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Sir-2.1 modulates ‘calorie-restriction-mediated’ prevention of neurodegeneration in Caenorhabditis elegans: Implications for Parkinson’s disease
Biochemical and Biophysical Research Communications 413 (2011) 306–310
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Sir-2.1 modulates ‘calorie-restriction-mediated’ prevention of neurodegeneration in Caenorhabditis elegans: Implications for Parkinson’s disease Pooja Jadiya a, Manavi Chatterjee b, Shreesh Raj Sammi a, Supinder Kaur a, Gautam Palit b, Aamir Nazir a,⇑ a b
Laboratory of Functional Genomics and Molecular Toxicology, Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow 226 001, UP, India Neuropharmacology Unit, Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow 226 001, UP, India
a r t i c l e
i n f o
Article history: Received 18 August 2011 Available online 26 August 2011 Keywords: Sir-2.1 Calorie restriction 6-OHDA Parkinson’s disease Caenorhabditis elegans Neurodegeneration
a b s t r a c t The phenomenon of aging is known to modulate many disease conditions including neurodegenerative ailments like Parkinson’s disease (PD) which is characterized by selective loss of dopaminergic neurons. Recent studies have reported on such effects, as calorie restriction, in modulating aging in living systems. We reason that PD, being an age-associated neurodegenerative disease might be modulated by interventions like calorie restriction. In the present study we employed the transgenic Caenorhabditis elegans model (Pdat-1::GFP) expressing green fluorescence protein (GFP) specifically in eight dopaminergic (DA) neurons. Selective degeneration of dopaminergic neurons was induced by treatment of worms with 6hydroxy dopamine (6-OHDA), a selective catecholaminergic neurotoxin, followed by studies on effect of calorie restriction on the neurodegeneration. Employing confocal microscopy of the dopaminergic neurons and HPLC analysis of dopamine levels in the nematodes, we found that calorie restriction has a preventive effect on dopaminergic neurodegeneration in the worm model. We further studied the role of sirtuin, sir-2.1, in modulating such an effect. Studies employing RNAi induced gene silencing of nematode sir-2.1, revealed that presence of Sir-2.1 is necessary for achieving the protective effect of calorie restriction on dopaminergic neurodegeneration. Our studies provide evidence that calorie restriction affords, an sir-2.1 mediated, protection against the dopaminergic neurodegeneration, that might have implications for neurodegenerative Parkinson’s disease. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Various dietary agents, environmental agents and lifestyles are suspected to be implicated in the development of a wide range of human diseases. Such alterations are known to act as extrinsic epigenetic factors that may have important consequences on many diseases. Amongst such diseases, neurodegenerative Parkinson’s Disease (PD) is a class of syndrome, known to have a multifactorial origin, depending not only on genetic but also on extrinsic factors [1]. PD is the second most severe debilitating age related disorder after Alzheimer’s disease (AD) with an average onset age of about 60 years [2,3]. This disease is governed by several genetic and environmental factors such as toxins, mitochondrial dysfunction, impairment of the ubiquitin–proteasome as well as of the endosomal-lysosomal system, oxidative damage. Genetic basis of the disease involves mutation in 6 genes viz. SNCA, LRRK2, PRKN,
Abbreviations: CR, calorie restriction; 6-OHDA, 6-hydroxy dopamine; PD, Parkinson’s disease; GFP, green fluorescence protein; DA, dopamine. ⇑ Corresponding author. Fax: +91 522 2623405. E-mail address: [email protected] (A. Nazir). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.08.092
DJ1, PINK1, and ATP13A2 [4]. PD is characterized by selective irreversible degeneration of dopaminergic neurons in the substantia nigra leading to a reduction of neurotransmitter dopamine (DA) in the target structure [5]. DA plays a modulatory role in the vertebrate central nervous system. The dopaminergic deficit results in motor disabilities, such as rigidity, akinesia, tremor and postural abnormalities as well as cognitive disturbances [4]. There is no cure to PD yet; patients are provided with symptomatic treatment in form of dopamine agonist drugs like levodopa, which initially help in bettering the motor abnormalities but after a prolonged usage of these drugs, a stage reaches when the patient stops responding to the medications as a result of loss of the dopaminergic neurons [6]. The absence of complete cure to the disease is mainly because of lack of understanding of the mechanistic aspects of the disease condition. On the other hand, there are enough evidences from recent studies that provide clues towards role of calorie restriction (CR) in longevity of model organisms as well as humans [7,8]. This makes it relevant to study effect of such factors, as calorie restriction, on the age associated neurodegenerative PD. Further, the advances in methodologies, including availability of ‘human’ alpha synuclein expressing transgenic invertebrate model systems, hold great promise in filling the existing lacunae.
