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The dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) is widely used as a therapeutic choice for the treatment of patients with Parkinson's disease. However, the long-term use of L-DOPA leads to the development of debilitating involuntary movements, called L-DOPA-induced dyskinesia (LID). The cAMP/protein kinase A (PKA) signaling in the striatum is known to play a role in LID. However, from among the nine known adenylyl cyclases (ACs) present in the striatum, the AC that mediates LID remains unknown. To address this issue, we prepared an animal model with unilateral 6-hydroxydopamine lesions in the substantia nigra in wild-type and AC5-knock-out (KO) mice, and examined behavioral responses to short-term or long-term treatment with L-DOPA. Compared with the behavioral responses of wild-type mice, LID was profoundly reduced in AC5-KO mice. The behavioral protection of long-term treatment with L-DOPA in AC5-KO mice was preceded by a decrease in the phosphorylation levels of PKA substrates ERK (extracellular signalregulated kinase) 1/2, MSK1 (mitogen- and stress-activated protein kinase 1), and histone H3, levels of which were all increased in the lesioned striatum of wild-type mice. Consistently, FosB/ΔFosB expression, which was induced by long-term L-DOPA treatment in the lesioned striatum, was also decreased in AC5-KO mice. Moreover, suppression of AC5 in the dorsal striatum with lentivirusshRNA- AC5 was sufficient to attenuate LID, suggesting that the AC5-regulated signaling cascade in the striatum mediates LID. These results identify the AC5/cAMP system in the dorsal striatum as a therapeutic target for the treatment of LID in patients with Parkinson's disease. [ABSTRACT FROM AUTHOR]

Stress is a potent risk factor for depression, yet the underlying mechanism is not clearly understood. In the present study, we explored the mechanism of development and maintenance of depression in a stress-induced animal model. Mice restrained for 2 h daily for 14 d showed distinct depressive behavior, and the altered behavior persisted for >3 months in the absence of intervention. Acute restraint induced a surge of oxidative stress in the brain, and stress-induced oxidative stress progressively increased with repetition of stress. In vitro, the stress hormone glucocorticoid generated superoxide via upregulation of NADPH oxidase. Consistently, repeated restraints increased the expression of the key subunits of NADPH oxidase, p47phox and p67phox, in the brain. Moreover, stressed brains markedly upregulated the expression of p47phox to weak restress evoked in the poststress period, and this molecular response was reminiscent of amplified ROS surge to restress. Pharmacological inhibition of NADPH oxidase by the NADPH oxidase inhibitor apocynin during the stress or poststress period completely blocked depressive behavior. Consistently, heterozygous p47phox knock-out mice (p47phox+/-) or molecular inhibition of p47phox with Lenti shRNA-p47phox in the hippocampus suppressed depressive behavior. These results suggest that repeated stress promotes depressive behavior through the upregulation of NADPH oxidase and the resultant metabolic oxidative stress, and that the inhibition of NADPH oxidase provides beneficial antidepression effects. [ABSTRACT FROM AUTHOR]

Adenylyl cyclase (AC) is the principal effector molecule in the β-adrenergic receptor pathway. ACV and ACVI are the two predominant isoforms in mammalian cardiac myocytes. The disparate roles among AC isoforms in cardiac hypertrophy and progression to heart failure have been under intense investigation. Specifically, the salutary effects resulting from the disruption of ACV have been established in multiple models of cardiomyopathy. It has been proposed that a continual activation of ACV through elevated levels of protein kinase C could play an integral role in mediating a hypertrophic response leading to progressive heart failure. Elevated protein kinase C is a common finding in heart failure and was demonstrated in murine cardiomyopathy from cardiac-specific overexpression of Gαq protein. Here we assessed whether the disruption of ACV expression can improve cardiac function, limit electrophysiological remodeling, or improve survival in the Gαq mouse model of heart failure. We directly tested the effects of gene-targeted disruption of ACV in transgenic mice with cardiac-specific overexpression of Gαq protein using multiple techniques to assess the survival, cardiac function, as well as structural and electrical remodeling. Surprisingly, in contrast to other models of cardiomyopathy, ACV disruption did not improve survival or cardiac function, limit cardiac chamber dilation, halt hypertrophy, or prevent electrical remodeling in Gαq transgenic mice. In conclusion, unlike other established models of cardiomyopathy, disrupting ACV expression in the Gαq mouse model is insufficient to overcome several parallel pathophysiological processes leading to progressive heart failure. [ABSTRACT FROM AUTHOR]

