Paper 2: AutophagyCell Recycling

IntroductionDeep down, we are cannibals. Although that may sound repulsive, practicing a little self-cannibalism, paradoxically, is the key to living a healthy and long life. Our cells are perpetually eating themselves, breaking down their own organelles, and recycling them to build new parts and energy. This self-eating process is known as autophagy; where cells destroy and recycle cellular components. Autophagy has a lot of profound effects on our bodies that are just now being discovered. For example, without autophagy humans would die right after birth. When babies are born, they need a huge amount of energy to begin their lives without their mothers. However, this demand for energy comes right at the time when the umbilical cord is cut, preventing them from receiving any nutrients (Zimmer). Japanese scientists have also found that lysosomes in mice start working more more efficiently after they are born. Then after a couple of days, the rate of autophagy drops back down to normal. When the scientists genetically engineered the mice to prevent autophagy from occurring, the newborn mice died immediately of starvation (Zimmer).

BackgroundThe term, Autophagy was derived from Greek meaning “eating of self”. This term was first coined by Christian de Duve over 40 years ago, when he observed degradation of mitochondria and other intracellular structures within lysosomes of rat liver perfused with pancreatic hormone, glucagon, a pancreatic peptide hormone that raises the concentration of glucose in the bloodstream (Glick).In recent years the scientific world has rediscovered autophagy and realized its importance for the survival of any organism. This year’s Nobel Prize in physiology went to Yoshinori Ohsumi, a Japanese cell biologist specializing in autophagy. Dr. Ohsumi was able to discover and locate genes that regulate this cellular self-eating process that helps our body stay healthy using budding yeast. Dr. Ohsumi focused on starvation-induced non-selective autophagy, while Daniel J Klionsky, a researcher at the University of Michigan, discovered the Cytoplasm-to-Vacuole Targeting (CVT) pathway, a form of selective autophagy ("Autophagy."). These scientists soon found that they were all looking at the same pathway from different angles. The Autophagy-related genes that Dr. Ohsumi and other scientists discovered were given a unified name, ATG. Dr. Ohsumi’s work is revolutionary because it explains what goes wrong in a range of illnesses from cancer to Parkinson’s (Roberts). Currently, the main theme of autophagy research is the relationship between cancer and autophagy and other health problems.

What exactly is Autophagy?Autophagy is a natural process that the body uses to clean itself by removing defective proteins and organelles, eating them, and then using the resulting molecules for energy or for ingredients to make new cell parts. This process can be seen as the recycling factory that also promotes energy efficiency through ATP generation (Glick).

There are three types of autophagy: Macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) (Glick). All three promote proteolytic degradation of cytosolic components at the lysosome; the breakdown of proteins into smaller polypeptides or its respective amino acids (Proteolytic Degradation). Macroautophagy is the main pathway used to remove cytoplasmic cargo such as damaged cell organelles or unused proteins. The first step begins in the isolated membrane, the phagophore. The phagophore elongates around the substrate that the cell wants to degrade, forming a double membrane

called the autophagosome. The autophagosome then carries the cargo to the lysosome1 where the autophagosome and lysosome fuse to form the autolysosome, where the substrates undergo degradation (Reader). There the contents of the autophagosome and its inner membrane are digested by the acidic lysosomal hydrolases 2.(Glick). Finally, the amino acids and other by-products of degradation get exported to the cytoplasm via lysosomal permeases and transporters. There they can be used for energy and building macromolecules. Unlike Macroautophagy, Microautophagy involves the direct engulfment of cytoplasmic waste into the lysosome through invagination of the lysosomal membrane. The process of degradation is identical to macroautophagy. The third form of autophagy, chaperone-mediated autophagy (CMA), involves specifically the degradation of soluble proteins. CMA exists only in mammalian cells and is extremely selective about what materials it will degrade (Cuervo). The lack of an oxygen containing molecule that has one or more unpaired electrons known as oxidative stress is known to induce CMA. CMA “is an intracellular catabolic pathway that mediates the degradation of a selective subset of cytosolic proteins in lysosomes,” (Cuervo).

Molecular Biology In order to understand where autophagy can go wrong, autophagy can be seen as three major parts: selection, sequestration, and lysosomal digestion of substrates. In this paper I will focus on the first two parts.

