Zheng-Hong Qin

The vanadium (IV) compound rescues septo-hippocampal cholinergic neurons from neurodegeneration in olfactory bulbectomized mice

The bilateral olfactory bulbectomy (OBX) mouse exhibits neurodegeneration of cholinergic neurons in the medial septum with concomitant cognitive deficits. Consistent with our previous observations, choline acetyltransferase (ChAT) protein levels in the medial septum decreased by 43.5% 2 weeks after OBX without changes in glutamic acid decarboxylase-65 (GAD65) levels. Interestingly, levels of the vesicular acetylcholine transporter (VAChT), which is localized at cholinergic neuron terminals, decreased both in hippocampal CA1 and CA3 regions following OBX. Confocal microscopy showed that VAChT expression was more severely reduced in CA3 14 days after OBX compared with CA1. Intriguingly, chronic treatment with a vanadium (IV) compound, VO(OPT) [bis(1-N-oxide-pyridine-2-thiolato)oxovanadium(IV)] (0.5-1 mg as vanadium (V)/kg/day, i.p.), significantly rescued cholinergic neurons in the medial septum in a dose-dependent manner. VO(OPT) treatment also prevented decreased VAChT immunoreactivity both in CA1 and CA3 regions in the hippocampus. Consistent with these findings, an impaired hippocampal long-term potentiation (LTP) and memory deficits seen in OBX mice were significantly prevented by VO(OPT) treatment. Taken together, OBX induces neurodegeneration of septo-hippocampal cholinergic neurons and impairment of memory-related behaviors. The neuroprotective effect of VO(OPT) could lead to novel therapeutic strategies to ameliorate cognitive deficits associated with cholinergic neuron degeneration in Alzheimers disease and other neurodegenerative disorders.

Accumulation of beta-amyloid in the brain microvessels accompanies increased hyperphosphorylated tau proteins following microsphere embolism in aged rats.

To define mechanisms underlying neurovascular injury following brain embolism-induced neurodegeneration, we investigated temporal and spatial pathological changes in brain microvessels up to 12 weeks after microsphere embolism (ME) induction in aged male rats. Mild ME upregulated endothelial nitric oxide synthase (eNOS) and protein tyrosine nitration in brain microvessels. Strong beta-amyloid immunoreactivity coincident with increased eNOS immunoreactivity was observed in microvessels. Immunoblotting of purified brain microvessels revealed that beta-amyloid accumulation significantly increased 1 week after ME induction and remained elevated for 12 weeks. Importantly, beta-amyloid accumulation in brain parenchyma was also observed in areas surrounding injured microvessels at 12 weeks. Levels of Alzheimers-related hyperphosphorylated tau proteins also concomitantly increased in neurons surrounding regions of beta-amyloid accumulation 12 weeks after ME induction, as did glycogen synthase kinase (GSK3beta) (Tyr-216) phosphorylation. Taken together, ME-induced aberrant eNOS expression and subsequent protein tyrosine nitration in microvessels preceded beta-amyloid accumulation both in microvessels and brain parenchyma, leading to hyperphosphorylation of neuronal tau proteins through GSK3beta activation.

Cobratoxin inhibits pain-evoked discharge of neurons in thalamic parafascicular nucleus in rats: Involvement of cholinergic and serotonergic systems

The present study investigated the inhibitory effect of cobratoxin (CTX) on pain-evoked discharge of neurons in thalamic parafascicular nucleus (Pf) of rats and analyzed some of the mechanisms involved in this effect. Intracerebroventricular injection (icv) of CTX at 0.56, 1.12 and 4.50 microg/kg resulted in a dose-dependent inhibitory effect on the pain-evoked discharges of Pf neurons. The inhibition of pain-evoked discharges of Pf neurons by CTX at high dose (4.50 microg/kg) persisted at least for 2h, while the inhibitory effect of morphine (40 microg) persisted no longer than 30 min. The inhibitory effect of CTX was reversed by pretreatment with atropine (icv, 5 microg). In contrast, icv injection of naloxone (4 microg) had no effect on CTX-induced inhibition. Furthermore, pretreatment with parachlorophenylalanine, a specific inhibitor of tryptophan hydroxylase, also significantly attenuated the inhibitory effect of CTX. The results suggested that: (a) CTX has a dose-dependent inhibitory effect on pain-evoked discharges of Pf neurons, confirming electrophysiologically the antinociceptive action of CTX; (b) the inhibitory effect of CTX has a longer duration compared to that of morphine; (c) central cholinergic and serotonergic systems, but not opioidergic system, are involved in the inhibitory effect of CTX.

