It is fall and the season for American football. For this football fan, watching the game is a bit less enjoyable than it used to be, as more and more information is available about the serious and permanent brain injuries suffered by football players.
In the introduction to a recent paper in the journal Cell, “P7C3 Neuroprotective Chemicals Function by Activating the Rate-Limiting Enzyme in NAD Salvage”, not a word about American football is mentioned.
However, the paper begins, “No substantive therapeutics are available for the treatment of almost any form of disease entailing nerve death” (1). The authors list a range of neurodegenerative disorders such as Huntington’s, Alzheimers and Parkinson’s diseases, as well as ALS or Lou Gherig’s disease. They also note that there are currently no effective treatments for trauma to the brain or peripheral nervous system.
The authors note that a chemical treatment that could interfere with nerve cell death would have a “transformative impact in modern medicine”.
Earlier Research: Compounds selected and administered to mice, searching for formation of new hippocampal nerve cells
Researchers looked for such a chemical treatment through use of a 250,000-compound high-throughput screening center at the University of Texas Southwestern Medical Center. They selected 1,000 chemicals that had drug-like qualities, that preserved chemical diversity and that had no reactive moieties.
The chemicals were then combined into pools of 10 compounds for initial screening, and administered directly into brains of adult mice. Drugs were administered along with daily injections of BrdU (bromodeoxyuridine), used to monitor formation of new hippocampal nerve cells.
Over two years, the 1,000 compounds were whittled down to “a handful” of pooled compounds that augmented neurogenesis. Further division of the pools into individual compounds eventually revealed eight pro-neurogenic chemicals (2). However, pharmacological examination narrowed the pool to only one compound with favorable pharmacological properties.
This one compound was named for Pool seven (P7). The pool contained an aminopropyl carbazole as its active third compound (C3); hence the P7C3 compound name.The researchers found that this compound, whether administered intraperitoneally, intravenously or by oral routes, had favorable half-life and brain penetration. Mice and rats could be safely dosed with P7C3 over long periods of time at concentrations higher than what was needed to stimulate hippocampal neurogenesis, indicating low toxicity to rodents.
It was initially assumed that proneurogenic compounds acted by stimulating the birth of new nerve cells, this was not observed with administration of P7C3. BrdU-positive neurons that increased twofold in number over a week of P7C3 administration were not seen after 24 hours of P7C3 administration. This and other results led to the conclusion that rather than creating new nerve cells by stimulating mitotic division, P7C3 was increasing the number of nerve cells by decreasing their rate of death.
Typically in these animals models, only 10–20% of new nerve cells survived the month-long differentiation process to become properly wired hippocampal neurons. P7C3 administration preserved nearly 50% of the new nerves, increasing survival of the transition to terminal nerve cell differentiation.
In this Report
Here the authors sought to learn the mechanism by which P7C3 acts in preserving new nerve cells, using medicinal chemisty techniques and cultured cells. Their results showed that active variants of P7C3 protected cultured cells from doxorubicin-mediated toxicity selectively, as demonstrated by three findings: 1) doxorubicin reduced intracellular NAD levels, most likely by activation of PARP; 2) active variants of P7C3 protected cultured cells from dox:tox presumably by stimulating a rebound in intracellular levels of NAD; and 3) active P7C3 variants enhanced activity of purified NAMPT enzyme.
The authors concluded that the dox:tox protective effect is directly connected to the ability of P7C3 to enhance the salvage pathway by which nicotinamide is converted to NAD. Their findings are supported by earlier reports that programmed expression of NAMPT enzyme protected mammalian cells from genotoxic stress (3) as well as extending life span of budding yeast (4).
This work adds to a body of exciting research that we hope brings a ray of hope to those suffering from any damage- or disease-related neurological syndrome. It will be interesting to watch the progress in this area of research.
Promega Reagents Used in this Report
Cellular survival assays were performed with the CellTiter-Glo® Luminescent Cell Viability Assay , which was used to measure cellular ATP levels, as a signal of cell viability. Intracellular NAD amounts were determined using the NAD/NADH-Glo™ Assay. Briefly, in the presence of NADH, the enzyme reductase assay reagent reduces the proluciferin substrate to luciferin, and the resulting luminescence allowed quantitation of the NAD analyte.
(These Promega products are labeled For Research Use Only. Not for Use in Diagnostic Procedures.)
References
- P7C3 neuroprotective chemicals function by activating the rate-limiting enzyme in NAD salvage.
- Discovery of a proneurogenic, neuroprotective chemical.
- Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival.
- Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels.
Kari Kenefick
