Biologic basis of Longevity

Researches Biologic Basis of Longevity

The Biologic basis for longevity A Study on Stem cells, Telomerase and Gene Expression following IA

Prof .Dr.Krishna Shama Rao MBBS, FRCS ( Ed ), MDS, FDSRCS(Eng) Director, Clinical Research and Translational Medicine, Nithyananda University Bangalore

Its All In The Music

• In MY FAIR LADY, Prof.Higgins asks:

• Why can't a Woman be more like a Man? • When it comes to longevity, the question should be the other way round.

Is Defying Time, Defying Age

• Masoro defines ageing as deteriorating changes with time during post maturational life that under lie ,increasing vulnerability to challenge, thereby , decreasing the ability of the organism to survive.

What Is Time?

• Although it is customary to view ageing as a time dependent process, time itself runs on different clocks,

• For e.g: • Biological • Circadian • light dependent etc.

Deleting Time

• It is important therefore to remove time from the analysis of ageing

• Now we come to Time independent processes of physiology and physiological ageing, such as, Neuro endocrine mechanism and Gene expression changes etc.

• Thus ageing has a significant molecular signature and is not tied to a clock or a calendar .

Defining Age

• Ageing is a time independent series of cumulative, progressive , intrinsic, and dilitereous functional and structural changes culminating in Death.

Ageing Body Or Ageing Cell ?

• The other terminology one needs to define is Senescence, which includes all the processes that bring about changes in an organism's Gene expression patterns or other physiological bio markers known to be related to health and somatic maintainence.

Can One live forever?

• In other words are there any non ageing systems in the universe?

• At a cosmological level , the universe is supposed to have a mathematically finite time.At a subatomic level, most particles decay and are reborn

• However, there are some subatomic particles that possibly exist as time independent phenomena.

• It is therefore possible that time independent ageing systems do or can exist in the universe.

Is The Whole A Sum Of The Parts ?

• At the level of an organism, like a human being, life span and cell death are often not related as several cells have extremely short life spans and some continue to exist until the death of the entire organism.

Genotype Versus Phenotype

• In fact, even within the same species one cannot generalise the ageing process, and there are a huge number of confounding variables which could be either intrinsic e.g: Sex or extrinsic e.g : environmental signals, both of which contribute to the plasticity of the longevity phenotype.

Gender Bias in Longevity

• Over the past centuries , longevity in both Men and Women has increased significantly

• Though the overall life span is different in different countries, there is still a gender gap in life expectancy

• For example, even in the U.S, half of all women older than 65 are widow’s, and they out number the men by 3:1

• By age 85, there are only 38 men for every 100 women.

Fecundity and longevity- relationship between reproduction and life span

• Estrogen versus Testosterone:

• Male fetuses are more likely to miscarry than females • Estrogen reduces LDL and increases HDL • Present data on testosterone is incomplete regarding its effect on the cardiovascular system

• Life style differences in terms of smoking and alcohol can be contributory

• Finnish studies linked Iron levels to longevity • Though obesity is common, visceral fat is higher in men

Men Are From Mars

• In other words:

• Macho • Alcoholic • Reclining • Smokers

Molecular Basis of Gender Bias

• Role of estrogen receptors:Mitochondria contain estrogen receptors which exerts a protective effect on the cell

• Estrogen - Estrogen receptor MAP Kinase phosphorylation -NF-kB activation - up regulation of anti oxidant genes.

Molecular Bio markers in Ageing

• Estrogen • Mitochondria • Signalling pathways • Up regulation of beneficial genes • Down regulation of deleterious genes • Telomere and Telomerase • Stem cells

Mitochondria

• Mitochondria are essentially the energy producing batteries of the cell

• Mitochondria is inherited from the X gene

• Mitochondrial DNA is naked in contrast to nuclear DNA , which is protected by Histones

• Thus , Mitochondrial DNA is more susceptible to damage leading to production of ROS, which in turn , damages the cell

• Mitochondria have estrogen receptors, which are protective indirectly by activating antioxidant pathways.

Mitochondria assay Study 1

• Mitochondria assay was done on IA Participants in Dec 2010 • The technique used was the MTT Assay, and was performed at Department of Biotechnology, St Alloycious College, Mangalore

• This was a quantitative analysis of Mitochondria • There was an increase of Mitochondria by about 1000% , at the end of the IA Program.

