Just when researchers think that a venerable theory of aging is dead, it often springs back to life. This is what has happened with Dr. Johan Bjorksten’s Crosslinkage Theory of Aging. Dr. Bjorksten had a gift for describing complex molecular functions using clear, easy-to-understand graphics. One of his favorite ways of portraying the crosslinkage theory was to describe an office or factory in which several people were handcuffed together (Fig. 1). Every day more people would be handcuffed to each other, and over time these couples would be handcuffed to other couples. Eventually so many people in the enterprise are handcuffed to each other (crosslinked), that all work grinds to a halt. Prof. Bjorksten used this example to describe how crosslinking in the body damages (and renders useless) important macromolecules, proteins, lipids and nucleic acids.
Crosslinks occur in the body in a number of different ways: individual strands of DNA can become crosslinked to each other (intra-strand crosslinking) (Fig. 2); proteins can fold over and become crosslinked to themselves; and proteins can become crosslinked to other proteins (Fig. 3). In time, these crosslinked strands and proteins will attach with other crosslinked structures to form large aggregates joined with multiple crosslinks.
The Aluminum Connection
One important discovery that Dr. Bjorksten introduced in the 1930s was the role of aluminum in crosslinking. At the time, Bjorksten worked for a company that made emulsions for the hectograph -- a predecessor of modern photocopy machines. The hectograph used gelatin plates to make a master of an original document. This impression was then used to make additional copies. Aluminum is a trivalent substance, meaning that it forms three crosslinks to occur simultaneously in a polymer. This made aluminum the agent of choice for forming tough gelatin emulsion plates for use in the hectograph. Bjorksten made his discovery when he noted that the effects of polymer crosslinking in the gelatin emulsions reminded him of wrinkled skin.
Enzymatic Crosslinking
There are a number of types of crosslinks that occur during aging. We just alluded to aluminum-induced cross-links. Enzymes can also induce crosslinks. Enzymatic crosslinking does not generally have a detrimental effect on health. In fact, we need enzymatic crosslinking for normal biological functions. The best example of this is triple collagen. The precursor proteins that make up collagen (procollagen) must be crosslinked to form a triple helix to keep our tissues intact and to act as a cellular glue to hold our tissues and organs intact. Otherwise, our bodies would simply collapse into pools of liquid.
Non-enzymatic Crosslinking
Another form of crosslinking -- non-enzymatic crosslinking -- has come to the forefront in the last two decades. Non-enzymatic crosslinking results from interactions between sugars and proteins that lead to the formation of macromolecules. These reactions (glycation and glycosylation) occur when a sugar (fructose, glucose, etc.) reacts with a primary amino group on a protein molecule. Once those two molecules get together they form a Schiff base. This is a reversible reaction. If it’s not reversed within a certain time period, the Schiff base can then form what is called an Amadori product. Here it is slightly reversible. However, a percentage of Amadori products do not reverse, and these products then undergo a number of oxidative and non-oxidative reactions that form crosslinked macromolecules that have been designated AGEs (Advanced Glycosylation End products).
Advanced Glycosylation Endproducts (AGEs)
Once an Amadori product has progressed to form an AGE, the crosslinking process becomes totally irreversible. But AGEs don’t just sit there -- they go on to generate free radicals and to form more crosslinks with other proteins (Fig. 4.). AGEs also create a whole host of catalytic problems for the cell during aging. AGEs form on macromolecules, such as proteins, lipids, and many nucleic acids. They form aggregates that cause problems with the proteolytic machinery of the cell. The lipids that react with AGEs form reactive oxygen species (ROS). Interestingly enough, AGEs then activate nitric oxide formation through nitric oxide inhibition.
AGEs are immunogenic to the system, so the immune system also comes into play. AGE products interact with lymphocytes, which in turn produce interferons. Interferons react with macrophages or directly interact with monocytes. Cytokines such as interleukin and tumor necrosis factor are also released, causing abnormal cell proliferation and growth.
AGEs are also inflammatory agents. They form aggregates, age pigments, and lipofuscin. Being mutagenic, they also contribute to carcinogenesis. We now think they may be involved in Alzheimer’s disease and other diseases where amyloid deposits and protein aggregation occur, such as Parkinson’s disease.
