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Activation of the IL-17/TRAF6/NF-κB pathway is implicated in Aβ-induced neurotoxicity

BMC Neuroscience volume 24, Article number: 14 (2023) Cite this article

Abstract

Background

Neuroinflammation plays a critical role in Amyloid-β (Aβ) pathophysiology. The cytokine, interleukin-17A (IL-17) is involved in the learning and memory process in the central nervous system and its level was reported to be increased in Alzheimer's disease (AD) brain, while the effect of IL-17 on the course of Aβ has not been well defined.

Methods

Here, we used APP/PS1 mice to detect the IL-17 expression level. Primary hippocampal neurons were treated with IL-17, and immunofluorescence was used to investigate whether IL-17 induced neuron damage. At the same time, male C57BL/6 mice were injected with Aβ42 to mimic the Aβ model. Then IL-17 neutralizing antibody (IL-17Ab) was used to inject into the lateral ventricle, and the Open field test, Novel Objective Recognition test, Fear condition test were used to detect cognitive function. LTP was used to assess synaptic plasticity, molecular biology technology was used to assess the IL-17/TRAF6/NF-κB pathway, and ELISA was used to detect inflammatory factors.

Results

Altogether, we here found that IL-17 was increased in APP/PS1 mice, and it induced neural damage by the administration to primary hippocampal neurons. Interestingly, Using Aβ42 mice, the results showed that the level of IL-17 was increased in Aβ42 model mice, and IL-17Ab could ameliorate Aβ-induced neurotoxicity and cognitive decline in C57BL/6 mice by downregulation the TRAF6/NF-κB pathway.

Conclusion

These findings highlight the pathogenic role of IL-17 in Aβ induced-synaptic dysfunction and cognitive deficits. Inhibition of IL-17 could ameliorate Aβ-induced neurotoxicity and cognitive decline in C57BL/6 mice by downregulation of the TRAF6/NF-κB pathway, which provides new clues for the mechanism of Aβ-induced cognitive impairments, and a basis for therapeutic intervention.

Peer Review reports

Introduction

Alzheimer’s disease (AD) is the most common type of dementia and a rising threat to public health [1,2,3,4]. Amyloid beta (Aβ) is a hallmark of Alzheimer's disease, and its accumulation in the brain is thought to play a key role in the molecular pathology of AD [5,6,7], Aβ is thought to have the strongest toxicity to synaptic damage, neuronal death, and cognitive impairments[8, 9]. Neuroinflammation plays a critical role in Aβ pathophysiology [10, 11], but its etiopathogenesis is still unclear. The interleukin 17 (IL-17) family of cytokines contains 6 structurally related cytokines, IL-17A through IL-17F. Although less is known about IL-17B–F, IL-17A (the prototypical member of this family, commonly known as IL-17) has received much attention for its pro-inflammatory role [12]. IL-17 is a pro-inflammatory cytokine produced by various types of cells including CD4 T cells which are categorized as a new subset called Th17 cells, acting on its specific receptor (IL-17R) highly expressed in the CA1 region of the hippocampus [13, 14]. After binding to IL-17, IL-17R could recruit NF-κB activator (ACT1) with the same domain through its SEFIR domain. ACT1 in turn recruits tumour necrosis factor receptor-associated factor 6 (TRAF-6), which is an adapter protein that mediates a wide array of protein–protein interactions via its TRAF domain and a RING finger domain that possesses non-conventional E3 ubiquitin ligase activity. TRAF6 was identified as a mediator of interleukin-1 receptor (IL-1R)-mediated activation of NF-κB, which plays a vital role by regulating certain functions like neuronal plasticity and neuronal growth. [15,16,17]. It is interesting to note that plasma IL-17 level has been identified as one of the plasma biomarkers for AD diagnosis and neocortical Aβ load [18, 19]. In addition to this, it was reported that IL-17 triggers the onset of cognitive and synaptic deficits in the early stages of Alzheimer's disease [20]. We here found that IL-17 was increased in APP/PS1 mice, but the effect of IL-17 on the course of Aβ has been unclear. Therefore, we further wanted to investigate whether IL17 is involved in Aβ neurotoxicity.

In this study, we here found that the level of IL-17 was increased in Aβ42 model mice, and IL-17Ab could ameliorate Aβ-induced neurotoxicity and cognitive decline in C57BL/6 mice by downregulating the TRAF6/ NF-κB pathway, which provides new clues for the mechanism of Aβ-induced cognitive impairments, and a basis for therapeutic intervention.

Materials and methods

Ethics statement

All methods were carried out in accordance with relevant ARRIVE guidelines. All methods were approved by the Animal Care Committees of the Ethics Committee of Renmin Hospital, Wuhan University (IACUC Issue No. WDRY2018-K033).

Animals and reagents

Male C57BL/6 mice (2 months old, 20 ± 2 g) were purchased from the Center for Animal Experiment of Wuhan University. 6 months Male APP/PS1 mice (APPswe, PSEN1dE9 and 85Dbo/MmJNju mice) were purchased from Model Animal Research Center of Nanjing University (Nanjing, China). The animals were housed in Experimental Animal Central of Renmin Hospital of Wuhan University under standard laboratory conditions: natural lighting for 12 h then total darkness of another 12 h with water and ad libitum food. Mice were randomly divided into groups and treated as explained in different parts of the study.

