Therapeutic potential of 6BIO and DKK1-LRP6 inhibitor in Wnt/β-catenin pathway modulation for amyloid-β-induced Alzheimer's disease model

Abstract

Background

The Wnt/β-catenin signaling pathway plays a crucial role in central nervous system development, with emerging evidence linking its dysregulation to the progression of Alzheimer's disease (AD).

Objective

This study investigates the activation of Wnt signaling by targeting GSK3β and the DKK1/LRP6 interaction using a combination of 6BIO (6Bromoindirubin-3-oxime) and a novel gallocyanine derivative (8e) modulator.

Methods

We identified the interaction energy scores of both modulators with target proteins through an in-silico approach. Furthermore, the effects of 6BIO (10 µM) and 8e (20 µM) were assessed in SH-SY5Y cells treated with Aβ_1–42 (20 µM). The efficacy of these modulators was also evaluated in male Wistar rats through dose-ranging studies. An Alzheimer's disease model was established via intracerebroventricular injection of Aβ_1–42, followed by treatment with 6BIO (23.8 µg/kg/day, i.p.) and 8e (4.2 mg/kg/day, i.p.).

Results

Both modulators demonstrated favorable binding energy scores and dynamic simulation results against the targeted proteins. In Aβ_1–42-treated SHSY5Y cells, the combination of 6BIO and 8e significantly reduced reactive oxygen species production and apoptotic activity while modulating protein expression. In vivo study, rats treated with combination of 6BIO and 8e modulators exhibited improved neurobehavioral activity compared to AD model rats, along with altered expression of DKK1, β-catenin, p-tau, and pGSK3β. Additionally, decreased oxidative stress and apoptosis markers.

Conclusions

These findings suggest that the combined targeting of GSK3β and LRP6 represents a promising therapeutic strategy for AD. The combination of 6BIO and 8e shows potential as a novel modulator and warrants further investigation in clinical trials to assess its therapeutic efficacy.

Keywords

Alzheimer's disease amyloid-β DKK1 GSK3β tau protein Wnt signaling

Introduction

Alzheimer's disease (AD) is the most common neurodegenerative disorder, characterized by cognitive decline, memory loss, and disorientation.¹ Its progression is primarily driven by amyloid-β (Aβ_1–42) plaque deposition and tau hyperphosphorylation, which form neurotoxic aggregates in the hippocampus and amygdala.² While genetic factor account for about 10% of AD cases, the majority are sporadic and influenced by risk factors such as head injury, depression, and vascular disorders. Faulty processing of amyloid-β protein precursor (AβPP) by β- and γ-secretase enzymes leads to Aβ accumulation, which promotes tau dysfunction and neurofibrillary tangle formation.^3,4 These pathological changes trigger oxidative stress, mitochondrial dysfunction, and neuroinflammation, ultimately leading to neuronal degeneration.^5–8 As of 2021, AD affects approximately 57 million people globally, with cases expected to reach 153 million by 2050. Current FDA-approved treatments provide only symptomatic relief with no cure, highlighting the urgent need for more effective therapeutic strategies.^9–13 The Wnt/β-catenin signaling pathway, crucial for neurogenesis, synapse formation, learning and memory, is disrupted in AD, making it a promising therapeutic target.^14,15

Wnt signaling has a key role in regulation of tau phosphorylation by maintaining the inactive state of kinase proteins such as GSK3β (glycogen synthase kinas-3 beta).¹⁵ Inhibiting these kinases during AD progression could potentially reduce the pre-aggregation of neurofibrillary tangles. Additionally, GSK3β and DKK1 (Dickkopf-1) negatively regulate the Wnt/LRP6 signaling cascade. Increased DKK1 levels in AD cases¹⁶ interact with the LRP6 receptor, activating the cytoplasmic destruction complex (GSK3β, Axin, CK1, APC). This process inhibits β-catenin nuclear entry, leading to its ubiquitination. Since β-catenin is essential for expressing neuronal survival genes, its inhibition contributes to neurodegeneration. Moreover, activated GSK3β further hyper-phosphorylates tau protein, primarily targeted by CDK5, ultimately causing neuronal cell death as illustrated in Figure 1.

Figure 1.

Hypothesis of canonical Wnt signaling pathway begins when Wnt molecules bind to frizzled receptor and the co-receptor LRP6, triggering downstream signaling. However, elevated levels of amyloid-β (Aβ_1–42), stimulate the expression of the DKK1 protein, which binds to the LRP6 receptor and block downstream signaling, contributing to the development of AD.

In this study, we utilized two modulators to target the Wnt signaling pathway in AD: 6BIO (6Bromoindirubin-3-oxime) as a well-known inhibitor of GSK3β, and 8e is a novel gallocyanine derivative that inhibits the DKK1/LRP6 interaction. This combination strategy represents the first comprehensive attempt to address Wnt signaling dysregulation in AD by simultaneously targeting both intracellular and extracellular regulatory mechanisms. 6BIO modulates intracellular signaling by inhibiting GSK3β, thereby reducing phosphorylated β-catenin levels.¹⁷ In parallel, 8e modulator can restore extracellular Wnt signaling by blocking the interaction between DKK1 (a Wnt antagonist) and the LRP6 receptor. While previous studies have explored these mechanisms individually, our study is the first to integrate both approaches, addressing a critical gap in Wnt pathway modulation.

This dual-target approach tackles complementary mechanisms of pathway dysfunction, offering enhanced neuroprotection compared to individual treatments. Previous studies have demonstrated the neuroprotective benefits of both extracellular and intracellular targeting mechanisms.^18–20 We hypothesize that their combination will lead to a synergistic effect, resulting in improved restoration of Wnt signaling and better therapeutic outcomes in AD.

As this specific dual targeting approach in Wnt signaling modulation has not been previously explored in the context of AD, our study provides novel insights into Wnt signaling modulation and offers a promising therapeutic strategy for AD and related neurodegenerative disorders.

6BIO is a bisindole alkaloid derivative of indirubin, a compound found in plants and mollusks. Indirubins and their derivatives have shown efficacy as inhibitors of GSK3β, with therapeutic potential in diabetes, neurodegeneration, and anti-aging.^21–24 On the other hand “Cyclopropylmethyl 4-(cyclopropylmethoxy)-7-(dimethyl amino)-3-oxo-3H-phenoxazine-1-carboxylate” or 8e, is a derivative of gallocyanine compound which itself is a weak inhibitor of DKK1-LRP6 interaction²⁵ Among its derivatives, 8e has demonstrated significant potential in inhibiting DKK1 binding to LRP6.²⁵ This study highlights the combined use of 6BIO and 8e as a novel strategy to restore downregulated Wnt signaling in AD, demonstrating their synergistic neuroprotective role in combating the disease.

Methods

In silico analysis

The molecular docking (kJ/mol) and molecular dynamic simulation (MDS) was run by using of Schrödinger maestro software^26,27 to access the interaction energy of modulatory compounds with target protein GSK3β (PDB:1UV5) and LRP6 (PDB:3S8V) and analyzed the stability score by RMSD (Root Mean Square Deviation) and RMSF (Root Mean Square Fluctuations) metrics of the selected protein-compound complex. Ther detail methodology mentioned in the Supplemental Material.

Chemicals and drug preparation

The AD model inducing agent Aβ_1–42 fragment (Sigma-Aldrich Cat. A9810) were acquired from Sigma-Aldrich. Aβ_1–42 was prepared with 1 mg/ml stock solution in sterile 0.1 M PBS (phosphate buffer saline) with pH of 7.4. The solution was aliquoted and stored at −20 °C. To induce aggregation, the Aβ_1–42 stock was incubated at 37°C for 4 days before to use it further, according to the protocol by Souza et al. (2013).²⁸ Aggregated Aβ_1–42 (20 µM) was prepared for in vitro study to mimic the AD like model development.²⁹ However, 10 µg/rodent was then administrated via intracerebroventricular (icv) injection. 6BIO (TCI Cat.B4006) and 8e (synthesized from Department of Chemistry, Kurukshetra university) was prepared with DMSO (Sigma-Aldrich Cat. 276855) at a concentration of 10 mg/ml, further stored it at −20˚C.

In vitro study

Neuroblastoma cells (SHSY5Y) were purchased from NCCS Pune. SHSY5Y cells were cultured in DMEM (Gibco, LOT;2368006) with 10% FBS (Thermo fisher) Cat.no. 5256801) and used the Amp-B (0.25 µg/ml) antibiotics. Furthermore, incubated the cells on 37°C, humidity was maintained at 5% CO₂. For cell differentiation, a 10 µM retinoic acid solution was prepared by diluting a 3 mg/ml (0.01 M) stock solution in DMSO (R2625-Sigma Aldrich) and applied to the cells for 5 days.

