3,4,5-Tricaffeoylquinic acid induces adult neurogenesis and improves deficit of learning and memory in aging model senescence-accelerated prone 8 mice

Abstract

Caffeoylquinic acid (CQA) is a natural polyphenol with evidence of antioxidant and neuroprotective effects and prevention of deficits in spatial learning and memory. We studied the cognitive-enhancing effect of 3,4,5-tricaffeoylquinic acid (TCQA) and explored its cellular and molecular mechanism in the senescence-accelerated mouse prone 8 (SAMP8) model of aging and Alzheimer's disease as well as in human neural stem cells (hNSCs). Mice were fed with 5 mg/kg of TCQA for 30 days and were tested in the Morris water maze (MWM). Brain tissues were collected for immunohistochemical detection of bromodeoxyuridine (BrdU) to detect activated stem cells and newborn neurons. TCQA-treated SAMP8 exhibited significantly improved cognitive performance in MWM compared to water-treated SAMP8. TCQA-treated SAMP8 mice also had significantly higher numbers of BrdU+/glial fibrillary acidic protein (GFAP+) and BrdU+/Neuronal nuclei (NeuN+) cells in the dentate gyrus (DG) neurogenic niche compared with untreated SAMP8. In hNSCs, TCQA induced cell cycle arrest at G0/G1, actin cytoskeleton organization, chromatin remodeling, neuronal differentiation, and bone morphogenetic protein signaling. The neurogenesis promoting effect of TCQA in the DG of SAMP8 mice might explain the cognition-enhancing influence of TCQA observed in our study, and our hNSCs in aggregate suggest a therapeutic potential for TCQA in aging-associated diseases.

Keywords: BMP signaling; SAMP8; TCQA; neurogenesis; spatial learning and memory.

Figure 1

Effect of ethanol extract of 3,4,5-triCaffeoylquinic acid (TCQA) on the spatial learning and memory as determined by escape latency of senescence-accelerated resistant mouse 1 (SAMR1) mice, senescence-accelerated prone mouse 8 (SAMP8) mice and SAMP8 TCQA-treated group determined by Morris water maze test (A). Effect of ethanol extract of TCQA on the time spent in the target quadrant (B). Effect of ethanol extract of TCQA on numbers of crossings of platform by SAMR1 untreated and SAMP8 treated or untreated mice (C). * P < 0.05, ** P < 0.01 Compared with SAMP8 + water group.

Figure 2

Effect of oral administration of 3,4,5-triCaffeoylquinic acid (TCQA) on anterior (A–C) and posterior (D–F) DG stem cell activation and neurogenesis. SAMP8 mice were administrated with TCQA (5 mg/kg) for 30 days. Photomicrographs show adult mouse brain in coronal sections containing the anterior (A) and posterior (D) DG processed for immunohistochemical detection of proliferating BrdU+ cells (red) and GFAP, a protein expressed by stem cells in the DG (green). Graphs represents the number of BrdU+ cells that co-express GFAP in anterior (B) and posterior (E) DG. Graphs represent the number of BrdU+ cells that co-express the mature neuronal marker NeuN in the anterior (E) and posterior (F) DG. Each bar represents the mean ±SEM * p < 0.05, ** p < 0.01 vs. SAMP8+TCQA group.

Figure 3

Effect of oral administration of 3,4,5-tricaffeoylquinic acid (TCQA) on subventricular zone (SVZ) proliferation. SAMP8 mice were administrated with TCQA (5 mg/kg) for 30 days (A) Photomicrograph shows adult mouse brain coronal sections containing the SVZ processed for immunohistochemical detection of proliferating BrdU⁺ cells (red) and GFAP+ (green), an astrocyte marker found in SVZ NSC. (B, C) Graphs represent the number of BrdU+/GFAP+ and BrdU⁺ cells, respectively in the different treatment groups.

Figure 4

The effect of 3,4,5-triCaffeoylquinic acid (TCQA) on cell viability of human neural stem cells (hNSCs). hNSCs in undifferentiated state (A), induced differentiation for 96 h (B) and induced differentiation and treated with TCQA for 96 h (C). Time after differentiation, hNSCs were treated with TCQA (1, 5, 10, 20 μM) for 24, 48, 72, and 96 h (D). After the treatment, cell viability was measured by MTT assay. Data was set as % of control. Data were presented as mean ± SD. ** P < 0.01 Compared with control group.

