Δ133p53α, a human/primate-specific p53 protein isoform, delays or inhibits the induction of the cellular senescence and associated secretory phenotype (SASP) in various types of human cells, including astrocytes from neurodegenerative diseases, Hutchinson-Gilford progeria syndrome (HGPS) fibroblasts, and exhausted CD8+ T-cells. A major regulatory mechanism of Δ133p53α expression in these human cells is protein degradation via chaperone-assisted selective autophagy (CASA). In this study, we describe a novel cell-based quantitative high-throughput screening (qHTS) assay using fluorescently labeled Δ133p53α to screen large chemical libraries for the identification of compounds that upregulate the Δ133p53α protein level. qHTS enabled the comprehensive and reliable profiling of over 10,000 small-molecule compounds. It also provided an opportunity to repurpose compounds and gain new mechanistic insights into the pathways regulating Δ133p53α expression. We successfully identified two candidate compounds, AZD1981 and celastrol, which were shown to upregulate the fluorescently labeled Δ133p53α protein, as well as the endogenous Δ133p53α protein in primary human astrocytes and the normal lung fibroblasts MRC-5, leading to reduced induction of cellular senescence and SASP factor secretion. The identification of celastrol, an inducer of heat shock protein 70 (HSP70) chaperones, is consistent with the CASA-mediated regulation of Δ133p53α protein turnover. Together with our previous findings of Δ133p53α overexpression-induced restoration of cell proliferation and function in cells otherwise approaching senescence, we propose that these two compounds may have therapeutic potential in senescence- and aging-associated diseases. The qHTS assay developed in this study could be used to identify new therapies for these diseases.

Cellular senescence is an important contributor to aging and age-related diseases such as Alzheimer's disease (AD). Senescent cells are characterized by a durable cell proliferation arrest and the acquisition of a proinflammatory senescence-associated secretory phenotype (SASP), which participates in the progression of neurodegenerative disorders. Clearance of senescent glial cells in an AD mouse model prevented cognitive decline suggesting pharmacological agents targeting cellular senescence might provide novel therapeutic approaches for AD. Δ133p53α, a natural protein isoform of p53, was previously shown to be a negative regulator of cellular senescence in primary human astrocytes, with clinical implications from its diminished expression in brain tissues from AD patients. Here we show that treatment of proliferating human astrocytes in culture with amyloid-beta oligomers (Aβ), an endogenous pathogenic agent of AD, results in reduced expression of Δ133p53α, as well as induces the cells to become senescent and express proinflammatory SASP cytokines such as IL-6, IL-1β and TNFα. Our data suggest that Aβ-induced astrocyte cellular senescence is associated with accelerated DNA damage, and upregulation of full-length p53 and its senescence-inducing target gene p21WAF1. We also show that exogenously enhanced expression of Δ133p53α rescues human astrocytes from Aβ-induced cellular senescence and SASP through both protection from DNA damage and dominant-negative inhibition of full-length p53, leading to inhibition of Aβ-induced, astrocyte-mediated neurotoxicity. The results presented here demonstrate that Δ133p53α manipulation could modulate cellular senescence in the context of AD, possibly opening new therapeutic avenues.

Tau accumulation is a core component of Alzheimer's disease and other neurodegenerative tauopathies. While tau's impact on neurons is a major area of research, the effect of extracellular tau on astrocytes is largely unknown. This article summarizes our recent studies showing that astrocyte senescence plays a critical role in neurodegenerative diseases and integrates extracellular tau into the regulatory loop of senescent astrocyte-mediated neurotoxicity. Human astrocytes in vitro undergoing senescence were shown to acquire the inflammatory senescence-associated secretory phenotype (SASP) and toxicity to neurons, which may recapitulate aging- and disease-associated neurodegeneration. Here, we show that human astrocytes exposed to extracellular tau in vitro also undergo cellular senescence and acquire a neurotoxic SASP (e.g. IL-6 secretion), with oxidative stress response (indicated by upregulated NRF2 target genes) and a possible activation of inflammasome (indicated by upregulated ASC and IL-1β). These findings suggest that senescent astrocytes induced by various conditions and insults, including tau exposure, may represent a therapeutic target to inhibit or delay the progression of neurodegenerative diseases. We also discuss the pathological activity of extracellular tau in microglia and astrocytes, the disease relevance and diversity of tau forms, therapeutics targeting senescence in neurodegeneration, and the roles of p53 and its isoforms in astrocyte-mediated neurotoxicity and neuroprotection.

