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We demonstrate that even mild systemic inflammation shifts signaling mechanisms giving rise to BDNF-induced phrenic motor plasticity. This finding has important experimental, biological, and translational implications, particularly since BDNF-dependent spinal plasticity is being translated to restore breathing and nonrespiratory movements in diverse clinical disorders, such as spinal cord injury (SCI) and amyotrophic lateral sclerosis (ALS).

Systemic inflammation associated with a wide range of clinical disorders elicits neuroinflammation in the CNS, exacerbating neurodegeneration or traumatic/ischemic injuries. Neuroinflammation also impairs important forms of neuroplasticity associated with cognitive and motor learning. Several mechanisms propagate systemic inflammation into the CNS, although important details are not yet understood. Reports suggest propagation via vagal pro—inflammatory sensory afferent neurons, and/or cytokines crossing the blood-brain barrier. Microglia are the resident CNS immune cells, and are key contributors to CNS neuroinflammation, producing cytokines, chemokines, NO and ROS. Important contributions are also known from other CNS cells ( e.g. astrocytes, pericytes and neurons). Here, we investigated CNS responses to systemic inflammation induced by systemic LPS injection (1mg/kg, i.p.) in 3 months old Sprague-Dawley rats to test an early stage in the neuroinflammatory process. Three hours post-LPS injection, rats were perfused with PBS followed by microglia isolation from cortex and cervical spinal cord via immunomagnetic separation; the microglia-free fraction (neurons, astrocytes, pericytes, and other cells) was also collected for analysis. The efficacy of microglial isolation was verified by flow cytometry, and both microglial and non-microglial mRNAs were analyzed via qRT-PCR. CD200 and CX3CL1 (fractalkine) are two proteins expressed exclusively by neurons that mediate neuron-microglial communication by interacting with microglial receptors. Post-LPS, both CD200 and CX3CL1 increased 2.5 and 8 fold, respectively. Colony stimulating factor 1 (CSF-1), a protein produced by astrocytes and oligodendrocytes that is crucial for microglial survival, increased 5-fold. Surprisingly, inflammatory cytokines IL6, IL1β, CCL2, iNOS and TNFα were significantly elevated in non-microglial cells, but only IL6 and iNOS were increased in microglia 3 hours post-LPS. Thus, non-microglial cell responses to systemic inflammation appear to occur prior to full microglial activation. We hypothesize that peripheral pro-inflammatory signals propagate into CNS via pericytes and/or astrocytes that are in close contact with blood vessels. Further studies are needed to identify specific cellular sources of pro-inflammatory cytokines. Since CD200 prevents excessive inflammation in peripheral tissues, we also hypothesize that neuronal CD200 upregulation limits microglial immune responses, preventing inappropriate over-activation. Supported by: R01 HL149800 & HL147554 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PurposeThe Survey of the Health of Wisconsin (SHOW) was established in 2008 by the University of Wisconsin (UW) School of Medicine and Public Health (SMPH) with the goals of 1) providing a timely and accurate picture of the health of the state residents; and 2) serving as an agile resource infrastructure for ancillary studies. Today SHOW continues to serve as a vital population health research infrastructure.ParticipantsSHOW currently includes 5,846 adult and 980 minor participants recruited between 2008-2019 in four primary waves. WAVE I (2008-2013) includes annual statewide representative samples of 3,380 adults ages 21 to 74 years. WAVE II (2014-2016) is a triannual statewide sample of 1957 adults (age ≥18 years) and 645 children. WAVE III (2017) consists of follow-up of 725 adults from the WAVE I and baseline surveys of 222 children in selected households. WAVEs II and III include stool samples collected as part of an ancillary study in a subset of 784 individuals. WAVE IV consist of 517 adults and 113 children recruited from traditionally under-represented populations in biomedical research including African Americans and Hispanics in Milwaukee county, WI.Findings to DateThe SHOW provides extensive data to examine the intersectionality of multiple social determinants and population health. SHOW includes a large biorepository and extensive health data collected in a geographically diverse urban and rural population. Over 60 studies have been published covering a broad range of topics including, urban and rural disparities in cardio-metabolic disease and cancer, objective physical activity, sleep, green-space and mental health, transcriptomics, the gut microbiome, antibiotic resistance, air pollution, concentrated animal feeding operations and heavy metal exposures.Future PlansThe SHOW cohort is available for continued longitudinal follow-up and ancillary studies including genetic, multi-omic and translational environmental health, aging, microbiome and COVID-19 research.Article SummaryStrengths and limitationsThe Survey of the Health of Wisconsin (SHOW) is an infrastructure to advance population health sciences including biological sample collection and broader data on individual and neighborhood social and environmental determinants of health.The extensive data from diverse urban and rural populations offers a unique study sample to compare how socio-economic gradients shape health outcomes in different contexts.The objective health data supports novel interdisciplinary research initiatives and is especially suited for research in causes and consequences of environmental exposures (physical, chemical, social) across the life course on cardiometabolic health, immunity, and aging related conditions.The extensive biorepository supports novel omics research into common biological mechanisms underlying numerous complex chronic conditions including inflammation, oxidative stress, metabolomics, and epigenetic modulation.Ancillary studies, such as the Wisconsin Microbiome Study, have expanded the utility of the study to examine human susceptibility to environmental exposures and opportunities for investigations of the role of microbiome in health and disease.Long-standing partnerships and recent participation among traditionally under-represented populations in biomedical research offer numerous opportunities to support community-driven health equity work.No biological samples were collected among children.The statewide sampling frame may limit generalizability to other regions in the United States.