Enzyme-linked ImmunoSorbent assays (Elisa)

The hippocampi were lysed in RIPA buffer and centrifuged at 3000 × g for 10 min at 4 °C and the supernatant was collected. Anti-mouse IL-17 Elisa kit from Elabscience Biotechnology (Wuhan, China) was used to assay hippocampus IL-17 level, according to the manufacturer’s instructions.

Primary hippocampal neuron Culture

Primary hippocampal neurons were dissected from the brains of E18 C57BL/6 mice embryos according to previously described procedures with minor modifications [21, 22]. Neurons were cultured in 12-well plates coated with 100 μg/mL poly-D-lysine and supplemented with 2% (v/v) B-27 and 1 × GlutaMAX. The neurons cultured for 9 days were used in experiments. In the experiment, the cells were divided into different groups: control group, IL-17 group (recombinant IL-17, 10 ng/mL, R&D Systems, Cat# 421-M). After treatments, cells were collected and lysed in RIPA buffer for further biological detection or fixed with 4% paraformaldehyde for immunofluorescence imaging. All cell culture reagents were purchased from Thermo Fisher Scientific.

Stereotactic surgery

The intracerebroventricular surgery was performed as follows: anterior–posterior: −0.3 mm; mediolateral: -1 mm; dorsoventral: −2.3 mm (from bregma and dura, flat skull). After injection, the needle was kept in place for 10 min to avoid solution flowback.

Aβ42 (Qiangyao Biotechnology) was oligomerized according to the procedure described previously [23, 24]. In brief, the Aβ42 was dissolved in 1% (vol/vol) DMSO and diluted in physiological saline to a final concentration of 2.0 μg/μL. Then, the solution was incubated at 37 °C in darkness for 1 week before use. The mice were anesthetized with isoflurane and placed in a stereotaxic apparatus.Then the mice were injected through the brain lateral ventricle, with the solution with Aβ42 (5 μL), and the control group was injected with sterile normal saline containing DMSO (1%) of the same volume for 7 consecutive days.

IL-17Ab (BioXCell, clone 17F3) was dissolved in saline and injected into the lateral ventricle at 1 mg/kg in 3 μL on 12 h prior to Aβ42 injection and the 6th-day post-Aβ42 injection. The subsequent experiments were conducted 24 h after the injection of the IL-17Ab.

Western blotting

The hippocampus was homogenized in a buffer (pH 7.4) containing 50 mmol/L Tris–HCl, 150 mmol/L NaCl, 10 mmol/L NaF, 1 mmol/L Na3VO4, 5 mmol/L EDTA, 2 mM benzamidine, and 1 mM PMSF. The supernatants were collected after centrifugation of the tissue homogenates or cell lysate at 12000 rpm/min. Protein concentrations were determined with the bicinchoninic acid protein kit (Pierce, Rockford, USA). The proteins were loaded onto 10% gel (Invitrogen, Bis–Tris), separated by electrophoresis, and then transferred to a NC membrane. The images were visualized with an Odyssey infrared imaging system (LI-COR Biosciences, USA). For western blotting, the primary antibodies used were anti-IL-17 (Cell Signaling, #13838, 1:1000), anti-synapsinI (SYN, Millipore, AB1543, 1:1000), anti-postsynaptic density protein 95 (PSD-95, Cell Signaling, #2507, 1:1000), TRAF6 (Santa Cruz, sc-8409, 1:500), p-NF-κB p65 (Ser536) (Cell Signaling, #3033, 1:1000), anti-actin (actin, Abcam, ab6276, 1:10 000), and Lamin B1 (Abcam, ab16048, 1:1000).

The nuclear and cytoplasmic proteins preparation kit (P1200, Pulilai) was used to separate the nuclear and cytoplasmic NF-κB p65 components according to the manufacturer’s procedures for subsequent experiments.

Immunofluorescence staining

Hippocampal neuronal cells were rinsed with phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde for 8 min and permeabilized with 0.1% Triton X-100 in PBS for 30 min. After being blocked with 5% milk for 30 min, cells were incubated with primary antibodies conjugated to Alexa-fluo®488 or 594 against microtubule-associated protein-2 (MAP2, Abcam, 1:250), PSD-95 (Cell signaling, 1:250) at 4 °C overnight. The second antibody was then incubated at room temperature for 1 h and then rinsed in PBS three times. The nucleus was stained by DAPI (Sigma) for 5 min. Fluorescence images were obtained using the BX53 Olympus fluorescence microscope at a 20 × magnification and captured using the Olympus CellSens Standard software. Sholl analysis was applied to measure dendritic complexity. The length of dendritic arborization was analyzed and measured using a semi-automatized protocol via Imaris software (Bitplane, Inc.).

Behavioral tests

Open-Field test

The open field is used to assess the tension, anxiety, and exploration activities of the mice. In brief, experimental mice were placed in a typical open field, and movements inside the field were tracked over a 5 min period. The total distance covered and central zone crossing were tracked and measured. The chamber was sanitized with 70% ethanol after each trial.

