User:Kinkreet/SCRA/Aging and Regeneration

Identifying factors involved in regeneration
Lesion tissues, and screen for differences in the proteome, glycome, transcriptome, metabolome and epigenomics between the lesioned tissues and unlesioned tissues. We can also carry out these omics screens for wild type organisms as opposed mutants which has deficiency in regeneration, of the same tissue between two different species which age differently, and between the same tissue but at different developmental stage. Once these factors are identified, a functional gene assay can be carried out where we knockout a gene of interest, injure the organism/tissue, and observe for the lack (hopefully) of regeneration.

Limited fibroblasts, low oxygen environment (hypoxia), immature immunoinflammatory response. Cell cultures derived from normal limb and early limb regenerate (blastema) of the newt Notophthalmus viridescens can be cultured without signs of senescence for more than a year, in contrast to heart and liver cell cultures of newt. These culture cells are called A1 cells, and expresses regeneration-associated antigen 22/18, which is intracellular and filamentous. Its expression depends on cell density, and is turned off after differentiation has occurred.

Serum
A1 cells can be induced to differentiate into myotubes (expressing muscle-specific myosin heavy chain) using low (0.5%) serum medium. Differentiation requires the withdrawal from the cell cycle, because it is usual for cell that differentiate to lose its ability to self-renew. Thus, terminally differentiated do not enter the cell cycle and divide, apart from some cancers. Cells are prevented in going into the cell cycle by an active retinoblastoma (Rb) protein; myotubes derived from Rb-KO mice was able to reenter the cell cycle after serum stimulation. But urodeles limbs have the natural ability to reenter the cell cycle after injury, and so the Rb-KO might not be required. Indeed, myotubes derived from A1 cells are able to reenter the cell cycle after high (5-20%)-serum stimulation; however, polypeptide grow factors such as FGF, PDGF, EGF and IGF stimulated division of mononucleate A1 cells, but did not induce reentry into the cell cycle. If this factor/these factors in the serum can be identified, it could potentially induce cells at the site of the wound to re-enter the cell cycle, and replicate and divide to make up the missing parts of the body.

Tanaka identified a factor in the serum called cyclin-dependent kinase (CDK) 4 or 6, that can phosphorylate Rb to make it inactive. E2F becomes unbound from Rb and can go on to drive expression of genes that drive cell cycle progression. To confirm this hypothesis, both A1-derived myotubes halted at G1 phase when injected with CDK4/6 inhibitor, or when serum is used to stimulate a Rb mutant (D34 Rb), which cannot be phosphorylated and inactivated. Thus, Rb is responsible for regeneration in newt myotubes. It was also found that cell cycle reentry was inhibited with cell contact with mononucleate cells, and more division was observed in low-density cultures. Thus, when an injury occurs, the cells at the plane of the injury loses contact with the now displaced cells, and this loss of cell contact stopped the inhibition to reenter the cell cycle. Thrombin was used to pre-treat serum, and then 1.5% of the treated serum was used to incubate myotubes; serum-free media was used as a control. Only the 1.5% serum treated myotubes reentered the cell cycle. It is found that thrombin cleaves the S-phase reentry factor (SPRF), a large and highly resistant to chemical denaturation, although is thermolabile; the exact characteristics of the SPRF has not yet been identified. SPRF is part of the serum, and when cleaved, directly acted on the myotubes to promote reentry. The SPRF did not work on mononucleated precursor cells or on cultured mouse myotubes.

Nerves
Nerves are not required during embryonic development, but is required for blastema division and growth. Without nerves, ectopic limbs with smaller skeletons are derived. A member of the anterior gradient protein family called nAG is secreted by distal (from the wound plane) Schwann cells of axons to drive proliferation of the blastema. Denervation prevented blastema growth and local expression of nAG induces blastema growth and the regeneration of distal structures. nAG is a ligand for a small GPI-lined surface protein called Prod 1. Prod 1 is expressed in proximodistal gradient, with Meis homeoproteins mediating this differential expression

Prod 1 is only found in salamanders and can be altered by retinoic acid. It also interacts with epidermal growth factor receptor (EGFR) and phosphorylates extracellular regulated kinase 1 and 2 (ERK1/2), leading to the induction of the transcription and expression of matrix metalloproteinase 9 (MMP9). M atrix metalloproteinase 9 is involved in breakdown of extracellular matrix allowing keratinocytes to detach from the basement membrane and migrate to cover the exposed connective tissue after injury.

Integrin signalling
Mice with a specific knockout of integrin β1 in fibroblasts shows delayed wound closure, reduced production of the ECM, and reduced production of α-smooth muscle actin (α-SMA). These fibroblasts expresses less type I collagen and connective tissue growth factor. This reduction in cell adhesion means the fibroblasts cannot migrate effectively to the wound site, and once there do not have enough structure to contract the wound. This KO of integrin reduced TGFβ signalling, but when TGFβ is introduced exogenously, wound healing was once again effective.

Methods
The chick chorioallantoic membrane (CAM) assay is performed by implanting a membrane or coverslip containing the compound of interest on the chick embryo chorioallantoic membrane through a hole cut in the egg shell. The subsequent incubation period ranges from 1-3 days, depending on the compound, after which time angiogenesis can be quantified via image analysis or colorimetric detection methods.

Model organisms
Regeneration without scarring in mammals are limited to the tips of fingers, liver , muscle, epidermis and bones. However, regeneration can only occur after injury, and not after the entire tissue is amputated. For example, no replacement is observed if the whole muscle is excised, or if the dermis is damaged along with the epidermisin contrast, some fish and amphibians are able to regenerate large areas of limb, tail, eye lens and parts of the heart. Some organisms such as urodeles (salamander, which includes newts and axolotl, falls into this group) and teleost fish (inc. zebrafish) are used to study regeneration for different tissues due to their ability to regenerate effectively. Urodeles are mostly used to study limb regeneration whereas zebrafish is used to study heart regeneration.

Urodeles
Urodeles are the only vertebrates known which is able to perfectly regenerate limbs after injury or loss of whole limbs, even after differentiation of the cells nearby. It has been shown to regenerate limbs, jaws, tail, skin, apex of the heart, heart ventricle, retina, lens, jaws and components of the central nervous system (CNS) - spinal cord, fore brain, midbrain dopaminergic neurons, retinotectal projection. Their ability to regenerate do not seem to be affected by their age, and the rate of regeneration is similar when normalized to their size. In addition to being able to regenerate, urodeles are resistant to cancer. After injury, the immune response is also known to be attenuated, and this might be important for the regenerative properties of urodeles.

The lack of scarring means no loss of original function of the tissue. Glial scarring following injury to the nervous system prevents growing axons from reaching their target The use of sutures to close the wound prevented regeneration, leading to the notion that maybe the lack of scarring is the essential component lacking in humans which prevents regeneration. Humans, during the ﬁrst trimester, is able to heal injuries scarlessly. The mechanism behind this ability to regenerate even after whole limb amputation is likely to be dedifferentiation of cells next to the wound, followed by differentiation. This process has been termed epimorphic regeneration This process can be broken down into two phases - preparation followed by redevelopment. After amputation, a sheet of wound epithelium covers the wound to stop signalling and infections, and will eventually become the apical ectodermal cap (AEC). The upregulation of matrix metalloproteinase (MMP) leads to the reorganization of the extracellular matrix. Then cells within a few milimeters of the amputation plane dedifferentiate and migrate and proliferate towards the AEC. Using tritiated thymidine to follow cell proliferation and migration. it was shown that the blastema comes from dedifferentiating internal tissues (a few milimeters from the amputation plane) and not from the epidermis. The preparation phase is complete by the formation of the blastema, cells at the end of the stump. In the redevelopmental stage the blastema redifferentiate to form all the tissues required for the formation of a new limb. The redevelopmental stage is similar to the embryonic developmental stage in terms of gene expression and its independence of signals from nerves.

The lateral ectoderm, neural fold, presomitic mesoderm, lateral plate mesoderm were obtained from different transgenic axolotls ubiquitously expressing GFP or nuclear Cherry, and transplanted into different host embryos and the fate of the different cells tracked. After transplantation, the axolotl are permitted to grow to a length of 8cm. The limbs are then amputated and the proliferation and migration of the fluorescently-labelled cells are tracked. The results show that each tissue produces progenitors with limited potency, only able to regenerate its own tissue. Thus, the blastema is a heterogeneous collection of restricted progenitor cells, and thus the dedifferentiation process during the preparation phase do not need to go as far back as the pluripotent stage of stem cells, but only to multipotency of progenitors.

In the blastema cells, Sox2, Klf4 and c-myc are expressed. Sox2 is probably the transcription factor promoting stemness-genes, Klf4 might function to keep the chromatin states and histones acetylated, while c-myc might function to aid proliferation and differentiation of progenitor cells.

Retinoblastoma protein (Rb) controls the entry of cells into the cell cycle; since regeneration requires extensive proliferation, the Rb protein is highly studied, not least because of its tie with cancer studies as well. Active, hypophosphorylated Rb (pRb) binds to eukaryotic transcription factor E2F and prevent E2F-dependent G1 to S phase transition; Rb can be hyperphosphorylated (ppRb) and do not E2F, allowing E2F to mediate G1 to S phase transition. An active Rb protein prevent cell cycle progression, and an inactive Rb allows cell cycle progression, thus, Rb can simply be viewed as an inhibitor of G1 to S phase transition.

So in theory, the deactivation of Rb by hyperphosphorylation should lead to cell progression and enable regeneration. This is a far too simplistic view because regeneration is not merely proliferation, but also directed differentiation. Knockouts or inactivation of Rb in myotubes in mammals do not lead to regeneration, but shows promise in hair and liver regeneration.

Other factors such as Piwi-like 1 (PL1) and Piwi-like 2 (PL2), which are said to regulate FGF8 signalling, is required for successful regeneration.

Different locations
The axolotl can have amputations anywhere between the shoulder and the hand, and will be able to regenerate perfectly.

Urodeles can regenerate limbs and part of the CNS (e.g. spinal cord, forebrain, midbrain DA neurons) without scarring

The lens of larval or adult newt can be removed and regenerated. In adult salamanders, the pupillary margin of the mid-dorsal iris contains pigmented epithelial cells (PEC) that reenters the cell cycle and transdifferentiate into lens. This regeneration is not observed in axolotl. It is hypothesized that a tissue factor colocalize at the pupillary margin of the mid-dorsal iris and activates thrombin to cleave SPRF, ultimately to encourage cell cycle reentry, with subsequent factors such as Wnt and FGF-2 promoting differentiation.

The newt can also regenerate the heart after 30-50% of it has been removed. Cardiomyocytes reenter the cell cycle and regenerates the ventricle; full dedifferentiation is not involved. About a third of dissociated newt cardiomyocytes reenter at S-phase in culture.

Urodele brain, as well as other fish, amphibians and reptiles, cells divide a lot postnatally, and can regenerate.

Zebrafish
Zebrafish (and newts) are able to regenerate a damaged heart with no fibrosis, whereas in humans, almost all the tissues being regenerated are fibroblasts. Mouse hearts are able to regenerate without scarring at or before E14, and exhibits increasing scarring after E18.

The zebrafish has many self-renewing neural precursor cells throughout the brain.

Turtles
Turtles are also long-living organisms that do not exhibit aging past maturity, although they have not been shown to regenerate after injury.

Deer antlers
The antlers on deer are shed after each mating season and are only used to fight and display for prowess. The increase in day length leads to a drop in testosterone levels which ultimately leads to the shedding of the antlers. Thus, regeneration of the deer antler is seasonally activated, and thus appears to be stem cell based and not the dedifferentiation process as it is with urodeles. This regeneration do not require nerves and although it requires much proliferation, cancers are not observed.

Acomys
African spiny mouse can regrow skin without scarring. After a skin wound, the wound quickly contracts and hair follicles regenerate, including sebaceous glands, dermis and cartilage. This gives the spiny mouse an evolutionary advantage as they can easily shed their skin to escape from a predator, only to repair it soon after.

Regeneration in humans
Regeneration in humans are largely suppressed. This may be due to an inability to adapt from the evolutionary point of view, or there is simply no adaptive advantage or enough evolutionary pressure for mammals to have regenerative abilities. Regeneration consumes a lot of energy and time, especially when regenerating whole limbs. Warm blooded mammals are susceptible to bacterial infection and regeneration takes a lot of time, scarring is a faster process and can prevent bacterial infection.

Dedifferentiation often see the expression of embryonic antigens, only amphibians with a weak immune system is able to tolerate these antigens.

Homeostasis
Our body is able to maintain homeostasis

Signalling
transforming growth factor and beta and the tumor suppressor p53 pathways are essential in axolotl regeneration.

Notch
Loss of ability to repair is due to transforming growth factor beta signalling via Smad3 which interferes with Notch function.

Wnt
Wnt has been shown to be required for regeneration.

Aging
Aging is a complicated field due to the fact that it is a gradual and heterogenous process, there is not a clearcut definition as to whether something is aged or not, because of this, there is also a lack of biomarkers that allows for the identification of 'aged' cells from young cells. Furthermore, different organs of the same organism age at different rates, and the aging of one organ will affect the aging of another organ. Lifestyles can also affect aging. With a mixture of genetic and extrinsic influences aging, there are not one master aging process, the study of aging is not as clear cut as other fields. A further set back is that aged organisms that can be used for research needs to be maintained for a long period of time, which costs a lot of money. An aged organ is defined as an organ which lost its ability to maintain homeostasis. This can produces symptoms as muscle weakness, anaemia, lack of wound healing and a weaker immune system. It can also make diseases such as cancer, cardiovascular diseases, arthritis, type 2 diabetes and pulmonary diseases easier and quicker to manifest.

