Sida cordifolia is efficacious in models of Huntington's disease by reducing ER stress

Abstract

Neurodegenerative disorders (NDs) are a major class of diseases where modern science has not succeeded in providing solutions to the desired levels. ER stress pathway is implicated in pathophysiology of several neurodegenerative disorders, especially those classified as proteinopathies. Several traditional medicines are used to treat neurodegeneration and Sida cordifolia (SC) is one of the common ingredients in formulations used for treating NDs and neuropathic pain. However, the mode of action is not clear. We studied the effectiveness of SC in Huntington's Disease (HD) model using Caenorhabditis elegans and mammalian cells. We used a transgenic C. elegans that expresses mutant huntingtin protein tagged with Yellow Fluorescent Protein (YFP) in their body wall muscle. In C. elegans, SC not only improved motility but also substantially increased the life span. Cell-based studies using inducible mutant Huntingtin protein (mHTT) with a long polyQ tail tagged with EGFP showed that SC profoundly modulates ER stress, reducing the stress caused by mHTT protein. The study showed that the mode of action of SC, at least partially, is through modulation of ER stress pathway, thereby normalizing the changes brought about by overexpression of mHTT.

Keywords: ER stress; Huntington’s disease; Sida cordifolia; ayurveda biology; neurodegeneration.

FIGURE 1

SC reduces polyQ aggregates in transgenic HD model of C. elegans (AM141). (A) Representative fluorescent microscopy images (day 1 adult worms) of poly Q aggregation in AM141 worms treated with SC or AC extracts (B) quantification of number of poly-Q puncta per worm (day1) (n = 3; for all the conditions) (C) average fluorescence intensity of poly-Q per worm (D) average fluorescence intensity of poly-Q in Day 1, Day3 and Day5 adult worms. (In all the graphs, each dot represents the mean of one experiment and a minimum of 3 means were considered for each graph. For each experiment 10 worms were used per group. Data represented as mean ± SEM, *p < 0.05, **p < 0.01).

FIGURE 2

SC improves motility in HD model of C. elegans. (A) Representative images of thrashing of Day 1 adult AM141 worms Thrashing assay of N2 (wild type) and AM141 (HD model) worms treated with SC or AC extracts. Quantification of thrashing of (B) Day 1, (C) Day 3, and (D) Day 5 adult AM141 worms. (In all the graphs, each dot represents the mean of one experiment and a minimum of 3 means were considered for each graph. For each experiment 10 worms were used per group. Data represented as mean ± SEM, **p < 0.01 *p < 0.05).

FIGURE 3

SC improves lifespan of HD model of C. elegans. Survival curves of AM141 (HD model) worms (A) treated with SC extract (B) treated with AC extract. (C) Quantification of percentage change in lifespan of AM141 worms treated with SC or AC extracts n = 3 for all the condition). (D) Lifespan assay data of AM141 worms treated with SC or AC extract. n = 25 per group. Data are mean ± SD. *p < 0.05 **p < 0.01 and n-Individual Biological replicates).

FIGURE 4

SC reduces protein aggregate in the poly-Q-EGFP expressing cells (A) Treatment protocol (B) Representative images of SC treated cells (C) Quantification of GFP-puncta per cell. (NIC, n = 3; IC, n = 4 SC 1 μg, n = 2; SC 5 μg, n = 4; SC 10 μg, n = 3) (D) Semi-quantitative PCR of GFP mRNA expression (E) Immunoblot of GFP protein (F) Quantification of immunoblots of GFP (n = 4 for all conditions). All data represented as mean ± SEM, *p < 0.05, **p < 0.01, ****p < 0.001 and ns is statistically non-significant and n = Individual biological replicate).

FIGURE 5

SC modulates ER stress pathway. (A) immunoblots of pIRE1α and IRE1α (Β) and its quantification (n = 4 for all conditions) (C) Relative expression of XBP1s and XBP1us (n = 8 for all groups except SC10µg where n = 9) (D) Relative expression of bip (n = 4, for all conditions) (E) Representative Immunoblot of pEif2a and total Eif2a, (F) its quantification n = 3 for all condition), (G) Relative expression of ATF4 (n = 4 for all conditions) and (H) CHOP. (n = 4 for all conditions) (All data represented as mean ± SEM, *p < 0.05. **p < 0.01, ****p < 0.001 and ns is statistically non-significant).

FIGURE 6

Sida cordifolia is efficacious in HD models by reducing ER stress. Reduces the aggregates in transgenic HD worm model expressing mutant protein (Q40::YFP) in their body wall muscle. This improves motility and enhances the lifespan of these worms. In neuroblastoma cells (N2a) expressing polyQ-EGFP SC treatment reduces the amount of mutant protein and thereby reducing the number of aggregates. This is accompanied by reduction in levels of key ER stress markers - Spliced XBP1s, pEIF2α and pIRE1α.