AD induction of AD model in neuroblastoma cells (SH-SY5Y) and treatment

Neuroblastoma cells (SHSY5Y) were seeded into a 6-well plate and treated with 10 µM of retinoic acid (RA) for a continuous period of 5 days. Neurite outgrowth was observed on the 5th day. In contrast, the untreated cells stayed in their original, undifferentiated state (sup.2). Following this differentiation, the RA-treated neuroblastoma cells (SHSY5Y) was exposed to Aβ_1–42 (20 μM) for 24 h which further pronounced morphological changes, including the degeneration of neurites, suggesting a detrimental effect on the differentiated cells. Further, AD induced cells treated with 6BIO and 8e with different concentration of 5 µM, 10 µM, 15 µM, and 20 μM for 24 h. This concentration was prepared from a stock solution of 10 mg/mL.

MTT assay for cell viability

The cell viability assay was performed by using MTT (CAS.no. 298931). The 96-well plate (5X10⁴ cells/wall) and 6-well plate (5X10⁵ cell/well) were used for further experimental analysis. Aβ_1–42 treated cells were incubated with MTT (0.5 mg/ml) for 3 h at 37˚C. After discarding the media, DMSO was added to solubilize the formazan crystals. Absorbance (Ab) was measured at 570 nm by using a Tecan-microplate-reader. The control group was considered to have 100% cell viability.

Reactive oxygen species analysis using flow cytometry

Reactive oxygen species (ROS) production was analyzed by using DCFDA kit (ab113851). Treated and control SHSY5Y cells were trypsinized and washed with sterile PBS. The pallet was re-suspended in PBS and DCFDA was added to each tube. Fluorescence intensity was analyzed via flow cytometry after 30-min incubation period. FACS Calibur flowcytometric and Cell quest software (BD biosciences) was used for data acquisition.

Real time PCR

The SHSY5Y cells (1 × 10⁶) and brain tissue were stored in triazole at −80°C. RNA was extracted and quantified through absorbance at 260/280 nm by using of nanodrop instrument. cDNA was synthesized from RNA sample by using of manufacturer's protocol which was given in iScript^TM cDNA kit (Bio-Rad, USA). mRNA expression of target gene was evaluated by using of RT-PCR (real time PCR). All the experiments were performed in triplicated, and data was calculated by using of 2-ΔΔct. For in-vitro study, the targeted gene was normalized by 18 s mRNA expression. For in-vivo study, beta-actin was used for normalization of gene expression. The detailed sequence of primer mentioned in the Supplemental Material.

ELISA (enzyme linked immunosorbent assay)

For the precise quantitative determination of β-catenin (CTNNB1), Phospho-β-catenin, GSK3β, GSK3β (p-216), and P-tau in in vitro and in vivo study used ELISA kit. The 96-well plates were pre-coated with antibodies. All regents of the kit were prepared as given in instructions. The OD value (optical density) was determined of each well by using the ELISA microplate reader at 450 nm. ELISA kit of GSK3 beta (cat. no. E0989Ra), p216GSK3 beta (cat.no. E2509Ra and BT-AP06543), β-catenin (cat.no. E1153Ra and E-EL-H0666), P-β catenin; (Cat No. 28853-1-AP), phospho-tau (cat. no. E1320Ra and E5874Hu) were purchased.

Confocal microscopy

SHSY5Y cells were seeded in 24-well plate with the confluence of 70% (0.05 × 10⁶ cells per wells) Firstly, the SHSY5Y cells differentiation expressed were evaluated by the NeuN expression for that we have used anti human NeuN antibody. In next, cells were treated with Aβ_1–42 (20 μM) for 24 h then treated with 6BIO and 8e for 24 h. In next, plated cells fixed with paraformaldehyde (4%, PFA) for 15 min and further used triton-X100 for permeabilization process for 3 min. The plated cells were blocked by 2.5% bovine serum albumin (BSA), kept for overnight (4°C). The proteins were detected by using primary antibodies such as tau ((cat.no. A1103 from ABclonal), 1:200 dilution), pTau-Ser396 (AP1028 ABclonal, 1:200 dilution), FasL (cat.no.AF5333 Affinity, 1:200 dilution), Caspase-3 (cat no. A0214 ABclonal,1:200 ratio dilution) followed by secondary antibody (Alexa Fluor^®488labelled lot no.21),1:1000 dilution). After 24-h cells stained with DAPI (1 mg/ml). The confocal microscope was used to get cells images, using a confocal microscope and analyzed by using ImageJ software.

In vivo study

This experimental study was performed on Male Wistar (n = 82) rat 200–250 g were procured by the Advanced Small Animal Research Facility of the PGIMER institute, Chandigarh, India. Rodents were kept in colony cages in the institutional animal house room having an adaptable temperature of 23 ± 2°C, relative humidity (RH) of 70 ± 10% and day-night cycle of 12 h. Food and water were provided ad libitum to the rodents. The animals were allowed to acclimate to the laboratory environment for one week prior to experiments. The study was initiated following approval of the protocol by the institute's animal ethics committee (IAEC) ref. No./108/97/IAEC/676, institutional biosafety committee (IBC) clearance ref. No.000591/IBC/2018.

Experimental design and treatment strategy

All animals were randomly assigned into 12 groups (each group, n = 6). Group 1 served as the control rats, Group 2 as the Sham group (intra-cerebroventricular injected saline), Group 3 as the Disease model (intra-cerebroventricular injected Aβ_1–42, 10 µg/2 µl volume), Group 4 as the Disease group treated with DMSO 0.5% (Vehicle). Groups 5 to 10 were disease groups treated with 6BIO at doses of 5.95 µg/kg/day, 11.9 µg/kg/day, and 23.8 µg/kg/day, and 8e at doses of 0.7 mg/kg/day, 2.1 mg/kg/day, and 4.2 mg/kg/day, respectively. Group 11 received a combination treatment of 6BIO (23.8 µg/kg) and 8e (4.2 mg/kg), while Group 12 was treated with the standard drug donepezil at a dose of 3 mg/kg/day, orally.³⁰ The doses of 6BIO and 8e were selected from previous in-vivo studies and extrapolated from mouse doses to the corresponding rat doses.^31,32 Aβ_1–42 was injected into the brain on day 1 to induce the AD model. Following 14 days post-injection, the treatment phase began with 6BIO and 8e administered intraperitoneally (i.p) from day 15 to day 28. Behavioral analysis was conducted on day 29 to assess treatment effects. Following this, the animals were sacrificed for molecular, biochemical and histopathological study as depicted in Figure 2.

Figure 2.

Timeline of events in experimental study.

Aβ1–42 induced AD model in rats

The ICV injection procedure were adopted by Ishrat et al.³³ Briefly, the male Wistar rats were anesthetized using ketamine (60 mg/kg) and xylazine (10 mg/kg) and placed on a stereotaxic apparatus. The skull was opened and exposed to locate the bregma. The holes were drilled in the skull according to the Paxinos and Watson atlas coordinates (−0.8 mm AP, 1.5 mm ML, 0.4 mm DV), the Aβ_1–42 were administered bilaterally in lateral ventricle. The 10 μg/rodent was then administrated via icv injection. The total volume of injection is 2 μl, either of Aβ_1–42 or saline was injected at the rate of 1 µl/1 min, by using a Hamilton syringe. The Sham group received saline 2 μl per site by icv. After the injection, the scalp was sutured, and the rats were allowed to recover from anesthesia.³⁴ The AD model was evaluated by using neurobehavioral assessment, biochemical analyses, and histo-morphological changes studies 14 days after icv injection.³⁵

Neurobehavior parameter study

Morris water test (MWT)

This test was performed as per standard protocol to evaluate spatial memory. The water filled (temperature = 28 ± 1°C) cylindrical pool (diameter = 150 cm, height = 45 cm) was located in a dimly lit room with visual cues at far-off distances to help in recognition of orientation of themselves rats. The water pool was divided into four equal quadrants, with a target quadrant containing a hidden platform (10 × 10 cm) submerged 1 cm below the water surface.³⁶ Over the four following days (−5 to −2 days), the rat underwent daily 120-s training sessions, during which they were assigned to each quadrant in turn to explore and familiarize themselves with the MWT setup. A probe test was conducted one day after the final training session to assess spatial memory (represented as a day 0). The time taken by each rodent to locate the hidden platform (as the escape latency time) was recorded by Etho-vision software.

