Modulatory Effect of Natural Antioxidants on Tissue Transglutaminase Levels in Olfactory Ensheathing Cells Exposed to Amyloid‐β: Integrated Biochemical and Computational Analysis
Rosalia Pellitteri, Cristina Tomasella, Maria Assunta Chiacchio, Matteo Pappalardo, Laura Legnani, Michela Spatuzza, Alessandro Massaro, Salvatore Guccione, Agatina Campisi
TL;DR
This study shows that natural antioxidants like berberine and curcumin can reduce harmful effects of amyloid-beta in brain cells, offering potential for treating Alzheimer's disease.
Contribution
The study integrates computational and biological methods to identify berberine and curcumin as promising neuroprotective agents.
Findings
Berberine and curcumin counteract Aβ-induced upregulation of TG2 in OECs.
Antioxidant pretreatment restores cell viability and normalizes cytoskeleton markers.
These compounds reduce ROS and prevent apoptosis in Aβ-exposed cells.
Abstract
Herein, we adopted a dual approach combining molecular modeling and biological studies, in order to assess the interaction between four selected natural antioxidants (NAs; berberine, curcumin, astaxanthin, indicaxanthin) and tissue transglutaminase (TG2) levels both in the absence and in the presence of full native peptide of amyloid‐β (Aβ). Docking studies were performed to ascertain the binding affinity of NAs against the TG2 closed, Ca2+‐bound closed, and open forms. In the biological investigation, the effect of berberine and curcumin treatment on TG2 in Olfactory Ensheathing Cells (OECs) exposed to Aβ(1–42) or to Aβ(25–35), a Aβ toxic fragment, or to reverse‐sequence fragment Aβ(35–25), an Aβ not toxic fragment, was tested. In addition, their effect on the percentage of cell viability and cytoskeleton marker (GFAP, vimentin and nestin) levels were evaluated. The role of berberine…
Genes, proteins, chemicals, diseases, species, mutations and cell lines named across the full text — each resolved to its canonical identifier and authoritative record.
Click any figure to enlarge with its caption.
FIGURE 1
FIGURE 2
FIGURE 3
FIGURE 4
FIGURE 5
FIGURE 6
FIGURE 7
FIGURE 8
FIGURE 9
FIGURE 10
FIGURE 11
FIGURE 12| Berberine (1) score | Curcumin (2) score | Indicaxanthin (3) score | Astaxanthin (4) score | |
|---|---|---|---|---|
| TG2 “Closed conformation” | 53.50 | 76.99 | 58.31 | 47.89 |
| TG2 “Closed conformation with calcium” | 53.05 | 66.60 | 48.31 | 47.29 |
| TG2 “Open conformation” | 41.74 | 53.40 | 70.30 | 32.16 |
| Treatment | nmoles of dichlorofluoresceine produced per mg protein/30 min |
|---|---|
| Control: | 5.95 +/− 1.04 |
| +10 mM Aβ(1–42) | 17.22 +/− 1.02 |
| +10 mM Aβ(25–35) | 19.29 +/− 0.99 |
| +10 mM Aβ(35–25) | 6.19 +/− 1.09 |
| +10 mM Berberine | 5.3 +/− 0.88 |
| +10 mM Berberine + 10 mM Aβ(1–42) | 8.79 +/− 0.56 |
| +10 mM Berberine + 10 mM Aβ(25–35) | 9.39 +/− 0.97 |
| +10 mM Berberine + 10 mM Aβ(35–25) | 6.01 +/− 0.81 |
| +10 mM Curcumin | 4.9 +/− 0.78 |
| +10 mM Curcumin + 10 mM Aβ(1–42) | 6.01 +/− 0.89 |
| +10 mM Curcumin + 10 mM Aβ(25–35) | 7.22 +/− 1.03 |
| +10 mM Curcumin + 10 mM Aβ(35–25) | 5.21 +/− 0.94 |
- —PIA.CE. RI 2020‐2022
Peer Reviews
No public reviews on file for this paper yet. If you reviewed it on a platform where reviews are public (OpenReview, ICLR, NeurIPS, ICML), you can paste yours below so the community can read it here.
Videos
No videos yet. Explain this paper in a talk, walkthrough, or lecture? Add one.
Taxonomy
TopicsBlood properties and coagulation · Skin and Cellular Biology Research · Nerve injury and regeneration
Introduction
1
Alzheimer's Disease (AD) is a neurodegenerative disease characterized by progressive cognitive impairment associated with reduced activity of daily life. Numerous evidence indicates that oxidative stress is responsible for brain dysregulation due to amyloid‐beta (Aβ) toxicity, which contributes to the key events involved in AD [1]. In particular, Aβ induces an increase in the levels of both intracellular calcium ions (Ca^2+^) and reactive oxygen species (ROS), mitochondrial dysfunctions and ultimately neuronal death.
