Investigation of the Neuroprotective effects of Haskap Berry (Lonicera caerulea) extract on Caenorhabditis elegans Aisa Dobie, Dr. Nathan Bialas Department of Biology University of the Fraser Valley Introduction • Alzheimer’s Disease (AD) is the most common form of neurodegenerative dementia in Canada1. • The pathogenesis of AD is characterized by the biochemical event of amyloid-β (Aβ) plaque accumulation in the brain2. • Many researchers are investigating therapeutic options for the prevention and treatments of AD using natural plant-based sources i.e. superfoods. • Haskap berries (HBs) (Fig. 1) are originally native to Siberia and northeastern Asia but have recently expanded to the Canadian market3. • The compounds found in HBs, such as the anthocyanin cyanidin-3-glucoside (C3G), have demonstrated in purified in vitro studies to have beneficial effects on lifespan, development, and neuronal growth4. • Age-synchronous worms were grown on experimental (HBE) and control (NGM) conditions and then β-amyloid protein production was initiated at L3 life stage by temperature upshift from 16°C to 23°C. • Onset of paralysis between the conditions was recorded every 2 hours from 24-40 hours after temperature upshift. • Analysis of the paralysis phenotype was determined by the ability of the worm to move their posterior segment, complete a full body-wave movement, and respond to manual stimulus. • Statistical analysis was conducted by OASIS 2, a surviorship analysis tool10. Fig. 3: Phenotypic movement patterns of strains. Scan QR. B C Fig. 4: (A) N2 strain showing S-like movement (B) CL2006/CL4176 showing rolling movement (C) Paralyzed CL2006/CL4176. β-Amyloid Accumulation X-34 Assay Fig. 1: (A) Close-up HBs morphology5. (B) HBs growing morphology6. • Due to the presence of C3G in HBs, there is the potential for the fruit to have neuroprotective effects against the accumulation of amyloid-β (Aβ) plaques. • HBE is known to increase expression and nuclear translocation of DAF-16 transcription factor (TF) (Fig. 2C) which is involved in the stress response pathway7. • In addition, DAF-16 upregulation is associated with delayed Aβ toxicity8. • This research supports a causative link between the known upregulation of DAF-16 by HBE and the effects of DAF-16 on delayed Aβ toxicity. • Transgenic C.elegans strain CL2006 was used, which constitutively produce β-amyloid protein, and toxic accumulation of β-amyloid protein in the C.elegans result in total paralysis. • CL2006 strain also has the characteristic rolling phenotype (Fig. 4B). • Age-synchronous worms were grown on experimental and control conditions and then stained with β-amyloid plaque sensitive X-34 dye. • Qualitative β-amyloid plaque accumulations were recorded via fluorescence microscopy. Quantitative normalized fluorescence was compared between the groups via ImageJ, a image analysis software, and statistically analyzed by t-tests. Results ER IGF-1 FOXO NUCLEUS C. ELEGANS INT DAF-2 D Experimental Conditions N Value P-value NGM vs. HBE (replicate one) 100 0.0035 NGM vs. HBE (replicate two) 100 0.0042 NGM vs. HBE (replicate three) 100 0.0004 ME DIA TES ER ME DIA TES DAF-16 NUCLEUS Fig 2: (A) Three designations of DAF-16 localization. Represents cytoplasmic DAF-16 localization (B) Represents intermediate DAF-16 localization, (C) Represents nuclear DAF-16 localization. (D)DAF-16 pathway and the homologous pathway found in mammals. A Research Goals • Continue previous work into the investigation of the effects of Haskap Berry Extract (HBE) on C. elegans. • Determine if HBE has neuroprotective effects in C. elegans, in terms of delaying the onset of phenotypic symptoms, and reducing the accumulation of Aβ plaques. B Fig. 5 (A). Survivorship curve that demonstrates the percentage of HBE and NGM worms affected by paralysis. The x-axis shows the number of hours since temperature upshift, and the y-axis shows the percent of each treatment not paralyzed at that time. Data was processed using OASIS 210. (B) Statistical analysis of the data of three replicates of the neuroprotection assay analyzed by OASIS 210. Significance is found at p<0.05. NGM HBE P-value 27.24 a.u. 21 2.58653E-05 9.52 a.u. F Fig. 6: (A). A qualitative look at HBE worms stained with X-34. (B). A qualitative look at NGM control worms stained with X-34. All photos were taken at the 565ms exposure. (C) A dead NGM control worm’s internal accumulation (arrows) of β-amyloid. (D)(E) An NGM control worm’s internal accumulation of β-amyloid. (F) The mean normalized fluorescence and p-values for the accumulation assay as analyzed by one tailed t-test. Data was replicated in triplicate studies and demonstrated consistency. A B NGM X-34 Neuroprotection Assay C D • Results of the neuroprotective assay