Mello, D.F. et al. (2019) International Worm Meeting "Linking mitochondrial dysfunction during development with pathogen resistance later in life."

Baugh, R.L., Mello, D.F., Meyer, J.N., Bergemann, C.M.

[International Worm Meeting, 2019]

Growing evidence has shown that mitochondrial dysfunction not only compromises the energetic metabolism of cells, but also plays key roles in other physiological processes such as immunomodulation. We hypothesize that mitochondrial toxicity can be a common link between increased prevalence in immune-related disorders and toxic environmental exposures. To test this hypothesis, we are using the pesticide rotenone -a widely known complex I inhibitor- and the model organism Caenorhabditis elegans. Synchronized N2 eggs were exposed to rotenone (0-0.5 M) or 0.25% DMSO (control) in liquid with food (HB101) and harvested 52h later (L4 stage). After a further 48h depuration period, worm survival was followed in the presence of the pathogens Pseudomonas aeruginosa strain PA14, and Salmonella enterica serovar Typhimurium strain SL1344. Our first finding was that rotenone caused a dose-dependent decrease in worm size, which was associated to developmental delay. Worm vulval development was assessed to precisely determine the hours of developmental delay. Stage-synchronized worms exposed to 0.5 M rotenone had a longer median survival in SL1344 than control animals (~40%); but were more susceptible to PA14 (~15%). To validate whether these altered pathogen responses were due to rotenone-induced mitochondrial dysfunction, we analyzed different mitochondrial parameters. No significant differences were observed in preliminary measurements of worm basal oxygen consumption rate (OCR), spare capacity and ATP-linked OCR, or the ratio of mitochondrial to nuclear DNA copy number. This apparent lack of mitochondrial dysfunction after a developmental rotenone exposure may be due to a metabolic compensation in the worms, most likely through upregulation of complex II activity and the glyoxylate cycle, according to previous work. Thus, this appears to be a great model to study signaling between mitochondria and the immune system caused by metabolism shifts, without the detrimental effects of overt mitochondrial dysfunction. Now we are investigating the mechanisms by which mitochondrial signaling might be regulating the observed shifts in resistance to pathogens.

Mello DF et al. (2022) Front Immunol "Rotenone Modulates Caenorhabditis elegans Immunometabolism and Pathogen Susceptibility."

Bergemann CM, Chitrakar R, Mello DF, Bijwadia SR, Fisher K, Baugh LR, Wang Y, Caldwell A, Meyer JN

[Front Immunol, 2022]

Mitochondria are central players in host immunometabolism as they function not only as metabolic hubs but also as signaling platforms regulating innate immunity. Environmental exposures to mitochondrial toxicants occur widely and are increasingly frequent. Exposures to these mitotoxicants may pose a serious threat to organismal health and the onset of diseases by disrupting immunometabolic pathways. In this study, we investigated whether the Complex I inhibitor rotenone could alter <i>C. elegans</i> immunometabolism and disease susceptibility. <i>C. elegans</i> embryos were exposed to rotenone (0.5 M) or DMSO (0.125%) until they reached the L4 larval stage. Inhibition of mitochondrial respiration by rotenone and disruption of mitochondrial metabolism were evidenced by rotenone-induced detrimental effects on mitochondrial efficiency and nematode growth and development. Next, through transcriptomic analysis, we investigated if this specific but mild mitochondrial stress that we detected would lead to the modulation of immunometabolic pathways. We found 179 differentially expressed genes (DEG), which were mostly involved in detoxification, energy metabolism, and pathogen defense. Interestingly, among the down-regulated DEG, most of the known genes were involved in immune defense, and most of these were identified as commonly upregulated during <i>P. aeruginosa</i> infection. Furthermore, rotenone increased susceptibility to the pathogen <i>Pseudomonas aeruginosa</i> (PA14). However, it increased resistance to <i>Salmonella enterica</i> (SL1344). To shed light on potential mechanisms related to these divergent effects on pathogen resistance, we assessed the activation of the mitochondrial unfolded protein response (UPR^mt), a well-known immunometabolic pathway in <i>C. elegans</i> which links mitochondria and immunity and provides resistance to pathogen infection. The UPR^mt pathway was activated in rotenone-treated nematodes further exposed for 24 h to the pathogenic bacteria <i>P. aeruginosa</i> and <i>S. enterica</i> or the common bacterial food source <i>Escherichia coli</i> (OP50). However, <i>P. aeruginosa</i> alone suppressed UPR^mt activation and rotenone treatment rescued its activation only to the level of DMSO-exposed nematodes fed with <i>E. coli</i>. Module-weighted annotation bioinformatics analysis was also consistent with UPR^mt activation in rotenone-exposed nematodes consistent with the UPR being involved in the increased resistance to <i>S. enterica</i>. Together, our results demonstrate that the mitotoxicant rotenone can disrupt <i>C. elegans</i> immunometabolism in ways likely protective against some pathogen species but sensitizing against others.

