NEK7 inhibition attenuates Aβ₄₂-induced cognitive impairment by regulating TLR4/NF-κB and the NLRP3 inflammasome in mice

Abstract

NEK7 is a serine/threonine kinase that regulates cell mitosis and the activation of the nucleotide-binding oligomerization domain-like (NOD-like) receptor thermal protein domain associated protein 3 (NLRP3) inflammasome, and is related to neuroinflammation and neuronal damage. The purpose of this study was to explore the role and mechanism of NEK7 in cognitive impairment in Alzheimer’s disease (AD). BV2 cells, a microglia cell line, was treated with Aβ₄₂. NEK7 expression was measured with reverse transcription–quantitative polymerase chain reaction and Western blotting. An apoptosis kit was used to determine the apoptotic rate. APPswe/PS1dE9 (APP/PS1) transgenic mice were used as an in vivo AD model. The experimental mice were infected with sh-NEK7 lentivirus to downregulate NEK7. The Morris water maze was conducted to explore the effect of NEK7 downregulation on cognitive ability. The results showed that Aβ₄₂ significantly upregulated NEK7 in BV2 cells. Silencing NEK7 suppressed the decrease in BV2 viability and the increase in inflammation, oxidative stress and apoptosis induced by Aβ₄₂. NEK7 mediated it effects through the TLR4/NF-κB signalling pathway and the NLRP3 inflammasome. Finally, inhibition of NEK7 alleviated the cognitive impairment in APP/PS1 mice. In conclusion, Silencing NEK7 suppresses Aβ₄₂-induced cell apoptosis, inflammation and oxidative stress, and improves cognitive performance in AD mice. NEK7 may be a potential target for AD treatment.

Introduction

Alzheimer’s disease (AD) is a slow, progressive neurodegenerative disease characterised by amyloid plaques, neurofibrillary tangles and nerve cell death.⁽¹⁾ Patients with AD initially show mild memory loss, followed by more severe memory loss, mood changes and eventually develop cognitive impairment and the inability to perform activities of daily living.⁽²⁾ Age is an important risk factor for AD, and given the ageing of the global population, AD will continue to be serious financial and emotional burden on society and families.⁽³⁾ Researchers have proposed several pathological mechanisms related to AD, including the amyloid cascade hypothesis, the tau hypothesis, the cholinergic hypothesis, excitotoxicity, vascular disease and neuroinflammation.⁽⁴⁾ Currently, there are two main types of drugs used to treat the symptoms of AD, including cholinesterase inhibitors such as donepezil and non-competitive N-methyl-d-aspartate receptor antagonists such as memantine. These drugs can delay the development of the disease, but cannot cure the disease.⁽⁵⁾ Therefore, an in-depth understanding of the pathological mechanism of AD and finding effective targets to control the course of the disease are crucial to the development of new drugs.

Neuroinflammation involves the interaction between multiple cells in the central nervous system, and plays an important role in the homeostasis of cells and tissues and neuronal function.⁽⁶⁾ Studies have found that neuroinflammation is involved in AD pathogenesis. Inflammation caused by acute injury has little effect on the survival of neurons, but long-term chronic inflammation keeps the levels of cytokines and chemokines high, which increases the expression and production of type I transmembrane amyloid precursor protein (APP) and beta amyloid (Aβ).⁽⁷⁾ The aggregation and extracellular deposition of Aβ oligomers can lead to the death of neurons and promote AD progression.⁽⁸⁾ In addition, neuroinflammation caused by amyloid plaques and neurofibrillary tangles can lead to neurodegeneration, and inhibiting neuroinflammation by targeting related genes can attenuate the pathological state of AD.⁽⁹⁾

NEK7, a member of the NEK family, is a serine/threonine kinase involved in different cellular processes.⁽¹⁰⁾ The main function of NEK7 is to regulate mitosis and to activate the NLRP3 inflammasome. In addition, NEK7 is distributed in the hippocampus and may be beneficial to the neurotransmitter system and immune regulation.⁽¹¹⁾ NEK7 bridges adjacent NLRP3 subunits through dichotomous interactions and activates the NLRP3 inflammasome.⁽¹²⁾ Activated NLRP3 may rely on interleukin (IL)-1β to induce tau hyperphosphorylation and aggregation.⁽¹³⁾ This is of great significance in the development of AD.

