Neuroprotective effects of huperzine A. A natural cholinesterase inhibitor for the treatment of Alzheimer's disease

Huperzine A (HupA), isolated from Chinese herb Huperzia serrata, is a potent, highly specific and reversible inhibitor of acetylcholinesterase. It has been found to reverse or attenuate cognitive deficits in a broad range of animal models. Clinical trials in China have demonstrated that HupA significantly relieves memory deficits in aged subjects, patients with benign senescent forgetfulness, Alzheimer's disease (AD) and vascular dementia (VD), with minimal peripheral cholinergic side effects compared with other AChEIs in use. HupA possesses the ability to protect cells against hydrogen peroxide, beta-amyloid protein (or peptide), glutamate, ischemia and staurosporine-induced cytotoxicity and apoptosis. These protective effects are related to its ability to attenuate oxidative stress, regulate the expression of apoptotic proteins Bcl-2, Bax, P53 and caspase-3, protect mitochondria, and interfere with APP metabolism. Antagonizing effects on NMDA receptors and potassium currents may contribute to the neuroprotection as well. It is also possible that the non-catalytic function of AChE is involved in neuroprotective effects of HupA. The therapeutic effects of HupA on AD or VD are probably exerted via a multi-target mechanism.     

In vitro production of huperzine A, a promising drug candidate for Alzheimer's disease

Alzheimer's disease (AD) is growing in impact on human health. With no known cure, AD is one of the most expensive diseases in the world to treat. Huperzine A (HupA), a anti-AD drug candidate from the traditional Chinese medicine Qian Ceng Ta (Huperzia serrata), has been shown to be a powerful and selective inhibitor of acetylcholinesterase and has attracted widespread attention because of its unique pharmacological activities and low toxicity. As a result, HupA is becoming an important lead compound for drugs to treat AD. HupA is obtained naturally from very limited and slowly growing natural resources, members of the Huperziaceae. Unfortunately, the content of HupA is very low in the raw plant material. This has led to strong interest in developing sources of HupA. We have developed a method to propagate in vitro tissues of Phlegmariurus squarrosus, a member of the Huperziaceae, that produce high levels of HupA. The in vitro propagated tissues produce even higher levels of HupA than the natural plant, and may represent an excellent source for HupA.     

Effect of Huperzine A on Aβ-induced p65 of astrocyte in vitro

Alzheimer’s disease (AD) is the most common cause of dementia. Its pathology often accompanies inflammatory action, and astrocytes play important roles in such procedure. Rela(p65) is one of significant message factors in NF-κB pathway which has been reported high expression in astrocyte treated by Aβ. HupA, an alkaloid isolated from Chinese herb Huperzia serrata, has been widely used to treat AD and observations reflected that it improves memory and cognitive capacity of AD patients. To reveal its molecular mechanisms on p65, we cultured astrocytes, built Aβ-induced AD model, treated astrocytes with HupA at different concentrations, assayed cell viability with MTT, and detected p65 expression by immunohistochemistry and PCR. Our results revealed that treatment with 10 μM Aβ1–42 for 24 h induced a significant increase of NF-κB in astrocytes; HupA significantly down-regulated p65 expression induced by Aβ in astrocytes. This study infers that HupA can regulate NF-κB pathway to treat AD.     

Levels of Amyloid Beta-42, Interleukin-6 and Tumor Necrosis Factor-Alpha in Alzheimer’s Disease and Vascular Dementia

Amyloid β42 (Aβ42) and proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) have been suggested to contribute to the pathogenesis of Alzheimer's disease (AD) and vascular dementia (VaD). Our aim was to examine whether the changes in these parameters would be able to discriminate the patients with AD from those with VaD and from healthy individuals. We have analyzed the levels of Aβ42, IL-6 and TNF-α in the serum of newly diagnosed 28 AD patients, 16 VaD patients and 26 healthy non-demented controls. We also investigated whether there is an association between Aβ42, IL-6 and TNF-α levels and mini-mental state examination (MMSE) scores and body mass indexes (BMI) of patients. Our data showed a significant decrease in serum Aβ-42 levels in AD patients compared to VaD patients and controls. Levels of IL-6 and TNF-α were not different between AD patients, VaD patients and controls. We observed a correlation between Aβ-42 levels and MMSE scores and BMI levels in both AD and VaD patients. However, Aβ-42 levels were not correlated with IL-6 and TNF-α levels. Significantly lower levels of Aβ42 found in the serum of AD patients than that of VaD patients and controls suggests that it can be a specific biochemical marker for AD.     

