Effect of alternating nicotinamide phosphoribosyltransferase expression levels on mitophagy in Alzheimer's disease mouse models.

AD is the abbreviation for Alzheimer's Disease, which is a neurodegenerative disorder that features progressive dysfunction in cognition. Previous research has reported that mitophagy impairment and mitochondrial dysfunction have been crucial factors in the AD's pathogenesis. More recently, literature has emerged which offers findings suggesting that the nicotinamide adenine dinucleotide (short for NAD+) augmentation eliminates the defective mitochondria and restores mitophagy. Meanwhile, as an enzyme which is rate-limiting, the Nicotinamide phosphoribosyltransferase, or NAMPT, is part of the salvage pathway of NAD+ synthesis. Therefore, the aim of the research project has been to produce proof for how the NAMPT-NAD +-silent information-regulated transcription factors1/3 (short for SIRT1/3) axis function in mediating mitophagy in APP/PS1 mice aged six months. The results revealed that the NAMPT-NAD+-SIRT1/3 axis in the APP/PS1 mice's hippocampus was considerably declined. Surprisingly, P7C3 (an NAMPT activator) noticeably promoted the NAD+-SIRT1/3 axis, improved mitochondrial structure and function, enhanced mitophagy activity along with the ability of learning and memory. While FK866 (an NAMPT inhibitor) reversed the decreased NAD+-SIRT1/3 axis, and even exacerbated Aß plaque deposition level in the APP/PS1 mice's hippocampus. The findings observed in this study indicate two main points: avoiding downregulation of the NAMPT activity can prevent AD-related mitophagy impairment; on the other hand, NAMPT characterizes a potential therapeutic intervention regarding AD pathogenesis.

A Multifunctional Therapeutic Strategy Using P7C3 as A Countermeasure Against Bone Loss and Fragility in An Ovariectomized Rat Model of Postmenopausal Osteoporosis.

By 2060, an estimated one in four Americans will be elderly. Consequently, the prevalence of osteoporosis and fragility fractures will also increase. Presently, no available intervention definitively prevents or manages osteoporosis. This study explores whether Pool 7 Compound 3 (P7C3) reduces progressive bone loss and fragility following the onset of ovariectomy (OVX)-induced osteoporosis. Results confirm OVX-induced weakened, osteoporotic bone together with a significant gain in adipogenic body weight. Treatment with P7C3 significantly reduced osteoclastic activity, bone marrow adiposity, whole-body weight gain, and preserved bone area, architecture, and mechanical strength. Analyses reveal significantly upregulated platelet derived growth factor-BB and leukemia inhibitory factor, with downregulation of interleukin-1 R6, and receptor activator of nuclear factor kappa-B (RANK). Together, proteomic data suggest the targeting of several key regulators of inflammation, bone, and adipose turnover, via transforming growth factor-beta/SMAD, and Wingless-related integration site/be-catenin signaling pathways. To the best of the knowledge, this is first evidence of an intervention that drives against bone loss via RANK. Metatranscriptomic analyses of the gut microbiota show P7C3 increased Porphyromonadaceae bacterium, Candidatus Melainabacteria, and Ruminococcaceae bacterium abundance, potentially contributing to the favorable inflammatory, and adipo-osteogenic metabolic regulation observed. The results reveal an undiscovered, and multifunctional therapeutic strategy to prevent the pathological progression of OVX-induced bone loss.

Higher concentration of P7C3 than required for neuroprotection suppresses renal cell carcinoma growth and metastasis.

