Huan Zhang

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.

Protective, antioxidative and antiapoptotic effects of 2-methoxy-6-acetyl-7-methyljuglone from Polygonum cuspidatum in PC12 cells.

Much correlative evidence indicates that the oxidative modification of protein by reactive oxygen species (ROS) is involved in normal aging as well as the pathogenesis of neurodegenerative diseases such as Alzheimers disease. In this study, we explored the antioxidative and neuroprotective effects of a naphthoquinone, 2-methoxy-6-acetyl-7-methyljuglone (MAM), purified from the dried rhizome of POLYGONUM CUSPIDATUM (Chinese name Hu-Zhang). Pretreatments with MAM (24 h) were investigated for their protective effects against apoptosis induced by the oxidizing agent TERT-butyl hydroperoxide ( T-BHP) in PC12 cells. The results indicated that MAM pretreatments could effectively protect PC12 cells against cytotoxicity induced by T-BHP in a dose-dependent manner. Cell viability was determined by both MTT and LDH assays. Increasing concentrations of MAM enhanced cell viability significantly and completely prevented cell death induced by T-BHP at 2.5 µM. The corresponding extracellular lactate dehydrogenase (LDH) levels were also attenuated significantly by various concentrations of MAM. In addition, it was found that the antioxidative effect of MAM was stronger than those of resveratrol and lipoic acid. The antiapoptotic property of MAM was further investigated with Hoechst 33342 nuclear staining and TUNEL assay. Pretreatments of MAM were able to prevent the T-BHP-induced nucleus fragmentation and accumulation of apoptotic bodies (commonly accepted as markers of apoptosis) inside the cells in a dose-dependent manner. T-BHP induced the phosphorylation of ERK 1/2, JNK and p38 MAPK, which were all impeded by pretreatments with MAM, indicating that MAM may act as a potent antioxidant which significantly interferes with the MAPK apoptotic cascades, probably rescuing cells by inhibiting the death pathways.

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.

Preventing H2O2-induced apoptosis in cerebellar granule neurons by regulating the VEGFR-2/Akt signaling pathway using a novel dimeric antiacetylcholinesterase bis(12)-hupyridone

Oxidative stress-induced apoptosis plays a critical role in the pathogenesis of various neurodegenerative disorders. In this study, the neuroprotective properties of bis(12)-hupyridone (B12H), a novel dimeric acetylcholinesterase (AChE) inhibitor modified from a naturally occurring monomeric analogue, huperzine A, on H₂O₂-induced neurotoxicity were investigated in cerebellar granule neurons (CGNs). Exposure of CGNs to H₂O₂ resulted in apoptosis which could be attenuated by the pre-treatment of B12H (0.3-5 nM) in a concentration-dependent manner. Moreover, tacrine and neostigmine failed to prevent neurotoxicity, indicating that the neuroprotection of B12H might not be due to its inhibitory property of AChE enzymatic activity. Increased activation of extracellular signal-regulated kinase (ERK) and decreased activation of glycogen synthase kinase (GSK) 3β were observed after H₂O₂ exposure, and B12H reversed the altered activation of GSK3β, but not that of ERK. Furthermore, using vascular endothelial growth factor (VEGF), phospho-VEGF receptor-2 (VEGFR-2) antibody, a specific VEGFR-2 inhibitor (PTK787/ZK222584) and specific phosphoinositide 3-kinase inhibitors (LY294002 and wortmannin), it was found that VEGF prevented H₂O₂-induced neuronal loss from activating the VEGF/VEGFR-2 system and that the observed B12H neuroprotective effects might share the same signaling pathway. These findings strongly suggest that B12H prevents H₂O₂-induced neuronal apoptosis independent of inhibiting AChE, but through regulating VEGFR-2/Akt/GSK3β signaling pathway.

Neuroprotection against excitotoxic and ischemic insults by bis(12)-hupyridone, a novel anti-acetylcholinesterase dimer, possibly via acting on multiple targets

The activation of N-methyl-d-aspartate (NMDA) receptors by excessive release of glutamate is involved in the pathogenesis of ischemic stroke. Thus the NMDA receptor has become an attractive therapeutic target for the development of neuroprotectants, especially from antagonists with moderate to low affinity. In the current study, NMDA receptor blockage and neuroprotective effects of bis(12)-hupyridone (B12H), a novel dimeric acetylcholinesterase inhibitor derived from a naturally occurring monomeric analog huperzine A, were investigated in vitro and in vivo. In primary rat cerebellar granule neurons, B12H (0.1 nM to 1 μM) prevented glutamate-induced apoptosis in a concentration- and time-dependent manner. Receptor-ligand binding analysis showed that B12H competed with [(3)H]MK801 with a K(i) value of 7.7 μM. In the 2-hour middle cerebral artery occlusion rat model, B12H (0.4 and 0.8 mg/kg, 30 min before-ischemia and 15 min post-ischemia, i.p.) significantly attenuated ischemia-induced apoptosis in the penumbra region, improved neurological behavior impairment, and decreased cerebral infarct volume, cerebral edema and neuronal apoptosis in the stroke model. Together, these results showed that B12H moderately blocks NMDA receptors at MK801 site and exerts neuroprotection against excitotoxic and ischemic insults in vitro and in vivo. Combined with our previous publications, we conjecture that B12H might exert neuroprotection via acting on multiple targets.

Two new anthraquinone malonylglucosides from Polygonum cuspidatum.

Two new anthraquinone malonylglucosides polygonins A and B were isolated from Polygonum cuspidatum along with seven known compounds. The structures of 1 and 2 were elucidated based on their spectroscopic data.

Tanshinone II A, a multiple target neuroprotectant, promotes caveolae-dependent neuronal differentiation

Neuron loss is one fundamental features of neurodegenerative diseases. Stimulating endogenous neurogenesis, especially neuronal differentiation, might potentially provide therapeutic effects to these diseases. In this study, tanshinone II A (TIIA), a multiple target neuroprotectant, was demonstrated to promote dose-dependent neuronal differentiation in three cell models of immortalized C17.2 neuronal stem cells, rat embryonic cortical neural stem cells (NSCs) and rat PC12 pheochromocytoma cells. In particular, TIIA exerted promising effects on NSCs even at the dose of 3 nM. In PC12 cells, TIIA activated mitogen-activated protein kinase 42/44 (MAPK42/44) and its downstream transcription factor, cAMP response element-binding protein (CREB). In addition, TIIA up-regulated the expressions of brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF). The MEK inhibitor and the antagonist to the receptors of NGF and BDNF could partially attenuate the differentiation effects, indicating that MAPK42/44 mediated BDNF and NGF signals were involved in TIIAs differentiation effects. Caveolin-1 (CAV-1), the major functional protein of membrane caveolae, plays critical roles in the endocytosis of exogenous materials. CAV1, which was activated by TIIA, might help TIIA transport across cell membrane to initiate its differentiation effects. It was proven by the evidences that suppressing the function of caveolin inhibited the differentiation effects of TIIA. Therefore, we concluded that TIIA promoted neuronal differentiation partially through MAPK42/44 mediated BDNF and NGF signals in a caveolae-dependent manner.