Increased oxidative stress is associated with a number of neurodegenerative diseases, including AD. It has been well documented that Aβ treatment can generate oxidative stress that eventually triggers a state of neurotoxicity and cell death (Xiao et al., 2002). Studies have demonstrated that HupA could enhance the cell viability and the activities of antioxidant enzymes including glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT); decrease the level of malondialdehyde (MDA) in PC12 (neuron-like rat pheochromocytoma) cells and cultured rat primary cortical neurons (Xiao et al., 2000a,b); and markedly reduced the MDA level in chronic cerebral hypo-perfusion rats (Wang et al., 2000) and aged rats (Shang et al., 1999). The protective effect of HupA on Aβ-induced cell lesion was also observed in NG108–15 cells (Zhang et al., 2002a). These results indicate that HupA can function as an antioxidant in Aβ-induced oxidative stress model by increasing the activities of antioxidant enzymes.

The neuroprotective effects of HupA against Aβ-induced oxidative injury are at least partly associated with its anti-apoptotic action. There is considerable evidence showing that Aβ can activate intracellular apoptosis pathways, which lead to neuronal death (Saille et al., 1999). Activation of caspase-3 is a key event in the execution of apoptotic cascade in central nervous system diseases. It has been demonstrated that HupA could attenuate the increase of caspase-3 activity induced by Aβ in cultured primary cortical neurons (Xiao et al., 2002) and inhibit Aβ-induced apoptosis 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., 2001a). 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., 2001b).

Perturbations in mitochondrial function have long been observed in samples derived from clinically confirmed patients with AD, including altered mitochondrial morphology, compromised enzyme complexes in the tricarboxylic acid cycle, and reduced cytochrome c oxidase activity (Cardoso et al., 2001). In addition, accumulated evidence showed that mitochondria are direct targets of Aβ (Yao et al., 2011). Aβ could be accumulated within mitochondria and interact with a mitochondrial protein, Aβbinding-alcohol-dehydrogenase, resulting in decreased cytochrome c oxidase activity and increased oxidative stress (Reddy and Beal, 2008). The neurotoxicity induced by Aβ will trigger a vicious cycle in which excessive Aβ accumulation and sustained mitochondrial dysfunction synergize to activate a cascade of neurodegenerative pathways (Silva et al., 2012).

Studies have provided evidence that HupA has the ability to effectively ameliorate the mitochondrial malfunction. Aβ(25–35) treatment could lead to a rapid decline of adenosine 5’-triphosphate (ATP) level, an obvious disruption of mitochondrial membrane homeostasis and integrity, a reduction in key enzyme activities in the electron transport chain and the tricarboxylic acid cycle, and an increase in intracellular reactive oxygen species (ROS) in PC12 cells, while HupA not only attenuated these signs of cellular stress caused by Aβ, but also enhanced ATP concentration and decreased ROS accumulation (Gao and Tang, 2006). In isolated rat brain mitochondria, HupA was able to effectively prevent Aβ-induced mitochondrial swelling, ROS increase, and cytochrome c release. Furthermore, it could also ameliorate Aβ-induced decreases in mitochondrial respiration, ATP synthesis, mitochondrial respiratory chain enzyme activity, and transmembrane potential (Gao et al., 2009). There is no evidence for the existence of cholinergic system in isolated brain mitochondria. Therefore, the mitochondria-targeted effects of HupA are clearly independent of cholinergic system. In addition, HupA could inhibit the penetration of Aβ into mitochondria and ameliorated Aβ-induced dysfunction of tricarboxylic acid cycle in isolated brain cortical mitochondria (Yang et al., 2012).

