The γ-Adducin 1–357 fragment promotes tau pathology

Background Tau phosphorylation is a pathological hallmark of Alzheimer’s disease (AD). Previously, we reported that the γ-adducin 1–357 fragment is present in the brains of AD patients. However, it remains unknown how γ-adducin regulates tau phosphorylation. Objective The aim of this project is to investigate the effects of the γ-adducin 1–357 fragment on tau phosphorylation and the kinases involved in this process. Methods Full-length γ-adducin or the γ-adducin 1–357 fragment was expressed in HEK293 cells, SH-SY5Y cells, and primary neurons. The phosphorylation of tau Ser396 was determined using Western blot and immunofluorescence. Tau P301S transgenic mice were injected with adeno-associated virus encoding full-length γ-adducin or γ-adducin 1–357 fragment to determine the phosphorylation of tau. Results The γ-adducin 1–357 fragment enhances tau phosphorylation at Ser396. Additionally, the expression of the γ-adducin 1–357 fragment leads to the activation of glycogen synthase kinase-3β (GSK-3β). This effect was mitigated by the GSK-3β inhibitor 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8). Conclusion The γ-adducin 1–357 fragment enhances tau phosphorylation by activating GSK3β. These results support that the fragmentation of γ-adducin may play a pivotal role in tau pathology.


Introduction
Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by memory loss and cognitive impairment (Scheltens et al., 2021).One of its key features is the presence of neurofibrillary tangles (NFTs), abnormal accumulations of tau protein with excessive phosphorylation (Metaxas and Kempf, 2016).Tau, a microtubule-associated protein (MAP), plays a central role in orchestrating the assembly, stabilization, and modulation of microtubules, which are vital for neuronal and cognitive functions (Wang and Mandelkow, 2016).Under pathological conditions, tau undergoes complex changes such as truncation and phosphorylation, forming harmful oligomers and fibrils (Li et al., 2022).Aspartic endopeptidase (AEP) has been reported to cleave tau and promote its aggregation (Zhang et al., 2014).Phosphorylation is one of the most prevalent post-translational modifications of tau in AD (Johnson and Stoothoff, 2004).Phosphorylation of tau facilitates the creation of pathological tau oligomers, which trigger neurotoxicity (Shafiei et al., 2017; OPEN ACCESS EDITED BY Bin Xu, North Carolina Central University, United States Yu et al. 10.3389/fnagi.2023.1241750Frontiers in Aging Neuroscience 02 frontiersin.orgCarroll et al., 2021).Understanding the molecular mechanisms underlying tau pathology will facilitate the development of novel therapeutic strategies.
Previously, we reported that the γ-adducin 1-357 fragment induces neurite damage and synaptic dysfunction, promoting cognitive impairments in AD (Xiong et al., 2021).However, how the γ-adducin 1-357 fragment regulates tau phosphorylation remains unclear.Glycogen synthase kinase-3β (GSK-3β) is one of the primary kinases for the tau protein and is capable of phosphorylating tau at multiple serine and threonine residues (Sayas and Ávila, 2021).Here, we established cellular and animal models and observed that the γ-adducin 1-357 fragment significantly enhances tau phosphorylation at Ser396 by activating GSK-3β.

Animals
Tau P301S transgenic mice on a C57BL/6 J background were obtained from Jackson Laboratory and housed at the Animal Experiment Center of Renmin Hospital, Wuhan University.The mice were maintained under specific pathogen-free (SPF) conditions with a 14-h light and 10-h dark cycle and had ad libitum access to food and water.All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Renmin Hospital, Wuhan University (IACUC issue number: WDRM animal [welfare] 202,200,084).

