Impact of Two Neuronal Sigma-1 Receptor Modulators, PRE084 and DMT, on Neurogenesis and Neuroinflammation in an Aβ1-42-Injected, Wild-Type Mouse Model of AD

Abstract

Alzheimer's disease (AD) is the most common form of dementia characterized by cognitive dysfunctions. Pharmacological interventions to slow the progression of AD are intensively studied. A potential direction targets neuronal sigma-1 receptors (S1Rs). S1R ligands are recognized as promising therapeutic agents that may alleviate symptom severity of AD, possibly via preventing amyloid-β-(Aβ-) induced neurotoxicity on the endoplasmic reticulum stress-associated pathways. Furthermore, S1Rs may also modulate adult neurogenesis, and the impairment of this process is reported to be associated with AD. We aimed to investigate the effects of two S1R agonists, dimethyltryptamine (DMT) and PRE084, in an Aβ-induced in vivo mouse model characterizing neurogenic and anti-neuroinflammatory symptoms of AD, and the modulatory effects of S1R agonists were analyzed by immunohistochemical methods and western blotting. DMT, binding moderately to S1R but with high affinity to 5-HT receptors, negatively influenced neurogenesis, possibly as a result of activating both receptors differently. In contrast, the highly selective S1R agonist PRE084 stimulated hippocampal cell proliferation and differentiation. Regarding neuroinflammation, DMT and PRE084 significantly reduced Aβ_1-42-induced astrogliosis, but neither had remarkable effects on microglial activation. In summary, the highly selective S1R agonist PRE084 may be a promising therapeutic agent for AD. Further studies are required to clarify the multifaceted neurogenic and anti-neuroinflammatory roles of these agonists.

Keywords: Alzheimer’s disease; Aβ1–42-induce mouse model; PRE084; dimethyltryptamine; neurogenesis; neuroinflammation; sigma-1 receptor.

Figure 1

(A) Results for 5-Bromo-2′-Deoxyuridine (BrdU) immunolabeling. We observed significant differences in the quantity of stem cells between the six groups (ANOVA: p ≤ 0.0001). Significantly fewer BrdU+ cells were detected in the Aβ_1–42-PBS, PBS-DMT, and in the Aβ_1–42-DMT treated animals compared to the PBS-PBS group (PBS-PBS vs. Aβ_1–42-PBS p = 0.001, vs. PBS-DMT p = 0.001, vs. Aβ_1–42-DMT p ≤ 0.0001). The difference between the Aβ_1–42-PBS and Aβ_1–42-DMT treatment groups was also significant (p = 0.005). PRE084-treatment increased the number of stem cells detected in the SGZ; this change was significant in the Aβ_1–42-administered group compared to its vehicle-treated control (Aβ_1–42-PBS vs. Aβ_1–42-PRE084 p ≤ 0.0001). The differences between the following groups in pairwise comparisons also reached significance: PBS-PRE084 vs. Aβ_1–42-PBS p ≤ 0.0001, vs. PBS-DMT p ≤ 0.0001, vs. Aβ_1–42-DMT p ≤ 0.0001; Aβ_1–42-PRE084 vs. PBS-DMT p ≤ 0.0001, vs. Aβ_1–42-DMT p ≤ 0.0001. (B–G) Representative images of BrdU staining: (B) PBS-PBS, (C) Aβ_1–42-PBS, (D) PBS-PRE084, (E) Aβ_1–42-PRE084, (F) PBS-DMT, (G) Aβ_1–42-DMT. Scale bars represent 100 μm. *: p ≤ 0.05.

Figure 2

(A) Results for doublecortin (DCX) immunostaining. Detected DCX densities significantly differed among the six groups (ANOVA: p ≤ 0.0001). Compared to the control (PBS-PBS) animals, a significantly higher amount of DCX+ cells were detected in the Aβ_1–42-PBS, PBS-PRE084 and Aβ_1–42-PRE084-treated groups (PBS-PBS vs. Aβ_1–42-PBS p = 0.037, vs. PBS-PRE084 p ≤ 0.0001, vs. Aβ_1–42-PRE084 p ≤ 0.0001). Similarly, a significant difference was detected between the groups treated with Aβ_1–42-PBS and Aβ_1–42-PRE084 (p = 0.007). DMT treatment did not alter the number of immature neurons in the SGZ. Furthermore, significant differences were found when the groups were compared to the PBS-PRE084-treated group: PBS-PRE084 vs. Aβ_1–42-PBS p = 0.023, vs. PBS-DMT p = 0.001, vs. Aβ_1–42-DMT p = 0.001. Additional significant results were detected: Aβ_1–42-PRE084 vs. PBS-DMT p ≤ 0.0001, vs. Aβ_1–42-DMT p ≤ 0.0001. (B–G) Representative images of DCX immunolabeling: (B) PBS-PBS, (C) Aβ_1–42-PBS, (D) PBS-PRE084, (E) Aβ_1–42-PRE084, (F) PBS-DMT, (G) Aβ_1–42-DMT. Scale bars represent 100 μm. *: p ≤ 0.05.