Novel objective recognition test (NORT)

The mice were taken to the new object recognition room 24 h before the test, and then we put the mouse into a 100 cm × 100 cm × 100 cm plastic container for 5 min without objects before the test. The next day, the mice reentered the container at the same starting point and were allowed for 5 min to familiarize themselves with A and B objects. After each period, the arena and objects were cleaned with 75% ethanol. Two hours after the familiarization period, the B object was replaced by the C object, and the mice were granted 5 min to explore the A object and C object. After 24 h, the C object was replaced with the D object, and the mice were also given 5 min to explore. The exploring time on each object was recorded.

Fear conditioning

The scene memory function of the mice was further detected by the fear conditioning test. Mice were placed into a square chamber (40 cm × 40 cm × 50 cm) with white board walls, a transparent front door, and a grid floor. On the day of training, the mice were allowed to explore in an enclosed training chamber for 180 s. The mice were then exposed to a pure tone for 30 s, followed by a 2 s foot shock (0.8 mA). At 60 s after delivery of the second shock, the mice were taken back to their home cages. 24 h later, mice were sent into the same chamber for 3 min without foot shock for fear memory tests. The time of freezing was measured using the Contextual NIR Video Fear Conditioning System (Med Associates).

Long-term potentiation (LTP)

After the mice were sacrificed, whole brains were immediately resected and soaked in ice-cold artificial cerebrospinal fluid (aCSF) saturated with 95% O2 and 5% CO2. Following sectioning at 300-μm thickness, the slices were incubated in oxygenated aCSF at 32 °C to recover for 40 min and at 20–25 °C to recover for 1 h. Then slices were transferred to a recording chamber and submerged in aCSF perfusion. Slices were laid in a chamber with an 8 × 8 microelectrode array (Parker Technology, Beijing, China) in the bottom planar (each 50 × 50 mm in size, with an interelectrode distance of 150 μm) and kept submerged in aCSF. Signals were acquired using the MED64 System (Alpha MED Sciences, Panasonic). The field excitatory postsynaptic potentials (fEPSP) in CA1 neurons were obtained by stimulating CA3 neurons. LTP was induced by applying three trains of high-frequency stimulation (100 Hz for 1 s, delivered 30 s apart). The LTP magnitude was quantified as the percentage change in the fEPSP slope (10–90%) taken during the 60-min interval after LTP induction [25].

Transmission electron microscopy (TEM)

After perfusion with fixatives hippocampus was dissected and slices were approximately 150 μm thick. The slices were fixed further by immersion in 0.1 M Na-cacodylate buffer containing 2.5% glutaraldehyde for 1 h at room temperature. Postfix with 1% OsO4 in 0.1 M PBS for 2 h at room temperature. Then dehydrate and infiltrate. Sections were photographed under a light microscope and then serially cut into semithin (2 μm thick) sections. The semithin sections were stained with 1% toluidine blue in 1% sodium borate and examined under the light microscope to locate the CA1 region. Selected semithin sections were further cut into serial ultrathin sections by using a Leica ultramicrotome. The ultrathin sections were examined under a HITACHI HT7800 TEM by an electron microscopy specialist from the Department of Ultrastructural Pathology Center, Renmin Hospital of Wuhan University. Synaptic densities were expressed as the number of synapses (identified via PSDs), per 100 μm2 of tissue.

Golgi staining

FD Rapid Golgi Staining Kit PK 401(FD NEUROTECHNOLOGIES, INC, Columbia MO, USA) was used to measure the morphology of neuronal dendrites and dendrites’ spines. The mice were anesthetized by isoflurane and transcardially perfused with proximately 400 mL of normal saline containing 0.5% sodium nitrite, followed by 400 ml 4% formaldehyde solution and then 500 ml Golgi dye solution (5% chloral hydrate, 5% potassium dichromate, and 4% formaldehyde) over 2 h. And then, the brains were dissected into 5 mm × 5 mm sections and incubated in the staining solution for 3 days and in 1% silver nitrate solution for another 3 days in the dark. Finally, the brains were sliced using a vibrating microtome (Leica, Wetzlar, Germany) at a thickness of 100 μm. Images were observed under the microscope (BX53 Olympus fluorescence microscope, Japan).

Statistical analysis

All experiments were repeated at least three times. Results are expressed as mean ± standard deviation (SD) and analyzed using GraphPad Prism 8.0 statistical software. The normality heterogeneity of the data was tested. The differences were analyzed by the unpaired t-test or one-way ANOVA followed by the Bonferroni post hoc test. If the data were not normally distributed, the nonparametric test (Mann–Whitney test) was used to analyze the differences. The analyses of the quantification of the different proteins by western-blot (ratio) were performed on long-transformed data. p < 0.05 was considered statistically significant between groups.

Results

The IL-17 level is increased in APP/PS1 mice

The cytokine, IL-17Ais involved in the learning and memory process in the central nervous system and its level was reported to be increased in Alzheimer's disease (AD) brain, while the effect of IL-17 on the course of Aβ has not been well defined. To investigate the role of IL-17 in neuropathological changes and memory deficits associated with Aβ pathophysiology, we used the transgenic mouse model of APP/PS1 mice, a progressive model of the amyloid plaques. The Elisa kit of IL-17 was used to detect the IL-17 level and the result showed that IL-17 was increased in the hippocampus of APP/PS1 mice when compared with the control mice (Fig. 1A). Furthermore, we performed western blotting and the result showed a significant increase in the protein levels of IL-17 from the APP/PS1mice (Fig. 1B, C), strongly supporting that the IL-17 level was increased in APP/PS1 mice.

