Mitofusin-2 mediates cannabidiol-induced neuroprotection against cerebral ischemia in rats.

Cannabidiol (CBD) reportedly exerts protective effects against many psychiatric disorders and neurodegenerative diseases, but the mechanisms are poorly understood. In this study, we explored the molecular mechanism of CBD against cerebral ischemia. HT-22 cells or primary cortical neurons were subjected to oxygen-glucose deprivation insult followed by reoxygenation (OGD/R). In both HT-22 cells and primary cortical neurons, CBD pretreatment (0.1, 0.3, 1 µM) dose-dependently attenuated OGD/R-induced cell death and mitochondrial dysfunction, ameliorated OGD/R-induced endoplasmic reticulum (ER) stress, and increased the mitofusin-2 (MFN2) protein level in HT-22 cells and primary cortical neurons. Knockdown of MFN2 abolished the protective effects of CBD. CBD pretreatment also suppressed OGD/R-induced binding of Parkin to MFN2 and subsequent ubiquitination of MFN2. Overexpression of Parkin blocked the effects of CBD in reducing MFN2 ubiquitination and reduced cell viability, whereas overexpressing MFN2 abolished Parkin's detrimental effects. In vivo experiments were conducted on male rats subjected to middle cerebral artery occlusion (MCAO) insult, and administration of CBD (2.5, 5 mg · kg-1, i.p.) dose-dependently reduced the infarct volume and ER stress in the brains. Moreover, the level of MFN2 within the ischemic penumbra of rats was increased by CBD treatment, while the binding of Parkin to MFN2 and the ubiquitination of MFN2 was decreased. Finally, short hairpin RNA against MFN2 reversed CBD's protective effects. Together, these results demonstrate that CBD protects brain neurons against cerebral ischemia by reducing MFN2 degradation via disrupting Parkin's binding to MFN2, indicating that MFN2 is a potential target for the treatment of cerebral ischemia.

Roflumilast, a cyclic nucleotide phosphodiesterase 4 inhibitor, protects against cerebrovascular endothelial injury following cerebral ischemia/reperfusion by activating the Notch1/Hes1 pathway.

The loss of tight junction (TJ) and adherens junction (AJ) proteins leads to the damage of the blood-brain barrier (BBB) during cerebral ischemia. Inhibition of cyclic nucleotide phosphodiesterase 4 (PDE4) by roflumilast (Roflu) protects against ischemic stroke-induced neuronal damage. However, the effects of Roflu on vascular endothelial injury and BBB integrity remain unknown. Here, we investigated whether and how Roflu protects against cerebrovascular endothelial injury caused by cerebral ischemia/reperfusion. We demonstrated that PDE4B knocking-down increased the expression of TJ and AJ proteins in human brain microvascular endothelial cells (HBMECs) subjected to oxygen-glucose deprivation reperfusion (OGD/R). Inhibition of PDE4 by Roflu (1.0 µM) showed similar effects as PDE4B knocking-down. We then found that Roflu activated Notch1/Hairy and enhancer of split 1 (Hes1) signaling. Consistently, the effects of Roflu on TJ and AJ proteins were reversed by the γ-secretase inhibitor DAPT or Hes1 knocking-down. Furthermore, Roflu (1.0 mg/kg) improved neurobehavioral outcomes and ameliorated BBB disruption in rats following ischemic stroke. Roflu also increased the levels of TJ proteins and AJ proteins in vivo. Collectively, these data suggest that Roflu is a promising compound for the prevention of BBB damage. The protective effects of Roflu are mediated through activation of the Notch1/Hes1 pathway.

Roflupram protects against rotenone-induced neurotoxicity and facilitates α-synuclein degradation in Parkinson's disease models.

We have previously shown that roflupram (ROF) protects against MPP+-induced neuronal damage in models of Parkinson's disease (PD). Since impaired degradation of α-synuclein (α-syn) is one of the key factors that lead to PD, here we investigated whether and how ROF affects the degradation of α-syn in rotenone (ROT)-induced PD models in vivo and in vitro. We showed that pretreatment with ROF (10 µM) significantly attenuated cell apoptosis and reduced the level of α-syn in ROT-treated SH-SY5Y cells. Furthermore, ROF significantly enhanced the lysosomal function, as evidenced by the increased levels of mature cathepsin D (CTSD) and lysosomal-associated membrane protein 1 (LAMP1) through increasing NAD+/NADH and the expression of sirtuin 1 (SIRT1). Pretreatment with an SIRT1 inhibitor selisistat (SELI, 10 µM) attenuated the neuroprotection of ROF, ROF-reduced expression of α-syn, and ROF-increased expression levels of LAMP1 and mature CTSD. Moreover, inhibition of CTSD by pepstatin A (20 µM) attenuated ROF-reduced expression of α-syn. In vivo study was conducted in mice exposed to ROT (10 mg·kg-1·d-1, i.g.) for 6 weeks; then, ROT-treated mice received ROF (0.5, 1, or 2 mg·kg-1·d-1; i.g.) for four weeks. ROF significantly ameliorated motor deficits, which was accompanied by increased expression levels of tyrosine hydroxylase, SIRT1, mature CTSD, and LAMP1, and a reduced level of α-syn in the substantia nigra pars compacta. Taken together, these results demonstrate that ROF exerts a neuroprotective action and reduces the α-syn level in PD models. The mechanisms underlying ROF neuroprotective effects appear to be associated with NAD+/SIRT1-dependent activation of lysosomal function.

