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. Author manuscript; available in PMC: 2021 May 15.
Published in final edited form as: Neuropharmacology. 2020 Feb 27;168:108017. doi: 10.1016/j.neuropharm.2020.108017

Characterization of the effects of calcitonin gene-related peptide receptor antagonist for Alzheimer’s disease

Hana Na a, Qini Gan a, Liam Mcparland a, Jack B Yang a, Hongbo Yao a,d, Hua Tian a,d, Zhengrong Zhang a, Wei Qiao Qiu a,b,c,*
PMCID: PMC7199095  NIHMSID: NIHMS1574747  PMID: 32113968

Abstract

Calcitonin gene-related peptide (cGRP) receptor antagonists effectively treat migraine through reducing neuroinflammation, vasoconstriction and possibly neruogenesis. Since neuroinflammation is also involved in the pathogenesis of Alzheimer’s diseases (AD), we hypothesized and tested if a cGRP receptor antagonist, BIBN 4096 BS (BIBN), has effects on AD pathology. Using an AD mouse model, 5XFAD, with different ages, here we report that the BIBN treatment significantly increased the brain expression of PSD95, a postsynaptic protein, in both young and old AD mice. In parallel, BIBN improved learning and memory in the behavior test in the young, but not old, AD mice. The BIBN treatment reduced α-synuclein aggregation in both young and old AD mice. BIBN significantly decreased neuroinflammatory markers of ionized calcium binding adapter molecules-1 (Iba-1) and the p38 MAPK and NFκB signaling pathways in young, but not old, AD mice. The treatment also reduced the accumulation of amyloid-β (Aβ), and decreased tau phosphorylation through the pathway of CDK5/p25 in young mice only. Our study provides the evidence and suggests that the cGRP antagonists might be a therapeutic target to attenuate the pathological cascade and delay cognitive decline of AD in humans.

Keywords: BIBN (BIBN4096), cGRP receptor, Alzheimer’s disease, Amyloid-beta, Neuroinflammation, Therapeutic

1. Introduction

Alzheimer’s disease (AD) has multiple pathological components in the brain including amyloid-β (Aβ) plaques, tau tangles, neuroinflammation, and the damage to synaptic terminals (Hardy and Selkoe, 2002). Although Aβ probably drives the formation of the pathological cascade leading to synaptic damage and neuronal death (Castillo-Carranza et al., 2018; Marsh and Blurton-Jones, 2012), the clinical trials targeting Aβ only had modest or no effects for AD so that searching alternative targets, especially those downstream of Aβ, are necessary for AD drug development (Gold, 2017). Recent studies have shown that neuroinflammation may play a key role in AD pathogenesis leading to cognitive decline (Heneka et al., 2015). In addition, α-synuclein aggregation is shown to be a common pathological factor shared by the neurodegenerative disorders such as Parkinson’s disease, Lewy body disease, and some AD cases (Goedert, 2001). As α-synuclein protein itself is involved in regulation of synaptic plasticity and dopamine neurotransmitter release (Burré, 2015), this could be another drug target for AD. Most importantly, synaptic loss in AD correlate most with cognitive function decline (Koffie et al., 2011; Scheff et al., 2006), but there are no drugs yet to increase synapses in AD.

Calcitonin gene-related peptide (cGRP) is a neuropeptide with 37 amino acids and produced in both peripheral and central neurons to be a potent vasodilator (Rosenfeld et al., 1983; Russell et al., 2014). cGRP is able to regulate smooth muscle cells of the peripheral vasculature, inducing vasodilation through a nonendothelial mechanisms by the activation of adenylate cyclase and generation of intracellular cyclic adenosine monophosphate (Iyengar et al., 2017). cGRP can act on neuronal activity in the trigeminocervial complex (Storer et al., 2004) and transmission of pain signals to the thalamus and cortical brain regions (Benemei et al., 2009; Schou et al., 2017). This peptide has effects on inducing neuroinflammation in neurologic disorders, especially in migraine (Cernuda-Morollón et al., 2013; Malhotra, 2016). Thus the cGRP receptor, mainly calcitonin-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) complex, is targeted for the treatment of migraine through reducing neuroinflammation (Edvinsson, 2015; Lennerz et al., 2008; Nikitenko et al., 2006). BIBN 4096 BS (BIBN; Olcegepant) is a nonpeptide cGRP receptor antagonist. It has high affinity and specificity with cGRP receptor as a treatment for migraines (Durham and Vause, 2010; Olesen et al., 2004). cGRP receptor-deficient (RAMP1−/−) mice significantly ameliorates leukocyte infiltration (Glowka et al., 2015). cGRP inhibition is also involved in the infiltration of macrophages and the expression of inflammatory mediators such as Interleukin-1β, Tumor necrosis factor (TNF)-α, and cell adhesion molecules like Intercellular adhesion molecule-1 (Singh et al., 2017).

