CDK5-mediated hyperphosphorylation of Tau217 impairs neuronal synaptic structure and exacerbates cognitive impairment in Alzheimer’s disease

Abstract

Numerous studies have demonstrated that tau phosphorylated at threonine 217 (p-T217) in cerebrospinal fluid (CSF) or plasma is a potential biomarker for Alzheimer’s disease (AD). However, the detailed pathological effects of elevated p-T217 and the mechanisms underlying T217 phosphorylation remain incompletely understood. In this study, we revealed a role of tau phosphorylated at T217 in AD. In 5 × FAD mice, increased p-T217 levels, correlated with CDK5 activation, were associated with neurite damage and neuronal apoptosis. Mice expressing a phospho-mimetic T217E mutant in the hippocampus exhibited significant learning impairments in the Morris water maze and Y-Maze test, along with reduced levels of the synaptic proteins Drebrin and PSD95. Electron microscopy revealed severe synaptic and microtubules damage in these mice, along with disrupted axonal structures confirmed by Golgi staining. Additionally, hyperactivation of CDK5 through p25 overexpression increased T217 phosphorylation, whereas CDK5 inactivation reduced it. The study concludes that CDK5 mediated Tau phosphorylation at T217 contributes to synaptic damage and cognitive deficits, highlighting it as a potential therapeutic target for AD.

Introduction

Alzheimer’s disease (AD) is the leading cause of dementia, and the number of dementia-related death is projected to rise to 4.91 million by 2050, including 0.18 million cases under the age of 70 [1]. Despite its growing prevalence, treatment options for AD remains limited, making it one of the most burdensome and lethal diseases worldwide [2]. Although the precise pathological mechanisms of AD have yet to be fully elucidated, recent studies have yielded valuable insights. Among these promising achievements, significant attention has been directed toward the tau protein and its hyperphosphorylation.

Numerous studies have identified tau phosphorylated at threonine 217 (p-T217) in CSF or plasma as a promising diagnostic and prognostic biomarker for AD [3, 4]. Elevated p-T217 levels have been positively correlated with brain atrophy and cognitive impairment in AD patients [5, 6]. Notably, some studies have also linked p-T217 with tauopathies, demonstrating that hyperphosphorylation at Tau T217 exacerbates tau pathologies and cognitive impairment [7]. These findings indicate that P-T217 may not only serve as a biomarker for AD but could also play a neurotoxic role in AD progression. However, regulatory mechanisms underlying Tau217 phosphorylation still remain unclear and require further investigation. An in vitro mass spectrometry assay conducted by Lund ET et al. [8] identified T217 as a potential phosphorylation sites for CDK5. Furthermore, the amino acid sequences of Tau is conserved across species-including mice (Mus musculus), chickens (Gallus gallus), zebrafish (Danio rerio)-and contains a conserved proline-directed phosphorylation site, supporting the hypothesis that T217 could be a substrate of CDK5 [9]. Based on these findings, we hypothesize that the CDK5-T217 pathway is involved in the pathophysiological processes of AD and may contribute to the progression of cognitive decline in AD.

Materials and methods

Mice

p35 knockout mice on a C57BL/6J background were obtained from Jackson Laboratory (stock number: # 004163, USA). 5 × FAD mice on a C57BL/6J background and C57BL/6J mice were obtained from Gene&Peace CO Ltd. (stock number: # N000013, # 008730, Jiangsu, China). All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Fujian Medical University (FJMU IACUC 2024-0056). Tail samples were genotyped using specific sets of PCR primers for p35 knockout mice (Table S1 and Fig. S1).

Primary neuronal culture

The procedures for primary neuronal culture were adapted from previous reports [10]. In brief, pregnant C57BL/6J mice were anesthetized, and embryos at E16.5–17.5 were removed from the uterus and immediately transferred to Neurobasal Medium (CAT#: 21103049; Gibco, USA) on ice in a biological safety cabinet. The hippocampi and cortex were dissected and digested with 2 μg/mL papain (CAT#: LS003126; Worthington, USA) and 5 U/mL DNase I (CAT#: D8071; Solarbio, Beijing, China) at 37 °C for 30 min. After digestion, the neurons were cultured in Neurobasal supplemented with 2% B27 (CAT#: 17504044; Gibco), 1% N2 (CAT#: 17502048; Gibco), and 1 × GlutaMAX™-I (CAT#: 35050061; Gibco). The cells seeded onto 10 μg/mL Poly-D-Lysin (CAT#: C02-01001; Bioss, Beijing, China) coated plates.

