Iyer, Sangeetha et al. (2017) International Worm Meeting "Identification of a small molecule modulator of a nematode model of Niemann Pick Type C disease."

Hartl, Tom, Iyer, Sangeetha, Perlstein, Ethan, Tsang, Hillary, DiPrimio, Nina

[International Worm Meeting, 2017]

Perlara PBC, is a public benefit company committed to discovering small molecule therapeutics for rare genetic diseases. Our approach consists of the use of simple, whole animal models such as yeast, worm, flies in drug discovery screens. Molecules that pass successfully through our screens are validated in patient cells before advancing into higher animal models of the disease. Niemann Pick Type C (NPC) is a devastating lysosomal storage disease with high mortality rates. The nematode gene, ncr-1, was engineered to generate a nematode model lacking functional ncr-1 protein. The resulting animal showed slowed development, small brood size and sensitivity to cholesterol deprivation. We capitalized on the growth defect observed in the absence of cholesterol to conduct a discovery screen. 50000 molecules were screened at an initial concentration of 25 M to identify candidates that rescued the growth defect. 'Hits' resulting from this screen were validated in a secondary assay on worms as well as on patient-derived cells. Perl101 was identified as a lead candidate for a class of molecules that rescued the NPC phenotype in worms and changed the cellular fingerprint of NPC cells. RNA sequencing of nematodes exposed to incremental concentration of Perl101 also provided insight into its mechanism of action. Furthermore, as an unoptimized screening hit, Perl101 demonstrated high oral bioavailability, blood brain barrier permeability and minimal adverse effects in a 90 day mouse tolerability study. Additional studies to probe the mechanism of action of Perl101 and its efficacy in a mouse model of NPC are underway. In summary, utilization of simple animal models such as nematodes, flies and yeast present an untapped opportunity to discover therapuetic candidates with greater translational potential for rare genetic diseases. A parallel, multi-model whole animal screening approach leverages shared evolutionary linkages and increases the probability of identifying potential therapeutics. This approach can lead to the discovery of treatments for a class of diseases with high unmet needs. </em>

Kara Chin et al. (2005) International Worm Meeting "Who needs mitochondrial mtDNA anyway? Fitness effects of the 3.1kb UaDf-5 mtDNA deletion."

Cleonique Deshommes, Aidyl Gonzalez-Serricchio, Craig LaMunyon, Kara Chin

[International Worm Meeting, 2005]

Mitochondrial chromosomes have very little non-coding DNA, and non-mutated chromosomes are generally considered to be associated with increased metabolic fitness. In C. elegans, the UaDf5 mtDNA deletion removes 3.1kb encompassing 11 genes, and yet this deletion can be maternally transmitted for at least 100 generations in a stable heteroplasmic state at all developmental stages (Tsang and Lemire , 2002). Why should a mitochondrial deletion persist? We investigated this question by looking at several measures of metabolism and fitness in relation to the proportion of UaDf-5 deleted mtDNA. Our data support Tsang and Lemires (2002) findings of stable heteroplasmy between wild-type and deleted mtDNA, even though the proportion of deleted mtDNA can vary greatly among siblings and over the course of several generations. We also looked at individual tissues of worms and found no mosaicism among the gut, testes and other tissues for this deletion. We found a positive correlation between the proportion of deleted mtDNA and measures of metabolism (defecation, feeding, and egg-laying rates), indicating that worms bearing this deletion of the mitochondrial chromosome are more fit than wild-type, at least under laboratory conditions.Tsang WY, Lemire BD, 2002. Biochem Cell Biol 80:645-54.

Peterson, Sagen et al. (2015) International Worm Meeting "Elucidating the mechanisms for purifying selection of mitochondrial DNA."

Goyal, Sidhartha, Torres Cleuren, Yamila, Peterson, Sagen, Rothman, Joel

[International Worm Meeting, 2015]

The mitochondrial genome (mtDNA) is present in many copies within each cell and follows a maternal pattern of inheritance. mtDNA experiences a high mutation rate which leads to heteroplasmy, in which a mixed population of both wildtype and altered mtDNA exist within the same cell. While each mtDNA can act as a "selfish DNA" element, purifying selection is used to eliminate from the germline mtDNAs that are defective and debilitate mitochondrial function. We are investigating the mechanisms used in purifying selection in the germline. One level at which purifying selection might act is during the early embryonic divisions that establish the germline (P lineage). We have obtained preliminary evidence that the most active mitochondria (those with the highest membrane potential) are enriched in the germline lineage during the early embryonic cell divisions. Following this early phase, we found that germline mitochondria are then fully quiescent (no detectable membrane potential) until after the initiation of larval development. We have taken advantage of a 3 kb mtDNA deletion, uaDf5 [Tsang et al 2002], to investigate further the mechanisms that remove defective mtDNAs. This deletion exhibits stable heteroplasmy, raising the suggestion that it is maintained as a result of complementing mutations on the non-deleted mtDNAs in the strain [Tsang et al 2002]. However, we found that the uaDf5-bearing strain appears to contain fully intact, wild-type mtDNA, suggesting that the deletion may be maintained as a result of replicative advantage. Further, we found that the uaDf5 strain gives rise to arresting embryos with many cell corpses, reminiscent of cells that have undergone programmed cell death. Lines that generate arresting embryos at highest frequency also show extended lifespan in the survivors, consistent with a trade-off between mitochondrial function required for survival and lifespan extension. Finally, we have obtained preliminary results suggesting that physiological state of worms influences the rate of removal of mtDNAs encoding defective proteins. We are investigating the trans-generational dynamics of this apparent purifying selection.