• It is well known that damaged Mitochondria leads to ageing and disease

• Following these unexpected results, further tests were initiated to study the effects of IA, on a molecular level of Gene Expression, signaling pathways of the body, stem cells and the Telomerase enzyme, which preserves the Chromosome, allowing cell division to continue , thus preventing or delaying disease and death

Signaling Pathways

• p38 signaling mediated by MAPKAP kinases[19] • P38 MAPK signaling pathway[20-25 • Validate targets of C-MYC transcriptional activation[26] • VEGFR1 specific signal[27-28] • TNF alpha/NF-kB[29-31] • TGFBR[32] • Hypoxic and oxygen homeostasis regulation of HIF-1-alpha[33-36]

• Endogenous TLR signaling[37] • Direct Interactions[38-39] • Cellular roles of Anthrax toxin[40]

Signaling Pathways

• C-MYC pathway[41] • BMP receptor signaling[42-43] • Androgen Receptor[44-46] • Androgen-Mediate signaling[47-49] • ATF-2 transcription factor network[50-51] • Signaling mediated by p38-alpha and p38-beta[52-53] • Regulation of p38-alpha and p38-beta[54] • Regulation of Androgen receptor activity[43-44] • N0cadherin signaling events[55] • IL27-mediated signaling events[56] • IL12-mediated signaling events[57-58]

Explanation

• There are very definitive signaling pathways in the body which tell the cells what kind of proteins to produce and thus, protect the cell and total organism from damage, by allowing repair to take place easily

• Some pathways are beneficial in repair and were shown to be up regulated ( activated ) in the gene expression studies.

• Other pathways which were not beneficial were down regulated ( de-activated )

• Thus, the study of the signaling pathways in the Gene Expression Study, clearly demonstrated significant improvements in the relevant protective signaling pathways, and suppression of non beneficial pathways

Gene Expression changes Study 2

• An Intensive Mind and Body Therapeutic Program Leads to Gene Expression Changes in Peripheral Blood Mononuclear cells

• Krishna S Rao, MD, FRCS (Ed)1, Swarup K Chakrabarti PhD 2, Vaishali S DongareMSc 2, Sharath BS MSc 2, Vikas HM MSc 2, Chetana K MDS 1, Kaushik D Deb* MD(AM) PhD FRHS 2

MATERIALS & METHODS

• Study design & Labeling :40 Healthy adult male and female volunteers were enrolled for the study, over a period of 21 days

• Blood samples with informed consent for differential gene expression study before and after the program was collected

• Samples were collected from volunteers before program were names as BIAY samples and after theprogram were named as AIAY samples.

Materials and Methods

• BIAY were considered as controls and AIAY were considered as test samples

• Sample numbers 1-10 were pooled in as BIAY1 matched against sample numbers 1-10 of after samples as AIAY1

• Pooled samples were similarly labeled for 11-20, 21-30 and 31-40 as:

BIAY2 vs AIAY2, BIAY3 vs AIAY3, BIAY4 vs AIAY4.

Process

• Human whole genome HG-U133_Plus_2 chips form Affymatrix (Santa Clara, CA, USA) wasused for the study

• Microarray Data AnalysisFor microarray Data Analysis latest version best software GeneSpring 11.5 has been used.The CEL files are imported in Affymetrix (Santa Clara, CA, USA) expression workflow usingTechnology for HG-U133_Plus_2.

Gene Expression Data

• Critical pathways such as TNF alpha/NF-kB, Hypoxic and oxygen homeostasis regulation of HIF-1-alpha, C-MYC transcriptional activation, P38 MAPK signaling pathway, IL-12 signaling pathway that are known to be associated with aging were dysregulated.

• Apart from pathway analysis, 420 upregulated and 165 downregulated entities (genes) were obtained from microarray data analysis.

Results

• Significant genes either 2-fold up or 2-fold down were (EIF4E, BMI1, TMEF2) that are known to be key players in C-MYC pathway. Image for C-MYC pathway with fold change overlaid for additional key genes (EP300, PKN2, HSP90AA1, TMEFF2, IREB2, CREB1) were found to be significantly altered (more than 2-fold) as well.

Results

• Importantly, expression of a potent pro-inflammatory cytokine interferon-gamma went down 2.51-fold in the meditated group compared to unmeditated group, thereby suggesting a reduction of chronic inflammation in the meditated group. Chronic inflammation has been shown to increase during aging.

Results

• Moreover, Activating Transcription Factor-2 (ATF2), which is implicated in DNA damage response, cell cycle and apoptosis59, went up 6.86-fold in the study group. • Furthermore, ATF2 has been shown to downregulate c-Jun N -terminal kinases (JNK) pathway that activates inflammation59. • STAT 2 (Signal transducer and activator of transcription factor 2) which augments proinflammatory response60 was downregulated (2.47-fold) in thestudy group.

Results

• NDUFA5, an important component of the mitochondrial electron transport chain (Complex 1), was found to be upregulated (4.26-fold) in thestudy group that may improve cognitive function in older age • In addition, expression of RSP11 (Ribosomal protein, small subunit) was 6.5-fold down regulated in the study group

Results • It has been reported that knockdown to RSP 11 increased lifespan in mice. • SLC25A24 (Solute carrier family 25 (mitochondrial carrier, phosphate carrier), member 24) was augmented significantly (7-fold) in the study group. • This may indicate lessened mitochondrial dysfunction in the study group that can lead to improved cognitive function in the older age.

