Acetylcholine suppresses microglial inflammatory response via α7nAChR to protect hippocampal neurons.

Neuroinflammation is principally linked to glial function and has been demonstrated to participate in the pathogenesis of Alzheimer's disease, a neurodegenerative disorder characterized by beta-amyloid ccumulation and neurotransmission disruption. Previous findings suggest acetylcholine exerts anti-inflammatory and neuroprotective properties in several neurodegenerative disorders. However, the underlying mechanisms remain elusive. Here evaluation of the influence of acetylcholine on neuroinflammation and neurodegeneration in Alzheimer's disease is reported and further neuroprotective mechanisms are investigated. Investigation of microglia in lipopolysaccharide-induced hippocampal neuronal toxicity employed α7nAChR gene silencing and demonstrated that both the anti-inflammatory and neuroprotective effects of acetylcholine rely on α7nAChR pathways. As expected, in neuron-microglia co-cultures lipopolysaccharide induced an increase in expression of pro-inflammatory factors, including inducible nitric oxide synthase, interleukin-1α, and tumor necrosis factor-α, and decreased expression of neurotrophic factors such as insulin-like growth factor-1, and neuronal apoptosis. Acetylcholine protects against lipopolysaccharide-elicited neuronal injury by inhibiting the microglial inflammatory response and promoting microglial neurotrophic factor production via the action of α7nAChR on microglia. These findings establish that ACh activates α7nAChR in microglia, which in turn protects hippocampal neurons.

[Acetylcholine suppresses microglial inflammatory response in rats via acting on microglial α7nAChR].

Microglia are the main immune cells in the central nervous system. In the present study, the mechanism for acetylcholine (ACh) inhibiting microglial inflammatory response was investigated. Primary culture of microglia was isolated from cerebral cortex of Sprague-Dawley (SD) rats. Lipopolysaccharide (LPS) was used to activate the microglia to induce inflammatory response, and then the microglia were treated with ACh for 24 h. Protein expressions of several inflammatory factors, insulin-like growth factor 1 (IGF-1) and α7 nicotinic acetylcholine receptor (α7nAChR) were detected by Western blot. Release of inflammatory factors and IGF-1 into media was detected by ELISA. After α7nAChR gene silence was achieved by lentivirus-transfection of α7nAChR-shRNA, the change of ACh effect was observed. The results showed that LPS induced microglial activation, up-regulated inducible nitric oxide synthase (iNOS) protein expression, increased the expressions and release of IL-1ß and TNF-α, and decreased the expression and release of the neurotrophic factor, IGF-1. ACh could reverse these effects of LPS. Meanwhile, LPS reduced the protein expression of α7nAChR on the microglial cells, whereas ACh could reverse the effect. Silencing of α7nAChR gene in microglia abolished the ability of ACh to inhibit LPS-induced inflammatory responses. These results suggest that ACh exerts its protection against LPS-induced microglial inflammation via acting on α7nAChR on microglia, which may provide a novel target for the treatment of neuro-inflammatory diseases.

Cerebellar ataxia induced by 3-AP affects immunological function.

OBJECTIVE: We previously showed that the cerebellum modulates the immune system. Here we determined whether cerebellar ataxia alters immunological function to further demonstrate an involvement of the cerebellum in immune modulation. METHODS: Neurotoxin 3-acetylpyridine (3-AP) was intraperitoneally injected in rats to induce cerebellar ataxia. Behavior and motor coordination were tested on day 7 following 3-AP injection. Nissl staining and high-performance liquid chromatography (HPLC) were used to determine neuronal loss and neurotransmitter contents, respectively, in all the three cerebellar nuclei, fastigial nucleus (FN), interposed nucleus (IN) and dentate nucleus (DN). T and B lymphocyte differentiation and function were measured by flow cytometry, Western blot and ELISA. RESULTS: 3-AP induced motor discoordination and locomotor reduction. In all the three cerebellar nuclei, FN, IN and DN, there was a neuronal loss and a decrease in contents of glutamate and GABA (but not glycine) after 3-AP injection. Importantly, CD4+ T cells, but not CD8+ T cells, were increased by the 3-AP treatment. Moreover, interferon (IFN)-γ-producing cells and interleukin (IL)-17-producing cells were decreased in cerebellar ataxia rats, but IL-4-producing cells and CD25-expressing cells were increased. Expression of the T helper (Th)1- and Th17-related cytokines, IFN-γ, IL-2, IL-17 and IL-22, was downregulated in CD4+ cells in cerebellar ataxia rats, while expression of the Th2 and regulatory T (Treg)-related cytokines, IL-4, IL-5, IL-10 and transforming growth factor (TGF)-ß, was upregulated. Furthermore, B lymphocyte number and anti-bovine serum albumin (BSA) IgM and IgG antibody levels were elevated in cerebellar ataxia. CONCLUSION: Cerebellar ataxia alters cellular and humoral immunity.

Cerebellar fastigial nuclear glutamatergic neurons regulate immune function via hypothalamic and sympathetic pathways.

We previously have shown that cerebellar fastigial nucleus (FN) modulates immune function, but pathways or mechanisms underlying this immunomodulation require clarification. Herein, an anterograde and retrograde tracing of nerve tracts between the cerebellar FN and hypothalamus/thalamus was performed in rats. After demonstrating a direct cerebellar FN-hypothalamic/thalamic glutamatergic projection, 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of glutaminase that catalyzes glutamate synthesis, was injected bilaterally in the cerebellar FN and simultaneously, D,L-threo-ß-hydroxyaspartic acid (THA), an inhibitor of glutamate transporters on cell membrane, was bilaterally injected in the lateral hypothalamic area (LHA) or the ventrolateral (VL) thalamic nucleus. DON treatment in the FN alone decreased number of glutamatergic neurons that projected axons to the LHA and also diminished glutamate content in both the hypothalamus and the thalamus. These effects of DON were reduced by combined treatment with THA in the LHA or in the VL. Importantly, DON treatment in the FN alone attenuated percentage and cytotoxicity of natural killer (NK) cells and also lowered percentage and cytokine production of T lymphocytes. These DON-caused immune effects were reduced or abolished by combined treatment with THA in the LHA, but not in the VL. Simultaneously, DON treatment elevated level of norepinephrine (NE) in the spleen and mesenteric lymphoid nodes, and THA treatment in the LHA, rather than in the VL, antagonized the DON-caused NE elevation. These findings suggest that glutamatergic neurons in the cerebellar FN regulate innate and adaptive immune functions and the immunomodulation is conveyed by FN-hypothalamic glutamatergic projections and sympathetic nerves that innervate lymphoid tissues.