The Cell’s Positive Spin on Adversity
Autophagy — a subcellular recycling mechanism
By Jyothi Devakumar and Brandon Simmons
Many of us appreciate Norman Vincent Peale’s “The Power of Positive Thinking” or find inspiration in the teachings of people like Sadhguru and Tony Robbins on the impact of a positive outlook in life. As science pushes boundaries in the study of cellular biology, we’ve increased our understanding of how effectively and efficiently cells use their molecular machinery to convert every adversity they face into a positive outcome. I can’t help but appreciate how life, be it at the cellular level or the organismal level, thrives on linking adversity to rejuvenation. For cells, positivity is “The way of life.”
For this month’s Longevity Blog, I teamed up with my Prime Movers Lab partner, Brandon Simmons, to explain cellular autophagy, how it fits into the longevity strategy, and how you can stimulate autophagy in your own body via the much-talked-about practice of intermittent fasting.
What is Autophagy?
Cannibals don’t just exist in dark fairy tales. We live with them in our bodies every day, as cells (the fundamental units of the body) indulge in cannibalizing their own parts. When dysfunctional damaged parts and molecules accumulate in the cell, they perform the ultimate form of recycling, a process called autophagy. We can define autophagy in simple terms as the process of intracellular (within the cell) recycling.
Cells and their components (proteins, organelles, DNA, RNA, lipids, and even parts of the nucleus, etc) are under constant turnover (they live fast; they die fast; like characters in an Eagles song) and this process of recycling involves breaking down these damaged, dysfunctional macromolecules and using the building blocks generated (nucleotides, nucleosides, amino acids, etc) to resynthesize functional macromolecules. Be it organelles such as mitochondria or macromolecules such as proteins, there is constant recycling and it is conservation at its best at the cellular level!
The unwanted cargo is delivered to the recycling plant, i.e. the destination of this cargo, which is the seat of degradation, called the lysosomes. This process, which effectively clears the cellular junk — when the cell degrades the damaged and dysfunctional parts and uses the raw materials obtained to create new ones- is the process of autophagy. It is similar to cannibalism at the cellular level. The end products of degradation — amino acids, nucleotides and nucleosides, simple sugars — are all redirected to biosynthetic routes and maintenance creating a robust rejuvenation environment.
There are three types of autophagy: macroautophagy, chaperone-mediated autophagy (CMA), and microautophagy. Macroautophagy, as the name suggests, involves larger amounts of cargo (macromolecules, parts of the cytoplasm, organelles) being picked up (as seen in figure 1.0 below, a membranous structure is formed around the specific cargo to segregate and transport) by structures called autophagosomes which then deliver the cargo to lysosomes where degradation followed by recycling happens. In CMA, selected proteins are picked up by other proteins called chaperones and targeted to lysosomes for recycling. In microautophagy, cargo is directly taken up by lysosomes. Autophagy takes place in all cells under normal conditions. Younger cells are better at it than older cells. Aging diminishes the efficiency of all three types of autophagy.
Types and science of Autophagy
This classification is based on the mechanism involved in the process. Autophagy pathways are highly conserved through evolution indicating their importance. Likewise, genes associated with autophagy are not a common target of somatic mutations, indicating its fundamental role in survival.
Fig 1.0- Three different types of autophagy and their mechanism
(Adapted from — PMC3894687)
Macroautophagy is when a part of the cytoplasm including organelles is sequestered into a cup-shaped structure referred to as the phagophore, which then closes up to form a double-membrane bounded structure termed the autophagosome. This, in turn, fuses with another organelle that is capable of degrading its contents, called the lysosome, thereby eliminating damage or dysfunctional burden on the cell. This is a highly selective process with an elaborate set of protein complexes taking part.
Chaperone-mediated autophagy (CMA), on the other hand, is a highly specific process that involves degrading proteins with a specific subsequence of amino acids (a “motif”), KFERQ. About 30% of cytosolic proteins are recycled by CMA. The process involves the recognition of proteins with this specific motif by chaperone proteins, such as HSC70. (Chaperones are the proteins that help other proteins to obtain and maintain the right shape and also help them move to the right cellular compartment for optimal function.) Upon binding to the chaperone, the protein is delivered to the lysosome and is internalized through a lysosomal membrane protein called LAMP2A.
Microautophagy differs from the above as it involves the topological arrangement of membranes where the autophagic cargo is directly taken up by the lysosome. Initially, there is a morphological transformation where the membrane of the lysosome invaginates and forms a cup-shaped indent that sequesters the cargo. This then undergoes a topological transformation and pinches off into the interior of the lysosome, becoming a structure called the microautophagic body, and degradation of the engulfed cargo begins.
