Phosphorylated peptide Tau-pT181 induced tau aggregation and cognitive dysfunction

Abstract

Background

Clinically, phosphorylation of Tau protein at threonine 181 (p-Tau181) acts as a crucial biomarker for AD detection. However, the mechanisms through which Tau phosphorylation at threonine (Thr)181 site leads to Tau aggregation and corresponding neuropathological changes remain unclear.

Objective

To investigate the effect of the phosphorylated tau peptide at Thr181 on tau aggregation, synaptic and cognitive impairments.

Methods

We synthesized the phosphorylated tau peptide Tau-pT181 and the non-phosphorylated tau peptide Tau-nT181, and verified their effects on the aggregation of the Tau repeat domain R3 fragment peptide and Tau pathology.

Results

Thioflavin S assay showed that Tau-pT181 significantly promoted the aggregation of R3, whereas Tau-nT181 did not induce R3 aggregation. Moreover, Tau-pT181 not Tau-nT181 led to the aggregation of Tau protein in 293/tau cells and decreased synapse-associated proteins in primary hippocampal neurons. One month after injecting Tau-pT181 and Tau-nT181 respectively into the rat CA1 hippocampal region, we found that exclusively the phosphorylated peptide Tau-pT181 induced endogenous Tau aggregation, synaptic damage, neuronal loss, while Tau-pT181 group exhibited significant cognitive impairment compared with the normal saline rats. Transcriptome analysis of neurons differentiated from iPSCs treated with Tau-pT181/Tau-nT181 suggest that phosphorylated peptides had a greater impact on axonogenesis, neuronal development, and Wnt signaling pathway.

Conclusions

The present study offers the first direct evidence that Tau phosphorylation at Thr181 induces Tau aggregation, and Tau-pT181 directly leads to neuropathological alterations and cognitive impairments, and establishes a new theoretical foundation for Tau Thr181 phosphorylation site as a core diagnostic marker and therapeutic target for AD.

Keywords

Alzheimer's disease phosphorylation Tau aggregation Tau-pT181

Introduction

As a primary progressive neurodegenerative disorder and a leading health burden in an aging world, Alzheimer's disease (AD) clinically manifests as memory impairment, cognitive dysfunction, behavioral changes, and functional decline.^1,2 The abnormal hyperphosphorylation of Tau protein is a fundamental requirement for these clinical manifestations.³ Notably, Tau pathology alone is sufficient to induce dementia independently of amyloid-β (Aβ), and the levels of neurofibrillary tangles (NFTs) strongly predict the severity of clinical dementia in AD patients.^4,5 Therefore, Tau pathology is central to the pathogenesis of AD. This process involves the aggregation of hyperphosphorylated Tau into NFTs, which triggers neuronal degeneration and subsequent clinical manifestations such as memory loss and cognitive deficits.^6,7 However, the precise molecular mechanisms driving this pathology remain incompletely understood.

Tau protein contains multiple phosphorylation sites, such as Thr181, Ser202, Thr205, Thr231, and Ser396, among others.⁸ The phosphorylation at different sites exhibits distinct biological functions and pathological significance in the development and progression of AD.⁹ Notably, the phosphorylation of Thr181 (p-Tau181), due to its detectability in cerebrospinal fluid (CSF) and peripheral blood,^10,11 has become a crucial biomarker for the clinical diagnosis of AD and has been approved by the FDA (Food and Drug Administration) for auxiliary diagnosis.^12,13 However, despite the significant clinical utility of p-tau181 in AD detection, its precise molecular mechanisms and pathological roles remain incompletely understood. Current research primarily focuses on several key questions: First, is p-tau181 a driver of neurodegeneration, or merely a byproduct of disease progression? Second, how does phosphorylation at this site interact with other critical sites (e.g., Ser202/Thr205) to collectively promote the pathological aggregation of Tau protein? Additionally, does p-tau181 influence the conformational changes of Tau protein, thereby enhancing its ability to form NFTs? These questions await further investigation through advanced molecular and cellular biology studies.

Naturally, addressing these questions fundamentally requires clarifying the precise impact of p-tau181 on Tau aggregation and tau pathology. In this study, we synthesized phosphorylated Tau fragment Tau-pT181 and non-phosphorylated Tau fragment Tau-nT181, and systematically investigated their effects on Tau aggregation, synaptic morphology and function, as well as cognitive performance through both in vitro and in vivo experiments.

Methods

Reagents

The polypeptides were generously supplied by Wuhan Baiyixin Biotechnology Co., Ltd All antibodies employed in this study are listed in Table 1.

Table 1.

Antibodies employed in this study.

