Adverse outcome pathways and linkages to transcriptomic effects relevant to ionizing radiation injury.

BACKGROUND: In the past three decades, a large body of data on the effects of exposure to ionizing radiation and the ensuing changes in gene expression has been generated. These data have allowed for an understanding of molecular-level events and shown a level of consistency in response despite the vast formats and experimental procedures being used across institutions. However, clarity on how this information may inform strategies for health risk assessment needs to be explored. An approach to bridge this gap is the adverse outcome pathway (AOP) framework. AOPs represent an illustrative framework characterizing a stressor associated with a sequential set of causally linked key events (KEs) at different levels of biological organization, beginning with a molecular initiating event (MIE) and culminating in an adverse outcome (AO). Here, we demonstrate the interpretation of transcriptomic datasets in the context of the AOP framework within the field of ionizing radiation by using a lung cancer AOP (AOP 272: https://www.aopwiki.org/aops/272) as a case example. METHODS: Through the mining of the literature, radiation exposure-related transcriptomic studies in line with AOP 272 related to lung cancer, DNA damage response, and repair were identified. The differentially expressed genes within relevant studies were collated and subjected to the pathway and network analysis using Reactome and GeneMANIA platforms. Identified pathways were filtered (p < .001, ≥3 genes) and categorized based on relevance to KEs in the AOP. Gene connectivities were identified and further grouped by gene expression-informed associated events (AEs). Relevant quantitative dose-response data were used to inform the directionality in the expression of the genes in the network across AEs. RESULTS: Reactome analyses identified 7 high-level biological processes with multiple pathways and associated genes that mapped to potential KEs in AOP 272. The gene connectivities were further represented as a network of AEs with associated expression profiles that highlighted patterns of gene expression levels. CONCLUSIONS: This study demonstrates the application of transcriptomics data in AOP development and provides information on potential data gaps. Although the approach is new and anticipated to evolve, it shows promise for improving the understanding of underlying mechanisms of disease progression with a long-term vision to be predictive of adverse outcomes.

Characterizing the protracted neurobiological and neuroanatomical effects of paraquat in a murine model of Parkinson's disease.

The primary motor symptoms of Parkinson's disease (PD) result from the degeneration of dopamine-producing neurons of the substantia nigra pars compacta (SNc), and often, the loss is asymmetrical, resulting in unilateral tremor presentation. Notably, age is the primary risk factor for PD, and it is likely that the disease ultimately stems from the impact of environmental factors, which interact with the aging process. Recent research has focused on the role of microglia and pro-oxidative responses in dopaminergic neuronal death. In this study, we sought to examine the neurodegenerative, inflammatory, and stress effects of exposure to the etiologically relevant pesticide, paraquat, over time (up to 6 months after injections). We also were interested in whether a high-resolution, 7-Tesla animal magnetic resonance imaging would be sensitive enough to detect the degenerative impact of paraquat. We found that paraquat induced a loss of dopaminergic SNc neurons and activation of microglia that surprisingly did not change over 6 months after the last injection. A long-lasting reduction was evident for body weight, and alterations in organ (lung and heart) weight were evident, which reflect the peripheral impact of the toxicant. The microglial proinflammatory actin-remodeling factor, WAVE2, along with the inflammatory transcription factor, nuclear factor kappa B were also elevated within the brain. Remarkably, the stress hormone, corticosterone, was still significantly elevated 1 month after paraquat, whereas the inflammasome factor, caspase-1, and antigen presentation factor, MFG-E8, both displayed delayed rises after the 6-month time. Using high-resolution magnetic resonance imaging, we detected no striatal changes but modest hemispheric differences in the SNc and time-dependent volumetric enlargement of the ventricles in paraquat-treated mice. These data suggest that paraquat induces long-term nigrostriatal pathology (possibly asymmetric) and inflammatory changes and stress and trophic/apoptotic effects that appear to either increase with the passage of time or are evident for at least 1 month. In brief, paraquat may be a useful nonspecific means to model widespread stress and inflammatory changes related to PD or age-related disease in general, but not the progressive nature of such diseases.

Leucine-rich repeat kinase-2 (LRRK2) modulates microglial phenotype and dopaminergic neurodegeneration.

