Autophagy and pluripotency: self-eating your way to eternal youth

Abstract

Pluripotent stem cells (PSCs) can self-renew indefinitely in culture while retaining the potential to differentiate into virtually all normal cell types in the adult animal. Due to these remarkable properties, PSCs not only provide a superb system to investigate mammalian development and model diseases, but also hold promise for regenerative therapies. Autophagy is a self-digestive process that targets proteins, organelles, and other cellular contents for lysosomal degradation. Here, we review recent literature on the mechanistic role of different types of autophagy in embryonic development, embryonic stem cells (ESCs), and induced PSCs (iPSCs), focusing on their remodeling functions on protein, metabolism, and epigenetics. We present a perspective on unsolved issues and propose that autophagy is a promising target to modulate acquisition, maintenance, and directed differentiation of PSCs.

Keywords: autophagy; chaperone-mediated autophagy; differentiation; embryonic stem cells; induced pluripotent stem cells; macroautophagy; microautophagy; self-renewal.

Figure 1.. Types and characteristics of autophagy.

(A) Macroautophagy. The process of macroautophagy involves multiprotein complexes and signaling pathways. Macroautophagy is regulated by AMPK and mTOR signaling pathways, which control autophagosome initiation via modulating the assembly of ULK1 protein complex (ATG13, ATG101, FIP200 and ULK1) at the ATG9-containing membrane sites. Upon activation, ULK1 phosphorylates ATG9 and initiates the elongation of autophagosome through recruiting the PI3K protein complex (Beclin1, VPS34, VPS15 and ATG14L). VPS34 generates phosphatidylinositol 3-phosphate (PI(3)P) to extend the phagophore membranes with the help of WIPI family proteins. Two ubiquitin-like conjunction systems – ATG12 system (ATG5~ATG12-ATG16L1) and LC3-phosphatidylethanolamine (PE) system – are necessary for the completion of the autophagosome. The autophagosome is fused with lysosome to form autolysosome, and the hydrolases inside then degrade the components for recycling. (B) Chaperone-mediated autophagy (CMA). CMA is a multiple-step process that selectively degrades cytoplasmic soluble proteins. A target protein containing a KFERQ-like motif is recognized by chaperone protein Hsc70. The target protein-Hsc70 complex translocates to the lysosomal surface and docks to the cytosolic tail of monomeric lysosomal membrane protein LAMP2A. This binding promotes LAMP2A multimerization, which helps transport the target protein to the lysosome lumen for degradation. (C) Three types of microautophagy. Lysosomal microautophagy (types 1 and 2): cytoplasmic components are wrapped in small vesicles through protrusion (type 1) or invagination (type 2) of lysosomal membrane and are subsequently degraded inside the lysosome, this process is partially regulated by ATG proteins. Endosomal microautophagy (type 3): invagination of endosomal membrane with the help of endosomal sorting complex required for transport (ESCRT) machinery generates multi-vesicular bodies (MVB) that eventually delivers bulk or selective (the latter requires Hsc70 or NBR1 to recognize target proteins) cytosolic cargos into the lysosomal lumen for degradation.

Figure 2.. Role of autophagy in embryonic development and the maintenance and acquisition of pluripotency.

(A) Upon fertilization, macroautophagy is rapidly induced to enable the oocyte-to-embryo transition. Atg5-deficient zygotes are developmentally arrested at the four- to eight-cell stages. Macroautophagy is also required for the generation of blastocyst cavity by facilitating the removal of apoptotic cells. Mice lacking macroautophagy-related genes die at various stages during development. Homozygous mTOR knockout embryos die shortly after implantation and mice lacking one of the phagophore-forming genes Becn1, Ambra1, and Fip200 are embryonic lethal. Mice lacking genes involved in the downstream process of macroautophagy, such as Atg3, Atg5, Atg7, Atg9 and Atg16l1, all die shortly after delivery. In addition, canonical and non-canonical autophagy-related pathways are involved in the regulation of ESC maintenance and differentiation, as well as somatic reprogramming. TFs, transcription factors. (B) Macroautophagy and chaperone-mediated autophagy (CMA) regulate stemness, differentiation, and reprogramming by remodeling protein, metabolism, and epigenetics. Various forms of autophagy such as canonical and noncanonical non-selective macroautophagy, selective macroautophagy (e.g., midbophagy and mitophagy), and CMA, as well as their regulatory factors, are involved in remodeling protein, metabolism, and epigenetics. They maintain protein quality, regulate global protein turnover rates, and fine-tune levels of pluripotency factors. They also balance cellular oxidative stress, regulate mitochondrial functions, and fulfill energy requirements. Through modulating metabolites that support various forms of histone/DNA modifications, certain forms of autophagy can also influence epigenetic and transcriptional states, thus determining fate decisions of pluripotent stem cells (PSCs). Abbreviations: αKG, α-ketoglutarate; AMPK, 5′-AMP-activated protein kinase; ATG, autophagy-related; EPG5, ectopic P-granules macroautophagy protein 5 homolog; IDH, isocitrate dehydrogenase; iPSCs, induced pluripotent stem cells; mTOR, mechanistic target of rapamycin; PGC1, peroxisome proliferator-activated receptor coactivator 1; TFs, transcription factors; Ulk1, unc-51-like kinase 1; USP8, ubiquitin-specific peptidase 8.