Antibodies	Specificity	Type	Dilution	Sources
Tau5	Total tau	mAb	1:1500	Zen-bio
Anti-pT181	p-tau (T181)	mAb	1:500	Zen-bio
Anti-pT231	p-tau (T231)	mAb	1:1500	Zen-bio
Anti-pS262	p-tau (S262)	mAb	1:1500	Zen-bio
Synaptophysin	Synaptophysin	mAb	1:800	Abcam
Synapsin-1	Synapsin-1	pAb	1:800	Abcam
PSD95	Post Synaptic Density95	mAb	1:800	Cell Signaling Technology
β-actin	β-actin	mAb	1:1000	Santa-Cruz

mAb: monoclonal antibody; pAb: polyclonal antibody.

Animals

Male Sprague Dawley (SD) rats (2 months old) were obtained from the Animal Colony of Tongji Medical College. All animals were housed under ideal/controlled conditions with a natural circadian rhythm (ambient temperature: 18–22°C; 12 h light/12 h dark cycle) and were provided ad libitum access to food, water, and freedom of movement. The rats were randomly assigned to three groups: the Tau-pT181 treatment group, the Tau-nT181 treatment group, and the normal saline control group. All animal experiments were approved by the Animal Care and Use Committee of Huazhong University of Science and Technology and performed in compliance with the NIH Guide for the Care and Use of Laboratory Animals.

Th S fluorescence spectrometric

Thioflavin S (Th-S) is a fluorescent dye that emits at 490 nm upon excitation at 440 nm when bound to the β-sheet structure of protein fibrils. The Th-S assay was used for quantitative detection of amyloid fibril formation.¹⁴ Following reagent mixing as per the experimental protocol, reaction mixtures were prepared according to the experimental protocol (the multi-reaction system is shown in Table 2), and Th-S fluorescence was monitored using a fluorescence spectrometer under the following conditions: constant temperature of 37°C, excitation wavelength of 440 nm (slit width 10 nm), emission wavelength of 490 nm (slit width 10 nm), with final concentrations of 2.4 mM R3 peptide, 2.4 mM Tau-pT181 or Tau-nT181 peptide, 320 μM Th-S, 50 mM Tris-HCl buffer (pH 7.4), and 10 mg/mL heparin sodium. Fluorescence was measured continuously for 12 h with data recorded every 5 min.

Table 2.

Reaction system in th S fluorescence spectrometric assay.

Name	B2	B3	B4	B5	B6
R3-normal	10 μL	10 μL	10 μL	0 μL	0 μL
Tau-pT181	0 μL	10 μL	0 μL	10 μL	0 μL
Tau-nT181	0 μL	0 μL	10 μL	0 μL	10 μL
Tris-Hcl	190 μL	180 μL	180 μL	190 μL	190 μL
Th S	4 μL	4 μL	4 μL	4 μL	4 μL
Heparinsodium	10 μL	10 μL	10 μL	10 μL	10 μL

Primary neuron culture of hippocampus

Pregnant rats (embryonic day 16–18) were anesthetized and euthanasia. Hippocampal tissues were dissected from embryos, digested with 0.25% (vol/vol) trypsin. Neurons were plated and cultured in Neurobasal medium supplemented with 2% (v/v) B-27 and 1× GlutaMAX. Cultures were subsequently used for polypeptide treatment.

Cell culture and polypeptide treatment

HEK293 with stable expression of wild-type full-length human tau (termed as HEK293/hTau)¹⁵ were cultured in 90% DMEM/High Glucose medium containing 10% FBS, in a humidified atmosphere of 5% CO₂ at 37°C. Neofect (transfection reagent, TF20121201) was used for peptides enter the cells. HEK293/hTau cells were incubated with Tau-pT181, Tau-nT181, or normal saline solutions for 48 h, respectively. Then the cells were lysed with RIPA buffer, and total protein concentration was quantified by bicinchoninic acid (BCA) assay.

Intracerebral stereotaxic injection

Rats were anesthetized via intraperitoneal (ip) injection of 3% sodium pentobarbital. The scalp was incised along the midline between the eyes (detached from the skull, and fascia excised to expose the cranial fontanel) and connective tissue was removed to expose the cranial sutures. The surgical site was sterilized with 3% hydrogen peroxide. Stereotaxic coordinates were established using the fontanel as the origin and skull sutures as reference lines, targeting the hippocampal CA1 region according to the rat brain atlas (A/P + 1.3 mm, M/L ± 2.0 mm, D/V −4.0 mm). Bilateral burr holes were drilled, and a Hamilton syringe connected to a microinjection pump was used to deliver 2 μL injections (120 nL/min) into each hemisphere: experimental groups received 2.4 mM Tau-pT181 or 2.4 mM Tau-nT181, while controls received PBS. The injection needle was left in place for 8 min post-injection to minimize reflux or diffusion. Injections were performed sequentially, beginning with the right CA1 region followed by the left.

Behavioral test

Open field test

Exploratory behavior was evaluated using the open field test. Rats were placed individually into a square open-field chamber and allowed to acclimate. Animal movements were recorded for 5 min using a video-tracking system, and locomotor activity was analyzed.

