A expressão aumentada de GSK3b em pacientes da doença de Alzheimer (DA), aparentemente está relacionada com a formação de placas senis e emaranhados neurofibrilares. Utilizamos qPCR para avaliar os efeitos do lítio sobre os níveis do mRNA de GSK3a e GSK3b em culturas primárias de neurônios corticais e hipocampais de rato tratados com lítio. Nossos resultados, sugeriram que lítio é capaz de reduzir os níveis de mRNA de Gsk3b hipocampal de modo específico e dose-dependente. Estes dados foram subsequentemente comprovados in vivo em amostras cerebrais de ratos tratados com LiCl. Utilizamos hibridização subtrativa de cDNA e eletroforese bidimensional de culturas de neurônios hipocampais tratados com o lítio. Nossos estudos sugeriram a expressão diferencial de 88 genes, além de oito proteínas com cerca de 50% de alteração de expressão. Estas observações sugerem novos efeitos regulatórios do lítio sobre GSK3b e fornecem potenciais alvos da ação do lítio. / The increased systemic expression of active GSK3b in Alzheimers disease (AD) patients apparently is associated with the formation of senile plaques and neurofibrillary tangles, the hallmarks of this disease. In this study we used qPCR to evaluate the transcriptional regulation of GSK3a and GSK3b in lithium-treated primary cultures of rat cortical and hippocampal neurons. We found a significant and dose-dependent reduction in the mRNA levels of GSK3b, which was specific to hippocampus. This same effect was further confirmed in vivo by measuring GSK3 expression in different brain regions of adult rats treated with lithium. We used subtractive hybridizations and two-dimensional electrophoresis patterns of culture of hippocampus neurons treated with lithium. Our study suggested 88 genes differently expressed besides 8 proteins identified with variation expression. These observations suggest new regulatory effects of lithium over GSK3b and suggesting new potential lithium action targets.

A expressão aumentada de GSK3b em pacientes da doença de Alzheimer (DA), aparentemente está relacionada com a formação de placas senis e emaranhados neurofibrilares. Utilizamos qPCR para avaliar os efeitos do lítio sobre os níveis do mRNA de GSK3a e GSK3b em culturas primárias de neurônios corticais e hipocampais de rato tratados com lítio. Nossos resultados, sugeriram que lítio é capaz de reduzir os níveis de mRNA de Gsk3b hipocampal de modo específico e dose-dependente. Estes dados foram subsequentemente comprovados in vivo em amostras cerebrais de ratos tratados com LiCl. Utilizamos hibridização subtrativa de cDNA e eletroforese bidimensional de culturas de neurônios hipocampais tratados com o lítio. Nossos estudos sugeriram a expressão diferencial de 88 genes, além de oito proteínas com cerca de 50% de alteração de expressão. Estas observações sugerem novos efeitos regulatórios do lítio sobre GSK3b e fornecem potenciais alvos da ação do lítio. / The increased systemic expression of active GSK3b in Alzheimers disease (AD) patients apparently is associated with the formation of senile plaques and neurofibrillary tangles, the hallmarks of this disease. In this study we used qPCR to evaluate the transcriptional regulation of GSK3a and GSK3b in lithium-treated primary cultures of rat cortical and hippocampal neurons. We found a significant and dose-dependent reduction in the mRNA levels of GSK3b, which was specific to hippocampus. This same effect was further confirmed in vivo by measuring GSK3 expression in different brain regions of adult rats treated with lithium. We used subtractive hybridizations and two-dimensional electrophoresis patterns of culture of hippocampus neurons treated with lithium. Our study suggested 88 genes differently expressed besides 8 proteins identified with variation expression. These observations suggest new regulatory effects of lithium over GSK3b and suggesting new potential lithium action targets.

Myelin pallor is frequently reported in HIV patients, and can occur in the CNS prior to other evidence of disease process. Our exploratory studies showed that oligodendrocytes (OLs) are direct targets of HIV-1 Tat (transactivator of transcription). Tat induces a dose-dependent increase of intracellular Ca2+ level ([Ca2+]i) in cultured murine OLs, which can be attenuated by ionotropic glutamate receptor (iGluR) antagonists MK801 and CNQX. The Tat-induced [Ca2+]i increase leads to increased death in immature (O4+, MBP-), but not mature (O4+, MBP+) OLs, over 96 h. In addition, Tat-induced [Ca2+]i increase also reduced myelin-like membrane production by mature OLs. Calcium/Calmodulin dependent kinase IIβ (CaMKIIβ) and glycogen synthase kinase 3β (GSK3β) have been known to regulate differentiation, myelination, and apoptosis in OLs. Since both CaMKIIβ and GSK3β are important downstream modulators of [Ca2+]i change, we hypothesized that the detrimental effects of Tat on immature/mature OL viability and function are mediated via CaMKIIβ and GSK3β activation. Our results showed that Tat activates both CaMKIIβ and GSK3β in immature OLs, but only activates CaMKIIβ in mature OLs. MK801 completely blocks Tat-induced CaMKIIβ and GSK3β activation in both immature and mature OLs, while CNQX blocks GSK3β activation, but has only a partial effect on CaMKIIβ activity. Blocking iGluRs or inhibiting GSK3β both rescue Tat-induced immature OL death, but only MK801 reverses the membrane injury in mature OLs. Together, these data strongly suggest that 1) activity of CaMKIIβ and GSK3β in OLs can be regulated by Tat-induced iGluRs activation and 2) OLs at different developmental stages show different responses to Tat, possibly due to activation of different signaling pathways.

