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. 2007 Jul 27;28(2):173–183. doi: 10.1007/s10571-007-9163-z

Non-cholinergic Effects of Huperzine A: Beyond Inhibition of Acetylcholinesterase

Hai Yan Zhang 1, Han Yan 1, Xi Can Tang 1,✉
PMCID: PMC11516533  PMID: 17657601

Abstract

The use of acetylcholinesterase inhibitors to decrease the breakdown of the neurotransmitter acetylcholine has been the main symptomatic therapy for mild to moderate Alzheimer’s patients, though the etiology of Alzheimer’s disease remains unclear and seems to involve multiple factors. Further evidence has indicated that some of these acetylcholinesterase inhibitors also have non-cholinergic functions on the pathogenesis of Alzheimer’s disease including the formation and deposition of β-amyloid. Huperzine A, a potent and reversible inhibitor of acetylcholinesterase that was initially isolated from a Chinese herb, has been found to improve cognitive deficits in a broad range of animal models and has been used for Alzheimer’s disease treatment in China. The novel neuroprotective effects of huperzine A might yield beneficial effects in Alzheimer’s disease therapy and provide a potential template for the design of new selective and powerful anti-Alzheimer’s drugs. The present paper gives an overview on the neuroprotective effects of huperzine A beyond its acetylcholinesterase inhibition. These effects include regulating β-amyloid precursor protein metabolism, protecting against β-amyloid-mediated oxidative stress and apoptosis. The structure–function relationship of huperzine A is also discussed.

Keywords: Alzheimer’s disease, Huperzine A, Acetylcholinesterase, β-amyloid, Oxidative stress, Apoptosis

Introduction

Alzheimer’s disease (AD) is an irreversible, progressive neurodegenerative disease accompanied by three main structural hallmarks in the brain: diffuse loss of neurons, extracellular deposition of the β-amyloid (Aβ) in senile plaques, and the appearance of intracellular neurofibrillary tangles (NFT) consisting of hyperphosphorylated tau protein (Selkoe 1996). Among the large array of strategies that have been developed to palliate AD symptoms, the use of acetylcholinesterase (AChE) inhibitors continues to be the mainstay of the symptomatic therapy for mild to moderate AD. Recent data suggest that certain AChE inhibitors may also have neuroprotective effects by preventing the generation of Aβ or protecting against Aβ-induced neurotoxicity. Understanding the neuroprotective mechanisms of AChE inhibitors will greatly expand the opportunities for successful development of drugs that halt or slow AD progression beyond symptomatic therapy.

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 AChE. A large number of studies have shown that HupA has mild-to-no side effects or toxicity in mice, rats, rabbits and dogs, without fasciculation or other cholinergic hyperactivity symptoms, and shows no histopathological changes after subacute administration (reviewed by (Wang et al. 2006a)). In contrast to the mild side effects, HupA has been proven to effectively reverse or attenuate cognitive deficits in the passive footshock avoidance task in chickens, and in the Morris water maze performance, eight-arm radial maze performance in rats and mice, as well as in the delayed response performance in monkeys (reviewed by (Wang et al. 2006a)).

The anti-dementia effects of HupA have been well evaluated by double-blind, placebo-controlled clinical trials in China. A large number of clinical studies have shown that HupA administration can significantly improve the memory, cognitive skills, and daily life abilities of AD patients while showing no severe side effects. HupA can also markedly improve the memory impairment in vascular dementia (VD) patients. In addition, HupA was reported to ameliorate the memory deficits in schizophrenia patients and improve sleep in insomniacs. The clinical studies mentioned here have been discussed in details in our previous review (Wang et al. 2006a).

HupA is currently in the phase IV clinical trial for AD treatment in China and in clinical trials for the treatment of age-related memory deficiency in the United States. Recent data indicate that HupA has multiple neuroprotective effects aside from its AChE inhibition, which include regulating β-amyloid precursor protein (APP) metabolism and counteracting Aβ-associated neurotoxicity. This may provide adequate evidence for the clinical and experimental improvement gained from HupA (reviewed by (Zhang and Tang 2006)). In the present paper, the classic AChE inhibitory effect of HupA and its new neuroprotective targets on AD pathology will be reviewed.

Classic Cholinergic Effect of Huperzine A

A decrease of ACh was found in the brains of patients with AD and was proven to be a critical element in the development of dementia (DeKosky and Scheff 1990). Among the many approaches aimed at enhancing the dwindling amount of cholinergic neurotransmitters to improve the learning and memory deficiency in AD, AChE inhibitors were considered the most promising class of drugs for symptomatic therapy. The principle of this indirect cholinomimetic therapy is to reduce ACh breakdown in the central nervous system by inhibiting AChE, which results in increased extracellular ACh concentration, potentially reversing the central cholinergic hypofunction in AD.