Activation of M3 muscarinic receptors inhibits T-type Ca2+ channel currents via pertussis toxin-sensitive novel protein kinase C pathway in small dorsal root ganglion neurons

Cobrotoxin (CbT), a short-chain postsynaptic α-neurotoxin, has been reported to play a role in analgesia. However, to date, the detailed mechanisms still remain unknown. In the present study, we identify a novel functional role of CbT in modulating T-type Ca(2+) channel currents (T-currents) in small dorsal root ganglia (DRG) neurons as well as pain behaviors in mice. We found that CbT inhibited T-currents in a dose-dependent manner. CbT at 1μM reversibly inhibited T-currents by ~26.3%. This inhibitory effect was abolished by the non-selective muscarinic acetylcholine receptor (mAChR) antagonist atropine, or the selective M3 mAChR antagonist 4-DAMP, while naloxone, an opioid receptor antagonist had no effect. Intracellular infusion of GDP-β-S or pretreatment of the cells with pertussis toxin (PTX) completely blocked the inhibitory effects of CbT. Using depolarizing prepulse, we found the absence of direct binding between G-protein βγ subunits and T-type Ca(2+) channels in CbT-induced T-current inhibition. CbT responses were abolished by the phospholipase C inhibitor U73122 (but not the inactive analog U73343). The classical and novel protein kinase C (nPKC) antagonist chelerythrine chlorid or GF109203X abolished CbT responses, whereas the classical PKC antagonist Ro31-8820 or inhibition of PKA elicited no such effects. Intrathecal administration of CbT (5μg/kg) produced antinociceptive effects in mechanical, thermal, and inflammatory pain models. Moreover, CbT-induced antinociception could be abrogated by 4-DAMP. Taken together, these results suggest that CbT acting through M3 mAChR inhibits T-currents via a PTX-sensitive nPKC pathway in small DRG neurons, which could contribute to its analgesic effects in mice.

Alpha-cobratoxin inhibits T-type calcium currents through muscarinic M4 receptor and Go-protein βγ subunits-dependent protein kinase A pathway in dorsal root ganglion neurons

The long-chain neurotoxic protein, alpha-cobratoxin (α-CTx), has been shown to have analgesic effects. However, the underlying mechanisms still remain unclear. In this study, we examined the effects of α-CTx on T-type calcium channel currents (T-currents) and elucidated the relevant mechanisms in mouse dorsal root ganglion (DRG) neurons. Our results showed that α-CTx reversibly inhibited T-currents in a dose-dependent manner. This inhibitory effect was blocked by the selective muscarinic M4 receptor antagonist tropicamide, while methyllycaconitine, a specific antagonist for the α7 subtype of nicotinic receptor had no effect. siRNA targeting the M4 receptor in small DRG neurons abolished α-CTx-induced T-current inhibition. Intracellular application of GDP-β-S or a selective antibody against the G(o)α-protein, as well as pretreatment of the cells with pertussis toxin, abolished the inhibitory effects of α-CTx. The M4 receptor-mediated response was blocked by dialyzing cells with QEHA peptide or anti-G(β) antibody. Pretreatment of the cells with protein kinase A (PKA) inhibitor H89 or intracellular application of PKI 6-22 abolished α-CTx-induced T-current inhibition in small DRG neurons, whereas inhibition of phosphatidylinositol 3-kinase or PKC elicited no such effects. In addition, α-CTx significantly increased PKA activity in DRG neurons, whereas pretreatment of the cells with tropicamide abolished this effect. In summary, our results suggest that activation of muscarinic M4 receptor by α-CTx inhibits T-currents via the G(βγ) of G(o)-protein and PKA-dependent pathway. This article is part of a Special Issue entitled Post-Traumatic Stress Disorder.

