Gintonin Binds to Reduced LPA4 Receptor Subtype in Human Cortical Neurons in Alzheimer's Disease Brains

Abstract

Ginseng, a traditional herbal medicine with a long history of use, is known to support human health, particularly by influencing brain function. Recent studies have identified gintonin, a lysophosphatidic acid (LPA) receptor ligand derived from ginseng, as a key bioactive. However, the specific LPA receptor subtypes targeted by gintonin in the human brain to exert its anti-Alzheimer's (AD) effects remain unclear. This study aimed to elucidate the LPA receptor subtype targeted by gintonin in the human cortex. Using a fluorescent gintonin conjugate, we investigated receptor binding in cortical samples from healthy individuals (n = 4) and AD patients (n = 4). Our results demonstrated that fluorescent gintonin selectively binds to human cortical neurons rather than glial cells and that gintonin-binding sites are co-localized with the LPA4 receptor subtype. Furthermore, the expression of LPA4 receptors was significantly reduced in the cortical neurons of AD patients. These results suggest that the LPA4 receptor may serve as a novel histopathological marker for AD and represent a promising therapeutic target for gintonin-based prevention and treatment strategies.

Keywords: AD prevention; LPA4 receptor; ginseng; gintonin; human cortex.

Figure 1

Identification of gintonin (GT)-binding cells in the cortices of the healthy controls (HCs) and Alzheimer’s disease (AD) patients. (A) Representative confocal images showing DAPI (blue), NeuN (a neuronal marker, green), and GT (red) in the brains of the HCs and patients with AD. Scale bar = 200 μm. (B) Quantitative analysis of the co-localization between NeuN and GT and (C) NeuN positive cell number. (D) Representative confocal images showing DAPI (blue), Iba1 (a microglia marker, green), and GT (red) in the brains of the HCs and patients with AD. Scale bar = 200 μm. (E) Quantitative analysis of the co-localization between Iba-1 and GT and (F) integrated density of Iba1. (G) Representative confocal images showing GFAP (an astrocyte marker, green) and GT (red) in the brains of the HCs and patients with AD. (H) Quantitative analysis of the co-localization between GFAP and GT and (I) integrated density of GFAP. Scale bar = 200 μm. Statistical significance was determined using a t-test. Data are presented as mean $\pm$ SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 2

Co-localization of the gintonin (GT)-binding site with the LPA4 receptor subtype and a selective reduction in LPA4 receptor subtype expression levels in patients with AD. (A) Representative confocal images of the LPA receptor subtypes (LPAR1, LPAR2, LPAR3, LPAR4, LPAR5, and LPAR6; green) and GT (red) in the cortices of the HCs and patients with AD. Scale bar = 200 μm. The insert images focus on the co-localization between gintonin (GT) and the LAP receptor subtypes. Scale bar = 100 μm. (B) Quantitative analysis of the co-localization between the GT and LPA receptor subtypes. (C–F) Western blot analysis of the LPA receptor subtypes in brain tissue from the HCs and patients with AD. The results indicate a significant decrease in LPAR4 expression in patients with AD, whereas no significant differences were observed for the other LPA receptor subtypes. Statistical significance was determined using a t-test. Data are presented as mean $\pm$ SEM. * p < 0.05, ** p < 0.01. Original Western blot images can be found in Figure S1.

Figure 2

Co-localization of the gintonin (GT)-binding site with the LPA4 receptor subtype and a selective reduction in LPA4 receptor subtype expression levels in patients with AD. (A) Representative confocal images of the LPA receptor subtypes (LPAR1, LPAR2, LPAR3, LPAR4, LPAR5, and LPAR6; green) and GT (red) in the cortices of the HCs and patients with AD. Scale bar = 200 μm. The insert images focus on the co-localization between gintonin (GT) and the LAP receptor subtypes. Scale bar = 100 μm. (B) Quantitative analysis of the co-localization between the GT and LPA receptor subtypes. (C–F) Western blot analysis of the LPA receptor subtypes in brain tissue from the HCs and patients with AD. The results indicate a significant decrease in LPAR4 expression in patients with AD, whereas no significant differences were observed for the other LPA receptor subtypes. Statistical significance was determined using a t-test. Data are presented as mean $\pm$ SEM. * p < 0.05, ** p < 0.01. Original Western blot images can be found in Figure S1.

Figure 3

The relationship between LPA4 receptor subtype expression in the cortices of HCs and patients with AD. (A) Representative confocal images showing DAPI (blue), LPAR4 (green), and NeuN (red) in the cortices of HCs and patients with AD. Scale bar = 100 μm. (B) Quantitative analysis of the co-localization between the LPA4 receptor subtype and NeuN. (C) Representative confocal images showing DAPI (blue), LPAR4 (green), and GFAP (red) in the brains of HCs and patients with AD. Scale bar = 100 μm. (D) Quantitative analysis of the co-localization between the LPA4 receptor subtype and GFAP. (E) Representative confocal images showing DAPI (blue), LPAR4 (green), and Iba1 (red) in the cortices of HCs and patients with AD. Scale bar = 100 μm. (F) Quantitative analysis of the co-localization between the LPA receptor subtype and Iba 1. Statistical significance was determined using a t-test. Data are presented as mean $\pm$ SEM. * p < 0.05.

Figure 3

The relationship between LPA4 receptor subtype expression in the cortices of HCs and patients with AD. (A) Representative confocal images showing DAPI (blue), LPAR4 (green), and NeuN (red) in the cortices of HCs and patients with AD. Scale bar = 100 μm. (B) Quantitative analysis of the co-localization between the LPA4 receptor subtype and NeuN. (C) Representative confocal images showing DAPI (blue), LPAR4 (green), and GFAP (red) in the brains of HCs and patients with AD. Scale bar = 100 μm. (D) Quantitative analysis of the co-localization between the LPA4 receptor subtype and GFAP. (E) Representative confocal images showing DAPI (blue), LPAR4 (green), and Iba1 (red) in the cortices of HCs and patients with AD. Scale bar = 100 μm. (F) Quantitative analysis of the co-localization between the LPA receptor subtype and Iba 1. Statistical significance was determined using a t-test. Data are presented as mean $\pm$ SEM. * p < 0.05.