Synaptic plasticity, the variable efficacy of neurotransmission at synapses, is thought to underlie the ability of the brain to store memories (Martin et al., 2000). The forms of synaptic plasticity that are most likely to be involved in memory storage require gene transcription and protein synthesis to stabilize synaptic changes over time (Adams and Dude, 2005). Glutamatergic synapses mediate virtually all excitatory neurotransmission in mammalian brains (Sucher et al., 1996). Glutamate released from presynaptic terminals activates several types of glutamate-gated ion channels on postsynaptic membranes, including N-methyl-d-aspartate (NMDA) receptors (Rao and Finkbeiner, 2007). Permeability to Ca²⁺ is a feature of all NMDA receptors (NMDA-R), which are composed of an essential NR1 subunit and multiple NR2 subunits (Rao and Finkbeiner, 2007). Excitotoxicity caused by disturbances of glutamatergic neurotransmission in the brain has been shown to be involved in the pathogenesis of AD (Emilien et al., 2000). NMDA receptor antagonists have been used as neuroprotective agents to ameliorate the cognition deficits of patients with AD (Emilien et al., 2000; Marx, 2000).

Huperzine A could inhibit NMDA-induced toxicity in a dose-dependent way in cultured primary neuronal cells (Marx, 2000). HupA interacts with the NMDA ion channel by inhibition of [³H]MK-801 and [³H]TCP binding in brain synaptosomal plasma membranes but not the glycine or NMDA ligand-specific sites. The non-competitive binding results suggest that HupA inhibits the NMDA-induced toxicity most likely by blocking NMDA ion channels and the subsequent Ca²⁺ mobilization at or near the PCP and MK-801 ligand sites (Gordon et al., 2001). HupA reversibly inhibited NMDA-induced current in acutely dissociated rat hippocampal pyramidal neurons and blocked specific [³H]MK-801 binding in synaptic membranes from rat cerebral cortex (Wang et al., 1999) and the inhibitory effect is non-competitive (Zhang and Hu, 2001). HupA acts as antagonist of the NMDA-R, acting at one of the polyamine binding sites on the NMDA-R (Zhang et al., 2002b).

Nerve growth factor (NGF), a neurotrophin, plays a trophic role both during development and in adulthood (Aloe et al., 2012), and exerts its biological action by interacting with the specific receptor tropomyosin kinase receptor A (TrkA) (Huang and Reichard, 2003). Studies on rodents (Fischer et al., 1987) and primates (Tuszynski et al., 1991) have demonstrated that exogenous NGF was able to protect basal forebrain cholinergic neurons (BFCNs) from both traumatic insults and age-related cholinergic decline. Also, NGF is able to regulate both amyloid gene expression and protein processing (Rossner et al., 1998) and to counteract tau hyperphosphorylation (Zhang et al., 2010), acting directly at the two classical hallmarks of AD. A decreased NGF immunoreactivity in the BFCNs of patients with AD suggested that impaired NGF supply via retrograde transport could be the effective cause of cholinergic neurodegeneration in AD (Scott et al., 1995). These findings plus the experimental studies on NGF deficit-induced neurodegeneration in transgenic mice demonstrated a novel causal link between neurotrophic signaling deficits and Alzheimer’s neurodegeneration (Cattaneo and Calissano, 2012). Alteration in the homeostasis and equilibrium of NGF processing and NGF/TrkA signaling in target neurons has been considered as an upstream driver of all the cellular and molecular central hallmarks of AD (Tang et al., 2005).

Therefore, it will be very important to re-establish a correct homeostatic balance between ligands, and receptors, of the NGF pathway (Tang et al., 2005). Wang et al. (2006) investigated the effects of HupA on secretion of NGF in cultured rat cortical astrocytes and neurite outgrowth in rat PC12 cells and demonstrated that HupA treatment induced a significant increase in both mRNA and protein levels of NGF in astrocytes. They also found that treatment of PC12 cells with HupA led to a significant increase in the number of neurite-bearing cells without significant alteration in cell viability or other signs of cytotoxicity. The positive effect of HupA on the mRNA and protein levels of NGF, accompanied by the inhibitory effects on the memory deficits and neuronal damage, have also been reported in mice treated with transient cerebral ischemia and re-perfusion (Wang et al., 2006). These findings suggested that HupA has a direct or indirect neurotrophic activity, which might be beneficial in treatment of neurodegenerative disorders such as AD (Tang et al., 2005).