There is an urgent need to track the early and ongoing impact of the COVID-19 pandemic on population health from local to global scales. At the same time, there is an overall lack of U.S. state-specific surveillance data tracking social determinants of health (SDOH) and associations with population well-being, individual mitigation and coping strategies, family dynamics and other economic shocks of the pandemic in populations. Statewide data can offer important insights into how SDOH shape the long-term effects of COVID-19 in the population since implementation of many policies and programs varied widely early on in the pandemic. In May of 2020, the Survey of the Health of Wisconsin (SHOW) program launched a statewide online/phone survey of early and ongoing impacts of COVID-19 on health and well-being across diverse communities and families. The goal of this study is to provide descriptive data including perceived COVID-19 risks, access to and results of COVID-19 antigen testing, individual mitigation and coping strategies, family dynamics and other economic shocks of the pandemic on health and mental health in populations. Key findings include higher rates of testing and perceived past infection from COVID-19 among non-white respondents. Higher economic shifts and job changes in female vs male respondents. Families with children reported overall higher levels of stress, and stress from the pandemic. There were urban and rural differences in changes to access to care. Rural regions, which had a lower prevalence of infections early in the pandemic as compared to urban areas, also reported fewer delays or missed appointments due to COVID-19. Key findings show that SDOH are shaping impacts of health and well-being early on in the pandemic and future longitudinal follow-up will be important to shape policies and programs well into the future.

Antibody surveillance provides essential information for public health officials to work with communities to discuss the spread and impact of COVID-19. At the start of the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in the United States, diagnostic testing was limited with many asymptomatic and thus undetected cases. Irrespective of symptom severity, antibodies develop within two to three weeks after exposure and may persist 6 months or more.; Thus, antibody surveillance is an important tool for tracking trends in past infections across diverse populations. This study includes adults and children (≥12 years old) recruited from a statewide sample of past 2014-2020 Survey of the Health of Wisconsin (SHOW) participants. SHOW, an ongoing population-based health examination study including a randomly selected sample of households, partnered with the Wisconsin Department of Health Services and the Wisconsin State Laboratory of Hygiene to conduct longitudinal antibody surveillance using the Abbott Architect SARS-CoV-2 IgG antibody test, which detects antibodies against the nucleocapsid protein. Three WAVES of sample collection were completed in 2020-2021, tracking mid-summer, late fall, and early spring COVID-19 trends prior to vaccine availability. Crude estimates of seroprevalence in the total study population increased ten-fold from 1.4% during WAVE I to 11.5% in WAVE III. Within the statewide probability sample, weighted estimates increased from 1.6% (95% CI:0.6-2.5%), to 6.8% (95% CI:4.3-9.4%) in WAVE II and to 11.4% (95% CI:8.2, 14.6%) in WAVE III. Longitudinal trends in seroprevalence match statewide case counts. Local seroprevalence showed variation by state health region with increasing prevalence among higher income (>200% poverty income ratio), and rural health regions of the state seeing the highest increase in COVID-19 prevalence over time. Significant disparities in prevalence by racial and ethnic groups also exist, with greater than two times seroprevalence among Latino and black participants compared to non-Hispanic whites. This public health and academic partnership provides critical data for the ongoing pandemic response and lays the foundation for future research into longer-term immunity, health impacts and population-level disparities.

Background Obesity has been shown to alter response to air pollution and smoking but underlying biological mechanisms are largely unknown and few studies have explored mechanisms by which obesity increases human sensitivity to environmental exposures. Objective Overall study goals were to investigate whole blood gene expression in smokers and non-smokers to examine associations between cigarette smoke and changes in gene expression by obesity status and test for effect modification. Methods Relative fold-change in mRNA expression levels of 84 genes were analyzed using a Toxicity and Stress PCR array among 50 21–54 year old adults. Data on smoking status was confirmed using urinary cotinine levels. Adjusted models included age, gender, white blood cell count and body-mass index. Results Models comparing gene expression of smokers vs. non-smokers identified six differentially expressed genes associated with smoking after adjustments for covariates. Obesity was associated with 29 genes differentially expressed compared to non-obese. We also identified 9 genes with significant smoking/obesity interactions influencing mRNA levels in adjusted models comparing expression between smokers vs non-smokers for four DNA damage related genes (GADD45A, DDB2, RAD51 and P53), two oxidative stress genes (FTH1, TXN), two hypoxia response genes (BN1P3lL, ARNT), and one gene associated with unfolded protein response (ATF6B). Conclusions Findings suggest that obesity alters human sensitivity to smoke exposures through several biological pathways by modifying gene expression. Additional studies are needed to fully understand the clinical impact of these effects, but risk assessments should consider underlying phenotypes, such as obesity, that may modulate sensitivity of vulnerable populations to environmental exposures.

Low-protein diets promote metabolic health in rodents and humans, and the benefits of low-protein diets are recapitulated by specifically reducing dietary levels of the three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine. Here, we demonstrate that each BCAA has distinct metabolic effects. A low isoleucine diet reprograms liver and adipose metabolism, increasing hepatic insulin sensitivity and ketogenesis and increasing energy expenditure, activating the FGF21-UCP1 axis. Reducing valine induces similar but more modest metabolic effects, whereas these effects are absent with low leucine. Reducing isoleucine or valine rapidly restores metabolic health to diet-induced obese mice. Finally, we demonstrate that variation in dietary isoleucine levels helps explain body mass index differences in humans. Our results reveal isoleucine as a key regulator of metabolic health and the adverse metabolic response to dietary BCAAs and suggest reducing dietary isoleucine as a new approach to treating and preventing obesity and diabetes.

