Pereira, Flavia, Quines, Caroline, Neto, Manoel, Avila, Daiana, Rodrigues, Alisson, Haas, Sandra
[
International Worm Meeting,
2021]
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by the progressive loss of motor neurons. There are several mutations that can lead to the development of this disease, including the SOD-1 gene. Riluzole is the only approved drug to slow the progression of the disease and to promote longer survival. However, its effects are limited. Therefore, it is necessary to develop new therapies for ALS treatment. Previous studies have suggested trehalose and vitamin E, individually, as good candidates for ALS treatment. In order to associate these molecules, we developed nanocapsules and nanoemulsions of trehalose and vitamin E and exposed to Caenorhabditis elegans (C. elegans) ALS model and evaluated the pharmacological potential of these nanoparticles. Pregnant AM263 [
unc-54p::
Hsa-sod-1 (WT)::YFP] and AM265 [Punc-54::hSOD1(G93A)::YFP] worms were synchronized. The eggs obtained from these nematodes were transferred to Petri dishes previously dried containing NGM medium and UV-inactivated E.coli OP50 with or without treatment. Trehalose/vitamin E nanomaterials were provided by a partner laboratory. The treatment was performed with nanoparticles pipetted on NGM surface. When the worms reached L4 stage, they were transferred to new plates containing or not nanoparticles. This process was performed from day 1 adult until 4th day adult. Nematode paralysis was assessed along longevity; nematodes were considered paralyzed if they did not move when prodded with a wire pick. SOD-::GFP aggregation was observed at 5th day adult through fluorescence microscope. Our data showed that the nanoparticles decreased the number of paralyzed worms and increased their longevity. Besides that, the treatment with Vit. E+ trehalose nanoparticles provided a slight modification in the SOD1 aggregation profile. ALS promote motor neuron disabling, leading to muscular paralysis and death. This way, these results suggest a great advantage of nanoparticles in mitigating the main effects caused by ALS. There are studies reporting the protective effect of sugars, such as sucrose, however, these carbohydrates have many disadvantages. Trehalose is more cost-effective than other sugars, in addition to having neuroprotective and autophagosome modulating activity. In turn, vitamin E is an antioxidant widely used in several pharmacological area including in neuronal studies. In conclusion, the association of these two compounds proved to be efficient in the ALS treatment.
[
International Worm Meeting,
2021]
Nano-sized drug delivery systems have been the subject of intense research in recent years because polymeric materials allow the absorption and release of active substances in a controlled manner. The use of polymeric nanoparticles as a drug carrier has emerged as an alternative. Despite the benefits, the safety of nanoparticulate systems is an aspect to be understood, particularly in in vivo systems. Caenorhabditis elegans is a very useful alternative model for nanotoxicology and has been recently applied in this field. The aim of this study was to evaluate toxicological endpoints in worms exposed to nanomaterials prepared with different polymers: polyethylene glycol (PEG), chitosan (CH), eudragit (EU) and polysorbate 80 (P80) in C. elegans. First larval staged worms were obtained by a synchronization process and treated with nanomaterials at concentrations of 0.015, 0.225 and 0.45 mg/mL for 30 minutes in liquid medium (acute exposure). Soon after, they were washed to remove the treatments and transferred to Petri dishes containing NGM and E. coli OP50 for 48 h. Survival rate, brood size and worms length were determined. Data were expressed as mean ± standard error, and statistical analysis were done by one-way ANOVA followed by Tukey post-hoc test. We observed that the EU nanoparticles did not cause any significant change in toxicological endpoints when compared to control. On the other hand, exposure to CH, PEG and P80 nanoparticles decreased worms survival, reduced their progeny and significantly altered worms size. This work demonstrates the toxicological differences between polymers and a potential for EU to be used in new formulations for future drug vectoring and targeting systems.
