Monitoring Autophagy in Human Aging: Key Cell Models and Insights

Abstract

Autophagy, a key cellular degradation and recycling pathway, is critical for maintaining cellular homeostasis and responding to metabolic and environmental stress. Evidence for age-related autophagic dysfunction and its implications in chronic age-related diseases including neurodegeneration is accumulating. However, as a complex, multi-step process, autophagy can be challenging to measure, particularly in humans and human aging- and disease-relevant models. This review describes the links between macroautophagy, aging, and chronic age-related diseases. We present three novel human cell models, peripheral blood mononuclear cells (PBMCs), primary dermal fibroblasts (PDFs), and induced neurons (iNs), which serve as essential tools for studying autophagy flux and assessing its potential as a biomarker for aging. Unlike traditional models, these cell models retain age- and disease-associated molecular signatures, enhancing their relevance for human studies. The development of robust tools and methodologies for measuring autophagy flux in human cell models holds promise for advancing our understanding of autophagy's role in aging and age-related diseases, ultimately facilitating the discovery of therapies to enhance health outcomes.

Keywords: aging; autophagy; biomarkers; chronic age-related diseases; human cell models; induced neurons (iNs); peripheral blood mononuclear cells (PBMCs); primary dermal fibroblasts (PDFs).

Fig. 1.. Overview of autophagy: mechanism, assays, and age-related changes in humans.

(A) The multi-step autophagy process is depicted in stages: initiation, elongation and closure, transport and fusion, and degradation. In the initiation phase, the Unc-51 Like Autophagy Activating Kinase 1 (ULK1) and Phosphatidylinositol 3-Kinase (PI3K) complexes activate autophagy by recruiting key autophagy-related proteins (ATGs) to the pre-autophagosomal structure (PAS), where phagophore formation begins. During elongation and closure, ATG proteins, such as ATG3, ATG7, and ATG8, facilitate phagophore growth and closure around cargo. ATG8 is represented by green circles attached to both the inner and outer autophagosomal membranes. Selective autophagy involves the association of selective autophagy receptors (SARs) and their cargo with the ULK1 complex. SARs can interact with ATG8 proteins on the inner autophagosomal membrane. The autophagosome (AP) is then transported to the lysosome (LY), where SNAREs and tethers facilitate fusion to form an autolysosome (AL), where cargo is degraded. (B) Various biochemical, imaging, and structural assays are used to investigate different stages of the autophagy process and overall autophagy flux. These include ATG-gene transcription assays (RT-qPCR, RNA-seq) to measure changes in gene expression, protein expression assays (Western blot, ELISA, LC-MS) to quantify levels of autophagy proteins like ATG8 or SARs like p62, and additional proteomics methods (e.g., co-immunoprecipitation, IP) for investigating post-translational modifications, such as ULK1 phosphorylation. Imaging techniques, including fluorescent reporters, immunofluorescence (IF), stains, and dyes, allow visualization of autophagosomes, while structural studies (e.g., electron microscopy, EM) provide detailed views of autophagy structures, subcellular localization, and morphology. (C) Experimental evidence indicates that autophagy becomes dysregulated in aging and age-related diseases, with effects seen across different stages of autophagy. Dysregulation may manifest as changes in ATG-gene transcription, autophagosome morphology, lysosomal pH, and overall autophagy flux, contributing to waste accumulation, impaired proteostasis, and cellular dysfunction. Phenotypes that increase with age are indicated with a red arrowhead, and phenotypes that decrease with age are indicated with a blue arrowhead. Some phenotypes can change in either or both directions with age. Figure created in BioRender (https://www.biorender.com/).

Fig. 2.. Key human cellular models for aging- and age-related disease-relevant autophagy research.

Advances in human cell collection and culture have yielded cell models that largely retain aging- and chronic age-related disease-associated signatures of donors. Three key human cell models are induced neurons (iNs) generated via direct reprogramming, primary dermal fibroblasts (PDFs), and fresh peripheral blood mononuclear cells (PBMCs). Age-related changes in some autophagy readouts have been observed across human tissues (see right panels); the human cell models shown can be used to validate and thoroughly investigate the molecular mechanisms underlying age-related autophagy dysfunction, and test autophagy-enhancing interventions. ATG, autophagy-related. Figure created in BioRender (https://www.biorender.com/).