Oxidative stress and proinflammatory cytokines contribute to demyelination and axonal damage in a cerebellar culture model of neuroinflammation

Background: Demyelination and axonal damage are critical processes in the pathogenesis of multiple sclerosis (MS). Oxidative stress and pro-inflammatory cytokines elicited by inflammation mediates tissue damage. Methods/Principal Findings: To monitor the demyelination and axonal injury associated with microglia activation we employed a model using cerebellar organotypic cultures stimulated with lipopolysaccharide (LPS). Microglia activated by LPS released pro-inflammatory cytokines (IL-1β, IL-6 and TNFα), and increased the expression of inducible nitric oxide synthase (iNOS) and production of reactive oxygen species (ROS). This activation was associated with demyelination and axonal damage in cerebellar cultures. Axonal damage, as revealed by the presence of non-phosphorylated neurofilaments, mitochondrial accumulation in axonal spheroids, and axonal transection, was associated with stronger iNOS expression and concomitant increases in ROS. Moreover, we analyzed the contribution of pro-inflammatory cytokines and oxidative stress in demyelination and axonal degeneration using the iNOS inhibitor ethyl pyruvate, a free-scavenger and xanthine oxidase inhibitor allopurinol, as well as via blockage of pro-inflammatory cytokines using a Fc-TNFR1 construct. We found that blocking microglia activation with ethyl pyruvate or allopurinol significantly decreased axonal damage, and to a lesser extent, demyelination. Blocking TNFα significantly decreased demyelination but did not prevented axonal damage. Moreover, the most common therapy for MS, interferon-beta, was used as an example of an immunomodulator compound that can be tested in this model. In vitro, interferon-beta treatment decreased oxidative stress (iNOS and ROS levels) and the release of pro-inflammatory cytokines after LPS stimulation, reducing axonal damage. Conclusion: The model of neuroinflammation using cerebellar culture stimulated with endotoxin mimicked myelin and axonal damage mediated by the combination of oxidative stress and pro-inflammatory cytokines. This model may both facilitate understanding of the events involved in neuroinflammation and aid in the development of neuroprotective therapies for the treatment of MS and other neurodegenerative diseases

Fetal microglial phenotype in vitro carries memory of prior in vivo exposure to inflammation

Objective. Neuroinflammation in utero may result in life-long neurological disabilities. The molecular mechanisms whereby microglia contribute to this response remain incompletely understood. Methods. Lipopolysaccharide (LPS) or saline were administered intravenously to non-anesthetized chronically instrumented near-term fetal sheep to model fetal inflammation in vivo. Microglia were then isolated from in vivo LPS and saline (naïve) exposed animals. To mimic the second hit of neuroinflammation, these microglia were then re-exposed to LPS in vitro. Cytokine responses were measured in vivo and subsequently in vitro in the primary microglia cultures derived from these animals. We sequenced the whole transcriptome of naïve and second hit microglia and profiled their genetic expression to define molecular pathways disrupted during neuroinflammation.Results. In vivo LPS exposure resulted in IL-6 increase in fetal plasma 3 h post LPS exposure. Even though not histologically apparent, microglia acquired a pro-inflammatory phenotype in vivo that was sustained and amplified in vitro upon second hit LPS exposure as measured by IL-1β response in vitro and RNAseq analyses. While NFKB and Jak-Stat inflammatory pathways were up regulated in naïve microglia, heme oxygenase 1 (HMOX1) and Fructose-1,6-bisphosphatase (FBP) genes were uniquely differentially expressed in the second hit microglia. Microglial calreticulin/LRP genes implicated in microglia-neuronal communication relevant for the neuronal development were up regulated in second hit microglia.Discussion. We identified a unique HMOX1down and FBPup phenotype of microglia exposed to the double-hit suggesting interplay of inflammatory and metabolic pathways as a memory of prior inflammatory insult. These findings suggest new therapeutic targets for early postnatal intervention to prevent brain injury

Activating mGlu3 Metabotropic Glutamate Receptors Rescues Schizophrenia-like Cognitive Deficits Through Metaplastic Adaptations Within the Hippocampus

Background: Polymorphisms in GRM3, the gene encoding the mGlu3 metabotropic glutamate receptor, are associated with impaired cognition and neuropsychiatric disorders such as schizophrenia. Limited availability of selective genetic and molecular tools has hindered progress in developing a clear understanding of the mechanisms through which mGlu3 receptors regulate synaptic plasticity and cognition. Methods: We examined associative learning in mice with trace fear conditioning, a hippocampal-dependent learning task disrupted in patients with schizophrenia. Underlying cellular mechanisms were assessed using ex vivo hippocampal slice preparations with selective pharmacological tools and selective genetic deletion of mGlu3 receptor expression in specific neuronal subpopulations. Results: mGlu3 receptor activation enhanced trace fear conditioning and reversed deficits induced by subchronic phencyclidine. Mechanistic studies revealed that mGlu3 receptor activation induced metaplastic changes, biasing afferent stimulation to induce long-term potentiation through an mGlu5 receptor–dependent, endocannabinoid-mediated, disinhibitory mechanism. Selective genetic deletion of either mGlu3 or mGlu5 from hippocampal pyramidal cells eliminated effects of mGlu3 activation, revealing a novel mechanism by which mGlu3 and mGlu5 interact to enhance cognitive function. Conclusions: These data demonstrate that activation of mGlu3 receptors in hippocampal pyramidal cells enhances hippocampal-dependent cognition in control and impaired mice by inducing a novel form of metaplasticity to regulate circuit function, providing a clear mechanism through which genetic variation in GRM3 can contribute to cognitive deficits. Developing approaches to positively modulate mGlu3 receptor function represents an encouraging new avenue for treating cognitive disruption in schizophrenia and other psychiatric diseases