Exploring the Biological Nature of Brown and Beige Fat

Over two years ago this blog discussed the possibility of incorporating a specialized preparation routine before exercise in an attempt to stimulate both brown and beige adipose tissue in order to increase the efficiency and overall calorie and fat burning potential of standard exercise. However, that post did not seek to fully understand or discuss the specific biological mechanisms that govern the behavior of brown or beige adipose tissue. This lack of knowledge limits the efficiency for exercise programs as individuals could either be consuming certain foods or performing certain warm-up tasks to increase exercise potential in addition to those suggested in the past blog post. Increasing exercise efficiency could be an easy means to increase the overall health of society without having to devote more precious time to exercise; therefore it would prove useful to better understand the processes that activate these types of fat.

At the most basic level there are two key elements to the fat burning capacity of brown fat. First, brown fat has multiple mitochondria versus the single mitochondria possessed by white fat; these additional mitochondria allow for greater rates of metabolism along with an increased lipid concentration. Also brown fat releases norepinephrine which reacts with lipases to breakdown fat into triglycerides and later to glycerol and non-esterified fatty acids finally producing CO2 and water, which can lead to a positive feedback mechanism.1,2 Second, brown fat contains significant expression rates of uncoupling protein 1 (UCP-1).1 UCP-1 is responsible for dissipating energy, which leads to the decoupling of ATP production and mitochondrial respiration.1 Basically UCP-1 returns protons after they have been pumped out of the mitochondria by the electron transport chain where these protons are released as heat instead of producing energy (i.e. leaking).

It is important to understand that there are two types of brown fat: natural brown fat and intermediate brown fat commonly known as beige fat. Natural brown is typically exemplified by the fat located in the interscapular region and contains cells from muscle-like myf5+ and pax7+ lineage.3 Natural brown fat is typically isolated from white fat and almost entirely synthesized in the prenatal stage of development as a means to produce heat apart from shivering.4 Beige fat is commonly interspaced within white fat, do not have these muscle-like cells (although Myh11 could be involved),5 and can be activated by thermogenic pathway and the strain of exercise. Beige fat also has the potential to influence the conversion of white fat to beige fat through a process commonly called “browning”.6,7

Natural brown fat is thought to have larger concentrations of UCP1-expression because they constitutively express it after differentiation versus beige, which expresses large amounts of UCP-1 in response to thermogenic or exercise cues.1,5 Therefore, natural brown fat is more effective at energy expenditure. However, it may not be possible to develop more natural brown fat after development; therefore, any positive progression in brown fat development will come from beige fat.

Early understanding of brown fat activation involved non-discriminate increases in the activity of the sympathetic nervous system (SNS). The standard pathway governing brown fat activation uses a thermogenic response involving the release of norepinephrine, which initiates cAMP-dependent protein kinase (PKA) and p38-MAPK signaling leading to the production of free fatty acids (FFA) through lipolysis due to UCP-1 induced proton uncoupling.4 UCP-1 concentrations are further increased through secondary pathways involving the phosphorylation of PPAR-gamma co-activator 1alpha (PGC1alpha), cAMP response element binding protein (CREB) and activating transcription factor 2 (ATF2).8 Among these three elements PGC1alpha appears to be the most important co-activating many transcription factors and playing an important role in linking oxidative metabolism and mitochondrial action.9

However, due to the complicated nature of SNS activation and its other downstream activators the attempt to replicate it in the form of weight loss drugs like Fenfluoramine or Ephedra resulted in severe negative cardiovascular side effects like elevated blood pressure and heart rate.10 While some argue that either increasing the sensitivity or the rate of simulation to the SNS can improve upon these results, the underlying elements associated with downstream activation of the SNS makes facilitating direct influence too complicated. Therefore, from a biological perspective it makes more sense to focus on a downstream element that interacts with brown fat at a more localized level.

Just a side note based on the differing interactivity between brown/beige and white fat from the SNS, white fat appears to represent long-term energy storage and brown fat is shorter-term energy, an unsurprising conclusion. However, frequent energy expenditure, like exercise, may condition the body to produce more beige fat versus white fat viewing short-term energy needs as more valuable than long-term energy needs. Basically if the above point is accurate then it stands to reason that a person would see more benefit from 20 minutes of exercise 6 days a week versus 40 minutes of exercise 3 days a week.

Moving away from direct SNS stimulation perhaps the appropriate method of increasing browning involves increasing transcription and translation of UCP1. Interestingly enough empirical evidence exists to support the idea that reinoic acid could be an effective inducer of UCP-1 gene transcription in mice and operates through a non-adrenergic pathway.11,12 However, a more focused study using loss of function techniques involving retinaldehyde dehydrogenase, which is responsible for converting retinal to retinoic acid, determined that retinal, not retinoic acid is the major inducer of brown fat activity.13 Unfortunately there is no direct understanding regarding the proportional response of brown fat to retinal or retinoic acid. Therefore, the general fat-soluble nature of vitamin A will probably make it difficult to utilize its derivatives as biological stimulants for brown fat activation or browning.

Another possible strategy to stimulate browning is through activated (type 2/M2) macrophages induced by eosinophils which are commonly triggered by IL-4 and IL-13 signaling. When activated this way these macrophages recruit around subcutaneous white fat and secrete catecholamines to facilitate browning in mice.14,15 A secondary means by which both IL-4 and IL-13 may influence fat conversion is their direct interaction with Th2 cytokines.16 Unfortunately while on its face this strategy looks promising, in a similar vein to vitamin A, it might not be effective due to unknown long-term side effects associated with IL-4 and IL-13 activation. Due to this lack of knowledge, if IL-4 or 13 is thought to be a viable biochemical strategy for inducing weight loss, long-term proper time lines for effects and dosages must be explored in humans, not just short-term studies in mice.

A more controversial agent in browning is fibronectin type III domain-containing protein 5 or more frequently known as irisin. Due to its significantly increased rate of secretion from muscle under the strain of exercise, some individuals believe that irisin is a key mediator in browning acting as a myokine;17 if this characterization is accurate then irisin could be a significant player in the biological benefits produced by exercise including weight loss, white fat conversion and reduced levels of inflammation.18,19 However, other parties believe that because human studies with irisin have produced results that do not demonstrate benefits similar to those studies using mice, irisin is another molecule that cannot scale-up its effectiveness when faced with the added biological complexity of humans versus a mouse.20-22

The key element within this controversy could be that irisin expression is augmented by the increased expression of PGC1alpha, but PGC1alpha increases the expression of many different proteins and other molecules, so the expression of irisin may not be relevant to the positive changes associated with exercise. Another factor may be that a key difference between mice and humans is the mutation in the start codon of the human gene involved in the production of irisin, which significantly reduces irisin availability.23 Thus this mutation could be the limiting factor to why despite a very conserved genetic sequence, humans do not see anywhere near the benefit mice do. If this explanation is correct it does potentially still leave the door open to directly inject irisin into the body to increase concentrations in an attempt to aid exercise derived results, but if PGC1alpha is the key, then this increased concentration of irisin could be of minimal consequence.

Another potential element that demonstrates a significant concentration increase in accordance to increased PGC1alpha is a hormone known as meteorin-like (Metrnl).24 The concentration of this hormone increases in both skeletal muscle and adipose tissue during exercise and exposure to cold temperatures in accordance to increases in PGC1alpha concentrations. When Metrnl circulates in the blood it seems to produce a widespread effect that induces browning resulting in a significant increase in energy expenditure.24 The influence of Metrnl on white fat does not appear due to direct interaction with the fat, but instead indirect action on various immune cells most notably M2 macrophages via the eosinophil pathway, which then interact with the fat through activation of various pro-thermogenic actions.24 As discussed above this interaction with eosinophil appears to function through IL-4 and IL-13 signaling indicating a common pathway purpose between IL-4/IL-13 and the original SNS pathway. Not surprisingly blocking Metrnl has a negative effect on the biological thermogenic response.24

Another potential strategy for browning may be targeting appropriate receptors instead of specific molecules; with this strategy in mind one potential target could be transient receptor potential vanilloid-4 (TRPV4). TRPV4 acts as a negative regulator for browning through its negative action against PGC1a and the thermogenic pathway in general.25 In addition TRPV4 appears to activate various pro-inflammatory genes that interact with white adipose tissue making it more difficult to facilitate browning even if the appropriate signals are present. TRPV4 inhibition and genetic ablation in mice significantly increase resistance to obesity and insulin resistance.25 The link between inflammation and thermogenesis is highlighted by the activity of TRPV4, which is one of the early triggers for immune cell chemoattraction.25

Obesity may also produce a positive feedback effect through TRPV4 by increasing cellular swelling and stretching through the ERK1/2 pathway, which increases the rate of TRPV4 activation.26,27 However, the validity of TRPV4 as a therapeutic target remains questionable for TRPV4 expression not only influences fat/energy expenditure, but also osmotic regulation, bone formation and plays some role in brain function.25,28,29 Fortunately a number of the issues with TRPV4 mutations/mis-function appear to be developmental in influence versus post-development, thus TRPV4 therapies could still be valid.

Natriuretic peptides (NPs) are hormones typically produced in the heart on two different operational capacities: atrial and ventricular. Both of these hormones appear to play a role in browning through association with the adrenergic pathway.30 The most compelling evidence for supporting this behavior is that a lack of NP clearance receptors demonstrated significant enhanced thermogenic gene expression in both white and brown adipose tissue.30 Also direct application of ventricular NP in mice increased energy expenditure.30 In addition to the above results, NPs are an inherent attractive therapeutic possibility because appropriate receptors are located in white and brown fat of both rats and humans31,32 and these receptors go through periods of significant decline in expression when exposed to fasting,33 which may account for some of the benefits seen from low calorie diets.

Atrial NPs increase lipolysis in human adipocytes similar to catecholamines (increasing cAMP levels and activation of PKA) although whether or not this increase is induced through interaction with beta-adrenergic receptors is unclear.34 Some believe that NPs activate the guanylyl cyclase containing NPRA producing the second messenger cGMP activating cGMP-dependent protein kinase (PKG).35,36 PKA and PKG have similar mechanisms for substrate phosphorylation including similar targets in adipocytes,36 thus this interaction may explain why atrial NPs act similar to catecholamines.

Recall from above that one of the means of inducing browning, especially for those tissues that are distant from SNS-based neurons, is macrophage recruitment. This recruitment appears to be initiated by CCR2 and IL-4 for when either is eliminated from mice models the conversion no longer occurs.15 Tyrosine hydroxylase (Th) is also important in this process facilitating the biosynthesis of catecholamines and later PKA levels.

With respects to producing a biomedical agent to enhance browning there appear to be three major pathways in play: 1) the SNS pathway producing a direct activation response; 2) macrophage recruitment pathway potentially involving Metrnl, which activates IL-4 and IL-13 eventually leading to PKA activation and an indirect activation response; 3) NPs activation pathway, which eventually leads to PKG activation and an indirect activation response. As mentioned earlier SNS pathway enhancement has already been attempted by at least two drugs and failed miserably, so that method is probably out. In addition the SNS pathway does not appear to have as much browning potential as the PKA or PKG pathways due to the reliance on the location of certain nerve fibers.

Enhancing macrophage recruitment could be a good strategy, but there appears to be little information regarding negative effects associated with short-term high frequency enhancement of IL-4 or IL-13 concentrations. Some reports have suggested an increase in allergic symptoms, but any more severe consequences are unknown. This is not to say that enhancing IL-4 or IL-13 is not a valid therapeutic strategy, but its overall value is unknown. In contrast enhancement of NPs appear to be a more stable choice due to positive results in initial exploration of both the application and the expected negative side effects. First, NPs can be administrated via the nose-brain pathway enabling access to the brain avoiding some potential systemic side effects.37 Second, there appear to be few, if any significant side effects to intranasal NP application, at least in the short-term.38

Overall the above discussion has merely identified some of the more promising candidates to enhance browning white fat. One could argue that resorting to drugs to enhance the overall health of an individual versus simple diet and exercise is a regretful strategy. Unfortunately the reality of modern society is that more and more people seem to have less available time to exercise or eat right. In addition to a mounting negative weight external environment (increased pollution and industrial chemicals like BPAs) this drug enhancement strategy may be the most time and economically efficient means to ensure proper weight control and overall health for the future.

Citations –

1. van Marken Lichtenbelt, W, et Al. “Cold-activated brown adipose tissue in healthy men.” The New England Journal of Medicine. 2009. 360:1500-08.

2. Lowell, B, and Spiegelman, B. “Towards a molecular understanding of adaptive thermogenesis.” Nature. 2000. 404:652-60.

3. Seale, P, et Al. “PRDM16 controls a brown fat/skeletal muscle switch.” Nature. 2008. 454:961–967.

4. Sidossis, L and Kajimura, S. “Brown and beige fat in humans: thermogenic adipocytes that control energy and glucose homeostasis.” J. Clin. Invest. 2015. 125(2):478-486.

5. Long, J, et Al. “A smooth muscle-like origin for beige adipocytes.” Cell Metab. 2014. 19(5):810–820.

6. Kajimura, S, and Saito, M. “A new era in brown adipose tissue biology: molecular control of brown fat development and energy homeostasis.” Annu Rev Physiol. 2014. 76:225–249.

7. Harms, M, and Seale, P. “Brown and beige fat: development, function and therapeutic potential.” Nat Med. 2013. 19(10):1252–1263.

8. Collins, S. “β-Adrenoceptor signaling networks in adipocytes for recruiting stored fat and energy expenditure.” Front Endocrinol (Lausanne). 2011. 2:102.

9. Handschin, C, and Spiegelman, B. “Peroxisome proliferatoractivated receptor gamma coactivator 1 coactivators, energy homeostasis, and metabolism.” Endocr. Rev. 2006. 27:728–735.

10. Yen, M, and Ewald, M. “Toxicity of weight loss agents.” J. Med. Toxicol. 2012. 8:145–152.

11. Alvarez, R, et Al. “A novel regulatory pathway of brown fat themogenesis, retinoic acid is transcriptional activator of the mitochondrial uncoupling protein gene.” J. Biol. Chem. 270:5666-5673.

12. Mercader, J, et Al. “Remodeling of white adipose tissue after retinoic acid administration in mice.” Endocrinology. 2006. 147:5325–5332.

13. Kiefer, F, et Al. “Retinaldehyde dehydrogenase 1 regulates a thermogenic program in white adipose tissue.” Nat. Med. 2012. 18:918–925.

14. Nguyen, K, et Al. “Alternatively activated macrophages produce catecholamines to sustain adaptive thermogenesis.” Nature. 2011. 480(7375):104–108.

15. Qiu, Y, et Al. “Eosinophils and type 2 cytokine signaling in macrophages orchestrate development of functional beige fat.” Cell. 2014. 157(6):1292–1308.

16. Stanya, K, et Al. “Direct control of hepatic glucose production by interleukins-13 in mice.” The Journal of Clinical Investigation. 2013. 123(1):261-271.

17. Pedersen, B, and Febbraio, M “Muscle as an endocrine organ: focus on muscle-derived interleukin-6.” Physiological Reviews. 2008. 88(4):1379–406.

18. Bostrom, P, et Al. “A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.” Nature. 2012. 481(7382):463–468.

19. Lee, P, et Al. “Irisin and FGF21 are cold-induced endocrine activators of brown fat function in humans.” Cell Metab. 2014. 19(2):302–309.

20. Erickson, H. “Irisin and FNDC5 in retrospect: An exercise hormone or a transmembrane receptor?” Adipocyte. 2013. 2(4):289-293.

21. Timmons, J, et Al. “Is irisin a human exercise gene?” Nature. 2012. 488(7413):E9-11.

22. Albrecht, E, et Al. “Irisin - a myth rather than an exercise-inducible myokine.” Scientific Reports. 2015. 5:8889.

23. Ivanov, I, et Al. “Identification of evolutionarily conserved non-AUG-initiated N-terminal extensions in human coding sequences.” Nucleic Acids Research. 2011. 39(10):4220-4234.

24. Rao, R, et Al. “Meteorin-like is a hormone that regulates immune-adipose interactions to increase beige fat thermogenesis.” Cell. 2014. 157:1279-1291.

25. Ye, L, et Al. “TRPV4 is a regulator of adipose oxidative metabolism, inflammation, and energy homeostasis.” Cell. 2012. 151:96-110.

26. Gao, X, Wu, L, and O’Neil, R. “Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathways.” J. Biol. Chem. 2003. 278:27129–27137.

27. Thodeti, C, et Al. “TRPV4 channels mediate cyclic strain-induced endothelial cell reorientation through integrin-to-integrin signaling.” Circ. Res. 2009. 104:1123–1130.

28. Masuyama, R, et Al. “TRPV4-mediated calcium influx regulates terminal differentiation of osteoclasts.” Cell Metab. 2008. 8:257–265.

29. Phelps, C, et Al. “Differential regulation of TRPV1, TRPV3, and TRPV4 sensitivity through a conserved binding site on the ankyrin repeat domain.” J. Biol. Chem. 2010. 285:731–740.

30. Bordicchia, M, et Al. “Cardiac natriuretic peptides act via p38 MAPK to induce the brown fat thermogenic program in mouse and human adipocytes.” The Journal of Clinical Investigation. 2012. 122(3):1022-1036.

31. Sarzani, R, et Al. “Comparative analysis of atrial natriuretic peptide receptor expression in rat tissues.” J Hypertens Suppl. 1993. 11(5):S214–215.

32. Sarzani, R, et Al. “Expression of natriuretic peptide receptors in human adipose and other tissues.” J Endocrinol Invest. 1996. 19(9):581–585.

33. Sarzani, R, et Al. “Fasting inhibits natriuretic peptides clearance receptor expression in rat adipose tissue.” J Hypertens. 1995. 13(11):1241–1246.

34. Sengenes, C, et Al. “Natriuretic peptides: a new lipolytic pathway in human adipocytes.” FASEB J. 2000. 14(10):1345–1351.

35. Potter, L, and Hunter, T. “Guanylyl cyclase-linked natriuretic peptide receptors: structure and regulation.” J Biol Chem. 2001. 276(9):6057–6060.

36. Sengenes, C, et Al. “Involvement of a cGMP-dependent pathway in the natriuretic peptide-mediated hormone-sensitive lipase phosphorylation in human adipocytes.” J Biol Chem. 2003. 278(49):48617–48626.

37. Illum, L. “Transport of drugs from nasal cavity to the central nervous system.” Eur. J. Pharm. Sci. 11:1-18.

38. Koopmann, A, et Al. “The impact of atrial natriuretic peptide on anxiety, stress and craving in patients with alcohol dependence.” Alcohol and Alcoholism. 2014. 49(3):282-286.

Recovery from Coma?

While the number of individuals suffering from long-term unconscious events (comas and coma similar states) is proportionally small relative to the population, the family members and friends of those in comas frequently suffer from significantly negative financial and psychological effects. One of the more prevalent negative effects is the uncertainty associated with comas. Patients and their loved ones can deal with most diseases and similar conditions because they know the cause, the available treatment options and how long to expect before recovery, if recovery is possible; unfortunately these elements are lacking for those in a coma. In addition most people tend to be optimistic and the idea that a person they care about will never regain consciousness is a significant psychological burden as well as a financial one due to resources required for care. Developing a treatment to increase the probability that one recovers from a coma will not produce the overall medical benefits of a cancer or Alzheimer’s cure, but it will produce a treatment for another serious condition that is sufficiently prevalent.

The classic definition of a coma is an individual who exhibits a complete absence of wakefulness and is unable to consciously feel, speak, hear, or move. Traditionally it is believed that consciousness is maintained through two separate components: the cerebral cortex and the reticular activating system (RAS).1 The cerebral cortex is the outermost layer covering the cerebrum and plays a key role in numerous functions including memory, attention, awareness, language, thought and consciousness. RAS is located within the brainstem in a tight association with the reticular formation (RF) and is composed of two tracts, the ascending and descending tract. The ascending tract is principally comprised of acetylcholine-producing neurons, which focus on arousal sending neuronal signals through the RF, then the thalamus and finally the cerebral cortex. The descending tract feeds into the reticulospinal tract, which acts on motor neurons mainly influencing movement and postural control. Basically RAS coordinates the arousal signal and the cerebral cortex acts upon it.