Substrate sequestration and autophagosome formationAutophagy is induced through changes in the phosphorylation states3 of stable autophagy-related proteins. The phosphorylation of ULK1, a protein essential for autophagy, is regulated by the inhibition of TORC1, a protein that “functions as a nutrient/energy/redox sensor and controls protein synthesis,”. Activation of the TORC1 promotes protein synthesis instead of autophagy. “Various stress on cells such as lowered concentrations of specific amino acids, ATP, protein aggregates, and endoplasmic reticulum stress, suppresses TORC1, thus activating ULK1 and ULK2 and turning on autophagy,” (Nixon). This

1 A lysosome is an organelle in eukaryotic cells with a very low PH that holds a variety of enzyme that are used to digest things within the cell. 2 Acidic hydrolase is an enzyme that works best acidic PH, commonly located in lysosomes.3 Phosphorylation is the addition of a phosphate group (PO4

3−) to a molecule of individual components.

affects many neuronal functions such as dendritic arborization4, myelination5, and synaptic plasticity6. The phosphorylation of ULK1 induces translocation of a multiprotein complex containing beclin-1 and class III phophoinositie 3-kinase forms a pre-autophagosomal structure that will later form the autophagosome (Nixon).

Substrate Selection The general consensus now is that autophagy can engulf cargo selectively and non-selectively (Glick). Selective autophagy is induced by a malfunction of an organelle or protein; when the cell needs maintenance. A damaged organelle or protein will release a protein degrading signal (PDS) called a degran so that the cell can identify what to degrade (Autophagy). These signals can be found on the surface of the proteins in need of degradation and can be simple amino acid modifications on the surface. For example, if there is a malfunction in the protein folding, hydrophobic residue will sometimes come out of the protein structure (Autophagy). This is usually a signal for degradation because normal protein structures have hydrophobic residue inside and hydrophilic residue on the outside. Once the signal is read, the cell begins the autophagy process. Another way the cell identifies what to degrade is through the use of the autophagic receptor, p62, also recognizes and facilitates the elimination of damaged proteins. PINK1 and Parkin are two proteins that target damaged mitochondria for selective autophagy (Nixon). PINK1 is activated when the mitochondria is damaged and the membrane is depolarized (Nixon). This allows parkin to bind and ubiquitinate 7 exposed membrane proteins, thereby inducing p62 and LC3 (Nixon). Non-selective autophagy is induced by starvation. Instead of targeting specific parts, the cell will wrap up random cargo, take it to the lysosome and use it as energy until the conditions have improved (Nixon).

Autophagic receptors attach the targeted substrate to core autophagic machinery, such as microtubule-associated protein light chain 3(LC3). .

Autophagy and Neurodegenerative diseases Mutations in genes that are involved in regulating autophagy and endosomal pathway are directly linked to neurodegenerative disorders. Many neurodegenerative diseases are the result of specific types of malfunctioning proteins in neuronal tissues such as the brain, forming clumps of proteins. Experiments using mice and other organisms suggest that these misfolded neural proteins form aggregates that the lysosomes can devour to prevent them from causing damage, slowing down the progression of the disease. For example, when autophagy was suppressed in flies, they began to accumulate abnormal clumps of proteins in their cells, especially in the neurons, and the cells died as a result (Zimmer). Neurons in the brain are particularly vulnerable to defects in the autophagy process due to the neurons’ “unusually large expanses of dendritic and axonal cytoplasm…” (Nixon). This characteristic of neurons makes it much more difficult for the neuron to prevent dysfunctional organelles and cellular waste from accumulating. Although young neurons perform this task with great efficiency, old neurons do not. This is the reason Alzheimer's and Parkinson’s are late onset disorders.

