Lanzo, A. et al. (2015) International Worm Meeting "Identification and characterization of new genes involved in Dopaminergic neurons function by cell specific knock-down."

Martinelli, S., Lanzo, A., Bazzicalupo, P., Pannone, L., Tartaglia, M., Safratowich, B., Di Schiavi, E., Carvelli, L.

[International Worm Meeting, 2015]

Dopamine (DA) is a neurotransmitter that plays very important roles in humans and other animals, such as regulating movement, behavior and cognition. Excess or deficiency of DA have been associated with a number of neurological diseases, including Parkinson's disease and drug addiction. To date the molecular mechanisms involved in these disorders have not completely been discovered. We are interested in identifying new players which, when depleted, may cause an alteration in DA system function, a less explored field of investigation compared to the use of chemical treatments (e.g. 6-OHDA) and of engineered transgenes (e.g. a-syn overexpression). In C. elegans genetic mutations have been largely used to study neuronal function, but in some cases they may not be useful when gene loss causes pleiotropic or lethal phenotypes, thus hampering the possibility to study its role in a specific neuronal system. To overcome these limitations, we took advantage of a powerful variant of RNAi (Esposito et al., Gene, 2007), to dissect the role played specifically in DAergic system by candidate genes. We initially confirmed, by silencing control genes (gfp, dat-1, cat-2), that the silencing is restricted to DA-neurons and highly efficient. We then started by silencing the first candidate gene, unc-64, which encodes a SNARE protein involved in synaptic vesicle fusion, and whose full depletion causes uncoordinated locomotion. Using an in vivo behavioral assay, the SWimming-Induced Paralysis (SWIP), we showed that animals lacking unc-64 in the DAergic neurons presented a SWIP defect, showing its importance in DAergic circuit for a correct DA reuptake. Moreover we revealed that the SWIP effect produced after treatment with the psychostimulant Amphetamine (AMPH), strongly depends on the presence of unc-64 specifically in DAergic circuit. We then investigated the role played in DAergic neurons by unc-63 gene, the ortholog of the mammalian Alpha 6 CHolinergic Nicotinic Receptor (CHRNA6), using the resistance assay to lethal effect of the nicotinic agonist DMPP (Ruaud and Bessereau, Development, 2006). With the same approach we silenced unc-63 and we exposed interfered animals to DMPP, demonstrating that unc-63 presence in the DAergic system is important to mediate the toxic action produced by DMPP. We are now interested in characterizing the molecular pathways in which these genetic modifiers of DAergic function are involved, by genetic epistasis and pharmacological approaches.

Lanzo A et al. (2018) Front Physiol "Silencing of Syntaxin 1A in the Dopaminergic Neurons Decreases the Activity of the Dopamine Transporter and Prevents Amphetamine-Induced Behaviors in C. elegans."

Safratowich BD, Gallotta I, Lanzo A, Carvelli L, Kudumala SR, Zampi G, Di Schiavi E

[Front Physiol, 2018]

The dopamine transporter (DAT) is a cell membrane protein whose main function is to reuptake the dopamine (DA) released in the synaptic cleft back into the dopaminergic neurons. Previous studies suggested that the activity of DAT is regulated by allosteric proteins such as Syntaxin-1A and is altered by drugs of abuse such as amphetamine (Amph). Because <i>Caenorhabditis elegans</i> expresses both DAT (DAT-1) and Syntaxin-1A (UNC-64), we used this model system to investigate the functional and behavioral effects caused by lack of expression of <i>unc-64</i> in cultured dopaminergic neurons and in living animals. Using an inheritable RNA silencing technique, we were able to knockdown <i>unc-64</i> specifically in the dopaminergic neurons. This cell-specific knockdown approach avoids the pleiotropic phenotypes caused by knockout mutations of <i>unc-64</i> and ensures the transmission of dopaminergic specific <i>unc-64</i> silencing to the progeny. We found that, similarly to <i>dat-1</i> knockouts and <i>dat-1</i> silenced lines, animals with reduced <i>unc-64</i> expression in the dopaminergic neurons did not respond to Amph treatment when tested for locomotor behaviors. Our <i>in vitro</i> data demonstrated that in neuronal cultures derived from animals silenced for <i>unc-64</i>, the DA uptake was reduced by 30% when compared to controls, and this reduction was similar to that measured in neurons isolated from animals silenced for <i>dat-1</i> (40%). Moreover, reduced expression of <i>unc-64</i> in the dopaminergic neurons significantly reduced the DA release elicited by Amph. Because in <i>C. elegans</i> DAT-1 is the only protein capable to reuptake DA, these data show that reduced expression of <i>unc-64</i> in the dopaminergic neurons decreases the capability of DAT in re-accumulating synaptic DA. Moreover, these results demonstrate that decreased expression of <i>unc-64</i> in the dopaminergic neurons abrogates the locomotor behavior induced by Amph. Taken together these data suggest that Syntaxin-1A plays an important role in both functional and behavioral effects caused by Amph.