[
Food Funct,
2023]
&#
x3b2;-Carbolines norharman and harman, belonging to the class of heterocyclic aromatic amines (HAAs), are typical hazardous substances produced during the thermal processing of food. Compared to other HAAs, there have been limited reports on the toxicity of &#
x3b2;-carbolines. Nevertheless, the current studies are concerned with the neurotoxic effects of norharman and harman at high doses. It is still unknown whether the relatively low dose of &#
x3b2;-carbolines in foods induces neurotoxicity and the mechanism of the toxicity. In this study, <i>C. elegans</i> was exposed to a series of gradients of norharman and harman (0, 0.05, 5, and 10 mg L<sup>-1</sup>). The survival rate and indicators of ethology (locomotor behaviors, foraging behavior, and chemotaxis ability) were assessed. The antioxidant system and the contents of neurotransmitters, as well as the activity of acetylcholinesterase (AChE), were evaluated. Additionally, the RNA-seq screening of differentially expressed genes (DEGs) revealed the potential molecular mechanisms of norharman- and harman-induced toxic effects. Our results indicated that the risk of long-term exposure to norharman and harman at low doses (food-related doses) should be emphasized. Moreover, &#
x3b2;-carbolines might induce neurotoxicity by causing oxidative damage, regulating the content of neurotransmitters, and interfering with cytochrome P450 metabolism. This study would provide a toxicological basis for the neurotoxicity of &#
x3b2;-carbolines and lay the foundation for the risk assessment of endogenous pollutants in food.
[
International Worm Meeting,
2017]
Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.
[
Mol Biol Cell,
2007]
Monitoring Editor: Sandra Schmid Intersectin is a multifunctional protein that interacts with components of the endocytic and exocytic pathways and is also involved in the control of actin dynamics. Drosophila intersectin is required for viability, synaptic development and synaptic vesicle recycling. Here, we report the characterization of intersectin function in C. elegans. Nematode intersectin (ITSN-1) is expressed in the nervous system, and it is enriched in presynaptic regions. The C. elegans intersectin gene (
itsn-1) is nonessential for viability. In addition,
itsn-1-null worms do not display any evident phenotype, under physiological conditions. However, they display aldicarb-hypersensitivity, compatible with a negative regulatory role of ITSN-1 on neurotransmission. ITSN-1 physically interacts with dynamin and EHS-1, two proteins involved in synaptic vesicle recycling. We have previously shown that EHS-1 is a positive modulator of synaptic vesicle recycling in the nematode, likely through modulation of dynamin or dynamin-controlled pathways. Here, we show that ITSN-1 and EHS-1 have opposite effects on aldicarb sensitivity, and on dynamin-dependent phenotypes. Thus, the sum of our results identifies dynamin, or a dynamin-controlled pathway, as a potential target for the negative regulatory role of ITSN-1.
[
Mol Biol Cell,
2006]
Monitoring Editor: Sandra Schmid Sodium-dependent neurotransmitter transporters participate in the clearance and/or recycling of neurotransmitters from synaptic clefts. The
snf-11 gene in C. elegans encodes a protein of high similarity to mammalian GABA transporters (GATs). We show here that
snf-11 encodes a functional GABA transporter; SNF-11-mediated GABA transport is Na(+)- and Cl(-)-dependent, has an EC50 of 168 microM, and is blocked by the GAT1 inhibitor SKF89976A. The SNF-11 protein is expressed in seven GABAergic neurons, several additional neurons in the head and retrovesicular ganglion, and three groups of muscle cells. Therefore, all GABAergic synapses are associated with either presynaptic or post-synaptic (or both) expression of SNF-11. Although a
snf-11 null mutation has no obvious effects on GABAergic behaviors, it leads to resistance to inhibitors of acetylcholinesterase. In vivo, a
snf-11 null mutation blocks GABA uptake in at least a subset of GABAergic cells; in a cell culture system, all GABA uptake is abolished by the
snf-11 mutation. We conclude that GABA transport activity is not essential for normal GABAergic function in C. elegans; and the localization of SNF-11 is consistent with a GABA clearance function rather than recycling.