Mello DF et al. (2022) Environ Sci Technol "In Vivo Effects of Silver Nanoparticles on Development, Behavior, and Mitochondrial Function are Altered by Genetic Defects in Mitochondrial Dynamics."

Mello DF, Meyer JN, Jiang C, Wiesner MR, Ryde IT, Songr DH, Maurer LL, Hsu-Kim H, Marinakos SM

[Environ Sci Technol, 2022]

Silver nanoparticles (AgNPs) are extensively used in consumer products and biomedical applications, thus guaranteeing both environmental and human exposures. Despite extensive research addressing AgNP safety, there are still major knowledge gaps regarding AgNP toxicity mechanisms, particularly in whole organisms. Mitochondrial dysfunction is frequently described as an important cytotoxicity mechanism for AgNPs; however, it is still unclear if mitochondria are the direct targets of AgNPs. To test this, we exposed the nematode<i>Caenorhabditis elegans</i> to sublethal concentrations of AgNPs and assessed specific mitochondrial parameters as well as organismal-level endpoints that are highly reliant on mitochondrial function, such as development and chemotaxis behavior. All AgNPs tested significantly delayed nematode development, disrupted mitochondrial bioenergetics, and blocked chemotaxis. However, silver was not preferentially accumulated in mitochondria, indicating that these effects are likely not due to direct mitochondria-AgNP interactions. Mutant nematodes with deficiencies in mitochondrial dynamics displayed both greater and decreased susceptibility to AgNPs compared to wild-type nematodes, which was dependent on the assay and AgNP type. Our study suggests that AgNPs indirectly promote mitochondrial dysfunction, leading to adverse outcomes at the organismal level, and reveals a role of gene-environment interactions in the susceptibility to AgNPs. Finally, we propose a novel hypothetical adverse outcome pathway for AgNP effects to guide future research.

Perez, L. et al. (2019) International Worm Meeting "Caenorhabdidtis elegans mitochondrial bionergetics during development: advances using the Seahorse Extracellular Flux Analyzer."

Meyer, J.N., Perez, L., Mello, D.F.

[International Worm Meeting, 2019]

Mitochondrial biology has become an area of intense research owing to its unique role in several physiological processes and pathologies. The model organism Caenorhabditis elegans has been extensively used to study ageing and stress responses, which are often tightly associated with mitochondrial metabolism, highlighting the importance of improving our basic understanding about this organelle throughout worm lifespan. The Seahorse Extracellular Flux (XF) Analyzer measures oxygen consumption rates (OCR) of different biological samples in real-time, and has been a great asset to study mitochondrial function with relatively high throughput. The aim of this study was to characterize different mitochondrial parameters in all larval stages, as well as in young and older adult worms using the 24-well XF analyzer. Besides measuring whole worm basal OCR, with the use of different mitochondrial inhibitors we can also assess mitochondrial bioenergetics such as mitochondrial OCR, spare capacity, maximal OCR, ATP-linked OCR, and proton-leak, as well as non-mitochondrial OCR. In order to perform these measurements, we are first optimizing worm numbers and mitochondrial inhibitors concentrations for each life stage. During the optimization process we are also testing different worm media and possible drug interactions. Our preliminary results revealed that (i) the complex IV inhibitor sodium azide response seems to be affected if injected subsequently to the mitochondrial uncoupler FCCP and the ATPase inhibitor DCCD. We have also found that (ii) although L1 worms have very low OCR, their relative spare capacity can be even greater than in adult worms, which are known to have a higher metabolic rate. Moreover, (iii) we found significant differences between basal OCR in worms within a same larval stage. Late L3 worms had approximately a 2-fold increase in basal OCR when compared to early L3s that were only 3h younger. And finally, (iv) OCR measurements in adult worms appear to be much more variable than in larval stages, therefore slight changes in mitochondrial responses of adult worms might not be easily detectable. The careful characterization of the worm mitochondrial metabolism throughout different life stages using the XF analyzer should be a useful asset to the C. elegans research field, and so far has revealed that care must be taken with possible drug interactions, when choosing which life stage to analyze, and when treatments might affect the worm development.

