The Therapeutic Effects of Seven Lycopodium Compounds on Cell Models of Alzheimer’s Disease

Abstract

Background:

As an acetylcholinesterase inhibitor (AChEI), Huperzine-A (Hup-A) is marketed for treatment of mild to moderate Alzheimer’s disease (AD) for decades in China. However, Hup-A causes some side effects. To search for new analogs or derivatives of Hup-A, we produced five Lycopodium alkaloids and two analogues by chemical synthesis: Lyconadins A-E, H-R-NOB, and 2JY-OBZ4.

Objective:

To systematically evaluate the therapeutic effects of the seven compounds on AD cell models.

Methods:

We assessed the effects of the seven compounds on cell viability via CCK-8 kit and used HEK293-hTau cells and N2a-hAPP cells as AD cell models to evaluate their potential therapeutic effects. We examined their effects on cholinesterase activity by employing the mice primary neuron.

Results:

All compounds did not affect cell viability; in addition, Lyconadin A and 2JY-OBZ4 particularly increased cell viability. Lyconadin D and Lyconadin E restored tau phosphorylation at Thr231, and H-R-NOB and 2JY-OBZ4 restored tau phosphorylation at Thr231 and Ser396 in GSK-3β-transfected HEK293-hTau cells. 2JY-OBZ4 decreased the level of PP2Ac-pY307 and increased the level of PP2Ac-mL309, supporting that 2JY-OBZ4 may activate PP2A. Lyconadin B, Lyconadin D, Lyconadin E, H-R-NOB, and 2JY-OBZ4 increased sAβPPα level in N2a-hAPP cells. 2JY-OBZ4 decreased the levels of BACE1 and sAβPPβ, thereby reduced Aβ production. Seven compounds exhibited weaker AChE activity inhibition efficiency than Hup-A. Among them, 2JY-OBZ4 showed the strongest AChE inhibition activity with an inhibition rate of 17% at 10μM.

Conclusion:

Among the seven Lycopodium compounds, 2JY-OBZ4 showed the most expected effects on promoting cell viability, downregulating tau hyperphosphorylation, and Aβ production and inhibiting AChE in AD.

Keywords

Alzheimer’s disease beta-Secretase 1 cholinesterase inhibitor Lycopodium alkaloid protein phosphatase-2A

INTRODUCTION

Alzheimer’s disease (AD) is an incurable, neurodegenerative disease characterized by progressive dysfunction of memory and cognition. Intracellular neurofibrillary tangles formed by hyperphosphorylated tau and extracellular senile plaques made by accumulated amyloid-β (Aβ) are thought to be neuropathological hallmarks of AD [1]. The World Health Organization (WHO) estimates that around 55 million individuals are living with dementia, with AD being the most common form, accounting for 60–70% of all cases [2]. It is alarming that the worldwide dementia epidemic is expected to reach 82 million people by 2030 and 152 million people by 2050 [2]. In 2019, AD and other forms of dementia were ranked as the 7th leading cause of death worldwide [2]. However, AD not only causes serious and irreversible damage to patients’ life, but also increases the economic and medical burden on their families and society [3]. Unfortunately, there is still lack of efficient drug to reverse the progression of AD. It is imperative to find a valid drug to treat the disease.

For the complexity of this neurodegenerative disease, there are many hypotheses for AD pathogenesis, which are cholinergic hypothesis, Aβ deposition hypothesis, tau protein hypothesis, and neuroinflammation hypothesis. Of these hypotheses, the cholinergic hypothesis is the earliest one and the most common therapies for approved AD drugs [4]. Galantamine, tacrine, rivastigmine, and donepezil were four acetylcholinesterase inhibitors (AChEI) approved for AD worldwide. But they cause annoying gastrointestinal side effects and peripheral cholinergic side effects, and tacrine was even discontinued due to its severe liver toxicity [5, 6].

Currently, chemicals derived from natural plants to treat AD are considered a promising field of medicine. Huperziaceae and Lycopodiaceae (Lycopodium s. l., club mosses), have a long history of use in Chinese folk medicine for the treatment of memory deficits, contusions, strains, swellings, schizophrenia, myasthenia gravis, and organophosphate poisoning [7]. Huperzine A (Hup-A) is a natural Lycopodium alkaloid extracted and isolated from the herb Huperzia Serrata, which is a member of Huperziaceae [8]. Hup-A was found to be a potent, reversible, and selective AChEI and be able to penetrate the blood–brain barrier better, so it has been approved for treatment of mild to moderate AD in China since 1996, and is marketed in the US as dietary supplement [8]. It is reported that Hup-A possesses additional pharmacological actions except for affecting the hydrolysis of synaptic acetylcholine (ACh), for instance, an antagonist effect on the N-methyl-D-aspartate (NMDA) receptor and the protection of neuronal cells against Aβ, free radicals, and hypoxia-ischemia induced injury [9 –13]. However, the peak concentrations of drug in the plasma (Cmax) are high and the time to reach the Cmax (tmax) is relatively short after oral administration, inducing annoying cholinergic side effects such as nausea, vomiting, and diarrhea [14]. So, psychiatrists cannot enhance therapeutic effects by increasing the dosage, making its efficacy restricted. Searching for new analogs or derivatives of Hup-A with higher activity has caught our attention.