Subclassifications of autophagy exist which are dependent on the targeted cargo, namely lipophagy (lipids), glycophagy (glycogen), mitophagy (mitochondria), nucleophagy (parts of the nucleus), xenophagy (pathogens) and lysophagy (lysosomes themselves). Irrespective of the mechanism involved in cargo recognition or the type of cargo in different types of autophagy, the final destination of cargo digestion and associated recycling remains the same and is the lysosome.
It is important here to distinguish between ubiquitination and autophagy. Ubiquitination is a process by which proteins are marked by copies of a small protein called ubiquitin. Monoubiquitination (attachment of just one ubiquitin molecule) marks the protein for membrane trafficking, while polyubiquitination marks a protein for degradation. There is a partial overlap between the autophagy and ubiquitin pathways. The major difference is that organelles and large protein aggregates can only be cleared by autophagy.
Autophagy and Longevity
Hey, wait, why is all this relevant? Why is this worth my time going through what a measly cell is doing to get rid of damage at the macromolecular or organelle level? If the cell has evolved an efficient mechanism to handle this, why should you or I meddle with it? Let’s answer that in the following section by addressing the physiological significance of autophagy and the role it plays in diseases especially in the context of aging. We have in past blog posts defined aging as the accumulation of damage over time that disturbs homeostasis at various levels including the molecular mechanisms at the subcellular level, and let’s now dive into how autophagy is disrupted as well with aging.
Autophagy pathways are considered to be protective pathways whose function is to maintain homeostasis, differentiation, development, and survival at the cellular level. It is important to understand the triggers of autophagy to better understand how disruption of autophagy results in disease and precisely the types of diseases the disruption results in. There are two very important known inducers of autophagy: inhibition of mTOR and activation of AMPK. Both respond to stress (elevated temperature or starvation) and/or exercise. mTOR is a serine-threonine kinase which is a component of the complex called mTORC. This comes in two flavors viz., mTORC1 and mTORC2. Under normal conditions, mTORC1 negatively regulates autophagy while mTORC2 positively regulates autophagy. Other metabolites that affect autophagy (mTOR-independent) include calcium inositol phosphates. A large number of studies have been carried out on autophagy with inhibitors of mTOR such as rapamycin; these have been paramount in leading to elucidation of the role autophagy plays in the maintenance of homeostasis between health and disease. Since mTOR has a pleiotropic role and inhibition/activation can touch multiple important processes within the cells, autophagy interventions are increasingly focussing on mTOR-independent pathways that affect autophagy.
Let’s now understand the connection between autophagy and aging. Several studies have shown a negative correlation between age and autophagic activity. The expression of several genes associated with the autophagy machinery (Atg5, Atg7 and NECN1) decreases with age compromising autophagic efficiency. The proteolytic function of the lysosomes has also been shown to decline with age. Studies have been conducted (such as knockdown experiments of the key genes involved in autophagy) in animal models to reduce the efficiency of autophagy and the resulting tissue and organismal level function decline is highly corroborative to what is observed in aging. Conversely, other experiments (genetic manipulations in animal models) with increased autophagy have resulted in healthspan and lifespan extension. One noteworthy example here is that of rapamycin, a known inhibitor of mTOR that is shown to extend both median and maximum lifespan in both male and female rats. Autophagy is known to impact the adaptive immune system’s memory, which declines with age, and intervention with spermidine that restores autophagy restores immune memory and also intervenes effectively with cognitive decline in mouse models. In addition to its overall contribution to aging and age-associated functional decline, there exists a significant body of evidence to link cell/tissue-specific loss of autophagy to neurodegenerative diseases, metabolic defects, and cancers.
Let’s try and understand the link between neurodegeneration and autophagy. Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis are all associated with aggregates of misfolded proteins. The notion here is that the very fact that these misfolded aggregates accumulate is evidence of inefficient clearance and therefore impaired autophagy. Observations that depleting the proteins associated with autophagosome formation (atg7 or atg5) has been shown to cause neurodegeneration in the mouse brain supports this association.