P. Jadiya et al. / Biochemical and Biophysical Research Communications 413 (2011) 306–310
We employ transgenic Caenorhabditis elegans models towards understanding various mechanistic aspects of neurodegenerative diseases. Previously, C. elegans has successfully been employed to study various aspects related to alpha synuclein aggregation in transgenic lines expressing human alpha synuclein [9]. C. elegans also displays all the mammalian enzyme activity involved in dopamine biosynthesis, storage transport and signaling [10–12]. C. elegans nematodes, being transparent and genetically accessible, can be made transgenic for fusion proteins expressed in the desired subset of cells, provide a very unique environment for in vivo visualization of an entire population of specific subset of neurons within a living organism [13]. Taking advantage of this system, we investigated the role of calorie restriction to ameliorate the effects of DA-specific toxicity as it is known from previous studies that calorie restricted diet attenuates the risk of developing neurodegenerative disease and increases resistance to toxicity and stress [14]. Of late, studies have extensively related the effect of calorie reduction with a family of proteins known as sirtuins, which are a class of NAD dependent deacetylases. Of the seven mammalian sirtuins, SIRT1 is most notable for its involvement in anti-aging effects of calorie restriction [15] and for its protective role in several models of neurodegenerative disease [16]. Hence the present study was carried out to study the role of CR in preventing DA neurodegeneration in C. elegans and to investigate the role of sir-2.1 in the process.
2. Materials and methods 2.1. C. elegans culture and maintenance Standard conditions were followed for C. elegans propagation as described previously [17,18]. All experiments were carried out at 22 °C and worms were raised on the standard Nematode Growth Medium (NGM). For isolation of age synchronized worms, gravid nematode populations were subjected to axenization on the day of initiation of treatment [19]. The isolated embryos were then transferred to OP50 seeded NGM plates. In this study, wild type Bristol N2 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).
2.2. Treatment of worms with 6 hydroxy dopamine (6-OHDA) Selective degeneration of dopaminergic neurons was induced by treatment of worms with 6-hydroxy dopamine (6-OHDA; Sigma, St. Louis, MI; catalog no - H4381) as previously described [9]. Briefly, 6-OHDA, at desired concentrations, was mixed with OP50 and seeded onto NGM plates (treatment plates). We first carried out dose titration studies employing three different concentrations (2.5 mM, 25 mM and 50 mM) of 6-OHDA in order to deduce the concentration that induces neurodegeneration without causing any gross phenotypic effects. Age synchronized worms were then grown on these treatment plates for 48 h.
2.3. Dietary restriction of worms Several methods of dietary restriction have been used in C. elegans [20], most widely used method being that of reduction of the amount of available E. coli, that we followed for this study. For dietary restriction, bacterial density was reduced from 5 1011 cells/ ml to 5 107 cells/ml (10,000 fold dilution) [21].
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2.4. Assay for analysis of dopaminergic neurodegeneration Degeneration of dopaminergic neurons was induced by exposing worms to 25 mM 6-OHDA pre-mixed with OP50, as described previously. Worms were raised either on normal diet (control) or on a reduced calorie diet (10,000 times dilution) and each experiment was carried out in triplicates. Treatment was terminated after 48 h by washing the worms of the treatment plates. Any adhering bacteria were removed by washing the worm pellet three times before mounting the worms onto an agar padded glass slide using 100 mM sodium azide (Sigma, cat No. 71289) to immobilize the worms under coverslip. Imaging of live (anesthetized) worms was carried out to monitor the dopaminergic neurodegeneration in control and experimental conditions using laser scanning confocal microscope (Carl Zeiss). The analyses were carried out by an investigator who was unaware of the group names, to avoid observer bias. Fluorescence intensity was quantified using Image J software (Image J, National Institute of health, Bethesda, MD). 2.5. Estimation of dopamine We wanted to check whether the effect of 6-OHDA on dopaminergic neurons, was also reflected by its effect on the content of neurotransmitter dopamine, hence we quantified the levels of dopamine in the exposed nematodes, using HPLC with electrochemical detector [22]. In brief, worms (1000 per sample) were washed thrice with filtered M9 buffer to remove adhering bacteria and pellets of worms (30–40 ll) were weighed. 