Increased lifespan in transgenic Caenorhabditis elegans overexpressing human α-synuclein

doi:10.1016/j.exger.2006.05.005

Experimental Gerontology

Volume 41, Issue 9, September 2006, Pages 871-876

https://doi.org/10.1016/j.exger.2006.05.005 Get rights and content

Abstract

α-Synuclein is a short 14-kDa protein found in pathological lesions of age-related neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, and multiple system atrophy. Its overexpression in transgenic mice, rats, Drosophila melanogaster, and Caenorhabditis elegans recapitulates many of the pathologic features observed in human Parkinson’s disease including loss of dopaminergic neurons and motor deficits. Integrated transgenic C. elegans lines were generated that overexpress either human wildtype (WT) or mutant (A53T) forms. These transgenic lines demonstrated ∼25% increase in lifespan (p < 0.0001) compared to controls. When the transgenes were crossed into long-lived daf-2 (m577) or daf-2 (e1370) genetic backgrounds, the lifespan increase was also ∼25% in comparison to the corresponding daf-2 strains (p < 0.05). Pharyngeal pumping and egg laying were significantly decreased in the overexpressing transgenic lines, and lifespan increases were attenuated when lines were grown on thick bacterial lawns, suggesting that caloric restriction may explain some of the effects on lifespan. These studies provide initial evidence for a beneficial role of human α-synuclein in influencing lifespan.

Introduction

α-Synuclein is a 14-kDa protein found in neuropathological lesions in age-related neurodegenerative disorders including Alzheimer’s disease, Parkinson’s disease (PD), and various synucleopathies (Spillantini et al., 1997, Spillantini and Goedert, 2000). Physiological functions attributed to α-synuclein include development of neurons, response to neurotoxin exposure, regulation of neurotransmitter release, and regulation of nuclear histone proteins (Norris et al., 2004). A considerable body of literature suggests that α-synuclein becomes toxic and contributes to neuropathology through increasing oxidative stress or by formation of annular structures and protein aggregation (reviewed in Volles and Lansbury, 2003). Mutations in the α-synuclein gene, including A53T, lead to an early-onset familial form of PD that is transmitted through a dominant mode of inheritance (Polymeropoulos et al., 1997). Moreover, PD patients have a shortened life expectancy that can be overcome with pharmacological treatment (Diamond et al., 1990, Marttila and Rinne, 1991).

Transgenic animal studies in mice, rats, Drosophila melanogaster, and Caenorhabditis elegans have demonstrated that overexpression of α-synuclein recapitulates many of the features of PD including neuronal degeneration and motor deficits which were age-related depending on the animal model used (Feany and Bender, 2000, Lakso et al., 2003, Lo Bianco et al., 2002, van der Putten et al., 2000). We previously produced a transgenic C. elegans model of PD by overexpression of human WT and A53T mutant α-synuclein (Lakso et al., 2003). This model displays neuronal loss and movement impairment. Since α-synuclein is involved in multiple age-related neurodegenerative disorders, we asked whether ectopic expression of the human gene in C. elegans could influence lifespan. Our results suggest that α-synuclein increases lifespan that is independent of WT or mutant form, and genetic background. Furthermore, we demonstrate the utility of C. elegans as a model experimental system to investigate human neurodegenerative disease genes in aging processes.

Section snippets

Materials and methods

Transgenic worm lines were produced by integration of extrachromosomal arrays: dat-1::GFP, aex-3::GFP, WT [dat-1::GFP, aex-3::human α-synuclein], and A53T [dat-1::GFP, aex-3::human α-synuclein mutant A53T] in the N2 strain using X-ray irradiation followed by selection (Lakso et al., 2003). Selection was performed using the coinjection marker GFP. Integrated transgenic lines were backcrossed to N2 10 times. Integration of α-synuclein transgenes was confirmed by Western blots (data not shown).

Results

Transgene expression of human α-synuclein under a pan-neuronal promoter causes an increase in lifespan. The effect is seen in N2 (Fig. 1A), daf-2 (m577) (Fig. 1B) and daf-2 (e1370) (Fig. 1C) genetic backgrounds. Both the wildtype and mutant A53T forms of the transgene increased lifespan. The increases were proportional to the lifespans of the animals in the non-transgenic genetic background. Thus, the increases were ∼25% when WT α-synuclein was the transgene in N2 (20.9 ± 0.5 vs. 15.6 ± 0.3), the

Discussion

In this study, we compared the lifespans of transgenic C. elegans overexpressing human α-synuclein under control of a pan-neuronal promoter (Iwasaki et al., 1997) in different genetic backgrounds. Transgenic worms expressing human α-synuclein had a significantly longer lifespan compared to control or non-transgenic worms. We then investigated factors that may have contributed to the observed increases in lifespan.

