quantification-insoluble-protein-aggregation-caenorhabditis-elegans

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

Reactive α-dicarbonyls (α-DCs), like methylglyoxal (MGO), accumulate with age and have been implicated in aging and various age-associated pathologies, such as diabetic complications and neurodegenerative disorders like Alzheimer's and Parkinson's diseases. Evolutionarily conserved glyoxalases are responsible for α-DC detoxification; however, their core biochemical regulation has remained unclear. We have established a Caenorhabditis elegans model, based on an impaired glyoxalase (glod-4/GLO1), to broadly study α-DC-related stress. We show that, in comparison to wild-type (N2, Bristol), glod-4 animals rapidly exhibit several pathogenic phenotypes, including hyperesthesia, neuronal damage, reduced motility, and early mortality. We further demonstrate TRPA-1/TRPA1 as a sensor for α-DCs, conserved between worms and mammals. Moreover, TRPA-1 activates SKN-1/Nrf via calcium-modulated kinase signaling, ultimately regulating the glutathione-dependent (GLO1) and co-factor-independent (DJ1) glyoxalases to detoxify α-DCs. Interestingly, this pathway is in stark contrast to the TRPA-1 activation and the ensuing calcium flux implicated in cold sensation in C. elegans, whereby DAF-16/FOXO gets activated via complementary kinase signaling. Finally, a phenotypic drug screen using C. elegans identified podocarpic acid as a novel activator of TRPA1 that rescues α-DC-induced pathologies in C. elegans and mammalian cells. Our work thus identifies TRPA1 as a bona fide drug target for the amelioration of α-DC stress, which represents a viable option to address aging-related pathologies in diabetes and neurodegenerative diseases.

A Caenorhabditis elegans Model Elucidates a Conserved Role for TRPA1-Nrf Signaling in Reactive α-Dicarbonyl Detoxification.

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.

Alzheimer's disease (AD) is a neurodegenerative disorder and the leading cause of dementia in aged populations worldwide. The deposition of toxic protein aggregates such as amyloid beta (Aβ) is a hallmark of AD, and there is growing awareness that a key driver of AD pathogenesis is the neuroinflammatory cascade triggered and sustained by these proteins. Consequently, interventions that suppress prolonged neuroinflammation represent viable therapeutic approaches for AD. In this context, we tested the natural product gedunin which is an anti-inflammatory molecule, found in the seeds of the neem tree (Azadirachta indica), whose mechanism of action remains to be fully elucidated. Using a mouse microglia cell line (IMG), we show that gedunin suppresses neuroinflammation arising from Aβ oligomer exposure. Our results demonstrate that gedunin suppresses Aβ-induced NF-κB activation and its targets, including nitric oxide (NO) and IL-1β, known proinflammatory molecules. Further, we show that gedunin inhibits neuroinflammation by activating nuclear factor 2 erythroid-related factor 2 (Nrf2) and its downstream targets γ-glutamylcysteine synthetase, heme oxygenase 1, and NADPH quinone dehydrogenase 1, which are involved in quenching reactive oxygen and nitrogen species (NO) generated by NF-κB activation. Nrf2 activation appears essential for the anti-inflammatory effect because when silenced, the proinflammatory effects of Aβ are enhanced and the protective effect of gedunin against NO production is reduced. Additionally, using human neuronal cells (SH-SY5Y), we show that gedunin prevents neurotoxicity secondary to Aβ-induced microglial activation. In conclusion, our findings highlight a potential therapeutic role of gedunin in neurodegenerative diseases.

Gedunin Inhibits Oligomeric Aβ-Induced Microglia Activation Via Modulation of Nrf2-NF-κB Signaling.

The goal of the Caenorhabditis Intervention Testing Program is to identify robust and reproducible pro-longevity interventions that are efficacious across genetically diverse cohorts in the Caenorhabditis genus. The project design features multiple experimental replicates collected by three different laboratories. Our initial effort employed fully manual survival assays. With an interest in increasing throughput, we explored automation with flatbed scanner-based Automated Lifespan Machines (ALMs). We used ALMs to measure survivorship of 22 Caenorhabditis strains spanning three species. Additionally, we tested five chemicals that we previously found extended lifespan in manual assays. Overall, we found similar sources of variation among trials for the ALM and our previous manual assays, verifying reproducibility of outcome. Survival assessment was generally consistent between the manual and the ALM assays, although we did observe radically contrasting results for certain compound interventions. We found that particular lifespan outcome differences could be attributed to protocol elements such as enhanced light exposure of specific compounds in the ALM, underscoring that differences in technical details can influence outcomes and therefore interpretation. Overall, we demonstrate that the ALMs effectively reproduce a large, conventionally scored dataset from a diverse test set, independently validating ALMs as a robust and reproducible approach toward aging-intervention screening.

Automated lifespan determination across Caenorhabditis strains and species reveals assay-specific effects of chemical interventions.

Mutations in the human ATP13A2 gene are associated with an early-onset form of Parkinson's disease (PD) known as Kufor Rakeb Syndrome (KRS). Patients with KRS show increased iron deposition in the basal ganglia, suggesting iron toxicity-induced neurodegeneration as a potential pathogenesis associated with the ATP13A2 mutation. Previously we demonstrated that functional losses of ATP13A2 disrupt the lysosomes ability to store excess iron, leading to reduce survival of dopaminergic neuronal cells. To understand the possible mechanisms involved, we studied a Caenorhabditis elegans mutant defective in catp-6 function, an ortholog of human ATP13A2 gene. Here we show that catp-6 mutant worms have defective autophagy and lysosomal function, demonstrate characteristic PD phenotypes including reduced motor function and dysregulated iron metabolism. Additionally, these mutants have defective mitochondrial health, which is rescuable via iron chelation or mitophagy induction.

Dysregulated iron metabolism in C. elegans catp-6/ATP13A2 mutant impairs mitochondrial function.

Cell senescence is a process that causes growth arrest and the release of a senescence associated secretory phenotype (SASP), characterized by secretion of chemokines, cytokines, cell growth factors and metalloproteases, leading to a tissue condition that may precipitate cancers and neurodegenerative processes. With the recent pandemic of coronavirus, senolytic drugs are being considered as possible therapeutic tools to reduce the virulence of SARS-CoV-2. In the last few years, our research group showed that lithium carbonate at microdose levels was able to stabilize memory and change neuropathological characteristics of Alzheimer's disease (AD). In the present work, we present evidence that low-dose lithium can reduce the SASP of human iPSCs-derived astrocytes following acute treatment, suggesting that microdose lithium could protect cells from senescence and development of aging-related conditions. With the present findings, a perspective of the potential use of low-dose lithium in old patients from the "high risk group" for COVID-19 (with hypertension, diabetes and chronic obstructive pulmonary disease) is presented.

Manish Chamoli

Buck Institute for Research on Aging

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