Autophagy (cellular self-degradation) plays a major role in maintaining the functional integrity (homeostasis) of essentially all eukaryotic cells. During the process, superfluous and damaged cellular constituents are delivered into the lysosomal compartment for enzymatic degradation. In humans, age-related defects in autophagy have been linked to the incidence of various age-associated degenerative pathologies (e.g., cancer, neurodegenerative diseases, diabetes, tissue atrophy and fibrosis, and immune deficiency) and accelerated ageing. Muscle mass decreases at detectable levels already in middle-aged patients, and this change can increase up to 30–50% at age 80. AUTEN-67 and -99, two small-molecule enhancers of autophagy with cytoprotective and anti-ageing effects have been previously identified and initially characterized. These compounds can increase the life span in wild-type and neurodegenerative model strains of the fruit fly Drosophila melanogaster. Adult flies were treated with these AUTEN molecules via feeding. Fluorescence and electron microscopy and Western blotting were used to assess the level of autophagy and cellular senescence. Flying tests were used to measure the locomotor ability of the treated animals at different ages. In the current study, the effects of AUTEN-67 and -99 were observed on striated muscle cells using the Drosophila indirect flight muscle (IFM) as a model. The two molecules were capable of inducing autophagy in IFM cells, thereby lowering the accumulation of protein aggregates and damaged mitochondria, both characterizing muscle ageing. Furthermore, the two molecules significantly improved the flying ability of treated animals. AUTEN-67 and -99 decrease the rate at which striated muscle cells age. These results may have a significant medical relevance that could be further examined in mammalian models.

Autophagy (cellular self-eating) is a tightly regulated catabolic process of eukaryotic cells during which parts of the cytoplasm are sequestered and subsequently delivered into lysosomes for degradation by acidic hydrolases. This process is central to maintaining cellular homeostasis, the removal of aged or damaged organelles, and the elimination of intracellular pathogens. The nematode Caenorhabditis elegans has proven to be a powerful genetic model for investigating the regulation and mechanism of autophagy. To date, the fluorescent autophagy reporters developed in this organism have predominantly relied on multi-copy, randomly integrated transgenes. As a result, the interpretation of autophagy dynamics in these models has required considerable caution due to possible overexpression artifacts and positional effects. In addition, starvation-induced autophagy has not been characterized in detail using these reporters. Here, we describe the development of two endogenous autophagy reporters, gfp::mCherry::lgg-1/atg-8 and gfp::atg-5, both inserted precisely into their endogenous genomic loci. We demonstrate that these single-copy reporters reliably track distinct stages of the autophagic process. Using these tools, we reveal that (i) the transition from the earliest phagophore to the mature autolysosome is an exceptionally rapid event because the vast majority of the detected fluorescent signals are autolysosome-specific, (ii) starvation triggers autophagy only after a measurable lag phase rather than immediately, and (iii) the regulation of starvation-induced autophagy depends on the actual life stage, and prevents excessive flux that could otherwise compromise cellular survival. We anticipate that these newly developed reporter strains will provide refined opportunities to further dissect the physiological and pathological roles of autophagy in vivo.

Autophagy is a lysosome-mediated self-degradation process of eukaryotic cells which is critical for the elimination of cellular damage. Its capacity progressively declines with age, and this change can lead to the development of various neurodegenerative pathologies including Spinocerebellar ataxia type 1 (SCA1). SCA1 is mainly caused by mutations in the polyglutamine region of Ataxin 1 protein. In patients affected by the disease, Purkinje neurons of the cerebellum frequently undergo demise and eventually become lost. Here we tested whether two well-characterized autophagy-enhancing small molecules, AUTEN-67 and -99, which antagonize the autophagy complex Vps34 through blocking the myotubularin-related lipid phosphatase MTMR14/EDTP, have the capacity to ameliorate SCA1 symptoms. We found that in a Drosophila model of SCA1, only AUTEN-67 exerts positive effects including improvement in climbing ability and extending life span. Based on these results, we hypothesized that the two compounds influence autophagy in the brain in a neuron-specific manner. Indeed, according to data we obtained, AUTEN-67 and -99 exhibit shared and unique functional domains in the Drosophila brain. AUTENs enhance autophagy in GABAergic and dopaminergic neurons. In addition, AUTEN-67 also affect autophagy in cholinergic neurons, while AUTEN-99 trigger the process in glutaminergic neurons and motoneurons. We also observed varying efficiencies between the two AUTENs among different subtypes of cultured hippocampal neurons of mice. These data suggest that the two compounds display neuron-specific differences in exerting autophagy-enhancing effects, and may lead to a better understanding of which types of neurons autophagy could potentially be activated to treat SCA1 in human patients.