Novel objective recognition test

Recognition memory was assessed using the novel object recognition test. Rats were first habituated to an open-field box (100 × 100 × 70 cm). During the familiarization phase, two identical objects were presented for exploration. After a 5 min retention interval, one familiar object was replaced with a novel object. Exploration was recorded for 5 min using a video-tracking system, and recognition memory was evaluated based on the preference for the novel object.

Fear conditioning test (FCT)

To assess the impact of Tau-pT181 or Tau-nT181 administration on episodic memory in rats, FCT was performed as previously described.¹⁴ Rats were first subjected to a 10 s auditory stimulation followed by a 3 s foot shock (0.8 mA). After 24 h, freezing behavior was recorded, and the duration of immobility was quantified as an index of associative memory.

Morris water maze test

After a continuous experimental training of 5 d × 4 times/day, the time for rats to reach the platform position for the first time and the number of crossings were recorded to assess the impact on memory function. Training was performed for 5 consecutive days, with four trials per day. Escape latency (time to reach the platform) and swimming paths were recorded using a video-tracking system.

Sarkosyl-insoluble Tau fractionation

HEK293/Tau cells were incubated with Tau-pT181 or Tau-nT181 for 48 h, washed with PBS and lysed in RIPA lysis buffer. Protein concentration was quantified using BCA method. Subsequently, sarkosyl was added to the lysate to a final concentraiton of 1% (v/v). Samples were centrifuged at 100,000 × g for 1 h at 4°C. The supernatant (sarkosyl-soluble fraction) and pellet (sarkosyl-insoluble fraction) were collected separately for further analysis.¹⁶

Western blotting

Rat brain tissue was homogenized in RIPA strong lysis buffer. The protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were blocked with 5% non-fat milk and incubated with primary antibody overnight at 4°C, followed by incubation with secondary antibody. Bands were visualized using enhanced chemiluminescence (ECL).

Golgi staining

Solutions A and B from the kit were mixed 24 h in advance to prepare the AB mixture. After rinsing the rat brain tissue with sterile PBS buffer, it was immersed in the AB mixture for 6 h, then it was replaced with fresh AB mixture and stored at room temperature away from light for 2 weeks. The tissue was transferred to solution C and immersed for 24 h, then replaced with fresh C solution and stored at room temperature away from light for 7 days. Using a vibratome, 100 μm thick sections were prepared and then dried completely at room temperature. The slides were rinsed twice with sterile distilled water for 4 min each. The DE mixture was prepared (solutions D, E and water in a ratio of 1:1:2 were mixed). The slides were immersed in the DE mixture for 10 min, then rinsed twice with sterile distilled water for 4 min each. The sections were dehydrated in 50%, 75%, 95% ethanol and absolute ethanol, each concentration gradient for 3 times, 4 min each. After dehydration, the sections were made transparent in xylene for 3 times, 4 min each. The sections were sealed with neutral gum. Images were collected using an upright microscope and the number of dendritic spines were counted and quantitatively analyzed using Image J software.

Nissl staining

Rats were anaesthetized and sacrificed. Brains were collected, post-fixed, and dehydrated in a sucrose gradient. Coronal brain sections were subjected to Nissl staining according to the manufacturer's protocol (Nissl Staining Kit, C0117, Beyotime). The brain tissues were fixed with paraformaldehyde and dehydrated with sucrose solution following the protocol of the Beyotime Nissl Staining Kit. The staining of Nissl bodies within the cytoplasm of neurons was strictly completed accordance with the manufacturer's instructions (Wuhan Good-bio Technology CO, LTD, Wuhan, Hubei, China).

Statistical analysis

Data were presented as mean ± SEM and analyzed using GraphPad prism9 (GraphPad Software, San Diego, CA, USA). One-way ANOVA was applied for multiple group comparisons. Student's t-test was used for western blot analyses and assessment of behavioral tests. Significance was accepted at the 95% confidence level (p < 0.05). A p-value < 0.05 was considered statistically significant.

Results

The phosphorylated peptide Tau-pT181 promotes the aggregation of the R3 fragment

Although the phosphorylation of Tau protein at the Thr181 site is currently used as a blood-based biomarker for predicting AD pathology in clinical practice,^11,17 there is no direct evidence that phosphorylation at this site directly promotes AD pathology, particularly Tau pathology like Tau aggregation. Therefore, we first synthesized and obtained the phosphorylated peptide Tau-pT181 (containing the phosphorylated Thr181 site) and the non-phosphorylated peptide Tau-nT181, respectively (Figure 1A).

Figure 1.

Investigating the impact of phosphorylated peptides Tau-pT181 on R3 fragment aggregation. (A) The amino acid sequences of three polypeptides were synthesized in vitro. (B) Th S fluorescence spectrometric results of R3 peptide fragment aggregation during concubation with Tau-pT181 +R3 or Tau-nT181 +R3 (the specific reaction system in Table 2).