However, on a biological level simply defining unconsciousness, and indirectly a coma, as “the absence of consciousness” does little to facilitate a treatment. There are different gradients of unconsciousness between blows to the head, focal deficits (blindsight), epilepsy, chloroform and other chemical exposure (like anesthesia) and comas/vegetative states.2 Some believe that comas are an emergency response by the body to brain injury to create a better therapeutic environment for self-recovery. Within the context of this theory any damage that is not permanent should eventually be repaired and increase the probability of a return of consciousness.

The general biological methodology of a coma is that a form of injury damages or kills a certain number of neurons, which reduces their ability to send action potentials to other neurons within the range of their synapse. Without consistent action potential activation the otherwise healthy neurons that previously bound neurotransmitters released from these damaged neurons down-regulate their dendritic and post-synaptic receptors limiting their ability to produce action potentials creating a negative feedback across entire networks of neurons. Natural recovery is thought to occur as the damaged neurons repair themselves and once again start sending action potentials (remember that these neurons are essential for consciousness, so consistent action potential generation is the norm) causing adjacent neurons to up-regulate their receptors “rebooting” the previously lost network. The problem, even if this belief is correct, is that there is no timeline for identifying when that recovery will be completed.

In order to achieve an accurate and consistent assessment of the possibility an individual will regain consciousness from an unconscious state (i.e. maximize treatment expectations) each general stage of unconsciousness must be identified. Consciousness itself is divided into two main features: arousal and awareness with arousal incorporating wakefulness and awareness incorporating acknowledgement of environment and oneself.3,4 Note that arousal is a necessary condition for awareness. For the purpose of this discussion four states will be identified: coma, vegetative state, minimum conscious state (MCS) and locked-in syndrome. Brain death is not considered because there is no reasonable and consistent path to recovery.

A coma is principally defined as the absence of arousal, thus also the lack of awareness and the lack of consciousness. In a coma the patient is unresponsive unable to open his/her eyes. Stimulation does not produce spontaneous periods of arousal.3 A coma requires at least one hour of arousal absence to separate it from concussion or syncope (fainting). Fortunately most individuals tend to move beyond a coma state into either a vegetative state or MCS, but after this progression further advancement is less certain.

A vegetative state is defined as sporadic, yet existing arousal with a complete lack of awareness. The term “vegetative” is typically defined as “living merely a physical life devoid of intellectual activity or social intercourse”.5 This state can be acute, persistent or permanent where a persistent vegetative state is one that is prolonged for at least 1 month after brain damage be it acute traumatic or non-traumatic.6 Not surprisingly permanent vegetative states are believed to be irreversible and require at least 3 months after a non-traumatic brain injury or 12 months after a traumatic one for such classification.

A MCS was created as a form of middle ground between full consciousness and a vegetative state, thus it is defined as an individual who has consistent arousal, but inconsistent awareness. Inconsistent awareness is defined as the temporary ability to follow simple commands, gesture or verbally reply “yes or no”, engage in intelligible speech, or produce purposeful behavior.3 Not surprisingly individuals in a MCS have a much higher probability of returning to full consciousness versus individuals in a vegetative state.

Finally locked-in syndrome is defined through sustained arousal and eye opening with awareness of the environment, but the inability to verbally communicate that awareness due to a form of muscle paralysis. Usually communication with other parties is limited to blinking or rarely appendage movements. Typically locked-in syndrome, unlike vegetative states and MCS, originate from neurological damage to the lower portion of the brain versus upper portions of the brain.3 For example one common method of occurrence is derived from quadriplegia and anarthria due to the disruption of corticospinal and corticobulbar pathways.7 Fortunately locked-in syndrome is easy to diagnose, but there is no real treatment.

Initial assessment of the type of lack of consciousness involves the observation of spontaneous exhibited actions as well as responses to vocal and painful stimuli commonly known as AVPU (alert, vocal stimuli, painful stimuli and unresponsive) scale. However, distinguishing between vegetative and a MCS is the real importance of coma evaluation because it is the difference between these two states that largely determines whether or not one should expect the patient to recover using current treatments. Unfortunately, but not surprisingly, these specific elements of voluntary and reactionary behavior can be easily missed or inappropriately linked or dismissed to consciousness making differentiation between different states tricky. Some previously studies indicate that 37-43% of patients diagnosed with the vegetative state later manifested goal-directed behaviors that could be interpreted as a MCS state.8-10

The Glasgow Coma Scale (GCS) is the most widely used method for diagnosing the type of coma state. The GCS defines severity through visual cues like observing the oculocephalic reflex to test the integrity of the brainstem through witnessing opposing movement between a patient’s eyes and their head.11 If both eyes fail to move in the opposite direction (i.e. head turns left eyes move right) then there is more than likely some damage to the affected side. Caloric reflex tests also produce insight to cortical and brainstem function where eye deviation towards an ear that is injected with cold water is anticipated. If no direct eye movement occurs a high probability exists for brainstem damage and no real probability for recovery.12 For example one study identified 47 of 111 patients with at least 1 absent brainstem reflex (pupillary light responses, corneal reflexes, or oculocephalic reflex) where only 2 eventually had a significant improvement over time.12,13

While GCS is popular some believe that there are better evaluation scales like Full Outline of UnResponsiveness (FOUR), Wessex Head Injury Matrix (WHIM) or Coma Recovery Scale-Revised (CRS-R).14 FOUR focuses on detecting and distinguishing between vegetative state, locked-in syndrome, MCS and brain death through the use of a 17-point scale characterizing motor response, eye response, breathing and brainstem reflexes.15-17 The chief strength of FOUR is that it can be applied to patients with endotracheal tubes where GCS cannot. WHIM focuses on the empirically derived sequence of recovery through a 62-point scale among 6 different categories (communication, attention, social behavior, concentration, visual awareness, and cognition) and can effectively distinguish between different awareness levels from vegetative state, MCS and partial recovery.14,18

CRS-R focuses exclusively on vegetative state and MCS and the prospects of transitioning between those states by evaluating 29 hierarchical items categorized in auditory, visual, oromotor/verbal, communication, motor, and arousal.19,20 Some believe that the statistical nature of CRS-R makes it the superior evaluation scale because score summation among the 29 criteria items can be used to track changes in consciousness over time (i.e. linear estimates of ability over time).

However, like GCS these other evaluation scales have their own drawbacks. One of the biggest drawbacks for CRS-R is its limited diagnostic utility due to its lack of diagnostic criteria.11,21 Basically CRS-R develops a diagnosis directly from the rating system. WHIM seems to have a problem measuring recovery as its progression via WHIM is probabilistic and lacking in precision.14 FOUR and GCS have problems measuring the importance of visual fixation.14 This mischaracterization of visual fixation can lead to a misdiagnosis rate of 24% for FOUR and 38% for GCS respectively, typically defining a patient as having a vegetative state versus MCS.22 Elements surrounding the mischaracterization of visual cues in general seem to be the factor that produces the most misdiagnosis.23

It is also widely regarded that recovery from unconsciousness is extremely unlikely in the absence of pupillary light responses, corneal reflexes or bilaterially absent cortical N20 responses 72 hours after unconsciousness.12 Absence of somatosensory-evoked potentials (SEP) after CPR is also a reliable predictor for negative coma outcomes.24,25 A little more controversial is that some believe that high (> 33 ug/liter) neuron-specific enolase (NSE) serum levels also effectively predict low recovery probabilities, but this correlation is questionable in its significance as recovery has been seen in patients with 90+ ug/liter values.26,27 The debate involving the prediction reliability of NSE serum levels is further clouded by the lack of a standard measurement methodology (different laboratories use different methods to determine NSE levels) and outside factors like hemolysis, which increases NSE levels, but does not affect brain function.28,29

One of the problems with evaluating the reliability of biological tests or even the aforementioned scales is the concept of “self-fulfilling prophecy”. For a number of individuals there is a subconscious intent to restrict treatment for patients with characteristics that indicate a low recovery probability, thereby creating a positive feedback loop that further lowers their ability to recover. This problem is compounded by the double-edged sword of experimental testing between required resources and the significance of the result.

For a study to draw significant conclusions there needs to be a large enough number of patients in order to account for outliers; however, the more patients that are enrolled in the study increases the resources required and the overall costs of the study both in manpower and money. Coma studies also have the problem of a lack of reproducibility due to the unique nature behind the origins of the coma both in the event(s) leading to their loss of consciousness and the biological changes that produced it. Overall the best hope is to simply conduct double blind studies separating those doing the initial and future probability evaluations from those applying the actual treatments.

Not surprisingly the advent of modern technology has lead to the use of imaging modalities to attempt to evaluate unconsciousness on a more tiered level. The two most popular strategies to measure consciousness, both in conscious and unconscious patients, are functional magnetic resonance imaging (fMRI) and electro-encephalography (EEG)/magneto-encephalography (MEG).30 Note that some researchers produce a wSMI, which is an analysis technique to determine the shared information between multiple, usually two, EEG signals.31 Both EEG and fMRI information is typically compiled during visual (usually with a bright light), auditory or pain stimulation as well as command following instructions, all of which are designed to produce strong conscious processing reactions. Event-related potentials (ERPs) can also provide insight into improper brain function as they have short latency periods typically reflect activation in low-level sensory receptive structures of the brain.34

Not surprisingly an increasing wSMI (greater synchrony between EEGs) is directly proportional to an increasing probability for coma recovery.30 Increases across centroposterior areas and across medium and long interchannel distances appear especially predictive.31 Another advantage of wSMI over EEGs alone is the comparison reduces the probability of common source artifacts that could create erroneous conclusions about conscious standing.30 EEGs are typically favored versus fMRI due to cost and required procedure.35,36

The advancement of modern imaging technology has provided improvements in navigating the nuances of characterizing a patient as either in a vegetative state or a MCS in that across various studies anywhere from 24%-33% of patients that were originally classified in a vegetative state were reclassified as being in a MCS after EEG analysis.30,35 However, whether or not this new diagnosis was due to missed behavior signs signifying consciousness or a secondary VS subset where neuronal patterns change before outward behavior changes is unclear. This secondary explanation does make sense because neuronal plasticity leads to brain repair from traumatic damage, which would manifest internally before reestablishing external conscious behaviors.
Overall both the inclusion of behavior measures as well as neuroimaging will increase diagnostic accuracy and increase successful treatment probability.

One of the possibly tricky issues surrounding the evaluation of potential conscious signals is that subconscious/non-conscious processing is more advanced than historically thought. For example the brain can subconsciously recognize certain abstractions in pictures, words and faces,37,38 interpret the relationship between similar words,39,40 and the social context of certain objects like money.41-43 There are even questions regarding whether long-distance synchrony can be produced between prefrontal and occipital cortex through long-term potentiation under unconscious conditions.44,45 Fortunately these subconscious triggers rarely manifest into actionable streams, so while subconscious activity can produce behavior priming and small levels of activity in certain networks the rate of their existence is ephemeral. Therefore, despite these concerns, attributing general consciousness cues to conscious brain activity in a currently unactionable state appears more appropriate than attributing these signals to subconscious brain activity.

Another question when using neuroimaging to diagnosis a state of unconsciousness is when it is ideal to measure the “signal of consciousness”. There is a question to whether or not it is best to focus on early or late neuronal responses to sensory stimulation; i.e. how long does it take before the brain produces a conscious response and is everything else signal chatter?46-50 This question is largely contingent on if conscious action can emerge solely from regional reverberating activity and can skip integration or processing. This concern becomes somewhat academic because neuroimaging a patient in a coma-like state typically collects numerous samples to accurately determine whether or not consciousness was demonstrated; therefore, checking late signals should be preferred due to the belief that a majority of conscious thought does require integration. Also integration is essential for consistency of awareness and significant prospects for recovery.

As previously alluded to when determining an existing conscious state the most important distinction is between a vegetative state and a MCS. Both states demonstrate a similar form of preserved arousal, but MCS patients have an additional layer of intentional behavior associated awareness accompanying this arousal. The problem is whether this intentional behavior is absent or the patient is unable to communicate it to the testers. fMRI data has detected blood flow patterns characteristic of consciousness in some vegetative patients.35,36 Both stand-alone EEG and wSMI have also produced certain patterns characterizing consciousness in vegetative patients.51,52 Taking consideration of the above concern regarding unconscious processing, these results could imply that there needs to be an intermediate stage between vegetative and MCS. However, even if this intermediate stage does exist the question is what does it change regarding treatment and conscious awareness?

Another characteristic feature that is used to distinguish vegetative and MCS patients is an EEG of MCS patients typically have increased alpha (at parietal and occipital sources) and theta wave number and a reduced delta wave frequency.30,53 Alpha waves are neural oscillations at a frequency between 7.5 to 12.5 Hz. They originate from the occipital lobe, or possibly the thalamus, when a subject is awake, but resting with closed eyes. Alpha waves are reduced when the subject has open eyes or is asleep. Biologically during alpha wave activity it appears that areas of the cortex not in use are inhibited and there is a non-visual network coordination and communication.54 A second form of alpha wave occurs during REM sleep originating from the frontal lobe area of the brain and has a generally unknown influence, but is thought to have an inverse relationship to REM sleep pressure.54

Delta waves are neural oscillations typically at a frequency between 0 to 4 Hz although some narrow that range to between 0.5 to 2 Hz. They are the slowest waves, but have the highest amplitude and are a common occurrence during deep stages 3 and 4 of sleep (a.k.a. slow-wave sleep (SWS)). Delta waves also indicate an unconscious state with an enhancement of information iteration, which is why this state is thought to increase the probability that declarative and explicit memories are formed.

Theta waves are neural oscillations at a frequency between 4 to 7 Hz. There are two types of theta waves: hippocampal and cortical. Hippocampal are more common to non-human mammals while cortical are more common to humans. Hippocampal theta waves occur through the medial septal area and flow to both the hippocampus and neocortex.55 These waves are related to learning and memory formation and could be related to arousal, sensorimotor processing or even environmental position.56

Interestingly most theta waves involve GABAergic or glutaminergic signals to drive inhibition and excitation versus cholinergic signals.57 Cortical theta waves are common in young children, but lessen in frequency and potency with age occurring later only during meditative or drowsy states. Theta frequencies are especially important as they are thought to mediate a serial stream of consciousness from the fronto-parietal networks.58-60 For a vegetative state these changes are not surprising as increases in low-frequency oscillations like delta waves are classical elements of deep sleep or coma.

One of the key newer elements in judging coma recovery probability is the influence of the posterior cingulated cortex (PCC). The PCC is the central node in the default mode network (DMN) model and along with the precuneus appears to govern wakefulness and awareness, especially relative to anesthetized and various coma-like states.58 Correlation of mesioparietal activity occurs in the PCC as well as pain and episodic memory retrieval.58,59,61 The DMN is quick to activate and deactivate when thoughts are internally directed

Note that the DMN is the active regions of the brain during periods that lack specific attention or focus (i.e. daydreaming, etc.). Its typical characteristic is coherent neuronal oscillations under 0.1 Hz. DMN may also drive self-referential thought and is at optimal function when an individual’s eyes are closed.63 This self-referential thought can manifest in spontaneous inspiration that embodies creativity. It also could have some connection to tying an emotion to a given memory or event. However, the DMN is criticized for its inability to effectively explain the large amounts of processing that occur in a “resting” brain.62

Not surprisingly as one of the critical elements to wakefulness the PCC is one of the most metabolically active regions in the brain with blood flow and consumption rates significantly higher than other brain regions.63 Aside from driving consciousness the PCC is also important to spatial memory, autobiographical memory, configural learning and maintenance of discriminative avoidance learning.31 There is some debate on the role of PCC in triggering internal and external attention and thereby controlling arousal and focus making the PCC a dynamic network over a static brain element.63

A strong associated activation element with the PCC is the precuneus, which is located near the two cerebral hemispheres between the somatosensory cortex and forward of the cuneus. Historically little information has been collected on the precuneus because of its position in the brain, in part it was previously thought to be a homogeneous structure, but now is known to have three subdivisions.64 The precuneus in posterior areas aids episodic and source memory while a second subdivision aids visuospatial imagery. This aid has sometimes been described as “providing context clues” for the hippocampus in memory retrieval.64

With regards to consciousness, similar to the PCC, the precuneus has much higher average metabolic levels and is “deactivated” or compromised during SWS, loss of conscious events during epilepsy, specific brain lesions and vegetative states.63,64 One means to drive rapid activation of the precuneus is to induced language learning through brief flashes attaining supraliminal instead of subliminal characterization.

The idea that the PCC and precuneus are focal points of importance for consciousness also makes sense within the context of corticocortical and thalamocortical degradation, including among medium spiny neurons,65 for these two areas have been functionally linked to thalamus nuclei.66,67 This influence on the synchronization of these cortical networks also appears to correlate to global workspace theory (GWT).68

GWT is a theory designed to describe how the conscious and unconscious mind interact to produce cognitive thought and was first applied to the concept of working memory. Most analogize GWT with a play at a theater where the active consciousness is the actor currently speaking (i.e. the “spotlight” of attention, which has limited reach/range)while other actors compete for the spotlight.69 The seating in the theater along with the attending audience represents the unconscious mind, aware of what is consciously occurring, but not providing any direct influence to the behavior of the actors and of great capacity. Finally the non-actors like the director, stage hands, etc. act like executive processes in that they influence actor behavior, but are not directly witnessed.69 One of the major boons of GWT is that it successfully models certain characteristics of consciousness like managing novel situations, working with capacity limits, and incorporating unconscious processes to conscious processes, a characteristic seen in brain elements like how the dorsal cortical stream influences the visual system.69

This model also applies a competition-cooperation parameter to form a “stream of consciousness” where if two elements are received within 100 ms of each other they will be sensory cooperative vs. being sensory competitive, i.e. when the video and audio of a movie are in or out of synch. Alpha, theta and gamma brain waves correspond to this 100 ms threshold whereas ERPs are in the 200-300 ms domain.70 Most argue that the “stream of consciousness” is not an actual stream with events falling perfectly in place with one another, but instead are “edited” together by conscious and unconscious processes similar to how a movie is put together after various scenes and takes. Overall the chief problem with the GWT is that it does not actually explain consciousness, but instead places boundary conditions on theories that do attempt to explain consciousness.71

One of the initial strategies to increase the probability of recovering from a coma, regardless of its specific classification, involves application of mild hypothermia after patient stabilization, especially those suffering from loss of consciousness related to cardiac arrest. The patient’s body is cooled intravascularly at 32-34 degrees C for 24 hours, which typically lowers core body temperature by 2-3 degrees C.12 Fortunately this strategy has become commonplace for many patients, thus reducing the worst-case scenarios for most individuals who lose consciousness in the long-term.72,73 While the specifics of why hypothermia is a successful deterrent of increased future neuronal damage is unclear, there are theories, which involve the reduction of both electrophysiologic and homeostatic energy use,74 reduction of extracellular concentration of excitatory neurotransmitters like glutamate,75 or the reduction of the post-traumatic inflammatory response.76,77

It must be noted that even when individuals recover from comas or coma-like conditions there will be a transition period where the individual will have reduced cognitive and physical ability. Most individuals who recover from comas required physical therapy, speech therapy and some psychological counseling before they are able to continue with their normal lives, that is assuming that they are able to recover fully at all.

Regarding the treatment of any neurological condition some will note the potential of Deep Brain Stimulation (DBS). DBS involves attaching electrodes to specific portions of the brain and applying an electric current in an attempt to initiate excitatory action potentials, typically in the forebrain neurons. It has already drawn interest in treating degenerative neurological conditions like Parkinson’s and dystonia along with psychiatric disorders like depression, obsessive compulsive disorder and various additions.78 The one major general drawback to DBS is that it is an invasive procedure that comes with standard surgical risks and potential complications.

