Sex-dependent astrocyte reactivity: Unveiling chronic stress-induced morphological changes across multiple brain regions.

Chronic stress is a major precursor to various neuropsychiatric disorders and is linked with increased inflammation in the brain. However, the bidirectional association between inflammation and chronic stress has yet to be fully understood. Astrocytes are one of the key inflammatory regulators in the brain, and the morphological change in reactive astrocytes serves as an important indicator of inflammation. In this study, we evaluated the sex-specific astrocyte response to chronic stress or systemic inflammation in key brain regions associated with mood disorders. We conducted the unpredictable chronic mild stress (UCMS) paradigm to model chronic stress, or lipopolysaccharide (LPS) injection to model systemic inflammation. To evaluate stress-induced morphological changes in astrocyte complexity, we measured GFAP fluorescent intensity for astrocyte expression, branch bifurcation by quantifying branch points and terminal points, branch arborization by conducting Sholl analysis, and calculated the ramification index. Our analysis indicated that chronic stress-induced morphological changes in astrocytes in all brain regions investigated. The effects of chronic stress were region and sex specific. Notably, females had greater stress or inflammation-induced astrocyte activation in the hypothalamus (HYPO), CA1, CA3, and amygdala (AMY) than males. These findings indicate that chronic stress induces astrocyte activation that may drive sex and region-specific effects in females, potentially contributing to sex-dependent mechanisms of disease.

Chronic Stress-Induced Neuroinflammation: Relevance of Rodent Models to Human Disease.

The brain is the central organ of adaptation to stress because it perceives and determines threats that induce behavioral, physiological, and molecular responses. In humans, chronic stress manifests as an enduring consistent feeling of pressure and being overwhelmed for an extended duration. This can result in a persistent proinflammatory response in the peripheral and central nervous system (CNS), resulting in cellular, physiological, and behavioral effects. Compounding stressors may increase the risk of chronic-stress-induced inflammation, which can yield serious health consequences, including mental health disorders. This review summarizes the current knowledge surrounding the neuroinflammatory response in rodent models of chronic stress-a relationship that is continually being defined. Many studies investigating the effects of chronic stress on neuroinflammation in rodent models have identified significant changes in inflammatory modulators, including nuclear factor-κB (NF-κB) and toll-like receptors (TLRs), and cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, and IL-6. This suggests that these are key inflammatory factors in the chronic stress response, which may contribute to the establishment of anxiety and depression-like symptoms. The behavioral and neurological effects of modulating inflammatory factors through gene knockdown (KD) and knockout (KO), and conventional and alternative medicine approaches, are discussed.

Voluntary wheel running promotes resilience to the behavioral effects of unpredictable chronic mild stress in male and female mice.

Besides significant benefits to physical health, exercise promotes mental health, reduces symptoms of mental illness, and enhances psychological development. Exercise can offset the impact of chronic stress, which is a major precursor to the development of mental disorders. The effects of exercise on chronic stress-induced behaviors are contradictory in preclinical studies, primarily due to the lack of data and sex-specific investigations. We sought to evaluate the effects of exercise on chronic stress-induced behavioral changes in both male and female mice. Mice were subjected to an Unpredictable Chronic Mild Stress (UCMS) paradigm with accessibility to running wheels for 2 h daily. Physiological and behavioral evaluations were conducted throughout the stress paradigm to determine if exercise blunts the effects of UCMS. Chronic stress induced voluntary wheel running (VWR) and weight loss in male and female mice. Compared to males, increased VWR was reported in females who also regained their weight lost by the end of the UCMS protocol. Exercise promoted resilience to stress-induced hyponeophagia in the novelty-suppressed feeding test and increased sucrose consumption. Exercise induced a sex-specific reduction in immobility and avoidance behavior in the tail suspension and open field tests and increased exploratory behavior in the light-dark test. These results indicate that exercise can promote resilience to the behavioral effects of chronic stress in males and females, and can affect behavior independent of chronic stress.

Adolescent oxycodone exposure inhibits withdrawal-induced expression of genes associated with the dopamine transmission.