Infection of the primary neurons

The following plasmids were used for AAV production by OBIO Technology (Shanghai, China), pAAV-CMV-MCS-EF1a-EGFP-tWPA (empty vector, referred to as GFP), pAAV-CMV-p25 (CDK5R1: 99–307aa)-3 × FLAG-EF1a-EGFP-tWPA (referred to as p25), pAAV-CMV -MAPT- 3 × FLAG-EF1a-EGFP-tWPA (referred to as TauWT), pAAV-CMV-MAPT-(T217A) 3 × FLAG-EF1a-EGFP-tWPA (referred to as TauT217A), and pAAV-CMV-MAPT-(T217E)-3 × FLAG-EF1a-EGFP-tWPA (referred to as TauT217E). The human Tau (MAPT) gene (NCBI GeneID: 4137) was cloned from human cDNA encoding the 441-amino acid isoform of microtubule-associated protein tau (RefSeq: NM_005910.6). The cultured neurons were transduced with the purified AAVs 12 h after plating, and the medium was replaced with fresh culture medium after 24 h of incubation.

Stereotaxic injection of the virus

The following plasmids were used for AAV production by OBIO Technology, pAAV-hSyn-EGFP-P2A-3 × FLAG-WPRE (empty vector, referred to as GFP), pAAV-hSyn-EGFP-P2A-TAU-3 × FLAG-WPRE (referred to as TauWT), pAAV-hSyn-EGFP-P2A-MAPT(T217A)-3 × FLAG-WPRE (referred to as TauT217A), and pAAV-hSyn-EGFP-P2A-MAPT(T217E)-3 × FLAG-WPRE (referred to as TauT217E). 5 × FAD mice were randomly assigned to experimental groups using a random number table prior to experimentation.

5 × FAD mice (3 months old, males, n = 10–11 each group) were anesthetized using isoflurane and injected with 1 μL of AAV-GFP, AAV-Tau, AAV-TauT217A, or AAV-TauT217E into the CA1 region of the hippocampus in each hemisphere using a stereotactic instrument (RWD Life Science, Shenzhen, China). Injections were performed with a Hamilton syringe using the following coordinates (relative to bregma): anterior = +2 mm, lateral = ±1.45 mm from midline, depth = 1.35 mm (from dura).

Y-maze test

The procedures for Y-Maze and Morris water maze tests were adapted from previous reports [11]. 5 × FAD mice (n = 10–11) were tested for the Y-maze (Noldus, Netherlands) at 2 and 5 months pos-stereotaxic injection. Each test began with the mouse being placed at the end of one arm, and the trial lasted for 8 min. The number of times the mice visited a new arm was recorded. Spontaneous alternation rate (%)=(number of spontaneous alternations / total number of times entering the arm - 2) × 100%. Different researchers were assigned to conduct the experimental procedures and data analysis in the behavioral experiments.

Morris water maze test and tissue preparation

8-month-old 5 × FAD mice injected with different viruses (n = 10–11) were trained from random starting location in the four quadrants of the water maze apparatus (XR-XM101-M, Shxinruan, Shanghai, China). Each mouse underwent 4 trials per day over 6 consecutive days, each lasting up to 60 s. If the mouse failed to find the hidden platform within this time, it was manually guided to the platform and allowed to stay there for 15 s. After 6 days, the platform was removed, and the relevant data were recorded over 60 s. All trials were analyzed in real time using an automatic tracking system (VisuTrack, Shxinruan, China).

Immunostaining

The procedures for immunostaining were adapted from previous reports [12]. Both the brain sections (n = 3) and cells slides (n = 4–6) were blocked with a blocking buffer, followed by overnight incubation with Phospho-Tau (Thr217) (1:1000, CAT#: 44–744, Invitrogen, USA), β-III Tubulin antibody (1:1000, CAT#: ab18207, Abcam, USA), and DYKDDDDK Tag (1:1000, CAT#: 8146T; Invitrogen) at 4 °C. After washing, the brain slices or cells coverslips were incubated with secondary antibodies: anti-mouse IgG, Alexa Fluor® 488 (1:1000, CAT#: 4408; Cell Signaling Technology, USA), anti-mouse IgG, Alexa Fluor® 594 (1:1000, CAT#: 8890; Cell Signaling Technology), anti-rabbit IgG, Alexa Fluor® 488 (1:1000, CAT#: 4412; Cell Signaling Technology), or anti-rabbit IgG, Alexa Fluor® 594 (1:1000, CAT#: 8889; Cell Signaling Technology) for 1 h in the dark. The cell nuclei were counterstained with DAPI (CAT#: C1005; Beyotime, China) for 3 min. Fluorescence images were acquired using confocal microscopy (TCS SP8; Leica, Germany) and analyzed using ImageJ software (Fiji, ImageJ 1.47, NIH, Bethesda, MD, USA).