Elevated plus maze test (EPM)

The EPM was made of white plywood and had two open and two closed arms. The closed arm was 50 cm long, 10 cm wide and 40 cm high, while the central square was 10 cm×10 cm and left open. The maze was elevated 80 cm above ground. Initially, during the acquisition trial, the rat was placed at the end of open arms and allowed to explore for 2 min, with a food reward placed in one of the closed arms. On the 3rd day, a probe test was conducted and asses the time spent in the food containing arms was recorded. The rat had 30 s to reach the food reward in the closed arm.

Assessment of biochemical parameters and molecular estimation in brain tissue

Estimation of glutathione concentration

GSH (glutathione) level were evaluated in rat brain. In this assay, the total volume of mixture is 3 ml which was made up phosphate buffer (0.1 M) and DTNB (10 mM). After then, it has been given a yellow color and took reading at 412 nm on spectrophotometer (PGI/EPL/4004/ICMR/UV-01). The concentration of GSH was calculated by using of GSH standard curve.³⁷

Estimation of superoxide dismutase (SOD)

The SOD activity in the rat brain was calculated using of NBT (nitro blue tratrazolium) method. 500 μl brain homogenate was taken and added 100 μl a NBT solution, and 20 μl hydroxylamine, mixed all and vortex it for 10 s. The autoxidation was calculated every 30 s to 120 s at 560 nm under spectrophotometry. The SOD activity was measured in U/mg protein.

Estimation of catalase activity

For catalase activity, 50 μl supernatant from brain homogenate was used. 1.0 ml PBS (0.1 M) and H₂O₂ were added. The absorbance was calculated at 30 s to 120 s at 240 nm. The catalase activity was quantified as the amount of H2O2 consumed (in nmol) per minute per mg protein.

Real time PCR

The method was the same as mentioned above. The detailed sequence of primer mentioned in the Supplemental Material.

ELISA

The method was the same as mentioned above.

Western blot

The brain homogenate was assessed by using western blotting (WB) to determine the protein expression. Samples including Control, Disease (Aβ), Combination, and Donepezil were put into SDS-PAGE, and the resulting protein bands were transferred to PVDF membrane. The transfer process involved assembling a sandwich with the gel facing the anode and the PVDF membrane facing the cathode.³⁸ Protein transfer was conducted at 70 V for 2 h using a Mini Blot Module (Thermo Fisher Scientific, USA) and chilled transfer buffer. After the transfer, 5% bovine serum albumin (BSA) was used to block the PVDF membrane in Tris Buffer Saline (TBS) with 0.1% tween for 4 h at 4°C. The PVDF membrane was then washed four times with TBST to remove any unbound blocking agent. Primary antibodies for GSK3 beta p216 (Cat. PAB12624 Abnova 1:1000), β-catenin (Cat. log 9562 CST (1:1000), and p-Tau (AP1028 ABclonal (1:1000) were prepared in a BSA solution and incubated with the PVDF membrane at RT (room temperature) with 3 h of shaking. The membrane was subsequently washed multiple times with TBS-T. A secondary antibody, labeled with streptavidin and HRP-conjugated peroxidase, was prepared in a BSA solution and incubated with the PVDF membrane at room temperature with shaking for 3 h. The membrane was again washed multiple times with TBS-T. Protein bands were detected using Western ECL substrate (Bio-RAD, USA). A working substrate solution was prepared by mixing equal volumes of reagent 1 and reagent 2, and it was carefully poured onto the membrane to avoid bubble formation. The blot was visualized by exposing it to a dark room using the protein simple FluorChem M (Bio-Techne, USA) imaging system.

Brain morphology detection

Under deep anesthesia, Wistar rats were trans-cardinal perfused for 10 min with 0.9% saline, followed by 20 min of cold PFA 4% in 0.1 M PBS. The brain tissue was sectioned and post fixed in the same PFA for 2 days. The sample were embedded in parafilm, took sections at 3–4 µm thickness, stained with hematoxylin and eosin, and imaged at Evos inverted microscope. The hippocampal region was chosen for analysis. Degenerative neurons were identified by their shrunken or swollen cell bodies and eosinophilic cytoplasm.

Immunohistochemistry for apoptotic marker: Fas-L and Caspase 3

The IHC procedure used in this study was based on a previous protocol.³⁹ The 3–4 μm brain sections were used for IHC. The Antigen retrieval was carried out using sodium citrate buffer, followed by blocking of endogenous peroxidase with H₂O₂. Primary (1:200) and secondary antibodies GenTex (Cat. No. GTX26013) were incubated with the sections, followed by staining with DAB and counterstaining with hematoxylin. The sections were then dehydrated, cover slipped and observed under the microscope using EVOS Flauto imaging system. Further, Image J software was used to quantify the Fas-L and Caspase 3 percentage.⁴⁰ From each group, 5 randomly selected sections from hippocampus were analyzed.

Statistical analysis

All the experimental data were evaluated by using Graph Pad prism (version 9, Graph Pad software, Inc., USA). The Results were presented as mean ± SD. To assess the normality of data distribution, the Shapiro Wilk test was performed. Further, data set that followed a normal distribution, one way Analysis of Variance (ANOVA) was performed. For comparing the multiple groups, one way ANOVA test was used. For repeated measure data such as neurobehavioral parameters, a repeated measure ANOVA test was applied. The post-hoc Tukey's test was conducted for inter-group comparisons. The p-value at the level of p less than 0.05 (p < 0.05) was considered as statistical significance. When compared to control group, * represent the p value less than 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and when compared with disease group, ^# represent p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001.

Results

In silico results

The activated GSK3β and DKK1-LRP6 interaction play a significant role in the inactivation of Wnt signaling. In the current study, potent inhibitor compounds against the target proteins LRP6 (PDB: 3S8V) and GSK3 beta (PDB:1UV5) were identified using an in silico approach. The 3D structures of both proteins were prepared using the Protein Wizard tool provided by Maestro Schrodinger software. Thtsiadis et al. synthesized various derivative molecules of gallocyanine, which demonstrated good binding affinity at the DKK1 binding site in the LRP6 protein.²⁵ Among these derivatives, 8e or Cyclopropylmethyl 4-(cyclopropylmethoxy)-7-(dimethylamino)-3-oxo-3H-phenoxazine-1-carboxylate and 11b or Ethyl 7-(dimethylamino)-4-((4-methylbenzyl)oxy)-3-oxo-3H-phenoxazine-1-carboxylate exhibited significant properties such as molecular weight, LogD, logP, Fsp3, and lower DKK1 binding percentage with LRP6 (Supplemental Table 1). To analyze the binding energy score, molecular docking studies were performed against the LRP6 protein (at the DKK1 binding site). The docking result of LRP6 with 8e molecules is −4.714, and physiological interaction residues are LRP6 chain A: His834, Trp850, Ser851, and LRP6 chain B: Asn794, Tyr875, Glu708, and Asn794, Tyr706 (Supplemental Material). The docking score of the gallocyanine compound is −4.787, showing interactions with chain A: Tyr850, Ser851, and chain B: Trp850, Tyr875. Additionally, molecular dynamic simulation was performed for 50 ns to evaluate the stability of the protein-ligand complex. We compared the MD results of the control complex (LRP6-gallocyanine) with the (LRP6-8e complex) and LRP6-11b complex. The gallocyanine compound showed a higher RMSD (35 Å) and RMSF (32 Å), whereas the 8e molecules showed a lower RMSD (4 Å) and RMSF (2.4 Å). The gallocyanine compound demonstrated less stability and more fluctuation with the LRP6 receptor compared to 8e molecules, which showed greater stability with the LRP6-DKK1 binding site as mentioned Supplemental Table 1 and Figure 1.

Secondly, 6BIO is potent GSK3β inhibitor was screened through our previous in silico study.⁴¹ We observed a significant docking score (−10.451) and molecular dynamic simulation results (RMSD:1.8 Å) and (RMSF:1 Å). Based on in silico experimental study, 6BIO and 8e compound demonstrated significant interaction and stability with targeted protein. Next, the efficacy of both modulators was evaluated by in vitro and in vivo study.