Leucine-rich repeat kinase 2 (LRRK2) is a common gene implicated in Parkinson's disease and many inflammatory processes. Thus, we assessed the role of LRRK2 in the context of endotoxin (lipopolysaccharide, LPS)-induced inflammation of the substantia nigra together with the environmental toxicant, paraquat, that has been implicated in PD. Here we found that LRRK2 ablation prevented the loss of dopaminergic neurons and behavioral deficits (motor) induced by LPS priming followed by paraquat exposure. The LRRK2 ablation also provoked a phenotypic shift in LPS-primed microglia cells. The LRRK2 deficiency reduced their "activated" morphology and upregulation of the inflammatory phagocytic regulator, WAVE2 (critical for actin remodeling), while the chemokine receptor, CX3CR1, was elevated in isolated CD11b+ myeloid cells. Furthermore, LRRK2 knockout attenuated the signs of oxidative stress and morphological changes induced in primary microglia by LPS treatment. However, induced WAVE2 expression together with LPS exposure in microglia overcame the inhibitory effects of LRRK2 knockout, suggesting WAVE2 may be acting downstream of LRRK2. Neither WAVE2 nor did LRRK2 knockout influence LPS-induced cytokine elevations in the microglia. We are the first to show the importance of LRRK2 in neurodegenerative and inflammatory processes in this multi-hit toxin model of PD. These data are consistent with the proposition that LRRK2 and WAVE2 are useful therapeutic targets for PD or other conditions with a prominent neuroinflammatory component.

Differential Effects of Short-term Environmental Enrichment in Juvenile and Adult Mice.

Environmental enrichment has been shown to increase cognitive abilities and accelerate recovery from a number of disease states. Typically, enrichment protocols last from four to eight weeks, however, it has previously been shown that two weeks of environmental enrichment is sufficient to increase cognitive abilities and the proliferation of the astroglial stem cell pool in juvenile mice. The current study examines whether a short-term enrichment protocol can induce similar effects in adults as compared to juveniles. Using juvenile and adult wild-type mice, we examined the effects of short-term environmental enrichment (including a running wheel) on cognitive abilities, anxiety-like behaviour, and the stem cell potential of sub-ventricular neural stem cells (NSC's) in vitro using neurosphere assays. We found that short-term environmental enrichment decreased anxiety behaviour and increased overall memory abilities similarly in juveniles and adults. However, the rate of acquisition on the Morris water maze, hippocampal Sox2 and Ki67 expression, and neurosphere potential increased in response to enrichment only in juveniles, suggesting that the effects of enrichment on these measures are age dependant. Together, these data suggest that the potential beneficial effects of environmental manipulations decrease with age.

Microglia depletion prior to lipopolysaccharide and paraquat treatment differentially modulates behavioral and neuronal outcomes in wild type and G2019S LRRK2 knock-in mice.

BACKGROUND: Substantial data have implicated microglial-driven neuroinflammation in Parkinson's disease (PD) and environmental toxicants have been long expected as triggers of such inflammatory processes. Of course, these environmental insults act in the context of genetic vulnerability factors and in this regard, leucine rich repeat kinase 2 (LRRK2), may play a prominent role. METHODS: We used a double hit, lipopolysaccharide (LPS; endotoxin) followed by paraquat (pesticide toxicant) model of PD in mice with the most common LRRK2 mutation G2019S, knockin mice and wild type littermates. In order to assess the contribution of microglia, we depleted these cells (through 14 days of the CSF-1 antagonist, PLX-3397) prior to LPS and paraquat exposure. RESULTS: We found that the G2019S mice displayed the greatest signs of behavioral pathology, but that the PLX-3397 induced microglial depletion at the time of LPS exposure diminished toxicity and weight loss and blunted the reduction in home-cage activity with subsequent paraquat exposure. However, neither the PLX-3397 pre-treatment nor the G2019S mutation affected the LPS â€‹+ â€‹paraquat induced loss of substantia nigra pars compacta (SNc) dopamine neurons or elevation of circulating immune (IL-6) or stress (corticosterone) factors. Intriguingly, microglial morphological ratings were basally enhanced in G2019S mice and the PLX-3397 pre-treatment reversed this effect. Moreover, PLX-3397 pre-treatment selectively elevated soluble a-synuclein and SIRT3 levels, while reducing SNc caspase-1 and 3, along with CX3CR1. Hence, the re-populated "new" microglia following cessation of PLX-3397 clearly had an altered phenotype or were immature at the time of sacrifice (i.e. after 11 days). CONCLUSIONS: Collectively, these findings suggest that G2019S knock-in and PLX-3397 microglial depletion at the time of LPS exposure affects behavioral, but not neurodegenerative responses to subsequent environmental toxin exposure.

