The research, published in Frontiers in Aging, analyzed autophagy flux in primary skin fibroblasts from apparently healthy male donors aged 20, 38, 74, and 96 years. Fibroblasts are a widely adopted aging cell system because they retain the cellular and molecular signatures of their donors: the skin is directly exposed to ultraviolet radiation, pollutants, and genotoxic agents over a lifetime, so fibroblasts from older individuals carry the in vivo wear of their chronological age into a controlled culture dish. By comparing cells across this age range, the team could trace how each phase of autophagy changes as human cells grow old.

Autophagy is a multi-step assembly line. Under starvation signaling, inhibition of mTOR activates the ULK1/2 kinase complex, which nucleates a phagophore. Two ubiquitin-like conjugation systems, ATG12/ATG5/ATG16L and LC3, elongate and seal the membrane into an autophagosome. The SNARE protein syntaxin 17 is then recruited to mature autophagosomes, licensing their fusion with lysosomes to form autolysosomes, where hydrolases degrade the luminal content. Because each step can fail independently, measuring flux across the whole pathway is essential for designing therapies that reactivate the specific phase that is actually broken.

The master switch of this pathway is transcription factor EB, or TFEB, which controls the expression of autophagy and lysosomal genes. Under nutrient-rich conditions, phosphorylated TFEB sits inert in the cytoplasm; during stress, calcineurin-mediated dephosphorylation drives it into the nucleus. When the researchers examined fibroblasts by confocal microscopy, TFEB activation looked surprisingly intact, even enhanced, in aging. Starvation pushed TFEB into the nuclei of cells from all donors, and the 96-year-old fibroblasts showed the strongest nuclear signal, even accumulating TFEB in the nucleus under normal, non-starved conditions.

That nuclear buildup translated into transcription. Quantitative RT-PCR showed that the TFEB target genes LAMP1, SQSTM1, and MAP1LC3B responded to starvation in cells from all ages, with SQSTM1 and MAP1LC3B transcripts rising markedly higher in the 96-year-old fibroblasts than in the 20-year-old controls. The authors interpret this premature nuclear accumulation of TFEB in old cells as a compensatory alarm: a signaling mechanism mobilized to counteract the impaired flux and lysosomal dysfunction discovered downstream. Alternative explanations, such as altered mTORC1- or ERK-mediated phosphorylation trapping TFEB in the nucleus, or an age-dependent decline in CRM1-mediated nuclear export, remain to be tested.

Autophagosome formation also held up. Treating cells with chloroquine, which blocks autophagosome-lysosome fusion, revealed LC3-positive puncta in fibroblasts from all donors, and Western blotting of the LC3-I to LC3-II conversion showed that autophagosomes formed with similar efficiency across the age spectrum. Flow cytometry with Cyto-ID dye even found a greater accumulation of autophagic vacuoles in the older cultures, implying that the early machinery operates at least as vigorously in old cells, likely as an active coping mechanism against age-related cellular stress.

The picture changed when the team probed the end of the line. The selective autophagy receptor p62, which is itself degraded in autolysosomes, normally accumulates when flux is blocked. Yet when fibroblasts were starved and treated with the lysosomal inhibitors E64D and pepstatin A, young and middle-aged cells accumulated LC3-II and p62 robustly, while cells from the 74- and 96-year-old donors showed no significant accumulation at all. The lysosome-dependent turnover of the canonical autophagy markers had decayed with age, a result the authors confirmed with bafilomycin A1, an inhibitor of the V-ATPase pump that arrests autophagosome-lysosome fusion.

Confocal colocalization assays pinpointed where the assembly line jams. In young fibroblasts, starvation significantly increased the overlap between LC3 and syntaxin 17, indicating enhanced autophagosome maturation, with Pearson correlation coefficients rising from 0.35 to 0.63. In old fibroblasts, starvation produced no such increase, with coefficients of 0.42 versus 0.39. Similarly, the colocalization of LC3 with the lysosomal membrane protein LAMP1, an indicator of autophagosome-lysosome fusion, rose sharply in young cells under starvation but only marginally in aged cells. The final two steps of autophagy, maturation and fusion, had quietly failed.

The culprit appears to be the lysosome itself. LysoBrite staining revealed that fibroblasts from the oldest donor carried a significantly increased abundance of lysosomes, and starvation enlarged lysosomes in all cultures, with the most expanded organelles in the 74- and 96-year-old cells. These enlarged lysosomes were unresponsive to autophagy induction: the 96-year-old fibroblasts showed the greatest basal lysosomal activity but failed to activate it further under starvation, unlike cells from younger donors. Consistent with overloaded, dysfunctional organelles, lipofuscin, the non-degradable autofluorescent pigment formed by incomplete lysosomal digestion, accumulated heavily in the oldest fibroblasts, and starvation could clear it in young cells but not in old ones.

The findings carry a clear therapeutic message. Rather than simply switching autophagy on, interventions in aging may need to target the final steps of the pathway: TFEB activators to stimulate lysosomal biogenesis through the CLEAR gene network, mTOR inhibitors such as rapamycin to restore flux, and lysosomal acidification enhancers, from small molecules to acidic nanoparticles, to boost enzymatic activity. The authors acknowledge that a single donor per age group limits generalizability, and future studies with additional donors will be needed to confirm whether late-stage autophagy impairment is a universal hallmark of fibroblast aging. But the core conclusion stands: in old human cells, the recycling plant’s opening hours are fine; it is the crusher at the end of the line that has broken down.

Subject of Research: Impaired autophagy flux and lysosome dysfunction in aged human fibroblasts

Article Title: Impaired autophagy and lysosome function in human fibroblast aging

Article References: Unzueta-Mendoza, J. J., García-Aguirre, I., Königsberg, M., Arrieta-Cruz, I., Lizcano-Meneses, S., Magaña, J. J., Alarcón-Aguilar, A., & Cisneros, B. (2026). Impaired autophagy and lysosome function in human fibroblast aging. Frontiers in Aging, 7, Article 1971981. https://doi.org/10.3389/fragi.2026.1971981

Image Credits: AI Generated

DOI: 10.3389/fragi.2026.1971981

Keywords: autophagy, lysosomes, TFEB, fibroblast aging, lipofuscin, autophagosome-lysosome fusion, LC3, p62, cellular recycling, aging biology, Impaired, lysosome