There are also cases in which people do not appear to age, and no known genetic syndrome or chromosomal abnormality have been attributed to this. The girl has normal telomere lengths, but her anthropometric measurements are that of an 11-month-old. Furthermore, her systems are not fully developed resulting in difficulty breathing and digesting, and infantile brain structure and thinking ability. Her corpus callosum is largely missing, meaning the left and right cerebral hemisphere are not connected.

Skeletal muscle
Generally, people over 50 loses 1-2% of their muscle mass, and 0.5% of ATP production every year, even with exercise. Aging is characterized by increase fiber size variability (usually myotubes are of the same size), disorganization of the fibers, larger extracellular spaces and more ECM deterioration, increased fibrosis and non-contractile material such as fats and other connective tissues, and protein aggregates within the interstitial matrix. The mitochondria of aged muscle cells are decreased in numbers, and have larger vacuoles and shorter cristae. ATP production decreases and the complexes encoded by mtDNA (Complex I, III and IV) also decrease in numbers. The proportion of damaged mitochondria increases.

The infiltration of fats into the muscles can lead to metabolic diseases such as insulin resistance. The fat cells secrete signals which may dampen the insulin signalling, which leads to high blood glucose levels, as well as reduced protein synthesis in these muscles cells, which allows further fat tissues to infiltrate and replace the muscles. The fats can also induce inflammation and generates ROS that breaks down fatty acids and release them into the blood.

Skeletal muscles contains stem/progenitor cells called satellite cells (marked by paired box proteins 3 and 7, Pax3/7) which lie beneath the basal lamina that surrounds each myofibre, and are able to divide and provides a source of myogenic cells which are able to replace damaged tissues. These satellite cells fuse with the damaged muscle fibres to give them a myonuclei and thus allowing them to regenerate. In aged mice and humans, the number of satellite cells decrease and those that remain are able to divide less. The myotubes that aged satellite cells produce tend to be more disorganized and fragile (apoptose more often), presumably due to mutations in its genetic material. The increase of adipocytes and fibroblasts also gives unwanted signalling to the satellite cell niche, which might have an detrimental effect on their self-renewal, proliferative and potency properties. Evidence supporting the role of the niche in muscle maintenance includes transplantation and parabiosis experiments. Transplantation of aged hind limb muscles into a young host allows the old muscle to be regenerated; and in contrast, young hind limb muscle in an aged host is less able to regenerate. There are factors which are present in the blood which promotes regenerate which are in young mice but not aged mice, or it is also possible that factors which inhibit regeneration which are present in aged mice and not in young mice. Indeed, Wnt and Notch signalling is attributed to ageing in both the transplantation and parabiosis experiments, whereby a young system, lacking in Wnt ligands and high in Notch, is able to rejuvenate old muscles. Recently, it has been found that FGF2 expressed by aged muscle fibers, possibly in an effort to aid regeneration, actually drives satellite cells out of quiescence and depletes them. . Differences in the niche between young and aged samples have also been observed in the haematopoietic lineages. Also recently, a component of the classical complement pathway, C1qa, have been identified to inhibit regeneration.

Aging in skeletal muscle is easy to study because it is easy to take biopsies and measure changes.

C1qa
Wnt is a large family of secreted proteins involved in development and the self-renewal and differentiation of stem cells. Canonical Wnt signalling molecules binds to Wnt receptors Frizzled family of serpentine proteins, and low-densitiy lipoprotein receptor related protein 5/6 (LRP5/6); once activated, cytoslic beta-catenin is no longer degraded but stabilized, and translocates into the nucleaus where it binds to T cell factor/Lymphoid enhancer factor (Tcf/Lef) and induces Tcf/Lef transcription.

Complement C1q (in the serum) activates the canonical Wnt pathway by binding to the extracellular cysteine-rich domain (CRD) of the Wnt receptor Frizzled (Fz), inducing C1s-dependent cleavage of the ectoderm of Wnt coreceptor low-densitiy lipoprotein receptor-related protein 6 (Lrp6). Activation of Wnt decrease the ability of cells to regenerate (sign of aging). Further evidence to suggest this includes:


 * Serum C1q expression and concentration increases with aging (as determined using ELISA and Western blot analysis ), this coincides with increased Wnt signalling during aging.
 * During aging in wild-type mice, Wnt signalling is augmented. Wnt signalling is not augmented in Cq1-deficient mice.
 * Administration of exogenous Cq1 inhibited muscle regeneration. Disruption of the C1qa gene or inhibition of C1s leads to retoration of regeneration.
 * Canonical Wnt signalling is augmented in mouse model of accelerated aging
 * Inhibition of Wnt signalling reverse age-associated impairment of skeletal muscle regeneration..

Method for measuring canonical Wnt signalling
A TOPFLASH reporter gene assay is used to determine the level of canonical Wnt activation. The version used here involves a vector containing a single luciferase gene regulated by 8 Tcf/Lef binding sites (AGATCAAAGG), with each site separated by a spacer (GGGTA). Stabilized β-catenin translocate to the nucleus and binds to and activate the Tcf/Lef transcription factor, which in turn binds to Tcf/Lef binding sites and induce the expression of the gene it regulates, which in this case is the luciferase gene. Thus, the higher the activation of Wnt, the higher the population of cytosolic stabilized β-catenin, the higher the level of Tcf/Lef transcription, and the high expression and thus concentration of luciferase in Wnt-active cells.

Cells would have been plated and transfected with the TOPFLASH vector. 2 days after transfection, the cells are lysed and the levels of luciferase is measured by luciferase reporter assay kit, which makes use of the fact that luciferase is bioluminescent, thus increase in Wnt signalling can be monitored by the light intensity emitted from the cells.

(need to criticize this method - not precise because transfection have a low success rate, background bioluminescence. The transfection process itself may induce the cell to respond. The group said so themselves, that TOPFLASH is carried out a late time points, and therefore be affected by other factors which indirectly modulate Tcf/Lef.

C1q binds specifically to Frizzled
It has already been previously described that Wnt signalling is increased in aging samples, and thus the level of Wnt ligands associated with aging is likely to increase in aging samples compared to young samples. The Komuro group used the TOPFLASH reporter gene assay and found that canonical Wnt signalling is also increased in mice with heart disease (specifically pressure overload and dilated cardiomyopathy).

Frizzled (Fz) is part of the Fz/low density lipoprotein (LDL) receptor-related protein (LRP) complex to which Wnt proteins bind to; thus, any Wnt ligands is likely to bind to Fz (more specifically, the cysteine-rich domain, or CRD). Using a Fz8CRD-IgG/Fc fusion proteins to precipitate Fz-binding proteins (using protein G; IgG/Fc was used as a control), the Komuro group was able to identify a 26kDa protein upregulated in aging mice, and even more so in mice with heart disease. Mass spectrometry identified the band as C1qa, a major constituent of the complement C1q. The experiment was repeated for Fz1, -2, -4 and -7, and the results was the same.

C1q is a Wnt ligand which binds specifically to Frizzled
Because there is a strong correlation between the levels of C1qa and Wnt signalling, it has led to the hypothesis that C1qa is a Wnt ligand particularly involved in aging. To confirm this hypothesis, Komuro and group administered C1qa to mice and observed an increased level of Tcf/Lef transcription (as determined by TOPFLASH) and a corresponding increased level of stabilized cytosolic β-catenin (the population which induces Tcf/Lef transcription. This binding of C1qa is thought to be specific and in competition with Wnt (as determined by a heterologous competition assay), with similar binding affinities but with a 200-fold effective dose.

C1qa is unique within the complements as C3 or C5 depleted serum did not reduce Wnt activity nor reduce β-catenin levels, only C1q depleted serum reduced Wnt activity and β-catenin levels. Adding Fz8/Fc into C3 or C5 depleted serum also reduced Wnt activity, but Fz8/Fc addition into C1q depleted serum did not reduce Wnt activity further.

C1q is a Wnt ligand which binds specifically to Frizzled and increases Wnt signalling by C1s-dependent cleavage of the extracellular domain of LRP6
The complement system is part of the innate immunity; the classical pathway is triggered by the activation of C1, which consists of C1q and two proenzymes C1r and C1s. C1q binds to aggregated immunoglobulins which alters its conformation and activates it; this causes the autoactivation of C1r and consequently C1s. C1s then cleaves C2 and C4 to activates subsequent steps of the complement pathway. C1r and C1s is expressed and secreted by target cells, and is involved in C1qa-dependent canonical Wnt activation. Knockdown of C1r and C1s using siRNA or inhibition of C1r/C1s using antibodies and inhibitors led to a decrease in C1qa-dependent canonical Wnt activation. Induction of C1qa led to the appearance of cleaved LRP6 N-terminal fragments in the media, where the LRP6 is cleaved between residues Arg792-Ala793. The C-terminal domain seems be be autophagocytosed after cleavage as chloroquine treatment (which prevents autophagosome-lysosome fusion and thus inhibits autophagy) saw an accumulation of undegraded LRP6 ICD.

The cleavage of LRP6 is C1qa-dependent, N-terminally myc-tagged LRP6 was transfected into HepG2 cells (a human liver carcinoma cell line). Immunoblotting using anti-myc antibody showed that upon normal serum treatment, the N-terminal (containing the myc tag) is cleaved and found in large numbers in the culture medium; while with C1qa-depleted serum, the N-terminal remains with the cell. This is observed in mice models also.

It is likely that the C-terminal domain is responsible for Wnt signalling transduction. Transfection of DEL-LRP6 (a deletion mutant lacking the N-terminus prior to the cleavage point) into the cell saw a 47-fold increase in Wnt signalling compared to WT-LRP6. Phosphorylation of the cleaved fragment was also increased after C1qa treatment of the wildtype or transfected mutant. A mutant C1qa which has glycine residues substituted for Arg792-Ala793 saw a lower Wnt activation rate compared to wildtype after C1qa stimulation. These suggest that the cleaved C-terminus of LRP6 is sufficient for Wnt signalling activation, and phosphorylation may have a role in this process.

C1qa activates Wnt signalling in skeletal muscle and decrease regenerative capacity
Treatment of satellite cells and fibroblasts, derived from young mice, with C1q and Wnt3a resulted in the stabilization of beta-catenin and thus increased Wnt signalling; this is marked by an increase in Axin2 mRNA expression, which has previously been linked to increased Wnt signalling. They repeated this experiment in aged mice, and found that the increase in Wnt signalling is even more prominent in both cell populations. Thus, C1qa induces Wnt activation in both satellite cells and fibroblasts in vitro.

In vivo, using hydrogel containing C1q for the treatment, it was shown that C1q alone did not induce Wnt activation; it is only when the tissue is cryoinjured did Wnt signalling activity dramatically increased. This is probably because after injury, the expression of the C1rand C1s gene is upregulated (as determined by RT-PCR), providing a higher sensitivity to C1q-mediated Wnt activation.

The physiological effect of C1q treatment in vivo after cryoinjury, presumably through increased Wnt signalling, is increase fibrosis and decreased satellite cell proliferation. Thus, C1q impairs skeletal muscle regeneration. To confirm this, the level of collagen, a marker of fibroblasts, is increased after C1q treatment, or in aged mice. These effects were quenched using M241, an antibody against C1q.

C1qa-mediated promotion of age-related phenotypes are independent of the classical complement pathway
Canonical Wnt signalling was activated when the gastrocnemius muscle of young mice were cryoinjured and then treated with C1qa. The administration of C1qa or the activation of canonical Wnt impaired regeneration and resulted in more fibroblasts in the tissue. In C3-deficient mice, whose classical complement pathway is nulled, Wnt activation and levels of regeneration in control and C1qa treated mice saw no difference with wildtype mice. This means that the impairment of regeneration mediated by C1qa acts independently of the classical complement pathway.

The gastrocnemius muscle of aged wildtype and C1qa-deficient (C1qaKO) mice were cryoinjured, a hydrogen containing M241 or BB5.1 was inserted. BB5.1 is an antibody against C5 and inhibits the classical complement pathway without affecting C1; whereas M241 inhibits the classical complement pathway by inhibiting C1s and C1q. In M241 treated mice, the activation of Wnt was at comparable levels to C1qaKO mice, further indicating C1qa/Wnt-mediated impairment of regeneration is independent of the classical complement pathway.

Sarcopenia
Sarcopenia is the loss of muscle mass/cells, leading to the loss of strength and function of the skeletal muscle. It can be caused by many factors.

Heart and the cardiovascular system
The aging of the heart and cardiovascular system comes in two components - the endogenous aging of the heart, and the stresses the rest of the body exerts on the CV system, given that the CV system carries much of the endocrine signallings and some ROS.

