Nass, Richard et al. (2015) International Worm Meeting "The identification and characterization of SKN-1-associated acetylation pathways involved in dopamine neurodegeneration in Parkinson's disease models."

Shi, Jing, Trinidad, Jonathan, Ren, Karen, Nass, Richard

[International Worm Meeting, 2015]

Background: Idiopathic Parkinson's disease (PD) is an oxidative stress-related disorder that result in abnormal dopamine (DA) signaling and cell death. Although the origin of the pathogenesis in PD remains unclear, corollary evidence suggests both genetic and environmental contributions. The overlapping molecular determinants involved in the neuropathology in both genetic- and toxicant-associated PD models are largely ill defined. Aims: In this study we asked what are the common genes, molecular pathways and mechanisms involved in PD-associated DA neuron vulnerability. Methods: We utilized reverse genetics, biochemical assays, immunofluorescence, transgenic C. elegans, RT-PCR, Western analysis, LC/MS, and neuronal morphology analysis to characterize expression and localization of the PD-associated transcription factor SKN-1 and a gene involved in protein acetylation play in several genetic- and toxicant-associated C. elegans PD models. Results: In this study we demonstrate that a gene involved in protein acetylation renders the DA neurons up to 15-fold more resistant to PD-associated genetic mutations or neurotoxicants, and that overexpression results in a 2-fold increase in DA neurodegeneration. We have also generated a C. elegans mutant that results in highly dysfunctional protein acetylation and an age dependent complete loss of DA neurons in young adult nematodes. The generation of the first C. elegans acetylome as well as reverse genetics and biochemical assays indicates SKN-1 modulates the protein acetylation that effect PD-associated DA neuron vulnerability. Conclusions: This study identifies novel genes and molecular pathways involved in DA neuron vulnerability, and provides in vivo evidence that a common epigenetic mechanism likely plays a significant role in PD-associated neurodegeneration. Support: NIH, BRG, Alzheimer's Disease Foundation, and FNDR Fund to RN, IUCRG to RN and JT.

Nass, Richard M. et al. (2013) International Worm Meeting "The C. elegans acetylome identifies genes and molecular pathways involved in dopamine neuron vulnerability."

VanDuyn, Natalia, Nass, Richard M., Trinidad, Jonathan

[International Worm Meeting, 2013]

Background: Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder, and is characterized by the selective loss of dopamine neurons (DA) in the substantia nigra. Recent studies suggest that epigenetic dysregulation may play a significant role in neurodegenerative diseases. Histone deacetylases (HDACs) are a class of enzymes that remove acetyl groups from histone and non-histone proteins that play critical roles in cell regulation, longevity, and neuropathological disorders. Statement of purpose: To generate the first reported genome-wide identification of acetylated proteins in C. elegans in WT and acetylation-associated mutants, and determine whether these modulate DA neuron vulnerability to PD-associated proteins or neurotoxicants. Methods: We utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS), biochemical assays, immunofluorescence, reverse genetics, transgenic C. elegans, RT-PCR, Western and neuronal morphology analysis to characterize expression, localization and the role that acetylation plays in PD-associated DA neurodegeneration. Results: Our results indicate that lysine acetylation is abundant in a wide variety of proteins including those involved in transcription, translation, metabolism, and stress responses. We also demonstrate that an HDAC mutant inhibits DA neurodegeneration up to 15-fold relative to WT, and acetylation of specific proteins modulates DA neuron vulnerability and induces specific stress response pathways. Conclusion: This study defines the first C. elegans acetylome and identifies acetylation-associated proteins involved in neuroprotection that will likely yield novel insight into the molecular basis of DA neuron vulnerability and associated diseases. Support: NIEHS ES014459 and ES003299 to RN; and EPA STAR Graduate Fellowship to NVD.