Hedges K A et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Copper Exposure Damages Neurons and Induces Paralysis at Far Sublethal Levels in the Nematode, Caenorhabditis elegans"

Hedges K A, O'Gara B A

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

Copper is essential to life at low levels as a cofactor for many enzymes that harness its ability to transfer electrons. This redox activity can catalyze the formation of reactive oxygen species when copper ions are unbound to proteins, so excessive levels are toxic. Neurotoxicity has been observed in all species observed except the nematode, Caenorhabditis elegans. This study examined the effects of copper exposure on neurons compared to the excretory cell in C. elegans, using transgenic strains with specific green fluorescent protein (GFP)-tagged cells. Exposure to CuSO4 damaged dopaminergic (DOP) neurons in a time- and concentration-dependent manner and induced paralysis at sublethal concentrations. The concentrations at which 50% of the animals had DOP neuron damage (EC50) were 0.94, 0.67, and 0.49 mM CuSO4 after exposure for 10, 12, and 14 hr; EC50 values for the excretory cell were 7.02, 4.28, and 3.74 mM. Therefore, the excretory cell resisted damage by a factor of seven compared to neurons. A two way ANOVA indicated significant differences in neuron damage due to exposure time (P&lt;0.001, F=14.947, d.f.=2,36), copper concentration (P&lt;0.001, F=332.787, d.f.=5,36), and the interaction (P&lt;0.001, F=4.214, d.f.=10,36). A two way ANOVA indicated a significant difference in excretory cell damage due to exposure time (P&lt;0.001, F=38.570, d.f.=2, 36), copper concentration (P&lt;0.001, F=476.945, d.f.=5,36) and the interaction (P&lt;0.001, F=11.416, d.f.=10,36). Animals paralyzed by copper were misidentified as dead by previous researchers. Live/dead testing with the cell-impermeant nucleic acid stain, SYTOX Orange, identified dead animals more accurately. Since cations interfere with dye fluorescence, SYTOX Orange was validated in animals treated with 2-32 mM CuSO4 before heat-killing. The highest usable copper concentration was 16 mM. Subsequent live/dead testing found fewer than 5% of animals died after copper treatment up to 16 mM for 14 hr. Therefore, the LC50 for CuSO4 is far greater than 16 mM; even this lower limit is approximately 25 times the EC50 for neuron damage. Therefore, copper exposure damages neurons at far sublethal levels. The difference in toxicity for neurons vs. the excretory cell, and the difference between the neuron EC50 and the LC50, refute the claim that copper is not neurotoxic in C. elegans.

Mello C (2007) Cell Death Differ "Return to the RNAi world: rethinking gene expression and evolution."

Mello C

[Cell Death Differ, 2007]

Thanks to the Nobel Foundation for permission to publish this Lecture (Copyright((c)) The Nobel Foundation 2006). Here we report the transcript of the lecture delivered by Professor Craig C Mello at the Nobel Prize ceremony. Professor Mello vividly describes the years of research that led to the discovery of RNA interference and the molecular mechanisms that regulate this fundamental cellular process. The turning point of discoveries and the role played by all his colleagues and collaborators are described, making this a wonderful report of the adventure of research. The lecture explains in simple language the importance of this discovery that has added a great level of complexity to the way cells regulate protein levels; moreover, it points out the beauty and importance of Caenorhabditis elegans as a model organism and how the use of this model has greatly contributed to the advance of science. Finally, Professor Mello leaves us with a number of questions that his research has raised and that will require years of future research to be answered.Cell Death and Differentiation (2007) 14, 2013-2020; doi:10.1038/sj.cdd.4402252.

