Substantial protection against MPTP-associated Parkinson's neurotoxicity in vitro and in vivo by anti-cancer agent SU4312 via activation of MEF2D and inhibition of MAO-B.

We have previously demonstrated the unexpected neuroprotection of the anti-cancer agent SU4312 in cellular models associated with Parkinson's disease (PD). However, the precise mechanisms underlying its neuroprotection are still unknown, and the effects of SU4312 on rodent models of PD have not been characterized. In the current study, we found that the protection of SU4312 against 1-methyl-4-phenylpyridinium ion (MPP+)-induced neurotoxicity in PC12 cells was achieved through the activation of transcription factor myocyte enhancer factor 2D (MEF2D), as evidenced by the fact that SU4312 stimulated myocyte enhancer factor 2 (MEF2) transcriptional activity and prevented the inhibition of MEF2D protein expression caused by MPP+, and that short hairpin RNA (ShRNA)-mediated knockdown of MEF2D significantly abolished the neuroprotection of SU4312. Additionally, Western blotting analysis revealed that SU4312 potentiated pro-survival PI3-K/Akt pathway to down-regulate MEF2D inhibitor glycogen synthase kinase-3beta (GSK3ß). Furthermore, using the in vivo PD model of C57BL/6 mice insulted with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we found that intragastrical administration of SU4312 (0.2 and 1 mg/kg) greatly ameliorated Parkinsonian motor defects, and restored protein levels of MEF2D, phosphorylated-Ser473-Akt and phosphorylated-Ser9-GSK3ß. Meanwhile, SU4312 effectively reversed the decrease in protein expression of tyrosine hydroxylase in substantia nigra pars compacta dopaminergic neurons, inhibited oxidative stress, maintained mitochondrial biogenesis and partially prevented the depletion of dopamine and its metabolites. Very encouragingly, SU4312 was able to selectively inhibit monoamine oxidase-B (MAO-B) activity both in vitro and in vivo, with an IC50 value of 0.2 µM. These findings suggest that SU4312 provides therapeutic benefits in cellular and animal models of PD, possibly through multiple mechanisms including enhancement of MEF2D through the activation of PI3-K/Akt pathway, maintenance of mitochondrial biogenesis and inhibition of MAO-B activity. SU4312 thus may be an effective drug candidate for the prevention or even modification of the pathological processes of PD.

Protection against ß-amyloid-induced synaptic and memory impairments via altering ß-amyloid assembly by bis(heptyl)-cognitin.

ß-amyloid (Aß) oligomers have been closely implicated in the pathogenesis of Alzheimer's disease (AD). We found, for the first time, that bis(heptyl)-cognitin, a novel dimeric acetylcholinesterase (AChE) inhibitor derived from tacrine, prevented Aß oligomers-induced inhibition of long-term potentiation (LTP) at concentrations that did not interfere with normal LTP. Bis(heptyl)-cognitin also prevented Aß oligomers-induced synaptotoxicity in primary hippocampal neurons. In contrast, tacrine and donepezil, typical AChE inhibitors, could not prevent synaptic impairments in these models, indicating that the modification of Aß oligomers toxicity by bis(heptyl)-cognitin might be attributed to a mechanism other than AChE inhibition. Studies by using dot blotting, immunoblotting, circular dichroism spectroscopy, and transmission electron microscopy have shown that bis(heptyl)-cognitin altered Aß assembly via directly inhibiting Aß oligomers formation and reducing the amount of preformed Aß oligomers. Molecular docking analysis further suggested that bis(heptyl)-cognitin presumably interacted with the hydrophobic pockets of Aß, which confers stabilizing powers and assembly alteration effects on Aß. Most importantly, bis(heptyl)-cognitin significantly reduced cognitive impairments induced by intra-hippocampal infusion of Aß oligomers in mice. These results clearly demonstrated how dimeric agents prevent Aß oligomers-induced synaptic and memory impairments, and offered a strong support for the beneficial therapeutic effects of bis(heptyl)-cognitin in the treatment of AD.

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

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: MPP(+) -treated neurons and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated zebrafish were used to study neuroprotection by SU4312. NOS activity was assayed in vitro to examine direct interactions between SU4312 and NOS isoforms. KEY RESULTS: SU4312 unexpectedly prevented MPP(+) -induced neuronal apoptosis in vitro and decreased MPTP-induced loss of dopaminergic neurons, reduced expression of mRNA for tyrosine hydroxylase and impaired swimming behaviour in zebrafish. In contrast, PTK787/ZK222584, a well-studied VEGFR-2 inhibitor, failed to prevent neurotoxicity, suggesting that the neuroprotective actions of SU4312 were independent of its anti-angiogenic action. Furthermore, SU4312 exhibited non-competitive inhibition of purified neuronal NOS (nNOS) with an IC(50) value of 19.0 µM but showed little or no effects on inducible and endothelial NOS. Molecular docking simulations suggested an interaction between SU4312 and the haem group within the active centre of nNOS. CONCLUSIONS AND IMPLICATION: SU4312 exhibited neuroprotection against MPP(+) at least partly via selective and direct inhibition of nNOS. Because SU4312 could reach the brain in rats, our study also offered a support for further development of SU4312 to treat neurodegenerative disorders, particularly those associated with NO-mediated neurotoxicity.

Bis(12)-hupyridone, a novel acetylcholinesterase inhibitor, protects against glutamate-induced neuronal excitotoxicity via activating α7 nicotinic acetylcholine receptor/phosphoinositide 3-kinase/Akt cascade.

Bis(12)-hupyridone (B12H), derived from the Chinese medicinal component huperzine A, was originally designed as a novel acetylcholinesterase (AChE) inhibitor. In this paper, we report that B12H (24-h pretreatment) effectively blocked glutamate-induced neuronal excitotoxicity in cerebellar granule neurons (CGNs). However, the huge discrepancy between the EC50 value and IC50 value of B12H, to protect against neuronal toxicity (0.09 µM) and to block the NMDA receptor (21.8 µM) respectively, suggests that the neuroprotection of B12H might be not primarily due to the blockade of the NMDA receptor. Pretreatment by specific antagonists of alpha7-nicotinic acetylcholine receptor (α7nAChR), but not muscarinic acetylcholine receptor (mAChR) or α4ß2nAChR, decreased the neuroprotection of B12H. The neuroprotection of B12H could also be abolished by the pretreatment of specific PI3-K inhibitors. Furthermore, B12H restored the suppressed activation of the Akt pathway caused by glutamate as evidenced by the decreased expressions of pSer473-Akt and pSer9-GSK3ß. All these results suggest that B12H substantially protected CGNs against glutamate-induced neuronal excitotoxicity via activating α7nAChR/PI3-K/Akt cascade.

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 IC(50) 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.