We synthesized seven Lycopodium compounds chemically. They are Lyconadin A, Lyconadin B, Lyconadin C, Lyconadin D, Lyconadin E, H-R-NOB, and 2JY-OBZ4. All of them are structurally similar to Hup-A. Lyconadins A-E share the same pyridone ring motif with Hup-A, H-R-NOB, and 2JY-OBZ4 possess the same Nitrogen tricyclic skeleton with Hup-A. It has been shown that Lyconadins A and B could upregulate the mRNA expression of neurotropic growth factor in 1,321 human astrocytoma cells [15], which indicates that Lyconadins may possess bioactivity to nerve cells. However, there is still lack of research on Lyconadins’ effects on ameliorating AD pathology.

According to the recent data for anti-AD drugs, AChEIs have a moderate efficacy in the treatment of AD dementia, but the effect is not sustained. Most clinically used cholinesterase inhibitors have achieved limited clinical results [16]. In addition, the current single-target drugs under research targeting tauopathy or Aβ toxicity almost have not been marketed due to their limited efficacy in clinical trials [17, 18]. Only the Aβ oligomer-targeting antibody Aducanumab has recently obtained a tentative FDA approval to be in the phase IV trial. However, this decision is still controversial, and the efficacy of Aducanumab is still needed to be proven in future clinical studies [19, 20]. Since AD is a multifactorial disorder, researchers have turned their attention to developing drugs following multi-target strategy on AD [21]. These multiple targets include Aβ aggregation, tau phosphorylation, metal dyshomeostasis, oxidative stress, and the decreased ACh levels. To explore whether the seven compounds have therapeutic effects on AD, in the present study, we focused on their pharmacological effects on tauopathy, Aβ toxicity, and ACh activity in AD cell models.

MATERIALS AND METHODS

Chemicals and antibodies

According to the molecular formulas, Lyconadin A, Lyconadin B, Lyconadin C, Lyconadin D, and Lyconadin E are synthesized chemically by Prof. Yang’s laboratory (School of Pharmacy, Huazhong University of Science and Technology, China) [22]. H-R-NOB and 2JY-OBZ4 are intermediates in the synthesis of Lyconadin C and Lyconadin E [22]. Huperzine A (purity: 98%) was purchased from MedChemExpress, USA.

The primary antibodies used in this study include the following: GSK-3β (21002, Signal way Antibody), tau (R25863, Zen-Bioscience), tau-pS199 (382945, Zen-Bioscience), tau-pT231 (381181, Zen-Bioscience), tau-pS396 (381213, Zen-Bioscience), PP2Ac (R25422, Zen-Bioscience), PP2Ac-pY307 (380708, Zen-Bioscience), PP2A-mL309 (ab66597, Abcam), APP (R22718, Zen-Bioscience), BACE1 (A11533, Abclonal), sAβPPα (11088, Immuno-Biological Laboratories), sAβPPβ (18957, Immuno-Biological Laboratories), β-actin (AC026, Abclonal). The secondary antibodies include Goat anti-Rabbit IgG (Li-Cor Biosciences) and Goat anti-Mouse IgG (Li-Cor Biosciences).

Cells cultures and plasmids transfection

Human embryonic kidney 293 cell line with stable expression of full-length human tau (termed as HEK293-hTau) and mouse neuroblastoma cell line with stable expression of full-length human AβPP (termed as N2a-hAPP) [23, 24] were cultured in a humidified incubator aerated with 5% CO2 at 37°C. Cells were cultured in DMEM (Gibco, USA) media supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and antibiotics (100 U/mL penicillin and 100μg/mL streptomycin). For western blot, cells were seeded into 6-well plates and cultured until the cell density reached 80%. Then changed the medium with DMEM dissolved with corresponding Lycopodium compound or the same volume of DMSO. The Lycopodium compound powder was dissolved in DMSO and diluted in DMEM to the final concentration of 16μM. After incubating for 24 h, the cell protein extract was collected.

