Voisine C et al. (2010) Neurobiol Dis "Chaperone networks: tipping the balance in protein folding diseases."

Pedersen JS, Morimoto RI, Voisine C

[Neurobiol Dis, 2010]

Adult-onset neurodegeneration and other protein conformational diseases are associated with the appearance, persistence, and accumulation of misfolded and aggregation-prone proteins. To protect the proteome from long-term damage, the cell expresses a highly integrated protein homeostasis (proteostasis) machinery to ensure that proteins are properly expressed, folded, and cleared, and to recognize damaged proteins. Molecular chaperones have a central role in proteostasis as they have been shown to be essential to prevent the accumulation of alternate folded proteotoxic states as occurs in protein conformation diseases exemplified by neurodegeneration. Studies using invertebrate models expressing proteins associated with Huntington's disease, Alzheimer's disease, ALS, and Parkinson's disease have provided insights into the genetic networks and stress signaling pathways that regulate the proteostasis machinery to prevent cellular dysfunction, tissue pathology, and organismal failure. These events appear to be further amplified by aging and provide evidence that age-related failures in proteostasis may be a common element in many diseases.

Voisine C et al. (2004) Methods Mol Biol "Caenorhabditis elegans as a model system for triplet repeat diseases."

Hart AC, Voisine C

[Methods Mol Biol, 2004]

Common features underlie the generation and function of neurons in multicellular animals. It is likely that conserved pathways and genes also are involved in neuronal degeneration and malfunction. To address the molecular mechanisms of complex human neurological disorders, many investigators are choosing to study these diseases in simpler organisms. The nematode Caenorhabditis elegans provides an excellent model system to address genetically the mechanisms of triplet repeat diseases. Advantages of using C. elegans as a model system include the ease of genetic manipulation, the sequenced genome, and a short life cycle. Furthermore, researchers can precisely identify specific neurons and follow their development or survival throughout the animal's lifetime. This chapter describes the tools and approaches for modeling triplet repeat diseases in C. elegans with a specific emphasis on polyglutamine (polyQ) diseases. Although the bulk of the chapter is devoted to generating a polyQ disease model in C. elegans, it also addresses potential avenues for assessing the impact of specific candidate genes/pathways on the disease process, including cell death and aging.

Voisine C et al. (2007) PLoS One "Identification of potential therapeutic drugs for huntington's disease using Caenorhabditis elegans."

Stockwell BR, Varma H, Hart AC, Walker N, Bates EA, Voisine C

[PLoS One, 2007]

BACKGROUND: The prolonged time course of Huntington's disease (HD) neurodegeneration increases both the time and cost of testing potential therapeutic compounds in mammalian models. An alternative is to initially assess the efficacy of compounds in invertebrate models, reducing time of testing from months to days. METHODOLOGY/PRINCIPAL FINDINGS: We screened candidate therapeutic compounds that were identified previously in cell culture/animal studies in a C. elegans HD model and found that two FDA approved drugs, lithium chloride and mithramycin, independently and in combination suppressed HD neurotoxicity. Aging is a critical contributor to late onset neurodegenerative diseases. Using a genetic strategy and a novel assay, we demonstrate that lithium chloride and mithramycin remain neuroprotective independent of activity of the forkhead transcription factor DAF-16, which mediates the effects of the insulin-like signaling pathway on aging. CONCLUSIONS/SIGNIFICANCE: These results suggest that pathways involved in polyglutamine-induced degeneration are distinct from specific aging pathways. The assays presented here will be useful for rapid and inexpensive testing of other potential HD drugs and elucidating pathways of drug action. Additionally, the neuroprotection conferred by lithium chloride and mithramycin suggests that these drugs may be useful for polyglutamine disease therapy.

Voisine, C. et al. (2019) International Worm Meeting "Cell to cell spreading of TDP-43 may be linked to toxicity in Caenorhabditis elegans."

Rendleman, E., Figueroa, Z., Nussbaum-Krammer, C., Voisine, C., Nguyen, Q.

