Alexandre Benedetto

Caenorhabditis elegans: An Emerging Model in Biomedical and Environmental Toxicology

The nematode Caenorhabditis elegans has emerged as an important animal model in various fields including neurobiology, developmental biology, and genetics. Characteristics of this animal model that have contributed to its success include its genetic manipulability, invariant and fully described developmental program, well-characterized genome, ease of maintenance, short and prolific life cycle, and small body size. These same features have led to an increasing use of C. elegans in toxicology, both for mechanistic studies and high-throughput screening approaches. We describe some of the research that has been carried out in the areas of neurotoxicology, genetic toxicology, and environmental toxicology, as well as high-throughput experiments with C. elegans including genome-wide screening for molecular targets of toxicity and rapid toxicity assessment for new chemicals. We argue for an increased role for C. elegans in complementing other model systems in toxicological research.

The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans.

Polarized intracellular trafficking in epithelia is critical in development, immunity, and physiology to deliver morphogens, defensins, or ion pumps to the appropriate membrane domain. The mechanisms that control apical trafficking remain poorly defined. Using Caenorhabditis elegans, we characterize a novel apical secretion pathway involving multivesicularbodies and the release of exosomes at the apical plasma membrane. By means of two different genetic approaches, we show that the membrane-bound V0 sector of the vacuolar H+-ATPase (V-ATPase) acts in this pathway, independent of its contribution to the V-ATPase proton pump activity. Specifically, we identified mutations in the V0 “a” subunit VHA-5 that affect either the V0-specific function or the V0+V1 function of the V-ATPase. These mutations allowed us to establish that the V0 sector mediates secretion of Hedgehog-related proteins. Our data raise the possibility that the V0 sector mediates exosome and morphogen release in mammals.

Manganese transport in eukaryotes: The role of DMT1

Manganese (Mn) is a transition metal that is essential for normal cell growth and development, but is toxic at high concentrations. While Mn deficiency is uncommon in humans, Mn toxicity is known to be readily prevalent due to occupational overexposure in miners, smelters and possibly welders. Excessive exposure to Mn can cause Parkinsons disease-like syndrome; patients typically exhibit extrapyramidal symptoms that include tremor, rigidity and hypokinesia [Calne DB, Chu NS, Huang CC, Lu CS, Olanow W. Manganism and idiopathic parkinsonism: similarities and differences. Neurology 1994;44(9):1583-6; Dobson AW, Erikson KM, Aschner M. Manganese neurotoxicity. Ann NY Acad Sci 2004;1012:115-28]. Mn-induced motor neuron diseases have been the subjects of numerous studies; however, this review is not intended to discuss its neurotoxic potential or its role in the etiology of motor neuron disorders. Rather, it will focus on Mn uptake and transport via the orthologues of the divalent metal transporter (DMT1) and its possible implications to Mn toxicity in various categories of eukaryotic systems, such as in vitro cell lines, in vivo rodents, the fruitfly, Drosophila melanogaster, the honeybee, Apis mellifera L., the nematode, Caenorhabditis elegans and the bakers yeast, Saccharomyces cerevisiae.

Extracellular dopamine potentiates mn-induced oxidative stress, lifespan reduction, and dopaminergic neurodegeneration in a BLI-3-dependent manner in Caenorhabditis elegans

Parkinsons disease (PD)-mimicking drugs and pesticides, and more recently PD-associated gene mutations, have been studied in cell cultures and mammalian models to decipher the molecular basis of PD. Thus far, a dozen of genes have been identified that are responsible for inherited PD. However they only account for about 8% of PD cases, most of the cases likely involving environmental contributions. Environmental manganese (Mn) exposure represents an established risk factor for PD occurrence, and both PD and Mn-intoxicated patients display a characteristic extrapyramidal syndrome primarily involving dopaminergic (DAergic) neurodegeneration with shared common molecular mechanisms. To better understand the specificity of DAergic neurodegeneration, we studied Mn toxicity in vivo in Caenorhabditis elegans. Combining genetics and biochemical assays, we established that extracellular, and not intracellular, dopamine (DA) is responsible for Mn-induced DAergic neurodegeneration and that this process (1) requires functional DA-reuptake transporter (DAT-1) and (2) is associated with oxidative stress and lifespan reduction. Overexpression of the anti-oxidant transcription factor, SKN-1, affords protection against Mn toxicity, while the DA-dependency of Mn toxicity requires the NADPH dual-oxidase BLI-3. These results suggest that in vivo BLI-3 activity promotes the conversion of extracellular DA into toxic reactive species, which, in turn, can be taken up by DAT-1 in DAergic neurons, thus leading to oxidative stress and cell degeneration.

