Mitochondrial integrity and antioxidative enzyme efficiency in fischer rats effects of ageing and epigallocatechin 3 gallate intervention 1 2

1 Introduction 1.1 Definition of aging Aging is traditionally regarded as the process of becoming older A broader definition of aging is offered in the "Handbook of the Biology of Aging" [1], to encompass the process of system's deterioration with time Sometimes, the term senescence is used to describe cellular aging, where normal diploid differentiated cells lose the ability to divide This phenomenon is also known as "replicative senescence", the "Hayflick phenomenon", or the Hayflick limit When referring to the organismal senescence, which is the aging of the whole organism, the term “aging” is always used interchangeably with “senescence” Aging does not result from diseases and it is not necessarily related with diseases, however aging increases the risk of disease happening Several typical changes occur with age including presbyopia, cataracts, loss of the ability to hear and taste, reduced body weight, muscle strength, thymus mass, reduced sensitivity to growth factors and hormones, decline of reaction times, and increased pathological conditions and disabilities Other serious degenerative diseases might also develop such as prostatitis, osteoporosis, diabetes, cancer, atherosclerosis, heart disease, Alzheimer's disease, Parkinson's disease, etc Under very rare abnormal circumstances, people suffer from certain accelerated aging diseases such as Werner's syndrome, Cockayne syndrome, Hutchinson-Gilford Progeria syndrome and so on Such symptoms are sometimes used as models to study the mechanisms of aging Aging and senescence has become a globalized issue Recently, the aging population has been growing at a considerably faster rate than that of the world’s total population, particularly in the developed countries where the aging population occupies a larger proportion of the total population, although the aged group is growing more rapidly in the less developed regions It has been pointed out that “Population ageing is unprecedented, pervasive, enduring and has profound implications for many facets of human life.” in a report prepared by the Population Division, the United Nation With the increasing aging population in society, study the mechanisms of aging and age-related diseases/disorders has become an intriguing research area 1.2 Mechanisms of aging In the process of exploring the mechanisms of aging, more than 300 theories have been proposed However, it's important to note, many of the theories of aging are not mutually exclusive, but intrinsically related and sometimes supportive of each other 1.2.1 The free radical theory of aging The free radial theory was first proposed by Denham Harman in 1956 [2] According to this theory, the reactive oxygen species (ROS) which includes superoxide radicals, hydrogen peroxide, and hydroxyl radicals, is capable of causing oxidative damages to many biological macromolecules such as DNA, RNA, proteins and lipids [3] The accumulation of such oxidative damages in the cells and tissues is probably a direct cause of aging (More details will be discussed in the following sections.) 1.2.2 The mitochondrial theory of aging Later in 1972, Dr Harman developed the ‘mitochondrial theory of aging’ based on the free radial theory of aging, where he specifically pointed out the sideeffects of mitochondrial respiration as the main cause of ROS formation [4] Meanwhile, mitochondria are susceptible to ROS attack due to their close approximation to the ROS producing site on the mitochondrial electron transport chain (ETC) and dysfunctional mitochondria in turn cause more leakage of ROS, thus forming a vicious cycle Moreover, the mitochondrial theory of aging was further refined and developed by Dr Miquel in 1980 where some additional attention was given to mitochondrial genetics, membranes, and bioenergetics besides the ROS production [5] (More details will be discussed in the following section.) 1.2.3 The cross-linking theory of aging In the aging process, proteins are damaged by both free-radicals and glycation Glycation also known as Maillard reaction, or non-enzymatic glycosylation, is a reaction in which reducing sugars such as glucose and fructose bind to free amino groups in proteins The glycated proteins also known as Amadori product then react with other proteins, finally resulting in the irreversible 'cross-linking' [6] The representative reaction between glucose and a lysine amino acid in a protein molecule is demonstrated as follows (Figure 1) The cross-linked proteins become impaired and are unable to function efficiently However, the cross-linkages inhibit the activity of proteases from breaking down the damaged proteins which accumulate in the tissue and cause a series of problems [6] The glycated proteins inhibit cellular transport processes, stimulate cells to