Background: P7C3 is a novel compound that has been widely applied in neurodegenerative diseases and nerve injury repair. Here, we show that higher concentrations of P7C3 than are required for in vivo neuroprotection have the novel function of suppressing renal cell carcinoma (RCC) proliferation and metastasis. Methods: Colony formation, CCK-8 and EdU assay were applied to evaluate RCC cell proliferation. Wound healing and transwell assay were used to measure RCC cell migration and invasion. Flow cytometry assay was employed to detect RCC cell apoptosis and cell cycle. qRT-PCR assay was carried out to measure ribonucleotide reductase subunit M2 (RRM2) mRNA expression level, while western blot assay was utilized to detect the expression level of target proteins. RCC cell growth in vivo was determined by xenografts in mice. Results: We observed that high concentrations of P7C3 could restrain the proliferation and metastasis of RCC cells and promote cell apoptosis. Mechanistically, this new effect of higher dose of P7C3 was associated with reduced expression of RRM2, and the beneficial efficacy of P7C3 in RCC was blocked when suppression of RRM2 was prevented. When RRM2 suppression was permitted, the cGAS-STING pathway was activated by virtue of RRM2/Bcl-2/Bax signaling. Lastly, intraperitoneal injection of this high level of P7C3 in mice potently inhibited tumor growth. Conclusion: In conclusion, we show here that P7C3 that exerts an anti-cancer effect in RCC. Our study indicated that P7C3 might act as a novel drug for RCC in the future. The regulatory signal pathway RRM2/Bcl-2/BAX/cGAS-STING might present novel insight to the potential mechanism of RCC development.

P7C3-A20 treats traumatic brain injury in rats by inhibiting excessive autophagy and apoptosis.

Traumatic brain injury is a severe health problem leading to autophagy and apoptosis in the brain. 3,6-Dibromo-beta-fluoro-N-(3-methoxyphenyl)-9H-carbazole-9-propanamine (P7C3-A20) can be neuroprotective in various diseases, including ischemic stroke and neurodegenerative diseases. However, whether P7C3-A20 has a therapeutic effect on traumatic brain injury and its possible molecular mechanisms are unclear. Therefore, in the present study, we investigated the therapeutic effects of P7C3-A20 on traumatic brain injury and explored the putative underlying molecular mechanisms. We established a traumatic brain injury rat model using a modified weight drop method. P7C3-A20 or vehicle was injected intraperitoneally after traumatic brain injury. Severe neurological deficits were found in rats after traumatic brain injury, with deterioration in balance, walking function, and learning memory. Furthermore, hematoxylin and eosin staining showed significant neuronal cell damage, while terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining indicated a high rate of apoptosis. The presence of autolysosomes was observed using transmission electron microscope. P7C3-A20 treatment reversed these pathological features. Western blotting showed that P7C3-A20 treatment reduced microtubule-associated protein 1 light chain 3-II (LC3-II) autophagy protein, apoptosis-related proteins (namely, Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 [BNIP3], and Bcl-2 associated x protein [Bax]), and elevated ubiquitin-binding protein p62 (p62) autophagy protein expression. Thus, P7C3-A20 can treat traumatic brain injury in rats by inhibiting excessive autophagy and apoptosis.

P7C3 Ameliorates Bone Loss by Inhibiting Osteoclast Differentiation and Promoting Osteogenesis.

Bone homeostasis, the equilibrium between bone resorption and formation, is essential for maintaining healthy bone tissue in adult humans. Disruptions of this process can lead to pathological conditions such as osteoporosis. Dual-targeted agents, capable of inhibiting excessive bone resorption and stimulating bone formation, are being explored as a promising strategy for developing new treatments to address osteoporosis. In this study, we investigated the effects of P7C3 on bone remodeling and its potential therapeutic role in osteoporosis treatment in mice. Specifically, P7C3 can remarkably suppress receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast differentiation in bone marrow macrophages via the Akt-NF-κB-NFATc1 signaling pathway. Additionally, RNA sequencing (RNAseq) analysis revealed that P7C3 promoted osteoblast differentiation and function through the Wnt/ß-catenin signaling pathway, thereby enhancing bone formation. Furthermore, µCT analysis and histological examination of bone tissues from P7C3-treated mice showed attenuation of both Ti-induced bone erosion and ovariectomy (OVX)-induced bone loss. These findings suggest that P7C3 may have a novel function in bone remodeling and may be a promising therapeutic agent for the treatment of osteoporosis. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Development and testing of nanoparticles delivery for P7C3 small molecule using injury models.