200 ll of 0.2 N perchloric acid and 2.5 ll 30 4-dihydroxy benzylamine DHBA (10 lg/ ml) were added to each sample followed by vortexing at room temperature and sonication on ice at 15 s intervals using 25% amplitude. Samples were then centrifuged at 14,000 rpm for 15 min to remove any insoluble residue. Fourty Microlitre acid supernatant was injected into an HPLC loading tube via HPLC pump (Model 1525, Binary Gradient Pump, Waters, Milford, MA, USA) into a column (Spherisorb, RP C18, 5 lm particle size, 4.6 mm i.d. 250 mm at 30 C) connected to a Electrochemical detector (Model 2465, Waters, Milford, MA, USA) at a potential of +0.8 V with glassy carbon working electrode vs. Ag/AgCl reference electrode. Mobile phase consisted of 32 mM citric acid, 1.4 mM sodium octyl sulfonate, 12.5 mM disodium hydrogen orthophosphate, 0.05 mM EDTA and 16% (v/v) methanol (pH 4.2) at a flow rate of 1.2 ml/min [23]. 2.6. RNAi induced gene silencing RNAi induced gene silencing was achieved using standard feeding protocol as described previously [24] by raising worms on Escherichia coli expressing dsRNA corresponding to the target gene. We used bacterial clones from the Ahringer RNAi library that was purchased from SA Biosciences (Cambridge, UK). Briefly, the bacteria expressing dsRNA for specific gene-of-interest were cultured for 6–8 h in LB containing 50 lg/ml ampicillin, then seeded directly onto NGM plates with 5 mM IPTG and 25 mg/L carbenicillin and incubated overnight at 37 C to induce expression. The synchronous aged embryos were first transferred onto NGM plates containing eri-1 seeded bacteria to enhance RNAi sensitivity [25] for 24 h and then transferred onto bacteria expressing dsRNA for desired gene and also for eri-1 gene. Incubation was carried out at 22 °C for 24 h. 2.7. Statistical analysis All data presented are expressed as mean ± SEM. Statistical analysis was carried out using Graph Pad prism 5 software pack-
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age; calculation of statistical significance between various groups was carried out employing Student’s t test. 3. Results and discussion 3.1. 6–hydroxy dopamine (6-OHDA) caused selective degeneration of dopaminergic neurons Nematode C. elegans has four bilaterally symmetric pairs of dopaminergic (DA) neurons that include two pairs of CEP neurons and one pair of anterior deirid (ADE) neurons, located within head region (Fig. 1A) Another pair is that of posterior deirid (PDE) neurons, located in a posterior lateral position. These DA neurons are known to get degenerated in presence of 6-OHDA [9]. We carried out dose kinetics studies to figure out the most optimal concentration for inducing DA neurodegeneration in nematodes. Our studies with 2.5 mM, 25 mM and 50 mM of 6-OHDA revealed that 25 mM concentration was the most effective as it resulted in complete GFP loss within processes of CEP and ADE neurons. PDE neurons showed a moderate reduction in GFP expression (Fig. 1C). 6-OHDA, a selective catecholaminergic neurotoxin, is already established as one of the most potent neurodegenerative agent, that leads to physiological conditions mimicking PD in various model systems such as mice [26], rats [26] and C. elegans [9]. 3.2. Calorie restriction provided protection against 6-OHDA induced neurodegeneration It was observed that worms raised on the reduced calorie diet exhibited a protective effect in dopaminergic neurons with ADE and PDE neurons exhibiting an enhanced and complete expression of GFP; CEP neurons exhibited a marginal increase in GFP expression (Fig. 1D); worms grown on 10,000 fold diluted bacterial food showed no significant effect on DA neurons in healthy condition. We quantified the images for fluorescence intensity in DA neurons using Image J software (Image J, National Institutes of Health, Bethesda, MD; n = 5). The mean fluorescence (GFP) intensity was 7.084 ± 0.76 arbitrary units in control worms, 6.270 ± 0.3800 arbitrary units in worms raised on reduced calorie diet; CR alone did
not induce any significant effect when compared to control worms (Figs. 1B and 2A). Treatment of worms with 6-OHDA showed significantly reduced fluorescence intensity (p < 0.05) as compared to that of untreated worms. The mean fluorescence (GFP) intensity, was 2.135 ± 0.23 arbitrary units in 6-OHDA treated subjects (a 3.3fold reduction vs. control) and 6.199 ± 0.74 arbitrary units in case of 6-OHDA treated worms fed with a 10,000 fold diluted OP50, a 2.9-fold increase (p < 0.05) as compared to 6-OHDA treated subjects (Figs. 1C and D 2A). Previous epidemiological studies suggest that excessive calorie intake poses an increased risk for age-related pathologies in humans. Therefore, individuals with low-calorie intake may be at reduced risk of PD. Many studies have focused on beneficial effects of CR on specific cognitive functions in various species, including mammals [27,28]. CR has also been shown to slow aging in Rhesus Monkeys by delaying the onset of age-associated diseases [29]. Such beneficial effects of CR on aging could be because of the reduced accumulation of oxidation products of proteins, lipids and DNA as correlated previously [30]. Similarly, the neuroprotection observed in the present study could be because of such beneficial effects of CR.
