Characterization of novel mouse models to study the role of necroptosis in aging and age-related diseases

Authors: SELVARANI, RAMASAMY - University Of Oklahoma Health Sciences Center; NGUYEN, HOANG VAN - University Of Oklahoma Health Sciences Center; THADATHIL, NIDHEESH - University Of Oklahoma Health Sciences Center; WOLF, ROMAN - Oklahoma Veteran Affairs Medical Center; FREEMAN, WILLARD - Oklahoma Medical Research Foundation; WILEY, CHRISTOPHER - Jean Mayer Human Nutrition Research Center On Aging At Tufts University; DEEPA, SATHYASEELAN - University Of Oklahoma Health Sciences Center; RICHARDSON, ARL - University Of Oklahoma Health Sciences Center

Submitted to: GeroScience
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/20/2023
Publication Date: 10/4/2023
Citation: Selvarani, R., Nguyen, H., Thadathil, N., Wolf, R.F., Freeman, W.M., Wiley, C., Deepa, S.S., Richardson, A.N. 2023. Characterization of novel mouse models to study the role of necroptosis in aging and age-related diseases. GeroScience. https://doi.org/10.1007/s11357-023-00955-7.
DOI: https://doi.org/10.1007/s11357-023-00955-7

Interpretive Summary: Necroptosis is a form of programmed cell death that results in the release of cell contents into the surrounding tissue, which ultimately activates inflammation and potentially causes degenerative disease. Necroptosis increases with age, but whether it actually drives age-associated disease is still unclear. Here, we characterized two mouse models of increased necroptosis in the liver. Both models showed increased inflammation, along with increases in fatty liver and markers of fibrosis in response to aging or liver damage. Furthermore, markers of cellular aging were increased in these models. Together, these models confirm a role for necroptosis as a driver of aging and degenerative diseases of the liver.

Technical Abstract: To study the impact of necroptosis-induced chronic inflammation on age-related diseases and aging, two knockin mouse models (Ripk3-KI and Mlkl-KI) were generated that overexpress two genes involved in necroptosis (Ripk3 or Mlkl) when crossed to Cre transgenic mice. Crossing Ripk3-KI or Mlkl-KI mice to albumin-Cre transgenic mice produced hepatocyte specific hRipk3-KI or hMlkl-KI mice, which express the two transgenes only in liver. Ripk3 and Mlkl proteins were overexpressed 10- and 4-fold, respectively in the livers of the hRipk3-KI or hMlkl-KI mice. Treating young (2-months) hRipk3-KI or hMlkl-KI mice with carbon tetrachloride (CCl4), a chemical inducer of oxidative stress, resulted in increased necroptosis (Mlkl-oligomers) and inflammation in the liver compared to control mice receiving CCl4. Mlkl-oligomerization also was significantly increased in old (18-months) hRipk3-KI and hMlkl-KI mice compared to old control (Cre negative, Ripk3-KI and Mlkl-KI) mice. The increase in necroptosis was associated with an increase in inflammation, e.g., inflammatory cytokines (TNFalpha, IL-6) and macrophage markers (F4/80, CD68). Importantly, steatosis (triglycerides) and fibrosis (e.g., picrosirius red staining, hydroxyproline levels, and transcripts for TGF beta, Col1 alpha 1, and Col3 alpha 1) that increase with age were significantly higher in the livers of the old hRipk3-KI or hMlkl-KI mice compared to old control mice. In addition, markers of cellular senescence were significantly increased in the livers of the old hRipk3-KI and hMlkl -KI mice. Thus, the first mouse models have been developed that allow researchers to study the impact of inducing necroptosis in specific cells/tissues on chronic inflammation in aging and age-related diseases.