Mycobacterium tuberculosis (Mtb) kills infected macrophages by inhibiting apoptosis and promoting necrosis. The tuberculosis necrotizing toxin (TNT) is a secreted nicotinamide adenine dinucleotide (NAD+) glycohydrolase that induces necrosis in infected macrophages. Here, we show that NAD+ depletion by TNT activates RIPK3 and MLKL, key mediators of necroptosis. Notably, Mtb bypasses the canonical necroptosis pathway since neither TNF-α nor RIPK1 are required for macrophage death. Macrophage necroptosis is associated with depolarized mitochondria and impaired ATP synthesis, known hallmarks of Mtb-induced cell death. These results identify TNT as the main trigger of necroptosis in Mtb-infected macrophages. Surprisingly, NAD+ depletion itself was sufficient to trigger necroptosis in a RIPK3- and MLKL-dependent manner by inhibiting the NAD+ salvage pathway in THP-1 cells or by TNT expression in Jurkat T cells. These findings suggest avenues for host-directed therapies to treat tuberculosis and other infectious and age-related diseases in which NAD+ deficiency is a pathological factor.
DNA repair is essential for cell vitality, cell survival, and cancer prevention, yet cells’ ability to patch up damaged DNA declines with age for reasons not fully understood.
Now, research led by scientists at Harvard Medical School (HMS) reveals a critical step in a molecular chain of events that allows cells to mend their broken DNA.
The findings, to be published March 24 in Science, offer a critical insight into how and why the body’s ability to fix DNA dwindles over time and point to a previously unknown role for the signaling molecule NAD as a key regulator of protein-to-protein interactions in DNA repair. NAD, identified a century ago, is already known for its role as a controller of cell-damaging oxidation.
Additionally, experiments conducted in mice show that treatment with the NAD precursor NMN mitigates age-related DNA damage and wards off DNA damage from radiation exposure.
The scientists caution that the effects of many therapeutic substances are often profoundly different in mice and humans owing to critical differences in biology. However, if affirmed in further animal studies and in humans, the findings can help pave the way to therapies that prevent DNA damage associated with aging and with cancer treatments that involve radiation exposure and some types of chemotherapy, which, along with killing tumors, can cause considerable DNA damage in healthy cells. Human trials with NMN are expected to begin within six months, the researchers said.
“Our results unveil a key mechanism in cellular degeneration and aging, but beyond that they point to a therapeutic avenue to halt and reverse age-related and radiation-induced DNA damage,” said senior author David Sinclair, professor in the Department of Genetics at HMS, co-director of the Paul F. Glenn Center for the Biology of Aging, and professor at the University of New South Wales School of Medicine in Sydney.
A previous study led by Sinclair showed that NMN reversed muscle aging in mice.
A plot with many characters
The investigators started by looking at a cast of proteins and molecules suspected to play a part in the cellular aging process. Some of them were well-known characters, others more enigmatic figures.
The researchers already knew that NAD, which declines steadily with age, boosts the activity of the SIRT1 protein, which delays aging and extends life in yeast, flies, and mice. Both SIRT1 and PARP1, a protein known to control DNA repair, consume NAD in their work.
Another protein, DBC1, one of the most abundant proteins in humans and found across life forms from bacteria to plants and animals, was a far murkier presence. Because DBC1 previously had been shown to inhibit vitality-boosting SIRT1, the researchers suspected DBC1 may also somehow interact with PARP1, given the similar roles PARP1 and SIRT1 play.
“We thought if there is a connection between SIRT1 and DBC1, on one hand, and between SIRT1 and PARP1 on the other, then maybe PARP1 and DBC1 were also engaged in some sort of intracellular game,” said Jun Li, first author on the study and a research fellow in the Department of Genetics at HMS.
They were.
To get a better sense of the chemical relationship among the three proteins, the scientists measured the molecular markers of protein-to-protein interaction inside human kidney cells. DBC1 and PARP1 bound powerfully to each other. However, when NAD levels increased, that bond was disrupted. The more NAD was present inside cells, the fewer molecular bonds PARP1 and DBC1 could form. When researchers inhibited NAD, the number of PARP1-DBC1 bonds went up. In other words, when NAD is plentiful, it prevents DBC1 from binding to PARP1 and meddling with its ability to mend damaged DNA.