In aged mice and humans, the left ventricle walls are thicker, probably due to the fact that with decreased function, more muscle fibres are required to provide enough force of that part of the heart to pump blood to the rest of the body. Increased fibrosis is observed, similar to that seen in aged skeletal muscle. Other changes include vacuolization of the cytoplasm, variable and hypertrophic myocyte fibre size, disorganization of and collapse of sarcomeres, mineralization, arteriolosclerosis, mitochondria damage and permeability. In aged mice, the level of the mitochondria protein sarcoplasmic reticulum adenosine triphosphatase (SERC2) is decreased. SERC2 usually regulates calcium to ensure the heart cells are coupled in their contraction.

Stem cells in aged mice and humans shows classic signs of ageing - shorter telomeres, more apoptosis, lower turnover rate Arteries and capillaries are less flexible (due to crosslinked ECM) and more dilated), and are less able to regenerate (due to decreased levels of endothelial progenitor cells) Insulin and growth hormone are said to improve cardiac function, likely because it encourages fats and cholesterol to be taken up from the blood and metabolized, reducing hyperlipidemia and subsequent atherosclerosis-dependent cardiovascular diseases.

Brain
Nearly half of people over 85 have Alzheimer's disease. It is a disease (although more likely a syndrome) which leads to cognitive decline.

The aged brain, even without disease, appears to coordinate activity between different parts of the brain less well, and so the decline of cognitive function is systemic, and so the aged person have reduced higher-order cognitive function. This might be compensated by the remyelination of the brain, allowing different regions to connect and communicate with each other.

Also observed is that activity in the pre-frontal cortex (PFC) is reduced in older individuals than young individuals. Furthermore, higher-performing old adults have high activity only on the right PFC, whereas high-performing old adults are able to utilize both sides of the PFC, a feat not even found in young adults. It is thus believed that in the brains of high-performing old adults, although their cognitive function is decreased, they are able to remodel the neurocognitive network to utilize more of the brain.

Neurons require a lot of energy, especially dopamineuric neurons which act as pacemaker cells. However, it has been found that a decline mitochondria activity (reduced ETC and Mclk1 [for ubiquinone synthesis] ) might induces a stress response that protects the brain.

Parkinson's disease begins developing in the 40's, DJ-1, PINK (PTEN-induced putative kinase 1) and Parkin E3 ligase are involved.

Autophagy declines in the old human brain.

DNA repair
DNA damage theory of aging states that DNA damage caused by ROS, chemical reactions, radiation that leads to strand breaks, depurination, deamination and other DNA damage, ultimately is the cause of aging. Some DNA damage are more harmful than others; UV radiation induces cyclobutane pyrimidine dimers which are often ignored because it resembles normal DNA. These DNA damage causes dysregulation in transcription and alter cell function. It can also lead to cell cycle arrest and apoptosis; if this occurs in stem and progenitor cells, then the tissue cannot maintain homeostasis, leading to weakening and aging.

The cell has DNA repair mechanisms which repairs DNA and hypothetically delay aging. Single stranded breaks can be repaired either via base excision repair (BER) or nucleotide excision repair (NER). In BER, DNA glycosylate excises the mismatched base; 5' AP endonuclease then introduces a chain break by cleaving the phosphodiester bond at the cleavage site. dRpase removes the CH group to allow a new base to be added on using DNA polymerase and DNA ligase.

Autophagy
And ubiquitin-proteosome system (UPS)

Autophagy
Autophagy is the process where organelles and portions of the cytosol are sequestered into double-membraned autophagosomes, which is later merged with lysosomes or vacuoles, where the resident acid hydrolases will degrade the contents of the autophagosome.

Autophagy is required for the homeostasis of cell, where biosynthesis of new material is balanced with the degradation of old material. Autophagy is upregulated in unfavourable or stressful situations such as nutrient deprivation, cytosolic infection. It can be stimulated in the laboratory by using hormones as well as drugs.

The dis- or un-regulation of autophagy means this homeostasis maintenance and possible defence mechanism is malfunctioned, and can cause disease such as cancer, neurodegeneration, cardiovascular disorders and infectious diseases.

There are three types of autophagy - macroautophagy, micropinoautophagy and chaperone-mediated autophagy. In chaperone mediated autophagy, pentapeptide on the substrate is recognized and bound by hsc70 chaperone, which binds to the lysosome membrane, where the substrate unfolds and is bound by membrane-bound LAMP-2, which translocates the substrate into the lysosome to be degraded.

Autophagy can also be linked with aging. The levels of LAMP-2a decreases with age, and so dysfunctional proteins cannot be degraded as efficiently, preventing normal cell function if not causing cellular damage. In long-lived mutants of C. elegans, autophagy-related genes are induced; Drosophila with mutation in the adaptor protein Atg8 leads to reduced lifespan. On the contrary, overexpression of Atg8a in the nervous system increases lifespan by 56%

Modulators
Induction of Autophagy


 * Rheb inhibition by TSC1 and TSC2 	(Tuberous sclerosis) - lower activation of TOR and activates 	autophagy


 * Starvation (amino acids - TOR - or 	glucose - Ras/PKA)


 * Glucagon - increase blood sugar 	levels and decrease cell sugar level


 * (Hydroxychloroquine - upregulates 	Beclin-1 - also induces cell death)


 * PTEN (reverse PIP3 	production and decreases downstream PKB/Akt)


 * Lithium chloride (LiCl) - inhibits IMPase (inositol monophophatase), reduces inositol and Ins(1,4,5)P3 levels; mTOR-independent


 * Trehalose - a dissacharide; mTOR-independent


 * cAMP-reducing drugs (cloidine, Rilmenidine, adenylate cyclase inhibitors)

Inhibition of Autophagy


 * 3-Methyladenine - inhibits class 	III Phosphatidylinositol 3-kinases (PI-3K) - probably have 	non-specific effects


 * Bafilomycin - pH pump inhibitor


 * Monensin - inhibits protein 	transport to the Golgi


 * Chloroquine (CQ)


 * Hydroxychloroquine - upregulates 	Beclin-1 - also induces cell death


 * Lys01 	and Lys05 - deacidifies the lysosome

Mitochondrial damage
Histone deacetylase-6, an ubiquitin-binding deacetylase, is a central component of basal autophagy that target protein aggregates and damaged mitochondria, but dispensable in starvation-induced autophagy.

HDAC6 promotes autophagy by binding to ubiquitinated misfolded proteins using its intrinsic ubiquitin-binding BUZ finger, while using another domain to recruit acetylated cortactin. Once recruited, HDAC6 deacetylates cortactin leading to the formation of actin cytoskeleton network

HDAC6 controls the fusion of autophagosomes to lysosomes.

HDAC6 has an intrinsic ubiquitin-binding BUZ finger that binds to ubiquitinated misfolded proteins and transport them via microtubule networks to form aggresomes.

HDAC6 associates with both microtubules and F-actin

HDAC6 is a regulatory component of aggresome, the MTOC (microtubule-organizing centre)-localized inclusion body, where protein aggregates are depositied and processed by autophagy.

HDAC6 also plays a role in aggresome clearance

Nutrient Deprivation
Nutrient deprivation can be detected by at least two pathways - TOR and Ras-cAMP-PKA pathways - and causes an upregulation of autophagy.

TOR pathway
SLC1A5 (solute carrier family 1 member 5) and SLC7A5 are transporters which transport extracellular amino acids into the cell. Rag proteins (Ras-related small ATPases) senses the amino acids and, in response, transport TORC1 to a Rheb-containing subcellular compartment. Rheb (Ras homolog enriched in brain) is a GTP-binding protein and part of the Ras superfamily, Rheb activates (probably through phosphorylates) TOR.

Class III PtdIns3K (hVps34) have also been shown to activate mTOR. However, as you will see, Class III PtdIns3K (hVps34) is also required for autophagosome nucleation, and so appears to serve contradictory roles. A possible explanation for this is that hVps34 is localized at different cellular locations, where they have different functions.

As you will see, the TOR complex I (TORC1) down-regulates autophagy by phosphorylating Atg1 and Atg13, and thus preventing the Atg1-Atg13-Atg17 scaffold from forming. Furthermore, TORC1 also phosphorylates Tap42 and activates it. Tap42 have a PP2A (serine/threonine protein phosphatase 2A) catalytic subunit which down-regulates autophagy.

Ras/PKA pathway
Whereas the TOR pathway is known to sense the level of amino acids, the Ras/PKA (cAMP-dependent protein kinase A) signalling pathway senses the level of glucose. In yeast, PKA is made up of a heterotetramer of 3 catalytic subunits (Tpk1, Tpk2 and Tpk3) and 1 inhibitory/regulatory subunit (Bcy1).

When the levels of glucose is high, Ras1 and Ras2 GTPases activates adenylyl cyclase, which produces cAMP. cAMP binds to the Bcy1 regulatory subunit and stops its inhibition on the catalytic subunits of PKA. PKA is then activated and suppresses autophagy.

PKA also phosphorylates Atg1 in the presence of glucose, this causes Atg1 to move into the cytosol and away from the PAS, thus suppressing autophagy. When glucose levels are low, Atg1 is dephosphorylated and can localize at the PAS again. PKA and Ras are likely to regulate more autophagy proteins.

Hormones
Growth factors are required for the suppression of autophagy. Different organisms may have different methods of sensing for hormones and its downstream signalling, but all pathways have found to converge on TOR.

Insulin and insulin-like growth factors signals for high blood-sugar levels and encourages the uptake of sugars into the cell, during these high-nutrient conditions, autophagy should be suppressed. Insulin and insulin-like growth factors bind to the insulin receptor, which autophosphorylates on tyrosine residues and recruits IRS1 and IRS2 (insulin receptor substrate 1 and 2, respectively); this creates a docking site for adaptor proteins such as p85 of class I PtdIns3K. Activated class I PtdIns3K then induces the synthesis of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) on the lipid membrane. PIP3 forms a docking site for protein kinase B (PKB)/Akt and its activator phosphinositide-dependent protein kinase 1 (PDK1). PDK1 then activates (by phosphorylation) PKB/Akt.

PKB/Akt encourages the phosphorylation of the protein encoded by the tumour suppressor gene TSC2 (named because mutation in this gene causes tuberous sclerosis complex tumour syndrome. This phosphorylation means it cannot associate with TSC1 and form the TSC1/TSC2 complex. This complex is required for the inactivation of Rheb. So when insulin or insulin-like growth factors are present, the TSC1/TSC2 complex cannot form, and Rheb is in its GTP-bound active form. Active Rheb then activates TORC1 and suppresses autophagy.

PTEN (Phosphatase and tensin homolog), a 3’-phosphoinositide phosphatase reverses PIP3 production, and thus decreases downstream PKB/Akt signalling and de-suppresses autophagy.

Metabolic stress
During periods of metabolic stress, the ratio of ATP:AMP would be relatively low. This increase proportion of AMP is sensed by LKB1 kinase and transducted to the 5’-AMP-activated protein kinase (AMPK). Activated AMPK activates (by phosphorylation) the TSC1/TSC2 complex, which in turns inhibits TOR via Rheb, resulting in the de-repression of autophagy by the TOR pathway. Furthermore, AMPK also activates (by phosphorylation) p27kip1, a cyclin-dependent kinase inhibitor leading to cell cycle arrest. p27kip1 induces autophagy and inhibit apoptosis. The degraded products from autophagy are then used to fuel ATP production.

ER stress
The ER is a compartment of the cell responsible for facilitating the correct folding of newly-synthesized proteins as well as package them into vesicles destined for different cellular locations.

ER stress can be induced by the expression of aggregate-prone proteins, glucose deprivation leading to reduced glycosylation of proteins and decreased energy available for chaperons, hypoxia and oxidative stress leading to reduced disulphide bond formation.

In mammalian cells, the ER is also a reservoir for calcium ions, Ca2+, and so ER stress can also be induced by an efflux of Ca2+ from the ER. This increase in cytosolic calcium concentration simulates calmodulin-dependent kinase kinase-β (CaMKKβ), which further activates AMPK used to mediate the metabolic stress response. Furthermore, increased cytosolic calcium also activates (by phosphorylation) protein kinase Cθ (PKCθ) that induces LC3-I conversion to LC3-II, activating autophagy.

Thus reducing agents such as DTT, and glycosylation-inhibitors such as tunicamycin, plus other chemical stressors which prevents the correct folding of proteins, will induce autophagy.

Unfolded proteins also induces the unfolded protein responses (UPR). Grp78/BiP is a ER-specific heat shock protein of heat shock protein 70 (Hsp70) family; when Grp78/BiP senses an accumulation of unfolded proteins, itactivates the endoribonuclease domain of Ire1. Ire1 (inositol-requiring kinase 1) is a trans-ER-membrane protein with a stress-sensing domain at the luminal side, and an endoribonuclease domain at the cytosolic side. When the endoribonuclease domain of Ire1 is activated, it splices the RNA Hac1 (XBP1 in mammals). Hac1 encodes for a transcription factor involved in activating protein modification/folding, vesicle transport, phospholipid biosynthesis and ER-associated degradation (ERAD). UPR seems to be required for induction of autophagy, as the knockdown of Grp78/BiP using siRNA resulted in the inhibition of autophagosome formation. However, UPR is not responsible for the upregulation of ATG genes. Grp78/BiP knockdown does not affect soluble LC3-I’s conversion to lipid bound LC3-II, which is thought to be required for autophagosome formation; thus the UPR seem to promote autophagosomal membrane expansion, rather than induce its formation..

