Chronic, low-grade inflammation (inflammaging) is a hallmark of aging and is a major risk factor for both morbidity and mortality in the elderly humans. In addition, inflammation is a major risk factor for a variety of age-related diseases, e.g. type 2 diabetes, cardiovascular disease, cancer, neurodegenerative diseases, frailty, etc. Despite the link between inflammation, aging, and age-associated diseases, we do not know the molecular mechanism(s)/pathway(s) responsible for the chronic, low-grade inflammation seen in old animals and whether the increase in chronic inflammation is a causative factor in aging and age-related diseases. Necroptosis is a recently identified pathway of programmed necrosis that induces cell death through the lysis of cells, resulting in the release of damage-associated molecular patterns (DAMPs) from the dead cells. DAMPs are potent inducers of inflammation, and circulating DAMPs have been shown to increase with age in humans and to be strongly correlated to the level of inflammation in the individual. We hypothesize that necroptosis plays a role in chronic, low-grade inflammation, which occurs with age, and preventing necroptosis will attenuate inflammation, leading to increased lifespan, improved healthspan, and reduced age-related pathology. We will use genetically modified mouse models and a pharmacological intervention to block/reduce apoptosis by targeting the receptor-interacting protein kinases (RIPK) 1 or 2 and mixed lineage kinase domain like (MLKL) protein, which are involved in the initiation of necroptosis.
Aim1. Determine the role of necroptosis in chronic inflammation that occurs with age and the types of cells undergoing necroptosis. We will determine the effect of reducing necroptosis either genetically (using Ripk3+/-, Ripk3-/-, Mlkl+/-, and Mlkl-/- mice) or pharmacologically (treating with necrostatin-1s, a RIPK1 inhibitor) on inflammation. We will also identify the cell type(s) that undergo necroptosis in selected tissues of old mice. Based on our hypothesis, we predict that those tissues showing an up-regulation in necroptosis with age will show an increase in production of proinflammatory cytokines and reducing/blocking necroptosis will decrease inflammation in these tissues as well as circulating levels of proinflammatory cytokines.
Aim2. Identify the pathways that increase necroptosis with age and determine the mechanism(s) that mediates the age-related increase in inflammation arising from necroptosis. TNF?, oxidative stress, and mTOR signaling have been proposed to initiate necroptosis, and these pathways increase with age.
In Aim 2, we determine the role of these pathways play in the age-related increase in necroptosis using specific inhibitors to each of the pathways. DAMPs released by necroptotic cells induce inflammation through activation of three pathways: the IKK-NF-?B, MAPK-AP1 and inflammasome pathways.
In Aim 2, we will also assess the effect of age on the activation of these pathways in various tissues of WT mice and determine which of the pathways are reduced when necroptosis is reduced/blocked in Ripk3 and Mlkl knockout mice or by necrostatin-1s treatment.
Aim3. Determine the role of necroptosis in aging.
In Aim 3, two groups of mice in which necroptosis has been blocked/reduced will be compared to wildtype mice. Mouse models (e.g., Ripk3 and/or Mlkl knockout mice) will be used that we find block inflammaging in Aim 1. The lifespan, healthspan (e.g., activity, rotarod performance, grip strength, insulin sensitivity, cognition, etc.), and age-associated pathology of these mice will be compared to WT mice. Based on our hypothesis, we predict that reducing/blocking necroptosis will retard aging as shown by improved healthspan, reduced pathology, and increased lifespan. These data would provide the first direct evidence that necroptosis induced inflammation is directly involved in aging and that chronic, low-grade inflammation plays a role in aging and the increase in pathology and age-related diseases.
The demographics of the veteran population clearly demonstrate that illnesses afflicting the elderly are a major concern of the Department of Veterans Affairs. Currently, the number of veterans over 50 years of age represents over 70% of the total veteran population. It is noteworthy that the number of veterans 80 years and older is projected to increase 500% over the next 20 years. Research has shown that chronic, low-grade inflammation increases dramatically with age and that inflammation is a major risk factor for a variety of age-related diseases that affect elderly veterans. The research in this application will study a novel mechanism, necroptosis that might be responsible for the chronic inflammation that occurs with age in elderly veterans. Using genetically modified mice and a pharmacological agent that block necroptosis, we will determine if inflammation arising from necroptosis plays a role in aging by measuring the lifespan, healthspan, and pathology of the mice.