Fig. 1
figure 1

IL-17 is increased in APP/PS1 mice. Results from Elisa tests, the level of IL-17 in APP/PS1 mice and control mice hippocampus (A). N = 4. Western blotting was used to detect the IL-17 level (B) and quantification of IL-17 (C) N = 3. Data are presented as mean ± SD. p value significance is calculated from a T-test. **p < 0.01 and ***p < 0.001 vs control group

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IL-17 induces neuronal toxicity in primary hippocampal neurons

The above result showed that the IL-17 level was increased in APP/PS1 mice. However, the effect of IL-17 on neurodegenerative disease is still unclear. The hippocampus is an important part of the limbic system and is known to play a crucial role in memory function which is the reflection of synaptic plasticity [26, 27]. We then try to investigate the IL-17 effect on neuron in primary hippocampal neurons. The hippocampal primary neurons were divided into the control group and the IL-17 group (IL-17, 10 ng/mL). To investigate the underlying effect based on morphology,we examined the dendritic morphology of hippocampal primary neurons following treatment with IL-17 by using anti-MAP2 and PSD95 antibodies (Fig. 2A). When compared with the control, IL-17 resulted in an obvious decreased dendritic arborization complexity at all points farther than 50 μm from the cell body (Fig. 2B), as well as the total dendritic length (Fig. 2C). These findings suggest that IL-17 induced hippocampal neural damage.

Fig. 2
figure 2

IL-17 induces neuronal toxicity in primary hippocampal neurons. Mice primary hippocampal neurons were treated with DMSO for control and IL-17 for the IL-17 group for 9 d. The neuronal morphology changes were measured by immunofluorescent staining of anti-PSD95 and anti-MAP2 antibodies. Representative images after treatment (A), Sholl analysis (B), quantitative analyses of dendritic length (C), N = 20 hippocampal neurons. Data are presented as mean ± SD. p value significance is calculated from a T-test. ***p < 0.001 vs control group

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Inhibition of IL-17 alleviates Aβ42-induced cognitive deficits and synaptic dysfunction

We confirmed that the IL-17 level was increased in APP/PS1 mice and cloud induce hippocampal neuronal damage. We therefore hypothesized that the IL-17 might been a target for intervention of Aβ42 induced-neurotoxicity. To test my hypothesis, Aβ42 was used to inject in lateral ventricle to induce the Aβ model. Then we explored whether IL-17Ab could ameliorate Aβ42-induced cognitive impairment and synaptic dysfunction. We divided our experiments into three groups and showed the flow chart for the experiments (Fig. 3A). The control group were injected with saline (5 μL) in the brain unilateral ventricle, and the Aβ42 model group were established after injecting in the brain unilateral ventricle with the solution Aβ42 (2.0 μg/μL, 5 μL). While the Aβ42 + IL-17Ab group was injected with Aβ42 (2.0 μg/μL, 5 μL), then IL-17Ab was injected into the lateral ventricle (1 mg/kg, 3 μL) on 12 h prior to Aβ42 injection and the 6th-day post-Aβ42 injection (Fig. 3A). We performed western blotting and the result showed a significant decrease in the protein levels of IL-17 from the Aβ42 + IL-17Ab mice (Fig. 3B, C) when compared with the Aβ42 mice. Furthermore, Elisa kit of IL-17 was used to detect the IL-17 level and the result showed that it was decreased in the hippocampus of Aβ42 + IL-17Ab mice (Fig. 3D).

Fig. 3
figure 3

Inhibition of IL-17 alleviates Aβ42-induced cognitive deficits. The flow chart for the experiments (A). IL-17 level was detected by western blotting using specific antibodies, and actin was used as a loading control (B). Intensity analysis of IL-17 level (C). N = 3. ELISA assays to measure levels of IL-17 (D), N = 3. The open-field test showed the total distance covered (E), and the time of center duration (F) of the three groups. NORT showed the time spent exploring new objects 2 h (G) and 24 h (H). The contextual fear conditioning test figured out the freezing time in 2 h (I) and 24 h (J). N = 10 for independent experiments. Data are presented as mean ± SD. p value significance is calculated from a one-way ANOVA test, *p < 0.05, **p < 0.01 and ***p < 0.001 vs Aβ group

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Healthy C57BL/6 mice were randomly divided into 3 groups. Following the treatment, we again performed a couple of behavioral tests. In the open-field test, the total distance showed no significant differences among the three groups (Fig. 3E), indicating that the locomotion activity was not influenced by Aβ42 and IL-17Ab treatment, but the time of center duration was reduced in the Aβ42 group compared with the control, and ameliorated with the administration of IL-17Ab (Fig. 3F), implicating that inhibition of IL-17 might reduce anxiety. The novelty recognition experiment allows to explore short-term memory capacity, the results from this experiment showed that in the Aβ42 + IL-17Ab group, the curiosity of exploring new things was significantly higher when compared with the Aβ42 group, as the time spent exploring new object in 2 h and 24 h tests were significantly increased (Fig. 3G, H). And then, in the fear conditioning test, the Aβ42 + IL-17Ab group showed a significantly increase of freezing time when compared with the Aβ42 group, during 2 h and 24 h test (Fig. 3I, J), suggesting that inhibition of IL-17 could rescue the memory function. Taken together, these data demonstrate that inhibition of IL-17 attenuates Aβ42-induced cognitive anxiety, social ability impairments, and learning and memory.