Crotoxin enhances the antitumor activity of gefinitib (Iressa) in SK-MES-1 human lung squamous carcinoma cells

Crotoxin (CrTX), a neurotoxin, is isolated from the venom of South American rattlesnakes and has potent antitumor activity. Here, we investigated the antitumor effect of CrTX on the SK-MES-1 human lung squamous cell carcinoma cell line that has acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors. CrTX caused G1 arrest and p-JNK protein upregulation that resulted in apoptosis of SK-MES-1 cells. SP600125, a specific inhibitor of p-JNK, could rescue SK-MES-1 cells from CrTX-induced apoptosis. CrTX and gefinitib (Iressa) both inhibited the viability and proliferation of SK-MES-1 cells in a dose- and time-dependent manner. The combination of CrTX and Iressa significantly enhanced the antitumor activity of Iressa. Inxa0vivo studies revealed that CrTX caused increased damage to blood vessels and reduced tumor size when combined with Iressa. The present study showed that the JNK signal transduction pathway mediated the anti-apoptotic effect of CrTX, and furthermore, CrTX could enhance the antitumor effect of tyrosine kinase inhibitors in cells with acquired resistance.

Modulation of A-type K+ channels by the short-chain cobrotoxin through the protein kinase C-delta isoform decreases membrane excitability in dorsal root ganglion neurons

A-type K(+) channels are crucial in controlling neuronal excitability, and their regulation in sensory neurons may alter pain sensation. In this study, we identified the functional role of cobrotoxin, the short-chain α-neurotoxin isolated from Naja atra venom, which acts in the regulation of the transient A-type K(+) currents (IA) and membrane excitability in dorsal root ganglion (DRG) neurons via the activation of the muscarinic M3 receptor (M3R). Our results showed that cobrotoxin increased IA in a concentration-dependent manner, whereas the sustained delayed rectifier K(+) currents (IDR) were not affected. Cobrotoxin did not affect the activation of IA markedly, however, it shifted the inactivation curve significantly in the depolarizing direction. The cobrotoxin-induced IA response was blocked by the M3R-selective antagonists DAU-5884 and 4-DAMP. An siRNA targeting the M3R in small DRG neurons abolished the cobrotoxin-induced IA increase. In addition, dialysis of the cells with the novel protein kinase C-delta isoform (PKC-δ) inhibitor δv1-1 or an siRNA targeting PKC-δ abolished the cobrotoxin-induced IA response, whereas inhibition of PKA or classic PKC activity elicited no such effects. Moreover, we observed a significant decrease in the firing rate of the neuronal action potential induced by M3R activation. Pretreatment of the cells with 4-aminopyridine, a selective blocker of IA, abolished this effect. Taken together, these results suggest that the short-chain cobrotoxin selectively enhances IA via a novel PKC-δ-dependent pathway. This effect occurred via the activation of M3R and might contribute to its neuronal hypoexcitability in small DRG neurons.

NADPH ameliorates MPTP-induced dopaminergic neurodegeneration through inhibiting p38MAPK activation

Parkinson’s disease (PD) is the second most common neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in substantia nigra pars compacta (SNpc). Although the pathogenic mechanism underlying PD remains largely unknown, decreased nigral glutathione (GSH) in postmortem brains of PD patients supports the presence of oxidative stress in PD. We found that Nicotinamide adenine dinucleotide phosphate (NADPH), which is important for maintaining the level of GSH, protected dopaminergic (DA) neurons from neurotoxicity of MPTP/MPP+. In the present study, NADPH prevented DA neurons from MPTP toxicity with increased GSH and decreased reactive oxygen species (ROS) levels in the ventral midbrain of mice, and improved motor activity. Our present results demonstrated that NADPH inhibited the phosphorylation of p38MAPK, decreased the level of TP53 protein, and inhibited TP53 nuclear translocation in DA neurons of SNpc and in MES23.5 cells. Furthermore, NADPH decreased the protein level of TP53 target gene, Bax, cleavage of PARP, and nuclei condensation. Taken together, NADPH abrogated MPTP-induced p38MAPK phosphorylation, TP53 nuclear translocation, and Bax induction, and finally, MPTP/MPP+-induced apoptosis of DA neurons. This study suggests that NADPH may be a novel therapeutic candidate for PD.