Figure 3

(A) Results for neuronal nuclei (NeuN) immunostaining. Significant differences were detected among the groups as follows (ANOVA: p = 0.001): in DMT-treated animals, significantly lower NeuN densities were evident compared to the PBS-PBS and Aβ_1–42-PBS groups (PBS-DMT vs. PBS-PBS p = 0.001, vs. Aβ_1–42-PBS p ≤ 0.0001; Aβ_1–42-DMT vs. PBS-PBS p = 0.022, vs. Aβ_1–42-PBS p = 0.003). Furthermore, significant differences were found when the groups were compared to the PBS-DMT-treated group: PBS-DMT vs. PBS-PRE084 p = 0.001, vs. Aβ_1–42-PRE084 p = 0.006; Aβ_1–42-DMT vs. PBS-PRE084 p = 0.024. (B–G) Representative photomicrographs of NeuN immunolabeling: (B) PBS-PBS, (C), Aβ_1–42-PBS (D) PBS-PRE084, (E) Aβ_1–42-PRE084, (F) PBS-DMT, (G) Aβ_1–42-DMT. Scale bars represent 200 μm. *: p ≤ 0.05.

Figure 4

(A) Results for ionized calcium-binding adapter molecule 1 (Iba1) immunolabeling. Significant differences were observed among the groups (ANOVA: p = 0.002). Aβ_1–42 increased the density of Iba1+ microglia significantly compared to PBS-PBS, PBS-PRE084, and PBS-DMT treated mice, respectively (PBS-PBS vs. Aβ_1–42-PBS p = 0.015; PBS-PRE084 vs. Aβ_1–42-PRE084 p = 0.035; PBS-DMT vs. Aβ_1–42-DMT p = 0.039). The difference between the PBS-PBS and PBS-DMT groups was also significant (PBS-PBS vs. PBS-DMT p = 0.031). Moreover, significant differences were detected between the following groups: Aβ_1–42-PBS vs. PBS-PRE084 p = 0.005, vs. PBS-DMT p ≤ 0.0001; Aβ_1–42-PRE084 vs. PBS-DMT p = 0.002. (B–G) Representative images of Iba1 immunostaining: (B) PBS-PBS, (C) Aβ_1–42-PBS, (D) PBS-PRE084, (E) Aβ_1–42-PRE084, (F) PBS-DMT, (G) Aβ_1–42-DMT. Scale bars represent 100 μm. *: p ≤ 0.05.

Figure 5

(A) Results of glial fibrillary acidic protein (GFAP) immunostaining. The densities of GFAP+ astrocytes differed among the groups (ANOVA: p ≤ 0.0001). A significantly higher GFAP+ density was detected in the Aβ_1–42-PBS group compared to those treated with PBS-PBS (p ≤ 0.0001), PBS-PRE084 (p = 0.013), Aβ_1–42-PRE084 (p = 0.013), PBS-DMT (p ≤ 0.0001), and Aβ_1–42-DMT (p = 0.001). (B–G) Representative images of GFAP immunolabeling: (B) PBS-PBS, (C) Aβ_1–42-PBS, (D) PBS-PRE084, (E) Aβ_1–42-PRE084, (F) PBS-DMT, (G) Aβ_1–42-DMT. Scale bars represent 100 μm. *: p ≤ 0.05.

Figure 6

(A) Results for the western blot (WB) analysis. Significant differences were observed in the S1R levels among the groups (ANOVA: p ≤ 0.0001). Compared to PBS-PBS-treated mice, the S1R protein levels were significantly elevated in the Aβ_1–42-PBS (p ≤ 0.0001), PBS-PRE084 (p = 0.018), Aβ_1–42-PRE084 (p ≤ 0.0001), and Aβ_1–42-DMT (p ≤ 0.0001) groups. In PBS-DMT-treated mice, the S1R protein expression remained close to the control level (p = 0.540), while S1R levels were somewhat higher in the PRE084-treated groups (PBS-PBS vs. PBS-PRE084 p = 0.018; Aβ_1–42-PBS vs. Aβ_1–42-PRE084 p = 0.004; PBS-PRE084 vs. Aβ_1–42-PRE084 p ≤ 0.0001). In contrast, the co-administration of Aβ_1–42 and DMT induced a significant increase in the quantity of S1R (PBS-DMT vs. Aβ_1–42-DMT p ≤ 0.0001). Furthermore, significant differences were detected in the S1R expression upon the pairwise comparisons of the following groups: Aβ_1–42-PBS vs. PBS-PRE084 p = 0.032, vs. PBS-DMT p = 0.001; Aβ_1–42-PRE084 vs. PBS-DMT p ≤ 0.0001, vs. Aβ_1–42-DMT p ≤ 0.0001, respectively. (B) WB gel electrophoresis images of S1R and GAPDH lines of the experimental groups. *: p ≤ 0.05.