Although it is unknown whether cGRP or its receptor is involved in AD pathogenesis, neuroinflammation is certainly a key element in AD pathological process. We thus hypothesized and aimed to investigate if BIBN has any effects on AD pathogenesis. In this study, 5XFAD mice were injected with BIBN to test whether the antagonist could influence on the pathological formation of AD. Here we report that while the BIBN treatment enhanced the expression of a postsynaptic protein, PSD95, in the brain of the AD mouse model with different ages, it effectively decreased the other components of AD pathological cascade in young, but not old, AD mice.

2. Material and methods

2.1. Mice and experimental treatments

Female 5XFAD mice which are amyloid precursor protein (APP)/Presnilin 1(PS1) double transgenic mice with five familial AD mutations (Oakley et al., 2006) and female wild-type (WT) mice were purchased from MMRRC and Jackson Laboratory (Bar Harbor, ME, USA). BIBN 4096 (BIBN; #4561) was purchased from Tocris bio-techne brand (Minneapolis, MN, USA). Age -matched WT and 5XFAD mice were treated with intraperitoneal (i.p) injections of 400 μg/kg BIBN vs. phosphate-buffered saline (PBS) once daily for 6 weeks. 5XFAD mice with 3.5 months old (n = 8 for PBS and n = 10 for BIBN) and 7–10 months old (n = 8 of PBS and n = 10 BIBN) were used. Each injection volume was 150 μl. Test and control groups were matched for age and body weight before injection. The mice were checked body weight before and after treatment. All animal procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Boston University Animal Care and Use Committee.

2.2. Immunohistochemistry of mouse brains

Immunohistochemistry by floating method was used to evaluate the pathology in mouse brains. The brains were obtained after perfusion, that were fixed with 4% paraformaldehyde in PBS for 48 h and incubated in 30% sucrose buffer for 48 h. Serial coronal cryosections (30 μm) were cut and stored in the PBS with 0.05% sodium azide at 4 °C. The brain sections were incubated in PBS for 10 min and incubated to 0.3% hydrogen peroxide in methanol for 15 min for quenching of endogenous peroxidase activity. The sections were incubated in blocking buffer (5% [vol/vol] goat serum and 1% BSA [g/vol] in Tris-buffered salin with 0.1% Tween 20] for 30 min at room temperature. The sections were incubated with primary antibodies (Aβ (6E10), 1:2000, #803001, Biolegend; Iba-1, 1:2000, #019–19741,Wako; alpha-synuclein, 1:2000, #4179, Cell signaling) overnight. The secondary antibodies were used with biotinylated mouse antibodies (1:4000, #PK-6101, −6102, Vector Labs, Inc.) for 1 h. A vectastain ABC kit and DAB (#PK-4100, Vector Lab, Inc.) kits were used for reacting with substrate and counter-staining. The images of brain sections were visualized using SPOT 5.2 software (Sterling Heights, MI, USA) at a total magnification of ×10 and ×20.

To confirm the PSD95 expression in specific brain region, the sections were incubated in PBS for 10 min and incubated in blocking buffer (5% [vol/vol] horse serum and 5% BSA [g/vol] in Tris-buffered salin with 0.1% Tween 20] for 1 h at room temperature. The brains were incubated with PSD95 antibody (#3450; Cell signaling; 1:200) over night and secondary antibodies were stained with anti-rabbit antibody conjugated with Alexa Fluor® 488 (Life Technology, A11008; 1:700) for 1 h. The brain sections were mounted with ProLong Gold antifade reagent with DAPI for nuclear staining (#P36935; Thermo Fisher Scientific). The stained brains were observed under the fluorescence microscopy (Carl Zeiss, Germany).

