4. Salameh, Y., Bejaoui, Y., & El Hajj, N. (2020). DNA
methylation biomarkers in aging and age-related
diseases. Frontiers in Genetics, 11, 171.
DNA
methylation
(DNAm)
influences
biological
age
Epigenetic
clocks
measure
DNAm
based age
5. AIM
• Heterochronic Parabiosis is already used in short-term aging research
and mice phenotypically show rejuvenation pattern, but they
extended attachment period to 3 months, then 2 months later mice
were separated from each other and they examined whether
heterochronic parabiosis old mice rejuvenated by using epigenomic
and transcriptomic analysis.
6. Fig. 1 | Prolonged parabiosis followed by detachment leads to extended life span and health span. a, Overview of the parabiosis
and detachment model. All pairs were anastomosed for 3 months, starting at 20 months of age for old mice and 3 months of age
for young mice. At 23 months, old mice were detached and the remaining life span was assessed. b, Schematic of the molecular
profiling methods applied to mouse tissues.
RESULTS
7. 1.c, Survival curves of detached old mice from isochronic (red, n = 23) and
heterochronic (purple, n = 20) pairs. 1.d, Body weight, lean mass, fat mass and cage
activity measurements before parabiosis and at monthly time points starting 1 month
after detachment.
8. Fig. 2 | Persistent epigenetic age reversal in blood and liver on HPB assessed by RRBS-based aging clocks.
a, DNA methylation age of old heterochronic blood before and after detachment plotted with isochronic
young and old controls assessed by the Meer multi-tissue clock, Petkovich blood clock, Thompson
multitissue clock and scAge blood clock.
9. 1.b, DNA methylation age of old heterochronic liver before and after detachment plotted with isochronic
young and old controls assessed with the RRBS-based aging clocks which are Meer multi-tissue clock,
Petkovich blood clock, Thompson multi-tissue clock and scAge liver clock.
10. Fig. 3 | Sustained epigenetic age reversal in liver on HPB assessed by microarray-based aging clocks. a,b
Epigenetic age of long-term (3-month) and short-term (5-week) parabiosis liver samples from old
heterochronic attached or detached mice, plotted with young and old isochronic controls, based on the
universal relative age mammalian, universal log-linear-transformed age mammalian, liver and liver
development clocks
11. 3. c,d, Epigenetic age of short-term (5-week) mock parabiosis blood (c) and liver samples from old
heterochronic attached mice, plotted with young and old isochronic controls, based on the same clocks as
in a.
12. Fig. 4 | Transcriptomic analyses of HPB reveal unique pathway enrichment and differential expression patterns. a, GSEA of
old isochronic versus old heterochronic mice (left), as well as of detached old isochronic versus detached old
heterochronic mice (right). Hallmark gene sets (n = 50) were used as input to the GSEA. The gene sets shown in green
signify pathways enriched in the heterochronic samples, and orange those enriched in the isochronic samples
13. 4. b,c, Volcano plot of differentially expressed genes in attached (b) and detached (c) old heterochronic
(n = 3) and old isochronic (n = 3) mice. Several highly significant genes are shown in each plot. Genes
shown in red are significantly downregulated in heterochronic mice, while genes shown in blue are
significantly upregulated in heterochronic mice
14. Fig. 5 | Dimensionality reduction highlights rejuvenated molecular profiles after HPB. a, PCA of liver RNA-
seq data of mice after long-term parabiosis or 2 months after detachment. b, PCA of liver RNA-seq data of
mice after short-term parabiosis.
15. 5.c,d PCA of liver and blood RRBS data across all highly covered common CpGs. e,f PCA of liver and blood RRBS
data across gene promoters. g,h PCA of liver and blood RRBS data across gene bodies.
16. Fig. 6 | The transcriptomic signatures of HPB align with longevity interventions and oppose aging. a, Association between
gene expression changes induced by long-term parabiosis with (blue) and without (red) a 2-month detachment period, and
signatures of aging and life span extension. The latter include gene signatures of individual interventions (CR and GH
deficiency), common intervention signatures (interventions: common) and signatures associated with an effect on life span
(maximum and median life span). b, Association between gene expression changes induced by long-term (red) and short-
term (yellow) parabiosis without detachment and signatures of aging and life span extension. The signatures analyzed are
the same as in a.