Cerebrospinal fluid markers in differential diagnosis of Alzheimer's disease and vascular dementia

Alzheimer's disease (AD) and vascular dementia (VaD) are the two most common causes of dementia in old people. They remain difficult to differentiate in practice because of lack of sensitivity and specificity of current clinical diagnostic criteria. Recent molecular and cellular advancements indicate that the use of cerebrospinal fluid markers may improve early detection and differential diagnosis of AD. Our objective in this study was to determine diagnostic accuracy of three cerebrospinal (CSF) markers: total tau protein (t-tau), tau protein phosphorylated on threonine 181 (p-tau181) and tau protein phosphorylated on serine 199 (p-tau199). Using commercially available ELISA kits concentrations of t-tau, p-tau181 and p-tau199 were analyzed in 12 patients with probable AD, 9 patients with VaD and 12 NC subjects. The median levels of all three markers were significantly higher in AD group versus VaD and NC groups. However, when the sensitivity levels were set to 85% or higher, only t-tau and p-tau199 satisfied consensus recommendations (specificity more than 75%) when differentiating AD from VaD. In conclusion, our preliminary data on a small group of selected subjects suggest that the CSF t-tau and p-tau199 levels are useful markers for differentiating AD from VaD.     

Altered expression of claudin family proteins in Alzheimer’s disease and vascular dementia brains

Claudins (Cls) are a multigene family of transmembrane proteins with different tissue distribution, which have an essential role in the formation and sealing capacity of tight junctions (TJs). At the level of the blood–brain barrier (BBB), TJs are the main molecular structures which separate the neuronal milieu from the circulatory space, by a restriction of the paracellular flow of water, ions and larger molecules into the brain. Different studies suggested recently significant BBB alterations in both vascular and degenerative dementia types. In a previous study we found in Alzheimer’s disease (AD) and vascular dementia (VaD) brains an altered expression of occludin, a molecular partner of Cls in the TJs structure. Therefore in this study, using an immunohistochemical approach, we investigated the expression of Cl family proteins (Cl‐2, Cl‐5 and Cl‐11) in frontal cortex of aged control, AD and VaD brains. To estimate the number of Cl‐expressing cells, we applied a random systematic sampling and the unbiased optical fractionator method. We found selected neurons, astrocytes, oligodendrocytes and endothelial cells expressing Cl‐2, Cl‐5 and Cl‐11 at detectable levels in all cases studied. We report a significant increase in ratio of neurons expressing Cl‐2, Cl‐5 and Cl‐11 in both AD and VaD as compared to aged controls. The ratio of astrocytes expressing Cl‐2 and Cl‐11 was significantly higher in AD and VaD as compared to aged controls. The ratio of oligodendrocytes expressing Cl‐11 was significantly higher in AD and the ratio of oligodendrocytes expressing Cl‐2 was significantly higher in VaD as compared to aged controls. Within the cerebral cortex, Cls were selectively expressed by pyramidal neurons, which are the ones responsible for cognitive processes and affected by AD pathology. Our findings suggest a new function of Cl family proteins which might be linked to response to cellular stress.     

Huperzine A Alleviates Oxidative Glutamate Toxicity in Hippocampal HT22 Cells via Activating BDNF/TrkB-Dependent PI3K/Akt/mTOR Signaling Pathway