Assessment of modulators by cytotoxicity in SHSY5Y cells

To determine the effective dose of the selected modulator (6BIO and 8e),we performed an MTT cell viability assay.⁴² Differentiated SHSY5Y cells treated with Aβ_1–42 were further exposed to 6BIO and 8e at 5 µM, 10 µM, 15 µM, AND 20 µM doses for 24 h. As reported in previous study, Aβ_1–42 at 20 µM concentration induced significant cell toxicity.⁴³

Our results showed that 6BIO at 10 µM increased cell viability, while 8e exhibited a dose dependent effect. A significant difference was found between control groups and Aβ_1–42 treated groups, with a mean difference of 49.56442 (p < 0.0001). Following treatment with 6BIO at different concentrations (5 µM, 10 µM, 15 µM, and 20 µM) revealed that both the lowest dose (5 µM) and highest dose (20 µM) resulted in approximately 50% cell viability, where the 10 µM concentration demonstrated a more significant difference compared to the disease group and showed cell viability. However, at higher concentrations 6BIO exhibited toxicity by reducing cell viability. Our findings are consistent with previous studies indicating that 6BIO promote cell proliferation and differentiation in a dose-dependent manner.^21,44 In contrast, the 8e modulator did not significantly enhance cell viability after treatment at lower doses (5 µM) but showed a positive effect at higher concentration. This aligns with prior research showing that 8e does not induce detectible toxicity in neuronal cells at concentration up to 100 µM after 24 h of treatment.²⁵ When comparing the Aβ_1–42 treated (disease group) with the 10 µM 6BIO and 20 µM 8e groups, significant differences were observed (41.52 and 48.91, respectively, p < 0.0001).

Based on the cell viability assay, we selected the 10 µM concentration of 6BIO and the 20 µM concentration of 8e for further treatment. Additionally, Aβ_1–42-treated cells were exposed to 10 µM 6BIO, 20 µM 8e, and a combination of both (6BIO + 8e) for 24 h. We found that SH-SY5Y cells showed improved cell viability, as demonstrated in Figure 3. These findings highlight the efficacy of both in vitro treatments, and the synergistic effect observed with the combination of 6BIO and 8e.

Figure 3.

Effect of modulator on cell-viability was evaluated using MTT assay. SHSY5Y cells were treated with 10 µM RA (Retinoic Acid), 20 µM Aβ_1–42, and 6BIO and 8e at concentrations of 5 µM, 10 µM, 15 µM, and 20 µM. The values represent a summary of three separate trials and are presented as mean ± SD. Representative images of SH-SY5Y cells treated with Aβ, 6BIO (10 µM), and 8e (20 µM) are shown. (a) illustrates the degeneration of neurons and cell clumping in Aβ-treated cells. (b–d) represents the ameliorative effect of compounds. ^####p value of less than 0.0001 was observed in the comparison of Aβ_1–42 (20 µM) treated differentiated cells with 6BIO and 8e. Compared to control groups, ***p = 0.001, and ****p < 0.0001.

Assessment of modulators on ROS production

Aβ contribute to excessive ROS production, leading to oxidative stress, neuronal damage, and cognitive decline in AD.⁴⁵ This oxidative imbalance, driven by mitochondrial dysfunction and reduced antioxidant defense, directly affects synaptic activity and neurotransmission. Importantly, lowering ROS level reduces oxidative damage, preserves neuronal integrity, and improves synaptic function, which are critical for maintaining cognitive abilities.^46–48 Clinically reduced ROS levels have been associated with neuroprotection, prevention of neuronal apoptosis, and enhanced synaptic plasticity, all of which can slow disease progression. Since high ROS level accelerate Aβ aggregation and tau hyperphosphorylation, key drivers of AD pathology, lowering ROS may help delay cognitive decline. Furthermore, antioxidant-based interventions targeting ROS reduction have shown promise in preclinical studies, supporting their potential therapeutic relevance.^47,49,50

In this study, the production of ROS in SH-SY5Y cells was measured using 2,7-dichlorodihydrofluorescein diacetate (DCFDA) labelling. This assay evaluates the cell cytosolic oxidation index. ROS production was increased by 60% after treatment with Aβ_1–42 in SH-SY5Y cells. The effects of 6BIO (10 µM), 8e (20 µM), and a combination of 6BIO (10 µM) + 8e (20 µM) on ROS were determined through flow cytometry. We observed a significant increase in ROS production after Aβ_1–42 treatment (20 µM), with a mean difference of 1149 (p = 0.005) compared to the control group. When comparing the Disease group (Aβ) with 6BIO (10 µM), a mean difference of 1238 was found (p = 0.0023). Similarly, 8e (20 µM) treatment showed a significant difference (804.8, p = 0.0078) with the Aβ_1–42 group. The combination of 6BIO and 8e also showed a mean difference with the Disease group of 861.0 (p = 0.0024) (Figure 4). The ROS production was significantly reduced after treatment with 6BIO and 8e (F (1.712, 6.850) = 41.10; p < 0.001). These findings highlight the significant reduction in ROS production with 6BIO and 8e treatment, shown in cell culture.

Figure 4.

Effect of modulator on ROS production. The production of ROS was estimated using DCF-DA flow cytometry. 6BIO, 8e, and their combination (6BIO + 8e) effectively reduced ROS in a dose-dependent manner. (a) Treatment with 6BIO (10 µM), 8e (20 µM), and the combination of 6BIO + 8e significantly decreased ROS production inSH-SY5Ycells compared to Aβ_1–42 (20 µM) treated cells. A significant difference was observed between the control and Aβ-treated cells (p = 0.005). The combination of 6BIO + 8e also showed a significant difference with Aβ (p = 0.0024). Data shown as representative of 3 experiments; values are given as mean ± SD. (b) Cells treated with Aβ_1–42 (20 µM), 6BIO (10 µM), 8e (20 µM), and the combination (6BIO + 8e).

Evaluate the effect of modulators in expression of downstream molecules of Wnt signaling

The regulation of Wnt signaling in AD was evaluated by examining the expression of specific genes and proteins. In AD, β-catenin is phosphorylated by activated GSK3β, which subsequently leads to its ubiquitination, preventing it from translocating to the nucleus. The molecules 6BIO and 8e effectively increased β-catenin levels in the cytoplasm, allowing it to move into the nucleus. Inside the nucleus, β-catenin triggered the expression of Wnt3a and further enhanced its own production. Simultaneously, it reduced the expression of the DKK1 protein through a feedback loop mechanism. Wnt3a and DKK1 compete for binding to the LRP6 receptor. In this feedback loop, as β-catenin levels rise, DKK1 expression decreases, facilitating Wnt3a's binding to LRP6 to positively regulate Wnt signaling. This positive regulation helps to reduce activated GSK3β (p216). This reduction is crucial because GSK3β is known to hyperphosphorylated tau protein, which lead to the progression of neurofibrillary tangles.

The data presented in Figure 5 demonstrated that exposure of Aβ_1–42 in differentiated SHSY5Y cells significantly increase the mRNA ratio of DKK1, GSK3β, CDK5 expression compared to control groups, with the mean difference of 7.128, 1.07, and 12.87, respectively (p < 0.005). The treatment with 6BIO (10 µM) significantly reduced CDK5 and GSK3β expression (p = 0.001) but did not significantly affect the DKK1 expression. While Wnt3a and β-catenin levels significantly decreased in the disease group (p < 0.0001); however, 6BIO treatment significantly restore expression of Wnt3a (F (4, 10) = 24.93; p < 0.005)] and β-catenin F (4, 10) = 10.84; p = 0.001].

Figure 5.

Effect of modulator on Wnt signaling pathway at mRNA level and protein level in Aβ treated SHSY5Y cells. (a–e) mRNA expression of DKK1, Wnt3a, GSK3beta, CDK5, β-catenin in all groups. (f–i) Protein expression of GSK3β (p216), β- catenin, Phosphorylated β-catenin, Phospho-Tau in Control, RA (Retinoic Acid), Aβ_1–42 (Disease group), 6BIO, 8e and combination (6BIO + 8e) treatment groups by ELISA technique. All data measured by mean ± sd and, when compared with control represents by *p < 0.05, ** p = 0.001, *** p = 0.0001. # Represent when compared with disease group (Amyloid beta), p value should be less than 0.05, ## represent p < 0.005.

