Shengquan Hu

Protection against 1-methyl-4-phenylpyridinium ion (MPP+)-induced apoptosis by water extract of ginseng (Panax ginseng C.A. Meyer) in SH-SY5Y cells.

ETHNOPHARMACOLOGICAL RELEVANCE The present study investigates the protective effects of water extract of ginseng (Panax ginseng C.A. Meyer) against 1-methyl-4-phenylpyridinium ion (MPP(+))-induced cytotoxicity in SH-SY5Y human neuroblastoma cells and explores the underlying mechanisms. The approach may be used for screening therapeutic agents for degenerative disorders such as Parkinsons disease. MATERIALS AND METHODS SH-SY5Y human neuroblastoma cells were used to analyze the protective effects of water extract of ginseng (WEG) against multiple parameters such as MPP(+)-induced viability, oxidative injury, expression of Bax, Bcl-2, cytochrome c and cleaved caspase-3. RESULTS WEG exerted inhibitory effect on cell death, overproduction of ROS, elevated Bax/Bcl-2 ratio, release of cytochrome c and activation of caspase-3 expression in MPP(+)-treated SH-SY5Y cells. CONCLUSIONS WEG exhibited significant protective effects against MPP(+)-induced cytotoxicity in SH-SY5Y cells possibly through the suppression of ROS generation and the inhibition of mitochondria-dependent apoptotic pathway.

Unexpected neuronal protection of SU5416 against 1- methyl-4-phenylpyridinium ion-induced toxicity via inhibiting neuronal nitric oxide synthase

SU5416 was originally designed as a potent and selective inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) for cancer therapy. In this study, we have found for the first time that SU5416 unexpectedly prevented 1-methyl-4-phenylpyridinium ion (MPP+)-induced neuronal apoptosis in cerebellar granule neurons, and decreased 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced loss of dopaminergic neurons and impairment of swimming behavior in zebrafish in a concentration-dependent manner. However, VEGFR-2 kinase inhibitor II, another specific VEGFR-2 inhibitor, failed to reverse neurotoxicity at the concentration exhibiting anti-angiogenic activity, strongly suggesting that the neuroprotective effect of SU5416 is independent from its anti-angiogenic action. SU5416 potently reversed MPP+-increased intracellular nitric oxide level with an efficacy similar to 7-nitroindazole, a specific neuronal nitric oxide synthase (nNOS) inhibitor. Western blotting analysis showed that SU5416 reduced the elevation of nNOS protein expression induced by MPP+. Furthermore, SU5416 directly inhibited the enzyme activity of rat cerebellum nNOS with an IC50 value of 22.7 µM. In addition, knock-down of nNOS expression using short hairpin RNA (shRNA) abolished the neuroprotective effects of SU5416 against MPP+-induced neuronal loss. Our results strongly demonstrate that SU5416 might exert its unexpected neuroprotective effects by concurrently reducing nNOS protein expression and directly inhibiting nNOS enzyme activity. In view of the capability of SU5416 to cross the blood-brain barrier and the safety for human use, our findings further indicate that SU5416 might be a novel drug candidate for neurodegenerative disorders, particularly those associated with NO-mediated neurotoxicity.

The anti-cancer agent SU4312 unexpectedly protects against MPP(+) -induced neurotoxicity via selective and direct inhibition of neuronal NOS.

SU4312, a potent and selective inhibitor of VEGF receptor‐2 (VEGFR‐2), has been designed to treat cancer. Recent studies have suggested that SU4312 can also be useful in treating neurodegenerative disorders. In this study, we assessed neuroprotection by SU4312 against 1‐methyl‐4‐phenylpyridinium ion (MPP+)‐induced neurotoxicity and further explored the underlying mechanisms.

Bis(propyl)-cognitin protects against glutamate-induced neuro-excitotoxicity via concurrent regulation of NO, MAPK/ERK and PI3-K/Akt/GSK3β pathways

