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?7 Nicotinic Acetylcholine Receptor Signaling Modulates Ovine Fetal Brain Astrocytes Transcriptome in Response to Endotoxin.

ABSTRACT: Neuroinflammation in utero may result in lifelong neurological disabilities. Astrocytes play a pivotal role in this process, but the mechanisms are poorly understood. No early postnatal treatment strategies exist to enhance neuroprotective potential of astrocytes. We hypothesized that agonism on ?7 nicotinic acetylcholine receptor (?7nAChR) in fetal astrocytes will augment their neuroprotective transcriptome profile, while the inhibition of ?7nAChR will achieve the opposite. Using an in vivo-in vitro model of developmental programming of neuroinflammation induced by lipopolysaccharide (LPS), we validated this hypothesis in primary fetal sheep astrocytes cultures re-exposed to LPS in the presence of a selective ?7nAChR agonist or antagonist. Our RNAseq findings show that a pro-inflammatory astrocyte transcriptome phenotype acquired in vitro by LPS stimulation is reversed with ?7nAChR agonistic stimulation. Conversely, ?7nAChR inhibition potentiates the pro-inflammatory astrocytic transcriptome phenotype. Furthermore, we conducted a secondary transcriptome analysis against the identical ?7nAChR experiments in fetal sheep primary microglia cultures. Similar to findings in fetal microglia, in fetal astrocytes we observed a memory effect of in vivo exposure to inflammation, expressed in a perturbation of the iron homeostasis signaling pathway (hemoxygenase 1, HMOX1), which persisted under pre-treatment with ?7nAChR antagonist but was reversed with ?7nAChR agonist. For both glia cell types, common pathways activated due to LPS included neuroinflammation signaling and NF-?B signaling in some, but not all comparisons. However, overall, the overlap on the level of signaling pathways was rather minimal. Astrocytes, not microglia-the primary immune cells of the brain, were characterized by unique inhibition patterns of STAT3 pathway due to agonistic stimulation of ?7nAChR prior to LPS exposure. Lastly, we discuss the implications of our findings for fetal and postnatal brain development.

SUBMITTER: Cao M

PROVIDER: S-EPMC6520997 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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α7 Nicotinic Acetylcholine Receptor Signaling Modulates Ovine Fetal Brain Astrocytes Transcriptome in Response to Endotoxin.

Cao Mingju M MacDonald James W JW Liu Hai L HL Weaver Molly M Cortes Marina M Durosier Lucien D LD Burns Patrick P Fecteau Gilles G Desrochers André A Schulkin Jay J Antonelli Marta C MC Bernier Raphael A RA Dorschner Michael M Bammler Theo K TK Frasch Martin G MG

Frontiers in immunology 20190509

Neuroinflammation <i>in utero</i> may result in lifelong neurological disabilities. Astrocytes play a pivotal role in this process, but the mechanisms are poorly understood. No early postnatal treatment strategies exist to enhance neuroprotective potential of astrocytes. We hypothesized that agonism on α7 nicotinic acetylcholine receptor (α7nAChR) in fetal astrocytes will augment their neuroprotective transcriptome profile, while the inhibition of α7nAChR will achieve the opposite. Using an <i>i ...[more]

PMID: 31143190

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Project description:PurposeThe α7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of α7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer's disease (AD) and schizophrenia , the α7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for α7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the α7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the α7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an α7 nAChR KO mouse when compared to a wild-type (WT) mouse.MethodsTo examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and α7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes.ResultsIn the whole retina, there was a statistically significant upregulation of α2, α9, α10, β4, nAChR subunit, and m1 and m4 mAChR subtype transcripts in the α7 nAChR KO mice. However, the retinal layers showed complex patterns of transcript expression. In the ganglion cell layer (GCL), m2 and m4 mAChR subtype transcripts were significantly upregulated, while β3 and β4 nAChR subunit transcripts were significantly downregulated. In the inner portion of the inner nuclear layer (iINL), α2, α9, β4, nAChR subunit, and m3 and m4 mAChR subtype transcripts were significantly downregulated. In the outer portion of the inner nuclear layer (oINL), β2, β4, and m4 AChR subunit transcripts were significantly upregulated. Western blot experiments confirmed the protein expression of α3-α5 and α9-containing nAChR subunits and m1-m2 mAChR subtypes in mouse retinas. IHC results supported many of the mRNA changes observed. Finally, this is the first report of α9 and α10 nAChR subunit expressions in the retina of any species.ConclusionsRather than a simple upregulation of a single AChR subunit or subtype, the absence of the α7 nAChR in the KO mice was associated with complex layer-specific changes in the expression of AChR subunits and subtypes.

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?7 Nicotinic Acetylcholine Receptor Signaling Modulates Ovine Fetal Brain Astrocytes Transcriptome in Response to Endotoxin.

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α7 Nicotinic Acetylcholine Receptor Signaling Modulates Ovine Fetal Brain Astrocytes Transcriptome in Response to Endotoxin.

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