The use of nanoparticles (NPs) has emerged as a potential tool for safe and effective drug delivery. In the present study, we developed small molecule P7C3-based NPs and tested its efficacy and toxicity along with the tissue specific aptamer-modified P7C3 NPs. The P7C3 NPs were prepared using poly (D, L-lactic-co-glycolic acid) carboxylic acid (PLGA-COOH) polymer, were conjugated with skeletal muscle-specific RNA aptamer (A01B P7C3 NPs) and characterized for its cytotoxicity, cellular uptake, and wound healing in vitro. The A01B P7C3 NPs demonstrated an encapsulation efficiency of 30.2 ± 2.6%, with the particle size 255.9 ± 4.3 nm, polydispersity index of 0.335 ± 0.05 and zeta potential of + 10.4 ± 1.8mV. The FTIR spectrum of P7C3 NPs displayed complete encapsulation of the drug in the NPs. The P7C3 NPs and A01B P7C3 NPs displayed sustained drug release in vitro for up to 6 days and qPCR analysis confirmed A01B aptamer binding to P7C3 NPs. The C2C12 cells viability assay displayed no cytotoxic effects of all 3 formulations at 48 and 72 h. In addition, the cellular uptake of A01B P7C3 NPs in C2C12 myoblasts demonstrated higher uptake. In vitro assay mimicking wound healing showed improved wound closure with P7C3 NPs. In addition, P7C3 NPs significantly decreased TNF-α induced NF-κB activity in the C2C12/NF-κB reporter cells after 24-hour treatment. The P7C3 NPs showed 3-4-fold higher efficacy compared to P7C3 solutions in both wound-closure and inflammation assays in C2C12 cells. Furthermore, the P7C3 NPs showed 3-4-fold higher efficacy in reducing the infarct size and protected mouse hearts from ex vivo ischemia-reperfusion injury. Overall, this study demonstrates the safe and effective delivery of P7C3 NPs.

Application of P7C3 Compounds to Investigating and Treating Acute and Chronic Traumatic Brain Injury.

Traumatic brain injury (TBI) is a leading worldwide cause of disability, and there are currently no medicines that prevent, reduce, or reverse acute or chronic neurodegeneration in TBI patients. Here, we review the target-agnostic discovery of nicotinamide adenine dinucleotide (NAD+)/NADH-stabilizing P7C3 compounds through a phenotypic screen in mice and describe how P7C3 compounds have been applied to advance understanding of the pathophysiology and potential treatment of TBI. We summarize how P7C3 compounds have been shown across multiple laboratories to mitigate disease progression safely and effectively in a broad range of preclinical models of disease related to impaired NAD+/NADH metabolism, including acute and chronic TBI, and note the reported safety and neuroprotective efficacy of P7C3 compounds in nonhuman primates. We also describe how P7C3 compounds facilitated the recent first demonstration that chronic neurodegeneration 1 year after TBI in mice, the equivalent of many decades in people, can be reversed to restore normal neuropsychiatric function. We additionally review how P7C3 compounds have facilitated discovery of new pathophysiologic mechanisms of neurodegeneration after TBI. This includes the role of rapid TBI-induced tau acetylation that drives axonal degeneration, and the discovery of brain-derived acetylated tau as the first blood-based biomarker of neurodegeneration after TBI that directly correlates with the abundance of a therapeutic target in the brain. We additionally review the identification of TBI-induced tau acetylation as a potential mechanistic link between TBI and increased risk of Alzheimer's disease. Lastly, we summarize historical accounts of other successful phenotypic-based drug discoveries that advanced medical care without prior recognition of the specific molecular target needed to achieve the desired therapeutic effect.

Anti-inflammatory Therapy Protects Spiral Ganglion Neurons After Aminoglycoside Antibiotic-Induced Hair Cell Loss.