Western blot

Samples (n = 3–4) were collected using RIPA lysis buffer (CAT#: P0013B; Beyotime). Proteins were denatured at 100 °C and then transferred onto polyvinylidene difluoride (PVDF) membranes (CAT#: IPVH00010; Millipore, Germany). The membranes were blocked with 5% dry milk (CAT#: 9999; Cell Signaling Technology) and incubated with primary antibodies against Phospho-Tau (Thr217) (1:1000, CAT#: 44–744, Invitrogen), TAU-5 (1:1000, CAT#: ab80579; Abcam), p35 (1:1000, CAT#: sc-820, Santa Cruz Biotechnology, USA), Anti-FLAG M2 (1:1000, CAT#: F1804, Sigma), Cdk5 (1:1000, CAT#: sc-6247, Santa Cruz Biotechnology), Drebrin (1:1000, CAT#: ab60933; Abcam), PSD95 (1:1000, CAT#: ab18258; Abcam) or β-Actin (1:2500, CAT#: GB15001; Servicebio, Wuhan, China) overnight at 4 °C. The membranes were incubated with the horseradish peroxidase (HRP)-conjugated goat anti-mouse (1:5000, CAT#: AP123P; Millipore) or anti-rabbit (1:5000, CAT#: AP132P; Millipore) secondary antibodies.

Transmission electron microscopy

Mouse brains (n = 3) were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde, dehydrated through a gradient of ethanol solutions, processed in a 1:1 ratio of acetone to EPON618, and gradually embedded in EPON618. The samples were sectioned at a thickness of 10 nm using a Leica UC7 ultramicrotome system (Leica Microsystems, Germany). Cellular ultrastructure were visualized using a transmission electron microscopy (Tecnai G2 20 S-TWIN TEM).

Golgi staining

Golgi staining was performed following the protocol of the Hito Golgi‐Cox OptimStain™ Kit (CAT#: HTKNS1125; HitoBiotec Corp, USA). The mouse brain (n = 3) was incubated in a mixture of solution 1 and solution 2 for two weeks, then in solution 3 for 72 h. After cooling in isopentane at −70 °C for 60 s, the brains were sectioned at 100 μm sections. The brain slices were treated with a mixture of solutions 4 and 5 for 10 min, dehydrated in 50–100% ethanol, cleared in xylene, and imaged using a Leica TCS SP8 confocal microscope (Germany).

TUNEL staining

Apoptotic cells were detected using the One-step TUNEL In Situ Apoptosis Kit (CAT#: E-CKA322; Elabscience, Wuhan, China). Cell slides (n = 11–12) were fixed in methanol for 20 min, permeabilized with Triton X-100 for 10 min, treated with equilibration buffer for 30 min, and incubated with TdT enzyme reaction working solution for 4 h. DAPI was used to counterstain nuclei. Fluorescence images were captured using a Leica DMi8 fluorescence microscope.

Statistical analysis

All experiments were performed at least 3 times. Data were presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 10.0 (La Jolla, CA, USA). The first 6 days of water maze testing were analyzed by repeated-measure two-way ANOVA. For other experiments, Student’s t-test was used for two groups analyses, and one-way ANOVA with Tukey’s post-hoc multiple comparison test was used for comparisons among three or more groups. A p-values < 0.05 was considered statistically significant.

Results

Phosphorylated Tau217 and p25 are increased in 5×FAD mice

In AD, abnormal phosphorylation of Tau protein and dysregulation of kinases such as CDK5 are key pathological features. To investigate the involvement of Tau217 phosphorylation and CDK5 overactivation in AD progression, the 5 × FAD mouse model was employed. As shown in Fig. 1A, B, the ratio of phosphorylated Tau217 to total Tau (detected using Tau5 antibody) in cortical tissues of 8-month-old 5 × FAD mice was significantly higher (p < 0.05) compared to age-matched WT mice (p < 0.05). p25, a truncated form of p35 (Fig. 1C) and CDK5 (Fig. 1D) protein levels, but not p35 (Fig. 1E), were significantly higher in 5 × FAD mice relative to WT control (p < 0.05). These results indicate that Tau217 hyperphosphorylation and abnormal CDK5 overactivation are present in 8-month-old 5 × FAD mice.

