nAChRs gene expression and neuroinflammation in APPswe/PS1dE9 transgenic mouse

Abstract

An evaluation of the APPswe/PS1dE9 transgenic AD mouse, presenting with the toxic Aβ1-42 deposition found in human AD, allowed us to characterize time-dependent changes in inflammatory and cholinergic markers present in AD. Astrogliosis was observed in cortex and hippocampus, with cellular loss occurring in the same areas in which Aβ plaques were present. In this setting, we found early significantly elevated levels of IL-1β and TNFα gene expression; with the hippocampus showing the highest IL-1β expression. To investigate the cholinergic anti-inflammatory pathway, the expression of nicotinic receptors (nAChRs) and cholinesterase enzymes also was evaluated. The anti-inflammatory nAChRα7, α4, and β2 were particularly increased at 6 months of age in the hippocampus, potentially as a strategy to counteract Aβ deposition and the ensuing inflammatory state. A time-dependent subunit switch to the α3β4 type occurred. Whether α3, β4 subunits have a pro-inflammatory or an inhibitory effect on ACh stimulation remains speculative. Aβ1-42 deposition, neuronal loss and increased astrocytes were detected, and a time-dependent change in components of the cholinergic anti-inflammatory pathway were observed. A greater understanding of time-dependent Aβ/nAChRs interactions may aid in defining new therapeutic strategies and novel molecular targets.

Figure 1

Spatial distribution of Aβ-deposits in hemibrain from APPswe/PS1dE9 mice at different ages, compared to an representative image from a 24-month old WT mouse. Aβ-deposits in (A) 6-month old, (B) 12-month old and (C) 24-month old APPswe/PS1dE9 mouse. (D) 24-month old WT mouse, stained for but without any evident hemibrain Aβ-deposits. Coronal section 50 µm. (×10). Anti-Aβ (green) and bismencimide (blue). Bar 1 cm.

Figure 2

Distribution in cortex and hippocampus of Aβ deposits and GFAP, in WT and APPswe/PS1dE9 mice at 6-, 12- and 24-months of age (representative images). (A1) Aβ deposits in 24-month old WT mouse. (B1,C1,D1) time-dependent Aβ deposits in APPswe/PS1dE9 Tg mice. (A2) GFAP in 24-month old WT mouse. (B2,C2,D2) time-dependent GFAP in APPswe/PS1dE9 Tg mice. (A3,B3,C3,D3) previous merged images. Coronal section 50 µm (×20). Anti-Aβ (green), anti-GFAP (red). Bar 1 cm.

Figure 3

Distribution in cortex and hippocampus of Aβ deposits and neuN, in WT and APPswe/PS1dE9 mice at 6-, 12- and 24-months of age (representative images). (A1) Aβ deposits in 24-month old WT mouse. (B1,C1,D1) age-dependent Aβ deposits in APPswe/PS1dE9 Tg mice. (A2) neuN in 24-month old WT mouse. (B2,C2,D2) age-dependent neuN in APPswe/PS1dE9 Tg mice. (A3,B3,C3,D3) previous merged images. Coronal section 50 µm (×20). Anti-Aβ (green), anti-neuN (red). Bar 1 cm.

Figure 4

Presence of clusters of reactive astrocytes surrounding Aβ-deposits in APPswe/PS1dE9 mouse cortex and hippocampus (representative images). (A) Distribution of Aβ-deposits in cortex coronal section 50 µm of 24-months old AD (APPswe/PS1dE9) mouse (×10). (A1) Reactive astrocytes (×40). (A2) Distribution of Aβ-deposits, (×40). (A3) Cell nuclei (×40), arrows indicate the loss of cells in the same locality where there are Aβ-deposits, and the presence of reactive astrocytes around and between the Aβ plaques. (A4) Merged (A1,A2,A3). (B) Distribution of Aβ-deposits in hippocampus coronal section 50 µm of 24-month old AD (APPswe/PS1dE9) mouse (×10). (B1) Reactive astrocytes in granular layer of the gyrus dentate of hippocampus (×40). (B2) Distribution of Aβ-deposits, (×40). (B3) Cell nuclei (×40), arrows indicate the loss of cells in the same location where there are Aβ-deposits, and the localization of reactive astrocytes around and between the Aβ plaques. (B4) Merged (B1,B2,B3). Coronal section 50 µm. Anti-GFAP (red), anti-Aβ (green), bismencimide (blue). Bars 1 cm (A,B) and 0.5 cm (A1–A4, B1–B4).

Figure 5

Cytokine/chemokine expression levels in cortex and hippocampus at different postnatal ages in AD (APPswe/PS1dE9) Tg mice with respect to age-matched WT mice. Bar plots graphically depict the mean and SEM of pro-inflammatory cytokine/chemokine gene expression levels in cortex and hippocampus at different postnatal ages. Statistically significant p-values, after adjustment according to the FDR methods, are graphically depicted too.

Figure 6

nAChRs expression levels in cortex and hippocampus at different postnatal ages. Bar plots graphically depict the mean and SEM of nAChR α7, α4, β2, α3, and β4 gene expression levels in cortex and hippocampus at different postnatal ages. Statistically significant p-values, after adjustment according to the FDR methods, are graphically depicted too.

Figure 7

Graphical representation of age-related IL-1β and nAChRα7 expression levels and Aβ deposition in cortex and hippocampus. Bar plots graphically depict the mean and SEM of IL-1β and nAChRα7 gene expression levels and the percentage of Aβ deposition in cortex and hippocampus at different postnatal ages. The trend lines for the same parameters are depicted too with β coefficient ± SEM for IL-1β: 1.8 ± 1.1, for nAChRα7: − 0.1 ± 0.1, and for Aβ: 0.4 ± 0.1 in cortex, and with β coefficient ± SEM for IL-1β: 0.5 ± 1.4, for nAChRα7: − 2.2 ± 0.8, and for Aβ: 0.3 ± 0.1 in hippocampus.

Figure 8

Cholinesterase enzymes expression levels in cortex and hippocampus at different postnatal ages. Bar plots graphically depict the mean and SEM of AChE and BuChE gene expression levels in cortex and hippocampus at different postnatal ages, in comparison to WT mice. Statistically significant p-values, after adjustment according to the FDR methods, are graphically depicted too.