The transgenic mouse Tg2576 is widely used as a murine model of Alzheimer's disease (AD) and exhibits plaque pathogenesis in the brain and progressive memory impairments. Here we report that Tg2576 mice also have severe spinal cord deficits. At 10 months of age, Tg2576 mice showed a severe defect in the hindlimb extension reflex test and abnormal body trembling and hindlimb tremors when suspended by the tail. The frequency and severity of these abnormalities were overt at 10 months of age and became gradually worsened. On the foot-printing analysis, Tg2576 mice had shorter and narrower strides than the non-transgenic control. Histological analyses showed that neuronal cells including cholinergic neurons in the lumbar cord of Tg2576 mice were severely reduced in number. At 16 months of age, Tg2576 mice showed high levels of amyloid-β accumulation in the spinal cord. Consistent with this, Tg2576 mice showed that lipid peroxidation levels were increased and mitochondrial metabolic activity were significantly reduced in the spinal cord. Administration of curcumin, a natural compound that has antioxidant properties, notably reversed motor function deficits of Tg2576 mice. The enhanced lipid peroxidation and neuronal loss in the lumbar cord was also partially suppressed by curcumin. Electron microscopic analysis revealed that the sciatic nerve fibers were severely reduced in number and were demyelinated in Tg2576 mice, which were partially rescued by curcumin. These results showed that Tg2576 mice display severe degeneration of motor neurons in the spinal cord and associated motor function deficits. [ABSTRACT FROM AUTHOR]

Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to act as an anti-inflammatory molecule in various pathological conditions, which include sepsis or hemorrhagic shock. Recently, we showed that ethyl pyruvate has a neuroprotective effect in the postischemic brain and also in KA-induced pathogenesis in the brain. In this study, we examined whether aspirin augments neuroprotective effect of ethyl pyruvate in transient focal ischemia model by complementing the neuroprotective effects of ethyl pyruvate. Although, most of neuroprotective effect of aspirin has been attributed to the anti-platelet action, aspirin also has direct neuroprotective effects, including NF-κB inhibition. Ethyl pyruvate dose-dependently suppressed infarct formation in the postischemic brain, wherein intravenous administration of 5 mg/kg ethyl pyruvate 30 min after the occlusion reduced infarct volume to 34.5 ± 15.5% ( n = 6, P < 0.01) of that of the untreated control. In combination with aspirin (5 mg/kg, i.v.), the neuroprotective effect was enhanced, resulting in 16.0 ± 5.9% ( n = 6, P < 0.01) infarct volume. The time window for synergistic neuroprotection by ethyl pyruvate and aspirin extended to 9 h post-MCAO. The synergistic reduction in infarct volume was accompanied by suppression of the clinical manifestations associated with cerebral ischemia including motor impairment and neurological deficits. Inflammatory processes including microglial activation and proinflammatory cytokine expression were notably suppressed by the combination treatment in the postischemic brain and in primary microglia cultures, wherein ethyl pyruvate and aspirin modulate NF-κB signaling differentially. Aspirin interferes with IκB phosphorylation and degradation in the cytoplasm, possibly by specifically inhibiting IκB kinase-beta, whereas, the effect of ethyl pyruvate seems to occur in the nucleus, where it may interfere with the binding of NF-κB to responsive promoter elements in the target genes. Similar enhancement in neuroprotective effect was also observed in primary cortical cultures after NMDA or Zn2+ treatment or oxygen–glucose deprivation. Together, these results indicate that combination treatment of ethyl pyruvate and aspirin affords synergistic neuroprotection in the postischemic brain with a wide therapeutic window, in part via differential modulation of the NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease (AD) is a progressive neurodegenerative disease caused by genetic and non-genetic factors. Most AD cases may be triggered and promoted by non-genetic environmental factors. Clinical studies have reported that patients with AD show enhanced baseline levels of stress hormones in the blood, but their physiological significance with respect to the pathophysiology of AD is not clearly understood. Here we report that AD mouse models exposed to restraints for 2 h daily on 16 consecutive days show increased levels of β-amyloid (Aβ) plaque deposition and commensurable enhancements in Aβ(1–42), tau hyperphosphorylation, and neuritic atrophy of cortical neurons. Repeated restraints in Tg2576 mice markedly increased metabolic oxidative stress and down-regulated the expression of MMP-2, a potent Aβ-degrading enzyme, in the brain. These stress effects were reversed by blocking the activation of the hypothalamus-pituitary-adrenal gland axis with the corticotropin-releasing factor receptor antagonist NBI 27914, further suggesting that over-activation of the hypothalamic-pituitary-adrenal axis is required for stress-enhanced AD-like pathogenesis. Consistent with these findings, corticosteroid treatments to cultured primary cortical neurons increased metabolic oxidative stress and down-regulated MMP-2 expression, and MMP-2 down-regulation was reversed by inhibition of oxidative stress. These results suggest that behavioral stress aggravates AD pathology via generation of metabolic oxidative stress and MMP-2 down-regulation. [ABSTRACT FROM AUTHOR]