Some proteins are known to interact with AGEs, such as plasma, collagen, and beta amyloid in brain tissues. And there are actually receptors in the cell surface for the AGE molecule, which brings into play a whole new realm of problems. AGE products have been shown to interact with AGE receptors on endothelial cells. This increases the permeability of the cells and results in vascular leakage and coagulation, and increases the incidence of strokes. This process also increases cell proliferation of smooth muscle cells, which is thought to enhance -- and be part of -- the atherosclerotic mechanism. Fibroblasts also have receptors that react with growth factors that contribute to the accumulation of abnormal collagens and elastins.
Alzheimer’s and RAGEs
It is now recognized that AGEs are involved in Alzheimer’s disease for a couple of reasons. One is that senile plaques and neurofibrillary tangles are the two pathological hallmarks of Alzheimer’s disease. AGES decrease cellular signals, adversely affecting ion transport, glucose transport and glutamate signals. This results in a cytotoxic effect on the neurons. Most interestingly though, is that AGEs bind to RAGEs (Receptors of AGEs) on the neurons. This binding of AGEs to receptors sets off a cascade of abnormal, adverse events. When an AGE binds to a RAGE, it increases the oxidative stress of the cell, and also increases the binding affinity for beta amyloid, which we know is very toxic to the cells. Beta amyloid is one of the first proteins discovered in Alzheimer’s disease. AGEs also increase the release of proteases, which start chewing up the neurons themselves. In addition, we have pro-inflammatory cytokines, such as NF Kappa Beta, which turns on a cascade of inflammatory growth factors, such as tumor necrosis factor (TNF).
When AGEs bind to RAGEs, many really scary things occur. For example, gene expression is altered, growth factors are produced in abnormal quantities and qualities, transcription factors are altered, and oxidative stress is increased. I’m sure we will find out other things that result as well in the next few years. Equally as scary is that once RAGEs are formed, they are autocatalytic in that there’s a positive feedback loop that makes more RAGEs, and more receptors for more AGE products. Once this process starts, it doesn’t stop. It ends up in abnormal cell proliferation and inflammation, which ultimately ends up in tissue dysfunction and disease, and is an intimate part of the aging process. Aging-related diseases that have been attributed to this complex process include cardiovascular problems (mainly due to atherosclerosis) that also results in blood pressure changes, cardiomyopathies, blood clotting, Alzheimer’s, stroke, neuropathies, retinopathies, cataracts, renal failure, diabetes and skin aging.
Preventing and Reversing Crosslinking
But what can we do about AGEs? There are a number of potential anti-cross linking therapies that have come about recently, as well as some others that have regained appreciation (Fig. 5). These are in three general categories: (1) glucose lowering agents to stop the problem in the beginning to remove the sugars; (2) cross linking inhibitors, which do not reverse crosslinks, but can stop the crosslinks from occurring; and (3) crosslink breakers which actually get rid of AGEs by breaking the crosslinks, something that Dr. Bjorksten dedicated his life to.
Lowering Glucose
One of the most effective ways to lower glucose -- a regimen that almost no one can adhere to (but monkeys can, by coercion) -- is caloric restriction. Caloric restriction can increase the life span by about 30 percent. Caloric restriction lowers glucose levels by 20-30 percent, triglycerides by three fold, and restores insulin sensitivity. Caloric restriction is thus the prime model for the beneficial effects of insulin receptor-sensitizing/glucose-lowering agents. Agents that have been available for a long time that have moderate insulin receptor sensitizing effects include chromium, vanadyl sulfate, and adaptogens like ginseng and Siberian ginseng (Eleutherococcus sinensis).
Pharmaceuticals like phenformin and Metformin are among the most effective insulin sensitizing agents. ALT-4037 is a compound under development by a pharmaceutical company called Alteon that mimics the benefits of caloric restriction. Other substances that lower insulin and glucose by acting as starch blockers include the drug Acarbose, and the natural substance, 2-deoxy glucose.
Inhibiting Crosslinks
The next class is specific crosslinkage inhibitors. Alteon has identified nine chemical classes and over 852 compounds that have potential crosslinkage inhibiting effects. The most effective ones to date include: ALT-946, carnosine, aminoguanidine, vitamins B1 and B6 (there are reports that B6, as pyridoxal-5-phosphate [P5P] is as effective as aminoguanidine), caloric restriction, antioxidants, glutathione, metal chelators (including EDTA and carnosine), and perhaps ascorbic acid and anti-inflammatory analgesics (but they are weaker inhibitors).
