Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.
Department of Pharmacology, University of São Paulo, São Paulo, Brazil.
Methods Mol Biol. 2022;2549:103-136. doi: 10.1007/7651_2021_420.
Autophagy is an evolutionarily conserved catabolic pathway for the degradation of cytoplasmic constituents in eukaryotic cells. It is the primary disposal route for selective removal of undesirable cellular materials like aggregation-prone proteins and damaged organelles for maintaining cellular homeostasis, and for bulk degradation of intracellular macromolecules and recycling the breakdown products for providing energy homeostasis during starvation. These functions of autophagy are attributed to cellular survival and thus pertinent for human health; however, malfunction of this process is detrimental to the cells, particularly for post-mitotic neurons. Thus, basal autophagy is vital for maintaining neuronal homeostasis, whereas autophagy dysfunction contributes to neurodegeneration. Defective autophagy has been demonstrated in several neurodegenerative diseases wherein pharmacological induction of autophagy is beneficial in many of these disease models. Elucidating the mechanisms underlying defective autophagy is imperative for the development of therapies targeting this process. Disease-affected human neuronal cells can be established from patient-derived human induced pluripotent stem cells (hiPSCs) that provide a clinically relevant platform for studying disease mechanisms and drug discovery. Thus, modeling autophagy dysfunction as a phenotypic readout in patient-derived neurons provides a more direct platform for investigating the mechanisms underlying defective autophagy and evaluating the therapeutic efficacy of autophagy inducers. Toward this, several hiPSC-derived neuronal cell models of neurodegenerative diseases have been employed. In this review, we highlight the key methodologies pertaining to hiPSC maintenance and neuronal differentiation, and studying autophagy at an endogenous level in hiPSC-derived neuronal cells.
自噬是真核细胞中一种进化上保守的溶酶体降解途径，用于降解细胞质成分。它是选择性去除聚集倾向蛋白和受损细胞器等不需要的细胞物质的主要途径，以维持细胞内稳态，并在饥饿时批量降解细胞内大分子和回收降解产物以提供能量内稳态。这些自噬功能归因于细胞的生存，因此与人类健康有关；然而，该过程的功能障碍对细胞有害，尤其是对有丝分裂后神经元。因此，基础自噬对于维持神经元内稳态至关重要，而自噬功能障碍则导致神经退行性变。在几种神经退行性疾病中已经证明了自噬的缺陷，其中药理学诱导自噬在许多这些疾病模型中是有益的。阐明自噬缺陷的机制对于开发针对该过程的治疗方法至关重要。可以从患者来源的人诱导多能干细胞（hiPSC）中建立受疾病影响的人神经元细胞，为研究疾病机制和药物发现提供了一个临床相关的平台。因此，将自噬功能障碍作为患者来源神经元的表型读数进行建模，为研究自噬缺陷的机制和评估自噬诱导剂的治疗效果提供了一个更直接的平台。为此，已经采用了几种神经退行性疾病的 hiPSC 衍生神经元细胞模型。在这篇综述中，我们重点介绍了与 hiPSC 维持和神经元分化以及在 hiPSC 衍生神经元细胞中研究内源性自噬相关的关键方法。
