Smilagenin Protects Dopaminergic Neurons in Chronic MPTP/Probenecid-Lesioned Parkinson's Disease Models.

Current therapies for Parkinson's disease (PD) only offer limited symptomatic alleviation but fail to hamper the progress of the disease. Thus, it is imperative to establish new approaches aiming at protecting or reversing neurodegeneration in PD. Recent work elucidates whether smilagenin (abbreviated SMI), a steroidal sapogenin from traditional Chinese medicinal herbs, can take neuroprotective effect on dopaminergic neurons in a chronic model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) conjuncted with probenecid mice. We reported for the first time that SMI significantly improved the locomotor ability of chronic MPTP/probenecid-lesioned mice. SMI increased the tyrosine hydroxylase (TH) positive and Nissl positive neuron number in the substantia nigra pars compacta (SNpc), augmented striatal DA and its metabolites concentration and elevated striatal dopamine transporter density (DAT). In addition, dopamine receptor D2R not D1R was down-regulated by MPTP/probenecid and slightly raised by SMI prevention. What's more, we discovered that SMI markedly elevated striatal glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) protein levels in SMI prevented mice. And we found that SMI increased GDNF and BDNF mRNA level by promoting CREB phosphorylation in 1-methyl-4-phenylpyridimium (MPP+) treated SH-SY5Y cells. The results illustrated that SMI could prevent the impairment of dopaminergic neurons in chronic MPTP/probenecid-induced mouse model.

Catalpol protects synaptic proteins from beta-amyloid induced neuron injury and improves cognitive functions in aged rats.

Synapse loss is one of the common factors contributing to cognitive disorders, such as Alzheimer's disease (AD), which is manifested by the impairment of basic cognitive functions including memory processing, perception, problem solving, and language. The current therapies for patients with cognitive disorders are mainly palliative; thus, regimens preventing and/or delaying dementia progression are urgently needed. In this study, we evaluated the effects of catalpol, isolated from traditional Chinese medicine Rehmannia glutinosa, on synaptic plasticity in aged rat models. We found that catalpol markedly improved the cognitive function of aged male Sprague-Dawley rats and simultaneously increased the expression of synaptic proteins (dynamin 1, PSD-95, and synaptophysin) in the cerebral cortex and hippocampus, respectively. In beta-amyloid (Aß) injured primary rat's cortical neuron, catalpol did not increase the viability of neuron but extended the length of microtubule-associated protein 2 (MAP-2) positive neurites and reversed the suppressive effects on expression of synaptic proteins induced by Aß. Additionally, the effects of catalpol on stimulating the growth of MAP-2 positive neurites and the expression of synaptic proteins were diminished by a PKC inhibitor, bisindolylmaleimide I, suggesting that PKC may be implicated in catalpol's function of preventing the neurodegeneration induced by Aß. Altogether, our study indicates that catalpol could be a potential disease-modifying drug for cognitive disorders such as AD.

[Synaptic vesicle recycling and Alzheimer's disease].

Loss of synapses correlates well with cognitive decline in Alzheimer's disease (AD). However, the molecular mechanisms underlying the synaptic dysfunction and loss are not well understood. Synaptic vesicle (SV) recycling is a key process for synaptic transmission. A body of evidences suggested that malfunction or loss of the machinery for SV recycling occurred in AD, which could result in disruption of neuronal circuitry. In this article, we summarized the recent progress in the research of synaptic proteins for SV recycling and the pathological changes of some proteins in AD.

Memory defect induced by ß-amyloid plus glutamate receptor agonist is alleviated by catalpol and donepezil through different mechanisms.