17. 6.c, Spearman correlation matrix of gene expression signatures associated with aging (red labels), life span
extension (green labels) and HPB (blue labels). 6.d, Functional enrichment analyses of gene expression signatures.
Only functions significantly associated with at least one signature are shown.
18. Fig. 7 | Gene expression analyses hint at putative rejuvenation mechanisms of long-term HPB. a–c, Expression
of Sirt3 (a), Gstt2 (b) in mice subjected to parabiosis by RNA-seq analyses (left), RT–qPCR analyses (middle) and
in response to established life span-extending interventions and aging (right)
19. 7.c, Expression of C1qb in mice subjected to parabiosis by RNA-seq analyses (left), RT–qPCR analyses (middle)
and in response to established life span-extending interventions and aging (right).
20. CONCLUSIONS
• exposure to the young circulatory system leads to persistent and systemic slowing
of epigenetic aging. This effect correlates with a longer life span, improved
physiological parameters and a globally rejuvenated epigenome and
transcriptome.
• they observed multi-omic, systemic and sustained (after detachment) biological
rejuvenation.
• long-term (3-month) parabiosis is much more effective at rejuvenating the
transcriptome and epigenome of mice compared to short-term (5-week)
parabiosis.
Editor's Notes
First of all, ageing is time-related accumulation of a wide variety of molecular and cellular damage. Factors that cause aging are genomic instability, mitochondrial malfunction, accumulation of senescent cells ((cell cycle arrest, it’s like a Differentiated cell in G0 phase, metabolically active and secrete pro-inflammatory cytokine and trigger inflammation, immuno senecent.)), stem cell exhaustion. Rejuvenation is such as reversing these, providing genomic stability, mitochondrial biogenesis, reducing inflammation, and self-renewal of stem cells. And this is achieved epigenetically, by the balance between heterochromatin and euchromatin (transcriptionally active part) with DNA methylation, histone modifications and chromatin remodelling.//
-(?what is difference btw ) Rejuvenation means that the aging markers return to their yongest state. regeneration is the capacity of a tissue to renew itself after damage (regrowing). Erasing epigenetic mark that accumulate with age.
-Epigenetic occur through modifications on the DNA and its associated proteins that affect gene expression, without changing DNA sequence. post-translational modifications such as lysine methylation occurs on histone. ((modulating the availability of DNA to transcriptional complexes)).
Loss of heterochromatin
There are many attempts to extend the life span through Interventions such as reprogramming, heterochronic parabiosis, caloric restriction.//
Epigenetic clock estimates biological age based on change.
-Methylome instability can be realted to Hypomethylation: reduction in DNA methylation increase genomic instability, as it may result in the activation of normally silenced gene.
In this article Parabiosis mice models were used to rejuvenate old mice. Parabiosis is a surgical joing of two animals so that they share blood circulation. When the skins of two mice are surgically attached, new blood vessels (angiogenesis) are formed at the sites of attachment, allowing the exchange of blood and circulating factors. Heterochronic Parabiosis is between old animal and a young animal. Isochronic mice use as a control between same aged mice.
-One of the strong speculation is young blood dilute harmful factors in old blood.
DNA methylation levels (the pattern of 5-methylcytosine) are altered during ageing
This DNA methylation level used to predict biological age in a variety of tissues, such as blood and liver ,and DNA methylation based epigenetic age is measured by epigenetic clock.
In this article, they use petkovich, thompson and meer DNA methylation pattern (based on different number of CpG sites) to demonstrate rejuvenation.
CpG islands (often found near promoter region) are associated with the regulation of gene expression. When they are unmethylated, genes tend to be transcriptionally active. However, when methylated, can lead to the repression of gene expression.
And they try to find gene related to rejuvenation and aging.