3.3. Calorie restriction enhanced dopamine levels in 6-OHDA treated worms Our observations of protective effect of CR on dopaminergic neurodegeneration, led us to quantify the levels of neurotransmitter dopamine in order to ascertain that CR was indeed bettering the dopaminergic neurophysiology in 6-OHDA treated worms. We quantified the dopamine content using HPLC and observed that our results were corresponding to the confocal microscopy studies wherein we had observed prevention of dopaminergic neurodegeneration by CR. As depicted in Fig. 2B, worms raised on 6-OHDA had a 6.5-fold (p < 0.05) reduced dopamine content as compared to the worms of control group. When raised on reduced calorie diet, worms exhibited a 4.1-fold increase in dopamine content as compared to the worms from 6-OHDA treated group (p < 0.05), thus leading us to conclude that calorie restriction indeed helps in bettering the dopaminergic neurophysiology, not only by preventing
Fig. 1. GFP expression pattern in dopaminergic neurons of transgenic C. elegans strain (Pdat-1::GFP). Control (A), worms raised on reduced calorie diet (B), 6-OHDA treated (C), 6-OHDA treated worms raised on reduced calorie diet (D), Worms with RNAi induced gene silencing of sir-2.1 (E), sir-2.1 silenced worms raised on reduced calorie diet (F), sir-2.1 silenced worms exposed to 6-OHDA (G), and sir-2.1 silenced worms exposed to 6-OHDA, raised on reduced calorie diet (H). Scale bar, 50 lm.
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Fig. 2. Effect of various treatment conditions on GFP expression pattern in dopaminergic neurons of transgenic C. elegans strain (Pdat-1::GFP) as quantified by Image J analysis (A) and Effect of various treatment conditions on the content of neurotransmitter dopamine in C. elegans. (B) ⁄p < 0.05, ⁄⁄p < 0.001, NS – Not significant.
DA neurodegeneration, but also by increasing the content of neurotransmitter dopamine. 3.4. Calorie-restriction-mediated protection is Sir-2.1 dependent SIRT1, from the silent information regulator protein 2 (Sir2) family of histone deacetylases (HDACs) is a homolog of the yeast gene, that plays a central role in the regulation of cellular physiological processes such as survival, apoptosis, aging and neuroprotection [31,32]. Further, the anti-aging function of Sir2 has also been reported [33]. sir-2.1, the C. elegans ortholog of SIRT1 has also been extensively studied for its role in calorie-restriction mediated effect on aging process [34]. Considering the role of sir-2.1 in aging process, we reasoned that sir-2.1 might have role in the protective effect that calorie restriction affords against the DA neurodegeneration. We, hence, employed the RNAi methodology and silenced sir-2.1 in control, calorie restricted, 6-OHDA treated and 6-OHDA + CR treated conditions. We observed that sir-2.1 silenced worms raised on control diet and on reduced calorie diet showed no significant effect on GFP expression in DA neurons of the (Pdat1::GFP) strain (Figs. 1E and F and 2A).The GFP expression pattern exhibited a 2-fold reduction (p < 0.001 as compared to sir-2.1 silenced worms) when sir-2.1 knocked down worms were exposed to 6-OHDA (Figs. 1E–H and 2A). When the sir-2.1 knocked down worms, exposed to 6-OHDA were raised on a reduced calorie diet, the degeneration of DA neurons was not prevented as the GFP expression was statistically insignificant between the two groups (6-OHDA/sir-2.1 vs 6-OHDA/sir-2.1 CR). The observations were reflected in the dopamine levels too as the content of dopamine in sir-2.1 knocked down nematodes exposed to 6-OHDA, exhibited a 10-fold reduction as compared to their control counterparts (Fig. 2B). When raised on a reduced calorie diet, the dopamine depletion was not prevented as was observed under the conditions when sir-2.1 was not knocked down. These findings are in agreement with previous studies where the role of sir-2.1 has been described in neuroprotection probably mediated by the larger effects of reducing oxidative stress as reasoned for the theory of aging effects.
Our studies, employing genetically powerful model system C. elegans, prove with precision, the preventive effect of CR on dopaminergic cell death in the 6-OHDA treated nematodes. The 6-OHDA model, which presents with selective dopaminergic cell death, serves as a pharmacological model of PD; hence the beneficial effects of CR as observed in the study, has implications for the neurodegenerative PD. Further, the role of sir-2.1 in mediating the protective effect of calorie restriction on dopaminergic neurodegeneration, as observed in the study, presents with an opportunity to carry out further studies so as to ascertain its potential towards drawing any therapeutic outcome in case of PD. 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: 8111. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2011.08.092. References [1] H.M. Gao, J.S. Hong, Gene-environment interactions: Key to unraveling the mystery of Parkinson’s disease, Prog. Neurobiol. 94 (2011) 1–19. [2] G. Alves, E.B. Forsaa, K.F. Pedersen, M. Dreetz Gjerstad, J.P. Larsen, Epidemiology of Parkinson’s disease, J. Neurol. 255 (5) (2008) 18–32. [3] D. Weintraub, C.L. Comella, S. Horn, Parkinson’s disease–Part 1: Pathophysiology, symptoms, burden, diagnosis, and assessment, Am. J. Manag. Care 14 (2008) S40–S48. [4] L.M. Bekris, I.F. Mata, C.P. Zabetian, The genetics of Parkinson disease, J. Geriatr. Psychiatry Neurol. 23 (2010) 228–242. [5] R. Hosono, S. Nishimoto, S. Kuno, Alterations of life span in the nematode Caenorhabditis elegans under monoxenic culture conditions, Exp. Gerontol. 24 (1989) 251–264.
