Part 2:Huperzine A And Its Neuroprotective Molecular Signaling in Alzheimer’s Disease

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3. Neuroprotective Molecular Signaling of HupA against AD

The neuroprotective effects of HupA treatment result from cholinergic signaling, which increases neurotrophic factor expression, synaptic activity, antagonism of the N- methyl-D-aspartate (NMDA) receptor (NMDAR), modulation of ROS, and neuronal survival [3,9].

Synaptic vesicle release is one of Hupa's neuroprotective mechanisms. During the synaptic vesicle cycle in CNS synapses, synaptic vesicles are filled with neurotransmitters via active transport and form a vesicle pool. Filled vesicles dock at the active zone, where they undergo a priming reaction that makes them competent for Ca2+-triggered fusion-pore opening [47]. In this step, synaptotagmins function as Ca2+ sensors; in fact, synaptotagmin- 1 knockout lacks a synchronized and rapid release of neurotransmitters, showing how relevant their function is [47]. HupA promotes synaptotagmin expression, improving synaptic vesicle exocytosis and neurotransmitter release [48] (see Table 1).

As previously mentioned, HupA inhibits AChE, so, after HupA treatment, acetylcholine will accumulate. Acetylcholine signaling acts mainly through α7nAChRs and α4β2nAChRs, producing an anti-inflammatory response [39]. Acetylcholine signaling modulates the function of neural progenitor cells through the mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) pathway, which is also activated as a response to HupA, exerting neuroprotective functions [49]. The same happens with brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) signaling, which in turn activates the phosphatidylinositol 3-kinase (PI3K)/Akt serine-threonine kinase pathway (see Table 1). Activation of the BDNF/TrkB-dependent PI3K/TrkB/mammalian target of rapamycin (mTOR) signaling pathway suppresses apoptosis and contributes to neuronal survival [50]. The cholinergic increase in BDNF in basal forebrain cholinergic neurons improves the induction and maintenance of long-term potentiation (LTP), synaptic transmission (by acting at pre-and postsynaptic sites), and the presynaptic release of the excitatory neurotransmitter glutamate [51], thus preventing cholinergic neuron loss and preserving cognitive functions [52,53]. Finally, HupA increases the levels of phosphorylated cyclic adenosine monophosphate responsive element-binding protein (CREB) in the hippocampus, which interacts with BDNF in the CREB/BDNF pathway, essential in multiple gene-cognition-environment interactions [54]. One of the main neuroprotective effects of HupA is the amelioration of Aβ accumulation and neurotoxicity through different cholinergic mechanisms [55]. First, through its AChEI activity, HupA disrupts AChE-Aβ interaction, as described in the previous section (see Figure2) [19,56,57], attenuating p53-mediated neuronal death [37]. HupA exerts a similar action with the post-synaptic cell adhesion molecule neuroligin 1 (NL1) [48]. NL1 has an extracellular domain with a high degree of similarity to cholinesterases (ChEs) [58,59]. Interaction between NL1 and Aβ oligomers has been reported. The purified extracellular ChE-like domain of NL1 binds directly to Aβ oligomers [60], and Aβ oligomers associate with NL1 at excitatory hippocampal synapses [61]. As with AChE, HupA interacts with the ChE-like domain of NL1 [46], disrupting the Aβ-NL1 interaction and downregulating oligomerization-induced excitotoxicity (see Table 1). Besides disrupting the interaction with Aβ oligomers, HupA also alleviates Aβ accumulation. Even though HupA has no effect on the production of the APP, it does modulate its cleavage.

The APP is cleaved by one of two possible pathways. In the α-secretase nonamyloidogenic pathway, the APP is cleaved by an α-secretase such as disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), producing the soluble ectodomain of APP (sAPPα) and a membrane-tethered intracellular C-terminal fragment (C83). Further processing by a y-secretase cleaves the C83, yielding a 3-kDa peptide (P3) and an APP intracellular domain (AICD) [62]. P3 and AICD are both neurotrophic and neuroprotective [26]. On the other hand, in the β/y-secretase amyloidogenic pathway, the cleavage is made by BACE1, and it yields the slightly shorter soluble APPβ fragment (sAPPβ) and a C99 fragment. Further processing by a y-secretase cleaves C99 and releases the AICD and the Aβ peptide, a hallmark of AD pathology [62].