Destruction of cochlear hair cells by aminoglycoside antibiotics leads to gradual death of the spiral ganglion neurons (SGNs) that relay auditory information to the brain, potentially limiting the efficacy of cochlear implants. Because the reasons for this cochlear neurodegeneration are unknown, there are no neuroprotective strategies for patients. To investigate this problem, we assessed transcriptomic changes in the rat spiral ganglion following aminoglycoside antibiotic (kanamycin)-induced hair cell destruction. We observed selectively increased expression of immune and inflammatory response genes and increased abundance of activated macrophages in spiral ganglia by postnatal day 32 in kanamycin-deafened rats, preceding significant SGN degeneration. Treatment with the anti-inflammatory medications dexamethasone and ibuprofen diminished long-term SGN degeneration. Ibuprofen and dexamethasone also diminished macrophage activation. Efficacy of ibuprofen treatment was augmented by co-administration of the nicotinamide adenine dinucleotide-stabilizing agent P7C3-A20. Our results support a critical role of neuroinflammation in SGN degeneration after aminoglycoside antibiotic-mediated cochlear hair cell loss, as well as a neuroprotective strategy that could improve cochlear implant efficacy.

Nampt activator P7C3 ameliorates diabetes and improves skeletal muscle function modulating cell metabolism and lipid mediators.

BACKGROUND: Nicotinamide phosphoribosyltransferase (Nampt), a key enzyme in NAD salvage pathway is decreased in metabolic diseases, and its precise role in skeletal muscle function is not known. We tested the hypothesis, Nampt activation by P7C3 (3,6-dibromo-α-[(phenylamino)methyl]-9H-carbazol-9-ethanol) ameliorates diabetes and muscle function. METHODS: We assessed the functional, morphometric, biochemical, and molecular effects of P7C3 treatment in skeletal muscle of type 2 diabetic (db/db) mice. Nampt+/- mice were utilized to test the specificity of P7C3. RESULTS: Insulin resistance increased 1.6-fold in diabetic mice compared with wild-type mice and after 4 weeks treatment with P7C3 rescued diabetes (P < 0.05). In the db-P7C3 mice fasting blood glucose levels decreased to 0.96-fold compared with C57Bl/6J wild-type naïve control mice. The insulin and glucose tolerance tests blood glucose levels were decreased to 0.6-fold and 0.54-folds, respectively, at 120 min along with an increase in insulin secretion (1.76-fold) and pancreatic ß-cells (3.92-fold) in db-P7C3 mice. The fore-limb and hind-limb grip strengths were increased to 1.13-fold and 1.17-fold, respectively, together with a 14.2-fold increase in voluntary running wheel distance in db-P7C3 mice. P7C3 treatment resulted in a 1.4-fold and 7.1-fold increase in medium-sized and larger-sized myofibres cross-sectional area, with a concomitant 0.5-fold decrease in smaller-sized myofibres of tibialis anterior (TA) muscle. The transmission electron microscopy images also displayed a 1.67-fold increase in myofibre diameter of extensor digitorum longus muscle along with 2.9-fold decrease in mitochondrial area in db-P7C3 mice compared with db-Veh mice. The number of SDH positive myofibres were increased to 1.74-fold in db-P7C3 TA muscles. The gastrocnemius and TA muscles displayed a decrease in slow oxidative myosin heavy chain type1 (MyHC1) myofibres expression (0.46-fold) and immunostaining (6.4-fold), respectively. qPCR analysis displayed a 2.9-fold and 1.3-fold increase in Pdk4 and Cpt1, and 0.55-fold and 0.59-fold decrease in Fgf21 and 16S in db-P7C3 mice. There was also a 3.3-fold and 1.9-fold increase in Fabp1 and CD36 in db-Veh mice. RNA-seq differential gene expression volcano plot displayed 1415 genes to be up-regulated and 1726 genes down-regulated (P < 0.05) in db-P7C3 mice. There was 1.02-fold increase in serum HDL, and 0.9-fold decrease in low-density lipoprotein/very low-density lipoprotein ratio in db-P7C3 mice. Lipid profiling of gastrocnemius muscle displayed a decrease in inflammatory lipid mediators n-6; AA (0.83-fold), and n-3; DHA (0.69-fold) and EPA (0.81-fold), and a 0.66-fold decrease in endocannabinoid 2-AG and 2.0-fold increase in AEA in db-P7C3 mice. CONCLUSIONS: Overall, we demonstrate that P7C3 activates Nampt, improves type 2 diabetes and skeletal muscle function in db/db mice.