With regards to the ability of DBS to treat coma and coma-like patients the results are not overwhelmingly positive. Most DBS successes are single isolated MCS patients with no positive correlative trend for improved recovery time.65 While DBS does produce behavioral arousal including widening of the palpebral fissure, increased heart rate and blood pressure along with scattered fragmentary movements these improvements are not sustained.65,79 In vegetative state patients there is almost no positive benefit as DBS triggers a local and slow response that does not facilitate synchronization.

Some may argue that the Yamamoto 2010 study demonstrated a significant impact of DBS on vegetative state patients. However, this study appeared to have some serious sampling bias, especially in the old control group where none of the untreated patients recovered from their vegetative states, which mitigates its usefulness.65,80 The second major problem for the credibility of this study is that a number of the “biggest gainers” from the DBS actually had MCS at the beginning of the DBS treatment.81

The reason reclassification of vegetative state patients as MCS patients is a big concern is that the probability that an individual spontaneously regains consciousness from a MCS is thought to be much higher than a vegetative state. For example about 80% of patients in a MCS after 6 months recover spontaneously after 10 months.82,83 Therefore, there is confusion regarding whether or not the patients naturally recovered or recovered due to DBS.

To be fair populating and controlling a significant study to determine improvements in recovery times for coma patients is difficult. Currently there has been only one such clinical trial involving 200 patients and 200 controls spread over 11 participating institutions and 7 years of data collection.65,84 However, currently there is no evidence that DBS facilitates a significant increased probability of recovery for coma patients that are not already significantly through the process of recovery.65

In addition to DBS, there has been exploration regarding pharmaceutical agents for increasing the probability of coma recovery that has produced inconsistent results from L-dopa, Amantadine, and Zolpidem (Ambient).65,84,85 Amantadine is a mixture of a dopaminergic agonist and NMDA antagonist, which seems to have a strong influence on medium spiny neurons triggering greater action potential firing, which then leads to greater mesial cortical neuron firing stimulating conscious activation.65,84 L-dopa is the precursor to the neurotransmitter dopamine, which supposedly acts on neurons in the striatum and frontal cortex to stimulate action potentials. Zolpidem is an alpha-subtype selective positive allosteric modulator of GABA-A receptors. This pathway interaction seems perplexing to why it could help coma patients, but there is a thought that increased GABA-A activity can inhibit the inhibition of thalamocortical outflow, which can increase awakefulness.85 However, none of these methods appear to be consistent enough to be an effective treatment for coma.

As noted above with DBS, one of the major treatments for individuals in a coma or coma-like condition is brain stimulation. Interestingly enough there is significant evidence that focus/attention can be produced even in an unconscious individual.86,87 One common experiment demonstrating this point is orthogonally manipulating visibility and attention through the use of masked images at the edge of conscious perception (some conscious other subconsciously presented).88 From these types of experiments it was theorized that attention over visibility modulated early occipital activity where visibility over attention modulated late temporal and parieto-frontal activity.88 However, there is a changing structure to when the brain reacts to the external stimuli and when the individual becomes conscious of it.89,90

In addition it is recognized that conscious realization of a stimulus requires exceeding a threshold that separates subliminal and supraliminal processing. Exceeding this threshold demands the consistent accumulation of sensory evidence. However, the brain does have a limited capacity to process external stimuli, which is one of the reasons why multi-tasking produces a significant reduction in efficiency between the applied events. Conscious processing of one element creates a bottleneck resulting in either significant reduction of secondary element processing (psychological refractory period (PRP)) or inhibition of the origin of the secondary element (attentional blink or inattentive blindness).91 There is also competition between different stimuli during processing which can make it less likely that any conscious realization occurs.

Finally the adult brain has significant plasticity to allow for repair, but must be primed to truly maximize the efficiency of that repair. This priming element should explain why a number of individuals do not recover from coma states. Similar to the common psychological adage of “use it or lose it” coma/coma-like patients need to “use it” to drive repair recovery. At a biological level this concept involves the activation of positive feedback systems for given neurological pathways, which reinforce certain neurological thoughts/actions versus the termination of neurological pathways that are not utilized or oppose these thoughts/actions. Taking all of these elements into account and tying it to what is known about the PCC and precuneus and their roles in consciousness another potential stimulation strategy emerges.

The first step is to initiate a visual signal cascade to trigger arousal and focus in the patient. This initiation could trigger through the use of a stroboscope (preferable) or general strobe light, which uses high frequency light pulses at various phases and speeds to produce excitatory reactions in the visual processing regions of the brain. Whether or not sounds should also be included in the stroboscope application is questionable. On one hand it can be argued that the addition of sounds should increase arousal probability and recognition of changes in the environment. On the other hand the addition of sound may create some connective confusion, as noted above, and limit the overall efficiency of producing arousal synchronization.

The second step is to request the patient visualize a significant emotional moment in the past. One of the key operational characteristics of the PCC is that it acts as a central integration center for episodic memory, especially those with emotional overtones. Asking the patient to recall, through visualization, an emotional memory should facilitate significant activation of the PCC and trigger the initialization of consciousness recollection, which could initiate further downstream elements of consciousness.

A third optional step would be to ask the patient to visualize themselves on a field running to catch a football or baseball. This visualization should trigger visuospatial areas of the brain, which would aid in triggering precuneus activity. After a seven-minute period (starting with step 1: 2 minutes, step 2: 3 minutes, step 3: 2 minutes), the stimulation is ended and repeated again multiple times after a ten-minute break. The exact amount is unknown but for the moment three times in an hour period over a 24-hour period seems intuitively appropriate.

The above treatment is simply thought to be a potential new therapy option based on understanding the general biological elements associated with how the body retains remedial consciousness. Currently there is no empirical evidence to support the capability of the proposed theory to aid coma recovery beyond the visual activation elements associated with a stroboscope. However, it stands to reason that testing this method should be rather simple due to the lack of known negative elements like invasive surgery or pharmaceutical side effects. One possible side effect could be an increased probability to invoke a seizure due to the action of the stroboscope, but this possibility appears incredibly unlikely. Overall there are certainly no guarantees that this new proposed method will develop into an effective treatment for vegetative state and MCS patients, but there appears to be little reason not to attempt to study its effectiveness.



Citations –

1. Wikipedia Entry - Coma

2. Noirhomme, Q, and Laureys, S. “Consciousness and unconsciousness: an EEG perspective.” Clinical EEG and Neuroscience. 2014. 45(1):4-5.

3. Laureys, S, Owen, A, and Schiff, N. “Brain function in coma, vegetative state, and related disorder.” The Lancet: Neurology. 2004. 3:537-546.

4. Zeman, A, Grayling A, and Cowey, A. “Contemporary theories of consciousness.” J Neurol Neurosurg Psychiatry. 1997. 62:549–52.

5. Jennett, B, and Plum, F. “Persistent vegetative state after brain damage: a syndrome in search of a name.” Lancet. 1972. 1:734–37.

6. The Multi-Society Task Force on Persistent Vegetative State. Medical aspects of the persistent vegetative state. N Engl J Med. 1994. 330:1499–508.

7. Plum, F, and Posner, J. The diagnosis of stupor and coma (3rd edn). Philadelphia: FA Davis, 1983.

8. Andrews, K, et Al. “Misdiagnosis of the vegetative state: retrospective study in a rehabilitation unit.” BMJ. 1996. 313:13-6.

9. Childs, N, and Mercer, W. “Misdiagnosing the persistent vegetative state. Misdiagnosis certainly occurs.” BMJ. 1996. 313:944.

10. Schnakers, C, et Al. “Diagnostic accuracy of the vegetative and minimally conscious state: clinical consensus versus standardized neurobehavioral assessment.” BMC Neurol. 2009. 9:35.

11. Porta, F, et Al. “Can we scientifically and reliably measure the level of consciousness in vegetative and minimually conscious states? Rasch analysis of the coma recovery scale-revised.” Archives of Physical Medicine and Rehabilitation 2013;94:527-35

12. Bouwes, A, et Al. “Prognosis of coma after therapeutic hypothermia: a prospective cohort study.” Ann Neurol. 2012. 71:206–212.

13. Rossetti, A, et Al. “Prognostication after cardiac arrest and hypothermia: a prospective study.” Ann Neurol. 2010. 67:301–307.

14. Schnakers, C, et Al. “A French validation study of the coma recovery scale-revised (CRS-R).” Brain Injury, September 2008; 22(10):786–792.

15. Giacino, J, et Al. “The minimally conscious state: Definition and diagnostic criteria.” Neurology. 2002. 58:349–353.

16. American Congress of Rehabilitation Medicine. Recommendations for use of uniform nomenclature pertinent to patients with severe alterations of consciousness. Archives of Physical Medicine and Rehabilitation. 1995. 76:205–209.

17. Wijdicks, E. “The diagnosis of brain death.” N Engl J Med. 2001. 344:1215–1221.

18. Shiel, A, et Al. “The Wessex Head Injury Matrix (WHIM) main scale: A preliminary report on a scale to assess and monitor patient recovery after severe head injury.” Clinical Rehabilitation. 2000. 14:408–416.

19. Giacino, J, Kalmar, K, and Whyte, J. “The JFK Coma Recovery Scale-Revised: measurement characteristics and diagnostic utility.” Arch Phys Med Rehabil. 2004. 85:2020-9.

20. Seel, R, et Al. “Assessment scales for disorders of consciousness: evidence-based recommendations for clinical practice and research.” Arch Phys Med Rehabil. 2010. 91:1795-813.

21. Lombardi, F, et Al. “The Italian version of the Coma Recovery Scale-Revised (CRS-R).” Funct Neurol. 2007. 22:47-61.

22. Schnakers, C, et Al. “Does the FOUR correctly diagnose the vegetative and minimally conscious states?” Annals of Neurology. 2006. 17:744–745.

23. Childs, N, Mercer, W, and Childs, H. “Accuracy of diagnosis of persistent vegetative state.” Neurology. 1993. 43:1465–1467.

24. Tiainen, M, et Al. “Somatosensory and brainstem auditory evoked potentials in cardiac arrest patients treated with hypothermia.” Crit Care Med. 2005. 33:1736–1740.

25. Bouwes, A, et Al. “Somatosensory evoked potentials during mild hypothermia after cardiopulmonary resuscitation.” Neurology. 2009. 73:1457–1461.

26. Rundgren, M, et Al. “Neuron specific enolase and S-100B as predictors of outcome after cardiac arrest and induced hypothermia.” Resuscitation. 2009. 80:784–789.

27. Steffen, I, et Al. “Mild therapeutic hypothermia alters neuron specific enolase as an outcome predictor after resuscitation: 97 prospective hypothermia patients compared to 133 historical non-hypothermia patients.” Crit Care. 2010. 14:R69.

28. Stern, P, et Al. “Performance characteristics of seven neuron-specific enolase assays.” Tumour Biol. 2007. 28:84–92.

29. Johnsson, P, et Al. “Neuron-specific enolase increases in plasma during and immediately after extracorporeal circulation.” Ann Thorac Surg. 2000. 69:750–754.

30. Sitt, J, et Al. “Large scale screening of neural signatures of consciousness in patients in a vegetative or minimally conscious state.” Brain. 2014. doi:10.1093/brain/awu141

31. King, J, et Al. “Information sharing in the brain indexes consciousness in non-communicative patients.” Current Biology. 2013. 23:1–6.

32. Gutling, E, et Al. “EEG reactivity in the prognosis of severe head injury.” Neurology. 1995. 45:915-918.

33. Logi, F, Pasqualetti, P, and Tomaiuolo, F. “Predict recovery of consciousness in post-acute severe brain injury: the role of EEG reactivity.” Brain Inj. 2011. 25:972-979.

34. Vanhaudenhuyse, A, Laureys, S, and Perrin, F. “Cognitive event-related potentials in comatose and post-comatose states.” Neurocrit Care. 2008. 8:262-270.

35. Owen, A, et Al. “Detecting awareness in the vegetative state.” Science. 2006. 313:1402.

36. Monti, M, et Al. “Willful modulation of brain activity in disorders of consciousness.” N. Engl. J. Med. 2010. 362:579–589.

37. Dehaene, S, et Al. “Cerebral mechanisms of word masking and unconscious repetition priming.” Nat Neurosci. 2001. 4:752-758.

38. Nakamura, K, et Al. “Universal brain systems for recognizing word shapes and handwriting gestures during reading.” PNAS. 2012. 109:20762-20767.

39. Luck, S, Vogel, E, and Shapiro, K. “Word meanings can be accessed but not reported during the attentional blink.” Nature. 1996. 383:616-618.

40. Devlin, J, et Al. “Morphology and the internal structure of words.” PNAS. 2004. 101:14984-14988.

41. Schmidt, L, et Al. “Splitting motivation: unilateral effects of subliminal incentives.” Psychol Sci. 2010. 21:977-983.

42. Pessiglione, M, et Al. “Subliminal instrumental conditioning demonstrated in the human brain.” Neuron. 2008. 59:561-567.

43. Pessiglione, M, et Al. “How the brain translates money into force: a neuroimaging study of subliminal motivation.” Science. 2007. 316:904-906.

44. Dehaene, S, et Al. “Toward a computational theory of conscious processing.” Current Opinion in Neurobiology. 2014. 25:76–84

45. Cohen, M, et Al. “Unconscious errors enhance prefrontal-occipital oscillatory synchrony.” Front Hum Neurosci. 2009. 3:54.

46. Pins, D. “The neural correlates of conscious vision.” Cereb Cortex. 2003. 13:461–74.

47. Koivisto, M, Revonsuo, A, and Lehtonen, M. “Independence of visual awareness from the scope of attention: an electrophysiological study.” Cereb Cortex. 2006. 16:415–24.

48. Melloni, L, et Al. “Synchronization of neural activity across cortical areas correlates with conscious perception.” J Neurosci. 2007. 27:2858–65.

49. Gaillard, R, et Al. “Converging intracranial markers of conscious access.” PLoS Biol. 2009. 7: e1000061.

50. Sergent, C, Baillet, S, and Dehaene, S. “Timing of the brain events underlying access to consciousness during the attentional blink.” Nat Neurosci. 2005. 8:1391–400.

51. Cruse, D, et Al. “Bedside detection of awareness in the vegetative state: a cohort study.” Lancet. 2011. 378:2088–94.

52. Goldfine, A, et Al. “Determination of awareness in patients with severe brain injury using EEG power spectral analysis.” Clin Neurophysiol. 2011. 122:2157–68.

53. Posner, J, et Al. “Plum and Posner’s diagnosis of stupor and coma.” New York, NY: Oxford University Press. 2007.

54. Palva, S, and Plava, J. “New vistas for alpha-frequency band oscillations.” Trends Neurosci. 2007. 30:150-158.

55. Stewart, M, and Fox, S “Do septal neurons pace the hippocampal theta rhythm?.” Trends Neurosci. 1990. 13(5):163-8.

56. Hasselmo, M. “What is the Function of Hippocampal Theta Rhythm? Linking Behavioral Data to Phasic Properties of Field Potential and Unit Recording Data.” Hippocampus. 2005. 15(7):936-49.

57. Balazs, U, and Kiss, T. “How do glutamatergic and GABAergic cells contribute to synchronization in the medial septum?.” Journal of computational neuroscience. 2006. 21(3):343-357.

58. Dehaene S, and Changeux, J. “Experimental and theoretical approaches to conscious processing.” Neuron. 2011. 70:200–27.

59. Laureys, S, and Schiff, N. “Coma and consciousness: paradigms reframed by neuroimaging.” Neuroimage. 2012. 61:478–91.

60. Vanhaudenhuyse A, et Al. “Default network connectivity reflects the level of consciousness in non-communicative brain-damaged patients.” Brain. 2010. 133(Pt 1):161–71.

61. Alkire, M, Hudetz, A, Tononi, G. “Consciousness and anesthesia.” Science. 2008. 322:876–880.

62. Buckner, R, Andrews-Hanna, J, and Schacter, D. “The brain’s default network: anatomy, function, and relevance to disease.” Ann N Y Acad Sci. 2008. 1124:1–38.

63. Champfleur, N, et Al. “Disrupting posterior cingulated connectivity disconnects consciousness from the external environment.” Neuropsychologia. 2014. 56:239-244.

64. Wikipedia Entry - Precuneus

65. Schiff, N. “Moving toward a generalizable application of central thalamic deep brain stimulation for support of forebrain arousal regulation in the severely injured brain.” Ann. N.Y. Acad. Sci. 2012. 1265:56-68.

66. Parvizi, J, et Al. “Neural connections of the postromedial cortex in the macaque.” PNAS. 2006. 103:1563-1568.

67. Cauda, F, et Al. “Functional connectivity of the posteromedial cortex.” PloS One. 2010. 5.

68. Lambert, I, et Al. “Alteration of global workspace during loss of consciousness: a study of parietal seizures.” Epilepsia. 2012. 53(12):2104-2110.

69. Baars, B “The conscious access hypothesis: Origins and recent evidence.” Trends in Cognitive Sciences. 2002. 6(1):47-52.

70. Robinson, R. “Exploring the “Global Workspace” of consciousness.” PLoS Biol. 2009. 7(3):e1000066. doi:10.1371/journal.pbio.1000066

71. Dalton, J. W. “The unfinished theatre.” JCS. 1997. 4(4):316-18.

72. Deakin, C, et Al. “Advanced Life Support Chapter Collaborators.” Part 8: Advanced life support: 2010 International Consensus on Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Science with Treatment Recommendations. Resuscitation. 2010. 81(Suppl 1):e93–e174.

73. Peberdy, M, et Al. Part 9: Post-cardiac arrest care: 2010 American Heart Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Circulation. 2010. 122:S768–S786.

74. Nemoto, E, et Al. “Suppression of cerebral metabolic rate for oxygen (CMRO2) by mild hypothermia compared with thiopental.” J Neurosurg Anesthesiol. 1996. 8:52-9.

75. Busto, R, et Al. “Effect of mild hypothermia on ischemia-induced release of neurotransmitters and free fatty acids in rat brain.” Stroke. 1989. 20:904-10.

76. Clark, R, et Al. “Neutrophil accumulation after traumatic brain injury in rats: comparison of weight drop and controlled cortical impact models.” J Neurotrauma. 1994. 11:499-506.

77. Dietrich, W, et Al. “Delayed posttraumatic brain hyperthermia worsens outcome after fluid percussion brain injury: a light and electron microscopic study in rats.” Neurosurgery. 1996. 38:533-41.

78. Patuzzo, S, and Manganotti, P. “Deep brain stimulation in persistent vegetative states: ethical issues governing decision making.” Behavioural Neurology. 2014. Article ID: 641213. http://dx.doi.org/10.1155/2014/641213

79. Schiff, N, et Al. “Behavioural improvements with thalamic stimulation after severe traumatic brain injury.” Nature. 2007. 448:600–603.

80. Yamamoto, T, et Al. “Deep brain stimulation for the treatment of vegetative state.” Eur J Neurosci. 2010. 32:1145–1151.

81. Yamamoto, T, and Katayama, Y. “Deep brain stimulation therapy for the vegetative state. 2005. Neuropsychol Rehabil. 15:406–413.

82. Giacino, J, and Kalmar, K. “The vegetative and minimally conscious states: a comparison of clinical features and functional outcome.” J Head Trauma Rehabil. 1997. 12: 36–51.

83. Lammi, M, et Al. “The minimally conscious state and recovery potential: a follow-up study 2 to 5 years after traumatic brain injury.” Arch Phys Med Rehabil. 2005. 86:746–754.

84. Giacino, J, et Al. “Placebo-controlled trial of amantadine for severe traumatic brain injury.” N Engl J Med. 2012. 366:819–826.

85. Brefel-Courbon, C, et Al. “Clinical and imaging evidence of zolpidem effect in hypoxic encephalopathy.” Ann Neurol. 2007. 62:102–105.

86. Kentridge, R, Nijboer, T, and Heywood, C. “Attended but unseen visual attention is not sufficient for visual awareness.” Neuropsychologia. 2008. 46:864-869.

87. Naccache, L, Blandin, E, and Dehaene, S. “Unconscious masked priming depends on temporal attention.” Psychol Sci. 2002. 13:416-424.