HupA increases the expression of the sAPPa and C83 fragments [26,32], and it dose-dependently restores the constitutive expression of ADAM10, while downregulating the membrane translocation of BACE1, which hinders BACE1-APP association (see Figure 3).

Table 1. Molecular targets of HupA’s cholinergic mechanisms in dementia-related pathologies and effects of treatment.

(AChE: acetylcholinesterase; nAChRs: nicotinic acetylcholine receptors; Bcl-2: B-cell lymphoma-2; Bax: Bcl-2-like protein 4; Gsk-3β: glycogen synthase kinase-3β: mTOR: mammalian target of rapamycin; BDNF: brain-derived neurotrophic factor; TrkB: tropomyosin receptor kinase B).

Figure 3. HupA regulates APP processing. HupA dose-dependently increases the activity of the nonamyloidogenic pathway through the cleavage of amyloid precursor protein (APP) by an α-secretase, producing soluble α-APP fragments, P3 peptide and a C83 fragment. These are both neurotrophic and neuroprotective and are upregulated after HupA treatment. HupA also reduces the levels of BACE1, a β/y-secretase that produces soluble β-APP fragments in what is known as the amyloidogenic pathway, in addition to the C99 fragment that is cleaved to form the Aβ peptide. These products trigger apoptotic responses, such as neuronal membrane blebbing and cell shrinkage, leading to cell death (APP: amyloid precursor protein; BACE1: beta-site amyloid precursor protein cleaving enzyme 1; sAPPβ: soluble amyloid precursor protein-β; sAPPα: soluble amyloid precursor protein-α).

Activation of mAChRs and protein kinase C (PKC)/MAPK pathways have been proposed as a mechanism for this effect [15,23,46], as well as HupA mediated increase of phosphorylation of ERK1/2, that can also regulate ADAM10 transcription. Another mechanism for the cholinergic modulation of APP processing is the activation of Wnt signaling. Wnt signaling plays a crucial role in presynaptic assembly, postsynaptic differ- entiation at vertebrate peripheral neuromuscular synapses, stem cell differentiation, and the development and maintenance of the CNS [63–65]. Recent evidence suggests that it also regulates vascular stability, BBB integrity, and inflammation [66]. In the canonical Wnt pathway or Wnt/β-catenin pathway, Wnt ligands bind to receptors of the Frizzled (FZD) and low-density lipoprotein-related protein (LRP) families on the cell surface and activate several cytoplasmic relay components, including GSK-3β and adenomatous polyposis coli (APC), finally signaling to β-catenin. β-catenin enters the nucleus and forms a complex with lymphoid enhancer-binding factor 1 (LEF1) to activate the transcription of Wnt target genes. Wnt signaling pathways that are independent of β-catenin are referred to as non- canonical pathways, which include the planar cell polarity (PCP) pathway, also known as the Wnt/Jun N-terminal kinase (JNK) pathway, and the Wnt/ Ca2+ pathway [63] (see Figure 4).

Figure 4. HupA affects Wnt signaling. HupA affects both canonical and noncanonical Wnt signaling. Regarding canonical signaling, HupA modulates the Wnt/β-catenin pathway by downregulating GSK-3β activity. HupA increases the phosphorylation levels of GSK-3α/β, which stabilizes β-catenin and upregulates expression of its target genes, related to synaptic plasticity and neuronal survival while preventing tau hyperphosphorylation and Aβ neurotoxicity. HupA also interacts with the noncanonical pathway via PKC/MAPK signaling. PKC activation also inhibits GSK-3β activity and plays a role in upregulating the expression of the α-secretase, preventing Aβ accumulation and neuronal apoptosis (Wnt: wingless-related integration site; GSK3: glycogen synthase kinase 3; PKC: protein kinase C; MAPK: mitogen-activated protein kinase).