In mammalian cells, the UPR signalling pathways includes two additional distinct pathways: ATF6 (activating transcription factor 6) and PERK (RNA-dependent protein kinase-like ER kinase). Together with IRE1 (equivalent to yeast Ire1), these three distinct pathways senses the levels of misfolded proteins in the ER and trigger the transcription of different target genes. For example, the IRE1 pathway activates the transcription of c-Jun N-terminal kinase (JNK), essential for the conversion of LC3-I to LC3-II.

In addition, as found in murine cells, expression of misfolded expanded polyglutamine 72 repeat (polyQ72) aggregates or mutant dysferlin proteins lead to ER stress which led to the phosphorylation at Ser51 of eukaryotic initiation factor 2α (eIF2α) by PERK, a eIF2α kinase; this phosphorylation is required for LC3-I conversion to LC3-II. There is one such eIF2α kinase in yeast - Gcn2 - and four in mammals - GCN2, PKR, PERK, and HRI - activated by amino acid starvation, viral infection, ER stress and heme deprivation, respectively.

Hypoxia
Hypoxia is the condition where cells are exposed to <1% oxygen (as opposed to the usual 2-9%); it is the condition in which developing embryos and tumour cells are in. Cells respond to hypoxia primarily by activating the hypoxia-inducible factor-1 (HIF-1), a transcription factor which promotes hundreds of genes that promote erythropoiesis, angiogenesis, and decrease mitochondrial biogenesis and respiration, often inducing mitophagy via Bcl-2 adenovirus E1a nineteen kDa interacting protein 3 (BNIP3) and BNIP3-like protein (BNIP3L, a.k.a. NIX) activated by HIF-1. BNIP3 is a competitive binding partner to Bcl-2, thus able to displace Beclin 1, releasing its inhibition and allowing it to form the PtdIns3K complex and take part in mitophagy.

Oxidative Stress
Formation of reactive oxygen species (ROS) by agents such as hydrogen peroxide (H2O2), methoxyestradiol and any other agents which disrupt the electron transport chains, will induce autophagy. Normally, the levels of ROS is kept at a tolerable level using mechanisms such as superoxide dismutase (SOD), catalase and the redox system.

Pathogen Infection

Pathogens such as bacteria, viruses can induce autophagy. This sort of autophagy appears to be TOR-independent.

Immune cells have on their surface peptidoglycan-recognition protein (PGRP) receptor family which recognizes the peptidoglycans on the surface of bacteria, and activate the production of antimicrobial peptides.

Toll-like receptors (TLRs) are on the membrane of endosomes and the cell surface, they can recognize and bind to pathogen-associated molecular patterns (PAMPs) and activate a signal transduction pathway that leads to the upregulation of transcription of genes responsible for T cell activation, inflammation, and antiviral responses. The specific nature of the response depends on the type of PAMP and the type of TLR which recognizes it. For example, TLR7 binds to viral ssRNA and induces autophagy, TLR2 binds to zymosan and encourage the conversion of cytosolic LC3I to membrane-bound LC3II, and TLR4 binds to lipopolysaccharides (LPS) found on the cell walls of Gram-negative bacteria and triggers autophagy.

After TLR binding to its PAMP, the signal which triggers autophagy is transducted through downstream effectors; myeloid differentiation factor 88 (a.k.a. myeloid differentiation primary-response protein 88, MyD88) is a very common adaptor protein. Another one is the Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-β (TRIF), which is thought to be specific to TLR4, which recognizes LPS on Gram-negative bacteria. MyD88 and TRIF can, together, interact and bind with Beclin strongly when the TLR has bound its ligand; this removes Beclin 1 from Bcl-2 and allow the PtdIns3K complex to form, inducing autophagy.

Mycobacteria is represented by IFN-γ and IRGM1/LRG-47 to activate autophagy, whereas virus activates the eIF2 kinase pathway to activate autophagy.

Common Theme
Stimulation of autophagy by one stimulus can activate different pathways in different cell types, and can also lead to different consequences also. If cells are stimulated too much, it is likely that the cell will induce cell death by apoptosis and autophagy.

Mechanism
The process of autophagy is divided into induction, cargo recognition and selection (for selective autophagy), vesicle formation, autophagosome-vacuole/lysosome fusion and the release of breakdown products. These steps are mediated primarily by a set of autophagy-related (Atg) proteins; as of 2009, there has been more than 32 unique Atg proteins identified.

Induction
The basal level of autophagy is very low. It is only upregulated in response to intra- or extracellular stress (e.g. starvation, growth factor deprivation, ER stress, pathogen infection etc).

In yeast in nutrient-rich conditions, TOR (target of rapamycin, a serine/threonine kinase) integrates signals from multiple upstream signalling pathways and negatively regulate, by phosphorylation, Atg1 and Atg13. Atg1 is another serine/threonine kinase which when activated, have a higher affinity Atg13 and Atg17, which causes them to associate strongly with each other, forming a stable Atg1-Atg13-Atg17 scaffold complex which localizes at the phagophore assembly site (PAS) and recruits other Atg proteins to the PAS and initiates autophagosome formation.

The Unc-51-like kinase 1 (ULK1) and the Unc-51-like kinase 2 (ULK2) are both human homologs of Atg1. FIP200 (focal adhesion kinase family-interacting protein of 200kDa) is a homolog of Atg17. Atg13 retains the same name in mammals.

It is suggested that Atg1 (ULKs) autophosphorylates as well as phosphorylate Atg13 and Atg17 (FIP200). Phosphorylation causes a conformational change and induces autophagy. The phosphorylation of TOR and Atg1/ULK are on different residue, which gives opposite effects.

In mammals, an additional Atg protein, Atg101, is required to stabilize Atg13, and is required for autophagy.

Cargo recognition (in selective autophagy)
Macromolecules

There are two types of autophagy: bulk and selective. In bulk autophagy, a portion of the cytosol is sequestered, whereas in selective autophagy a specific receptor binds to a specific cargo.

An example of cargo recognition can be found in the cytoplasm-to-vacuole (Cvt) pathway of yeast. The Cvt pathway uses Atg19 to bind to a vacuolar-targetting signal on precursor aminopeptidase 1 (preApe1), a resident vacuolar protease. An adaptor protein, Atg11, then binds to Atg19-prApe1 as well as Atg8 (located at the PAS); prApe will then be packaged into Cvt vesicles (analogous to autophagosomes) and delivers it to the vacuole where it becomes the mature enzyme Ape1.

Ubiquitinated or aggregate-prone proteins can be selectively degraded through p62/sequestosome 1 (SQSTM1) in mammals and Ref(2)P in Drosophila. p62 bind to poly- or mono-ubiquitin of the protein to be degraded using its ubiquitin-associated (UBA) domain; p62 also binds to the Atg8 homolog, LC3 (microtubule-associated protein 1 light chain 3), at the PAS.

In C. elegans, P granule proteins are present only in germ cells and must be degraded by selective autophagy in somatic cells. SEPA-1 (Suppressor of ectopic P granule in autophagy mutants 1) interacts with P granules and LGG-1 (homolog of Atg8) and induces autophagy.

A common mechanism is observed, where a cargo binding protein (Atg19, p62 or SEPA) binds to the cargo and, either directly or indirectly, associates with Atg8 or its homologs and induces vesicle formation and eventual fusion with the vacuole/lysosome.

Organelles

Cargo degraded by autophagy is not limited to molecules alone, damaged and/or superfluous organelles are also degraded through autophagy. Different terms are used to refer to the different types of organelles being degraded: mitophagy (mitochondria), ribophagy (ribosome), pexophagy (Peroxisomes) and reticulophagy (ER).

Peroxisomes are required in large numbers in organisms which uses methanol as its sole carbon source, such as the yeast Pichia pastoris. When methanol is replaced with glucose or ethanol, the high quantities of peroxisomes are now obsolete and needs to be selectively degraded using the autophagy mechanism to delivery to the vacuole. PpAtg30 binds to both the peroxisomal membrane proteins PpPex14 and PpPex3, and autophagy proteins PpAtg11 and PpAtg16 at the PAS, to induce autophagy.

Autophagyosome Formation
Autophagosome formation appears unconventional in comparison with the typical vesicle formation; where as others appear to form from budding from a pre-existing membrane, or by sealing a continuous membrane, it appears new membrane are added to the PAS. The mechanism of this ‘addition’ is not clear.

What is clear is that many Atg proteins must be recruited to the phagophore for autophagosome formation; this recruitment is highly regulated.

The Class III phosphatidylinositol 3-kinase (PtdIns3K) complex is required for nucleation and assembly of phagophore membrane. The PtdIns3K complex consists of the PtdIns3K Vps34 (vacuolar protein sorting 34), a myristoylated serine/threonine kinase Vps15 (p150 in mammals), Atg14 (Darkor or mAtg14 in mammals) and Atg6/Vps30 (Beclin 1 in mammals).

The PtdIns3K complex produces PtdIns3P (phosphatidylinositol 3-phosphate) at the PAS, and PtdIns3P recruits Atg proteins which are able to bind to it, namely Atg18, Atg20, Atg21 and Atg24. Atg20 and Atg24 interacts with the Atg1-Atg13-Atg17 scaffold required for induction; this is observed in yeast but less well characterized for autophagy in mammals.

The PtdIns3K complex plus the associated Atg proteins further recruits Atg12-Atg5-Atg16 and Atg8-PE (Phosphatidylethanolamine), two interrelated ubiquitin-like (Ubl) conjugation system required for the elongation and expansion of the phagophore membrane.

Atg12 conjugation

The ubiquitin-like (Ubl) proteins Atg12 or Atg8 are added to the substrate protein in a similar mammber as ubiquitin conjugation. Atg12 is first activated by Atg7 (similar to E1 activating enzyme), then transferred on to Atg10 (E2 conjugating system) and then covalently attached to an internal lysine residue of Atg5. The Atg12-Atg5 complex then binds to Atg16, a coiled-coiled protein, which allows it to autotetramerize and attach to the phagophore. Atg12 conjugation is constitutive.

Atg8 conjugation

The C-terminal glycine on Atg8 is exposed after processing by Atg4, a cysteine protease. Atg7 (same as in the Atg12 conjugation system) activates Atg8 (LC3-II in mammals) and transfer Atg8 onto Atg3 (the E2 equivalent), and then conjugated onto PE via an amide bond, facilitated by an Atg12-Atg5 complex (E3 equivalent). Atg12-Atg5 complex lacks at the HECT and RING domains found conserved in E3 conjugating enzymes, and is not essential in conjugation.

As we have read, Atg8 binds indirectly to cargo and induces autophagy. In nutrient-rich conditions, Atg8 is mostly cytosolic; when autophagy is induced, Atg8 becomes lipid conjugated (with PE) and localizes on both sides of the phagophore membrane. There, it is theought to determine the membrane curvature and thus control the size of the autophagosome.

After the autophagosome is formed, and before or soon after autophagosome-lyososome fusion, Atg8 on the outer membrane is cleaved by Atg4 and returns to the cytosol.

Other Atg proteins

Atg9 is a transmembrane protein required for autophagosome formation, although its mechanism is unclear. It self-multimerize and binds to the Atg1-Atg13 scaffold. Atg11, Atg23 and Atg27 are essential for anterograde transport of vesicles, and Atg1-Atg13, Atg2, Atg18 and PtdIns3K complex are required for retrograde transport.

Regulation

Nucleation of membrane requires the whole PtdIns3K complex to form, if any part of the PtdIns3K complex is missing, autophagy would be down-regulated; this is a form of regulation. Regulation of phagophore formation lies with Bcl-2 (B-cell lymphoma/leukaemia 2), an antiapoptotic protein. In nutrient-rich conditions, Bcl-2 binds Beclin 1 and prevents it from forming the PtdIns3K complex and thus inhibits autophagy.

Autophagosome-lysosome fusion
In mammals, fusion requires the lysosomal membrane protein LAMP-2 and the small GTPase Rab7. In yeast, Ypt7 (homolog of Rab7), Sec18 (NSF homolog) and the SNARE proteins Vam3, Vam7, Vti1 and Ykt6, the class C Vps/HOPS complex proteins and Ccz1 and Mon1 are required for fusion.

Degradation
The phagosomes is double-membraned. The fusion event uses the outer membrane of the phagophore with the lysosomal membrane; this leaves the inner membrane still to be degraded. Degradation is mediated by a series of lysosomal/vacuolar acid hydrolases (including PEP4 and PRB1 gene products) and lipase Atg15 (in yeast) or cathepsin B, D and C in mammals.

The molecules derived from the degradation, such as amino acids, are transported to the cytosol to be reused. This is mediated by Atg22, Avt3, Avt4 and other vacuolar permeases and vacuolar amino acid effluxers.

Secretory and endocytic pathways. - (PROVIDE MEMBRANES?)
A functional ER and Golgi is required for autophagy and the Cvt pathway.

A subset of GTP exchange factors are required for biogenesis of autophagosomes, they include Sec12, Sec16, and Sec23/Sec24 heterodimer (components of the COPII coat complex). Biogenesis of autophagosomes and Cvt vesicles, two compartments involved in autophagy, have common aspects as well as unique aspects.

Cytoskeleton
Autophagosomes, as well as the required proteins required in autophagy, needs to be transported to the correct location; this is presumed to be carried out by the cytoskeleton network.

Atg9 is a transmembrane protein required for autophagosome formation. It needs to be anterograde transported to the PAS in order to function, and this transportation requires a functional actin cytoskeleton as well as Arp(actin-related protein) 2/3 complex, which nucleates branching of actin filaments.