HupA is a mixed-competitive and reversible AChE inhibitor, which shows higher potency and selectivity of AChE inhibition both in vitro and in vivo as compared with galanthamine, donepezil, tacrine, and rivastigmine (reviewed by (Wang et al. 2006a)). Our recent study also showed that HupA has an 8- and 2-fold higher potency for increasing cortical ACh level than donepezil and rivastigmine, respectively, with a longer duration of action and fewer side effects (Liang and Tang 2004). Moreover, we investigated the selectivity of five AChE inhibitors for monomeric AChE (G1) and tetrameric AChE (G4) isoforms from the cortex and hippocampus of rats. In this study, HupA was demonstrated to have an overall preference on G4 isoform in both brain regions; tacrine and rivastigmine preferentially inhibited G1 isoform; donepezil showed pronounced selectivity for G1 isoform in hippocampus but not in cortex; while physostigmine showed no isoform selectivity (Zhao and Tang 2002). Ectocellular G4 isoform is the major form for metabolizing ACh (Taylor and Radic 1994), and this form is selectively depleted in AD patients (Siek et al. 1990). Therefore, potent inhibitors of AChE G4 isoform in cortex and hippocampus, such as HupA, would be expected to have a greater effect in ameliorating cognitive deficits in AD patients.

In order to understand the mechanisms of the highly potent and selective AChE inhibition of HupA, computer-aided docking study (Pang and Kozikowski 1994; Dvir et al. 2002) and X-ray crystallography (Raves et al. 1997) approaches were performed. The X-ray crystallographic structure of Torpedo californica AChE (TcAChE) indicated that the active site of AChE exists at the bottom of the gorge, and the peripheral site lies at the entrance of the gorge near the surface of AChE, with Ser200 and Trp84 at the active site and Trp279 at the peripheral (Sussman et al. 1991). The structure of HupA–TcAChE complex measured by 3D X-ray suggested that HupA has a tighter binding and greater specificity to the enzyme than other known AChE inhibitors including tacrine and edrophonium (Raves et al. 1997). There are several principle protein-ligand interactions involved in the potent AChE inhibition of HupA. The direct strong hydrogen bonds are formed between the carbonyl group of HupA and the hydroxy oxygen of Tyr130 (located at the peripheral site of the enzyme), as well as between the thylidene methyl group and the main chain oxygen of His440. The indirect hydrogen bonds, mediated by one or two water molecules, are formed between HupA and residues of the enzyme, which constitute the active center (e.g.,–NH+3 group of HupA and the aromatic rings of Trp84 and Phe330 at the choline site) (Raves et al. 1997; Dvir et al. 2002; Jiang et al. 2003).

Moreover, the most striking observation seen in the TcAChE–HupA complex is the peptide bond flip between Gly117 and Gly118. The carbonyl oxygen of HupA can form short hydrogen bond and interact with the hydroxyl of Tyr130, which in turn causes the flip. In consequence, the carbonyl oxygen of Gly117 is stabilized by forming hydrogen bonds with Gly119N and Ala201N (Dvir et al. 2002). It has been conjectured that the peptide flip is responsible for the low on-rates observed for HupA inhibition on AChE (Raves et al. 1997). Meanwhile, its stabilization may also contribute to the low rates of dissociation (Ashani et al. 1992).

On the other hand, previous study have proven that HupA had no direct effect on the amplitude or kinetics of nAChRs (Fayuk and Yakel 2004). However, HupA can slow the rate of recovery from desensitization of the α7 containing nAChRs in rat hippocampal interneurons and increased both the amplitude and decay time of non-α7 nAChRs-mediated responses through an indirect mechanism involving the inhibition of the breakdown of ACh, resulting in an enhanced amount of duration of ACh exposed to these channels (Fayuk and Yakel 2004). Therefore, the effect of HupA on ACh diffusion through nictonic receptor could also contribute to the symptomatic therapy for AD.

