Optogenetic stimulation of basal forebrain cholinergic neurons prevents neuroinflammation and neuropsychiatric manifestations in pristane induced lupus mice

Abstract

Background: Neuroinflammation has been identified as one of the primary pathogenic factors of neuropsychiatric systemic lupus erythematosus (NPSLE). However, there are no dedicated treatments available in clinics to alleviate neuroinflammation in NPSLE. It has been proposed that stimulating basal forebrain (BF) cholinergic neurons may provide potent anti-inflammatory effects in several inflammatory diseases, but its potential role in NPSLE remains unexplored. This study aims to investigate whether and how stimulating BF cholinergic neurons has a protective effect on NPSLE.

Results: Optogenetic stimulation of BF cholinergic neurons significantly ameliorated olfactory dysfunction and anxiety- and depression-like phenotype in pristane induced lupus (PIL) mice. The increased expression of adhesion molecules (P-selectin and vascular cell adhesion molecule-1 (VCAM-1)), leukocyte recruitment, blood-brain barrier (BBB) leakage were significantly decreased. Notably, the brain histopathological changes, including the elevated levels of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β), IgG deposition in the choroid plexus and lateral ventricle wall and lipofuscin accumulation in the cortical and hippocampal neurons, were also significantly attenuated. Furthermore, we confirmed the colocalization between the BF cholinergic projections and the cerebral vessels, and the expression of α7-nicotinic acetylcholine receptor (α7nAChR) on the cerebral vessels.

Conclusion: Our data indicate that stimulation of BF cholinergic neurons could play a neuroprotective role in the brain through its cholinergic anti-inflammatory effects on cerebral vessels. Therefore, this may be a promising preventive target for NPSLE.

Keywords: Basal forebrain; Behavioral deficits; Cholinergic anti-inflammatory effect; Neuroinflammation; Neuropsychiatric lupus; Optogenetics; α7nAChR.

Fig. 1

Experimental schedule Mice received microinjection of viruses and surgical implantation of optical fibers in the BF. Pristane or PBS was administered to mice via intraperitoneal injection. Optogenetic stimulation of BF cholinergic neurons was applied to mice for 4 months. Following a series of behavioral tests and intravital microscopy, mice were sacrificed and brain tissues were collected for further assays

Fig. 2

Effects of optogenetic stimulation of BF cholinergic neurons on behavioral deficits in PIL mice A Olfactory sensitivity test. Quantitative analysis of total time spent sniffing male feces, female feces, vinegar or alcohol. B Elevated zero maze test. Left panel showing representative track plots of the path. Right panel showing quantitative analysis of total track distance and percentage of time spent in the open arms (%). C Forced swim test. Quantitative analysis of immobility time. The data are expressed as the mean ± SEM (n = 12 in each group). One-way ANOVA followed by Tukey’s post hoc test: ^## p < 0.01 compared to the control group, ^* p < 0.05 or ^** p < 0.01 compared to the PIL group

Fig. 3

Effects of optogenetic stimulation of BF cholinergic neurons on endothelium activation, leukocyte recruitment and BBB leakage in PIL mice (A) and (C) Representative images of P-selectin and VCAM-1 expression. (B) and (D) Quantitative analysis of P-selectin and VCAM-1 expression by MFI and normalized to the control group. (E) Representative images and quantitative analysis of leukocyte rolling and adhesion. (F) Quantitative analysis of Evans blue dye extravasation. The data are expressed as the mean ± SEM (n = 12 in each group). One-way ANOVA followed by Tukey’s post hoc test: ^## p < 0.01 compared to the control group, ^* p < 0.05 or ^** p < 0.01 compared to the PIL group

Fig. 4

Effects of optogenetic stimulation of BF cholinergic neurons on cytokine expression, IgG deposition and lipofuscin accumulation in the brain of PIL mice A Quantitative analysis of the levels of brain cytokines (TNF-α, IL-6, IL-1β and IL-10). B and C Representative images of IgG deposition in the choroid plexus and lateral ventricular wall (green). DAPI staining for nuclei (blue). D and E Quantitative analysis of IgG deposition in the choroid plexus and lateral ventricular wall by MFI and normalized to the control group. F and G Representative images of autofluorescent lipofuscin at 480 nm exciting light (green) in the cortex and hippocampus. DAPI staining for nuclei (blue). H and I Quantification analysis of lipofuscin foci in the cortex and hippocampus and normalized to the control group. The data are expressed as the mean ± SEM (n = 12 in each group). One-way ANOVA followed by Tukey’s post hoc test: ^## p < 0.01 compared to the control group, ^* p < 0.05 or ^** p < 0.01 compared to the PIL group

Fig. 5

Examination for the relationship between BF cholinergic projection and cerebral vessels, and α7nAChR expression on cerebral vessels A Representative images of BF cholinergic projection (red) and lectin immunoreactive cerebral vessels (green). White arrow showing the co-localization between cholinergic projection (red) and cerebral vessels (green). B Representative images and quantitative analysis of α7nAChR expression in the cerebral cortex and normalized to the control group. The data are expressed as the mean ± SEM (n= 12 in each group). One-way ANOVA followed by Tukey’s post hoc test