Aging is considered a risk factor for developing many chronic diseases which includes cardiovascular diseases, cancer, and neurodegenerative diseases. Researchers are now showing that many of these diseases are associated with cellular senescence. Cellular senescence is an important process for the removal of damaged cells, but if senescent cells are not removed a chronic pro-inflammatory environment ensues, increasing the risk of many age-related diseases. These cells accumulate as a result of cancer chemotherapy, are associated with age-related diseases, and are important for development. Recent work has shown that specific targeting of senescent cells results in increased life expectancy in a progeroid Ercc1-/Δ mouse model. As such there is great interest in identifying, characterizing and targeting senescent cells. Senescent cells are identified by the release of pro-inflammatory cytokines and chemokines, increased beta galactosidase activity (b-gal), senescence associated heterochromatin foci (SAHF), and changes in morphology which are increased size and a flattened appearance. There is no single good marker for determining senescence, so these cells are identified through the collective interpretation of multiple markers. Senescence associated b-gal (SA-b-gal) is considered the “gold standard” for identifying senescent cell, and it is detected with the blue-green precipitating colorimetric x-gal substrate. However, this substrate is limited use in that it cannot be used in flow cytometry or other fluorescence based platforms. Even though C12FDG, a fluorescein based b-gal substrate, has been used since the mid 90’s, it has limited utility restricted by its lack of fix-ability. Newer b-gal substrates are demonstrated here which do not leak out of cell; enabling researchers to more easily identify and evaluate cellular senescence. In this study we use a fluorescence approach to assess the senescence phenotype based on a combination of western blots, fluorescence based imaging and ICC, flow cytometry, cell sorting, and RT-qPCR. We examined several cell-based models include replicative senescence characterized by telomere shortening in primary fibroblasts, cellular senescence characterized through DNA damage pathway by doxorubicin, oxidative stress senescence induced by confluency in atmospheric oxygen environment (SIPS), and senescence induced with palbociclib treatment (a CDK 4/6 inhibitor). Overall, we found that phenotypic characteristics of senescence vary depending on the cellular model and that a multiplex fluorescence based approach to identifying senescent cells will contribute to the understanding of cellular senescence and the age-related diseases. Citation Format: Scott T. Clarke, Taryn Jackson, Amanda Kelley, Quentin Low, Timothy Huang, Yi-Zhen Hu, Veronica Calderon, Rong Wang, Michael O'Grady. Evaluation of cellular senescence through fluorescence characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 898.

The dicistronic Drosophila stoned mRNA produces two proteins, stonedA and stonedB, that are localized at nerve terminals. While the stoned locus is required for synaptic-vesicle cycling in neurons, distinct or overlapping synaptic functions of stonedA and stonedB have not been clearly identified. Potential functions of stoned products in nonneuronal cells remain entirely unexplored in vivo. Transgene-based analyses presented here demonstrate that exclusively neuronal expression of a dicistronic stoned cDNA is sufficient for rescue of defects observed in lethal and viable stoned mutants. Significantly, expression of a monocistronic stonedB trangene is sufficient for rescuing various phenotypic deficits of stoned mutants, including those in organismal viability, evoked transmitter release, and synaptotagmin retrieval from the plasma membrane. In contrast, a stonedA transgene does not alleviate any stoned mutant phenotype. Novel phenotypic analyses demonstrate that, in addition to regulation of presynaptic function, stoned is required for regulating normal growth and morphology of the motor terminal; however, this developmental function is also provided by a stonedB transgene. Our data, although most consistent with a hypothesis in which stonedA is a dispensable protein, are limited by the absence of a true null allele for stoned due to partial restoration of presynaptic stonedA by transgenically provided stonedB. Careful analysis of the effects of the monocistronic transgenes together and in isolation clearly reveals that the presence of presynaptic stonedA is dependent on stonedB. Together, our findings improve understanding of the functional relationship between stonedA and stonedB and elaborate significantly on the in vivo functions of stonins, recently discovered phylogenetically conserved stonedB homologs that represent a new family of “orphan” medium (μ) chains of adaptor complexes involved in vesicle formation. Data presented here also provide new insight into potential mechanisms that underlie translation and evolution of the dicistronic stoned mRNA.

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