Fig. 1. Tau217 is hyperphosphorylated and CDK5 is overactivated in 8-month-old 5×FAD mice.

A, B P-Tau217 and total Tau5 protein levels were measured by western blot, and the ratio of P-Tau217 to Tau5 was analyzed. A, C p25 protein levels were measured by western blot, and the ratio of p25 to β-actin was calculated. A, D CDK5 protein expression was detected by western blot, and the ratio of CDK5 to β-actin was analyzed. A, E p35 protein levels were detected by western blot, and the ratio of p35 to β-actin was carried out. The images were analyzed using ImageJ software. Data were presented as mean ± SEM. n = 3 mice per group. Student’s t-test. *p < 0.05.

Hyperphosphorylation of Tau217 leads to neuronal death and exacerbates cognitive dysfunction in 5×FAD mice

Primary cortical neurons from C57BL/6J mice were infected with AAV-GFP, AAV-TauWT-GFP, AAV-TauT217A-GFP or AAV-TauT217E-GFP viruses at optimal MOIs (Fig. S2), and cultured in vitro for 3, 5 and 7 days. FLAG-tagged protein and P-Tau217 levels were measured by western blot (Fig. S3A–D). As shown in Fig. 2A, at 3 DIV (Days In Vitro), neurons in all groups exhibited normal morphology with synaptic outgrowth and outward extension. By 5 DIV, neurons infected with AAV-GFP or AAV-TauT217A-GFP maintained clear cellular boundaries with distinct nuclei and nucleoli. However, neurons infected with AAV-TauT217E-GFP showed restricted growth, cell shrinkage, and synaptic damage. By 7 DIV, neurons infected with AAV-GFP or AAV-TauT217A-GFP groups displayed abundant synaptic networks, whereas AAV-TauT217E-GFP infected neurons exhibited significant neuronal loss, severe synaptic atrophy, and cell body shedding.

Fig. 2. Hyperphosphorylation of Tau217 leads to neuronal death and exacerbated cognitive dysfunction in 5×FAD mice.

A The morphology of primary cortical neurons infected with AAV was observed by light microscopy at 3, 5 and 7 days post-infection. B Neuronal viability was measured using CCK-8 assay at 3, 5 and 7 days post-infection with AAV-GFP, AAV-Tau^WT, AAV-Tau^T217A and AAV-Tau^T217E, (n = 6). C Illustration of the experimental procedure. D Image showing stereotaxic injection of the AAV-GFP, AAV-Tau^WT, AAV-Tau^T217A and AAV-Tau^T217E into the hippocampal CA1 region of 2-month-old 5 × FAD mice. E, F Y Maze behavioral test, assessing short term memory, were carried out using the 5 × FAD mice at the age of 5 and 8 months, (n = 10–11 per group). G, H Mice overexpressing TauT217E group showed longer escape paths during 6 days of training and shorter times spent on the platform or target quadrant during probe test, (n = 10–11 per group). I–K A 60 s probe test was carried out on day 7 after the hidden platform was removed. Mice overexpressing Tau^T217E showed significantly impaired spatial learning and memory, (n = 10–11 per group). Data were presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA. Scale bars = 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001.

Given that mitochondrial dehydrogenase activity, assessed using the Cell Counting Kit (CCK-8), has been prevalently used to measure cell viability, this method was used here for evaluating neuronal viability. As shown in Fig. 2B, no significant difference in absorbance at 450 nm (OD450) were observed across groups at 3 DIV post AAV infection, indicating similar viability. However, at 5 DIV post AAV infection, OD450 value in the AAV-TauWT-GFP, AAV-TauT217A-GFP, and AAV-TauT217E-GFP groups were significantly lower than in the AAV-GFP control group (p < 0.05), though no significant differences were observed among AAV-Tau expression groups. At 7 DIV post AAV infection, the OD450 in the AAV-TauT217E-GFP group was significantly lower than in both the AAV-TauWT-GFP and AAV-TauT217A-GFP groups (p < 0.001). Together, these data indicate that Tau217 hyperphosphorylation significantly reduces neuronal viability in a time-dependent manner.