Autophagy (cellular self-eating) is a tightly regulated catabolic process of eukaryotic cells in which parts of the cytoplasm are sequestered and subsequently degraded within lysosomes by acidic hydrolases. This process is central to maintaining cellular homeostasis, the removal of aged or damaged organelles, and the elimination of intracellular pathogens. The nematode Caenorhabditis elegans has proven to be a powerful genetic model for investigating autophagy. To date, the fluorescent autophagy reporters developed in this organism have predominantly relied on multi-copy, randomly integrated transgenes. As a result, the interpretation of autophagy dynamics in these models has required considerable caution due to possible overexpression artifacts and positional effects. Here, we describe the development of two endogenous autophagy reporters, engineered using CRISPR-Cas9 genome editing: gfp::mCherry::lgg-1/atg-8 and gfp::atg-5, both inserted precisely into their endogenous genomic loci. These single-copy reporters reliably track distinct stages of the autophagic process. Using these tools, we demonstrate that (i) the transition from the earliest phagophore to the mature autolysosome is an exceptionally rapid event, (ii) starvation triggers autophagy only after a measurable lag phase, rather than immediately, and (iii) autophagy in C. elegans is subject to strict regulatory control, preventing excessive flux that could otherwise compromise cellular survival. We anticipate that these newly developed reporter strains will provide refined opportunities to dissect the physiological and pathological roles of autophagy in vivo.

Autophagy is a tightly regulated catabolic process essential for cellular homeostasis, stress adaptation, and regeneration. In the nematode Caenorhabditis elegans, with its short lifespan, transparent body, and well-defined genetics, the process can be investigated at tissue- and age-specific manner, making it an excellent model to study the connection between autophagy and longevity. While autophagy is indispensable for development and homeostasis, recent studies have revealed that its role in aging is more complex than previously thought. During post-reproductive life, autophagic flux and the degradative capacity of lysosomes decline, resulting in the accumulation of undegraded material and cellular stress. Several studies have demonstrated that the experimental modulation of core autophagy in aged or post-reproductive C. elegans, particularly in neurons, can improve proteostasis, preserve tissue integrity, and extend lifespan. Here we review the current results obtained using the genetic model system Caenorhabditis elegans that link autophagy to lifespan regulation. We focus on studies that investigate unexpected, context-dependent, or deleterious effects of inhibiting autophagy-related genes during aging. We also discuss how age- and tissue-specific modulation of autophagy could define the most effective strategies for promoting healthy aging. This could provide relevant insights for the therapeutic targeting of autophagy in humans.

Autophagy is a tightly regulated catabolic process essential for cellular homeostasis, stress adaptation, and regeneration. In the nematode Caenorhabditis elegans, with its short lifespan, transparent body, and well-defined genetics, the process can be investigated in a tissue- and age-specific manner, making it an excellent model to study the connection between autophagy and longevity. While autophagy is generally protective—promoting cellular maintenance and longevity—its dysregulation or hyperactivation during aging can be deleterious, leading to cellular stress, tissue damage, and cell death. In this context, autophagy can act as a double-edged sword: its beneficial effects can become harmful if hyperactivated or improperly controlled, particularly in post-reproductive or stressed tissues. Here, we review studies in C. elegans that link autophagy to lifespan regulation, with a focus on unexpected, context-dependent, or harmful effects of modulating autophagy-related genes during aging. We highlight how age- and tissue-specific regulation of autophagy can optimize its protective role and discuss the implications of these findings for designing strategies to promote healthy aging, potentially providing insights for the therapeutic targeting of autophagy in humans.