Our previous studies demonstrate that a non-cholinesterase inhibitor (AChEI) compound catalpol, purified from a traditional Chinese medicinal herb Rehmannia glutinosa, could improve the symptoms and pathological changes in animal and cellular models of memory related neurodegenerative diseases. In this study, we compared catalpol with the most commonly used AChEI donepezil in respect to their mechanism of action on the neurodegenerative changes in an animal model induced by beta-amyloid (Aß) plus glutamate receptor agonist. It was found that the model mice showed significant deficit in the learning ability and memory in Y maze avoidance test, and meanwhile both donepezil and catalpol greatly improve the learning ability and memory after 2 to 3 months' administration. At the selected doses, donepezil only partially raised the declined brain muscarinic acetylcholine receptor (M receptor) density and choline acetyltransferase (ChAT) activity resulting in these levels still lower than normal control, while catalpol completely retrieved these two parameters. ELISA revealed that catalpol, instead of donepezil, possessed the capability of elevating the declined brain BDNF level of the animal model. The ELISA results on the BDNF protein level was confirmed by quantitative RT-PCR measurement of BDNF mRNA in Aß25₋35-treated primary culture of forebrain neurons. In combination with our previous work, we think the neuroprotective effects of donepezil and catalpol are mediated through different mechanisms. Since BDNF has been proved to be an important intrinsic factor in protecting neurodegenerative diseases, catalpol may be a hopefully effective compound against neurodegenerative changes induced by Aß and glutamate receptor agonist.

Harpagoside attenuates MPTP/MPP⁺ induced dopaminergic neurodegeneration and movement disorder via elevating glial cell line-derived neurotrophic factor.

Parkinson's disease is a chronic neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. New therapeutic approaches aiming at delaying or reversing the neurodegenerative process are under active investigations. In this work, we found that harpagoside, an iridoid purified from the Chinese medicinal herb Scrophularia ningpoensis, could not only prevent but also rescue the dopaminergic neurodegeneration in MPTP/MPP(+) intoxication with promising efficacy. Firstly, in cultured mesencephalic neurons, harpagoside significantly attenuated the loss of TH-positive neuron numbers and the shortening of axonal length. Secondly, in a chronic MPTP mouse model, harpagoside dose-dependently improved the loco-motor ability (rotarod test), increased the TH-positive neuron numbers in the substantia nigra pars compacta (unbiased stereological counting) and increased the striatal DAT density ((125) I-FP-CIT autoradiography). Thirdly, harpagoside markedly elevated the GDNF mRNA and GDNF protein levels in MPTP/MPP(+) lesioned models. However, the protecting effect of harpagoside on the dopaminergic degeneration disappeared when the intrinsic GDNF action was blocked by either the Ret inhibitor PP1 or the neutralizing anti-GDNF antibody. Taken together, we conclude that harpagoside attenuates the dopaminergic neurodegeneration and movement disorder mainly through elevating glial cell line-derived neurotrophic factor.

Preservation of TSPO by chronic intermittent hypobaric hypoxia confers antiarrhythmic activity.

Abnormal activation of mitochondrial translocator protein (TSPO) contributes to arrhythmogenesis during cardiac metabolic compromise; however, its role in the antiarrhythmic activities of chronic hypoxia adaptation remains unclear. Our results demonstrated that 80% of normoxic rats developed ischaemic VF, whereas this condition was seldom observed in rats with 14 days of chronic intermittent hypobaric hypoxia (CIHH). TSPO stimulation or inhibition affected the arrhythmias incidence in normoxic rats, but did not change the CIHH-mediated antiarrhythmic effects. Abrupt and excessive elevation of TSPO activity was positively linked to ischaemic VF, and CIHH preserved TSPO activity during ischaemia. The preservation of TSPO activity by CIHH also contributed to the maintenance of intracellular Ca homeostasis. These results suggest that the blunt sensitivity of TSPO to ischaemic stress may be responsible for the antiarrhythmic effects by CIHH.

Role of CREB in the regulatory action of sarsasapogenin on muscarinic M1 receptor density during cell aging.

This work studied the role of cyclic AMP responsive element binding protein (CREB) in the up-regulation of M(1) muscarinic acetylcholine receptor (M(1) receptor) density by sarsasapogenin (ZMS) in CHO cells transfected with M(1) receptor gene (CHOm1 cells). During cell aging, sarsasapogenin elevated M(1) receptor density as well as CREB and phosphor-CREB (pCREB) levels. CREB peaked earliest, followed by pCREB and M(1) receptor density peaked last. When CREB synthesis was blocked by antisense oligonucleotides, the elevation effect of sarsasapogenin on M(1) receptor density was abolished. These results suggest that sarsasapogenin up-regulates M(1) receptor density in aged cells by promoting CREB production and phosphorylation. Furthermore, the results support the hypothesis that pCREB regulates M(1) receptor gene expression through heterodimer formation.

Regulation of M1-receptor mRNA stability by smilagenin and its significance in improving memory of aged rats.