The Neurogenic Compound P7C3 Regulates the Aerobic Glycolysis by Targeting Phosphoglycerate Kinase 1 in Glioma.

BACKGROUND: P7C3 is a neurogenic compound that exhibits neuroprotective properties in neural cells. However, its target proteins and effects in glioma are unknown. METHODS: The candidate P7C3 target proteins were analyzed using a human protein microarray containing 23136 human proteins. A streptavidin agarose affinity assay was used to verify the direct interaction between P7C3 and phosphoglycerate kinase 1 (PGK1). Mass spectrometry was used to identify the binding sites of PGK1 for P7C3 binding. Seahorse XF96 extracellular flux analyzer was used to measure the cell oxygen consumption rate and extracellular acidification rate. Glycolytic metabolites were measured using the related kits. Protein level was detected by western blotting and immunohistochemical staining. Autophagy was analyzed using a transmission electron microscope and western blotting. The malignancy of tumor progression in vitro and in vivo was analyzed based on cell viability, apoptosis and proliferation, migration and invasion, and xenograft model. Glial cells were marked by antibodies via immunohistochemical staining. RESULTS: The human protein microarray identified 577 candidate P7C3 target proteins. The global profile of P7C3 target proteins indicated that P7C3 regulates glycolysis. Metabolic experiments confirmed that P7C3 regulates aerobic glycolysis in glioma cells. The underlying mechanism of P7C3 was found to be direct targeting PGK1 at lysine residues and asparagine residues, and the specific P7C3-PGK1 interaction led to decreased protein level and total intracellular kinase activity of PGK1. The Cancer Genome Atlas and Chinese Glioma Genome Atlas databases indicated that the mRNA level of PGK1 is significantly increased in high-grade glioma, and the abnormally high mRNA level of PGK1 is associated with a poor prognosis in patients with glioma, suggesting that PGK1 is a promising target for glioma therapy. The inhibition of PGK1 and the subsequent suppression of aerobic glycolysis caused by P7C3 inhibited the malignant growth of glioma in vitro and in vivo. Furthermore, P7C3 did not damage normal glial cells under concentration, which exhibit an inhibitory effect on gliomas. CONCLUSIONS: This study revealed that P7C3 suppresses glioma by regulating aerobic glycolysis via directly targeting PGK1. Furthermore, we identified the P7C3 target proteins for the first time which is expected to provide scientific clues for future studies.

Reducing acetylated tau is neuroprotective in brain injury.

Traumatic brain injury (TBI) is the largest non-genetic, non-aging related risk factor for Alzheimer's disease (AD). We report here that TBI induces tau acetylation (ac-tau) at sites acetylated also in human AD brain. This is mediated by S-nitrosylated-GAPDH, which simultaneously inactivates Sirtuin1 deacetylase and activates p300/CBP acetyltransferase, increasing neuronal ac-tau. Subsequent tau mislocalization causes neurodegeneration and neurobehavioral impairment, and ac-tau accumulates in the blood. Blocking GAPDH S-nitrosylation, inhibiting p300/CBP, or stimulating Sirtuin1 all protect mice from neurodegeneration, neurobehavioral impairment, and blood and brain accumulation of ac-tau after TBI. Ac-tau is thus a therapeutic target and potential blood biomarker of TBI that may represent pathologic convergence between TBI and AD. Increased ac-tau in human AD brain is further augmented in AD patients with history of TBI, and patients receiving the p300/CBP inhibitors salsalate or diflunisal exhibit decreased incidence of AD and clinically diagnosed TBI.

Nicotinamide Phosphoribosyltransferase as a Key Molecule of the Aging/Senescence Process.

