Lithium induces follicular atresia in rat ovary through a GSK‐3β/β‐catenin dependent mechanism

Lithium chloride (LiCl) is a drug used to treat bipolar disorder, but has side effects in the female reproductive system. Although lithium is known to decrease folliculogenesis and induce follicular atresia in rodent ovaries, its cellular and molecular effects in the ovary have not yet been addressed. To investigate these effects, 23‐day‐old immature female rats were injected with 10 IU pregnant mare serum gonadotropin (PMSG), followed by injections of 250 mg/kg LiCl every 12 hr for four doses. Ovaries were removed 40 and 48 hr after PMSG administration and prepared for histology, immunohistochemistry, Western blotting, and DNA laddering analysis. Our results showed that in the ovaries of LiCl‐treated rats, few antral but more atretic follicles were present compared to those of the control rats. The induction of atresia by LiCl was further confirmed by the presence of DNA fragmentation, accompanied by a reduced level of 17β‐estradiol in the serum. At the cellular level, lithium significantly decreased the number of proliferating cell nuclear antigen (PCNA)‐positive cells and conversely increased the number of TUNEL‐positive cells in the granulosa layer of the antral follicles. At the molecular level, lithium increased the level of phosphorylated glycogen synthase kinase‐3β, and unexpectedly decreased the expression of active (stabilized) β‐catenin. Altogether, our results indicate that lithium disrupts the balance between proliferation and apoptosis in granulosa cells, leading to follicular atresia possibly through the reduction in both the stabilized β‐catenin and 17β‐estradiol synthesis. Mol. Reprod. Dev. 80: 286–296, 2013. © 2013 Wiley Periodicals, Inc.     

TGF-β1 regulation of dendritic cells

Dendritic cells (DCs) represent antigen-presenting cell (APC) populations in lymphoid and nonlymphoid organs which are considered to play key roles in the initiation of antigen-specific T-cell proliferation. According to current knowledge, the net outcome of T-cell immune responses seems to be significantly influenced by the activation stage of antigen-presenting DCs. Several studies have shown that transforming growth factor-beta 1 (TGF-β1) inhibits in vitro activation and maturation of DCs. TGF-β1 inhibits upregulation of critical T-cell costimulatory molecules on the surface of DCs and reduces the antigen-presenting capacity of DCs. Thus, in addition to direct inhibitory effects of TGF-β1 on effector T lymphocytes, inhibitory effects of TGF-β1 at the level of APCs may critically contribute to previously characterized immunosuppressive effects of TGF-β1. In contrast to these negative regulatory effects of TGF-β1 on function and maturation of lymphoid tissue type DCs, certain subpopulations of immature DCs in nonlymphoid tissues are positively regulated by TGF-β1 signaling. In particular, epithelial-associated DC populations seem to critically require TGF-β1 stimulation for development and function. Recent studies established that TGF-β1 stimulation is absolutely required for the development of epithelial Langerhans cells (LCs) in vitro and in vivo. Furthermore, TGF-β1 seems to enhance antigen processing and costimulatory functions of epithelial LCs.     

Involvement of α2‐antiplasmin in dendritic growth of hippocampal neurons

The α2‐Antiplasmin (α2AP) protein is known as a principal physiological inhibitor of plasmin, but we previously demonstrated that it acts as a regulatory factor for cellular functions independent of plasmin. α2AP is highly expressed in the hippocampus, suggesting a potential role for α2AP in hippocampal neuronal functions. However, the role for α2AP was unclear. This study is the first to investigate the involvement of α2AP in the dendritic growth of hippocampal neurons. The expression of microtubule‐associated protein 2, which contributes to neurite initiation and neuronal growth, was lower in the neurons from α2AP^?/? mice than in the neurons from α2AP^+/+ mice. Exogenous treatment with α2AP enhanced the microtubule‐associated protein 2 expression, dendritic growth and filopodia formation in the neurons. This study also elucidated the mechanism underlying the α2AP‐induced dendritic growth. Aprotinin, another plasmin inhibitor, had little effect on the dendritic growth of neurons, and α2AP induced its expression in the neurons from plaminogen^?/? mice. The activation of p38 MAPK was involved in the α2AP‐induced dendritic growth. Therefore, our findings suggest that α2AP induces dendritic growth in hippocampal neurons through p38 MAPK activation, independent of plasmin, providing new insights into the role of α2AP in the CNS.     

