A study of glial associated changes in the complexity of primary cortical neurons
Citation:O'REILLY, KATE AILEEN, A study of glial associated changes in the complexity of primary cortical neurons, Trinity College Dublin.School of Pharmacy & Pharma. Sciences, 2021
Neuroinflammation and disruptions in the integrity of neuronal circuitry are hallmarks of a range of CNS disorders. There is extensive evidence linking pro-inflammatory signalling and glial dysfunction with neuronal atrophy and impairments in synaptic transmission. As neuronal atrophy and loss of synaptic connections precede cell death, a greater understanding of the neurobiological mechanisms and mediators underlying inflammatory-driven glial cell dysfunction would pave the way for the identification of novel drug targets and drug development strategies for neuroinflammatory and degenerative disorders. An in vitro model of glial neuronal interaction composed of rat mature primary cortical neurons, microglia and astrocytes was used in this project. Neuronal complexity was determined by microtubule associated protein 2 (MAP2) immunocytochemistry and subsequently by Sholl analysis. Synapse number was measured by determination of the co-localised expression of the presynaptic protein synaptophysin and the postsynaptic protein postsynaptic density 95 (PSD-95). Treatment of enriched primary astrocyte cultures with the astrocyte toxin L-alpha-aminoadipic acid (L-AAA, 0.05, 0.5 mM) induced changes in astrocyte morphology, reduced immunoreactivity of the astrocytic marker glial fibrillary acidic protein (GFAP), reduced expression of the astrocytic markers GFAP and S100β and mitochondrial respiration. Treatment of mature primary cortical neurons at days in vitro 21 (DIV 21) with conditioned media from L-AAA treated astrocytes reduced neuronal complexity and synapse formation. Administration of L-AAA into the prelimbic cortex of mice increased the number of thin and stubby spines as measured by Golgi-Cox staining. Stimulation of primary mixed glial and enriched microglial cells with IFN&γ (10 ng/mL) induced changes in cell morphology reflecting a reactive state, and increased expression of pro-inflammatory factors including tumor necrosis factor-‐α alpha (TNF--‐α) and interleukin- alpha (IL-1-‐α). Conditioned media from IFNγ; (10 ng/mL) treated glial cells reduced the complexity and co-localised expression of synaptic markers in mature primary cortical neurons. Treatment of enriched astrocytic cultures with TNF--‐α; (30 ng/mL) and IL-1-‐α (3 ng/mL) induced the expression and release of interleukin-6 (IL-6) and produced a conditioned media which reduced neuronal complexity and synapse formation in primary cortical neurons. Immunoneutralisation of IL-6 blocked these effects demonstrating a role for IL-6 in mediating reactive astrocyte associated neuronal atrophy. L-AAA attenuated the release of IL-6 from mixed glial cultures induced by IFNγ and partially attenuated reductions in neuronal complexity and synapse formation induced by conditioned media from IFNγ treated mixed glia. L-AAA also attenuated the expression and release of astrocytic IL-6 following treatment with TNF--‐α (30 ng/mL) and IL-1-‐α (3 ng/mL). Notably, L-AAA provided greater protection against reductions in neuronal complexity and synapse formation induced by conditioned media from TNF--‐α and IL-1-‐α treated astrocytes. Previous studies have shown that IFNγ increases the expression of Indoleamine 2, 3-dioxygenase (IDO), decreases the expression of Kynurenine amino transferase II (KAT II) and has no effect on the expression of Kynurenine monooxygenase (KMO) or Tryptophan 2, 3-dioxygenase (TDO) in mixed glia and enriched microglial cultures. This study further demonstratetd that TNF--‐α (30 ng/mL) and IL-1-‐α (3 ng/mL) induced the expression of kynurenine pathway enzymes KAT II, KMO, IDO and TDO in enriched primary cortical astrocytic cultures indicating pathway induction. Conversely, IL-6 increased the expression of TDO, reduced the expression of KAT II and KMO and had no effect on KYNU or IDO in enriched primary cortical microglial cultures. Treatment with the IDO inhibitor 1-methyltryptophan (1-MT) protected against reductions in neuronal complexity induced by conditioned media from IFNγ treated mixed glia and conditioned media from IL-6 treated microglia. This indicates a role for the KP in driving atrophy affiliated with IFNγ and IL-6 activation of primary glia. Kynurenic acid (0.03, 0.1 and 0.3 ?M) increased neuronal complexity and synapse formation and protected against reductions in neuronal atrophy and synapse loss induced by conditioned media from IFNγ treated mixed glia and by the neurotoxic microglial metabolite quinolinic acid (1 ?M). Results of this thesis highlight the importance of healthy astrocytes, and the implications of astrocytic dysfunction, glial activation and pro-inflammatory signalling on neuronal complexity and synapse formation in vitro. In particular, they point to IL-6 signalling and the KP as attractive targets for the development of novel therapeutics aimed at restoring synaptic connections and promoting neuronal remodelling in CNS disorders affiliated with inflammatory-induced, reactive glial-associated neuronal atrophy.
Author: O'REILLY, KATE AILEEN
Publisher:Trinity College Dublin. School of Pharmacy & Pharma. Sciences. Discipline of Pharmacy
Type of material:Thesis
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