Neurons are postmitotic cells (cells that have lost the ability to divide) and therefore, they have also lost the ability to dilute away the harmful load of toxic or dysfunctional macromolecules and organelles. They rely heavily on autophagy to maintain protein homeostasis. The long axonal structure also adds on to this complexity. Amyloid plaques and neurofibrillary tangles are the hallmarks of Alzheimer’s disease. Without going into the molecular details, intracellular neurofibrillary tangles that contain aggregates of hyperphosphorylated tau are suggested to result from impaired clearance of autophagosome vesicles. Rapamycin has been shown to decrease neurofibrillary tangles and therefore ameliorate Alzheimer’s. These observations have also been extended to Parkinson’s disease (PD): postmortem analysis of PD brains shows the accumulation of autophagosome vesicles and dysfunctional lysosomes. PD is associated with the loss of dopaminergic neurons that have accumulated in Lewy bodies (which are intracellular inclusions of alpha-synuclein and polyubiquitinated proteins). Overexpression of TFEB, an important regulator of autophagy, has been shown to ameliorate alpha-synuclein pathology. Resveratrol, trehalose, nilotinib, and lithium have all been shown to decrease the alpha-synuclein burden through induction of autophagy.
The link between autophagy and neurodegeneration is incomplete without touching upon the impact of mitophagy. Aging is associated with the accumulation of large deletions in mitochondrial DNA molecules, causing compromise both in function and number of functional mitochondria. Accumulation of damaged mitochondria is implicated in PD. Disruption of the mitochondrial gene PINK1 is known to disrupt mitophagy and has been shown to play an important role in familial PD.
One of the most intriguing aspects is the link between autophagy and cancer. Several studies have assigned a cancer-preventive role to autophagy, while an equal number or more have assigned a cancer-promoting role to autophagy. In general, the tumor-suppressor role of autophagy stems from its protective role against oxidative stress, inflammation, DNA damage, and accumulation of dysfunctional organelles. Unfortunately, once the tumor is established, autophagy increases in the hypoxic regions of the solid tumors which helps in coping with the high growth factor- and nutrient-deficient tumor environment through increased rates of turnover. Studies have also shown that autophagy is commonly induced as a survival mechanism against cytotoxic therapies such as chemotherapy and radiotherapy and emergence of resistance and survival of tumor cells.
Intermittent Fasting and Autophagy
How can understanding autophagy impact us today? Like most aspects of science, is this also something that only scientists get excited about to work in the lab to elucidate the underlying molecular pathways and discover intervention strategies to create drugs for the most dreaded diseases of the century such as neurodegeneration and cancer? Should we wait for another decade to enjoy the fruits of this labor? Since 2016 when Yoshinori Ohsumi was awarded the Nobel prize for elucidating the morphological and molecular mechanisms of autophagy, this field has captured everyone’s imagination. No cocktail dinners, biohacking summits, longevity meetups, or health club discussions are complete without a discussion around induction of autophagy. All these discussions center around the linkage between intermittent fasting and the ability to induce autophagy.
Let’s begin by asking what is intermittent fasting (IF) and why is it attracting attention? A feeding pattern with prolonged periods of lack of food intake (6–8 hours fasting each day, or fasting every other day, or prolonged periods of low food intake) on a recurring basis is intermittent fasting or restricted feeding. Various studies show that IF leads to a reduction in insulin resistance and several beneficial effects on chronic conditions such as cardiovascular indications, diabetes type 2, cancers, neurological disorders, and stroke. This is because animals, including humans, evolved in environments where availability of food was episodic, with numerous adaptations to survive and exhibit both high levels of physical and cognitive abilities under food-deprived states.
As we understand, autophagy is regulated by nutrient-sensing master regulators such as mTOR and AMPK.
(Ref- 30032222)
Amino acid abundance activates mTOR, which decreases macroautophagy. In contrast, nutrient depletion (glucose) decreases cellular ATP and activates AMPK, which in turn activates macroautophagy. The AMP/ATP or ADP/ATP ratio impacts the activity of AMPK in a positive correlation.
The link between autophagy and intermittent fasting is being studied with great interest and is being established both from a mechanistic point of view as well as the kinetics of autophagy induction. IF is being heralded as one of the most potent known inducers of autophagy. In rats starved for 24 to 46 hours, most cells in every vital tissue had an increased number of autophagosomes. Similar studies in animal models have been reported in many types of tissues and cells linking longevity and extension of healthspan with autophagy and IF. Cessation of fasting also abrogated the beneficial effects of IF. Lots of empirical evidence also exists in the literature linking the two to several types of beneficial outcomes. This in a nutshell represents how cells convert a lack of nutrients into a regenerative cue. That’s what we call a completely positive spin!
While we all believe that longevity is the future of medicine, the problem faced today is that there are very few treatments available or that can even be considered a step in that direction. The understanding of autophagy — and its ability to improve chronic age-related conditions such as cardiovascular health, diabetes, cancer, and neurological disorders by simply adjusting eating habits — is one of many revolutionary developments that will shape how healthcare is practiced in the coming years.
