Specific Aim 1: Investigate p53 and microRNAs as molecular nodes in replicative stress and stem cell biology Hypothesis: p53 isoforms contribute to neurodegeneration through the regulation of cellular senescence. Neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), are major medical challenges of the 21st century. Astrocytes are the most abundant cell type in the brain and play multiple key roles in providing structural, functional, and metabolic support to neurons. Astrocytes can exert both neuroprotective and neurotoxic roles. Their neurotoxic effect is mediated via senescence-associated secretory phenotype (SASP), including the production and secretion of pro-inflammatory cytokines IL-6 and IL-8, which increases with aging and neurodegenerative diseases. We have revealed that human astrocytes express p53 isoforms, delta133p53 and p53beta, and that these p53 isoforms regulate SASP in astrocytes and their protective and toxic effects on neurons. ALS and AD brain tissues, as well as senescent astrocytes in vitro after serial passaging, showed decreased delta133p53 and increased p53beta, which are the senescence-associated expression signature observed in other cell types including fibroblasts, CD8+ T-lymphocytes and pre-malignant colon adenoma. These changes were attributed to selective autophagy-mediated degradation of delta133p53 and SRSF3-mediated alternative mRNA splicing generating p53beta, which are again conserved across different cell types. Early-passage astrocytes with delta133p53 knockdown or p53beta overexpression (which reproduces the expression signature in neurodegenerative diseases and senescent astrocytes) were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Importantly, restored expression of delta133p53 in near-senescent, neurotoxic astrocytes resulted in repressed SASP, elevated neurotrophic growth factors (NGF and IGF-1) and enhanced neuroprotection in co-culture (i.e., increased neuronal survival and decreased neuronal apoptosis). These findings, especially the delta133p53-induced reversion of neurotoxic astrocytes to neuroprotective ones, indicate that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases. We are initiating a high-throughput screening to identify small molecule compounds that modulate the expression or activity of delta133p53 and p53beta. Hypothesis: Delta133p53 rescues progeria cells from premature senescence and DNA damage. Werner syndrome (WS) and Hutchinson-Gilford progeria syndrome (HGPS) are human disorders characterized by aging-associated phenotypes that occur earlier in life than normal. Cells derived from WS or HGPS patients have decreased cell proliferation potential, show impaired DNA damage response, and are prematurely induced into cellular senescence. We have found that in vitro cultured WS and HGPS fibroblasts show the senescence-associated expression signature (i.e., decreased delta133p53 and increased p53beta) at an earlier passage than normal fibroblasts, prompting us to examine whether this p53 isoform expression signature contributes to premature senescent phenotypes in these progeria-derived cells. Our current data indicate that restored expression of delta133p53 in WS and HGPS fibroblasts rescues them from premature senescence and extends their replicative lifespan at least by 15 population doubling levels. These effects of delta133p53 are attributed to the downregulation of p21WAF1 and microRNA-34a through its dominant-negative inhibition of full-length p53. In addition, delta133p53 is suggested to function independently of full-length p53 to upregulate a set of DNA repair genes including Rad51. We have also found that delta133p53 exerts similar effects (i.e., inhibition of premature senescence and extension of replicative lifespan) in mouse fibroblasts derived from a mouse model of HGPS (Lmna G609G/G609G knock-in mice), which provides a basis towards generating a new mouse model to examine in vivo effects of delta133p53 on progeria pathologies. Senescence inhibition and lifespan extension in HGPS fibroblasts can be used as cellular phenotypes to identify small molecule compounds that upregulate or activate delta133p53. Hypothesis: p53 isoforms are physiological regulators of human pluripotent stem cells. We previously showed that delta133p53 increases the efficiency of reprogramming from human fibroblasts to iPS cells likely through repression of p53 target genes involved in cellular senescence (e.g., p21WAF1 and microRNA-34a). Our new data show that delta133p53, which is abundantly expressed in undifferentiated iPS, becomes downregulated during differentiation to neural stem cells and to mature neurons, further supporting a functional link between this p53 isoform and the acquisition and maintenance of an undifferentiated, self-renewing state in human pluripotent stem cells. To elucidate the role of abundant levels of endogenous delta133p53 in undifferentiated ES and iPS cells, we have successfully optimized the conditions for efficient transfection of siRNA specifically knocking down delta133p53. Our preliminary data showed that the siRNA-mediated knockdown of delta133p53 primed an ES cell line for neural differentiation and induced an iPS cell line into cellular senescence. Further experiments are ongoing to examine the molecular mechanisms by which delta133p53 controls differentiation and senescence in ES and iPS cells.
Specific Aim 2 : Define the Role of p53 Isoforms and Mutant Variants in Control of Cellular Division of Normal and Cancer Cells Hypothesis: p53 isoforms and mutants have gain-of-function activities. Cancer-associated mutants of full-length p53 can promote tumorigenesis in the absence of wild-type p53 (so-called gain-of-function mutants). Because of a possible mutant p53 conformation of delta133p53, we hypothesize that this p53 isoform and its mutant versions also have gain-of-function activities. We have generated p53-null cell lines (fibroblast and lung cancer cell lines) that inducibly express wild-type delta133p53 and mutants (V157F, R175H, R249S and R275H). We performed a microarray-based expression profiling of mRNA and a Nanostring-based expression profiling of microRNA upon induction of these wild-type and mutant delta133p53. The data are being analyzed to identify genes and signaling pathways regulated by their gain-of-function activities. Because gain-of-function mutants of full-length p53 can function by interacting with and inhibiting the p53 family members p63 and p73, we are examining whether wild-type and mutant delta133p53 also interact with p63 and p73.
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