To evaluate cognitive function, 3-month-old male 5 × FAD transgenic mice were stereotactically injected with AAV-GFP, AAV-TauWT-GFP, AAV-TauT217A-GFP, or AAV-TauT217E-GFP into the hippocampal CA1 region (Fig. 2D). Spontaneous alternation rates in the Y-maze were used to evaluate short-term memory at 5 and 8 months of age (Fig. 2E, F). At 5 months, no significant differences were observed among groups. However, at 8 months, spontaneous alternation was significantly reduced in the TauWT and TauT217E groups compared to the GFP controls (P < 0.01, P < 0.0001 respectively), with TauT217E group showing the greatest decline. Conversely, the TauT217A group showed significantly better spontaneous alternation rate than TauT217E group (P < 0.01). These findings indicate that hyperphosphorylation of Tau217 impairs short-term working memory in 5 × FAD mice.

To evaluate the effect of Tau217 hyperphosphorylation on visuospatial learning and memory in animals, 5 × FAD transgenic mice stereotaxically injected with four viruses were subjected to water maze detection at 8 months of age. As shown in Fig. 2G, H, after training, the swimming trajectory of TauT217E group remained chaotic, and the time to find the platform was significantly prolonged (P < 0.05). After the platform was evacuated on the 7th day, as shown in Fig. 2I–K, the escape latency in the TauT217E group was significantly increased, and the number of crossings of the platform and the time in target quadrant in the TauT217E group were significantly reduced. These results suggest that hyperphosphorylation of Tau217 impairs visuospatial learning and memory in 5 × FAD transgenic mice memory.

Hyperphosphorylation of Tau217 leads to synaptic disorder and downregulation of synapse-associated proteins

Since neuronal communication relies on synapses, axon length and dendrite branching are indicative of synaptic transmission efficiency [13–19]. To assess the impact of Tau217 hyperphosphorylation on synapses, neuronal damage caused by Tau217 hyperphosphorylation was initially observed using light microscopy, followed by an examination of synaptic morphology with laser confocal microscopy. To this end, primary cortical neurons expressing FLAG-tagged TauWT, TauT217A or TauT217E were stained with anti-FLAG antibody (green) to confirm AAV infection, and β-III Tubulin was labeled (red) to visualize neuronal morphology (Fig. 3A). As shown in Fig. 3B, C, axons in the TauT217E treatment group were significantly shorter (p < 0.01), and the number of dendrites was significantly reduced (p < 0.0001) compared to the TauT217A group. These findings suggest that Tau217 hyperphosphorylation leads to primary cortical neuronal damage, while dephosphorylation of Tau217 has a protective effect against such injury.

Fig. 3. Hyperphosphorylation of Tau217 results in synaptic damage in primary neurons.

A–C After 5 days of AAV infection in primary cortical neurons, synapses were measured by confocal microscopy. Axon length was quantified using ImageJ / Fiji for analysis (n = 4). D–F After 5 and 7 days of AAV infection, the levels of PSD95 and Drebrin protein were measured by western blot. Representative Western blot images of PSD95 and Drebrin are shown, with β-actin used as a loading control. Data were presented as mean ± SEM, n = 3–4 per group. Significance was determined using one-way ANOVA. Scale bars = 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001.

Given that synapse-associated proteins such as PSD95 and Drebrin are important indicators of synaptic plasticity, their expression in primary cortical neurons cultured for 5 or 7 DIV expressing GFP, TauWT, TauT217A, or TauT217E was assessed to evaluate changes in synaptic plasticity [20–22]. As shown in Fig. 3D–F, Drebrin protein levels were reduced in the TauWT, TauT217A and TauT217E groups (p < 0.05, p < 0.01, p < 0.0001 respectively) on 5 DIV compared to the GFP group. Of note, Drebrin expression in the TauT217E group was significantly lower than in the TauT217A group (p < 0.0001). Compared to the GFP and TauT217A groups, the expression of PSD95 protein in the TauT217E group was significantly lower (p < 0.0001). On 7 DIV, Drebrin expression in the TauT217E group was significantly lower than in the TauWT and TauT217A groups (p < 0.05, p < 0.01 respectively). Additionally, PSD95 expression in the TauT217E group was significantly lower than in the TauT217A group (p < 0.001). Taken together, these data suggest that hyperphosphorylation of Tau217 downregulates the expression of postsynaptic and dendritic spinous proteins in primary cortical neurons, resulting in severe damage to postsynaptic neurons and spines, thereby impairing synaptic plasticity. In contract, dephosphorylation of Tau217 mitigates these deleterious effects.