Oxidative glutamate toxicity is involved in diverse neurological disorders including epilepsy and ischemic stroke. Our present work aimed to assess protective effects of huperzine A (HupA) against oxidative glutamate toxicity in a mouse-derived hippocampal HT22 cells and explore its potential mechanisms. Cell survival and cell injury were analyzed by MTT method and LDH release assay, respectively. The production of ROS was measured by detection kits. Protein expressions of BDNF, phosphor-TrkB (p-TrkB), TrkB, phosphor-Akt (p-Akt), Akt, phosphor-mTOR (p-mTOR), mTOR, phosphor-p70s6 (p-p70s6) kinase, p70s6 kinase, Bcl-2, Bax, and β-actin were assayed via Western blot analysis. Enzyme-linked immunosorbent assay was employed to measure the contents of nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Our findings illustrated 10 μM HupA for 24 h significantly protected HT22 from cellular damage and suppressed the generation of ROS. Additionally, after treating with LY294002 or wortmannin [the selective inhibitors of phosphatidylinositol 3 kinase (PI3K)], HupA dramatically prevented the down-regulations of p-Akt, p-mTOR, and p-p70s6 kinase in HT22 cells under oxidative toxicity. Furthermore, it was observed that the protein levels of BDNF and p-TrkB were evidently enhanced after co-treatment with HupA and glutamate in HT22 cells. The elevations of p-Akt and p-mTOR were abrogated under toxic conditions after blockade of TrkB by TrkB IgG. Cellular apoptosis was significantly suppressed (decreased caspase-3 activity and enhanced Bcl-2 protein level) after HupA treatment. It was concluded that HupA attenuated oxidative glutamate toxicity in murine hippocampal HT22 cells via activating BDNF/TrkB-dependent PI3K/Akt/mTOR signaling pathway.     

Mutation screening of EXT genes in Chinese patients with multiple osteochondromas

Since vascular risk factors commonly act for susceptibility to Alzheimer's disease (AD) and vascular dementia (VaD) by declining cognitive abilities, we conducted a genetic association study to identify their common underlying genetic factors. We selected single nucleotide polymorphisms (SNPs) which had been previously discovered for association with AD, and case and control associations of VaD were examined with the individual SNPs using 207 patients with VaD and 207 sex- and age-matched control subjects. As a result, no significant associations of susceptibility to VaD with 13 selected SNPs were observed even without employing a multiple test (P>0.05). This study suggests that genetics of VaD might be quite different from that of AD, and cautions should be taken especially when inferences about genetic factors are made with patients with mixed dementia.     

Vascular Cognitive Disorder. A Biological and Clinical Overview

Although vascular dementia (VaD) represents the second most common cause of dementia after Alzheimer’s disease (AD) in the elderly, and is referred as the “silent epidemic of the twenty-first century”, there is still a controversy on terminology, classification and diagnostic criteria of VaD. The diagnosis of VaD resides in clinical criteria determining a cognitive impairment, the presence of cerebrovascular disease and, only in the case of post-stroke dementia or multi-infarct dementia, a temporal relationship between these. The search for a reliable biochemical tests helping in the diagnosis of VaD is so far not available. Several vascular risk factors have a role in the development of VaD and their identification and treatment are among the major aspects of management of VaD. A new line of research in this field is the study of genetic factors underlying vascular cognitive impairment which are: (1) genes predisposing to cerebrovascular disease, and (2) genes that influence brain tissue responses to cerebrovascular lesions. Evidence in favour of a coexistence of vascular and degenerative components in the pathogenesis of dementia in an elderly population comes from neuropathological and epidemiological studies. There is now a great debate whether VaD and AD are more than common coexisting unrelated pathologies and, instead, represent different results of synergistic pathological mechanisms. Preventive approaches aiming at reducing incident VaD by targeting patients at risk of cerebrovascular disease (primary prevention), or acting on patients after a stroke (secondary prevention) to prevent stroke recurrence and the progression of brain changes associated with cognitive impairment are mandatory therapeutic strategies.     

Statins prevent cholinesterase inhibitor blockade of sympathetic alpha7-nAChR-mediated currents in rat superior cervical ganglion neurons

