Limiting the debilitating consequences of ageing is a major medical challenge of our time. Robust pharmacological interventions that promote healthy ageing across diverse genetic backgrounds may engage conserved longevity pathways. Here we report results from the Caenorhabditis Intervention Testing Program in assessing longevity variation across 22 Caenorhabditis strains spanning 3 species, using multiple replicates collected across three independent laboratories. Reproducibility between test sites is high, whereas individual trial reproducibility is relatively low. Of ten pro-longevity chemicals tested, six significantly extend lifespan in at least one strain. Three reported dietary restriction mimetics are mainly effective across C. elegans strains, indicating species and strain-specific responses. In contrast, the amyloid dye ThioflavinT is both potent and robust across the strains. Our results highlight promising pharmacological leads and demonstrate the importance of assessing lifespans of discrete cohorts across repeat studies to capture biological variation in the search for reproducible ageing interventions.

Mitochondria are the primary source of ATP needed for the steps of the synaptic vesicle cycle. Dynamin-related protein (DRP) is involved in the fission of mitochondria and peroxisomes. To assess the role of mitochondria in synaptic function, we characterized a Drosophila DRP mutant combination that shows an acute temperaturesensitive paralysis. Sequencing of the mutant reveals a single amino acid change in the guanosine triphosphate hydrolysing domain (GTPase domain) of DRP. The synaptic mitochondria in these mutants are remarkably elongated, suggesting a role for DRP in mitochondrial fission in Drosophila. There is a loss of neuronal transmission at restrictive temperatures in electroretinogram (ERG) recordings. Like stress-sensitive B (sesB), a mitochondrial adenosine triphosphate (ATP) translocase mutant we studied earlier for its effects on synaptic vesicle recycling, an allele-specific reduction in the temperature of paralysis of Drosophila synaptic vesicle recycling mutant shibire was seen in the DRP mutant background. These data, in addition to depletion of vesicles observed in electron microscopic sections of photoreceptor synapses at restrictive temperatures, suggest a block in synaptic vesicle recycling due to reduced mitochondrial function.

Background: Mechanisms that regulate plasma membrane expression of neuronally toxic DEG/ENaC channels are unclear. Results: Disruption of ER NRA-2, a Nicalin homolog, enhances C. elegans MEC-10(d) DEG/ENaC neurotoxicity. Immunocytochemistry, TIRF imaging, and electrophysiological assays support that NRA-2 controls relative MEC-10(d) distribution between ER and cell surface to regulate channel activity levels. Conclusion: NRA-2 regulates surface expression of a mutant DEG/ENaC channel. Significance: NRA-2 Nicalin can modulate DEG/ENaC pathophysiology.

The Caenorhabditis Intervention Testing Program (CITP) seeks to identify compounds that robustly improve lifespan and/or attenuate age-dependent declines in health across a genetically diverse set of Caenorhabditis species and strains. A core mission of the CITP is to collect data that will inform on the reproducibility of our experimental results. To achieve this, multiple replicates of each experiment are performed by each of three groups at separate laboratory sites. In an effort to limit non-biological confounding factors that may influence the reproducibility of these results, we sought to minimize differences in experimental conditions across the individual laboratories. This led to the development and utilization of a set of standardized protocols that describe specific methods for nematode maintenance and culture as well as detailed experimental procedures. All laboratories of the CITP are tasked with strict adherence to protocols. Here we provide several protocols that describe the CITP standard operating practices for bacterial cultures, media preparation and strain handing as well as assays for quantifying the development rate, fertility and lifespan of hermaphroditic Caenorhabditis strains. Individual components of these methods were not tested for their effects on outcomes and are not suggested to be best practices. However our consideration of the methodological variability available for even these simple assays does highlight the potential for differences in methodology to influence experimental reproducibility.