Results • In addition, KDM6A (Lysine (K)-specific demethylase 6A), a modifier of chromatin, has been shown to be key modulator of aging process, was found to be upregulated (3.16-fold) in the study group, thereby indicating a possible underlying chromatin mechanism in aging.

Results • Expression of CREB1 (cAMP response element binding protein 1) was up (2.19-fold) in the study group which is known to regulate a key aging-related gene ATM (ataxia telangiectasia mutated), an early-onset disease some argue is characterized by signs of premature aging.

Explanation

• A normal human being has approximately 20,000 Genes • At any given time, about 10,000 Genes are expressed ( active) • The role of the Gene is mainly to induce the production of proteins, which are essential for cellular function, repair and survival • In the above study, during IA : 420 additional were expressed, through up regulation ( activated ), and165 Gene entities were suppressed through down regulation (de activated ) • A close look at the Gene entities show that, pro inflammatory ( damage causing ), were down regulated and Genes which induced formation of stem cells and other beneficial actions were up regulated • This included Genes and pathways related to Auto-Immune Disorders, Psychiatric illness, Ageing and Malignancies

Telomerase and Stem cell Study 3 • Antiaging Effects of an Intensive Mind and Body Therapeutic Program through Enhancement of Telomerase activity and Adult Stem cell Counts • Krishna S Rao, MD, FRCS (Ed)1, Swarup K Chakrabarti, PhD 2, Vaishali S Dongare, MSc2, Vikas HM, MSc 2, Sharath BS, MSc2, Chetana K, MDS1 Kaushik D Deb*, MD (AM), PhD, FRHS21 • The individual values of Stem cells and Telomerase for each participant is available

Telomere and Stem cell

Explanation

• A human being has 46 pairs of Chromosomes , the end of each being protected by a cap like structure called the Telomere

• As cells age, the length of the Telomere reduces and when it reaches a critical shortening ( about 50 kilo base pairs ) , the cells stop dividing and enter senescence and death

• The Telomere can be maintained or produced only by the enzyme Telomerase

• A normal adult, has 0 – very minimal Telomerase activity

Explanation

• Stem cells are reserve cells of the body • In the embryo, the stem cells can become any part of the body and is called Totipotent stem cells

• As we get older, the stem cells mature to a degree and are now able to become a large number of possibilities , but not everything , and are now called Pluripotent

• In the adult, the stem cells , can become only some components of the body , depending on their origin and location and are called Multipotent

• By this time , the number of stem cells are reduced to a small holding areas called Stem cell Niches

Materials and Methods • The study determined effect of telomerase enzyme activity on aging by detecting peripheral blood adult regenerative stem cell (ARSS) and telomerase activity in peripheral blood mononuclear cells (PBMC). • All samples were collected at the site of the study. • The study included 110 participants.

Process • The stem cells were also analyzed for expression of pluripotency marker like OCT4, SOX2, NANOG by reverse transcriptase –polymerase chain reaction (RT-PCR) Process • Telomerase activity measurement in PBMCs: • Telomerase relative activity assessed were done as per the instruction of Millipore’s TRAPeze telomerase detection kit (Billerica, MA, USA). • These assays were done in the peripheral blood mononuclear cell (PBMC) counts. • Protein estimation assay was done by using Lowry method [35]and equal amount of (0.4µg cell lysate per assay).

Results • Study group showed higher fold changes in stem cell numbers, indicating a role of stem cells on the well-being of the subjects undergoing the program •Stem cells reside in relative hypoxic niche in the body( Processes like Pranayama with Bandhas induce relative hypoxia , leading to increase in Stem cells) •The process of IA has already shown to up regulate Hypoxia Inducing Factor-alpha in earlier gene expression studies.

Normal Adults Have Negligible to Zero Telomerase Activity • The next process was to investigate a key molecular parameter of human-well being; telomerase activity. • 27% participants showed more than 2-fold( 200%) increase in Telomerase, 45% showed more than 1 fold (100%) increase in Telomerase activity in 21 days • Other published data indicate that it takes up to 3 – 4 months for enzyme activity to increase, leading to increased Telomere length

Conclusion • There is a small but distinct advantage in longevity in women due to Estrogen receptor activation • This gender inequality is decreasing due to llife style changes and other advances in medical therapy. • There is ample evidence that intense processes like IA, involving completion , Yoga, Meditation and Energisation , induces epigenetic changes that alters gene expression and there by altering the molecular basis of ageing to our benefit.