ImageJ software was used to analyze the immunostaining results (Zhu et al., 2017). Data were pooled from 8 to 10 mice which has 5 images in each mouse. The images were changed to eight-bit RBG images and adjusted the threshold. The software was used to measure the count of plaques, total area, and average size.

2.3. Western blots

Mouse brain proteins from the cortex including the regions of frontal, temporal, pariental lobes and hippocampus were extracted with radioimmunoprecipitation assay (RIPA) buffer for total protein and 1% triton X-100 buffer (TBS-x) for soluble fractions (Zhu et al., 2017). 50 μg protein were used to evaluate the protein levels of amyloid-β (Aβ), and p25-CDK5 and regarding tau phosphorylation. For the expression of Aβ and amyloid precursor protein, the proteins were incubated with mouse 6E10 antibody (1:1000; Biolegend) and anti-APP-C99 (1:2000; #MABN380; MilliporeSigma). The soluble proteins were detected with specific α-synuclein and PSD95 antibodies (#2642, #3450; Cell signaling), and synaptopysin (sc-17750; santa cruz). Phosphorylated p38 (sc-166182; santa cruz), p38α/β (sc-7972; santa cruz), and IκBα (#9242) antibodies were used for inflammatory pathways. Total proteins were detected using the pTau (s199, s202, 1:1000, #29957, #39357), PHF-1 (1:500), total tau (1:1000, #46687, cell signaling) for tau phosphorylation to identify the CDK5-p35-p25 pathway, the brain extracts were detected with specific antibodies (p35/p25; 1:2000; #2680, CDK5; 1:1000; #2506; Cell signaling). The amounts of proteins were normalized with total protein or actin (1:200; sc-81178; Santa cruz), and quantified by ImageJ software.

2.4. Morris water maze test

Morris water maze test with spatial learning and memory performance was used with 5XFAD mice (PBS; n = 8, BIBN; n = 10) after the completion of treatment (Zhu et al., 2017). All mice underwent reference memory training with a hidden platform in one quadrant of the pool for 7 or 8 days with four trials per day. The mice were trained every day during 8 days and tested probe trials after skipping for 2 days. After the last trial, the platform was removed, and each mouse received one 60 s swim probe trial. Escape latency (seconds) is reported. Other indices, including length of swim path, swim speed, percent of time in the outer zone, and percent of time and path in each quadrant of the pool were also recorded using an HVS image video tracking system.

2.5. Statistical analysis

The outcomes in mouse experiments, including synaptic proteins, α-synuclein, amyloid plaques, tauopathy and microglia cells as well as Morris water maze behavior data, were compared either between WT and different aged 5XFAD or between PBS and BIBN by using ANOVA, followed with posthoc analysis by using a Tukey test. Mean ± SE and p value statistical significance were presented.

3. Results

3.1. BIBN enhances the expression of synaptic protein and improves memory and learning in an AD mouse model

Using western blots, compared to wild type (WT) mice, 5XFAD mice had the age-dependently decreased expression of PSD95 (F(2,16) = 1167; p ˂ 0.0001), a postsynaptic protein, as the old 5XFAD mice tended to have even lower expression of PSD95 than the young mice (Fig. 1A, Supplementary Fig. 1), but did not show the difference in the expression of presynaptic protein, synaptophysin, in the cortex region. The BIBN treatment significantly increased the PSD95 expression in both young (F(1,16) = 4.79; p = 0.05) and old (F(1,6) = 7.68; p = 0.05) 5XFAD mice (Fig. 1B and C), and had no effect on the synaptophysin expression. The treatment had no effects on body weight (Supplementary Fig. 2).

Fig. 1. BIBN increases PSD95 expression and improves cognitive function in the AD mouse model.

Fig. 1.

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WT mice, 5XFAD mice aged 3.5 months (young) and aged 7–10 months (old) were used. 5XFAD mice with two different ages were treated by i.p. injection of PBS or BIBN (400 μg/kg) daily for 6 weeks. (A) The expressions of PSD95 and synaptophysin in the extracted brain proteins of WT, young and old 5XFAD mice were detected and compared. The comparison between WT and 5XFAD mice with ***p < 0.001 for statistical significance are shown. (B) The brain proteins from young 5XFAD mice treated with PBS vs. BIBN were detected for the expressions of PSD95 and synaptophysin and compared. (C) The brain proteins from old 5XFAD mice treated with PBS vs. BIBN were detected for the expressions of PSD95 and synaptophysin and compared. For the statistical analyses of B and C, the comparisons between PBS and BIBN treatments are shown with *p < 0.05 for statistical significance. (D) Brain sections in cortex were examined for PSD95 expression by using immunohistochemistry. Compared to WT, 5XFAD mice had decreased the expression in the brain with statistical significance *p < 0.05. BIBN treated young 5XFAD mice had a significantly increased the expression in cortex with statistical significance #p < 0.05. Scale bar is shown: 100 μm. (E and F) Morris water maze test was conducted in 5XFAD mice. Compared to the PBS treatment, the BIBN treatment improved cognition in young (E), but not old (F), 5XFAD mice by showing shortened times in finding the hidden platform at day 6–8 (D6–8) and in memory after skipping the training for 2 days (Mean ± SE was used with *p < 0.05, **p < 0.01). For all comparisons, the mean ± SE is shown, and statistical analyses were done on different conditions (ANOVA followed by Tukey test) to compare different groups.

Using immunostaining, compared to WT mice, 5XFAD mice were also shown to have decreased PSD95 expression in cortex in both young (F(1,16) = 5.56, p = 0.012) and old (F(1,16) = 4.49, p = 0.034) ages (Fig. 1D). The BIBN treatment increased the expression of PSD95 compared to the PBS treatment (F(1,16) = 7.28, p = 0.014) in young, but did not reach statistical significance in old, 5XFAD mice (Fig. 1D). While 5XFAD mice tended to have decreased PSD95 expression in hippocampus (both CA3 and CA1) compared to WT, the BIBN treatment did not have effects on the expression of PSD95 in these regions (Supplementary Fig. 3).

In parallel, the water maze test was conducted to investigate the BIBN’s effects on cognitive ability (Fig. 1E and F). In young 5XFAD mice, the BIBN treatment had reduced escaping time at day 6 to day 8 (F(1,16) = 5.48; p = 0.04; day 6, F(1,16) = 286.8; p = 0.0002; day 7, F(1,16) = 9.29; p = 0.02; day 8) (Fig. 1E) and only tended to decrease the escaping time at day 11 in the probe trial with statistical significance. However, in old 5XFAD mice the BIBN treatment did not reach statistical significance (Fig. 1F). The BIBN treatment had no such effects in WT mice (Supplementary Fig. 4).

3.2. BIBN decreases α-synuclein expression and aggregation in the AD mouse model

Compared to WT mice, young 5XFAD mice had a higher expression level (Fig. 2A) and aggregated pathology (Fig. 2B) of α-synuclein, but older 5XFAD mice only tended to do so (F(2,16) = 3.16; p = 0.08) (Fig. 2A, Supplementary Fig. 5A). The BIBN treatment significantly reduced the α-synuclein pathology including the number and the average size of α-synuclein positive cells, and total α-synuclein positive area to 40% in the brain of both young and old mice compared with the PBS treatment (Fig. 2B, Supplementary Fig. 6). Using western blots, we also found that the BIBN treatment significantly reduced the expression of α-synuclein protein in the brains of both young (F(1,16) = 296.6; p = 0.008) and old (F(1,6) = 7.39; p = 0.05) mice (Fig. 2C and D, Supplementary Figs. 5B and C).

Fig. 2. BIBN decreases the cellular expression of alpha-synuclein in the brain of the AD mouse model.

Fig. 2.