Whereas age increases microglial inflammatory activities and impairs their ability to effectively regulate their immune response, it is unclear at what age these exaggerated responses begin. We tested the hypotheses that augmented microglial responses to inflammatory challenge are present as early as middle age and that repeated stimulation of primed microglia in vivo would reveal microglial senescence. Microglial gene expression was investigated in a mouse model of repeated systemic inflammation induced by intraperitoneal injection of bacterial lipopolysaccharide (LPS). Following LPS, microglia from middle-aged mice (9–10 mo) displayed larger increases in Tnfα, Il-6, and Il-1β gene expression compared with young adults (2 mo). Similar results were observed in the spleens of middle-aged mice, indicating that exaggeration of both central and peripheral immune responses are already evident at early middle age. Interestingly, despite greater proinflammatory responses to the first LPS challenge in the aged mice, there were no age-dependent differences in either microglia or spleen following a subsequent LPS dose, suggesting that animals at this age retain the ability to effectively control their immune response following repeated challenge. The exacerbated microglial immune response to systemic inflammation at early middle age suggests that the CNS may be vulnerable to age-dependent alterations earlier than previously appreciated.

Background/Aim: Obesity may increase human sensitivity to environmental toxicants through multiple mechanisms including inflammation. Obesity has been shown to increase susceptibility of asthma sym...

Activation of microglia, CNS resident immune cells, is a pathological hallmark of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder affecting motor neurons. Despite evidence that microglia contribute to disease progression, the exact role of these cells in ALS pathology remains unknown. We immunomagnetically isolated microglia from different CNS regions of SOD1(G93A) rats at three different points in disease progression: presymptomatic, symptom onset and end-stage. We observed no differences in microglial number or phenotype in presymptomatic rats compared to wild-type controls. Although after disease onset there was no macrophage infiltration, there were significant increases in microglial numbers in the spinal cord, but not cortex. At disease end-stage, microglia were characterized by high expression of galectin-3, osteopontin and VEGF, and concomitant downregulated expression of TNFα, IL-6, BDNF and arginase-1. Flow cytometry revealed the presence of at least two phenotypically distinct microglial populations in the spinal cord. Immunohistochemistry showed that galectin-3/osteopontin positive microglia were restricted to the ventral horns of the spinal cord, regions with severe motor neuron degeneration. End-stage SOD1(G93A) microglia from the cortex, a less affected region, displayed similar gene expression profiles to microglia from wild-type rats, and displayed normal responses to systemic inflammation induced by LPS. On the other hand, end-stage SOD1(G93A) spinal microglia had blunted responses to systemic LPS suggesting that in addition to their phenotypic changes, they may also be functionally impaired. Thus, after disease onset, microglia acquired unique characteristics that do not conform to typical M1 (inflammatory) or M2 (anti-inflammatory) phenotypes. This transformation was observed only in the most affected CNS regions, suggesting that overexpression of mutated hSOD1 is not sufficient to trigger these changes in microglia. These novel observations suggest that microglial regional and phenotypic heterogeneity may be an important consideration when designing new therapeutic strategies targeting microglia and neuroinflammation in ALS.

Macrophage colony stimulating factor (CSF1) is a cytokine that is upregulated in several diseases of the central nervous system (CNS). To examine the effects of CSF1 overexpression on microglia, transgenic mice that overexpress CSF1 in the glial fibrillary acidic protein (GFAP) compartment were generated. CSF1 overexpressing mice have increased microglial proliferation and increased microglial numbers compared with controls. Treatment with PLX3397, a small molecule inhibitor of the CSF1 receptor CSF1R and related kinases, decreases microglial numbers by promoting microglial apoptosis in both CSF1 overexpressing and control mice. Microglia in CSF1 overexpressing mice exhibit gene expression profiles indicating that they are not basally M1 or M2 polarized, but they do have defects in inducing expression of certain genes in response to the inflammatory stimulus lipopolysaccharide. These results indicate that the CSF1 overexpression observed in CNS pathologies likely has pleiotropic influences on microglia. Furthermore, small molecule inhibition of CSF1R has the potential to reverse CSF1-driven microglial accumulation that is frequently observed in CNS pathologies, but can also promote apoptosis of normal microglia.

Although microglial activation is associated with all CNS disorders, many of which are sexually dimorphic or age-dependent, little is known about whether microglial basal gene expression is altered with age in the healthy CNS or whether it is sex dependent. Analysis of microglia from the brains of 3-day (P3)- to 12-month-old male and female C57Bl/6 mice revealed distinct gene expression profiles during postnatal development that differ significantly from those in adulthood. Microglia at P3 are characterized by relatively high iNOS, TNFα and arginase-I mRNA levels, whereas P21 microglia have increased expression of CD11b, TLR4, and FcRγI. Adult microglia (2-4 months) are characterized by low proinflammatory cytokine expression, which increases by 12 months of age. Age-dependent differences in gene expression suggest that microglia likely undergo phenotypic changes during ontogenesis, although in the healthy brain they did not express exclusively either M1 or M2 phenotypic markers at any time. Interestingly, microglia were sexually dimorphic only at P3, when females had higher expression of inflammatory cytokines than males, although there were no sex differences in estrogen receptor expression at this or any other time evaluated here. Compared with microglia in vivo, primary microglia prepared from P3 mice had considerably altered gene expression, with higher levels of TNFα, CD11b, arginase-I, and VEGF, suggesting that culturing may significantly alter microglial properties. In conclusion, age- and sex-specific variances in basal gene expression may allow differential microglial responses to the same stimulus at different ages, perhaps contributing to altered CNS vulnerabilities and/or disease courses.