88. Wyart, V, Dehaene, S, and Tallon-Baudry, C. “Early dissociation between neural signatures of endogenous spatial attention and perceptual awareness during visual masking.” Front Hum Neurosci. 2012. 16:1-14.

89. Wyart, V, and Tallon-Baudry, C. “Neural dissociation between visual awareness and spatial attention.” J Neurosci. 2008. 28:2667-2679.

90. Watanabe, M, et Al. “Attention but not awareness modulates the BOLD signal in the human V1 during binocular suppression.” Science. 2011. 334:829-831.

91. Marti, S, Sigman, M, and Dehaene, S. “A shared cortical bottleneck underlying attentional blink and psychological refractory period.” Neuroimage. 2012. 59:2883-2898.

Putting the Breaks on Aging

The idea of finding a means to extend and improve the quality of existing life has captured the human consciousness for as long as humans have had societal stability. Early attempts focused on the short-term like surviving the elements, wildlife or even each other whereas modern times have given rise to tackling biological and biochemical obstacles. The process of aging is evident in almost all living organisms and is characterized by an increased probability of disease acquisition, decline in fertility, loss of physiological integrity, increased physical impairment and obviously increased probability of death.

Aging is not principally viewed as a genetically programmed process, but instead an entropic process; it is also typically categorized into either primary or secondary categories where primary aging is the reduced ability to maintain tissue homeostasis without external assistance and secondary aging involves the symptoms derived from this reduction in homeostasis that have an increased probability of occurrence as an individual ages.1 The term “senescence” is frequently used to refer to primary aging. Primary aging is the focus of most research on life extension because it entails the potential ceiling/maximum lifespan for a given organism. The principle challenge of this research is differentiating between causes of cellular aging and the associated effects of that aging.

Numerous elements have been characterized as keys to aging and critical to developing therapies to reduce or even halt aging: telomere length, reactive oxidative species (ROS), mitochondrial dysfunction, macromolecule accumulation, stem cell depletion, inflammatory cascades, calorie restriction, etc.2,3 Unfortunately it is unlikely that focusing only on one of these elements will result in a treatment that will arrest aging in humans; therefore, an important question is how are these different systems interconnected and how do they contribute to aging in a way that can be addressed through pharmaceutical and/or dietary changes with few detrimental side effects?

One of the most discussed issues in aging is the role of ROS damage. The general idea behind the influence of ROS damage in aging, commonly regarded as the free radical theory of aging (FRTA), is that during the course of metabolism and general function cells accumulate free radical damage from oxidative species like superoxides (O2--), hydrogen peroxide (H2O2-) and hydroxyl radicals (OH-), which lead to accelerated breakdown in cellular communication and mitochondrial dysfunction.4-7 Simply stated FRTA suggests that under normal physiological conditions the majority of the time there is a slight imbalance between prooxidants (elements like free radicals) and antioxidants (elements that can be oxidized without biological detriment) that leads to the accumulation of oxidative damage in various cells and pathway essential molecules that increase the rate of function loss seen in aging.

Free radicals create damage by stripping an electron off another nearby molecule in order to pair its lone electron; however, this process has a high probability of creating a substitute free radical in the molecule that lost the electron. This catalytic-like behavior increases the probability that an important molecule loses an electron changing its characteristics and eliminating its functionality. If enough of these molecules are damaged then the biological pathway they are associated with is also damaged. In addition free radical damage can also lead to cross-linking DNA inducing inaccurate replication producing another avenue for cellular damage.8

Some evidence suggests that the central nervous system is most acutely vulnerable to oxidative stress due to increases in lipfuscin and bcl-2 concentrations, reductions in redox active iron and glutamine synthetase expression and increases in oxidized glutathione to total glutathione ratios.9-13 In addition increased oxidative stress is thought to increase glial fibrillary acidic protein expression as an individual ages along with increased probability for the initiation of the inflammatory response without an initial stimulus trigger.14-16

The origins of FRTA stem from Max Rubner’s work on how oxygen consumption within metabolism correlated with longevity in eutherian mammals.17,18 However, overall interest in the validity of the theory was limited due to the belief that even if oxygen free radicals were produced their existence was transient enough that they could not react with other elements. Later though it was discovered that oxygen free radicals were formed endogenously through standard metabolic processes and their resultant activity induced cellular damage and disrupted various pathways.17,19 Denham Harman modified FRTA later to include a central role for the mitochondria due to their production of the vast majority of ROS in cells, especially because as mentioned above ROS damage can induce mutations to create a positive feedback effect that can fosters even greater concentrations of ROS from the mitochondria.20

Further modifications have been made to include other ROS like aldehydes or peroxides despite not being free radicals because they can damage cells through oxidative reactions.21 In fact some now bifurcate FRTA into “strong” and “weak” versions where the strong version associates oxidative damage to lifespan and the weak version associated oxidative damage to age-related diseases, similar to the categorical split in aging itself.6 Very few individuals dispute the “weak” version of FRTA because numerous studies exist showing a correlation between oxidative damage and age-related disease.17,22

Among various model organisms strong FRTA is supported by work with Podospora anserine (P. anserine), Drosophila melanogaster and various transgenic mice, is questionable in Caenorhabditis elegans (C. elegans) and does not appear important in Saccharomyces cerevisiae (yeast).17,23-27 Other research has demonstrated increased aging and age-related symptoms when superoxides are overproduced in humans.28,29

It has been demonstrated in yeast that blocking mitochondrial free radical production at Complex III, overexpression of methionine oxidation repair enzyme (MsrB) and retaining CuZnSOD or MnSOD versus knocking either one out all increase life span.27-30 However, the biggest problem with accepting strong FRTA in yeast is that research has also demonstrated that growing it under complete anaerobic conditions (which would heavily limit oxygen availability and thus reduce the concentration of available ROS) results in a decreased clonal life span.28 The reasoning behind this apparent contradiction may be due to the role of glycolysis. Most yeast require a significant level of glycolysis for energy to drive growth, especially in anaerobic environments, which produce reactive aldehydes and protein carbonyls.31 These metabolic elements may be the chief cause of lifespan shortening in yeast versus oxygen derived ROS agents, especially because the accumulation of protein carbonyls is a strong indicator of age.17,32

C. elegans are probably the most perplexing organisms when trying to draw support for FRTA. One of the more controversial pieces of support for FRTA in C. elegans is a study by Lithgow demonstrating that superoxide dismutase/catalase mimetics increased life spans, but this result has been difficult to replicate instead sometimes demonstrating toxicity depending on the level of expression.33-35 However, even if the Lithgow work is excluded oxygen tension still modulates life span36 and RNAi screens demonstrate that knocking out most of the proteins in the electron transfer chain increase lifespan by approximately 30%,37,38 and decreased mitochondrial superoxide production also increases lifespan.39

Despite the results of the above studies most opponents of FRTA also find evidence for their opposition in C. elegans studies.40,41 However, there are two important separate issues to consider when studying FRTA in C. elegans. First, there is a significant difference between C. elegans and mammals regarding energy metabolism and oxidative stress mechanisms. Obviously mammals rely largely on aerobic mechanisms to produce energy (most notably through the mitochondrial electron transport chain) for any study of glycolysis reveals the paltry amount of ATP it produces. For C. elegans glycolysis and the glyoxylate cycle are more efficient producing more ATP allowing for multiple day survival in anaerobic conditions.42 More than likely this difference stems from evolution as most C. elegans reside in topsoil, which can have low-oxygen content over certain periods of time. Second, the living environment is important for C. elegans for lifespan is directly associated with ambient temperature, thus living temperature must be controlled across comparative studies. Both of these issue can influence the amount of ROS created over a given time period and create replication problems with the results.17

The chief enzymatic defense utilized by the body against free radicals, especially superoxides, is super oxide dismutase (SOD) augmented by catalase, glutathione peroxidase, peroxiredoxins, glutathione (GSH), thioredoxin, ascorbate, uric acid and alpha-tocopherol.43 There are three major family classifications for SOD characterized by the associated metal cofactor: Cu/Zn (binds copper and zinc), Fe/Mn (iron or manganese) and Ni (nickel). Humans possess three different versions of SOD: SOD1 that contains copper and zinc (family 1), SOD2 that resides in the mitochondria and uses manganese and SOD3 that also contains copper and zinc.

The role of SOD in aging has been somewhat controversial because some studies have demonstrated that increasing SOD activity increases longevity while other studies have demonstrated that increasing SOD activity does not increase longevity. There are two explanations for this apparent contradiction. First, there are numerous types of SOD in both model organisms and humans and these different types interact with ROS in different ways. Also because most ROS cannot freely cross cellular membranes they create three distinct extracellular, cytosolic and mitochondrial pools of free radicals and SOD activity is also typically relegated to these pools.44

Therefore, not only do studies need to factor in the type of SOD they need to focus on the compartmentalization of that particular SOD or the other oxidant/antioxidant being studied as well as the concentrations of other ROS. For example two different studies involving the over-expression of catalase produced contradictory results until it was determined that one study over-expressed catalase in the mitochondria showing lifespan extension in transgenic mice versus the other study that over-expressed catalase in peroxisomes showing no lifespan extension.45,46

Second, there may be an element of avoided detriment due to lifespan restrictions. In some organisms free radical damage may have a greater effect on the maximum lifespan than the average lifespan, but in most experiments the studied organisms die before reaching their maximum lifespan. Therefore, if a model organism never lives long enough to reach the point where the damage significantly increases the probability of death then neutralizing free radicals is irrelevant. It would be similar to developing a cure for Alzheimer’s and then giving it to a population that does not live past the age of 40. Realistically to determine whether or not free radical damage significantly influences aging in humans, studies must be performed on humans or other long-lived primates rather than model organisms like C. elegans and mice.

Another one of the issues with testing the role of free radicals in aging is that there is no standard measure for assessing oxidative damage. For example some measure protein oxidation from ROS by observing carbonyl groups in serum47 while others measure the amount of lipid peroxidation or the number of isoprostanes in plasma and urine.48,49 Clearly the best method would be to directly measure ROS, but many molecules within the ROS “family” are unstable, thus difficult to accurately measure directly. Without a standardized measurement to determine ROS induced damage, experimental replication will have an inherent issue for concern.

Another common evidentiary citation by opponents of FRTA focuses on studies that conclude antioxidant therapies have no significant positive effect in reducing mortality or reducing most detrimental outcomes (symptoms) that are derived from aging and in some cases can even increase rates of mortality.50-53 These studies can lead to the seemingly understandable conclusion that if increasing antioxidant concentrations does not significantly retard aging or the symptoms of aging ROS must not have a significant influence on aging.

One explanation for this outcome that could salvage FRTA is the belief that due to basic biological operations and signaling humans will have a certain level of oxidative stress from metabolism and other triggers and produce a certain amount of antioxidants to manage that stress.54 However, based on these concentrations, generated through expression and signaling, cells may not have sufficient receptors to interact with large concentrations of external antioxidants from either diet or supplements. Therefore, it is not that free radicals fail to significantly impact aging or that antioxidants fail to neutralize those free radicals, but due to certain levels of receptor expression cells are not able to internalize these additional concentrations of antioxidants to retard aging, which is why anti-aging therapies using large concentrations of antioxidants do not appear to be effective. Basically compartmentalization is reducing the effectiveness of the treatment.

Another key element to the utilization of antioxidants in the body may be the involvement of iron and other chelators.43,55 Some believe that transitional metal ions like iron and copper aid in the formation of O2-- and H2O2-. Furthermore these metal ions can also produce OH- through additional reactions with O2-- and H2O2 increasing damage probability.56 The most notable reaction born from this possibility is the Haber-Weiss reaction coupled to Fenton chemistry.56

Some compounds contribute to antioxidant defense by chelating these transition metals and preventing them from catalyzing the production of free radicals in the cell. Metal chelating antioxidants such as transferrin, albumin, and ceruloplasmin avoid radical production by inhibiting the Fenton reaction.56,57

The natural low biological iron concentration is supported by a functional metal redox cycling mechanism where antioxidants can actually become detrimental. For example O2-- or various specific antioxidants (like ascorbate) act as the reducing agent in the Haber-Weiss reaction converting Fe(III) to Fe(II), which can then reenter the Fenton reaction, and convert free O2 to OH-.58 Therefore, increases in O2--, H2O2-, redox active metal ions or certain antioxidants can create a positive feedback environment that can create more OH- leading to further cellular damage. Unfortunately the cellular pools of low-molecular weight iron are not characterized well leaving questions to the prominence of this effect.59

It has also been reported that the iron content of cells increases as the cells age normally, which can enhance the above effect.60 Some excessive iron can be removed from the body by regular blood transfusions like in the case of haemochromatosis.58 Additionally, certain polyphenols inhibit the absorption of iron with flavonoids acting as antioxidant agents through free radical scavenging and metal chelation.61 Therefore, if FRTA is a valid driver of aging one promising treatment strategy could be increasing the concentration of metal chelating elements with smaller increases in antioxidants like vitamin C and E.

One of the most important molecules affecting the aging process appears to be NF-kB (quick reminder NF-kB = nuclear factor kappa beta). In a recent meta-analysis comparing age-related genetic expression profiles of mice, rats and humans the most common signature involved the over-expression of inflammation and immune response genes and those tied to lysosomal system function.62 NF-kB is an essential element in the inflammation and immune response; therefore it stands to reason that even without additional evidence supporting its role in aging, NF-kB would be viewed as an important element in the aging process.

Five different molecules make up the NF-kB transcription factor family: NF-kB1 (p105/p50), NF-kB2 (p100/p52), Rel A (p65), c-Rel, and Rel B.63,64 These different molecules commonly associate with one another to form various heterodimeric and homodimeric elements. The formation of these hetero and homodimeric elements is normally required to induce receptor activation as the NF-kb molecules typically remain inactive in the cytoplasm before compound formation through additional interaction of ankyrin-containing inhibitor-kBs (I-kB).63 The I-kB inhibition complex for NF-kB members is typically made up of three different inhibitor elements (IKKa, IKKb, and IKKg).64 Also note that only the Rel members of the family (Rel A, Rel B and c-Rel) have transactivation domains which can activate transcription.64,65

The most common compound element among the NF-kB family is NF-kB1/REL A or (p50/p65), which is commonly regarded as the “classic pathway” and is activated by tumor necrosis factor alpha (TNFa).65,66 This classic pathway has also been identified in having a role in the promotion and pathogenesis of cancer where activation is largely induced by cytotoxic agents and maintained by oncogenic activation of various tyrosine kinases. An alternative to the classic pathway is induced by binding of other TNF family members and processes p100/RelB to p52/RelB.65,66 The alternative pathway components, which includes IKKa/IKKa homodimers, largely regulate survival of premature B lymphocytes and development of peripheral lymphoid tissues.64,67 Both pathways are complex with various cofactors such as CK2 or Akt influencing whether NF-kB compounds will have a gene inducing or gene suppressing effect including multiple overlaps.64 This complexity makes a straight application of “agent that inhibits element x in the NF-kB pathway” as a therapy difficult because in some pathways it will promote survival and in others it will promote death.

Of the five members of the NF-kB family, fully processed NF-kB1 (p50) seems to be the most important. Not only can it form a heterodimer with p65 which can then activate the classic pathway it appears that its formation of a homodimer (p50/p50) can actually inhibit classic pathway activation.68 While the formation of homodimer (p50/p50) can still bind at the kB binding sites, only those Rel family members have the necessary transactivation domains to begin gene transcription. Therefore, homodimer (p50/p50) binding instead of heterodimer (p65/p50) prevents gene transcription and the activation of the classic pathway.68,69

The influence of NF-kB on aging has been studied most often in skin cells where it is cell autonomous because visible signs of aging in various skin cell cultures can be neutralized or even reversed after exposure to NF-kB inhibitors like 4-OHT.70 NF-kB binding activity increases with age in mice in various tissues including the skin, heart, kidney, liver, spleen and possibly even stem cells.70-72 This increased binding is thought to regulate senescence, but only replicative and oncogene induced senescence have been observed.73,74 In addition SIRT1 and FOXO, which are widely regarded as strong longevity signals, seem to inhibit NF-kB.75 Interestingly because of the increased binding, the effect of NF-kB on aging acts as an age-dependent positive feedback effect.70

Although aging is not perceived as genetic the closest thing to an “aging trigger” at the moment could be NF-kB. It has been hypothesized that aging is not actually the natural state of cells instead the aging phenotype is achieved through the continuous presence of sufficient concentrations of NF-kB and expansion of its influence through its positive feedback effect.70 The elimination of NF-kB through inhibitors removed markers of cell senescence like cell-cycle inhibitor protein p16 and enhanced proliferation of progenitor cells in skin. Overall, at least relative to skin, NF-kB activity appears to be continuously required to facilitate aging.

One of the most telling issues regarding NF-kB and general aging is that p52 and p65 expression increases significantly in older organisms versus young ones, yet p50 expression along with IkB inhibitors IKKa and IKKb remain at similar levels regardless of age.76,77 Also while p52 and p65 expression increase, their mRNA expression levels demonstrate no significant increases.77 The most probable explanation for this result is that NF-kB protein retention in the nuclei increases with age. This additional NF-kB also increases DNA-binding activity, especially in major lymphoid tissue including constitutive activation of NF-kB within T and B-lymphocytes and macrophages.78

There also appears to be a relationship between ROS and NF-kB in that ROS can induce NF-kB signaling after their production by pro-inflammatory cytokines like IL-1beta and TNFa and lipopolysaccharide stimulation.79 However, not surprisingly this interaction is complicated by timing where early ROS production can act as important messengers for NF-kB activation ROS produced after TNFR1 engagement only facilitates cross-talk between NF-kB and JNK with respects to inducing pro or anti-apoptotic pathway activation.80 Of course because JNK acts as a pro-apoptotic trigger, depending on the situation it can be a pro-aging or an anti-aging trigger meaning the relationship of ROS and NF-kB is even more complicated. This complication could be why there is some evidence that demonstrates dietary therapies with antioxidants down-regulating the age-related increase DNA-binding activity of NF-kB in addition to neutralizing the increased IL-6 and IL-12 expression.78

As alluded to above NF-kB also plays a role with various important aging-related genetic elements including SIRT (mammalian homolog family for silent information regulator (SIR)) and FOXO.81 SIRT1 physically interacts with p65/RelA protein complex to deacetylate lysine-310 on p65 inducing an inhibitor effect on NF-kB transcription.81 It appears that SIRT6 can also inhibit NF-kB activity by modifying the chromatin structure of promoters that interact with various NF-kB genes, which appears to reduce speed of aging.82,83 This SIRT interaction with NF-kB is one of the explanations to why calorie restricted diets could reduce aging and the expression of pro-inflammatory elements or reducing calories appear to increase expression of SIRT1 and NAD+, which is required to activate SIRT1.84,85

Forkhead transcription factors (FOXO) are the mammalian homolog to the famous DAF-16 protein in C. elegans. FOXO3a seems to induce inhibition of NF-kB by limiting the length of activation for unnecessary inflammation, which reduces cellular damage thereby decelerating aging.86 Whether or not this inhibitory effect is driven by direct inhibition of the NF-kB complex or indirect inhibition of elements that activate the NF-kB complex like TNFa or even both is not completely clear.86,87

One of the unclear issues regarding NF-kB inhibition is whether or not it actually affects the overall lifespan of a cell. Intuitively it would stand to reason that if inhibiting NF-kB causes cells to revert to a younger biochemistry and behavior then their lifespan would increase as well; however, this may not be the case. There could be other age-related signals that correspond to senescence that NF-kB has no effect on, thus NF-kB inhibition may not change cell lifespan, but simply create younger cell activity over the course of that lifespan. It would be akin to individual A living 80 years and aging normally versus individual B living 80 years, but remaining as a 30 year-old biologically for the last 50 years, but still dying at a chronological age of 80.

Another question is how would inhibiting NF-kB affect the immune system because NF-kB has an important role in influencing immune response, especially c-Rel and its initiation of IL-12 production. Among other things kappa light chains are critical components to immunoglobulins.70 One interesting experiment would be to vaccinate a mouse then apply a NF-kB inhibitor and then determine whether or not the vaccination is still effective against the target infection.