Inhibition of Wnt signaling causes cognitive deficits, increases tau phosphorylation, increases Aβ1-42 peptide concentrations and the Aβ42/Aβ40 ratio in the hippocampus, and decreases Aβ1-42 concentrations in the cerebrospinal fluid, indicating insufficient clearance across the BBB [18]. Thus, activation of Wnt signaling by HupA ameliorates Aβ burden and cognitive deficits [23,25,32].

Wnt signaling also modulates the nonamyloidogenic processing of APP, as transcriptional activation of Wnt target genes by β-catenin translocation directs APP processing towards ADAM10 while repressing BACE1 transcription, thus linking Wnt signaling and Aβ accumulation [67], as mentioned earlier. GSK-3 is one of Wnt's cytoplasmic relay components, and its increased activation causes increased phosphorylation of β-catenin, which inhibits canonical Wnt signaling [63], and AD hallmarks such as excessive tau protein phosphorylation and Aβ generation. In addition, it has been shown that Aβ impairs PI3K/Akt signaling, leading to the activation of GSK-3β. This creates a feedback loop that induces tau hyperphosphorylation, neurofibrillary tangle formation, synaptotoxicity, synaptic loss, and eventually neuronal death [64]. However, as was previously mentioned, HupA increases PKC signaling, and PKC inhibits GSK-3β activity, preventing tau phosphorylation and Aβ production (see Figure 4) [32]. Through the activation of Wnt and PKC signaling, HupA treatment rescues the nonamyloidogenic processing of APP, indicating that these pathways might be central to HupA's neuroprotective effects against Aβ burden [32] (see Table 1). Furthermore, through these mechanisms, HupA protects the presynaptic structure and synaptic plasticity [26,32,61,68] (see Table 1).

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HupA also acts via non-cholinergic mechanisms, targeting mitochondrial structure and function as well as Fe2+homeostasis. Mitochondrial perturbations have been described in AD pathology, including changes in morphology, a compromised tricarboxylic acid (TCA) cycle, reduced cytochrome C (CytoC) oxidase activity, and oxidative stress [69]. Aβ can accumulate in the mitochondria, interacting with the mitochondrial protein Aβ- binding alcohol dehydrogenase (ABAD), causing perturbations that lead to neurotoxicity and apoptosis. Aβ accumulation and mitochondrial dysfunction synergize to activate neurodegenerative pathways involved in cognitive loss [52,53]. Mitochondria initiate apoptosis by releasing proteins such as CytoC from intermembrane and Intracoastal spaces, followed by the activation of caspases and apoptosis-inducing factor (AIF) (a caspase-independent apoptosis pathway). Outer membrane permeabilization leads to mitochondrial metabolic failure with subsequent mitochondrial swelling and neuronal death [69]. In turn, APP overexpression may affect mitochondrial function by disrupting NIPSNAP1 protein localization in the mitochondrial matrix through direct interaction, altering the binding of the dihydrolipoyl transacylase and dihydrolipoyl transacetylase components of the branched-chain ketoacids complex and pyruvate dehydrogenase complex in the mitochondrial interior membrane [37] (see Figure 5). HupA inhibits the cytoplasmic translocation of CytoC and caspase-3 cleavage [69], attenuating apoptotic-inducing signals (see Table 2), and possibly prevents APP-induced mitochondrial dysfunction by reversing the disruption of NIPSNAP1 protein localization in the mitochondrial matrix [37]. HupA also upregulates glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT), thus preventing oxidative stress [70]. HupA mediated neuroprotection possibly involves restoration of an intact double membrane, which restores the membrane potential and ATP production, meaning preserved energy homeostasis [35,52] (see Table 2). Proteomic analysis confirms that HupA increases the expression of proteins involved in oxidative phosphorylation [37].

