Regulation of acetylcholinesterase during the lipopolysaccharide-induced inflammatory responses in microglial cells.

The non-classical function of acetylcholine (ACh) has been reported in neuroinflammation that represents the modulating factor in immune responses via activation of α7 nicotinic acetylcholine receptor (α7 nAChR), i.e., a cholinergic anti-inflammatory pathway (CAP). Acetylcholinesterase (AChE), an enzyme for ACh hydrolysis, has been proposed to have a non-classical function in immune cells. However, the involvement of AChE in neuroinflammation is unclear. Here, cultured BV2 cell, a microglial cell line, and primary microglia from rats were treated with lipopolysaccharide (LPS) to induce inflammation and to explore the regulation of AChE during this process. The expression profiles of AChE, α7 nAChR, and choline acetyltransferase (ChAT) were revealed in BV2 cells. The expression of AChE (G4 form) was induced significantly in LPS-treated BV2 cells: the induction was triggered by NF-κB and cAMP signaling. Moreover, ACh or α7 nAChR agonist suppressed the LPS-induced production of pro-inflammatory cytokines, as well as the phagocytosis of microglia, by activating α7 nAChR and followed by the regulation of NF-κB and CREB signaling. The ACh-induced suppression of inflammation was abolished in AChE overexpressed cells, but did not show a significant change in AChE mutant (enzymatic activity knockout) transfected cells. These results indicate that the neuroinflammation-regulated function of AChE may be mediated by controlling the ACh level in the brain system.

Tacrine Induces Endoplasmic Reticulum-Stressed Apoptosis via Disrupting the Proper Assembly of Oligomeric Acetylcholinesterase in Cultured Neuronal Cells.

Acetylcholinesterase inhibitors (AChEIs), the most developed treatment strategies for Alzheimer's disease (AD), will be used in clinic for, at least, the next decades. Their side effects are in highly variable from drug to drug with mechanisms remaining to be fully established. The withdrawal of tacrine (Cognex) in the market makes it as an interesting case study. Here, we found tacrine could disrupt the proper trafficking of proline-rich membrane anchor-linked tetrameric acetylcholinesterase (AChE) in the endoplasmic reticulum (ER). The exposure of tacrine in cells expressing AChE, e.g., neurons, caused an accumulation of the misfolded AChE in the ER. This misfolded enzyme was not able to transport to the Golgi/plasma membrane, which subsequently induced ER stress and its downstream signaling cascade of unfolded protein response. Once the stress was overwhelming, the cooperation of ER with mitochondria increased the loss of mitochondrial membrane potential. Eventually, the tacrine-exposed cells lost homeostasis and underwent apoptosis. The ER stress and apoptosis, induced by tacrine, were proportional to the amount of AChE. Other AChEIs (rivastigmine, bis(3)-cognitin, daurisoline, and dauricine) could cause the same problem as tacrine by inducing ER stress in neuronal cells. The results provide guidance for the drug design and discovery of AChEIs for AD treatment. SIGNIFICANCE STATEMENT: Acetylcholinesterase inhibitors (AChEIs) are the most developed treatment strategies for Alzheimer's disease (AD) and will be used in clinic for at least the next decades. This study reports that tacrine and other AChEIs disrupt the proper trafficking of acetylcholinesterase in the endoplasmic reticulum. Eventually, the apoptosis of neurons and other cells are induced. The results provide guidance for drug design and discovery of AChEIs for AD treatment.

Promising tacrine/huperzine A-based dimeric acetylcholinesterase inhibitors for neurodegenerative disorders: From relieving symptoms to modifying diseases through multitarget.

Neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, are devastating diseases in the elderly world, which are closely associated with progressive neuronal loss induced by a variety of genetic and/or environmental factors. Unfortunately, currently available treatments for neurodegenerative disorders can only relieve the symptoms but not modify the pathological processes. Over the past decades, our group by collaborating with Profs. Yuan-Ping Pang and Paul R. Carlier has developed three series of homo/hetero dimeric acetylcholinesterase inhibitors derived from tacrine and/or huperzine A. The representative dimers bis(3)-Cognitin (B3C), bis(12)-hupyridone, and tacrine(10)-hupyridone might possess disease-modifying effects through the modulation of N-methyl-d-aspartic acid receptors, the activation of myocyte enhancer factor 2D gene transcription, and the promotion of neurotrophic factor secretion. In this review, we summarize that the representative dimers, such as B3C, provide neuroprotection against a variety of neurotoxins via multiple targets, including the inhibitions of N-methyl-d-aspartic acid receptor with pathological-activated potential, neuronal nitric oxide synthase, and ß-amyloid cascades synergistically. More importantly, B3C might offer disease-modifying potentials by activating myocyte enhancer factor 2D transcription, inducing neuritogenesis, and promoting the expressions of neurotrophic factors in vitro and in vivo. Taken together, the novel dimers might offer synergistic disease-modifying effects, proving that dimerization might serve as one of the strategies to develop new generation of therapeutics for neurodegenerative disorders.

Interacting with α 7 nAChR is a new mechanism for AChE to enhance the inflammatory response in macrophages.

Acetylcholine (ACh) regulates inflammation via α7 nicotinic acetylcholine receptor (α7 nAChR). Acetylcholinesterase (AChE), an enzyme hydrolyzing ACh, is expressed in immune cells suggesting non-classical function in inflammatory responses. Here, the expression of PRiMA-linked G4 AChE was identified on the surface of macrophages. In lipopolysaccharide-induced inflammatory processes, AChE was upregulated by the binding of NF-κB onto the ACHE promotor. Conversely, the overexpression of G4 AChE inhibited ACh-suppressed cytokine release and cell migration, which was in contrast to that of applied AChE inhibitors. AChEmt, a DNA construct without enzymatic activity, was adopted to identify the protein role of AChE in immune system. Overexpression of G4 AChEmt induced cell migration and inhibited ACh-suppressed cell migration. The co-localization of α7 nAChR and AChE was found in macrophages, suggesting the potential interaction of α7 nAChR and AChE. Besides, immunoprecipitation showed a close association of α7 nAChR and AChE protein in cell membrane. Hence, the novel function of AChE in macrophage by interacting with α7 nAChR was determined. Together with hydrolysis of ACh, AChE plays a direct role in the regulation of inflammatory response. As such, AChE could serve as a novel target to treat age-related diseases by anti-inflammatory responses.

Shexiang Baoxin Pill, a Traditional Chinese Herbal Formula, Rescues the Cognitive Impairments in APP/PS1 Transgenic Mice.

BACKGROUND: Shexiang Baoxin Pill (SBP), a formulated traditional Chinese medicine (TCM), has been widely used to treat cardiovascular diseases for years. This herbal mixture has been shown to promote differentiation of cultured neuronal cells. Here, we aimed to investigate the effects of SBP in attenuating cognitive impairment in APP/PS1 transgenic mice. METHODS: Ethanol and water extracts of SBP, denoted as SBPEtOH and SBPwater, were standardized and applied onto cultured rat pheochromocytoma PC12 cells. The potential effect of SBPEtOH extract in attenuating the cognitive impairments in APP/PS1 transgenic mice was shown by following lines of evidence: (i) inhibition of Aß fibril formation, (ii) suppression of secretions of cytokines, and (iii) improvement of behavioral tests by Morris water maze. RESULTS: SBPwater and SBPEtOH inhibited the formation of ß-amyloid fibrils and protected the Aß-induced cytotoxicity in cultured PC12 cells. In APP/PS1 transgenic mice, the treatment of SBPEtOH inhibited expressions of NO, NOS, AChE, as well as aggregation of Aß. Besides, the levels of pro-inflammatory cytokines were suppressed by SBP treatment in the transgenic mice. Importantly, the behavioral tests by Morris Water maze indicated that SBP attenuated cognitive impairments in APP/PS1 transgenic mice. CONCLUSION: The current result has supported the notion that SPB might ameliorate the cognitive impairment through multiple targets, suggesting that SBP could be considered as a promising anti-AD agent.