produce more free radicals, and activate pro-inflammatory cytokines such as Tumor Necrosis Factor alpha (TNF-α) and interleukin [7] In addition, some glycated proteins are immunogenic or mutagenic, whereas others reduce cell proliferation and induce apoptosis, resulting in excessive loss of cells and further contributing to the risk of degeneration [8] The known cross-linking disorders include diabetics, age-related cataracts, renal disorders, cardiac enlargement, the hardening of collagen and so on [8] Figure Cross-linking between glucose and a lysine amino acid in a protein molecule 1.2.4 The immunological theory of aging "Immunosenescence" was first used by Dr Roy Walford to describe the ageassociated immune deficiency, and such concept was later developed into the immunological theory of aging [9] Briefly, he hypothesized that many aging effects are related to the declining ability of the immune system, especially the function of T-cells in the aging process due to the decay of the thymus gland It is known that the effectiveness of the immune system peaks at puberty and gradually declines thereafter with advance in age As a result, immune system becomes less capable of resisting infection and cancer, so that a variety of infectious and non infectious diseases, such as arthritis, psoriasis and other autoimmune diseases become more prominent with age 1.2.5 The telomere theory of aging The telomere theory of aging suggests that cell death is caused by the shortening of telomeres Telomeres are sequences of nucleic acids extending from the ends of chromosomes and they shorten with each cell division It is believed that telomere is a biological clock that decides aging because the number of times that cells can divide is limited by the length of telomere For some species there is a correlation between maximum lifespan and the number of fibroblast doublings for that species [10] However, adverse evidence comes from the observation of mice, which have very long telomeres and show no reduction of telomere length with age [11], but most mouse cells stop dividing after only 10−15 doublings Only a few cells that rapidly proliferate such as endothelial cells or immune system cells show decreased function with age that could be associated with telomere shortening, other post-mitotic cells like neurons and muscle cells survive but never divide Thus the aging in fruit flies or nematodes that comprised entirely of post-mitotic cells is hardly relevant to telomeres 1.2.6 The wear and tear theory of aging The wear and tear theory of aging considers aging as the effect of progressive accumulation of damages to biomacromolecule due to radiation, chemical toxins, metal ions, free-radicals, hydrolysis, glycation and disulfide-bond cross-linking Such damages can affect genes, proteins, cell membranes and enzyme functions The organisms have limited capacity to repair damages so that when the total damages accumulate and finally reach a critical level, the cells become senescent 1.2.7 Longevity genes It is also believed that aging can be regulated at the gene expression level In the process of studying the mechanisms of aging, investigators have identified numerous promising regulatory pathways and longevity genes in model animals of flies, worms and mice On top of the longevity genes, it is probably the well characterized genetic regulatory networks that involve the function of insulin/insulin-like growth factor (IGF)-1 signaling axis Lifespan regulation by IGF-1 signaling appears to be evolutionarily conserved over a wide range of organisms including yeast, fruit fly, nematode as well as mouse [12] In the C elegans for example, the binding of insulin-like molecules to the IGF-1 receptor DAF-2 initiates a cascade of protein kinases, including AGE-1, DAF-2/AGE-1, AKT-1, AKT-2, etc., and the IGF-1 level is proved to be reversely correlated with the lifespan of the worms Some of the confirmed longevity genes from model animals are listed in Table S cerevisiae C elegans D melanogaster M musculus lag1 daf-2 sod1 prop-1 lac1 age-1/daf-23 cat1 p66shc ras2 akt-1/akt-2 ras1 phb1 daf-16 daf-18 phb2 daf-12 mth cdc7 ctl-1 mclk1 bud1 old-1 rtg2 spe-26 rpd3 clk-1 hda1 mev-1 sir2 sir4-42 uth4 ygl023 sgs1 rad52 fob1 Table The identified longevity genes in the S cerevisiae, C elegans, D melanogaster and M musculus Saccharomyces cerevisiae, bakers' yeast; Caenorhabditis elegans, the soil roundworm; Drosophila melanogaster, the fruit fly; and Mus musculus, the mouse 1.3 The free radial theory of aging Among the great diversity of the aging theories, the free radical theory of aging is one of the most popular and strongly supported theories Considering that the current project is based on the free radical theory of aging and the closely related mitochondrial theory of aging, relevant concepts will be introduced with more details in this section and the following section 1.4 to clarify the