To investigate the underlying the role of IL-17 in preventing behavioral alterations, we wonder whether IL-17 play a role in synaptic plasticity, and carried out some electrophysiology tests from hippocampal slices after behavioral tests. We explored whether hippocampal-dependent synaptic plasticity was detected by recording long-term potentiation (LTP). The LTP test showed that inhibition of IL-17 enhanced the slope of field excitatory postsynaptic potential (fEPSP) after high-frequency stimulation (HFS) compared with the Aβ42 group (Fig. 4A, B). We also observed the number of synapses in the hippocampus with TEM, and the results showed that the number of synapses per 100 μm2 CA1 area increased significantly after the IL-17Ab supplement compared with the Aβ42 model group mice (Fig. 4C, D). In addition, we further examined the spine density of hippocampal neurons (Fig. 4E). The Golgi staining showed a significant increase in the dendritic spine density of the Aβ42 + IL-17Ab rats compared with the Aβ42 group (Fig. 4F). We examined molecular changes in synapse-related proteins in the hippocampus. Western blotting (Fig. 4G) result showed a significant increase in the levels of SYN (Fig. 4H) and PSD95 (Fig. 4I) in the Aβ42 + IL-17Ab group. These results indicate that the inhibition of IL-17 alleviates Aβ42-induced cognitive deficits and synaptic dysfunction.

Fig. 4
figure 4

Inhibition of IL-17 alleviates Aβ42-induced synaptic dysfunction. Normalized CA3-CA1 fEPSP mean slope recorded from CA1 dendritic region in hippocampal slices (A). Quantitative analysis of normalized fEPSP slops (B). N = 3, brain slice per mouse was recorded. TEM showed the structure of synapses. Red arrows indicated the structure of the presynaptic and postsynaptic membranes, and the synaptic cleft (C). Quantitative analyses of the number of synapses (D), N = 12, scale bar = 1 μm. Representative images of dendritic spines of neurons from Golgi staining hippocampus (E), averaged spine density (mean spine number per 10-mm dendrite segment) was measured in mice (F), N = 90, scale bar = 2 μm. PSD95 and SYN expression levels were detected by western blotting using specific antibodies, and actin was used as a loading control (G). Intensity analysis of SYN (H) and PSD95levels (I). N = 3. Data are presented as mean ± SD.p value significance is calculated from a one-way ANOVA test, *p < 0.05, **p < 0.01 and ***p < 0.001 vs Aβ group

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Inhibition of IL-17 attenuates Aβ42-induced neuronal damage by inactivation TRAF6-NF-κB signaling

To explore mechanism of restorative effect of IL-17Ab on learning and memory in Aβ42 + IL-17Ab mice. We performed western blotting to detect the levels of TRAF6. The results showed that the level of TRAF6 was increased in Aβ42 model mice compared with control mice, and IL-17Ab decreased the levels of TRAF6 compared with the Aβ42 group (Fig. 5A, B). Therefore, in order to explore NF-κB activation. We separate cytosolic and nuclear proteins, western blotting results showed that Aβ42 treatment upregulated the translocation of p65 from the cytoplasm to the nucleus (Fig. 5C–F). IL-17Ab obviously decreased Aβ42-induced nucleus phospho-NF-κB p65 (Ser536) translocation (Fig. 5C–F). Thus, these results suggested that IL-17Ab attenuates Aβ42-induced neuronal damage by inactivation TRAF6/NF-κB signaling.

Fig. 5
figure 5

Inhibition of IL-17 attenuates Aβ42-induced neuronal damage by inactivation TRAF6/NF-κB signaling. TRAF-6 expression levels was detected by western blotting using specific antibodies, and actin was used as a loading control (A). Intensity analysis of TRAF-6 (B). Western blotting showed the expression of phospho-NF-κB p65 in the cytoplasm (C) and nuclear (E) in the hippocampus of mice. Intensity analysis of the phospho-NF-κB p65 levels in the cytoplasm (D) and nuclear (F). N = 5 for independent experiments. Data are presented as mean ± SD. p value significance is calculated from a one-way ANOVA test, **p < 0.01 and ***p < 0.001 vs Aβ group

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Discussion

Neuroinflammation plays a critical role in the pathophysiology of Aβ-induced neurotoxicity and cognitive decline [28,29,30]. In the present study, we here found that IL-17 was increased in APP/PS1 mice, we showed that IL-17 is involved in Aβ-induced neurotoxicity.

IL-17 is a pro-inflammatory cytokine produced by various types of cells including CD4 T which are categorized as a new subset called Th17 cells [31]. Th17 cells are the main cellular mediators responsible for immune-mediated damages that polarize to the site of inflammation in presence of noxious or inflammatory stimuli [31, 32]. However, the effect of cytokine IL-17 remains poorly understood and very little is known about its pathophysiological role in the regions of the CNS usually compromised in disease [33]. To investigate the role of IL-17 in neuropathological changes and memory deficits, the IL-17 (10 ng/mL) was treated in hippocampal primary neurons, and the dendrintic morphology of hippocampal primary neurons was stained by using anti-MAP2 and PSD95 antibodies. And results showed that when compared with the control, IL-17 resulted in an obvious decreased dendritic arborization complexity at all points farther than 50 μm from the cell body, as well as the total dendritic length. These findings suggest that IL-17 induced hippocampal neural damage.