Chronic unpredictable stress influenced the behavioral but not the neurodegenerative impact of paraquat.

The impact of psychological stressors on the progression of motor and non-motor disturbances observed in Parkinson's disease (PD) has received little attention. Given that PD likely results from many different environmental "hits", we were interested in whether a chronic unpredictable stressor regimen would act additively or possibly even synergistically to augment the impact of the toxicant, paraquat, which has previously been linked to PD. Our findings support the contention that paraquat itself acted as a systemic stressor, with the pesticide increasing plasma corticosterone, as well as altering glucocorticoid receptor (GR) expression in the hippocampus. Furthermore, stressed mice that also received paraquat displayed synergistic motor coordination impairment on a rotarod test and augmented signs of anhedonia (sucrose preference test). The individual stressor and paraquat treatments also caused a range of non-motor (e.g. open field, Y and plus mazes) deficits, but there were no signs of an interaction (neither additive nor synergistic) between the insults. Similarly, paraquat caused the expected loss of substantia nigra dopamine neurons and microglial activation, but this effect was not further influenced by the chronic stressor. Taken together, these results indicate that paraquat has many effects comparable to that of a more traditional stressor and that at least some behavioral measures (i.e. sucrose preference and rotarod) are augmented by the combined pesticide and stress treatments. Thus, although psychological stressors might not necessarily increase the neurodegenerative effects of the toxicant exposure, they may promote co-morbid behaviors pathology.

Leucine-rich repeat kinase-2 (LRRK2) modulates paraquat-induced inflammatory sickness and stress phenotype.

BACKGROUND: Leucine-rich repeat kinase 2 (LRRK2) is a common gene implicated in Parkinson's disease (PD) and is also thought to be fundamentally involved in numerous immune functions. Thus, we assessed the role of LRRK2 in the context of the effects of the environmental toxicant, paraquat, that has been implicated in PD and is known to affect inflammatory processes. METHODS: Male LRRK2 knockout (KO) and transgenic mice bearing the G2019S LRRK2 mutation (aged 6-8 months) or their littermate controls were exposed to paraquat (two times per week for 3 weeks), and sickness measures, motivational scores, and total home-cage activity levels were assessed. Following sacrifice, western blot and ELISA assays were performed to see whether or not LRRK2 expression would alter processes related to plasticity, immune response processes, or the stress response. RESULTS: Paraquat-induced signs of sickness, inflammation (elevated IL-6), and peripheral toxicity (e.g., organ weight) were completely prevented by LRRK2 knockout. In fact, LRRK2 knockout dramatically reduced not only signs of illness, but also the motivational (nest building) and home-cage activity deficits induced by paraquat. Although LRRK2 deficiency did not affect the striatal BDNF reduction that was provoked by paraquat, it did blunt the corticosterone elevation induced by paraquat, raising the possibility that LRRK2 may modulate aspects of the HPA stress axis. Accordingly, we found that transgenic mice bearing the G2019S LRRK2 mutation had elevated basal corticosterone, along with diminished hippocampal 5-HT1A levels. CONCLUSION: We are the first to show the importance of LRRK2 in the peripheral neurotoxic and stressor-like effects of paraquat. These data are consistent with LRRK2 playing a role in the general inflammatory tone and stressor effects induced by environmental toxicant exposure.

Paraquat Exposure Increases Oxidative Stress Within the Dorsal Striatum of Male Mice With a Genetic Deficiency in One-carbon Metabolism.