HEK293-hTau cells were seeded into 6-well plates at least 24 h before transfection with neofect DNA transfection reagent (Neofect biotech Corporation, China). When the cell density reached 60% –80%, it is appropriate to do the transfection following the manufacturer’s instruction. After 36–48 h, when plasmid expression reached its peak, HEK293-hTau cells were treated with Lycopodium compounds or DMSO. The plasmid pCDNA3.0-GSK-3β WT was a generous gift from Dr. Zhou Qiuzhi (Hubei Key Laboratory of Education Ministry of China, Wuhan, China) [25].

Mice primary neurons were isolated from embryonic day E17 to E18 C57 mice and cultured as previous described [26]. Neurons were cultured in neurobasal medium supplemented with B27, GlutaMAX, and antibiotics. On Day 8, Lycopodium compounds or the same volume of DMSO were diluted in complete medium and respectively treated to the neurons. After 24 h, the AChE activity was checked with Acetylcholinesterase assay kit. All reagents needed for primary neuron culture were purchased from Gibco, USA.

CCK-8 cell viability assay

The viability of HEK293-hTau cells treated with Lycopodium compounds was determined by the CCK-8 cell viability assay kit (Biosharp, China, BS350B). Cells were seeded at a concentration of 5,000 per well in a 96-well plate and then treated with various concentration of Lycopodium compounds (0, 250 nM, 500 nM, 1μM, 2μM, 4μM, 8μM, 16μM, 32μM, 64μM) for 24 h. After the treatments, the medium was removed, and 10μl of CCK-8 reagent in 100μl of medium was added. After incubating for 30 min at 37°C, the absorbance was measured using microplate reader (BioTek, 250058) at 450 nm.

Western blot

Cells were washed twice with cold PBS. Then the cells were homogenized in RIPA lysis buffer (Beyotime Biotechnology, China, P0013D) and 4×buffer containing PMSF (Thermo Scientific, 36978) and cocktail (MCE, HY-K0010). The homogenates were boiled for 10 min and then centrifuged (12000×g, 15 min, 4°C). The supernatants were collected, and the protein concentration was assessed by BCA Protein Assay Kit (Beyotime, P0011). If fresh proteins were not used immediately, they were stored in –80°C.

Cell proteins were boiled for 5 min after taking from –80°C. The proteins were loaded in 10% SDS-polyacrylamide gel. We used 5μg proteins to detect the level of β-actin, 10μg for GSK-3β, tau-pS199, tau-pT231, tau-pS396, PP2Ac, PP2Ac-pY307, and AβPP, 15μg for PP2Ac-mL309, sAβPPα, sAβPPβ, and BACE1. Then the proteins were electrophoresed for 1–1.5 h. Then the proteins were transferred to nitrocellulose membranes (Amersham Biosciences). After blocking with 5% non-fat milk dissolved in TBS at room temperature for 1 h, the membranes were incubated in primary antibody overnight at 4°C. Then the proteins were incubated with secondary antibodies at room temperature for 1 h and visualized using the Odyssey Infrared Imaging System (LI-COR Bioscience, USA). Image J software (Rawak Software, Germany) was utilized to quantitatively analyze the protein bands.

Quantification of soluble Aβ₄₂

The concentrations of Aβ₄₂ in the culture supernatants of N2a-hAPP cells were quantified using the Human Aβ1-42 ELISA Kit (Elabscience, China). Cell culture supernatants were collected and centrifuged at 2,000 g for 20 min, the supernatant was collected and detected according to the manufacturer’s instructions.

Acetylcholinesterase activity assay

The AChE activity of primary neurons lysates was carried out following the protocol in the Acetylcholinesterase assay kit (Abcam, ab138871). This kit detects AChE activity using DTNB to quantify the thiocholine produced from hydrolysis of acetylthiocholine by AChE. The absorption intensity of DTNB adduct (410 nm) is used to measure the amount of thiocholine formed, which is proportional to the AChE activity. The assay can detect as little as 0.1 mU AChE in a 100μL assay volume (1 mU/mL), which served as a reliable and sensitive test for this study. In brief, neurons were cultured for 9 days before respectively treatment with 10μM Lycopodium compounds. After 24 h, cells were collected with RIPA lysis buffer. Then 50μl cell lysates samples (test samples) were added to a 96-well plate; 50μl assay buffer was used as blank control; 50μl serial dilutions of standard AChE were utilized to construct standard curve. Acetylthiocholine-reaction mixture was prepared and 50μl of which was added to each well of the test samples, blank control, and the AChE standard. After incubation for 10 to 30 min at room temperature under the dark condition, the each well absorbance was measured with microplate reader at 410 nm. The protein concentration of test samples was assessed by BCA Protein Assay Kit (Beyotime, P0011), then the protein value of each sample well was calculated. The sample well absorbances were compared to the standard curve values and the AChE amounts (mU) were normalized to protein values (mU/ mg protein).

