Targeting lysosome/RPE heterogeneity in AMD pathobiology as a novel therapy

针对 AMD 病理学中的溶酶体/RPE 异质性作为一种新疗法

• 批准号: 10636943
• 负责人: James T Handa
• 金额: $ 60.78万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10636943
• 关键词: Actins; Address; Affect; Age; Age related macular degeneration; Aging; Antioxidants; Autoantibodies; Automobile Driving; Autophagocytosis; Autophagosome; Behavior; Biogenesis; Blindness; Cell Death; Cell Nucleus; Cell physiology; Cells; Cessation of life; Characteristics; Chronic; Crystallins; Cytoskeletal Modeling; Cytosol; Defect; Disease; Dose; E-Cadherin; Elderly; Endosomes; Environment; Epidermal Growth Factor Receptor; Epithelium; Exhibits; Eye; Functional disorder; Gene Activation; Genetically Engineered Mouse; Health; Heterogeneity; Homeostasis; Human; Impairment; Injections; Knockout Mice; Laboratories; Link; Lysosomes; Mesenchymal; Messenger RNA; Micronutrients; Minority; Molecular Analysis; Nonexudative age-related macular degeneration; Nuclear; Oxidative Stress; Pathogenesis; Pathologic; Pathology; Patients; Phagocytosis; Phenotype; Prevention; Process; Proteins; Receptor Signaling; Rejuvenation; Resistance; Retina; Rodent Model; Stains; Stress; Structure of retinal pigment epithelium; Testing; Therapeutic Agents; Time; Tissues; Vascular Diseases; Vimentin; Vision; Work; activating transcription factor; adeno-associated viral vector; age related; aging population; bafilomycin A1; bevacizumab; cigarette smoke; conditional knockout; epithelial to mesenchymal transition; gene network; gene therapy; in vivo; induced pluripotent stem cell; mouse model; mutant; neovascular; novel; novel therapeutics; pre-clinical; preservation; prevent; promoter; response; targeted treatment; therapeutic target; transcription factor; transcriptome

SUMMARY: Vision loss from age-related macular degeneration (AMD) is a major, expanding problem due to the aging population. While treatment is available for intermediate AMD and the advanced wet form, no prevention or treatment is available for early AMD. While cellular heterogeneity can help tissue adapt to stress, degenerative heterogeneity from aging is associated with disease. The current perspective is that RPE (retinal pigmented epithelium) degeneration is a central, chronic process that inevitably leads to cell death. Thus, prior studies have focused on the impact of RPE cell death on AMD pathobiology. However, a subset of RPE cells exhibit features of epithelial-mesenchymal transition (EMT), an adaptive process that allows cells to survive a harsh environment. RPE heterogeneity has been recognized for some time, but the impact of disease driving cell subsets on AMD pathogenesis has not been considered. Lysosomal dysfunction is clearly involved in several age-related diseases including AMD, but the impact of heterogeneity of lysosomal impairment, which can cause disease, on RPE cellular function or heterogeneity has not been considered. This proposal is a departure from the norm because it proposes that RPE heterogeneity including a subset of cells with EMT in early AMD is initiated by heterogeneity of lysosomal clearance defects. RPE in EMT are resistant to death, and thus, could drive AMD pathobiology for an extended period. On the other hand, since EMT can be reversed, RPE in EMT are a compelling treatment target because its vision preserving functions can be restored. Lysosomal function and autophagy are modulated by the master regulator transcription factor EB (TFEB). Our laboratory has demonstrated reduced nuclear TFEB staining in the RPE of early AMD globes compared to age-matched controls, thus identifying TFEB as a potential therapeutic target for AMD. Given the possibility that heterogeneity of lysosomal dysfunction drives RPE heterogeneity, from EMT to degeneration, the objective of this proposal is to determine the impact of restoring lysosomal function and autophagy on the AMD-associated RPE heterogeneity in our validated Cryba1 cKO rodent model of lysosomal impairment that develops a dry AMD-like phenotype, and in human-iPSC derived RPE cells. The central hypothesis of “rejuvenating lysosomal and autophagic function by activating TFEB will maintain RPE cell function and prevent pathologic RPE heterogeneity including EMT, in early AMD” will be addressed with the following aims: Aim 1: Test the extent that TFEB activation rejuvenates lysosomal/autophagy function, and modulates RPE heterogeneity and RPE cell health. Aim 2: Test the degree that TFEB activation in the RPE of Cryba1 cKO mice rescues its AMD-like phenotype. Aim 3: Test the degree that lysosomal heterogeneity leads to RPE heterogeneity in AMD. Successful completion of this proposal will establish the novel concept of lysosome/autophagy dysfunction as a key driver of RPE heterogeneity in early AMD and identify TFEB as a novel therapeutic agent.