Prescription opioid misuse is a major public health concern among children and adolescents in the United States. Opioids are the most commonly abused drugs and are the fastest growing drug problem among adolescents. In humans and animals, adolescence is a particularly sensitive period associated with an increased response to drugs of abuse. Our previous studies indicate that oxycodone exposure during adolescence increases morphine reward in adulthood. How early drug exposure mediates long-term changes in the brain and behavior is not known, but epigenetic regulation is a likely mechanism. To address this question, we exposed mice to oxycodone or saline during adolescence and examined epigenetic modifications at genes associated with dopamine activity during adulthood at early and late withdrawal, in the ventral tegmental area (VTA). We then compared these with alterations in the VTA of adult-treated mice following an equivalent duration of exposure and withdrawal to determine if the effects of oxycodone are age dependent. We observed persistence of adolescent-like gene expression following adolescent oxycodone exposure relative to age-matched saline exposed controls, although dopamine-related gene expression was transiently activated at 1 day of withdrawal. Following prolonged withdrawal enrichment of the repressive histone mark, H3K27me3, was maintained, consistent with inhibition of gene regulation following adolescent exposure. By contrast, mice exposed to oxycodone as adults showed loss of the repressive mark and increased gene expression following 28 days of withdrawal following oxycodone exposure. Together, our findings provide evidence that adolescent oxycodone exposure has long-term epigenetic consequences in VTA of the developing brain.

CREB deletion increases resilience to stress and downregulates inflammatory gene expression in the hippocampus.

The transcription factor CREB (cyclic AMP response element (CRE)-binding protein) is implicated in the pathophysiology and treatment of depression. Structural and functional studies in both animals and humans suggest that abnormalities of the hippocampus may play a role in depression. CREB regulates thousands of genes, yet to date, only a handful that mediate depression or antidepressant response have been identified as relevant CREB targets. In order to comprehensively identify genes regulated by CREB in the hippocampus, we employed translating ribosome affinity purification (TRAP) to detect actively translating mRNAs in wild type and CREB-deficient mice. Using CrebloxP/loxP; RosaLSL-GFP-L10a mice, we conducted whole genome sequencing to identify transcripts only in cells that lack CREB, as introduction of Cre-recombinase simultaneously deleted CREB and expressed GFP-tagged L10a ribosomes that enabled TRAP. We identified over 200 downregulated genes predominantly associated with inflammation and the immune system, including toll-like receptor 1 (TLR1). To determine if baseline disruption in gene expression in the hippocampus of CREB-deficient mice can modulate behavior, we used unpredictable chronic mild stress (UCMS) to produce a set of behavioral alterations with strong validity for depression. We found that CREB-deficient mice demonstrated resilience to the physiological effects of UCMS and also showed changes in affective behaviors specifically in the presence of stress. TLR1 expression was increased following UCMS in control but not in CREB-deficient mice. The results suggest that CREB-mediated regulation of immune system and inflammatory factors may provide additional targets for the treatment of depression.

STIMs and Orai1 regulate cytokine production in spinal astrocytes.

BACKGROUND: Our previous study demonstrated that a store-operated calcium channel (SOCC) inhibitor (YM-58483) has central analgesic effects. However, the cellular and molecular mechanisms of such effects remain to be determined. It is well-known that glial cells play important roles in central sensitization. SOC entry (SOCE) has been implicated in many cell types including cortical astrocytes. However, the role of the SOCC family in the function of astrocytes has not been determined. Here, we thoroughly investigated the expression and the functional significance of SOCCs in spinal astrocytes. METHODS: Primary cultured astrocytes were prepared from neonatal (P2-P3) CD1 mice. Expressions of mRNAs and proteins were respectively assessed by real-time PCR and Western blot analysis. SOCE was measured using a calcium imaging system. Live-cell STIM1 translocation was detected using a confocal microscope. Cytokine levels were measured by the enzyme-linked immunosorbent assay. RESULTS: We found that the SOCC family is expressed in spinal astrocytes and that depletion of calcium stores from the endoplasmic reticulum by cyclopiazonic acid (CPA) resulted in a large sustained calcium entry, which was blocked by SOCC inhibitors. Using the siRNA knockdown approach, we identified STIM1 and Orai1 as primary components of SOCCs in spinal astrocytes. We also observed thapsigargin (TG)- or CPA-induced puncta formation of STIM1 and Orai1. In addition, activation of SOCCs remarkably promoted TNF-α and IL-6 production in spinal astrocytes, which were greatly attenuated by knockdown of STIM1 or Orai1. Importantly, knockdown of STIM2 and Orai1 dramatically decreased lipopolysaccharide-induced TNF-α and IL-6 production without changing cell viability. CONCLUSIONS: This study presents the first evidence that STIM1, STIM2, and Orai1 mediate SOCE and are involved in cytokine production in spinal astrocytes. Our findings provide the basis for future assessment of SOCCs in pain and other central nervous system disorders associated with abnormal astrocyte activities.

Novel antifungal drug discovery based on targeting pathways regulating the fungus-conserved Upc2 transcription factor.