AAV packaging and stereotaxic injection
Adeno-associated virus (AAV) particles encoding full-length γ-adducin and the γ-adducin 1-357 fragment under the control of the human synapsin I promoter were prepared by BrainVTA (Wuhan) Co., Ltd.Bilateral intracerebral injection of AAVs was performed stereotactically in two-month-old Tau P301S mice.The mice were first prepared by removing the fur on their head, followed by disinfection of the scalp using iodine.An incision along the midline exposed the skull, and the mice were then carefully positioned with their head secured horizontally within the stereotactic injection apparatus.This method involved targeting specific coordinates relative to the bregma.The injection site was precisely located at posterior 2.5 mm, lateral 2.0 mm, and ventral 1.7 mm.Perioperative care and anesthesia received meticulous attention during the surgical procedure.The mice were anesthetized using isoflurane.Before the incision, the surgical site was thoroughly disinfected.Crucial vital signs, including heart and respiratory rates, were carefully monitored throughout the procedure.To prevent hypothermia, the mice were maintained at an appropriate temperature.Following surgery, the mice recuperated in a quiet, sterile environment and received post-operative care to ensure their well-being during the recovery period.

Primary neuronal culture
Primary cortical neurons were isolated from tau P301S mouse embryos at embryonic day 18 (E18).The embryos were dissected from the uterine horns and sterilized with 75% ethanol and sterile PBS.The brains were then transferred to a fresh dish with PBS, the meninges were removed, and the cortex tissue was extracted.The extracted cortex tissue was incubated with trypsin/EDTA at 37°C for 15 min and halted with DMEM-HS-FS (DMEM supplemented with horse serum and fetal serum).After centrifugation, the cell pellet was resuspended in 3 mL of DMEM-HS-FS to obtain a single-cell suspension, which was plated in 6-well plates.The primary neurons were cultured in neurobasal medium supplemented with B-27, 0.5 mM L-glutamine, penicillin, and streptomycin at 37°C in a 5% CO 2 /95% air environment.To overexpress γ-adducin in primary neurons, AAVs encoding GFP-vector, GFP-γ-adducin, and GFP-γ-adducin 1-357 fragment were introduced.After seven days, neurons were fixed with 4% PFA and subjected to immunostaining.

Western blot analysis
The mice were anesthetized and perfused transcardially with saline.The brain was carefully removed from the skull.The brain was dissected to isolate the specific region of the hippocampus.Protein extracts were prepared from either cultured cells or the mouse hippocampus using lysis buffer containing a phosphatase and protease inhibitor cocktail.The protein extracts were separated on a 12% SDS-PAGE gel and transferred to nitrocellulose membranes using a semidry system.After blocking with 5% skim milk for 1 h, the membranes were incubated with primary antibodies overnight at 4°C.After undergoing three washes with Tris-buffered saline with Tween 20 (TBST), membranes were incubated with HRP-conjugated antibodies.Subsequently, the membranes underwent three additional TBST washes.Protein bands were then visualized using enhanced chemiluminescence (ECL) within a Western blotting system (Bio-Rad).The antibodies used in the Western blot experiment are listed in Table 1.

Immunofluorescence
The anesthetized mice were transcardially perfused using saline and 4% paraformaldehyde.Extracted brain tissues were dehydrated and then sliced into 4-μm sections after embedding in paraffin.Initially, the paraffin-embedded sections were treated with xylene to remove paraffin and gradually rehydrated using a series of ethanol solutions.Next, the sections underwent heat-induced antigen retrieval at 94°C for 20 min in 10 mM sodium citrate buffer (pH 6.0).After washing with phosphate-buffered saline (PBS), endogenous peroxidase activity was neutralized with a 0.3% hydrogen peroxide solution for 10 min.After blocking with 3% bovine serum albumin (BSA), the sections were incubated with primary antibodies overnight at 4°C.After a PBS rinse, goat antirabbit IgG (H + L) Alexa Fluor™ 594 secondary antibodies were applied for 2 h at room temperature.Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).Finally, fluorescence images were captured using a fluorescence microscope from Olympus.
For immunocytofluorescence, cultured cells were initially fixed and permeabilized using a solution of 4% paraformaldehyde and 1% Triton X-100 for 10 min.Subsequently, the slides were washed with PBS, followed by blocking with 3% BSA for 30 min.The cells were then incubated with primary antibodies overnight at 4°C.After another round of washing with PBS, the cells were exposed to goat anti-mouse IgG (H + L) Alexa Fluor™ 488 and goat anti-rabbit IgG (H + L) Alexa Fluor™ 594 secondary antibodies for 2 h at room temperature in the dark.Then, the cells were washed with PBS and subjected to nuclear staining with DAPI.After a quick rinse in PBS, the cells were examined using a fluorescence microscope from Olympus.