We therefore hypothesized that the IL-17 might been a target for intervention of Aβ42 induced-neurotoxicity. To test my hypothesis, we explored whether IL-17Ab could ameliorate Aβ42-induced cognitive impairment and synaptic dysfunction. Interestingly, Using Aβ42 mice, the results showed that the level of IL-17 was increased in Aβ42 model mice, andIL-17Abcould ameliorate Aβ-induced neurotoxicity and cognitive decline in C57BL/6 mice.

Then, to explore mechanism of restorative effect of IL-17Ab on learning and memory. Mechanistically, we show that IL-17Ab ameliorates the neuronal damage in Aβ42 model mice which is mediated by inhibition of IL-17 signaling. The cellular levels of TRAF6 and nuclear phospho-NF-κB p65 (Ser536) were elevated in Aβ42 model mice, and administration of IL-17Ab reduced the levels. IL-17 is reported to activate NF-κB signaling through IL-17R by recruiting TRAF6. Our study showed that IL-17Ab may inactivate NF-κB and reduce its translocation from the cytoplasm to the nucleus. NF-κB signaling in the central neurons system has a vital role by regulating certain functions like neuronal plasticity and neuronal growth [34], and the inhibition of the NF-κB plays a novel target in Alzheimer's disease therapy [35]. IL-17A binded ACT1 to IL-17-RA allows incorporation of tumour necrosis factor receptor-associated factor 6 (TRAF-6) adaptor protein into the complex, which in turn leads to activation of inhibitory κB kinase, liberating the transcription factors nuclear factor κB (NF-κB), our results showed that Aβ42 treatment upregulated the translocation of p65 from the cytoplasm to the nucleus. IL-17Ab obviously decreased Aβ42-induced nucleus phospho-NF-κB p65 translocation. Thus, these results imply that IL-17 induces neuronal damage by activation of IL-17/ TRAF6/ NF-κB pathway.

The key finding in our study was that Aβ mediated neurotoxicity via IL-17 inflammatory factor. Our results showed that inhibition of IL-17 could reverse the Aβ-induced neurotoxicity. In the future, we will apply this basic research to the clinical application of IL-17 inhibition to improve cognitive impairment in patients with clinical Alzheimer's disease. That will be a new challenge.

Conclusion

Conclusively, we have described the pathogenic role of IL-17 in Aβ induced-synaptic dysfunction and cognitive deficits. Using Aβ42 mice, the results showed that the level of IL-17 was increased in Aβ42 model mice, and IL-17Ab could ameliorate Aβ-induced neurotoxicity and cognitive decline in C57BL/6 mice by downregulation the TRAF6/ NF-κB pathway (Fig. 6), which provides new clues for the mechanism of Aβ-induced cognitive impairments, and a basis for therapeutic intervention.

Fig. 6
figure 6

Schematic diagram of hypothesis that activation of the IL-17/TRAF6/NF-κB pathway is implicated in Aβ-induced neurotoxicity. It showed that the level of IL-17 was increased in Aβ42 model mice, and IL-17Ab could ameliorate Aβ-induced neurotoxicity and cognitive impairments in C57BL/6 mice by downregulation the TRAF6/ NF-κB pathway

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Availability of data and materials

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

Abbreviations

AD:

Alzheimer’s disease

Aβ:

Amyloid-β

CTR:

Control

DMSO:

Dimethyl sulfoxide

Elisa:

Enzyme-linked ImmunoSorbent assays

fEPSP:

Field excitatory postsynaptic potential

IL-17A:

Interleukin-17A

IL-17Ab:

Interleukin-17A antibody

LTP:

Long-term potentiation

NF-κB:

Transcription factors nuclear factor κB

NORT:

Novel objective recognition test

PSD-95:

Postsynaptic density protein 95

SYN:

SynapsinI

TEM:

Transmission electron microscopy

TRAF-6:

Tumour necrosis factor receptor-associated factor 6

PBS:

Phosphate buffered saline

aCSF:

Artificial cerebrospinal fluid

References

  1. Mantzavinos V, Alexiou A. Biomarkers for Alzheimer’s disease diagnosis. Curr Alzheimer Res. 2017;14(11):1149–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Graff-Radford J, Yong KXX, Apostolova LG, Bouwman FH, Carrillo M, Dickerson BC, Rabinovici GD, Schott JM, Jones DT, Murray ME. New insights into atypical Alzheimer’s disease in the era of biomarkers. Lancet Neurol. 2021;20(3):222–34.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Hodson R. Alzheimer’s disease. Nature. 2018;559(7715):S1.

    Article  CAS  PubMed  Google Scholar 

  4. Brody H. Alzheimer’s disease. Nature. 2011;475(7355):S1.

    Article  CAS  PubMed  Google Scholar 

  5. Gallardo G, Holtzman DM. Amyloid-beta and Tau at the crossroads of Alzheimer’s disease. Adv Exp Med Biol. 2019;1184:187–203.

    Article  CAS  PubMed  Google Scholar 

  6. Lesne S, Koh MT, Kotilinek L, Kayed R, Glabe CG, Yang A, Gallagher M, Ashe KH. A specific amyloid-beta protein assembly in the brain impairs memory. Nature. 2006;440(7082):352–7.

