The Effects of Retinoic Acid on Lifespan and Protein Aggregation in C . elegans

1. The Effects of Retinoic Acid on Lifespan and Protein Aggregation in C. elegans Michael A. Stolberg, Karla A. Mark, and Gordon J. Lithgow The Buck Institute for Research on Aging, Novato, CA Introduction Specific Aims ER Stress Model C. elegans  are great model organisms and are often used for compound screens. Retinoic Acid (RA) is a metabolite of Vitamin A, Retinol. It is often considered to be the most biologically active metabolite. RA is the ligand for both the Retinoic Acid Receptor (RAR) and the Retinoid X Receptor(RXR). In mammalian cells, RAR and RXR are nuclear hormone receptors. Each receptor has 3 isoforms α, b, andg. RXR and RAR can heterodimers with each other or the Vitamin D receptor (VDR) to regulate gene transcription (Fig. 1). A screen preformed with over 640 natural compounds found Vitamin D as one of the most interesting hits for Amyloid Beta suppression. Much research is currently focused on Vitamin D in the lab, however little is know as to the possible role of another natural compound, RA on C. elegan’shealth benefits. Additionally, we examined the role of RA on Vitamin D signaling. All-trans-RA has a higher affinity to the RAR, but can also activate RXR. In contrast, 9-cis-RA can only activate the RXR. Therefore, the goal of this project is to test and analyze the health benefits of both all-trans-RA and 9-cis-RA on C. elegans. We tested the following specific aims: Determine the effect of RA on lifespan Determine the effect of RA on worm protein aggregation models Determine the effect of RA on molecular protein chaperones D1. D2. Results Lifespans Control 50µM A1. A2. D3. D4. 75µM 100µM A1. N2 C. elegans show significant increase in lifespan when on both 50µM and 100µM all-trans-RA, Log-rank test gives p= <.0001. Median Lifespan increases 2 fold from control to 50µM. Each concentration had 2 technical replicates. A2. N2 C. elegansshow significant lifespan increase at all concentrations of all-trans-RA, Log-rank test gives p= <.0001. Median lifespan is almost 2 fold increase in all concentrations from control. Each concentration had 2 technical replicates. D5. D1-4. Images ofPhsp-4::gfpexpression in worms exposed to all-trans-RA. Worms have definite increase in brightness with increase in concentration of all-trans-RA. This shows that RA could be increasing activity of the chaperone. However, western blots to measure the amount of GFP, as shown in D5.do not show an increase in the amount of protein. We attribute the increase in brightness to the yellow color of RAthat is being ingested causing the worms to appear brighter. Protein Aggregation Models GFP B1. B2. Beta Actin Lifespan Extension Fig. 1 Control 75µM 100µM 50 µM Strains and Protocols Conclusions and Future Directions Lifespans Using wild-type(N2) worms on 35mm plates with NGM agar + FUdR and OP-50 B1. HE250 worms on Retinoic Acid show significantly less paralysis, one way ANOVA shows p= .0001 and 3 biological replicates each with 2 technical replicates. B2. RA shows less paralysis compared to control. D3 also shows significantly less paralysis than the control consistent with previous experiments. When combined HE250 worms show slightly less paralysis than D3 or RA alone, and significantly less paralysis than the double control which has the same final concentration of DMSO. ANOVA reveals that this change in paralysis is significant p= .0008 against control, but between groups only 50µM D3 and 50µM RA+ 50µM D3 are significantly different form the control. Each treatment has 5 technical replicates. Error bars represent mean ± SEM Overall, Retinoic Acid has some positive health effects for C. elegans. Retinoic Acid has been shown to give significant lifespan extension. In order to try to find out how Retinoic acid gives this lifespan extension we looked into possible pathways. One pathway was protein aggregation. The accumulation of bad proteins is one of the key pathways of aging. Clearing these proteins has been shown to extend lifespan. The HE250 (perlecan) model showed that RA does help in the clearing of these clumped proteins. Another model of protein aggregation is the