Previous studies have shown that caloric or dietary restriction (DR) lengthens

Acknowledgements

S.V. was supported by the A.I. Virtanen Institute graduate school program. V.A. was supported by a grant from the Sigrid Juselius Foundation. The authors thank Dr. Josh McElwee for daf-2 strains and advice. The Caenorhabditis Genetics Center, supported by the National Institute of Health National Center for Research Resources, is acknowledged for providing strains.

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This evidence led to the suggestion that CR might also protect against the development of age-related diseases, including neurodegenerative disease, such as Parkinson’s disease (PD). Consistently, CR has been shown to increase lifespan of different PD models (Duan and Mattson, 1999; Maswood et al., 2004; Vartiainen et al., 2006). To the best of our knowledge, CR effects on yeast model for synucleinopathies [yeast cells expressing α-synuclein (syn)] were never addressed.
Alpha-synuclein (syn) is the main component of proteinaceous inclusions known as Lewy bodies (LBs), which are implicated in the pathogenesis of the neurodegenerative diseases known as synucleinopathies, like Parkinson’s disease (PD). Aging is a major risk factor for PD and thus, interventions that delay aging will have promising effects in PD and other synucleinopathies. Caloric restriction (CR) is the only non-genetic intervention shown to promote lifespan extension in several model organisms. CR has been shown to alleviate syn toxicity and herein we confirmed the same effect on the yeast model for synucleinopathies during chronological lifespan. The data gathered showed that TOR1 deletion also results in similar longevity extension and abrogation of syn toxicity. Intriguingly, these interventions were associated with decreased autophagy, which was maintained at homeostatic levels. Autophagy maintenance at homeostatic levels promoted by CR or TOR1 abrogation in syn-expressing cells was achieved by decreasing Sir2 levels and activity. Furthermore, the opposite function of Tor1 and Sir2 in autophagy is probably associated with the maintenance of autophagy activity at homeostatic levels, a central event linked to abrogation of syn toxicity promoted by CR.
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These phenotypes could be exploited using forward and reverse chemical genetic approaches to identify drugs that reverse the dpy phenotype. Additionally, even if a disease gene ortholog is not present, relevant information can still be obtained by expressing human transgenes in C. elegans [42,43]. For example, mutations in α1-antitrypsin (AT) lead to lung and liver diseases via loss-of-function and gain-of-function mechanisms, respectively [44,45].
Caenorhabditis elegans has been proven to be a useful model organism for investigating molecular and cellular aspects of numerous human diseases. More recently, investigators have explored the use of this organism as a tool for drug discovery. Although earlier drug screens were labor-intensive and low in throughput, recent advances in high-throughput liquid workflows, imaging platforms and data analysis software have made C. elegans a viable option for automated high-throughput drug screens. This review will outline the evolution of C. elegans-based drug screening, discuss the inherent challenges of using C. elegans, and highlight recent technological advances that have paved the way for future drug screens.
Differences between normal and alpha-synuclein overexpressing SH-SY5Y neuroblastoma cells after Aβ(1-42) and NAC treatment
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Several experimental data suggest that programmed cell death could contribute to Parkinson's disease neuropathology [30] and deficits in αSN regulation per se may elicit apoptosis [21]. On the contrary, our data and some recent reports [8,26,40] show the anti-apoptotic or neuroprotective property of wild-type αSN overexpression. To explain this phenomenon, da Costa et al. [8] pointed out the possible chaperon-like property of αSN, as αSN exhibits a 40% homology with members of the 14-3-3 chaperone protein family [31].
Alpha-synuclein (αSN) plays a major role in numerous neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease. Intracellular inclusions containing aggregated αSN have been reported in Alzheimer's and Parkinson's affected brains. Moreover, a proteolytic fragment of αSN, the so-called non-amyloid component of Alzheimer's disease amyloid (NAC) was found to be an integral part of Alzheimer's dementia related plaques. Despite the extensive research on this topic, the exact toxic mechanism of αSN remains elusive. We have taken the advantage of an αSN overexpressing SH-SY5Y cell line and investigated the effects of classical apoptotic factors (e.g. H₂O₂, amphotericin B and ruthenium red) and aggregated disease-related peptides on cell viability compared to wild type neuroblastoma cells. It was found that αSN overexpressing cells are more sensitive to aggregated peptides treatment than normal expressing counterparts. In contrast, cells containing elevated amount of αSN were less vulnerable to classical apoptotic stressors than wild type cells. In addition, αSN overexpression is accompanied by altered phenotype, attenuated proliferation kinetics, increased neurite arborisation and decreased cell motility. Based on these results, the αSN overexpressing cell lines may represent a good and effective in vitro model for Alzheimer's and Parkinson's disease.
Caenorhabditis Elegans Models of Parkinson's Disease. A Robust Genetic System to Identify and Characterize Endogenous and Environmental Components Involved in Dopamine Neuron Degeneration
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This chapter presents a robust genetic system to identify and characterize endogenous and environmental components involved in dopamine neuron degeneration for the Caenorhabditis elegans (C. elegans) models of Parkinson's disease (PD). C. elegans is a powerful genetic model system for exploring the molecular mechanisms of neuronal function and disease. One of the most signiﬁcant advantages of C. elegans is the ease of generating genetic knockdown or deletion mutants to identify gene function. C. elegans is sensitive to the mitochondria complex I inhibitor rotenone, the insecticide that has been weakly associated with the propensity to develop PD. It is found that of the genes identiﬁed to date that have been associated with PD, only α-synuclein does not have a clear ortholog in C. elegans. C. elegans overexpression of human α-synuclein may also increase overall ﬁtness. It is suggested in vertebrates α-synuclein may also function as a chaperone and to protect against proapoptotic stimuli, and C. elegans could be a useful genetic model to begin to identify the molecular components involved in the α-synuclein neuroprotection.
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This chapter presents a robust genetic system to identify and characterize endogenous and environmental components involved in dopamine neuron degeneration for the Caenorhabditis elegans (C. elegans) models of Parkinson's disease (PD). C. elegans is a powerful genetic model system for exploring the molecular mechanisms of neuronal function and disease. One of the most signiﬁcant advantages of C. elegans is the ease of generating genetic knockdown or deletion mutants to identify gene function. C. elegans is sensitive to the mitochondria complex I inhibitor rotenone, the insecticide that has been weakly associated with the propensity to develop PD. It is found that of the genes identiﬁed to date that have been associated with PD, only α-synuclein does not have a clear ortholog in C. elegans. C. elegans overexpression of human α-synuclein may also increase overall ﬁtness. It is suggested in vertebrates α-synuclein may also function as a chaperone and to protect against proapoptotic stimuli, and C. elegans could be a useful genetic model to begin to identify the molecular components involved in the α-synuclein neuroprotection.