Illiano P et al. (2016) Eur J Neurosci "A Caenorhabditis elegans model to study Dopamine Transporter Deficiency Syndrome."

Gainetdinov RR, Lanzo A, Illiano P, Schiavi ED, Leo D, Paglione M, Zampi G

[Eur J Neurosci, 2016]

Dopamine Transporter Deficiency Syndrome (DTDS) is a novel autosomal recessive disorder caused by mutations in the Dopamine Transporter (DAT), which leads to the partial or total loss-of-function of the protein. DTDS is a pharmacoresistant syndrome and very little is known about its neurobiology, in part due to the lack of relevant animal models. The objective of this study was to establish the first animal model for DTDS with strong construct validity, using Caenorhabditis elegans, and to investigate the in vivo role played by DTDS-related mutations found in human DAT (hDAT). We took advantage of a C. elegans knockout for the hDAT orthologue, cedat-1, to obtain genetically humanized animals bearing hDAT, in the wild type and in two mutated forms (399delG and 941C&gt;T), in a null background. In C. elegans transgenic animals expressing the human wild type form we observed a rescue of the knockout phenotype, as assessed using two well-established paradigms, known to be regulated by the endogenous uptake of Dopamine or 6-Hydroxydopamine (6-OHDA) by DAT. The less severe mutation (941C&gt;T) was able to partially rescue only one of the knockout phenotypes, while the 399delG mutation impaired DAT function in both phenotypic paradigms. Our in vivo phenotypic findings demonstrate a functional conservation between human and nematode DAT and validate previous in vitro indications of the loss-of-function of hDAT in carriers of DTDS-related mutations. Taken together, these observations establish C. elegans as a novel animal model for fast and unexpensive screening of hDAT mutations in functional and in vivo tests. This article is protected by copyright. All rights reserved.

Martinelli S et al. (2020) Parkinsonism Relat Disord "Co-occurring WARS2 and CHRNA6 mutations in a child with a severe form of infantile parkinsonism."

Galosi S, Di Schiavi E, Ng J, Palma E, Fucile S, Bocchinfuso G, Tartaglia M, Leuzzi V, Ciolfi A, Cogo S, Greggio E, Stella L, Pannone L, Farrotti A, Di Nottia M, Paglione M, Lanzo A, Traversa A, Carrozzo R, Venditti M, Martinelli S, Limatola C, Roseti C, Kurian MA, Carducci C, Sciaccaluga M, Bentivoglio A, Rizza T, Trettel F, Civiero L, Cordeddu V, Bernardini L, Caputo V

[Parkinsonism Relat Disord, 2020]

OBJECTIVE: To investigate the molecular cause(s) underlying a severe form of infantile-onset parkinsonism and characterize functionally the identified variants. METHODS: A trio-based whole exome sequencing (WES) approach was used to identify the candidate variants underlying the disorder. In silico modeling, and in vitro and in vivo studies were performed to explore the impact of these variants on protein function and relevant cellular processes. RESULTS: WES analysis identified biallelic variants in WARS2, encoding the mitochondrial tryptophanyl tRNA synthetase (mtTrpRS), a gene whose mutations have recently been associated with multiple neurological phenotypes, including childhood-onset, levodopa-responsive or unresponsive parkinsonism in a few patients. A substantial reduction of mtTrpRS levels in mitochondria and reduced OXPHOS function was demonstrated, supporting their pathogenicity. Based on the infantile-onset and severity of the phenotype, additional variants were considered as possible genetic modifiers. Functional assessment of a selected panel of candidates pointed to a de novo missense mutation in CHRNA6, encoding the 6 subunit of neuronal nicotinic receptors, which are involved in the cholinergic modulation of dopamine release in the striatum, as a second event likely contributing to the phenotype. In silico, in vitro (Xenopus oocytes and GH4C1 cells) and in vivo (C. elegans) analyses demonstrated the disruptive effects of the mutation on acetylcholine receptor structure and function. CONCLUSION: Our findings consolidate the association between biallelic WARS2 mutations and movement disorders, and suggest CHRNA6 as a genetic modifier of the phenotype.

Quartey MO et al. (2021) Sci Rep "The A(1-38) peptide is a negative regulator of the A(1-42) peptide implicated in Alzheimer disease progression."