Different regulation of Purkinje cell dendritic development in cerebellar slice cultures by protein kinase Cα and ‐β

Activity of protein kinase C (PKC), and in particular the PKCγ‐isoform, has been shown to strongly affect and regulate Purkinje cell dendritic development, suggesting an important role for PKC in activity‐dependent Purkinje cell maturation. In this study we have analyzed the role of two additional Ca²⁺‐dependent PKC isoforms, PKCα and ‐β, in Purkinje cell survival and dendritic morphology in slice cultures using mice deficient in the respective enzymes. Pharmacological PKC activation strongly reduced basal Purkinje cell dendritic growth in wild‐type mice whereas PKC inhibition promoted branching. Purkinje cells from mice deficient in PKCβ, which is expressed in two splice forms by granule but not Purkinje cells, did not yield measurable morphological differences compared to respective wild‐type cells under either experimental condition. In contrast, Purkinje cell dendrites in cultures from PKCα‐deficient mice were clearly protected from the negative effects on dendritic growth of pharmacological PKC activation and showed an increased branching response to PKC inhibition as compared to wild‐type cells. Together with our previous work on the role of PKCγ, these data support a model predicting that normal Purkinje cell dendritic growth is mainly regulated by the PKCγ‐isoform, which is highly activated by developmental processes. The PKCα isoform in this model forms a reserve pool, which only becomes activated upon strong stimulation and then contributes to the limitation of dendritic growth. The PKCβ isoform appears to not be involved in the signaling cascades regulating Purkinje cell dendritic maturation in cerebellar slice cultures. © 2003 Wiley Periodicals, Inc. J Neurobiol 57: 95–109, 2003     

Regulation of interleukin‐1β by interferon‐γ is species specific,limited by suppressor of cytokine signalling 1 and influences interleukin‐17 production

Reports describing the effect of interferon‐γ (IFNγ) on interleukin‐1β (IL‐1β) production are conflicting. We resolve this controversy by showing that IFNγ potentiates IL‐1β release from human cells, but transiently inhibits the production of IL‐1β from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL‐1β and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNγ and IFNβ are anti‐inflammatory. We observed that these cytokines suppress IL‐1β production in response to MTB, resulting in a reduced number of IL‐17‐producing cells. In human cells, IFNγ increased IL‐1β production, and this might explain why IFNγ is detrimental for multiple sclerosis. In mice, IFNγ decreased IL‐1β and subsequently IL‐17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.     

Amyloid β‐induced astrogliosis is mediated by β1‐integrin via NADPH oxidase 2 in Alzheimer's disease

Astrogliosis is a hallmark of Alzheimer′s disease (AD) and may constitute a primary pathogenic component of that disorder. Elucidation of signaling cascades inducing astrogliosis should help characterizing the function of astrocytes and identifying novel molecular targets to modulate AD progression. Here, we describe a novel mechanism by which soluble amyloid‐β modulates β1‐integrin activity and triggers NADPH oxidase (NOX)‐dependent astrogliosis in vitro and in vivo. Amyloid‐β oligomers activate a PI3K/classical PKC/Rac1/NOX pathway which is initiated by β1‐integrin in cultured astrocytes. This mechanism promotes β1‐integrin maturation, upregulation of NOX2 and of the glial fibrillary acidic protein (GFAP) in astrocytes in vitro and in hippocampal astrocytes in vivo. Notably, immunochemical analysis of the hippocampi of a triple‐transgenic AD mouse model shows increased levels of GFAP, NOX2, and β1‐integrin in reactive astrocytes which correlates with the amyloid β‐oligomer load. Finally, analysis of these proteins in postmortem frontal cortex from different stages of AD (II to V/VI) and matched controls confirmed elevated expression of NOX2 and β1‐integrin in that cortical region and specifically in reactive astrocytes, which was most prominent at advanced AD stages. Importantly, protein levels of NOX2 and β1‐integrin were significantly associated with increased amyloid‐β load in human samples. These data strongly suggest that astrogliosis in AD is caused by direct interaction of amyloid β oligomers with β1‐integrin which in turn leads to enhancing β1‐integrin and NOX2 activity via NOX‐dependent mechanisms. These observations may be relevant to AD pathophysiology.     