demonstrate that HBE significantly delays the effects of β-amyloid protein toxicity in vivo. • Results of the β-amyloid accumulation X-34 assay demonstrate that HBE significantly reduced the amount of β-amyloid protein accumulation in vivo. Future Directions • Future studies using larger sample size studies of the β-amyloid accumulation X-34 assay is necessary to ensure replicability of the study. • Integration of RT-PCR work would allow us to elucidate if DAF-16 is the gene responsible for the neuroprotective effects seen in the study. • Thank you to Eric Gerbrandt for supplying the HBs essential to this research. Many thanks to the UFV lab technicians, especially Avril Alfred, Valentina Jovanovic, Fabiola Rojas, Natallia Varankovich and Jenny Hamilton for their knowledge and help. Big thanks to Erik Hayes for his help and support, and Chris Kernel for his graphic designing skills. Model Organism • C. elegans were chosen for this experiment due to our vast knowledge of cellular pathways, the availability of inducible amyloid (Aβ) transgenic strains, and the ease of care within the laboratory9. • In the first assay, triplicate studies were conducted as to determine the effect of HBE on delaying the paralysis brought on by the accumulation of β-amyloid protein in vivo. • All three replicates resulted in consistent results and p-values <0.05 via statistical analysis by OASIS 2 (Fig. 5B)10. • Interestingly, onset of paralysis in both control and experimental conditions was delayed by 4 hours across all replicates as compared to literature expectations. • The delay in paralysis could be due to inconsistent upshift temperature, developmental stage, or differing lawn size shifting the kinetics of paralysis. • The β-amyloid accumulation X-34 assay was conducted in triplicate to determine if the presence of HBE would significantly reduce the accumulation of the Aβ protein in vivo. • Due to the lack of available procedures for the use of X-34 dye, a procedure for live staining was designed and carried out to effectively stain the β-amyloid in the live worms without sacrificing. • The analysis via ImageJ excluded the body walls to isolate fluorescence by the protein accumulation. • The three replicates resulted in consistent results and p-values <0.05 via statistical analysis by one tailed t-test assuming equal variance (Fig. 6F). Acknowledgments β-Amyloid Accumulation X-34 Assay Materials and Methods • A transgenic C.elegans strain CL4176 was used as they produce β-amyloid protein under a temperature sensitive (23°C) repressible mRNA surveillance system. • Accumulation of toxic levels of Aβ protein in the C.elegans results in total paralysis. • CL4176 strain also has a characteristic rolling phenotype to differentiate the worms, (Fig. 3) from regular N2 worms that have S-shaped movement (Fig. 4). Mean Normalized Fluorescence Conclusion Neuroprotection Assay MAMMALS INT N-value Discussion A B Experimental Conditions E Literature Cited 1. “Statistics Canada,” Government of Canada, 2016. [Online]. Available: https://www.statcan.gc.ca/eng/start. [Accessed October 2018]. 2. Friedrich, R. P., Tepper, K., Ronicke, R., Soom, M., Westermann, M., Reymann, K., … Fandrich, M. (2010). Mechanism of amyloid plaque formation suggests an intracellular basis of A pathogenicity. Proceedings of the National Academy of Sciences, 107(5), 1942–1947. doi:10.1073/pnas.0904532106 3. Bors, B., Thomson, J., Sawchuk, E., Reimer, P., Sawatzky, R., & Sander, T. (2012). Haskap breeding and production—final report (pp. 1–142). Saskatchewan Agriculture: Regina 4. Rupasinghe, H. P. V., Yu, L. J., Bhullar, K. S., & Bors, B. (2012). Haskap (Lonicera caerulea): A new berry crop with high antioxidant capacity. Canadian Journal of Plant Science, 92(7), 1311–1317. 5. Photo courtesy of Bryce Wylde, 2014, http://p3health.net/natural-health-product-of-the-month-haskap-berry/ 6. Photo courtesty of Opioła Jerzy, 2006, https://commons.wikimedia.org/wiki/File:Lonicera_coerulea_a3.jpg 7. Ogg S, Paradis S, Gottlieb S, Patterson GI, Lee L, Tissenbaum HA, Ruvkun G. The fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature. 1997;389:994–999. 8. Cohen, E., Bieschke, J., Perciavalle, R. M., Kelly, J. W., & Dillin, A. (2006). Opposing Activities Protect Against Age-Onset Proteotoxicity. Science, 313(5793), 1604–1610. doi:10.1126/science.1124646. 9. Dostal, V., & Link, C. D. (2010). Assaying β-amyloid Toxicity using a Transgenic C. elegans Model. Journal of Visualized Experiments, (44). doi:10.3791/2252 10. Han, S. K., Lee, D., Lee, H., Kim, D., Son, H. G., Yang, J. S., ... & Kim, S. (2016). OASIS 2: online application for survival analysis 2 with features for the analysis of maximal lifespan and healthspan in aging research. Oncotarget, 7(35), 56147.