Paraquat is an herbicide that is commonly used worldwide. Exposure to paraquat results in Parkinson's disease (PD)-like symptoms including dopaminergic cell loss. Nutrition has also been linked in the pathogenesis of PD, such as reduced levels of folic acid, a B-vitamin, and component of one-carbon metabolism. Within one-carbon metabolism, methylenetetrahydrofolate reductase (MTHFR) catalyzes the irreversible conversion of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. A polymorphism in MTHFR (677 C&→T) has been reported in 5%-15% of North American and European human populations. The MTHFR polymorphism is also prevalent in PD patients. The goal of this study was to investigate the impact of paraquat-induced PD-like pathology in the context of reduced levels of MTHFR. Three-month-old male Mthfr+/- mice, which model the MTHFR polymorphism observed in humans, were administered intraperitoneal injections of paraquat (10 mg/kg) or saline 6 times over 3 weeks. At the end of paraquat treatment, motor and memory function were assessed followed by collection of brain tissue for biochemical analysis. Mthfr+/- mice treated with paraquat showed impaired motor function. There was increased microglial activation within the substantia nigra (SN) of Mthfr+/- mice treated with paraquat. Additionally, all Mthfr+/- mice that were treated with paraquat showed increased oxidative stress within the dorsal striatum, but not the SN. The present results show that paraquat exposure increases PD-like pathology in mice deficient in one-carbon metabolism.

Perinatal antibiotic exposure alters composition of murine gut microbiota and may influence later responses to peanut antigen.

BACKGROUND: Accumulating evidence suggests that the gut microbiota shapes developmental processes within the immune system. Early life antibiotic use is one factor which may contribute to immune dysfunction and the recent surge in allergies by virtue of its effects on gut microbiota. OBJECTIVE AND METHODS: As a first step towards determining whether a relationship exists between perinatal antibiotic induced changes in the gut microbiota and the later development of a peanut allergy, we exposed newborn mice to either the broad-spectrum antibiotic vancomycin or to a vehicle for 6 weeks and then used a novel murine model of peanut allergy. RESULTS: Early-life treatment with vancomycin resulted in a significant shift in the gut microbiota community characterized by a reduction in the abundance of firmicutes and preponderance of inflammatory proteobacteria. Mice with an antibiotic-altered microbiota, showed a localized allergic-like response characterized by ear swelling and scratching following intra-dermal peanut antigen challenge. Likewise, circulating IgE levels were increased in antibiotic-treated mice, but no evidence of a systemic allergic or anaphylactic-like response was observed. Importantly, we utilized the naturally occurring pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α), rather than the more commonly used cholera toxin, as an adjuvant together with the peanut antigen. CONCLUSION: Our data suggest that early antibiotic exposure promotes a shift in the gut microbiota community that may in turn, influence how mice later respond to a TNF-α + antigen challenge. However, further studies verifying the capacity of microbiota restoration to protect against allergic responses will be needed to confirm a causal role of antibiotic-induced microbiota variations in promoting allergic disease phenotypes.

The impact of murine LRRK2 G2019S transgene overexpression on acute responses to inflammatory challenge.

The most common Parkinson's disease (PD) mutation is the gain-of-function LRRK2 G2019S variant, which has also been linked to inflammatory disease states. Yet, little is known of the role of G2019S in PD related complex behavioral or immune/hormonal processes in response to inflammatory/toxicant challenges. Hence, we characterized the behavioral, neuroendocrine-immune and central monoaminergic responses in G2019S overexpressing mutants following systemic interferon-gamma (IFN-γ) or lipopolysaccharide (LPS) administration. Although LPS markedly (and IFN-γ modestly in some cases) increased cytokine and corticosterone levels, while inducing pronounced sickness and home-cage activity deficits, the G2019S mutation had no effect on these parameters. No differences were observed with regards to brain microglia with the acute LPS injection, regardless of genotype. Nor did the G2019S mutation influence neurotransmitter levels within the medial prefrontal cortex or paraventricular nucleus of the hypothalamus. However, the LRRK2 G2019S transgenic mice did have altered monoamine levels within the striatum and hippocampus. Indeed, G2019S mice had altered basal levels and turnover of dopamine within the striatum, along with changes in hippocampal serotonin and norepinephrine activity in response to LPS and IFN-γ. The present findings suggest the importance of murine G2019S in hippocampal and striatal neurotransmission, but that the transgene didn't appear to be involved in functional behavioral and stress-like hormonal and cytokine changes provoked by inflammatory insults.