    Article  CAS  PubMed  Google Scholar 

  7. Pinheiro L, Faustino C. Therapeutic strategies targeting amyloid-beta in Alzheimer’s disease. Curr Alzheimer Res. 2019;16(5):418–52.

    Article  CAS  PubMed  Google Scholar 

  8. Benilova I, Karran E, De Strooper B. The toxic Abeta oligomer and Alzheimer’s disease: an emperor in need of clothes. Nat Neurosci. 2012;15(3):349–57.

    Article  CAS  PubMed  Google Scholar 

  9. Brouillette J, Caillierez R, Zommer N, Alves-Pires C, Benilova I, Blum D, De Strooper B, Buee L. Neurotoxicity and memory deficits induced by soluble low-molecular-weight amyloid-beta1-42 oligomers are revealed in vivo by using a novel animal model. J Neurosci. 2012;32(23):7852–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Mangalmurti A, Lukens JR. How neurons die in Alzheimer’s disease: implications for neuroinflammation. Curr Opin Neurobiol. 2022;75:102575.

    Article  CAS  PubMed  Google Scholar 

  11. Kleffman K, Levinson G, Rose IVL, Blumenberg LM, Shadaloey SAA, Dhabaria A, Wong E, Galan-Echevarria F, Karz A, Argibay D, et al. Melanoma-secreted amyloid beta suppresses neuroinflammation and promotes brain metastasis. Cancer Discov. 2022;12(5):1314–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. McGeachy MJ, Cua DJ, Gaffen SL. The IL-17 Family of cytokines in health and disease. Immunity. 2019;50(4):892–906.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Moynes DM, Vanner SJ, Lomax AE. Participation of interleukin 17A in neuroimmune interactions. Brain Behav Immun. 2014;41:1–9.

    Article  CAS  PubMed  Google Scholar 

  14. Cai Y, Shen X, Ding C, Qi C, Li K, Li X, Jala VR, Zhang HG, Wang T, Zheng J, et al. Pivotal role of dermal IL-17-producing gammadelta T cells in skin inflammation. Immunity. 2011;35(4):596–610.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Akbar M, Crowe LAN, McLean M, Garcia-Melchor E, MacDonald L, Carter K, Fazzi UG, Martin D, Arthur A, Reilly JH, et al. Translational targeting of inflammation and fibrosis in frozen shoulder: molecular dissection of the T cell/IL-17A axis. Proc Natl Acad Sci U S A. 2021. https://doi.org/10.1073/pnas.2102715118.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Draberova H, Janusova S, Knizkova D, Semberova T, Pribikova M, Ujevic A, Harant K, Knapkova S, Hrdinka M, Fanfani V, et al. Systematic analysis of the IL-17 receptor signalosome reveals a robust regulatory feedback loop. EMBO J. 2020;39(17):e104202.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wang X, Yang L, Huang F, Zhang Q, Liu S, Ma L, You Z. Inflammatory cytokines IL-17 and TNF-alpha up-regulate PD-L1 expression in human prostate and colon cancer cells. Immunol Lett. 2017;184:7–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Burnham SC, Faux NG, Wilson W, Laws SM, Ames D, Bedo J, Bush AI, Doecke JD, Ellis KA, Head R, et al. A blood-based predictor for neocortical Abeta burden in Alzheimer’s disease: results from the AIBL study. Mol Psychiatry. 2014;19(4):519–26.

    Article  CAS  PubMed  Google Scholar 

  19. Hampel H, O’Bryant SE, Molinuevo JL, Zetterberg H, Masters CL, Lista S, Kiddle SJ, Batrla R, Blennow K. Blood-based biomarkers for Alzheimer disease: mapping the road to the clinic. Nat Rev Neurol. 2018;14(11):639–52.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Brigas HC, Ribeiro M, Coelho JE, Gomes R, Gomez-Murcia V, Carvalho K, Faivre E, Costa-Pereira S, Darrigues J, de Almeida AA, et al. IL-17 triggers the onset of cognitive and synaptic deficits in early stages of Alzheimer’s disease. Cell Rep. 2021;36(9):109574.

    Article  CAS  PubMed  Google Scholar 

  21. Latt HM, Matsushita H, Morino M, Koga Y, Michiue H, Nishiki T, Tomizawa K, Matsui H. Oxytocin inhibits corticosterone-induced apoptosis in primary hippocampal neurons. Neuroscience. 2018;379:383–9.

    Article  CAS  PubMed  Google Scholar 

  22. Turlova E, Bae CYJ, Deurloo M, Chen W, Barszczyk A, Horgen FD, Fleig A, Feng ZP, Sun HS. TRPM7 regulates axonal outgrowth and maturation of primary hippocampal neurons. Mol Neurobiol. 2016;53(1):595–610.

    Article  CAS  PubMed  Google Scholar 

  23. Do KV, Kautzmann MI, Jun B, Gordon WC, Nshimiyimana R, Yang R, Petasis NA, Bazan NG. Elovanoids counteract oligomeric beta-amyloid-induced gene expression and protect photoreceptors. Proc Natl Acad Sci U S A. 2019;116(48):24317–25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zeng J, Jiang X, Hu XF, Ma RH, Chai GS, Sun DS, Xu ZP, Li L, Bao J, Feng Q, et al. Spatial training promotes short-term survival and neuron-like differentiation of newborn cells in Abeta1-42-injected rats. Neurobiol Aging. 2016;45:64–75.