Knapp-Wilson, A. et al. (2017) International Worm Meeting "Investigating Complex I activity and mitochondrial protein import in the multi-cellular model organism C. elegans."

Collinson, I., Knapp-Wilson, A., Kuwabara, P.

[International Worm Meeting, 2017]

Mitochondria are small cytoplasmic organelles essential for meeting the majority of cellular energy demands via the oxidative phosphorylation (OXPHOS) pathway. [1, 2]. They feature a double membrane that separates the organelle into four distinct compartments [3]. The vast majority of the work on mitochondria has been carried out in Saccharomyces cerevisiae, a single-celled genetically tractable eukaryote [4]. Although biochemically competent; the model has limitations in its applicability to humans. The mitochondrial structure, metabolism and bioenergetics of the nematode are very similar to the mammalian counterpart [5], this conservation of mitochondrial function and the technological and anatomical advantages offered by the nematode make it an excellent model organism to explore mitochondrial function and activity. [1, 6] My project is multidisciplinary, using a broad range of techniques including whole animal physiology, biochemical and structural analysis, and molecular dynamic simulations to gain a better understanding of the mitochondrial respiratory machinery, and the impact dysfunction of this machinery can have. Mitochondrial dysfunction leads to many serious human diseases and a better understanding of the mechanisms involved is essential in future medical applications. [1] Maglioni, S. & Ventura, N., 2016. C. elegans as a model organism for human mitochondrial associated disorders. Mitochondrion, 30, pp.117-125. [2] Lemire, B. Mitochondrial genetics (September 14, 2005), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.25.1, [3] McBride et al, 2006. Mitochondria: more than just a powerhouse., 16(14), pp.R551-60 [4] Tokatlidis, K., et al. (2000). "Membrane protein import in yeast mitochondria." Biochem Soc Trans 28(4): [5] Murfitt, R.R., Vogel, K. & Sanadi, D.R., 1976. Characterization of the mitochondria of the free-living nematode, Caenorhabditis elegans. Comparative biochemistry and physiology. B, Comparative biochemistry, 53(4), [6] Tsang, W.Y. & Lemire, B.D., 2003. The role of mitochondria in the life of the nematode, Caenorhabditis elegans. Biochimica et biophysica acta, 1638(2)

Aidyl S Gonzalez-Serricchio et al. (2005) International Worm Meeting "Paternal mitochondrial inheritance: No longer restricted to Moms"

Craig LaMunyon, Aidyl S Gonzalez-Serricchio

[International Worm Meeting, 2005]

A vast majority of eukaryotic organisms solely inherit maternal mitochondrial DNA (mtDNA) with the exception of mussels (males inherit paternal mtDNA and females inherit maternal mtDNA) (Zouros, 2000; Zouros et al., 1994). Paternal mtDNA disappear early in embryogenesis either due to selective destruction, inactivation or simple dilution by the numerous of oocyte mitochondria. Is there a deleterious effect if offspring inherits paternal mtDNA? Our aim is to further understand the mechanism of mitochondrial inheritance. We were able to isolate a strain with the capability to pass onto its offspring its paternal mtDNA. UadDf-5 is a stable heteroplasmic strain with a 3kb deletion on its mt genome (removes 11 genes; Tsang and Lemire, 2002). UaDf-5; him-8 (e1489) worms were mutagenized with ethylmethanesulfonate (EMS). F1 worms were isolated and allowed to clone in order to isolate F2 males. Next, the F2 males were crossed into fer-1 (hc13ts) hermaphrodites. Finally, F3 worms were isolated and underwent a nestedPCR assay to detect the mutant UaDf-5 band inherited from the Po: UaDf-5; him-8 (e1489) males. As we further confirm, homozygosis (if stable), and characterize the DNA lesion of the mutagenized strain(s), we will have a strong tool to use in explaining the mechanism that eliminates the sperm mitochondria from the embryo. Also, we will observe offspring that inherit some paternal mitochondria to see if it results in reduced fitness. Either initially, or after numerous generations, understand the evolution of maternal inheritance.Zouros E, 2000. The exceptional mitochondrial DNA system of the mussel family Mytilidae. Genes Genet Syst 75:313-8.Zouros E, Oberhauser Ball A, Saavedra C, Freeman KR, 1994. An unusual type of mitochondrial DNA inheritance in the blue mussel Mytilus. Proc Natl Acad Sci U S A 91:7463-7Tsang WY, Lemire BD, 2002. Stable heteroplasmy but differential inheritance of a large mitochondrial DNA deletion in nematodes. Biochem Cell Biol 80:645-54.