Remarkably, Aβ is also a substrate of tissue transglutaminase (TG2), a ubiquitous Ca^2+^‐dependent protein that belongs to enzyme families which catalyze cross‐linking reactions responsible for the formation of oligomers and Aβ aggregates. TG2 structure shows four functional domains that are modulated differently depending on GTP/GDP or Ca^2+^ intracellular levels [2]. TG2, an injury‐responsive protein, also changes the functions depending on many factors, including its conformation and cellular localization. In particular, it presents two conformational states: “closed” and “open”, depending on GTP/GDP and/or Ca^2+^ level concentration. When TG2 is bound to GTP/GDP, it adopts a “closed” conformation catalytically inactive, without transamidating activity, while when the Ca^2+^ levels increase the protein changes its conformation becoming catalytically active (TG2‐Long, TG2‐L). In addition, the further enhancement of Ca^2+^ levels is responsible for TG2 β‐barrel domain loss, assuming a short conformation, TG2‐S [3]. Furthermore, the protein functions change based on its localization: when TG2 is localized in the nucleus, it exerts a protective effect against cell damage and apoptosis (TG2‐L); when it is localized in the cytosol and mitochondria, it possesses transamidating activity and is involved in apoptosis (TG2‐Short, TG2‐S) [3].
It has been reported that several inhibitors of TG2 overexpression are able to maintain the inactive closed form of TG2. Interestingly, several findings report the protective effect of natural antioxidants (NAs) for the treatment of several neurodegenerative diseases, including AD [4, 5, 6]. Accordingly, their regular dietary intake could contribute to the maintenance of human health and the prevention of degenerative conditions. However, the interactions between NAs and TG2 have not been clarified [7].
In previous studies, we demonstrated that TG2 was overexpressed in Olfactory Ensheathing Cells (OECs) exposed to Aβ(1–42) and Aβ(25–35) that some growth factors were able to reduce its expression levels [8]. In addition, we found that the pre‐treatment of OECs exposed to Aβ isoforms with either indicaxanthin, a phytochemical produced by Opuntia ficus‐indica fruit, or astaxanthin, a carotenoid from microalga Haematococcus pluvialis , exerted a remarkable protective effect by reducing the Aβ‐induced TG2 upregulation [9, 10]. In particular, OECs represent a glial population of the olfactory system involved in several neurodegenerative diseases, including AD [11]. These cells exhibit morphological characteristics of both Schwann cells and astrocytes and are capable of producing various growth factors [12, 13] and express a wide range of specific markers [14]. In addition, OECs can promote continuous self‐renewal also supporting axonal regeneration following nervous system injury [15, 16].
Within this scenario, the aim of this study was to elucidate the interactions between NAs and TG2, either in the absence or presence of Aβ. To address these questions, we adopted a dual approach combining molecular modeling and biological studies to assess the effects of NAs on TG2 expression and on its localization in OECs exposed to Aβ(1–42), its toxic fragment Aβ(25–35), and the non‐toxic reverse fragment Aβ(35–25). Docking studies were performed with berberine, curcumin, indicaxanthin and astaxanthin (Figure 1) against the closed, Ca^2+^‐bound closed, and open forms of TG2 using GOLD 2025.1.0, a high‐accuracy protein–ligand docking software based on a genetic algorithm [17]. These in silico analyses provide insights into domain‐specific binding affinities, ligand‐contact residues, and conformational preferences. Computational findings were then integrated with experimental data to explore how specific interactions between ligand and TG2 may influence enzyme conformation and downstream cellular responses under Aβ toxicity. The biological approach allowed us to evaluate TG2 levels and its localization, specific markers of both glial reactivity and neural progenitor state (vimentin, GFAP, nestin), intracellular ROS, cell viability and apoptotic pathway activation, focusing on the modulatory effects of berberine and curcumin. In particular, we used MTT test in order to monitor the percentage of cell viability following the different treatments, immunocytochemical analysis to detect TG2 localization and expression and specific marker levels (vimentin, GFAP, nestin). ROS levels production was evaluated to assess the oxidative stress status. In addition, the apoptotic pathway activation was studied through immunocytochemical analysis testing caspase‐3 cleavage.
Selected NAs: Berberine 1, Curcumin 2, Indicaxanthin 3, and Astaxanthin 4.
Experimental Procedures
2
Modeling Studies
2.1
Ligands were built and minimized using Flare, version V10.0.1. Crystal structures of TG2, in the “closed” state with (pdb code: 6KZB)/without Ca^2+^ complexed (pdb code: 4pyg) or in the “open” state co‐crystallized with gluten‐peptide mimic or other similar inhibitors (pdb code: 3S3J), were retrieved from the Protein Data Bank (PDB). The binding site was defined through the co‐crystallized structure of the enzyme with GTP (closed conformation of TG2) and a L‐Peptide linking (“open” conformation of TG2). The retrieved protein structure was carefully checked and refined. Missing hydrogens or residues were added to the structure, and the obtained protein was geometrically refined through minimization using the XED force field as implemented in Flare version V10.0.1. The possible protonation states of ionizable residues were established to avoid the lack of electrostatic interactions during docking [18, 19, 20].
Docking of the minimized conformers of compounds 1–4 into the binding site of TG2 identified as above reported was performed using the software GOLD version 2025.1.0 with default values [17].
The binding residues were treated as flexible, and their side chains were rotated in 10° increments and scanned over 360°. Two docking scoring functions were used, namely CHEMPLP fitness (for docking), which is an empirical fitness function optimized for pose prediction, and ChemScore (for rescoring). This latter scoring function takes into account hydrophobic‐hydrophobic contacts, hydrogen bonding, and ligand flexibility by incorporating ΔG°, protein‐ligand atom clash term, and an internal energy term. The scoring functions were used both with default parameters. For each of the 10 GA runs, a number of 100.000 GA operations were performed on a set of five groups with a population size of 100 individuals; crossover, mutation, and migration were set to 95, 95, and 10, respectively; default cutoff values of 2.5 Å for hydrogen bonds and 4.0 Å for van der Waals distance.