We have previously reported that bis(propyl)-cognitin (B3C), similar to memantine (MEM), is an uncompetitive N-methyl-d-aspartate receptor antagonist with fast off-rate property. In the current study, we further demonstrated that in primary cultures of rat cerebellar granule neurons (CGNs), 2h pretreatment of B3C (IC50, 0.45μM) prevented glutamate-induced excitotoxicity 10 times more potently than memantine (IC50, 4.58 μM), as evidenced by cell viability and lactate dehydrogenase release assays. Additionally, B3C pretreatment could inhibit the increase of intracellular nitric oxide (NO) and the activation of phosphorylated ERK, and reverse the suppression of phosphorylated Akt and GSK3β caused by glutamate. Furthermore, the neuroprotection of B3C was abolished by phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002. Meanwhile, pharmacological inhibition showed that neither the single specific inhibitors of nitric oxide synthase (L-NMMA), MEK1/2 (U0126) and GSK3β (SB415286 and LiCl) nor the combinations of any two of them could fully protect against glutamate-induced apoptosis. However, the co-application of these three inhibitors produced nearly 100% inhibition of glutamate-induced apoptosis. These results taken together suggest that B3C elicits neuroprotection against glutamate-induced neurotoxicity in CGNs via concurrent inhibition of NO, MAPK/ERK pathways and activation of PI3-K/Akt/GSK3β pathway. Combining these and our previous publications, it is conjectured that the dimer might be an ideal candidate drug in delaying the course of neurodegeneration related with Alzheimers disease.

Tacrine(2)–ferulic acid, a novel multifunctional dimer, attenuates 6-hydroxydopamine-induced apoptosis in PC12 cells by activating Akt pathway

Oxidative stress is closely related to the pathogenesis of neurodegenerative disorders such as Parkinsons disease (PD). In this study, we investigated the neuroprotective effect of tacrine-ferulic acid dimers linked by an alkylenediamine side chain (TnFA, n=2-7), a series of novel acetylcholinesterase inhibitors, against 6-hydroxydopamine (6-OHDA)-induced apoptosis in PC12 cells. Among these dimers, pre-treatment of tacrine(2)-ferulic acid (T2FA, 3-30 μM) attenuated 6-OHDA-induced apoptosis in a concentration-dependent manner. The activations of glycogen synthase kinase 3β (GSK3β) and extracellular signal-regulated kinase (ERK) were observed after the treatment of 6-OHDA. Both SB415286 (an inhibitor of GSK3β) and PD98059 (an inhibitor of ERK kinase) reduced the neurotoxicity induced by 6-OHDA, indicating that GSK3β and ERK are involved in 6-OHDA-induced apoptosis. T2FA was able to inhibit the activation of GSK3β, but not ERK, in an Akt-dependent manner. Furthermore, LY294002, a phosphoinositide 3-kinase inhibitor, abolished the neuroprotective effect of T2FA. Collectively, these results suggest that T2FA prevents 6-OHDA-induced apoptosis possibly by activating the Akt pathway in PC12 cells.

PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP+-induced neuronal apoptosis.

Vascular endothelial growth factor (VEGF), a specific pro-angiogenic peptide, has shown neuroprotective effects in the Parkinsons disease (PD) models, but the underlying mechanisms remain elusive. In this study, the neuroprotective properties of VEGF on 1-methyl-4-phenylpyridinium ion (MPP(+))-induced neurotoxicity in primary cerebellar granule neurons were investigated. Pretreatment of VEGF prevented MPP(+)-induced neuronal apoptosis in a concentration- and time-dependent manner. And this prevention was blocked by PTK787/ZK222584, a VEGF receptor-2 specific inhibitor. Both inhibition of the Akt pathway and activation of the extracellular signal-regulated kinase (ERK) pathway contribute to MPP(+)-induced neuronal apoptosis. VEGF reversed the inhibition of phosphoinositide 3-kinase (PI3-K)/Akt pathway caused by MPP(+), but further enhanced the activation of ERK induced by MPP(+). Interestingly, VEGF and PD98059 (an ERK kinase inhibitor) play a synergistic role in protecting neurons from MPP(+)-induced toxicity. Collectively, these findings suggest that the PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP(+)-induced neuronal apoptosis. This finding offers not only a new and clinically significant modality as to how VEGF exerts its neuroprotective effects but also a novel therapeutic strategy for PD by differentially regulating PD-associated signaling pathways.

