Quantification of Insoluble Protein Aggregation in <em>Caenorhabditis elegans</em> during Aging with a Novel Data-Independent Acquisition Workflow

We and others have shown that the aging process results in a proteome-wide accumulation of insoluble proteins. Knocking down genes encoding the insoluble ...

Buck Institute for Research on Aging

Loss of germline precursor cells in C. elegans has previously been shown to improve protein homeostasis and extend lifespan, possibly due to reallocation of resources to somatic cells. In contrast, mutants that are sterile simply due to loss of sperm or oocyte production have a normal lifespan, often leading to the conclusion that loss of reproduction per se may have minor effects on C. elegans. We have found that inhibiting reproduction in C. elegans via the DNA synthesis inhibitor 5-fluoro-2-deoxyuridine (FUdR) improves protein homeostasis, stress resistance, and healthspan in wild-type animals. We find that FUdR is dependent on oogenesis and oocytic maturation. The effects of FUdR are dependent on FEM pathways, which regulate initiation of spermatogenesis. Loss of FEM expression leads to feminized animals that maintain arrested oocytes and are refractory to the effects of FUdR. FUdR-dependence is restored by spermatogenic signals, which trigger oocytic maturation and ovulation. Further, loss of FEM-3, a novel protein required for spermatogenesis, is sufficient to improve aspects of proteostasis. These effects are independent of previously described germline signals, including the DAF-16/FOXO, DAF-12/VDR, and HSF-1 pathways. These findings suggest that genetic or chemical inhibition of oocyte production can improve protein homeostasis in C. elegans. 

A DNA synthesis inhibitor is protective against proteotoxic stressors via modulation of fertility pathways in Caenorhabditis elegans.

Parkinson's disease (PD) is a debilitating motor and cognitive neurodegenerative disorder for which there is no cure. While aging is the major risk factor for developing PD, clear environmental risks have also been identified. Environmental exposure to the manganese (Mn) metal is a prominent risk factor for developing PD and occupational exposure to high levels of Mn can cause a syndrome known as manganism, which has symptoms that closely resemble PD. In this study, we developed a model of manganism in the environmentally tractable nematode, Caenorhabditis elegans. We find that, in addition to previously described modes of Mn toxicity, which primarily include mitochondrial dysfunction and oxidative stress, Mn exposure also significantly antagonizes protein homeostasis, another key pathological feature associated with PD and many age-related neurodegenerative diseases. Mn treatment activates the ER unfolded protein response, severely exacerbates toxicity in a disease model of protein misfolding, and alters aggregate solubility. Further, aged animals, which have previously been shown to exhibit decreased protein homeostasis, are particularly susceptible to Mn toxicity when compared to young animals, indicating that the aging process sensitizes animals to metal toxicity. Mn exposure also significantly alters iron (Fe) and calcium (Ca) homeostasis, which is important for mitochondrial and ER health and which may further compound toxicity. These findings indicate that modeling manganism in C. elegans can provide a useful platform for identifying therapeutic interventions for ER stress, proteotoxicity, and age-dependent susceptibilities, key pathological features of PD and other related neurodegenerative diseases.

Manganese disturbs metal and protein homeostasis in Caenorhabditis elegans.

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.

Impact of genetic background and experimental reproducibility on identifying chemical compounds with robust longevity effects.

Glial activation and subsequent release of neurotoxic proinflammatory factors are believed to play an important role in the pathogenesis of several neurological disorders including Parkinson's disease (PD). Inhibition of glial activation and inflammatory processes may represent a therapeutic target to alleviate neurodegeneration. Securinine, a major natural alkaloid product from the root of the plant , has been reported to have potent biological activity and is used in the treatment of neurological conditions such as amyotrophic lateral sclerosis, poliomyelitis, and multiple sclerosis. In this study, we explored the underlying mechanisms of neuroprotection elicited by securinine, particularly its anti-inflammatory effects in glial cells. Our results demonstrate that securinine significantly and dose-dependently suppressed the nitric oxide production in microglia and astrocytic cultures. In addition, securinine inhibited the activation of the inflammatory mediator NF-B, as well as mitogen-activated protein kinases in lipopolysaccharide- (LPS-) stimulated BV2 cells. Additionally, securinine also inhibited interferon-- (IFN--) induced nitric oxide levels and iNOS mRNA expression. Furthermore, conditioned media (CM) from securinine pretreated BV2 cells significantly reduced mesencephalic dopaminergic neurotoxicity compared with CM from LPS stimulated microglia. These findings suggest that securinine may be a potential candidate for the treatment of neurodegenerative diseases related to neuroinflammation.

Suzanne Angeli

Buck Institute for Research on Aging

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow