Potential therapeutic targets of huperzine A for Alzheimer's disease and vascular dementia

Huperzine A (HupA), a novel Lycopodium alkaloid isolated from Chinese folk medicine Huperzia serrata (Qian Ceng Ta), is a potent, selective and well-tolerated inhibitor of acetylcholinesterase (AChE). It has been proven to significantly improve the learning and memory impairment in Alzheimer's disease (AD) and vascular dementia (VaD) patients in China. Interestingly, our recent data indicate that HupA also possesses other protective functions. This paper will give an overview on the protective effects of HupA, which includes regulating beta-amyloid precursor protein (APP) metabolism, protecting against Abeta-mediated oxidative stress, apoptosis and mitochondrial dysfunction, as well as anti-inflammation. The multiple neuroprotective effects of HupA might yield additional beneficial effects in AD and VaD therapy.     

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.     

The association of a cystatin C gene polymorphism with late-onset Alzheimer's disease and vascular dementia

A polymorphism in the cystatin C (CST3) gene was suggested to associate with Alzheimer's disease (AD). In the present study we attempted to determine the association between CST3 polymorphism and AD or vascular dementia (VD), and whether such effects are dependent of the APOE4 allele. The polymorphisms of CST3 genotype were determined using polymerase chain reactions (PCR) followed by gel electrophoresis in 124 AD, 70 VD, and 115 control individuals. No statistical difference in CST3B allele frequencies was observed among all three groups. Associations between CST3B/B genotype and AD patients older than 75-year-old, or VD patients younger than 75-year-old were evident. The APOE4 allele alone significantly increased the odds for the developing AD, but not VD. A logistic regression analysis revealed that either CST3 or its interaction with APOE4 were not significant predictors of AD. However, a synergistic association of CST3 and APOE4 alleles was observed in predicting VD patients. These results suggest that CST3 might interact with APOE4 on conferring vascular pathologies.     

A combination of lycopene and human amniotic epithelial cells can ameliorate cognitive deficits and suppress neuroinflammatory signaling by choroid plexus in Alzheimer's disease rat

Neuroinflammation characterized by glial activation and release of proinflammatory mediators is considered to be correlated with cognitive deficits in Alzheimer's disease (AD). Previously, some studies have demonstrated that lycopene (LYCO) or human amniotic epithelial cells (HAECs) could attenuate inflammation in AD. Specifically, the choroid plexus (CP), an epithelial layer that forms the blood-cerebrospinal fluid barrier, is able to modulate the cognitive function, through changes in the neuroinflammatory response and in brain immune surveillance. However, it is unclear if LYCO can interact with HAECs to improve neuroinflammation at the CP. Thus, this study chose the region of interest, considered the feasibility of using a combination of LYCO and HAECs, as a therapeutic agent for immunomodulatory effects at the CP in an acutely induced AD rat model. Results showed that oral administration of LYCO, HAECs transplantation, and their combination significantly improved cognitive deficits in water maze test, decreased the level of proinflammatory mediators (TNF-α and IL-1β), increased the level of anti-inflammatory mediators (IL-10 and TGF-β1) in the cerebro-spinal fluid, and hippocampal tissue. Interestingly, LYCO administration, HAECs transplantation and their combination reversed the Aβ_1–42 induced up-regulation of Toll like receptor 4 and nuclear factor-κB p65 mRNA and protein expressions at the CP. This study provided the novel experimental evidence for the influence of co-treatment with LYCO and HAECs on immunomodulatory capabilities of CP. It could also warrant therapeutic window for the pathophysiology of AD and the associated underlying mechanisms at the CP.     

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.     

Lipid Peroxidation and Antioxidant Enzyme Activities in Vascular and Alzheimer Dementias

It has been reported that oxidative stress may play a role in the pathogenesis of dementia of the Alzheimer type (AD) and the cerebral ischemia which causes vascular dementia (VD). We measured malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) activities in blood samples from patients with AD and VD and in healthy non-demented controls (CTR) which similar ages to the patients, in order to evaluate the degree of oxidative stress in patients with AD and VD. A sample of 150 subjects consisting of 50 patients with AD; 50 patients with VD and 50 CTR, aged from 65 to 85 years on, was analyzed. Most of the changes observed were in SOD activity and MDA levels. Catalase activity were least affected. Significant differences were observed in SOD and GR activity between males and females in CRT and in patients with AD, but not in VD. We have found a decrease in antioxidant enzymes activities (SOD, CAT, GPx and GR) in patients with AD and VD and significant differences were observed between CRT and AD patients for ages from 65 to 74, 75 to 84 and from 85 years to 94 years in SOD activity and MDA levels (P < 0.001). MDA levels increase with age in VD, AD and CTR. No significant variation with respect to sex were detected, but significant variations in MDA levels were detected between CRT and patients with VD and AD (P < 0.001). We conclude that oxidative stress plays an important role in the brain damage for both AD and VD, being observed higher levels of oxidative stress for AD that for VD.     

The mitochondria-targeted antioxidant MitoQ extends lifespan and improves healthspan of a transgenic Caenorhabditis elegans model of Alzheimer disease

β-Amyloid (Aβ)-induced toxicity and oxidative stress have been postulated to play critical roles in the pathogenic mechanism of Alzheimer disease (AD). We investigated the in vivo ability of a mitochondria-targeted antioxidant, MitoQ, to protect against Aβ-induced toxicity and oxidative stress in a Caenorhabditis elegans model overexpressing human Aβ. Impairment of electron transport chain (ETC) enzymatic activity and mitochondrial dysfunction are early features of AD. We show that MitoQ extends lifespan, delays Aβ-induced paralysis, ameliorates depletion of the mitochondrial lipid cardiolipin, and protects complexes IV and I of the ETC. Despite its protective effects on lifespan, healthspan, and ETC function, we find that MitoQ does not reduce DCFDA fluorescence, protein carbonyl levels or modulate steadystate ATP levels or oxygen consumption rate. Moreover, MitoQ does not attenuate mitochondrial DNA (mtDNA) oxidative damage. In agreement with its design, the protective effects of MitoQ appear to be targeted specifically to the mitochondrial membrane and our findings suggest that MitoQ may have therapeutic potential for Aβ- and oxidative stress-associated neurodegenerative disorders, particularly AD.     

Alzheimer病患者APP及PS1基因表达量的研究

为了检测Alzheimer病（Alzheimer’s disease,AD）患者外周血中淀粉样前体蛋白（Amyloid Precursor Protein, APP）基因及早老素1（Presenilin 1, PS1）基因的表达情况,进而探讨APP及PS1基因的表达与AD的相关性,采用SYBRGreenⅠ的方法对45例AD患者、25例血管性痴呆（vascular dementia, VD）患者及60名正常对照组样本的mRNA进行绝对定量,检测得到APP基因及PS1基因在对照组中的表达水平分别为0.026±0.005 amol/μg cDNA和0.026±0.004 amol/μg cDNA;在AD患者组中的表达量分别为0.044±0.006 amol/μg cDNA和0.051±0.011 amol/μg cDNA;,在VD患者组中的表达水平分别为0.072±0.013 amol/μg cDNA和0.039±0.005 amol/μg cDNA 。经显著性检验,AD患者组APP基因的表达水平上调,t=2.639, P<0.01;PS1基因的表达水平同样呈上调趋势,t=2.173,P<0.05,差异均具有统计学意义。VD患者组APP基因的表达水平上调,t=3.028,P<0.01;PS1基因的表达水平也同样呈上调趋势,t=2.012,P<0.05,均有显著性差异。因此,APP及PS1基因的表达水平的增高并不一定与AD发生特异性关联,而可能与多种导致痴呆的脑部病变发生关联。     

Oridonin Attenuates Aβ1–42-Induced Neuroinflammation and Inhibits NF-κB Pathway

Neuroinflammation induced by beta-amyloid (Aβ) plays a critical role in the pathogenesis of Alzheimer’s disease (AD), and inhibiting Aβ-induced neuroinflammation serves as a potential strategy for the treatment of AD. Oridonin (Ori), a compound of Rabdosia rubescens, has been shown to exert anti-inflammatory effects. In this study, we demonstrated that Ori inhibited glial activation and decreased the release of inflammatory cytokines in the hippocampus of Aβ_1–42-induced AD mice. In addition, Ori inhibited the NF-κB pathway and Aβ_1–42-induced apoptosis. Furthermore, Ori could attenuate memory deficits in Aβ_1–42-induced AD mice. In conclusion, our study demonstrated that Ori inhibited the neuroinflammation and attenuated memory deficits induced by Aβ_1–42, suggesting that Ori might be a promising candidate for AD treatment.     

Acupuncture Prevents the Impairment of Hippocampal LTP Through β1-AR in Vascular Dementia Rats

It is widely accepted that the synaptic dysfunction and synapse loss contribute to the cognitive deficits of vascular dementia (VD) patients. We have previously reported that acupuncture improved cognitive function in rats with VD. However, the mechanisms involved in acupuncture improving cognitive ability remain to be elucidated. The present study aims to investigate the pathways and molecules involved in the neuroprotective effect of acupuncture. We assessed the effects of acupuncture on hippocampal long-term potentiation (LTP), the most prominent cellular model of memory formation. Acupuncture enhanced LTP and norepinephrine (NE) levels in the hippocampus. Inhibition of the β-adrenergic receptor (AR), but not the α-AR, was able to block the effects of acupuncture on hippocampal LTP. Furthermore, inhibition of β1-AR, not β2-AR, abolished the enhanced LTP induced by acupuncture. The expression analysis revealed a significant upregulation of β1-AR and unchanged β2-AR with acupuncture, which supported the above findings. Specifically, increased β1-ARs in the dentate gyrus were expressed on neurons exclusively. Taken together, the present data supports a beneficial role of acupuncture in synaptic plasticity challenged with VD. A likely mechanism is the increase of NE and activation of β1-AR in the hippocampus.     

