Anti-Parkinsonian effects of Bacopa monnieri- insights from transgenic and pharmacological...

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 413 (4), 605-610

Anti-Parkinsonian effects of Bacopa monnieri:

insights from transgenic and pharmacological

Caenorhabditis elegans models of Parkinson’s

disease.

Pooja Jadiya1, Asif Khan

2, Shreesh Raj Sammi

1, Supinder Kaur

1, Snober S. Mir

2 and Aamir

Nazir1*

Abstract

Neurodegenerative Parkinson‟s Disease (PD) is associated with aggregation of protein alpha synuclein and selective death of

dopaminergic neurons, thereby leading to cognitive and motor impairment in patients. The disease has no complete cure yet; the

current therapeutic strategies involve prescription of dopamine agonist drugs which turn ineffective after prolonged use. The present

study utilized the powerful genetics of model system Caenorhabditis elegans towards exploring the anti-parkinsonian effects of a

neuro-protective botanical Bacopa monnieri. Two different strains of C. elegans; a transgenic model expressing “human” alpha

synuclein [NL5901 (Punc-54::alphasynuclein::YFP+unc-119)], and a pharmacological model expressing green fluorescent protein

(GFP) specifically in the dopaminergic neurons [BZ555 (Pdat-1::GFP)] treated with selective catecholaminergic neurotoxin 6-hydroxy

dopamine (6-OHDA), were employed for the study. Bacopa monnieri was chosen for its known neuroprotective and cognition

enhancing effects. The study examined the effect of the botanical, on aggregation of alpha synuclein, degeneration of dopaminergic

neurons, content of lipids and longevity of the nematodes. Our studies show that Bacopa monnieri reduces alpha synuclein

aggregation, prevents dopaminergic neurodegeneration and restores the lipid content in nematodes, thereby proving its potential as a

possible anti-Parkinsonian agent. These findings encourage further investigations on the botanical, and its active constituent

compounds, as possible therapeutic intervention against Parkinson‟s disease.

Keywords: Bacopa Monnieri, Alpha synuclein, Parkinson‟s disease, Caenorhabditis elegans, Neurodegeneration.

Affiliations: 1Laboratory of Functional Genomics and Molecular Toxicology, Division of Toxicology, CSIR-Central Drug Research

Institute, 2Department of Biotechnology, Integral University, Lucknow

Abbreviations:

BM - Bacopa Monnieri; 6-OHDA - 6-hydroxy dopamine; PD – Parkinson‟s disease; GFP – Green Fluorescence Protein; DA -

Dopamine

*Corresponding Author:

Dr. Aamir Nazir

Scientist

Division of Toxicology

CSIR-Central Drug Research Institute

Lucknow, 226001, (U.P.) India

Tel: +91-522-2612411

Fax: +91-522-2623405

E-mail: [email protected]


1. Introduction

Neurodegenerative diseases like Alzheimer‟s, Parkinson‟s, Huntington‟s and multiple sclerosis are associated with the

process of memory loss and cognitive decline which results from selective degeneration of particular neuronal cells and the

accretion of aggregated proteins [1]. Parkinson‟s disease (PD) is predominantly characterized as a movement disorder but

non motor symptoms are also involved. It affects dopamine-producing neurons in the brain, and the loss of these neurons

induces the symptoms of PD. Since dopamine is associated with motor activity, therefore the progressive loss of

dopaminergic neurons leads to muscle rigidity, tremors and bradykinesia as well as cognition, mental, sleeping, personality

and behaviour disorders including depression and anxiety [2]. It is an age related disorder because the incidence for PD

increases rapidly in the population cohort exceeding 60 years of age [2,3]. At present, the etiology of PD is still not clearly

known. Alpha synuclein becomes the major protein in Lewy bodies which has a key role in pathogenesis of both familial

and sporadic PD [4].

Currently, there is no cure for PD and the drugs used for treatment are dopamine agonists and monoamine oxidase-B

(MAO-B) inhibitors, which provide only symptomatic relief. Considering the promise that several natural products have

yielded in treating various ailments, such products as herbs, medicinal plant extracts and other such botanicals are being

explored for their therapeutic potentials in neuronal disorders. In this quest, some herbs have been found to be effective

neuroprotectants. Bacopa monnieri (BM), commonly known as Brahmi, an ayurvedic medicinal plant acting as anti-

oxidant [5], anti-depressant [6], anti – inflammatory and anti microbial [6,7,8], is the most popular neurotonic and a well

known memory booster [9], which, we reasoned, could be tested for its beneficial effects on the neurodegenerative PD.