Hyperphosphorylation of Tau217 results in ultrastructural damage in hippocamp of 5×FAD mice

To explore the morphological changes associated with cognitive decline induced by Tau217 hyperphosphorylation, transmission electron microscopy (TEM) was employed to analyze the ultrastructure of microtubules and synapses in the hippocampal CA1 region of 8-month-old 5 × FAD mice. We injected AAV-GFP, AAV-TauWT-GFP, AAV-TauT217A-GFP, and AAV-TauT217E-GFP into the hippocampal CA1 region of 5 × FAD mice. FLAG-tagged protein and P-Tau217 levels were measured by western blot (Fig. S3E–H). As shown in Fig. 4A, axonal microtubules in the GFP and TauT217A groups were dense and arranged in parallel bundles, reflecting healthy ultrastructure, while axonal microtubules in the TauWT and TauT217E groups appeared loose, disordered, fragmented, and dissolved. Notably, the TauT217E group showed extensive microtubule disintegration, indicating severe structural damage.

Fig. 4. Hyperphosphorylation of Tau217 impairs synaptic and microtubule structure in 5×FAD mice.

A Transmission electron microscopy images of the hippocampus microtubules following stereotaxic injection of AAV virus. Scale bars = 1 μm. Insets show detailed images of microtubules. Scale bars = 3 μm. B–E Compared to the Tau^T217E overexpression group, the synaptic structures in the other three groups were more intact. Scale bars = 1 μm. Insets show detailed images of synaptic structures. Scale bars = 3 μm. Overexpression of Tau^T217E resulted in a significant decrease in synaptic plasticity. Width of synaptic cleft and maximal PSD thickness were quantified using ImageJ / Fiji for comparison. F, G Golgi staining showed that axon length was significantly shortened in the Tau217 hyperphosphorylation group. Scale bars = 25 μm. H, I Golgi staining showed a significant decrease in dendritic spine density in the Tau217 hyperphosphorylation group. Scale bars = 5 μm. Data are presented as means ± SEM, n = 3, and were analyzed using one-way ANOVA. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001.

The postsynaptic dense substance (PSD) is a dense layer of material with a uniform texture on the medial cytoplasmic surface of the postsynaptic membrane, serving as a marker of postsynaptic components [23]. PSD thickness, along with synaptic cleft width and synaptic density, are important indicators of synaptic plasticity [24–26]. As shown in Fig. 4B, compared to the TauWT and TauT217E groups, hippocampal synapses in the GFP and TauT217A groups exhibited more regularity; the presynaptic membrane and postsynaptic membrane were more clearly visible, and the synaptic cleft was more intact. In addition, in the GFP or TauT217A groups, the presynaptic terminal was rich in round synaptic vesicles, and electron-dense substances were visible in the postsynaptic membrane. However, the synaptic ultrastructure in the TauT217E group showed significant damage, characterized by disrupted synaptic membranes and impaired synaptic clefts.

As shown in Fig. 4C–E, the TauT217E group exhibited a significantly wider synaptic cleft (p < 0.001) and significantly increased maximum PSD thickness (p < 0.001) compared to the TauT217A group. As shown in Fig. 4F–I, Golgi staining results showed that the axon length in the TauT217E group was significantly shortened. Similarly, density of dendritic spines in the TauT217E group was significantly reduced. Together, these results suggest that Tau217 hyperphosphorylation may impair cognitive function by disrupting microtubules and compromising synaptic plasticity. Dephosphorylation of Tau217 ameliorates this pathological effect.

Aberrant activation of CDK5 by increased p25 leads to primary cortical neurons death

Numerous studies have demonstrated that abnormal activation of CDK5 is a key pathological process in AD. This abnormal activation, through the conversion of p35 into p25, plays an essential role in AD pathology [27–29]. However, the detailed mechanisms underlying this pathological process remain incompletely elucidated. To determine the specific effects of CDK5 overactivation via increased p25 on neurons, TUNEL staining was employed. As shown in Fig. 5A–D, TUNEL staining revealed that the number of TUNEL-positive cells in the AAV-p25 group was significantly higher than in the AAV-GFP group at all time points (3 DIV, 5 DIV, or 7 DIV). Furthermore, the number of TUNEL-positive cells in the p25 group increased over time, peaking at 7 DIV. As shown in Fig. 5E, F, at 5 DIV, neuronal survival was significantly improved in the group pretreated for 24 h with the CDK5 inhibitor Roscovitine. These results suggest that p25/CDK5 is neurotoxic and leads to neuronal death.