Bis(12)-hupyridone, a novel multifunctional dimer, promotes neuronal differentiation more potently than its monomeric natural analog huperzine A possibly through alpha7 nAChR

The cause of many neurodegenerative disorders can be ascribed to the loss of functional neurons, and thus agents capable of promoting neuronal differentiation may have therapeutic benefits to patients of these disorders. In this study, the effects and underlying mechanisms of bis(12)-hupyridone (B12H), a novel dimeric acetylcholinesterase inhibitor modified from huperzine A (HA), on neuronal differentiation were investigated using both the rat PC12 pheochromocytoma cell line and adult rat hippocampus neural stem cells. B12H (3-30 μM), characterized by morphological changes and expression of GAP-43, induced neurite outgrowth in a concentration- and time-dependent manner, with almost 3-fold higher efficacy than that of HA in PC12 cells. Furthermore, B12H (2.5-10 μM), but not HA, promoted neuronal differentiation as shown by the percentage increase of βIII-tubulin positive neurons in neural stem cells. The activities of extracellular signal-regulated kinase (ERK), as well as its downstream transcription factors Elk-1 and cAMP response element-binding protein (CREB) were elevated in the B12H-treated PC12 cells. Mitogen-activated protein kinase kinase inhibitors and alpha7-nicotinic acetylcholine receptor (α7nAChR) antagonist blocked the neurite outgrowth and the activation of ERK induced by B12H. All these findings suggest that B12H potently induces pro-neuronal cells into differentiated neurons by activating the ERK pathway possibly via regulating α7nAChR. These findings support the recent proposition that α7nAChR is required for the neuronal dendritic arborization and differentiation in the adult mice hippocampus, and provide insights into the possible therapeutic potential of B12H in treating neurodegenerative disorders.

Sunitinib Produces Neuroprotective Effect Via Inhibiting Nitric Oxide Overproduction

Sunitinib is an inhibitor of the multiple receptor tyrosine kinases (RTKs) for cancer therapy. Some sunitinib analogues could prevent neuronal death induced by various neurotoxins. However, the neuroprotective effects of sunitinib have not been reported.

Reversal of scopolamine-induced spatial and recognition memory deficits in mice by novel multifunctional dimers bis-cognitins.

Our previous reports indicated that bis(propyl)-cognitin (B3C) and bis(heptyl)-cognitin (B7C), as novel dimers derived from tacrine, may be potential multifunctional drugs for treating Alzheimers disease. There is little knowledge on the cognitive function of B3C while B7C appeared to reverse learning and memory impairments. In this study, for the first time, we evaluated the anti-amnesic effects of B3C and B7C on learning and memory deficits induced by scopolamine using both Morris water maze and novel object recognition tasks in mice. Under the same experimental condition, the anti-amnesic effect of tacrine was also compared. Briefly, in both tasks, scopolamine (0.1-0.6 mg/kg, ip) dose-dependently impaired learning and memory functions. B3C (1.5-2.5 μmol/kg), B7C (0.4-0.6 μmol/kg) or tacrine (8-12 μmol/kg), each administered ip, dose-dependently mitigated scopolamine-induced learning and memory impairments in both tasks. Our present results show, for the first time, that B3C and B7C reverse cognitive impairment resulted from scopolamine in both water maze and object recognition tasks; and under the same condition, the relative potency of B3C and B7C to improve cognitive capacity was 5-20 folds over that of tacrine. These novel in vivo findings further demonstrate that both B3C and B7C may potentially be developed as Alzheimers therapeutic drugs for different severities of neurodegenerations.

Indirubin-3-Oxime Effectively Prevents 6OHDA-Induced Neurotoxicity in PC12 Cells via Activating MEF2D Through the Inhibition of GSK3β

Indirubin-3-oxime (I3O), a synthetic derivative of indirubin, was originally designed as potent inhibitors of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3β (GSK3β) for leukemia therapy. In the current study, we have shown, for the first time, that I3O prevented 6-hydroxydopamine (6OHDA)-induced neuronal apoptosis and intracellular reactive oxygen species accumulation in PC12 cells in a concentration-dependent manner. GSK3β inhibitors but not CDK5 inhibitors reduced the neurotoxicity induced by 6OHDA. Moreover, the activation of GSK3β was observed after 6OHDA treatment. Furthermore, 6OHDA substantially decreased the transcriptional activity of myocyte enhancer factor 2D (MEF2D), a transcription factor that plays an important role in dopaminergic neuron survival, and reduced nuclear localized MEF2D expression. Interestingly, indirubin-3-oxime and GSK3β inhibitors prevented 6OHDA-induced dysregulation of MEF2D. In addition, short hairpin RNA-mediated decrease of MEF2D expression significantly abolished the neuroprotective effects of indirubin-3-oxime. Collectively, our results strongly suggested that indirubin-3-oxime prevented 6OHDA-induced neurotoxicity via activating MEF2D, possibly through the inhibition of GSK3β. In view of the capability of indirubin-3-oxime to cross the blood–brain barrier, our findings further indicated that indirubin-3-oxime might be a novel drug candidate for neurodegenerative disorders, including Parkinson’s disease in particular.