Neuroinflammation mediated by microglia is a pathological hallmark of many CNS disorders. Cell lines derived from inbred C57Bl/6 and outbred ICR/CD1 mice (BV2 and N9 respectively), are often used to study microglial inflammatory activities. Although many studies demonstrate different responses of these cell lines to the same stimulus, no comparisons have been done in vivo. Because inbreeding reduces resistance to pathogens and parasites, we hypothesized that microglia from outbred ICR/CD1 mice would have a stronger response to centrally administered LPS than microglia from inbred C57Bl/6 mice. The evaluation of gene expression in freshly-isolated CD11b+ cells from brain revealed that microglia from ICR/CD1 mice were more pro-inflammatory than those from C57Bl/6 mice, although these differences did not appear to result from alterations in the expression levels of the LPS receptors TLR4 or CD14. Notably, the timing of inflammatory gene expression did not correlate with CD11b+ cell proliferation/infiltration. The highest expression of TNFα, IL-6 and iNOS occurred 3 hours after LPS injection when the number of CD11b+ cells was not changed. Whereas the expression of these pro-inflammatory genes had returned to basal by 48 hours when the highest number of CD11b+ cells in the brain was found, the expression of the anti-inflammatory cytokine IL-10 was still significantly up-regulated. This is important because the increased presence of CD11b+ cells in the CNS is often used as an indicator of neuroinflammation. While LPS did not affect the expression of the growth factors VEGF or BDNF, we observed that mechanical injury (caused by intraparenchymal injection) induced distinct patterns of microglial activation characterized by increased expression of VEGF and down-regulation of BDNF. It remains to be determined which type of microglia is more beneficial/detrimental to the CNS, but our data suggest that genetic traits determining microglial properties may have profound effect on many CNS pathologies.

We investigated the anti-inflammatory effects of minocycline in EAE, an animal model of MS. Minocycline, administered for two weeks after the clinical onset, significantly decreased the cumulative and mean clinical scores of EAE. This was associated with the reduction of both CD4(+) and CD8(+) T cell numbers in the spinal cord and the downregulation of LFA-1 on T cells without affecting the cytokine production profile. The predominant cytokine produced by T cells in the spleen was IFN-gamma whereas in the CNS it was IL-17. Our results indicate that minocycline regulates T cell infiltration into the CNS without modifying the dominant cytokine production.

Posttranscriptional mechanisms play an important role regulating pituitary levels of vasopressin V1b receptors (V1bR) during adaptation to stress. This study investigates the involvement of an internal ribosome entry site (IRES) in the 5'untranslated region (5'UTR) on V1bR translation. Transfection of bicistronic luciferase constructs into MCF-7 cells showed marked increases in translation of the second cistron after insertion of a 499-bp fragment of the V1bR 5'UTR in the intercistronic region, independently of cap-mediated translation, indicating the presence of IRES activity. IRES-mediated translation was potentiated by the protein kinase C activators, 12-O-tetradecanoylphorbol 13-acetate (PMA) and bryostatin 1, and appears to involve phosphorylation of amino terminus of eIF4G. In Chinese hamster ovary cells transfected with pV1bR-green fluorescent protein (pV1bR-GFP), PMA increased V1bR-GFP protein levels when cap-mediated translation was inhibited by rapamycin. The effect of PMA was due to increased translation because it persisted under transcriptional blockade by actinomycin D, and it was completely abolished by cycloheximide. In addition, PMA stimulated [35S]methionine incorporation into V1bR-GFP but not beta-actin in the absence of mRNA changes. The data show that regulation of IRES activity in the 5'UTR of the V1bR mRNA probably through phosphorylation of eIF4G may serve as a mechanism for rapid changes in V1bR translation to meet physiological demands.

Corticotropin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH-expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus-mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH-like immunoreactivity (CRH-LI) and expressed CRH, VP and CRH1 receptor RNA via the reverse transcriptase-polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule-associated protein-2. We verified that the CRH-LI from IVB cell lysates coeluted with CRH standard via reversed-phase high-performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC profile to that identical with oxidized CRH standard. In addition, IVB cells exhibited high affinity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration-dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho-cyclic-AMP response element binding protein (CREB) immunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB-mediated gene expression as determined via the PathDetect CREB trans-reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH-R1-mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.

The regulation of CRH promoter activity by cAMP was studied in two cell lines, the pituitary corticotroph cell line AtT-20 and the immortalized hypothalamic cell line 4B, which expresses CRH and vasopressin. In 4B cells transfected with a CRH promoter-luciferase construct, the adenylyl cyclase stimulator, forskolin, increased luciferase activity in parallel with increases in intracellular cAMP. In 4B cells, however, the phosphodiesterase inhibitor, isobutylmethylxanthine, potentiated forskolin-stimulated cAMP without affecting further increases in luciferase activity. In AtT-20 cells, forskolin plus isobutylmethylxanthine elevated cAMP only slightly, but increased luciferase activity to levels similar to those observed in 4B cells. AtT-20 cells were also unresponsive to 8-bromo-cAMP, due in part to higher phosphodiesterase (PDE) activities. Although both cells contained PDE1, -3, and -4, inhibition of either PDE4 or PDE1 potentiated luciferase activity stimulated by submaximal forskolin concentrations in 4B cells, while only simultaneous inhibition of PDE3 and PDE4 was effective in AtT-20 cells. The data show that minor elevations in intracellular cAMP are sufficient for full stimulation of CRH promoter activity regardless of the cell line. Furthermore, poor CRH promoter activation in AtT-20 cells appears to result from deficient cAMP production and rapid cAMP degradation by PDE.