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Short ReportIncreased lifespan in transgenic Caenorhabditis elegans overexpressing human α-synuclein

Abstract

Introduction

Section snippets

Materials and methods

Results

Discussion

Acknowledgements

Regulation of lifespan by sensory perception in Caenorhabditis elegans

Nature

An examination of male–female differences in progression and mortality of Parkinson’s disease

Neurology

Structural and functional conservation of the Caenorhabditis elegans timing gene clk-1

Science

A Drosophila model of Parkinson’s disease

Nature

A systematic RNAi screen for longevity genes in C. elegans

Genes Dev.

Molecular genetics of life span in C. elegans: how much does it teach us?

Trends Genet.

Alterations of life span in the nematode Caenorhabditis elegans under monoxenic culture conditions

Exp. Gerontol.

Life extension via dietary restriction is independent of the Ins/IGF-1 signalling pathway in Caenorhabditis elegans

Exp. Gerontol.

DAF-2 pathway mutations and food restriction in aging Caenorhabditis elegans differentially affect metabolism

Neurobiol. Aging

Measurements of age-related changes of physiological processes that predict lifespan of Caenorhabditis elegans

Proc. Natl. Acad. Sci. USA

Aex-3 encodes a novel regulator of presynaptic activity in C. elegans

Neuron

Aging in the nematode Caenorhabditis elegans: major biological and environmental factors influencing life span

Mech. Ageing Dev.

The genetics of caloric restriction in Caenorhabditis elegans

Proc. Natl. Acad. Sci. USA

Short Report
Increased lifespan in transgenic Caenorhabditis elegans overexpressing human α-synuclein