Phosphodiesterase 10A deactivation induces long-term neurological recovery, Peri-infarct remodeling and pyramidal tract plasticity after transient focal cerebral ischemia in mice.

The phosphodiesterase (PDE) superfamily comprises enzymes responsible for the cAMP and cGMP degradation to AMP and GMP. PDEs are abundant in the brain, where they are involved in several neuronal functions. High PDE10A abundance was previously observed in the striatum; however its consequences for stroke recovery were unknown. Herein, we evaluated the effects of PDE10A deactivation by TAK-063 (0.3 or 3 mg/kg, initiated 72 h post-stroke) in mice exposed to intraluminal middle cerebral artery occlusion. We found that PDE10A deactivation over up to eight weeks dose-dependently increased long-term neuronal survival, angiogenesis, and neurogenesis in the peri-infarct striatum, which represents the core of the middle cerebral artery territory, and reduced astroglial scar formation, whole brain atrophy and, more specifically, striatal atrophy. Functional motor-coordination recovery and the long-distance plasticity of pyramidal tract axons, which originate from the contralesional motor cortex and descend through the contralesional striatum to innervate the ipsilesional facial nucleus, were enhanced by PDE10A deactivation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed a set of dopamine receptor-related and neuronal plasticity-related PDE10A targets, which were elevated (e.g., protein phosphatase-1 regulatory subunit 1B) or reduced (e.g., serine/threonine protein phosphatase 1α, ß-synuclein, proteasome subunit α2) by PDE10A deactivation. Our results identify PDE10A as a therapeutic target that critically controls post-ischemic brain tissue remodeling and plasticity.

Phosphodiesterase 10A Is a Critical Target for Neuroprotection in a Mouse Model of Ischemic Stroke.

Phosphodiesterase 10A (PDE10A) hydrolyzes adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP). It is highly expressed in the striatum. Recent evidence implied that PDE10A may be involved in the inflammatory processes following injury, such as ischemic stroke. Its role in ischemic injury was unknown. Herein, we exposed mice to 90 or 30-min middle cerebral artery occlusion, followed by the delivery of the highly selective PDE10A inhibitor TAK-063 (0.3 mg/kg or 3 mg/kg) immediately after reperfusion. Animals were sacrificed after 24 or 72 h, respectively. Both TAK-063 doses enhanced neurological function, reduced infarct volume, increased neuronal survival, reduced brain edema, and increased blood-brain barrier integrity, alongside cerebral microcirculation improvements. Post-ischemic neuroprotection was associated with increased phosphorylation (i.e., activation) of pro-survival Akt, Erk-1/2, GSK-3α/ß and anti-apoptotic Bcl-xL abundance, decreased phosphorylation of pro-survival mTOR, and HIF-1α, MMP-9 and pro-apoptotic Bax abundance. Interestingly, PDE10A inhibition reduced inflammatory cytokines/chemokines, including IFN-γ and TNF-α, analyzed by planar surface immunoassay. In addition, liquid chromatography-tandem mass spectrometry revealed 40 proteins were significantly altered by TAK-063. Our study established PDE10A as a target for ischemic stroke therapy.

Inhibition of Fatty Acid Synthesis Aggravates Brain Injury, Reduces Blood-Brain Barrier Integrity and Impairs Neurological Recovery in a Murine Stroke Model.

Inhibition of fatty acid synthesis (FAS) stimulates tumor cell death and reduces angiogenesis. When SH-SY5Y cells or primary neurons are exposed to hypoxia only, inhibition of FAS yields significantly enhanced cell injury. The pathophysiology of stroke, however, is not only restricted to hypoxia but also includes reoxygenation injury. Hence, an oxygen-glucose-deprivation (OGD) model with subsequent reoxygenation in both SH-SY5Y cells and primary neurons as well as a murine stroke model were used herein in order to study the role of FAS inhibition and its underlying mechanisms. SH-SY5Y cells and cortical neurons exposed to 10 h of OGD and 24 h of reoxygenation displayed prominent cell death when treated with the Acetyl-CoA carboxylase inhibitor TOFA or the fatty acid synthase inhibitor cerulenin. Such FAS inhibition reduced the reduction potential of these cells, as indicated by increased NADH2 +/NAD+ ratios under both in vitro and in vivo stroke conditions. As observed in the OGD model, FAS inhibition also resulted in increased cell death in the stroke model. Stroke mice treated with cerulenin did not only display increased brain injury but also showed reduced neurological recovery during the observation period of 4 weeks. Interestingly, cerulenin treatment enhanced endothelial cell leakage, reduced transcellular electrical resistance (TER) of the endothelium and contributed to poststroke blood-brain barrier (BBB) breakdown. The latter was a consequence of the activated NF-κB pathway, stimulating MMP-9 and ABCB1 transporter activity on the luminal side of the endothelium. In conclusion, FAS inhibition aggravated poststroke brain injury as consequence of BBB breakdown and NF-κB-dependent inflammation.