The study aimed to explore the role and mechanism of NEK7 in Aβ₄₂-treated microglia and AD mice. We found that silencing NEK7 reduced Aβ₄₂-induced neuroinflammation in BV2 cells by inhibiting the activation of TLR4/NF-κB pathway and NLRP3 inflammasome. In addition, silencing NEK7 alleviated cognitive impairment in APP/PS1 transgenic mice. Targeting NEK7 may provide a potential strategy to treat AD.

Materials and Methods

Animal model

Twenty 6-month-old APPswe/PS1dE9 (APP/PS1) double transgenic male mice and 10 C57BL/6J wild-type male mice of the same age were purchased from the Model Animal Research Center of Nanjing University. All mice were kept in a specific pathogen free animal laboratory at 23 ± 3°C and 60–65% relative humidity. APP/PS1 mice were randomly divided into two groups, one group was the control group and the other was the experimental group. Mice in the experimental group were injected with sh-NEK7 lentivirus through the tail vein. At the end of the experiment, the mice were fasted overnight, euthanised, blood was collected and hippocampal tissue was dissected for subsequent experiments. This research was performed according with the guidelines for the use of laboratory animals and approved by the institutional research animal committee of Shaanxi Provincial People’s Hospital.

Cell culture and transfection

Immortalised mouse BV2 microglia were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA) medium supplemented with 10% foetal bovine serum (Gibco) and 100 U/ml penicillin and streptomycin at 37°C and 5% CO₂. NEK7 small interfering RNA (siRNA) was used to knock down NEK7 in BV2 cells, and pcDNA3.1 was used to overexpress TLR4 and NLRP3 in BV2 cells. BV2 cells were transfected with control siRNA, NEK7 siRNA, pcDNA3-TLR4 or pcDNA3.1-NLRP3 following the manufacturer’s instructions. The efficiency of knockdown or overexpression of BV2 cells was confirmed by reverse transcription–quantitative polymerase chain reaction (RT-qPCR) and Western blotting.

Cell viability determination

The cell viability was measured using the CCK-8 method. BV2 cells were seeded in 96-well culture plates (2 × 10³ cells per well). After 24 h of culture, 10 μl of CCK-8 solution was added to each well and incubated for 4 h. The absorbance was measured at 450 nm using a microplate reader.

Apoptosis

The Annexin V-FITC/PI cell apoptosis detection kit (Solarbio, Beijing) was used to determine the apoptotic rate. Cells (1 × 10⁶ cells per time point) were collected and washed with cold phosphate-buffered saline (PBS), centrifuged and resuspended in the binding buffer. Then, 5 μl of Annexin V-FITC was added for every 1 × 10⁵ cell, and the mixture was incubated in the dark at room temperature for 10 min. Next, 5 μl of propidium iodide (PI) was added and the mixture was incubated for 5 min. The proportion of apoptotic cells was detected by flow cytometry.

Enzyme-linked immunosorbent assay

The content of IL-6, IL-8, tumour necrosis factor (TNF)-α, glutathione peroxidase (GPx), and superoxide dismutase (SOD) was determined by using commercially available ELISA kits (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China). The level of reactive oxygen species (ROS) was detected by the Total ROS Detection Kit (Thermo Fisher Scientific). The level of NAD/NADH was detected with the NAD/NADH kit (Beyotime, Beijing, China), Finally, the level of ATP was detected with the ATP Detection Kit (Beyotime, Beijing, China). Each assay was completed in accordance with the manufacturers’ instructions. The measurements were performed in triplicate.

RT-qPCR

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). RNA was reverse transcribed to cDNA using Qiagen One-Step RT-PCR kit (Qiagen Gmbh, Dusseldorf, Germany). cDNA, primers and SYBR^TM Green PCR Master Mix (Thermo Fisher) were mixed and qRT-PCR was performed. GAPDH was used as an internal reference. The reaction conditions in the experiments were performed according to the manufacturer’s instructions. The expression levels of target genes were determined using the 2^−ΔΔCT method. The sequences of each primer are: NEK7: F 5'-CCGTTACTCAGTTCCAGCCA-3', R 5'-CTACCGGCACTCCCATCCAAG-3'; GAPDH: F 5'-GTT ACC AGG GCT GCC TTC TC-3', R 5'-GTG ATG GCA TGG ACT GTG GT-3'.