[International Worm Meeting, 2019]

Many neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and prion diseases are characterized by abnormal accumulation of disease proteins in nerve cells leading to selective neurotoxicity. Moreover, a prion-like spreading mechanism might play a role in disease progression, where misfolded disease proteins spread from affected to unaffected neurons. Interestingly, ALS exhibits a focal clinical onset followed by a regional spreading of protein misfolding and cell death. Evidence points towards TAR DNA-binding protein 43 (TDP-43) as the major pathological protein in sporadic and certain familial forms of ALS where aggregates in affected neurons contain full length and fragmented forms of TDP-43. Despite recent advances in biomedical research on ALS disease associated proteins like TDP-43, a mechanistic explanation that links toxicity with cell to cell transmission remains unclear. To examine TDP-43 related toxicity, we have established C. elegans models that express human TDP-43 fused to fluorescent proteins. Using well-established behavioral assays, we found that neuronal expression of full length TDP-43 decreased motility and lowered chemosensory and mechanosensory detection compared to wild type suggesting defects in neuronal functionality. In addition to neuronal dysfunction associated with TDP-43 expression, we observed a decline in organismal health as measured by a reduction in fecundity, embryogenesis, larval development and lifespan. These results demonstrate both a cell autonomous and non-autonomous toxic effect associated with TDP-43 neuronal expression. To explore whether spreading of TDP-43 could contribute to organismal decline, we employed high-resolution time-lapse imaging and observed the intercellular movement of a C-terminal fragment of TDP-43 from body wall muscle cells to the hypodermis, intestinal cells and gonad in living animals. These results confirm that at least certain fragments of TDP-43 are released from expressing cells into neighboring non-expressing cells, which may contribute to the decline in organismal health. Our in vivo evidence for the spreading of TDP-43 supports the model that cell to cell transmissibility of toxic forms of TDP-43 contribute to disease progression.

Voisine C et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Distinct Co-Chaperones Prevent Age-related Aggregation and Toxicity in C. elegans Protein Misfolding Models"

Orton K, Voisine C, Schieber M, Pedersen J, Morimoto R

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Misfolded and damaged proteins challenge homeostasis and the capacity of molecular chaperones and clearance machineries to restore the health of the cell. The chronic expression of aggregation-prone proteins can result in irreversible toxicity as occurs in human neurodegenerative disease. Although molecular chaperones play a critical role in managing proteotoxic insults encountered in both normal and pathological processes, it remains unclear which chaperone networks protect cells from misfolding and toxicity. To address this question, we systematically evaluated the C. elegans chaperome to identify those that maintain protein homeostasis, delaying protein-specific aggregation and cellular dysfunction. Using well-established C. elegans models for protein misfolding diseases, we identified a limited number of chaperones that exacerbate the functional deficits of polyglutamine and Abeta models upon RNAi knock-down. Furthermore, these chaperones cooperate to maintain normal physiology in aging animals. While the major folding machines, Hsc70, Hsp90, and the chaperonin complex, protect cells from both aggregation and toxicity, distinct subsets of co-chaperones specialize either in preventing aggregation or ameliorating toxicity. Illustrating this difference, we find that increasing the folding capacity does not restore cellular function after loss of specific co-chaperones. Our comparative analyses of chaperone requirements suggest that co-chaperone function separates aggregation and toxicity related to chronic expression of aggregation-prone proteins.

Cindy Voisine et al. (2001) International C. elegans Meeting "Mutations in the C elegans pqe-1 gene enhance polyglutamine-mediated ASH neurodegeneration"

Cindy Voisine, Anne C Hart, Daphne C King, Peter W Faber

[International C. elegans Meeting, 2001]

At least eight hereditary neurodegenerative disorders, including Huntington's Disease, have been identified in which the disease locus expresses a protein that contains an expanded glutamine tract. The mechanism by which these polyglutamine (polyQ) repeats cause neurodegeneration and cell death is unknown. We have established a Caenorhabditis elegans model system to identify proteins involved in polyglutamine (polyQ) neurotoxicity. (PNAS 96, 179-184, 1999). N-terminal fragments of the Huntington's Disease protein huntingtin (Htn) containing polyQ tracts of 2, 23, 95 or 150 residues are expressed in the ASH sensory neurons. Expression of Htn-Q150, but not Htn-Q2, Htn-Q23 or Htn-Q95, cause age dependent ASH degeneration without ASH cell death in aged (8-day-old) animals. ASH neurodegeneration is determined by the ability of the exposed sensory endings of ASH neurons to uptake lipophilic dyes. ASH cell death is assessed by immunohistochemical techniques or GFP expression. PolyQ-mediated ASH neurodegeneration is partially dependent on ced-3 caspase function suggesting the involvement of the apoptotic cell death pathway. To identify genes that normally protect neurons from polyQ-mediated neurodegeneration, we performed an F2 EMS screen for mutations in genes that exacerbate polyQ-mediated ASH neurodegeneration. After screening approximately 30,000 mutagenized animals, 7 mutant strains were identified that carried mutations in the polyQ enhancer-1 ( pqe-1 ) gene. Overt phenotypes have not been observed in pqe-1 mutant animals other than enhancement of polyQ neurotoxicity. In pqe-1 mutant animals, ASH neuronal cell death is dependent upon the presence of expanded polyQ tracts (Htn-Q95, Htn-Q150), as ASH neurons expressing N-terminal fragments of huntingtin with shorter polyQ tracts (Htn-Q2, Htn-Q23) are unaffected. In addition, pqe-1 mutant animals are wild-type in two ASH mediated behavioral assays (nose-touch and osmotic avoidance). Differential splicing of pqe-1 results in three nuclear localized PQE-1 proteins. PQE-1A and PQE-1B contain a putative exonuclease domain. PQE-1A and PQE-1C contain a large glutamine/proline-rich domain which is essential for pqe-1 function. PQE-1 proteins may interact with expanded polyQ tracts or their pathological targets via the glutamine/proline domain to protect neurons from polyQ insults. Molecular and biochemical experiments are underway to address the connection between PQE-1 function and polyQ neurotoxicity. To identify genes that are required for pqe-1 function or poly-Q mediated neurodegeneration, we have initiated a genetic screen to isolate mutations that suppress this polyQ-mediated ASH neurodegeneration. Identification and characterization of genes isolated from these genetic screens will provide insight into pathogenic mechanisms underlying polyQ-induced neurodegeneration and cell death.

