Substantial Neuroprotective and Neurite Outgrowth-Promoting Activities by Bis(propyl)-cognitin via the Activation of Alpha7-nAChR, a Promising Anti-Alzheimer's Dimer.

The cause of Alzheimer's disease (AD) could be ascribed to the progressive loss of functional neurons in the brain, and hence, agents with neuroprotection and neurite outgrowth-promoting activities that allow for the replacement of lost neurons may have significant therapeutic value. In the current study, the neuroprotective and the neurite outgrowth-promoting activities and molecular mechanisms of bis(propyl)-cognitin (B3C), a multifunctional anti-AD dimer, were investigated. Briefly, B3C (24 h pretreatment) fully protected against glutamate-induced neuronal death in primary cerebellar granule neurons with an IC50 value of 0.08 µM. The neuroprotection of B3C could be abrogated by methyllycaconitine, a specific antagonist of alpha7-nicotinic acetylcholine receptor (α7-nAChR). In addition, B3C significantly promoted neurite outgrowth in both PC12 cells and primary cortical neurons, as evidenced by the increase in the percentage of cells with extended neurites as well as the up-regulation of neuronal markers growth-associated protein-43 and ß-III-tubulin. Furthermore, B3C rapidly upregulated the phosphorylation of extracellular signal-regulated kinase (ERK), a critical signaling molecule in neurite outgrowth that is downstream of the α7-nAChR signal pathway. Specific inhibitors of ERK and α7-nAChR, but not those of p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase, blocked the neurite outgrowth as well as ERK activation in PC12 cells induced by B3C. Most importantly, genetic depletion of α7-nAChR significantly abolished B3C-induced neurite outgrowth in PC12 cells. Taken together, our results suggest that B3C provided neuroprotection and neurite outgrowth-promoting activities through the activation of α7-nAChR, which offers a novel molecular insight into the potential application of B3C in AD treatment.

Robust Neuritogenesis-Promoting Activity by Bis(heptyl)-Cognitin Through the Activation of alpha7-Nicotinic Acetylcholine Receptor/ERK Pathway.

AIMS: Neurodegenerative disorders are caused by progressive neuronal loss in the brain, and hence, compounds that could promote neuritogenesis may have therapeutic values. In this study, the effects of bis(heptyl)-cognitin (B7C), a multifunctional dimer, on neurite outgrowth were investigated in both PC12 cells and primary cortical neurons. METHODS: Immunocytochemical staining was used to evaluate the proneuritogenesis effects, and Western blot and short hairpin RNA assays were applied to explore the underlying mechanisms. RESULTS: B7C (0.1-0.5 µM) induced robust neurite outgrowth in PC12 cells, as evidenced by the neurite-bearing morphology and upregulation of growth-associated protein-43 expression. In addition, B7C markedly promoted neurite outgrowth in primary cortical neurons as shown by the increase in the length of ß-III-tubulin-positive neurites. Furthermore, B7C rapidly increased ERK phosphorylation. Specific inhibitors of alpha7-nicotinic acetylcholine receptor (α7-nAChR) and MEK, but not those of p38 or JNK, blocked the neurite outgrowth as well as ERK phosphorylation induced by B7C. Most importantly, genetic depletion of α7-nAChR significantly abolished B7C-induced neurite outgrowth in PC12 cells. CONCLUSION: B7C promoted neurite outgrowth through the activation of α7-nAChR/ERK pathway, which offers novel insight into the potential application of B7C in the treatment of neurodegenerative disorders.

Inhibiting ß-amyloid-associated Alzheimer's pathogenesis in vitro and in vivo by a multifunctional dimeric bis(12)-hupyridone derived from its natural analogue.

Fibrillar aggregates of ß-amyloid protein (Aß) is the main constituent of senile plaques and considered to be one of the causative events in the pathogenesis of Alzheimer's disease (AD). Compounds that could inhibit the formation of Aß fibrils and block Aß fibrils-associated toxicity may have therapeutic potential to combat AD. Bis(12)-hupyridone (B12H) is a multifunctional homodimer derived from huperzine A, which is an anti-AD drug in China. In the current study, the inhibitory effect of B12H on the formation of Aß fibrils and their associated toxicity was investigated both in vitro and in vivo. By using Thioflavin T fluorescence assay, we found that B12H (0.3-3 µM) directly inhibited Aß fibrils formation following co-incubation of B12H and Aß1-40 at 37 °C for 6 days in vitro. However, huperzine A, at the same concentrations, did not show significant inhibitory effect on Aß1-40 fibrils formation. Moreover, B12H markedly reduced Aß1-40-induced cytotoxicity in cultured SH-SY5Y cells, as evidenced by the increase in cell viability, the decrease in lactate dehydrogenase release, and the reduction of apoptotic nuclei. Most importantly, B12H (0.2 and 0.4 mg/kg) reduced intracerebroventricular Aß1-40 infusion-induced cognitive and memory impairments in rats, as evidenced by the decrease in escape latency and the increase in the spatial bias in Morris water maze test along with increasing choline acetyltransferase activity and decreasing acetylcholinesterase activity. Collectively, our study provided novel sights into the potential application of B12H in AD treatment.

