Ashby MN et al. (1992) J Biol Chem "COQ2 is a candidate for the structural gene encoding para-hydroxybenzoate:polyprenyltransferase."

Ackerman S, Ashby MN, Tzagoloff A, Edwards PA, Kutsunai SY

[J Biol Chem, 1992]

Coenzyme Q functions as a lipid-soluble electron carrier in eukaryotes. In Saccharomyces cerevisiae, the enzymes responsible for the assembly of the polyisoprenoid side chain and subsequent transfer to para-hydroxybenzoate (PHB) are encoded by the nuclear genes COQ1 and COQ2, respectively. Yeast mutants defective in coenzyme Q biosynthesis are respiratory defective and provide a useful tool to study this non-sterol branch of the isoprenoid biosynthetic pathway. We isolated a 5.5-kilobase genomic DNA fragment that was able to functionally complement a coq2 strain. Additional complementation analyses located the COQ2 gene within a 2.1-kilobase HindIII-BglII restriction fragment. Sequence analyses revealed the presence of a 1,116-base pair open reading frame coding for a predicted protein of 372 amino acids and a molecular mass of 41,001 daltons. The amino acid sequence exhibits a typical amino-terminal mitochondrial leader sequence and six potential membrane-spanning domains. Primer extension and Northern analyses indicate the gene is transcriptionally active. Transformation of a coq2 strain with the 2.1-kilobase HindIII-BglII genomic restriction fragment on a multicopy plasmid restores PHB:polyprenyltransferase activity to wild-type levels. Disruption of the chromosomal COQ2 gene indicates the gene is not essential for viability, yet is required for PHB:polyprenyltransferase activity and respiratory function. In addition, the deduced amino acid sequence of PHB:polyprenyltransferase contains a putative allylic polyprenyl diphosphate-binding site. The presence of this aspartate-rich domain in a number of functionally distinct proteins which utilize polyprenyl diphosphate substrates is reported.

Ashby MN et al. (1990) J Biol Chem "Elucidation of the deficiency in two yeast coenzyme Q mutants. Characterization of the structural gene encoding hexaprenyl pyrophosphate synthetase."

Ashby MN, Edwards PA

[J Biol Chem, 1990]

The assembly of a polyisoprenoid side chain and its transfer to para-hydroxybenzoate are the first two steps of coenzyme Q biosynthesis. In yeast these reactions are catalyzed by hexaprenyl pyrophosphate synthetase and PHB:polyprenyltransferase, respectively. We have screened nine complementation groups of yeast coenzyme Q mutants for the activities of these two enzymes and found two strains deficient in either activity. The strain deficient in hexaprenyl pyrophosphate synthetase activity, C296-LH3, is complemented by the plasmid pG3/T1. When C296-LH3 was transformed with a shuttle vector containing a 2,187-base pair fragment from the genomic insert of pG3/T1, both glycerol growth and hexaprenyl pyrophosphate synthetase activity were restored. The activity of the latter enzyme was higher than that seen in wild-type yeast. The increase in activity could be attributed to a gene dosage effect of the multi-copy plasmid. A 1,419-base pair open reading frame encoding a 52,560-dalton protein was found on the genomic fragment. The size of the RNA transcript and the location of transcriptional initiation indicate that the entire open reading frame is contained within the mRNA. Comparison of the hexaprenyl pyrophosphate synthetase amino acid sequence with amino acid sequences from the related enzyme farnesyl pyrophosphate synthetase show the presence of three highly conserved domains. Within two of the domains is an aspartate-rich motif found invariantly in the amino acid sequences of farnesyl pyrophosphate synthetase from three species and the hexaprenyl pyrophosphate synthetase amino acid sequence reported here. These aspartic acid motifs may comprise binding sites for the allylic and homoallylic substrates. The hydrophobicity profiles of the hexaprenyl pyrophosphate synthetase sequence and the farnesyl pyrophosphate synthetase sequence from rat appear similar. Furthermore, the hydrophobicity correlation coefficient of the comparison of these two sequences indicate with a high degree of confidence (p less than 0.001) that the two proteins will fold into similar three-dimensional structures.

