Oxidative stress induces cell death partially by decreasing both mRNA and protein levels of nicotinamide phosphoribosyltransferase in differentiated PC12 cells

Background. Multiple studies have indicated crucial roles of NAD+ deficiency in several neurological diseases and aging. It is critical to discover the mechanisms underlying the NAD+ deficiency. A decreased level of Nicotinamide phosphoribosyltransferase (Nampt)—an important enzyme in the salvage pathway of NAD+ synthesis—has been found under certain pathological conditions, while the mechanisms underlying the Nampt decrease are unclear. The purpose of this study is to test the hypothesis that oxidative stress can produce decreased Nampt, and to investigate the biological effects of Nampt on NAD+ synthesis and cell survival under both basal and oxidative stress conditions. Methods. We used differentiated PC12 cells as a cellular model to investigate the effects of oxidative stress on the levels of Nampt. Multiple assays, including flow cytometry-based cell death assays and NAD+ assays were conducted. Results. First, oxidative stress can decrease the levels of Nampt mRNA and Nampt protein; second, Nampt plays significant roles in NAD+ synthesis under both basal conditions and oxidative stress conditions; third, Nampt plays critical roles in cell survival under both basal conditions and oxidative stress conditions; and fourth, oxidative stress produced decreased NAD+ levels and cell survival partially by decreasing Nampt. Collectively, our study has indicated that oxidative stress is a pathological factor leading to decreased Nampt, which plays important roles in oxidative stress-produced decreases in NAD+ levels and cell survival. Our findings have indicated major roles of Nampt in maintaining NAD+ levels and cell survival under both basal and oxidative stress conditions.

Hydrogen Gas Attenuates Hypoxic-Ischemic Brain Injury via Regulation of the MAPK/HO-1/PGC-1a Pathway in Neonatal Rats

Neonatal hypoxic-ischemic encephalopathy (HIE) is a leading cause of death in neonates with no effective treatments. Recent advancements in hydrogen (H2) gas offer a promising therapeutic approach for ischemia reperfusion injury; however, the impact of this approach for HIE remains a subject of debate. We assessed the therapeutic effects of H2 gas on HIE and the underlying molecular mechanisms in a rat model of neonatal hypoxic-ischemic brain injury (HIBI). H2 inhalation significantly attenuated neuronal injury and effectively improved early neurological outcomes in neonatal HIBI rats as well as learning and memory in adults. This protective effect was associated with initiation time and duration of sustained H2 inhalation. Furthermore, H2 inhalation reduced the expression of Bcl-2-associated X protein (BAX) and caspase-3 while promoting the expression of Bcl-2, nuclear factor erythroid-2-related factor 2, and heme oxygenase-1 (HO-1). H2 activated extracellular signal-regulated kinase and c-Jun N-terminal protein kinase and dephosphorylated p38 mitogen-activated protein kinase (MAPK) in oxygen-glucose deprivation/reperfusion (OGD/R) nerve growth factor-differentiated PC12 cells. Inhibitors of MAPKs blocked H2-induced HO-1 expression. HO-1 small interfering RNA decreased the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and sirtuin 1 (SIRT1) and reversed the protectivity of H2 against OGD/R-induced cell death. These findings suggest that H2 augments cellular antioxidant defense capacity through activation of MAPK signaling pathways, leading to HO-1 expression and subsequent upregulation of PGC-1α and SIRT-1 expression. Thus, upregulation protects NGF-differentiated PC12 cells from OGD/R-induced oxidative cytotoxicity. In conclusion, H2 inhalation exerted protective effects on neonatal rats with HIBI. Early initiation and prolonged H2 inhalation had better protective effects on HIBI. These effects of H2 may be related to antioxidant, antiapoptotic, and anti-inflammatory responses. HO-1 plays an important role in H2-mediated protection through the MAPK/HO-1/PGC-1α pathway. Our results support further assessment of H2 as a potential therapeutic for neurological conditions in which oxidative stress and apoptosis are implicated.

Simulation of the Gαq /Phospholipase Cβ1 Signaling Pathway Returns Differentiated PC12 Cells to a Stem-like State

ABSTRACTPhospholipase Cβ1 is activated by Gαq to generate calcium signals in response to hormones and neurotransmitters, and is found at high levels in mammalian neuronal tissue. Besides carrying out this key plasma membrane function, PLCβ1 has a cytosolic population that helps, in part, to drive the differentiation of PC12 cells by inhibiting a nuclease that promotes RNA-induced silencing (C3PO). Here, we show that down-regulating PLCβ1 or reducing its cytosolic population by activating Gαq to drive it to the plasma membrane, returns differentiated PC12 cells to an undifferentiated state. In this state, the cells return to a spherical morphology, resume proliferation and express the stem cell transcription factors nanog and Oct4. Similar changes are seen with C3PO down-regulation. This return to a stem-like state is accompanied by shifts in multiple miR populations, such as increased levels of rno-miR-21 and rno-miR-26a. Surprisingly, we find that de-differentiation can also be induced by extended stimulation of the Gαq. In this case, the neurites completely retract over a 10-minute period, and while levels of nanog remain unchanged, the levels of some miRs begin to return to their undifferentiated values. In complementary studies, we followed the real time hydrolysis of a fluorescent-tagged miR in cells where PLCβ1 or C3PO were down-regulated. These samples showed substantial differences in miR processing in cells both the undifferentiated and differentiated states. Taken together, our studies suggest that PLCβ1, through its ability to regulate C3PO and endogenous miR populations, plays a key role in mediating PC12 cell differentiation.

Cocaine- and amphetamine-regulated transcript promoter regulated by nicotine in nerve growth factor-treated PC12 cells

Nicotine and cocaine- and amphetamine-regulated transcripts (CART) have several overlapping functions, such as the regulation of reward, feeding behavior, stress response, and anxiety. Previous studies showed that nicotine regulates CART expression in various brain regions. However, the molecular mechanisms underlying this regulation are not known. This study investigated the regulatory effect of nicotine on promoter activity of the CART gene in PC12 cells, which were differentiated into a neuronal phenotype by nerve growth factor (NGF) treatment. Two vectors containing reporter genes (Gaussia luciferase or mCherry) and the 1,140-bp upstream of the transcriptional start site of the mouse CART gene are used to analyze the CART promoter activity. Transient transfection of PC12 cells with either vector displayed strong promoter activity in both undifferentiated and differentiated PC12 cells. CART promoter activity in the PC12 cell line is increased by forskolin or NGF treatment. In differentiated PC12 cells, exposure to 50 nM nicotine for 6 h increased CART promoter activity. However, treatment with higher nicotine doses for 6 h and treatment with all nicotine doses for 24 h showed no effect. A nicotine concentration of 50 nM is comparable to brain nicotine levels experienced by chronic smokers over long periods of time. Taken together, these data indicate that nicotine may exert some of its actions through the regulation of CART transcription in the brain.