Microtubules are required in higher eukaryotes. Nicodazole, a drug which depolymerises microtubules, inhibited autophagosome formation. Other microtubules-related proteins such as tubulin deacetylase HDAC6 and dynein are required for clearance of autophagic substrates.

It has been observed that autophagosomes in mammals are formed at random locations and then move towards the nucleus after its completion. Because microtubules extend from the nucleus to the cell periphery, it has been suggested that the completed autophagosome associates with microtubule tracks and move towards the nucleus using the dynein motor.

Transcription
The transcription of Atg8/LC3 is upregulated during starvation. Forkhead box transcription factor class O (FoxO3) is a transcriptional factor that binds to the promoter region of, and induces the expression of LC3B, Gabarapl1, atg12, atg4B, vps34, ulk2, beclin 1, Bnip3 and Bnip3l.

It makes sense that during the induction of autophagy, the synthesis of proteins should be arrested, this is achieved because transcription factors for genes involved in cell growth and proliferation, such as eIF4GI, also inhibits autophagy. In contrast, transcription factors which arrests protein synthesis, such as eukaryotic elongation factor-2 (eEF-2) kinase, positively regulate autophagy.

Insulin/IGF-1 signalling
The insulin/IGF-1 signalling pathways is the first to be discovered to have ties with lifespan. Insulin is a hormone that is secreted by the pancreas and signals to other cells that there is a high level of glucose in the blood, and that cells should take into glucose. Thus, insulin signals for an abundance of energy. Common across species (yeast, C. elegans, Drosophila, mammals), mutations that lower insulin/IGF-1 signalling leads to longer lifespan. Linking insulin/IGF-1 signalling with dietary restriction, if cells are told that the body is low on energy, they tend to live longer also. This could partly be due to the fact that insulin is also a stimuli that activates autophagy, and autophagy is able to degrade dysfunctional and superfluous cell components and organelles to ensure all the components in the cell are function, which prevents aging.

Calorie restriction by about 30% (not malnutrition, the body still receives a complete set of nutrients, just not excessive) improves lifespan. Excessive nutrients can cause insulin to be irresponsive, leading to obesity and other metabolic problems. A reduction in blood glucose level induces the transcription of stress response transcription factor genes. In yeast, glucose is sensed by the receptor Gpr1, which activates the Ras pathway leading to activation of PKA, which inhibit stress resistance transcription factor genes. The deletion of Ras2 leads to an doubles the chronological life span in yeast; Ras2 signals for the abundance of glucose, so its deletion mimics a signal for the lack of glucose. TOR is also another pathway that senses glucose, and its down regulation, by the same prinicples, also increases CLS and RLS. Alternatively, the TOR target Sch9 when knocked down, increase CLS by 3 times and increases RLS. Furthermore, a low glucose level will halt cell cycle progression.

In C. elegans, there are 37-38 insulin signalling family members, but only one insulin receptor - Daf-2. Daf-2 activation leads to PI3K activation, which generates PIP3 that activates downstream kinases. These kinases phosphorylates FOXO transcription factor DAF-16, which keeps it out of the nucleus and prevents it promoting any genes. Daf-16 is involved in inducing ROS scavenging proteins such as SOD2 and catalase, heat shock proteins that mediate protein breakdown and stop transcription, help switch to hypometabolic state - storing excess nutrients like fats and glycogen, ultimately leading to longevity. Thus the downregulation of the insulin signalling pathway is likely to lead to longevity without behavioral decline. Daf2 deletion mutants are not able to sense insulin, and the worms have 60-100% longer life.

In Drosophila, there are 7 Drosophila insulin-like peptides (DILPs) and one insulin-like receptor (InR). In a similar mechanism as yeast and C. elegans, the sensing of insulin by InR activates a set of kinases and their products including CHICO, P13K, PIP3 and PKB, which inhibit the the forkhead transcription factor dFOXO transcription factor through phosphorylation. dFOXO

When the insulin-producing cells are ablated, or when InR or CHICO is knockout, the lifespan of flies increased by more than 50%. Specifically mutating InR leads to more than 85% longer life, reduced cardiac ageing, higher storage of nutrients and upregulated SOD proteins; mutation of the receptor ligand CHICO lead to 48% longer life, which is less tha that of the InR mutation, suggesting that InR also transduce signals through other ligands apart from CHICO.

In mammals, there are three inslin receptors - IGF-1R, IR-1 and IRB - but all three pretty much share the same downstream signalling pathways. Activation of the insulin receptor leads to an activation of kinases, leading to an inhibition of FoxO transcription factors (FoxO1, 3, 4 and 6)

Organisms require energy in the form of glucose for them to grow, and yet glucose sensing seems to correlate negatively with anti-aging. Thus, an idea is that growth and anti-aging compete for a common effector, so a cell can either grow or keep away from senescence, but not both at the same time. The proposed common effect is β-catenin. β-catenin is able to form a complex with FOXO and promote FOXO targets, but it can also bind with TCF in the TCF/Lef complex to promote Wnt targets that can signal for growth and development.

However, these studies have to be treated with caution when applied to mammals, as mice which have their insulin receptor knocked out have much shorter lives; this is because the KO of IRec not only has the positive effect of allowing the FOXO trasncription factors to function, but also causes the cells to use alternate sources of energy even though glucose is in abundance. The break down of fatty acids and amino acids leads to an excessive keto acid level in the blood, causing the blood to be acidic, in a condition called ketoacidosis. More well known in humans, the disruption of insulin signalling leads to diabetes and cancer. Thus, it is clear that there must be a balance in the scale of insulin sensitivity that must be reached to prevent ageing, but not cause metabolic diseases.

Insulin/IGF-1 signalling might have different effects in different tissues. The knock out of Insulin receptor substrate 2 (IRS-2) in the brain of mice (using floxed IRS-2 crossed with nestin-Cre; nestin is a protein specifically expressed in nerve cells) extended lifespan. However, the increase is not dramatic, only 18%. . The insulin receptor gene FIRKO is knocked out in adipocytes in mice using the Cre-loxP system ; these knockouts shows 20% longer lifespan than controls, are leaner and showed more sensitivity to insulin.

Comparing the offspring of long-lived individuals with the offspring of normal-life individual might elucidate genetic factors that leads to longevity. In a study of Ashkenazi Jewish centenarians and their offspring, Suh identified insulin-like growth factor I receptor to be central to longevity. In yeasts, worms, flies, and mice, the loss-of-function mutations or knockout of insulin receptors or other components of the insulin/IGF pathway leads to a longer life. Female offsprings of these centurions have 35% higher serum IGF1 levels than normal, which is likely to be due a mutation in the IGF1 receptor. Thus, a reduction in insulin/IGF1 signalling might increase longevity. Other studies have also shown links between insulin/IGF signalling and longevity.

Growth hormones
When the pituitary gland is removed in mice, aging phenotypes decreased. The pituitary gland produces the growth hormone (GH), thus GH might act on cells directly to encourage aging, or it might cause insulin-like growth factor (Igf-1) to be produced and released from the liver, which works via paracrine signalling to signal for high glucose levels to neighbouring cells, leading to aging. In Ames dwarf mice, where the Prop1 mutation affects the pituitary gland differentiation, meaning are no GH or Igf-1 in the circulation, these dwarf mice live 49% longer in males and 68% longer in females. Likewise, Snell dwarf mice with their Pit-1 (downstream of Prop-1) mutation also have no GH and Igf-1 in their circulation and live longer. In Laron dwarf mice, the GH receptor/binding protein is knocked out, this results in longer life span (16% in males and 33% in females); in these mice, the level of GH in the plasma is still high, but there are almost no plasma Igf-1. Insulin and growth hormone signalling generally reduces metabolism, mimicking a nutrition-deprived scenario, leading the cells to induce stress responses. This might be a factor which allows for the prevention of aging. In humans, however, GH treatment during aging humans can prevent some aging symptoms, but do not increase lifespan.

Dietary restriction
Dietary restriction means a reduction of one or a few nutrient types. Generally reducing the amount of nutrients taken in is called calorie restruction (CR). Calorie restriction by about 30% (not malnutrition, the body still receives a complete set of nutrients, just not excessive) improves lifespan by up to 50%, when compared to mice which can eat when they want.

DR effect on lifespan might function via different pathways, including the forementioned insulin/Igf pathway, and/or the TOR pathway.

TOR signalling
TOR is an integration hub of many different signals. Mostly it senses the abundance of nutrients available to a cell, which includes amino acid levels as well as glucose levels. Inhibition of TOR (by RNAi)mimics a signal for malnutrition, and the cell will undergo a stress response, although maybe not using the same stress response genes as by the insulin pathway. TOR is thought to be key to DR, as TOR KO mutants are no longer affected by DR.

As an active TOR signals for an abundance of nutrients, cells with an active TOR will want to grow and divide. TOR upregulates the ribosomal subunit S6K and inhibits the translation of translation inhibitor 4E BP, leading to higher levels of translation. During dietary restriction, levels of translation is decreased, because the cell no longer needs new proteins to be made, but instead degrade dysfunctional and superfluous proteins for energy.

CR reduces mitochondria ROS production, because it forces the catabolism of fats and amino acids which bypasses complex I of the ETC, where much of the ROS is generated from. CR also encourages more new mitochondria to be made, a non-leaky mitochondria might prevent aging, although we known that some ROS leakage might be beneficial as it signals for a mild stress response.

CR trials in young, non-obeses humans shows the CR by 25% over six months led to upregulation of Sirt1, increased mitochondria numbers and reduced DNA damage markers in muscle. However, whether this is true for aging individuals or individuals with obesity or diabetes, is not clear.

Sirts
Sirtuins stands for silent information regulator proteins. They are highly conserved NAD+-dependent deacetylases. Sirtuins are required for calorie restriction-dependent increase in lifespan, possibly because when under DR, more NAD+ are present rather than NADH, which allows Sirtuins to carry out its function.

Sirtuins can directly deacetylate proteins including histones to remodel chromatin. Sirt1 deacetylate PGC-1a transcription factor, which is part of the pathway in mitochondriogenesis, and allows it to induce the transcription of mitochondrial genes.

The general function of Sirtuins is diverse. Sirt1 generall promotes survival of starvation, induces increase bile acid synthesis and fatty acid degradation, which encourage the breakdown of lipids; increase food intake and decrease leptin levels (leptin inhibits appetite). Sirt3, 4 and 5 functions in the mitochondria.

However, the effect of sirtuins on aging have to be dealt with caution, because not all strains are affected by differing levels of sirtuins.

In humans, calorie restriction induces Sirt1 expression and nuclear localization and these two are thought to be interrelated.

ER stress
The endoplasmic reticulum is the primary site for protein synthesis and maturation, for transmembrane, secretoary and ER-resident proteins. Proteins get 3 to 5 shots at folding correctly. Dysregulation of ER homeostasis, or simply the synthesis of proteins at very high levels, for example due to very high insulin-signalling, can lead to the accumulation of unfolded proteins (UPs). UPs can cause dysfunction or malfunctions in the cell because the proteins can either not function or have a detrimental function in the cell.

To get rid of these unfolded proteins using the unfolded protein response (UPR). The UPR includes different signalling pathways which notifies the cytoplasm and the nucleus of the accumulation of UPs. These signalling pathways leads to the transcription of UPR genes in the nucleus, attenuation of global protein synthesis in the cytoplasm, and the degradation of misfolded proteins via the process called endoplasmic-reticulum-associated protein degradation (ERAD). These are the three primary pathways in which a cell deals with ER stress. If these pathways prove to be insufficient in dealing with the ER stress, the cell undergoes apoptosis.

Three transducers are important in UPR - PERK, IRE1 and ATF6. All three proteins are transmembrane proteins which lumenal domain binds to a master chaperone protein called BiP (a.k.a. hsp70). BiP binds to these transducers and inhibit their function. Upon the accumulation of misfolded proteins, ADP-BiP binds to these UPs and releases the transducers. BiP binding to UP sequester the hydrophobic regions of the UPs to prevent them from aggregating. The released IRE1 and PERK can then homodimerize and activates themselves; and ATF6 can now be transported to the Golgi apparatus where it is cleaved to activate its transcription activation activity. All produce transcription factors when under ER stress which converge in the nucleus to activate transcription of UPR genes. Once the misfolded proteins are dealt with, ATP replaces ADP, which induces a conformational change in BiP, allowing it to bind to the transducers and inhibit their function once more.

ATF6 is translated on the ER membrane and remains at the ER membrane until ER stress, where ATF6α and ATF6β migrate to the Golgi apparatus where they are cleaved by S1P and S2P into fragments which are released into the cytosol. Once in the cytosol, they further migrate into the nucleus where they act as activating transcription factors for the UPR genes. ATF6 also induces XBP1 transcription, giving a positive feedback mechanism that promotes UPR gene transcription.