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.     

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.     

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。     

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.     

Neuroglobin attenuates Alzheimer-like tau hyperphosphorylation by activating Akt signaling

Neuroglobin (Ngb) is a recently identified member of hemoglobin family, distributed mainly in central and peripheral nervous systems. Recent studies suggest that Ngb can protect neural cells from β-amyloid-induced toxicity in Alzheimer disease (AD). Hyperphosphorylation of tau is another characterized pathological hallmark in the AD brains; however, it is not reported whether Ngb also affects tau phosphorylation. In this study, we found that the level of Ngb was significantly reduced in Tg2576 mice (a recognized mouse model of AD) and TgMAPt mice, and the level of Ngb was negatively correlated with tau phosphorylation. Over-expression of Ngb attenuates tau hyperphosphorylation at multiple AD-related sites induced by up-regulation of glycogen synthase kinase-3β (GSK-3β), a crucial tau kinase. While Ngb activates Akt and thus inhibits GSK-3β, simultaneously inhibition of Akt abolishes the effects of Ngb on GSK-3β inhibition and tau hyperphosphorylation. Our data indicate that Ngb may attenuate tau hyperphosphorylation through activating Akt signaling pathway, implying a therapeutic target for AD.     

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。     

Alterations in calcium homeostasis as biological marker for mild Alzheimer's disease?

The calcium hypothesis of neurodegenerative disorders such as Alzheimer's disease (AD) suggests that altered cytosolic Ca(2+) levels ( Ca(2+) (i)) and/or disturbances in Ca2+ homeostasis concern cellular mechanisms underlying neuronal pathology. To search for a diagnostic marker of Alzheimer's disease, we measured cytosolic calcium concentrations in platelets of AD patients, age-matched control subjects (AMC), and vascular dementia (VD) patients. The ( Ca(2+) (i)) was determined using long wavelength indicator Fluo-3AM in 21 mild AD patients, 17 AMC, and 23 patients with VD. The basal values of [Ca(2+)](i) were significantly lower in AD compared to AMC. After the addition of 1 mM calcium, the [Ca(2+)](i) markedly increased in platelets of AD compared to AMC and VD. Measurement of calcium homeostasis could provide a very sensitive, but less specific biological marker of AD. These results support the hypothesis that influencing calcium homeostasis may provide a therapeutic strategy in dementia.     

Neuroprotective and neurotoxic properties of glial cells in the pathogenesis of Alzheimer's disease

Alzheimer's disease (AD) affects more than 18 million people worldwide and is characterized by progressive memory deficits, cognitive impairment and personality changes. The main cause of AD is generally attributed to the increased production and accumulation of amyloid-beta (Abeta), in association with neurofibrillary tangle (NFT) formation. Increased levels of pro-inflammatory factors such as cytokines and chemokines, and the activation of the complement cascade occurs in the brains of AD patients and contributes to the local inflammatory response triggered by senile plaque. The existence of an inflammatory component in AD is now well known on the basis of epidemiological findings showing a reduced prevalence of the disease upon long-term medication with anti-inflammatory drugs, and evidence from studies of clinical materials that shows an accumulation of activated glial cells, particularly microglia and astrocytes, in the same areas as amyloid plaques. Glial cells maintain brain plasticity and protect the brain for functional recovery from injuries. Dysfunction of glial cells may promote neurodegeneration and, eventually, the retraction of neuronal synapses, which leads to cognitive deficits. The focus of this review is on glial cells and their diversity properties in AD.     

Huperzine A from <Emphasis Type="Italic">Huperzia serrata:</Emphasis> a review of its sources,chemistry, pharmacology and toxicology

The use and popularity of herbal medicines has been increasing worldwide. In fact, today, the traditional Chinese medicine offers a vast repertory for pharmaceutical research, as is the case of Huperzia serrata, a member of Huperziaceae family. This review reports the Lycopodium alkaloids that have been isolated from this plant. However, it was mainly focused on the huperzine A (HupA), a promising therapeutic option in several acute and chronic disorders. The major therapeutic interest described for HupA has been directed to the treatment of acetylcholine-deficit dementia, including Alzheimer’s disease. However, HupA was also shown to be effective on cerebrovascular dementia and other neurodegenerative disorders with an ischemic component, as well as on other kind of cognitive impairments; the value of HupA on myasthenia gravis, organophosphate poisoning and schizophrenia has also been described. In addition, many other pharmacological properties have been ascribed to HupA, namely its anti-inflammatory, antinociceptive and anticonvulsant properties, which was recently identified, promoting a growing interest on HupA research. Furthermore, its particular chemical structure and the fact that HupA is well tolerated in humans, even at doses well above those clinically required, along with its favorable pharmacokinetics, also boosted an intense research in the pharmaceutical industry. Therefore, several HupA-related features are addressed in this review, including not only its therapeutic properties, but also its chemistry, biological and chemical sources, structure–activity relationship, pharmacokinetics and toxicology, which are discussed in detail covering the literature published from 1962 to 2014.     

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.