Western blot

BV2 cells and hippocampal tissue were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer. After incubation and centrifugation, the supernatant was collected. The BCA method was used to determine the protein concentration in the supernatant, and sodium dodecyl sulphate (SDS) loading buffer was added to denature the protein. Protein was loaded on a 10% or 12% polyacrylamide gel and separated by electrophoresis. The separated protein was transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with 5% non-fat milk for 1 h, incubate with the primary antibody overnight at 4°C and then incubated with the secondary antibody at room temperature for 2 h. An enhanced chemiluminescence (ECL) reagent was used to visualise the protein bands. ImageJ software was to quantify the density of the bands. Blots were probed with the following antibodies: rabbit polyclonal to GAPDH-Loading Control (1:1,000, abcam, ab8245), rabbit polyclonal anti-NEK7 antibody (1:2,000, abcam, ab133514), rabbit monoclonal anti-caspase-3 antibody (1:2,000, abcam, ab32351), rabbit monoclonal anti-caspase-9 antibody (1:10,000, abcam, ab65608), rabbit monoclonal anti-Bax antibody (1:1,000, abcam, ab32503), rabbit monoclonal anti-Bcl-2 antibody (1:2,000, abcam, ab32124), rabbit monoclonal anti-COX-2 antibody (1:1,000, abcam, ab120295), rabbit polyclonal anti-iNOS antibody (1:1,000, abcam, ab178945), rabbit polyclonal anti-BDNF antibody (1:1,000, abcam, ab108319), rabbit polyclonal anti-NGF antibody (1:1,000, abcam, ab52987), rabbit polyclonal anti-NLRP3 antibody (1:1,000, abcam, ab263899), rabbit polyclonal anti-TLR4 antibody (1:1,000, abcam), rabbit polyclonal anti-NF-kB p65 antibody (1:1,000, abcam).

Morris water maze test

The Morris water maze test was performed on the mice 1 week before the end of the experiment. The maze is divided into four quadrants. The platform is placed in one 1 cm below the surface of the water. For each trial, the swimming path and the escape latency, the time it takes the mouse to find the platform, are recorded. Before training, all mice were allowed to swim freely in the pool for 1 min to familiarise themselves with the environment. For each training trial, the mouse was put into a different position in the pool, and the escape latency and path were recorded by a computer tracking system. The interval between each trial was 15 min. After 5 days of training, each mouse was subjected to the space exploration experiment. For this endeavour, the underwater platform was removed and the percentage of time the mouse spent in the target quadrant (which had contained the platform), the number of times it passed over the original platform location and the path were recorded.

Statistical analysis

The results of three independent replicate experiments were expressed as mean ± SD. One-way analysis of variance and Student’s unpaired t-test were used for statistical comparison of the groups. P<0.05 was considered statistically significant. All analyses were performed with SPSS Statistics ver. 20.0 (IBM Corp., Armonk, NY).

Results

Different Aβ₄₂ concentrations promote the expression of NEK7 in BV2 cells

We first treated BV2 cells with Aβ₄₂; it significantly and dose-dependently inhibited BV2 viability (Fig. 1A). Compared with the control group, Aβ₄₂ significantly and dose-dependently promoted NEK7 messenger RNA (mRNA) and protein expression (Fig. 1B and C). Taken together, Aβ₄₂ significantly inhibits the viability of BV2 cells and promotes NEK7 expression.

Fig. 1.

NEK7 expression is induced by Aβ₄₂ in BV2 cells. BV2 cells were treated with different concentrations (0, 100, 200, 500 ng/ml) of Aβ₄₂ for 12 h. (A) The cell viability of BV2 cells treated with different doses of Aβ₄₂. (B) The mRNA expression of NEK7 in BV2 cells after treated with different doses of Aβ₄₂. (C) The protein expression of NEK7 in BV2 cells after treated with different doses of Aβ₄₂. * means compared untreated group p<0.05. ** means compared with untreated group p<0.01. GAPDH was used as an invariant internal control for calculating protein-fold changes.