The treatment with 8e (20 µM) significantly reduced the gene expression of DKK1 (p < 0.0001) but did not significantly reduce CDK5 and GSK3β level. However, 8e (20 μM) treatment increased Wnt3a (p = 0.01) and β-catenin (p = 0.05) levels compared to the disease group. Combination treatment with 6BIO and 8e (10 µM + 20 µM) effectively reduced overexpression of DKK1, GSK3β and CDK5 mRNA [F (4,10) = 8.283; p = 0.003], [F (4,10) = 7.540; p < 0.005], [F (4,10) = 7.800; p < 0.005] and significantly increase Wnt3a [F (4, 10) = 24.93; p < 0.0001] and β-catenin expression [F (4, 10) = 12.94; p = 0.0006], suggesting a synergizing effect in positively regulating the Wnt signaling pathway (Figure 5(a)–(e)). Furthermore, protein analysis revealed that β-catenin level decrease significantly in the Aβ-treated group but restored with 6BIO,8e, or in combination, with mean differences of 3.108 (p = 0.001), 2.284 (p = 0.01), and 2.519 (p < 0.005), respectively. Both 6BIO and 8e reduced phosphorylated β-catenin level (p = 0.01), while phosphorylated GSK3β (p216) was elevated in the disease group (mean difference: 0.92, p < 0.0001) and reduced significantly by 6BIO (0.7120, p = 0.01), 8e (0.4346, p = 0.02), and their combination (0.5113, p < 0.0001). Moreover, phosphorylated tau protein level was significantly elevated in the AD (mean difference: 10.06, p = 0.003), but treatment with 6BIO, 8e and their combination significantly reduced P-tau level, with mean differences of 7.580, 7.317, and 10.17, respectively [F (5,12) = 6.868; p < 0.005]. these finding indicate that 6BIO and 8e, especially in combination, effectively regulate Wnt signaling in AD by decreasing GSK3β activity, and mitigating hyperphosphorylation of tau pathology.

Assessment of hyperphosphorylated tau and apoptosis markers by confocal microscopy

SH-SY5Y cells were treated with 20 μM Aβ_1–42 to induce the AD model, followed by treatment with 6BIO, 8e, and a combination of both. After fixation and blocking, cells were incubated with anti-tau antibody and subsequently treated with Alexa Fluor^® 488-labelled goat anti-rabbit IgG secondary antibody. Hyperphosphorylation of tau protein is a key pathological hallmark of AD. In the disease condition tau protein is phosphorylated at the Ser396 amino acid residue, and its expression is significantly reduced after treatment with the combination of 6BIO (10 µM) + 8e (20 µM) [F (4, 132) = 17.61; p < 0.0001]. The mean difference between Aβ_1–42 treated and combination treatment (6BIO + 8e) was 11.9 (p < 0.0001) (Figure 6). Reducing phosphorylated tau level could have significant clinical benefits in AD and other neurodegenerative diseases. Since pTau accumulation led to neurofibrillary tangle formation, which disrupt neuronal function and accelerates cognitive decline, lowering pTau may help preserve neuronal integrity. By stabilizing microtubules and maintaining intracellular transport, reduced pTau levels could prevent synaptic dysfunction and support brain function.^51–53

Figure 6.

Effect of modulators on expression of phosphorylated tau in Aβ_1–42 treated SHSY5Y cells. (a) To assess the expression of Tau (Ser396) protein in Aβ (20 µM) treated SHSY5Y cells, followed by treatment with 6BIO (10 µM), 8e (20 µM), combination group (10 µM + 20 µM) for 24 h. Quantification of fluorescence staining was estimated by ImageJ software. Data shown are representative of 4 separate values given as mean ± SD. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 when Aβ_1–42 (20 μM) treated differentiated cells compared with three treatment group.

Furthermore, to examine the extrinsic pathway of apoptosis in AD, the Fas Ligand (Fas-L) and caspase 3 expression were analyzed using confocal microscopy. The expression Fas Ligand was found to be upregulated in disease conditions. Treatment with 8e significantly reduced Fas-L expression, with mean difference that is 12.19 (p = 0.0002) compared to the disease group (Aβ_1–42 treated). Moreover, the combination treatment of 6BIO and 8e further reduced Fas-L expression, showing a highly significant mean difference is 11.25 (p < 0.0001). Similarly, Caspase-3 expression, which was increased in the Aβ_1–42 treated group, was showed less expressed in the 8e (20 µM) treatment, mean difference 29.99 (p < 0.001). The combination treatment led to a more substantial decrease in caspase 3 expression, with a mean difference of 35.69 (p < 0.005). These findings indicate that the apoptotic activity is decreased in combination with 8e, effectively mitigating extrinsic apoptotic markers in the AD model (Figures 7 and 8).

Figure 7.

(a) Effect of modulators on Fas-L expression in Aβ_1–42 treated SHSY5Y cells. Asses the expression of the apoptotic marker FasL expression evaluate in Aβ_1–42 (20 µM) treated SHSY5Y cells, followed by treatment with 6BIO (10 µM), 8e (20 µM), combination group (10 µM + 20 µM) for 24 h. The quantitative analysis of fluorescence staining was estimated by ImageJ software. Data shown are representative of 4 separate values given as mean ± SD. #p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 when Aβ_1–42 (20 μM) treated differentiated SHSY5Ycells compared to treatment group, ns represent the non-significant difference with disease group (Amyloid beta).

Figure 8.

Effect of modulators on caspase 3 in Aβ_1–42 treated SHSY5Y cells. Assess the expression of apoptotic marker Caspase 3 protein in SHSY5Y cells which treated with Aβ (20 µM) and further treated with 6BIO (10 µM), 8e (20 µM), combination group (10 µM + 20 µM) for 24 h. The quantification of fluorescence staining was estimated by ImageJ software. Data shown are representative of 4 separate values given as mean ± SD. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 when Aβ_1–42 (2 0 μM) treated differentiated cells compared three treatment group, ns represent the non-significant difference with disease group (Amyloid beta).

Evaluate the effect of 6BIO and 8e in Aβ_1–42 induced AD model by neurobehavior parameter

Morris water test (MWT)

Initially, all Wistar rats underwent training for 3 days (−4, −3, −2) in the MWT, followed by probe trial on day 0 to establish baseline performance. there is no significant difference observed in Escape latency time among all groups on day 0 [(F (11, 60) = 1.305; p = 0.24]. Further, the probe test was performed on day 15. The groups of rat that received an ICV injection of Aβ_1–42 on day 1 showed significantly longer escape latency time(second) comparison of control and Sham group [F (11, 60) = 75.72; p < 0.0001]. Following the development of AD model, the treatment with the modulators 6BIO and 8e, either individually or in combination, was given for 14 days, consecutively (from day 15 to 28). The treatment groups showed a decrease in latency time in seconds compared to disease group [F (11, 60) = 49.27; p < 0.0001]. Notable, the group treated with 6BIO (23.8 µg/kg/day) and 8e (4.2 mg/kg/day) in combination exhibited a higher significant difference compared to disease group (p < 0.0001) (Figure 9(a)—(c)).

Figure 9.

Assessment of memory and learning behavior in male Wistar rats by Morris water maze test (a–c) and elevated plus maze (d–f). Morris Water Maze Test: (a) Baseline Reading or pre-induction model (day 0); (b) Result of the post-induction model; (c) Treatment group on day 29. (d–f) Elevated Plus Maze Test was performed for memory and learning behavior assessment in male Wistar rats. (d) Baseline Reading or pre-induction model (day 0); (e) Result of the post-induction model; (f) Treatment group, which was evaluated on day 29. Data are expressed as mean ± SD, (N = 6 rats in each group) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 when compared with control. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 compared to Aβ_1–42 (One-Way ANOVA).

Elevated plus maze test

This test was conducted to evaluate memory in rats. During the training phase, which lasted two days, the rats were conditioned to locate food in the closed arm of the maze. On day 0, after training, all groups successfully reached the food pellet within 30 s, with no significant differences observed between groups [F (11, 60) = 1.305; p = 0.25]. However, following the induction of the AD model, the disease group demonstrated a significant difference compared to the control group [F (11, 60) = 75.72; p < 0.0001]. In contrast, no significant differences were observed between the Sham and Control groups. When the disease group (Aβ_1–42 treated) was compared with the low dose of 6BIO (5.95 µg/kg/day) and the lowest dose of 8e (0.7 mg/kg/day), the differences were statistically significant (p = 0.0005, p < 0.01). However, the 11.9 µg/kg/day dose of 6BIO and the 2.1 mg/kg/day dose of 8e showed more pronounced differences compared to disease group, with mean differences of 43.16 (p < 0.001) and 36.17 (p < 0.0001) [F (11, 60) = 50.74], respectively. The highest doses of 6BIO (23.8 µg/kg/day) and 8e (4.2 mg/kg/day) exhibited significant differences compared to the disease group, with mean differences of 44.70 (p < 0.0001) and 44.72 (p < 0.0001), respectively. Notably, the combination of modulators treatment group showed a marked improvement in latency time compared to the disease group, with a mean difference of 46.17 (p < 0.0001). These results indicate that the 23.8 µg/kg/day dose of 6BIO and the 4.2 mg/kg/day dose of 8e, alone and in combination, effectively ameliorated memory deficits in the rats (Figure 9(d)–(f)).