The canonical Wnt signaling pathway has an important role in development and maintenance of the nervous system and is also associated with neurodegenerative diseases (De Ferrari et al., 2003). Loss of Wnt signaling function is involved in Aβ-dependent neurodegeneration in the AD brain and two key components of the canonical Wnt signaling pathway, glycogen synthase kinase (GSK)-3β and β-catenin, are altered in the AD model mouse brain (Pei et al., 1999). It has also been reported that activation of Wnt signaling can prevent neurodegeneration induced by Aβ fibrils (De Ferrari et al., 2003) and that inhibition of GSK-3β and enhancement of β-catenin activity could prevent the loss of function of the Wnt signaling pathway caused by Aβ toxicity (Farias et al., 2005).

Wang et al. (2011) showed that HupA has a role in the regulation of Wnt signaling in APPswe/PS1dE9 (APP/PS1) transgenic mouse and APPsw cell models and could significantly inhibit GSK-3β activity and stabilize β-catenin protein level in vivo and in vitro. They also found that HupA induced a significant increase in the phosphorylation levels of both GSK-3α and GSK-3β proteins in APP/PS1 mouse brain and APPsw-overexpressing cells, while activation of both GSK-3α and GSK-3β through their autophosphorylation has been implicated in AD pathogenesis (Caricasole et al., 2004). Their findings suggested that HupA can inhibit the activity of GSK-3α/β and, hence, may inhibit Aβ generation and tau phosphorylation. HupA reverses protein kinase C (PKC)- and Wnt-inhibitor-induced inhibition of non-amyloidogenic processing of amyloid precursor protein (APP), paralleled by an inactivation of GSK-3 and a reversal of the level of β-catenin (Zhang et al., 2004; Wang et al., 2011). These findings imply that HupA is involved in regulation of the Wnt signaling pathway and that HupA regulates APP processing to the non-amyloidogenic pathway at least partly through activation of PKC and Wnt/β-catenin signaling.

Misregulation in brain iron has been considered to be one of the primary causes of neuronal death in neurodegenerative disorders (Qian et al., 1997; Qian and Shen, 2001; Lei et al., 2012). Evidence has also been gathered to imply that Aβ production, precipitation, and toxicity in AD are caused by abnormal interactions with neocortical iron (Zhu et al., 2004; Zhao et al., 2008; Smith et al., 2010). In a recent study (Huang et al., 2014), we demonstrated for the first time that HupA was able to reduce significantly the contents of insoluble and soluble A(β-40 and A(β-42 and hyperphosphorylated tau in the brain of APP/PS1 transgenic mice. Also, HupA could decrease the deposition of amyloid plaques and the levels of oligomeric Aβ. It also suppressed APP695 expression and increase ADAM10 contents in APP/PS1 mice brain. However, all of these beneficial effects of HupA could be largely abolished by high iron diet. In addition, the iron levels in the hippocampus and cortex of the APP/PS1 mice were found to be elevated while quantitative analysis revealed that the abnormal increase in brain iron contents in these two regions were almost completely normalized by the chronic treatment with HupA. Our findings provided direct evidence for the inhibitory effect of HupA on brain iron, and implied that the beneficial effects of HupA on AD is caused by the reduction in brain iron in the APP/PS1 mice.

Furthermore, we showed that mutation of APPswe/PS1dE9 could lead to a significant increase in transferrin receptor 1 (TfR1) expression and that HupA was able to induce a significant reduction in TfR1 expression in the brain of APP/PS1 mice in vivo, and a remarkable reduction in TfR1 expression as well as transferrin-bound iron (TBI) uptake in the cultured neurons in vitro. Two AChE inhibitors donepezil or galantamine did not induce any changes in TfR1 expression in the brain and had no effect on TBI uptake by the neurons, indicating that effects of HupA on TfR1 expression and TBI uptake were independent of its anti-AChE action. Based on the above findings, we propose that HupA has the ability to inhibit TfR1 expression and then reduce TBI uptake by the neurons or other brain cells, leading to a progressive reduction in iron contents and also iron-induced oxidative stress in the brain (Huang et al., 2014).