Silencing of NEK7 attenuates the apoptosis of Aβ₄₂-induced BV2 cells

To explore the effect of NEK7 on BV2 cells, we inhibited NEK7 expression with siRNA (Fig. 2A). We used caspase inhibitor (Z-VAD) to evaluate the effect of NEK7 on cell apoptosis. si-NEK7 significantly counteracted the reduced BV2 viability induced by Aβ₄₂ (Fig. 2B). Aβ₄₂ enhanced BV2 apoptosis, while NEK7 silencing attenuated this effect, consistent with the effect of Z-VAD. (Fig. 2C). Consistently, Aβ₄₂ increased the expression of the pro-apoptotic proteins Bax and caspase-3 and decreased the expression of the anti-apoptotic protein Bcl-2. On the contrary, NEK7 silencing significantly reversed these effects (Fig. 2D–F). These results indicate that silencing NEK7 inhibits BV2 apoptosis.

Fig. 2.

Silencing of NEK7 attenuates the apoptosis of Aβ₄₂-induced BV2 cells. BV2 cells were transfected with control si-RNA or si-NEK7 for 24 h, and then treated with Aβ₄₂ (200 ng/ml) for 12 h. Caspase inhibitor (Z-VAD) was used to evaluate the effect of NEK7 on cell apoptosis. (A) The protein expression of NEK7 was detected by Western blot. Untreated cells were performed as control. (B) The cell viability of BV2 cells was detected by CCK-8 assay. (C) The apoptosis rate of Aβ₄₂-induced BV2 cells was measured by fluorescence flow cytometry. (D, E) The protein expression of caspase-3, caspase-9, Bax, and Bcl-2 was detected by Western blot. (F) Western blot was used to measure the protein expression of NEK7, caspase-3, caspase-9, Bax, and Bcl-2. * means compared with untreated group p<0.05, and ^# means compared with si-RNA group p<0.05. GAPDH was used as an invariant internal control for calculating protein-fold changes.

Silencing NEK7 attenuates Aβ₄₂-induced BV2 inflammation

We next explored the role of NEK7 on inflammation of Aβ₄₂-treated BV2 cells. Aβ₄₂ induced IL-6, IL-8, and TNF-α production compared with the untreated group, and increased the expression of COX-2 and iNOS, while silencing of NEK7 significantly reversed these effects (Fig. 3A–D). The study found that Aβ42 inhibited the expression of BNDF and NGF compared with the untreated group, while the silence of NEK7 significantly promoted the expression of BNDF and NGF (Fig. 3E and F). These results indicated that silencing of NEK7 inhibited the inflammation of Aβ₄₂-induced BV2 cells and promotes the release of protective factors.

Fig. 3.

Silencing of NEK7 attenuates the inflammation of Aβ₄₂-induced BV2 cells. BV2 cells were transfected with si-RNA or si-NEK7 for 24 h, and then treated with Aβ₄₂ (200 ng/ml) for 12 h. (A–C) The levels of IL-6, IL-8, and TNF-α in BV2 cells were measured by ELISA assay. (D) The protein expression of iNOS and COX-2 was detected by Western blot. (E) The protein expression of BDNF and NGF was detected by Western blot. (F) Western blot was used to measure the protein expression of iNOS, COX-2, BDNF, and NGF. * means compared with untreated group p<0.05, ** means compared with untreated group p<0.01, and ^# means compared with si-RNA group p<0.05. GAPDH was used as an invariant internal control for calculating protein-fold changes.

Silencing NEK7 attenuates the oxidative stress of Aβ₄₂-treated BV2 cells

As expected, Aβ₄₂ significantly elevated ROS production in BV2 cells compared with the untreated group, and si-NEK7 reduced ROS production in these cells (Fig. 4A). Interestingly, silencing NEK7 increased ATP and NAD⁺ in Aβ₄₂-stimulated BV2 cells (Fig. 4B and C). In addition, si-NEK7 enhanced the activity of SOD and GPx compared with the control si-RNA group. These results indicate that si-NEK7 inhibits Aβ₄₂-induced oxidative stress in BV2 cells.

Fig. 4.