Discovery of a novel small molecule PT109 with multi-targeted effects against Alzheimer's disease in vitro and in vivo.

Alzheimer's disease (AD), which is characterized by impairment of cognitive functions, is a chronic neurodegenerative disease that mainly affects the elderly. Currently available anti-AD drugs can only offer limited symptom-relieving effects. "One-compound-Multitargeted Strategy" have been recognized as the promising way to win the war against AD. Herein we report a potential anti-AD agent PT109 with multi-functions. First, an 81-kinase screening was carried out and results showed that PT109 potently inhibited c-Jun N-terminal kinases and Serum and glucocorticoid-inducible kinase 1, which are the important signaling molecules involved in neurogenesis, neuroprotection and neuroinflammation and mildly inhibit glycogen synthase kinase-3ß as well as protein kinase C gamma, both are involved in AD pathological processes. In addition, invitro studies of immunofluorescent staining and Western blot showed that PT109 might promote the neurogenesis of C17.2 cells and induce synaptogenesis in primary cultured rat hippocampal neurons. We detected and confirmed the neuroprotective effect of PT109 in cultured HT22 cells by MTT assay, dehydrogenase assay, glutathione assay and reactive oxygen species assay. Furthermore, the results of Western blot, ELISA assay and immunofluorescent staining indicated that PT109 attenuated lipopolysaccharide-induced inflammation in BV2 cells and primary astrocytes. The results of Morris water maze and Step-through test indicated that PT109 improved the spatial learning ability in APP/PS1 mice. More importantly, the invivo pharmacokinetic parameters indicated that PT109 had better medicinal properties. Taken together, our findings suggest that PT109 may be a promising candidate for treating AD through multiple targets although further studies are ought to be conducted.

Significant combination of Aß aggregation inhibitory and neuroprotective properties in silico, in vitro and in vivo by bis(propyl)-cognitin, a multifunctional anti-Alzheimer's agent.

Inhibition of Aß aggregation and neurotoxicity has been developed as an attractive therapeutic strategy to combat Alzheimer's disease (AD). Bis(propyl)-cognitin (B3C) is a multifunctional dimer derived from tacrine. Herein, the anti-aggregation and disassembly effects of B3C on Aß, together with the neuroprotective effects and underlying mechanisms of B3C against Aß-induced neurotoxicity were investigated in silico, in vitro and in vivo. Data from Thioflavin-T fluorescence and atomic force microscopy assays indicated that B3C (1-10 µM), but not its monomer tacrine, greatly inhibited the formation of Aß fibrils and disaggregated pre-formed mature Aß fibrils. Comparative molecular dynamics simulation results revealed a possible binding mode that prevented Aß fibrils formation, showing that B3C favorably bound to Aß via hydrophobic interactions. Additionally, B3C was able to block the neurotoxicity caused by Aß fibrils in cultured PC12 cells. Very encouragingly, B3C (0.3 and 0.45 mg/kg) markedly alleviated the cognitive impairments in rats insulted by intra-hippocampal injection of Aß1-42 fibrils, more potently than tacrine (1 and 2 mg/kg). Furthermore, mechanistic studies demonstrated that B3C reversed the inhibition of phospho-GSK3ß at Ser9 site in vitro and in vivo caused by Aß, suggesting the neuroprotection of B3C was achieved through the inhibition of GSK3ß pathway. These findings indicate that B3C could serve as an effective inhibitor of Aß aggregation and neurotoxicity, and provide novel molecular insights into the potential application of B3C in AD prevention and treatment.

Multifunctional memantine nitrate significantly protects against glutamate-induced excitotoxicity via inhibiting calcium influx and attenuating PI3K/Akt/GSK3beta pathway.