Figure 5. HupA rescues mitochondrial integrity after exposure to Aβ. Aβ crosses the mitochondrial double membrane and aggregates in the matrix. Mitochondrial perturbations include changes in morphology, compromised TCA cycle and reduced CytoC oxidase activity. Cell death is initiated by releasing proteins from intermembrane and Intracoastal spaces which activate caspases and AIF (caspase-independent apoptosis pathway). Outer membrane permeabilization leads to mitochondrial metabolic failure with subsequent mitochondrial swelling, ROS accumulation, and neuronal death. HupA treatment prevents Aβ accumulation in the matrix as well as Aβ interaction with mitochondrial proteins. HupA prevents the release of CytoC and subsequent apoptosis. It also rescues overall membrane stability, thus protecting mitochondrial enzymatic activity, membrane potential, and ATP formation. HupA also enriches proteins involved in oxidative phosphorylation, improving mitochondrial respiration (ABAD: amyloid beta-binding alcohol dehydrogenase; CAT: catalase; GSH-PX: glutathione peroxidase; SOD: superoxide dismutase; ROS: reactive oxygen species; IMS: intermembrane space; CytoC: cytochrome C; Cas-3: caspase-3; AIF: apoptosis-inducing factor).

Table 2. Molecular targets of HupA’s noncholinergic mechanisms in dementia-related and effects of treatment.

Cytotec: cytochrome C; Cas-3: caspase-3; AIF: apoptosis-inducing factor; CAT: catalase; GSH-PX: glutathione peroxidase; SOD: superoxide dismutase; TFR1: transferrin receptor 1; ROS: reactive oxygen species; APP: Amyloid precursor protein; IRP: iron regulatory protein).

Regarding Fe2+ homeostasis, it has been reported that AD mice models have elevated levels of Fe2+ in the cortex and hippocampus compared with WT mice. Accordingly, the expression of the transferrin receptor protein 1 (TFR1) is upregulated in APPswe/PS1dE9 mice, which explains the increase in Fe2+ uptake [34]. Fe2+-mediated neurodegeneration might originate in the downregulation of α-secretase activity, inhibiting the nonamylaaaoidogenic pathway, with a concomitant increase of APP expression due to a functional iron-responsive element (IRE) that has been identified in the APP promoter, which would explain why APP levels increase after Fe2+ treatment and decrease after chelator treatment. An alternative explanation could be that the translational expression of APP could be directed via the release of a classic repressor interaction between APP mRNA and iron regulatory protein-1 (IRP-1) [34]. HupA might act directly as a Fe2+ chelator, reducing the capacity of IRP-1 to induce APP translation [34]. HupA also downregulates TFR1 expression in mice in vivo, which reduces the uptake of transferrin-bound iron (TBI) in neurons [34] (see Figure 6) (see Table 2).

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Finally, it is noteworthy that HupA reduces proinflammatory cytokines expression through activation of nAChRs. Activating the cholinergic anti-inflammatory pathway, the efferent arc of the inflammatory reflex in the CNS results in suppressed inflammatory cytokine expression and systemic inflammatory responses. The activation of α7nAChR downregulates macrophages, microglia, and astrocytes activation inhibiting the production of proinflammatory cytokines while anti-inflammatory cytokine expression is not altered [10,46,71]. The cholinergic anti-inflammatory effects of HupA downregulate IL-1β, IL-6, and TNF-α secretion and NF-kB signaling, attenuating neuroinflammatory response [72].

Figure 6. HupA regulates Fe2+ homeostasis. HupA prevents the accumulation of Fe2+ inside the neurons. It might act as a chelator, while also downregulating the expression of TFR1 and preventing Fe2+ uptake into the cell. By reducing Fe2+ concentration inside the neuron, HupA reduces APP translation. Fe2+ binds the IRP, releasing it from APP mRNA which can then get translated. In the presence of HupA, Fe2+ concentration drops, and less IRP is released, meaningless APP is translated. HupA indirectly reduces ROS through all these mechanisms (TFR1: transferrin receptor 1; Tf: transferrin; TBI: transferrin bound iron; ROS: reactive oxygen species; APP: Amyloid precursor protein; IRP: iron regulatory protein).

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