During postnatal development, microglia, CNS resident innate immune cells, are essential for synaptic pruning, neuronal apoptosis and remodeling. During this period microglia undergo morphological and phenotypic transformations; however, little is known about how microglial number and density is regulated during postnatal CNS development. We found that after an initial increase during the first 14 postnatal days, microglial numbers in mouse brain began declining in the third postnatal week and were reduced by 50% by 6 weeks of age; these “adult” levels were maintained until at least 9 months of age. Microglial CD11b levels increased, whereas CD45 and ER-MP58 declined between P10 and adulthood, consistent with a maturing microglial phenotype. Our data indicate that both increased microglial apoptosis and a decreased proliferative capacity contribute to the developmental reduction in microglial numbers. We found no correlation between developmental reductions in microglial numbers and brain mRNA levels of Cd200, Cx3Cl1, M-Csf or Il-34. We tested the ability of M-Csf-overexpression, a key growth factor promoting microglial proliferation and survival, to prevent microglial loss in the third postnatal week. Mice overexpressing M-Csf in astrocytes had higher numbers of microglia at all ages tested. However, the developmental decline in microglial numbers still occurred, suggesting that chronically elevated M-CSF is unable to overcome the developmental decrease in microglial numbers. Whereas the identity of the factor(s) regulating microglial number and density during development remains to be determined, it is likely that microglia respond to a “maturation” signal since the reduction in microglial numbers coincides with CNS maturation.

Inflammation is a hallmark of spinal cord injury (SCI), mediated in part by CD11b+ resident microglia (MG) and infiltrating macrophages (MP). Although neuroinflammation is well-characterized at the...

Increased microglial numbers and activated microglial phenotypes are associated with many central nervous system (CNS) disorders including gliomas, neurodegeneration and brain injury. Thus, microglia are being tested as potential drug targets. Macrophage Colony Stimulating Factor (CSF1), an important cytokine for microglia and macrophages, is upregulated in these CNS disorders, but the effects of CSF1 on microglial phenotypes have not been well-characterized in vivo. To study the effects of CSF1 over-expression on microglia in the unperturbed CNS in vivo, we generated a transgenic mouse model in which CSF1 is inducibly over-expressed in the GFAP compartment. We found that CSF1 over-expression results in increased microglial proliferation, increased microglial numbers and an altered microglial phenotype compared to control mice. Microglia in CSF1 over-expressing mice do not exhibit gene expression profiles typical of either M1 or M2 polarization, however they are defective in their response to the inflamma...

The influence of an upstream open reading frame (ORF) in the 5'-untranslated region (UTR) of the mRNA on corticotropin-releasing hormone receptor type 1 (CRHR1) translation was studied in constructs containing the 5'-UTR of CRHR1, with or without an ATG-to-ATA mutation in the upstream ORF, and the main ORF of luciferase or CRHR1. Upstream mutation in luciferase constructs increased luciferase activity when transfected into COS-7 or AtT20 cells compared with the native 5'-UTR. Transfection of CRHR1 constructs containing the upstream mutation into AtT20 or LVIP2.0zc reporter cells, resulted in higher (125)I-Tyr-oCRH binding and corticotropin-releasing hormone-stimulated cAMP production, without changes in CRHR1 mRNA levels (measured by RNase protection assay). In vitro translation of luciferase or CRHR constructs with or without mutation of the upstream ATG, and Western blot analysis with anti-luciferase and anti-CRHR1 antibodies confirmed that mutation of the upstream ATG increases translation of the main ORF. The mechanism by which the upstream ORF inhibits translation may involve translation of the upstream peptide, because in vitro translation, or transfection into LVIP2.0zc cells of a fusion construct of the upstream ORF and green fluorescent protein (GFP) yielded a band consistent with the molecular size of GFP protein. The study shows that the upstream AUG in 5'-UTR of CRHR1 mRNA inhibits receptor expression by inhibiting mRNA translation and suggests the short open reading frame in the 5'-UTR plays a role in regulating translation of the CRH receptor.

Summary Ethanol exerts profound effects on the endocrine and exocrine pancreas. Some effects of chronic alcohol consumption on insulin secretion in response to glucose load are similar to those of TRH gene disruption. TRH is present in insulin-producing B-cells of the islets of Langerhans; its role in this location is still not fully explored. To examine the possible effect of long-term in vivo ethanol treatment on pancreatic TRH we compared three groups of rats: a 10% (wt:vol) ethanol-drinking group (E), absolute controls (AC) and pair-fed (PF) group with solid food intake corresponding to that of E. The fluidity of pancreatic membranes was not affected by chronic in vivo exposure of rats to ethanol, but was significantly decreased in PF group. Four-week treatment resulted in significantly higher TRH content in isolated islets of the E group and increased basal and 80 mM isotonic ethanol-induced secretion compared to AC and PF. Plasma levels of insulin, C-peptide, IGF-I, and glycemia were, however, not affected by ethanol treatment. Cell swelling, which can be induced by the presence of permeants (e.g. ethanol) in an isotonic extracellular medium, is a strong stimulus for secretion in various types of cells. In the present study, isosmotic ethanol (40, 80, and 160 mM) induced dose-dependent release of TRH and insulin from adult rat pancreatic islets in vitro. The same concentrations were not effective when applied in a hyperosmotic medium (addition of ethanol directly to the medium), thus indicating the participation of cell swelling in the ethanol-induced secretion. In conclusion, chronic ethanol treatment significantly affected pancreatic TRH and this effect might be mediated by cell swelling. The role of these changes in the profound effect of ethanol on the endocrine and exocrine pancreas remains to be established.

Macrophage colony stimulating factor (CSF1) is a cytokine that is upregulated in several diseases of the central nervous system (CNS). To examine the effects of CSF1 over-expression on microglia, transgenic mice that over-express CSF1 in the glial fibrillary acidic protein (GFAP) compartment were generated. CSF1 over-expressing mice have increased microglial proliferation and increased microglial numbers compared to controls. Treatment with PLX3397, a small molecule inhibitor of the CSF1 receptor CSF1R and related kinases, decreases microglial numbers by promoting microglial apoptosis in both CSF1 over-expressing and control mice. Microglia in CSF1 over-expressing mice exhibit gene expression profiles indicating that they are not basally M1 or M2 polarized, but they do have defects in inducing expression of certain genes in response to the inflammatory stimulus lipopolysaccharide (LPS). These results indicate that the CSF1 over-expression observed in CNS pathologies likely has pleiotropic influences on microglia. Furthermore, small molecule inhibition of CSF1R has the potential to reverse CSF1-driven microglial accumulation that is frequently observed in CNS pathologies, but can also promote apoptosis of normal microglia.