Statistical analysis

All data were expressed as mean±SEM and analyzed using Graph Pad Prism 8 software (San Diego, CA, USA). Difference between groups were assessed using one-way ANOVA, or student’s t-test. In all cases, a value of p < 0.05 was considered statistically significant.

RESULTS

All of seven compounds are structurally similar to Huperzine A

Structurally, Lyconadins A and B possess a pentacyclic skeleton, whereas Lyconadin C features a tetracyclic ring system, and all contain a (dihydro-) pyridone motif (Fig. 1). Lyconadins D and E share a cage-shaped tetracyclic frame that highlights two contiguous quaternary centers at the C4 and C13 positions, with the fifth ring either fused onto the bridged N or the N atom outside of the tetracycle (Fig. 1). H-R-NOB has a 6/6/7-azacyclic frame and a free hydroxyl, while 2JY-OBZ4 has a complex caged tetracyclic frame and nitrone functional group (Fig. 1). The seven compounds share a common heterocyclic frame and carbonyl structure [22]. All of the seven compounds share a common heterocyclic frame with anti-AD drug, Hup-A. In addition, Lyconadins A-E feature a same pyridone ring with Hup-A. Thus, it can be seen that all of seven compounds are structurally similar to Hup-A.

Fig. 1

All of seven compounds are structurally similar to Huperzine A.

All of seven compounds had no adverse effects on cell viability in HEK293-hTau cells

To evaluate the effects of these Lycopodium compounds on cell viability, the compounds were diluted to nine gradient concentrations (250 nM –64μM) firstly, and then treated to HEK293-hTau cells. We detected cell viability via CCK-8 cell viability assay kit. As illustrated in Fig. 2A-G, all of seven compounds showed no adverse effects on cell viability at gradient concentrations. Moreover, Lyconadin A increased cell viability at 1μM (Fig. 2A, p < 0.05), and 2JY-OBZ4 showed increased cell viability at 1μM and 8μM (Fig. 2G, p < 0.05). These data indicated that all of seven compounds had no adverse effects on cell viability in HEK293-hTau cells. Furthermore, Lyconadin A and 2JY-OBZ4 increased cell viability at specific concentrations.

Fig. 2

All of seven compounds had no harmful effects on cell viability in HEK293-hTau cells. A-G) HEK293-hTau cells were respectively treated with gradient concentrations of Lyconadins A-E, H-R-NOB, or 2JY-OBZ4 (0, 250 nM, 500 nM, 1μM, 2μM, 4μM, 8μM, 16μM, 32μM, 64μM) for 24 h, then cell viability was detected via CCK-8 assay. n = 3 per group. *p < 0.05.

Lyconadin D, Lyconadin E, N-R-NOB, and 2JY-OBZ4 resisted tau hyperphosphorylation induced by overexpression of GSK-3β in HEK293-hTau cells

CCK-8 assay illustrated that gradient concentrations of seven Lycopodium compounds did not exhibit any decrease on cell viability (Fig. 2). We wanted to choose a relatively high concentration to detect the maximal effect. However, 32μM H-R-NOB showed the tendency of decrease on cell viability, and 64μM Lyconadin A and H-R-NOB also exhibited the tendency of decrease on cell viability. Accordingly, we choose the concentration of 16μM to evaluate the compounds’ effects on AD cell models.

Glycogen synthase kinase-3β (GSK-3β) is a prominent kinase which regulates tau phosphorylation in the brain [27, 28]. To explore whether these compounds have potential effects on ameliorating tau hyperphosphorylation, we constructed a cell model of GSK-3β-mediated tau hyperphosphorylation through transient transfection of GSK-3β in HEK293-hTau cells. The results were evaluated by western blot after treatment with Lycopodium compounds respectively for 24 h, which showed that GSK-3β was increased for more than one time between groups transfected with GSK-3β and negative control (Fig. 3A, B). Furthermore, there was no difference on tau level among all groups (Fig. 3A, C). It can be observed from Fig. 3A–E that overexpression of GSK-3β induced hyperphosphorylation of tau at Ser199, Thr231, and Ser396. However, Lyconadin D, Lyconadin E, H-R-NOB, and 2JY-OBZ4 decreased tau phosphorylation level of Thr 231 site (Fig. 3A, E); Lyconadin B had tendency to decrease tau phosphorylation level of Thr 231 site, but there was no statistical difference (Fig. 3A, E). H-R-NOB and 2JY-OBZ4 decreased tau phosphorylation level of Ser 396 site (Fig. 3A, F). The phosphorylation level of tau-S199 did not change after treatments (Fig 3A, D). These findings suggested that Lyconadin D, Lyconadin E, H-R-NOB, and 2JY-OBZ4 resisted tau hyperphosphorylation in HEK293-hTau cells transfected with GSK-3β plasmid.