Age and Chronicity of Administration Dramatically Influenced the Impact of Low Dose Paraquat Exposure on Behavior and Hypothalamic-Pituitary-Adrenal Activity.

Little is known of the age-dependent and long-term consequences of low exposure levels of the herbicide and dopaminergic toxicant, paraquat. Thus, we assessed the dose-dependent effects of paraquat using a typical short-term (3 week) exposure procedure, followed by an assessment of the effects of chronic (16 weeks) exposure to a very low dose (1/10th of what previously induced dopaminergic neuronal damage). Short term paraquat treatment dose-dependently induced deficits in locomotion, sucrose preference and Y-maze performance. Chronic low dose paraquat treatment had a very different pattern of effects that were also dependent upon the age of the animal: in direct contrast to the short-term effects, chronic low dose paraquat increased sucrose consumption and reduced forced swim test (FST) immobility. Yet these effects were age-dependent, only emerging in mice older than 13 months. Likewise, Y-maze spontaneous alternations and home cage activity were dramatically altered as a function of age and paraquat chronicity. In both the short and long-term exposure studies, increased corticosterone and altered hippocampal glucocorticoid receptor (GR) levels were induced by paraquat, but surprisingly these effects were blunted in the older mice. Thus, paraquat clearly acts as a systemic stressor in terms of corticoid signaling and behavioral outcomes, but that paradoxical effects may occur with: (a) repeated exposure at; (b) very low doses; and (c) older age. Collectively, these data raise the possibility that repeated "hits" with low doses of paraquat in combination with aging processes might have promoted compensatory outcomes.

Individual and interactive sex-specific effects of acute restraint and systemic IFN-γ treatment on neurochemistry.

Compelling evidence supports the involvement of the pro-inflammatory cytokines, IL-6, IFN-α and TNF-α in depression and related stress-associated pathologies. A role has also been suggested for the Th1-type cytokine, IFN-γ, with most mechanistic accounts focusing on the cytokine's capacity to induce indoleamine 2,3-dioxygenase (IDO), leading to diminished tryptophan and the generation of kynurenine metabolites. Beyond these IDO-dependent routes, there is surprisingly little evidence directly linking IFN-γ to alterations of brain regional monoamine activity and HPA axis functioning. Our specific aims in the present study were twofold: 1) assess the behavioural, plasma corticosterone and brain regional monoamine effects of acute systemic IFN-γ, with or without short duration restraint stress (15 min), and 2) determine the sex-specific nature of these effects. As predicted, IFN-γ stimulated monoaminergic activity within a number of stressor-sensitive limbic brain regions, most notably the paraventricular nucleus of the hypothalamus, central amygdala and prefrontal cortex. While several of these effects were sex-specific, there was little in the way of synergism between the cytokine and stressor treatments. Nonetheless, IFN-γ did synergistically interact with acute restraint stress to increase plasma corticosterone concentrations, and this effect was most pronounced in the male mice. These data are among the first to show that systemically administered IFN-γ can alone or in conjunction with psychologically relevant stressor, modify brain regional monoamine activity and the plasma corticosterone response.

The interactive effects of ketamine and magnesium upon depressive-like pathology.

Approximately one-third of patients with major depressive disorders (MDDs) are resistant to current treatment methods, and the majority of cases relapse at some point during therapy. This has resulted in novel treatments being adopted, including subanesthetic doses of ketamine, which affects aberrant neuroplastic circuits, glutamatergic signaling, and the production of brain-derived neurotrophic factor. Ketamine rapidly relieves depressive symptoms in treatment-resistant major depressive disorder patients with effects that last for up to 2 weeks even after a single administration. However, it is also a drug with an abusive potential and can have marked side effects. Hence, this study aimed at enhancing the antidepressant-like effects of ketamine (allowing for lower dosing regimens) by coadministering magnesium hydroaspartate (Mg(2+) normally affects the same receptors as ketamine) and also assessed whether an Mg(2+)-deficient diet would modify the impact of ketamine. It was found that a single 15 mg/kg dose of ketamine did indeed induce rapid antidepressant-like effects in the forced swim test but did not affect brain levels of the brain-derived neurotrophic factor. Contrary to our hypothesis, magnesium administration or deficiency did not influence the impact of ketamine on these outcomes. Thus, these data do not support the use of magnesium as an adjunct agent and instead suggest that further research involving other antidepressant and animal models is required to confirm the present findings.