Overactivation of N-methyl-D-aspartate (NMDA) receptors has been associated with neurodegenerative disorders such as Alzheimer's disease (AD), cerebral vascular disorders and amyotrophic lateral sclerosis (ALS). We have previously designed and synthesized a series of memantine nitrate and some of them have shown vessel dilatory effects and neuroprotective effects; however, the detailed mechanisms have not been elucidated. In this study, we further demonstrated that memantine nitrate-06 (MN-06), one of the novel compounds derived from memantine, possessed significant neuroprotective effects against glutamate-induced excitotoxicity in rat primary cerebellar granule neurons (CGNs). Pretreatment of MN-06 reversed the activation of GSK3b and the suppression of phosphorylated Akt induced by glutamate. In addition, the neuroprotective effects of MN-06 could be abolished by LY294002, the specific phosphatidylinositol 3-kinase (PI3-K) inhibitor. Ca2+ imaging shown that pretreatment of MN-06 prevented Ca2+ influx induced by glutamate. Moreover, MN-06 might inhibit the NMDA-mediated current by antagonizing NDMA receptors, which was further confirmed by molecular docking simulation. Taken together, MN-06 protected against glutamate-induced excitotoxicity by blocking calcium influx and attenuating PI3-K/Akt/GSK-3b pathway, indicating that MN-06 might be a potential drug for treating neurodegenerative disorders.

Pharmacological Characterizations of anti-Dementia Memantine Nitrate via Neuroprotection and Vasodilation in Vitro and in Vivo.

We have previously designed and synthesized a series of novel memantine nitrates, and some of them have shown neuroprotective effects; however, the detailed mechanisms remain unknown. In this study, we demonstrated that MN-12, one of the memantine nitrates, concentration-dependently protected against glutamate-induced neurotoxicity in rat primary cultured cerebellar granule neurons (CGNs). Western blotting assays revealed that MN-12 might possess neuroprotective effects through the inhibition of ERK pathway and activation of PI3K/Akt pathway concurrently. Moreover, MN-12 concentration-dependently dilated precontracted rat middle cerebral artery through activation of NO-cGMP pathway ex vivo. In the 2-vessel occlusion (2VO) rat model, MN-12 alleviated the impairments of spatial memory and motor dysfunction possibly via neuroprotection and improvement of the cerebral blood flow. Furthermore, the results of preliminary pharmacokinetic studies showed that MN-12 might quickly distribute to the major organs including the brain, indicating that MN-12 could penetrate the blood-brain barrier. Taken together, MN-12 might provide multifunctional therapeutic benefits for dementia associated with Alzheimer's disease, vascular dementia, and ischemic stroke, via neuroprotection and vessel dilation to improve the cerebral blood flow.

The dual-functional memantine nitrate MN-08 alleviates cerebral vasospasm and brain injury in experimental subarachnoid haemorrhage models.

BACKGROUND AND PURPOSE: Cerebral vasospasm and neuronal apoptosis after subarachnoid haemorrhage (SAH) is the major cause of morbidity and mortality in SAH patients. So far, single-target agents have not prevented its occurrence. Memantine, a non-competitive NMDA re3ceptor antagonist, is known to alleviate brain injury and vasospasm in experimental models of SAH. Impairment of NO availability also contributes to vasospasm. Recently, we designed and synthesized a memantine nitrate MN-08, which has potent dual functions: neuroprotection and vasodilation. Here, we have tested the therapeutic effects of MN-08 in animal models of SAH. EXPERIMENTAL APPROACH: Binding to NMDA receptors (expressed in HEK293 cells), NO release and vasodilator effects of MN-08 were assessed in vitro. Therapeutic effects of MN-08 were investigated in vivo, using rat and rabbit SAH models. KEY RESULTS: MN-08 bound to the NMDA receptor, slowly releasing NO in vitro and in vivo. Consequently, MN-08 relaxed the pre-contracted middle cerebral artery ex vivo and increased blood flow velocity in small vessels of the mouse cerebral cortex. It did not, however, lower systemic blood pressure. In an endovascular perforation rat model of SAH, MN-08 improved the neurological scores and ameliorated cerebral vasospasm. Moreover, MN-08 also alleviated cerebral vasospasm in a cisterna magna single-injection model in rabbits. MN-08 attenuated neural cell apoptosis in both rat and rabbit models of SAH. Importantly, the therapeutic benefit of MN-08 was greater than that of memantine. CONCLUSION AND IMPLICATIONS: MN-08 has neuroprotective potential and can ameliorate vasospasm in experimental SAH models.

