Modulation of neuroinflammation and pathology in the 5XFAD mouse model of Alzheimer's disease using a biased and selective beta-1 adrenergic receptor partial agonist

Abstract

Degeneration of noradrenergic neurons occurs at an early stage of Alzheimer's Disease (AD). The noradrenergic system regulates arousal and learning and memory, and has been implicated in regulating neuroinflammation. Loss of noradrenergic tone may underlie AD progression at many levels. We have previously shown that acute administration of a partial agonist of the beta-1 adrenergic receptor (ADRB1), xamoterol, restores behavioral deficits in a mouse model of AD. The current studies examined the effects of chronic low dose xamoterol on neuroinflammation, pathology, and behavior in the pathologically aggressive 5XFAD transgenic mouse model of AD. In vitro experiments in cells expressing human beta adrenergic receptors demonstrate that xamoterol is highly selective for ADRB1 and functionally biased for the cAMP over the β-arrestin pathway. Data demonstrate ADRB1-mediated attenuation of TNF-α production with xamoterol in primary rat microglia culture following LPS challenge. Finally, two independent cohorts of 5XFAD and control mice were administered xamoterol from approximately 4.0-6.5 or 7.0-9.5 months, were tested in an array of behavioral tasks, and brains were examined for evidence of neuroinflammation, and amyloid beta and tau pathology. Xamoterol reduced mRNA expression of neuroinflammatory markers (Iba1, CD74, CD14 and TGFβ) and immunohistochemical evidence for microgliosis and astrogliosis. Xamoterol reduced amyloid beta and tau pathology as measured by regional immunohistochemistry. Behavioral deficits were not observed for 5XFAD mice. In conclusion, chronic administration of a selective, functionally biased, partial agonist of ADRB1 is effective in reducing neuroinflammation and amyloid beta and tau pathology in the 5XFAD model of AD.

Keywords: 5XFAD; Alzheimer's disease; Amyloid beta; Beta-1 adrenergic receptor; Betaxolol hydrochloride (PubChem CID: 107952); CGP 20712A (PubChem CID: 2685); ICI-118551 (PubChem CID: 5484725); Isoproterenol (PubChem CID: 3779); Neuroinflammation; Xamoterol; Xamoterol (PubChem CID: 155774).

Fig. 1

Timeline of in vivo studies indicates age, drug administration and behavioral testing schedules for Experiment 1 and Experiment 2. Two independent cohorts of 5XFAD and wildtype mice were chronically administered a selective partial ADRB1 agonist, xamoterol, or vehicle, 6 mg/kg oral gavage from 4.0 to 6.5 months of age or 3 mg/kg/day subcutaneous pump from 7.0 to 9.5 months of age. Experiment 1 is referred to as “6 month - oral gavage” as behavior and tissue collection occurs during the 6^th month. Experiment 2 is likewise referred to as “9 month - pump”. Behavioral testing was performed in the indicated order during the final 4 to 5 weeks of administration. At the end of testing, mice were perfused and brains collected for neurobiochemical endpoints. Sample size = 10 to 11 per group for behavior and 4–6 per group for neurobiological endpoints. Experiments were analyzed independently. Abbreviations: OF, open field, AC, activity chamber; YM, Y-maze; SD, social discrimination; NOR, novel object recognition; MWM, Morris Water Maze; EPM, elevated plus maze; FC, fear conditioning; s.c., subcutaneous.

Fig. 2

Xamoterol is a selective ADRB1 partial agonist with functional bias at the cAMP signaling pathway at the cloned human ADRB1 receptor. A) Dose-response curves for cAMP show xamoterol (S-enantiomer) with an efficacy of 50% relative to isoproterenol through the ADRB1 receptor and an EC₅₀ of 2.2 nM (0.3–14.6 nM). Xamoterol has no cAMP activity through ADRB2 or ADRB3 up through 100 μM. B) Dose-response curves for β-arrestin activity show minimal (<10%) action of xamoterol on β-arrestin through ADRB1 or ADRB2. Data for cAMP represent 5 experiments performed in singlet or duplicate, and for β-arrestin, technical replicates within a single experiment.

Fig. 3

ADRB1 modulates LPS-induced TNF-α production in rat primary microglia. Both xamoterol (A; 1 μM), a selective partial agonist of the ADRB1 receptor, and isoproterenol (B; 1 μM), a non-selective full agonist of the ADRB1 receptor, reduced TNF-α production following challenge of primary rat microglia with LPS (10 ng/mL). A) Effects of xamoterol were reversed by pretreatment with either the selective ADRB1 antagonist, CGP 20712A (0.1 μM) or betaxolol (10 μM), but not the selective ADRB2 antagonist ICI-118551 (0.1 μM). B) Effects of isoproterenol, were partially reversed by pretreatment with the selective ADRB1 antagonist, betaxolol (10 μM) or the selective ADRB2 antagonist ICI-118551 (0.1 μM), but not CGP 20712A (0.1 μM). Data represent means ± SEM from multiple experiments (n= 4–9 per group) normalized to internal LPS controls. *p < .05, **p < .01, ***p < .001; ns, not significant; Dunnett’s multiple comparisons against LPS exposure alone, following one-way ANOVA.