The minimal aggregation motifs of Tau protein are VQIINK located in the R2 repeat region and VQIVYK in the R3 repeat region. Among these, the VQIVYK motif in R3 is present in all six isoforms of Tau protein, whereas the VQIINK motif in R2 exists only in 4R-Tau isoforms.¹⁸ Notably, VQIVYK serves as the primary sequence responsible for pathological Tau aggregation in NFTs found in the brains of AD patients.¹⁹ To establish a standardized peptide for in vitro Tau aggregation assays, we employed solid-phase peptide synthesis technology to produce a 31-amino acid residue peptide containing the VQIVYK hexapeptide motif (Figure 1A). This synthesized peptide corresponds to the R3 sequence of Tau protein (Repeat Domain R3 fragment, R3-normal) and serves as a reference standard for studying Tau aggregation in vitro.

Thioflavin S assay revealed that during incubation, the R3 peptide gradually formed self-aggregates over time, whereas neither Tau-pT181 nor Tau-nT181 could form aggregates independently. However, when co-incubated with R3 peptide, Tau-pT181 significantly enhanced R3 aggregation, while Tau-nT181 showed no such effect (Figure 1B). These results suggest that phosphorylation at T181 may promote Tau protein aggregation.

Tau-pT181 promoted Tau aggregation in vitro

To further elucidate whether phosphorylation at the T181 site directly induces Tau aggregation, we treated HEK293/hTau cells with either Tau-pT181 or Tau-nT181 peptides in different concentrations. After 48 h of treatment, western blot analysis demonstrated that neither the phosphorylated nor non-phosphorylated peptides affected Tau phosphorylation status or total Tau protein levels in these cells (Figure 2A-C). Strikingly, the phosphopeptide (Tau-pT181) significantly increased the ratio of insoluble to soluble Tau. Meanwhile, The higher the concentration of Tau-pT181, the stronger their effect on the aggregation of endogenous Tau protein. while the non-phosphorylated peptide (Tau-nT181) showed no such effect (Figure 2A, D). We obtained consistent results in primary neurons, further strengthening our findings (Figure 2E-H). These results collectively suggest that phosphorylation at T181 promotes the aggregation of endogenous Tau protein in cells.

Figure 2.

In Vitro promotion tau aggregation by phosphorylated polypeptide Tau-pT181. (A) Western blotting were performed for phosphorylated Tau, soluble Tau and insoluble Tau proteins in HEK293-hTau cells. (B-D) Quantitative analysis of the bands in (A). n = 3 per group. (E) Western blotting were performed for phosphorylated Tau, soluble Tau and insoluble Tau proteins in primary neurons. (F-H) Quantitative analysis of the bands in (E). ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 versus group 1. *p < 0.05, **p < 0.01, ***p < 0.001 versus group 2. All data represent mean ± SEM.

Tau-pT181 impaired synapse-associated proteins in primary hippocampal neurons

The primary biological function of Tau protein is to promote microtubule stabilization and assembly.²⁰ Tau pathology, including tau aggregation, leads to the loss of its biological function, resulting in synaptic damage and even the reduction of synapse-associated proteins.²¹ To determine whether Tau-pT181-induced Tau aggregation causes the loss of synaptic proteins, we treated primary hippocampal neurons with Tau-pT181 and Tau-nT181 for 48 h, followed by protein homogenate extraction and western blot analysis to measure the levels of synaptic proteins (Figure 3A). The results showed that Tau-pT181 reduced the levels of synaptophysin (Figure 3B), Synapsin1 (Figure 3C), and PSD-95 (Figure 3D) compared to the control group, The higher the concentration of Tau-pT181, the stronger the damage to the synaptic-related proteins, whereas Tau-nT181 had no effect on these proteins (Figure 3 A-D). This finding indicates that, in primary hippocampal neurons, phosphorylation of Tau at T181 site damages synapses probably by promoting tau aggregation.

Figure 3.

Reduced synapse associated proteins levels in primary hippocampal neurons induced by phosphorylated polypeptide Tau-pT181. (A) Western blots and B-D) quantitative analysis for Synaptophysin, Synapsin-1 and PSD-95 in primary hippocampal neurons. n = 3 per group. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 versus group 1. **p < 0.01, ***p < 0.001, ****p < 0.0001 versus group 2.

Tau-pT181 induced cognitive impairments in rats

Since synaptic impairment compromises cognitive function,²² we further investigated whether the Tau-pT181 peptide could induce cognitive deficits in rats. Using stereotaxic intracerebral microinjection, we administered either phosphorylated or non-phosphorylated peptides into the CA1 hippocampal subregion of rats, with sterile saline serving as the vehicle control group. Behavioral tests were performed one-month post-injection (Figure 4A). The open-field test revealed that both Tau-pT181 and Tau-nT181 injected rats exhibited similar patterns of movement and exploratory activity comparable to saline-treated control animal (Figure 4B, C). The novel object recognition test demonstrated a significantly lower discrimination index in the phosphorylated peptide-treated group compared to controls (Figure 4D), indicating impaired learning and memory function in rats administered with the Tau-pT181 peptide. Fear conditioning test revealed significantly reduced the immobility time in Tau-pT181-treated rats compared to saline controls, while there was no effect on rats treated with non-phosphorylated peptides (Figure 4E), indicating that phosphorylation of Tau at T181 impairs contextual memory formation.