Figure 5

The effect of 3,4,5-tricaffeoylquinic acid (TCQA) on fate, protein expression levels of differentiation markers, and cell proliferation of human neural stem cells (hNSCs). Three differentiation markers (β3-tubulin: neuron, myelin basic protein (MBP): oligodendrocyte, glial fibrillary acidic protein (GFAP): astrocyte) were used. hNSCs were treated with differentiation medium with or without 10 μM TCQA. Expression levels of each differentiation marker were observed using confocal microscopy. Immunofluorescence images demonstrating the expression of β3-tubulin (A), MBP (B), and GFAP (C). hNSCs were treated with differentiation medium with or without 10 μM TCQA for 24 - 96 h. The expression level of each differentiation marker was determined by western blotting. Immunopositive bands of β3-tubulin (D), MBP (E), and GFAP (F) were quantified and expressed as a normalized value compared to glyceraldehyde-3-phosphate dehydrogenase (Gapdh). The cell number and cell viability were measured by ViaCount assay (G). * P < 0.05, ** P < 0.01 significance compared with control group.

Figure 6

The effect of 3,4,5-tricaffeoylquinic acid (TCQA) on cell cycle and phosphorylation of tumor protein p53 in human neural stem cells (hNSCs). Cell cycle was determined by labeling cellular DNA. hNSCs were treated with growth medium or differentiation medium with or without 10 μM TCQA for 24 h. (A) Ratio of each cell cycle phase as a percentage of total cells. (B) The histograms show the cells in G0/G1 (pink peak on left), S (green center peak) and G2/M (blue peak on right). (C) hNSCs were treated with growth medium or differentiation medium with or without 10 μM TCQA for 24 - 96 h. Data was set as % of undifferentiated control. Data were presented as mean ± SD. * P < 0.05, ** P < 0.01 Compared with undifferentiated control cells. # P < 0.05 significance by student’s t test.

Figure 7

The effect of 3,4,5-triCaffeoylquinic acid (TCQA) on intracellular Ca²⁺ levels, mitochondrial function, and intracellular reactive oxygen species (ROS) levels at very early phase of human neural stem cell (hNSCs) differentiation. hNSCs were pre-treated with Fluo4 AM for 30 min followed by treatment with growth medium or differentiation medium with or without TCQA 10 μM for 1–30 min. Time after differentiation and TCQA treatment, intracellular Ca²⁺ level was detected by measurement of fluorescence intensity (A). hNSCs were treated with growth medium or differentiation medium with or without 10 μM TCQA for 30–180 min. TCQA was treated with rhodamine 123 and detected mitochondrial function by measuring the fluorescence intensity (B). Intracellular ROS levels were detected by measuring fluorescence intensity of DCF oxidized by ROS. hNSCs were pre-treated with DCFH-DA for 1 h followed by treatment with growth medium or differentiation medium with or without 10 μM TCQA for 15–180 min (C). Data was set as % of undifferentiated control. Data were presented as mean ± SD. * P < 0.01, ** P < 0.01 Compared with undifferentiated control cells.

Figure 8

The effect of 3,4,5-tricaffeoylquinic acid (TCQA) on gene expressions related to bone morphogenetic protein (BMP) signaling pathway. Human neural stem cells (hNSCs) were treated with differentiation medium with or without 10 μM TCQA for 24 h. Genes expressing BMP ligand BMP5, BMP receptor 2 and SMAD5 downstream BMP signaling pathway as well as the neuronal differentiation transcription factor NEUROD1 were increased by TCQA (A). Genes related to p38–p53 signaling pathway regulating G0/G1 cell cycle arrest of hNSCs activated by the BMP signaling pathway were increased by TCQA (B). Genes related to the Cdc42 signaling pathway regulating neurite extension and activated by the BMP signaling pathway were increased by TCQA (C). Data was set as % of undifferentiated control. Data were presented as mean ± SD. ** P < 0.01 Compared with undifferentiated control.