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WT mice, 5XFAD mice aged 3.5 months (young; n = 8 PBS, n = 10 BIBN) and aged 7 months (old; n = 3 PBS, n = 5 BIBN) were used. 5XFAD mice with two different ages were treated by i.p. injection of PBS or BIBN (400 μg/kg) daily for 6 weeks. (A) Brain homogenates by TBS-x in WT, young and old 5XFAD mice were detected for the expression of α-synuclein by using western blots. The expression levels of α-synuclein were quantified mean ± SE. Compared to WT, both young old 5XFAD brains had higher expression levels of α-synuclein with *p < 0.05. (B) Brain sections in cortex (III-V layer) were examined for the α-synuclein pathology by using immunohistochemistry. Compared to WT, 5XFAD mice had increased aggregated α-synuclein pathology in the brain with statistical significance *p < 0.05, **p < 0.01, ***p < 0.001. Both BIBN treated young and old 5XFAD mice had a significant reduction in the cellular burden of α-synuclein in cortex quantitated by average amyloid intensity × plaque size with statistical significance #p < 0.05, ##p < 0.01,###p < 0.001. Scale bar is shown: 100 μm. (C and D) Detection of α-synuclein in western blots in brain homogenates from young (C) and old (D) 5XFAD mice treated with PBS or BIBN and quantified with Mean ± SE and statistical significance *p < 0.05 and **p < 0.01. For all comparisons, statistical analyses were done on different conditions (ANOVA followed by Tukey test) to compare different groups.

3.3. BIBN reduces neuroninflammation in young AD mouse model

We examined whether BIBN had any effects on neuroinflammation in the AD brain, specifically the inflammatory signal pathway of p38 MAPK and Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). Compared to WT mice, 5XFAD mice had age-dependent increased expression of phosphorylated p38 (F(2,16) = 20.9; p = 0.006) and decreased expression of IκBα (F(2,16) = 37.9; p = 0.009), and the old 5XFAD mice had higher level of phosphorylated p38 expression than the young mice (Fig. 3A, Supplementary Fig. 7A).

Fig. 3. BIBN reduces neuroinflammation in the young AD mouse model.

Fig. 3.

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WT mice, 5XFAD mice aged 3.5 months (young; n = 8 PBS, n = 10 BIBN) and aged 7 months (old; n = 3 PBS, n = 5 BIBN) were used. 5XFAD mice with two different ages were treated by i.p. injection of PBS or BIBN (400 μg/kg) daily for 6 weeks. (A) Brain homogenates by TBS-x in WT, young and old 5XFAD mice were detected for the expressions of phosphorylated p38 (p-p38), total p38, and IκBα by using western blots. Compared to WT, 5XFAD brains had higher expression levels of p-p38 and lower levels of IκBα correlating with age with *p < 0.05 and **p < 0.01. (B and D) Detection of phosphorylated p38 (p-p38), total p38, and IκBα in western blots in brain homogenates from young (B) and old (D) 5XFAD mice treated with PBS or BIBN and quantified with Mean ± SE and statistical significance *p < 0.05 and **p < 0.01. (C and E) Brain sections were examined for the Iba-1 by using immunohistochemistry. Cortex and hippocampus sections were stained with Iba-1 antibody (scale bar: 200 μm). Compared to the PBS treatment, the BIBN treatment reduce the Iba-1 expression in young (C), but not old (E), 5XFAD mice with statistical significance *p < 0.05. For all comparisons, statistical analyses were done on different conditions (ANOVA followed by Tukey test) to compare different groups.

The BIBN treatment decreased the level of phosphorylated p38 (F(1,16) = 348.7; p = 0.003) and increased the level of IκBα ((F(1,16) = 5.23; p = 0.04) proteins (Fig. 3B, Supplementary Fig. 7B), as well as significantly reduced the expression level of ionized calcium-binding adaptor (Iba)-1 in cortex and hippocampus regions (Fig. 3C) in young, but not in old, 5XFAD mice (Fig. 3D and E, Supplementary Fig. 7C).

3.4. BIBN reduces amyloid burden in the brain of young AD mouse model

We examined the BIBN effects on amyloid beta (Aβ) pathology including analyzed full-length APP (fAPP), secreted APPα (sAPPα), C99 and Aβ oligomers in the 5XFAD mice brain (Fig. 4). Compared to WT mice, 5XFAD mice brains significantly increased APP, C99, Aβ oligomers expressions (Fig. 4A, Supplementary Fig. 8A), but did not show statistical significance between young and old ones. The BIBN treatment only significantly decreased C99 (F(1,16) = 5.16; p = 0.05) and Aβ oligomers (F(1,16) = 10.94; p = 0.03) expression in young, but not old, 5XFAD mice (Fig. 4B and 4D, Supplementary Figs. 8B and 8C). Consistently, the BIBN treatment reduced the Aβ burden (50%) in the cortex and hippocampus areas in young, but not old, 5XFAD mice (Fig. 4C and 4E).