Aging is a phenomenon underlined by complex molecular and biochemical changes that occur over time. One of the metabolites that is gaining strong research interest is nicotinamide adenine dinucleotide, NAD+, whose cellular level has been shown to decrease with age in various tissues of model animals and humans. Administration of NAD+ precursors, nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), to supplement NAD+ production through the NAD+ salvage pathway has been demonstrated to slow down aging processes in mice. Therefore, NAD+ is a critical metabolite now understood to mitigate age-related tissue function decline and prevent age-related diseases in aging animals. In human clinical trials, administration of NAD+ precursors to the elderly is being used to address systemic age-associated physiological decline. Among NAD+ biosynthesis pathways in mammals, the NAD+ salvage pathway is the dominant pathway in most of tissues, and NAMPT is the rate limiting enzyme of this pathway. However, only a few activators of NAMPT, which are supposed to increase NAD+, have been developed so far. In this review, we will focus on the importance of NAD+ and the possible application of an activator of NAMPT to promote successive aging.

Antinociceptive effect of intrathecal P7C3 via GABA in a rat model of inflammatory pain.

The recently identified molecule P7C3 has been highlighted in the field of pain research. We examined the effect of intrathecal P7C3 in tissue injury pain evoked by formalin injection and determined the role of the GABA system in the activity of P7C3 at the spinal level. Male Sprague-Dawley rats with intrathecal catheters implanted for experimental drug delivery were studied. The effects of intrathecal P7C3 and nicotinamide phosphoribosyltransferase (NAMPT) administered 10 min before the formalin injection were examined. Animals were pretreated with bicuculline, a GABA-A receptor antagonist; saclofen, a GABA-B receptor antagonist; L-allylglycine, a glutamic acid decarboxylase (GAD) blocker; and CHS 828, an NAMPT inhibitor; to observe involvement in the effects of P7C3. The effects of P7C3 alone and the mixture of P7C3 with GABA receptor antagonists on KCl-induced calcium transients were examined in rat dorsal root ganglion (DRG) neurons. The expression of GAD and the concentration of GABA in the spinal cord were evaluated. Intrathecal P7C3 and NAMPT produced an antinociceptive effect in the formalin test. Intrathecal bicuculline, saclofen, L-allylglycine, and CHS 828 reversed the antinociception of P7C3 in both phases. P7C3 decreased the KCl-induced calcium transients in DRG neurons. Both bicuculline and saclofen reversed the blocking effect of P7C3. The levels of GAD expression and GABA concentration decreased after formalin injection and were increased by P7C3. These results suggest that P7C3 increases GAD activity and then increases the GABA concentration in the spinal cord, which in turn may act on GABA receptors causing the antinociceptive effect against pain evoked by formalin injection.

Maternal P7C3-A20 Treatment Protects Offspring from Neuropsychiatric Sequelae of Prenatal Stress.

Aims: Impaired embryonic cortical interneuron development from prenatal stress is linked to adult neuropsychiatric impairment, stemming in part from excessive generation of reactive oxygen species in the developing embryo. Unfortunately, there are no preventive medicines that mitigate the risk of prenatal stress to the embryo, as the underlying pathophysiologic mechanisms are poorly understood. Our goal was to interrogate the molecular basis of prenatal stress-mediated damage to the embryonic brain to identify a neuroprotective strategy. Results: Chronic prenatal stress in mice dysregulated nicotinamide adenine dinucleotide (NAD+) synthesis enzymes and cortical interneuron development in the embryonic brain, leading to axonal degeneration in the hippocampus, cognitive deficits, and depression-like behavior in adulthood. Offspring were protected from these deleterious effects by concurrent maternal administration of the NAD+-modulating agent P7C3-A20, which crossed the placenta to access the embryonic brain. Prenatal stress also produced axonal degeneration in the adult corpus callosum, which was not prevented by maternal P7C3-A20. Innovation: Prenatal stress dysregulates gene expression of NAD+-synthesis machinery and GABAergic interneuron development in the embryonic brain, which is associated with adult cognitive impairment and depression-like behavior. We establish a maternally directed treatment that protects offspring from these effects of prenatal stress. Conclusion: NAD+-synthesis machinery and GABAergic interneuron development are critical to proper embryonic brain development underlying postnatal neuropsychiatric functioning, and these systems are highly susceptible to prenatal stress. Pharmacologic stabilization of NAD+ in the stressed embryonic brain may provide a neuroprotective strategy that preserves normal embryonic development and protects offspring from neuropsychiatric impairment. Antioxid. Redox Signal. 35, 511-530.