Alcoholism is a complex neuropsychiatric disease that is characterized by compulsive alcohol use and intensifying cravings and withdrawals, often culminating in physiologic dependency. Fundamental alterations in brain chemistry underlie the transition from initial ethanol exposure to repetitive excessive use. Key mediators of this adaptation include changes in gene expression and signal transduction. Here we investigated gene expression pathways in prefrontal cortex and nucleus accumbens following acute or chronic ethanol treatment, to identify genes with potentially conserved involvement in the long-term response of the corticolimbic system to repeated ethanol exposure. We investigated Gsk3b, which encodes glycogen synthase kinase 3-beta, as a highly ethanol responsive gene associated with risk for long-term maladaptive responses to ethanol. On the level of the protein, we found that GSK3B and to a lesser extent the GSK3A isoform showed robust increases in inhibitory phosphorylation following acute ethanol. This inhibition may underlie aspects of the behavioral response to acute ethanol, as pre-treatment with a GSK3B inhibitor (tideglusib) augmented ethanol’s locomotor effects. Following long term ethanol exposure, we re-tested GSK3B phosphorylation and found that its ethanol response is blunted, consistent with molecular tolerance as a corollary to increased consumption. As the prefrontal cortex (PFC) plays a vital role in the reward pathway via its glutamatergic projections to the nucleus accumbens, we investigated the role of the Gsk3b gene specifically in PFC and in glutamatergic neurons. Overexpression of Gsk3b in the PFC robustly increased ethanol consumption, while deletion in Camk2a-positive neurons significantly attenuated ethanol consumption. Pharmacologic antagonism of GSK3B also decreased drinking in a model of binge-like consumption. Collectively this data implicates GSK3B as a mediator of excessive ethanol intake via its kinase activity, wherein inhibition of the kinase via phosphorylation exerts a protective effect in the context of acute ethanol, but desensitizes with repeated exposure.

Stem-cell antigen 1-positive (Sca-1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5'-azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. b-arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of b-arrestin2 in Sca-1+ CSC differentiation, we used b-arrestin2-knockout mice and overexpression strategies. Real-time PCR revealed that b-arrestin2 promoted 5'-azacytizine-induced Sca-1+ CSC differentiation in vitro. Because the microRNA 155 (miR-155) may regulate b-arrestin2 expression, we detected its role and relationship with b-arrestin2 and glycogen synthase kinase 3 (GSK3β), another probable target of miR-155. Real-time PCR revealed that miR-155, inhibited by b-arrestin2, impaired 5'-azacytizine-induced Sca-1+ CSC differentiation. On luciferase report assay, miR-155 could inhibit the activity of b-arrestin2 and GSK3β, which suggests a loop pathway between miR-155 and b-arrestin2. Furthermore, b-arrestin2-knockout inhibited the activity of GSK3β. Akt, the upstream inhibitor of GSK3β, was inhibited in b-arrestin2-Knockout mice, so the activity of GSK3β was regulated by b-arrestin2 not Akt. We transplanted Sca-1+ CSCs from b-arrestin2-knockout mice to mice with myocardial infarction and found similar protective functions as in wild-type mice but impaired arterial elastance. Furthermore, low level of b-arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3β, similar to in vitro findings. The β-arrestin2/miR-155/GSK3β pathway may be a new mechanism with implications for treatment of heart disease.

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The breadth of genetic diversity in the mammalian immune response stands out amongst the ubiquity of variation seen in the genome, evidence that microbial infections have been a major driver of evolution. As technology has facilitated an understanding of the etiology of immunological diversity, so too has it enabled the assessment of its varied functions. Functional genomics, with its ability to assess both cause and effect, has revolutionized our understanding of fundamental biological phenomena and recalibrated our hypotheses. We build upon the model of host immunity established by rare genetic variants that are causative of immunodeficiencies, but that incompletely consider the complexities of the genome. To expand our understanding, we performed a series of forward genetic screens to identify regulators of distinct functions of the innate immune system. Our studies discovered genes with novel functions in antigen presentation and immunoregulation, including several involved in central metabolism. Studies in macrophages and dendritic cells identified mitochondrial respiration as a positive regulator of the interferon-gamma response, and cells incapable of respiration failed to activate T cells. Notably, human mutations in several of these genes are responsible for immune dysfunction. In summary, this work uses new methods in genetic engineering to systematically assess the regulation of innate immunity. Our results suggest that variation in these regulatory pathways is likely to alter immunity in states of health and disease. Thus, our work validates a new approach to identify candidate genes relevant to immune dysfunction.

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