Reduced nicotinamide adenine dinucleotide phosphate inhibits rat platelet aggregation and p38 phosphorylation

Previous studies found that reduced nicotinamide adenine dinucleotide phosphate (NADPH) protected neurons against ischemia/reperfusion-induced injury. In addition to ROS reduction and ATP increment, preliminary data suggested that NADPH inhibited ADP and thrombin-induced platelet aggregation. As the effect of NADPH on platelet function was not reported by other investigators, the actions of NADPH on platelet function and mechanisms of actions were investigated in the present study. In vitro studies, the effects of different concentrations of NADPH on platelet aggregation induced by ADP (10u202fμM), thrombin (0.05u202fU/mL) or AA (50u202fμM) were determined. The results showed that NADPH could inhibit platelet aggregation induced by ADP, thrombin or AA in a concentration dependent manner. When the inhibitory effects of NAD+, NADH, NADP+ and NADPH on platelet aggregation were compared, NADPH demonstrated the relatively best effect on platelet aggregation. In vivo studies, the effects of NADPH on platelet aggregation, tail bleeding time, coagulation response and ferric chloride-induced thrombosis were determined in mice or rats. The maximum aggregation rate of platelets of rats injected with NADPH (5u202fmg/kg) was lower than platelets from control rats. NADPH transiently prolonged tail bleeding time in mice at 30u202fmin after the injection of NADPH (7.5u202fmg/kg), while aspirin (15u202fmg/kg) significantly prolonged the tail bleeding time in mice at all time points examined. NADPH (5u202fmg/kg), as well as aspirin (10u202fmg/kg), had no effect on coagulation response in rats. Using a FeCl3-induced abdominal aorta injury thrombosis model, administration of NADPH (5u202fmg/kg) significantly delayed the onset of vessel occlusion, while aspirin (10u202fmg/kg) almost completely prevented the vessel occlusion. With microscopic examination the thrombi in injured vessel sections of rats received NADPH were much smaller and less dense than that of rats received vehicle treatment. ADP induced an increase in phosphorylation of p38 and the effect was markedly inhibited by the p38 inhibitor SB203580. Similarly, NADPH also inhibited ADP-induced phosphorylation of p38. Similar to NADPH, SB203580 robustly inhibited ADP- and thrombin-induced platelet aggregation. In addition, NADPH also reduced ADP-induced increases in ROS in platelets. The current results demonstrated that NADPH inhibited platelet aggregation, oxidative stress and p38 phosphorylation, suggesting that NADPH might be a novel compound for management of high risk of cardiovascular disease.

Reduced Nicotinamide Adenine Dinucleotide Phosphate Inhibits MPTP-Induced Neuroinflammation and Neurotoxicity

It is generally believed that oxidative stress and neuroinflammation are implicated in the pathogenesis of Parkinsons disease (PD). Reduced nicotinamide adenine dinucleotide phosphate (NADPH) has been demonstrated to have potent neuroprotective effects against oxidative stress. In the present research, we investigated if NADPH could offer neuroprotection by inhibiting glia-mediated neuroinflammation induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a mechanism contributing to PD pathogenesis. The current data demonstrated that MPTP/MPP+ increased levels of reactive oxygen species (ROS), activated glial cells, and inflammasome proteins in the substantia nigra (SNpc), in addition to inducing the nuclear translocation of nuclear factor-κB (NF-κB) and phosphorylation of p38 MAPK. These responses were inhibited by supplementation of exogenous NADPH. Moreover, NADPH effectively decreased MPP+-induced excessive production of ROS, p38 phosphorylation and inflammatory protein of Cyclooxygenase2 (COX2) in cultured microglial BV-2 cells in vitro studies. Similarly, the p38 MAPK inhibitor SB203580 suppressed the upregulation of MPP+-induced p38 phosphorylation and COX2 protein levels. Co-culture of neuronal cells with MPP+-primed BV-2 cells increased the levels of tumor necrosis factor-alpha (TNF-α) and induced cell death of neuronal cells. These effects were diminished by TNF-α neutralizing antibody and NADPH. NADPH reduced motor dysfunction and the loss of dopaminergic (DA) cells induced by MPTP. Therefore, the present study demonstrates that NADPH protects DA neurons by inhibiting oxidative stress and glia-mediated neuroinflammation both in vitro and in vivo, thus suggesting a potential of clinical application for PD and other neurodegenerative diseases.