Richard Nass et al. (2001) International C. elegans Meeting "A Novel Pharmacogenetic Model for Parkinson's"

Richard Nass, David H Hall, Randy D Blakely, David M Miller

[International C. elegans Meeting, 2001]

Current animal models for idiopathic Parkinson's Disease rely on inducing nigrostriatal damage in mammals with the neurotoxin 6-OHDA or MPTP. The dopamine transporter DAT, which is the target for many psychoactive drugs, provides the cellular gateway for the accumulation of the neurotoxin that evokes neuronal death and Parkinson-like syndrome. We have previously cloned the C. elegans DAT (DAT-1), and have shown that it is functionally similar to mammalian DATs and expressed exclusively in the dopamine (DA) neurons (Jayanthi et al. 1998, Nass et al. 1999, 2000). We have also developed WT and dat-1 knockout (see abstract by Duerr et al.) transgenic lines which target a dat-1 promotor-gfp fusion to all 8 DA neurons in the hermaphrodite. Exposure of the reporter line to 6-OHDA results in DA neuronal blebbing, soma deformation, and loss of GFP expression. DA transport by DAT-1 is found to be blocked by 6-OHDA, antidepressants, and amphetamine in vitro. These latter agents block the effects of 6-OHDA on DA neurons, and a dat-1 deletion line is insensitive to the neurotoxin. Ultrastructural analysis of the worm DA neurons show significant signs of neurodegeneration including small, dark, and rounded cell bodies, as well as vacuolated and loss of neuronal processes following 6-OHDA exposure. Interestingly, although these phenotypes are characteristic of apoptosis both in mammals and worms, the effect appears to be independent of the classic apoptotic pathway, since the caspase-deficient ced-3 and ced-4 backgrounds still display the toxin-induced neuronal blebbing and loss of GFP expression. Finally, studies with DA deficient lines suggest that endogenous DA production plays a role in the neurotoxicity. These studies as well as our early progress on toxin-based genetic screens for regulators of DAT function, localization, and the toxin-mediated cell death will be presented. Supported by P01 DK58212 (RN, DMM, RDB), RR12596 (DHH), PhRMA (RN)

Richard Nass et al. (1999) International C. elegans Meeting "Pharmacology and Regulation of the C. elegans Dopamine Transporter"

Jim Rand, Janet Duerr, David M Miller, Richard Nass, Randy D Blakely

[International C. elegans Meeting, 1999]

The neurotransmitter dopamine (DA) plays a significant role in a number of physiological processes including motor control, cognition, emotion, and reward. Abnormal DA signaling has been implicated in a variety of neuropathological illnesses such as Parkinson's disease, schizophrenia, depression, and drug addiction. A primary mechanism for regulating the strength and duration of dopamine signalling involves the Na + -dependent dopamine transporter (DAT). Following DA release at the synapse, DAT mediates the reuptake of DA into the presynaptic neurons. DATs are targeted by psychoactive drugs such as methylphenidate (Ritalin), cocaine and amphetamines. Although DAT pharmacology has been extensively characterized, endogenous regulators of DAT expression, localization, and activity are as yet unknown. DA is an important neurotransmitter in C. elegans . To provide an opportunity for genetic evaluation of DAT regulators, we have cloned the C.elegans dopamine transporter (CeDAT) 1 . The CeDAT gene encodes a 615 amino acid polypeptide with high sequence identity to the mammalian dopamine transporters (43%). Mammalian cells expressing CeDAT exhibit saturable and high affinity Na + and Cl - dependent DA transport, and are inhibited by the mammalian DAT antagonist cocaine and amphetamines, as well as the antidepressant imipramine. These structural and pharmacological profiles indicate that CeDAT is likely to function as the dopamine transporter in C. elegans and is similar to its mammalian counterpart. We are currently exploring the expression of CeDAT using CeDAT-GFP and peptide antibodies. Several transgenic lines have been developed which target CeDAT transgenes to dopaminergic neurons and processes. We are also investigating the role of CeDAT in response to cocaine and amphetamines in N2 and CeDAT knockout worms. These studies as well as psychostimulant-induced phenotypes suitable for genetic screens of CeDAT regulators will be presented. 1. Jayanthi et al. (1998) Mol. Pharm. 54 :601-609.

VanDuyn, Natalia et al. (2013) International Worm Meeting "The ABC transporter MRP-7 inhibits methylmercury-induced whole animal and dopamine neuron pathology."