Overexpression of Toll-Like Receptor 4 Amplifies the Host Response to Lipopolysaccharide and Provides a Survival Advantage in Transgenic Mice

Toll-like receptors are transmembrane proteins that are involved in the innate immune recognition of microbial constituents. Among them, Toll-like receptor 4 (Tlr4) is a crucial signal transducer for LPS, the major component of Gram-negative bacteria outer cell membrane. The contribution of Tlr4 to the host response to LPS and to infection with virulent Salmonella typhimurium was studied in four transgenic (Tg) strains including three overexpressing Tlr4. There was a good correlation between the level of Tlr4 mRNA expression and the sensitivity to LPS both in vitro and in vivo: Tg mice possessing the highest number of Tlr4 copies respond the most to LPS. Overexpression of Tlr4 by itself appears to have a survival advantage in Tg mice early during infection: animals possessing more than two copies of the gene survived longer and in a greater percentage to Salmonella infection. The beneficial effect of Tlr4 overexpression is greatly enhanced when the mice present a wild-type allele at natural resistance-associated macrophage protein 1, another critical innate immune gene involved in resistance to infection with Salmonella. Tlr4 and natural resistance-associated macrophage protein 1 exhibit functional epistatic interaction to improve the capacity of the host to control bacterial replication. However, this early improvement in disease resistance is not conducted later during infection, because mice overexpressing Tlr4 developed an excessive inflammatory response detrimental to the host.

Manganese-induced dopaminergic neurodegeneration: insights into mechanisms and genetics shared with Parkinson's disease.

Manganese (Mn) is an abundant, naturally occurring element in the Earth’s crust. It is most frequently found in the form of oxides, carbonates, and silicates.1 It is also one out of seven essential metals for animal physiology. Mn is a cofactor for many enzymes, such as transferases, hydrolases, lyases, arginase, glutamine synthetase, and superoxide dismutase, and it is also found in integrins.2,3 The well-studied Mn-containing proteins are arginase, an enzyme present in lipids that is required for ammonia elimination, and Mn-containing superoxide dismutase (Mn-SOD), a principal antioxidant enzyme typically found in the mitochondria. Given the dependence of multiple enzymes on Mn, it is essential for various physiological processes, such as modulation of the immune system, stellate process production in astrocytes, cell adhesion, and protein and carbohydrate metabolism.4-8 Mn also plays an important role in the development and functioning of the brain and skeletal structures.9,10 Mn deficiency may result in birth defects, poor bone formation and increased susceptibility to seizures.11,12 Despite being essential for metabolic functions, excessive exposure to Mn is a well-recognized occupational hazard, and inhalation of particulate Mn compounds is associated with lung inflammation, characterized by cough, bronchitis, pneumonitis, and impaired pulmonary function in human, primates,13-19 and nasal epithelium inflammation in rodents.20 Impotence and loss of libido have also been reported in male workers with high Mn exposures,21,22 possibly due to the importance of arginase in those functions.23 Though most Mn is obtained through the diet, Mn toxicity from dietary intake is rare,24,25 because Mn balance is tightly regulated by both the enterocytes (intake) and the biliary duct cells (excretion). In contrast, pulmonary uptake and particulate transport via the olfactory bulb26-28 can lead to deposition of Mn within the striatum and cerebellum, and inflammation of the nasal epithelium.20 Occupational exposure to Mn for periods from 6 months to 2 years can cause an extrapyramidal syndrome, referred to as manganism, closely resembling idiopathic Parkinson’s disease (IPD, see below), at both the molecular and clinical levels.29-31 Manganism represents a progressive Parkinsonism syndrome with a dystonic gait disorder (“cock gait”). Patients suffering from manganism exhibit a signature biphasic mode of physical decline, which comprises of an initial phase of psychiatric disturbance including rare cases of emotional lability, and neurological deficits which are followed by motor defects such as akinetic rigidity, dystonia, and bradyskinesia.29,31 Mn exposure represents a significant public health matter due to the use of Mn as a catalyzer in countless * To whom correspondence should be addressed. 2215-B Garland Avenue, 11425 MRB IV, Vanderbilt University Medical Center, Nashville, TN 37232-0414. Telephone: 615-322-8024. Fax: 615-936-4080. E-mail: michael.aschner@vanderbilt.edu. † Department of Pediatrics. ‡ Center for Molecular Neuroscience. § Department of Pharmacology. | Kennedy Center for Research on Human Development. Chem. Rev. 2009, 109, 4862–4884 4862

SMF-1, SMF-2 and SMF-3 DMT1 orthologues regulate and are regulated differentially by manganese levels in C. elegans.