Fig. 5. Aberrant activation of CDK5 by increased p25 leads to primary cortical neurons death.

A–D Primary cortical neurons were infected with AAV-GFP or AAV-p25 infection for 3, 5 and 7 days. Neuronal death was assessed using fluorescence microscope with the TUNEL staining kit. E, F On day 5 post-infection, neurons were preincubated with CDK5 inhibitor Roscovitine for 24 h, and neuronal death was assessed by fluorescence microscope using the TUNEL staining kit. Data were presented as mean ± SEM, n = 11–12 mice for each group. Significance was determined using one-way ANOVA. Scale bar = 0.75 mm. *p ＜ 0.05, ****p ＜ 0.0001.

Hyperphosphorylation of Tau217 is mediated by CDK5 overactivation in primary neurons in vitro

Our study found that the amino acid sequence surrounding Tau217 is conserved across species and matches the typical substrate motif for CDK5, suggesting that threonine at 217 site of Tau is an optimal phosphorylation site for CDK5 (Fig. 6A). In Fig. 6B, C, Tau217 phosphorylation increased with the duration of p25 infection in primary neurons. At 7 DIV, phosphorylation of Tau217 in neurons infected with p25 was significantly higher than at 3 DIV or 5 DIV. In Fig. 6D, E, Tau217 phosphorylation in neurons overexpressing p25 and pretreated with roscovitine was significantly lower than in neurons overexpressing p25 alone at 5 DIV. Moreover, to assess the effects of CDK5-mediated T217 phosphorylation in vitro, a mixture of purified CDK5 and p25 was incubated with Tau protein, and Tau217 phosphorylation was assessed over time (0, 0.5 h, 1 h, 1.5 h, 2 h) at 30 °C (Fig. 6F); Tau217 phosphorylation peaked at 1.5 h (Fig. 6G). As shown in Fig. 6H, I, in the presence of CDK5 inhibitor Roscovitine, Tau217 phosphorylation was significantly decreased to near baseline levels, indicating CDK5 directly phosphorylates Tau at threonine 217 site. These data confirm that CDK5 mediates Tau217 phosphorylation. In addition, as shown in Fig. S4, Tau217 phosphorylation in the cortex and hippocampus of p35 knockout mice (p35^−/−) was significantly lower compared to wild-type mice, consistent with immunofluorescence results. These findings indicate that deletion of p35/CDK5 inhibits Tau217 phosphorylation, providing further evidence of CDK5’s role in Tau217 regulation.

Fig. 6. CDK5 regulates Tau phosphorylation at T217.

A Sequence alignment of the Tau217 region across different animal species. B–E AAV-p25 upregulates the phosphorylation level of Tau217 in primary neurons at various time points, while Roscovitine significantly inhibits Tau217 phosphorylation. n = 3 per group. F–I In vitro incubation of purified CDK5 and p25 with purified Tau protein for 1.5 h resulted in the highest level of Tau T217 phosphorylation, which was significantly inhibited by Roscovitine. n = 3 per group. Data are presented as means ± SEM and analyzed using one-way ANOVA. *p < 0.05, **p < 0.01, ****p < 0.001.

Discussion

AD is a complex and multifactorial neurodegenerative disorder, with the amyloid-β (Aβ) pathway long considered central to its pathophysiology [30–33]. Although the detailed molecular mechanisms by which Aβ contributes to synaptic failure, neurodegeneration, and the onset of clinical symptoms remain under active investigation, it is increasingly clear that other pathways also play critical roles. Among these, neurofibrillary tangles caused by abnormal phosphorylation of Tau protein have gained significant attention [34–36]. Emerging evidence suggests Aβ pathology, emphasizing the importance of Tau in the disease’s progression [37]. Tau is a microtubule-associated protein (MAP) with well-recognized functions in promoting microtubule assembly and maintaining stability, primarily regulated through its phosphorylation state [38, 39]. Recent studies have highlighted the significance of Tau phosphorylation, particularly at the threonine 217 (Tau217) residue, which has become a focus in AD research [6, 40, 41]. For instance, Campese et al. identified phosphorylated Tau217 (P-Tau217) as a novel diagnostic and prognostic biomarker for AD, with potential utility in monitoring disease progression [42]. Similarly, Milà-Alomà et al. demonstrated a strong correlation between plasma p-Tau217 levels and early Aβ accumulation in AD patients, with changes detectable long before significant Aβ plaque deposition [43]. These findings suggest that Tau217 phosphorylation may not only serve as a biomarker for AD but also play a role in AD pathogenesis.