Statins are reported to be beneficial in treating a multitude of disorders including dementia due to Alzheimer disease (AD) and vascular dementia (VaD) with varying, yet-to-be determined mechanisms of actions. Although cholinesterase inhibitors (ChEIs) are still recommended as the primary drug of choice for AD and related diseases, their efficacy is frequently questioned. We recently reported that alpha7-neuronal acetylcholine nicotinic receptor (alpha7-nAChR)-mediated neurogenic vasodilation of porcine cerebral arteries was blocked by ChEIs, and this blockade was prevented by statin pretreatment. The exact mechanism of interaction between ChEIs and statins remains unclear. Activation of alpha7-nAChRs located on perivascular postganglionic sympathetic nerve terminals releases norepinephrine, which then acts on presynaptic beta(2)-adrenoceptors located on neighboring nitrergic nerve terminals, resulting in nitric oxide release and vasodilation. The present study, therefore, was designed to determine whether interaction of ChEIs and statins occurs at the alpha7-nAChR level. We examined effects of concurrent application of ChEIs and statins on alpha7-nAChR-mediated inward currents in primary neuronal cultures of rat superior cervical ganglion cells, the origin of the perivascular sympathetic innervation to the cerebral arteries. The results indicated that physostigmine, neostigmine, and galantamine inhibited choline- and nicotine-induced whole cell currents in a concentration-dependent manner. This inhibition, which was noncompetitive in nature, was prevented by concurrent application of mevastatin and lovastatin in a concentration-dependent manner. These results suggest that statins protect alpha7-nAChR function directly at the receptor level. Since alpha7-nAChR is neuroprotective, having beneficial effects on memory and cerebral vascular function, its functional inhibition by ChEIs may explain in part the limitation of its effectiveness in AD and VaD therapy. Protection of alpha7-nAChR function from ChEI inhibition by concurrent administration of statins may provide an alternative strategy in improving the efficacy of AD and VaD therapy.     

蛇足石杉中产石杉碱甲内生真菌的分离和鉴定

【目的】从野生蛇足石杉(Huperzia serrata)中分离筛选产石杉碱甲的内生真菌。【方法】采用薄层层析及高效液相色谱法对内生真菌代谢产物进行测定和分析以期分离获得产石杉碱甲菌株,运用形态及ITS序列分析方法对产石杉碱甲菌株进行鉴定,并利用连续传代方法考察菌株遗传稳定性。【结果】经筛选获得一株产石杉碱甲内生真菌NSH-5,经形态学鉴定及ITS序列分析鉴定为轮枝镰孢菌(Fusarium verticillioides),其石杉碱甲产量为11.76 mg/100 m L,菌株经20次连续传代后遗传稳定。【结论】NSH-5菌株为一株具有产石杉碱甲能力的轮枝镰孢菌,该菌株的发现为生物合成石杉碱甲提供了新的菌种资源。     

Smoking Is Associated with an Increased Risk of Dementia: A Meta-Analysis of Prospective Cohort Studies with Investigation of Potential Effect Modifiers

BackgroundPrevious studies showed inconsistent results on the association of smoking with all-cause dementia and vascular dementia (VaD), and are limited by inclusion of a small number of studies and unexplained heterogeneity. Our review aimed to assess the risk of all-cause dementia, Alzheimer’s disease (AD) and VaD associated with smoking, and to identify potential effect modifiers.ConclusionsSmokers show an increased risk of dementia, and smoking cessation decreases the risk to that of never smokers. The increased risk of AD from smoking is more pronounced in apolipoprotein E ε4 noncarriers. Survival bias and competing risk reduce the risk of dementia from smoking at extreme age.     

Huperzine A protects isolated rat brain mitochondria against β-amyloid peptide

Our previous work in cells and animals showed that mitochondria are involved in the neuroprotective effect of huperzine A (HupA). In this study, the effects of HupA on isolated rat brain mitochondria were investigated. In addition to inhibiting the Aβ_25-35 (40 μM)-induced decrease in mitochondrial respiration, adenosine 5′-triphosphate (ATP) synthesis, enzyme activity, and transmembrane potential, HupA (0.01 or 0.1 μM) effectively prevented Aβ-induced mitochondrial swelling, reactive oxygen species increase, and cytochrome c release. More interestingly, administration of HupA to isolated mitochondria promoted the rate of ATP production and blocked mitochondrial swelling caused by normal osmosis. These results indicate that HupA protects mitochondria against Aβ at least in part by preserving membrane integrity and improving energy metabolism. These direct effects on mitochondria further extend the noncholinergic functions of HupA.     