In the last five years rapamycin and the mTOR pathway [mechanistic (formally mammalian) target of rapamycin] has become a promising candidate for life extension and recapturing youth. Rapamycin is an anti-fungal agent utilized by various soil bacteria that was used as a possible tumor suppressor and immunosuppressor. The TOR pathway (note that in invertebrates it is normally referred to as TOR not mTOR) plays a large role in nutrient sensing and growth through lipid biosynthesis and storage and is highly conserved among various different species. TOR is activated by glucose, insulin, free radicals and growth factors.88 TOR first gained significant recognition as a potential factor in aging when it was reported that inhibition of TOR complex 1 (TORC1) in invertebrates increased lifespans in yeast, C. elegans and Drasophila.88 Overall mTOR interacts with numerous proteins to form mTOR complex 1 and 2, which have different upstream and downstream activation pathways. Note that rapamycin only legitimately inhibits mTORC1 and not mTORC2, although it can disrupt the structure of mTORC2 after long-term high concentration treatments.88

The two major pathways that TORC1 interacts with that influence aging are first global up-regulation of mRNA translation, including ribosome synthesis by direct phosphorylation of S6 kinase and eukaryotic initiation factor 4E binding proteins.89 Second the down-regulation of autophagy, although this regulation is complicated by compartmental segregation between elements of the TOR pathway and autophagy initiating factors like ULK-1.89-91 Also there is some belief that TORC1 interacts with other dietary/nutrition pathways like insulin signaling pathway, hypoxic response transcription factor Gcn4 in yeast and Sirtuins (SIRT family).92,93 Note that autophagy involves the degradation of proteins and organelles via the lysosomal pathway and has been shown to decline during aging, which may lead to the increase of misfolded proteins and ROS in older cells. These effects have also been tied to the anti-aging results seen from calorie restriction.94,95

Due to an initial belief that rapamycin might have a therapeutic effect against some forms of cancer the National Institute on Aging Interventions Testing Program (NIAITP) conducted studies in cancer susceptible genetically modified mice. The result of these studies identified that the addition of rapamycin treatment in 600-day (approximately 20 months) old mice significantly increased the lifespan of both male and female mice, noting that the increase in females was significantly larger than the increase in males.96 Another unassociated study also produced similar findings of 16% and 13% maximum lifespan extensions in 9-month old male and female mice respectively.97

Another important initial study with rapamycin observed its influence on S6K1 (mice equivalent of S6) knockout mice and concluded that these knockout mice had significantly increased lifespans.98 This study indirectly increased the viability of mTOR as a pathway for aging because it demonstrated a longevity increase in a second genetically distinct mouse class versus the NIAITP study. The supposed pathway of operation was not identified, but the activation of adenosine monophosphate (AMP) activated protein kinase (AMPK) was thought to be an important element. While the importance of its activation is still unclear some believe that AMPK negatively regulates TORC1 through phosphorylation of Tsc2, a TORC1 inhibitor.99

However, it must be noted that the increase in lifespan was only commonly significant in female mice not males similar to the NIAITP study and in contrast to the Miller study.98 Also the knockout mice were significantly smaller in body mass than the non-knockouts, which is understandable due to the growth elements controlled by S6K1. Finally the activation of AMPK may not be a requirement for life extension as rapamycin application to wild type flies resulted in life extension without AMPK activation,100 thus raising questions about the role of AMPK.

One possibility is that the activation of AMPK may simply be a secondary effect of rapamycin with the primary lifespan expanding effect being its interaction with stem cells in aged animals as it enhanced the in vivo replicatory capacity of hematopoietic stem cells in aged animals.101,102 In addition there is some question to whether or not rapamycin is immuno-active or immunosuppressive. If it has a positive effect on the immune system then this interaction may be how rapamycin affects longevity. For example TORC1 inhibition prevents the secretion of “pro-aging” cytokines IL-6 and IL-8 by Ras-transduced cells.94 Another possibility could be that the enhanced autophagy allows the body to eliminate damaged cells before they can secrete negative feedback molecules that would create a damage cascade negatively affecting other healthy cells in the local area including inducing aggregating proteins that could result in misfolded proteins.

Overall it appears that TOR dependent changes with regards to aging at a cellular or tissue level are hypertrophic in contrast to the atrophic degeneration that is thought to occur through ROS driven damage. These changes also support the idea that rapamycin is able to increase lifespan beyond its apparent tumor suppression effect because aging is the result of multiple pathways activating under certain boundary conditions and cannot be significantly prevented by only stopping a single disease or pathology. It must be noted though that rapamycin influence on stem cells is not entirely positive as it can impair pluripotency through a reduction of proliferation and promotion of differentiation of human, and to a lesser extent mouse, embryonic stem cells.103,104

As mentioned above while rapamycin has demonstrated an empirically valid potential as an anti-aging element due to its interaction with mTOR, the biggest concern surrounding its application is its influence on the immune system. Originally rapamycin was viewed as an immunosuppressive element and used thusly,105,106 at times in attempted treatment of cancer, generating the conclusion that giving it to aged individuals, especially in the era of antibiotic resistant pathogens, would be inappropriate because it would increase the probability of infection and the probability of death from infection. However, rapamycin supporters have attempted to counter this claim stating that rapamycin may bolster immune system activity.101,107,108 So which side is correct on this important issue?

Unfortunately the role of mTOR and rapamycin in immune system functionality is quite complicated and time dependent. mTOR is a part of the phosphoinositide 3-hydroxy kinase (PI3K) related kinase family and the PI3K-Akt-mTORC1 complex can be co-stimulated, which leads to activation by molecules like the Cluster of Differentiation 28 (CD28) in addition to various interleukins (IL-x).109-111 One of the initial results from mTOR activation is an increased activation time for CD8+ cells and developmental enhancement of T-helper 17 (Th17) cells.112-114 Therefore, one would expect that inhibiting mTOR via an agent like rapamycin will reduce these effects.

Recall that there are two distinct types of immune responses: innate and adaptive. The innate immune system is the older of the two, much less specific in its assault and activates upon the entry of almost any foreign pathogen. Elements of the innate system include epithelial and mucosal membrane obstructions that aid phagocytosis and lysis, various phagocytes, natural killer cells along with other leukocytes, dendritic cells to begin recruitment of more specialized cells and the eventual release of cytokines, and the beginning of the inflammation response.108,115 If it is determined, through signaling, that the innate response will not be sufficient to neutralize the pathogenic threat dendritic cells can act as antigen presenting cells (APC) to activate naïve T cells to initiate an adaptive immune response.

The role of these dendritic cells in relation to the adaptive immune response is critical to the influence of mTOR on the immune system as a whole. mTOR appears to measure the standing of the immune environment and influences antigen recognition similar to its nutrient-sensing ability.110,111 This influence affects how the dendritic cells act as APC, thus determining their interaction with naïve T cells.116,117 However, this influence is affected by the types of dendritic cells activated and the length of time that mTOR is activated (i.e. the amount of rapamycin utilized).118,119 Short-term treatment of rapamycin increases the concentration of cytokines IL-12 and IL-1beta and reduces Toll-like receptor induction of IFN alpha and beta, which act as one of the first defenses against viral infections.118

Long-term treatment decreases the innate immunity of monocyte-derived dendritic cells both via the conventional method and the plasmacyoidal method utilizing cytokine Flt3 as well as reducing the up-regulation of dendritic co-stimulatory molecules, thus suppressing mature dendritic function.120-125 Also knocking out the raptor component of mTORC1, which generally mimics inhibition, leads to a change in the ability to initiate anti-inflammation through the production of additional phenotypes of splenic CD8+ and intestinal CD11+ cells.126 Finally long-term rapamycin treatment reduced allogeneic T-cell response while increasing regulatory T-antigen specific Foxp3+ response.127

The adaptive immune response is highly specific and is dependent on the type of pathogen present and its characteristics. The principle agents involved in the adaptive response are B cells, which govern the humoral (antibodies) response and T lymphocytes, which govern the cell (white blood) response. The adaptive response is so named because it changes, normally increasing effectiveness, each time a pathogen triggers it. The best example of the adaptive response is the specific antibiotic response to a specific pathogen, primed through vaccination.

In vitro it has been demonstrated that rapamycin reduces B lymphocyte proliferation and plasma cell differentiation, which would also reduce antibody production reducing adaptive immune response efficiency.128-130 Also a hypomorphic mouse model with a disrupted mTOR transcript demonstrated reduced B-cell development, reduced cell proliferation and reduce B-cell and T-cell antibody formation.131 mTOR also appears to be required for B-cell differentiation to plasma cells as well as LPS-induced B-cell proliferation and differentiation.131,132 However, interestingly enough only one part of the mTOR pathway, mTORC2, may be principally responsible for B-cell development due to the activation of AKT through phosphorylation and progressive inhibition of FOXO1.133 Therefore, it could be that mTORC1 governs dendritic cell maturation and development and mTORC2 governs B-cell maturation and development, thus short-term rapamycin treatment only disrupts dendritic cells, but long-term treatment could disrupt both dendritic and B-cells.

With regards to T-cells mTOR plays a critical role in the differentiation of certain T-helper cells, most notably 1 and 17.134,135 Also mTORC2 activates PKC theta which helps promote T-helper 2 differentiation, although it does not appear necessary.136 Also mTORC1 regulates hypoxia inducible factor 1 (HIF1) and is required for glycolysis, related enzyme activity and glucose homeostasis in activated CD8+ cytolytic cells.137,138

While some evidence exists that rapamycin treatment can induce immunostimulation of CD8+ T-cells,139-141 the effect may be derived from enhanced cytokine production by macrophages and may not be induced directly by mTOR inhibition.142 Also in most studies rapamycin treatments have been low dose and short-term, thus there is no evidence that any “enhancement” effect will persist over the long-term.140-141 Additionally most rapamycin experiments are conducted in environments that do not tend to have pathogens, thus eliminating real-world examination of how the immune system may be augmented or compromised. Finally there is some evidence that increased used of rapamycin fosters various inflammatory events like lymphocytic alveolitis, glomerulonephritis and interstitial pneumonitis.143-145 Surprisingly the best feature associated rapamycin may be its CD8+ anti-tumor training ability versus its potential anti-aging effects or other immunosuppressive effects.146-148 However, that anti-tumor effect may come from the reduction in IL-10 concentration born from mTOR inhibition for the IL-10 cytokine is thought to have an immuno-masking effect.

Currently existing results support the position that short-term rapamycin treatment has an overall negative effect on the innate immune system with some positive attributes and more than likely a negative effect on the adaptive immune system; long-term rapamycin treatment has negative effects on both the innate and adaptive immune system. Most rapamycin proponents seem to focus on the niche enhancement of the innate system from short-term treatments; however, clearly short-term treatments will do very little to ward off the negative outcomes of aging, thus as it stands the use of rapamycin as an anti-aging tool comes with an immunosuppressive trade-off.

One of the more interesting factors of the mTOR pathway is its dependency on other signaling factors, which appear to make it a general feedback catalyst. For example mTOR increases the secretion of aging elements from damaged and/or senescent cells, but also increases the secretion of anti-aging elements from younger undamaged/unstressed cells. This feature is most notable in muscle tissue where mTOR promotes secretion of trophic factors like IGF-1 from young cells and cytokines like IL-6 from old cells.149 Also rapamycin may have a negative effect on wound healing as it interferes with the ability of p38alpha to activate mTOR to balance the synthesis of IL-12 and IL-10 to properly regulate CD4+ Th1 response to wounds and tissue damage.150 Specifically inhibition of mTOR promotes IL-12 production and reduces IL-10 more than likely through its cross interaction with PI3K, which is a IL-10 enhancing signal.145

mTOR is not the only major element that can both influence aging and the immune system. As mentioned above NF-kB also plays a significant role in activating immune system elements to initiate an appropriate response.151,152 The major signaling pathway that is utilized to connect NF-kB to the immune system is the toll-like receptors (TLRs) along with IKKb kinase and various cytokines, which influences inflammatory signaling.151-153 As most know inflammation can be a useful element in combating infection and other anomalies, but chronic inflammation can increase the damage probability for various cells through increasing oxidative stress and lipid peroxidation, increasing unnecessary cytokines and inducing matrix degradation through the production of metalloproteases.154,155 Cytokinese play an important role in creating a balanced and appropriate immune and inflammatory response. IL-10 and IL-12 have been previously discussed due to their association with mTOR; however, other cytokines appear to influence aging outside of the mTOR pathway including IL-2, IL-4 and most importantly IL-6 all of which are influenced by NF-kB. The most important of the three is IL-6, which is a pro-inflammatory cytokine that has enhanced expression as an organism ages and is thought to contribute to numerous pathophysiologic conditions.156,157

One of the major reasons suspected for the success of rapamycin in reducing aging and its effects is increased autophagy potential positively affecting cellular senescence. NF-kB influences cellular senescence through changes in apoptotic resistance and autophagy augmentation. The chief role of NF-kB signaling relative to apoptosis is to increase the expression of apoptosis inhibitors like Bcl-xL, and the IAPs along with repressing expression of apoptosis activators like JNK and elements in the Fas pathway.158,159 NF-kB elements IKKa and IKKb activate the mTOR complex to inhibit autophagy.160 So NF-kB and rapamycin actually compete in their influence of mTOR and its associated elements like cellular autophagy. With respects to NF-kB mTOR interaction appears to have negative feedback structure where continued activation decreases NF-kB concentrations and increases STAT3 concentrations to reduce the rate of inflammation and increase autophagy at least in younger individuals.

With the principal causes of aging born from environmental and genetic factors there is great interest in producing various biomarkers to detect and track aging in response to treatments. Officially biomarkers are defined by the National Institute of Health as “features objectively measured and evaluated as an indicator of normal biologic, pathogenic or pharmacologic responses to a therapeutic intervention.”161 The chief problem with developing biomarkers for aging is that the deterioration associated with aging occurs over multiple systems with unknown levels of interdependency. This problem is magnified when considering biomarkers that can be compared across different species. Despite these problems researchers have attempted to define biomarkers among elements associated with oxidative stress, inflammatory markers, telomere shortening and hormones, but these markers have not been supported by longitudinal studies.161,162

In addition none of these biomarkers can be viewed as genuine biomarkers to describe aging, but instead are related to disease where age is the biggest risk factor for their appearance. Also interesting is that these biomarkers tend to be expressed in primary elderly populations (65-80 years old), but not secondary elderly populations (80+ years old).163.164 Basically when individuals exceed 80 years old standard age-related “biomarkers” like blood pressure and various metabolic syndromes do not associated significantly with mortality, which of course is unexpected.163-165 The only “biomarker” that seems to retain its predictability at some level is telomere length.166

This important aging element was discovered in the 1930s when both Barbara McClintock and Hermann Muller identified specialized repeating structures at the end of chromosomes.167,168 These structures were later labeled telomeres and were implicated as critically important for cell division preventing chromosome fusion and an incomplete chromosomal copy. Telomeres are important because of how DNA polymerase operates during DNA replication due to the opposing leading and lagging strands of replication. The lagging strand requires a RNA primer to attach a short distance ahead of the initiation site. However, the genetic material behind that new starting point is not replicated; this is fine if the fragment consists of the telomere, but can damage the cell if there critical information is left behind.

A possible role for telomeres in aging was not identified until the 1960s when Leonard Hayflict famously observed that human cells could only undergo a limited number of cell divisions before death, a behavior now referred to as replicative senescence.169,170 Soon after the identification of replicative senescence Alexei Olovnikov proposed that telomeres acted as a buffer of sorts that “sacrificed” itself during replication so the whole chromosome could remain intact.171 This process was also viewed as irreversible because of the one-directional nature of DNA replication. In the late 70s Elizabeth Blackburn and Joseph Gall formally identified telomeres confirming both McClintock/Muller’s and Olovnikov’s theories.

It was eventually determined that somatic cells were unable to maintain telomere length after a specific number of cell divisions leading to cell death. While somatic cells are unable to maintain telomere length, stem cells are able to replicate at various levels of frequency due to the expression of telomerase, an enzyme derived from the telomerase reverse transcriptase (hTERT) gene172,173 that is responsible for “rebuilding” telomeres so the telomere is never completely lost eliminating any inherent ceiling to cellular lifespan through replicative senescence.

Telomerase expression is high during embryonic development, but is down-regulated almost entirely soon after birth in almost all differentiated adult tissues with the exception of specialized stem cell compartments and down-regulated significantly, but not entirely in cell types that have rapid division frequencies like lymphocytes or skin keratinocytes.174

It is somewhat difficult to draw conclusions from telomere research in vivo because of the differences in fidelity of DNA repair and replication pathways between humans and various model organisms. In most model organisms like mice, yeast and C. elegans the elimination of telomerase is irrelevant for several generations of cellular replication whereas in humans after cutting telomerase concentration in half numerous negative symptoms arise like aplastic anemia, immune system deficiencies and pulmonary fibrosis after a few generations.175-177 Also genetic linkage analysis is rather muddled due to difficult to identify relationships between clinical phenotypes and telomere-related genes.

The belief that telomeres are important in aging is supported by short telomere association with numerous premature aging syndromes such as Werner syndrome, Ataxia telangiectasia, Bloom syndrome, Nijmegen breakage syndrome, Fanconi anemia, bone marrow failure, dyskeratosis congenita, aplastic anemia, pulmonary fibrosis, etc, due to cross-sectional and longitudinal cohort studies.178-181 There was some question regarding the role of telomeres and aging in that some research supports an inverse relationship between telomere length and lifespan,182 but this relationship did not seem to exclude tumor/cancer related deaths and most of the differentiation between telomere length and lifespan occurs between life forms with > 1 kg mass and < 1 kg mass. Also clearly aging is a more complicated process than simply looking at telomeres especially when considering average lifespan, not maximum lifespan the element that telomere influence, in normal functioning creatures is studied more in laboratory tests.

Also in vitro studies have tied telomere length to oxidative stress and damage, which has some compelling anecdotal evidence in that the estimated telomere loss per cell division is 50-100 base pairs, but lagging end-replication only seems to account for a loss of 20 base pairs.183 Therefore, it is reasonable to suggest that the additional loss is derived from oxidative damage.183 If this association between telomere length and oxidative stress is accurate then one may be able to better manage telomere length through anti-oxidative stress strategies. However, as mentioned above these strategies must be more specific and rational than consuming a large quantity of Vitamin C and E.

Previously some argued that the role telomeres play in aging could be neutralized by simply activating telomerase in somatic cells. However, this strategy is complicated because the deactivation of telomerase in somatic cells acts as a form of tumor suppression limiting clonal proliferation and dominance. This reality is demonstrated in actual cancer cells where one general aspect of their enhanced ability to replicate is dictated by the reactivation of dedicated telomerase gene expression. Also there is some question to whether the activation of telomerase can create a dysfunctional telomere, which can lead to a malfunction in the DNA damage response for a cell creating a pseudo-tumor cell.175

Telomerase deactivation is not necessarily a bad thing as restrictions in the proliferation of somatic cells pose a barrier for the growth of aspiring tumor cells. Unfortunately, the telomere mechanism that limits the growth of pre-malignant cells also provides strong selection for cells that no longer respond to the DNA damage signals originating from short telomeres. Such cells are genetically unstable and have greatly increased ability to acquire genetic rearrangements that provide further growth advantages. The intricate involvement of telomeres in both aging and cancer ensures that pathways involving telomeres and telomerase will remain subject to intensive studies for many years to come.

Interestingly it appears that after reaching adulthood telomere length changes very little between different cells with different frequencies of replication (leukocytes, muscle, skin and fat) due solely to the influence of time passage.184 The difference in telomere length between these cells seems to occur during the first two decades of life creating an intra-individual synchrony among telomere length and cell types.185-188 For leukocytes it is thought that this two tiered telomere length behavior is born from the expansion of hematopoietic stem and hematopoietic progenitor cell pools.189

While it can be difficult to extrapolate it stands to reason that early symmetric stem cell divisions from the progenitor pool versus asymmetric divisions also define telomere length for the other cell types. Therefore, it seems that stem cell division among somatic tissues for maintenance purposes proceeds at similar rates in adults despite their inherent proliferation status. This information could prove useful because if there is a similar reduction of telomere length in various cell types for adults any treatment that increases telomerase concentrations and thereby increase telomeres will not have an imbalanced influence among various cell types. Basically there should not be a higher probability of developing cancer born from a specific cell type over another cell type.

