p53, Aging, and Cancer

假设：p53同工异构体通过调控细胞衰老参与神经退行性变。神经退行性疾病，如阿尔茨海默病（AD）和肌萎缩侧索硬化症（ALS），是21世纪的主要医学挑战。星形胶质细胞是大脑中最丰富的细胞类型，在为神经元提供结构、功能和代谢支持方面发挥着多种关键作用。星形胶质细胞可以发挥神经保护和神经毒性作用。它们的神经毒性作用是通过衰老相关分泌表型（SASP）介导的，包括促炎细胞因子IL-6和IL-8的产生和分泌，IL-6和IL-8随着衰老和神经退行性疾病而增加。我们发现人类星形胶质细胞表达p53亚型，delta133p53和p53beta，这些p53亚型调节星形胶质细胞中的SASP及其对神经元的保护和毒性作用。在体外连续传代的ALS和AD脑组织以及衰老的星形胶质细胞中，delta133p53减少，p53beta增加，这是在成纤维细胞、CD8+ t淋巴细胞和恶性前结肠腺瘤等其他细胞类型中观察到的衰老相关表达特征。这些变化归因于选择性自噬介导的delta133p53降解和srsf3介导的替代mRNA剪接产生p53β，这在不同的细胞类型中也是保守的。delta133p53敲低或p53beta过表达的早期星形胶质细胞（再现神经退行性疾病和衰老星形胶质细胞的表达特征）在与神经元共培养时被诱导表现出SASP并发挥神经毒性。重要的是，在近衰老的神经毒性星形胶质细胞中，恢复delta133p53的表达导致SASP受到抑制，神经营养生长因子（NGF和IGF-1）升高，并在共培养中增强神经保护（即增加神经元存活和减少神经元凋亡）。这些发现，特别是delta133p53诱导的神经毒性星形细胞向神经保护性星形细胞的逆转，表明p53亚型及其调控机制是神经退行性疾病治疗干预的潜在靶点。我们正在启动一项高通量筛选，以确定调节delta133p53和p53beta表达或活性的小分子化合物。假设：Delta133p53拯救早衰细胞免于过早衰老和DNA损伤。Werner综合征（WS）和Hutchinson-Gilford早衰综合征（HGPS）是人类疾病，其特征是与衰老相关的表型，发生时间比正常情况更早。来自WS或HGPS患者的细胞增殖能力下降，DNA损伤反应受损，并过早诱导细胞衰老。我们发现体外培养的WS和HGPS成纤维细胞比正常成纤维细胞在更早的传代中表现出衰老相关的表达特征（即delta133p53减少和p53beta增加），这促使我们研究这种p53同型表达特征是否有助于这些早衰性细胞的过早衰老表型。我们目前的数据表明，在WS和HGPS成纤维细胞中恢复delta133p53的表达可以使它们免于过早衰老，并将它们的复制寿命延长至少15倍。delta133p53的这些作用是通过其对全长p53的显性阴性抑制，下调p21WAF1和microRNA-34a。此外，delta133p53可能独立于全长p53，上调包括Rad51在内的一系列DNA修复基因。我们还发现，delta133p53在HGPS小鼠模型（Lmna G609G/G609G敲入小鼠）衍生的小鼠成纤维细胞中具有类似的作用（即抑制过早衰老和延长复制寿命），这为建立新的小鼠模型来研究delta133p53对早衰病理的体内影响提供了基础。HGPS成纤维细胞的衰老抑制和寿命延长可以作为细胞表型来鉴定上调或激活delta133p53的小分子化合物。假设：p53亚型是人类多能干细胞的生理调节因子。我们之前的研究表明，delta133p53可能通过抑制参与细胞衰老的p53靶基因（如p21WAF1和microRNA-34a），提高了从人成纤维细胞到iPS细胞的重编程效率。我们的新数据显示，在未分化的iPS中大量表达的delta133p53在向神经干细胞和成熟神经元的分化过程中变得下调，进一步支持了这种p53亚型与人类多能干细胞获得和维持未分化、自我更新状态之间的功能联系。为了阐明丰富的内源性delta133p53在未分化的ES和iPS细胞中的作用，我们成功地优化了高效转染特异性敲除delta133p53的siRNA的条件。我们的初步数据显示，sirna介导的delta133p53敲低启动了ES细胞系的神经分化，诱导了iPS细胞系的细胞衰老。进一步的实验正在进行中，以研究delta133p53控制胚胎干细胞和iPS细胞分化和衰老的分子机制。特异性目标2：确定p53同工型和突变变体在控制正常细胞和癌细胞分裂中的作用假设：p53同工型和突变体具有功能获得活性。全长p53的癌症相关突变可以在缺乏野生型p53的情况下促进肿瘤发生（所谓的“功能获得”突变）。由于delta133p53可能存在突变型p53构象，我们假设该p53异构体及其突变型也具有功能获得活性。我们已经产生了p53-null细胞系（成纤维细胞和肺癌细胞系），诱导表达野生型delta133p53和突变体（V157F, R175H， R249S和R275H）。我们对这些野生型和突变型delta133p53进行了基于微阵列的mRNA表达谱分析和基于纳米链的microRNA表达谱分析。正在分析这些数据，以确定由其功能获得活动调节的基因和信号通路。由于全长p53的功能获得突变体可以通过与p53家族成员p63和p73相互作用并抑制其功能，因此我们正在研究野生型和突变型delta133p53是否也与p63和p73相互作用。

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.