Fig. 3

The effects of seven compounds on tau pathology in HEK293-hTau cells. A) Western blots and B-F) quantitative analysis for GSK-3β, tau, tau-pS199, tau-pT231, and tau-pS396 in HEK293-hTau cells overexpressed with GSK-3β. MW, molecular weight. n = 3 per group. NS, not significant. *p < 0.05, **p < 0.01, ***p < 0.001.

2JY-OBZ4 directly decreased the phosphorylation level of PP2Ac and increased the methylation level of PP2Ac in HEK293-hTau cells

PP2A, which is a member of the protein phosphatase family and an omnipresent Ser/Thr phosphatase in mammalian cells, accounts for more than 70% of Tau Ser/Thr dephosphorylation in human brain [29]. Compromised PP2A activity is believed to induce tau hyperphosphorylation and tau phosphatase activity is decreased by 30% in brains of AD patients compared with controls [30 –32], therefore, increasing PP2A activity has become a therapeutic strategy for AD. As phosphorylation of PP2Ac Tyr307 negatively regulates PP2A activity [29], and methylation of PP2Ac Leu309 positively regulate the activity of PP2A holoenzyme, PP2Ac phosphor-Tyr³⁰⁷ level and methyl-Leu³⁰⁹ were examined by western blot after treatments with Lycopodium compounds in HEK293-hTau cells. Lyconadin E and H-R-NOB were observed to decrease the PP2A protein level (Fig. 4A, B); Lyconadin A increased phosphorylation of PP2A Tyr307 (Fig. 4A, C), both of which indicated they facilitated downregulation of PP2A activity. 2JY-OBZ4 decreased the phosphorylation of PP2Ac Tyr307 (Fig. 4A, C), suggested that PP2A activity was upregulated through treatment of 2JY-OBZ4. As for methylation of PP2Ac Leu309, Lyconadin B, Lyconadin E, H-R-NOB, and 2JY-OBZ4 increased the methylation level, which indicated that they may activate PP2Ac (Supplementary Figure 1A, C). To conclude, 2JY-OBZ4 at the same time decreased the phosphorylation level of PP2Ac and increased the methylation level of PP2Ac in HEK293-hTau cells, which strongly suggested that 2JY-OBZ4 may activate PP2A in HEK293-hTau cell line.

Fig. 4

2JY-OBZ4 decrease the phosphorylation level at Tyr 307 site of PP2Ac. A) Western blots and B, C) quantitative analysis for PP2Ac and PP2Ac-pY307. MW, molecular weight. n = 3 per group. *p < 0.05, **p < 0.01.

Effects of seven Lycopodium compounds on AβPP cleavages in N2a-hAPP cells

Excessive production of neurotoxic Aβ by amyloid-β protein precursor (AβPP) proteolysis is a critical step in AD progression. AβPP is mainly metabolized by a series of secretases, which are α-secretase, β-secretase (BACE1), and γ-secretase [34]. BACE1 conducts the first cleavage event to produce AβPP secreted β fragment (sAβPPβ) and C-terminal fragment (C99), the latter is sequentially cleaved by an enzymatic complex of four proteins (presenilin, nicastrin, anterior pharynxdefective1, and presenilin enhancer 2), collectively termed γ-secretase, to produce Aβ peptide and a cell-membrane-bound fragment [35]. As the catalytic subunit of γ-secretase, presenilin can be encoded by either the PSEN1 or the PSEN2 gene [18]. The process is called the amyloidogenic pathway of AβPP. First cleavage by BACE1 is a prime therapeutic target for preventing or reverting initial biochemical events involved in AD [36]. However, when AβPP is firstly cleaved by α-secretase, AβPP secreted α segment (sAβPPα) and C83 are generated, which prevents Aβ production and accumulation [37], so this process is called the non-amyloidogenic pathway of AβPP.