Biological Studies
2.2
OEC Cultures
2.2.1
Experiments were carried out on 2‐day‐old mouse pups P2; Envigo RMS s.r.l. (Italy) in compliance with the Italian law on animal care no. 116/1992 and no. 26/2014 and in accordance with the European Community Council Directive (86/609/EEC) and were approved (authorization no. 174/2017‐PR) by the Ethical Committee at the University of Catania (Italy).
Olfactory bulbs were removed from pups and digested in medium, containing collagenase and trypsin mixture. Subsequently, trypsinization was stopped by adding DMEM supplemented with 10% FBS. Cells were re‐suspended and plated in flasks, fed with medium. In order to reduce the number of dividing fibroblasts, cytosine arabinoside, an antimitotic agent, was added 24 h after initial plating. When OECs were confluent, they were detached by trypsinization, and some were plated on 14 mm diameter glass coverslips for immunocytochemical analysis and others on 96‐multi‐wells flat bottomed for MTT test and incubated at 37°C in a humidified 5%–95% CO_2_ air mixture [21].
Treatment of OECs
2.2.2
In preliminary experiments, to establish the optimal concentration of ANs, OECs were treated with different concentrations of berberine or curcumin (0.5, 1.0, 10, 20, 40 μM) for 24 h. Once the optimal concentration of each ANs was chosen, the cell cultures were divided in four different groups: (1) cultures were incubated only with the medium or with the corresponding volume of DMSO (final concentration 0.01% v/v) used to solubilize Aβ peptides (control cultures, CTR); (2) cultures were exposed for 24 h to 10 μM Aβ(1–42) or Aβ(25–35) or Aβ(35–25), as reported in our previous work [10]. (3) cultures were treated with 10 μM of berberine (cod. 14,050, 90% purity, Sigma‐Aldrich, Milan, Italy) or 10 μM of curcumin solubilized in DMSO (cod. C‐138, 99% purity, Sigma‐Aldrich, Milan, Italy) for 30 min; (4) OEC cultures pretreated with berberine or curcumin for 30 min were exposed to Aβ(1–42), or its fragments Aβ(25–35) or Aβ(35–25) for 24 h.
MTT Test
2.2.3
Cellular viability was evaluated for each condition by 3‐[(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl] tetrazolium bromide (MTT) reduction assay [10] on OECs grown in 96‐multi‐wells plates. Briefly, MTT (1.0 mg/mL) was added to each well for 2 h in the incubator, then the supernatant was gently removed and acid‐isopropanol/SDS (MTT solvent) was added; the cells were shaken for 10 min. A microplate spectrophotometer reader (Titertek Multiskan; Flow Laboratories, Helsinki, Finland) at λ = 570 nm was used to evaluate the optical density for each sample well. For each experimental condition, four replicates were carried out, and data were expressed as the percentage MTT absorbance reduction, when compared with control cells.
Immunocytochemistry
2.2.4
Immunocytochemical techniques were used to detect the expression of some antibodies in OECs grown in all conditions. Cells were fixed by 4% PFA (0.1 M PBS) for 30 min, washed in PBS and incubated for 30 min at room temperature with 5% normal goat serum in PBS containing 0.1% Triton X‐100 to permeabilize the cell membranes. Afterwards cells were incubated overnight at 4°C with the following primary antibodies: mouse monoclonal antibody against TG2 (1:200; Neomarkers), mouse monoclonal antibody against caspase‐3 (1:500; Becton‐Dickinson (Milan, Italy)), mouse monoclonal antibody against GFAP (1:1000; DAKO), mouse monoclonal antibody against vimentin (1:50; DAKO), mouse monoclonal antibody against nestin (1:200; Abcam, Milan, Italy). Then, after washing with PBS, OECs were incubated with following secondary antibodies conjugated to different fluorochromes: FITC anti‐mouse (1:200; Jackson ImmunoResearch, Laboratories Inc) to detect caspase‐3 and GFAP, and Cy3 anti‐mouse (1:500; Jackson ImmunoResearch, Laboratories Inc) to detect TG2, vimentin and nestin for 1 h at room temperature and in dark conditions. Subsequently, the coverslips were analyzed on a Zeiss fluorescent microscope (Zeiss, Germany) and captured with an Axiovision Imaging System. The imagines for TG2 were obtained using a Confocal Laser Scanning Microscope (CLSM) 510 Meta (Zeiss, Germany) and captured with an Axiovision Imaging System. For the acquisition with CLSM, we utilize an Apo 63 X/1.4 oil immersion objective and the Argon (λ = 488 nm) and HeNe (λ = 543 nm). The software ZEN2009 (version no 5.5.0.452) was used to enhance brightness and contrast. In all OECs, no specific staining was observed when primary antibodies were omitted.
Total ROS Levels
2.2.5
Cellular ROS Detection Assay was used to assess the production of total ROS in all experimental conditions. To quantify ROS levels production dichlorofluorescein diacetate assay (DCFH‐DA; Sigma‐Aldrich, Milan, Italy) was used. The intensity of fluorescence emitted from each sample was evaluated through fluorescence spectrophotometry (excitation λ = 488 nm; emission λ = 525 nm). Data are expressed as intensity of fluorescence for 30 min for mg protein. Protein content was detected using the Bio‐Rad Protein Assay kit (Bio‐Rad Labs Srl, Milan, Italy) and evaluated the protein concentration at λ = 595.
