Kynurenine metabolic balance is disrupted in the hippocampus following peripheral lipopolysaccharide challenge.

BACKGROUND: Inflammation increases the risk of developing depression-related symptoms, and tryptophan metabolism is an important mediator of these behavior changes. Peripheral immune activation results in central up-regulation of pro-inflammatory cytokine expression, microglia activation, and the production of neurotoxic kynurenine metabolites. The neuroinflammatory and kynurenine metabolic response to peripheral immune activation has been largely characterized at the whole brain level. It is unknown if this metabolic response exhibits regional specificity even though the unique indoleamine 2,3-dioxygenase (IDO)-dependent depressive-like behaviors are known to be controlled by discrete brain regions. Therefore, regional characterization of neuroinflammation and kynurenine metabolism might allow for better understanding of the potential mechanisms that mediate inflammation-associated behavior changes. METHODS: Following peripheral immune challenge with lipopolysaccharide (LPS), brain tissue from behaviorally relevant regions was analyzed for changes in mRNA of neuroinflammatory targets and kynurenine pathway enzymes. The metabolic balance of the kynurenine pathway was also determined in the peripheral circulation and these brain regions. RESULTS: Peripheral LPS treatment resulted in region-independent up-regulation of brain expression of pro-inflammatory cytokines and glial cellular markers indicative of a neuroinflammatory response. The expression of kynurenine pathway enzymes was also largely region-independent. While the kynurenine/tryptophan ratio was elevated significantly in both the plasma and in each brain regions evaluated, the balance of kynurenine metabolism was skewed toward production of neurotoxic metabolites in the hippocampus. CONCLUSIONS: The upstream neuroinflammatory processes, such as pro-inflammatory cytokine production, glial cell activation, and kynurenine production, may be similar throughout the brain. However, it appears that the balance of downstream kynurenine metabolism is a tightly regulated brain region-dependent process.

Low-Level Stress Induces Production of Neuroprotective Factors in Wild-Type but Not BDNF+/- Mice: Interleukin-10 and Kynurenic Acid.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) deficiency confers vulnerability to stress, but the mechanisms are unclear. BDNF(+/-) mice exhibit behavioral, physiological, and neurochemical changes following low-level stress that are hallmarks of major depression. After immune challenge, neuroinflammation-induced changes in tryptophan metabolism along the kynurenine pathway mediate depressive-like behaviors. METHODS: We hypothesized that BDNF(+/-) mice would be more susceptible to stress-induced neuroinflammation and kynurenine metabolism, so BDNF(+/-) or wild-type littermate mice were subject to repeated unpredictable mild stress. Proinflammatory cytokine expression and kynurenine metabolites were measured. RESULTS: Unpredictable mild stress did not induce neuroinflammation. However, only wild-type mice produced the neuroprotective factors interleukin-10 and kynurenic acid in response to repeated unpredictable mild stress. In BDNF(+/-) mice, kynurenine was metabolized preferentially to the neurotoxic intermediate 3-hydroxykynurenine following repeated unpredictable mild stress. CONCLUSIONS: Our data suggest that BDNF may modulate kynurenine pathway metabolism during stress and provide a novel molecular mechanism of vulnerability and resilience to the development of stress-precipitated psychiatric disorders.

Intracerebroventricular administration of lipopolysaccharide induces indoleamine-2,3-dioxygenase-dependent depression-like behaviors.

BACKGROUND: Activation of the tryptophan degrading enzyme indoleamine-2,3-dioxygenase 1 (IDO1) is associated with the development of behavioral signs of depression. Systemic immune challenge induces IDO1 in both the periphery and the brain, leading to increased circulating and brain concentrations of kynurenines. However, whether IDO1 activity within the brain is necessary for the manifestation of depression-like behavior of mice following a central immune challenge remains to be elucidated. METHODS: We investigated the role of brain IDO1 in mediating depression-like behavior of mice in response to intracerebroventricular injection of saline or lipopolysaccharide (LPS, 10 ng). RESULTS: LPS increased the duration of immobility in the tail suspension test and decreased preference for a sucrose solution. These effects were associated with an activation of central but not peripheral IDO1, as LPS increased brain kynurenine but had no effect on plasma concentrations of kynurenine. Interestingly, genetic deletion or pharmacological inhibition of IDO1, using 1-methyl-tryptophan, abrogated the reduction in sucrose preference induced by intracerebroventricular LPS. 1-Methyl-tryptophan also blocked the LPS-induced increase in duration of immobility during the tail suspension test. CONCLUSIONS: These data indicate that activation of brain IDO1 is sufficient to induce depression-like behaviors of mice in response to central LPS.