Fig. 4. BIBN treatment reduces amyloid pathology in the young AD mouse model.

Fig. 4.

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WT mice, 5XFAD mice aged 3.5 months (young; n = 8 PBS, n = 10 BIBN) and aged 7 months (old; n = 3 PBS, n = 5 BIBN) were used. 5XFAD mice with two different ages were treated by i.p. injection of PBS or BIBN (400 μg/kg) daily for 6 weeks. (A) Brain homogenates by TBS-x in WT, young and old 5XFAD mice were detected for the expressions of full-APP, secreted APPα, C99, Aβ oligomers by using western blots. Compared to WT, 5XFAD brains had higher expression levels of all these proteins correlating with age with *p < 0.05 and **p < 0.01. (B and D) Detection of full-APP, secreted APPα, C99, Aβ oligomers in western blots in brain homogenates from young (B) and old (D) 5XFAD mice treated with PBS or BIBN and quantified with Mean ± SE and statistical significance *p < 0.05. (C and E) Brain sections were examined by using immunohistochemistry. Cortex and hippocampus sections were stained with Aβ antibody (6E10) (scale bar: 200 μm). Compared to the PBS treatment, the BIBN treatment amyloid burden in the brain of young (C), but not old (E), 5XFAD mice with statistical significance **p < 0.01. For all comparisons, statistical analyses were done on different conditions (ANOVA followed by Tukey test) to compare different groups.

3.5. BIBN reduces tau phosphorylation mainly by p25-CDK5 signaling pathway

Compared to WT mice, 5XFAD mice had significantly increased phosphorylated Tau (s199 and s202) and PHF-1 expressions (Fig. 5A, Supplementary Fig. 9A), but there was no difference between young and old ones. Interestingly, 5XFAD mice had increased p25 expression in old (F(2,16) = 14.05; p = 0.04) but only tended to be so in young mice, while having age-dependent decreased p35 expression levels in both young (F(2,16) = 92.93; p = 0.002) and old mice (F(2,16) = 100; p = 0.0001) (Fig. 5B, Supplementary Fig. 9B). We investigated whether the BIBN treatment has any effect on tau phosphorylation in 5XFAD mice. The BIBN treatment decreased tau phosphorylation at three sites of tau protein in young (F(1,16) = 404.8; p = 0.0005; pTau (s199), F(1,16) = 424.6; p = 0.0005; pTau (s202); F(1,16) = 6.72; p = 0.04; PHF-1), but not old, 5XFAD mice (Fig. 5C and 5E, Supplementary Figs. 9C and 9E). In parallel, the BIBN treatment significantly reduced the p25 expression in young (F(1,16) = 56.99; p = 0.01), but not old, 5XFAD mice (Fig. 5D and F, Supplementary Figs. 9D and 9F).

Fig. 5. BIBN treatment decreases tau phosphorylation in the young AD mouse model.

Fig. 5.

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WT mice, 5XFAD mice aged 3.5 months (young; n = 8 PBS, n = 10 BIBN) and aged 7 months (old; n = 3 PBS, n = 5 BIBN) were used. 5XFAD mice with two different ages were treated by i.p. injection of PBS or BIBN (400 μg/kg) daily for 6 weeks. (A and B) Brain homogenates by TBS-x in WT, young and old 5XFAD mice were detected for the expressions of tau phosphorylation (A) and the p25-CDK5 signaling pathway m (B) by using western blots. Compared to WT, 5XFAD brains had higher expression levels of phosphorylated tau protein (pTau) detected by three antibodies, pTau (s199), pTau (s202) and PHF-1, and correlating with age. Compared to WT, 5XFAD brains had higher levels of p25 and CDK5, but not p35, correlating with age. Statistical significance in the comparison between WT and 5XFAD are shown with *p < 0.05, **p < 0.01 and ***p < 0.001; between young and old 5XFAD are shown with #p < 0.05. (C and E) Detection of pTau (s199), pTau (s202) and PHF-1 in western blots in brain homogenates from young (C) and old (E) 5XFAD mice treated with PBS or BIBN were quantified with Mean ± SE and statistical significance *p < 0.05, **p < 0.01 and ***p < 0.001. (D and F) Detection of p25, p35 and CDK5 in western blots in brain homogenates from young (E) and old (F) 5XFAD mice treated with PBS or BIBN were quantified with Mean ± SE and statistical significance *p < 0.05. For all comparisons, statistical analyses were done on different conditions (ANOVA followed by Tukey test) to compare different groups.