Statistical Analysis
2.2.6
The data analysis of different groups was carried out through a one‐way ANOVA, followed by post hoc Holm–Sidak test. Data represent the mean ± S.D. of five separated experiments performed in triplicate. Values were considered statistically significant with *p < 0.05 vs. control.
Results
3
Modeling Results
3.1
Based on the GOLD v2025.1.0 dockings, berberine 1 (Table 1), binding with the closed form of TG2 (score: 53.50, Figure 2B), shows hydrophobic interactions of the benzyl group with PHE174, VAL479 and a π‐σ bond with GLN 481. The π‐σ interactions enhanced the ligand and receptor binding [22]. Although the π‐σ bond lacks in the interaction of berberine with the “no Ca^2+^” closed form of TG2, the score value (53.05) is similar (Figure 2C). A slight preference is determined for the closed form. The overall weak binding suggests limited efficacy in modulating TG2 conformational changes and activity. The interaction with the open form (Figure 2A) shows a score value of 41.74 and interactions with Lys 202, Lys 205 Arg 222 and Tyr 388. Berberine shares with curcumin a good affinity for the open form of TG2, even smaller than indicaxanthin (70.30), as below reported.
Binding mode of 1 with TG2 in open (A), closed (B) and closed with Ca2+ (C) conformations. Amino acid residues involved in berberine (green) binding are in blue sticks.
Regarding curcumin (2), we found that it shows a higher score than the other NAs (1–3–4) with both the “closed” and the “open” form of TG2 (Table 1). Curcumin 2 (Table 1) binds to TG2 closed conformation (score: 76.99) giving hydrogen bond interactions with LYS173, PHE174 and VAL479; hydrophobic interactions with GLN481, SER482, ASP581, LEU582, TYR583 and GLU585 and a Sulfur‐O bond involving the oxygen of the phenolic rings and the sulfur group of MET483 (Figure 3B). Different interactions are established by curcumin 2 (Table 1) with the “open” form of TG2 (score: 53.40) (Figure 3A). A hydrophobic interaction between a phenolic ring and PRO201 was detected. In addition, a π‐Sulfur interaction with MET227 and hydrogen bonds with residues ARG201, LYS202, LYS205, TYR219, GLY226, VAL233 and THR368 are established [23, 24, 25]. Curcumin 2 (Table 1), that demonstrated a strong preference for the “closed” conformation of TG2, showed reduced binding in the calcium‐bound state (docking score 66.60) and, to some extent, to the “open” state (docking score 53.40). In the calcium‐bound state, curcumin interacts by its phenolic ring establishing hydrophobic interactions with VAL479, MET483, PHE174. Hydrogen bonds are formed by carbonyl oxygen and phenolic oxygens with ARG580 and TYR583, respectively.
Binding mode of 2 with TG2 in open (A), close (B) and close with calcium ion (C) conformations, respectively. Amino acid residues involved in curcumin (green) binding are highlighted with blue sticks.
These data suggest that curcumin might effectively stabilize or induce the “closed” state of the protein, while its interaction is negatively influenced by calcium, potentially due to conformational constraints or competitive binding.
Indicaxanthin 3 (Table 1) showed good affinity for both the closed (score: 58.31, Figure 4A) and open (score: 70.30, Figure 4B) conformations of TG2. A less favorable interaction was observed in the calcium‐bound closed state (score: 48.31, Figure 4C). This pattern suggests a preference for the open or intermediate conformations of the protein, rather than for the calcium‐stabilized closed state. The interaction with the closed state of TG2 is characterized by four hydrogen bonds (involving LYS202, LYS205, ARG222, and TYR388) via the carboxyl group. However, the overall binding affinity might be reduced by the electrostatic repulsion between the positively charged nitrogen atom of indicaxanthin and LYS202.
Binding mode of 3 with TG2 in closed (A), open (B) and open with Ca2+ (C) conformations. Amino acid residues involved in astaxanthin (green) binding are in blue sticks.
In comparison, astaxanthin 4 (Table 1) exhibited the lowest docking scores across all the TG2 conformations: 47.89 for the closed (Figure 5A), 47.29 for the calcium‐bound closed (Figure 5C), and 32.16 for the open state (Figure 5B). Its binding appears largely unaffected by the presence of calcium, suggesting a weak and conformationally independent binding. Only a few hydrophobic contacts were identified with ARG418, ARG476, ARG478, and VAL479 in the closed form, and with PRO201, LYS202, and TYR388 in the open form, mostly involving the methyl groups in the β‐carotene spacer of the molecule. This weak binding profile indicates limited potential of astaxanthin to influence TG2 activity or conformation.
Binding mode of 4 with TG2 in Closed (A), open (B) and open with Ca2+ (C) conformations. Amino acid residues involved in indicaxanthin (green) binding are highlighted in blue sticks.
Based on the docking data, curcumin 2 emerges as the most promising candidate for stabilizing or inducing the closed conformation of TG2, which is sensitive to calcium‐mediated conformational changes; indicaxanthin 3 exhibits reduced binding with respect to curcumin 2. The binding seems to be less influenced by conformation. Berberine 1 and astaxanthin 4 show minimal interaction across all the conformations (Table 1).