Figure 4.

Cognitive impairment induction in rats by phosphorylated polypeptide tau-pT181. (A) Two-month-old SD rats were respectively subjected to open field, new object recognition, Morris water maze, and fear conditioning test after being injected with polypeptides in CA1 brain region. (B-C) In open field test, the movement speed and total distance of the rats were recorded. (D) In new object recognition test, the recognition index of phosphorylated polypeptide treated rats was reduced when compared to that of control rats. (E) In fear conditioning test, the percentage freezing time of rats in the phosphorylated polypeptide group was lower than control group. (F) In Morris water maze, rats learned and memorized the latency time of each day. (G) In test day, the movement time of rats in the target quadrant. (H) The time when rats crossed the target position for the first time. (I) The number of times rats crossed the target position. ns, not signiﬁcant. **p < 0.01, ***p < 0.001.

Morris water maze analysis showed Tau-pT181 rats demonstrated significantly prolonged escape latency in locating the hidden platform compared to saline animals, whereas non-phosphorylated peptide-treated rats showed no significant difference in acquisition latency relative to controls (Figure 4F). On day 6 of testing, Tau-pT181 rats exhibited significantly lower performance than controls in target quadrant occupancy time, first platform-crossing latency and the number of times crossing the target platform when compared to saline rats (Figure 4G-1), indicating that the Tau-pT181 peptide impaired the hippocampal-dependent spatial memory consolidation of rats. Together, these findings suggest that phosphorylation of Tau at T181 site damage learning and memory abilities.

Tau-pT181 induced Tau aggregation, synaptic damage, and neuronal loss in vivo

To evaluate whether the learning and memory deficits in Tau-pT181 rats were associated with Tau phosphorylation and aggregation, we performed immunoblotting analysis. Consistent with the in vitro western blotting results, Tau-pT181 and Tau-nT181 did not alter Tau phosphorylation levels at Thr181, Thr231, Ser262, or total Tau compared to control animals (Figure 5A-C). However, Tau-pT181 significantly increased the ratio of insoluble to soluble Tau in the hippocampus, whereas Tau-nT181 exhibited no effect on Tau solubility (Figure 5A, D). These findings demonstrate that Thr181 phosphorylation specifically promotes Tau aggregation without influencing phosphorylation at other epitopes.

Figure 5.

Phosphorylated polypeptide Tau-pT181 induces tau aggregation, synaptic damage and neuron loss in vivo. (A) Western blotting were conducted for phosphorylated Tau, soluble Tau and insoluble Tau proteins in rat hippocampus. (B-D) Quantitative analysis of the bands in (A). (E) Western blotting of Syn-1, PSD-95, and β-actin. (F-G) Quantitative analysis of the bands in (E). (H) Representative images of Golgi staining of dendritic spines in the rat hippocampus, scale bar 10 μm. (I) Quantitative analysis of dendritic spine density, 7–9 intact dendrites per group were selected and counted. (J) Representative images of Nissl staining of rat brain sections with scale bars of 500 μm and magnification scale of 200 μm. (K) The quantitative analysis of the number of neurons in DG, CA1 and CA3 regions of hippocampus. The cell numbers in the DG, CA1 and CA3 area were counted by Image software in 2–3 brain sections from each rat hippocampus. All data represent mean ± SEM. ns, not signiﬁcant. *p < 0.05, **p < 0.01, ***p < 0.001.

To further elucidate the synaptic effects of phosphorylated peptides, we examined synaptic-related proteins in the hippocampi of peptide-treated rats. Consistent with observations in primary neurons, Tau-pT181 treatment significantly downregulated hippocampal levels of synaptophysin-1 and PSD-95 compared to controls, whereas Tau-nT181 showed no detectable alterations in synaptic proteins (Figure 5E-G). These data further support that Tau-pT181 selectively impairs synaptic integrity.

Since Tau phosphorylation and aggregation are associated with neuronal loss, we examined the effects of Tau peptides on hippocampal neuronal morphology and viability. Golgi Staining showed that Tau-pT181 treatment induced a significant reduction in dendritic spine density, suggesting impaired synaptic connectivity, while no morphological alterations were observed in Tau-nT181-treated neurons (Figure 5H, 1). Nissl Staining revealed that Tau-pT181 caused marked neuronal depletion in the hippocampus, particularly in CA1 and CA3 subfields, while Tau-nT181 showed no significant differences versus controls (Figure 5J, K).

Collectively, our data demonstrate that Tau-pT181 directly contributes to neurodegeneration through both synaptic impairment and neuronal death, while non-phosphorylated Tau exhibits no detectable toxicity.