Nass, Richard, VanDuyn, Natalia

[International Worm Meeting, 2013]

Background: Methylmercury (MeHg) exposure from occupational, environmental, and food sources is a significant threat to public health. Recent epidemiological and vertebrate studies suggest that MeHg exposure may contribute to dopamine (DA) neuron vulnerability and the propensity to develop Parkinson's disease (PD). We have developed a novel Caenorhabditis elegans (C. elegans) model of MeHg toxicity and have shown that low, chronic exposure confers embryonic defects, developmental delays, and DA neuron degeneration, and that the toxicity is partially dependent on the phase II antioxidant transcription factor SKN-1/Nrf2. Statement of Purpose: In this study we asked what genes and molecular pathways are involved in MeHg-induced whole animal and DA neuron pathology. Methods: We utilized a reverse genetic screen, biochemical assays, immunofluorescence, transgenic C. elegans, RT-PCR, ICP-MS, Western analysis, and neuronal morphology analysis to characterize expression, localization and the role that SKN-1 and MRP-7 play in MeHg-induced whole animal and DA neuronal death. Results: Over 17,000 genes were screened for whole animal sensitivity to MeHg, and 92 genes were identified (90% have strong human homologues) that affect whole animal and/or DA neuron pathology. Here we report detailed analysis of a previously uncharacterized ABC transporter, MRP-7. MeHg confers increased mortality in mrp-7(RNAi) animals relative to WT. Furthermore, exposure of the genetic knockdown animals to extremely low MeHg levels results in 40% of animals displaying DA neuron degeneration. Transgenic animals expressing mrp-7 transcriptional fusions indicate that mrp-7 is expressed in DA neurons as well as other cell types. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) studies indicate that mrp-7(RNAi) animals contain 2-fold higher Hg levels relative to WT. Conclusions: This study describes a novel whole genome reverse genetic screen that identified MRP-7 as well as a number of other genes involved in MeHg-associated resistance that will likely prove useful in identifying therapeutic targets to inhibit MeHg-induced cellular toxicity in humans. Support: NIEHS ES014459 and ES003299 to RN; and EPA STAR Graduate Fellowship to NVD.

Townsend SR et al. (1994) Worm Breeder's Gazette "C. elegans Molecular Genetics and Long PCR."

Finelli AL, Savage C, Xie T, Padgett RW, Townsend SR

[Worm Breeder's Gazette, 1994]

C. elegans Molecular Genetics and Long PCR Scott R. Townsend, Cathy Savage, Alyce L. Finelli, Ting Xie, and Richard W. Padgett, Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08855

LeVora, Jennifer et al. (2009) International Worm Meeting "Biochemical and Genetic Analysis of Parkinson's Disease-associated and Stress Response Proteins in C. elegans Models of Manganism."

Settivari, Raja, Nass, Richard, LeVora, Jennifer

[International Worm Meeting, 2009]

Parkinson''s disease (PD) and manganism are characterized by motor deficits and damage to the substantia nigra, and dopamine or its metabolites are believed to contribute to both disorders. Proteins in which mutations have been linked with familial Parkinson''s disease have also been proposed to contribute to the pathogenesis. In these studies we show that manganese (Mn2+) causes dopamine (DA) neuronal death in C. elegans, and most of the known PD-associated genes, as well as specific glutathione transferases (GSTs) and heat shock proteins, are significantly upregulated following a 30 min exposure to Mn2+. DA neuron vulnerability to Mn2+ is partially dependent on the divalent metal transporter-1 (DMT-1) orthologue SMF-1, since knockdown or deletion of the putative Mn2+ transporter partially protects against the neurodegeneration. Indirect immunofluorescence (IF) studies indicate that SMF-1 is expressed in DA neurons (as well as other cells), consistent with its role in contributing to Mn2+-induced DA neurodegeneration. SMF-1 also significantly contributes to Mn2+-induced reduction of mitochondrial membrane potential as determined by the fluorescent dye Tetramethylrhodamine ethyl ester (TMRE). IF studies also indicate that putative C. elegans GSTs play a significant role in inhibiting Mn2+- or PD-associatiated neurotoxin-induced DA neurodegeneration, as knockdown or deletion of the GSTs increase DA neuron vulnerability in our PD models. Overall these studies suggest that C. elegans is a powerful model system to explore the molecular basis of DA neuron vulnerability in human manganism. Support Contributed By: NIH R011ES014459, NIH R01ES010563, and DOD (RN).

Settivari, Raja S et al. (2009) International Worm Meeting "Pharmacogenetic and biochemical analysis of Parkinson's disease-associated proteins in C.elegans: effects on mitochondrial function and dopamine neuron vulnerability."