Manganese (Mn) is an essential metal that can exert toxic effects at high concentrations, eventually leading to Parkinsonism. A major transporter of Mn in mammals is the divalent-metal transporter (DMT1). We characterize here DMT1-like proteins in the nematode C. elegans, which regulate and are regulated by Mn and iron (Fe) content. We identified three new DMT1-like genes in C. elegans: smf-1, smf-2 and smf-3. All three can functionally substitute for loss of their yeast orthologues in S. cerevisiae. In the worm, deletion of smf-1 or smf-3 led to an increased Mn tolerance, while loss of smf-2 led to increased Mn sensitivity. smf mRNA levels measured by QRT-PCR were up-regulated upon low Mn and down-regulated upon high Mn exposures. Translational GFP-fusions revealed that SMF-1 and SMF-3 strongly localize to partially overlapping apical regions of the gut epithelium, suggesting a differential role for SMF-1 and SMF-3 in Mn nutritional intake. Conversely, SMF-2 was detected in the marginal pharyngeal epithelium, possibly involved in metal-sensing. Analysis of metal content upon Mn exposure in smf mutants revealed that SMF-3 is required for normal Mn uptake, while smf-1 was dispensable. Higher smf-2 mRNA levels correlated with higher Fe content, supporting a role for SMF-2 in Fe uptake. In smf-1 and smf-3 but not in smf-2 mutants, increased Mn exposure led to decreased Fe levels, suggesting that both metals compete for transport by SMF-2. Finally, SMF-3 was post-translationally and reversibly down-regulated following Mn-exposure. In sum, we unraveled a complex interplay of transcriptional and post-translational regulations of 3 DMT1-like transporters in two adjacent tissues, which regulate metal-content in C. elegans.

In vivo collective cell migration requires an LPAR2-dependent increase in tissue fluidity

Neural crest epithelial–mesenchymal transition (EMT) and collective cell migration rely on a solid-to-liquid-like transition triggered by internalization of N-cadherin downstream of lysophosphatidic acid receptor 2.

Genes Required for Osmoregulation and Apical Secretion in Caenorhabditis elegans

Few studies have investigated whether or not there is an interdependence between osmoregulation and vesicular trafficking. We previously showed that in Caenorhabditis elegans che-14 mutations affect osmoregulation, cuticle secretion, and sensory organ development. We report the identification of seven lethal mutations displaying che-14-like phenotypes, which define four new genes, rdy-1–rdy-4 (rod-like larval lethality and dye-filling defective). rdy-1, rdy-2, and rdy-4 mutations affect excretory canal function and cuticle formation. Moreover, rdy-1 and rdy-2 mutations reduce the amount of matrix material normally secreted by sheath cells in the amphid channel. In contrast, rdy-3 mutants have short cystic excretory canals, suggesting that it acts in a different process. rdy-1 encodes the vacuolar H+-ATPase a-subunit VHA-5, whereas rdy-2 encodes a new tetraspan protein. We suggest that RDY-1/VHA-5 acts upstream of RDY-2 and CHE-14 in some tissues, since it is required for their delivery to the epidermal, but not the amphid sheath, apical plasma membrane. Hence, the RDY-1/VHA-5 trafficking function appears essential in some cells and its proton pump function essential in others. Finally, we show that RDY-1/VHA-5 distribution changes prior to molting in parallel with that of actin microfilaments and propose a model for molting whereby actin provides a spatial cue for secretion.

Anthranilate Fluorescence Marks a Calcium-Propagated Necrotic Wave That Promotes Organismal Death in C. elegans

Death of the nematode Caenorhabditis elegans involves a conserved necrotic cell death cascade which generates endogenous blue anthranilate fluorescence, allowing death to be visualized.