Sunday, June 14, 2009

Autophagy, Aging, ATP, and AMD

Over recent months I have being trying to deepen my understanding of autophagy. For a good overview of autophagy check out this site. The nutshell goes like this:

Autophagy is an intra cellular process whereby cellular contents are degraded and then either recycled or expelled from the cell. Autophagy is mediated by organelles called lysosomes, which have a high internal acidity of 4.5 so as to allow the degradation of many molecules and organelles such as mitochondria; the latter process being mediated via macroautophagy, the former through chaperone mediated autophagy.

Autophagy can induce cell death in cancer cell lines though exactly how this happens is not understood. This issue has been bugging me for some time now, I'll come back to it later.

Under most circumstances however autophagy is protective for many cell types and improving autophagy, particularly in the aging cell, is fundamental to preserving cellular health. Autophagy tends to decrease with age, probably concurrently with the increase in lipofuscin waste products in cells. Lipofuscin is undegraded waste products that accumulate in the lysosomes. Lipofuscin can occupy a very large intra cellular volume and will inhibit lysosomal function. This can create a vicious feedback cycle as more lipofuscin will acccumulate, eventually causing the lysosome to rupture and spill the contents into the cytoplasm. Lysosomes used to be known as "suicide vesicles" and with good reason, the degradative enzymes and a pH of 4.5 do a lot of damage in the cytoplasm, potentially killing the cell.

Why does autophagy decrease with age? Speculations:

To maintain an internal pH of 4.5 lysosomes have proton pumps which are ATP dependent. "ATP" is an acronym for a molecule the provides energy in various chemical reactions. It is produced predominantly by mitochondria. ATP levels tend to fall with age and strategies to promote ATP production are vital in maintaining cellular health. If lysosomes cannot maintain an appropriate pH then lipofuscin accumulation will be accelerated.

An aging cell requires more house keeping. Protein production is not as good as it used to be, thereby placing more work on lysosomes and the ubiquiotin - proteasome processes to degrade damaged or dangerous molecules. These two degradative pathways must also contend with the problems created by various irritants that have entered the cell. While these irritants may never actually kill a cell there is the potential for the disruption of any number of processes that could perturb cellular functions.

Nuclear DNA Damage. Again, essentially the aging cell is confronted with increased maintenance. More aberrant proteins are transcribed which requires more degradation by the ubiquitin - proteasome system.

Mitochondrial DNA damage. Mitochondria have DNA more typical of bacteria than mammals. It is single stranded and circular, encoding only 13 proteins, the other proteins required by mitochondria are provided by nuclear DNA. Mitochondrial DNA is much more susceptible to damage than nuclear DNA and lacks the repair enzyme capacity of nuclear DNA. This is unfortunate because mitochondria are the major source of all oxidants created in our cells. The consequent of this is reduced ATP production and all the downstream consequences that entails.

Before Autophagy There Must be ATP

While small amounts of ATP are produced in the cytoplasm it is the ATP production of mitochondria that can determine the health of a cell. Following the ideas of Lynn Marguilis, mitochondria appear to be ancient cells that took up residence in another cell type, thereby allowing a powerful symbiosis that underlies the increasingly complexity of cells through evolution. Various studies have indicated that targeting mitochondrial function could have considerable clinical value.

Professor Bruce Ames was so impressed with his results that he started a company, Juvenon, to market a product specifically aimed at "rejuvenating" mitochondria. The active components of this product are Alpha Lipoic Acid and Acetyl L Carnitine. For an overview of their research refer to this page where you can download various scientific articles. Alpha Lipoic Acid is a very powerful antioxidant that can "regenerate" vitamins C and E. Acetyl L Carnitine transports fatty acids into mitohondria, thereby providing the essential substrates for ATP production.

In the work of Ames et al there is a striking recovery of mitochondrial structure. The internal cristae are like those from the mitochondria of young cells. There are a number of issues here that baffle me. I find it hard to believe that mitochondria, with a genome encoding only 13 proteins, can initiate repair processes. Alternatively this recovery of structure may reflect a simple reiterative process of molecular dynamics that gives rise to the structure. Chaperones and heat shock proteins, typically providing a protective effect for cells, often play important roles in folding proteins and unfolding misfolded proteins. As many of these functions are ATP dependent, increased ATP availability will enhance the protective effects of chaperones and heat shock proteins.

As the principal mode of house keeping for lysosomes is chaperone mediated autophagy, an important chaperone here being HSC70, and the ATP dependent proton pump in lysosomes, improved ATP levels will enhance autophagic processes. This raises the question as to whether the improved mitochondrial structure arose because the increased "house keeping" activities removed various misfolded proteins and irritants thereby allowing mitochondrial structure to become more like that associated with healthy mitochondria. This suggests a complex interplay between mitochondrial and lysosomal functions. Aging and rejuvenation studies clearly indicate that mitochondrial and lysosomal functions are linchpins of health.