Marsili, J. et al. (2017) International Worm Meeting "The nematode Caenorhabditis elegans displays a chemotaxis behavior to tuberculosis-specific odorants."

Nguyen , Q.H., Voisine, C., McFall , S.M., Marsili, J., Neto, M.F., Aldakeel , S.

[International Worm Meeting, 2017]

The World Health Organization's Millennium Development Goal of halting and reversing the tuberculosis (TB) epidemic by 2015 has been met; on average, incidence of TB has fallen 1.5% per year and is now 18% lower than in 2000. Despite these gains, TB remains one of the leading causes of morbidity and mortality globally-in 2014, 9.6 million people fell ill with TB and 1.5 million died from the disease. Each year, approximately one third of the TB infected population remain undiagnosed, equating to roughly 3 million people. A simple, affordable diagnostic test for pulmonary tuberculosis (TB) is urgently needed to improve detection of active Mycobacterium tuberculosis. Recently, it has been suggested that animal behavior can be used as a biosensor to signal the presence of human disease. For example, the giant African pouched rats can detect tuberculosis by sniffing sputum specimens while honeybees respond to three of the four tuberculosis-specific volatile organic compounds (TB-VOCs) detected in the breath of TB positive patients by proboscis extension. However, both rats and honeybees require animal housing facilities and professional trainers, where few laboratories in low-income, high-burden TB settings possess the needed infrastructure. Rather than using trained animals, we propose exploiting the innate sensing capabilities of the nematode Caenorhabditis elegans, which has demonstrated reproducible, behavioral responses to over 100 VOCs. Our data shows that the innate olfactory behavioral response of C. elegans can be used to detect the TB-specific VOCs methyl p-anisate, methyl nicotinate, methyl phenylacetate and o-phenylanisole, in chemotaxis assays. It is important to note, the breath of a TB patient contains a heterogeneous mixture of TB-VOCs, although the proportionality of this blend remains unknown. To further evaluate the efficacy of C. elegans as a potential TB biosensor, we also investigated C. elegans' behavioral response to combinations of TB-VOCs, both pairwise and collectively. We found that C. elegans detects a mixture of all TB-VOCs, exhibiting a significant avoidance behavior. In addition, dauer larvae, a long-lived stress resistant alternative development state of C. elegans in which the animals can survive for extended periods of time in dry conditions with no food, were also demonstrated to detect the VOCs. Taken together, we propose a potential work-flow for a noninvasive diagnostic test for TB that incorporates collecting VOCs from the breath of the patient, exposing dauer larvae housed in a microfluidic device to the VOCs, and recording their response. These results showcase the potential for C. elegans to be incorporated into a new and affordable diagnostic test device for TB and, perhaps, other disease states, capitalizing upon its innate olfactory responses.

Faber PW et al. (2002) Proc Natl Acad Sci U S A "Glutamine/proline-rich PQE-1 proteins protect Caenorhabditis elegans neurons from huntingtin polyglutamine neurotoxicity."