In the age of genomic research a holy grail of sorts for aging would be to identify a single gene that has significant control over the aging process. In the pursuit of this goal along with a better understanding of aging in general numerous potential candidates have been researched among various model organisms and humans. As previously mentioned the Sir2 family of NAD+-dependent lysine deacetylases are viewed as a high quality genetic candidate for regulating aging due to its influence in extending the lifespan of numerous model organisms including C. elegans, yeast and Drosophila. In humans there are seven different Sir2 homologues (Sirtuins) most of which appear to also have influential roles in governing aging with SIRT1, SIRT2 and SIRT6 playing the most prominent roles. The reason for this prominence is that SIRT1 plays a role in metabolism and inflammation, SIRT2 plays a role in cell cycle and tumor development and SIRT6 plays a role in DNA repair, metabolism and TNFa secretion.190-192

SIRT1 is somewhat unique because it tends to become mobile in response to stress relocating to sites of DNA damage where it helps initiate DNA repair.193 However, this movement may increase the probability of gene expression that are enhanced during aging; basically increased stress can indirectly trigger changes in chromatin state due to SIRT1 influence.193 SIRT6 is an important positive element in promoting replicative capacity through maintaining telomeric chromatin.194 It also interacts with NF-kB subunit RelA as a form of negative feedback through the deacetylation of H3K9Ac to reduce NF-kB signaling.195-197 SIRT6 influence on aging may also involve reducing levels of insulin-like growth factor 1 (IGF-1).195 Finally note that because Sirtuins are dependent on NAD+, insufficient concentrations will result in reduced influence and increase aging potential.

Another popular area of aging study focuses on the relationship between aging and insulin interaction. Numerous studies in model organisms have demonstrated that increasing insulin sensitivity results in a significant increase in longevity.198-201 Not surprisingly though while the insulin receptor substrate (IRS)/PI3 kinase pathway influences aging in C. elegans and Drosophila in a rather straightforward manner, insulin pathways are more complicated in mammals with many more receptors thus elimination of insulin-like growth factor (IGF) and its respective receptors (IGFR) can result in perinatal lethality, diabetes, hyperlipidemia, obesity and liver dysfunction.198,201 Also the insulin/IGF-1 signaling (IIS) pathway in mammals has a strong interaction with growth hormones (GH), an element not utilized in non-mammalian model organisms, further complicating conclusions. Despite this increased complexity there is strong evidence that the IIS pathway does play a role in aging in mammals in that significant increases in average and maximal lifespan occur in mice with reduced plasma levels of IGF-1 and insulin.199-202

A study using Fat Insulin Receptor Knockout (FIRKO) mice further supported the idea that insulin signaling was important for longevity where despite having a normal appearance, appetite and fertility the knockouts had higher insulin sensitivity and less fat and outlived controls by 18%.203 This type of research also produced greater understanding behind a potential influencing mechanisms within the insulin pathway demonstrating that increased mitochondrial oxidative metabolism and white adipose tissue (WAT) metabolism play a role in FIRKO increased longevity.204 Unfortunately there is no specific understanding to how this increased metabolism influences inflammatory adipolines beyond the suspicion that there is a positive (increased anti-inflammatory) effect. Some point to adiponectin as a highly influential insulin sensitivity element that is derived from adiopocytes, but there is no definite evidence for the appropriate mechanism.205,206

The influence of IIS in aging is further supported by the detrimental effects of increased insulin resistance on lifespan and the positive effects of increased insulin sensitivity on lifespan. One of the more interesting real-world studies identified lower insulin resistance and more preserved beta cell function in centenarians versus other individuals who were only seventy to ninety years old.207 Some also link the increased life expectancy seen in calorie restricted diets to reduced plasma insulin levels and increased insulin sensitivity.208

While the influence of IIS in aging appears well supported,209,210 the currently understood involvement of GH is more controversial. Mice deficient in growth hormone like Ames dwarf, which do not secrete GH, prolactin or TSH, outlive controls by 35-70% dependant on other environmental factors.202,211 In addition GH receptor (GHR) knockout mice also demonstrated increased longevity.212,213 However, when GH antagonists are used to reduce GH signaling without a corresponding change in insulin levels there is no significant change in lifespan.213

Such a contradiction seems strange in that if GH signaling is eliminated through lack of substrate or lack of receptor then life is significantly extended, but if receptor activation is eliminated through antagonist binding life is not significantly extended. It stands to reason that despite the antagonist some GH does bind to the appropriate receptor initiating the GH pathway. This result seems to imply a very high initial sensitivity to GH binding that reaches activation saturation rather quickly. Think a Michaelis-Menten graph with a sharp initial slope. If this is true then it is difficult to conclude that increased longevity can be acquired through interaction with GH due to some of the negative effects associated with GH knockouts in humans.

The body types produced by GH or GHR knockout mice are somewhat interesting because they appear to have a lean body type whereas humans who suffer from Laron syndrome (the lack of GHRs) develop an obese body type due to increased fat and significant losses in bone density and muscle mass. While some studies have reported a loss of bone density in GHR knockouts this result does not explain the difference in fat content.214,215 From an evolutionary standpoint GH may be required to differentiate pre-adipocytes into adipocytes, a majority of which form white adipose tissue.216,217 Therefore, the loss of the GH pathway eliminates a principle formation pathway for white adipose tissue. However, GH has also been reported to be an important element in suppressing fat accumulation and increase muscle mass, a seemingly contradictory behavior.213

The explanation for this dual behavior is that during development GH is important in initiating the fat storage pathway and after maturity is used to regulate this pathway in a negative manner.213 This behavior is similar to the neurotransmitter GABA, which is excitatory during development and later becomes inhibitory in mature brains. Thus without white adipose tissue, more than likely the lean body type in Ames and GHR knockout mice is born from the development of brown fat and the increased use of lipids as a source of energy over carbohydrates reducing glucose production leading to suppression of gluconeogenesis and increasing insulin sensitivity.218-220 The reason that GH concentrations appear negative to aging is that aging is not advantageous from an evolutionary standpoint in that reproductive success is favored over longevity so rapid growth, early sexual maturation and strong levels of fertility, characteristics directly related to GH pathway activation, would be supported by evolution.

Another element that may support a role for insulin is that the development of hyperglycemia from high levels of insulin resistance appear to increase the synthesis rate of advanced glycation end products (AGEs) and glycation of proteins.198 AGEs typically form when reducing sugars react with carbohydrates and free amino groups and accumulate in structural proteins like elastin and collagen.221 However, there is no clear evidence that increased insulin resistance leads to increased synthesis of AGEs.

For those who have accepted the cause of aging to involve nutrient sensitive signaling elements like IGF-1 and mTOR the effect of these signaling mechanism induce aging through the facilitation of excessive macromolecule build-up within cells. The chief cause of this build-up is largely regarded as the decline in effectiveness and frequency of cellular autophagy removing damaged and unnecessary molecules and the continued expression of these nutrient sensitivity systems after their usefulness has ended.

Not surprisingly one analogy used to encapsulate this aging methodology, largely because it is used for numerous other things as well like global warming, is that of a bathtub with a running faucet. Think of IGF-1 and mTOR as the agents that control the faucet, autophagy as the drain and the amount of water in the tub being the biological age where too much water leads to flooding (i.e. death). If IGF-1 and mTOR expression exceed autophagy then an individual will experience biological aging as water fills the tub. Therefore, to accelerate aging one must either reduce autophagy or increase IGF-1 and/or mTOR expression; to decelerate aging one must increase autophagy or decrease IGF and/or mTOR expression. Proponents of this mindset believe that while ROS may induce genetic damage, cells die from an unbalanced growth/damage correction mechanism long before the genetic damage is sufficient to induce death.

If one is to believe this series of events then between the two treatment avenues enhancing or maintaining autophagy appears to be the superior one due to the negative side effects and quality of life elements associated with inhibiting mTOR or IGF-1/insulin interactions. Therefore, it may be a better strategy for individuals seeking an anti-aging therapy to focus on autophagy enhancement rather than further evaluating rapamycin or similar agents.

Finally one of the most robust and reproducible methods for positively altering lifespan is calorie restriction (CR) that excludes malnutrition or nutrient deprivation. CR is usually studied with non-control animals receiving a diet that is about 10-40% (normally 30% is standard) less caloric intake than control animals. CR-derived life expansion has been demonstrated in a variety of species including C. elegans, Drosophila and mice.222-225 Numerous rationalities have been hypothesized for why calorie restriction is able to achieve such success.

Supporters of ROS argue that reduced calorie consumption results in reduced metabolism and a reduced probability of synthesizing greater concentrations of ROS thereby extending life and youth due to less DNA damage, especially 8-OHdG damage.226-228 Supporters of insulin dependent aging argue that reduced calorie consumption results in a reduced concentration of circulating insulin and a reduced activation of the IIS pathway through IGF-1 binding thereby extending life and youth, although a reduction of protein is also required to achieve this particular effect.225,228-230 Supporters of NF-kB aging argue that CR down-regulates PI3K and AKT transcription which reduces NF-kB activation removing consistent activation of the “aging” phenotype.231 Others still think that CR influences mitochondrial biogenesis and recycling methods like autophagy to increase longevity.222,232 One of the chief benefits of CR is that there appears to be no age floor in which the methodology must start. Basically a 60-year old individual can begin a CR diet and still develop similar positive biological outcomes similar to that of a 40-year old on a CR diet for decades.

While there is significant evidence to suggest that CR plays an important role in influencing aging in various life forms there are some outstanding issues. First, CR has never definitely demonstrated lifespan extension in humans or non-human primates. In fact the one major concluded study that focused on non-human primates, the 1987 National Institute on Aging (NIA) study using rhesus monkeys reported improved health benefits and possible lower mortality rates, but no ceiling life extension.233 A University of Wisconsin study that started in 1989 that is also utilizing rhesus monkeys is still ongoing and has not released final conclusions, but has released studies in 2009 that concluded 13% of the CR group died from age-related causes versus 37% from the control group, which is in contrast to the NIA study.234 However, there are concerns that the diet fed to the controls in the Wisconsin study is too unhealthy (28.5% sucrose versus 3.9% for the NIA study) creating an inappropriate environment for comparison because the monkeys in the Wisconsin study were healthier simply from not eating the unhealthy diet not from the calorie restriction.235

Second, there are some significant drawbacks associated with CR in humans including reduced sex hormone production, reduced immune response, reduced muscle mass and lower bone mineral density.236-238 Whether or not the decrease in bone mineral density will increase the probability of fractures is unknown because bone quality appears retained despite this reduced mineral density.239 Note that all of these results are short-term, thus these issues could exacerbate with time or self-correct at a new type of homeostasis.

Third, the extension of ceiling lifespan in yeast utilizing a CR protocol relies on enhanced respiratory rates.240,241 This result may help support mitochondrial hormesis as an important element in the life extension effects of CR.242,243 However, hormesis is a somewhat controversial concept in biology, thus there is significant skepticism regarding its validity. This methodology could also tie into the reduction of ROS damage, but not on a reduced production level, but increased neutralization efficiency. This hormesis “priming” has some level of support in that induction of endogenous ROS production can extend life in some organisms.243-245 The change in methodology is unknown, but it may have some influence in increasing antioxidant efficiency.

Fourth, there are no good studies where CR is compared against a healthy Mediterranean type diet. Thus, the benefits from CR may be derived not from excluding calories, but from excluding “bad” calories. For example when one consumes additional calories those calories, to a point obviously, would not be negative if the appropriate vitamins and minerals are contained within maintaining a “nutrient/calorie” ratio relative to exercise level versus smaller calorie consumption strategies. This mindset also coincides with the idea that controls isolated solely to a laboratory are not effective wild-type mimics, thus the lifespan increases seen in CR organisms cannot be genuinely regarded as an increase versus wild-type organisms. Overall there is a lot of promise that CR restriction can be utilized as a means to reduce most of the negative age-related conditions, but whether or not CR is an effective means to extend ceiling lifespan is currently unknown because of these concerns.

With respects to anti-aging treatment, despite the positive results seen from treatments with rapamycin, the biggest problem with focusing on rapamycin as an anti-aging therapeutic is there are numerous side effects not simply those associated with immune system disruption. Not surprisingly rapamycin also induces metabolic alterations like hyperlipidemia, decreased insulin sensitivity (which should increase the probability for developing diabetes and may conflict with IGF-1 anti-aging) and glucose intolerance.246,247. Also there are questions regarding how it influences the gastrointestinal tract due to frequent diarrhea events in patients.248 While individuals suffering from illnesses like cancer and transplant recovery could look past these side effects, would it be responsible to expose healthy individuals to them?

Also most of the rapalogs (chemical agents that are derivatives of rapamycin) that have been developed to avoid or limit rapamycin side effects and/or increase pharmacokinetics for treatment like ridaforolimus, 32-deoxo-rapamycin, temsirolimus, etc. have significantly underpreformed relative to expectations both as anti-cancer agents and anti-aging agents.248 Some believe that these rapalogs focus too much on interacting with mTOR and have a lack of interaction with either PI3K or AKT, which facilitates the failure to properly mimic the biological effects of rapamycin.248

Of the numerous studies performed to investigate the causes of aging, a large amount of support has been developed for numerous different pathways. One of the key elements to developing a therapy is to define what is plausible and what is not. For example the expectation should not be to repair all damage incurred through aging because there will always be elements like epigenomic drift. While major players have been identified developing strategies to take advantage of this knowledge must be taken with caution, as shown above with rapalogs, because of the complexity and interactivity of the pathways involved and the natural aging aspect of organized life, which generates a progression towards random patterns of gene expression that may supercede pharmacological intervention.

Some of the key questions with establishing a lifespan extending methodology are as followed:

1) As discussed above there are numerous elements that are responsible for advancing biological age and it stands to reason that none of these elements as a standalone therapy will produce significant results. One might object to this statement citing significant life extension in various model organisms, but it is important to acknowledge that these gains are exclusive to these respective model organisms and as organisms increase in biological complexity the influence of these life extending changes wane as seen in studies of humans and non-human primates. For example genetic dampening of IIS in C. elegans increase average lifespan by 100%, in Drosophila by 25-30%, in mice by 20% and in non-human primates and humans <5%.249-252

2) There is significant overlap and relationship between cancer and various aging mechanisms where most of the mechanisms that appear to retard cancer formation induce aging; how will anti-aging therapies be reconciled with the possibility of higher rates of cancer?

3) There is a difference between extending ceiling/maximum lifespan and extending average lifespan. It can be rationalized that almost all life extension achieved by society throughout human history, the development of antibiotics, surgical procedures, better and balanced diets, etc., has been of the latter category; does human plasticity even allow for the extension of maximum lifespan and how could such a possibility be tested?

4) If maximum lifespan extension is achieved how will society manage the increased population, especially when most of the members of this population will consume more resources than they produce? Or will lifespan extension simply be a commercial industry that only the rich are able to utilize?

5) Clearly there are complexity issues that must be addressed for just because increasing the concentration of Compound A increases lifespan in C. elegans does not mean that increasing it in all situations will induce the same result. For example for the IIS pathway invertebrates have a single receptor that binds molecules that biologically represent insulin or IGF-1 and reducing binding efficiency leads to life extension. However, mammals have distinct specific receptor for binding both insulin and IGF-1 with different and overlapping function (IGF-1 controls growth and insulin controls metabolism). In addition mouse average lifespan is only increased when IIS is influenced in the right tissue with the right signaling elements because of coinciding requirements for an increase in insulin sensitivity. Similar complexity increases are seen in important longevity genes – FOXO and SIR(SIRT) where mammals have multiple genes per family that have a variety of influences.

Overall there are some key molecules that have an important role in aging. Of these molecules mTOR and NF-kB seem to govern youthfulness more than maximum lifespan while telomeres and ROS seem to have a greater influence on maximum lifespan. Therefore, society must develop a strategy to address both of these influencing categories otherwise life extension will either not be plausible or will be a rather torturous experience of extended old age. Also the balance between these categories must be considered in their interaction and consequence. For example with the rise of antibiotic resistant pathogens the loss of immune system functionality to increase youthful lifespan of cells does not appear to be a beneficial tradeoff for individuals or society, thus both mTOR and NF-kB strategies must be carefully studied and applied. However, between the two a pursuit of a NF-kB neutralizing treatment appears to be the better strategy.




Citations -

1. Hughes, L. “The Curious Concept of Ageing.” Scottish Universities Medical Journal. 2013. Electronically Published 2:1. http://sumj.dundee.ac.uk/data/uploads/epub-article/2001-sumj.epub.pdf

2. Hayflick, L. “Biological aging is no longer an unsolved problem.” Ann. N Y Acad. Sci. 2007. 1100:1–13.

3. Lopez-Otin, C, et Al. “The Hallmarks of Aging.” Cell 2013. 153(6):1194-1217.

4. Harman, D. “Aging: a theory based on free radical and radiation chemistry.” Journal of Gerontology. 1956. 11(3):298–300.

5. Beckman, K, and Ames, B. “The free radical theory of aging matures.” Physiological Reviews. 1998. 78(2):547–581.

6. Trifunovic, A, and Larsson, N. “Mitochondrial dysfunction as a cause of ageing.” Journal of Internal Medicine. 2008. 263:2167–178.

7. Bratic, I, and Trifunovic, A. “Mitochondrial energy metabolism and ageing.” Biochimica et Biophysica Acta. 2010. 1797(6-7):961–967.

8. Crean, C, Geacintov, N, and Shafirovich, V. “Intrastrand G-U cross-links generated by the oxidation of guanine in 5’-d(GCU) and 5’-r(GCU).” Free Radical Biology and Medicine. 2008. 45(8):1125-34.

9. Gilissen, E, Jacobs, R, and Allman, J. “Magnetic resonance microscopy of iron in the basal forebrain cholinergic structures of the aged mouse lemur.” J Neurol Sci. 1999. 168:21–7.

10. Sadoul, R. “Bcl-2 family members in the development and degenerative pathologies of the nervous system.” Cell Death Differ. 1998. 5:805–15.

11. Carney, J, et Al. “Aging- and oxygeninduced modifications in brain biochemistry and behavior.” Ann NY Acad Sci. 1994. 738:44 –53. 27.

12. Savory, J, et Al. “Age-related hippocampal changes in Bcl-2:Bax ratio, oxidative stress, redox-active iron and apoptosis associated with aluminum-induced neurodegeneration: increased susceptibility with aging.” Neurotoxicology. 1999. 20:805–17.

13. Olanow, C. “An introduction to the free radical hypothesis in Parkinson’s disease.” Ann Neurol. 1992. 32:S2–9.

14. Rozovsky, I, Finch, C, and Morgan, T. “Age-related activation of microglia and astrocytes: in vitro studies show persistent phenotypes of aging, increased proliferation, and resistance to down-regulation.” Neurobiol Aging. 1998. 19:97–103.

15. McGeer, P, and McGeer, E. “The inflammatory response system of the brain: implications for therapy of Alzheimer and other neurodegenerative diseases.” Brain Res Rev. 1995. 21:195–218.

16. Joseph, J, Shukitt-Hale, B, and Casadesus, G. “Reversing the deleterious effects of aging on neuronal communication and behavior: beneficial properties of fruit polyphenolic compounds.” Am. J. Clin. Nutr. 2005. 81:313S-6S.

17. Muller, F, et Al. “Trends in oxidative aging theories.” Free Radical Biology and Medicine. 2007. 43:477-503.

18. Austad, S, and Fischer, K. “Mammalian aging, metabolism, and ecology: evidence from the bats and marsupials.” J. Gerontol. 1991. 46:B47–B53.