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Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Sir-2.1 modulates ‘calorie-restriction-mediated’ prevention of neurodegeneration in Caenorhabditis elegans: Implications for Parkinson’s disease Pooja Jadiya a, Manavi Chatterjee b, Shreesh Raj Sammi a, Supinder Kaur a, Gautam Palit b, Aamir Nazir a,⇑ a b
Laboratory of Functional Genomics and Molecular Toxicology, Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow 226 001, UP, India Neuropharmacology Unit, Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow 226 001, UP, India
a r t i c l e
i n f o
Article history: Received 18 August 2011 Available online 26 August 2011 Keywords: Sir-2.1 Calorie restriction 6-OHDA Parkinson’s disease Caenorhabditis elegans Neurodegeneration
a b s t r a c t The phenomenon of aging is known to modulate many disease conditions including neurodegenerative ailments like Parkinson’s disease (PD) which is characterized by selective loss of dopaminergic neurons. Recent studies have reported on such effects, as calorie restriction, in modulating aging in living systems. We reason that PD, being an age-associated neurodegenerative disease might be modulated by interventions like calorie restriction. In the present study we employed the transgenic Caenorhabditis elegans model (Pdat-1::GFP) expressing green fluorescence protein (GFP) specifically in eight dopaminergic (DA) neurons. Selective degeneration of dopaminergic neurons was induced by treatment of worms with 6hydroxy dopamine (6-OHDA), a selective catecholaminergic neurotoxin, followed by studies on effect of calorie restriction on the neurodegeneration. Employing confocal microscopy of the dopaminergic neurons and HPLC analysis of dopamine levels in the nematodes, we found that calorie restriction has a preventive effect on dopaminergic neurodegeneration in the worm model. We further studied the role of sirtuin, sir-2.1, in modulating such an effect. Studies employing RNAi induced gene silencing of nematode sir-2.1, revealed that presence of Sir-2.1 is necessary for achieving the protective effect of calorie restriction on dopaminergic neurodegeneration. Our studies provide evidence that calorie restriction affords, an sir-2.1 mediated, protection against the dopaminergic neurodegeneration, that might have implications for neurodegenerative Parkinson’s disease. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Various dietary agents, environmental agents and lifestyles are suspected to be implicated in the development of a wide range of human diseases. Such alterations are known to act as extrinsic epigenetic factors that may have important consequences on many diseases. Amongst such diseases, neurodegenerative Parkinson’s Disease (PD) is a class of syndrome, known to have a multifactorial origin, depending not only on genetic but also on extrinsic factors [1]. PD is the second most severe debilitating age related disorder after Alzheimer’s disease (AD) with an average onset age of about 60 years [2,3]. This disease is governed by several genetic and environmental factors such as toxins, mitochondrial dysfunction, impairment of the ubiquitin–proteasome as well as of the endosomal-lysosomal system, oxidative damage. Genetic basis of the disease involves mutation in 6 genes viz. SNCA, LRRK2, PRKN,
Abbreviations: CR, calorie restriction; 6-OHDA, 6-hydroxy dopamine; PD, Parkinson’s disease; GFP, green fluorescence protein; DA, dopamine. ⇑ Corresponding author. Fax: +91 522 2623405. E-mail address: [email protected] (A. Nazir). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.08.092
DJ1, PINK1, and ATP13A2 [4]. PD is characterized by selective irreversible degeneration of dopaminergic neurons in the substantia nigra leading to a reduction of neurotransmitter dopamine (DA) in the target structure [5]. DA plays a modulatory role in the vertebrate central nervous system. The dopaminergic deficit results in motor disabilities, such as rigidity, akinesia, tremor and postural abnormalities as well as cognitive disturbances [4]. There is no cure to PD yet; patients are provided with symptomatic treatment in form of dopamine agonist drugs like levodopa, which initially help in bettering the motor abnormalities but after a prolonged usage of these drugs, a stage reaches when the patient stops responding to the medications as a result of loss of the dopaminergic neurons [6]. The absence of complete cure to the disease is mainly because of lack of understanding of the mechanistic aspects of the disease condition. On the other hand, there are enough evidences from recent studies that provide clues towards role of calorie restriction (CR) in longevity of model organisms as well as humans [7,8]. This makes it relevant to study effect of such factors, as calorie restriction, on the age associated neurodegenerative PD. Further, the advances in methodologies, including availability of ‘human’ alpha synuclein expressing transgenic invertebrate model systems, hold great promise in filling the existing lacunae.