respective theories 1.3.1 Free radicals Free radicals are the molecules that carry an impaired electron All free radicals are extremely reactive and are capable of catching an electron from other molecules, starting a chain reaction of free radical formation The main free radicals are superoxide radical (O2·-), hydroxyl radical (OH·), hydroperoxyl radical (HOO·), alkoxyl radical (LO·), peroxyl radical (LOO·) and nitric oxide radical (NO·) [13] Other molecules that are technically not free radicals, but act much like them or are readily converted into free radicals, are singlet oxygen (1O2), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl) [13] Collectively, the free radicals and non-free radical mimics that contain oxygen are called reactive oxygen species (ROS) Free radicals are able to damage virtually all biomolecules, including proteins, sugars, fatty acids and nucleic acids [3] However, free radicals are extremely short-lived because of their extreme reactivity [14] With the help of metal ions, free radicals are converted to H2O2 that can diffuse to the cellular membrane and mitochondrial membrane and oxidize the biomacromolecurs there Table shows the main ROS formation in biological systems ROS Formula Characteristics Hydroperoxyl HOO· Strong oxidant, lipid soluble Hydroperoxide LOOH Half-life time# Low reactivity, forms per/alkoxyl radicals in presence of transiton metal ions Peroxyl LOO· Low oxidant, lipid radical Ten millisecond Alkoxyl anion LO· Intermediate oxidant, lipid radical One microsecond Superoxide O2·- Good reductant, poor oxidant One microsecond OH· Extremely reactive, low diffusion One nanosecond Nitric oxide NO· Weak oxidant, no diffusion Few seconds Peroxynitrite ONOO- Product of NO·and O2·-, very strong anion Hydroxyl radical oxidant, no diffusion, very reactive Hydrogen H2O2 peroxide Singlet oxygen Hypochlorous acid Oxidant, high diffusion, low Stable reactivity O2 Strong oxidant, short half life One microsecond HOCl Found in neutrophil phagosomes, stable strong oxidant Table The major ROS formation in biological systems #, half-life time is determined at 37ºC (Table modified from the study of Abuja P.M and Albertini R 2001 [15] and Robert A., 1995 [14]) 10 software with the default settings as advised by Illumina Data analysis was performed using Genespring GX 7.3 software (Agilent Technologies, Santa Clara, CA) Normalization was performed according to Per Chip and Per Gene: Median polishing method After using filters to control low signal interference, volcano plot was used to identify the genes, which had at least 1.5-fold change and p value less than 0.05 The above selected genes were classified into various functional categories referred to as "Gene Ontology" 3.3.13 Statistics Statistical analysis of results was performed using one-way ANOVA with subsequent post hoc multiple comparison by Tukey’s method, and p value less than 0.05 was considered significant All statistical calculations were done with the SPSS 14.0 computer program Main results 4.1 Part one: Age-related changes in mitochondrial function and antioxidative enzyme activity in Fischer 344 rats (Paper I) 4.1.1 Introduction The free radical theory of aging reveals ROS such as superoxide radicals, hydrogen peroxide, and hydroxyl radicals as being capable of causing oxidative damages to many biological macromolecules [3, 133] There is strong evidence to link the unfavorable accumulation of ROS and the resulting oxidative damages with the aging process and human diseases [97, 134] In general, the majority of 64 ROS are released as by-products of respiration from the ETC located on the mitochondrial inner membrane Due to the close proximity of ROS generation site, mitochondria become the first potential targets of ROS attack Genetic changes at mitochondrial DNA that encodes a number of subunits of the ETC complexes [48, 135] result in the impairment of ETC activity and concomitantly cause even greater leaking of ROS, leading to the formation of a vicious cycle [136] RCR is an index of ETC activity, indicating the coupling between respiration and oxidative phosphorylation Since ETC is well coupled in intact cells of normal tissues, a decreased RCR is always related to mitochondrial malfunction The variations in the mitochondrial respiratory function and ETC activity have been reported in aging studies, although not many [45, 137] Furthermore, mitochondrial membrane is also subjected to oxidative damage, which results in a decreased mitochondrial membrane potential Such a mitochondrial dysfunction is a principal underlying event in aging [41] The age-related decrease in mitochondrial trans-membrane potential was demonstrated in different organs of several species [17, 138] The antioxidant system plays a vital role in the protection of cells against oxidative stress in aerobic organisms Both SOD1 and SOD2 convert O2·- to H2O2, which is then decomposed to