Targeting the brain 5-HT7 receptor to prevent hypomyelination in a rodent model of perinatal white matter injuries

Abstract

Approximately 15 million babies are born prematurely every year and many will face lifetime motor and/or cognitive deficits. Children born prematurely are at higher risk of developing perinatal brain lesions, especially white matter injuries (WMI). Evidence in humans and rodents demonstrates that systemic inflammation-induced neuroinflammation, including microglial and astrocyte reactivity, is the prominent processes of WMI associated with preterm birth. Thus, a new challenge in the field of perinatal brain injuries is to develop new neuroprotective strategies to target neuroinflammation to prevent WMI. Serotonin (5-HT) and its receptors play an important role in inflammation, and emerging evidence indicates that 5-HT may regulate brain inflammation by the modulation of microglial reactivity and astrocyte functions. The present study is based on a mouse model of WMI induced by intraperitoneal (i.p.) injections of IL-1β during the first 5 days of life. In this model, certain key lesions of preterm brain injuries can be summarized by (i) systemic inflammation, (ii) pro-inflammatory microglial and astrocyte activation, and (iii) inhibition of oligodendrocyte maturation, leading to hypomyelination. We demonstrate that Htr7 mRNA (coding for the HTR7/5-HT7 receptor) is significantly overexpressed in the anterior cortex of IL-1β-exposed animals, suggesting it as a potential therapeutic target. LP-211 is a specific high-affinity HTR7 agonist that crosses the blood-brain barrier (BBB). When co-injected with IL-1β, LP-211 treatment prevented glial reactivity, the down-regulation of myelin-associated proteins, and the apparition of anxiety-like phenotypes. Thus, HTR7 may represent an innovative therapeutic target to protect the developing brain from preterm brain injuries.

Keywords: Astrocyte; HTR7; Microglia; Myelination; Neurodevelopmental disorders; Preterm birth; Serotonin.

Fig. 1

Impact of IL-1β on the expression of Htr7 mRNA. A Recapitulative schematic representation of the perinatal inflammation model induced by IL-1β-exposure and its characteristics. B Relative expression of Htr7 mRNA in the forebrain at P5 and P10 after perinatal administration of PBS or IL-1β. t test (n = 9–14/group, mean SEM), (*p ≤ 0.05, **p ≤ 0.01, comparing IL-1β vs PBS). C Relative expression of Htr7 mRNA in sorted astrocytes (ACSA-2⁺) and microglia (CD11B⁺) at P3 after perinatal administration of PBS or IL-1β. ND = non-detectable signal. RT-qPCR results are presented as the fold change relative to PBS-exposed animals. T test (n = 8/group, mean SEM), (*p ≤ 0.05, comparing IL-1β vs PBS)

Fig. 2

Impact of the agonist LP-211 on plasma cytokine levels at P3 following IL-1β administration. Plasma concentrations of pro-inflammatory (IFN-γ and TNF-α), multi-functional (IL-1β, IL-5, GM-CSF), and anti-inflammatory (IL-4 and IL-10) cytokines, in pg/mL, at P3 after perinatal administration of PBS or IL-1β co-injected with LP-211. One-way analysis of variance (ANOVA) Kruskal–Wallis test corrected by Dunn’s test (n = 6/group, mean SEM), (*p ≤ 0.05, **p ≤ 0.01, in comparison to PBS)

Fig. 3

Impact of the agonist LP-211 on astrocyte reactivity at P3 following IL-1β administration. Relative expression of mRNA encoding pan-reactive (A), A1- (B), and A2-reactive markers (C) by astrocytes from P3 animals after perinatal administration of PBS or IL-1β co-injected with LP-211. Cells were obtained after ACSA-2⁺-cell magnetic sorting. RT-qPCR results are presented as the fold change relative to PBS-DMSO-exposed animals. One-way analysis of variance (ANOVA) Kruskal–Wallis test corrected by Dunn’s test (n = 9–12/group, mean SEM), (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, comparing IL-1β vs PBS, #p ≤ 0.05, comparing LP-211 vs DMSO)

Fig. 4

Impact of the agonist LP-211 on microglial reactivity at P3 following IL-1β administration. Relative expression of mRNA encoding pro-inflammatory (A), immune-regulatory (B), and anti-inflammatory markers (C) by microglia from P3 animals after perinatal administration of PBS or IL-1β co-injected with LP-211. Cells were obtained after CD11B⁺-cell magnetic sorting. RT-qPCR results are presented as the fold change relative to PBS-DMSO-exposed animals. One-way analysis of variance (ANOVA) Kruskal–Wallis test corrected by Dunn’s test (n = 9–12/group, mean SEM), (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, comparing IL-1β vs PBS, ^#p ≤ 0.05, comparing LP-211 vs DMSO)

Fig. 5

Impact of the agonist LP-211 on glial ROS production at P3 following IL-1β administration. Relative expression of reactive oxygen species (ROS) production by astrocytes (ACSA-2⁺) and microglia (CD11B⁺) from P3 animals after perinatal administration of PBS or IL-1β co-injected with LP-211. Results of the basal and PMA-stimulated states are presented as the fold change relative to PBS-DMSO-exposed animals. One-way analysis of variance (ANOVA) Kruskal–Wallis test corrected by Dunn’s test (n = 9–12/group, mean SEM) (*p ≤ 0.05, ***p ≤ 0.001, comparing IL-1β vs PBS, ^#p ≤ 0.05, comparing LP-211 vs DMSO)

Fig. 6

Impact of the agonist LP-211 on myelinization following IL-1β administration. A Relative expression of myelin protein mRNA in the forebrain of P10 animals after perinatal administration of PBS or IL-1β co-injected with LP-211. B Representative western blots and relative expression of myelin basic protein (MBP) isoforms in the forebrain at P10 after perinatal administration of PBS or IL-1β co-injected with LP-211. One-way analysis of variance (ANOVA) Kruskal–Wallis test corrected by Dunn’s test (n = 5–7/group, mean SEM) (*p ≤ 0.05, **p ≤ 0.01, in comparison to PBS). C Representative immunohistochemistry images and relative expression of PLP in the corpus callosum and somato-sensorial cortex from P30 animals after perinatal administration of PBS or IL-1β co-injected with LP-211. Results are presented as the fold change relative to PBS-DMSO-exposed animals. One-way analysis of variance (ANOVA) Kruskal–Wallis test corrected by Dunn’s test (n = 6/group, mean SEM for RT-qPCR, n = 5–7/group, mean SEM for histology) (*p ≤ 0.05, ***p ≤ 0.001, comparing IL-1β vs PBS, #p ≤ 0.05, comparing LP-211 vs DMSO). D Representative traces of CAP and measure of N1 and N2 amplitude under increasing stimulation intensity (0–4 mA) in the corpus callosum from P30-35 animals after perinatal administration of PBS or IL-1β co-injected with LP-211. Results are presented as the total area under the curve (AUC). Brown-Forsythe ANOVA test corrected by Dunnett’s T3 test (n = 6–8/group, mean SEM), (*p ≤ 0.05, **p ≤ 0.01, comparing IL-1β vs PBS, ^##p ≤ 0.01, comparing LP-211 vs DMSO)

Fig. 7

Impact of the agonist LP-211 on anxiety-like behavior following IL-1β administration. Two-month-old animals were submitted to a 20-min open-field test. Travel distance and time spent in the center stage of the arena were measured. One-way analysis of variance (ANOVA) Kruskal–Wallis test corrected by Dunn’s test (n = 15/group, mean SEM), (*p ≤ 0.05, comparing IL-1β vs PBS)