P. Jadiya et al. / Biochemical and Biophysical Research Communications 413 (2011) 306–310
We employ transgenic Caenorhabditis elegans models towards understanding various mechanistic aspects of neurodegenerative diseases. Previously, C. elegans has successfully been employed to study various aspects related to alpha synuclein aggregation in transgenic lines expressing human alpha synuclein [9]. C. elegans also displays all the mammalian enzyme activity involved in dopamine biosynthesis, storage transport and signaling [10–12]. C. elegans nematodes, being transparent and genetically accessible, can be made transgenic for fusion proteins expressed in the desired subset of cells, provide a very unique environment for in vivo visualization of an entire population of specific subset of neurons within a living organism [13]. Taking advantage of this system, we investigated the role of calorie restriction to ameliorate the effects of DA-specific toxicity as it is known from previous studies that calorie restricted diet attenuates the risk of developing neurodegenerative disease and increases resistance to toxicity and stress [14]. Of late, studies have extensively related the effect of calorie reduction with a family of proteins known as sirtuins, which are a class of NAD dependent deacetylases. Of the seven mammalian sirtuins, SIRT1 is most notable for its involvement in anti-aging effects of calorie restriction [15] and for its protective role in several models of neurodegenerative disease [16]. Hence the present study was carried out to study the role of CR in preventing DA neurodegeneration in C. elegans and to investigate the role of sir-2.1 in the process.
2. Materials and methods 2.1. C. elegans culture and maintenance Standard conditions were followed for C. elegans propagation as described previously [17,18]. All experiments were carried out at 22 °C and worms were raised on the standard Nematode Growth Medium (NGM). For isolation of age synchronized worms, gravid nematode populations were subjected to axenization on the day of initiation of treatment [19]. The isolated embryos were then transferred to OP50 seeded NGM plates. In this study, wild type Bristol N2 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).
2.2. Treatment of worms with 6 hydroxy dopamine (6-OHDA) Selective degeneration of dopaminergic neurons was induced by treatment of worms with 6-hydroxy dopamine (6-OHDA; Sigma, St. Louis, MI; catalog no - H4381) as previously described [9]. Briefly, 6-OHDA, at desired concentrations, was mixed with OP50 and seeded onto NGM plates (treatment plates). We first carried out dose titration studies employing three different concentrations (2.5 mM, 25 mM and 50 mM) of 6-OHDA in order to deduce the concentration that induces neurodegeneration without causing any gross phenotypic effects. Age synchronized worms were then grown on these treatment plates for 48 h.
2.3. Dietary restriction of worms Several methods of dietary restriction have been used in C. elegans [20], most widely used method being that of reduction of the amount of available E. coli, that we followed for this study. For dietary restriction, bacterial density was reduced from 5 1011 cells/ ml to 5 107 cells/ml (10,000 fold dilution) [21].
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2.4. Assay for analysis of dopaminergic neurodegeneration Degeneration of dopaminergic neurons was induced by exposing worms to 25 mM 6-OHDA pre-mixed with OP50, as described previously. Worms were raised either on normal diet (control) or on a reduced calorie diet (10,000 times dilution) and each experiment was carried out in triplicates. Treatment was terminated after 48 h by washing the worms of the treatment plates. Any adhering bacteria were removed by washing the worm pellet three times before mounting the worms onto an agar padded glass slide using 100 mM sodium azide (Sigma, cat No. 71289) to immobilize the worms under coverslip. Imaging of live (anesthetized) worms was carried out to monitor the dopaminergic neurodegeneration in control and experimental conditions using laser scanning confocal microscope (Carl Zeiss). The analyses were carried out by an investigator who was unaware of the group names, to avoid observer bias. Fluorescence intensity was quantified using Image J software (Image J, National Institute of health, Bethesda, MD). 2.5. Estimation of dopamine We wanted to check whether the effect of 6-OHDA on dopaminergic neurons, was also reflected by its effect on the content of neurotransmitter dopamine, hence we quantified the levels of dopamine in the exposed nematodes, using HPLC with electrochemical detector [22]. In brief, worms (1000 per sample) were washed thrice with filtered M9 buffer to remove adhering bacteria and pellets of worms (30–40 ll) were weighed. 