Could cannabinoids alter the aging process through epigenetic changes?
- We have discovered a new aging model.
- It is based on epigenetic evolution in living cells.
- Using this model, we seek to identify new therapeutics to mitigate aging.
- We currently have preliminary evidence that certain cannabinoid isolates may impact cellular aging.
Summary of Study. The AGE (AGing Epigenetic) Screen.
We have discovered a cell-based model to identify agents that impact the process of age-related epigenetic revision. The AGE Screen (AGing Epigenetic) is based on a neutral GFP reporter gene to rapidly track changes in DNA methylation (Me-C) in the context of a living cell giving high physiological relevance. Any forward (more DNA methylation) or backward (less methylation) alters gene expression in a permanent fashion. The quantitative readout we use is flow cytometry and a qualitative readout of single cell live imaging. Either read out presents reporter expression in real time and in living cells. These methods are well-validated by numerous publications in the epigenetics field in top peer reviewed research journals.
The AGE screen will detect virtually any small molecule, drug, synthetic compound, or natural product impact on gene expression or supression in a permanent way.
The AGE screen uses a heavily methylated GFP reporter (stably integrated into a human cell line model). The cells that express GFP poorly are referred to as being’ ‘Dim’ cells. After many population doublings (>20), Dim cells progressively lose Me-C residues as they age in a specific gene location in the DNA and convert to a hypo-methylated cell phenotype that we call ‘Bright’ cells. The shift from “Dim to Bright” in GFP expression can also be detected by live imaging. Interestingly, low doses of certain purified cannabinoids can inhibit Dim to Bright transition as the cells divide and grow. Our preliminary data shows that we can detect a natural process of DNA methylation reprogramming using this AGE platform. The importance of DNA methylation in human disease and aging is well established, but there are no suitable platforms to rapidly test factors that replicate the positive (or block the negative) epigenetic changes as we age, and our AGE Screen developed through support from CBxGEN is an answer to this crucial issue.
Where do Dim/Bright Cells Originate?
The AGE screen is based on a rich population of Dim Cells.
The live image time lapse shows how Dim cells were made:
Explanation
The cell in the middle turns bright green because the GFP gene in that one single cell becomes activated after DNA repair. This ‘progenitor’ cell is producing large amounts of GFP protein because the repaired gene is unmethylated. After (or during) cell division, the GFP gene is differentially methylated such that one daughter cell is mostly devoid of methyl- C DNA while the other daughter becomes more heavily methylated. Thus, dual populations of cells form: Dim cells (heavy methylation) and Bright cells (minimal methylation). We used Flow sorting to make an enriched population of Dim cells (90% Dim, 105 Bright).
The AGE SCREEN and how it works
Overview of the AGE Screen
Many weeks
In Culture
(20 Cell Passages)
As Me-C is removed from the reporter gene, over 21 days in culture, the cells shift from low GFP to higher GFP levels.
This is a progressive loss of Me-C as cells ‘age’ in culture.
Evolution of DNA Me-C Revisions in AGE
Dim cells (90% and 10% Bright) were grown by routine sub-culture approximately every 3 days to keep the cells healthy. At each sub-culture, cells were analyzed by FACS to measure % of Dim cells. A fraction of the cultures (2x106) were removed and treated with 5uM Aza-C for 36 hours before FACS to determine %DIM Cells after hypo-methylating drug treatment. The data show the following:
- Dim cells progressively lose Me-C with each round of replication aging.
- Over time, the Dim cells become resistant to Aza-C induced hypo-methylation.
Epigenetic changes detected by the AGE SCREEN are altered by some, but not all cannabinoids.
CBG is showing activity
Fig 3. AGE Screening of cannabinoids. GFP fluorescence was monitored by FACS before and after successive doublings to measure the fraction of Bright cells (%Brights) in the population. The cells were monitored at 0, 9, 14 and 26d in the presence of 0 drug or 6uM CBD or 6uM CBG (purified, courtesy of CBx Genomics and Therapeutics) as follows: Each plate was sub-cultured every 3-4 days and at the indicated passage numbers, a fraction (0.5ml) of cells were retained for FACS while the remaining cells were diluted with media+serum +or- drug into fresh plates. The cell viability was monitored by live imaging (not shown) to ensure cells were healthy, uncontaminated and growing well (as evidence by cell yields). Thus, the experiment was conducted on the same cells but at different passages.
This result shows that CBG (but not CBD) can alter epigenetic revisions in our cell context model.
The AGE Model: is it a real or a laboratory model with no physiologic relevance?
To Test this we did the following.
We used CRISPR to Integrate WT GFP into the Rosa26 Locus of Mouse ES Cells.
3 Clones (random)
After 3 weeks, we found clones that had different %Dim and showed different responses to Aza-C.
Based on this, we have good validation that our system is a natural, progressive epigenetic change with replicative aging.
REF
DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism
Cusl Russo, Rosaria Lanci, Antonio Pezone, Annellss Morano, Candida Zuchegns, Antonela Romano, Mark T. Muller, Max E. Gottesman, Antonio Porcelini & Cnrico V. Avvedimento
Scientific Reports 6. Article number: 33222 (2016) Cite this article
Practical Applications of a Cell-based Screen for Epigenetic Effectors in Aging
Application potential
- Epi-therapeutics: cancer and other diseases
- Powerful cell based screen
Targeted to specific genes - Limits collateral damage
Few FDA Approved Epi-therapeutics - Important new in-roads to prevent aging due to epigenetic shifts
Targeted to specific genes
Few FDA Approved Epi-therapeutics
Why these experiments are important.
Why these experiments are important.
- Biological Age = DNA Methylation is a biomarker for aging
- High Biological (Methylation) age relative to actual age is not heathy
- High Me-aging predicts disease susceptibility
You want to live to 100?
It is mandatory to acquire the right epigenetic variants through the environment or through natural products.
We believe cannabinoids, like CBG, contained in natural hemp
products have the potential to help mitigate age-linked diseases.