[
Mol Biol Cell,
2007]
Monitoring Editor: Sandra Lemmon Caenorhabditis elegans gut granules are intestine specific lysosome-related organelles with birefringent and autofluorescent contents. We identified
pgp-2, which encodes an ABC transporter, in screens for genes required for the proper formation of gut granules.
pgp-2(-) embryos mislocalize birefringent material into the intestinal lumen and are lacking in acidified intestinal V-ATPase containing compartments. Adults without
pgp-2(+) function similarly lack organelles with gut granule characteristics. These cellular phenotypes indicate that
pgp-2(-) animals are defective in gut granule biogenesis. Double mutant analysis suggests that
pgp-2(+) functions in parallel with the AP-3 adaptor complex during gut granule formation. We find that
pgp-2 is expressed in the intestine where it functions in gut granule biogenesis and that PGP-2 localizes to the gut granule membrane. These results support a direct role of an ABC transporter in regulating lysosome biogenesis. Previously,
pgp-2(+) activity has been shown to be necessary for the accumulation of Nile Red stained fat in C. elegans. We show that gut granules are sites of fat storage in C. elegans embryos and adults. Notably, levels of triacylglycerides are relatively normal in animals defective in the formation of gut granules. Our results provide an explanation for the loss of Nile Red stained fat in
pgp-2(-) animals as well as insight into the specialized function of this lysosome-related organelle.
[
International C. elegans Meeting,
1999]
In order to understand the processes governing the establishment of asymmetry within the early C. elegans embryo, our lab has begun an investigation of several conditional mutants isolated in a screen for temperature sensetive, embryonic lethal mutations (see abstract by D. Hamill). Two of these mutations,
or191ts and
or182ts disrupt different aspects of asymmetry normally observed at the two-cell stage. The
or191ts mutation results in random spindle orientation within the two-cell stage blastomeres AB and P 1 , which normally divide transversely and longitudinally, respectively. In addition to dividing along different axes, the cell cycle times of AB and P 1 are unequal in wild-type embryos, with AB dividing slightly ahead of P 1 . A second mutation we have identified increases this asymmetry. The mutation
or182ts , delays the cell cycle timing of both AB and P 1 . The AB cell cycle time, however, is increased only slightly, while the time of the P 1 cell cycle is doubled.
or191ts maps to the center of chromosome V while
or182ts maps to the far left arm of chomosome III. Phenotypic analysis of these mutants and progress on identifying the affected genes will be presented. For information concerning the analysis of other mutants affecting similar processes see posters presented by Danielle Hamill and Sandra Encalada in our laboratory.
[
Mol Biol Cell,
2008]
Monitoring Editor: Sandra Lemmon Following endocytosis, membrane proteins are often sorted between two alternative pathways: a recycling pathway, and a degradation pathway. Relatively little is known about how trafficking through these alternative pathways are differentially regulated. Here we identify UNC-108/Rab2 as a regulator of post-endocytic trafficking in both neurons and coelomocytes. Mutations in the C. elegans Rab2 gene
unc-108, caused the GFP-tagged glutamate receptor, GLR-1 (GLR-1::GFP), to accumulate in the ventral cord and in neuronal cell bodies. In neuronal cell bodies of
unc-108/Rab2 mutants, GLR-1::GFP was found in tubulovesicular structures that colocalized with markers for early and recycling endosomes, including Syntaxin-13 and Rab8. GFP-tagged Syntaxin-13 also accumulated in the ventral cord of
unc-108/Rab2 mutants. UNC-108/Rab2 was not required for ubiquitin-mediated sorting of GLR-1::GFP into the multivesicular body (MVB) degradation pathway. Mutations disrupting the MVB pathway and
unc-108/Rab2 mutations had additive effects on GLR-1::GFP levels in the ventral cord. In coelomocytes, post-endocytic trafficking of the marker Texas Red-BSA was delayed. These results demonstrate that UNC-108/Rab2 regulates post-endocytic trafficking, most likely at the level of early or recycling endosomes, and that UNC-108/Rab2 and the MVB pathway define alternative post-endocytic trafficking mechanisms that operate in parallel. These results define a new function for Rab2 in protein trafficking.