Roflumilast prevents ischemic stroke-induced neuronal damage by restricting GSK3ß-mediated oxidative stress and IRE1α/TRAF2/JNK pathway.

Inhibition of phosphodiesterase 4 (PDE4) protects against neuronal apoptosis induced by cerebral ischemia. However, the exact mechanisms responsible for the protection of PDE4 inhibition have not been completely clarified. Roflumilast (Roflu) is an FDA-approved PDE4 inhibitor for the treatment of chronic obstructive pulmonary disease. The potential protective role of Roflu against ischemic stroke-associated neuronal injury remains unexplored. In this study, we investigated the effect and mechanism of Roflu against ischemic stroke using in vitro oxygen-glucose deprivation reperfusion (OGD/R) and in vivo rat middle cerebral artery occlusion (MCAO) models. We demonstrated that Roflu significantly reduced the apoptosis of HT-22 cells exposed to OGD/R, enhanced the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2), and reduced oxidative stress. Treatment with Roflu increased the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3ß (GSK3ß) but decreased the level of phosphorylated inositol requiring enzyme 1α (IRE1α). Interestingly, constitutively active GSK3ß (S9A) mutation abolished the effects of Roflu on oxidative stress and IRE1α phosphorylation. Moreover, Roflu decreased the binding of IRE1α to tumor necrosis factor receptor-associated factor 2 (TRAF2) and attenuated the phosphorylation of c-Jun N-terminal kinase (JNK). We also found that PDE4B knockdown reduced the phosphorylation of both IRE1α and JNK, while overexpression of PDE4B antagonized the role of PDE4B knockdown on the activation of IRE1α and JNK. Besides, the inhibition of PDE4 by Roflu produced similar effects in primary cultured neurons. Finally, Roflu ameliorated MCAO-induced cerebral injury by decreasing infarct volume, restoring neurological score, and reducing the phosphorylation of IRE1α and JNK. Collectively, these data suggest that Roflu protects neurons from cerebral ischemia reperfusion-mediated injury via the activation of GSK3ß/Nrf-2 signaling and suppression of the IRE1α/TRAF2/JNK pathway. Roflu has the potential as a protective drug for the treatment of cerebral ischemia.

Roflupram, a novel phosphodiesterase 4 inhibitor, inhibits lipopolysaccharide-induced neuroinflammatory responses through activation of the AMPK/Sirt1 pathway.

Roflupram (ROF) is a novel phosphodiesterase 4 inhibitor. We previously found that ROF suppressed the production of pro-inflammatory factors in microglial cells; however, the underlying mechanisms are largely unknown. The present study aimed to elucidate the underlying molecular mechanisms of the anti-neuroinflammatory effects of ROF in lipopolysaccharide (LPS)-activated microglial cells and LPS-challenged mice. Treatment with ROF suppressed LPS-induced expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in BV-2 microglia cell line. Immunofluorescence and Western blotting analysis showed that ROF significantly inhibited the activation of microglia, as evidenced by decreased expression of ionized calcium binding adaptor molecule-1 (Iba1). Similar results were obtained in primary cultured microglial cells. ROF induced the phosphorylation of AMP-activated protein kinase (AMPK) and the expression of Sirtuin 1 (Sirt1). Interestingly, the AMPK inhibitor, compound C, blocked the role of ROF in both the phosphorylation of AMPK and the expression of Sirt1 in BV-2 cells stimulated with LPS. More importantly, the Sirt1 inhibitor, EX527, abolished the inhibitory role of ROF on the production of pro-inflammatory factors, and reactivated BV-2 cells. In mice challenged with LPS, ROF improved cognition and decreased the levels of IL-6 and TNF-α in both the cortex and hippocampus. In contrast, EX527 weakened the effects of ROF on cognitive enhancement and reduction of pro-inflammatory factors in the cortex and hippocampus. Furthermore, EX527 blocked the inhibitory role of ROF in the activation of microglial cells in both the hippocampus and cortex. Taken together, our results indicated that ROF attenuated LPS-induced neuroinflammatory responses in microglia, and the AMPK/Sirt1 pathway is essential for the anti-inflammatory effects of ROF.