The decline in ATP production with age is probably occurring through the gradual loss of mitochondrial numbers and efficiency. (And, I just stumbled upon this, for a laugh, read it.) However, according to this article at least, under the right conditions it does seem possible that mitochondrial structure can be repaired. Note the chaperone and heat shock protein involvement in these processes and also the "spontaneous" macromolecule assembly that occurs.
The article addresses mitochondrial replication, an ongoing process in our cells.

The Retina as a Model of Autophagy and Aging

While enhancing cellular house keeping functions is essential to warding off damage and maintaining optimal cellular metabolism there remains the problem that lipofuscin accumulation is almost inevitable. These are chemical processes after all and there are always residues that are not excreted as digested products from lysosomes. lipofuscin accumulates, not only in lysosomes but also in other cellular regions including mitochondria, where it directly impacts on ATP production. Lysosomal degradation results in the expelling of the digested products for recycling. There is also evidence for some of these products being "packaged", transported to the cell surface, and expelled.

In one hypothesis, what I consider to be an exquisite process that gives me a moment's sympathy with Intelligent Design, it is put forward that lysosomes and\or the waste products are transported to the cell surface, excreted, absorbed by microglia, which then move to the capillary and dump the contents into the general circulation. As hard as I find that to believe recently I read a study addressing the contents of drusen, the waste products that builds up in the retina and beyond a certain level is a key marker for Age Related Macular Degeneration, is composed of molecules that are related to autophagic processes, including mitochondrial related proteins. That finding suggests that the waste products of lysosomal processes are being excreted. In relation to the retinal pigment epithelium the finding is not surprising because these are probably the cells that do the most degradation work of any cells in the body; at least in terms of constancy of demand.

That study raises the interesting question: why are the RPE cells expelling these materials into Bruch's Membrane, which lies between the RPE cells and the choroid, the blood supply? I hate to so obviously invoke intentionality but I trust you'll understand my point: Bruch's Membrane is a two way transport membrane, allowing nutrients to reach the RPE and waste products to be excreted into the bloodstream. In one study I read it was found that with age the permeability of Bruch's Membrane decreases by many orders of magnitude. In many perhaps most people over 50 there are some traces of drusen. In AMD drusen is all over place and where drusen is present the underlying photoreceptors cells are either suffering or dying; probably both.

A very interesting aspect of that study is the finding of mitochondrial related proteins. Such contents may have been expelled from the cell in vesicles. Mitochondria are degraded by a complicated process called macroautophagy. from what I can presently understand this process requires a lot of energy and makes big demands on a lysosome. Thus any strategy which can reduce the overall rate of mitochondrial turnover could have value in retinal and neural degenerative conditions. This may explain some experimental and clinical results suggesting improving mitochondrial function can impede disease progression.

With the decreasing permeability of Bruch's Membrane larger particles may effectively become trapped in the membrane. However all is not lost for this is where the little appreciated aspects of immune function come into play. There are various types of immune cells that carry what is called a "scavenger receptor". These cells move through tissues and do the extra-cellular equivalent of house keeping. Extra cellular debri is removed, dead or dying cells are absorbed and digested, cell health in the tissues is monitored through MHC class I receptors.

When the Peace Corps Come Marching In

At this point we need to enter into immunology. Here's the nutshell. Cytokines are a class of molecular messengers with far ranging effects, mostly immunological but that can get difficult ... . The types of immune cells that are engaged in the above functions are helped along by cytokines il4 and il10. These are typically perceived as anti-inflammatory cytokines. It appears to be the case that inflammation and house keeping don't go together(males may disagree). That makes sense, you don't vacuum the carpet when there's a burgler in the house.

In times of peace the immune system is cleaning up the area. This is a natural extension of an important process in limiting inflammation. As the inflammatory process progresses there must eventually be an elimination of inflammatory signals. This will involve the elimination of various proteins and lipids that signals danger and so invites an inflammatory response. For example, heat shock protein 60, expressed at very high levels, can and will drive inflammatory signals. The more quickly proteins like hsp 60 are removed from the extra cellular environment the more quickly inflammation will recede and then the repair processes can begin.

It is important to remember that "inflammation" is a word describing a series of processes. Unfortunately it is a loaded word. "Inflammation" can play important roles in initiating cellular repair, providing nutrients, and some so called "inflammatory mediators" are important in regulating neural transmission. (That's a whole fascinating other subject, the many relationships between the the nervous systems and the immune "system".) Take heed of Wittgenstein's Wisdom: "Words are posts on which we hang meanings".

In studies of Age Related Macular Degeneration there is are clear genetic linkages with immunological function and susceptibility to AMD. In particular, Complement Factor H, an inhibitor of complement function, is strongly implicated. The general trend suggests that a persistently elevated level of systemic inflammation is a considerable risk factor for AMD. Systemic inflammation is driven by two principal cytokines: tnf a and il1. A current model popular in immunology is the Th1 - Th2 paradigm. Th 1 is typically perceived as inflammatory and driven by by tnf a and il1, whereas Th 2 is driven by il4 and il10. Echinacea can cause a distinct shift towards the Th 2 type of function, and vitamin D also plays an important role here. Even omega 3's can be important as these fats will, over time, alter the production of prostaglandins, the downstream effect of this being to inhibit the production of il1 and tnfa.

