Maria Nikodemova

Minocycline exerts inhibitory effects on multiple mitogen‐activated protein kinases and IκBα degradation in a stimulus‐specific manner in microglia

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‐κB (NF‐κB) 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 α of NF‐κB (IκBα) 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 IκBα implying a possible inhibitory role on NF‐κB transcriptional activity.

Microglia express distinct M1 and M2 phenotypic markers in the postnatal and adult central nervous system in male and female mice

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.

Minocycline Down-regulates MHC II Expression in Microglia and Macrophages through Inhibition of IRF-1 and Protein Kinase C (PKC)α/βII

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 interferonγ (IFNγ)-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 IFNγ and necessary for CIITA expression. Further experiments revealed that MHC II expression is down-regulated in the presence of the PKCα inhibitor Gö6976. Minocycline inhibited IFNγ-induced PKCα/βII phosphorylation and the nuclear translocation of both PKCα/βII and IRF-1 that subsequently inhibits CIITA expression. Our present data delineate a molecular pathway of minocycline action that includes inhibitory effects on PKCα/βII 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.

Efficient isolation of live microglia with preserved phenotypes from adult mouse brain.

BackgroundMicroglial activation plays a key role in the neuroinflammation associated with virtually all CNS disorders, although their role in normal CNS physiology is becoming increasingly appreciated. Neuroinflammation is often assessed by analyzing pro-inflammatory mediators in CNS tissue homogenates, under the assumption that microglia are the main source of these molecules. However, other cell types in the CNS can also synthesize inflammatory molecules. Hence, to enable direct analysis of microglial activities ex vivo, an efficient, reliable, and reproducible method of microglial isolation is needed.MethodsAfter enzymatic digestion of brain tissues and myelin removal, CD11b+ cells were isolated using immunomagnetic separation, yielding highly purified microglia without astrocyte or neuronal contamination. We used three methods of myelin removal (30% Percoll, 0.9 mol/l sucrose and anti-myelin magnetic beads), and compared their effects on microglial viability and yield. To determine whether the isolation procedure itself activates microglia, we used flow cytometry to examine microglial properties in brain-tissue homogenates and isolated microglia from control and lipopolysaccharide (LPS) -treated mice.ResultsThis method yielded a highly purified CD11b+ cell population with properties that reflected their in vivo phenotype. The viability and yield of isolated cells were significantly affected by the myelin removal method. Although the microglial phenotype was comparable in all methods used, the highest viability and number of CD11b+ cells was obtained with Percoll. Microglia isolated from LPS-treated mice displayed a pro-inflammatory phenotype as determined by upregulated levels of TNF-α, whereas microglia isolated from control mice did not.ConclusionsImmunomagnetic separation is an efficient method to isolate microglia from the CNS, and is equally suitable for isolating quiescent and activated microglia. This technique allows evaluation of microglial activities ex vivo, which accurately reflects their activities in vivo. Microglia obtained by this method can be used for multiple downstream applications including qRT-PCR, ELISA, Western blotting, and flow cytometry to analyze microglial activities in any number of CNS pathologies or injuries.

Minocycline downregulates MHC II expression in microglia and macrophages through inhibition of IRF-1 and PKCα/βII

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 interferonγ (IFNγ)-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 IFNγ and necessary for CIITA expression. Further experiments revealed that MHC II expression is down-regulated in the presence of the PKCα inhibitor Gö6976. Minocycline inhibited IFNγ-induced PKCα/βII phosphorylation and the nuclear translocation of both PKCα/βII and IRF-1 that subsequently inhibits CIITA expression. Our present data delineate a molecular pathway of minocycline action that includes inhibitory effects on PKCα/βII 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.

Expression of P2 nucleotide receptors varies with age and sex in murine brain microglia

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.

Association of sleep disordered breathing and cognitive deficit in APOE ε4 carriers.

STUDY OBJECTIVES The aim of the study was to determine whether apolipoprotein E epsilon 4 genotype (APOE4) modifies the association of sleep disordered breathing (SDB) with cognitive function in a middle-aged population. DESIGN Cross-sectional analysis of a community-dwelling cohort. SETTINGS Sleep laboratory at the Clinical Research Unit of the University of Wisconsin Hospitals and Clinics. PARTICIPANTS There were 755 adults from the Wisconsin Sleep Cohort who provided a total of 1,843 polysomnography and cognitive evaluations (most participants were assessed multiple times at approximately 4-y intervals); 56% males, average age 53.9 years (range 30-81 years). INTERVENTIONS None. MEASUREMENT AND RESULTS In-laboratory overnight polysomnography was used to assess SDB. Cognition was evaluated by a battery of six neurocognitive tests assessing memory and learning, attention, executive function, and psychomotor efficiency. The APOE4 genotype (ε3/ε4 or ε4/ ε4) was identified in 200 participants. Data were analyzed using linear mixed-effects models, accounting for multiple observations per participant. Cognitive test scores were regressed on SDB categories (AHI < 5, 5 ≤ AHI < 15, AHI ≥ 15); APOE4 and their interaction; and age, education, sex, and body mass index. There was no statistically significant association between SDB and cognitive performance among APOE4-negative individuals. However, in APOE4-positive individuals, those with AHI ≥ 15 had significantly worse performance on the Auditory Verbal Learning Test and the Controlled Oral Word Association Test. CONCLUSIONS In APOE4-positive individuals, moderate to severe sleep disordered breathing (AHI ≥ 15) was associated with poorer performance on cognitive tests that require both memory and executive function engagement.

Spinal but not cortical microglia acquire an atypical phenotype with high VEGF, galectin-3 and osteopontin, and blunted inflammatory responses in ALS rats.

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.

Minocycline attenuates experimental autoimmune encephalomyelitis in rats by reducing T cell infiltration into the spinal cord

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.

The P2X7-Egr pathway regulates nucleotide-dependent inflammatory gene expression in microglia

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.