Faber PW, Voisine C, Hart AC, King DC, Bates EA

[Proc Natl Acad Sci U S A, 2002]

Huntington's disease is a progressive neurodegenerative disease caused by a polyglutamine (polyQ) repeat expansion in the huntingtin protein [Huntington's Disease Collaborative Research Group (1993) Cell 72, 971-983]. To understand the mechanism by which polyQ repeats cause neurodegeneration and cell death, we modeled polyQ neurotoxicity in Caenorhabditis elegans. In our model, expression of N-terminal fragments of human huntingtin causes polyQ-dependent degeneration of neurons. We conducted a genetic screen to identify proteins that protect neurons from the toxic effects of expanded polyQ tracts. Loss of polyQ enhancer-1 (pqe-1) gene function strongly and specifically exacerbates neurodegeneration and cell death, whereas overexpression of a pqe-1 cDNA protects C. elegans neurons from the toxic effects of expanded huntingtin fragments. A glutamineproline-rich domain, along with a charged domain, is critical for PQE-1 protein function. Analysis of pqe-1 suggests that proteins exist that specifically protect neurons from the toxic effects of expanded polyQ disease proteins.

Neto MF et al. (2016) J Clin Tuberc Other Mycobact Dis "The nematode Caenorhabditis elegans displays a chemotaxis behavior to tuberculosis-specific odorants."

Voisine C, Marsili J, Nguyen QH, McFall SM, Neto MF

[J Clin Tuberc Other Mycobact Dis, 2016]

A simple, affordable diagnostic test for pulmonary tuberculosis (TB) is urgently needed to improve detection of active <i>Mycobacterium tuberculosis.</i> Recently, it has been suggested that animal behavior can be used as a biosensor to signal the presence of human disease. For example, the giant African pouched rats can detect tuberculosis by sniffing sputum specimens while trained honeybees respond to three of the volatile organic compounds (VOCs) detected in the breath of TB positive patients by proboscis extension. However, both rats and honeybees require animal housing facilities and professional trainers, which are outside the scope of most disease testing facilities. Here, we report that the innate olfactory behavioral response of the roundworm nematode <i>Caenorhabditis elegans</i> can be used to detect the TB-specific VOCs methyl <i>p</i>-anisate, methyl nicotinate, methyl phenylacetate and <i>o</i>-phenylanisole, in chemotaxis assays. Dauer larvae, a long-lived stress resistant alternative development state of <i>C. elegans</i> in which the animals can survive for extended periods of time in dry conditions with no food, were also demonstrated to detect the VOCs. We propose that exposing naive dauer larvae to TB-related VOCs and recording their response in this behavioral assay could lead to the development of a new method for TB diagnostics using breath as the sample type.

Cindy Voisine et al. (2005) International Worm Meeting "Assessing drug efficacy in a C. elegans model of polyglutamine toxicity"

Nicola Walker, Brent R Stockwell, Anne C Hart, Cindy Voisine, Hemant Varma

[International Worm Meeting, 2005]

Huntington's Disease (HD) is one of nine neurodegenerative disorders caused by polyglutamine (polyQ) domain expansion. There is currently no cure nor effective treatment available for HD. In our C. elegans model of polyQ (CGC # 3360), the N-terminus of the toxic expanded human huntingtin protein causes age dependent degeneration of ASH neurons. We are developing drug efficacy assays to test possible therapeutic agents and assess their impact on polyQ mediated neurodegeneration in C. elegans. Drug studies should also provide insight into the contributions of different cellular pathways to the disease process. Our first assay format used animals that also carry a mutation in pqe-1, a previously identified, genetic enhancer of polyQ toxicity (CGC # 5585). In pqe-1 mutant animals, the vast majority of ASH neurons undergo cell death before the animals reach adulthood. L1s were incubated with drugs and food for two or three days before the death of ASH neurons was evaluated. We identified several compounds, but some therapeutic doses also slowed growth. To assess the impact of slower growth on polyQ toxicity, we developed a second assay that was independent of growth. ASH cell death increases over time in arrested L1 pqe-1 animals. L1s were incubated with drugs in the absence of food for one or two days before the death of ASH neurons was evaluated. We tested several compounds in this new assay format and found that LiCl and mithramycin suppressed neurodegeneration. Both compounds have similar effects in other HD models. LiCl is a GSK-3 inhibitor that has been shown to reduce polyQ toxicity in cell culture (Carmichael J et al, 2002). Mithramycin binds to GC rich regions of DNA and has been shown to reduce polyQ toxicity in a mouse HD model (Ferrante et al, 2004). Currently, we are developing an additional assay utilizing aged animals with normal pqe-1 function to further evaluate what effect these compounds have on the age dependent degeneration associated with expression of the toxic expanded human huntingtin protein. We believe that our drug assays provide an efficient means of identifying therapeutic agents for further testing in vertebrate models of polyQ diseases.