To explore whether these compounds impact AβPP cleavage, we chose N2a-hAPP cell line, a mouse neuroblastoma cell line with stable expression of full-length human APP, to do the detection. After treatments with Lycopodium compounds for 24 h, western blot was used to assess AβPP, AβPP N-terminal fragments, and Aβ secretion rate-limiting enzyme, BACE1. As shown in Fig. 5A–F, 2JY-OBZ4 decreased the protein levels of BACE1 and sAβPPβ (Fig. 5C, E), suggesting that the amyloidogenic pathway of AβPP was weakened. To further confirm this result, we evaluate Aβ₄₂ level in the supernatants of N2a-hAPP cells through ELISA (Fig. 5F). The result showed that Aβ₄₂ secretion was decreased after treatment with 2JY-OBZ4. Lyconadin B, Lyconadin D, Lyconadin E, H-R-NOB, and 2JY-OBZ4 increased sAβPPα, which meant non-amyloidogenic pathway of AβPP was enhanced (Fig. 5D). Taken together, these results indicated that Lyconadin B, Lyconadin D, Lyconadin E, H-R-NOB, and 2JY-OBZ4 had effects on AβPP cleavages, all of them facilitated non-amyloidogenic of AβPP cleavage, and 2JY-OBZ4 particularly suppressed amyloidogenic of AβPP cleavage.

Fig. 5

Effects of seven Lycopodium compounds on AβPP cleavages in N2a-hAPP cells. A) Western blots and B-E) quantitative analysis for AβPP, BACE1, sAβPPα, and sAβPPβ. MW, molecular weight. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001. F) ELISA assessment of soluble Aβ₄₂ in the supernatants of N2a-hAPP cells after treatment with DMSO or 2JY-OBZ4 for 24 h. *p < 0.05.

2JY-OBZ4 had moderate AChE inhibitory activity in mice primary neuron

As cholinergic hypothesis of AD is the first hypothesis of AD pathogenesis, strengthening cholinergic delivery system has been an important target for therapeutic development of AD [38]. As the seven compounds are structurally similar to Hup-A, which is a potent AChE inhibitor [8], we speculated that these compounds may have the activity on inhibiting AChE. To address it, we detected the effects of seven Lycopodium compounds on AChE activity in mice primary neuron, with Hup-A as a positive control. As shown in Fig. 6, all of these compounds showed lower inhibitory effects than Hup-A. Among the seven compounds, 2JY-OBZ4 was observed to have the highest inhibitory effect. 2JY-OBZ4 inhibited 17% AChE activity compared to control, suggesting that 2JY-OBZ4 had moderate AChE inhibitory activity in mice primary neuron.

Fig. 6

Effects of seven Lycopodium compounds on AChE activity in mice primary neuron. A) The AChE release of compounds under study and Huperzine A in mice primary neuron. B) The inhibitory percentages of compounds under study and Huperzine A relative to control. n = 3 per group. #p < 0.05.

Summary of these compounds’ therapeutic effects on AD

After detection of these compounds’ cell viability, effects on tauopathy, toxic Aβ production, and AChE activity, we made summary of these results in Table 1. As shown in Table 1, most of the effects were positive and beneficial, among them, Lyconadins B and D exhibited effects on alleviating tau hyperphosphorylation and enhancing non-amyloidogenic pathway of AβPP metabolism; Lyconadin E and H-R-NOB showed effects on enhancing non-amyloidogenic pathway of AβPP metabolism. In particular, 2JY-OBZ4 exhibited the effects on ameliorating tau hyperphosphorylation, activating PP2A, weakening amyloidogenic pathway of AβPP and enhancing non-amyloidogenic of AβPP. In terms of the effects on AChE inhibition, 2JY-OBZ4 showed the most beneficial activity. Hence, we made the conclusion that among the seven Lycopodium compounds, 2JY-OBZ4 exhibited the most potent anti-AD effects.

Table 1

Summary of these compounds’ therapeutic effects on Alzheimer’s disease

Compounds	Lyconadin A	Lyconadin B	Lyconadin C	Lyconadin D	Lyconadin E	H-R-NOB	2JY-OBZ4
Cell viability	1μM						1μM, 8μM
PS199
PT231
PS396
PP2Ac
PP2Ac-pY307
PP2Ac-mL309
BACE1
sAβPPα
sAβPPβ
AChE activity inhibition	7.3%	7.4%	9.2%	-1.9%	5.5%	10.4%	16.7%

Red arrows reflect positive effects on anti-AD; blue arrows reflect negative effects on anti-AD.