Bacopa monnieri, a member of the Scrophulariaceae family [10] assists in heightening mental acuity and supports the

physiological processes involved in relaxation. Extracts of Bacopa monnieri have been reported to exert cognitive

enhancing effects in animals [11,12]. Research on anxiety, epilepsy, bronchitis and asthma, irritable bowel syndrome, and

gastric ulcers also support the Ayurvedic uses of Bacopa [13].

We employed transgenic Caenorhabditis elegans model to evaluate the effect of BM on Parkinson‟s disease. Since its 60-

80 % genes are homologous to human [14] and it also has orthologs of PD associated genes, model system C. elegans may

help in establishing an insight into therapeutic aspects of PD. Transgenic strain expressing „human‟ α-synuclein, helps in

studying the effect of its aggregation on the gross phenotype and provides a robust model to study the effect of any

potential therapeutic agent [4,15,16,17,18,19]. The transparent anatomy of the nematode helps in monitoring the

aggregation of alpha synuclein protein and degeneration of dopamine neurons. Further, dopaminergic neurodegeneration

can easily be induced by neurotoxins such as 6-hydroxydopamine (6-OHDA) [20] thus providing a pharmacological model

of PD. Hence, the present study took advantage of the model system C. elegans towards examining the anti-Parkinsonian

effects of neuro-protective botanical Bacopa monnieri.

Materials and Methods

1.1. C. elegans culture and maintenance

Maintenance of C. elegans was carried out as described previously by Brenner [21,22]. Worms were raised on the OP50

seeded standard Nematode Growth Medium (NGM) and grown at 220C. In this study, wild type Bristol N2, transgenic

strain NL5901 (Punc-54::alphasynuclein::YFP+unc-119; expressing human alpha synuclein protein with YFP expression in

the muscles) and transgenic strain BZ555 (Pdat-1::GFP; bright GFP observable in dopamine neuronal soma and processes)

were used. These strains were obtained from the Caenorhabditis Genetics Center (University of Minnesota).

1.2. Treatment of worms with Bacopa monnieri

Concentrated mother tincture of Bacopa monnieri extract, which is used as a homeopathic medicine in India, was obtained

(SBL Private Limited, Uttaranchal, India). The objective of using the mother tincture was to study this homeopathic drug

„as is‟, so as to evaluate the anti-Parkinsonian effects of the whole mother tincture of the botanical. Any encouraging

observations will lead to detailed studies on various active constituents of the extract.

The mother tincture was diluted tenfold in OP50 before seeding onto NGM plates. The plates were incubated overnight for

optimum growth of bacteria OP50 following which, age synchronized worms were grown on the plates, for further studies.


1.3. Treatment of worms with 6 hydroxy dopamine (6 – OHDA) and 6 –OHDA - BM

To generate selective degeneration of dopaminergic neurons, worms were exposed to 6-hydroxy dopamine (6-OHDA;

Sigma, St. Louis, MI; catalogue no - H4381) at concentrations of 25mM [20]. Briefly, 6-OHDA was mixed with OP50

and seeded onto NGM plates. To prepare 6-OHDA- BM treatment plates, BM was added along with OP50 at this stage.

Plates were then incubated overnight. Embryos of BZ555 strain of C. elegans were then transferred onto these prepared

treatment plates for 48 hours at 22ºC. Bacopa extract is known to be stable, as studies have reported beneficial effects of

the extract over long periods after extraction.

1.4. Assay for analysis of alpha synuclein protein aggregation

Aggregation of alpha synuclein protein was observed in control and BM treated NL5901 strain of C. elegans. After 48 hrs.

of treatment, worms were washed thrice with M-9 buffer to remove adhering bacteria and transferred to agar padded slides

(2% agarose) and sealed with a cover slip. Worms were immobilized with 100mM sodium azide (Sigma, cat no. 71289).

Imaging of live (immobilised) worms using confocal microscopy (Carl Zeiss) was carried out to monitor the aggregation of

alpha synuclein protein and aggregation was quantified using image J Software (Image J, National Institutes of Health,

Bethesda, MD) by measuring florescence intensity. Statistical analysis was carried out using Graph Pad software package;

calculation of statistical significance between various groups was carried out employing Student‟s t test.