4. Discussion

In this study, we have demonstrated that the BIBN treatment enhanced the expression of PSD95, which is a component of synapses and involved in synaptic plasticity (de Bartolomeis and Fiore, 2004), in both young and old AD mice, while it modified the AD pathological cascade by decreasing the typical AD pathological components in addition to neuroinflammation only when the mice were young. Neuroinflammation is also involved in AD pathogenesis and correlates with the typical AD pathological components, β-amyloid and tau aggregation (Heneka et al., 2015). BIBN and other cGRP receptor antagonists are a class of drugs for the treatment of migraine probably through modulating neuroinflammation (Benemei et al., 2009; Geppetti et al., 2005; Olesen et al., 2004) and reducing the recruitment of immune cells (Glowka et al., 2015). Our study suggests that cGRP antagonists have potential to be repurposed for AD treatment.

Like the AD brain in humans, the 5XFAD mice have reduced synapses as they were aged, and the BIBN treatment reversed the decreased expression level of postsynaptic protein, PSD95, in the brain even when the mice were old, but only paralleled with learning and memory improvement in young mice (Fig. 1). Because synaptic loss in AD correlate with cognitive function decline (Koffie et al., 2011; Scheff et al., 2006), BIBN may serve to be a drug candidate to delay cognitive decline in AD in humans. Two studies show that PSD95 levels are lower in the brains of AD (Whitfield et al., 2014) and MCI (Sultana et al., 2010) subjects than in control brains, and one study shows that both PSD95 and Synaptophysin levels are low in AD brains (Love et al., 2006). PSD95 levels and synapses negatively correlate with Aβ42 accumulation and with cognitive function in sporadic AD brains (Shinohara et al., 2014). In addition, the PSD95-NMDA receptor complex has been shown to regulate tau phosphorylation at the post-synaptic region (Mondragón-Rodríguez et al., 2012). While there is no drug yet targeting synapse loss in AD, current clinical trials targeting other components of AD pathology either failed or had minimum effects to delay cognitive decline (Holmes et al., 2009; Karran and De Strooper, 2016; Selkoe, 2013).

Research studies reported that α-synuclein is an another key player for cognitive decline in AD (Crews et al., 2009; Oikawa et al., 2016), and accumulated α-synuclein in the lipid vesicle and membranes is toxic to neuron function mainly in Parkinson Disease (PD) as well as in AD (Burré, 2015; Cookson, 2009). In our study, compared to WT, the expression of α-synuclein was increased in young 5XFAD mice but decreased when aged (Fig. 2A). Studies show that α-synuclein mRNA is age-dependently decreased in whole brain, cerebral cortex, hippocampus, striatum from α-synuclein TG mice and rats (Li et al., 2004; Malatynska et al., 2006; Petersen et al., 1999), after peaking at early postnatal development (Mak et al., 2009; Petersen et al., 1999), which is consistent with our finding (Fig. 2A). While our study also shows that the 5XFAD mice had accumulated form of α-synuclein pathology (Fig. 2B), the BIBN treatment reduced the numbers and size of α-synuclein aggregation (Fig. 2B), and decreased the expression of α-synuclein in both young and old AD mice (Fig. 2C and D). α-synuclein is mainly expressed in presynaptic terminals and it interacts with phospholipids and proteins (Burré, 2015), and regulates the release of neurotransmitters and vesicles from neurons (Bridi and Hirth, 2018; Gallegos et al., 2015). Because synapses were further reduced in aged 5XFAD mice (Fig. 1A), it may also explain why the expression of α-synuclein was reduced in the aging process (Fig. 2A). A lack of α-synuclein may result in neurotransmitter dysfunction and loss of synaptic plasticity, but α-synuclein overload causes harmful consequences such as the formation of oligomeric forms and impairment of protein degradation (Conway et al., 2001; Martinez-Vicente et al., 2008).