200 ll of 0.2 N perchloric acid and 2.5 ll 30 4-dihydroxy benzylamine DHBA (10 lg/ ml) were added to each sample followed by vortexing at room temperature and sonication on ice at 15 s intervals using 25% amplitude. Samples were then centrifuged at 14,000 rpm for 15 min to remove any insoluble residue. Fourty Microlitre acid supernatant was injected into an HPLC loading tube via HPLC pump (Model 1525, Binary Gradient Pump, Waters, Milford, MA, USA) into a column (Spherisorb, RP C18, 5 lm particle size, 4.6 mm i.d. 250 mm at 30 C) connected to a Electrochemical detector (Model 2465, Waters, Milford, MA, USA) at a potential of +0.8 V with glassy carbon working electrode vs. Ag/AgCl reference electrode. Mobile phase consisted of 32 mM citric acid, 1.4 mM sodium octyl sulfonate, 12.5 mM disodium hydrogen orthophosphate, 0.05 mM EDTA and 16% (v/v) methanol (pH 4.2) at a flow rate of 1.2 ml/min [23]. 2.6. RNAi induced gene silencing RNAi induced gene silencing was achieved using standard feeding protocol as described previously [24] by raising worms on Escherichia coli expressing dsRNA corresponding to the target gene. We used bacterial clones from the Ahringer RNAi library that was purchased from SA Biosciences (Cambridge, UK). Briefly, the bacteria expressing dsRNA for specific gene-of-interest were cultured for 6–8 h in LB containing 50 lg/ml ampicillin, then seeded directly onto NGM plates with 5 mM IPTG and 25 mg/L carbenicillin and incubated overnight at 37 C to induce expression. The synchronous aged embryos were first transferred onto NGM plates containing eri-1 seeded bacteria to enhance RNAi sensitivity [25] for 24 h and then transferred onto bacteria expressing dsRNA for desired gene and also for eri-1 gene. Incubation was carried out at 22 °C for 24 h. 2.7. Statistical analysis All data presented are expressed as mean ± SEM. Statistical analysis was carried out using Graph Pad prism 5 software pack-
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age; calculation of statistical significance between various groups was carried out employing Student’s t test. 3. Results and discussion 3.1. 6–hydroxy dopamine (6-OHDA) caused selective degeneration of dopaminergic neurons Nematode C. elegans has four bilaterally symmetric pairs of dopaminergic (DA) neurons that include two pairs of CEP neurons and one pair of anterior deirid (ADE) neurons, located within head region (Fig. 1A) Another pair is that of posterior deirid (PDE) neurons, located in a posterior lateral position. These DA neurons are known to get degenerated in presence of 6-OHDA [9]. We carried out dose kinetics studies to figure out the most optimal concentration for inducing DA neurodegeneration in nematodes. Our studies with 2.5 mM, 25 mM and 50 mM of 6-OHDA revealed that 25 mM concentration was the most effective as it resulted in complete GFP loss within processes of CEP and ADE neurons. PDE neurons showed a moderate reduction in GFP expression (Fig. 1C). 6-OHDA, a selective catecholaminergic neurotoxin, is already established as one of the most potent neurodegenerative agent, that leads to physiological conditions mimicking PD in various model systems such as mice [26], rats [26] and C. elegans [9]. 3.2. Calorie restriction provided protection against 6-OHDA induced neurodegeneration It was observed that worms raised on the reduced calorie diet exhibited a protective effect in dopaminergic neurons with ADE and PDE neurons exhibiting an enhanced and complete expression of GFP; CEP neurons exhibited a marginal increase in GFP expression (Fig. 1D); worms grown on 10,000 fold diluted bacterial food showed no significant effect on DA neurons in healthy condition. We quantified the images for fluorescence intensity in DA neurons using Image J software (Image J, National Institutes of Health, Bethesda, MD; n = 5). The mean fluorescence (GFP) intensity was 7.084 ± 0.76 arbitrary units in control worms, 6.270 ± 0.3800 arbitrary units in worms raised on reduced calorie diet; CR alone did
not induce any significant effect when compared to control worms (Figs. 1B and 2A). Treatment of worms with 6-OHDA showed significantly reduced fluorescence intensity (p < 0.05) as compared to that of untreated worms. The mean fluorescence (GFP) intensity, was 2.135 ± 0.23 arbitrary units in 6-OHDA treated subjects (a 3.3fold reduction vs. control) and 6.199 ± 0.74 arbitrary units in case of 6-OHDA treated worms fed with a 10,000 fold diluted OP50, a 2.9-fold increase (p < 0.05) as compared to 6-OHDA treated subjects (Figs. 1C and D 2A). Previous epidemiological studies suggest that excessive calorie intake poses an increased risk for age-related pathologies in humans. Therefore, individuals with low-calorie intake may be at reduced risk of PD. Many studies have focused on beneficial effects of CR on specific cognitive functions in various species, including mammals [27,28]. CR has also been shown to slow aging in Rhesus Monkeys by delaying the onset of age-associated diseases [29]. Such beneficial effects of CR on aging could be because of the reduced accumulation of oxidation products of proteins, lipids and DNA as correlated previously [30]. Similarly, the neuroprotection observed in the present study could be because of such beneficial effects of CR.