TRH is present throughout the central nervous system possessing many different functions. Only TRH synthesized in the hypothalamic PVN and transported to the ME regulates anterior pituitary secretion of thyrotropin. To investigate this hypophysiotropic system, we have developed a method using dissected rat PVN and ME, representing mostly cell bodies and nerve terminals respectively, of the same TRH system. Tissues were incubated for four 30 min periods each in Locke's medium with alternatively normal and high KCl concentration. Repeated KCl-induced membrane depolarization resulted in significantly increased TRH release from both tissues (15% of TRH content) which was dependent on Ca2+ influx. Some important differences were found in the regulation of TRH in the PVN and ME: Comparison of TRH content in the tissue before and after incubation with the amount of secreted TRH showed new production of TRH during incubation in the ME, but not in the PVN. Frequent medium replacement during depolarization revealed that TRH secretion at the level of the ME, but not the PVN, is probably inhibited by some substance released during incubation. These data suggest that there is a different regulation of TRH in the PVN and ME and that TRH secreted by the isolated PVN may come mostly from the perikarya and represent paracrine neurohormone secretion.

Microglial hyperactivity contributes to neuronal damage resulting from CNS injury and disease. Therefore, a better understanding of endogenous microglial receptor systems that can be exploited to modulate their inflammatory functions is important if better, neuroprotective therapeutics are to be designed. Previous studies from our lab and others have demonstrated that the P2X7 purinergic receptor agonist BzATP attenuates microglial inflammatory mediator production stimulated by lipopolysaccharide (LPS), suggesting that purinergic receptors may be one such receptor system that can be used for manipulating microglial activation. However, although P2X7 receptor activation is well recognized to regulate processing and release of cytokines, little is known concerning its role in regulating the transcription of inflammatory genes, nor the molecular mechanisms underlying these transcriptional effects. In the present studies, we identify that the transcription factors early growth response (Egr)-1, -2 and -3 are downstream signaling targets of P2X7 receptors in microglia, and that their activation is sensitive to MEK and p38 mitogen-activated protein kinase (MAPK) inhibitors. Moreover, using RNAi, we demonstrate that Egr factors and P2X7 receptors are necessary for BzATP-mediated attenuation of iNOS, and stimulation of TNF-α and IL-6 gene expression. BzATP also attenuates neuronal death induced by LPS conditioned medium, and P2X7 receptors are required for this effect. These studies are the first to identify Egr factors as regulators of inflammatory gene expression following P2X7 receptor activation, and suggest that P2X7 receptors may utilize the MAPK-Egr pathway to exert differential effects on microglial inflammatory activities which are beneficial to neuron survival.

Microglia are implicated in multiple neurodegenerative disorders, many of which display sexual dimorphisms and have symptom onsets at different ages. P2 purinergic receptors are critical for regulating various microglial functions, but little is known about how their expression varies with age or sex. Therefore, comprehensive information about purinergic receptor expression in normal microglia, in both sexes, over age is necessary if we are to better understand their roles in the healthy and diseased CNS. We analyzed the expression of all fourteen rodent P2X and P2Y receptors in CD11b+ cells freshly-isolated from the brains of C57Bl/6 mice at five different ages ranging from postnatal day 3 to 12 months, in males and females, using quantitative RT-PCR. We also compared purinergic receptor expression in microglia freshly-isolated from 3 day-old pups to that in primary neonatal microglial cultures created from mice of the same age. We observed patterns in P2 receptor expression with age, most notably increased expression with age and age-restricted expression. There were also several receptors that showed sexually dimorphic expression. Lastly, we noted that in vitro culturing of neonatal microglia greatly changed their P2 receptor expression profiles. These data represent the first complete and systematic report of changes in purinergic receptor expression of microglia with age and sex, and provide important information necessary for accurate in vitro modeling of healthy animals.

Multiple sclerosis is an immune-mediated demyelinating disease, with axonal loss underlying long-term progressive disability. In this study, we have analyzed axonal and myelin pathology in a chronic relapsing-remitting experimental autoimmune encephalomyelitis model in Biozzi ABH mice induced by immunization with a syngeneic spinal cord homogenate. The animals were followed for3 months; inflammation, T-cell infiltration, demyelination, and axonal loss were examined at various time points throughout the disease course. We found that macrophage infiltration and microglia activation preceded detectable T-cell infiltration. Axonal loss was first evident at the acute phase of disease before demyelination was detected. Demyelination and axonal loss occurred after each relapse and correlated with increasing residual motor deficits in remission. The resulting lesions displayed evidence of demyelination, remyelination, axonal degeneration, and axon loss. After a series of 3 relapses, animals entered a chronic progressive phase with permanent paralysis and a relative absence of inflammation. Axonal loss continued in this phase, although demyelinated axons persisted. These findings indicate that experimental autoimmune encephalomyelitis in Biozzi ABH mice has important similarities to multiple sclerosis with a relapsing-remitting disease course followed by a secondary progressive phase; it is thus a suitable model in which to explore remyelination and neuroprotective therapies for multiple sclerosis.