Settivari, Raja S, LeVora, Jennifer, Nass, Richard

[International Worm Meeting, 2009]

Parkinson''s disease (PD) is a progressive neurodegenerative disorder largely characterized by the loss of dopaminergic neurons. Although the etiology of the disease is unknown, it is believed to be multifactorial, with both genetic and environmental contributions that result in oxidative damage and mitochondrial dysfunction. We previously established novel C. elegans models for Parkinson''s disease that now allow for the evaluation of PD-associated orthologues and other proteins in their role in dopamine (DA) neuron vulnerability in response to endogenous and exogenous insults, and provides opportunities to identify and characterize novel PD therapeutic targets and leads. Here we describe our expression and biochemical investigations of WT and mutant PD- and DA neuron-associated proteins, and their effects on mitochondria function and DA neurons under basal conditions and following exposure to neurotoxins. qPCR gene expression studies of PD- and DA neuron-associated proteins show significant changes in expression of most of the familial PD genes. Biochemical analysis shows that mutations within some of the PD-associated genes in C. elegans affect mitochondrial membrane potential (as measured by the fluorescent dye TMRE), respiration capacity (as measured by oxygen consumption), and cellular dopamine and glutathione concentrations. Mutations within several PD- and DA neuron-associated proteins increase the vulnerability of DA neurons to mitochondria-targeted neurotoxins, and anti-oxidant flavonoids that may increase mitochondrial function significantly protect against DA neurodegeneration. We have also generated antibodies to over 40 C. elegans proteins, including PD- and DA neuron-associated proteins, and our Western blot analysis and indirect immunofluorescence suggest differential protein expression in our PD models. Overall these studies suggest that mutations within DA neuron- and PD-associated genes result in increases in oxidative stress and mitochondria dysfunction, and further validates C. elegans as a powerful genetic model to explore PD. Support Contributed By: NIH R011ES014459, NIH R01ES010563, DOD and Collaborative Research Grant (Purdue/IUSM) (RN).

VanDuyn, Natalia et al. (2011) International Worm Meeting "Identification and characterization of molecular modulators of methylmercury-induced whole animal and dopamine neuron pathology in C. elegans."

Settivari, Raja, Nass, Richard, Sinclair, Gary, VanDuyn, Natalia

[International Worm Meeting, 2011]

Methylmercury (MeHg) exposure from occupational, environmental, and food sources is a significant threat to public health. MeHg poisonings in adults may result in severe psychological and neurological deficits, and in utero exposures can confer embryonic defects and developmental delays. Recent epidemiological and vertebrate studies suggest that MeHg exposure may also contribute to dopamine (DA) neuron vulnerability and the propensity to develop Parkinson's disease (PD). We have developed a novel Caenorhabditis elegans (C. elegans) model of MeHg toxicity that shows that low, chronic exposure confers embryonic defects, developmental delays, decreases in brood size and animal viability, and DA neuron degeneration. Toxicant exposure results in the robust induction of the glutathione-S-transferases (GSTs) gst-4, gst-5, gst-12, gst-21, and gst-38, with some GSTs largely dependent on the PD-associated phase II antioxidant transcription factor SKN-1/Nrf2. We also demonstrate that the expression of SKN-1, a protein previously localized to a small subset of chemosensory neurons and intestinal cells in the nematode, is also expressed in the DA neurons, and a reduction in SKN-1 gene expression increases MeHg-induced animal vulnerability and DA neuron degeneration. We will also present our initial studies from two genome-wide genetic screens to identify mediators and suppressors of MeHg-induced DA neuron degeneration, as well as our investigations using DA-associated mutants to elucidate the role that the neurotransmitter may play in the toxicity. Support contributed by: NIEHS ES014459, ES003299 and Vanderbilt University Toxicology Program Pilot Grant to RN; PhRMA Pre Doctoral Fellowship in Pharmacology/Toxicology and EPA STAR Graduate Fellowship to NVD.

Martinelli SD et al. (1995) International C. elegans Meeting "ACEDB"

Durbin RM, Martinelli SD

[International C. elegans Meeting, 1995]

We hope to provide a demonstration of the current state of the ACeDB worm database on Unix workstations, and if possible Apple Macintosh, throughout the poster sessions. This will be based on the new version 4 release of the acedb software (Jean Thierry-Mieg, Richard Durbin and numerous others), which contains many new features for greater efficiency, more flexible printing, and display of new features.