PERK is an ER transmembrane protein kinase which phosphorylates the α-subunit of translation initiation factor 2 (eIF2α) under ER stress. The phosphorylation of eIF2α reduces the formation of the translation initiation complexes, and so less AUG initiation codons are recognized, leading to global reduction in protein synthesis. This is compensated by the cell because mRNAs encoding for components of the initiation complex, such as the activating transcription factor ATF4, is enhanced. ATF4 also induce the transcription of GADD34, which protein products recruits protein phosphatase 1 (PP1) to dephosphorylates and deactivates eIF2α to inhibit translational attenuation. This is a negative feedback mechanism to ensure translation is not permanently switched off and is remains dynamic. IRE1 is an ER transmembrane glycoprotein which has both a kinase and RNase activity on the cytosolic domain. When the ER is stressed, IRE1 autophosphorylates which leads to activation of the RNase domain, which specifically splices the mRNA for XBP1 to make it a mature mRNA. The mature XBP1 mRNA can then be translated into a basic leucine-zipper-containing transcription factor, which downstream effects enter the nucleus and induce transcription of UPR genes.

The mammalian ER stress element (ERSE) is present in many but not all UPR target genes. ATF6 fragments, XBP1 and CAAT-binding factors all bind to ERSE to promote UPR gene transcription.

UPR genes includes molecular chaperones and folding catalysts, which provides a quicker response to misfolded proteins, reducing the overall number of misfolded proteins. UPR genes also includes genes that mediate ERAD; ERAD ubiquitinylate misfolded proteins and transport it to the proteosome to be degraded, so an increase in ubiquinyltransferases and in protein degradation complexes clears misfolded proteins from the ER. Protein disulfide-isomerase (PDI) levels are also increased; PDI catalyse the breakage and formation of disulfide bonds between cysteine as the protein fold, allowing them to break away when misfolded and provide them with another chance to fold properly. Calreticulin and calnexin are related proteins that are part of the ER chaperone system which binds to misfolded glycoproteins and prevents them from being exported from the endoplasmic reticulum to the Golgi apparatus. In yeast, phosporylated Ire1 also induces transcription of INO1, which encourages the biosynethesis of phospholipids, increasing the overall volume of the ER, giving more space of misfolded proteins to roam and prevents aggregation.

If these mechanisms prove insufficient, IRE1 can recruit Jun N-terminal inhibitory kinase (JIK) and TRAF2 to activate apoptosis-signalling kinase 1, and induces apoptosis. ASK1 activates JNK and mitochondria/Apaf1-dependent caspases. TRAF2 usually binds to procaspase-12 (pCASP-12), which when released clusters and activates CSP-12. CSP-12 activates CSP-9, which activates CSP-3, leading to apoptosis. CSP-7 is also activated under ER stress and cleaves CSP-12 to activate it also.

PERK and ATF4 induces expression of CHOP/GADD153, which is a transcription factor activating proappototic genes. The attenuation of protein synthesis under ER stress includes the attenuation of cyclin D1, which is required for G1→S phase transition; its attenuation leads to cell cycle arrest, and the induction of apoptosis.

DNA damage
DNA damage theory of aging is a solid model because DNA damage accumulates as we age, and so there is a strong logical hypothesis backed by strong correlation. Furthermore, other types of cellular damage can be reversed, whereas mutations in the DNA are permanent. It has emerged recently that DNA damage and its repair process can lead to epigenetic changes, leading to alterations of gene transcription, leading to expression of genes related to aging.

ROS
ROS can cause formation of 8-oxo-guanine from guanine which can pair with cysteine and adenine, causing G to T and A to C substitutions.

HDAC
Histone deaceylases might act to prevent the formation of double-strand breaks (DSBs), as HDAC class I knock down (via RNAi or inhibition) led to an increase in DSB formation, and subsequently higher neuronal death and neurodegeneration. HDAC class III (Sirt1 and Sirt6) are also required for DSB repair; Sirt6-/- mice do not repair oxidatively damaged bases and the chromosomes can fuse end to end. In humans, Sirt6 can recruit Werner to telomeres to unwind and repair DNA and maintain telomeres. The Sirt6-/- mice die soon after weaning (changing from an infant diet to the adult diet) and show signs of progeroid diseases such as lower bone density, curved spine, lymphopenia (lack of white blood cells) and severe metabolic defects.

Telomere
Telomeres are TTAGGG repeats that are repeated over 10 kb and caps the end of chromosomes. This is to ensure that the whole genome is replicated and there is nothing left at the end, because DNA polymerase is not able to replicate the last sequences of DNA. Telomeres are maintained by telomerase, a ribonucleoprotein complex involving an RNA template (TERC) and a reverse transcriptase subunit (TERT). As telomerase are only required for cells which will remain in the body, it is not expressed in somatic cells but only in germline, stem and progenitor cells. The decline of telomerase expression in somatic cell can prevent tumours from forming and gaining immortality. Telomerase extends the telomere repeats and to ensure the telomere length is kept long to prevent gene deletions.

In TERC null mice, premature aging, poor organ maintenance, high cell turnover rate, and dysfunctional telomeres are observed. Exo1 is a nuclease involved in mismatch repair, DSB repair, stalled replication fork processing and in the DNA damage response triggered by dysfunctional telomeres. When it senses an uncapped telomere, it will synthesize ssDNA at the ends and this signals for cell cycle arrest. When Exo1 is knocked out, this sensing mechanism is no longer present, and DNA damage is not noticed, meaning the cell will progress through the cell cycle even though it is not fit.

Overexpression of TERT and tumour suppressor genes leads to mice having longer (26% more) lifespan. TERT and the tumour suppressor work against each other to promote and inhibit proliferation, effectively forming a buffer to which the cell can be maintained at. This encourages homeostasis and leads to longevity.

Another aspect of telomeres is that the subtelomere regions are methylated by DNA methyltransferases, the knockdown of which leads to recombination of telomeres.

Congestive heart failure patients have shorter telomeres and telomere length may be associated with the severity of CHF symptoms.

Telomeres and stem cells
A low telomerase activity in primitive haematopoietic stem cells saw shortening of telomeres when the stem cells proliferated. CD34+/CD38+ cells had higher telomerase activity than CD34+/CD38- and CD34- cells. HSCs grown in the presence of kit ligand ( KL), interleukin-3 (IL-3}, IL-6, erythropoietin and granulocyte colony-stimulating factor resulted in the upregulation of telomerase.

Progeroid syndromes (PSs)
Progeroid syndromes (PSs) is the name of a group of disorders that mimic aging at an early age. Some disorders shows hallmarks of aging, whereas others do not; it can affect one organ or many. Segmental progeroid syndromes are PSs which do not associate with all aspects of physiological aging; examples include Werner syndrome, Hutchinson-Gilford progeria, ataxia telangiectasia, dyskeratosis congenita and Bloom syndrome. Other PSs affects only one organ, and includes familial Alzheimer's disease, familial Parkinson's disease.

PS can be separated into sub-groups corresponding to the cause of the disorder, which includes mutations in the genes encoding for DNA repair, usually DNA helicase, and genes affecting the structure or post-translational maturation of lamin A, a nuclear intermediate filament constituting large parts of the nuclear lamina.

Different segmental progerias have suggested that maintaining a stable genome and nuclear environment is essential for keeping away from senescence. However, questions arise as to whether these Progeroid syndromes really represents normal aging.

Werner Syndrome
Werner syndrome is a very rare autosomal recessive disorder. WS clinical signs include altered distribution of subcutaneous fat, juvenile bilateral cataracts, a mask-like face and bird-like nose, trophic ulcers of the feet, diabetes mellitus, and premature atherosclerosis. The habitus is characteristic, with short stature, stocky trunk and slender extremities. WS frequency has been roughly estimated to be 1: 100,000 in Japan and 1: 1,000,000-1: 10,000,000 outside of Japan.

WS is caused by mutations in the WRN gene, which is located on the short arm of chromosome 8 (locus 8p12-p11.2) and encodes for a RecQ DNA helicase with 3' to 5' exonuclease activity (used to unwind and repair DNA, recombination and maintenance of telomeres). The mutation occurs between residues 949-1092, and causes the helicase to lose its nuclear localization signal (NLS) , which means it cannot bind to DNA to repair it. This results in mis-recombination and telomere shortening; ultimately, the cells will try to safeguard themselves by entering prematurely into senescence. Patients do not show signs of aging until puberty and late adolescence, when they'd stop growing and show premature aging. Symptoms includes cataracts, grey hair, osteoporosis, cancer, artherosclerosis, poor glucose regulation, skin atrophy and myocardial infarction. The median lifespan of a WS patient is 47-48 years old, and they usually die from myocardiac infarction and/or cancer. Fertility is greatly reduced for individuals with Werner syndrome, possibly due to accelerated loss of priomordial follicles in the ovaries, and testicular atrophy. There is currently no cure for this disease.

A mouse model with the Werner protein knocked out do not show degerenative properties.

Hutchinson-Gilford Progeria Syndrome (HGPS)
Hutchinson-Gilford Progeria is similar to Werner syndrome, only that it manifests usually before 12 months to 2 years of age. Patients have very thin skin, lose subcutaneous fat, alopecia (loss of body hair), stiff joints and osteoporosis. The median lifespan is 13 years, and patients usually die by stroke or heart attack, although cancer is not very prevalent.

HGPS is caused by a dominant mutation in lamin A which causes 50 amino acids to be incorrectly spliced out, and causes the progerin protein to remain farnesylated. Lamin A is the major constituent of the nuclear lamina, and its mutation leads to a distorted nucleus. A dysfunctional nuclear lamina may lead to a lack of β-catenin entering the nucleus, because it can no longer interact with the nuclear pore. This leads to reduced Lef1 activity, which reduces Wnt signalling. The lack of Wnt signalling leads to reduced ECM production. Fibroblasts require ECM to grow on, and thus the lack of ECM leads to a halt in the proliferation of fibroblast and this may be fatal. This also explains by HGPS patients have very thin skin and blood vessels, and also skeletal problems.

HGPS cells in culture have reduced lifespan, its nucleus looks distorted, have less heterochromatin and many binuclear cells exist. The chromosomes have shortened telomeres, as well as aggregation of telomeres. Chronic DNA-damage response where progerin signals to p53 and Rb to cause early cell senescence. Gene transcription are modified and DNA repair is reduced in HGPS cells.

Ataxia telangiectasia (A-T)
Ataxia telangiectasia (A-T) is another rare autosomal segmental progeroid synrome. A-T is caused by mitations in the ATM gene, a kinase that phosphorylates proteins involved in DNA damage signalling, DNA repair and telomere maintenance. A-T cells exhibits premature senescence, genome instability and telomere shortening. Symptoms includes progressive cerebellar degeneration (ataxia), skin abnormalities and immunodeficiency.

A mouse model with the ATM knocked out do not show degerenative properties.

Dyskeratosis congenita (DC)
Dyskeratosis congenita (DC) is a rare hereditary disorder usually present in males. It is caused by mutations in 6 DC genes which are normally involved in telomere maintenance; its mutation leads to genomic instability. Symptoms of DC includes cancer, grey hair and osteoporosis at an early age, and affects the mucous membranes, teeth, nails, skin pigmentation. The disease state of DC can be studied using iPSCs, to give an in vitro human model of the disease. When DC fibroblasts are transfected with OCT4, SOX2, KLF4 and c-MYC, the resulting iPSCs have longer telomeres, due to the induction of telomerase reverse transcriptase gene (TERT). The RNA component (TERC) were upregulated in these reprogrammed iPSCs. When these iPSCs differentiate, trasncription of TERT is silenced and the RNA component is deleted at the 3' end; the telomeres get shorter once more.

Cockayne syndrome (CS)
Cockayne syndrome (CS) is a rare segmental progeroid syndrome. CS is caused by a mutation in the Cockayne syndrome complementation group B (CSB) gene, which lies within the CSA gene. CSB encodes for a DNA-dependent ATPase, which dimer is wrapped around by DNA; CSA encodes for a component of a ubiquitin ligase complex. Their mutation means they cannot repair oxidation-induced damage to the DNA, and thus any mutations during replication is likely to remain. It also stalls the RNA Pol II as the TCR which normally removes the stall RNA Pol II can no longer perform its function, as a co-factor (CSB) is missing. Mutations in the CSA gene can be as detrimental, as CSB needs to be ubiquinated (by CSA) for repair to occur. This means CS patients are sensitve to DNA damage especially UV, and thus must remain in doors away from UV as much as possible. CS leads to progressive neurological degeneration, hearing loss and cataracts. The mean life span is 12 years.

Mitotic spindle assembly checkpoint (SAC)
During mitosis, sister chromatids attach to the spindle to be pulled apart into different cells. SAC effectors includes BubR1 and Bub3, of which older mice express less BubR1. BubR1 and Bub3 knock down leads to premature aging.

ROS
Because reactive oxygen species (ROS) can damage cell components, especially the mitochondria, it was initially thought to contribute to aging. This theory is known as the mitochondria free radical theory of ageing (MFRTA); this has now proven not be entirely true (though not false), and ROS is now viewed more as a signal for stress for the cell to respond to.

ROS can cause damage to the cell by oxidizing amino acids and co-factors, polyunsaturated fatty acids (peroxidation) - ROS removes hydrogens from the fatty acid chain, leaving the tail as a free radical; this free radical will bond with other fatty acid side chains and merge into one. Often these are seen as spots in the liver. Lysosomes can digest these damaged lipids, and the debris left over is known as lipofuscin granules, yellow-brown in colour.