Bis(propyl)-cognitin potentiates rehabilitation of treadmill exercise after a transient focal cerebral ischemia, possibly via inhibiting NMDA receptor and regulating VEGF expression.

Combination therapies may have greater efficacy compared with monotherapy in treating stroke. We investigated the molecular mechanisms by which the combination of bis(propyl)-cognitin, an uncompetitive antagonist of NMDA receptor, and treadmill exercise promote rehabilitation after ischemic stroke. Rats were distributed into 3 treatment groups: infarct/bis(propyl)-cognitin(drug only group, DO); infarct/treadmill exercise(exercise only group, EO); infarct/bis(propyl)-cognitin + treadmill exercise (drug + exercise group, DE). The DE group had further separated to 3 sub-groups to investigate the effects achieved by different time for drug administration (60 min before stroke (DE-60 m), 15 min (DE+15 m) and 60 min (DE+60 m) after stroke). Although all infarct groups improved over time, the combination of bis(propyl)-cognitin and treadmill exercise effectively enhanced motor recovery during 14-day intervention. Early drug intervention has a best recovery result, the DE+15 m group with drug intervention at 15-min after stroke had better motor recovery than DE+60 m, DO, EO and control groups. Both bis(propyl)-cognitin and treadmill exercise significantly elevated brain VEGF expression and decreased brain infarct volume at 14 day post-ischemia. Our study reveals that bis(propyl)-cognitin potentiated rehabilitation of treadmill exercise after ischemic stroke, possibly via regulating brain VEGF expression, indicating that the combination of NMDA receptor antagonists and exercise might be useful for stroke rehabilitation.

Tacrine(10)-Hupyridone Prevents Post-operative Cognitive Dysfunction via the Activation of BDNF Pathway and the Inhibition of AChE in Aged Mice.

Post-operative cognitive dysfunction (POCD) could cause short-term or long-term cognitive disruption lasting weeks or months after anesthesia and surgery in elderly. However, no effective treatment of POCD is currently available. Previous studies indicated that the enhancement of brain-derived neurotrophic factor (BDNF) expression, and the elevation the cholinergic system, might be effective to prevent POCD. In this study, we have discovered that tacrine(10)-hupyridone (A10E), a novel acetylcholinesterase (AChE) inhibitor derived from tacrine and huperzine A, could prevent surgery-induced short-term and long-term impairments of recognition and spatial cognition, as evidenced by the novel object recognition test and Morris water maze (MWM) tests, in aged mice. Moreover, A10E significantly increased the expression of BDNF and activated the downstream Akt and extracellular regulated kinase (ERK) signaling in the surgery-treated mice. Furthermore, A10E substantially enhanced choline acetyltransferase (ChAT)-positive area and decreased AChE activity, in the hippocampus regions of surgery-treated mice, indicating that A10E could prevent surgery-induced dysfunction of cholinergic system, possibly via increasing the synthesis of acetylcholine and the inhibition of AChE. In conclusion, our results suggested that A10E might prevent POCD via the activation of BDNF pathway and the inhibition of AChE, concurrently, in aged mice. These findings also provided a support that A10E might be developed as a potential drug lead for POCD.

Neuroprotection Against MPP+-Induced Cytotoxicity Through the Activation of PI3-K/Akt/GSK3ß/MEF2D Signaling Pathway by Rhynchophylline, the Major Tetracyclic Oxindole Alkaloid Isolated From Uncaria rhynchophylla.