Non-Classic Cholinergic Effects of Huperzine A

Interaction Between Aβ and AChE

Aβ, the main component of the senile plaques (Selkoe 1994), is a peptide derived from a larger polypeptide integral membrane protein named β-amyloid precursor protein (APP). According to the amyloid cascade hypothesis, the neurodegeneration in AD begins with the abnormal processing of APP and results in the production, aggregation, and deposition of the toxic Aβ peptide (Hardy and Selkoe 2002). Moreover, with the advent of new research on molecular mechanisms of AD pathogenesis, accumulative evidence has implicated that AChE not only plays an important role in cholinergic dysfunction, it is also involved in the Aβ cascade through the AChE peripheral anionic site, such as mediating the processing and deposition of Aβ (Inestrosa et al. 1996a; Inestrosa and Alarcon 1998; De Ferrari et al. 2001; Bartolini et al. 2003). AChE is one of the several proteins associated with the amyloid core of mature senile plaques, pre-amyloid diffuse deposits, and cerebral blood vessels in AD brain (Moran et al. 1993; Fuentealba et al. 2004). It is incorporated into Aβ aggregates in vitro by forming macromolecular complexes with the growing Aβ fibrils (Alvarez et al. 1997). These complexes have been shown to accelerate the aggregation of Aβ fibrils and maturation of Aβ plaques (Inestrosa et al. 1996b; Alvarez et al. 1997; Fuentealba et al. 2004), and turn out to be more toxic than the amyloid fibrils alone at the cellular level (Alvarez et al. 1998). These results suggest that AChE might contribute to Aβ-mediated neurotoxicity in vivo in those neuronal subpopulations that are more susceptible to neurodegeneration in AD.

The peripheral anionic site of AChE lies at the entrance to the active site gorge, which is composed of five residues—Tyr70, Asp72, Tyr121, Trp279, and Tyr334. Previous study by using the automated docking program predicted that HupA could have three possible orientations of binding with AChE (Pang and Kozikowski 1994), one of them only has very small difference with the X-ray crystal structure between HupA and TcAChE. HupA was also predicted to bind to the peripheral site (Jiang et al. 2003). Furthermore, the X-ray crystal graph also showed that HupA interfered with the hydrogen bonding networks formed by Tyr70, Asp72, and Tyr121 (located in the peripheral anionic site) (Raves et al. 1997). Since the peripheral anionic site of AChE was demonstrated to be involved in Aβ pathology (Inestrosa et al. 1996a; Inestrosa and Alarcon 1998; De Ferrari et al. 2001; Bartolini et al. 2003), the interaction of HupA with this site might be of great importance. However, further study will be needed to make the conclusion.

Neuroprotective Effects of HupA on APP Processing

A large body of evidence suggests that Aβ is central to the pathophysiology of AD and is likely to start this intractable neurodegenerative disorder (Hardy 1997). Aβ itself is also well proven to be neurotoxic in vivo and in vitro (Behl et al. 1994; Hensley et al. 1994; Wang et al. 2001b; Xiao et al. 2002). Therefore, targeting Aβ production and/or Aβ-mediated neurotoxicity may be a reasonable strategy for developing new AD drugs.

Two main pathways have been described for APP processing (Nitsch et al. 1992): the amyloidogenic pathway, which is mediated by β- and γ-secretase, creates Aβ peptide; and the non-amyloidogenic pathway, which is mediated by α- and γ-secretase, cleaves APP within Aβ sequence, and releases a soluble secretory amyloid precursor protein (sAPPα) (Esch et al. 1990; Haass and Selkoe 1993). A large number of studies have proven that sAPPα exhibits neuroprotective effects including promotion of cell proliferation and neurite outgrowth, as well as prevention of intracellular calcium accumulation (Rossner et al. 1998), which could be beneficial to delay AD progression. Previous data have shown that activation of M1 receptor leads to the promotion of α-secretase-generated products (Nitsch et al. 1992; Haring et al. 1998), which is mediated via two divergent transduction pathways that converge at a step of tyrosine phosphorylation: a PKC-dependent pathway and a Ras/MAPK dependent pathway (Haring et al. 1998). It has been widely reported that AChE inhibitors can affect APP processing by increasing sAPPα secretion in addition to its catalytic function (Mori et al. 1995; Giacobini et al. 1996). Consistent with these findings, we demonstrated that HupA enhanced the non-amyloidogenic pathway by increasing the levels of sAPPα and PKCα in intracerebroventricular Aβ-infused rats and human embryonic kidney 293 Swedish mutant cells (Zhang et al. 2004). Our recent study, using specific antagonists or inhibitors, indicated that M1-AChR, PKC, and MAPK might be involved in the non-amyloidogenic enhancing effect of HupA (Yan et al. 2007). Interestingly, the effects of HupA on NGF signaling have been proven in PC12 cells, SH-SY5Y and cultured rat cortical astrocytes, through the TrkA receptor-mediated MAPK/ERK pathway (Tang et al. 2005a, 2005b; Wang et al. 2006b). Taken together, our results suggest that the HupA can enhance non-amyloidogenic pathway through activating PKC and MAPK pathways, meanwhile decreasing amyloidogenic pathway, since increased sAPPα secretion is associated with the reduction of Aβ generation (Busciglio et al. 1993; Buxbaum et al. 1993; Wolf et al. 1995).