Statistical analysis
Statistical analyses were conducted using GraphPad Prism (version 8.0).All data were presented as means ± standard deviation (SD) from three or more independent experiments.One-way analysis of variance (ANOVA) was performed to determine significant main effects and differences among three or more groups, followed by Tukey's or least significant difference (LSD) multiple comparisons for post hoc tests.Differences with p values less than 0.05 were considered significant.

Discussion
Over phosphorylation and aggregation of Tau are widely considered pathological hallmarks in AD (Wegmann et al., 2021).The factors initiating tau phosphorylation in the AD brain remain unknown.Thus, tau phosphorylation has been extensively researched.Here we focused on the effect of the γ-adducin 1-357 fragment on tau phosphorylation.We found that the γ-adducin 1-357 fragment promotes the phosphorylation of tau at Ser396, which is one of the early pathological signs of AD (Mondragón-Rodríguez et al., 2014).Furthermore, the activation of GSK-3β by the γ-adducin 1-357 fragment underscores its role as a critical kinase involved in tau pathology.The inhibition of GSK-3β activity by the specific kinase inhibitor TDZD-8 suggests the Previous studies have indicated that the γ-adducin 1-357 fragment induces synaptic dysfunction and promotes cognitive impairments in AD.The γ-adducin 1-357 fragment inhibits neurite outgrowth by disrupting spectrin-actin assembly (Xiong et al., 2021).Building upon this foundation, our current study demonstrates that γ-adducin 1-357 fragment enhances tau phosphorylation.It has been reported that over phosphorylation of tau also affects neurite outgrowth (Morris and Brady, 2022).Thus, γ-adducin 1-357 fragment may not only directly influence the formation of actin filaments, but also interfere with neurite outgrowth by enhancing tau phosphorylation.
We demonstrate that the upregulation of the γ-adducin 1-357 fragment triggers tau phosphorylation by activating GSK-3β.
Targeting the aberrant phosphorylation of tau holds promise for potential clinical therapies.GSK3-β is considered a key player in AD pathophysiology since dysregulation of this kinase influences the major hallmarks of the disease, including tau phosphorylation, amyloid-β production, memory, neurogenesis, and synaptic function (Lauretti et al., 1867).Recent decades have witnessed concerted efforts to comprehend the role of GSK-3β, with the aim of designing potent and selective GSK-3β inhibitors.However, clinical and preclinical trials using GSK-3β inhibitors have exhibited suboptimal potency (Llorens-Martín et al., 2014;Xia et al., 2021).Likewise, a combinatorial approach involving multiple drugs may be necessary to counteract tau phosphorylation, given the involvement of numerous kinases that target multiple phosphorylation sites.Upon cleavage of γ-adducin to generate the 1-357 fragment, its actin-binding activity is compromised, leading to disruption of the cellular cytoskeleton (Xu et al., 2013).This structural alteration in the γ-adducin 1-357 fragment may expose specific regions or functional motifs, enhancing its interaction with GSK-3β and tau.Moreover, the γ-adducin 1-357 fragment might disrupt the regulatory mechanisms controlling GSK-3β activity and its interactions with downstream substrates like tau.In contrast to full-length γ-adducin, the γ-adducin 1-357 fragment exhibits enhanced activity in enhancing tau Ser396 phosphorylation and regulating GSK-3β Ser9 and Tyr216 phosphorylation.This phenomenon could be ascribed to underlying structural changes, altered binding affinity, and the potential disturbance of regulatory pathways.
The current findings lay a foundation for future investigations into the mechanisms and therapeutic strategies targeting the γ-adducin 1-357 fragment and GSK-3β in AD.However, it remains unknown how the γ-adducin 1-357 fragment activates possibility of targeted therapeutic interventions to modulate tau phosphorylation and its associated pathological consequences.