Hsiung, Kuei Ching et al. (2021) International Worm Meeting "Are levels of autophagy increased or decreased in daf-2 insulin/IGF-1 mutants?"

Gems, David, Chapman, Hannah, Hsiung, Kuei Ching

[International Worm Meeting, 2021]

According to one theory, molecular damage is the principal cause of ageing, while somatic maintenance mechanisms act against damage accumulation and ageing. One such maintenance mechanism is autophagy, which can remove damaged cellular constituents. Autophagy is considered a likely mechanism protecting against ageing. Various findings support the view that autophagy levels are elevated in daf-2 insulin/IGF-1 receptor mutants in C. elegans, and that autophagy contributes to their extended lifespan. We have been investigating the causes of intestinal atrophy, a major senescent pathology in C. elegans. Our evidence supports the view that gut atrophy is the result of the intestine consuming its own biomass to support yolk synthesis in sperm-depleted hermaphrodites [1], an example of a programmatic mechanism of ageing [2]. This yolk is vented via the vulva after reproduction and can support larval growth [3] (see presentation by C.C. Kern). Such intestinal biomass repurposing is facilitated by autophagy, and greatly reduced in daf-2 mutants [1,4]. This suggests that autophagy is decreased in daf-2 mutants. Consistent with this, it has been reported that protein turnover is also reduced in daf-2 mutants. To try to resolve the conflicting conclusions about the relationship between IIS, autophagy and ageing in C. elegans we are re-examining effects of knockdown of genes encoding the machinery of autophagy on pathology and ageing, taking into consideration issues of timing and severity of knockdown, daf-2 allele class, temperature, bacterial status and other issues. Our interim data will be described. [1] M. Ezcurra, et al (2018) Curr. Biol. 28: 2544. [2] A.A Maklakov, T. Chapman (2019) Proc. Biol. Sci. 286: 20191604. [3] C.C. Kern et al (2020) BioRxiv doi.org/10.1101/ 2020.11.15.380253. [4] A. Benedetto, D. Gems (2019) Autophagy 15: 731.

Hsiung, Kuei-Ching et al. (2017) International Worm Meeting "Loss of Caenorhabditis elegans W02B12.15, mammalian cisd1, alters iron homeostasis, mitochondrial electron transport complexes and lipid metabolism."

Lo, Szecheng, Hsiung, Kuei-Ching, Tan, Bertrand, Cheng, Mei-Ling, Liu, Kuan-yu

[International Worm Meeting, 2017]

Iron-sulfur cluster (ISC) has indispensable functions in many biological processes; including mitochondrial respiratory chain and numerous enzymatic functions, due to its unique molecular geometry. Three species of CDGSH iron sulfur domain-containing proteins, CISD1, CISD2, and CISD3 have been identified in mammals. CISD1 and CISD2 contain a CDGSH domain and a mitochondrial transmembrane domain. CISD1 might play a role in transferring ISC to an apo-ferredoxin in vitro and obesity-associated dysfunction adiposeness in human VAT while CISD2 mediates mitochondrial integrity and life span in mammals. CISD3 has two CDGSH domains but no transmembrane domain. In C. elegans, one CDGSH coding gene, W02B12.15, was found and based on a phylogenetic tree analysis, W02B12.15 is closer to the Cisd1 clade. Furthermore, deletion mutant (tm4993) showed no defects in life span, motility, and development. Thus, C. elegans W02B12.15 was designated as a Cecisd1 gene. CeCISD1 was found primarily in the mitochondrial membrane fraction and the CeCISD1::GFP was colocalized with Mitotracker Red. The deficiency of Cecisd1 showed the abnormal mitochondrial morphology. Although the basal level of oxygen consumption of tm4993 was higher than wild type, the spare respiratory capacity did not reflect this phenomenon. Noticeably, the ATP-linked respiration and ROS production were increased in tm4993. The mitochondrial electron transport chain complexes were altered in tm4993, which lead to proton leakage and loss of mitochondrial membrane potential (??m). It might cause the increasing of AMP/ATP ratio that activated the AMPK phosphorylation. Subsequently, fatty acid beta-oxidation genes acs-2, cpt-5 and ech-1 were activated by AMPK to react in decreasing of the total amount and the average size of lipid droplets. The lipidomic and metabolic sets enrichment analyses indicated the increasing of branched chain fatty acids oxidation and beta-oxidation of long chain fatty acids. Without the ISC transferring from CeCISD1 to cytosolic aconitase, the iron regulatory proteins increase the uptake of iron, which leads the overloading of cytosolic total iron ion (especially ferrous iron) in the tm4993. Collectively, our data suggest that depletion of CeCISD1 causes the imbalance of mitochondrial energy and lipid metabolism through interruption of cellular iron homeostasis.