Indoleamine 2,3-dioxygenase mediates anhedonia and anxiety-like behaviors caused by peripheral lipopolysaccharide immune challenge.

Upregulation of indoleamine 2,3-dioxygenase (IDO) by proinflammatory cytokines has been implicated as a biological mediator of inflammation-related mood disorders. Clinical reports on this neuro-immune interaction remain correlative, while mechanism-centered preclinical experiments have focused on a relatively narrow, and somewhat controversial, survey of depression-like behaviors that include the forced swim and tail suspension tests. Here, we sought to determine whether peripheral immune challenge with Escherichia coli, lipopolysaccharides (LPS) precipitates the development of translationally relevant depression-like behaviors and to investigate the role of IDO in mediating these LPS-induced behaviors. Intraperitoneal injection of C57BL/6J mice with LPS resulted in a robust, but transient, reduction in exploratory locomotor activity (eLMA) that returned to near baseline levels by 24h. Sucrose preference, a preclinical correlate of anhedonia, was diminished by more than 20% in LPS-treated compared to saline-treated control mice, and LPS induced a significant increase in anxiety-like behavior at 24h that was independent eLMA. Pretreatment of mice with an IDO inhibitor, 1-methyltryptophan (1MT), ablated the anxiogenic effects of LPS, while having no impact on sickness associated changes in body weight or eLMA. Additionally, 1MT pretreatment attenuated the LPS-induced reduction in sucrose preference, which was also confirmed in IDO-1 null mice. Interestingly, acute systemic administration of l-kynurenine, the enzymatic product of IDO, precipitated an anhedonic and anxiogenic effect in naïve mice without effect on eLMA. In a preclinical model, these data implicate IDO as a pivotal mediator of LPS-induced depression- and anxiety-like behavior.

Protocol for assessing phagocytosis activity in cultured primary murine microglia.

In this protocol, we describe steps to assess inflammation-induced cell response in cultured primary murine microglia through the analysis of fluorescent bead phagocytosis. We detail primary murine mixed glial cell culture preparation followed by microglia-specific isolation. Further, we describe treatment with lipopolysaccharide (LPS) to induce phagocytosis of fluorescent beads, followed by quantitative analysis using fluorescent imaging and Fiji - ImageJ software. For complete details on the use and execution of this protocol, please refer to Parrott et al.1.

Altered inflammatory response in FMRP-deficient microglia.

Fragile X syndrome (FXS) is an inherited intellectual disability with a high risk for comorbid autism spectrum disorders. Since FXS is a genetic disease, patients are more susceptible to environmental factors aggravating symptomatology. However, this confounding interaction between FXS environmental and genetic risk factors is under-investigated. Here, Fmr1 knock-out (KO) mice and the immune stimulus lipopolysaccharide (LPS) were used to explore this interaction between FXS development and inflammation in microglia, the brain's primary immune cell. Our results demonstrate that Fmr1 KO and wild-type (WT) microglia are not different in inflammatory outcomes without LPS. However, Fmr1 KO microglia produces an elevated pro-inflammatory and phagocytic response following LPS treatment when compared to WT microglia. Our experiments also revealed baseline differences in mitochondrial function and morphology between WT and Fmr1 KO microglia, which LPS treatment exaggerated. Our data suggest an altered inflammatory mechanism in Fmr1 KO microglia implicating a gene and environment interaction.

Brain derived neurotrophic factor deficiency exacerbates inflammation-induced anhedonia in mice.