Silencing of NEK7 attenuates the oxidative stress of Aβ₄₂-induced BV2 cells. BV2 cells were transfected with si-RNA or si-NEK7 for 24 h, and then treated with Aβ₄₂ (200 ng/ml) for 12 h. (A) The level of ROS of each group in BV2 cells. (B) The level of NAD⁺/NADH of each group in BV2 cells. (C) The level of ATP of each group in BV2 cells. (D) The level of SOD of each group in BV2 cells. (E) The level of GPx of each group in BV2 cells. * means compared with untreated group p<0.05, ** means compared with untreated group p<0.01, and ^# means compared with si-RNA group p<0.05.

Silencing NEK7 inhibits activation of the TLR4/NF-κB pathway and the NLRP3 inflammasome

We hypothesised that activation of TLR4/NF-κB pathway and the NLRP3 inflammasome promotes Aβ₄₂-induced injury to BV2 cells. Consistently, Aβ₄₂ significantly increased the expression of TLR4, phosphorylated p65 (p-p65) and NLRP3, while si-NEK7 inhibited the expression of these proteins (Fig. 5). These results suggest that silencing NEK7 inhibits activation of the TLR4/NF-κB pathway and the NLRP3 inflammasome.

Fig. 5.

Silencing of NEK7 inhibits activation of TLR4/NF-κB pathway and NLRP3 inflammasome. BV2 cells were transfected with si-RNA or si-NEK7 for 24 h, and then treated with Aβ₄₂ (200 ng/ml) for 12 h. (A) The protein expression of TLR4 was detected by Western blot. (B) The protein expression of p-p65 was detected by Western blot. (C) The protein expression of NLRP3 was detected by Western blot. (D) Western blot was used to measure the protein expression of TLR4, p-p65, and NLRP3. * means compared with untreated group p<0.05, ** means compared with untreated group p<0.01, and ^# means compared with si-RNA group p<0.05. GAPDH was used as an invariant internal control for calculating protein-fold changes.

The TLR4/NF-κB pathway and the NLRP3 inflammasome are involved in regulating the effects of NEK7 on Aβ_42-induced cell injury

Next, we explored the underlying mechanism by which si-NEK7 could inhibit the progression of AD. si-NEK7 significantly increased BV2 viability, while TLR4 or NLRP3 overexpression reduced viability (Fig. 6A). si-NEK7 suppressed BV2 cells apoptosis, while TLR4 or NLRP3 overexpression increased BV2 apoptosis (Fig. 6B). Meanwhile, si-NEK7 suppressed IL-6 production, while TLR4 or NLRP3 overexpression promoted IL-6 production (Fig. 6C). As expected, si-NEK7 significantly reduced ROS production compared with untreated group, and promoted NAD⁺ in Aβ₄₂-stimulated BV2 cells, while TLR4 or NLRP3 overexpression reversed these effects (Fig. 6D and E). These results suggest that the TLR4/NF-κB pathway and the NLRP3 inflammasome are involved in the mechanism by which NEK7 controls Aβ₄₂-induced AD progression.

Fig. 6.

TLR4/NF-κB pathway and NLRP3 inflammasome are involved in the regulation of NEK7 on Aβ₄₂-induced cell injury. BV2 cells were transfected with si-RNA, si-NEK7, pcDNA3.1-TLR4, or pcDNA3.1-NLRP3 for 24 h, and then treated with Aβ₄₂ (200 ng/ml) for 12 h. (A) The cell viability of BV2 cells was detected by CCK-8 assay. (B) The apoptosis rate of BV2 cells was measured by fluorescence flow cytometry. (C) The level of IL-6 in BV2 cells was measured by ELISA assay. (D) The level of ROS of each group in BV2 cells. (E) The level of NAD⁺/NADH of each group in BV2 cells. * means compared with untreated group p<0.05, and ^# means compared with si-NEK7 group p<0.05. GAPDH was used as an invariant internal control for calculating protein-fold changes.