Zhang, Aihan et al. (2021) International Worm Meeting "Testing the use of liposomes for drug delivery in C. elegans"

Hsiung, Kuei Ching, Zhang, Aihan, Gems, David

[International Worm Meeting, 2021]

While C. elegans is an excellent model organism for studying the genetics of aging, for testing effects of drugs on aging it is problematic, for two reasons in particular. First, many compounds administered to C. elegans are not taken up (1). Second, increases in lifespan may reflect drug effects on the E. coli food source (2). Further issues are bioconversion of drugs by E. coli (3), and the high cost of some compounds. One possible solution to all these problems is the use of liposome-mediate drug delivery, which can increase uptake into the worm, as shown previously using the fluorescent dye uranine (4). This may reflect better carriage by the pharynx of particulate matter into the gut lumen and/or better passage from the lumen into intestinal cells. Liposomes should also reduce interactions between drug and E. coli, and enable delivery of drugs to worms using much smaller quantities than required when administering them via the agar. With the ultimate aim of testing effects of anti-aging drugs on senescent pathology in C. elegans, we have been investigating the efficacy of liposome-mediated drug delivery, initially using fluorescent dyes. Our results thus far have confirmed that liposomes are an efficient means to deliver drugs into the intestinal lumen using very small quantities of compound. However, they appear not to improve passage across the apical intestinal membrane. We are currently testing effects on lifespan of liposome-mediated delivery of a range of putative anti-aging drugs, and will present the results at the meeting. (1)Nat. Chem. Biol. 2010 6: 549. (2) Cell 2013 153: 228. (3) Cell 2017 169: 442. (4) Mech. Ageing Dev. 2009 130: 652.

Yi, Yung-Hsiang et al. (2015) International Worm Meeting "Omega-6 polyunsaturated fatty acid is required for yolk endocytosis and lipid uptake in C. elegans oocytes examined by CARS microscopy."

Lin, Yi-Chun, Chen, Wei-Wen, Chang, Yu-Sun, Yi, Yung-Hsiang, Chang, Ta-Chau, Hsiung, Kuei-Ching, Liu, Kuan-yu, Ma, Tian-Hsiang, Lo, Szecheng J., Chien, Cheng-Hao

[International Worm Meeting, 2015]

The transportation of yolk lipoprotein, the major carrier of nutrition that brings fatty acids into oocytes, is critical for the maturation of oocytes in the nematode Caenorhabditis elegans (C. elegans). However, it is unclear how polyunsaturated fatty acids (PUFAs) regulate the transportation of yolk lipoprotein and maturation of oocytes in C. elegans. Here we utilize coherent anti-Stokes Raman scattering (CARS) and two-photon excitation fluorescence microscopy to examine the transportation of yolk lipoprotein and maturation of oocytes in wild-type C. elegans and PUFA-related fat mutants (fat-1 to fat-4) on single-cell level. We found higher percentage of worms containing large yolk lipoprotein accumulation in the psuedocoelom of all fat-1 to fat-4 mutants. To investigate the maturation of oocytes, we have established a quantification method by integrating CARS lipid signal in each oocyte in live C. elegans. We demonstrate that the integrated lipid signal has a strong linear correlation (R2>0.9) with the integrated fluorescent signal of GFP-tagged yolk lipoprotein in oocytes. Our further study shows that the supplement of omega-6 PUFA to fat-2 mutant, but not omega-3 PUFA, can rescue the transportation of yolk lipoprotein into oocyte and hence recover the ovulation rate of oocytes. This result suggests that omega-6 PUFA is essential for the transportation of yolk lipoprotein and maturation of oocytes in C. elegans. In summary, our work demonstrates that CARS imaging is a useful tool which can provide a fast, label-free, and quantitative means for examining the transportation of yolk lipoprotein and the maturation of oocyte in live C. elegans.

Martin Chalfie (2008) C.elegans Neuronal Development Meeting "A Protein-Lipid Complex Transduces Touch in C. elegans"

Martin Chalfie

[C.elegans Neuronal Development Meeting, 2008]

Little is known about the molecular basis of mechanical sensing in eukaryotes, in part because of the difficulty of biochemically isolating components needed for mechanosensory transduction. Genetic analysis, however, provides a way of identifying important molecules needed to sense a mechanical stimulus. In the nematode Caenorhabditis elegans, gentle touch to the head or the tail produces a withdrawal response that is sensed by six sensory neurons. The study of the genes identified in screens for touch-insensitive mutants has provided insights into both the differentiation of these cells and the molecular basis of mechanosensation. The genes needed for the function of the cells encode channel components, extracellular matrix proteins, cytoskeletal proteins, and possible enzymes that enable or modulate the touch response. Genetic interactions, molecular analysis of these genes and their products, and electrophysiological studies using heterologous cells as well as in situ recordings indicate that these cells sense touch through a DEG/ENaC channel complex. This complex is localized and, possibly, coupled to extracellular components at attachment sites to the body wall. The channel complex contains at least four proteins (MEC-2, MEC-4, MEC-6, and MEC-10) with MEC-4 and MEC-10, both DEG/ENaC proteins, forming the pore of the channel. Similar amino acid substitutions in MEC-4 and MEC-10 altered the properties of the transduction current, demonstrating that the proteins are part of the transduction machinery. MEC-2 and MEC-6 amplify channel activity 30-40 fold in frog oocytes. MEC-2 appears to act through its ability to bind to the DEG/ENaC proteins, itself, and cholesterol, which is needed for channel function. We have hypothesized that this binding alters the local lipid environment of the channel, allowing it to open when mechanically stimulated. MEC-6 may also affect the lipid microenvironment, since it is similar to the human protein paraoxonase, which has been implicated in the regulation of lipid peroxidation. Our current model for mechanosensory transduction is derived from the elevator model of Ching Kung. In this model the touch stimulus causes the membrane to approach or move away from the attachment sites, causing the tethered channel complexes to move perpendicular to the membrane. This displacement then leads to the opening of the channel. Such a model can explain the rapid response and adaptation seen in the mechanosensory current.