Peres TV et al. (2018) Biol Trace Elem Res "Small Molecule Modifiers of In Vitro Manganese Transport Alter Toxicity In Vivo."

Bornhorst J, Horning KJ, Bowman AB, Schwerdtle T, Peres TV, Aschner M

[Biol Trace Elem Res, 2018]

Manganese (Mn) is essential for several species and daily requirements are commonly met by an adequate diet. Mn overload may cause motor and psychiatric disturbances and may arise from an impaired or not fully developed excretion system, transporter malfunction and/or exposure to excessive levels of Mn. Therefore, deciphering processes regulating neuronal Mn homeostasis is essential to understand the mechanisms of Mn neurotoxicity. In the present study, we selected two small molecules (with opposing effects on Mn transport) from a previous high throughput screen of 40,167 to test their effects on Mn toxicity parameters in vivo using Caenorhabditis elegans. We pre-exposed worms to VU0063088 and VU0026921 for 30min followed by co-exposure for 1h with Mn and evaluated Mn accumulation, dopaminergic (DAergic) degeneration and worm survival. Control worms were exposed to vehicle (DMSO) and saline only. In pdat-1::GFP worms, with GFP labeled DAergic neurons, we observed a decrease of Mn-induced DAergic degeneration in the presence of both small molecules. This effect was also observed in an smf-2 knockout strain. SMF-2 is a regulator of Mn transport in the worms and this strain accumulates higher Mn levels. We did not observe improved survival in the presence of small molecules. Our results suggest that both VU0063088 and VU0026921 may modulate Mn levels in the worms through a mechanism that does not require SMF-2 and induce protection against Mn neurotoxicity.

Chen P et al. (2015) Toxicol Res (Camb) "Manganese-induced Neurotoxicity: From C. elegans to Humans."

Peres TV, Chakraborty S, Bowman AB, Aschner M, Chen P

[Toxicol Res (Camb), 2015]

Manganese (Mn) is one of the most abundant metals on the earth. It is required for normal cellular activities, but overexposure leads to toxicity. Neurons are more susceptible to Mn-induced toxicity than other cells, and accumulation of Mn in the brain results in Manganism that presents with Parkinson's disease (PD)-like symptoms. In the last decade, a number of Mn transporters have been identified, which improves our understanding of Mn transport in and out of cells. However, the mechanism of Mn-induced neurotoxicity is only partially uncovered, with further research needed to explore the whole picture of Mn-induced toxicity. In this review, we will address recent progress in Mn-induced neurotoxicity from C. elegans to humans, and explore future directions that will help understand the mechanisms of its neurotoxicity.

Taylor CA et al. (2020) J Nutr "Maintaining Translational Relevance in Animal Models of Manganese Neurotoxicity."

Mukhopadhyay S, Tuschl K, Bornhorst J, Nicolai MM, Aschner M, Taylor CA, Gubert P, Varao AM, Smith DR

[J Nutr, 2020]

Manganese is an essential metal, but elevated brain Mn concentrations produce a parkinsonian-like movement disorder in adults and fine motor, attentional, cognitive, and intellectual deficits in children. Human Mn neurotoxicity occurs owing to elevated exposure from occupational or environmental sources, defective excretion (e.g., due to cirrhosis), or loss-of-function mutations in the Mn transporters solute carrier family 30 member 10 or solute carrier family 39 member 14. Animal models are essential to study Mn neurotoxicity, but in order to be translationally relevant, such models should utilize environmentally relevant Mn exposure regimens that reproduce changes in brain Mn concentrations and neurological function evident in human patients. Here, we provide guidelines for Mn exposure in mice, rats, nematodes, and zebrafish so that brain Mn concentrations and neurobehavioral sequelae remain directly relatable to the human phenotype.

Gubert P et al. (2018) Neurotoxicology "Metabolic effects of manganese in the nematode Caenorhabditis elegans through DAergic pathway and transcription factors activation."