Hajjar, Connie et al. (2011) International Worm Meeting "Sperm Mitochondria are Associated with Ubiquitinated Vesicles After Fertilization."

Hajjar, Connie, Boyd, Lynn, Golden, Andy

[International Worm Meeting, 2011]

Studies in C. elegans and other organisms have indicated that mitochondria are maternally inherited.1 This has presented an interesting cell biological conundrum as to what becomes of sperm mitochondria that enter the egg upon fertilization. Studies in mammalian systems have suggested that sperm mitochondria are eliminated from the fertilized egg via a mechanism involving ubiquitination.2 We have investigated this possibility in the worm. Using strains with germline expression of GFP::ubiquitin, we have observed ubiquitinated vesicles that cluster around the sperm DNA after fertilization. We propose that these vesicles are sperm-derived based on three observations: 1) they are not seen in oocytes, 2) they are closely associated with the sperm DNA, and 3) they stain positive with a sperm-specific antibody (see below for more details). Using Mitotracker tagging of sperm, we show that the sperm mitochondria are closely associated with the ubiquitinated vesicles but are not ubiquitinated themselves. The sperm vesicle-mitochondria conglomerate remains compact and close to the sperm DNA throughout the maternal meiotic divisions. Starting around prophase of the first mitosis, the sperm vesicles and mitochondria begin to disperse and their numbers diminish. By the 8-cell stage, we no longer detect either. In order to learn more about the nature of the ubiquitinated vesicles, we have done additional labelling experiments. They stain with wheat germ agglutinin, indicating that they are indeed membrane bound vesicles. They do not stain with mitotracker or lysotracker. They stain positively with the 1CB4 antibody that recognizes the sperm-specific organelle, MO. 1CB4 stains the ubiquitinated vesicles, but not the sperm mitochondria in the embryo. Thus, the vesicles seen in the fertilized embryo may be derived from MO structures in the sperm. Linkage specific antibodies to polyubiquitin show that the vesicles harbor K63, but not K48, linked polyubiquitin chains. K63 chains can sometimes trigger autophagy suggesting that autophagic destruction may be involved in the elimination of the sperm-derived vesicles and possibly also the nearby sperm mitochondria. This interesting association between sperm mitochondria and these unusual ubiquitinated vesicles may reveal a novel mechanism for the elimination of paternal mitochondria from the fertilized egg. 1- Tsang and Lemire, (2002) Bioch. Cell Biol. 80 645-654; 2- Sutovsky et al., (2000) Biol Reprod. 63 582-90.

Norman Breuil et al. (2007) International Worm Meeting "Modeling mitochondrial heteroplasmy in the nematode Caenorhabditis elegans."

Norman Breuil, Emmanuel Culetto, Laure Rifault, Francesca Farina, Moise Pinto

[International Worm Meeting, 2007]

The mitochondrion plays a critical role in the cellular energy supply and performs other essential role in a variety of metabolic function. The mitochondrion possesses its own genome (mtDNA) coding for 13 polypeptides in the mitochondrial respiratory chain. Typically individuals harbour a single sequence variant of mtDNA. However, germline and somatic mutations can lead to the coexistence of a mixture of mutant and normal mtDNA. This state is defined as heteroplasmy. Some human mitochondrial diseases are often characterized by the coexistence of normal and mutated mtDNA molecules within the same cells. In effect, the disease-associated symptoms are clinically revealed once the mitochondrial mutated version overreach a certain threshold of heteroplasmy. We decided to address the molecular mechanisms controlling heteroplasmy and mtDNA maintenance using the model Caenorhabditis elegans. The C. elegans mtDNA is very similar to the human one, encoding for 12 proteins, is maternally inherited and the existence of heteroplasmic state have been demonstrated (Tsang and Lemire, 2002). We propose to generate heteroplasmic mutants that could be easily detected by drug resistance phenotype, then, to modify the genetic background of these worms by RNAi screening to assess whether there is any modification in the heteroplasmic status that could be detected by direct visual inspection. We aim to isolate animals harbouring point mutations in the mtDNA conferring a selective advantage to the worm: resistance to oligomycin. Actually, in yeast and human cells, point mutations in the mitochondrial gene coding for ATPase 6 subunit lead to oligomycin resistance. Therefore, we hypothesized that C. elegans mutant harbouring mtDNA mutation(s) in this gene could be recovered during the isolation of mutants resistant to the drug. We have shown that oligomycin effectively affect mitochondrial morphology and restricted C. elegans development and survival. In a forward genetic analysis we have screened 3000 F2 hermaphrodites for EMS induced mutations that allow worms to develop in presence of oligomycin. We have isolated 15 oligomycin resistant worms (oli-1 to oli-15) and started to study whether these resistance loci are maternally inherited. Genetic crosses have permitted to show that oli-1 is nuclear and recessive. We identified that a point mutation in the nuclear gene encoding ATP-9 subunit of the mitochondrial ATPsynthase complex is responsible for the oligomycin resistance. For 6 oli mutants, the genetic crosses indicate that the inheritance is nuclear dominant or semi-dominant, whereas oli-7 seems to show a cytoplasmic inheritance. We will present data showing further characterization of the latter mutant.