Brain-derived neurotrophic factor (BDNF) is implicated in the pathology of major depression and influences the inflammatory response. Prolonged immune system activation can cause depression symptoms, and individuals with low BDNF expression may be vulnerable to inflammation-induced depression. We tested the hypothesis that BDNF deficient mice are vulnerable to the induction of depressive-like behavior following peripheral immune challenge. BDNF heterozygous (BDNF+/-) or wild-type (BDNF+/+) littermate mice were injected intraperitoneally (i.p.) with endotoxin (lipopolysaccharide, LPS) to trigger an acute pro-inflammatory response. After resolution of the acute sickness response, central expression of inflammatory genes, kynurenine metabolites, and depressive-like behaviors across multiple dimensions (symptoms) were measured. BDNF+/- mice displayed an exaggerated neuroinflammatory response following peripheral immune challenge. Pro-inflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) were overexpressed in BDNF+/- mice relative to BDNF+/+ littermate control mice. While behavioral despair and anxiety-like behavior was not different between genotypes, LPS-induced anhedonia-like behavior was significantly more pronounced in BDNF+/- mice relative to BDNF+/+ mice. The kynurenine pathway mediates the many depressive-like behavioral effects of peripheral LPS, and similar to pro-inflammatory cytokine gene expression, indoleamine 2,3-dioxygenase (IDO) expression and kynurenine metabolism was exaggerated in BDNF+/- mice. Genetic BDNF deficiency results in a dysregulated neuroinflammatory and metabolic response to peripheral immune challenge and in a specific vulnerability to the development of inflammation-induced anhedonia.

Hypothesis-driven investigations of diverse pharmacological targets in two mouse models of autism.

Autism spectrum disorder is a neurodevelopmental syndrome diagnosed primarily by persistent deficits in social interactions and communication, unusual sensory reactivity, motor stereotypies, repetitive behaviors, and restricted interests. No FDA-approved medical treatments exist for the diagnostic symptoms of autism. Here we interrogate multiple pharmacological targets in two distinct mouse models that incorporate well-replicated autism-relevant behavioral phenotypes. Compounds that modify inhibitory or excitatory neurotransmission were selected to address hypotheses based on previously published biological abnormalities in each model. Shank3B is a genetic model of a mutation found in autism and Phelan-McDermid syndrome, in which deficits in excitatory neurotransmission and synaptic plasticity have been reported. BTBR is an inbred strain model of forms of idiopathic autism in which reduced inhibitory neurotransmission and excessive mTOR signaling have been reported. The GABA-A receptor agonist gaboxadol significantly reduced repetitive self-grooming in three independent cohorts of BTBR. The TrkB receptor agonist 7,8-DHF improved spatial learning in Shank3B mice, and reversed aspects of social deficits in BTBR. CX546, a positive allosteric modulator of the glutamatergic AMPA receptor, and d-cycloserine, a partial agonist of the glycine site on the glutamatergic NMDA receptor, did not rescue aberrant behaviors in Shank3B mice. The mTOR inhibitor rapamycin did not ameliorate social deficits or repetitive behavior in BTBR mice. Comparison of positive and negative pharmacological outcomes, on multiple phenotypes, evaluated for replicability across independent cohorts, enhances the translational value of mouse models of autism for therapeutic discovery. GABA agonists present opportunities for personalized interventions to treat components of autism spectrum disorder. Autism Res 2019, 12: 401-421 © 2019 The Authors. Autism Research published by International Society for Autism Research published by Wiley Periodicals, Inc. LAY SUMMARY: Many of the risk genes for autism impair synapses, the connections between nerve cells in the brain. A drug that reverses the synaptic effects of a mutation could offer a precision therapy. Combining pharmacological and behavioral therapies could reduce symptoms and improve the quality of life for people with autism. Here we report reductions in repetitive behavior by a GABA-A receptor agonist, gaboxadol, and improvements in social and cognitive behaviors by a TrkB receptor agonist, in mouse models of autism.

Kynurenine pathway metabolic balance influences microglia activity: Targeting kynurenine monooxygenase to dampen neuroinflammation.