Papp A et al. (1995) International C. elegans Meeting "INVESTIGATION OF C. ELEGANS ELECTROPORATION"

Jahani A, Papp A

[International C. elegans Meeting, 1995]

C. elegans can be transformed efficiently with exogenous genetic material using the technique of microinjection (Mello et. al, 1991). Although microinjection is becoming easier and more economical through the use of devices like our $425 microINJECTOR System, the procedure relies on a microscope system and accessories costing between $10,000 and $30,000, as well as some dexterity and knowledge of worm anatomy.

Guedes SF et al. (1997) International C. elegans Meeting "The C. elegans MEX-1 protein is a P granule component that is required for the proper localization of other P granule components"

Hill RJ, Guedes SF, Priess JR

[International C. elegans Meeting, 1997]

In the nematode C. elegans, germ cells arise from early embryonic cells called germline blastomeres. Cytoplasmic structures called P granules are present in the fertilized egg and are segregated into each of the germline blastomeres during the first few cleavages of the embryo. Mutations in the maternally-expressed gene mex-1 disrupt the segregation of P granules, prevent the formation of germ cells, and cause inappropriate patterns of somatic cell differentiation (Mello et al., 1992; Schnabel et al., 1996). The mex-1 gene can encode for a protein, MEX-1, that contains two copies of a CCCH-type "finger" domain also found in the PIE-1 (Mello et al., 1996) and POS-1 (Hiroaki et al., this meeting) proteins of C. elegans (Guedes and Priess, 1997). MEX-1 (Guedes and Priess, 1997), PIE-1 (Mello et al., 1996) and POS-1 (Hill et al., this meeting) are expressed in the germline blastomeres, and are components of P granules. We show that MEX-1 is required to restrict PIE-1 expression and activity to the germline blastomeres during the early embryonic cleavages. We further show that MEX-1 is required for the localization of PIE-1 and POS-1 to the P granules but MEX-1 is not required for the P granule localization of MEX-3, GLH-1, and GLD-1, other P granules components.

Grishok A et al. (2000) Science "Genetic requirements for inheritance of RNAi in C. elegans."

Mello CC, Grishok A, Tabara H

[Science, 2000]

In Caenorhabditis elegans, the introduction of double-stranded RNA triggers sequence-specific genetic interference (RNAi) that is transmitted to offspring. The inheritance properties associated with this phenomenon were examined. Transmission of the interference effect occurred through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-1 and rde-4 were required for the formation of this interfering agent but were not needed for interference thereafter. In contrast, the rde-2 and mut-7 genes were required downstream for interference. These findings provide evidence for germ line transmission of an extragenic sequence-specific silencing factor and implicate rde-1 and rde-4 in the formation of the inherited agent.AD - Program in Molecular Medicine, Department of Cell Biology, University of Massachusetts Cancer Center, Two Biotech Suite 213, 373 Plantation Street, Worcester, MA 01605, USA.FAU - Grishok, AAU - Grishok AFAU - Tabara, HAU - Tabara HFAU - Mello, C CAU - Mello CCLA - engID - GM58800/GM/NIGMSPT - Journal ArticleCY - UNITED STATESTA - ScienceJID - 0404511RN - 0 (DNA Transposable Elements)RN - 0 (Helminth Proteins)RN - 0 (RNA, Double-Stranded)RN - 0 (RNA, Helminth)RN - 0 (rde1 protein)SB - IM

Huang, George et al. (2021) MicroPubl Biol

Rettmann, Aubrie, Waterland, Skye, Huang, George, DeMott, Ella, Sylvester, Melynda, Dickinson, Daniel J, Rhodes, Anita, Flynn, Abbey, Alicea, Persephone, Blanco, Sara, Ren, Cassie, Koh, Alex, Doonan, Ryan, de Jesus, Bailey, Meng, Carrie

[MicroPubl Biol, 2021]

The self-excising cassette (SEC) knock-in approach uses hygromycin selection and a visible roller phenotype to identify knock-ins, followed by a heat-shock induced excision of these visible markers to yield a seamless insertion of a fluorescent protein into the genome (Dickinson et al. 2015). Compared to protocols that utilize Cas9 protein and linear DNA repair templates (Paix et al. 2015; Dokshin et al. 2018; Ghanta and Mello 2020), the plasmid-based SEC approach employs a simpler screening strategy but requires more worms to be injected (Dickinson and Goldstein 2016).