Sir-2.1 modulates ‘calorie-restriction-mediated’ prevention of neurodegeneration in Caenorhabditis elegans: Implications for Parkinson’s disease

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Abstract

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 6-hydroxy 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.

Highlights

► We employed Caenorhabditis elegans strain expressing GFP in dopaminergic neurons for this study. ► Calorie restriction prevents dopaminergic cell death in 6-OHDA treated C. elegans.Sir-2.1 is required for the protective effect of calorie restriction on cell death.

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, 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.

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], [11], [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.

Section snippets

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;

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

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.

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