The Small Molecule P7C3-A20 Exerts Neuroprotective Effects in a Hypoxic-ischemic Encephalopathy Model via Activation of PI3K/AKT/GSK3ß Signaling.

Hypoxic-ischemic encephalopathy (HIE) in neonates can lead to severe long-term disabilities including cerebral palsy and brain injury. The small molecule P7C3-A20 has been shown to exert neuroprotective effects in various disorders such as ischemic stroke and neurodegenerative diseases. However, it is unclear whether P7C3-A20 has therapeutic potential for the treatment of HIE, and the relationship between P7C3-A20 and neuronal apoptosis is unknown. To address these questions, the present study investigated whether P7C3-A20 reduces HI injury in vitro using a PC12 cell oxygen-glucose deprivation (OGD) model and in vivo in postnatal day 7 and 14 rats subjected to HI, along with the underlying mechanisms. We found that treatment with P7C3-A20 (40-100 µM) alleviated OGD-induced apoptosis in PC12 cells. In HI model rats, treatment with 5 or 10 mg/kg P7C3-A20 reduced infarct volume; reversed cell loss in the cortex and hippocampus and improved motor function without causing neurotoxicity. The neuroprotective effects were abrogated by treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. These results demonstrate that P7C3-A20 exerts neuroprotection by activating PI3K/protein kinase B/glycogen synthase kinase 3ß signaling and can potentially be used to prevent brain injury in neonates following HIE.

Neurotherapeutic capacity of P7C3 agents for the treatment of Traumatic Brain Injury.

Traumatic brain injury (TBI) is a significant public health problem around the world. A promising area of research is the characterization of small, drug-like molecules that have potent clinical properties. One pharmacotherapeutic agent in particular, an aminopropyl carbazole called P7C3, was discovered using an in vivo screen to identify new agents that augmented the net magnitude of adult hippocampal neurogenesis. P7C3 greatly enhanced neurogenesis by virtue of increasing survival rates of immature neurons. The potent neuroprotective efficacy of P7C3 is likely due to enhanced nicotinamide phosphoribosyltransferase (NAMPT) activity, which supports critical cellular processes. The scaffold of P7C3 was found to have favorable pharmacokinetic properties, good bioavailability, and was nontoxic. Preclinical studies have shown that administration of the P7C3-series of neuroprotective compounds after TBI can rescue and reverse detrimental cellular events leading to improved functional recovery. In several TBI models and across multiple species, P7C3 and its analogues have produced significant neuroprotection, axonal preservation, robust increases in the net magnitude of adult neurogenesis, protection from injury-induced LTP deficits, and improvement in neurological functioning. This review will elucidate the exciting and diverse therapeutic findings of P7C3 administration in the presence of a complex and multifactorial set of cellular and molecular challenges brought forth by experimental TBI. The clinical potential and broad therapeutic applicability of P7C3 warrants much needed investigation into whether these remedial effects can be replicated in the clinic. P7C3 may serve as an important step forward in the design, understanding, and implementation of pharmacotherapies for treating patients with TBI. This article is part of the Special Issue entitled "Novel Treatments for Traumatic Brain Injury".

P7C3 Inhibits LPS-Induced Microglial Activation to Protect Dopaminergic Neurons Against Inflammatory Factor-Induced Cell Death in vitro and in vivo.