Infections by Candida albicans and related fungal pathogens pose a serious health problem for immunocompromised patients. Azole drugs, the most common agents used to combat infections, target the sterol biosynthetic pathway. Adaptation to azole therapy develops as drug-stressed cells compensate by upregulating several genes in the pathway, a process mediated in part by the Upc2 transcription factor. We have implemented a cell-based high-throughput screen to identify small-molecule inhibitors of Upc2-dependent induction of sterol gene expression in response to azole drug treatment. The assay is designed to identify not only Upc2 DNA binding inhibitors but also compounds impeding the activation of gene expression by Upc2. An AlphaScreen assay was developed to determine whether the compounds identified interact directly with Upc2 and inhibit DNA binding. Three compounds identified by the cell-based assay inhibited Upc2 protein level and UPC2-LacZ gene expression in response to a block in sterol biosynthesis. The compounds were growth inhibitory and attenuated antifungal-induced sterol gene expression in vivo. They did so by reducing the level of Upc2 protein and Upc2 DNA binding in the presence of drug. The mechanism by which the compounds restrict Upc2 DNA binding is not through a direct interaction, as demonstrated by a lack of DNA binding inhibitory activity using the AlphaScreen assay. Rather, they likely inhibit a novel pathway activating Upc2 in response to a block in sterol biosynthesis. We suggest that the compounds identified represent potential precursors for the synthesis of novel antifungal drugs.

Novel Pharmacological Approaches to the Treatment of Depression.

Major depressive disorder is one of the most prevalent mental health disorders. Monoamine-based antidepressants were the first drugs developed to treat major depressive disorder. More recently, ketamine and other analogues were introduced as fast-acting antidepressants. Unfortunately, currently available therapeutics are inadequate; lack of efficacy, adverse effects, and risks leave patients with limited treatment options. Efforts are now focused on understanding the etiology of depression and identifying novel targets for pharmacological treatment. In this review, we discuss promising novel pharmacological targets for the treatment of major depressive disorder. Targeting receptors including N-methyl-D-aspartate receptors, peroxisome proliferator-activated receptors, G-protein-coupled receptor 39, metabotropic glutamate receptors, galanin and opioid receptors has potential antidepressant effects. Compounds targeting biological processes: inflammation, the hypothalamic-pituitary-adrenal axis, the cholesterol biosynthesis pathway, and gut microbiota have also shown therapeutic potential. Additionally, natural products including plants, herbs, and fatty acids improved depressive symptoms and behaviors. In this review, a brief history of clinically available antidepressants will be provided, with a primary focus on novel pharmaceutical approaches with promising antidepressant effects in preclinical and clinical studies.

Depression-like phenotype following chronic CB1 receptor antagonism.

Rimonabant was the first clinically marketed cannabinoid (CB)(1) receptor antagonist developed to treat obesity. Unfortunately, CB(1) receptor antagonism produced adverse psychiatric events in patients. To determine whether this occurs pre-clinically, we investigated the effects of rimonabant in rodent models of mood disorders. Chronic treatment with rimonabant increased immobility time in the rat forced swim test and reduced the consumption of sucrose-sweetened water in an assay postulated to model anhedonia. These responses were similar to the effects elicited by chronic mild stress in these behavioral models, which, taken together, are indicative of a depression-like phenotype. Additionally, chronic treatment with rimonabant produced decreases in frontal cortex serotonin levels, marked reductions in hippocampal cell proliferation, survival, and BDNF levels, and elevations in the concentrations of pro-inflammatory cytokines including interferon gamma and TNF alpha. These preclinical findings mimic clinical reports and implicate possible mechanisms responsible for the unfavorable psychiatric events reported following chronic rimonabant use.

Genome-wide redistribution of MeCP2 in dorsal root ganglia after peripheral nerve injury.

BACKGROUND: Methyl-CpG-binding protein 2 (MeCP2), a protein with affinity for methylated cytosines, is crucial for neuronal development and function. MeCP2 regulates gene expression through activation, repression and chromatin remodeling. Mutations in MeCP2 cause Rett syndrome, and these patients display impaired nociception. We observed an increase in MeCP2 expression in mouse dorsal root ganglia (DRG) after peripheral nerve injury. The functional implication of increased MeCP2 is largely unknown. To identify regions of the genome bound by MeCP2 in the DRG and the changes induced by nerve injury, a chromatin immunoprecipitation of MeCP2 followed by sequencing (ChIP-seq) was performed 4 weeks after spared nerve injury (SNI). RESULTS: While the number of binding sites across the genome remained similar in the SNI model and sham control, SNI induced the redistribution of MeCP2 to transcriptionally relevant regions. To determine how differential binding of MeCP2 can affect gene expression in the DRG, we investigated mmu-miR-126, a microRNA locus that had enriched MeCP2 binding in the SNI model. Enriched MeCP2 binding to miR-126 locus after nerve injury repressed miR-126 expression, and this was not mediated by alterations in methylation pattern at the miR-126 locus. Downregulation of miR-126 resulted in the upregulation of its two target genes Dnmt1 and Vegfa in Neuro 2A cells and in SNI model compared to control. These target genes were significantly downregulated in Mecp2-null mice compared to wild-type littermates, indicating a regulatory role for MeCP2 in activating Dnmt1 and Vegfa expression. Intrathecal delivery of miR-126 was not sufficient to reverse nerve injury-induced mechanical and thermal hypersensitivity, but decreased Dnmt1 and Vegfa expression in the DRG. CONCLUSIONS: Our study shows a regulatory role for MeCP2 in that changes in global redistribution can result in direct and indirect modulation of gene expression in the DRG. Alterations in genome-wide binding of MeCP2 therefore provide a molecular basis for a better understanding of epigenetic regulation-induced molecular changes underlying nerve injury.