Aminoguanidine
Aminoguanidine is a guanidine derivative. It stops crosslinks by (1) scavenging glucose by competing with amino groups of protein for the carbonyl group on glucose; or (2) binding and reacting to these groups, stopping the browning process (Maillard reaction). One of the most significant effects of aminoguanidine is its ability to prevent or reverse diabetic complications (Fig. 6), including diabetic nephropathy, retinopathy, and neuropathy. Aminoguanidine has been shown to increase the maximum life span of rodents by 20 to 30 percent. Guanidine is the active ingredient in the herb, Goat’s rue (Galega officinalis). Goat’s rue is, in fact, the herbal prototype of the anti-diabetic biguanide drugs, phenformin and Metformin, and presumably has similar effects and benefits as aminoguanidine, phenformin and Metformin (Fig. 7).
B1, B6 and ALT-946
The cyclic groups of vitamins B1 and B6 are involved in competing with the carbonyl groups of the sugars to prevent crosslinks -- or sometimes even break them. ALT-946 is another compound that Alteon claims is even more potent than aminoguanidine, and is pending phase II clinical trials.
Crosslink Breakers
I think the ultimate approach to preventing the adverse effects of crosslinkages is to develop crosslink breakers. If crosslinkages are truly a major cause of aging, and if we can break these crosslinks, we could potentially reverse the aging process. Alteon has come up with 4 chemical classes of crosslinkage breakers. ALT-711 is a crosslinkage breaker that is now being tested in clinical trials. Other crosslink breakers include carnosine, and to some extent, metal chelators like DMSA and EDTA.
Carnosine
Carnosine is a histidine di-peptide with a carbonyl group, which probably accounts for its mechanism of action. Carnosine inhibits carbonylation of proteins that tie up the carbonyl group, and inhibits glycation and fructation of proteins. It knocks out glucose and fructose interactions. It can stop AGEs from interacting with each other and it can stop the amyloid aggregation process. Carnosine has been shown in vitro to break crosslinks with serum albumin. It is also an antioxidant and a chelator for metallo-proteins, and it’s been shown to have some effect in vitro of disaggregating beta amyloid. Carnosine is also a preventive agent, able to stop the toxic effects of beta amyloid on cells (Fig. 8).
ALT-711
ALT-711 is a thiazolium derivative that is structurally similar to vitamin B1. Here again it has an effect of breaking covalent bonds on carbonyl groups. ALT-711 is in phase II trials, and has made some headlines because it’s improving cardiovascular function of both monkeys and dogs. For instance, in dogs, it’s been shown to decrease ventricular stiffness by 40 percent after only one month. Because of this reported breaking of crosslinks, other aspects of cardiac function are improved. These include soft tissue compliance, better elasticity of the cardiac muscle and surrounding aorta, increased arterial elasticity, improved blood pressure, and alleviation of congestive heart failure.
Summary
What seems to be occurring in AGE formation is that sugars are reacting with proteins, DNA or lipids. Sugars have a highly reactive carbonyl group that reacts with amino groups of these macromolecules. There are several potential preventive approaches that can be used. Caloric restriction and hypoglycemic agents, on one hand, can lower sugar, so there are fewer reactive molecules to begin with. Secondly, carnosine, ALT-946, or aminoguanidine can prevent crosslinks from occurring.
ROS (Reactive oxygen species) and free radicals can interact with molecules and macromolecules and increase age pigments and age-aggregated proteins. This can be inhibited by caloric restriction and antioxidants. Most importantly, science has shown that once the irreversible AGE-aggregates are formed, they can be reversed with carnosine and ALT-711.
Don Kleinsek, PhD, is CEO and senior scientist of GeriGene Medical Corporation of Madison, Wisconsin. GeriGene is a leader in the field of anti-aging applications of genetic research. Prior to his involvement in genetic research, Dr. Kleinsek was an associate of the late Dr. Johan Bjorksten, the leading proponent of the Crosslinkage Theory of Aging.