Firstly, Figure a and b shows experimental design. They used 3 different groups : young iso which blood exchange is between two young mice, old iso between old mice and they used as a control, and also old hetero between young and old mice. They connected the blood stream of the mice to each other for 3 months, then the mice were separated from each other and 2 months after separation, they did observation and data analysis with post-attachment and detacment mice. (they want to see significant difference in old hetero mice) İn the b part, they examine the liver and blood samples of mice. They did the RNA-seq as a transcriptome analysis ( want to see change in mRna level) and RRBS,Reduced Representation Bisulfite Sequencing, epigenomic clock analysis ( which detect change in methylation level at specific CpG sites.) (it targets some part of genome)
-RRBS examine methylation pattern at specific CpG sites.
-they observed blood circulation effect on solid tissue (why liver?)
İn this figure, to examine the effect of parabiosis on life span, parabiosis pairs are seperated from each other and allowed to live freely until their natural death.
As you can see, in general, old heterochronic mice ,which take blood from young mice, percent survival decreased slower than old isochronic mice. old iso groups survival rate drops faster, means that life span extension happened in old hetero.
After that they want to observe physiological improvement, As you know Younger mice are always heavier, faster, and have more fat and muscle mass. However, Old mice begin to lose weight, their movements become weaker, muscle and fat mass decreases. In old hetero compared to old iso mice, lean mass and distance rate increased as expected. But, in the body weight graph, initial drop was due to reduction in fat mass because mice are already old they dont tolarate initially, but, later time body weight sustained due to the maintenance of both lean and fat mass.
-İnitial reduction sustain later, when old iso gets weaker.
(It can still sustain, normally it gets weaker.)) ((Voluntary wheel running in mice is a widely used model to study exercise and behavior)) (At first, both body weight and fat mass decreased, but later on, it showed preservation in later life.)
For epigenetic clock analyses, they collected blood either immediately after the 3-months parabiosis procedure and 2 months after detachment.
To quantify the epigenetic age of the animals, they applied four RRBS-based epigenetic clocks (meer, petkovich, thompson and scAge which are all databases) to six different groups of mice as you can see the X-axis.
So, As i mentioned, These clocks’ databases contain DNA methylation profiles from multiple tissues and specifically blood based on previous epigenetic pattern analyses. So, they compare blood RRBS(bisulfide sequencing) (methylation pattern) data of parabiosis mice after attachment and deattachment.
*Compared to old isochronic DNA methylation age, heterochronic pairs DNA methylation age decreased and this effect sustained even after 2 months of detachment. Heterochronic parabiosis mice show a value between old and young iso as expected. (rejuvenated as a certain level.)
(Methyl-insensitive enzyme)
these same four clocks applied to liver RRBS data and showed similar result.
+some clocks showed marginal differences because the blood clock is not as precise in assessing epigenetic age in the liver.
((in heterochronic pairs DNA methylation age decreased and this effect sustained even after 2 months of detachment.
*comparable immediate and sustained epigenetic age reduction as observed in blood))
*epigenetic clocks demonstrate slowing of epigenetic age
To confirm this epigenetic age decrease, they used a completely independent analysis platform to estimate methylation array profiling of liver samples in three groups of animals (young isochronic, old isochronic and old heterochronic). They applied four different array-specific epigenetic clocks. Two universal clocks and two mouse-specific clocks. -as I showed in the previous figure, heterochronic old mice show significant reduction in epigenetic age for our long-term attachment protocol (3-month) and even if mice detached, methylation age of the liver is sustained. ((İts epigenetic age are between control old and young isochronic mice.)) Also in the b part, they tested the difference with commonly used 5-week, short-term parabiosis and its effect is smaller than in the long-term attachment (3 mounts) *This data suggests that while short-term parabiosis may induce some epigenetic remodeling leading to few rejuvenation, a longer-term treatment clearly show epigenetic remodeling, even if mice detached, rejuvenation of the liver epigenome sustained.
((except mouse (liver)) Only three of the four clocks applied detected a significant reduction in epigenetic age in heterochronic mice, stained.))