1.5. Assay for analysis of dopaminergic neurodegeneration

Study of dopaminergic neurodegeneration was carried out by exposing worms with 6-OHDA and 6-OHDA-BM as

described previously in section 1.3. Any adhering bacteria were removed after 48 hrs of treatment by washing the worm

pellet three times before mounting the worms onto agar padded glass slide using 100mM sodium azide. Imaging of live

(immobilised) worms was carried out to monitor the dopaminergic neurodegeneration in control and experimental

conditions using laser scanning confocal microscope (Carl Zeiss). Fluorescence intensity was quantified using Image J

software (Image J, National Institute of health, Bethesda, MD).

1.6. Nile red staining of lipid deposits

Lipid specific dye- Nile Red (Cat. no - N1142, Invitrogen) was mixed with E.coli before seeding it onto the NGM plates

[23]. Nile Red stock solution was prepared by dissolving 0.5mg Nile Red dye in 1 mL of acetone. Nile Red was fed to the

worms by mixing it with E. coli OP50 in the ratio 1: 250 as described previously [23].The synchronous aged embryos were

transferred onto the Nile-red containing plates and grown for 48 hrs at 22 o

C. Worms were washed off and mounted onto

2% agarose pads using sodium azide. The extent of fat staining was assessed using fluorescence upright microscope

(Nikon).

1.7. Life span study

Life span assay was performed by transferring adult worms every other day, from control as well as BM treatment

condition, to a fresh control or treated plate. This was done to avoid mixing of multiple generations. The number of live,

missing and dead worms was counted each day until the last worm was dead. Analyses were carried out by two different

investigators. Survival curves were plotted using the product-limit method of Kaplan and Meier; statistical analyses was

performed using SPSS software [24].

2. Results and Discussion

2.1. Bacopa monnieri reduced alpha synuclein protein aggregation

Worms of untreated and Bacopa monnieri treated groups were observed under confocal microscope for assaying their

alpha synuclein aggregation pattern. Phenotypically, the worms appeared normal and optimally fed. We observed a marked

and significant reduction in the aggregation of alpha synuclein in case of NL5901 worms treated with Bacopa monnieri

(Figure 1B) as compared to that of control group (Figure 1A). We quantified the images for fluorescence intensity of alpha

synuclein aggregation using Image J software. Treatment of worms with BM showed significantly reduced fluorescence


intensity of aggregation as compared to that of untreated worms. The mean fluorescence (GFP) intensity was 8.950 ±

0.6195 arbitrary units in control worms and 2.550 ± 0.2500 arbitrary units in BM treated subjects (Fig. 1C).

Figure 1: Alpha synuclein aggregation in NL5901 strain of C. elegans fed on OP50 (A, B), Bacopa monnieri (C, D).

Images A and C are fluorescent images, B and D are images grabbed using Differential Interference Contrast (DIC) optics.

Scale bar, 50 µm. Fig 1E is Graphical representation for fluorescence intensity of the nematodes as quantified using Image

J software *p < 0.05.

A 3.5 fold reduction (p<0.05) in alpha synuclein protein aggregation was observed in BM exposed worms. This preventive

effect on α-synuclein aggregation could be as a result of the effect of BM on protein aggregation mediated by expression of

stress proteins within the system. It is very well known that a suite of proteins within the living systems, called stress

proteins, buffer the cells from harm under stressful conditions [25,26]. Bacopa monnieri, has previously been reported to

induce such chaperoning protein called Hsp-70 [27]. Such an effect of BM on stress proteins, might be leading to dis-

aggregation of α-synuclein deposits or the unfolding of mis-folded proteins that otherwise are the part of toxic aggregates.

The protective effect of Bacopa could also be attributed to the antioxidant properties of the botanical, as it has been

reported that Bacopa scavenges free radicals and reduces lipid peroxidation in tissues [5,28]. Studies have also reported

enhancement of antioxidant enzyme levels as a result of treatment with Bacopa [5,28]. These protective properties of

Bacopa monnieri make it tempting for us to speculate that the extract might be decreasing the aggregation of α-synuclein

thereby reducing its toxic outcome in the cells, which, in case of our in vivo studies employing whole organismic

environment of model system C. elegans, makes the studies more relevant in overall disease condition.