Type 5 adenylyl cyclase (AC5) is highly concentrated in the dorsal striatum and nucleus accumbens (NAc), two brain areas which have been implicated in motor function, reward, and emotion. Here we demonstrate that mice lacking AC5 ( AC5−/−) display strong reductions in anxiety-like behavior in several paradigms. This anxiolytic behavior in AC5−/− mice was reduced by the D1 receptor antagonist SCH23390 and enhanced by the D1 dopamine receptor agonist, dihydrexidine (DHX). DHX-stimulated c- fos induction in AC5−/− mice was blunted in the dorso-lateral striatum, but it was overactivated in the dorso-medial striatum and NAc. The siRNA-mediated inhibition of AC5 levels within the NAc was sufficient to produce an anxiolytic-like response. Microarray and RT-PCR analyses revealed an up-regulation of prodynorphin and down-regulation of cholecystokinin (CCK) in the NAc of AC5−/− mice. Administration of nor-binaltorphimine (a kappa opioid receptor antagonist) or CCK-8s (a CCK receptor agonist) reversed the anxiolytic-like behavior exhibited by AC5−/− mutants. Taken together, these results suggest an essential role of AC5 in the NAc for maintaining normal levels of anxiety. [ABSTRACT FROM AUTHOR]

Opioid drugs produce their pharmacological effects by activating inhibitory guanine nucleotide-binding regulatory protein-linked μ, δ, and κ opioid receptors. One major effector for these receptors is adenylyl cyclase, which is inhibited upon receptor activation. However, little is known about which of the ten known forms of adenylyl cyclase are involved in mediating opioid actions. Here we show that all of the major behavioral effects of morphine, including locomotor activation, analgesia, tolerance, reward, and physical dependence and withdrawal symptoms, are attenuated in mice lacking adenylyl cyclase type 5 (AC5), a form of adenylyl cyclase that is highly enriched in striatum. Furthermore, the behavioral effects of selective μ or δ opioid receptor agonists are lost in AC5-/- mice, whereas the behavioral effects of selective κ opioid receptor agonists are unaffected. These behavioral data are consistent with the observation that the ability of a μ or δ opioid receptor agonist to suppress adenylyl cyclase activity was absent in striatum of AC5-/- mice. Together, these results establish AC5 as an important component of μ and δ opioid receptor signal transduction mechanisms in vivo and provide further support for the importance of the cAMP pathway as a critical mediator of opioid action. [ABSTRACT FROM AUTHOR]