Long lived mutant models of C. elegans have mutations in the isp-1 (component of complex III) and/or clk-1 (coenzyme for complex I and II), which dampens the electron transport chain, increases lifespan. Furthermore, RNAi screens which reduced different components of the ETC also resulted in longer life span. On the contrary, mev-1 (encodes for complex II subunit) activating mutant worms shows higher ROS and a shorter lifespan. However, these studies must be treated with caution as a short lifespan might not be due to aging, but due to pathology; and a longer lifespan does not mean the worms do not age, it might just mean that they are less prone to pathogens.

Damage to DNA by ROS is kept to a low level in a organism because in each cell there are DNA repair mechanisms (such as BER, NER, NHEJ). Furthermore, there are checkpoints in the cell cycle which notcies an stalled or damaged replication fork or double stranded break, and leads to cell cycle arrest and apoptosis. This is known as the DNA damage theory of ageing.

Most ROS are generated in the mitochondria, where nutrients are oxidized to produce energy. Electrons uses its energy to pump protons across from the matrix to the inter membrane space while they flow down the electron transport chain, carried by NADH and FADH2. The proton gradient is subsequently used to generate ATP.

The electrons, and oxygen represent in the mitochondria can generate superoxide radicals (•O2-), which can leak into the cytoplasm and cause damage. There are ROS scavenging systems which prevents the superoxides from causing damage; superoxide dismutase (SOD) onverts •O2- to H2O2, which is then deactivated by catalase or glutathione peroxidase or peroxiredoxins. There are two types of SODs - SOD1 works in the mitochondrial matrix, and SOD2 works in the cytosol. Other non-enzymatic ROS scavengers includes ascorbate, urate, glutathione, tocopherols, flavonoids, carotenoids, ubiquinol.

ROS causes the most damage inside the mitochondria, where it is derived from. MtDNA is located near the membrane, and thus is in close proximity to complex I and III of the ETC system, where most ROS are being genereated. ROS creates many lesions on the mtRNA such as oxidation of the bases, abasic sites, and single-/double-stranded breaks. 8-oxoG is a very common lesion used as a marker for oxidative stress. The mitochondria have DNA repair mechanisms to perform base excision repair (BER), mismatch repair etc.

The mitochondria is constantly producing ROS, and the scavenger systems are constantly on, and homeostasis sees a balance between the two. The scavenger system becomes less efficient with age and so the amount of ROS in a cell is likely to increase with age. ROS damage does increase with age, but no consensus can be reached as to whether the ROS damage causes ageing.

If ROS damage causes aging, then the removal of ROS should induce longevity, this is the case in invertebrates but not in mice, where over-expression of SOD1, SOD2 and catalase did not prolong the lives of mice. GPx4 is gene encoding for a mitochondria antioxidant enzyme that reduces membrane-bound lipid hydroperoxidases, and so if MFRTA is correct, its knockout should result in more oxidative damage and thus shorter lifespan. On the contrary, mice with GPx4 KO on one chromosome had a longer lifespan. Further evidence comes from the fact that long-lived animals can have high levels of ROS, such as the naked mole rat.

The Ames Dwarf mouse carries a recessive mutation in the Prop1 gene (Prop1df) which when present in two copies leads to a dwarf phenotype but ~40% longer lifespan. Likewise, the Snell Dwarf carries a recessive mutation in the Pit 1 gene and homozygotes are a-third of the heterozygotes' size, but exhibit a longer life span. This is correlated with a higher mitochondria ROS cardiovascular system. However, probably due to their small size, both the Ames and Snell Dwarf mice have trouble in maintaining body temperature.

Blockage of the mitochondria and the reduction of ETC actually leads to more mitochondria being synthesized, which is mediated by the PGC-1alpha-NRF1-TFAM pathway. This increase in mitochondria biogenesis and turnover reduces aging, possibly because more dysfunctional mitochondria are being degraded. In aging individuals, the number and turnover of mitochondria decreases possibly due to an impaired PGC-1alpha-NRF1-TFAM pathway.

The PGC-1alpha-NRF1-TFAM pathway can also be activated to induce mitochondriogenesis in young animals but not in older animals. , thus exercise might increase lifespan. Speculatively speaking, the increased mitochondria numbers also increases the amount of ROS, if aging is prevented by increase ROS, then one should not give antioxidants to athletes after exercise.

ROS also signals to release NF-κB, which is a transcription factor involved in regulating immune and inflammatory responses, developmental processes, cellular growth, and apoptosis genes.

These evidence suggests that there should be a balance in ROS level to prevent cellular damage but also delay aging. Mitochondria activity can be measured in vitro by biopsying tissue samples and uses luciferase to measure mt ATP production. Luciferase will oxidize luciferin to give a fluorescent product; this reaction is ATP-dependent and can be measured using a luminometer. The level of fluorescence correlates with mitochondria activity.

Metabolism
There is an observable trend that larger animals tend to live longer; they also seem to have a slower metabolic rate. This led to the notion that the aging process is sped up with increased rates of metabolism. This is known as the rate of living theory.

Reproduction
Reproduction drives aging, reproducible in C. elegans and other models.

Model organisms of aging
The choice of model organism is often a compromise between its resemblance to human aging, and the time frame and cost associated with the model organism. Yeast replicate very quickly, is easy to grow and its cheap, but factors and pathways identified in yeast might not be relevant in humans. Rodents are more closely related, but their it takes them 18 months to 4 years for them to be considered age; this brings considerable costs and requires a much longer time frame for experiments.

Chronological life span is defined as the time cells in a stationary phase culture remain viable

Aging yeast
The budding yeast Saccharomyces cerevisiae is a simple, cheap and easily genetically manipulated model organism. It is a good model organism because it has many orthologues in humans, and these can be easily knocked out or down.

Aged yeast are much larger in size, with closer cell cycle progression and protein synthesis, the surface of the yeast is loose and wrinkled.

Dietary restriction (reduce glucoase and amino acid) is thought to increase lifespan.

In a genome-wide screen of gene mutations found in long-living yeast strains reveals TOR (target of rapamycin) kinase. TOR inhibits autophagy and its inhibition increases the replicative life span as well as the chronological life span. TOR is upregulated during high nutrient environment, and so dietary restriction might inhibit TOR and promote longevity. TOR acts with other nutrient sensing kinases, such as Sch9 and PKA. Furthermore, when TOR is inhibited, stress response factors enter the nucleus, resist oxidative and temperature stresses. TOR promotes the transctiption of ribosomal proteins and rRNA process factors; when the cell is starved, TOR signalling is reduced and this decreases ribosome biogenesis and translation, allowing the cell to utilize its resources to remain alive without growth.

Sirtuins, or Sir2 (silent mating-type information regulation 2) family of protein deacetylases, prevents the recombination of rDNA and formation of extrachromosomal circles. Its over expression can lead to longer lifespan.

Caenorhabditis elegans
Caenorhabditis elegans is a well-characterized model organism, with its whole genome sequenced. The adult is transparent and have 959 somatic cells in the body. It is about 1mm in length and is cheap to maintain. The lifespan of C. elegans is short (12-20 days) and an aged sample can be taken at around day 18. It has orthalogues with humans but is missing major signalling pathways such as Shh.

Its aging phenotypes include muscle atrophy (sarcopenia), reduced skin elasticity and vulnerability to infections.

Genes can be knocked down in C. elegans by feeding them with bacteria expressing double stranded RNA of the target gene, which knocks down the gene via RNA interference. From these studies, the lifespan of C. elegans seem be be controlled by more than 300 genes, some of which are involved in insulin-like and germline signalling.

In C. elegans, if germline primordial cells are removed, C. elegans live 60% longer. The germline primordial cell population is small but can affect the whole organism.

Naked mole rat (Heterocephalus glaber)
The naked mole rat is extremely long-lived for a rodent of its size, exceeding 30 years. The naked mole rat do not shows signs of aging, and remains fertile throughout its adult life. They are resistant to cancers, both spontaneous and induced. Although the level of oxidative stress and ROS in the naked mole rat is high, they do not show any signs of damage to the proteome.

Drosophila
Dietary restriction and low fecundity (ability to reproduce) extend the life span of Drosophila. Similar to C. elegans, reducting insulin or IGF signalling 50-80 days

Rodents
18 months to 4 years

HSPG zebrafish
Zebrafish with lamin A/C KO exhibit muscular dystrophy and craniofacial abnormalities.

Heart
Human heart diseases are the leading cause of adult and childhood mortality. Diseases such as ischemia, inflammatory diseases, fatty plaques, infarction, developmental defects, can all lead to the death of the myocardium, which further triggers inflammation, fibrosis and ECM production, leading further to contractile dysfunction.

The heart, along with neurons and pancreas, are one of the hardest organs to regenerate; teleost fish are best at regenerating, followed by moderate regeneration in urodeles, and negligible regeneration in mammals. Cardiomyocytes in mammals withdraw from the cell cycle during late gestation, and will not enter the cell cycle again apart from after injury, and even then, the cardiomyocytes will not mitose. Consequently, an injured heart in mammals is 'healed' by scarring, and do not regain its full function.

One of the factors preventing cardiomyocyte's entry into the cell cycle is the active retinoblastoma (Rb) protein, preventing G1 to S phase transition. Thus, using an inhibitor of Rb, such as cyclin D2, will enable the myocardiocytes to enter back into the cell cycle, and improve cardiac function after injury. To ensure the inhibition is limited to the myocardiocytes and not other tissues, which may cause cancer by uncontrolled proliferation, cyclin D2 would be expressed using the cardiac-specific α-cardiac myosin heavy chain promoter.

However, simply re-entering the cell cycle might not be enough because although the cell mass can be regained, some might not be of the right cell type for that location. Dedifferentiation might be required.

Alternatively, exogenous stem cells can be introduced to support the heart to regenerate itself. Skeletal myoblasts, haematopoietic stem cells, mesenchymal stem cells and circulating endothelial progenitors may support cardiac cells, through paracrine signalling, to regenerate itself; exogenous stem cells do not integrate into the heart itself, or dedifferentiate and integrate into the heart, it simply provides support for the heart to regenerate itself. This eliminates the problems that can arise from graft survival and immune rejection.

isl1+ (a LIM-homeodomain TF) cells in rat, mouse and human myocardium are shown to be able to develop into mature cardiac cells which display myocytic markers in the absence of cell fusion, intact Ca2+-cycling, and the generation of action potentials, and contributes to the right ventricle, both atria, the outflow trac and regions of the left ventricle. Thus, isl1+ cells are progenitor cells which can be transplanted into hearts damaged by injury, or through aging, to regenerate by integrating into the heart. Thymosin β4 has been shown to induces adult embryonic epicardial progenitor cells (EPDCs) to form vascular precursors for neovascularization, and thus will induce any existing progenitor cells to proliferate. After this priming, the heart is injured. The epicardium is observed (using Wiln's tumor 1 (Wt1) as the marker) to regenerate the whole heart by triggering endogenous stem cells. Thus the epicardium (source of the whole heart) can be used to repair the whole heart.

Lung
As the lung has a large surface area and thus a large contact surface to the outside, it is prone to bacterial and viral infections and allergies. This can lead to inflammation, physical trauma (from coughing too much), cancer and idiopathic pulmonary fibrosis (IPF), resulting in a damaged lung.

The normal response to injury includes inflammation caused by activation of macrophages and neutrophils, the secretion of growth factors, cytokines, interleukins and matrix components. Other immune cells are recruited and nearby epithelial cells and fibroblasts secrete ECM and/or divides. These cells then secretes MMP, which breaks down the extracellular matrix to free the cells from the basal lamina to migrate up to close the wound. This type of injury response often leads to fibrosis, meaning that the lung is no longer as effective as before. However, selective apoptosis allows local tissues to remodel.

Growth factors secreted includes EGFs (stimulates mitosis, migration, spreading), HGF (stimulates proliferation, migration), KGF (aid repair), chemokines such as CCR3 (stimulates repair), interluekins such as IL-2 (stimulates migration and reduces apoptosis), and eicosanoid PGE2.

Cells from outside the lung may also contribute to the regeneration, including bone marrow cells.

Endogenous stem cells is likely to be present in the lung; it can be marked by ATP-binding cassette transporter breast cancer resistance protein (Bcrp) 1, which is a highly expressed in multiple stem cell populations including haematopoietic stem cells, and decreases its level by subsequent maturation.

iPSs

Human lung epithelial cell line and endothelial cells can be grown in 3D culture (matrigel), which mimics embryonic lung development, and is observed to form branching structures with alveoli-like structures at the end of branches. Thus, bioartificial tissues can be grow in culture and transplanted into the damaged lung.

Circulating stem and progenitor cells can home in on lung damage and integrate and differentiate. This process is highly inefficient, with very few cells that get there surviving. It is probably more effective by supporting the local cell populations to regenerate itself by secreting factors that act via the paracrine pathway. Other acts of support includes contributing to the ECM (such as producing collagen) during fibrosis, suppress proinflammatory response, induce angiogenesis and recruit and activate T-cells.

Mesenchymal stem cells can potentially be used for repair damaged lung, because it can home in on the injury, do no elicit an immune response as it is low on HLAI/II, co-stimulatory molecules, and can change cytokine secretion by immune cells.

The lung is an expandable organ which expands when inhaling, thus it is no surprise to see thatmechanical forces can influence lung regeneration. If the lung tissue is grown on rubber and this is stretched periodically (to mimic breathing) and have fluid flowed over it (to mimic blood flow), then it might change the cell biology in terms of cytoskeleton remodelling, adhesion, and generation of forces by the cell, and locomotion, possibly through altering gene expression. When primary cultures of human bronchial epithelial cells are grown on rubber and stretched, it stops migration loses focal adhesion but promotes division.

