Autophagy is the name given to a collection of recycling mechanisms involved in cellular maintenance. These processes clear out metabolic waste and break down damaged cellular components so that the parts, proteins and their constituents, can be used elsewhere. The better documented forms of autophagy involve the coordination of (a) systems that flag structures and molecules for recycling, (b) systems that engulf the flagged materials in membranes for delivery to cellular recycling centers, and (c) the recycling structures called lysosomes, packed with enzymes capable of dismantling up most of what they will encounter.
Autophagy fails with age, and this failure is thought to contribute to degenerative aging to some degree; certainly many of the methods of modestly slowing aging in laboratory species appear to at least involve - and in some cases rely upon - increased autophagy. Exactly why does autophagy falter with age, however? There are a lot of answers to that question, of varying degrees of incompleteness, speculation, and supporting evidence. The challenge here, as for everything that goes on inside a cell, is that autophagy is a highly dynamic, enormously complex chain of mechanisms. Failures could be subtle and hard to detect in any one component part, or they could be distributed throughout the system, and there are a lot of pieces to examine. It has taken decades for the modern research community to gather today's comparatively sophisticated, partial picture of what is going on under the hood, and the tools of biotechnology are only now gaining the capacity to do better than this given a reasonable amount of time and funding.
A further consideration is that autophagy is a large enough research space to develop specializations: teams will tend to have more experience in just one aspect of this set of processes. It is, like much of the life sciences, a case of the blind men and the elephant, and intensive, ongoing collaboration is required in order to gain any sort of holistic picture. Many autophagic mechanisms no doubt all become dysfunctional in their own particular ways across the course of aging, and each such chain of cause and effect reaches from the beginning of some form of fundamental molecular damage through numerous stages to reach whatever layer of the onion that any given scientist happens to be investigating. When people publish papers on the age-related decline of autophagy, it is always worth bearing this in mind: it is rare that anyone is working with more than a slice of the whole at one time.
That said, the research here is an example of the sort of approach needed to improve the present understanding of how autophagy works in detail, and thus build a better map of where it runs off the rails over the course of aging. You might compare the report here with, say, the standard SENS view of dysfunctional autophagy resulting from hardy metabolic waste accumulated in lysosomes, or the discovery that loss of autophagy can be restored at least partially through genetic engineering to add more receptors to lysosomes, increasing their ability to receive flagged materials for recycling. The lysosome is just one part of a much larger set of autophagic systems, however, and problems can certainly exist elsewhere - though it has to be said that the findings noted below are consistent with theories placing the whole of the problem in the lysosome, and thus supportive of the SENS approach to therapies.
"Autophagy," which means "self-eating" based on its Greek roots, is the normal physiological process the body's cells use to remove viruses, bacteria, and damaged material from the cell. Autophagy also helps cells "clean house" by recycling building blocks - similar to the way we recycle glass, plastic and metal. In recent years, defective autophagy has been linked to age-related diseases such as cancer, neurodegeneration and heart disease. "Increasingly, researchers are asking whether there is an age-related decline in autophagy and if it's connected to diseases that occur more frequently in older individuals. Exposing how autophagy becomes faulty with age may reveal opportunities for us to therapeutically intervene and correct the process to promote health aging."
Autophagy is a dynamic, multi-step process that starts with the formation of a double-membrane sac in the cell cytoplasm called the isolation membrane (IM). These structures engulf cellular material and debris, expanding in size to form vesicles called autophagosomes (APs). Finally, APs fuse with lysosomes to form autolysosomes (ALs) that digest and release the breakdown products for re-use, much like a recycling plant would repurpose incoming trash. "A major challenge with understanding how aging impacts autophagy is that researchers have been capturing a dynamic process with static measurements. Autophagy is most commonly monitored by counting the number of APs, which really only provides a snapshot of the process - similar to how counting the number of garbage trucks on the street doesn't tell you how much garbage is actually being recycled at the plant. And typically older organisms have an increased number of APs, but we don't know exactly why."
