Research

Efficacy of Plant Extracts in Alzheimer's Disease using Transgenic C. elegans

Sraavya (Aashi) Anne


Abstract

Alzheimer’s disease (AD) is an irreversible progressive neurodegenerative disease that slowly destroys the human memory There is an unfulfilled need to develop new treatments for AD, as the existing treatments only offer symptomatic benefits. In this experiment, the effect of four plant extracts: turmeric, ashwagandha, bitter melon, andgotukolaweretested using transgenic models of the nematode Caenorhabditiselegans(C.elegans). Specifically, CL4176 and CL2006 strains that express the Amyloid-β peptide in their muscles and the CL2355 strain that has pan-neuronal expression were used. The CL4176 and CL2006 strains were also made cuticle-deficient by crossing them over with DC19 bus-5 worms for better absorption of the compounds. The effects of these herbs were assessed in CL2355 using a chemotaxis assay and a starvation assay to test associative learning. A paralysis assay was performed in CL4176 to compare the efficacy of these extracts. In addition, CL2006 worms were observed under a fluorescent microscope to quantify the amount of amyloid-beta aggregation in the body wall muscles of these worms. For all the assays, ginkgobilobaextract was used as a positive control, and the results were also compared with wild-type N2 worms. In all the assays conducted, turmeric and ashwagandha performed superior to ginkgo and achieved statistically significant results (N = 50, p-value < 0.05) over bitter melon,gotukola, and untreated worms. Further studies into the mechanism of action, safety, and efficacy studies of these compounds in higher organisms could translate these into viable human treatments for AD.

 

Research

Alzheimer’s disease is a chronic neurodegenerative disorder and is the most common cause of dementia among older adults. Microscopic examination of the brain tissues from AD patients show intracellular neurofibrillary tangles of tau protein and extracellular amyloid-beta plaques {1}. The amyloid hypothesis states that amyloid-beta accumulation starts a series of downstream events exacerbating oxidative stress and damage to the mitochondria, thus leading to neuronal dysfunction and cell death. Oxidative damage and neuroinflammation are also found to play a role in the pathology of AD.

C. elegans is a transparent, 1-mm-long roundworm. With its rapid reproduction of offspring and a short life span of 23 days, C. elegans is a good model to study age-related diseases. Strains CL4176 and CL2006 express amyloid-beta peptides in their muscles. CL2355 has pan-neuronal expression of amyloid-beta protein. 

Turmeric is known to have antioxidant and anti-inflammatory properties, and both characteristics can slow the progression of AD {10}. Its anti-inflammatory effects lead to the inhibition of cyclooxygenase-2 enzymes (COX2), thus reducing the chronic inflammation that occurs in AD. Curcumin, the active ingredient in turmeric, is also known to decrease the formation of free radicals and increase the activity of superoxide dismutase, an enzyme that helps remove harmful free radicals from accumulating in cells {9}. Ashwagandha has been previously found to have neuroprotective effects through its ability to increase cholinergic activity and stimulate dendrite formation {10}. Gotu kola is thought to improve attention span and increase cognitive function as shown through studies in rats. Gotu kola exerts neuroprotective effects by potentiating naturally occurring enzymes that work as antioxidants, such as superoxide dismutase, catalase, and glutathione peroxidase {6}.Bitter melon is highly researched as having hypoglycemic properties but has also been found to protect against oxidative stress {8}. Due to the various attributes of these natural substances as investigated by previous studies, they were proposed as ways to alleviate symptoms of AD.

 

Methods

Crossover: CL4176 and CL2006 worms were crossed with DC19 worms to create new strains of cuticle-deficient C. elegans. A PCR/gel electrophoresis was performed to check for the presence of the mutation. 