Transcriptional analysis on human iPSC-derived neurons post-peptide intervention

To investigate the neuronal impact of these peptides, we conducted transcriptome analysis on human induced pluripotent stem cell (iPSC)-derived neurons following intervention with Tau-pT181 and Tau-nT181 peptides. Compared with the control group, the Tau-nT181 treatment group exhibited significant alterations in 7087 genes, among which 135 genes were upregulated (in red) and 481 genes were downregulated (in blue); while the Tau-pT181-treated group showed differential expression in 5013 genes, among which 224 genes were upregulated (in red) and 110 genes were downregulated (in blue). The above analysis results indicate that peptide treatment led to significant changes in the expression of some genes in the iPSCs-derived neurons. Compared with non-phosphorylated Tau-nT181, the differences in expression of genes (DEG) after Tau-pT181 peptide treatment were more significant. To explore the biological functions of the selected DEGs, the differential genes were subjected to GO functional annotation analysis. It was found that the differentially expressed genes mainly involved biological processes such as axonogenesis, neuronal development, forebrain development, and Wnt signaling pathway, and mainly participated in cellular components such as transport vesicles, focal adhesions, synapses, and membranes (Figure 6A-D). They mainly had molecular functions such as binding GTPase, calcium-binding protein, ubiquitin protein ligase, ubiquitin-like protein ligase, microtubule protein, and regulating GTPase, nucleotide triphosphatase. The transcriptome results suggest that phosphorylated peptides have a much greater effect on iPSCs-derived neurons than non-phosphorylated peptides.

Figure 6.

After peptide treatment with iPSCs, transcriptome sequencing analysis. Volcano plot of differentially expressed genes. (A) Tau-nT181 versus control (B) Tau-pT181 versus control. Go function annotation analysis. (C) Tau-nT181 versus control (D) Tau-pT181 versus control.

Discussion

With the acceleration of social modernization and population aging trends, Alzheimer's disease (AD) has experienced a rapid surge in incidence,²³ emerging as one of the most pressing global public health challenges.²⁴ However, current clinical practices face diagnostic uncertainties and lack effective therapeutic interventions, primarily due to the undefined core pathogenic mechanisms and actionable drug targets in AD. While Tau phosphorylation at Thr181 serves as a diagnostic biomarker in clinical settings,²⁵ current evidence insufficiently characterizes the causal relationship between hyperphosphorylation of Tau at Thr181 and subsequent Tau pathology,²⁶ with existing data being primarily correlative rather than mechanistic. In the current study, we synthesized the phosphorylated tau peptide Tau-pT181 and the non-phosphorylated tau peptide Tau-nT181, and evaluate the direct effect of these peptides on tau aggregation, synaptic and cognitive impairments.

Tau is a microtubule-associated protein whose C-terminal region contains 3 or 4 microtubule-binding repeat domains (MTBR, R1-R4), which are critical for regulating microtubule stability.²⁷ Among these, the third repeat domain (R3) holds unique pathological significance due to its β-sheet-prone sequences, making it a core fragment that promotes Tau misfolding and aggregation.²⁸ In vitro studies demonstrate that purified R3 peptides autonomously form amyloid fibrils without cofactors, exhibiting morphological features identical to Tau filaments isolated from AD brains. The hexapeptide motif 306VQIVYK311 within R3 serves as a critical β-sheet nucleation site, driving Tau protofilament assembly via hydrophobic interactions. Neurons overexpressing the R3 fragment exhibit pronounced Tau aggregation and neurotoxicity, confirming its central role in pathogenesis.²⁹ In this study, we synthesized repeat domain R3 fragment (R3-normal) served as a reference standard for studying Tau aggregation in vitro and found Tau-pT181 significantly enhanced R3 aggregation, suggesting that phosphorylation at T181 may promote Tau protein aggregation.

Tau aggregation is a typical feature of Tau pathology.³⁰ At the cellular level, we examined the effects of Tau-pT181 on Tau aggregation.⁹ Our findings demonstrate that the phosphorylated peptide (Tau-pT181) significantly increases the ratio of insoluble to soluble Tau in both HEK293/Tau cells and primary neurons, further supporting that phosphorylation at T181 promotes the aggregation of endogenous Tau protein within cells. Tau aggregation correlates with synaptic dysfunction and diminished levels of key synaptic proteins. Our experiments in primary neurons demonstrate that Tau-pT181 significantly reduces the abundance of synapse-associated proteins. These results provide direct evidence linking Tau phosphorylation at T181 to Tau aggregation and synaptic damage.

Cognitive decline is the primary clinical phenotype of AD.³¹ We investigated the effects of Tau-pT181 peptide on rat cognition and found that Tau-pT181 induced significant cognitive impairment, accompanied by Tau aggregation, synaptic damage, and neuronal loss. Tau-nT181 showed no effect, pinpointing Tau phosphorylation at T181-dependent toxicity and cognitive impairment.