Motivated by this, we investigated the neurotoxic effects of phosphorylation of Tau217 in the 5 × FAD mouse model of AD. Our study employed overexpression of wild-type Tau217 (TauWT), a phospho-mimic variant (TauT217E) and a non-phospho-mimic variant TauT217A (TauT217A). Behavioral experiments in 5 × FAD mice revealed that the tau217E, phosphor-mimic TauT217, exacerbated cognitive impairment, while non-phosphorylatable mimic TauT217A appeared to mitigate cognitive decline. These behavioral outcomes were supported by cellular data, where Tau217 hyperphosphorylation led to neurites shortening and disorder in primary cultured cortical neurons and exacerbated microtubule structure and synaptic damage in 5 × FAD mice. Furthermore, Tau217 hyperphosphorylation was associated with downregulation of synapse-associated proteins. These findings highlight the neurotoxic potential of Tau217 hyperphosphorylation and suggests that it may play a pivotal role in driving chronic neurodegeneration in AD.

Following confirmation of the neurotoxic effects of Tau217 hyperphosphorylation, we focused on understanding its regulatory mechanisms. Tau shares a conserved animo acid motif around threonine 217 across species, making it an optimal substrate for CDK5.

CDK5, a serine/threonine kinase primarily involved in neuronal development, has emerged as a key player in AD pathology, affecting synaptic plasticity and cytoskeletal dynamics [44–48]. In AD, abnormal activation of CDK5 is driven by the cleavage of its regulatory subunit p35 into p25, which serves as a pathological cofactor [49–52]. This abnormal activation has been implicated in the phosphorylation of multiple Tau sites, including Thr217 [53–55]. However, the specific role of CDK5 in Tau217 phosphorylation had not been fully elucidated. In our study, we confirmed that Tau217 is a direct substrate of CDK5. In vitro kinase assay demonstrated that purified CDK5/p25 phosphorylated Tau at Thr217, and this phosphorylation was significantly reduced in the presence of the CDK5 inhibitor Roscovitine. In addition, overexpression of p25 in cortical neurons resulted in Tau hyperphosphorylation at site 217 and subsequent neurite damage. In vivo, in 5 × FAD mice, Tau217 hyperphosphorylation was observed alongside increased p25 expression. Moreover, reduced Tau217 phosphorylation in the hippocampus of p35 KO mice further confirming the role of CDK5 in this process.

In summary, our findings established that Tau217 hyperphosphorylation significantly exacerbates cognitive decline in 5 × FAD mice, disrupts synaptic and microtubule structures, and downregulates synapse-related proteins. Importantly, CDK5 plays a pivotal role in regulating Tau217 phosphorylation. These findings underscore the importance of the CDK5-Tau217 axis in AD pathogenesis. Targeting this pathway offers a promising therapeutic strategy, potentially halting or reversing the neurodegenerative processes associated with Tau pathology. The development of specific inhibitors that modulate CDK5 activity or prevent its interaction with Tau could represent a significant advancement in AD treatment.

Author contributions

KF, TH, DQ, XC conceptualized and designed the research study. KF, YX conducted the experiments. Statistical analyses were performed by NL, EH, RH, ZW. KF wrote the manuscript with major edits from TH and DQ. NL, TH, RH supervised the study and obtained funding. All authors reviewed and approved the final manuscript.

Data availability

All data produced in this study is shown in manuscript and Supplementary Information, and unprocessed data are available from the corresponding author on reasonable request.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential competing interests.

Ethics approval and consent to participate

All methods were performed in accordance with the ARRIVE guidelines for animal research. All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Fujian Medical University (FJMU IACUC 2024-0056). The study was conducted in compliance with the NIH Guide for the Care and Use of Laboratory Animals. This study involved no human participants, and therefore informed consent was not applicable. No identifiable human images or data were used in this study.

Supplementary information

The online version contains supplementary material available at 10.1038/s41398-025-03551-9.