Minocycline, a tetracycline derivative with pleiotropic biological effects, exhibits anti-inflammatory properties in several models of CNS disease. In addition to reducing production of inflammatory mediators, it has been postulated that minocycline might also be directly neuroprotective under these circumstances. Therefore, we investigated the effect of minocycline on primary cortical neuronal cultures exposed to a nitric oxide (NO)-donor. Cultures were assessed for neuronal survival, axon survival and markers of intracellular signaling pathways. The NO donor significantly increased neuronal death and minocycline was protective under these conditions. Furthermore NO-induced reductions in axonal length were significantly attenuated by minocycline. Improvements in axonal length were dependent on mitogen-activated protein kinase (MAP kinase)/extracellular signal-related kinase (Erk) signaling, whereas phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling was important in neuronal survival. Further investigation into MAP kinase signaling pathways revealed inhibition of p38 MAP kinase and c-jun N-terminal kinase (JNK) signaling by minocycline. JNK pathways were activated by trophic factor-withdrawal and minocycline attenuated neuronal death induced by trophic withdrawal. These results indicate that, in addition to anti-inflammatory properties, minocycline has direct protective effects on neurons and provides further evidence for its use in disorders of the CNS.

Experimental allergic encephalomyelitis, an autoimmune disorder mediated by T cells, results in demyelination, inflammation, and axonal loss in the central nervous system (CNS). Microglia play a critical role in major histocompatibility complex class II (MHC II)-dependent antigen presentation and in reactivation of CNS-infiltrated encephalitogenic T cells. Minocycline, a tetracycline anti-biotic, has profound anti-inflammatory properties and is experimentally used for treatment of many CNS disorders; however, the mechanisms involved in minocycline effects remain unknown. We show that administration of minocycline for 2 weeks ameliorated clinical severity of experimental allergic encephalomyelitis, an effect that partially involves the down-regulation of MHC II proteins in the spinal cord. Therefore, we sought to elucidate the molecular mechanisms of minocycline inhibitory effects on MHC II expression in microglia. Although complex, the co-activator class II transactivator (CIITA) is a key regulator of MHC II expression. Here we show that minocycline inhibited interferongamma (IFNgamma)-induced CIITA and MHC II mRNA. Interestingly, however, it was without effect on STAT1 phosphorylation or IRF-1 expression, transcription factors that are activated by IFNgamma and necessary for CIITA expression. Further experiments revealed that MHC II expression is down-regulated in the presence of the PKC(alpha) inhibitor Gö6976. Minocycline inhibited IFNgamma-induced PKC(alpha/betaII) phosphorylation and the nuclear translocation of both PKC(alpha/betaII) and IRF-1 that subsequently inhibits CIITA expression. Our present data delineate a molecular pathway of minocycline action that includes inhibitory effects on PKC(alpha/betaII) and transcription factors that regulate the expression of critical inflammatory genes such as MHC II. Such a fundamental mechanism may underlie the pleiotropic effects of minocycline in CNS inflammatory disorders.

CNS inflammation mediated by microglial activation can result in neuronal and glial cell death in a variety of neurodegenerative and demyelinating diseases. Minocycline, a second-generation tetracycline, has profound anti-inflammatory properties in the CNS mediated, in part, by inhibition of microglia. MAPK and nuclear factor-kappaB (NF-kappaB) activation are hallmarks of activated microglia and they are critical for the expression of many inflammatory mediators. In the present study, we investigated minocycline effects on activation of p38, c-Jun-N-terminal activated protein kinase (JNK) 1/2 and extracellular signal regulated kinase (ERK) 1/2 MAPKs and inhibitor alpha of NF-kappaB (IkappaBalpha) degradation in BV-2 and primary microglial cells. Our results demonstrate that minocycline has the ability to inhibit all MAPKs but these effects strongly depend on the stimulus used for MAPK activation. Minocycline significantly decreased activation of all lipopolysaccharide-stimulated MAPKs but it was without effect on MAPKs activated by H2O2. Minocycline inhibited JNK1/2 and ERK1/2 but not p38 when stimulated by 2',3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate, indicating that minocycline affects only certain upstream signaling target(s) that are stimulus-specific. Our data also suggest that protein kinase C (PKC) inhibition may be partially involved in the minocycline mechanism of MAPK inhibition. In addition, minocycline attenuated lipopolysaccharide-stimulated degradation of IkappaBalpha implying a possible inhibitory role on NF-kappaB transcriptional activity.

ATP is abundant in the extracellular fluid following brain injury, and it exerts potent modulatory effects on microglia, whose hyperactivation is thought to exacerbate neuronal damage. We show here that ATP decreases LPS-stimulated iNOS and COX-2 expression and reduces NO release in BV-2 microglia by a mechanism involving p38 MAP kinase. Further, we demonstrate that the inhibitory effects of ATP on NO production occur within 30 min of exposure and correlate with activation of the transcription factor CREB. Together, these data suggest that ATP may exert neuroprotective effects in the brain via a mechanism involving augmented activation of the p38/CREB pathway.

Hypothalamic corticotropin releasing hormone (CRH) regulates pituitary ACTH secretion and mediates behavioral and autonomic responses to stress, through interaction with type 1 plasma membrane receptors (CRHR1) located in pituitary corticotrophs and the brain. Although the CHRI are essential for ACTH responses to stress, their number in the pituitary gland does not correlate with corticotroph responsiveness, suggesting that activation of a small number of receptors is sufficient for maximum ACTH production. CRH binding and hybridization studies in adrenalectomized, glucocorticoid-treated or stressed rats revealed divergent changes in CRH receptors and CRH1 mRNA in the pituitary, with a reduction in receptor binding but normal or elevated expression of CHR1 mRNA levels. Western blot analysis of CRHR1 protein in pituitary membranes from adrenalectomized rats showed unchanged receptor mRNA levels and increased CRHR1 protein, despite binding down-regulation, suggesting that decreased binding is due to homologous desensitization, rather than reduced receptor synthesis. In contrast, decreased CRH binding following glucocorticoid administration is associated with a reduction in CRHR1 protein, suggesting inhibition of CRH1 mRNA translation. The regulation of CRHR1 translation may involve binding of cytosolic proteins, and a minicistron in the 5'-UTR of the CRHR1 mRNA. It is likely that post-transcriptional regulatory mechanisms that permit rapid changes in CRH receptor activity are important for adaptation of corticotroph responsiveness to continuous changes in physiological demands.