DISCUSSION

AD is a multifactorial disorder, whose pathogenesis is characterized by cholinergic hypothesis, Aβ deposition hypothesis, tau protein hypothesis, and neuroinflammation hypothesis. Single-target anti-AD drugs under research were found to be ineffective on AD patients recently, and even the approved anti-AD drugs AChEI and memantine are reported to be able to reduce or slow down the symptoms of AD, but not be able to prevent brain damage [39]. Hence, it is quite necessary to develop therapies directed at multiple targets, such as hyperphosphorylated tau, Aβ production, decreased synaptic ACh level, oxidative stress, mitochondrial dysfunction, biometal dyshomeostasis, and neuroinflammation [40]. Lyconadins A-E are Lycopodium alkaloids which were originally isolated from Lycopodium complanatum. The latter is a member of Lycopodiaceae, which has a long history of use in treatment of memory deficits, contusions, strains, swellings, schizophrenia, myasthenia gravis, and organophosphate poisoning as Chinese folk medicine [7]. In our present research, we first assessed the effects of these compounds on cell viability and found that they were not toxic to HEK293-hTau cells even at high concentration (64μM). Then we evaluated their effects on hyperphosphorylated tau, Aβ production, and AChE activity, and the results strongly supported that 2JY-OBZ4, an intermediate product in the synthetic process of Lyconadin D, was the most desirable compound for AD therapy. 2JY-OBZ4 ameliorated tau hyperphosphorylation induced by overexpression of tau kinase GSK-3β in HEK293-hTau cells, reduced Aβ production via downregulating of Aβ production rate-limiting enzyme, BACE1 in N2a-hAPP cells and inhibited AChE activity at 10μM by 17% in mice primary neuron.

Intracellular abnormal neurofibrillary tangles, formed by hyperphosphorylated microtubule-associated protein tau, and extracellular aggregates of Aβ plaques are two critical pathological characteristics in AD brain [41, 42]. Tau is a highly soluble and natively unfolded protein, mainly expressed in neurons, which is involved in the stabilization and organization of microtubules [43]. The physiological functions of tau are regulated by a series of post-translational modifications, such as phosphorylation, glycation, acetylation, etc. In particular, hyperphosphorylation of tau induce its detachment from microtubules and pathological tau aggregation, leading to the formation of paired helical filaments and neurofibrillary tangles, and eventually results in tauopathies like AD [43]. In order to reduce tau pathology in AD, a variety of small molecules, including modulators of anti-tau immune therapy, posttranslational modifications, aggregation inhibitors of tau, and microtubule stabilizer, have been developed [17]. Most of them ended before phase III, and only tau aggregation inhibitor LMTM is currently under research in clinical phase III [20]. In addition to this, numerous research has focused on tau kinases or phosphatases to alleviate tau hyperphosphorylation, as a result, to attenuate cognition impairments in AD mice model [44 –47]. It has been reported that treatment with Hup-A could reduce Aβ levels and hyperphosphorylated tau in the cortex and hippocampus of APP/PS1 mice [48]. As a structural analog of Hup-A, small molecular 2JY-OBZ4 performed the effect of reducing hyperphosphorylated tau in HEK293-hTau cells overexpressed with tau kinase GSK-3β. The therapeutic benefit of 2JY-OBZ4’s effects on tau pathology in AD mice deserves further exploration and study.

AβPP is a transmembrane protein that plays a substantial part in the development and growth of neurons and anterograde axoplasmic trafficking [49]. Aβ peptides, which is the main components of amyloid plaque [50], is formed due to the progressive breakdown of AβPP by the activation of BACE1 and γ-secretase in the amyloidogenic pathway [34]. In particular, BACE1 is called rate-limiting enzyme in the amyloidogenic pathway [51]. On the other hand, proteolytic processing of AβPP by the α-secretase enzyme results in the production of soluble fragments of sAβPPα, in contrast to sAβPPβ. It is reported that under physiological condition, more than 90% of AβPP are cleaved by α-secretase, and the remaining 10% of AβPP is being cleaved and processed by β- and γ-secretases, thus producing Aβ peptides [52]. However, driven by a variety of factors, more AβPP is cleaved by β- and γ-secretases, producing plethoric Aβ peptide beyond brain’s ability to clear it. In addition, extracellular Aβ peptides not only aggregate, generate amyloid plaque, and induce neurodegeneration, but also potently impair synapse structure and function of neurons [53]. Hence, downregulating extracellular Aβ level turns into an important therapeutic target for AD. Anti-Aβ drugs under research are developed as BACE inhibitors, passive immunotherapy, active immunotherapy, γ-secretase inhibitors, γ-secretase modulators, Aβ aggregation inhibitors, and α-secretase [18]. A few of them were admitted into clinical phase III; moreover, Aducanumab was approved to be in clinical phase IV [18]. In our present study, 2JY-OBZ4 reduced the secretion of Aβ₄₂ of N2a-hAPP cells via decreasing BACE1 level and enhancing the non-amyloidogenic pathway; in addition, Lyconadin B, Lyconadin D, Lyconadin E, and H-R-NOB enhanced the non-amyloidogenic pathway of AβPP. In vitro experiments demonstrated that 2JY-OBZ4 showed the most potential effects on ameliorating Aβ pathology. Whether 2JY-OBZ4 could reduce Aβ plaque in AD brain still needs further study.