Rhynchophylline is a major tetracyclic oxindole alkaloid in Uncaria rhynchophylla, which has been extensively used as traditional herb medicine for the prevention of convulsions and hypertension. However, there is still little evidence about the neuroprotective effects of rhynchophylline for Parkinson's disease (PD), a neurodegenerative condition currently without any effective cure. In this present study, the neuroprotective molecular mechanisms of rhynchophylline were investigated in a cellular model associated with PD. It is shown that rhynchophylline (10-50 µM) greatly prevented neurotoxicity caused by 1-methyl-4-phenylpyridinium ion (MPP+) in primary cerebellar granule neurons, as evidenced by the promotion of cell viability as well as the reversal of dysregulated protein expression of Bax/Bcl-2 ratio. Very encouragingly, we found that rhynchophylline markedly enhanced the activity of the transcription factor myocyte enhancer factor 2D (MEF2D) at both basal and pathological conditions using luciferase reporter gene assay, and reversed the inhibition of MEF2D caused by MPP+. Additionally, pharmacological inhibition of PI3-Kinase or short hairpin RNA-mediated gene down-regulation of MEF2D abrogated the protection provided by rhynchophylline. Furthermore, Western blot analysis revealed that rhynchophylline could potentiate PI3-K/Akt to attenuate GSK3ß (the MEF2D inhibitor) in response to MPP+ insult. In conclusion, rhynchophylline inhibits MPP+-triggered neurotoxicity by stimulating MEF2D via activating PI3-K/Akt/GSK3ß cascade. Rhynchophylline is served as a novel MEF2D enhancer and might be a potential candidate for further preclinical study in the prevention of PD.

Tacrine(10)-hupyridone, a dual-binding acetylcholinesterase inhibitor, potently attenuates scopolamine-induced impairments of cognition in mice.

Tacrine(10)-hupyridone (A10E) was designed as a dual-binding acetylcholinesterase (AChE) inhibitor from the modification of tacrine and a fragment of huperzine A. We have found that A10E effectively inhibited AChE in a mixed competitive manner, with an IC50 of 26.4 nM, which is more potent than those of tacrine and huperzine A. Most importantly, we have shown, for the first time that A10E attenuated scopolamine-induced cognitive impairments without affecting motor function in mice. A10E effectively attenuated impairments of learning and memory to a similar extent as donepezil, an inhibitor of AChE used for treating Alzheimer's disease (AD). In addition, A10E significantly decreased AChE activity in the brain of mice, suggesting that A10E might cross the brain blood-barrier. Taken together, our results demonstrated that A10E, a designed dual-binding AChE inhibitor, could effectively reverse cognitive impairments, indicating that A10E might provide therapeutic efficacy for AD treatment.

Research and development of anti-Alzheimer's disease drugs: an update from the perspective of technology flows.

INTRODUCTION: Today, over 20 million people suffer from Alzheimer's disease (AD) worldwide. AD has become a critical issue to human health, especially in aging societies, and therefore it is a research hotspot in the global scientific community. The technology flow method differs from traditional reviews generating an informative overview of the research and development (R&D) landscape in a specific technological area. We need such an updated method to get a general overview of the R&D of anti-AD drugs in light of the dramatic developments in this area in recent years. AREAS COVERED: This study collects patent data from the Integrity database. A total of 399 patents with 821 internal citation pairs in the US from 1978 to 2017 were analyzed. Patent citation network analysis was used to visualize the technology relationship. EXPERT OPINION: For better production of anti-AD drugs, governments should emphasize the multi-target drug design, provide policy support for private companies, and encourage multilateral cooperation. The ß-amyloid peptide (Aß) theory leaves much to be desired; neurotransmitter and tau protein hypotheses are worth further examination. The use of old drugs for new indications is promising, as are traditional herbal medicines.

A Novel Tetramethylpyrazine Derivative Prophylactically Protects against Glutamate-Induced Excitotoxicity in Primary Neurons through the Blockage of N-Methyl-D-aspartate Receptor.