Fig. 4

Xamoterol modulates immune-related mRNA expression in 5XFAD transgenic mice. Bar graphs depict mRNA expression in homogenized cortical tissue from wildtype and 5XFAD vehicle-treated and xamoterol-treated mice normalized relative to wildtype vehicle-treated mice for each gene (A–P). In the 6 month – oral gavage study (A–H), all genes with the exception of CD74 were elevated in vehicle-treated 5XFAD mice relative to wildtype counterparts. Likewise, in the 9 month – pump study (I–P), all genes with the exception of IL6 were elevated in vehicle-treated 5XFAD mice relative to wildtype. Xamoterol treatment attenuated Iba1, CD74, CD14 and TGFβ mRNA expression in 5XFAD mice (I,M–O) in the 9 month – pump study. In the 6 month – oral gavage study, trends for attenuation of TNF-α and CD14 were observed (B,F; p < .1). Sample size, n = 4–6 per group, but n=3 for 5XFAD-VEH in 9 month – pump experiment. Data represent means +/− SEM. * p < .05, ** p < .01, *** p < .001, Bonferroni’s test for multiple comparisons following one-way ANOVA.

Fig. 5

Xamoterol reduces microglia/macrophage (Iba1) gliosis and amyloid beta (6E10) immunoreactivity (-ir). Chronic dosing with the selective partial ADRB1 agonist, xamoterol, attenuates Iba1-ir in all regions in 5XFAD transgenic mice and reduces 6E10-ir in DG and CA3 regions of the hippocampus. A) Atlas plates (top left) indicate one of multiple levels from which the regions of interest (green) were selected for analyses [1]. Black boxes indicate image capture frame. Numbers below atlas plates indicate mm bregma; black scale bar, 1 mm. Photomicrographs (5X magnification) illustrate representative immunostaining (DAPI, blue; 6E10, green; Iba1, red) from each region of interest in wildtype and 5XFAD vehicle- (VEH) and xamoterol-treated (XAM) mice, white scale bar, 100 μm. Dashed white lines indicate region of quantification. B) Quantitative immunohistochemistry revealed decreases in 6E10-ir (% area) in DG and CA3. C) Increases in Iba1-ir (mean intensity) were detected in all regions analyzed in VEH-treated 5XFAD transgenic mice relative to VEH-treated wildtype littermates. These increases were attenuated in each region in transgenic mice chronically dosed with XAM. Sample size, n=4–5 per group, with exception of wildtype-XAM (n=3 for DG/CA3/SUB and n=2 for RS). Data represent mean ± SEM. *p < .05, **p < .01, ***p < .001, Bonferroni’s test for multiple comparisons following one-way ANOVA. RS, retrosplenial cortex; LA/BLA, lateral/basolateral amygdala; SUB, subiculum; CA3, CA3 region of the hippocampus; DG, dentate gyrus.

Fig. 6

Xamoterol reduces astrocyte (GFAP) gliosis in the vicinity of amyloid beta (6E10) immunoreactivity (-ir). Chronic dosing with the selective partial ADRB1 agonist, xamoterol, attenuated GFAP-ir in the cingulate (Cg) and piriform/endopiriform (Pir/En) cortices in 5XFAD transgenic mice. A) Atlas plates (top left) indicate one of multiple levels from which the regions of interest (green) were selected for analyses [1]. Black boxes indicate image capture frame. Numbers below atlas plates indicate mm bregma; black scale bar, 1 mm. Photomicrographs (5X magnification) illustrate representative immunostaining (DAPI, blue; 6E10, green; GFAP, red) from each region of interest in wildtype and 5XFAD vehicle- (VEH) and xamoterol-treated (XAM) mice, white scale bar, 100 μm. Dashed white lines indicate region of quantification. B) Quantitative immunohistochemistry revealed non-significant trends for decreases in 6E10-ir (% area) in Cg and Pir/En. C) Increases in GFAP-ir (mean intensity) were detected in both Cg and Pir/En in VEH-treated 5XFAD transgenic mice relative to VEH-treated wildtype littermates. These increases in astrogliosis were attenuated in both regions in transgenic mice chronically dosed with XAM. Sample size, n=4–5 per group. *p < .05, **p < .01, ***p < .001, Bonferroni’s test for multiple comparisons following one-way ANOVA.

Fig. 7

Chronic dosing with the selective partial ADRB1 agonist, xamoterol, attenuated AT8-ir in the retrosplenial cortex (RS) in 5XFAD transgenic mice. A) Atlas plate indicates the level at which images were selected for analyses [1]. Black box indicates image capture frame. Number below atlas plate indicates mm bregma; black scale bar, 1 mm. Photomicrographs (10X magnification) illustrate representative AT8-immunostaining in wildtype and 5XFAD vehicle- (VEH) and xamoterol-treated (XAM) mice, white scale bar, 50 μm. B) Quantitative immunohistochemistry revealed increases in AT8-ir (% area) in VEH-treated 5XFAD transgenic mice relative to VEH-treated wildtype littermates. These increases were attenuated in transgenic mice chronically dosed with XAM. Sample size, n=5 per group. *p < .05, **p < .01, Bonferroni’s test for multiple comparisons following one-way ANOVA.