water by CAT and GPx Thus, factors that deteriorate the antioxidative enzyme activity tend to result in the accumulation of ROS [139] In some circumstances, the oxidative stress up-regulates antioxidative enzyme activity through various signaling pathways [56], whereas the excessive production of ROS is also able to decrease the efficiency of the antioxidant system [139] Several studies focus on the changes of antioxidative enzyme activity in 65 different organs and species in the process of aging, although there have been no conclusive results so far Total SOD activity decreased in relation to age in the kidney, brain and heart of rats and mice, specifically senescence-accelerated mice [111, 140] CAT activity was found to have an age-related decrease in the liver and kidney tissues from mice [141] Besides, a higher activity of SOD was observed in the cerebellum of aged rats [142], but in another study, both SOD and CAT activities were not altered in the old rats compared with young rats [143] The decreased GPx activity in the kidney was associated with increasing aging [144] However, higher levels of GPx activity were also found in the kidney, heart and testis of 19 months Sprague-Dawley rats and in the brain of 20 months Wistar rats [143] Thus, it is still not clear how aging affects the antioxidative enzyme activity The antioxidant enzymatic efficiency can be accounted for from two levels; one is the protein activity level which affects their antioxidant activities in a unit amount of protein The second level is the protein abundance level, which is also important to determine the overall antioxidant capacity [145], although these emphases have been missed unfortunately in most of the studies In the present study, we intend to emphasize the exact concept of determining the final antioxidant capacity by combining both protein expression and its activity levels Moreover, to date, there is no such research directly linking the changes in mitochondrial respiratory activity and mitochondrial membrane potential with the efficiency of antioxidant defense system in the aging process Therefore, we are interested in comparing the mitochondrial functional integrity that is one of the vital determinants of ROS accumulation in the major organs of young and old Fischer 344 rats The above findings were then related to the efficiency of 66 antioxidant defense system at both their enzyme activity and protein expression levels Thus, the present study suggests that the concurrent malfunctions of mitochondria and antioxidant enzymes most likely result in aging 4.1.2 Results Mitochondrial integrity by confocal microscopy In order to evaluate the effects of aging on the mitochondrial function, the mitochondrial membrane potential was determined using mitochondrial specific fluorescent cationic dye, Rho 123 Intact membrane correlates with a higher transmembrane potential and stronger fluorescent signal The fluorescent signal from the liver revealed that mitochondria in young rats (Figure 10A) gave stronger signal than that in the old rats (Figure 10B), which indicates more intact mitochondria in the young rats Similarly, the fluorescent signal was also stronger in the brain of young rats 67 Figure 10 Fluorescence image demonstrating mitochondrial membrane potential from liver of young and old rats Freshly prepared liver mitochondrial suspension from months rat (A) and 26 months rat (B) were incubated with µM Rhodamine123, mounted on slides and viewed under Zeiss laser confocal microscope with 63 x magnification Bar represents µm 68 Mitochondrial membrane potential by flow cytometry In order to determine mitochondrial membrane potential, the fluorescent signal of the Rho123-stained samples was quantified using flow cytometry Compared to the young rats, the old rats displayed significantly lower mitochondrial membrane potential in the liver Interestingly, it was observed that the membrane potential in the brain was much higher than that in the liver, whereas there was only a slight decreased potential (without significance) in the brain of old rats compared to the young rats (Figure 11) Mitochondrial membrane integrity by RCR test The RCR is positively correlated with the tightness of the coupling between mitochondria respiration and oxidative phosphorylation It is one of the indices used to evaluate mitochondrial membrane integrity The RCR decreased significantly in the liver of old rats (Figure 12), which means that the mitochondria are not well-coupled and the mitochondrial membrane loses its integrity during aging However, there was no detectable difference in the RCR values in the brain of young and old rats 69 Figure 11 Mitochondrial membrane potential in the liver and brain of young and old rats Median fluorescence intensity (MFI) was used as an indicator of the mitochondrial potential Data were expressed as mean ± SD, n=9 for the young and n=8 for the old, *p