Neuroprotective Effects of HupA on Aβ-associated Neurotoxicity

In agreement with previous studies (Behl et al. 1994; Hensley et al. 1994; Wang et al. 2001b; Xiao et al. 2002), our data proven that Aβ treatment can generate oxidative stress that eventually triggers a state of neurotoxicity and cell death. Pre-incubation of PC12 cells and cultured rat primary cortical neurons with HupA prior to Aβ exposure was found to enhance the cell survival and the activities of antioxidant enzymes including glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT), and decrease the level of lipid peroxidation product—malondialdehyde (MDA) (Xiao et al. 2000a, 2000b). Similarly, pretreatment of PC12 cells with HupA prior to hydrogen peroxide (H2O2) exposure also significantly elevated the cell survival and antioxidant enzyme activities, and decreased the level of MDA (Xiao et al. 1999). Moreover, HupA markedly reduced the MDA level in chronic cerebral hypo-perfusion rats (Wang et al. 2000) and aged rats (Shang et al. 1999). These results indicate that HupA has antioxidant effects in both Aβ and H2O2-induced oxidative stress models, which are mediated by increasing the activities of antioxidant enzymes and decreasing the lipid peroxidation products.

Apoptosis is believed to be an important contributor to the progression of AD. There is considerable evidence showing that Aβ can activate intracellular apoptosis pathways which leading to neuronal death (Anderson et al. 1995; Saille et al. 1999), and Aβ-induced oxidative stress may contribute to this event. The cellular commitment to apoptosis is regulated by the Bcl-2 family of proteins. Current understanding of the bcl-2 gene family indicates that the interactions and relative abundance of Bcl-2 and Bax can modulate the propensity of a cell to undergo apoptotic death (Knudson and Korsmeyer 1997), and that increased expression of P53 and Bax is associated with the initiation of apoptosis (Ko and Prives 1996). The anti-apoptotic effects of HupA have been well studied, in which HupA pretreatment was demonstrated to significantly alleviate apoptotic changes including DNA laddering, cell shrinkage, generation of nuclear apoptotic bodies, and TUNEL positive staining (Wang et al. 2001a, b; Xiao et al. 2002; Zhang and Tang 2003b). We have shown that the effects of HupA on Aβ-induced apoptosis were mediated by reversing the down-regulation of the expression of Bcl-2 and the up-regulation of the expressions of Bax and P53 (Wang et al. 2001b). Pretreatment with HupA had the same effects on H2O2-induced apoptosis with a down-regulation of the bax and p53 genes and up-regulation of the bcl-2 gene to normal levels (Wang et al. 2001a).

Previous studies have indicated that mitochondria are the target of the pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 (Er et al. 2006). In the mitochondria-mediated cell death pathway, cytochrome c binds to Apaf-1, and then polymerizes into an oligomer known as apoptosome. The apoptosome activates caspase-9, which in turn activates the apoptotic executive protein, caspase-3, and finally leads to apoptosis (Szalai et al. 1999; Zamzami and Kroemer 2001). HupA was found to attenuate the increase of caspase-3 activity induced by Aβ in cultured primary cortical neurons (Xiao et al. 2002), and the anti-apoptotic effects of HupA were further confirmed in apoptosis models of serum deprivation and staurosporine stimulation (Zhou and Tang 2002; Zhang and Tang 2003b). In addition, Aβ exposure can enhance the outward K+ current—I k (Yu et al. 1998), which mediates several forms of neuronal apoptosis. Recent studies found that HupA inhibited I k in CA1 pyramidal neurons (Li and Hu 2002a, 2002b), suggesting that the effects of HupA on potassium channels might also contribute to the neuroprotection against Aβ-induced apoptosis.