Compare to WT mice, the 5XFAD mice increased amyloid pathology, in addition to tauopathy, and neuroinflammation (Figs. 3–5). The BIBN treatment attenuated the development of these pathological components of AD only in young but not old mice. While BIBN reduced the typical AD pathology, amyloid pathology (Fig. 4), it also reduced the tau phosphorylation probably through both p25-CDK5 signaling pathways. (Fig. 5), when 5XFAD mice were young. It is possible that when the mice are aged, the typical aggregated AD pathology, including amyloids and tauopathy, is solidly formed and neuroinflammation is worsened so that the drug probably could not influence them anymore. BIBN decreased the expression of Iba-1 labeled of microglia in cortex and hippocampus area (Fig. 3C) and reduced the p38 MAPK and NFκB signaling pathway in young mice (Fig. 3B). Increased activation of microglia make severe inflammatory condition through the release of cytotoxic molecules including proinflammatory cytokines and reactive oxygen synthase mediates. Chronic inflammation can lead to neuronal damage in CNS (Dheen and Ling, 2007). The P38 MAPK signaling pathway is increased in microglia and astrocyte to increase inflammation by pro-inflammatory cytokines such as TNF-α and IL-1β, and in neuron to cause cell death (Lee and Kim, 2017).

The mechanism of BIBN’s effects on AD pathology in the brain is not clear. Although BIBN has vasodilation effect, it has been reported that a high dose of BIBN treatment had no effect on pulse rate, systolic and diastolic pressure (Iovino et al., 2004; Zeller et al., 2008). Although BIBN cannot cross the blood-brain barrier (BBB), BIBN have effects on the wall of meningeal arteries and luminal side of the BBB in the middle cerebral artery by binding of cGRP receptors (Edvinsson et al., 2007; Petersen et al., 2004). Since more and more studies have shown that cerebrovasculature may play an important role in AD pathogenesis (Arvanitakis et al., 2016; Jellinger, 2002; Santos et al., 2017), BIBN may reduce AD pathogenesis through affecting cerebrovasculature. Another possibility is more likely that BIBN targets neuroinflammation indirectly in the brain to reduce AD pathology. While the binding of Aβ to amylin receptors is controversial (Burns et al., 2014; Fu et al., 2012; Mietlicki-Baase, 2018), it is unknown if Aβ, especially Aβ oligomer, binds to cGRP receptor, which is in the same receptor family of amylin receptors (Burns et al., 2014). Although BIBN did not affect the typical AD pathology and neuroinflammation when the mice were old (Figs. 3–5), all clinical trials targeting these AD components failed to delay cognitive decline (Holmes et al., 2009; Karran and De Strooper, 2016; Selkoe, 2013). Thus we think that increased the expression of synaptic protein by the cGRP antagonists in the aging process in addition to their favorable safety profiles to treat migraine should be a rationale to consider the repurpose of the cGRP antagonists for AD in humans.

Supplementary Material

1

HIGHLIGHTS.

  • The cGRP receptor antagonist enhances the expression of synaptic protein in the AD mouse model.

  • cGRP antagonists reduce neuroinflammation and AD pathology, and leads to improving learning and memory in young AD mice.

  • cGRP antagonists may be a new therapeutic avenue to delay cognitive decline in AD.

Acknowledgements

This work was supported by grants from Alzheimer’s Disease Association, United States, IIRG-13-284238; National Institute on Aging, United States, R21AG045757 and RO1AG-022476; and Ignition Award (W.Q.Q). Support was provided through P30 AG13864 (N.K.) from National Institute on Aging, United States. Hua Tian was supported by Chinese National Foundation for Abroad Scholarship. The remaining authors have nothing to disclose.

Footnotes

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.neuropharm.2020.108017.

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