4 dendritic arborization is the signaling between dendrites (Byrne). 5 Myelination is the production of myelin, a fatty white substance that coats the axon, insulating the neuron from electricity. 6 Synaptic plasticity is the ability of synapses to strengthen or weaken over time, in response to increases or decreases in their activity (Byrne). 7Attaching ubiquitin to a protein inactivates it and acts as a signal for degradation

Errors in substrate sequestration and autophagosome formation can lead to many detrimental diseases. In Huntington’s disease, autophagosomes form properly and are cleared, despite slower-than-normal macro autophagic protein turnover. However, when scientists examined brain samples from people with Huntington’s disease, they noticed that the autophagosomes were unusually empty; the autophagosome membrane seemed to be inadequate at capturing substrates property during sequestration (Nixon). This could be due to the unplanned binding of beclin-1 by aggregates of mutant huntingtin leading to its depletion, resulting in the failure of sequestration. In Lafora's disease, a form of epilepsy associated with neurodegeneration, mutation in the protein phosphatase laforin abnormally actives TOR1, reducing the rate of autophagy (Nixon). The figure below shows in more detail each point at which each disability can be caused. (HD = Huntington’s disease, PD= Parkinson’s Disease)

Errors in substrate selection can also cause many problems. Mutant p62 may promote errors in different ways by disrupting autophagic clearance of damaged proteins. This could be an underlying reason for Alzheimer's. Parkinson’s disease can be credited to the mutation in the genes encoding PINK1 and parkin can impede autophagy, causing damaged mitochondria to accumulate, possibly initiating apoptosis8. CMA mediates the lysosomal degradation of a group of unique proteins, which includes a substantial number of proteins that cause diseases such as Parkinson’s Disease (Cuervo). In this process the Hsc70 protein assists the cargo to lysosome-associated membrane protein type 2a (LAMP2A) (Nixon). There the protein is unfolded and degraded in the lysosome. In many neurodegenerative diseases these altered proteins bind abnormally tightly to LAMP2A, preventing them for moving across the lysosomal membrane (Nixon). This decreases the rate of CMA, resulting in an accumulation of misfolded proteins (Nixon).

Other Health effects:

8 Apoptosis is programmed cell death.

Autophagy has many other profound implications on one’s health. It plays both a role in tumor suppression and tumor cell survival. In tumor cells, autophagy allows prolonged survival under metabolic stress. Inhibition of autophagy can induce apoptosis and tumor regression, curing the tumor (Autophagy). Lysosomes can also protect against cancer. As the mitochondria gets old, it can cast off charged molecules that can cause destruction in a cell and lead to potentially cancerous mutation. If the autophagy process was efficient it would degrade these defective organelles and decrease the chances of damaging its DNA. Doctors have found that breast cancer cells are often missing autophagy-related genes; this is most likely not a coincidence, although correlation doesn’t imply causation (Zimmer). In addition, autophagy plays important roles in immunity as well. Intracellular pathogens such as protozoans, viruses, and bacteria are eradicated through autophagy. Lastly, autophagy has an effect on other diseases such as cardiovascular conditions. For example, if one has plaque buildup in a coronary artery, the fat droplet can be engulfed by an autophagosome and then destroyed by the lysosome. The digestive enzyme then converts the cholesterol ester to free cholesterol and then enters it back into the bloodstream which is then metabolized to waste or reabsorbed. Defects in this process can cause atherosclerosis9.

Therapeutically enhancing autophagy: Many scientists are trying to manipulate autophagy directly and understand the genes that are behind autophagy. Dr. Cuervo, co-director of the Einstein Institute of Aging Research, and her colleagues have found that the CMA system, which removes around 30% of the cells damaged proteins, becomes less efficient with age due to the loss of receptors on the surface of the lysosomes. For the study, Dr. Cuervo engineered mice to have an extra gene for the lysosome receptors. The extra gene was only added to the liver cells and was only activated once the mice reached middle age (Edelson). Once the mice were 22-26 months of age (80 years old for a human), their liver cells were still functioning as if they were 20. “They were able to remove all the damages oxidized proteins,” said Dr. Cuervo.

The scientific direction of Telethon Institute of Genetics and Medicine Naples, Italy, Andrea Ballabio, found another way to raise the rate of autophagy. Dr. Ballabio found that 68 genes that build lysosomes are activated by a master protein known as TFEB (Andrea Ballabio). Her team then engineered cells to produce more TFEB, resulting in the production of more lysosomes. Each of these lysosomes became even more efficient. In addition when they injected the cells with huntingtin, a protein that clumps together to cause fatal brain disorder, Huntington’s disease, the cells did a much better job of destroying the huntingtin (Andrea Ballabio).