What this suggests, the researchers said, is that as NAD declines with age, fewer and fewer NAD molecules are around to stop the harmful interaction between DBC1 and PARP1. The result: DNA breaks go unrepaired and, as these breaks accumulate over time, precipitate cell damage, cell mutations, cell death, and loss of organ function.
Averting mischief
Next, to understand how exactly NAD prevents DBC1 from binding to PARP1, the team homed in on a region of DBC1 known as NHD, a pocket-like structure found in some 80,000 proteins across life forms and species whose function has eluded scientists. The team’s experiments showed that NHD is an NAD binding site and that in DBC1, NAD blocks this specific region to prevent DBC1 from locking in with PARP1 and interfering with DNA repair.
Sinclair said that since NHD is so common across species, the finding suggests that by binding to it, NAD may play a similar role averting harmful protein interactions across many species to control DNA repair and other cell survival processes.
To determine how the proteins interacted beyond the lab dish and in living organisms, the researchers treated young and old mice with the NAD precursor NMN, which makes up half of an NAD molecule. NAD is too large to cross the cell membrane, but NMN can slip across it easily. Once inside the cell, NMN binds to another NMN molecule to form NAD.
As expected, old mice had lower levels of NAD in their livers, lower levels of PARP1, and a greater number of PARP1 with DBC1 stuck to their backs.
After receiving NMN with their drinking water for a week, however, old mice showed marked differences both in NAD levels and PARP1 activity. NAD levels in the livers of old mice shot up to levels similar to those seen in younger mice. The cells of mice treated with NMN also showed increased PARP1 activity and fewer PARP1 and DBC1 molecules binding together. The animals also showed a decline in molecular markers that signal DNA damage.
In a final step, scientists exposed mice to DNA-damaging radiation. Cells of animals pre-treated with NMN showed lower levels of DNA damage. Such mice also didn’t exhibit the typical radiation-induced aberrations in blood counts, such as altered white cell counts and changes in lymphocyte and hemoglobin levels. The protective effect was seen even in mice treated with NMN after radiation exposure.
Taken together, the results shed light on the mechanism behind cellular demise induced by DNA damage. They also suggest that restoring NAD levels by NMN treatment should be explored further as a possible therapy to avert the unwanted side effects of environmental radiation, as well as radiation exposure from cancer treatments.
FDA Study conducted by the FDA highlights the benefits of NAD treatment specifically reduced stress, anxiety, depression, and decreased effects of drug withdrawls.
EDITORIAL Raising NAD in Heart Failure Time to Translate?
It has long been known that cellular NAD levels are a critical regulator of metabolism and bioenergetics. The intracellular NAD pool consists of both oxidized (NAD+) and reduced forms (NADH). NAD+ is the main hydride acceptor in intermediary metabolism. Electrons derived from substrate catabolism are carried by NADH and used for oxidative phosphorylation and biosynthetic reactions. These reduction-oxidation reactions are not only essential for mitochondrial function and cell metabolism but also serve as important modulators of cell signaling.1,2 NAD+ functions as a cosubstrate for sirtuin deacylases, ADP-ribose transferases, and cyclic ADP-ribose synthases that govern posttranslational modification of proteins, DNA repair, and inflammatory responses.
In a pre-clinical model, researchers find that NAD+ can trigger a response that protects against lethal infections
BRIGHAM AND WOMEN’S HOSPITAL
Researchers at Brigham and Women’s Hospital (BWH) have discovered a new cellular and molecular pathway that regulates CD4+ T cell response–a finding that may lead to new ways to treat diseases that result from alterations in these cells. Their discovery, published online in the Journal of Allergy and Clinical Immunology, shows that administering nicotinamide adenine dinucleotide (NAD+), a natural molecule found in all living cells, shuts off the capacity of dendritic cells and macrophages to dictate CD4+ T fate. Researchers found that NAD+ administration regulated CD4+ T cells via mast cells (MCs), cells that have been mainly described in the context of allergy, exclusively.
“This is a novel cellular and molecular pathway that is distinct from the two major pathways that were previously known. Since it is distinct and since it has the ability to regulate the immune system systemically, we can use it as an alternative to bypass the current pathways,” said Abdallah ElKhal, PhD, BWH Department of Surgery, senior study author.