19. Harman, D. “Aging: a theory based on free radical and radiation chemistry.” J. Gerontol. 1956. 11:298–300.

20. Harman, D. “The biologic clock: the mitochondria?” J. Am. Geriatr Soc. 1972. 20:145–147.

21. Sohal, R, and Weindruch, R. “Oxidative stress, caloric restriction, and aging.” Science. 1996. 273:59–63.

22. Beckman, K, and Ames, B. “The free radical theory of aging matures.” Physiol. Rev. 1998. 78:547–581.

23. Osiewacz, H. “Aging in fungi: role of mitochondria in Podospora anserina.” Mech. Ageing Dev. 2002. 123:755–764.

24. Le Bourg, E. “Oxidative stress, aging and longevity in Drosophila melanogaster.” FEBS Lett. 2001. 498:183–186.

25. Yu, B. “Aging and oxidative stress: modulation by dietary restriction.” Free Radic. Biol. Med. 1996. 21:651–668.

26. Barja, G. “Rate of generation of oxidative stress-related damage and animal longevity.” Free Radic. Biol. Med. 2002. 33:1167–1172.

27. Sasaki, T, et Al. “Age-related increase of superoxide generation in the brains of mammals and birds.” Aging Cell. 2008. 7(4):459-69.

28. Hamilton, C, et Al. “Superoxide excess in hypertension and aging: a common cause of endothelial dysfunction.” Hypertension. 2001. 37(2 Part 2):529-34.

29. Donato, A, et Al. “Direct evidence of endothelial oxidative stress with aging in humans: relation to impaired endothelium-dependent dilation and upregulation of nuclear factor-kappa B.” Circulation Research. 2007. 100(11):1659-66.

27. Longo, V, et Al. “Mitochondrial superoxide decreases yeast survival in stationary phase.” Arch. Biochem. Biophys. 1999. 365:131–142.

28. Koc, A, et Al. “Methionine sulfoxide reductase regulation of yeast lifespan reveals reactive oxygen species-dependent and -independent components of aging.” PNAS. 2004. 101:7999–8004.

29. Wawryn, J, et Al. “Deficiency in superoxide dismutases shortens life span of yeast cells.” Acta Biochim. Pol. 1999. 46:249–253.

30. Longo, V, Gralla, E, and Valentine, J. “Superoxide dismutase activity is essential for stationary phase survival in Saccharomyces cerevisiae. Mitochondrial production of toxic oxygen species in vivo.” J. Biol. Chem. 1996. 271:12275–12280.

31. Aguilaniu, H, et Al. “Asymmetric inheritance of oxidatively damaged proteins during cytokinesis.” Science. 2003. 299:1751–1753.

32. Stadtman, E. “Protein oxidation in aging and age-related diseases.” Ann. N. Y. Acad. Sci. 2001. 928:22–38.

33. Melov, S, et Al. “Extension of life-span with superoxide dismutase/catalase mimetics.” Science. 2000. 289:1567–1569.

34. Keaney, M, et Al. “Superoxide dismutase mimetics elevate superoxide dismutase activity in vivo but do not retard aging in the nematode Caenorhabditis elegans.” Free Radic. Biol. Med. 2004. 37:239–250.

35. Keaney, M, and Gems, D. “No increase in lifespan in Caenorhabditis elegans upon treatment with the superoxide dismutase mimetic EUK-8.” Free Radic. Biol. Med. 2003. 34:277–282.

36. Honda, S, et Al. “Oxygen-dependent perturbation of life span and aging rate in the nematode.” J. Gerontol. 1993. 48:B57–B61.

37. Dillin, A, et Al. “Rates of behavior and aging specified by mitochondrial function during development.” Science. 2002. 298:2398–2401.

38. Lee, S, et Al. “A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity.” Nat. Genet. 2003. 33:40–48.

39. Felkai, S, et Al. “CLK-1 controls respiration, behavior and aging in the nematode Caenorhabditis elegans.” EMBO J. 1999. 18:1783–1792.

40. Gems, D, and Doonan, R. “Antioxidant defense and aging in C. elegans: Is the oxidative damage theory of aging wrong?” Cell Cycle. 2009. 8:1681-7.

41. Pérez, V, et Al. “The overexpression of major antioxidant enzymes does not extend the lifespan of mice.” Aging Cell. 2009. 8:73-5.

42. Foll, R, et Al. “Anaerobiosis in the nematode Caenorhabditis elegans.” Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 1999. 124:269–280.

43. Missirlis, F, et Al. “Compartment-specific protection of iron-sulfur proteins by superoxide dismutase.” J. Biol. Chem. 2003. 278:47365–47369.

44. Finkel, T. “Oxygen radicals and signaling.” Curr. Opin. Cell Biol. 1998. 10:248–253.

45. Schriner, S, et Al. “Extension of murine life span by overexpression of catalase targeted to mitochondria.” Science. 2005. 308:1909–1911.

46. Chen, X, et Al. “Catalase transgenic mice: characterization and sensitivity to oxidative stress.” Arch. Biochem. Biophys. 2004. 422:197–210.

47. Chevion, M, Berenshtein, E, and Stadtman, E. “Human studies related to protein oxidation: protein carbonyl content as a marker of damage.” Free Radic Res. 2000. 33(suppl):S99–108.

48. Pratico, D, et Al. “IPF2alpha-I: an index of lipid peroxidation in humans.” PNAS. 1998. 95:3449–54.

49. Heilbronn, L, and Ravussin, E. “Calorie restriction and aging: review of the literature and implications for studies in humans.” Am. J. Clin. Nutr. 2003. 78:361-369.

50. Miller, E, et Al. “Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality.” Annals of Internal Medicine. 2005. 142(1):37-46.

51. Bjelakovic, G, et Al. “Antioxidant supplements for prevention of mortality in healthy participants and patients with various diseases.” Cochrane Database of Systematic Reviews. 2008. 2:Article ID CD007176.

52. Bardia, A, et Al. “Efficacy of antioxidant supplementation in reducing primary cancer incidence and mortality: systematic review and metaanalysis.” Mayo Clinic Proceedings. 2008. 83(1):23–34.

53. Poljsak, B, et Al. “Reproductive benefit of oxidative damage: an oxidative stress “malevolence”?” OxidativeMedicine and Cellular Longevity. 2011. 2011:Article ID 760978 – pp. 9.

54. Cutler, R, and Mattson, M. “Measuring oxidative stress and interpreting its relevance in humans.” in Oxidative Stress and Aging, R. G. Cutler and H. Rodriguez, Eds., World Scientific, Hackensack, NJ, USA, 2003.

55. Terman, A, and Brunk, U. “Oxidative stress, accumulation of biological “garbage”, and aging.” Antioxidants and Redox Signaling. 2006. 8(1-2):197–204.

56. Kell, D. “Iron behaving badly: inappropriate iron chelation as a major contributor to the aetiology of vascular and other progressive inflammatory and degenerative diseases.” BMC Medical Genomics. 2009. 2(2).

57. Imlay, J. “Pathways of oxidative damage.” Annual Review of Microbiology. 2003. 57. 395–418.

58. Poljsak, B, and Milisav, I. “The neglected significance of “antioxidative stress.” Oxidative Medicine and Cellular Longevity. 2012. Article ID 480895. 1-12.

59. Voogd, A, et Al. “Low molecular weight iron and the oxygen paradox in isolated rat hearts.” Journal of Clinical Investigation. 1992. 90(5):2050–2055.

60. Killilea, D, et Al. “Iron accumulation during cellular senescence in human fibroblasts in vitro.” Antioxidants and Redox Signaling. 2003. 5(5):507–516.

61. Glauce, V. et Al. “Role of plant extracts and polyphenolic compounds in oxidative stress-related diseases.” in Handbook of Free Radicals: Formation, Types and Effects, D. Kozyrev and V. Slutsky, Eds., Nova Science Publishers, Inc., New York, NY, USA, 2010.

62. De Magalhaes, J, Curado, J, and Church, G. “Meta-analysis of agerelated gene expression profiles identifies common signatures of aging.” Bioinformatics. 2009. 25:875–81.

63. Salminen, A, and Kaarniranta, K. “NF-kB signaling in the aging process.” J. Clin. Immunol. 2009. 29:397-405.

64. Van Waes, Carter. “Nuclear Factor-kB in Development, Prevention, and Therapy.” Clin Cancer Res. 2007. 13:1076-1082.

65. Baldwin, A. “Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB.” J. Clin Invest. 2001. 107:241-246.

66. Hayden, M, and Ghosh, S. “Signaling to NF-kappaB.” Genes Dev. 2004. 18:2195-2224.

67. Luo, J, Kamata, H, and Karin, M. “IKK/NF-kappaB signaling: balancing life and death-a new approach to cancer therapy.” J. Clin Invest. 2005. 115:2625-2632.

68. Hagemann, Thorsten, et, Al. “Regulation of macrophage function in tumors: the multifaceted role of NF-kB.” Blood. 2009. 113(14):3139-3146.

69. Bohuslav, J, et, Al. “Regulation of an essential innate immune response by the p50 subunit of NF-kappaB.” J. Clin. Invest. 1998. 102:1645-1652.

70. Adler, A, et Al. “Motif module map reveals enforcement of aging by continual NF-kB activity.” Genes Dev. 2007. 21:3244-3257.

71. Hayden, M, and Ghosh, S. “Signaling to NF-kB.” Genes & Dev. 2004. 18:2195–2224.

72. Chung, H, et Al. “The inflammation hypothesis of aging: Molecular modulation by calorie restriction.” Ann. N. Y. Acad. Sci. 2001. 928:327–335.

73. Hardy, K, et Al. “Transcriptional networks and cellular senescence in human mammary fibroblasts.” Mol. Biol. Cell. 2005. 16:943–953.

74. Bernard, D, et Al. “Involvement of Rel/nuclear factor-_B transcription factors in keratinocyte senescence.” Cancer Res. 2004. 64:472–481.

75. Yeung, F, et Al. “Modulation of NF-_Bdependent transcription and cell survival by the SIRT1 deacetylase.” EMBO J. 2004. 23:2369–2380.

76. Helenius, M, et Al. “Aging-induced up-regulation of nuclear binding activities of oxidative stress responsive NF-κB transcription factor in mouse cardiac muscle.” J Mol Cell Cardiol. 1996. 28:487–98.

77. Helenius, M, et Al. “Characterization of aging-associated up-regulation of constitutive nuclear factor-κB binding activity.” Antioxid Redox Signal. 2001. 3:147–56.

78. Spencer, N, et Al. “Constitutive activation of NF-κB in an animal model of aging.” Int Immunol. 1997. 9:1581–8.

79. Gloire, G, Legrand-Poels, S, and Piette, J. “NF-κB activation by reactive oxygen species: fifteen years later.” Biochem Pharmacol. 2006. 72:1493–505.

80. Kamata, H, et Al. “Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases.” Cell. 2005. 120(5):649-61.

81. Salminen, A, and Kaarniranta, K. “Genetics vs. entropy: longevity factors suppress the NF-kappaB-driven entropic aging process.” Ageing Res Rev. 2010. 9:298–314.

82. Mostoslavsky, R, et Al. “Genomic instability and aging-like phenotype in the absence of mammalian SIRT6.” Cell. 2006. 124:315–29.

83. Kawahara, T, et Al. “SIRT6 links histone H3 lysine 9 deacetylation to NF-κB-dependent gene expression and organismal life span.” Cell. 2009. 136:62–74.

84. Bordone, L, and Guarente L. “Calorie restriction, SIRT1 and metabolism: understanding longevity.” Nat Rev Mol Cell Biol. 2005. 6:298–305.

85. Weindruch, R, et Al. “Microarray profiling of gene expression in aging and its alteration by caloric restriction in mice.” J Nutr. 2001. 131:918S–23S.

86. Lin, L, Hron, J, Peng, S. “Regulation of NF-kappaB, Th activation, and autoinflammation by the forkhead transcription factor Foxo3a.” Immunity. 2004. 21:203–13.

87. Lee, H, et Al. “FOXO3a turns the tumor necrosis factor receptor signaling towards apoptosis through reciprocal regulation of c-Jun Nterminal kinase and NF-κB.” Arterioscler Thromb Vasc Biol. 2008. 28:112–20.

88. Stanfel, M, et Al. “The TOR pathway comes of age.” Biochim Biophys Acta. 2009. 1790:1067–1074.

89. Mehta, R, et Al. “Regulation of mRNA translation as a conserved mechanism of longevity control.” Adv Exp Med Biol. 2010. 694:14–29.

90. Narita, M, et Al. “Spatial coupling of mTOR and autophagy augments secretory phenotypes.” Science. 2011. 332:966-70.

91. Kim, J, et Al. “AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1.” Nat Cell Biol. 2011. 13:132-41.

92. Kaeberlein, M, and Kennedy, B. “Hot topics in aging research: protein translation and TOR signaling, 2010.” Aging Cell. 2011. 10(2):185-190.

93. Jia, K, Chen, D, and Riddle, D. “The TOR pathway interacts with the insulin signaling pathway to regulate C. elegans larval development, metabolism and life span.” Development. 2004. 131:3897–3906.

94. Kaeberlein, M. “Resveratrol and rapamycin: are they anti-aging drugs?” Bioessays. 2010. 32:96–99.

95. Kapahi, P, et Al. “With TOR, less is more: a key role for the conserved nutrient-sensing TOR pathway in aging.” Cell Metab. 2010. 11:453–465.

96. Harrison, D, et Al. “Rapamycin fed late in life extends lifespan in genetically heterogeneous mice.” Nature. 2009. 460:392–395.

97. Miller, R, et Al. “Rapamycin, but not resveratrol or simvastatin, extends life span of genetically heterogeneous mice.” J Gerontol A Biol Sci Med Sci. 2011. 66(2):191–201.

98. Selman, C, et Al. “Ribosomal protein S6 kinase 1 signaling regulates mammalian life span.” Science. 2009. 326:140–144.

99. Shaw, R. “LKB1 and AMP-activated protein kinase control of mTOR signalling and growth.” Acta Physiol (Oxf). 2009. 196:65–80.

100. Bjedov, I, et Al. “Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster.” Cell Metab. 2010. 11:35–46.

101. Chen, C, Liu, Y, and Zheng, P. “mTOR regulation and therapeutic rejuvenation of aging hematopoietic stem cells.” Sci Signal. 2009. 2:ra75.

102. Lamming, D, et Al. “Rapalogs and mTOR inhibitors as anti-aging therapeutics.” The Journal of Clinical Investigation. 2013. 123(3):980-989.

103. Zhou, J, et Al. “mTOR supports long-term self-renewal and suppresses mesoderm and endoderm activities of human embryonic stem cells.” PNAS. 2009. 106(19):7840–7845.

104. Murakami, M, et Al. “mTOR is essential for growth and proliferation in early mouse embryos and embryonic stem cells.” Mol Cell Biol. 2004. 24(15):6710–6718.

105. Kauffman, H, et Al. “Maintenance immunosuppression with target-of-rapamycin inhibitors is associated with a reduced incidence of de novo malignancies.” Transplantation. 2005. 80:883-889.

106. Martel, R, Klicius, J, and Galet, S. “Inhibition of the immune response by rapamycin, a new antifungal antibiotic.” Can J Physiol Pharmacol. 1977. 55(1):48–51.

107. Araki, K, et Al. “mTOR regulates memory CD8 T-cell differentiation.” Nature. 2009. 460:108-112.

108. Chen, C, “TSC-mTOR maintains quiescence and function of hematopoietic stem cells by repressing mitochondrial biogenesis and reactive oxygen species.” J Exp Med. 2008. 205(10):2397–2408.

109. Soliman, G. “The role of mechanistic target of rapamycin (mTOR) complexes signaling in the immune responses.” Nutrients. 2013. 5:2231-2257.

110. Powell, J, et Al. “Regulation of immune responses by mTOR.” Ann. Rev. Immunol. 2012. 30:39–68.

111. Powell, J, and Delgoffe, G. “The mammalian target of rapamycin: Linking T cell differentiation, function, and metabolism.” Immunity. 2010. 33:301-311.

112. Rao, R, Li, Q, and Shrikant, P. “Fine-tuning CD8(+) T cell functional responses: mTOR acts as a rheostat for regulating CD8(+) T cell proliferation, survival and differentiation?” Cell Cycle. 2010. 9:2996–3001.

113. Gulen, M, et Al. “The receptor SIGIRR suppresses Th17 cell proliferation via inhibition of the interleukin-1 receptor pathway and mTOR kinase activation.” Immunity. 2010. 32:54–66.

114. Xiao, H, et Al. “Loss of single immunoglobulin interlukin-1 receptor-related molecule leads to enhanced colonic polyposis in Apc(min) mice.” Gastroenterology. 2010. 139:574–585

115. Delves, P, et Al. Roitt’s Essential Immunology, 12th ed.; Wiley-Blackwell: Hoboken, NJ, USA, 2011.

116. Chi, H. “Regulation and function of mTOR signalling in T cell fate decisions.” Nat. Rev. Immunol. 2012. 12:325–338.

117. Iwasaki, A, and Medzhitov, R. “Regulation of adaptive immunity by the innate immune system.” Science. 2010. 327:291–295.

118. Haidinger, M, et Al. “A versatile role of mammalian target of rapamycin in human dendritic cell function and differentiation.” J. Immunol. 2010. 185:3919–3931.

119. Katholnig, K, et Al. “p38alpha senses environmental stress to control innate immune responses via mechanistic target of rapamycin.” J. Immunol. 2013. 190:1519–1527.

120. Saemann, M, et Al. “The multifunctional role of mTOR in innate immunity: Implications for transplant immunity.” Am. J. Transplant. 2009. 9:2655–2661.

121. Weichhart, T, and Saemann, M. “The PI3K/Akt/mTOR pathway in innate immune cells: Emerging therapeutic applications.” Ann. Rheum. Dis. 2008. 67(Suppl. 3):70–74.

122. Hackstein, H, et Al. “Rapamycin inhibits IL-4––Induced dendritic cell maturation in vitro and dendritic cell mobilization and function in vivo.” Blood 2003. 101:4457–4463.

123. Sathaliyawala, T, et Al. “Mammalian target of rapamycin controls dendritic cell development downstream of Flt3 ligand signaling.” Immunity. 2010. 33:597–606.

124. Hackstein, H, et Al. “Rapamycin inhibits macropinocytosis and mannose receptor-mediated endocytosis by bone marrow-derived dendritic cells.” Blood. 2002. 100:1084–1087.

125. Monti, P, et Al. “Rapamycin impairs antigen uptake of human dendritic cells.” Transplantation. 2003. 75:137–145.

126. Ohtani, M, et Al. “Cutting edge: mTORC1 in intestinal CD11c+ CD11b+ dendritic cells regulates intestinal homeostasis by promoting IL-10 production.” J. Immunol. 2012. 188:4736–4740.

127. Turnquist, H, et Al. “Rapamycin-conditioned dendritic cells are poor stimulators of allogeneic CD4+ T cells, but enrich for antigen-specific Foxp3+ T regulatory cells and promote organ transplant tolerance.” J. Immunol. 2007. 178:7018–7031.

128. Wicker, L, et Al. “Suppression of B cell activation by cyclosporin A, FK506 and rapamycin.” Eur. J. Immunol. 1990. 20:2277–2283.

129. Kay, J, et Al. “Inhibition of T and B lymphocyte proliferation by rapamycin.” Immunology. 1991. 72:544–549.

130. Donahue, A, and Fruman, D. “Distinct signaling mechanisms activate the target of rapamycin in response to different B-cell stimuli.” Eur. J. Immunol. 2007. 37:2923–2936.

131. Zhang, S, et Al. “Constitutive reductions in mTOR alter cell size, immune cell development, and antibody production.” Blood. 2011. 117:1228–1238.

132. Llorian, M, et Al. “The PI3K p110delta is required for down-regulation of RAG expression in immature B cells.” J. Immunol. 2007. 178:1981–1985.

133. Hess, K, et Al. “The p85alpha isoform of phosphoinositide 3-kinase is essential for a subset of B cell receptor-initiated signaling responses.” Eur. J. Immunol.2004. 34:2968–2976.