Chronic stress or inflammation increases tryptophan metabolism along the kynurenine pathway (KP), and the generation of neuroactive kynurenine metabolites contributes to subsequent depressive-like behaviors. Microglia regulate KP balance by preferentially producing oxidative metabolites, including quinolinic acid. Research has focused on the interplay between cytokines and HPA axis-derived corticosteroids in regulating microglial activity and effects of KP metabolites directly on neurons; however, the potential role that KP metabolites have directly on microglial activity is unknown. Here, murine microglia were stimulated with lipopolysaccharide(LPS). After 6 h, mRNA expression of interleukin(IL)-1ß, IL-6, tumor necrosis factor(TNF)-α and inducible nitric oxide synthase(iNOS) was dose-dependently increased along with the rate-limiting enzymes for oxidative KP metabolism, indoleamine-2,3-dioxygenase(IDO)-1 and kynurenine 3-monooxygenase(KMO). By 24 h post-LPS, kynurenine and quinolinic acid in the media was elevated. Inhibiting KMO with Ro 61-8048 during LPS challenge attenuated extracellular nitrite accumulation and expression of KMO and TNF-α in response to LPS. Similarly, primary microglia isolated from KMO-/- mice exhibited a significantly reduced pro-inflammatory response to LPS compared to WT controls. To determine whether the substrate (kynurenine) or end product (quinolinic acid) of KMO-dependent metabolism modulates the LPS response, microglia were treated with increasing concentrations of L-kynurenine or quinolinic acid in combination with LPS or saline. Interestingly, quinolinic acid did not impact the microglial LPS response. However, L-kynurenine had dose-dependent inhibitory effect on the LPS response. These data are the first to show an anti-inflammatory effect of KMO inhibition on microglia during immune challenge and suggest that KP metabolic balance may play a direct role in regulating microglia activity.

Kynurenine 3-Monooxygenase: An Influential Mediator of Neuropathology.

Mounting evidence demonstrates that kynurenine metabolism may play an important pathogenic role in the development of multiple neurological and neuropsychiatric disorders. The kynurenine pathway consists of two functionally distinct branches that generate both neuroactive and oxidatively reactive metabolites. In the brain, the rate-limiting enzyme for one of these branches, kynurenine 3-monooxygenase (KMO), is predominantly expressed in microglia and has emerged as a pivotal point of metabolic regulation. KMO substrate and expression levels are upregulated by pro-inflammatory cytokines and altered by functional genetic mutations. Increased KMO metabolism results in the formation of metabolites that activate glutamate receptors and elevate oxidative stress, while recent evidence has revealed neurodevelopmental consequences of reduced KMO activity. Together, the evidence suggests that KMO is positioned at a critical metabolic junction to influence the development or trajectory of a myriad of neurological diseases. Understanding the mechanism(s) by which alterations in KMO activity are able to impair neuronal function, and viability will enhance our knowledge of related disease pathology and provide insight into novel therapeutic opportunities. This review will discuss the influence of KMO on brain kynurenine metabolism and the current understanding of molecular mechanisms by which altered KMO activity may contribute to neurodevelopment, neurodegenerative, and neuropsychiatric diseases.

Hip displacement in spastic cerebral palsy: repeatability of radiologic measurement.

Radiologic measures of migration percentage (MP) and acetabular index (AI) taken from plain radiographs of the pelvis are the most commonly used tools for determining hip displacement and management options in children with cerebral palsy and spastic hip disease. This study determined interrater and intrarater reliability of MP and AI on pelvic radiographs chosen to represent a wide range of age (11 months to 8 years 5 months), MP (0%-56%), and AI (9 degrees -33 degrees ). The study demonstrated that an experienced rater would be expected to measure MP on a single radiograph to within +/-5.8% of the true value and a change in MP between two radiographs taken at different times to within +/-8.3% of the true value. Similarly for AI, the measurement error for a typical rater would be within +/-2.6 degrees on a single reading and +/-3.7 degrees if recording change between two occasions. The authors believe that the results indicating true change are acceptable in clinical practice, provided treatment decisions are based on a series of radiographs taken at 6-month intervals, methods and training are standardized, and consistent raters are used.