2.2. 6 – OHDA induced selective degeneration of dopaminergic neurons was prevented with Bacopa monnieri

The significance of using C. elegans as a model to study neuronal disorders lies in the fact that its nervous system is

simple, comprising of precisely 302 neurons wired by 7000 synapses, each neuron can be individually identified and the

entire connectivity of the nervous system has been reconstructed. Most significantly, all gene families involved in neuronal

function in mammals are present in the worm. Of importance to the present study, C. elegans contains precisely 8

dopaminergic neurons (Fig 2A, 2B); two pairs of CEP neurons and one pair of anterior deirid (ADE) neurons in head

region. In a posterior lateral position, it has another pair of posterior deirid (PDE) neurons. Selective degeneration of these

dopaminergic neurons was achieved through exposure to 6 –OHDA [20,29]. We found that processes of CEP and ADE


neurons showed complete GFP loss with moderate reduction in GFP expression in PDE neurons (Fig. 2C, 2D). When

worms were fed on 6 – OHDA- BM plates, significant protection was found in dopaminergic neurons with CEP, ADE and

PDE neurons exhibiting an enhanced expression of GFP; (Fig. 1E, 1F). We further quantified the images for fluorescence

intensity in DA neurons using Image J. In control worms, the mean fluorescence (GFP) intensity was 8.158 ± 0.2380

arbitrary units whereas it was reduced to 3.050 ± 0.05000 arbitrary units in 6-OHDA treated subjects (a 2.7 fold reduction

(p< 0.05) as compared to that of untreated worms). Worms raised on 6 – OHDA- BM plates exhibited a fluorescent

intensity of 6.900 ± 0.1000 arbitrary units (a 2.3 fold increase (p< 0.001) as compared to 6-OHDA treated subjects (Fig 2

G). This neuroprotective role of BM in 6 –OHDA induced dopaminergic neurodegeneration is probably associated with its

antioxidant [5,30] and anti apoptotic activity [31]. Since dopaminergic neurons are more susceptible for ROS and oxidative

stress plays an important mediator role in neuronal cell death and it consequently induces apoptosis. Although the

contribution of ROS and apoptosis in 6-OHDA-induced neuronal cell death have been fully elucidated [32,33]. BM may be

able to suppress oxidative stress of these neuronal cells. Furthermore, neuroprotective effect of BM against rotenone,

pharmacological model of PD, has also been reported in Drosophila melanogaster model [34]. It is also well known that

the central components of apoptotic pathways are the proteases of the caspase family. Caspase-3 is a potent effector

of apoptosis triggered via several different pathways in a variety of mammalian cell types, and is one of the most

important caspases in the cytochrome c-dependent apoptosis pathways [35]. Activation of caspase-3 appears to be a key

event in apoptosis. Furthermore, it has also been postulated that mitochondrial dysfunction triggered by 6-OHDA induces

release of cytochrome c, and the activation of caspase-3 [32,36,37]. The protective effect of Bacopa monnieri could

probably be associated with such events as activation of bcl-2, maintaining the stability of MMP, and decreasing the

activation of caspase-3 through the mitochondria-dependent pathway; however such mechanistic aspects need to be

explored in greater detail.

Figure 2: GFP expression pattern in dopaminergic neurons of transgenic C. elegans strain BZ555. Control (A, B), 6-

OHDA treated (C, D), 6-OHDA treated worms raised on BM (E, F). Images A, C and E are fluorescent images; B, D

and F are Differential Interference Contrast (DIC) images. Scale bar, 50 µm. Fig 2G is Graphical representation for

fluorescence intensity of GFP expression pattern in dopaminergic neurons of transgenic C. elegans strain as quantified

using Image J software *p < 0.05,


2.3. Bacopa monnieri restored lipid content in alpha synuclein expressing model of C. elegans

PD is known to be associated with altered levels of fatty acids and lipid content. We assayed the lipid levels in alpha

synuclein expressing worms, vis-a-vis normal (wild type) worms and worms treated with BM. Nile red staining was

carried out to fluorescently label the lipids within nematodes. The fluorescent intensity across various groups of worms

was analysed by fluorescent microscopy and quantified using Image J software. Worms of control group exhibited an

optimum level of lipids (Fig 3A), the mean fluorescent intensity of the group, as quantified by Image J, being 39.06 ±

4.158 arbitrary units. The lipid content in alpha synuclein expressing worms was reduced (Fig 3B) as the fluorescent

intensity of nile red staining was found to be 17.77 ± 1.804 arbitrary units, thereby exhibiting a 2.2 fold reduction (p<0.05)

as compared to the control group. When the alpha synuclein expressing worms, were treated with Bacopa monnieri, they

showed an increased staining pattern for nile red along the entire body of worms (Fig 3C) depicting an increase in lipid

content of the worms; the content in this group of worms appeared to be similar as seen for wild type worms; the mean

fluorescence intensity being 45.37 ± 4.676 arbitrary units, which is a 2.6 fold increase (p< 0.05) with respect to that of

untreated NL5901 worms.