Evaluation of the role of 6BIO and 8e on oxidative stress parameters

This study assessed oxidative stress markers, including GSH, SOD, and Catalase, across control, disease, and treatment groups (6BIO, 8e, and 6BIO + 8e). The disease group exhibited significantly reduced levels of all three markers compared to the control group (p < 0.0001). Following treatment with 6BIO and 8e, the 5.95 µg/kg/day and 11.9 µg/kg/day doses of 6BIO did not show a significant difference compared to the disease group. However, the 23.8 µg/kg/day dose significantly improved GSH levels (p = 0.0004). Similarly, the 0.7 mg/kg/day dose of 8e did not show a significant effect, but the 2.1 mg/kg/day and 4.2 mg/kg/day doses significantly increased GSH levels compared to the disease group, with mean differences of 2.70 (p = 0.0037) and 3.162 (p = 0.0005), respectively [F (11, 60) = 8.386; p < 0.0001] (Figure 10(a)). For SOD activity, the 5.95 µg/kg/day dose of 6BIO showed no significant effect, whereas the 11.9 µg/kg/day and 23.8 µg/kg/day doses significantly increased SOD activity, with mean differences of 1.0 (p = 0.0023) and 1.3 (p < 0.0001), respectively (Figure 10(b)). Likewise, the 2.1 mg/kg/day and 4.2 mg/kg/day doses of 8e significantly improved SOD activity compared to the disease group, with mean differences of 0.47 (p = 0.0006) and 1.2 (p < 0.0001), respectively [F (11, 60) = 12.19].

Figure 10.

Effect of modulators (6BIO and 8e) on the oxidative stress parameters (GSH, SOD, catalase). (a) GSH level in all groups. (b) SOD activity and (c) Catalase activity. The data expressed as Mean ± SD. (n = 6 rats in each group) Data expressed as a mean ± SD, ****p < 0.0001 when compared to control, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 when compared Aβ_1–42 (Amyloid beta).

Catalase activity significantly improved with the 4.2 mg/kg/day dose of 8e, while 6BIO showed no notable changes at low or medium doses (Figure 10(c)). However, the 23.8 µg/kg/day dose of 6BIO demonstrated a significant improvement in catalase activity (p = 0.006). Moreover, the combination treatment (6BIO 23.8 µg/kg/day + 8e 4.2 mg/kg/day) significantly enhanced catalase activity, with a mean difference of 2.547 (p = 0.0002) compared to the disease group [F (11, 60) = 10.87; p < 0.0001]. These findings underscore the efficacy of the combination treatment in significantly improving oxidative stress markers in vivo.

Evaluate the effect of 6BIO and 8e on downstream molecules of Wnt signaling through expression of gene and protein

The Wnt signaling pathway is negatively regulated by DKK1 and GSK3β proteins in AD, which leads to β-catenin degradation and an increase in phosphorylated tau protein expression. This study analyzed the effects of modulators on gene and protein expression in in vivo models using RT-PCR, ELISA, and Western blot techniques. The mRNA expression of the DKK1 gene was significantly higher in the disease group compared to the control group (p < 0.0001). Treatment with 6BIO showed no significant effect, whereas 8e at doses of 2.1 mg/kg (p < 0.001) and 4.2 mg/kg (p < 0.0001) significantly reduced DKK1 expression. The combination treatment (6BIO + 8e) demonstrated the most substantial reduction in DKK1 expression [F (11, 36) = 12.16; p < 0.0001] (Figure 11(a)).

Figure 11.

Effect of modulator on Wnt signaling pathway at mRNA and protein level in AD model. (a–e) mRNA expression of DKK1, Wnt3a, GSK3beta, CDK5, β- catenin in all groups. (f–i) Protein expression of GSK3β, GSK3β (p216), β catenin, Phospho-Tau in all groups by ELISA technique. (j–m) Protein expression of β-catenin, Phospho-Tau, GSK3β (p216) by western blot. (n = 6 rats in each group) All data expressed as mean ± sd. When compared to control group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 when compared with disease group (Amyloid beta).

The Wnt3a gene expression also notable increased in combination and individual treatments groups [F (11, 36) = 24.07; p < 0.0001]. The expression of GSK3β mRNA was increased in disease group as compared to control and sham group (p < 0.0001); however, all three dose of 6BIO reduced the expression of GSK3β (5.95 μg/kg (p < 0.001), 11.9 μg/kg (p = 0.0002), and 23.8 μg/kg (p < 0.0001) [F (11, 36) = 18.65]. Conversely, treatment with 8e showed no significant effect. the combination treatment group were significantly able to reduce the expression of GSK3β with mean difference 5.8 (p < 0.0001). Similarly, protein expression of GSK3β and phosphorylated GSK3β (p216) was reduced in combination treated rats as compared to disease group (F (11, 60) = 86.39; p < 0.001], [F (11, 60) = 26.13], respectively. The cdk5 expression was decrease in 6BIO treated group but no significant difference was found in 8e treated groups. The β-catenin (CTNNB1) gene expression was significantly reduced in the disease (Aβ_1–42) rat group compared to the control group. The higher dose of 6BIO (23.8 μg/kg) improved β-catenin expression (p < 0.01), while 8e treatment at 4.2 mg/kg also showed improvement compared to the Aβ_1–42 group (p < 0.01). The combination treatment (6BIO + 8e) further increased β-catenin expression (p = 0.007). Similarly, protein concentration of β-catenin in the combination treatment group significantly increased compared to the disease group (p < 0.0001) [F (11, 60) = 14.29]. The phosphorylated tau protein concentration was elevated in the Aβ_1–42 group compared to the control. The higher doses of 6BIO (23 μg/kg) and 8e (4.2 mg/kg), as well as their combination, significantly reduced tau expression [F (11, 60) = 14.29; p < 0.0001] (Figure 11(i) and (m)). collectively, these results indicate 6BIO does not reduce DKK1 level. Whereas 8e effectively decreases it. Conversely, 6BIO reduces the GSK3β level and CDK5 but 8e does not. Additionally, β-catenin level is significantly increased by either modulator treatment.

These results provide evidence that Wnt signaling is downregulated in AD, and the combination treatment of 6BIO and 8e effectively reduces DKK1, GSK3β, and phosphorylated tau while enhancing β-catenin expression, suggesting a synergistic therapeutic effect.

Assessment of histopathological changes in hippocampus by H&E staining in hippocampus

The hippocampus region usually affected in AD condition. In this study, cut the hippocampus section and further stained it with H&E staining to examine the morphological changes. The stained section of the rat hippocampus showing the layer of dentate gyrus with outer molecular layer, middle granular cells layer, and inner polymorphic layer. The neuronal damage was observed in Aβ_1–42 and DMSO (vehicle) group at 20x microscopic examination. Histopathological neuronal scores were used to assess the extent of injured and apoptotic neuron in the hippocampus region of the brain. A score of 0 was assigned for no evidence of neuronal injury or the presence of only occasional apoptotic neurons. A score of 1 indicated lesser than 5 clusters of apoptosis neuron, 2 represented the 5 to 15 clusters, 3 denoted more than 15 clusters, and 4 represent the diffuse neuronal injury.³⁵

In the Amyloid beta (diseased) group, shrinkage and darkening of pyramidal cells (indicative of apoptosis), disorganization, vacuolation (V), and a decreased population of granular cells were observed. Additionally, pycnotic cells were prominent in the Aβ_1–42 group. In contrast, the control and sham groups exhibited an intact granule cell layer of the dentate gyrus with oval-shaped, larger pyramidal cells. Treatment with three different doses of 6BIO and 8e, as well as their combination (6BIO + 8e), showed reduced neuronal damage, particularly at the 23.8 µg/kg/day dose of 6BIO and the 4.2 mg/kg/day dose of 8e (p < 0.001) in the dentate gyrus region of the hippocampus. The combination treatment (6BIO + 8e) demonstrated significant amelioration of neuronal damage compared to the disease group (p < 0.001). These results suggest that the modulators effectively reduce neuronal damage in the dentate gyrus region of the hippocampus [F (11, 24) = 13.12, p < 0.0001] (Figure 12).

Figure 12.