Biological Study
3.2
Based on the modeling results, we evaluated the effects of the NAs berberine and curcumin. Specifically, we focused on their impact on TG2 expression and localization in OECs exposed to Aβ and its fragments using immunocytochemical techniques.
Cell Viability
3.3
To monitor the percentage of cell viability in OECs unexposed and exposed to full native peptide Aβ(1–42) or to the fragments Aβ(25–35) and Aβ(35–25), both in the absence and in the presence of NAs (berberine and curcumin), the 3(4,5‐dimethyl‐thiazol‐2‐yl)2,5‐diphenyl‐tetrazolium bromide (MTT) test was performed. Firstly, to choose the optimal concentrations of NAs, OEC cultures were treated with different concentrations of berberine and curcumin (0.5, 1.0, 10, 20, 40 μM) for 24 h. As reported in the Figure 6A,B, we found that the optimal concentration of each NAs was 10 μM for 24 h. Subsequently, the cells were pretreated with 10 μM of berberine or curcumin and then exposed to 10 μM of Aβ(1–42) full native peptide or Aβ(25–35) or Aβ(35–25) fragments for 24 h.
*Percentage of OEC viability performed through MTT test. Cells were treated with berberine (A) and curcumin (B) at different concentrations for 24 h. Data represent the mean ± S.D. of three separate experiments performed in triplicate. p < 0.05 significant differences vs. CTR.
A significant decrease of the percentage of cell viability in the cells exposed to 10 μM Aβ(1–42) peptide or Aβ(25–35) fragments was found when compared with the untreated control (Figure 7). No significant changes were observed in Aβ(35–25) reverse sequence treated cells when compared with the control (Figure 7).
*Percentage of cell viability in OECs obtained through MTT test. OECs were exposed to Aβ(1–42) or Aβ(25–35) or Aβ(35–25) for 24 h; OECs pre‐treated with berberine or curcumin for 30 min and exposed to Aβ(1–42) or Aβ(25–35) or Aβ(35–25) for 24 h. Data represented the mean ± S.D. of five separate experiments in triplicate. p < 0.05 significant differences vs. CTR.
The pretreatment of OECs with berberine or curcumin was able to restore the percentage of cell viability to control values (Figure 7A,B).
Total Intracellular ROS Production
3.4
To monitor the intracellular oxidative status in the OECs exposed to Aβ and to its fragment both in the absence and in the presence of berberine and curcumin (10 μM) for 24 h, total intracellular ROS production was evaluated. As reported in the Table 2, the treatment of OECs with 10 μM Aβ(1–42) or Aβ(25–35) induced a significant increase in total ROS production when compared with the control. In contrast, no significant change in OECs exposed to the reverse sequence of 10 μM Aβ(35–25) for 24 h was found.
The treatment of the cells with 10 μM berberine or curcumin for 24 h did not induce any significant change of ROS production when compared with the control. The pre‐treatment of OECs with 10 μM berberine or curcumin for 30 min and successive exposure to Aβ(1–42) or to Aβ(25–35) for 24 h led to counteract the neurotoxic effect induced by Aβ, restoring ROS levels to those observed in the controls. When the cells were treated with 10 μM berberine or curcumin and then exposed to Aβ(35–25), ROS levels appeared comparable to control values.
Levels of GFAP, Vimentin and Nestin in OECs Exposed to Aβ and Its Fragments
3.5
The different levels of GFAP, vimentin, and nestin were investigated through immunofluorescence analysis in OECs exposed to Aβ(1–42), Aβ(25–35), and Aβ(35–25) both in the absence and in the presence of berberine or curcumin. In particular, we assessed the levels of: GFAP, which is a glial growth marker highly expressed during inflammatory processes and therefore more active in astrogliosis; vimentin, a cytoskeletal marker and TG2 substrate; and nestin, a neural marker also expressed in stem cells including OECs that present a certain stemness [12, 15].
The Figures 8 and 9 show the levels of GFAP and vimentin in OECs exposed to Aβ(10 μM) and its fragments for 24 h both in the absence and in the presence of berberine (10 μM) or curcumin (10 μM). Our results report in OECs exposed to the toxicity of Aβ(1–42) and Aβ(25–35) a notable increase in the levels of GFAP (Figure 8) and vimentin (Figure 9), when compared with the controls. When the cells were pre‐treated with berberine or curcumin, the levels of both markers decreased significantly and appeared similar to those observed in the control ones. These findings highlight that these natural compounds are able to counteract the toxic action of the peptide. OECs treated with either berberine or curcumin showed no change in the expression of GFAP and vimentin compared to controls (Figures 8 and 9).
Positive OECs for GFAP detected through immunocytochemistry. Images show the effect of berberine and curcumin in OEC treated to different conditions. No change related to GFAP positivity was observed in cells. The pre‐treatment with berberine or curcumin restored the levels of GFAP to control values, modified by the exposure to Aβ(1–42) or Aβ(25–35). Scale bar 20 μm.
Positive OECs for vimentin detected through immunocytochemistry. Images show the effect of berberine and curcumin in OEC treated to different conditions. No change related to vimentin positivity was observed in cells. The pre‐treatment with berberine or curcumin restored the levels of protein to control values, modified by the exposure to Aβ(1–42) or Aβ(25–35). Scale bar 20 μm.
