A paradoxical role of autophagy: promoting protein aggregates and hindering rejuvenation in a Caenorhabditis elegans L1 arrest model

During the selective process of aggrephagy, dynamic membrane structures engulf protein aggregates and deliver them to lysosomes for degradation; therefore, this is considered a major pathway for maintaining proteostasis. Along these lines, an age-related decline in autophagy function has been linked to the disruption of proteostatic regulation [1]. A C. elegans L1 arrest model has recently been established to study aging and rejuvenation [2]. In brief, following hatching, prolonged starvation leads to developmental arrest at the L1 larval stage. During this period of starvation, aging-like phenotypes – such as the accumulation of protein aggregates – are commonly observed. Reintroduction of food can reverse these features, a process regarded as rejuvenation. Although the majority of larvae survive starvation, only a subset is able to resume normal development and reach adulthood. Thus, the proportion of larvae that successfully continue development relative to the total number of survivors serves as a direct indicator of rejuvenation capacity. Previous studies have identified the unfolded protein response (UPR) pathway as essential for the clearance of protein aggregates and the resumption of development [2]; however, the underlying mechanisms remain largely unknown.

Murley et al. conducted a forward genetic screen in an ire-1 mutant strain, which is defective in the UPR pathway [3]. Through this screen, several atg (autophagy related) genes were identified and validated. Notably, the double knockout of ire-1 and atg-9 results in a partial restoration of rejuvenation activity in the L1 arrest model. This unexpected result suggests that autophagy activity plays a negative role in the rejuvenation process.

To further investigate the mechanistic role of autophagy in rejuvenation, Murley et al. quantified intra-lysosomal protein aggregates by microscopy using a combination of ProteoStat® (a protein aggregate-sensitive fluorescent dye) staining and a lysosome-localized, fluorescently tagged protein marker. Their results show that disruption of the autophagy pathway suppresses the accumulation of intra-lysosomal protein aggregates that are typically induced during starvation in the L1 larvae.

To elucidate the unexpected role of autophagy, the authors next examined lysosomal membrane integrity and found that autophagy also contributes to starvation-induced lysosomal membrane damage. Based on this observation, they proposed a hypothesis that prolonged starvation in L1 larvae enhances autophagic activity, leading to the excessive delivery of cellular cargo into lysosomes. This, in turn, results in the accumulation of intra-lysosomal protein aggregates and subsequent lysosomal membrane damage.

This hypothesis is further supported by experiments using HLH-30 overexpression strains. HLH-30, the functional ortholog of the human transcription factor TFEB, is a central regulator of lysosomal biogenesis [4]. While overexpression of HLH-30 is sufficient to enhance lysosomal activity, it does not rescue the rejuvenation defect in the ire-1 mutant. However, overexpression of HLH-30 in the ire-1 atg-9 double knockout background fully restores rejuvenation activity to a level comparable to that of the wild type. These findings suggest that insufficient lysosomal function is a key contributing factor – acting in parallel with autophagy – to the failure of rejuvenation in the ire-1 mutant.

This study delineates the L1 arrest model into two distinct phases. In the first phase – the starvation phase – prolonged nutrient deprivation activates autophagy, resulting in the accumulation of intra-lysosomal protein aggregates and damage to lysosomal membranes. In the second phase – the recovery phase – the UPR pathway plays a critical role in clearing protein aggregates and repairing organellar damage, thereby promoting rejuvenation.

This study further extends its findings from C. elegans to human cellular systems. Through the use of a CRISPR interference technique, the authors demonstrate that the underlying mechanisms are highly conserved across multiple human cell lines. However, the experiments in human cells primarily compare quiescent and proliferating states, which do not directly model aging or rejuvenation processes. These findings call for a reconsideration of the relationship between autophagy and proteostasis, particularly within the context of aging. Notably, under specific conditions, the activation of autophagy may paradoxically contribute to the accumulation of protein aggregates due to lysosomal damage.

Despite these insights, several key questions remain unresolved. For instance, during the starvation phase, how does autophagy exacerbate lysosomal damage? Does starvation-induced L1 arrest physiologically suppress lysosomal biogenesis? In the recovery phase, by what mechanisms does the UPR pathway facilitate lysosomal repair? Is UPR-dependent clearance of lysosomal aggregates contingent upon intact lysosomal function? What ultimately becomes of the lysosomal aggregates – are they degraded, retained, or partitioned? Does their clearance involve processes such as asymmetric cell division? Finally, can the findings from the C. elegans L1 arrest model extend to other aging and rejuvenation models?