The precise mechanisms of anti-oxidative and anti-apoptotic effects of HupA on Aβ-mediated neurotoxicity were further explored recently (Gao and Tang 2006). Consistent with previous studies (Casley et al. 2002; Kim et al. 2002), Aβ exposure was found to increase the intracellular levels of reactive oxygen species (ROS), and cause serious damage to the mitochondrial electron transport and the tricarboxylic acid (TCA) cycle as assessed by the decline of activities in complexes I, II/III, and especially IV, as well as in PDHC and α-KGDHC, the key components of the respiratory chain or glucose metabolism. Aβ can also directly interact with Aβ-binding alcohol dehydrogenase (ABAD) in the mitochondrial matrix, which in turn promotes leakage of ROS, mitochondrial dysfunction and cell death (Lustbader et al. 2004). Thus, it is suggested that the generation of free radicals is an early cellular response to Aβ, which in turn causes damage to mitochondrial enzymes, triggers impairment of glucose metabolism followed by a loss of cellular ATP and the collapse of mitochondrial membrane potential. This results in a vicious cycle, and ultimately leads to apoptotic insults and cell death (Blass 2001). Our studies demonstrated the cytoprotective effects of HupA on mitochondrial function in Aβ-exposed PC12 cells, which occur through reducing the levels of ROS, as well as increasing the levels of key components of the respiratory chain and the activities of key enzymes in TCA cycle (Gao and Tang 2006). These therapeutic mechanisms may enable HupA to attenuate neuronal apoptosis and aid in the treatment of neurodegenerative disease.

The potencies of (+)-HupA or (−)-HupA on AChE inhibition are very different based on in vitro and in vivo data (Tang et al. 1994). However, they have similar potencies against Aβ-induced toxicities (Zhang et al. 2002). In another study, we observed that AChE activity rose gradually with increasing time of exposure to Aβ in an AChE overexpressing mouse neuroblastoma cell line while cell viability decreased and DNA fragmentation became more apparent (Zhang et al. 2003a). There is a coincidence between the rise in AChE activity and the appearance of apoptosis. However, inhibiting the hydrolyzing activity of AChE cannot block the Aβ-induced apoptosis (Zhang et al. 2003a). Based on these data, there seems to be no direct correlation between the effects of HupA on Aβ-induced toxicities and the AChE inhibition. The interaction of HupA with the peripheral site of AChE, which may interrupt the formation of the AchE–Aβ complex, may partially explain the neuroprotective effects of HupA on reducing Aβ-induced toxicities. Additional work using realistic in vivo models of AD is needed to confirm this conclusion rigorously in order to further elucidate the precise mechanisms. Together with the action on AChE, other effects of HupA, such as modulating APP processing and enhancing NGF signaling, provide multiple targets for its neuroprotection, which will be beneficial for AD treatment (Fig. 1).

Fig. 1.

Fig. 1

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Multiple effects of HupA that involved in AD. Aβ is formed through β- and γ-secretase cleavage, while the nutritional sAPPα is created by α- and γ-secretase cleavage. α-secretase cleavage can be enhanced via two divergent transduction pathways that converge at a step of tyrosine phosphorylation: M1 muscarinic receptor-mediated PKC-dependent cascade and Trk receptor-mediated Ras/MAPK dependent cascade. The toxicities of Aβ could cause oxidative stress by increasing MDA and ROS levels while decreasing CAT and GSH-Px activities; induce apoptosis through up-regulating anti-apoptotic protein Bcl-2 expression and down-regulating pro-apoptotic proteins C-Jun, P53 and Bax expressions, as well as enhancing the apoptosis-related current, Ik, which in turn lead to mitochondrial dysfunction. HupA can modulate the AD progressing through increasing synaptic ACh levels by inhibiting AChE, modifying APP α-secretase-mediated pathway and decreasing Aβ-induced oxidative stress and apoptosis. Black thick arrows represent effects of enhancement; Gray thick arrows represent effects of inhibition; Dasheds represent possible effects

Conclusion

There is not one single cause, but several factors are involved in the pathology of AD disease. The notion of regarding AChE as a target for the palliative treatment of AD is renewed based on the new evidence about AchE–Aβ complexes. Both “classic” and “non-classic” roles of AChE have been demonstrated to be very important in AD pathology. In order to gain more successful clinical effects in AD therapy, novel dual inhibitors of AChE that target both the active site and the peripheral anionic site have been developed (Colletier et al. 2006), aiming at increasing the synapse ACh and disrupt the AChE/Aβ interaction, which consequently decreases the aggregation and deposition of Aβ. Interestingly, structural studies indicate that natural herbal medicine, HupA, may also act on the peripheral site, which might partly unravel its neuroprotective effects on APP abnormal processing and Aβ-induced neurotoxicity although further study will be needed for this conclusion. Therefore, HupA may not only ameliorate the cholinergic deficits, but also be capable of slowing down the progression of disease by protecting the neurons from toxic insults. This would be beneficial to the therapy of neurodegenerative diseases such as AD. In addition, understanding the mechanisms of HupA actions may also provide valuable clues to aid the development of new therapeutic agents for AD.

Acknowledgement

This work was supported by grants from the Ministry of Science and Technology of China (G199805110, G1998051115, 2004CB518907) and the National Natural Science Foundation of China (39170860, 39770846, 3001161954, 30123005, 30271494 and 30572169).

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