134. Delgoffe, G, et Al. “The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2.” Nat. Immunol. 2011. 12:295–303.

135. Delgoffe, G, et Al. “The mTOR kinase differentially regulates effector and regulatory T cell lineage commitment.” Immunity. 2009. 30:832–844.

136. Lee, K, et Al. “Mammalian target of rapamycin protein complex 2 regulates differentiation of Th1 and Th2 cell subsets via distinct signaling pathways.” Immunity. 2010. 32:743–753.

137. Finlay, D, et Al. “PDK1 regulation of mTOR and hypoxia-inducible factor 1 integrate metabolism and migration of CD8+ T cells.” J. Exp. Med. 2012. 209:2441–2453.

138. Sengupta, S, Peterson, T, and Sabatini, D. “Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress.” Mol. Cell. 2010. 40:310–322.

139. Araki, K, Youngblood, B, and Ahmed, R. “The role of mTOR in memory CD8 T-cell differentiation.” Immunol. Rev. 2010. 235:234–243.

140. He, S, et Al. “Characterization of the metabolic phenotype of rapamycin-treated CD8+ T cells with augmented ability to generate long-lasting memory cells.” PLoS One. 2011. 6:e20107

141. Pearce, E, et Al. “Enhancing CD8 T-cell memory by modulating fatty acid metabolism.” Nature. 2009. 460:103–107.

142. Li, Q, et Al. “A central role for mTOR kinase in homeostatic proliferation induced CD8+ T cell memory and tumor immunity.” Immunity. 2011. 34:541–553.

143. Dittrich, E, et Al. “Rapamycin-associated post-transplantation glomerulonephritis and its remission after reintroduction of calcineurin-inhibitor therapy.” Transpl. Int. 2004. 17:215–220.

144. Izzedine, H, Brocheriou, I, and Frances, C. “Post-transplantation proteinuria and sirolimus.” N. Engl. J. Med. 2005. 353:2088–2089.

145. Weichhart, T, et Al. “The TSC-mTOR signaling pathway regulates the innate inflammatory response.” Immunity. 2008. 29:565–577.

146. Rao, R, et Al. “The mTOR kinase determines effector versus memory CD8+ T cell fate by regulating the expression of transcription factors T-bet and Eomesodermin.” Immunity. 2010. 32:67–78.

147. Salcedo, R, et Al. “Human endothelial cells express CCR2 and respond to MCP-1: Direct role of MCP-1 in angiogenesis and tumor progression.” Blood. 2000. 96:34–40.

148. Guba, M, et Al. “Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: Involvement of vascular endothelial growth factor.” Nat. Med. 2002. 8:128–135.

149. Di Iorio, I, et Al. “Sarcopenia: age-related skeletal muscle changes from determinants to physical disability.” Int J Immunopathol Pharmacol. 2006:19:703-19.

150. Katholnig, K, et Al. “p38alpha senses environmental stress to control innate immune responses via mechanistic target of rapamycin.” The Journal of Immunology. 2013. http://www.jimmunol.org/content/early/2013/01/11/jimmunol.1202683

151. Perkins, N. “Integrating cell-signalling pathways with NF-κB and IKK function.” Nat Rev Mol Cell Biol. 2007. 8:49–62.

152. Trinchieri, G, and Sher, A. “Cooperation of Toll-like receptor signals in innate immune defence.” Nat Rev Immunol. 2007. 7:179–90.

153. Medzhitov, R, and Janeway, C Jr. “Innate immune recognition: mechanisms and pathways.” Immunol Rev. 2000. 173:89–97.

154. Gauldie, J. “Inflammation and the aging process: devil or angel.” Nutr Rev. 2007. 65:S167–9.

155. Libby, P. “Inflammatory mechanisms: the molecular basis of inflammation and disease.” Nutr Rev. 2007. 65:S140–6.

156. Spencer, N, et Al. “Constitutive activation of NF-kB in an animal model of aging.” International Immunology. 1997. 9(10):1581-1588.

157. Ershler, W, et Al. “Interleukin-6 and aging: blood levels and mononuclear cell production increase with advancing age and in vitro production is modifiable by dietary restriction.” Lymphokine Cytokine Res. 1993. 12:225.

158. Dutta, J, et Al. “Current insights into the regulation of programmed cell death by NF-κB.” Oncogene. 2006. 25:6800–16.

159. Papa, S, et Al. “Linking JNK signaling to NF-kappaB: a key to survival.” J Cell Sci. 2004. 117: 5197–208.

160. Dan, H, and Baldwin, A. “Differential involvement of IκBkinases α and ß in cytokine- and insulin-induced mammalian target of rapamycin activation determined by Akt. J Immunol.” 2008. 180:7582–9.

161. Sprott, R. “Biomarkers of aging and disease: introduction and definitions.” Exp Gerontol. 2010. 45:2–4.

162. Balistreri, C, et Al. “NF-kB pathway activators as potential ageing biomarkers: targets for new therapeutic strategies.” Immunity and Ageing. 2013. 10:24-40.

163. Simm, A, and Johnson, T. “Biomarkers of ageing: A challenge for the future.” Exp Gerontol. 2010. 45:731–732.

164. Euser, S, et Al. “The effect of age on the association between blood pressure and cognitive function later in life.” J Am Geriatr Soc. 2009. 57:1232–1237.

165. van Bemmel, T, et Al. “Markers of autonomic tone on a standard ECG are predictive of mortality in old age.” Int J Cardiol. 2006. 107:36–41.

166. Martin-Ruiz, C, et Al. “Telomere length predicts poststroke mortality, dementia, and cognitive decline.” Ann Neurol. 2006. 60:174–180.

167. McClintock, B. “The behavior in successive nuclear divisions of a chromosome broken at meiosis.” PNAS. 25:405–416, 1939.

168. Muller, H. “The re-making of chromosomes.” Collecting Net Woods Hole. 1938. 13:181–198.

169. Hayflick, L. “A brief history of the mortality and immortality of cultured cells.” Keio J Med. 1998. 47:174–182.

170. Hayflick, L. “The limited in vitro lifetime of human diploid cell strains.” Exp Cell Res. 1965. 37:614–636.

171. Olovnikov, A. “A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon.” J Theor Biol. 1973. 41:181–190.

172. Bodnar, A, et Al. “Extension of life-span by introduction of telomerase into normal human cells.” Science. 1998. 279:349–352.

173. Vaziri, H, and Benchimol, S. “Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span.” Curr Biol. 1998. 8:279–282.

174. Friedrich, U, et Al. “Telomere length in different tissues of elderly patients.” Mech. Ageing Dev. 2000. 119:89–99.

175. Aubert, G, and Lansdorp, P. “Telomeres and Aging.” Physiol. Rev. 2008. 88:557-579.

176. Riha, K, et Al. “Living with genome instability: plant responses to telomere dysfunction.” Science. 2001. 291:1797–1800.

177. Cheung, I, et Al. “High incidence of rapid telomere loss in telomerase-deficient Caenorhabditis elegans.” Nucleic Acids Res. 2006. 34:96–103.

178. Blasco, M. “Immunosenescence phenotypes in the telomerase knockout mouse.” Springer Semin Immunopathol. 2002. 24:75– 85.

179. O’Donnell, C, et Al. “Leukocyte telomere length and carotid artery intimal medial thickness: the Framingham Heart Study.” Arterioscler Thromb Vasc Biol. 2008. 28: 1165–1171.

180. Satoh, M, et Al. “Effect of intensive lipid-lowering therapy on telomere erosion in endothelial progenitor cells obtained from patients with coronary artery disease.” Clin Sci (Lond). 2009. 116:827– 835.

181. Brouilette, S, et Al. “Telomere length, risk of coronary heart disease, and statin treatment in the West of Scotland Primary Prevention Study: a nested case-control study.” Lancet. 2007. 369:107–114.

182. Gomes, N, et Al. “Comparative biology of mammalian telomeres: hypotheses on ancestral states and the roles of telomeres in longevity determination.” Aging Cell. 2011. 10(5):761-768.

183. Richter, T, and von Zglinicki, T. “A continuous correlation between oxidative stress and telomere shortening in fibroblasts.” Exp Gerontol. 2007. 42(11):1039-1042.

184. Daniali, L, et Al. “Telomeres shorten at equivalent rates in somatic tissues of adults.” Nature Communications. 2013. 4:1597-1603.

185. Youngren, K, et Al. “Synchrony in telomere length of the human fetus.” Hum. Genet. 1998. 102:640–643.

186. Kimura, M, et Al. “Synchrony of telomere length among hematopoietic cells.” Exp. Hematol. 2010. 38:854–859.

187. Granick, M, et Al. “Telomere dynamics in keloids.” Eplasty. 2011. 11:e15.

188. Gardner, J, et Al. “Telomere dynamics in macaques and humans.” J. Gerontol. A Biol. Sci. Med. Sci. 2007. 62:367–374.

189. Sidorov, I, et Al. “Leukocyte telomere dynamics and human hematopoietic stem cell kinetics during somatic growth.” Exp. Hematol. 2009. 37:514–524.

190. Ljubuncic, P and Reznick, A. “The evolutionary theories of aging revisited – a mini-review.” Gerontology 2009. 55:205–216.

191. Tissenbaum, H, and Guarente, L. “Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans.” Nature. 2001. 410: 227–230.

192. Rogina, B, and Helfand, S. “Sir2 mediates longevity in the fly through a pathway related to calorie restriction.” PNAS. 2004. 101: 15998–16003.

193. Oberdoerffer, P, et Al. “SIRT1 redistribution on chromatin promotes genomic stability but alters gene expression during aging.” Cell. 2008. 135:907–918.

194. Michishita, E, et Al. “SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin.” Nature. 2008. 452:492–496.

195. Rando, T, and Chang, H. “Aging, rejuvenation, and epigenetic reprogramming: resetting the aging clock.” Cell. 2012. 148:46-57.

196. Kawahara, T, et Al. “SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and organismal life span.” Cell. 2009. 136:62–74.

197. Tennen, R, and Chua, K. “Chromatin regulation and genome maintenance by mammalian SIRT6.” Trends Biochem. Sci. 2011. 36:39–46.

198. Bartke, A. “Insulin and aging.” Cell Cycle. 2008. 7(21):3338-3343.

199. van Heemst, D, et Al. “Reduced insulin/IGF-1 signaling and human longevity.” Aging Cell. 2005. 4:79–85.

200. Rincon, M, Rudin, E, and Barzilai, N. “The insulin/IGF-1 signaling in mammals and its relevance to human longevity.” Exp Gerontol. 2005. 40:873-7.

201. Yuan, R, et Al. “Aging in inbred strains of mice: Study design and interim report on median lifespans and circulating IGF1 levels.” Aging Cell. 2009. 8:277–87.

202. Brown-Borg, H, et Al. “Dwarf mice and the ageing process.” Nature. 1996. 384:33.

203. Blüher, M, Kahn, B, and Kahn, C. “Extended longevity in mice lacking the insulin receptor in adipose tissue.” Science. 2003. 299:572-4.

204. Katic, M, et Al. “Mitochondrial gene expression and increased oxidative metabolism: role in increased lifespan of fat-specific insulin receptor knock-out mice.” Aging Cell. 2007. 6:827-39.

205. Menzaghi, C, Trischitta, V, and Doria, A. “Genetic influences of adiponectin on insulin resistance, type 2 diabetes, and cardiovascular disease.” Diabetes. 2007. 56:1198-209.

206. Atzmon, G, et Al. “Adiponectin levels and genotype: a potential regulator of life span in humans.” J Gerontol A Biol Sci Med Sci. 2008. 62:447-53.

207. Paolisso, G, et Al. “Low insulin resistance and preserved beta-cell function contribute to human longevity but are not associated with TH-INS genes.” Exp Gerontol. 2001. 37:149-56.

208. Fontana, L, et Al. “Long-term calorie restriction is highly effective in reducing the risk for atherosclerosis in humans.” PNAS. 2004. 101:6659-63.

209. Dominici, F, et Al. “Compensatory alterations of insulin signal transduction in liver of growth hormone receptor knockout mice.” J Endocrinology. 2000. 166:579-90.

210. Bartke, A, et Al. “Longevity: Extending the lifespan of long-lived mice.” Nature 2001. 414:412.

211. Bartke, A, and Brown-Borg, H. “Life extension in the dwarf mouse.” Curr Top Dev Biol: Academic Press. 2004. 189-225.

212. Bartke, A. “Growth hormone, insulin and aging: The benefits of endocrine defects.” Exp. Gerontol. 2011. 46(2-3):108-111.

213. Coschigano, K, et Al. “Deletion, but not antagonism, of the mouse growth hormone receptor results in severely decreased body weights, insulin and insulin-like growth factor I levels and increased life span.” Endocrinology. 2003. 144:3799-810.

214. Deletion, but not antagonism, of the mouse growth hormone receptor results in severely decreased body weights, insulin and insulin-like growth factor I levels and increased life span. Endocrinology 2003; 144:3799-810. [20]

215. Deletion, but not antagonism, of the mouse growth hormone receptor results in severely decreased body weights, insulin and insulin-like growth factor I levels and increased life span. Endocrinology 2003; 144:3799-810. [39]

216. Deletion, but not antagonism, of the mouse growth hormone receptor results in severely decreased body weights, insulin and insulin-like growth factor I levels and increased life span. Endocrinology 2003; 144:3799-810. [44]

217. Deletion, but not antagonism, of the mouse growth hormone receptor results in severely decreased body weights, insulin and insulin-like growth factor I levels and increased life span. Endocrinology 2003; 144:3799-810. [45]

218. Alderman, J, et Al. “Neuroendocrine inhibition of glucose production and resistance to cancer in dwarf mice.” Exper Gerontol. 2009. 44:26–33.

219. Brooks, N, et Al. “Low utilization of circulating glucose after food withdrawal in snell dwarf mice.” J Biol Chem. 2007. 282:35069–35077.

220. Westbrook, R, et Al. “Alterations in oxygen consumption, respiratory quotient, and heat production in long-lived ghrko and ames dwarf mice, and short-lived bgh transgenic mice.” J Gerontol A Biol Sci Med Sci. 2009. 64A:443–451.

221. Ulrich, P, and Cerami, A. “Protein glycation, diabetes, and aging.” Recent Prog. Horm. Res. 2001. 56:1–21.

222. Anderson, R, and Weindruch, R. “Metabolic reprogramming in dietary restriction.”
Interdisciplinary topics in gerontology. 2007. 35:18-38.

223. Kennedy, B, Steffen, K, and Kaeberlein, M. “Ruminations on dietary restriction and aging.” Cell Mol Life Sci. 2007. 64:1323-1328.

224. Piper, M, and Bartke, A. “Diet and aging.” Cell metabolism. 2008. 8:99-104.

225. Mercken, E, et Al. “Calorie restriction in humans inhibits the PI3K/AKT pathway and induces a younger transcription profile.” Aging cell. 2013.

226. Kaneko, T, Tahara, S, and Matsuo, M. “Retarding effect of dietary restriction on the accumulation of 8-hydroxy-2'-deoxyguanosine in organs of Fischer 344 rats during aging.” Free Radic Biol Med. 1997. 23(1):76-81.

227. Hamilton, M, et Al. “Does oxidative damage to DNA increase with age?” PNAS. 2001. 98(18):10469-10474.

228. Bernstein, H., Payne, C.M., Bernstein, C., Garewal, H., Dvorak, K. (2008). “Cancer and aging as consequences of un-repaired DNA damage.” In: New Research on DNA Damages (Editors: Honoka Kimura and Aoi Suzuki) Nova Science Publishers, Inc., New York, Chapter 1, pp. 1-47.

229. Kenyon, C, et Al. “A C. elegans mutant that lives twice as long as wild type.” Nature. 1993. 366:461-464.

230. Fontana, L, et Al. “Long-term effects of calorie or protein restriction on serum IGF-1 and IGFBP-3 concentration in humans.” Aging cell. 2008. 7:681-687.

231. Mercken, E, et Al. “Calorie restriction in humans inhibits the PI3K/AKT pathway and induces a younger transcription profile.” 2013. doi: 10.1111/acel.12088

232. Wu, Z, et Al. “Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1.” Cell. 1999. 98:115-124.

233. Mattison, J, et Al. “Impact of caloric restriction on health and survival in rhesus monkeys from the NIA study.” Nature. 2012. 489:318-321.

234. Rezzi, S, et Al. “Metabolic shifts due to long-term caloric restriction revealed in nonhuman primates.” Experimental Gerontology. 2009. 44(5):356–62.

235. Maxmen, A. “Calorie restriction falters in the long run: Genetics and healthy diets matter more for longevity.” Nature News. August 29, 2012 http://www.nature.com/news/calorie-restriction-falters-in-the-long-run-1.11297

236. Morley, J, Chahla, E, and Alkaade, S. “Antiaging, longevity and calorie restriction.” Current Opinion in Clinical Nutrition and Metabolic Care. 2010. 13(1): 40–5.

237. Villareal, D, et Al. “Bone mineral density response to caloric restriction-induced weight loss or exercise-induced weight loss: a randomized controlled trial.” Archives of Internal Medicine. 2006. 166(22):2502–10.

238. Marzetti, E, et Al. “Cellular mechanisms of cardioprotection by calorie restriction: state of the science and future perspectives.” Clin. Geriatr. Med. 2009. 25(4):715-32.

239. Redman, L, et Al. “The effect of caloric restriction interventions on growth hormone secretion in nonobese men and women.” Aging Cell. 2010. 9(1):32-9.

240. Li, B, et Al. “Identification of potential calorie restriction-mimicking yeast mutants with increased mitochondrial respiratory chain and nitric oxide levels.” J Aging Res. 2011. 673185.

241. Tahara, E, et Al. “Calorie restriction hysteretically primes aging saccharomyces cerevisiae towards more effective oxidative metabolism.” PLoS ONE. 2013. 8(2): e56388.

242. Schulz, T, et Al. “Glucose Restriction Extends Caenorhabditis elegans Life Span by Inducing Mitochondrial Respiration and Increasing Oxidative Stress.” Cell Metabolism. 2007. 6(4): 280–293.

243. Ristow, M and Zarse, K. “How increased oxidative stress promotes longevity and metabolic health: the concept of mitochondrial hormesis (mitohormesis).” Experimental Gerontology. 2010. 45(6): 410–8.

244. Tapia, P. “Sublethal mitochondrial stress with an attendant stoichiometric augmentation of reactive oxygen species may precipitate many of the beneficial alterations in cellular physiology produced by caloric restriction, intermittent fasting, exercise and dietary phytonutrients: "Mitohormesis" for health and vitality.” Medical Hypotheses. 2007. 66(4): 832–43.

245. Bjelakovic, G, et Al. “Mortality in Randomized Trials of Antioxidant Supplements for Primary and Secondary Prevention: Systematic Review and Meta-analysis.” JAMA. 2007. 297(8): 842–57.

246. McCormack, F, et, Al. “Efficacy and safety of sirolimus in lymphangioleiomyomatosis.” N Engl J Med. 2011. 364(17):1595–1606.

247. Gyurus, E, Kaposztas, Z, and Kahan, B. “Sirolimus therapy predisposes to new-onset diabetes mellitus after renal transplantation: a long-term analysis of various treatment regimens.” Transplant Proc. 2011. 43(5):1583–1592.

248. Lamming, D, et Al. “Rapalogs and mTOR inhibitors as anti-aging therapeutics.” The Journal of Clinical Investigation. 2013. 123(3):980-989.

249. Vijg, J, and Campisi, J. “Puzzles, promises, and a cure for ageing.” Nature. 2008. 454(7208):1065-1071.

250. Selman, C, et Al. “Evidence for lifespan extension and delayed age-related biomarkers in insulin receptor substrate 1 null mice.” FASEB J. 2008. 22:807–818.

251. Ayyadevara, S, et Al. “Remarkable longevity and stress resistance of nematode PI3K-null mutants.” Aging Cell. 2007. 7:13–22.

252. Broughton, S, et Al. “Longer lifespan, altered metabolism, and stress resistance in Drosophila from ablation of cells making insulin-like ligands.” PNAS. 2005. 102:3105–3110.