Pennington PR, Heistad RM, Nyarko JNK, Barnes JR, Bolanos MAC, Parsons MP, Knudsen KJ, De Carvalho CE, Leary SC, Mousseau DD, Buttigieg J, Maley JM, Quartey MO

[Sci Rep, 2021]

The pool of -Amyloid (A) length variants detected in preclinical and clinical Alzheimer disease (AD) samples suggests a diversity of roles for A peptides. We examined how a naturally occurring variant, e.g. A(1-38), interacts with the AD-related variant, A(1-42), and the predominant physiological variant, A(1-40). Atomic force microscopy, Thioflavin T fluorescence, circular dichroism, dynamic light scattering, and surface plasmon resonance reveal that A(1-38) interacts differently with A(1-40) and A(1-42) and, in general, A(1-38) interferes with the conversion of A(1-42) to a -sheet-rich aggregate. Functionally, A(1-38) reverses the negative impact of A(1-42) on long-term potentiation in acute hippocampal slices and on membrane conductance in primary neurons, and mitigates an A(1-42) phenotype in Caenorhabditis elegans. A(1-38) also reverses any loss of MTT conversion induced by A(1-40) and A(1-42) in HT-22 hippocampal neurons and APOE 4-positive human fibroblasts, although the combination of A(1-38) and A(1-42) inhibits MTT conversion in APOE 4-negative fibroblasts. A greater ratio of soluble A(1-42)/A(1-38) [and A(1-42)/A(1-40)] in autopsied brain extracts correlates with an earlier age-at-death in males (but not females) with a diagnosis of AD. These results suggest that A(1-38) is capable of physically counteracting, potentially in a sex-dependent manner, the neuropathological effects of the AD-relevant A(1-42).

Danielle Thierry-Mieg et al. (2003) Worm Breeder's Gazette "www.wormgenes.org , a new gene centered database"

Vahan Simonyan, Michel Potdevin, Mark Sienkiewicz, Yuji KOHARA, Jean Thierry-Mieg, Danielle Thierry-Mieg, Tadasu SHIN-I

[Worm Breeder's Gazette, 2003]

Wormgenes is a new resource for C.elegans offering a detailed summary about each gene and a powerful query system.

Tangrodchanapong T et al. (2020) Front Pharmacol "Frondoside A Attenuates Amyloid- Proteotoxicity in Transgenic Caenorhabditis elegans by Suppressing Its Formation."

Tangrodchanapong T, Sobhon P, Meemon K

[Front Pharmacol, 2020]

Oligomeric assembly of Amyloid- (A) is the main toxic species that contribute to early cognitive impairment in Alzheimer's patients. Therefore, drugs that reduce the formation of A oligomers could halt the disease progression. In this study, by using transgenic <i>Caenorhabditis elegans</i> model of Alzheimer's disease, we investigated the effects of frondoside A, a well-known sea cucumber <i>Cucumaria frondosa</i> saponin with anti-cancer activity, on A aggregation and proteotoxicity. The results showed that frondoside A at a low concentration of 1 M significantly delayed the worm paralysis caused by A aggregation as compared with control group. In addition, the number of A plaque deposits in transgenic worm tissues was significantly decreased. Frondoside A was more effective in these activities than ginsenoside-Rg3, a comparable ginseng saponin. Immunoblot analysis revealed that the level of small oligomers as well as various high molecular weights of A species in the transgenic <i>C. elegans</i> were significantly reduced upon treatment with frondoside A, whereas the level of A monomers was not altered. This suggested that frondoside A may primarily reduce the level of small oligomeric forms, the most toxic species of A. Frondoside A also protected the worms from oxidative stress and rescued chemotaxis dysfunction in a transgenic strain whose neurons express A. Taken together, these data suggested that low dose of frondoside A could protect against A-induced toxicity by primarily suppressing the formation of A oligomers. Thus, the molecular mechanism of how frondoside A exerts its anti-A aggregation should be studied and elucidated in the future.

Hodgkin JA (1998) International Journal of Developmental Biology "Seven types of pleiotropy."

Hodgkin JA

[International Journal of Developmental Biology, 1998]

Pleiotropy , a situation in which a single gene influences multiple phenotypic tra its, can arise in a variety of ways. This paper discusses possible underlying mechanisms and proposes a classification of the various phenomena involved.

Tuck S (2011) Curr Biol "Extracellular vesicles: budding regulated by a phosphatidylethanolamine translocase."

Tuck S

[Curr Biol, 2011]

Recent work on a Caenorhabditis elegans transmembrane ATPase reveals a central role for the aminophospholipid phosphatidylethanolamine in the production of a class of extracellular vesicles.

Viney ME et al. (2004) Naturwissenschaften "Is dauer pheromone of Caenorhabditis elegans really a pheromone?"

Viney ME, Franks NR

[Naturwissenschaften, 2004]

Animals respond to signals and cues in their environment. The difference between a signal (e.g. a pheromone) and a cue (e.g. a waste product) is that the information content of a signal is subject to natural selection, whereas that of a cue is not. The model free-living nematode Caenorhabditis elegans forms an alternative developmental morph (the dauer larva) in response to a so-called 'dauer pheromone', produced by all worms. We suggest that the production of 'dauer pheromone' has no fitness advantage for an individual worm and therefore we propose that 'dauer pheromone' is not a signal, but a cue. Thus, it should not be called a pheromone.