Silencing of NEK7 alleviates cognitive impairment in mice

To evaluate the role of NEK7 in vivo, we used APP/PS1 mice, an established AD mouse model. We first examined the structure of the hippocampus in wild-type and APP/PS1 mice. In wild-type mice, the hippocampal neurons are banded. There are a large number of pyramidal neurons in CA1 and CA3, they appear orderly with a normal shape, and a large number of Nissl bodies in the cytoplasm. On the contrary, in APP/PS1 mice, the CA3 pyramidal neurons have obvious defects, including an incomplete cell structure, cellular swelling and rupture and disordered arrangement, and there are fewer Nissl bodies in the cytoplasm. In addition, Nissl bodies in DG region also decreased significantly. Infecting APP/PS1 mice with sh-NEK7 lentivirus markedly improved these defects, with a relatively complete and normal cellular structure, a much neater arrangement and more Nissl bodies in the cytoplasm (Fig. 7A). sh-NEK7 lentivirus infection downregulated NEK7 expression in the hippocampus (Fig. 7B). In the Morris water maze assay, the escape latency of untreated APP/PS1 mice significantly increased after 2–4 days of training compared with the control group. However, compared with the untreated APP/PS1 mice, the escape latency of APP/PS1 mice infected with sh-NEK7 lentivirus was significantly shorter, and the swimming speed was significantly improved (Fig. 7C and D). The untreated APP/PS1 mice spent significantly less time in the target quadrant and had significantly fewer platform crossings compared with wild-type mice (Fig. 7E and F). Infection of APP/PS1 mice with sh-NEK7 lentivirus markedly reduced these deficits, and these measures were not significantly different between the treated APP/PS1 mice and control mice (Fig. 7D and E).

Fig. 7.

Silencing of NEK7 alleviates cognitive impairment of mice. There were 30 mice, including 10 mice without any treatment (control group, n = 10), 10 6-month-old Alzheimer’s disease model mice (model group, n = 10), 6-month-old model mice injected with sh-NEK7 via tail vein (sh-NEK7 group, n = 10). (A) The pathological changes of each group were observed by Nissl staining (×400). Data were presented as mean ± SD with repeated for three times. (B) The protein expression of NEK7 was detected by Western blot. (C) The escape latency of each group mouse. (D) The swim speed of each group mouse. (E) The platform crossing times of each group mouse. (F) The times of platform crossing of each group mouse. * means compared with control group p<0.05, ** means compared with control group p<0.01, and ^# means compared with model group. GAPDH was used as an invariant internal control for calculating protein-fold changes.

Discussion

AD is a neurodegenerative disease that is closely related to pathological changes in neurons. It is the most common cause of dementia, followed by Lewy body dementia, and can be accompanied by vascular dementia.⁽¹⁴⁾ Microglia, as immune cells in the central nervous system, have two different roles in the pathogenesis of AD, and the specific mechanism remains to be resolved. Microglia can engulf Aβ and release anti-inflammatory factors and neuroprotective factors to protect neurons, but they also release pro-inflammatory mediators to aggravate inflammation and cause neuronal damage.⁽¹⁵⁾ This may be related to the activated phenotype of microglia.⁽¹⁶⁾ Neuroinflammation involving microglia is of great importance in the development of AD. Studies have shown that inhibiting neuroinflammation can improve the cognitive and memory functions of AD mice.⁽¹⁷⁾ In this study, we used Aβ₄₂ to induce damage in BV2 cells, a microglia cell line. In these cells, silencing NEK7 inhibited apoptosis and oxidative stress and significantly improved the state of inflammation. Silencing NEK7 also improved the cognitive impairment of APP/PS1 mice, a model of AD.

NEK7 is an activator of the NLRP3 inflammasome and thus essential for its key role in inflammatory diseases. Inhibition of the NLRP3 inflammasome improves the influence of peripheral immune stimulation on neuronal morphology and function, a phenomenon that underscores the relationship between NEK7 and neuroinflammation and neurological diseases.⁽¹⁸⁾ Studies have shown that NEK7 is expressed in parvalbumin-positive interneurons and regulates axon development in interneurons.⁽¹⁹⁾ Exogenous recombinant NEK7 can induce neuronal apoptosis and destroy the blood–brain barrier, leading to nervous system damage.⁽²⁰⁾ NEK7 knockdown inhibits activation of the NLRP3 inflammasome and its downstream activities, which reduces neuroinflammation and neuronal damage after traumatic brain injury.⁽²¹⁾ Therefore, we suspect that NEK7 may be related to the development of neurological diseases. In addition, NEK7 is a regulatory protein of the microtubule cytoskeleton, and alterations in microtubule regulatory factors are related to neurodegenerative diseases and neurodevelopmental disorders.⁽¹⁰⁾ We found that silencing NEK7 inhibited Aβ₄₂-induced inflammation and apoptosis of BV2 cells, findings in line with our expectations. In addition, we found that in APP/PS1 mice, silencing the expression of NEK7 improved the obvious defect of CA3 pyramidal neurons, and the Nissl bodies in the cytoplasm were significantly increased. The improvement is not obvious in CA1. This shows that silencing NEK7 in vivo can significantly improve the defect of neurons in AD mice.