CD4+ T helper cells and dendritic cells play a central role in immunity. Alterations or aberrant dendritic cells and T cell responses can lead to many health conditions including autoimmune diseases, infections, allergy, primary immunodeficiencies and cancer.
As of today, two major pathways have been described to regulate CD4+ T cell response. The first pathway was described by Peter C. Doherty and Rolf M. Zinkernagel (1996 Nobel prize winners) showing the requirement of MHC-TCR signaling machinery. More recently, a second mechanism involving the Pathogen or Damage Associated Molecular Patterns (PAMPs or DAMPs) was unraveled by Bruce A. Beutler and Jules A. Hoffmann (2011 Nobel Prize winners). Of importance, both pathways require antigen presenting cells (APCs) in particular dendritic cells (DCs) or macrophages (Mφ). Elkhal’s novel pathway is distinct from the two previous ones and may offer a path forward for novel therapeutic approaches.
For the current study, BWH researchers performed pre-clinical trials using an experimental infection model. They showed that mast cell-mediated CD4+ T cell response protects against lethal doses of infection (Listeria monocytogenes). Mice treated with NAD+ had a dramatically increased survival rate when compared to the non-treated group.
“Collectively, our study unravels a novel cellular and molecular pathway that regulates innate and adaptive immunity via MCs, exclusively, and underscores the therapeutic potential of NAD+ in the context of a myriad of diseases including autoimmune diseases, hemophilia, primary immunodeficiencies and antimicrobial resistance,” said Elkhal.
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This work was supported by the National Institutes of Health R01NS073635 and R01MH110438, R01 HL096795 and U01 HL126497, R01AG039449. Co-authors were supported by the Swiss Society of Cardiac Surgery, FIS-ISCIII (grant PI10/02 511) and Fundación Ramón Areces (CIVP16A1843).
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A natural molecule delays disease onset and reverses disease progression
By MARJORIE MONTEMAYOR-QUELLENBERG | October 14, 2014
The main function of the immune system is to protect against diseases and infections. For unknown reasons our immune system attacks healthy cells, tissues and organs in a process called autoimmunity, which can result in diseases such as multiple sclerosis, Type 1 diabetes, lupus or rheumatoid arthritis. There are currently no existing cures for these diseases.
Now, in a new study by researchers at Brigham and Women’s Hospital, a potential treatment may be on the horizon. Researchers found that NAD+, a natural molecule found in living cells, plants and food, protects against autoimmune diseases by altering the immune response and turning “destructive” cells into “protective” cells. The molecule is also able to reverse disease progression by restoring damaged tissue caused by the autoimmunity process.
“Our study is the first to show that NAD+ can tune the immune response and restore tissue integrity by activating stem cells,” said Abdallah ElKhal, HMS instructor in surgery at Brigham and Women’s Division of Transplant Surgery and Transplantation Surgery Research Laboratory and senior study author. “These findings are very novel and may serve for the development of novel therapeutics.”
The scientists performed preclinical trials using experimental autoimmune encephalomyelitis, a preclinical model for human multiple sclerosis. They showed that NAD+ can block acute or chronic inflammation by regulating how immune cells, called CD4+ T cells, differentiate. Mice receiving CD4+ T cells along with NAD+ present had a significant delayed onset of disease, as well as a less severe form, therefore demonstrating the molecule’s protective properties.
“This is a universal molecule that can potentially treat not only autoimmune diseases but other acute or chronic conditions such as allergy, chronic obstructive pulmonary disease, sepsis and immunodeficiency,” said Stefan G. Tullius, HMS professor of surgery, Brigham and Women’s Hospital’s chief of Transplant Surgery, director of Transplantation Surgery Research and lead study author.
Moreover, the researchers demonstrated that NAD+ can restore tissue integrity which may benefit patients that have advanced tissue damage caused by autoimmune diseases. In terms of next steps, ElKhal notes that the lab is currently testing additional pathways and the clinical potential of NAD+.
“Since this is a natural molecule found in all living cells, including our body, we hope that it will be well-tolerated by patients,” said ElKhal. “Thus, we hope that its potential as a powerful therapeutic agent for the treatment of autoimmune diseases will facilitate its use in future clinical trials.”
The Transplant Surgery Research Laboratory and Dr. ElKhal’s work is supported by the National Institutes of Health and the Carlos Slim Foundation.