MicroRNAs downregulated in neuropathic pain regulate MeCP2 and BDNF related to pain sensitivity.

Nerve injury induces chronic pain and dysregulation of microRNAs in dorsal root ganglia (DRG). Several downregulated microRNAs are predicted to target Mecp2. MECP2 mutations cause Rett syndrome and these patients report decreased pain perception. We confirmed MeCP2 upregulation in DRG following nerve injury and repression of MeCP2 by miRNAs in vitro. MeCP2 regulates brain-derived neurotrophic factor (BDNF) and downregulation of MeCP2 by microRNAs decreased Bdnf in vitro. MeCP2 T158A mice exhibited reduced mechanical sensitivity and Mecp2-null and MeCP2 T158A mice have decreased Bdnf in DRG. MeCP2-mediated regulation of Bdnf in the DRG could contribute to altered pain sensitivity.

Effect of histone deacetylase inhibitor JNJ-26481585 in pain.

Recent studies have shown that histone deacetylase (HDAC) inhibitors can alleviate inflammatory and neuropathic pain. We investigated the effects of JNJ-26481585, a pan-HDAC inhibitor on basal mechanical sensitivity. Unlike previous reports for HDAC inhibitors, JNJ-26481585 induced mechanical hypersensitivity in mice. This effect was reversible with gabapentin. Voltage-dependent calcium channel subunit alpha-2/delta-1, one of the putative targets for gabapentin, was upregulated in the spinal cord from JNJ-26481585-treated mice. Transcriptional profiling of spinal cord from JNJ-26481585-treated mice showed significant alterations in pathways involved in axon guidance, suggesting overlap in mechanisms underlying neurotoxicity caused by other known chemotherapeutic agents. To investigate the mechanisms underlying the development of pain, RAW 264.7 mouse macrophage cells were treated with JNJ-26481585. There was a dose- and time-dependent activation of nuclear factor-kappaB and interleukin-1ß increase. Thus, alterations in the axon guidance pathway, increase in voltage-dependent calcium channel alpha(2)delta-1 subunit, and the induction of proinflammatory mediators by JNJ-26481585 could all contribute to increased mechanical sensitivity. Our data indicate that the effect of HDAC inhibitors may be unique to the compound studied and highlights the potential to develop chemotherapy-induced peripheral neuropathy with the use of a pan-HDAC inhibitor for cancer treatment, and this pain may be alleviated by gabapentin.

Purinergic receptor-mediated morphological changes in microglia are transient and independent from inflammatory cytokine release.

Microglia are commonly described as existing in resting or active states based on morphology or level of cytokine production. Extracellular ATP is a physiologically-relevant activator of microglia, which express a number of purinergic receptors. As P2Y(12) has been linked to chemotaxis, we used a panel of purinergic compounds to understand the role of ATP receptors in morphological transformation and correlate this with TNFalpha production. We quantified activation of cultured microglia with LPS or purinergic receptor agonists by using automated image analysis of cell morphology and CD11b expression and correlated this with TNFalpha release measured by ELISA. Treatment with both ATP and the P2Y(12) receptor agonist, 2-methylthio adenosine diphosphate (2MeSADP), caused a transient increase in CD11b expression (EC(50)=1.2 microM and 187 nM, respectively) and a reduction in process count that reversed within 90 min later. These changes were not accompanied by the release of TNFalpha. Forskolin, IBMX, and pertussis toxin inhibited these changes, but the PLC inhibitor, U73122, did not. 2MeSAMP blocked the ATP response, while AP4A blocked the 2MeSADP response, implicating P2Y(12/13). Microglia activation by LPS also caused an increase in CD11b expression and a reduction in process count; however, in contrast to activation by ATP, morphological transformation was accompanied by a concentration-dependent increase in TNFalpha secretion These data demonstrate that morphological transformation and TNFalpha release are separable events mediated by different, or non-convergent pathways and that although ATP can initiate morphological changes, additional factors are required to maintain activation over sustained periods.