Plate Preparation: Plant extracts were diluted to a concentration of 0.5 mg/ml and pipetted onto NGM agar plates. A OP50 E. coli liquid culture was pipetted onto the plates, and the worms were grown on these plates. {2}

Paralysis Assay: The expression of amyloid-beta in CL4176 (smg-1dvIs2 - bus-5) depends on a temperature upshift from 15 °C to 25 °C. CL4176 was maintained at 15 °C on plates containing vehicle or drug. Then, the worms were egg-synchronized and were allowed to grow for 18 hours at 15 °C (n = 50). After 18 hours, the temperature was upshifted to 25 °C to induce amyloid-beta expression. Paralysis was scored at 1-hour intervals until all worms were paralyzed. A worm was considered paralyzed when it did not respond to a gentle touch from a platinum wire or if the worm was only able to move its head.

Fluorescent Staining: CL2006 (dvIs27 - bus-5) worms were egg-synchronized and plated onto seeded plates containing either drug or vehicle and propagated at 15 °C for 72 hours. L4 stage worms were transferred to NGM plates with extract or vehicle and incubated at 25 °C for 48 hours. The worms are then incubated in Methoxy X04 dye solution (1 mM) for 4 hours. They are allowed to destain for 12 hours on seeded NGM plates with no vehicle or drug, picked onto agar pads with sodium azide to be mounted, and observed under a fluorescent microscope. The mean numbers of amyloid-beta staining per head area were quantified using ImageJ software. {5}

Benzaldehyde Chemotaxis Assay: CL2355 worms that exhibit pan-neuronal expression of amyloid-beta protein has an impaired chemotaxis response. Age-synchronized L4 worms were cultured on plates containing vehicle or drug and pipetted into M9 buffer. The solution of worms was centrifuged and aspirated for a total of four times. Benzaldehyde, a well known chemoattractant, was used to test the worms’ chemosensation abilities. Benzaldehyde and water were placed into the appropriate quadrants of the plate, and the worm solution was pipetted in the center. A chemotaxis index was calculated for the worms. {2}

NaCl Associative Learning Assay {11}: Age-synchronized CL2355 worms in the L4 stage were cultured on NGM plates with extracts or vehicle then pipetted into M9 buffer. The worms were centrifuged, the supernatant was pipetted out, and the worms were placed into an unseeded (no food source) NGM plate containing NaCl and allowed to crawl for four hours. In the naive chemotaxis assay performed prior to this assay, it was found that NaCl by itself was neither an attractant nor a repellant. However, when NaCl is associated with starvation, N2 worms tend to be repelled by it. A chemotaxis assay was performed (as described for benzaldehyde) to test associative learning.

 

Results

For all graphs shown, standard error bars were calculated using a 95% confidence interval.

Paralysis Assay: Turmeric- and ashwagandha-treated worms had the most delayed onset of paralysis compared to other treatments, indicating that they were the most successful in this assay.

Benzaldehyde Chemotaxis Assay: All treatments showed a significantly stronger chemoattractant response. Turmeric- and ashwagandha-treated worms had a chemotaxis index that was significantly higher than all other treatments.  

NaCl Associative Learning Assay: Standard error bars as well as p-values with an alpha of 0.05 were used to establish significance in this assay. All treatments led to significant improvement in associative learning. Turmeric and ashwagandha performed superior, although they were not significantly better than all other treatments. Both ashwagandha- and turmeric-treated worms were not significantly different from N2 worms, showing how they were best able to restore the associative learning response.

Fluorescent Microscopy Assay: All treatments significantly decreased the number of plaques in the head muscle of the worms. Turmeric and ashwagandha were the most effective extracts, as they had significantly fewer plaques than worms grown on other extracts. 

 

Conclusion

Among all the extracts tested, turmeric and ashwagandha performed superior across all assays. This further warrants a possibility for these extracts to be tested in higher organisms for developing viable human treatments.

 

Acknowledgements

The research was conducted in a university lab at Cleveland State University. I would like to especially thank Dr. Aaron F. Severson for mentoring me and providing me with the opportunity to work in his lab.


 


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Sraavya (Aashi) Anne

Sraavya (Aashi) Anne


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