Assessment of modulator on histopathology of hippocampus in AD. (a) Coronal section of hippocampus (dentate gyrus) stained by H&E (20 x). H&E-stained section of the rat hippocampus showing the layer of dentate gyrus with outer molecular layer, middle granular layer and inner polymorphic layer. In the disease group, disorganization, vacuolation (V) and decreased population of granular cells are showing. (b) Score of damage in hippocampus. The data is represented as a mean ± SD. (n = 6 rats in each group) When compared to control, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 when compared with disease group (Amyloid beta).

Assessment of modulators on the apoptotic parameter (Fas-L and Caspase-3) by immunohistochemistry

Neuronal damage in AD is closely associated with the upregulation of apoptotic markers. Previous studies have demonstrated that Aβ_1–42 deposition increases the expression of Fas ligand (Fas-L) and Caspase-3, which are key mediators of apoptosis. Fas-L is located on the cell membrane, while Caspase-3 resides in the cytoplasm and further amplifies the apoptotic cascade.

In this study, the control group exhibited low expression levels of Fas-L and Caspase-3 in the hippocampal region, whereas the disease group showed significantly elevated levels of these markers (p < 0.0001). Treatment with 6BIO (23.8 µg/kg) and 8e (2.1 mg/kg) significantly reduced the expression of Fas-L and Caspase-3 compared to the disease group [F (11, 36) = 70.66; p < 0.001] and [F (11, 24) = 32.34; p < 0.001], respectively. However, lower doses of both modulators did not result in significant reductions in the expression of these apoptotic markers.

Notably, 6BIO (23.8 µg/kg/day) and 8e (4.2 mg/kg/day) alone and in combination demonstrated a significant ameliorative effect on the hippocampal region by markedly decreasing Fas-L and Caspase-3 levels. These findings suggest that combination therapy is particularly effective in mitigating apoptosis in AD by reducing the expression of these apoptotic markers (Figures 13 and 14).

Figure 13.

Effect of modulator on expression of Fas ligand (Fas-L) in hippocampus region was assessed by immunohistochemistry. Representative image showing brain hippocampus section immunostaining against apoptotic marker: Fas-L (magnification at 20X). (a) Aβ_1–42-treated group were shown significant expression of Fas ligand. the effect of modulators with different concentration was evaluated on Fas-L expression. (b) Quantification of Fas-L expression in hippocampus region. The data represented as a mean ± SD. (n = 6 rats in each group). Comparison to control group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 when compared with disease group (Amyloid beta).

Figure 14.

Effect of modulator on expression of Caspase 3 in hippocampus region evaluated by immunohistochemistry. Representative image showing brain hippocampus section immunostaining against apoptotic marker: Caspase 3 (magnification at 20X). (a) Aβ_1–42⁻treated group were shown significant expression of caspase 3. All treatment groups were shown ameliorative effect (b), quantification of caspase 3 expression in hippocampus region. The data represented that as a mean ± SD. (n = 6 rats in each group) When compared with control, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 when compared with disease group (Amyloid beta).

Discussion

This study aims to explore the canonical Wnt signaling pathway and identify potential targets within the pathway that serve as promising therapeutic approaches in AD. Modulating Wnt signaling with specific inhibitors may offer neuroprotection against Aβ induced neuronal damage and death.⁵⁴ AD is a major neurodegenerative disorder with limited treatment options, underscoring the need for novel therapeutic strategies.

Our study presents a unique dual-targeting strategy for restoring Wnt signaling in AD, combining intracellular GSK3β inhibition by 6BIO with extracellular blockade of DKK1–LRP6 interaction by 8e, a novel gallocyanine derivative molecules. This combination treatment strategy represents the first attempt to comprehensively address Wnt signaling dysregulation in AD by simultaneously targeting both intracellular and extracellular regulatory mechanisms. While previous studies have explored these mechanisms separately,^18–20 our study is the first to integrate both approaches, addressing a critical gap in Wnt pathway modulation. Our findings show that these modulators restore Wnt signaling, reduce oxidative stress, and mitigate neuronal damage, supporting their therapeutic potential.

Wnt signaling has been linked to early embryonic development, tissue homeostasis, and various disorders such as cancer, bone disease, cardiovascular disease, and neurodegenerative diseases.^55,56 In the nervous system, Wnt signaling plays a crucial role, from early neuronal patterning to supporting synaptic plasticity and memory function in the adult brain. Emerging evidence suggests that Aβ_1–42 disrupts Wnt signaling, weakening this vital cascade at the synapse and contributing to the progression of AD.⁵⁷ Parr et al. (2015) demonstrated that Wnt/β-catenin activation represses BACE1 transcription, reducing Aβ production and neurodegeneration.^56,58 Additionally, Wnt signaling regulates multiple neurodegenerative pathways, enhancing neurogenesis through Notch, supporting dopaminergic neurons via SHH, modulating neuroinflammation through TGF-β and NF-κB, and interacting with mTOR to regulate autophagy—key for clearing toxic aggregates in AD and Parkinson's disease.^15,59–61

To restore the Wnt signaling pathway in AD model, the current study explored two modulators: 6BIO and 8e. 8e (DKK1/LRP6 interaction inhibitor), is a derivative of NCI8642 (gallocyanine hydrochloride salt), a synthetic blue dye which belongs to the Phenoxazinone class, first synthesized by Otto in 1888. Phenoxazines have demonstrated as anticancer, antiviral, antimicrobial properties. Importantly, also been reported to inhibit heparin induced tau protein assembly, highlighting their potential relevance in neurodegenerative disease.^25,62,63 6BIO (6bromo- indirubin −3 oxime), is a semi-synthetic derivative of indirubins, naturally found in edible mollusks and plants. It is a potent inhibitor of GSK3β protein. 6BIO has demonstrated anti-tumor and neuroprotective properties, making it a promising therapeutic candidate.²¹ While this study provides valuable insights, a key limitation is the lack of experimental pharmacokinetic analysis. However, in silico pharmacokinetics predictions were performed for both 6BIO⁴¹ and 8e²⁵ modulator.

Our in vitro results provide significant insight into the neuroprotective effect of 6BIO and 8e on Aβ_1–42-induced neurotoxicity in SH-SY5Y cells which could lead to development of potential neuroprotective strategies for AD.^64,65 The choice of 20 µM Aβ_1–42 for AD induction was based on prior findings that this concentration induces significant neuronal degeneration in these cells.⁶⁶ Considering the well-established cytotoxicity effects of Aβ_1–42, the observed protective effects of 6BIO and 8e highlight their potential as neuroprotective agents. Consistent with previous reports, in our study, 6BIO exhibited no significant cytotoxicity (Martin et al.), while 8e remained non-toxic at lower concentrations (Thysiadis et al.).^20,25,44 These findings led to investigating the antioxidant capacity of these modulators.

Previous studies have shown that increased ROS generation plays a crucial role in the pathophysiology of AD by mitochondrial dysfunction and neuroinflammation, contributing to neuronal damage and progression of neurodegenerative processes.^67–70 Notably, 6BIO has been shown to reduce oxidative stress by activating antioxidant responses and modulating pathways such as GSK3β and mTOR, which help alleviate oxidative damage.^21,71,72 Additionally, 6BIO induces autophagy, which further reduces ROS accumulation and mitigates cellular stress, making it a promising candidate for treating neurodegenerative diseases.²² In agreement with these reports, our in vitro AD model exhibited elevated ROS production, that were effectively reduced by 6BIO and 8e treatment, suggesting their potential antioxidant properties in mitigating Aβ_1–42 induced oxidative stress. Likewise, we observed increased oxidative stress in the hippocampus region of AD rat brain corroborating with the a previous study.⁷³ Strikingly, both modulators, 6BIO (23.8 µg/kg/day) and 8e (4.2 mg/kg/day) effectively restored the activity of GSH, SOD, and catalase, improving oxidative stress levels. The reduction in ROS and oxidative stress levels observed in our study could thus be attributed to the modulation of key cellular mechanisms, including the activation of antioxidant defense pathways and the inhibition of ROS-producing enzymes. These findings suggest that both 6BIO and the 8e modulators could serve as potential therapeutic agents for AD by reducing oxidative stress.