Sunitinib produces neuroprotective effect via inhibiting nitric oxide overproduction.

BACKGROUND: 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. METHODS: Cerebellar granule neurons (CGNs) and SH-SY5Y cells were exposed to low-potassium and MPP(+) challenges, respectively. MTT assay, FDA/PI staining, Hoechst staining, DAF-FM, colorimetric nitric oxide synthase (NOS) activity assay, and Western blotting were applied to detect cell viability, NO production, NOS activity, and neuronal NOS (nNOS) expression. Short hairpin RNA was used to decrease nNOS expression. In vitro NOS enzyme activity assay was used to determine the direct inhibition of nNOS by sunitinib. RESULTS: Sunitinib prevented low-potassium-induced neuronal apoptosis in CGNs and MPP(+) -induced neuronal death in SH-SY5Y cells. However, PTK787, another RTK inhibitor, failed to decrease neurotoxicity in the same models. Sunitinib reversed the increase in NO levels, NOS activity, and nNOS expression induced by low potassium or MPP(+) . Knockdown of nNOS expression partially abolished the neuroprotective effects of sunitinib. Moreover, sunitinib directly inhibited nNOS enzyme activity. CONCLUSIONS: Sunitinib exerts its neuroprotective effects by inhibiting NO overproduction, possibly via the inhibition of nNOS activity and the decrease in nNOS expression.

Substantial neuroprotection against K+ deprivation-induced apoptosis in primary cerebellar granule neurons by novel dimer bis(propyl)-cognitin via the activation of VEGFR-2 signaling pathway.

BACKGROUND: Neuronal loss via apoptosis in CNS is the fundamental mechanism underlying various neurodegenerative diseases. Compounds with antiapoptotic property might have therapeutic effects for these diseases. In this study, bis(propyl)-cognitin (B3C), a novel dimer that possesses anti-AChE and anti-N-methyl-d-aspartate receptor activities, was investigated for its neuroprotective effect on K(+) deprivation-induced apoptosis in cerebellar granule neurons (CGNs). METHODS: Cerebellar granule neurons were switched to K(+) deprived medium with or without B3C. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, fluorescein diacetate (FDA)/propidium iodide (PI) staining, Hoechst staining, and DNA laddering assays were applied to detect cytotoxicity and apoptosis. Additionally, the expression of p-VEGFR-2, p-Akt, p-glycogen synthase kinase 3ß (GSK3ß), and p-extracellular signal-regulated kinase (ERK) was examined in CGNs. RESULTS: Switching CGNs to K(+) deprived medium resulted in remarkable apoptosis, which could be substantially blocked by B3C treatment (IC50 , 0.37 µM). Moreover, a rapid decrease in p-Tyr1054-VEGFR-2 was observed after the switch. B3C significantly reversed the inhibition of p-Tyr1054-VEGFR-2 as well as Akt and ERK pathways. VEGFR-2 inhibitor PTK787/ZK222584, as well as PI3-K inhibitor LY294002 and MEK inhibitor PD98059, each abolished the neuroprotective effect of B3C. CONCLUSIONS: Our results demonstrate that B3C blocks K(+) deprivation-induced apoptosis in CGNs through regulating VEGFR-2/Akt/GSK3ß and VEGFR-2/ERK signaling pathways, providing a molecular insight into the therapeutic potential of B3C for the treatment of neurodegenerative diseases.

Bis(propyl)-cognitin protects against glutamate-induced neuro-excitotoxicity via concurrent regulation of NO, MAPK/ERK and PI3-K/Akt/GSK3ß 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 GSK3ß 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 GSK3ß (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/GSK3ß 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 Alzheimer's disease.

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 Alzheimer's 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 Alzheimer's therapeutic drugs for different severities of neurodegenerations.

Bis(12)-hupyridone, a novel multifunctional dimer, promotes neuronal differentiation more potently than its monomeric natural analog huperzine A possibly through α7 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 ßIII-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.