摘要：与年龄相关的黄斑变性（AMD）导致的视力丧失是一个重大的，由于这个问题而扩大的问题 人口老龄化。虽然可用于中级AMD和高级湿式的治疗，但没有预防 或治疗可用于早期AMD。虽然细胞异质性可以帮助组织适应压力，但退化 衰老的异质性与疾病有关。当前的观点是RPE（视网膜色素） 上皮变性是一种中心的慢性过程，不可避免地导致细胞死亡。那是先前的研究 专注于RPE细胞死亡对AMD病理生物学的影响。但是，RPE单元的一个子集暴露了特征 上皮 - 间质转变（EMT），这是一种适应性过程，允许细胞在HARMSH环境中生存。 RPE异质性已被认可了一段时间，但是疾病驱动细胞子集对AMD的影响 尚未考虑发病机理。溶酶体功能障碍显然参与了几个与年龄有关的 包括AMD在内的疾病，但溶酶体损伤异质性的影响，可能导致疾病，对 尚未考虑RPE细胞功能或异质性。该提议与规范背道而驰 因为它提出了RPE异质性，包括早期AMD中EMT的一个子集，由AMD启动 溶酶体清除缺陷的异质性。 EMT中的RPE对死亡具有抵抗力，因此可以驱动AMD 病理生物学长期。另一方面，由于EMT可以逆转，因此EMT中的RPE是一个 令人信服的治疗目标是因为可以恢复其视力保留功能。溶酶体功能和 自噬是由主调节转录因子EB（TFEB）调节的。我们的实验室有 与年龄匹配相比 控制，从而将TFEB识别为AMD的潜在治疗靶点。考虑到异质性 溶酶体功能障碍驱动RPE异质性，从EMT到变性，该提案的目的是 确定恢复溶酶体功能和自噬对与AMD相关的RPE的影响 我们经过验证的Cryba1 CKO啮齿动物模型的溶酶体损伤模型的异质性 表型，在人类IPSC衍生的RPE细胞中。 “恢复溶酶体和 自噬功能通过激活TFEB将保持RPE细胞功能并防止病理RPE异质性 包括EMT在内，在AMD早期将以以下目的解决：目标1：测试TFEB的程度 激活使溶酶体/自噬功能恢复活力，并调节RPE异质性和RPE细胞健康。 AIM 2：测试Cryba1 CKO小鼠RPE中TFEB激活的程度，反应其类似AMD的表型。 AIM 3：测试溶酶体异质性导致AMD中RPE异质性的程度。成功完成 该提案将建立溶酶体/自噬功能障碍的新颖概念作为RPE的关键驱动力 早期AMD中的异质性，并将TFEB识别为一种新型的治疗剂。

项目成果

Thermal Shift Assay in Ferroptosis.

铁死亡中的热位移测定。

• DOI: 10.1007/978-1-0716-3433-2_16
• 发表时间: 2023
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Bammidi,Sridhar;Hose,Stacey;Handa,JamesT;Sinha,Debasish;Ghosh,Sayan
• 通讯作者: Ghosh,Sayan

Microglia-Neutrophil Interactions Drive Dry AMD-like Pathology in a Mouse Model.

• DOI: 10.3390/cells11223535
• 发表时间: 2022-11-09
• 期刊: Cells
• 影响因子: 6

A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for In Vitro Studies in AMD.

• DOI: 10.3390/ijms222111979
• 发表时间: 2021-11-05
• 期刊: International journal of molecular sciences
• 影响因子: 5.6
• 作者: Shang P;Stepicheva NA;Liu H;Chowdhury O;Franks J;Sun M;Hose S;Ghosh S;Yazdankhah M;Strizhakova A;Stolz DB;Zigler JS Jr;Sinha D
• 通讯作者: Sinha D