The purpose of this work is to study the effect of smilagenin on the mRNA stability of muscarinic receptor subtype 1 (M(1); m1 mRNA) in aged rat brains and its significance in improving memory. The Y-maze avoidance task showed that oral administration of smilagenin significantly improved spatial memory performance in aged rats. Mechanistic studies showed that smilagenin was neither a ligand of the M receptors nor a cholinesterase inhibitor, while radioligand binding assays revealed that smilagenin significantly increased the M(1)-receptor density. The increase of M(1)-receptor density correlated with memory improvement. Real-time polymerase chain reaction (RT-PCR) revealed that the m1 mRNA in m1 gene-transfected CHO cells increased significantly, and the average half-life of m1 mRNA was approximately doubled by smilagenin treatment. These results suggest that smilagenin improves memory of aged rats at least partially by increasing the stability of m1 mRNA. However since the ChAT activity in the cortex of aged rats was also elevated by smilagenin, it cannot be excluded that the increase of intrinsic acetylcholine excretion also plays a role in the memory-improvement effect of smilagenin.

Mitochondrial benzodiazepine receptors mediate cardioprotection of estrogen against ischemic ventricular fibrillation.

The cardioprotective effects of estrogen remain controversial in clinical practice. Previous reports have shown that cardioprotective mechanisms converge on the mitochondria, but the role of mitochondria in estrogen's actions on cardiac arrhythmias is unclear. Here, we report that stimulation or inhibition of mitochondrial benzodiazepine receptors (mBzR) affected ventricular fibrillation (VF) almost in an "all-or-none" manner in an in vitro rat heart model of ischemic VF. Low concentrations of estrogen did not provide antiarrhythmic effects; however, the combination of mBzR activator and estrogen reduced VF incidence in hearts from either gender. Such synergistic actions also enabled cardiomyocytes to resist metabolic stress-induced intracellular [Ca(2+)](i) overload. Ligand binding experiments revealed that estrogen itself did not affect mBzR activity under basal conditions but promoted its up-regulation under myocardial ischemia. Our results suggest that mBzR may be an important molecule for ischemic arrhythmia and may act as a molecular switch for estrogen's antiarrhythmic effects. This finding provides a clue for elucidating the conflicting results regarding estrogen's cardiac effects in clinical studies and also suggests potential new strategies for hormone treatment in the female population.

[Effects of Chinese herb compound Naoyikang on expression of choline acetyltransferase in brain of rats with Alzheimer's disease].

OBJECTIVE: To observe the effects of Naoyikang (NYK) on expression of choline acetyltransferase (ChAT) in brain of rats with Alzheimer' s disease (AD). METHOD: Bilateral infusions of Ibotenic acid (IBO) into nucleus basalis of Meynert (NBM) using hamilton syringe and stereotaxic apparatus were adopted to establish the rat model of AD. After intragastrically administrated with different solution for 28 days, immunohistochemistry and Western-blot were adopted to study the expression of ChAT in frontal cortex of AD rats. RESULT: NYK could improve the morphology and increase the number of ChAT immunoreactive neurons, and significantly promote ChAT protein expression. CONCLUSION: NYK may be able to increase the synthesis of acetylcholine (ACh) through elevating the expression of ChAT protein, thus improving the level of brain ACh so as to protect central cholinergic neurons.

Role of glial cell derived neurotrophic factor in the protective effect of smilagenin on rat mesencephalic dopaminergic neurons damaged by MPP+.

Tyrosine hydroxylase immunohistochemical analysis revealed that in cultured mesencephalic dopaminergic neurons smilagenin (SMI), added prior to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPP+), protected against the drop of neuron number and neurite outgrowth length caused by MPP+. Addition of anti-GDNF and/or anti-GFR alpha 1 functional antibodies to the medium prior to SMI, eliminated mostly, though incompletely, the action of SMI. The expression of glial cell derived neurotrophic factor (GDNF) mRNA, but not GDNF receptor alpha1 (GFR alpha 1) or receptor tyrosine kinase mRNA in MPP+ intoxicated neurons was markedly elevated as early as 2h after the addition of SMI with a peak at 24-48 h. Therefore, an important route of the protective action of SMI on dopaminergic neurons is to stimulate intrinsic GDNF expression.