Normobaric oxygen treatment improves neuronal survival functional recovery and axonal plasticity after newborn hypoxia-ischemia.

BACKGROUND: Newborn hypoxia ischemia (HI) is one of the most prevalent cases in the emergency and can result from fetal hypoxia during delivery. In HI, restricted blood supply to the fetal brain may cause epilepsy or mental disorders. METHODS: In the present study, seven-day-old pups were subjected HI and treated with different normobaric oxygen (NBO) concentrations (21%, 70% or 100%). In the acute phase, we analyzed infarct area, disseminate neuronal injury and surviving neurons. In addition, we studied the regulation of PTEN and MMP-9 proteins which were suggested to be activated by HI in the ischemic tissue. Moreover, long-term effects of NBO treatments were evaluated with open field, rotarod and Barnes maze tests. We also examined axonal plasticity with EGFP-AAV injection. RESULTS: Here, we demonstrate that hyperoxic NBO concentration causes an increase in cellular survival and a decrease in the number of apoptotic cells, meanwhile inhibiting the proteins involved in cellular death mechanisms. Moreover, we found that hyperoxia decreases anxiety, promotes motor coordination and improve spatial learning and memory. Notably that axonal sprouting was promoted by hyperoxia. CONCLUSION: Our data suggest that NBO is a promising approach for the treatment of newborn HI, which encourage proof-of-concept studies in newborn.

Interaction of melatonin and Bmal1 in the regulation of PI3K/AKT pathway components and cellular survival.

The circadian rhythm is driven by a master clock within the suprachiasmatic nucleus which regulates the rhythmic secretion of melatonin. Bmal1 coordinates the rhythmic expression of transcriptome and regulates biological activities, involved in cell metabolism and aging. However, the role of Bmal1 in cellular- survival, signaling, its interaction with intracellular proteins, and how melatonin regulates its expression is largely unclear. Here we observed that melatonin increases the expression of Bmal1 and both melatonin and Bmal1 increase cellular survival after oxygen glucose deprivation (OGD) while the inhibition of Bmal1 resulted in the decreased cellular survival without affecting neuroprotective effects of melatonin. By using a planar surface immunoassay for PI3K/AKT signaling pathway components, we revealed that both melatonin and Bmal1 increased phosphorylation of AKT, ERK-1/2, PDK1, mTOR, PTEN, GSK-3αß, and p70S6K. In contrast, inhibition of Bmal1 resulted in decreased phosphorylation of these proteins, which the effect of melatonin on these signaling molecules was not affected by the absence of Bmal1. Besides, the inhibition of PI3K/AKT decreased Bmal1 expression and the effect of melatonin on Bmal1 after both OGD in vitro and focal cerebral ischemia in vivo. Our data demonstrate that melatonin controls the expression of Bmal1 via PI3K/AKT signaling, and Bmal1 plays critical roles in cellular survival via activation of survival kinases.

Evidence that activation of P2X7R does not exacerbate neuronal death after optic nerve transection and focal cerebral ischemia in mice.

Conflicting data in the literature about the function of P2X7R in survival following ischemia necessitates the conductance of in-depth studies. To investigate the impacts of activation vs inhibition of the receptor on neuronal survival as well as the downstream signaling cascades, in addition to optic nerve transection (ONT), 30min and 90min of middle cerebral artery occlusion (MCAo) models were performed in mice. Intracellular calcium levels were assessed in primary cortical neuron cultures. Here, we show that P2X7R antagonist Brilliant Blue G (BBG) decreased DNA fragmentation, infarct volume, brain swelling, neurological deficit scores and activation of microglial cells after focal cerebral ischemia. BBG also significantly increased the number of surviving retinal ganglion cells (RGCs) after ONT and the number of surviving neurons following MCAo. Importantly, receptor agonist BzATP resulted in increased activation of microglial cells and induced phosphorylation of ERK, AKT and JNK. These results indicated that inhibition of P2X7R with BBG promoted neuronal survival, not through the activation of survival kinase pathways, but possibly by improved intracellular Ca2+ overload and decreased the levels of Caspase 1, IL-1ß and Bax proteins. On the other hand, BzATP-mediated increased number of activated microglia and increased survival kinase levels in addition to increased caspase-1 and IL-1ß levels indicate the complex nature of the P2X7 receptor-mediated signaling in neuronal injury.

