An aco-2::gfp knock-in enables the monitoring of mitochondrial morphology throughout C. elegans lifespan.

We used CRISPR/Cas9 gene editing in C. elegans in order to fluorescently tag endogenous aconitase-2 (ACO-2). ACO-2 is a mitochondrially localized protein, and the aco-2::gfp strain enabled the examination of native mitochondrial morphology in live animals. Here we validate that the aco-2::gfp strain displays the prototypic changes in mitochondrial morphology known to occur during aging and upon paraquat (PQ) induced mitochondrial stress. We also provide evidence that the ACO-2::GFP reporter can serve as a superior means for tracking mitochondrial morphology than conventional MitoTracker dyes-especially in aged-worms.

Screening Method for Identifying Toxicants Capable of Inducing Astrocyte Senescence.

Cellular senescence is a tumor-suppressive mechanism which leads to near irreversible proliferative arrest. However, senescent cells can cause tissue dysfunction, in large part because they express a senescence-associated secretory phenotype (SASP) involving secretion of, amongst other factors, proinflammatory cytokines known to compromise neuronal health. Therefore, established neurotoxicants may cause neurotoxicity in vivo, in part by triggering mitotic cells in the brain to undergo senescence and adopt an inflammatory SASP which in turn could cause deleterious effects to surrounding neurons. To begin to address this hypothesis, we examined whether we could screen known neurotoxicants for their ability to cause astrocytes (a mitotic cell type especially important for maintaining neuronal health) to undergo senescence. For this purpose, we utilized inducible pluripotent stem cell-derived human astrocytes and screened an 80 compound neurotoxicant library provided by the Biomolecular Screening Branch of the NIEHS National Toxicology Program. Here we present a screening method based on induction of the senescent marker, senescent-associated beta-galactosidase (SA-ß-gal). We describe in detail an automated method for the unbiased quantitation of percentage of SA-ß-gal + astrocytes. Although our results suggest that conducting an SA-ß-gal senescence screen using human inducible pluripotent stem cell-derived astrocytes may be feasible, they also highlight challenges that likely preclude its adaptation to high-throughput. We also explore the possibility of using primary mouse astrocytes for this purpose and explain why this platform is problematic and very unlikely to yield meaningful results, even in small screens with compound replicates.

Cellular Senescence Is Induced by the Environmental Neurotoxin Paraquat and Contributes to Neuropathology Linked to Parkinson's Disease.

Exposure to the herbicide paraquat (PQ) is associated with an increased risk of idiopathic Parkinson's disease (PD). Therapies based on PQ's presumed mechanisms of action have not, however, yielded effective disease therapies. Cellular senescence is an anticancer mechanism that arrests proliferation of replication-competent cells and results in a pro-inflammatory senescence-associated secretory phenotype (SASP) capable of damaging neighboring tissues. Here, we demonstrate that senescent cell markers are preferentially present within astrocytes in PD brain tissues. Additionally, PQ was found to induce astrocytic senescence and an SASP in vitro and in vivo, and senescent cell depletion in the latter protects against PQ-induced neuropathology. Our data suggest that exposure to certain environmental toxins promotes accumulation of senescent cells in the aging brain, which can contribute to dopaminergic neurodegeneration. Therapies that target senescent cells may constitute a strategy for treatment of sporadic PD, for which environmental exposure is a major risk factor.

Cellular senescence and the aging brain.

Cellular senescence is a potent anti-cancer mechanism that arrests the proliferation of mitotically competent cells to prevent malignant transformation. Senescent cells accumulate with age in a variety of human and mouse tissues where they express a complex 'senescence-associated secretory phenotype' (SASP). The SASP includes many pro-inflammatory cytokines, chemokines, growth factors and proteases that have the potential to cause or exacerbate age-related pathology, both degenerative and hyperplastic. While cellular senescence in peripheral tissues has recently been linked to a number of age-related pathologies, its involvement in brain aging is just beginning to be explored. Recent data generated by several laboratories suggest that both aging and age-related neurodegenerative diseases are accompanied by an increase in SASP-expressing senescent cells of non-neuronal origin in the brain. Moreover, this increase correlates with neurodegeneration. Senescent cells in the brain could therefore constitute novel therapeutic targets for treating age-related neuropathologies.