As a general rule the studies suggest that our physiology, particularly with age, is too inclined to shift towards an inflammatory state. The possible explanations for this, while plausible, are long winded and tedious so I'll avoid that aspect.

Our evolution predisposed towards a balance of fat intakes that is out of kilter in modern diets. There is too much omega 6 to omega 3, the consequence being that while omega 3's inhibit potentially inflammatory prostaglandins, omega 6 fats do the exact opposite. At the immunological level this involves a shift towards higher expression of il1 and tnfa. Hence there have been some very surprising results in treating early AMD with omega 3 fats and other nutrients. There is already one supplement out there that appears to even reverse early AMD, while another recent study suggest that even in juvenile retinal degenerative conditions nutrient support can delay disease progression.

With age there is a general increase for more house keeping functions. This arises as cell damage occurs and DNA damage, at both the nuclear and mitochondrial sites, induces aberrant protein production that places additional loads on the ubiquitin proteasome degradation pathway.

With age people can lose the capacity to produce vitamin D from sunlight exposure. Recent epidemiological studies suggest widespread vitamin D deficency. This has all sorts of worrying implications for general health but of particular relevance here is that vitamin D is very important in moving the immune balance towards a less inflammatory state.

Thus studies indicating the protective effects of vitamin D and omega 3's might be converging on the general metabolic effect this has and how this impacts of autophagy and immunological extra cellular house keeping functions. What is interesting about the successful supplement regimes is the specific targets the researchers had in mind: antioxidant strategy, omega 3s'(mininum of two functions here: DHA is a substrate for NPD1, an important neuroprotectant, and EPA works at the prostaglandin level), and mitochondrial enhancement.

And to Wrap Up ... .

This brings us all the way back to what I mentioned earlier about the susceptibility of mitochondrial DNA to oxidative damage. Mitochondria absorb nutrients and eventually divide but as the DNA is damaged the new mitochondria are effectively aged. Fortunately genes aren't everything, ongoing studies indicate that the astute use of nutrients can impact on cellular functions at such fundamental levels so as to provide cells with protective functions that may increase their lifespan and function.

Inducing autophagy is not easy but obviously a desired goal. Rapamycin, an antibiotic of all things, induces autophagy. Nutrient deprivation, as in Caloric Restriction where autophagy has long been recognised as an important antiaging component of this strategy, can induce autophagy but it is very difficult to know just how much nutrient deprivation ... . It might just be amino acid deprivation, or the deprivation of fuel. Indeed, it is known that rapamycin induces autophagy by inhibiting the receptor mTOR, which is a nutrient receptor. Interesting concordance with Caloric Restriction dynamics there. This is how in the earlier study I cited on cancer cells killed by autophagy the process unfolded. Now there's another fascinating mystery, why does autophagy kill cancer cells but not other cells; though it can kill other cells sometimes ... . It's just too difficult. At present, just today I managed to dream up at least one plausible approach to that challenge. "Plausible" don't mean that much though!

My other specific goal in relation to this is to find strategies that address the exocytosis of waste products from cells concurrently with a distinct immunological shift to induce a heightened state of extra cellular house keeping. It probably can be done to some decent extent but will require lots of discipline. Don't have much of that and besides I need to keep reading.


Anonymous said...

thanks , ur blog was easy to read and funny as well . keep things up!

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xlpharmacy said...

Unicellular Everyone is super interesting and complicated too, as well as it is impressive to expand our knowledge of its functionality

pSIVA said...


Thanks for providing these useful tips over here. Autophagy is a process of intracellular bulk degradation in which cytoplasmic components including organelles are sequestered within double membrane vesicles that deliver the contents to the lysosome...

Anonymous said...

Caloric restriction is always mentioned as a way to initiate autophagy, but a far more powerful strategy without the miserable life aspects inherent in caloric restriction, is intermittent fasting. Autophagy begins at 12 hrs since your last meal, peaks at 24 hrs , and then trails off after 36 hrs. There are also several autopnagy enhancers to multiply the effect during fasting like curcumin, resveratrol, hydroxycitric acid (Garcinia Cambogia is a good source) miacinimide (not nicotinic acid) , and EGCG (from green tea).
Trying to initiate autopnagy with just rapamycin is not nearly as powerful as intermittent fasting.
A long way third comes plain old long term caloric restriction - which is also dead boring.

John said...

Thanks Anon,

I follow the eat in a 6 hour window rule and like yourself think caloric restriction is too demanding in the real world. Additionally over the long term it can induce immunosuppression and I have concerns about cerebral function with long term CR. Another aspect to think about is restricting methionine consumption, it appears to play an important role in this context.