We tested the hypothesis that deletion of adenylyl cyclase type V (ACV) would be associated with decreased left ventricular (LV) contractile function and responsiveness to β–adrenergic receptor (βAR) stimulation. Absence of cardiac ACV expression was confirmed by RT–PCR and immunoblotting in ACV–deleted mice (ACV -/-). Compared to sibling mice with normal amounts of ACV (CON), basal and water–soluble forskolin derivative NKH477–stimulated cAMP production was reduced in both LV homogenates and in isolated cardiac myocytes. Basal LV +dP/dt (isolated perfused hearts) was increased (CON: 3649 ± 247 mmHg/s; ACV -/-: 4625 ± 350 mmHg/s; p = 0.035, n = 10), but the potency of dobutamine on LV +dP/dt was decreased by ACV deletion (log EC50: CON: –6.83 ± 0.14 M; ACV -/-: –5.99 ± 0.15 M; p = 0.0007, n = 10). The initial rates of ATP–dependent sarcoplasmic reticulum calcium uptake, assessed in LV homogenates, showed that ACV deletion increased SERCA2a affinity for Ca2+ (log EC50: CON: –5.94 ± 0.03 M; ACV -/-: –6.09 ± 0.02 M; p = 0.001, n = 8). ACV deletion is also associated with increased phospholamban phosphorylation, decreased type 1 protein phosphatase catalytic subunit content and activity, and reduced cardiac Gαs protein content. In conclusion, ACV deletion has a favorable effect on basal LV function despite reduced cAMP levels. Increased SERCA2a affinity for Ca2+ and increased phospholamban phosphorylation are contributing factors. However, ACV deletion is associated with reduced LV contractile responsiveness to βAR stimulation, an effect that is associated with reduced Gαs protein content and reduced cAMP generating capacity in cardiac myocytes. [ABSTRACT FROM AUTHOR]

The transmembrane protein Notch is cleaved by γ-secretase to yield an active form, Notch intracellular domain (Notch-IC), in response to the binding of ligands. such as Jagged. Notch-IC contributes to the regulation of a variety of cellular events, including cell fate determination during embryonic development as well as cell growth, differentiation, and survival. We now show that Notch1-IC suppresses the scaffold activity of c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP1) in the JNK signaling path-way. Notch1-IC physically associated with the JNK binding domain of JIP1 and thereby interfered with the interaction between JIPI and JNK. JIP1 mediated the activation of JNK1 induced by glucose deprivation in mouse embryonic fibroblasts, and ectopic expression of Notch1-IC inhibited INK activation and apoptosis triggered by glucose deprivation. Taken together, these findings suggest that Notch1-IC negatively regulates the JNK pathway by disrupting the scaffold function of JIP1. [ABSTRACT FROM AUTHOR]

Cerebral ischemic injury proceeds with excitotoxicity-induced acute neuronal death in the ischemic core and with delayed damage processes in the penumbra. However, knowledge concerning the direct mediators that connect these two processes is limited. Here, we demonstrate that high-mobility group box 1 (HMGB1), a nonhistone DNA-binding protein, is massively released into the extracellular space immediately after ischemic insult and that it subsequently induces neuroinflammation in the postischemic brain. Short hairpin (sh)RNA-mediated HMGB1 downregulation in the postischemic brain suppressed infarct size, microglia activation, and proinflammatory marker induction, indicating that HMGB1 plays a crucial role in the inflammatory process. The proinflammatory cytokine-like function of extracellular HMGB1 was further verified in primary cortical cultures and microglial cultures. HMGB1 was found to accumulate in NMDA-treated primary cortical culture media, and supernatants collected from these cultures were found to trigger microglia activation, the hallmark of brain inflammation. Moreover, treatment with recombinant HMGB1 also induced microglial activation, but HMGB1-depleted supernatant produced by anti-HMGB1 antibody treatment or by HMGB1 shRNA expression did not, thus demonstrating the essential role of HMGB1 in microglial activation. Together, these results indicate that HMGB1 functions as a novel proinflammatory cytokine-like factor that connects excitotoxicity-induced acute damage processes and delayed inflammatory processes in the postischemic brain. [ABSTRACT FROM AUTHOR]