The reduction of lipid content in alpha synuclein expressing worms is because of disturbed lipid composition caused as a

result of alpha synuclein toxicity within the worms. Further, the toxic nature of these protein aggregate species tends to

increase the lipid peroxidation in the tissues via increase in burden of reactive oxygen species [38]. The enormous

abundance of lipid molecules in the central nervous system (CNS) suggests that their role is not limited to be structural and

energetic components of cells. Some lipids in the CNS are known to function in neurotransmission. Perturbations in

cellular signalling have been reported in virtually every neurodegenerative disease. Spatial and temporal features of

cellular signalling are in part controlled by lipid components that can regulate protein location and scaffolding events

through a dynamic modulation of membrane microdomains. This protective effect of BM could be for the known

antioxidant properties of the botanical that leads to reduction in reactive oxygen species thus resulting in a reduced lipid

peroxidation [39]. Such a protective effect, thus, mediates the prevention of disturbed combination of lipids thereby

resulting in a rather efficient cellular signalling.


Figure 3: Nile Red staining of wild type C. elegans fed with OP50 (A), NL5901 strain of C. elegans treated with OP50

(B) , NL5901 strain of C. elegans fed on Bacopa monnieri (C). Scale bar, 50 µm. Fig. 3D is graphical representation for

fluorescence intensity of the nematodes as quantified using Image J software *p < 0.05

2.4. Bacopa monnieri increased the life span of wild type worms

The effect of Bacopa monnieri on the longevity of worms was studied. We observed that Bacopa monnieri increased the

life span of wild type worms marginally whereas the alpha synuclein expressing worms did not cause any significant effect

on the longevity of worms. The cumulative survival patterns, as calculated by Kaplan-Meier survival analysis of each

group are depicted in Figure 4.

Figure 1: Alpha synuclein aggregation in NL5901 strain of C. elegans fed on OP50 (A, B), Bacopa monnieri (C, D).

Images A and C are fluorescent images, B and D are images grabbed using Differential Interference Contrast (DIC) optics.

Scale bar, 50 µm. Fig 1E is Graphical representation for fluorescence intensity of the nematodes as quantified using Image

J software *p < 0.05

The mean survival for N2-BM group was 13.57 ± 0.03 days vs. 12.2 ± 0.17 days of N2-OP50 condition (significant at

p<0.05). However the life span in NL5901-BM group was 12.72 ± 1.54 days which was statistically insignificant at the

studied concentration as compared to that of control group. The subtle effect induced by BM on the longevity of nematodes

could be attributed to their antioxidant and anti-stress properties. Parkinson‟s disease is associated with ageing and in the


process of aging, accumulation of free radicals, cognitive impairment and memory loss has been proposed as the causal

factor. Further, the beneficial effects of Bacopa were observed in the control group too, which proves that the effects are

indeed as a result of absorption of Bacopa into the system and could not be because of any artifactual interaction in the

culture media. In Ayurvedic materia medica, Bacopa monniera is commonly mentioned as a rasayana which manifests its

effect to prevent ageing, increase longevity, impart immunity and improve mental functions. However, BM has already

been utilized for its anti-aging action [40].

Our studies provide strong evidence that Bacopa monnieri exhibits a significant ameliorative potential against alpha

synuclein effects. The degeneration of dopaminergic neurons, induced by 6-OHDA, is also prevented by BM, thus

warranting further studies regarding the identification of effective constituents of the botanical and regarding the study on

its mechanistic aspects by which it affords the amelioration. Our studies encourage further investigations on BM as a

possible therapeutic intervention against Parkinson‟s disease.

3. Acknowledgments

Confocal Microscopy facility of CDRI is acknowledged for their assistance in imaging; in particular Mr. Manish Singh is

gratefully acknowledged for his technical assistance. Nematode strains used in this work were provided by the C. elegans

Genetics Center (CGC) University of Minnesota, MN, USA, which is funded by the NIH National Center for Research

Resources (NCRR). CDRI communication no: 187/2011/AN

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