Neurogrit Gold Attenuates 6-OHDA-Induced Dopaminergic Neurodegeneration in Parkinson's Model of Caenorhabditis elegans by Reducing α-Synuclein Accumulation and Pink/Pdr-1 Driven Mitochondrial Dysfunction

Abstract

Introduction: Parkinson's disease (PD) is a neurodegenerative disorder majorly associated with movement and behavioral disturbances. Pathologically, the loss of dopaminergic (DA) neurons triggered by the deposition of α-synuclein (SNCA) leads to the decrease in dopamine levels affecting motor and cognitive functions of the brain. Current pharmacotherapy for PD only addresses its symptoms but is not able to halt its progression. Traditional medicines are being increasingly used for the treatment of neurodegenerative disorders.

Aim: The present study investigated the effects of Neurogrit Gold (NG), a herbo-mineral prescription medicine, on a Parkinson's model of Caenorhabditis elegans.

Methods: Chemical characterization of NG was performed on HPLC and GC-MS/MS platforms. Evaluation of NG was done in the neurotoxicant 6-OHDA-induced N2, BZ555, and NL5901 strains of C. elegans.

Results: It was observed that NG treatment did not hamper the lifespan, survival, and progeny development of C. elegans strains. The worms treated with NG were able to resist the deleterious effects of 6-OHDA on survival, progeny development, body bends, and chemotaxis in N2 and DA neuron degeneration in BZ555 worms. In NL5901 worms, NG treatment reduced SNCA aggregation, restored lipid content, as well as improved body bends, chemotaxis, and food uptake. Gene expression studies on 6-OHDA exposed and NG-treated N2 worms suggest that the neuroprotective effects of NG stem from its ability to regulate genes involved in mitochondrial autophagy (pink-1, pdr-1); dopamine synthesis (cat-2); redox (sod-3) and protein folding homeostasis (hsf-1, hsp-12.3).

Conclusion: Neurogrit Gold has robust neuroprotective effects, making it a suitable treatment option against etiologies of Parkinson's disease.

Keywords: Caenorhabditis elegans; 6‐OHDA; Neurogrit gold; Parkinson's disease; autophagy; levodopa; mitochondria.

FIGURE 1

Chemical analysis of Neurogrit Gold (NG) by HPLC. Overlap chromatogram of standard mix (blue line) and NG (pink line) at 247 and 270 nm wavelengths. Quantification of these compounds is mentioned in Table 3.

FIGURE 2

GC–MS/MS‐based chemical characterization of Neurogrit Gold (NG). (A) Overlap chromatogram of the identified fatty acids in NG (pink line) and standard mix (blue line). (B) Chromatogram of the identified compounds in NG (pink line). The identified squalene was further quantified by its standard (blue line). Quantification of these compounds is mentioned in Table 3. Further details of qualitative analysis have been mentioned in Table 4.

FIGURE 3

Lifespan analysis. Analysis of lifespan of (A) N2, (B) NL5901, and (C) BZ555 strain of C. elegans cultured with different doses of Neurogrit Gold (NG) (1, 3, 10, 30, and 100 μg/mL) as obtained by Kaplan–Meier survival analysis.

FIGURE 4

Neurogrit Gold (NG) treatment showed no adverse effect on adult survival and progeny development. Worms were treated with NG (3, 10, and 30 μg/mL) and the adult (%) and progeny development was assessed for (A,B) N2, (C,D) NL5901, and (E,F) BZ555 strain.

FIGURE 5

Neurogrit Gold (NG) treated N2 worms resisted the deleterious effects of the neurotoxicant 6‐OHDA. (A) Experimental design and treatment durations. NG (3, 10, and 30 μg/mL) normalized the toxic effects of 6‐OHDA (50 mM) on (B) Adult (%), (C) Progeny development, (D) Body bends, and (E) CI. L‐dopa (2 mM) was used as a positive control. Data represented as mean ± SEM. The significance of data with respect to the untreated (UN) is represented as ####p < 0.0001, and with respect to 6‐OHDA (50 mM) is represented as ****p < 0.0001; ***p < 0.001, and *p < 0.05.

FIGURE 6

Neurogrit Gold (NG) rescued dopaminergic (DA) neurons from 6‐OHDA‐induced degeneration in transgenic BZ555 strain of C. elegans . Post 24 h pre‐treatment with NG (3, 10, and 30 μg/mL) and later 5 days exposure of 50 mM 6‐OHDA along with NG (3, 10, and 30 μg/mL) or L‐dopa (2 mM) the BZ555 strain of worms were analyzed for changes in expression of DA neurons. (A) GFP expression pattern in DA neurons of BZ555. (B) Graphical representation of the fluorescence intensity of DA neurons as quantified by Image J. Data represented as mean ± SEM. The significance of data with respect to the untreated (UN) is represented as ####, p < 0.0001 and with respect to 6‐OHDA (50 mM) is represented as ****, p < 0.0001, and **, p < 0.01.

FIGURE 7

Neurogrit Gold (NG) arrested α‐synuclein (SNCA) aggregation and increased lipid deposition in NL5901 strain of C. elegans . (A) SNCA accumulation (GFP) and Nile red based evaluation of lipid deposition in NG (30 μg/mL) or L‐dopa (2 mM) treated NL5901 worms. Quantification of the (B) SNCA and (C) Nile red fluorescence intensity as observed by Image J. Data represented as mean ± SEM. The significance of data with respect to the untreated (UN) is represented as ****p < 0.0001; ***p < 0.001, and *p < 0.05.

FIGURE 8

Neurogrit Gold (NG) treatment enhanced mobility, chemosensory, and food uptake behavior of NL5901 strain of C. elegans . NG (3, 10, and 30 μg/mL) or L‐dopa (2 mM) treatment enhanced (A) Body bends, (B) Chemotaxis index, and (C) Food uptake parameters of NL5901 worms. Data represented as mean ± SEM. The significance of data with respect to the untreated (UN) is represented as ****p < 0.0001, and *p < 0.05.

FIGURE 9

Neurogrit Gold (NG) treatment modulated mRNA expression in 6‐OHDA‐exposed N2 strain of C. elegans . Post 24 h pre‐treatment with NG (3, 10, and 30 μg/mL) and later 5 days exposure of 50 mM 6‐OHDA along with NG (3, 10, and 30 μg/mL) or L‐dopa (2 mM), the mRNA expression of N2 worms was analyzed. Gene expression of (A) pink‐1, (B) pdr‐1, (C) cat‐2, (D) sod‐3, (E) hsf‐1, and (F) hsp‐12.3. Data represented as mean ± SEM. The significance of data with respect to the untreated (UN) is represented as ####p < 0.0001; ##p < 0.01 and #p < 0.05 and with respect to 6‐OHDA (50 mM) is represented as ****p < 0.0001; ***p < 0.001; **p < 0.01 and *p < 0.05.