The over-activation of NMDA receptor via the excessive glutamate is believed to one of the most causal factors associated with Alzheimer's disease (AD), a progressive neurodegenerative brain disorder. Molecules that could protect against glutamate-induced neurotoxicity may hold therapeutic values for treating AD. Herein, the neuroprotective mechanisms of dimeric DT-010, a novel derivative of naturally occurring danshensu and tetramethylpyrazine, were investigated using primary rat cerebellar granule neurons (CGNs) and hippocampal neurons. It was found that DT-010 (3-30 µM) markedly prevented excitotoxicity of CGNs caused by glutamate, as evidenced by the promotion of neuronal viability as well as the reversal of abnormal morphological changes. While its parent molecules did not show any protective effects even when their concentration reached 50 µM. Additionally, DT-010 almost fully blocked intracellular accumulation of reactive oxygen species caused by glutamate and exogenous oxidative stimulus. Moreover, Western blot results demonstrated that DT-010 remarkably attenuated the inhibition of pro-survival PI3K/Akt/GSK3ß pathway caused by glutamate. Ca2+ imaging with Fluo-4 fluorescence analysis further revealed that DT-010 greatly declined glutamate-induced increase in intracellular Ca2+. Most importantly, with the use of whole-cell patch clamp electrophysiology, DT-010 directly inhibited NMDA-activated whole-cell currents in primary hippocampal neurons. Molecular docking simulation analysis further revealed a possible binding mode that inhibited NMDA receptor at the ion channel, showing that DT-010 favorably binds to Asn602 of NMDA receptor via arene hydrogen bond. These results suggest that DT-010 could be served as a novel NMDA receptor antagonist and protect against glutamate-induced excitotoxicity from blocking the upstream NMDA receptors to the subsequent Ca2+ influx and to the downstream GSK3ß cascade.

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.

Indirubin-3-Oxime Prevents H2O2-Induced Neuronal Apoptosis via Concurrently Inhibiting GSK3ß and the ERK Pathway.

Oxidative stress-induced neuronal apoptosis plays an important role in many neurodegenerative disorders. In this study, we have shown that indirubin-3-oxime, a derivative of indirubin originally designed for leukemia therapy, could prevent hydrogen peroxide (H2O2)-induced apoptosis in both SH-SY5Y cells and primary cerebellar granule neurons. H2O2 exposure led to the increased activities of glycogen synthase kinase 3ß (GSK3ß) and extracellular signal-regulated kinase (ERK) in SH-SY5Y cells. Indirubin-3-oxime treatment significantly reversed the altered activity of both the PI3-K/Akt/GSK3ß cascade and the ERK pathway induced by H2O2. In addition, both GSK3ß and mitogen-activated protein kinase inhibitors significantly prevented H2O2-induced neuronal apoptosis. Moreover, specific inhibitors of the phosphoinositide 3-kinase (PI3-K) abolished the neuroprotective effects of indirubin-3-oxime against H2O2-induced neuronal apoptosis. These results strongly suggest that indirubin-3-oxime prevents H2O2-induced apoptosis via concurrent inhibiting GSK3ß and the ERK pathway in SH-SY5Y cells, providing support for the use of indirubin-3-oxime to treat neurodegenerative disorders caused or exacerbated by oxidative stress.

No synergism between bis(propyl)-cognitin and rasagiline on protecting dopaminergic neurons in Parkinson's disease mice.

Rasagiline, a monoamine oxidase-B inhibitor, and bis(propyl)-cognitin (B3C), a novel dimer are reported to be neuroprotective. Herein, the synergistical neuroprotection produced by rasagiline and B3C was investigated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice of Parkinsonism. By using neurobehavioural tests, high-performance liquid chromatography and western blot assay, we showed that B3C at 0.3 mg/kg, rasagiline at 0.02 mg/kg, as well as co-treatment with B3C and rasagiline prevented MPTP-induced behavioural abnormities, increased the concentrations of dopamine and its metabolites in the striatum, and up-regulated the expression of tyrosine hydroxylase in the substantia nigra. However, the neuroprotective effects of co-treatment were not significantly improved when compared with those of B3C or rasagiline alone. Collectively, we have demonstrated that B3C at 0.3 mg/kg and rasagline at 0.02 mg/kg could not produce synergistic neuroprotective effects.