    Article  CAS  PubMed  Google Scholar 

  25. Guo C, Liu Y, Fang MS, Li Y, Li W, Mahaman YAR, Zeng K, Xia Y, Ke D, Liu R, et al. omega-3PUFAs improve cognitive impairments through Ser133 phosphorylation of CREB upregulating BDNF/TrkB signal in schizophrenia. Neurotherapeutics. 2020;17(3):1271–86.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Lisman J, Buzsaki G, Eichenbaum H, Nadel L, Ranganath C, Redish AD. Viewpoints: how the hippocampus contributes to memory, navigation and cognition. Nat Neurosci. 2017;20(11):1434–47.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kutlu MG, Gould TJ. Nicotinic receptors, memory, and hippocampus. Curr Top Behav Neurosci. 2015;23:137–63.

    Article  CAS  PubMed  Google Scholar 

  28. Cai Z, Hussain MD, Yan LJ. Microglia, neuroinflammation, and beta-amyloid protein in Alzheimer’s disease. Int J Neurosci. 2014;124(5):307–21.

    Article  CAS  PubMed  Google Scholar 

  29. Nazem A, Sankowski R, Bacher M, Al-Abed Y. Rodent models of neuroinflammation for Alzheimer’s disease. J Neuroinflammation. 2015;12:74.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Minter MR, Taylor JM, Crack PJ. The contribution of neuroinflammation to amyloid toxicity in Alzheimer’s disease. J Neurochem. 2016;136(3):457–74.

    Article  CAS  PubMed  Google Scholar 

  31. Steinman L. A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage. Nat Med. 2007;13(2):139–45.

    Article  CAS  PubMed  Google Scholar 

  32. Martinez NE, Sato F, Kawai E, Omura S, Takahashi S, Yoh K, Tsunoda I. Th17-biased RORgammat transgenic mice become susceptible to a viral model for multiple sclerosis. Brain Behav Immun. 2015;43:86–97.

    Article  CAS  PubMed  Google Scholar 

  33. Ruiz de Morales JMG, Puig L, Dauden E, Canete JD, Pablos JL, Martin AO, Juanatey CG, Adan A, Montalban X, Borruel N, et al. Critical role of interleukin (IL)-17 in inflammatory and immune disorders: an updated review of the evidence focusing in controversies. Autoimmun Rev. 2020;19(1):102429.

    Article  CAS  PubMed  Google Scholar 

  34. Gutierrez H, Davies AM. Regulation of neural process growth, elaboration and structural plasticity by NF-kappaB. Trends Neurosci. 2011;34(6):316–25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Zhao Y, Bhattacharjee S, Jones BM, Hill J, Dua P, Lukiw WJ. Regulation of neurotropic signaling by the inducible, NF-kB-sensitive miRNA-125b in Alzheimer’s disease (AD) and in primary human neuronal-glial (HNG) cells. Mol Neurobiol. 2014;50(1):97–106.

    Article  PubMed  Google Scholar 

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Acknowledgements

The authors would like to thank Yingxia Jin and Lina Zhou from Central Laboratory, Renmin Hospital of Wuhan University, for their technical assistance.

Funding

This work was supported by the National Natural Science Foundation of China (No.8187032070) and the Fundamental Research Funds for the Central Universities (No.2042019kf0081).

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Author notes

  1. Yulan Liu and Yang Meng have contributed equally to this work and share the first authorship

Authors and Affiliations

  1. Department of Critical Care Medicine, Renmin Hospital of Wuhan University, Wuhan, China

    Yulan Liu, Chenliang Zhou, Juanjuan Yan & Cuiping Guo

  2. Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan, China

    Yulan Liu & Weiguo Dong

  3. Central Laboratory, Renmin Hospital of Wuhan University, Wuhan, China

    Yulan Liu, Yang Meng, Cuiping Guo & Weiguo Dong

  4. Department of Gastrointestinal Surgery II, Renmin Hospital of Wuhan University, Wuhan, China

    Yang Meng

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  1. Yulan Liu
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  4. Juanjuan Yan
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  5. Cuiping Guo
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Contributions

WD and CG planned, organized, and designed all experiments. YL and YM planned and performed all experiments, including the writing of the manuscript. CZ assisted with the manuscript preparation. YL, YM and JY analyzed the data. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Cuiping Guo or Weiguo Dong.

Ethics declarations

Ethics approval and consent to participate

This study was approved by the Animal Care Committees of T the Ethics Committee of Renmin Hospital, Wuhan University (IACUC Issue No. WDRY2018-K033), in accordance with international regulations. The study is reported in accordance with ARRIVE guidelines.

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Not applicable.

Competing interests

All authors declare no competing interest in this current study.

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Liu, Y., Meng, Y., Zhou, C. et al. Activation of the IL-17/TRAF6/NF-κB pathway is implicated in Aβ-induced neurotoxicity. BMC Neurosci 24, 14 (2023). https://doi.org/10.1186/s12868-023-00782-8

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  • DOI: https://doi.org/10.1186/s12868-023-00782-8

Keywords

  • IL-17
  • Amyloid-β
  • IL-17Ab
  • Synaptic dysfunction
  • Cognitive decline

BMC Neuroscience

ISSN: 1471-2202

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