Trindade LS, Aschner M, Lehmen T, Cheng P, Soares FAA, Fessel JP, Bornhorst J, Avila DS, Gubert P, Puntel B

[Neurotoxicology, 2018]

Manganese (Mn) is an essential trace element for physiological functions since it acts as an enzymatic co-factor. Nevertheless, overexposure to Mn has been associated with a pathologic condition called manganism. Furthermore, Mn has been reported to affect lipid metabolism by mechanisms which have yet to be established. Herein, we used the nematode Caenorhabditis elegans to examine Mn's effects on the dopaminergic (DAergic) system and determine which transcription factors that regulate with lipid metabolism are affected by it. Worms were exposed to Mn for four hours in the presence of bacteria and in a liquid medium (85mM NaCl). Mn increased fat storage as evidenced both by Oil Red O accumulation and triglyceride levels. In addition, metabolic activity was reduced as a reflection of decreased oxygen consumption caused by Mn. Mn also affected feeding behavior as evidenced by decreased pharyngeal pumping rate. DAergic neurons viability were not altered by Mn, however the dopamine levels were significantly reduced following Mn exposure. Furthermore, the expression of sbp-1 transcription factor and let-363 protein kinase responsible for lipid accumulation control was increased and decreased, respectively, by Mn. Altogether, our data suggest that Mn increases the fat storage in C. elegans, secondary to DAergic system alterations, under the control of SBP-1 and LET-363 proteins.

Leyva-Illades D et al. (2014) J Neurosci "SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism-causing mutations block its intracellular trafficking and efflux activity."

Mukhopadhyay S, Hutchens S, Morrisett RA, Mercado JM, Zogzas CE, Swaim CD, Bowman AB, Aschner M, Leyva-Illades D, Chen P

[J Neurosci, 2014]

Manganese (Mn) is an essential metal, but elevated cellular levels are toxic and may lead to the development of an irreversible parkinsonian-like syndrome that has no treatment. Mn-induced parkinsonism generally occurs as a result of exposure to elevated Mn levels in occupational or environmental settings. Additionally, patients with compromised liver function attributable to diseases, such as cirrhosis, fail to excrete Mn and may develop Mn-induced parkinsonism in the absence of exposure to elevated Mn. Recently, a new form of familial parkinsonism was reported to occur as a result of mutations in SLC30A10. The cellular function of SLC30A10 and the mechanisms by which mutations in this protein cause parkinsonism are unclear. Here, using a combination of mechanistic and functional studies in cell culture, Caenorhabditis elegans, and primary midbrain neurons, we show that SLC30A10 is a cell surface-localized Mn efflux transporter that reduces cellular Mn levels and protects against Mn-induced toxicity. Importantly, mutations in SLC30A10 that cause familial parkinsonism blocked the ability of the transporter to traffic to the cell surface and to mediate Mn efflux. Although expression of disease-causing SLC30A10 mutations were not deleterious by themselves, neurons and worms expressing these mutants exhibited enhanced sensitivity to Mn toxicity. Our results provide novel insights into the mechanisms involved in the onset of a familial form of parkinsonism and highlight the possibility of using enhanced Mn efflux as a therapeutic strategy for the potential management of Mn-induced parkinsonism, including that occurring as a result of mutations in SLC30A10.

Nicolai MM et al. (2021) Int J Mol Sci "Effects of Manganese on Genomic Integrity in the Multicellular Model Organism Caenorhabditis elegans."