3.3. Calorie restriction enhanced dopamine levels in 6-OHDA treated worms Our observations of protective effect of CR on dopaminergic neurodegeneration, led us to quantify the levels of neurotransmitter dopamine in order to ascertain that CR was indeed bettering the dopaminergic neurophysiology in 6-OHDA treated worms. We quantified the dopamine content using HPLC and observed that our results were corresponding to the confocal microscopy studies wherein we had observed prevention of dopaminergic neurodegeneration by CR. As depicted in Fig. 2B, worms raised on 6-OHDA had a 6.5-fold (p < 0.05) reduced dopamine content as compared to the worms of control group. When raised on reduced calorie diet, worms exhibited a 4.1-fold increase in dopamine content as compared to the worms from 6-OHDA treated group (p < 0.05), thus leading us to conclude that calorie restriction indeed helps in bettering the dopaminergic neurophysiology, not only by preventing
Fig. 1. GFP expression pattern in dopaminergic neurons of transgenic C. elegans strain (Pdat-1::GFP). Control (A), worms raised on reduced calorie diet (B), 6-OHDA treated (C), 6-OHDA treated worms raised on reduced calorie diet (D), Worms with RNAi induced gene silencing of sir-2.1 (E), sir-2.1 silenced worms raised on reduced calorie diet (F), sir-2.1 silenced worms exposed to 6-OHDA (G), and sir-2.1 silenced worms exposed to 6-OHDA, raised on reduced calorie diet (H). Scale bar, 50 lm.
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Fig. 2. Effect of various treatment conditions on GFP expression pattern in dopaminergic neurons of transgenic C. elegans strain (Pdat-1::GFP) as quantified by Image J analysis (A) and Effect of various treatment conditions on the content of neurotransmitter dopamine in C. elegans. (B) ⁄p < 0.05, ⁄⁄p < 0.001, NS – Not significant.
DA neurodegeneration, but also by increasing the content of neurotransmitter dopamine. 3.4. Calorie-restriction-mediated protection is Sir-2.1 dependent SIRT1, from the silent information regulator protein 2 (Sir2) family of histone deacetylases (HDACs) is a homolog of the yeast gene, that plays a central role in the regulation of cellular physiological processes such as survival, apoptosis, aging and neuroprotection [31,32]. Further, the anti-aging function of Sir2 has also been reported [33]. sir-2.1, the C. elegans ortholog of SIRT1 has also been extensively studied for its role in calorie-restriction mediated effect on aging process [34]. Considering the role of sir-2.1 in aging process, we reasoned that sir-2.1 might have role in the protective effect that calorie restriction affords against the DA neurodegeneration. We, hence, employed the RNAi methodology and silenced sir-2.1 in control, calorie restricted, 6-OHDA treated and 6-OHDA + CR treated conditions. We observed that sir-2.1 silenced worms raised on control diet and on reduced calorie diet showed no significant effect on GFP expression in DA neurons of the (Pdat1::GFP) strain (Figs. 1E and F and 2A).The GFP expression pattern exhibited a 2-fold reduction (p < 0.001 as compared to sir-2.1 silenced worms) when sir-2.1 knocked down worms were exposed to 6-OHDA (Figs. 1E–H and 2A). When the sir-2.1 knocked down worms, exposed to 6-OHDA were raised on a reduced calorie diet, the degeneration of DA neurons was not prevented as the GFP expression was statistically insignificant between the two groups (6-OHDA/sir-2.1 vs 6-OHDA/sir-2.1 CR). The observations were reflected in the dopamine levels too as the content of dopamine in sir-2.1 knocked down nematodes exposed to 6-OHDA, exhibited a 10-fold reduction as compared to their control counterparts (Fig. 2B). When raised on a reduced calorie diet, the dopamine depletion was not prevented as was observed under the conditions when sir-2.1 was not knocked down. These findings are in agreement with previous studies where the role of sir-2.1 has been described in neuroprotection probably mediated by the larger effects of reducing oxidative stress as reasoned for the theory of aging effects.
Our studies, employing genetically powerful model system C. elegans, prove with precision, the preventive effect of CR on dopaminergic cell death in the 6-OHDA treated nematodes. The 6-OHDA model, which presents with selective dopaminergic cell death, serves as a pharmacological model of PD; hence the beneficial effects of CR as observed in the study, has implications for the neurodegenerative PD. Further, the role of sir-2.1 in mediating the protective effect of calorie restriction on dopaminergic neurodegeneration, as observed in the study, presents with an opportunity to carry out further studies so as to ascertain its potential towards drawing any therapeutic outcome in case of PD. 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: 8111. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2011.08.092. References [1] H.M. Gao, J.S. Hong, Gene-environment interactions: Key to unraveling the mystery of Parkinson’s disease, Prog. Neurobiol. 94 (2011) 1–19. [2] G. Alves, E.B. Forsaa, K.F. Pedersen, M. Dreetz Gjerstad, J.P. Larsen, Epidemiology of Parkinson’s disease, J. Neurol. 255 (5) (2008) 18–32. [3] D. Weintraub, C.L. Comella, S. Horn, Parkinson’s disease–Part 1: Pathophysiology, symptoms, burden, diagnosis, and assessment, Am. J. Manag. Care 14 (2008) S40–S48. [4] L.M. Bekris, I.F. Mata, C.P. Zabetian, The genetics of Parkinson disease, J. Geriatr. Psychiatry Neurol. 23 (2010) 228–242. [5] R. Hosono, S. Nishimoto, S. Kuno, Alterations of life span in the nematode Caenorhabditis elegans under monoxenic culture conditions, Exp. Gerontol. 24 (1989) 251–264.
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