, or concentrates the added drug. It is therefore important that compound exposure tests employ standardized amounts of inviable (but not lysed) bacteria, and that C. elegans cultures are maintained monoaxenically.

16.5. REPRESENTATIVE DATA

16.5.1. Screening Compounds that Affect Aβ-Induced Pathological Behaviors

Using the model organism C. elegans and EGb 761 as a toll, we were able to associate Aβ species with Aβ-induced pathological behaviors. We reported that EGb 761 and one of its components, ginkgolide A, alleviates Aβ-induced pathological behaviors, including paralysis and chemotaxis behavior in a transgenic C. elegans model (Figure 16.3A and B). Interestingly, feeding the worms with antioxidants did not delay paralysis, suggesting that reducing oxidative stress is not the mechanism by which EGb 761 suppresses Aβ-induced toxicity [37].

16.5.2. Dose-Response of Epigallocatechin Gallate on Pharyngeal Pumping in C. elegans

In a series of experiments, EGCG and α-lipoic acid were administered to C. elegans, and the ability of these antioxidants to modulate several characteristic C. elegans behaviors was examined, including pharyngeal pumping, chemotaxis behavior, life span, and amyloid β–associated pathological behavior. We demonstrate that both antioxidants attenuate the levels of hydrogen peroxide in C. elegans, but their effects on the behaviors are different. EGCG, but not α-lipoic acid, enhances pharyngeal pumping behavior in C. elegans in a biphasic dose-dependent manner (Figure 16.3C). In contrast, α-lipoic acid, but not EGCG, facilitates the chemotaxis behavior in C. elegans. [34]

16.5.3. Life Span Extension by EGb 761 and Other Compounds

Given the extremely long life span (4000 years) of the Ginkgo biloba tree, the effect of EGb 761 and its constituents on life span of C. elegans was examined. In three independent experiments, with the total number of worms equal to 100, a significant difference (P = 0.033) between the survival curves in the worms fed with and without EGb 761 were observed (Figure 16.4A). EGb 761 moderately increased the median and maximum life span by 1 day each (less than 10%) [32].

FIGURE 16.4

(A) Survival curves of the wild-type adult worms, which were fed with E. coli (OP50) supplemented with either 100 μg/mL EGb 761 (filled circles), or with the vehicles (ethanol and Tween-80) at appropriate concentrations. Standard errors for each (more...)

16.5.4. Testing Compounds that Delay Age-Associated Muscle Degeneration

As a follow up study of life span extension by EGb 761, pharmacological modulation of age-dependent muscle degeneration, or sacropenia, was determined. Transgenic C. elegans strain (PD4251) expressing GFP-MYO-3 protein, localized in body wall muscles and vulval muscle nuclei, were fed with EGb 761 or Wisconsin Ginseng, and muscle integrity was analyzed by quantification of GFP fluorescence. Both EGb 761 and Wisconsin Ginseng significantly delayed sarcopenia (Figure 16.4C). Ginseng was more effective in worms of advanced age. Furthermore, both agents ameliorated age-associated decline of locomotive behaviors, including locomotion, body bend, and pharyngeal pumping, suggesting that pharmacological extension of life span is a consequence of maintaining functional capacity of the tissue, and that C. elegans is a valid model system for testing therapeutic intervention for delaying the progress of sarcopenia [35].

ACKNOWLEDGMENTS

We would like to thank National Institutes of Health grants for supporting the C. elegans work (AG012423 to Link lab, and AT001928 to Luo lab). We also thank Dr. Yves Christen (IPSEN, France) for inspiring the initial studies in the nematode.

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