Previously, one of the article said physical activity, especially in older populations, can affect epigenetic age. They want to eleminate effect of physical activity and also effect of surgery operation. They developed a ‘mock’ parabiosis model to connect mice without blood sharing for 5 weeks. (If one mouse (moves) is running, the other is also running). As seen in graphs, mock parabiosis did not alter epigenetic age in either blood or livers. (Old heterochronic and old isochronic methylation almost same.)
And then, They performed RNA-seq analyses (expression levels of genes) of liver tissue and calculated normalized enrichment scores (NES) comparing isochronic and heterochronic samples immediately after the 3-month parabiosis period, as well as after 2 months of detachment.NES used to assess enrichment of predefined specific gene sets (which share common biological functions and pathways) within a gene expression dataset in liver tissue.
There are Strong enrichment of the oxidative phosphorylation ((increase energy levels and decrease oxidative stress)), fatty acid metabolism ((integrity of cell membranes, source of energy)) and mitochondrial biogenesis (peroxisome) gene sets in heterochronic mice. As you know, these functions are disrupted during the aging process. These results indicate that Heterochronic parabiosis may reverse ! the age-associated decline of critical energy production pathway. They also observed depletion of inflammatory and interferon-gamma,alpha response gene sets in heterochronic mice and enrichment of inflammation in old isochronic mice. Inflamatory response protein level is known to increase with age to deal with ongoing stress. (Accumulation of Senecentcell cell which is metabolically active and secrete pro-inflammatory cytokine and trigger inflammation, immuno senecent.) So, they showed rejuvenating with decrease inflammation in heterochronic mice.
X((Increase in energy metabolism and decrease in inflammation are hallmarks of being young.))
(söylemesende olur) Also, enriched gene sets were generally decreased in the detached group and this indicate the gradual reduction of parabiosis effect after detachment.
The xenobiotic metabolism marked in red decreased after detachment, which may be due to the drugs given during surgery.
-------
They have database of predefined gene sets which share common biological functions and pathways. Also, they do Differential Expression Analysis to identifies genes that are significantly upregulated or downregulated in the liver tissue. After that, they evaluate predefined gene sets are enriched in the list of differentially expressed genes in liver tissue of attached and deattached hetero and iso mice.
Then, global differential gene expression analyses was performed with post-attachment (A) and detached (B) heterochronic parabiosis mice. Genes shown in red are significantly downregulated in heterochronic mice which are mostly immune response gene, while genes shown in blue are significantly upregulated in heterochronic mice mostly related to lipid metabolism or important cellular functioning gene.
A2M (plasma protein take role in immune system)
ATPase, GSTT2, LIPG They identified 1,044 significantly upregulated and 1,855 downregulated genes in the post-attachment (A) comparison in b, 876 upregulated and 1,034 downregulated genes in the detached (B) comparison in c part.
Then, they visualize samples epigenomic and transcriptomic data in two-dimensional space by using principal component analysis (PCA) (dimensionality reduction technique). They do basically data mining, detect similarityies of two data. In the a part, after long-term parabiosis and detachment, old heterochronic samples clustered between young and old isochronic samples, closer to the young samples than the old.
However,In the b part, clustering of short-term heterochronic parabiosis showed much closer to the old isochronic group. We can say that, long-term heterochronic parabiosis is a much more rejuvenate.
and demonstrate more shift to the young isochronic mice compared to short-term parabiosis.
They also performed principal component analysis(PCA) on liver and blood ((RRBS(bisulfide sequencing)) methylation data and compared methylation at different CpG sites. As seen in the figure, old heterochronic samples clustered in between old and young isochronic samples ((when analyzing over 1 million highly covered CpGs))Fig. 5c,d). especially in promoters and gene bodies methylation, heterochronic samples fall perfectly between the two isochronic groups (Fig. 5e–h).
** old heterochronic mice have transcriptomic and epigenetic profiles intermediate between young isochronic and old isochronic mice across both liver and blood.
(promoter: regions typically located near the transcription start site of a gene, when its metylated it leads to dowregulate gene expression) -Methylated promoters can hinder the binding of transcription factors and RNA polymerase, preventing the initiation of transcription and, at the same time, gene expression. -Promoter methylation is an epigenetic mark that control which genes are turned on or off -Gene body (exons and introns) methylation may play a role in regulating alternative splicing, which can result in different protein isoforms being produced from the same gene.