Parkinson's disease (PD) is the second most common neurodegenerative disorder. Although its pathogenesis remains unclear, growing evidencce suggests that microglia-mediated neuroinflammation contributes greatly to the progression of PD. P7C3, an aminopropyl carbazole, possesses significant neuroprotective effects in several neurodegenerative disease animal models, including PD. In this study, we designed to investigate the effects of P7C3 on neuroinflammation. We showed that P7C3 specially suppressed the expression of lipopolysaccharide (LPS)-induced pro-inflammatory factors but not influenced the anti-inflammatory factors in microglia. The inhibition of the nuclear factor κB (NF-κB) signaling pathway was involved in the mechanisms of the anti-inflammatory effects by P7C3. LPS-induced activation of IκB kinase (IKK), degradation of the inhibitory κB alpha (IκBα) and nuclear translocation of NF-κB can be attenuated by the pretreatment of P7C3 in microglia. Furthermore, in LPS-treated microglia, P7C3-pretreatment decreased the toxicity of conditioned media to MES23.5 cells (a dopaminergic (DA) cell line). Most importantly, the anti-inflammatory effects of P7C3 were observed in LPS-stimulated mouse model. In general, our study demonstrates that P7C3 inhibits LPS-induced microglial activation through repressing the NF-κB pathway both in vivo and in vitro, providing a theoretical basis for P7C3 in anti-inflammation.

The Protective effect of P7C3 against DNA and neuron damage in rat pups with congenital hypothyroidism.

Congenital hypothyroidism (CH) is defined as congenital thyroid hormone deficiency. The aim of this study was to examine the DNA and neuron damage in rat pups with CH and to evaluate the beneficial effects of 3.6-Dibromo-α-[(phenylamino) methyl]-9H-carbazole-9-ethanol (P7C3). Rat pups were assigned to four groups as Group 1: CH, Group 2: CH treated with P7C3, Group 3: CH treated with P7C3 and L-thyroxine, and Group 4: control group. Plasma 8-(OH)DG and neuron-specific enolase (NSE) concentrations were determined in all groups. For histopathological examinations haematoxylin-eosin staining was applied. Increased NSE concentrations were found in the CH group compared to the control group. The 8-(OH)DG concentrations were found to be higher in Group 2 and Group 3 than in the control group. Neuronal degenerations localized in the hippocampus and brain cortex were found in histopathological examinations in Group 1. The distribution of neuronal degeneration was less in Group 2 and Group 3 than Group 1 and lesser in Group 3 than in Group 2. DNA damage might have a role in CH pathogenesis. P7C3 compounds have a protective effect in CH.

Beneficial Effects of Delayed P7C3-A20 Treatment After Transient MCAO in Rats.

Despite ischemic stroke being the fifth leading cause of death in the USA, there are few therapeutic options available. We recently showed that the neuroprotective compound P7C3-A20 reduced brain atrophy, increased neurogenesis, and improved functional recovery when treatment was initiated immediately post-reperfusion after a 90-min middle cerebral artery occlusion (MCAO). In the present study, we investigated a more clinically relevant therapeutic window for P7C3-A20 treatment after ischemic stroke. MCAO rats were administered P7C3-A20 for 1 week, beginning immediately or at a delayed point, 6 h post-reperfusion. Delayed P7C3-A20 treatment significantly improved stroke-induced sensorimotor deficits in motor coordination and symmetry, as well as cognitive deficits in hippocampal-dependent spatial learning, memory retention, and working memory. In the cerebral cortex, delayed P7C3-A20 treatment significantly increased tissue sparing 7 weeks after stroke and reduced hemispheric infarct volumes 48 h after reperfusion. Despite no reduction in striatal infarct volumes acutely, there was a significant increase in spared tissue volume chronically. In the hippocampus, only immediately treated P7C3-A20 animals had a significant increase in tissue sparing compared to vehicle-treated stroke animals. This structural protection translated into minimal hippocampal-dependent behavioral improvements with delayed P7C3-A20 treatment. However, all rats treated with delayed P7C3-A20 demonstrated a significant improvement in both sensorimotor tasks compared to vehicle controls, suggesting a somatosensory-driven recovery. These results demonstrate that P7C3-A20 improves chronic functional and histopathological outcomes after ischemic stroke with an extended therapeutic window.