Inhibition of PDE4 protects neurons against oxygen-glucose deprivation-induced endoplasmic reticulum stress through activation of the Nrf-2/HO-1 pathway.

Inhibition of phosphodiesterase 4 (PDE4) produces neuroprotective effects against cerebral ischemia. However, the involved mechanism remains unclear. Augmentation of endoplasmic reticulum (ER) stress promotes neuronal apoptosis, and excessive oxidative stress is an inducer of ER stress. The present study aimed to determine whether suppression of ER stress is involved in the protective effects of PDE4 inhibition against cerebral ischemia. We found that exposing HT-22 cells to oxygen-glucose deprivation (OGD) significantly activated ER stress, as evidenced by increased expression of the 78-kDa glucose-regulated protein (GRP78), phosphorylated eukaryotic translation-initiation factor 2α (eIF2α), and C/EBP-homologous protein (CHOP). Overexpression of PDE4B increased ER stress, while knocking down PDE4B or treatment with the PDE4 inhibitor, FCPR03, prevented OGD-induced ER stress in HT-22 cells. Furthermore, FCPR03 promoted the translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2) from the cytoplasm to the nucleus. Importantly, the Nrf-2 inhibitor, ML385, blocked the inhibitory role of FCPR03 on OGD-induced ER stress. ML385 also abolished the protective role of FCPR03 in HT-22 cells subjected to OGD. Knocking down heme oxygenase-1 (HO-1), which is a target of Nrf-2, also blocked the protective role of FCPR03, enhanced the level of reactive oxygen species (ROS), and increased ER stress and cell death. We then found that FCPR03 or the antioxidant, N-Acetyl-l-cysteine, reduced oxidative stress in cells exposed to OGD. This effect was accompanied by increased cell viability and decreased ER stress. In primary cultured neurons, we found that FCPR03 reduced OGD-induced production of ROS and phosphorylation of eIF2α. The neuroprotective effect of FCPR03 against OGD in neurons was blocked by ML385. These results demonstrate that inhibition of PDE4 activates Nrf-2/HO-1, attenuates the production of ROS, and thereby attenuates ER stress in neurons exposed to OGD. Additionally, we conclude that FCPR03 may represent a promising therapeutic agent for the treatment of ER stress-related disorders.

FCPR03, a novel phosphodiesterase 4 inhibitor, alleviates cerebral ischemia/reperfusion injury through activation of the AKT/GSK3ß/ ß-catenin signaling pathway.

Inhibition of phosphodiesterase 4 (PDE4) is a promising strategy for the treatment of ischemic stroke. However, the side effects of nausea and vomiting from the current PDE4 inhibitors have limited their clinical applications. FCPR03 is a novel PDE4 inhibitor with little emetic potential. This study aimed to investigate the effects of FCPR03 on neuronal injury after cerebral ischemia/reperfusion and the underlying signaling pathway. The effects of FCPR03 on cellular apoptosis, intracellular accumulation of reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were evaluated in HT-22 neuronal cells and cortical neurons exposed to oxygen-glucose deprivation (OGD). The impact of FCPR03 on brain injury, neurological scores and behavioral performance was investigated in rats subjected to middle cerebral artery occlusion (MCAO). The protein kinase B (AKT) inhibitor MK-2206 and ß-catenin siRNA were used to investigate the underlying pathways. FCPR03 dose-dependently protected against OGD-induced cellular apoptosis in both HT-22 cells and cortical neurons. The levels of MMP and ROS were also restored by FCPR03. FCPR03 increased the levels of phosphorylated AKT, glycogen synthase kinase-3ß (GSK3ß), and ß-catenin. Interestingly, the role of FCPR03 was reversed by MK-2206 and ß-catenin siRNA. Consistently, FCPR03 reduced the infarct volume and improved neurobehavioral outcomes in rats following MCAO. Moreover, FCPR03 increased the levels of phosphorylated AKT, GSK3ß and ß-catenin within the ischemic penumbra of rats following cerebral ischemia-reperfusion. Taken together, FCPR03 has therapeutic potential in cerebral ischemia-reperfusion. The neuroprotective effects of FCPR03 are mediated through activation of the AKT/GSK3ß/ß-catenin pathway.