Regarding nestin levels, a notable decrease in OECs exposed to Aβ(1–42) and Aβ(25–35) was found when compared with the controls. In the OECs pretreated with berberine (10 μM) or curcumin (10 μM) for 24 h, the levels of nestin were similar to control ones (Figure 10). This result evidence the protective effect of the two natural substances against Aβ toxic. No marker expression was observed in OECs when all primary antibodies were omitted.
Nestin positive OECs detected through immunocytochemistry. Images show the effect of berberine and curcumin in OEC treated to different conditions. The pre‐treatment with berberine or curcumin restored the levels of protein to control values, altered by the exposure to Aβ(1–42) or Aβ(25–35). Scale bar 20 μm.
TG2 Levels by Immunofluorescence Assay
3.6
Figure 11 shows the positivity to TG2 and its localization in the OECs, highlighted with immunocytochemical techniques and CLSM analysis. In control cells, a very low positivity for TG2 was found, and the protein was predominantly localized in the cytosol. A more intense positive signal for TG2 was found in cells exposed to Aβ(1–42) and Aβ(25–35) when compared with the controls. In addition, the toxic fragment Aβ(25–35) induced a strong increase of TG2 levels.
Confocal laser scanning microscopy of labeling immunocytochemistry for anti‐TG2 in OECs exposed to different conditions. The pre‐treatment with berberine or curcumin restored the levels of protein to control values, modified by Aβ(1–42) or Aβ(25–35) exposure. Scale bar 20 μm.
Exposure to the non‐toxic fragment of Aβ(35–25) produced a slight increase in the positivity of TG2 cells, when compared with controls, showing a predominant localization of the protein in the cytosol.
In berberine or curcumin‐treated OECs, TG2 levels were similar to the control values, and the protein was prevalently localized in the cytosol. The pre‐treatment of OECs with berberine or curcumin and subsequently their exposure to Aβ(1–42) and Aβ(25–35) was able to restore TG2 levels to those found in the control. In addition, the NAs pretreatment induced a change in the protein localization that appeared prevalently in the nuclear compartment. In contrast, a weak staining for TG2 in the OECs pretreated with berberine or curcumin and then exposed to the fragment Aβ(35–25) was found. Furthermore, in this experimental condition TG2 was localized in the cytosol (Figure 11). No specific staining was revealed in control cells incubated in the absence of primary antibody against TG2.
Caspase‐3 Expression by Immunofluorescence
3.7
To verify the activation of the apoptotic pathway both in the absence and in the presence of NAs in OECs exposed to Aβ(1–42) or Aβ(25–35), the caspase‐3 cleavage through immunocytochemical techniques was evaluated. As shown in the Figure 12, in the control, cell positivity for caspase‐3 was almost absent. When cells were exposed to Aβ(1–42) or Aβ(25–35), strong activation of caspase‐3 positive cells was observed. The effect was particularly evident in Aβ(25–35), which is highly toxic to cells. No labeling for caspase‐3 was found in berberine or curcumin treated OECs (Figure 12). When the cells were pre‐treated with berberine or curcumin and then exposed to Aβ(1–42) or Aβ(25–35), the positivity for caspase‐3 appeared like that observed in the control (Figure 12). No positivity for caspase‐3 was detected in OECs pre‐treated with NAs and successively exposed to Aβ(35–25). These results highlight that berberine and curcumin pretreatment of OECs was able to counteract the toxicity of Aβ.
Immunocytochemistry for anti‐caspase‐3 in OECs treated at different conditions. Images show the effect of berberine and curcumin in OEC treated to different conditions. No positivity in controls, in pre‐treated cells with berberine and curcumin. The pre‐treatment with berberine or curcumin restored the levels of caspase‐3 to control values, modified by the exposure to Aβ(1–42) or Aβ(25–35). Scale bar 20 μm.
Discussion
4
In this work, for the first time, we assessed the interactions between NAs and TG2 in OECs exposed to Aβ, a neurotoxic protein involved in AD through docking and biological studies.
Several reports have demonstrated that oxidative stress represents one of the major factors contributing to AD, suggesting also that the treatment with antioxidants might be a tool for counteracting it [26]. Furthermore, Aβ induces several events, such as an intracellular increase in Ca^2+^ levels, autophagy, mitochondrial and blood–brain barrier dysfunctions, post‐translational modifications, including the aberrant activation of TG2 [27, 28, 29]. In particular, TG2 presents two conformational states: “closed” and “open”, depending on the cellular concentration of GTP/GDP or Ca^2+^: when TG2 is bound to GTP/GDP, it adopts a “closed” conformation, catalytically inactive, without transamidating activity. The intracellular Ca^2+^level increase induced the conformational change of the protein, which assumes an “open” conformation, catalytically active, promoting cell growth and cell survival [3]. The sustained enhancement of Ca^2+^levels is responsible for the loss of β‐barrel domain, adopting a short conformation, which is involved in apoptotic cell death [3].