The lung houses more than 40 cell types and different structures exists within the lung.

Different cells can be seeded on to a structure resembling the functional structure in terms of shape and constitution, and grown into a tissue which can then be implanted directly into the patient.

Trachea is removed from an deceased donor, washed with chemicals to remove all cellular components that can cause an immune response, such as cells and MHC molecules, leaving only non-cellular components as the 'scaffold'. The scaffold is then coated with epithelial cells and stem-cell-derived chondrocytes from the patient to be treated; the cells proliferate and differentiate until the whole cellular population is regenerated. The regenerated trachea can then be transplanted into the patient. The patient did not elicit an immune response and did not require immunosuppresants.

It is now possible to create an artificial windpipe from synthetic material, without the need of a donor. The synthetic scaffold is made and seeded with stem cells as with the 'natural' scaffold, but because it is synthetic, there is almost zero chance of it being rejected by the host.

The whole of an adult rat lung can be decellularized, but leaving the ECM intact resulting uin an acellular matrix scaffold. pulmonary epithelium and vascular endothelium cells are coated on top, to regenerate the whole lung. The mechanical properties of the engineered lung was similar to that of a native lung in vitro, and when transplanted into rats for a short period of time (45 to 120 minutes), the engineered lung was able to undergo gas exchange, without any visible air leaks. Note that the cells were cultured in a bioreactor which passes fluid to mimic blood flow and stretches to mimic breathing. This same technique has been repeated in human lung segments using A549 human epithelial carcinoma cells and human endothelial cells from cord blood progenitors. The A549 cells adhered well to the alveolar surfaces while the endothelial cells adhered to the vasculature, although the engineered lung is not as complete as the native lung. This technique has subsequently been achieved with the heart, liver and kidney in non-human models.

Actual implantation of the whole lung has not been achieved with humans, but have with adult Rhesus, an non-human primate model.

Brain
Neurogenesis in the brain of mammals are restricted to two telencephalic constitutively active zones - the dentate gyrus of the hippocampus and from the subventricular zone of the lateral ventricle, the rostral migratory stream to the olfactory bulb. This is despite the presence of neural stem cells and ongoing neurogenesis at the forementioned parts of the brain. After injury (stab wound, ischemia or amyloid deposits), non of the neurogenic factors such as Pax6, Mash1, Ngn2 was detected, but bHLH transcription factor Olig2 was expressed. Antagonizing Olig2 using a dominant negative retrotransposed Olig2, shows that its suppression led to neurogenesis of immature neurons. Thus, if we block Olig2 after injury it might allow for neurogenesis to occur.

Finger tip
The finger tip (distal to the last joint of fingers) is the only part of the mature mammalian limb that is able to regenerate, including skeleton and blood vessels). The finger tip is thought to be able to be regrow from the nail bed and thus the amputation needs to be within the nail organ. The nail tip is usually shorter than the original, and thus is not perfect regeneration, but it does have minimal scarring. Regeneration depends on the expression of Msx1, which is expressed by mesenchymal cells in the nail bed; but Msx1 do not mark a pluripotent stem cell population. Msx1 and Msx2 are turned on transiently during post-natal regeneration, and BMP4+ cells are found throughout the blastema. Different stuides also supports the notion at these cells are not homogenous pluripotent stem cells, as was first thought with blastema, but rather a pool of resident heterogenous progenitor cells.

Skin
The regenerative abilities of skin lies at the junction between the dermis and the epidermis, at a layer termed the basal layer. When the epidermis is injured, keratinocytes from this area, as well as from bulge and sebaceous stem cells population, proliferate and migrate to reconstitute the lost cells. When the injury is deeper and the dermis is damaged, neighbouring stem and progenitor cells might be able to proliferate into the wounded area and reconstitute the lost cells. But if the wound is large, then the center of the wound would have lost its regenerative ability, and the wound now heals by contraction, fibrogenesis leading to scarring.

The immune system is likely to play a part in skin regeneration because nude mice, which have no immune system, regenerate better. Human embryos also do not scar during the first third of gestation, at a time when inflammatory cells are not fully differentiated. Fibrin clots formation is reduced and increased platelet degranulation, which leads to the release of growth factors aiding regeneration, and chemoattractants leading to recruitment of macrophages to clean the wound. TGF-β signalling is also important in scarring.

Fibroblasts are often seen as detrimental to good regeneration, as fibroblasts are signs of fibrosis and scarring.

Bioscaffold and tissue engineering
Organs and tissues do not normally grow in two dimensions, as it is in a culture flask; they grow in three-dimensions and receive three dimensional signals. Bioscaffolds are artificial 3D structures which are implanted into the body for which cells and tissues can grow on. Bioscaffolds have the advantage of not inducing an immune response, is non-toxic (even break down products), is biodegradable so that eventually the tissue will contain only host-derived material (at the moment the life of a bioscaffold is 3 weeks), and be able to chemically and physically integrate with the endogenous system. With the onset of electrospinning and 3D printers, bioartificial tissues are becoming a more practical reality. Electrospinning, peptide self-assembly and biomineralisation allows the scaffold to mimic conditions found within tissues in vivo.

Collagen is a common material used for bioscaffolds. Collagen is the most common component of the ECM, both in soft and hard tissues. It is dynamic, mobile and flexible, but is weak, and so unprocessed collagen cannot be used to scaffold tissues which sustains large forces. Collagen is made stronger by plaiting, where collagen fibrils are electrohemically aligned in a process similar to isoelectric focusing used in chromatography techniques, giving electrochemically aligned collagen (ELAC) threads.

However, the scaffold should not be viewed as a static platform, it can also be used as a source of cell signalling, for example using integrins. One can seed integrin in a certain pattern on the scaffold to engineer different cell types to grow on it. Other signalling molecules can also be seeded at different locations. Thus, a good scaffold must support cell adhesion, proliferation and differentiation, through the signals the scaffold gives to the cells. Nanoparticles such as growth and differentiation factors can be integrated into the nanostructured scaffolds to aid in the regulation of cell behavior for optimal tissue regeneration.

The scaffold should be viewed as an interim extracellular matrix which will be gradually replaced as the seeded cells produce their own ECM.

Skin
Skin can be engineered in a dish and used to cover acute and chronic wounds. It is non-toxic (even break down products), non-immunogenic (as it can be derived fromt he patient's own cells), no excessive inflammation, and can be treated so that it is free from disease. However, this is often not enough because wounds and joints are acidic; this means that the engineered skin might not last very long. The bioengineered skin must also provide pain relief, prevent fluid and heat loss as well as form a tight physical barrier to prevent infections.

Future aims of bioengineered skin is to allow nerves and blood vessels to quickly integrate into the skin. Electronic skin is an important next step in generating prosthetic skin, as well as skin that allows AI robots to interpret human sensations. Mannsfield have developed a polydimethylsiloxane-based 'skin' covered with structured arrays of pixel-sized high-sensitive pressure sensors, which can ultimately be modified into electric skin. Most artificial skin are grow from allogenic cells onto a scaffold mimicking the dermis. Alpligraf supplies artificial skin with two layers - the bottom layer is composed of bovine type 1 collagen and human fibroblasts, mimicking the dermis, and the upper layer consists of human keratinocytes (epidermis). These layers deliver signals such as ECM components, cytokines, growth factors, inc.: interferons a & b, PDGF, interleukins 1, 6 & 8. However, these grafts do not contain melanocytes, Langerhans' cells, macrophages, and lymphocytes, or other structures such as blood vessels, hair follicles or sweat glands, and so are usually temporary, often requireing autologous epithelial cells.

The use of autologous cells reduces the risk of rejection but requires a second surgery site, adding pain, risk and possible longer aftercare. A 2-5cm2 section of skin is biopsied from the patient, the epidermis is separated from the dermis. Karatinocytes from the epidermis is released and expanded in culture, optionally using mitotically inactivated mouse fibroblasts and serum as support cells, The cultured cells can then be seeded onto the scaffold. Alternatively, the cells can be injected back into the wound site and allowed to reform the tissue in vivo.

Laserskin cultures autologous cells on a scaffold made out of hyaluronic acid polymer (part of the ECM) which is laser-perforated, allowing cells to migrate into the pores and create a skin layer with thickness and is structured. OrCel is a film of skin derived from the dermal fibroblasts and keratinocytes of neonatal foreskin. The fibroblasts are seeded on bovine type I collagen sponge to mimic the dermis, and then a non-porous collagen gel coating separates the confluent keratinocytes lying on top. These cells mimic the structure of the skin and is able to produce FGF, KGF, PDGF, VEGF, TGFa.

Other materials can potentially be used to form the scaffold, such as kertain-collagen, hyaluronan-fibronectin, polyurethane microfibers, collagen, silk and cellulose. Furthermore, using only fibroblasts and keratinocytes to mimic the skin layers do not allow for innervation, and formation of hair follicles. Thus, stem cells might need to be introduced to form these cells.

But at the moment, artificial skin can only act temporarily, to keep the wound clean and support the patient's own cells to heal itself.

Bone
The bone and cartilage are highly structured both at the nano and macro scale. The outermost layer of the bone is biocalcified. Inside the bone are round cylindrical structures termed osteons (a.k.a. Haversian systems), each with its own blood supply. Osteocytes lie in the osteons and is surrounded by aligned collagen I fibres. Osteocytes uses the collagen fibers as a scaffold and forms hydroxyapatite crystals on the junction between two adjacent collagen fibers. The cartilage does not have blood vessels and contains chondrocytes spaced 10–200 μm apart. Hydrophilic proteoglycans, such as aggrecan, form an interwoven network between collagen II fibres, providing a shock-absorbing matrix.

Cartilage is non-vascularized, and do not have the ability to self-renew; cartilage damage is thus the leading cause of joint pains. The bone contains different types of cells - osteoblasts (form bone), osteoclast (digest bones to release calcium), osteocytes (maintain bones) as well as resident macrophages, adiopocytes etc. The extracellular matrix is made up mostly of collagen fibers, with minerals in the form of hydroxyapatite crystals, as well as liquids containing factors.

Osteoporosis is often caused by over-digestion of bone tissues, or the lack of bone formation, and is a common condition found in the elderly; osteosarcoma is a bone-derived tumour often initiated during the rapid growth phase of bones during puberty and adolescence.

Regeneration of the bone requires putting forces on the cells, and the type of bone depends on the forces exerted on it; bone will develop abnormally if the wrong forces are applied.

Using artificial bone ECM such as ceramic and glass is not ideal because it can grind against endogenous tissues and cause injury; furthermore, body fluids can be corrosive and degrade away the metal nanoparticles, which can be toxic.

An alternate approach uses mesenchymal stem cells which will migrate to the site of the lack of osteogenesis, it will interact with epithelial cells and aggregate to fill in the sites, and differentiate into the osteogenic lineage. BMP is involved in signalling. Proteins engineered to contain a C-terminal cysteine can be chemisorpt onto gold. The scaffold can be coated with gold and be layered with osteopontin and BMP-2, promoting adhesion and preading of primary rat osteoblasts. BMP alone was able to maintain bone formation for 28 days without other osteogenic stimuli.

Decellularized organ matrix
The liver can be striped with detergent of all cellular components, leaving the ECM and other structures intact. Then cells are seeded onto the structure and allow it to recellularize the graft. The engineered liver shows albumin secretion, urea synthesis and cytochrome P450 expression similar to a normal liver.

Brain
Under normal physiological conditions, the distribution of active neurogenic niches in the red spotted newt and mammalian brain is highly similar, and so the regenerative properties of the newt brain cannot be wholly attributed to the enhanced regenerative abilities of newts. The newt brain can also only regenerate at certain areas, namely the forebrain, with other parts of the brain remaining in quiescence. However, ablation of the midbrain dopamine neurons induces hedgehog signalling of ependymoglia cells (in midbrain) to proliferate and and regenerate the dopaminergic neurons. Thus, neurons can be regenerated by activating neurogenesis at quiescent brain regions; furthermore, dopaminergic neurons regeneration can be done to help patients with Parkinson's disease.

Heart
The heart may also be regenerated in a method not dependent on endogenous stem cells. The human heart consists of cardiomyocytes, vascular cells, but the majority of the heart is made up of cardiac fibroblasts, which are connective cells with no cardiogenic potential. It is now, however, possible to reprogram these post-natal cardiac fibroblasts into cardiomyocytes in a process similar to the generation of iPS cells, using the developmental factors Gata4, Mef2c, and Tbx5. The resulting induced cardiomyocytes (iCMs) have a gene expression profile similar to that of endogenous cardiomyocytes, and contracted spontaneously in vitro, spontaneous Ca2+ flux and electrical activity similar to neonatal cardiomyocytes. It is unclear however the steps taken to go from fibroblast to myocardiocytes, or how well the iCMs complement the host heart in terms of electric signals and rhythm.

Interesting questions
What if you segregate the cells of an urodele, what factors are required for them to regenerate?

What changes (if any) are there to the lengths of telomeres after regeneration? Maybe this will provide clues as to the age of regenerated tissues - if it shortens, it means the regenerated tissues have aged post-regeneration; if it lengthens, it means that the tissue have dedifferentiated back to the embryonic state.