To explore the neuroprotective effects of 6BIO and 8e, we examined key proteins involved in the canonical Wnt signaling pathway both in vitro and in vivo, as this pathway plays a vital role in cellular plasticity and neurogenesis. Previous studies have reported that Wnt3a expression is reduced in AD, while levels of pGSK3β, DKK1, CDK5, p-β-catenin, and p-tau are elevated.^74–77 Additionally, overactivation of the AKT/GSK3β pathway has been also linked to tau hyperphosphorylation.⁷⁸ Notably, DKK1 infusion into the amygdala or hippocampus has been shown to impair memory function.^43,79 Consistent with these findings, Our comprehensive analysis of Wnt signaling pathway at the gene and protein levels revealed that the combination treatment with 6BIO and 8e significantly downregulated DKK1, pGSK3β, and CDK5, while upregulating Wnt3a and β-catenin levels compared to the Aβ-treated group.^80–84 Interestingly, 6BIO alone did not reduce DKK1 expression, indicating a synergistic effect when combined with 8e. Similarly, 8e alone was ineffective in reducing CDK5 expression, but the combination therapy successfully downregulated it. These results suggest that co-targeting GSK3β and DKK1 with 6BIO and 8e can synergistically activate Wnt signaling, offering a promising therapeutic strategy for AD.

Furthermore, we assessed the apoptotic activity induced by Aβ_1–42, a well-known trigger of apoptosis in AD models, through the activation of key apoptotic proteins such as Fas-L and Caspase 3.⁸⁵ Apoptotic pathways are fundamental in Aβ_1–42-induced neurodegeneration, contributing to neuronal cell death and cognitive decline.⁸⁶ As anticipated, treatment with combination resulted in a decline the apoptotic activity that was confirmed by reduced expression of key apoptotic markers Fas-L and Caspase 3. These results were supported by histopathological findings from our in vivo model. These findings suggests that both 6BIO and 8e may offer neuroprotective effects by inhibiting apoptotic cell death in AD.

The involvement of Wnt signaling in adult memory processes has been well established.⁸⁷ Several studies have highlighted its role in different aspects of memory regulation, particularly in the consolidation of fear memory and information storage within the amygdala and hippocampus.⁸⁸ In the present study, we investigated whether combination therapy of 6BIO and 8e could enhance cognitive recovery in Aβ-treated rats by evaluating their learning and memory performance using the Morris water maze and elevated plus maze tests.⁸⁹

Our results demonstrated that treatment with 6BIO and 8e, either individually or in combination, significantly mitigated cognitive deficits and improved memory function. This improvement is likely due to the modulators’ ability to activate Wnt signaling, which supports neurogenesis and reduces tau hyperphosphorylation, both of which are central to AD pathology.

Overall, these findings highlight the therapeutic potential of 6BIO and 8e as neuroprotective agents capable of alleviating key neurodegenerative processes in AD (Figure 15). Their effects extend beyond modulating Wnt signaling and reducing tau pathology—they also play a role in suppressing apoptotic pathways. Together, these modulators may offer a promising strategy to preserve neuronal integrity and cognitive function in AD. Further clinical studies are necessary to assess their therapeutic efficacy and determine their potential for treating AD. However, to advance toward clinical use, further rigorous investigation involving the bioavailability and pharmacokinetic profiling is required.

Figure 15.

A comprehensive graphical representation of the effect of 6BIO and 8e treatment observed across in in silico, in vitro, and in vivo study.

Conclusion

The present study offers a comprehensive evaluation of the effects of the DKK1-LRP6 inhibitor (8e), both as a standalone treatment and in combination with 6BIO. This combined approach demonstrated significant cytoprotective, anti-apoptotic, and antioxidant effects in both in-vitro and in-vivo models. Histopathological analysis indicated that both modulators were able to mitigate neuronal damage and modulate key Wnt signaling proteins, including Wnt3a, GSK3β, p-Tau, and β-catenin, highlighting their neuroprotective potential. Overall, 6BIO and 8e work synergistically to regulate the Wnt/β-catenin pathway, which could lead to promising therapeutic outcomes. However, clinical studies are required to evaluate their therapeutic efficacy and explore their potential for treating AD.

Supplemental Material

sj-docx-1-alz-10.1177_13872877251362787 - Supplemental material for Therapeutic potential of 6BIO and DKK1-LRP6 inhibitor in Wnt/β-catenin pathway modulation for amyloid-β-induced Alzheimer's disease model

Supplemental material, sj-docx-1-alz-10.1177_13872877251362787 for Therapeutic potential of 6BIO and DKK1-LRP6 inhibitor in Wnt/β-catenin pathway modulation for amyloid-β-induced Alzheimer's disease model by Manisha Prajapat, Phulen Sarma, GurjeetKaur, GajendraChoudhary, Shaveta Jain, Raj Kamal, Omkar Bains, Namrata Sangwan, Ajay Prakash and Bikash Medhi in Journal of Alzheimer's Disease

Footnotes

Acknowledgments

The authors are thankful to PGIMER, Chandigarh. The authors would like to express they’re thanks to CSIR-UGC institute for providing fellowship for pursuing PhD.

ORCID iDs

Gajendra Choudhary

Bikash Medhi

Ethical considerations

The study was initiated following approval of the protocol by the institute's animal ethics committee (IAEC) ref. No./108/97/IAEC/676, institutional bio safety committee (IBC) clearance ref.No.000591/IBC/2018. The rats were housed in a standard facility, fed with rat pellet diet and water ad libitum, and cared for by trained animal house personnel. They were sacrificed with thiopentone sodium and all chemicals were handled with care. The facilities for the procedures mentioned are available in various departments at PGIMER, Chandigarh.

Author contributions

Manisha Prajapat: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Validation; Visualization; Writing – original draft.

Phulen Sarma: Methodology; Resources; Visualization.

Gurjeet Kaur: Methodology; Supervision; Writing – review & editing.

Gajendra Choudhary: Formal analysis; Resources; Visualization.

Shaveta Jain: Data curation; Resources; Validation.

Raj Kamal: Methodology; Resources.

Omkar Bains: Methodology; Resources.

Namrata Sangwan: Data curation; Formal analysis; Writing – review & editing.

Ajay Prakash: Investigation; Project administration; Supervision; Visualization.

Bikash Medhi: Formal analysis; Funding acquisition; Investigation; Project administration.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statement

The corresponding author can provide the datasets generated and/or analyzed during the current study upon a reasonable request.

Supplemental material

Supplemental material for this article is available online.

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Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

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Therapeutic potential of 6BIO and DKK1-LRP6 inhibitor in Wnt/β-catenin pathway modulation for amyloid-β-induced Alzheimer's disease model

Abstract

Background

Objective

Methods

Results

Conclusions

Keywords

Introduction

Methods

In silico analysis

Chemicals and drug preparation

In vitro study

AD induction of AD model in neuroblastoma cells (SH-SY5Y) and treatment

MTT assay for cell viability

Reactive oxygen species analysis using flow cytometry

Real time PCR

ELISA (enzyme linked immunosorbent assay)

Confocal microscopy

In vivo study

Experimental design and treatment strategy

Aβ1–42 induced AD model in rats

Neurobehavior parameter study

Morris water test (MWT)

Elevated plus maze test (EPM)

Assessment of biochemical parameters and molecular estimation in brain tissue

Estimation of glutathione concentration

Estimation of superoxide dismutase (SOD)

Estimation of catalase activity

Real time PCR

ELISA

Western blot

Brain morphology detection

Immunohistochemistry for apoptotic marker: Fas-L and Caspase 3

Statistical analysis

Results

In silico results

Assessment of modulators by cytotoxicity in SHSY5Y cells

Assessment of modulators on ROS production

Evaluate the effect of modulators in expression of downstream molecules of Wnt signaling

Assessment of hyperphosphorylated tau and apoptosis markers by confocal microscopy

Evaluate the effect of 6BIO and 8e in Aβ1–42 induced AD model by neurobehavior parameter

Morris water test (MWT)

Elevated plus maze test

Evaluation of the role of 6BIO and 8e on oxidative stress parameters

Evaluate the effect of 6BIO and 8e on downstream molecules of Wnt signaling through expression of gene and protein

Assessment of histopathological changes in hippocampus by H&E staining in hippocampus

Assessment of modulators on the apoptotic parameter (Fas-L and Caspase-3) by immunohistochemistry

Discussion

Conclusion

Supplemental Material

sj-docx-1-alz-10.1177_13872877251362787 - Supplemental material for Therapeutic potential of 6BIO and DKK1-LRP6 inhibitor in Wnt/β-catenin pathway modulation for amyloid-β-induced Alzheimer's disease model

Footnotes

Acknowledgments

ORCID iDs

Ethical considerations

Author contributions

Funding

Declaration of conflicting interests

Data availability statement

Supplemental material

References

Supplementary Material

Evaluate the effect of 6BIO and 8e in Aβ_1–42 induced AD model by neurobehavior parameter