Translation of striatal-enriched protein tyrosine phosphatase (STEP) after beta1-adrenergic receptor stimulation.

The beta-adrenergic system is implicated in long-term synaptic plasticity in the CNS, a process that requires protein synthesis. To identify proteins that are translated in response to beta-adrenergic receptor stimulation and the pathways that regulate this process, we investigated the effects of isoproterenol on the translation of striatal-enriched protein tyrosine phosphatase (STEP) in both cortico-striatal slices and primary neuronal cultures. Isoproterenol stimulation induced a rapid dose-dependent increase in STEP expression. Anisomycin blocked the increase in STEP expression while actinomycin D had no effect, suggesting a translation-dependent mechanism. Isoproterenol-induced STEP translation required activation of beta1-receptors. Application of the MAPK/ERK kinase (MEK) inhibitor SL327 blocked both isoproterenol-induced activation of pERK and subsequent STEP translation. Inhibitors of PI3K (LY294002) or mTOR (rapamycin) also completely blocked STEP translation. These results suggest that co-activation of both the ERK and PI3K-Akt-mTOR pathways are required for STEP translation. As one of the substrates of STEP includes ERK itself, these results suggest that STEP is translated upon beta-adrenergic activation as part of a negative feedback mechanism.

The 73-kDa heat shock cognate protein is a CXCR4 binding protein that regulates the receptor endocytosis and the receptor-mediated chemotaxis.

The CXCR4 chemokine receptor is a G protein-coupled receptor that plays an important role in leukocyte homing, cancer metastasis, and human immunodeficiency virus infection. In response to ligand stimulation, chemokine receptors undergo endocytosis through clathrin-coated vesicle (CCV). Uncoating of CCV, a process involving heat shock cognate protein and several other proteins, is critical for fusion of CCV to endosomal compartments. The present study demonstrated that CXCR4 was associated with the 73-kDa heat shock cognate protein (Hsc73) in human embryonic kidney 293 cells in response to ligand stimulation. Truncation of the carboxyl terminal domain of CXCR4 reduced the association with Hsc73 and a glutathione S-transferase-CXCR4 carboxyl terminal fusion protein associated with Hsc73 in vitro, suggesting involvement of the carboxyl terminal domain of the receptor in the interaction. In response to ligand stimulation, CXCR4 underwent internalization and colocalization with Hsc73, but the receptor endocytosis was blocked by knockdown of Hsc73 with RNA interference. Moreover, Hsc73 knockdown significantly reduced the CXCR4-mediated chemotaxis of U87 glioma cell lines. These findings suggest that Hsc73 plays a role in chemokine receptor trafficking and the receptor-mediated chemotaxis.

A new approach to the pharmacological regulation of memory: Sarsasapogenin improves memory by elevating the low muscarinic acetylcholine receptor density in brains of memory-deficit rat models.

The purpose of this paper is to study the basic pharmacological action of sarsasapogenin, a sapogenin from the Chinese medicinal herb Rhizoma Anemarrhenae, (abbreviated as ZMS in this paper), on learning ability and memory of three animal models: aged rats and two neurodegeneration models produced either by single unilateral injection of beta-amyloid 1-40 (Abeta1-40) plus ibotenic acid (Ibot A) or by bilateral injection of Ibot A alone into nucleus basalis magnocellularis. Y-maze test and step-through test revealed that learning ability and memory were impaired in the three models and were improved by oral administration of ZMS. ZMS did not inhibit acetylcholinesterase nor did it occupy the binding sites of muscarinic acetylcholine receptor (M receptor), hence it is neither an cholinesterase inhibitor nor an agonist or antagonist of M receptors. On the other hand, the densities of total M receptor and its M1 subtype in the brain of the three models were significantly lower than control rats, and ZMS significantly raised the densities of total M receptors and its M1 subtype. Linear regression revealed significant correlation between the learning ability/memory and the density of either total M receptor or its M1 subtype. Autoradiographic study with 3H-pirenzipine showed that the M1 subtype density was significantly lowered in cortex, hippocampus and striatum of aged rats, and ZMS could reverse these changes towards normal control level. Interestingly, the M1 receptor density after ZMS administration only approached but did not exceed that of normal young control rats. Therefore, ZMS seems to represent a new approach to the pharmacological regulation of learning and memory and appears to be not simply palliative but may modify the progression of the disease.