Corticotropin releasing hormone (CRH) stimulates pituitary ACTH secretion through type-1 CRH (CRH1) receptors. Stimulation of the hypothalamic pituitary adrenal (HPA) axis as well as increased corticotroph responsiveness during stress and adrenalectomy are associated with marked pituitary CRH binding downregulation. The presence of CRH1 receptors in the pituitary are essential to maintain ACTH secretion. Downregulation of CRH binding is associated with normal or elevated levels of CRH1 receptor mRNA and this may contribute to the maintainence of permissive levels of CRH1 receptors in the pituitary. Injection of either CRH or glucocorticoids in rats in vivo induces CRH binding and CRH1 receptor mRNA downregulation, whereas their simultaneous administration causes only transient CRH1 receptor mRNA loss. Vasopressin increases CRH1 receptor mRNA levels. This suggest that interactions between CRH, vasopressin and glucocorticoids accounts for CRH1 receptor mRNA upregulation during stress. The lack of correlation between CRH binding and CRH1 receptor mRNA indicates that the major sites for pituitary CRH1 receptor regulation are at the post-transcriptional level.

The hypothalamic paraventricular nucleus, representing cell bodies in which thyrotropin-releasing hormone is synthesized, and the median eminence, representing nerve terminals, were incubated in vitro . Various hypo- and hyperosmotic solutions were tested to determine osmotic sensitivity of thyrotropin-releasing hormone secretion. High KCl (56 mM) causing membrane depolarization was used as a non-specific control stimulus to induce thyrotropin-releasing hormone secretion. A 30% decrease of medium osmolarity (from 288 to 202 mOsmol/l) increased thyrotropin-releasing hormone secretion from both the paraventricular nucleus and median eminence. A 30% decrease of medium NaCl content by its replacement with choline chloride did not affect basal thyrotropin-releasing hormone secretion. Increasing medium osmolarity with biologically inactive l -glucose did not affect basal or KCl-induced thyrotropin-releasing hormone secretion from either structure. Medium made hyperosmotic (350–450 mOsmol/l) by increasing the NaCl concentration resulted in a dose-dependent decrease of basal thyrotropin-releasing hormone secretion and abolished KCl-induced thyrotropin-releasing hormone secretion. If an osmotically equivalent amount of choline chloride was substituted for NaCl, there was no effect on thyrotropin-releasing hormone secretion, indicating a specific action of Na + . This study indicates a specific sensitivity to high concentrations of Na + ions of both thyrotropin-releasing hormone-producing parvocellular paraventricular neurons and of thyrotropin-releasing hormone-containing nerve terminals in the median eminence.

Because chronic ethanol ingestion may perturb thyroid function, we evaluated the effect of 4-wk of oral 10% ethanol ingestion on the hypothalamic-pituitary-thyroid (HPT) axis and septal thyrotropin-releasing hormone (TRH) in 200-g male Wistar rats. Animals were divided into three groups: absolute control receiving tap water and food ad libitum; ethanol group receiving food ad libitum and 10% ethanol as the sole source of drinking fluid; pair-fed group receiving tap water and an amount of food corresponding to the consumption of ethanol group. After 4-wk of treatment, the body weight of the ethanol group was 7% and of the pair-fed rats 19% lower than that of the absolute controls. Both chronic ethanol treatment and food deprivation produced a decrease in plasma thyroid-stimulating hormone (TSH). Pair-fed rats also had a lower plasma T3. Type I iodothyronine 5′-deiodinase activity in the liver was increased in the pair-fed and even more in the ethanol-treated group. The content and secretion in vitro of TRH from the hypothalamic paraventricular nucleus and median eminence were unchanged. TRH content in the septum was increased in both the ethanol and pair-fed groups. TRH secretion from the septum in vitro was lower in the pair-fed, but unchanged in the ethanol group. These data suggest that 4-wk of peroral ethanol intake affects thyroid function mostly at the extrahypothalamic level and that there is a contribution of concomitant food deprivation. Both ethanol treatment and food deprivation increased TRH content in the septum.

Previous studies have indicated that isosmolar, but not hyperosmolar, ethanol induces in vitro gonadotropin-releasing hormone secretion from the basal hypothalamus, presumably by causing cell swelling. Moreover, ethanol reduces secretion of another hypothalamic neuropeptide vasopressin. We have studied the acute effect of ethanol on specific hypophysiotropic basal and K + -stimulated thyrotropin-releasing hormone secretion in vitro especially in relation to cell swelling. Isosmotic 40–160 mM ethanol increased thyrotropin-releasing hormone release from the hypothalamic paraventricular nucleus and median eminence in a dose-dependent manner. Both a 30% decrease of osmolarity and isosmotic 80 mM ethanol induced 12% swelling of hypothalamic neurons. Hyperosmotic 80 mM or 160 mM ethanol induced release of thyrotropin-releasing hormone from both hypothalamic structures but did not cause cell swelling (80 mM) or even induced cell shrinkage (160 mM). Depletion of medium Ca 2+ did not affect thyrotropin-releasing hormone secretion caused by either isosmotic or hyperosmotic ethanol. Our data indicate that both iso- and hyperosmotic ethanol stimulated release of hypophysiotropic thyrotropin-releasing hormone despite opposite effects on neuron volume. The mechanism of ethanol action appears complex and variable depending on the type of cell and neuropeptide affected.