accumulation of Aßplaques mostly associated with Alzheimer’s disease. Lifespan extension has been shown when Aß is prevented from accumulating. In two different Aß models, RA appears to have no positive effect, and even in some cases worsens the paralysis. The final possible pathway we explored was the ER stress pathway. When the ER is stressed by aging it fails to properly fold proteins and can then lead to protein aggregation. Using GFP tagged to chaperones in the ER, we looked if RA had any effect. Although fluorescent images appeared to have an increase in GFP induction, western blots showed that there was not any significant increase in chaperone activity. It appears that the increased GFP is due to the yellow color of RA. Retinoic Acid is shown to give C. elegans significant lifespan extension. However, the possible reasons for this are still not fully understood. In the future, we would like to continue pursing the pathways of RA lifespan extension. By finding these pathways, it could give us a greater understanding of aging as a whole. As well as finding the pathway of RA lifespan extension we would also like to continue work into RA and Vitamin D3 interactions due to their link in receptors, and the positive results with the protein aggregation model. Protein Aggregation model HE250 carries a mutation in the unc-52 gene which encodes for the mammalian perlecan protein. When moved to 25°C, unc-52 proteins aggregate in the body wall muscle and causes paralysis. 35mm NGM agar with OP-50 bacteria Amyloid Beta Models C1. C2. Amyloid Beta models GMC101 accumulates Aß(1-42) proteins in the the body wall muscle of the worm when upshifted to 25°C. Using 12 well plates with NGM agar and OP-50 C3. C4. CL4176 accumulates Aß(3-42) proteins in the body wall muscle of the worm when upshifted to 25°C. Using 24 well plates with NGM agar and OP-50. CL4176 have a roller phenotype and when paralyzed have a “halo” around the head. References Endoplasmic Reticulum(ER) Stress Model hsp-4 encodes an ER chaperone that is tagged to GFP. hsp-4 is used as an indicator of unfolded proteins. Under ER stress hsp-4 induction occurs. 1Allenby, G et al. Retinoic acid receptors and retinoid X receptors: Interactions with endogenous retinoic acids(1993). Proc. Natl. Acad. Sci. USA 90(34):30-34. 2Bettoun, D et al. Retinoid X Receptor Is a NonsilentMajor Contributor to Vitamin D Receptor-Mediated Transcriptional Activation(2003). Molecular Endocrinology 17(11):2320-2328. 3Frankowski, H et al. Dimethyl sulfoxide and dimethyl formamide increase lifespan of C. elegans in liquid(2013). Mech Ageing Dev. 3(4):69-78. 4Lee, H et al. All-trans-Retinoic Acid as a Novel Therapeutic Strategy for Alzheimer’s Disease(2009). Expert Rev Neurother9(11):1615-1621. 5McColl, G et al Utility of an improved model of amyloid-beta (Aß1-42) toxicity in Caenorhabditiselegans for drug screening for Alzheimer's disease(2012). Molecular Neurodegeneration 7(57). C1. All concentrations of All-T-RA show significantly worse paralysis than the control with p= .0007 from 2-way ANOVA. Each treatment was done in 3 technical replicates. C2. Although data looks significantly better than the control, we attribute this to the low number of worms for the control (n=6) so random paralysis in the population caused high percentage of paralysis early on. The rest of the change is not significant p=.265 from 2-way ANOVA. We also believe the low levels of paralysis overall were mostly due to high concentrations of DMSO. C3. 2-way ANOVA shows that no concentrations were significantly different than the control, p= .328. Each concentration was done in 5 technical replicates. C4. 9-cis-RA does not have a significant effect on paralysis either, p= .349 from 2-way ANOVA. Although 100µM looks slightly worse, the deviation from the mean is too large to be considered significant. Each concentration was done with 4 technical replicates. Error bars represent mean ± SD Acknowledgments We would like to acknowledge the Buck Institute for Research on Aging for providing lab space and materials. As well we would like to thank Dr. Julie Mangada, PhD for organizing the High School Summer Scholars program at the Buck, and DipaBhaumik PhD.