ACh is an important excitatory neurotransmitter involved in learning and memory; however, the brains of patients with AD showed severe deficiency of ACh and impaired ACh transferase activity [54]. Among the regions innervated by cholinergic neurons, basic forebrain cholinergic neurons (BFCNs) play an important role [55]. The survival and differentiation of BFCNs can be regulated by nerve growth factor (NGF) [56]. Therapeutics targeting the cholinergic system include AChEI, improvement of deficiency in AChE, protection of BFCNs, and regulation of NGFs and brain derived neurotrophic factor and ACh receptor [54]. AChEI is designed to inhibit AChE from catalyzing the hydrolysis of ACh to generate choline and acetate ions, thereby to decrease the synaptic ACh level. At present, there are several drugs aimed at AChE, like tacrine, donepezil, rivastigmine, galantamine, and Hup-A, developed since 1990 s and were approved for AD [38]. Hup-A, as an approved drug in China, is more effective than galantamine in the inhibition of AChE; however, the inhibition concentration of Hup-A to butyrylcholinestrase is much higher than that to AChE [57]. According to Dvir et al.’s research, the α-pyridone moiety and carbonyl oxygen of Hup-A are indispensable for its inhibition on AChE [58]. The α-pyridone interacts with the active site of AChE via hydrogen bonding and possibly CH/π-interactions. On the other hand, the carbonyl oxygen of Hup-A repels the carbonyl oxygen of Gly117, causing the peptide bond between Gly118 and Gly117 flips, thereby affects the function of native enzyme. Bai et al. think the AChEI activity of Hup-A is related to its unsaturated bridge and fused pyridone ring [59]. It is observed that Lyconadins A-E possess the α-pyridone and carbonyl oxygen, whereas H-R-NOB and 2JY-OBZ4 possess the carbonyl oxygen. According to our data, all of the compounds except for Lyconadin D had effects on AChE inhibition; however, positive control Hup-A exhibited the most potent activation on AChE, which is consistent with previous studies [8]. It is worth noting that 2JY-OBZ4 possessed the most promising effect on AChE inhibition among the seven compounds, of which the inhibition rate reached 17% at 10μM in vitro. Although the AChE inhibitory activity of 2JY-OBZ4 is weaker than Hup-A, in the meanwhile, it indicated that the peripheral cholinergic side effects of 2JY-OBZ4 would be much weaker than Hup-A. However, 2JY-OBZ4’s effects on butyrylcholinestrase remain unknow. Furthermore, 2JY-OBZ4’s bioactivation on cholinesterase activity in AD mice models still needs further study.

Conclusions

Among the seven Lycopodium compounds, 2JY-OBZ4 showed the most expected effects on downregulating tau hyperphosphorylation, and Aβ production and inhibiting AChE in AD cell models. 2JY-OBZ4 appears to be a great promise to be a drug for AD and there is a need to do further research on its mechanisms and efficacy on AD cell and animal models.

Footnotes

ACKNOWLEDGMENTS

This work was supported by grants from the National Natural Science Foundation of China (31929002, 82071440, and 92049107), grants from the Innovative Research Groups of the National Natural Science Foundation of China (81721005), Science, Technology and Innovation Commission of Shenzhen Municipality (JCYJ20210324141405014), Guangdong Basic and Applied Basic Research Foundation (2020B1515120017) and the Academic Frontier Youth Team Project to Xiaochuan Wang from Huazhong University of Science and Technology.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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The Therapeutic Effects of Seven Lycopodium Compounds on Cell Models of Alzheimer’s Disease

Abstract

Background:

Objective:

Methods:

Results:

Conclusion:

Keywords

INTRODUCTION

MATERIALS AND METHODS

Chemicals and antibodies

Cells cultures and plasmids transfection

CCK-8 cell viability assay

Western blot

Quantification of soluble Aβ42

Acetylcholinesterase activity assay

Statistical analysis

RESULTS

All of seven compounds are structurally similar to Huperzine A

All of seven compounds had no adverse effects on cell viability in HEK293-hTau cells

Lyconadin D, Lyconadin E, N-R-NOB, and 2JY-OBZ4 resisted tau hyperphosphorylation induced by overexpression of GSK-3β in HEK293-hTau cells

2JY-OBZ4 directly decreased the phosphorylation level of PP2Ac and increased the methylation level of PP2Ac in HEK293-hTau cells

Effects of seven Lycopodium compounds on AβPP cleavages in N2a-hAPP cells

2JY-OBZ4 had moderate AChE inhibitory activity in mice primary neuron

Summary of these compounds’ therapeutic effects on AD

DISCUSSION

Conclusions

Footnotes

ACKNOWLEDGMENTS

References

Quantification of soluble Aβ₄₂