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Discovery and Basic Research

(M1130-04-19) Evaluation of Piperine Nanoparticles for FXTAS Treatment via CGG Repeat Drosophila Model

Monday, October 21, 2024
11:30 AM – 12:30 PM MT
Purpose: Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS) is a rare neurodegenerative disease caused by DNA mutation [1]. Recent studies have identified piperine as a potential therapeutic agent for FXTAS [2]. However, its low aqueous solubility (0.04 mg/mL) [3] limits its investigations at higher doses. Our previous study successfully formulated piperine into nanoparticles with a concentration of 50 mg/mL, enabling further evaluation of its effects in transgenic drosophila models of FXTAS. This drosophila model expressing 90 CGG repeats displays locomotor deficits [4] and retinal degeneration [5], allowing screening and mechanistic studies of potential drug candidates for the treatment of FXTAS. This project was designed aiming to evaluate the effects of piperine nanoparticles (PIP NPs) in CGG repeat drosophila on their behaviours and explore the possible mechanisms.

Methods: To assess the effects of PIP NPs on the behaviours of CGG repeat drosophila, climbing ability, lifespan, and pseudopupil were tested. To establish the CGG repeat model for evaluating climbing ability, parents of wild type (elav >W1118) and transgenic (elav >CGG90) flies were raised in an incubator at 25°C with cornmeal. After 0 day-post exclusion (dpe), the 1st filial generation (F1) with straight wings were collected and transferred to an incubator at 28°C. At 20 dpe, the climbing ability of elav >CGG90 flies was measured by comparing its climbing failure rate with that from elav >W1118 flies. To evaluate the effect of PIP NPs on the climbing ability of CGG repeat drosophila, another set of F1 flies with straight wings were used. At 0 dpe, F1 of elav >W1118 files were fed with cornmeal mixed with vehicle of nanoparticles (vehicle NPs, n=100), while F1 of elav >CGG90 flies were fed with cornmeal mixed with vehicle NPs (n=100) or PIP NPs at 5 μM/50 μM/100 μM (n=100 per treatment group) in an incubator at 28°C. At 20 dpe, the climbing failure rate of elav >CGG90 flies with different treatments and elav >W1118 flies were compared, followed by collecting the heads of all drosophila for monitoring the levels of FMR1 mRNA by RT-PCR, expression of FMR1 protein (FMRP) by western blot, and the presence of ubiquitin-positive intranuclear inclusions by fluorescence microscope. To further evaluate the effect of PIP NPs on the lifespan of CGG repeat drosophila, another set of F1 flies with straight wings were used to receive the same treatment as that mentioned above. The number of deaths were counted weekly until all flies are dead. To establish the CGG repeat model for evaluating pseudopupil, F1 flies (gmr >W1118 and gmr >CGG90, n=10 per group) were raised with cornmeal in an incubator at 21.5°C followed by counting the average number of rhabdomeres per ommatidium from total 200 ommatidia in the F1 of above flies at 18 dpe. To evaluate the effect of PIP NPs on pseudopupil of CGG repeat drosophila, another set of F1 flies with straight wings were used. At 0 dpe, F1 of gmr >W1118 files were fed with cornmeal mixed with vehicle NPs (n=10), while F1 of gmr >CGG90 flies were fed with cornmeal mixed with vehicle NPs (n=10) or PIP NPs at 5 μM/50 μM/100 μM (n=10 per treatment group) in an incubator at 21.5°C. At 18, 22, 26 and 30 dpe, the pseudopupil of gmr >CGG90 flies with different treatments and gmr >W1118 flies were compared.

Results: Treatment with PIP NPs significantly reduced the climbing failure rate in elav >CGG90 flies with a dose-dependency observed at 5 μM and 100 μM PIP NPs as shown in Figure 1A. In addition, it was noted that elav >CGG90 flies exhibited a trend of decrease in lifespan compared with that from elav >W1118, which was increased after the treatments of PIP NPs at 5 and 50 μM as shown in Figure 1B. Moreover, Figure 1C demonstrated that treatment with PIP NPs could significantly increase the numbers of rhabdomeres per ommatidium in gmr >CGG90 flies with no dose dependency observed. Further mechanism studies indicated i) a significant increase in FMR1 mRNA in all elav >CGG90 flies compared with that from elav >W1118 flies, which could be significantly reduced after treatment with 100 μM PIP NPs (Figure 2A); ii) a trend of decreased FMRP in elav >CGG90 flies compared with that from elav >W1118, which could be increased after treatment with all dose levels of PIP NPs (Figure 2B); iii) the presence of ubiquitin-positive intranuclear inclusions in elav >CGG90 flies, which could be reduced by the treatments of PIP NPs at all dosage levels as shown in Figure 2C.

Conclusion: Our study for the first time demonstrated that treatment of PIP NPs could significantly improve the climbing ability, extend the lifespan, increase the rhabdomeres numbers per ommatidium in CGG repeat drosophila, with reduced FMR1 mRNA level, a trend of increase in FMRP expression and decreased presence of ubiquitin-positive intranuclear inclusions, suggesting the great potential of PIP NPs in treating FXTAS.

References: [1]. Kong, H. E., Zhao, J., Xu, S., Jin, P., & Jin, Y. (2017). Fragile X-associated tremor/ataxia syndrome: from molecular pathogenesis to development of therapeutics. Frontiers in Cellular Neuroscience, 11, 128.
[2]. Verma, A. K., Khan, E., Mishra, S. K., Jain, N., & Kumar, A. (2019). Piperine modulates protein mediated toxicity in fragile X-associated tremor/ataxia syndrome through interacting expanded CGG repeat (r (CGG) exp) RNA. ACS chemical neuroscience, 10(8), 3778-3788.
[3]. O‘Neil MJ, The Merck index, 2013, 80:423-432
[4]. Sofola, O. A., Jin, P., Botas, J., & Nelson, D. L. (2007). Argonaute-2-dependent rescue of a Drosophila model of FXTAS by FRAXE premutation repeat. Human molecular genetics, 16(19), 2326-2332.
[5]. Jin, P., Zarnescu, D. C., Zhang, F., Pearson, C. E., Lucchesi, J. C., Moses, K., & Warren, S. T. (2003). RNA-mediated neurodegeneration caused by the fragile X premutation rCGG repeats in Drosophila. Neuron, 39(5), 739-747.

Acknowledgements: General Research Fund from University Grant Council of Hong Kong SAR (Reference number: 1145732).


Figure 1. Effect of PIP NPs treatment at 5, 10 and 100 uM on the behaviours of CGG repeat drosophila via monitoring the (A) climbing ability, (B) lifespan, (C) pseudopupil at 18 ~ 30 dpe (i) and 30 dpe (ii).


Figure 2. Effect of PIP NP treatment at at 5, 10 and 100 uM on (A) FMR1 mRNA level, (B) FMRP level and presence of (C) ubiquitin-positive intranuclear inclusions in the head of CGG repeat drosophila.