一株产石杉碱甲的蛇足石杉内生真菌的分离和鉴定

从药用蕨类植物蛇足石杉叶中分离得到53株内生真菌,在马铃薯葡萄糖液体培养基（PDB）中发酵后对内生真菌发酵提取物液进行TLC、HPLC 和ESI-MS检测,结果表明菌株LF40为产石杉碱甲内生菌株,含量达80.1μg/gdcw,并具有体外乙酰胆碱酯酶抑制活性;运用形态学及ITS-rDNA序列分析方法对菌株LF40进行鉴定,确定为黄曲霉Aspergillus flavus LF40。     

A novel endophytic Huperzine A–producing fungus,Shiraia sp. Slf14, isolated from Huperzia serrata

Aims: To characterize and identify a novel Huperzine A (HupA)‐producing fungal strain Slf14 isolated from Huperzia serrata (Thunb. ex Murray) Trev. in China. Methods and Results: The isolation, identification and characterization of a novel endophytic fungus producing HupA specifically and consistently from the leaves of H. serrata were investigated. The fungus was identified as Shiraia sp. Slf14 by molecular and morphological methods. The HupA produced by this endophytic fungus was shown to be identical to authentic HupA analysed by thin layer chromatographic, High‐performance liquid chromatography (HPLC), LC‐MS, ¹H NMR and acetylcholinesterase (AChE) inhibition activity in vitro. The amount of HupA produced by Shiraia sp. Slf14 was quantified to be 327·8 μg l^?1 by HPLC, which was far higher than that of the reported endophytic fungi, Acremonium sp., Blastomyces sp. and Botrytis sp. Conclusions: The production of HupA by endophyte Shiraia sp. Slf14 is an enigmatic observation. It would be interesting to further study the HupA production and regulation by the cultured endophyte in H. serrata and in axenic cultures. Significance and Impact of the Study: Although the current accumulation of HupA by the endophyte is not very high, it could provide a promising alterative approach for large‐scale production of HupA. However, further strain improvement and the fermentation process optimization are required to result in the consistent and dependable production.     

Indentification of huperzine A-producing endophytic fungi isolated from Huperzia serrata

This present study was designed to investigate the production of huperzine A (HupA), an acetylcholine inhibitor, which was produced by an endophytic fungi isolated from Huperzia serrata. Screening of 94 endophytic fungal isolates obtained from plant H. serrata was carried out for the production of HupA. Their morphological characteristics were studied and rDNA sequence analysis was carried out. The cultures were grown in liquid culture medium and the extracted metabolites were analyzed by thin layer chromatography and high performance liquid chromatograph for the presence of HupA. The DPPH scavenging ratio and inhibition ratio of acetylcholinesterase (AchE) of the same were determined. 3 out of 94 strains i.e. S29, L44 and S94 showed significant AchE-inhibitory activity and antioxidant activity. Strain L44 which exhibited maximum yield of HupA (37.63 μg/g on dry weight basis) was identified as Trichoderma species by ITS sequence analysis. In conclusion, endophytic fungi from H. serrata can be used as a new resource of HupA.     

Decreased thioredoxin and increased thioredoxin reductase levels in Alzheimer's disease brain

Increasing evidence supports the role of reactive oxygen species (ROS) in the pathogenesis of Alzheimer's disease (AD). Both in vivo and in vitro studies demonstrate that thioredoxin (Trx) and thioredoxin reductase (TR), the enzyme responsible for reduction of oxidized Trx, have protective roles against cytotoxicity mediated by the generation of ROS. The present study measured levels of Trx protein and activities of TR in the brain in AD compared with control subjects, and evaluated the possible protective role of TR and Trx against amyloid beta-peptide (Abeta) toxicity in neuronal cultures. Analysis of Trx protein levels in 10 AD and 10 control subjects demonstrated a general decrease in all AD brain regions studied, with statistically significant decreases in the amygdala (p <.05), hippocampus/parahippocampal gyrus (p <.05), and marginally significant (p <.10) depletions in the superior and middle temporal gryi. Thioredoxin reductase activity levels were increased in all AD brain regions studied with statistically significant increases occurring in AD amygdala (p =.01) and cerebellum (p =.007). To investigate the protective effects of Trx and TR against Abeta-induced toxicity, primary hippocampal cultures were treated with Trx or TR in combination with toxic doses of Abeta. Treatment of cultures with Trx led to a statistically significant concentration-dependent enhancement in cell survival against Abeta-mediated toxicity as did treatment with TR. Together, these data suggest that, although TR is protective against Abeta-mediated toxicity, the increase observed in AD brain offers no protection due to the significant decrease in Trx levels. This decrease in the antioxidant Trx-TR system may contribute to the increased oxidative stress and subsequent neurodegeneration observed in the brain in AD.     