Intriguingly, our study revealed a paradoxical phenomenon: while Tau-pT181 induced aggregation of endogenous Tau, it did not promote further phosphorylation of endogenous Tau at other sites. We speculate that (i) The Tau-pT181 peptide may not, in fact, influence Tau kinase or phosphatase activity—a conclusion that awaits further experimental validation, (ii) Tau phosphorylation at T181 indeed does not cause phosphorylation at other sites of tau, (iii) The phosphorylation sites detected need to be further expanded and increased, or (iv) The Tau-pT181 peptide may potentially act as a seed to promote the aggregation of endogenous Tau and further drive Tau pathology.

To further elucidate Tau-pT181's neuronal impact, we performed transcriptomic analysis on human iPSC-derived neurons treated with Tau-pT181/Tau-nT181 peptides. Notably, the phosphorylated peptide exerted significantly stronger effects than its non-phosphorylated counterpart, with pathway enrichment in axonogenesis, neuronal development, and Wnt signaling, providing crucial mechanistic leads for future research.

While pT181-Tau is established as a diagnostic biomarker, its functional contribution to Tau aggregation, microtubule destabilization,³² and synaptic toxicity requires rigorous experimental validation through (i) Site-specific phosphomimetic studies, (ii) Cryo-EM structural analysis of pT181-modified filaments, (iii) In vivo non-phosphorylation-rescue models.³³

In summary, this study establishes direct evidence linking Tau phosphorylation at T181 to Tau aggregation, its downstream neuro-pathological changes and cognitive deficits. Given that the Tau-pT181 can trigger Tau pathological events, blocking phosphorylation of Tau at Thr181 may provide a potential strategy for treating AD.

Footnotes

Acknowledgements

The authors are grateful to Mr Dan Ke and Ms. Qun Wang for helpful technical suggestions during the conduct of this study.

ORCID iDs

Yong Luo

Anqi Yin

Zhizhou Huang

Rong Liu

Xiaochuan Wang

Yuran Gui

Ethical considerations

No humans were used in this research. All animal experiments were approved by the Animal Care and Use Committee of Huazhong University of Science and Technology, and performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Consent to participate

Not applicable

Consent for publication

Not applicable

Author contribution(s)

Yong Luo: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Resources; Software; Supervision; Validation; Visualization; Writing – original draft; Writing – review & editing.

Gang Wu: Conceptualization; Data curation; Project administration; Resources; Validation; Writing – original draft.

Yi Liu: Conceptualization; Data curation; Writing – review & editing.

Anqi Yin: Conceptualization; Data curation; Writing – review & editing.

Zhizhou Huang: Conceptualization; Data curation; Funding acquisition; Resources; Writing – review & editing.

Jian-Zhi Wang: Funding acquisition; Investigation; Writing – review & editing.

Rong Liu: Conceptualization; Data curation; Writing – review & editing.

Xiaochuan Wang: Funding acquisition; Investigation; Writing – review & editing.

Yuran Gui: Funding acquisition; Investigation; Writing – review & editing.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported in parts by grants from National Natural Science Foundation of China (grant number 82330041), grant from Science and Technology Innovation Team project to Xiaochuan Wang from Department of Science and Technology of Hubei Province (grant number 2022-72-18) and Science, Technology and Innovation Commission of Shenzhen Municipality (grant number JCYJ20240813153427036).

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 datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.

References

Liu

Tan

Zhang

, et al. The interaction between ageing and Alzheimer's disease: insights from the hallmarks of ageing. Transl Neurodegener 2024; 13: 7.

Zhang

Wang

, et al. Recent advances in Alzheimer's disease: mechanisms, clinical trials and new drug development strategies. Signal Transduct Target Ther 2024; 9: 211.

Watamura

Foiani

Bez

, et al. In vivo hyperphosphorylation of tau is associated with synaptic loss and behavioral abnormalities in the absence of tau seeds. Nat Neurosci 2025; 28: 293–307.

van der Kant

Goldstein

LSB

Ossenkoppele

. Amyloid-β-independent regulators of tau pathology in Alzheimer disease. Nat Rev Neurosci 2020; 21: 21–35.

Zahra

Rubab Khakwani

Acosta

, et al. Neurofibrillary tangles predict dementia in patients with carotid stenosis. J Vasc Surg 2025; 81: 1381–1388.e1382.

Meng

Zhang

Saman

, et al. Hyperphosphorylated tau self-assembles into amorphous aggregates eliciting TLR4-dependent responses. Nat Commun 2022; 13: 2692.

Liu

Sui

Dexheimer

, et al. Hyperphosphorylation renders tau prone to aggregate and to cause cell death. Mol Neurobiol 2020; 57: 4704–4719.

Rawat

Sehar

Bisht

, et al. Phosphorylated tau in Alzheimer's disease and other tauopathies. Int J Mol Sci 2022; 23: 12841.

Parra Bravo

Naguib

Gan

. Cellular and pathological functions of tau. Nat Rev Mol Cell Biol 2024; 25: 845–864.

10.