APP and Aβ are some of the main markers of AD. Transgenic mice that overexpress mutated forms of APP (and thus produce and deposit Aβ) present a loss of neurons, which indicates that Aβ may act as a mediator in synaptic dysfunction and neuronal death.⁽²²⁾ Neuroinflammatory cytokines may cause Aβ accumulation by increasing APP expression.⁽²³⁾ High levels of Aβ would reduce glutamatergic synaptic transmission and cause synapse loss, which is closely related to cognitive decline.⁽²⁴⁾ In AD, the excessive production and deposition of Aβ leads to neuroinflammation, which includes the activation of microglia, the participation of the complement system and the production of cytokines.⁽²⁵⁾ Innate immune receptors such as TLR, Nod-like receptors, formyl peptide receptors, advanced glycation end product receptors, scavenger receptors, complement cascades and pentamers are activated after recognition of Aβ oligomers and aggregates, which in turn triggers neuroinflammation.⁽²⁶⁾ TLR4 is one of the key receptors of the innate immune system in microglia. The loss of TLR4 reduces neuronal death and improves cognitive impairment.⁽²⁷⁾ In addition to danger-associated molecular patterns (DAMPs), TLR4 activation in cells is regulated by Aβ oligomers.⁽²⁸⁾ TLR4 activates downstream signalling through MyD88-dependent and independent pathways, leading to the activation of NF-κB and interferon β (IFN-β).⁽²⁹⁾ Activation of this pathway intensifies inflammation and oxidative stress, which promotes the development of AD. Our research has shown that silencing NEK7 alleviates the cognitive impairment in mice by inhibiting the TLR4/NF-κB pathway.

Oxidative stress is a prominent early feature of AD and is closely related to inflammasome formation and inflammation. Activated microglia can also promote the release of ROS.⁽³⁰⁾ Increasing evidence shows that oxidative stress is related to the occurrence of AD. In addition to the combination of metal ions and Aβ peptides that will generate ROS, mitochondrial dysfunction can participate in AD pathogenesis by generating ROS.⁽³¹⁾ The NLRP3 inflammasome can mediate neuroinflammation and also plays an important role in the pathogenesis of AD. Inhibiting NLRP3 inflammasome activation can reduce the accumulation of Aβ and the deterioration of neuronal function.⁽³²⁾ Treatment with NLRP3 inhibitors alleviated AD-related deficiencies.⁽³⁰⁾ This indicates that inhibiting NLRP3 activity may be a relevant direction to ameliorate AD-related symptoms. We found that silencing NEK7 inhibited the activation of oxidative stress and the NLRP3 inflammasome in cells, which may be beneficial to control the progression of AD.

In conclusion, we have demonstrated for the first time the potential role of NEK7 in Aβ₄₂-induced microglia activation and AD mice. Silencing NEK7 reduced neuroinflammation by inhibiting activation of the TLR4/NF-κB pathway and the NLRP3 inflammasome, and further inhibited cognitive impairment in AD mice. NEK7 may be a potential target for AD treatment.

Author Contributions

PL and HG made substantial contribution to the conceptualization, investigation; NL involved in writing - original draft preparation; DZ, QY, NL and YH involved in methodology and formal analysis; HG involved in writing - review and editing.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of Interest

No potential conflicts of interest were disclosed.

Ethics Approval and Consent to Participate

This study was approved by the ethics committee of Shaanxi Provincial People’s Hospital. Prior to participating in the study, each participant signed a written informed consent form. The study was conducted according to the principles of the Declaration of Helsinki.

Availability of Data and Materials

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