Poloxamer P85 increases anticancer activity of Schiff base against prostate cancer in vitro and in vivo.

Prostate cancer is the second most common cancer among men and the leading cause of death after lung cancer. Development of hormone-refractory disease is a crucial step for prostate cancer progression for which an effective treatment option is currently unavailable. Therefore, there is a need for new agents that can efficiently target cancer cells, decrease tumor growth, and thereby extend the survival of patients in late-stage castration-resistant prostate cancer. In the current study, a novel heterodinuclear copper(II)Mn(II) Schiff base complex combined with P85 was used to evaluate anticancer activity against prostate cancer in vitro and in vivo. Cell proliferation and cytotoxicity were evaluated by cell viability, gene, and protein expression assays in vitro. Results showed that the heterodinuclear copper(II)Mn(II) complex-P85 combination decreased cell proliferation by upregulating the apoptotic gene expressions and blocking the cell proliferation-related pathways. Tramp-C1-injected C57/B16 mice were used to mimic a prostate cancer model. Treatment combination of Schiff base complex and P85 significantly enhanced the cellular uptake of chemicals (by blocking the drug transporters and increased life time), suppressed tumor growth, and decreased tumor volume steadily over the course of the experiments. Overall, heterodinuclear copper(II)Mn(II) complex-P85 showed remarkable anticancer activity against prostate cancer in in vitro and in vivo.

Effects of normobaric oxygen and melatonin on reperfusion injury: role of cerebral microcirculation.

In order to protect the brain before an irreversible injury occurs, penumbral oxygenation is the primary goal of current acute ischemic stroke treatment. However, hyperoxia treatment remains controversial due to the risk of free radical generation and vasoconstriction. Melatonin is a highly potent free radical scavenger that protects against ischemic stroke. Considering its anti-oxidant activity, we hypothesized that melatonin may augment the survival-promoting action of normobaric oxygen (NBO) and prevent brain infarction. Herein, we exposed mice to 30 or 90 min of intraluminal middle cerebral artery occlusion (MCAo) and evaluated the effects of NBO (70% or 100% over 90 min), administered either alone or in combination with melatonin (4 mg/kg, i.p.), on disseminate neuronal injury, neurological deficits, infarct volume, blood-brain barrier (BBB) permeability, cerebral blood flow (CBF) and cell signaling. Both NBO and particularly melatonin alone reduced neuronal injury, neurological deficits, infarct volume and BBB permeability, and increased post-ischemic CBF, evaluated by laser speckle imaging (LSI). They also improved CBF significantly in the ischemic- core and penumbra, which was associated with reduced IgG extravasation, DNA fragmentation, infarct volume, brain swelling and neurological scores. Levels of phosphorylated Akt, anti-apoptotic Bcl-xL, pro-apoptotic Bax and endothelial nitric oxide synthase (NOS) were re-regulated after combined oxygen and melatonin delivery, whereas neuronal and inducible NOS, which were increased by oxygen treatment, were not influenced by melatonin. Our present data suggest that melatonin and NBO are promising approaches for the treatment of acute-ischemic stroke, which encourage proof-of-concept studies in human stroke patients.

Sodium pentaborate pentahydrate and pluronic containing hydrogel increases cutaneous wound healing in vitro and in vivo.

After a disruption of skin integrity, the body produces an immediate response followed by a functional and comparable regeneration period, referred to as wound healing. Although normal wounds do not need much attention during the healing period, chronic (non-healing) wounds are the major challenge of current dermatological applications. Therefore, developing new, safe, and effective wound healing drugs has always been an attractive area of international research. In the current study, sodium pentaborate pentahydrate (NaB), pluronics (Plu; F68 and F127), and their combinations were investigated for their wound healing activities, using in vitro and in vivo approaches. The results revealed that NaB significantly increased migration capacity and superoxide dismutase activity in primary human fibroblasts. Combinations of optimized concentrations for pluronic block co-polymers further increased cell migration, and the messenger RNA (mRNA) expression levels of important growth factor and cytokines (vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-ß), and tumor necrosis factor alpha (TNF-α)). NaB containing hydrogel co-formulated with pluronics was also investigated for their wound healing activities using a full thickness wound model in rats. Macroscopic and histopathological analysis confirmed that wounds in combination gel-treated groups healed faster than those of control groups. NaB/Plu gel application was found to increase wound contraction and collagen deposition in the wound area. Therefore, our results suggest that NaB, and its pluronics combination, could be used in dermatological clinics and be a future solution for chronic wounds. However, further studies should be conducted to explore its exact action of mechanism and effects of this formulation on chronic wounds.