The failure to trigger mitophagy is implicated in the pathogenesis of familial Parkinson disease that is caused by PINK1 or Parkin mutations. According to the prevailing PINK1-Parkin signaling model, mitophagy is promoted by the mitochondrial translocation of Parkin, an essential PINK1-dependent step that occurs via a previously unknown mechanism. Here we determined that critical concentrations of NO was sufficient to induce the mitochondrial translocation of Parkin even in PINK1 deficiency, with apparent increased interaction of full-length PINK1 accumulated during mitophagy, with neuronal nitric oxide synthase (nNOS). Specifically, optimum levels of NO enabled PINK1-null dopaminergic neuronal cells to regain the mitochondrial translocation of Parkin, which appeared to be significantly suppressed by nNOS-null mutation. Moreover, nNOS-null mutation resulted in the same mitochondrial electron transport chain (ETC) enzyme deficits as PINK1-null mutation. The involvement of mitochondrial nNOS activation in mitophagy was further confirmed by the greatly increased interactions of full-length PINK1 with nNOS, accompanied by mitochondrial accumulation of phospho-nNOS (Ser1412) during mitophagy. Of great interest is that the L347P PINK1 mutant failed to bind to nNOS. The loss of nNOS phosphorylation and Parkin accumulation on PINK1-deficient mitochondria could be reversed in a PINK1-dependent manner. Finally, non-toxic levels of NO treatment aided in the recovery of PINK1-null dopaminergic neuronal cells from mitochondrial ETC enzyme deficits. In summary, we demonstrated the full-length PINK1-dependent recruitment of nNOS, its activation in the induction of Parkin translocation, and the feasibility of NO-based pharmacotherapy for defective mitophagy and ETC enzyme deficits in Parkinson disease. [ABSTRACT FROM AUTHOR]

Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to have antiinflammatory effects and to confer protective effects in various pathological conditions. Recently, a number of studies have reported EP inhibits high mobility group box 1 (HMGB1) secretion and suggest this might contribute to its antiinflammatory effect. Since EP is used in a calcium-containing balanced salt solution (Ringer solution), we wondered if EP directly chelates Ca2+ and if it is related to the EP-mediated suppression of HMGB1 release. Calcium imaging assays revealed that EP significantly and dose-dependently suppressed high K+-induced transient [Ca2+]i surges in primary cortical neurons and, similarly, fluorometric assays showed that EP directly scavenges Ca2+ as the peak of fluorescence emission intensities of Mag-Fura-2 (a low-affinity Ca2+ indicator) was shifted in the presence of EP at concentrations of ⩾7 mmol/L. Furthermore, EP markedly suppressed the A23187-induced intracellular Ca2+ surge in BV2 cells and, under this condition, A23187-induced activations of Ca2+-mediated kinases (protein kinase Cα and calcium/calmodulin-dependent protein kinase IV), HMGB1 phosphorylation and subsequent secretion of HMGB1 also were suppressed. (A23187 is a calcium ionophore and BV2 cells are a microglia cell line.) Moreover, the above-mentioned EP-mediated effects were obtained independent of cell death or survival, which suggests that they are direct effects of EP. Together, these results indicate that EP directly chelates Ca2+, and that it is, at least in part, responsible for the suppression of HMGB1 release by EP. [ABSTRACT FROM AUTHOR]