"We wanted to ask how age impacts autophagy - is it at the beginning of the process by increasing the rate at which APs are formed, or, by analogy, how many garbage trucks are rolling out on the street - or is it at the end of the process by blocking the conversion of APs to ALs, i.e., how much recycling is taking place at the recycling plant. Either one of these scenarios would cause an increased number of APs, but knowing which one would help pinpoint where interventions may be helpful. We found that there is indeed an age-dependent decline in autophagy over time in all tissues examined. We further provide evidence that the increase in APs results from an impairment at a step after APs are made. So basically the autophagy recycling process becomes incomplete with age by stopping somewhere after APs are formed. This research is important because it helps provide time- and site-of-action information for potential future interventions directed at sustaining autophagy to extend lifespan. Our next step will be to perform biochemical research to further pinpoint exactly how autophagy fails to complete its cycle, possibly providing targets to develop specific interventions."
Macroautophagy (hereafter referred to as autophagy) is a multistep cellular recycling process in which cytosolic components are encapsulated in membrane vesicles and ultimately degraded in the lysosome. As interest in this pathway and its pathophysiological roles has increased, it has become clear that measurement of autophagic vesicle levels at steady state, without monitoring the overall pathway flux, can lead to controversial results. Autophagy is commonly monitored by enumerating APs under steady-state conditions, also referred to as the AP pool size, using a GFP-tagged Atg8 marker. During AP formation, Atg8 is cleaved, conjugated to phosphatidylethanolamine, and inserted into the vesicle membrane, thus serving as a marker for IMs and APs. However, GFP-Atg8 only reports on the size of the IM and AP pools, not the rate by which IMs and APs are formed, or converted to ALs. For example, an increase in GFP-Atg8 could result from increased formation of APs or blockade of the downstream steps.
A tandem-tagged mCherry-GFP-Atg8 reporter, which separately monitors both IMs/APs and ALs can help distinguish between these possibilities.Specifically, when used in combination with chemical inhibitors of autophagy tandem-tagged reporters can assess autophagic activity in so called autophagic flux assays. Although tandem-tagged Atg8 markers have been used extensively to monitor autophagy in mammalian cells, as well as in adult Drosophila melanogaster and in Caenorhabditis elegans embryos, this reporter has not previously been used in adult C. elegans, and no comprehensive spatial or temporal analyses of autophagic activity have been reported in any animal thus far.
Autophagy plays important roles in numerous cellular processes and has been linked to normal physiological aging as well as the development of age-related diseases. Furthermore, accumulating evidence in long-lived species demonstrates that autophagy genes are required for extended longevity. In particular, autophagy is essential for lifespan extension by inhibition of the nutrient sensor mTOR. In C. elegans, autophagy genes are also required for the long lifespan induced by other conserved longevity paradigms, such as reduced insulin/IGF-1 signaling, germline ablation, and reduced mitochondrial respiration, and all these longevity mutants have increased transcript levels of several autophagy genes.
To better understand how aging affects autophagy in C. elegans, we employed a GFP-tagged and a novel tandem-tagged (mCherry/GFP) form of LGG-1 (a C. elegans ortholog of Atg8) to investigate the spatial and temporal autophagy landscape in wild-type (WT) and long-lived daf-2 mutants and germline-less glp-1 animals. Our data indicate that WT animals displayed an age-dependent increase in AP and AL numbers in all tissues, which flux assays suggest reflects a decrease in autophagic activity over time. In contrast, daf-2 and glp-1 mutants showed unique age- and tissue-specific differences consistent with select tissues displaying elevated, and in one case possibly reduced autophagic activity compared with WT animals. Moreover, tissue-specific inhibition of autophagy in the intestine significantly reduced the long lifespan of glp-1 mutants but not of daf-2 mutants, suggesting that autophagy in the intestine of daf-2 mutants may be dispensable for lifespan extension. Our study represents the first efforts to comprehensively analyze autophagic activity in a spatiotemporal manner of a live organism and provides evidence for an age-dependent decline in autophagic activity, and for a complex spatiotemporal regulation of autophagy in long-lived daf-2 and glp-1 mutants.