In our previous works, we found that astaxanthin and indicaxanthin pre‐treatment of OECs exposed to Aβ, inducing an overexpression of TG2, was able to restore its levels to control ones [9, 10]. In addition, we reported that, in an experimental model of AD, the administration of unloaded and curcumin‐loaded Solid Lipid Nanoparticles reduced TG2 upregulations and its isoforms [30]. Other authors demonstrated that the combinations of berberine and curcumin improved the cognitive function of AD mice by reducing oxidative stress and neuroinflammation [31]. In particular, berberine, a natural compound extracted from a variety of Chinese Medicinal herbs, shows anti‐inflammatory, antioxidant, antidiabetic, and cholesterol‐lowering activities [32], which are crucial aspects of several neurodegenerative diseases, including AD [33]. Berberine is able to down‐regulate TG2 overexpression induced by excitotoxicity in astroglial cell cultures, protecting them in the apoptosis [34, 35, 36]. Another promising active compound counteracting the formation of amyloid plaques [37], is curcumin, a natural antioxidant extracted from the rhizome of Curcuma longa . In particular, curcumin modulates AD‐associated epigenetic modifications through influencing methylation patterns and histone‐proteins [38]. Furthermore, it has been demonstrated that TG2 represents an important marker of neuroinflammation, and the treatment of curcumin in mouse microglial cells was able to reduce Aβ1‐42‐induced TG2 overexpression [39].
On the basis of these observations, we have here assessed the effects of berberine and curcumin on the TG2 levels in mouse OECs exposed to Aβ and its fragments. In particular, we used OECs as they represent peculiar glial cells of the olfactory system which play a fundamental role in olfactory performance, that is compromised in neurodegenerative diseases, among which AD and Parkinson's Disease [11].
To better understand the interaction of berberine, curcumin, indicaxanthin and astaxanthin with TG2, docking studies were performed. These helped to rationalize the interactions of the subject ligands with both the “closed” (also with Ca^2+^) and the “open” forms of TG2. This integrative strategy provides a coherent mechanistic framework linking structural determinants of TG2 modulation to cellular outcomes relevant to AD pathogenesis. By uncovering the domain‐level interactions responsible for allosteric regulation of TG2 by natural antioxidants, our work supports their potential application as modulators of neuroinflammation, oxidative stress, and neurodegenerative diseases, such as AD. In addition, to evaluate domain‐specific binding affinities, ligand‐contact residues and conformational preferences, we performed molecular docking studies using the GOLD v2025.1.0 software, targeting multiple TG2 states including the closed, Ca^2+^‐bound closed, and open forms. The computational results were then integrated by the biological findings to explore how compound‐specific ligand‐TG2 interactions may influence the protein conformation and downstream cellular responses in the context of Aβ toxicity.
Our data indicate that all four NAs engage TG2 at key interdomain interfaces to varying extents, thereby modulating its conformational equilibrium via an allosteric hinge mechanism rather than by direct active site inhibition. In particular, berberine demonstrates moderate, conformation‐sensitive binding (GOLD scores: closed 53.50; closed + Ca^2+^ 54.38; open 48.59), preferring closed forms and suggesting a stabilizing effect on the catalytically inactive conformation. Curcumin shows the highest affinity for apo–closed TG2 (score: 76.99), acting as a molecular staple that stabilizes the β‐sandwich/transamidating loop interface by forming H‐bonds with Lys173, Phe174, and Val479, potentially preventing opening under calcium stress. Astaxanthin exhibits similar binding across all conformations (scores: closed 63.89; closed + Ca^2+^ 63.35; open 63.73), targeting the β‐barrel 1 and catalytic core (Val315, Leu420, Tyr516) via hydrogen bonds and π–π interactions, suggesting a flexible allosteric engagement. Indicaxanthin shows favorable interaction in the open conformation (score: 58.00), engaging the catalytic core and β‐barrel 2 domain (Gly417, Cys421, Arg580), potentially modulating the active TG2 conformation.
These docking results align with our biological findings in OECs, where pretreatment with NAs restored TG2 expression altered by Aβ exposure, potentially by promoting conformational transitions toward the closed, nuclear‐protective form. The distribution of docking sites across the TG2 structure underscores the conformational selectivity of each antioxidant and offers mechanistic insight into their neuroprotective activity. Specifically, TG2 is structured in four domains: the N‐terminal β‐sandwich (residues 1–139), the catalytic core (140–460), and two C‐terminal β‐barrel domains (β‐barrel 1: 471–585; β‐barrel 2: 586–687). These domain‐specific interactions suggest a mode of action wherein NAs act as conformation‐selective stabilizers, capable of modulating TG2 structural flexibility and activity in the cellular context.
Consistently with the in silico findings, our experimental data demonstrated that pre‐treatment of OECs with NAs before exposure to Aβ or its toxic fragments significantly reduced intracellular Ca^2+^ levels, which in turn decreased TG2 expression to control values. This reduction supports the hypothesis that NAs modulate calcium‐dependent conformational changes of TG2, favoring its inactive, closed state. This effect was accompanied by an increase in the percentage of cell viability, which appeared at the same levels as the control. In addition, we found both a decrease in intracellular ROS production and the apoptotic pathway activation, as revealed by the low levels of caspase 3 cleavage. Furthermore, a downregulation of glial activation markers, such as GFAP and vimentin, indicating a protective role against Aβ‐induced gliosis, was observed [9, 10]. Additionally, NAs treatment increased nestin levels, implying the promotion of self‐renewal and neurogenic properties in OECs, a capacity closely linked to the nuclear localization of TG2 in its closed form, known to support reparative functions [40, 41]. These findings suggest that NAs not only mitigate TG2 upregulation but also shift the protein toward a neuroprotective conformation that favors nuclear functions, rather than extracellular crosslinking activity associated with neurodegeneration [41]. The data support a model in which differential binding of antioxidants to discrete TG2 domains induces distinct structural outcomes, thereby modulating its subcellular distribution and functional roles. Importantly, this dual approach combining molecular docking with cell‐based assays provides a powerful strategy to uncover the mechanistic basis of TG2 modulation in neuroinflammatory environments.