Paraquat and psychological stressor interactions as pertains to Parkinsonian co-morbidity.

A number of epidemiological and experimental studies have implicated the non-selective herbicide, paraquat, in the development of sporadic Parkinson's disease (PD). While preclinical research has focused mainly on elucidating the nigrostriatal effects of paraquat, relatively little data are available concerning non-motor brain systems and inflammatory immune processes (which have been implicated in PD). Hence, in the present study, we sought to take a multi-system approach to characterize the influence of paraquat upon extra-nigrostriatal brain regions, as well ascertain whether the impact of the pesticide might be enhanced in the context of chronic intermittent stressor exposure. Our findings support the contention that paraquat itself acted as a systemic stressor, with the pesticide increasing plasma corticosterone, as well as altering neurochemical activity in the locus coeruleus, paraventricular nucleus of the hypothalamus, nucleus accumbens, dorsal striatum, and central amygdala. However, with the important exception striatal dopamine turnover, the stressor treatment did not further augment these effects. Additionally, paraquat altered inter-cytokine correlations and, to a lesser extent, circulating cytokine levels, and concomitant stress exposure modulated some of these effects. Finally, paraquat provoked significant (albeit modest) reductions of sucrose preference and weight gain, hinting at possible anhendonic-like or sickness responses. These data suggest that, in addition to being a well known oxidative stress generator, paraquat can act as a systemic stressor affecting hormonal and neurochemical activity, but largely not interacting with a concomitant stressor regimen.

Use of induced pluripotent stem cell derived neurons engineered to express BDNF for modulation of stressor related pathology.

Combined cell and gene-based therapeutic strategies offer potential in the treatment of neurodegenerative and psychiatric conditions that have been associated with structural brain disturbances. In the present investigation, we used a novel virus-free re-programming method to generate induced pluripotent stem cells (iPSCs), and then subsequently transformed these cells into neural cells which over-expressed brain derived neurotrophic factor (BDNF). Importantly, the infusion of iPSC derived neural cells (as a cell replacement and gene delivery tool) and BDNF (as a protective factor) influenced neuronal outcomes. Specifically, intracerebroventricular transplantation of iPSC-derived neural progenitors that over-expressed BDNF reversed the impact of immune (lipopolysaccharide) and chronic stressor challenges upon subventricular zone adult neurogenesis, and the iPSC-derived neural progenitor cells alone blunted the stressor-induced corticosterone response. Moreover, our findings indicate that mature dopamine producing neurons can be generated using iPSC procedures and appear to be viable when infused in vivo. Taken together, these data could have important implications for using gene-plus-cell replacement methods to modulate stressor related pathology.

Antidepressant-like effects of erythropoietin: a focus on behavioural and hippocampal processes.

Depression is a chronic and debilitating condition with a significant degree of relapse and treatment resistance that could stem, at least in part, from disturbances of neuroplasticity. This has led to an increased focus on treatment strategies that target brain derived neurotrophic factor (BDNF), synaptic plasticity and adult neurogenesis. In the current study we aimed to assess whether erythropoietin (EPO) would have antidepressant-like effects given its already established pro-trophic actions. In particular, we assessed whether EPO would diminish the deleterious effects of a social stressor in mice. Indeed, EPO induced anxiolytic and antidepressant-like responses in a forced swim test, open field, elevated-plus maze, and a novelty test, and appeared to blunt some of the negative behavioural effects of a social stressor. Furthermore, EPO promoted adult hippocampal neurogenesis, an important feature of effective antidepressants. Finally, a separate study using the mTOR inhibitor rapamycin revealed that antagonizing this pathway prevented the impact of EPO upon forced swim performance. These data are consistent with previous findings showing that the mTOR pathway and its neurogenic and synaptogenic effects might mediate the behavioral consequences of antidepressant agents. Our findings further highlight EPO as a possible adjunct treatment for affective disorders, as well as other stressor associated disorders of impaired neuroplasticity.