Janelidze

Mattsson

Palmqvist

, et al. Plasma P-tau181 in Alzheimer's disease: relationship to other biomarkers, differential diagnosis, neuropathology and longitudinal progression to Alzheimer's dementia. Nat Med 2020; 26: 379–386.

11.

Chen

Huang

Shen

, et al. Longitudinal plasma phosphorylated tau 181 tracks disease progression in Alzheimer's disease. Transl Psychiatry 2021; 11: 356.

12.

Lantero Rodriguez

Karikari

Suárez-Calvet

, et al. Plasma p-tau181 accurately predicts Alzheimer's disease pathology at least 8 years prior to post-mortem and improves the clinical characterisation of cognitive decline. Acta Neuropathol 2020; 140: 267–278.

13.

Antonioni

Raho

Manzoli

, et al. Blood phosphorylated Tau181 reliably differentiates amyloid-positive from amyloid-negative subjects in the Alzheimer's disease continuum: a systematic review and meta-analysis. Alzheimers Dement (Amst) 2025; 17: e70068.

14.

Luo

Guo

, et al. Conformation pattern changes in R1-pS262 tau peptide induced endogenous tau aggregation, synaptic damage, and cognitive impairments. J Alzheimers Dis 2025; 103: 951–965.

15.

Wang

Zhou

Jiang

, et al. A novel small-molecule PROTAC selectively promotes tau clearance to improve cognitive functions in Alzheimer-like models. Theranostics 2021; 11: 5279–5295.

16.

Jiang

, et al. Phosphorylation of truncated tau promotes abnormal native tau pathology and neurodegeneration. Mol Neurobiol 2022; 59: 6183–6199.

17.

Karikari

Pascoal

Ashton

, et al. Blood phosphorylated tau 181 as a biomarker for Alzheimer's disease: a diagnostic performance and prediction modelling study using data from four prospective cohorts. Lancet Neurol 2020; 19: 422–433.

18.

Seidler

Boyer

Rodriguez

, et al. Structure-based inhibitors of tau aggregation. Nat Chem 2018; 10: 170–176.

19.

Ganguly

Larini

, et al. Tau assembly: the dominant role of PHF6 (VQIVYK) in microtubule binding region repeat R3. J Phys Chem B 2015; 119: 4582–4593.

20.

Barbier

Zejneli

Martinho

, et al. Role of tau as a microtubule-associated protein: structural and functional aspects. Front Aging Neurosci 2019; 11: 204.

21.

Chongtham

Ramakrishnan

Farinas

, et al. Neocortical tau propagation is a mediator of clinical heterogeneity in Alzheimer's disease. Mol Psychiatry 2025; 30: 4194–4213.

22.

Luan

Wang

Huang

, et al. Synaptic loss pattern is constrained by brain connectome and modulated by phosphorylated tau in Alzheimer's disease. Nat Commun 2025; 16: 6356.

23.

Luo

Zheng

. Trends and challenges for population and health during population aging – China, 2015-2050. China CDC Wkly 2021; 3: 593–598.

24.

Khan

HTA

Addo

Findlay

. Public health challenges and responses to the growing ageing populations. Public Health Chall 2024; 3: e213.

25.

Lleó

Zetterberg

Pegueroles

, et al. Phosphorylated tau181 in plasma as a potential biomarker for Alzheimer's disease in adults with Down syndrome. Nat Commun 2021; 12: 4304.

26.

Cheng

Fransson

Mani

. Modulation of pTau181 by glypican-1-derived heparan sulfate in human neural progenitor cells and ApoE4-expressing induced neurons. J Neurochem 2025; 169: e70162.

27.

Bhandare

Kumbhar

Kunwar

. Differential binding affinity of tau repeat region R2 with neuronal-specific β-tubulin isotypes. Sci Rep 2019; 9: 10795.

28.

Stöhr

Nick

, et al. A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells. Nat Chem 2017; 9: 874–881.

29.

Liu

Zhong

Liu

, et al. Disclosing the mechanism of spontaneous aggregation and template-induced misfolding of the key hexapeptide (PHF6) of tau protein based on molecular dynamics simulation. ACS Chem Neurosci 2019; 10: 4810–4823.

30.

Zhang

Wang

Zhang

, et al. Tau in neurodegenerative diseases: molecular mechanisms, biomarkers, and therapeutic strategies. Transl Neurodegener 2024; 13: 40.

31.

Ingram

Ocal

Halai

, et al. Graded multidimensional clinical and radiologic variation in patients with Alzheimer disease and posterior cortical atrophy. Neurology 2024; 103: e209679.

32.

Giacomucci

Mazzeo

Crucitti

, et al. Plasma p-tau181 as a promising non-invasive biomarker of Alzheimer's disease pathology in subjective cognitive decline and mild cognitive impairment. J Neurol Sci 2023; 453: 120805.

33.

Tagai

Tatebe

Matsuura

, et al. A novel plasma p-tau181 assay as a specific biomarker of tau pathology in Alzheimer's disease. Transl Neurodegener 2024; 13: 44.