Our results reinforce the therapeutic potential of berberine and curcumin, both of which showed high affinity for the closed form of TG2 and potent biological effects in OECs. The ability of these compounds to stabilize TG2 in its closed conformation, reduce calcium influx, and modulate glial markers provides a compelling rationale for their continued exploration as neuroprotective agents. Further studies are needed to clarify whether NAs also affect TG2 post‐translational modifications or nuclear shuttling in OECs and other glial cell types.
Conclusion
5
In this study we adopted a dual approach combining molecular modeling and biological studies, demonstrating that NA treatment, in particular curcumin, on OECs exposed to Aβ was able to promote the stabilization of TG2 in its closed, catalytically inactive conformation.
The conformational equilibrium of TG2 is tightly regulated by intracellular concentrations of calcium and nucleotides, and the closed form is maintained through specific intramolecular interactions, including hydrogen bonds [42]. Our combined wet‐in silico results show that NAs with a higher affinity for the closed form, such as berberine and curcumin, can enhance this stabilization, thus hindering the conformational transition to the enzymatically active state. By favoring the inactive conformation, these antioxidants effectively reduce or inhibit TG2's transamidating activity. Our results demonstrate how integrating computational and experimental approaches increases the likelihood of discovering potent therapeutic agents but also positions berberine and curcumin as candidates for the development of new drugs in neurodegeneration, including AD.
In addition, this mechanism offers a promising therapeutic avenue for conditions associated with dysregulated TG2 activity, including inflammatory and neurodegenerative diseases [43]. Further studies need to better clarify whether the NAs play an important role in the adoption of the “open” TG2 conformation, which has a key role in the self‐renewal ability of OECs, being cells capable of expressing and releasing neurotrophic receptors, representing a promising tool for neural regeneration in AD.
Funding
This research was partially supported by the University of Catania, Progetto PIA.CE. RI 2020–2022, Dotazione Ordinaria, Linea di intervento 2 (grant number 57722172121) and by Progetto NUTRAGE (FOE 2021)—DM MUR n. 844 del 16/07/2021—CUP B83C21001810005 (DBA.AD005.225).
Conflicts of Interest
The authors declare no conflicts of interest.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
- 1A. Safouris , G. Tsivgoulis , T. N. Sergentanis , and T. Psaltopoulou , “Mediterranean Diet and Risk of Dementia,” Current Alzheimer Research 12 (2015): 736–744, 10.2174/1567205012666150710114430.26159192 · doi ↗ · pubmed ↗
- 2L. E. Sima , D. Matei , and S. Condello , “The Outside‐In Journey of Tissue Transglutaminase in Cancer,” Cells 11 (2022): 1779, 10.3390/cells 11111779.35681474 PMC 9179582 · doi ↗ · pubmed ↗
- 3G. Singh , J. Zhang , Y. Ma , R. A. Cerione , and M. A. Antonyak , “The Different Conformational States of Tissue Transglutaminase Have Opposing Effects on Cell Viability,” Journal of Biological Chemistry 291 (2016): 9119–9132, 10.1074/jbc.M 115.699108.26893378 PMC 4861479 · doi ↗ · pubmed ↗
- 4P. Arulselvan , M. T. Fard , W. S. Tan , et al., “Role of Antioxidants and Natural Products in Inflammation,” Oxidative Medicine and Cellular Longevity 2016 (2016): 5276130, 10.1155/2016/5276130.27803762 PMC 5075620 · doi ↗ · pubmed ↗
- 5D. Krishnaiah , R. Sarbatly , and R. Nithyanandam , “A Review of the Antioxidant Potential of Medicinal Plant Species,” Food and Bioproducts Processing 89 (2011): 217–233, 10.1016/j.fbp.2010.04.008. · doi ↗
- 6V. Di Matteo and E. Esposito , “Biochemical and Therapeutic Effects of Antioxidants in the Treatment of Alzheimers Disease, Parkinsons Disease, and Amyotrophic Lateral Sclerosis,” Current Drug Targets ‐ CNS & Neurological Disorders 2 (2003): 95–107, 10.2174/1568007033482959.12769802 · doi ↗ · pubmed ↗
- 7M. A. E. Ventura , K. Sajko , M. Hils , et al., “The Oral Transglutaminase 2 (TG 2) Inhibitor Zed 1227 Blocks TG 2 Activity in a Mouse Model of Intestinal Inflammation,” Gastroenterology 154 (2018): S 490, 10.1016/S 0016-5085(18)31861-4. · doi ↗
- 8R. Pellitteri , R. Bonfanti , M. Spatuzza , et al., “Effect of Some Growth Factors on Tissue Transglutaminase Overexpression Induced by β‐Amyloid in Olfactory Ensheathing Cells,” Molecular Neurobiology 54 (2017): 6785–6794, 10.1007/s 12035-016-0152-4.27757835 · doi ↗ · pubmed ↗