Cellular senescence (CS) and senescence-associated secretory phenotype (SASP) contribute to aging and age-related diseases. Mechanistically, the stress sensor p53 plays a pivotal role in the initiation and maintenance of CS. In humans, TP53 is expressed as 12 isoforms that contribute to the fine-tuning of p53 activity. Delta133p53 is generated from an alternative promoter located in intron 4 and is therefore devoid of both the transactivation domain and part of the DNA binding domain. Delta133p53 is predominantly located in the nucleus and is largely regulated at the protein level through chaperone-assisted selective autophagic degradation. Delta133p53 counteracts p53-mediated replicative CS and reduces the secretion of SASP cytokines. Delta133p53 is expressed in most normal tissues, but its expression is deregulated in age-associated diseases such as cancer, neurodegenerative diseases, and premature aging disorders. Delta133p53 is downregulated in exhausted CD8+ T cells, nearly senescent fibroblasts from Hutchinson-Gilford progeria syndrome patients, and astrocytes from irradiated brains and Alzheimer’s disease and amyotrophic lateral sclerosis patients. Delta133p53 overexpression extends the replicative lifespan in normal cells but does not cause immortalization or malignant transformation. Hence, delta133p53 appears to be a safe, novel therapeutic target to regulate CS. The present study aims at identifying small-molecule compounds that stabilize or prevent the degradation of delta133p53 protein and inhibit CS. To screen for activators of delta133p53, we developed two cell-based high-throughput screening (HTS) assays using an inducible vector for the expression of an eGFP-tag recombinant protein, and a luminescent peptide tag (HiBiT) appended onto the endogenous gene. The biologic activity of the fusion proteins was confirmed by evaluating the i) nuclear localization, ii) autophagic degradation during CS, and iii) ability to delay replicative CS. In collaboration with the National Center for Advancing Translational Sciences (NCATS), we screened a collection of ~8,350 compounds that includes all drugs that have been approved for use by the US Food and Drug Administration. Selected compounds were then screened in a secondary assay to evaluate their efficacy at reducing SASP cytokines secretion. Several candidate compounds are currently being investigated to confirm their effect on endogenous delta133p53 expression and CS. We have established robust cell-based HTS assays to screen for activators of delta133p53 and identified candidate compounds that could potentially develop into novel therapeutic leads to treat major life-threatening diseases. Citation Format: Delphine Lissa, Kyra Ungerleider, Izumi Horikawa, Patricia Dranchak, Erin Oliphan, Jessica Beck, Sebastien Jo, James Inglese, Curtis C. Harris. Targeting Delta133p53 isoform with small-molecule compounds to modulate cellular senescence [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 19.

Cellular senescence (CS) is an important contributor to aging and age-related diseases such as Alzheimer's Disease (AD). Senescent cells are characterized by a durable cell cycle arrest and the acquisition of a senescence-associated secretory phenotype (SASP). Clearance of senescent astrocytes and microglia in an AD mouse model prevented tau-dependent pathology and cognitive decline suggesting pharmacological agents targeting CS might provide novel therapeutic approaches for AD. The tumor suppressor gene p53 plays a pivotal role in the initiation and maintenance of CS. Upregulation of p53 induces tau phosphorylation and inhibition of p53 has been shown to attenuate amyloid-beta mediated neuronal death. Our lab has shown delta133p53 regulates replicative CS by acting as a dominant-negative to full-length p53. We observe high levels of delta133p53 in proliferative cells, while conversely senescent cells, such as astrocytes from AD patients, have decreased levels of the isoform. Overexpression of delta133p53 restores the replicative capacity of near-senescent astrocytes and decreases the secretion of several SASP factors including interleukin 6 (IL-6). Our study aims at identifying small molecule compounds that increase delta133p53 expression to counteract CS. We developed a cell-based high-throughput screening assay using cell lines generated to express a translational reporter system, where the delta133p53 cDNA sequence is cloned in frame with the GFP. The biological activity of the fusion protein was confirmed by evaluating its nuclear localization, autophagic degradation and ability to inhibit CS. In collaboration with the National Center for Advancing Translational Sciences (NCATS), we screened two large chemical libraries consisting of approximately 8,500 compounds. Following the fluorescent-based primary screening assay, 95 compounds were screened in a secondary assay to evaluate their efficacy at reducing astrocyte secretion of IL-6 following irradiation. We are currently confirming the activity of 13 compounds on endogenous delta133p53 levels. We will further validate the candidate compounds by assessing their effect on DNA damage, cell proliferation, SASP and CS. Additionally, we plan to screen the Genesis Chemical Library (95,744 compounds), that contains novel chemotypes which have the potential to form new drugs. This first drug discovery program to target delta133p53 could potentially lead to the development of a first-in-class drug.

Traumatic brain injuries (TBIs) are a common and costly ongoing public health concern. Injuries that occur during childhood development can have particularly profound and long-lasting effects. One common consequence and potential mediator of negative outcomes of TBI is sleep disruption which occurs in a substantial proportion of TBI patients. These individuals report greater incidences of insomnia and sleep fragmentation combined with a greater overall sleep requirement meaning that many patients are chronically sleep-deprived. We sought to develop an animal model of developmental TBI-induced sleep dysfunction. Specifically, we tested the hypothesis that early (postnatal day 21), repeated closed head injuries in Swiss-Webster mice, would impair basal and homeostatic sleep responses in adulthood. Further, we asked whether environmental enrichment (EE), a manipulation that improves functional recovery following TBI and has been shown to alter sleep physiology, would prevent TBI-induced sleep dysfunction and alter sleep-modulatory peptide expression. In contrast to our hypothesis, the mild, repeated head injury that we used did not significantly alter basal or homeostatic sleep responses in mice housed in standard laboratory conditions. Sham-injured mice housed in enriched environments exhibited enhanced rapid eye movement (REM) sleep and expression of the REM-promoting peptide pro-melanin-concentrating hormone, an effect that was not apparent in TBI mice housed in enriched environments. Thus, TBI blocked the REM-enhancing effects of EE. This work has important implications for the management and rehabilitation of the TBI patient population.