As i mentioned in introduction part, some intervention extend life span. For example, life span get longer with growth hormon efficiency or calori restriction and they have gene expression data form this intervention which are known to extend life span. In the a part, They compare their heterochronic samples taken immediately after the 3-month parabiosis and after the 2-month detachment period, with this intervention and aging data. As you can see, there is a strong positive association in attachment and detachment mice with life span-extending interventions. ((including positive enrichment for median and maximum life span signatures and Calorie Resteiction and GH deficiency)). -NES (normalized enrichment score) decreased in detached samples compared to samples taken immediately after the long-term parabiosis period. -they observed a significant negative association with aging signatures, so these mice don’t express aging marker. ((including the mouse liver, rat multi-tissue and mouse multi-tissue aging signatures)) **This suggests that most transcriptomic changes are sustained after the detachment period, but the magnitude of the rejuvenating effect may be gradually decreased over time.
In b part, They did same with long-term (3-mounth) and short term (5-week) parabiosis mice, and see significant positive associations of Heterochronic parabiosis with several signatures of life span extension, while negative association with aging signatures. long-term parabiosis seems to induce more life span-extending effect and more negative correlation with aging marker.
// Also, So long-term parabiosis has more rejuvenating effects.
##Then, They investigate mutual associations (level of one variable is related to another variable) between changes induced by heterochronic parabiosis, life span extension interventions, and aging. Spearman correlation matrix was used to analyze relationships between gene expression changes in different gene sets or signatures. As seen in the right bottom (red square), the effects induced by Heterochronic parabiosis in old mice clustered together and positively correlated with life span-extending interventions. However, fold change of parabiosis mice negatively correlated with aging (blue square) ((Aging azalıyor, life span artıyor, fare gençleşiyor.)) For the d part, To understand which gene sets are responsible for the age reversal and life span-extending effects, they performed functional pathway analysis with previous result.((detect which genes are primarily responsible for specific functions )) -Functions upregulated in response to parabiosis, including, fatty acid metabolism, mitochondrial translation,oxidatice phosphorylation, respiratory electron transport chain and mitochondrial biogenesis, tricarboxylic acid cycle were upregulated by life span-extending interventions (upper red squares), but downregulated during aging (upper blue square). On the other hand, functions related to the immune response, such as interferon signaling and complement cascades, are significantly downregulated both, in response to Heterochronic parabiosis and life span-extending interventions, but upregulated with age.
*As a RESULT, gene set analyses of Heterochronic parabiosis resemble the effects of life span-extending interventions.
((oxidative phosphorylation is key to reducing the production of damaging free radicals.(ROS) Mitochondrial biogenesis,TCA provide energy demands of cells)) .
To uncover the potential gene underlying the rejuvenation effects, they investigated the individual genes regulated in response to long-term heterochronic parabiosis and their association with longevity and aging based on the gene expression signatures (Fig. 7) Among genes upregulated by long-term Heterochronic parabiosis in both attached and detached models, they identified Sirt3 sirtuin, histone deacetylases leads to gene repression, genomic stability) and Gstt2 (protect cells from oxidative stress ) and confirmed with RNA-seq, RT-qPCR analyses (expression analysis). in this right figure, Sirt3 and Gstt2 in liver are upregulated with many longevity interventions (in other words positively associated) but negatively associated with aging.
- Basically, Sirt3 maintain mitochondrial health, Gstt2 decrease of reactive oxygen species levels. -Gstt2 involved in glutathione metabolism, Glutathione is a critical antioxidant that helps **protect cells from oxidative stress by neutralizing harmful free radicals and toxins. ))
Conversely, Among genes downregulated by Heterochronic parabiosis, they identified immune response gene C1gb, which encodes for one of the complement (innate immunity) subcomponents which is related to immune responses against pathogens, This gene is positively associated with aging and negatively associated with life span extending interventions.
One of the strong speculation is young blood dilute harmful factors in old blood.