Ethanol and Methanol Can Improve Huperzine A Production from Endophytic Colletotrichum gloeosporioides ES026

Huperzine A (HupA) is a plant alkaloid that is of great interest as a therapeutic candidate for the treatment of Alzheimer''s disease. However, the current production of HupA from plants in large quantity is unsustainable because the plant resource is scarce and the content of HupA in plants is extremely low. Surprisingly, this compound was recently found to be produced by various endophytic fungi, which are much more controllable than the plants due to simpler genetics and ease of manipulation. However, it might be due to the innate properties of endophytic symbiosis, that production of this chemical in large quantity from endophytes has not yet been put into practice. Endophytic Colletotrichum gloeosporioides ES026 was previously isolated from a HupA producing plant and the fungi also proved to produce HupA. In this study, various fermentation conditions were tried to optimize the production of HupA from C. gloeosporioides ES026. Optimization of these parameters resulted in a 25.58% increase in HupA yield. Potato extracts supplemented with glucose or sucrose but not maltose facilitated HupA producing from the fungi. A final concentration of 0.5–2% ethanol stimulated the growth of fungi while methanol with the same treatment slightly inhibited the growth. However, both methanol and ethanol greatly increased the HupA production with the highest yield of HupA (51.89% increment) coming from ethanol treatment. Further analysis showed that both ethanol and methanol were strong inducers of HupA production, while ethanol was partially used as a carbon source during fermentation. It was noticed that the color of that ethanol treated mycelia gradually became dark while methanol treated ones stayed grey during fermentation. The present study sheds light on the importance of optimizing the fermentation process, which, combined with effective inducers, maximizes production of chemicals of important economic interest from endophytic fungi.     

Efficient strategy for maintaining and enhancing the huperzine A production of Shiraia sp. Slf14 through inducer elicitation

Huperzine A (HupA), a naturally occurring lycopodium alkaloid, is a potent, highly specific and reversible inhibitor of acetylcholinesterase and is a potential treatment for Alzheimer’s disease. However, isolating HupA from Huperziaceae plants is inefficient; thus, extracting this compound from endophytic fungi may be more controllable and sustainable. However, the large-scale production of this chemical from endophytes is limited by the innate instability of endophytic fungi. In this study, we maintained the stability and viability of the HupA-producing endophytic fungus Shiraia sp. Slf14 and enhanced the HupA titers during fermentation by adding Huperzia serrata extracts (HSE), l-lysine, and acetic acid into the culture as inducers. Adding trace amounts of HupA clearly improved the HupA production of Shiraia sp. Slf14, reaching a maximum content of approximately 40 μg g^?1. Moreover, the addition of HSE and l-lysine promoted HupA production in the flask fermentation. The aforementioned bioprocessing strategy may be potentially applied to other endophytic fungal culture systems for the efficient production of plant secondary metabolites.     

Endophytic fungus <Emphasis Type="Italic">Cladosporium cladosporioides</Emphasis> LF70 from <Emphasis Type="Italic">Huperzia serrata</Emphasis> produces Huperzine A

A strain LF70 endophytic fungus was isolated from the leaves of Huperzia serrata. The fungus was identified as Cladosporium cladosporioides LF70 according to its morphological characteristics and nuclear ribosomal DNA ITS sequence analysis. The strain could produce Huperzine A (HupA) identified through thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) with authentic HupA. The amount of HupA produced by this endophytic fungus was quantified to be 56.84 μg/L by HPLC, which was higher than that of other reported endophytic fungi, Acremonium sp., Blastomyces sp., and Botrytis sp. Acetylcholinesterase inhibition activity of HupA produced by strain LF70 was also similar to authentic HupA in vitro. Isolation of such a fungus may provide a promising alternative approach to producing HupA, which is used in treating Alzheimer’s disease and preventing further memory degeneration.