Improving Autophagy on your own:To induce autophagy one can exercise, lower carb intake, or fast; putting the body in discomfort can bring long term benefits. The cell is constantly searching its environment for amino acids, sugars, oxygen, and nitrogen. If the cell senses that one of these is deficient, instead of targeting specific parts, it will wrap up random cargo, that is taken to the vacuole and used as energy until the conditions have improved.

Exercising is an obvious way to improve health. Working your muscles creates microscopic tears that your body rushes to heal. This makes muscles stronger and more resistant to other stresses one might put on it (Worth All The Sweat). Regular exercise is the most popular way that people unintentionally

9 Atherosclerosis is disease in which plaque builds up inside arteries (What is Atherosclerosis?).

help their body to cleanse. Beth Levine, a Doctor at the University of Texas Southwestern Medical Centre, found evidence that exercise promotes autophagy in mice. In order to observe this clearly, Dr. Levine made the mice’s autophagosomes glow green. After these mice spent thirty minutes on the treadmill, the number of autophagosomes in their muscles increased, and it continued to increase until they ran for eighty minutes (Worth All The Sweat). Although this is a profound finding, determining the level of exercise needed to simulate autophagy in humans is still extremely difficult. However, there are a lot of other benefits that exercise brings so it’s probably in everyone's favor to exercise anyway.

Ketosis is another increasingly popular diet among people that are seeking a longer life span. The idea is to reduce carbohydrates in the body to such a low level that the body has to resort to using fat as a source for fuel, which helps people lose body fat while retaining muscle (Schachter). There is also some evidence that it helps the body fight cancerous tumors, lowers the risk of diabetes, and prevent some brain disorders, in particular epilepsy. Several studies have shown that over half of children who go on the ketogenic diet, have at least a 50% reduction in the number of seizures. Some even get rid of seizures forever (Schachter).

Skipping a few meals, it turns out, is another stressful act on the body that induces autophagy. Researches have shown that fasting has a tremendous amount of benefits such as lowering the risk of diabetes and heart diseases, which could be attributed to autophagy. Cells respond to famine by producing only critical molecules and using autophagy to destroy the rest (Zimmer). Similar to the Ketosis diet, the body will produce more cholesterol, “allowing it to utilize fat as a source of fuel, instead of glucose” decreasing the level of fat cells in the body (Intermountain). In one study, animals were put on a strict low-calorie diet. The results showed that the animals that ate less lived much longer than animals that ate a superfluous amount. Mice that were put on restrictive calorie diets increased their longevity by up to 40% by postponing the onset of age-related diseases (Wade). Another effect of intermittent fasting was the body’s responsiveness to insulin, the hormone that regulates blood sugar. When the body becomes insensitive to insulin, it’s likely to lead to obesity. A recent study at the Salk Institute of Biological studies, showed that mice that ate fatty foods for eight hours a day, and then fasted for the rest of each day, did not become obese (Stipp). Another extensive experiment performed by a group of scientists proved that food restriction induced autophagy in the brain. In this experiment six to seven year old engineered mice were on a food restricted diet for 24 or 48 hours. This process is already known to induce a rapid increase in the rate of autophagy in the liver, however, it was unclear whether it induced autophagy in the brain. The results of autophagy were as expected in the liver.

As the figure above shows, after 24 hours there are discrete regions of high fluorescence, indicating an increase in the autophagy. After 48 hours, the changes were magnified (Alirezaei). The scientists then applied this approach to the brain and found the same results.

Food restriction caused an increase in both the amount and size of neuronal autophagosomes specifically in the cell body of cortical neurons (Alirezaei). Although this is a very new area of research, the general consensus is that intermittent fasting has lots of profound benefits.

ConclusionThere is still much to be researched to fully understand more about Autophagy. As technology improves and more resources are put into this field, hopefully we will be able to prevent neurodegenerative diseases from occurring and create cures for it. Although there is a lot of evidence supporting fasting, it’s important to remember that there always new articles claiming they have found the new way to become healthy and skinny. Hopefully, this new research holds true under more scrutiny and improvement in technology, but it’s important to do be skeptical of everything you may find online.

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Picture Bibliography Figure 1: http://www.nexcelom.com/Applications/image-cytometry-method-for-autophagy.phpFigure 2: http://www.nature.com/nm/journal/v19/n8/full/nm.3232.html

Figure 3: http://www.nature.com/nm/journal/v19/n8/full/nm.3232.html