Brinkmann V, Gremme A, Winkelbeiner N, Baesler J, Schwerdtle T, Aschner M, Weishaupt AK, Fritz G, Bornhorst J, Nicolai MM, Wellenberg A

[Int J Mol Sci, 2021]

Although manganese (Mn) is an essential trace element, overexposure is associated with Mn-induced toxicity and neurological dysfunction. Even though Mn-induced oxidative stress is discussed extensively, neither the underlying mechanisms of the potential consequences of Mn-induced oxidative stress on DNA damage and DNA repair, nor the possibly resulting toxicity are characterized yet. In this study, we use the model organism <i>Caenorhabditis elegans</i> to investigate the mode of action of Mn toxicity, focusing on genomic integrity by means of DNA damage and DNA damage response. Experiments were conducted to analyze Mn bioavailability, lethality, and induction of DNA damage. Different deletion mutant strains were then used to investigate the role of base excision repair (BER) and dePARylation (DNA damage response) proteins in Mn-induced toxicity. The results indicate a dose- and time-dependent uptake of Mn, resulting in increased lethality. Excessive exposure to Mn decreases genomic integrity and activates BER. Altogether, this study characterizes the consequences of Mn exposure on genomic integrity and therefore broadens the molecular understanding of pathways underlying Mn-induced toxicity. Additionally, studying the basal poly(ADP-ribosylation) (PARylation) of worms lacking poly(ADP-ribose) glycohydrolase (PARG) <i>parg-1</i> or <i>parg-2</i> (two orthologue of PARG), indicates that <i>parg-1</i> accounts for most of the glycohydrolase activity in worms.

Raj V et al. (2021) Biochem Biophys Res Commun "Manganese exposure during early larval stages of C.elegans causes learning disability in the adult stage."

Nair A, Thekkuveettil A, Raj V

[Biochem Biophys Res Commun, 2021]

Manganese (Mn), even though an essential trace element, causes neurotoxicity in excess. In adults, over-exposure to Mn causes clinical manifestations, including dystonia, progressive bradykinesia, disturbance of gait, slurring, and stuttering of speech. These symptoms are mainly because of Mn-associated oxidative stress and degeneration of dopamine neurons in the central nervous system. Children with excessive Mn exposure often show learning disabilities but rarely show symptoms associated with dopaminergic neuron dysfunction. It is unclear why Mn exposure shows distinctive clinical outcomes in developing brains versus adult brains. Studies on nematode C.elegans have demonstrated that it is an excellent model to elucidate Mn-associated toxicity in the nervous system. In this study, we chronically exposed Mn to L1 larval stage of the worms to understand the effects on dopamine neurons and cognitive development. The worms showed modified behavior to exogenous dopamine compared to the control. The dopamine neurons showed resistance to neurodegeneration on repeated Mn exposure during the adult stage. As observed in mammalian systems, these worms showed significantly low olfactory adaptive learning and memory. This study shows that C.elegans alters adaptive developmental plasticity during Mn overexposure, modifying its sensitivity towards the metal ion and leads to remodeling in its innate learning behavior.

Avila DS et al. (2012) Toxicol Lett "Anti-aging effects of deuterium depletion on Mn-induced toxicity in a C. elegans model."

Avila DS, Somlyai I, Aschner M, Somlyai G

[Toxicol Lett, 2012]

Work with sub-natural levels of deuterium (D) in animals has demonstrated an anti-cancer effect of low D-concentration in water. Our objective was to investigate whether deuterium-depleted water (DDW) can overturn reverse manganese (Mn)-induced reduction in life span, using the Caenorhabditis elegans (C. elegans) as a model system. DDW per se had no effect on worm's life span 48 h after treatment; however, it reversed the Mn-induced decrease in C. elegans life span. Mn reduced DAF-16 levels, a transcription factor strongly associated with life-span regulation. Low D-concentration (90 ppm) restored the Mn-induced changes in DAF-16 to levels indistinguishable from controls, suggesting DDW can regulate the DAF-16 pathway. We further show that insulin-like receptor DAF-2 levels were unaltered by Mn exposure, tAKT levels increased, whilst superoxide dismutase (SOD-3) levels were decreased by Mn. DDW (90 ppm) restored the levels of tAKT and superoxide dismutase (SOD) to control values without changing DAF-2 levels. Treatment of Mn exposed worms with DDW (90 ppm) restored life-span, DAF-16 and SOD-3 levels to control levels, strongly suggesting that low D concentrations can protect against Mn toxic effects.