Roles for Intracellular pH Dynamics in Cancer

摘要 癌症和阿尔茨海默病相关痴呆（ADRD）越来越多地被认为是与阿尔茨海默病相关的疾病。 溶酶体失调虽然溶酶体功能严重依赖于约5.0的内腔酸性pH 对于所含的酸活化水解酶的活性，溶酶体pH（pHlys）是否失调， 癌症和ADRD中溶酶体功能受损的决定因素受到的关注有限。支持下 从我们关于细胞内pH动力学和癌症的母基金CA 197856中，我们产生并验证了一种新的 基因编码的pHlys生物传感器，pHLARE（pH溶酶体活性报告），其是唯一的pHlys传感器 可以在细胞中繁殖用于纵向研究。我们使用pHLARE来显示， 来自不同组织来源的人类癌细胞，与组织相比具有不同的突变特征， 匹配未转化的细胞，并确定新的药理学和遗传学方法， 更换pH值。在我们的父母资助下，我们正在使用这些新方法来确定如何 降低的pHlys使癌细胞的行为。我们的补充将使用这些新的方法开发 通过我们的父母补助金，以解决ADRD中尚未解决的pHlys问题，因此，非常适合 NOT-CA-20-019资金。我们的补充测试的假设，ADRD增加了pH值， 降低内腔水解酶和大分子催化剂的活性，导致增加 蛋白质聚集和神经变性。在目标1中，我们将定量表达ADRD模型中的pHlys， pHLARE，包括被工程化用于损失颗粒蛋白前体和早老素1的神经上皮细胞， ADRD中的决定因素和与受损的溶酶体功能相关，以及从ADRD分化的神经元 NHCDR产生的iPSC来源于具有确定的ADRD的患者和未受影响的家族突变 携带者和非携带者。在目标2中，我们将确定ADRD中pHlys失调的功能后果 细胞模型，通过使用药理学和遗传学方法，我们在我们的研究中发现， 更换pH值。使用目标1中描述的ADRD相关细胞模型，我们将确定pHlys在ADRD相关细胞中的作用。 蛋白质聚集的动力学，ADRD的标志，以及神经元形态和溶酶体 定位和移动性，在ADRD中受损，导致富含溶酶体的末端轴突扩大 sweetie。我们还将使用延时显微镜来确定细胞的存活率， 为纵向研究提供了新的工具。结果包括解决pHlys的功能后果 动力学和溶酶体功能失调。此外，我们对癌症中pHlys的平行研究 （父母补助）和ADRD（补充）有希望确定新的治疗方法， pHlys失调以限制这些疾病的进展。

Abstract Cancers and Alzheimer’s Disease Related Dementias (ADRDs) are increasingly recognized as diseases with dysregulated lysosomes. Although lysosome functions are critically dependent on a lumenal acidic pH of ~ 5.0 for the activity of contained acid-activated hydrolases, whether lysosome pH (pHlys) is dysregulated and a determinant for impaired lysosome functions in cancers and ADRDs has received limited attention. With support from our parent grant CA197856 on intracellular pH dynamics and cancer we generated and validated a new genetically encoded pHlys biosensor, pHLARE (pH Lysosomal Activity REporter), which is the only pHlys sensor that can be propagated in cells for longitudinal studies. We used pHLARE to show a significantly lower pHlys in human cancer cells from different tissue origins and with different mutational signatures compared with tissue- matched untransformed cells, and to identify new pharmacological and genetic approaches to experimentally change pHlys. With funding from our parent grant we are using these new approaches to determine how decreased pHlys enables cancer cell behaviors. Our supplement will use these new approaches developed through our parent grant to address unresolved questions on pHlys in ADRDs, and hence, is ideally suited for NOT-CA-20-019 funding. Our supplement tests the hypothesis that ADRDs have increased pHlys, which decreases the activity of lumenal hydrolases and macromolecular catabolism, leading to increased protein aggregation and neurodegeneration. In Aim 1 we will quantify pHlys in ADRD models expressing pHLARE, including neuroepithelial cells engineered for loss of progranulin and presenilin 1, which are determinants in ADRDs and associated with impaired lysosome function, and neurons differentiated from NHCDR-generated iPSCs derived from patients with identified ADRDs and from unaffected family mutations carriers and non-carriers. In Aim 2 we will determine functional consequences of dysregulated pHlys in ADRD cell models by using pharmacological and genetic approaches we identified in studies in our parent grant that change pHlys. Using the ADRD-associated cell models described in Aim 1 we will to determine the role of pHlys dynamics in protein aggregation, a hallmark of ADRDs, as well as neuronal morphology and lysosome localization and mobility, which are impaired in ADRDs resulting in lysosome-rich enlarged terminal axon swellings. We also will use time-lapse microscopy to determine cell survival, with genetically-encoded pHLARE allowing a new tool for longitudinal studies. Outcomes include resolving the functional consequences of pHlys dynamics and dysregulated lysosome functions in ADRDs. Additionally, our parallel studies on pHlys in cancer (parent grant) and ADRDs (supplement) have promise to identify new therapeutic approaches targeting dysregulated pHlys to limit the progression of these diseases.