Kappa opioids promote the proliferation of astrocytes via Gβγ and β‐arrestin 2‐dependent MAPK‐mediated pathways

GTP binding regulatory protein (G protein)‐coupled receptors can activate MAPK pathways via G protein‐dependent and ‐independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and β‐arrestin 2 pathways in kappa opioid receptor‐induced, extracellular signal‐regulated kinase 1/2 (ERK1/2)‐mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non‐nitrogenous agonist, C(2)‐methoxymethyl salvinorin B (MOM‐Sal‐B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gβγ subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of β‐arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM‐Sal‐B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM‐Sal‐B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gβγ subunits or β‐arrestin 2, suggesting that both G protein‐dependent and ‐independent ERK pathways are required for this outcome.     

Cholesterol‐β1AR interaction versus cholesterol‐β2AR interaction

Two 8‐µs all‐atom molecular dynamics simulations have been performed on the two highly homologous G protein‐coupled receptor (GPCR) subtypes, β₁‐ and β₂‐adrenergic receptors, which were embedded in a lipid bilayer with randomly dispersed cholesterol molecules. During the simulations, cholesterol molecules accumulate to different surface regions of the two receptors, suggesting the subtype specificity of cholesterol–β‐adrenergic receptor interaction and providing some clues to the physiological difference of the two subtypes. Meanwhile, comparison between the two receptors in interacting with cholesterols shed some new light on general determinants of cholesterol binding to GPCRs. Our results indicate that although the concave surface, charged residues and aromatic residues are important, neither of these stabilizing factors is indispensable for a cholesterol interaction site. Different combinations of these factors lead to the diversified binding modes of cholesterol binding to the receptors. Our long‐time simulations, for the first time, revealed the pathway of a cholesterol molecule entering the consensus cholesterol motif (CCM) site, and the binding process of cholesterol to CCM is accompanied by a side chain flipping of the conserved Trp4.50. Moreover, the simulation results suggest that the I‐/V‐/L‐rich region on the extracellular parts of helix 6 might be an alternatively conserved cholesterol‐binding site for the class‐A GPCRs. Proteins 2014; 82:760–770. © 2013 Wiley Periodicals, Inc.     

Experience‐dependent plasticity of dendritic spines of layer 2/3 pyramidal neurons in the mouse cortex

Previous studies have shown that sensory and motor experiences play an important role in the remodeling of dendritic spines of layer 5 (L5) pyramidal neurons in the cortex. In this study, we examined the effects of sensory deprivation and motor learning on dendritic spine remodeling of layer 2/3 (L2/3) pyramidal neurons in the barrel and motor cortices. Similar to L5 pyramidal neurons, spines on apical dendrites of L2/3 pyramidal neurons are plastic during development and largely stable in adulthood. Sensory deprivation via whisker trimming reduces the elimination rate of existing spines without significant effect on the rate of spine formation in the developing barrel cortex. Furthermore, we show that motor training increases the formation and elimination of dendritic spines in the primary motor cortex. Unlike L5 pyramidal neurons, however, there is no significant difference in the rate of spine formation between sibling dendritic branches of L2/3 pyramidal neurons. Our studies indicate that sensory and motor learning experiences have important impact on dendritic spine remodeling in L2/3 pyramidal neurons. They also suggest that the rules governing experience‐dependent spine remodeling are largely similar, but not identical, between L2/3 and L5 pyramidal neurons. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 277–286, 2016     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

The stress response neuropeptide CRF increases amyloid‐β production by regulating γ‐secretase activity

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid‐β (Aβ) production. In cells, CRF treatment increases Aβ production and triggers CRF receptor 1 (CRFR1) and γ‐secretase internalization. Co‐immunoprecipitation studies establish that γ‐secretase associates with CRFR1; this is mediated by β‐arrestin binding motifs. Additionally, CRFR1 and γ‐secretase co‐localize in lipid raft fractions, with increased γ‐secretase accumulation upon CRF treatment. CRF treatment also increases γ‐secretase activity in vitro, revealing a second, receptor‐independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ‐secretase activity. Unexpectedly, CRFR1 antagonists also increased Aβ. These data collectively link CRF to increased Aβ through γ‐secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aβ and in some cases preferentially increase Aβ42 via complex effects on γ‐secretase.     

Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

Therapeutic potential of α,β‐thujone through metabolic reprogramming and caspase‐dependent apoptosis in ovarian cancer cells

The therapeutic potential of α,β‐thujone, a functional compound found in many medicinal plants of the Cupressaceae, Asteraceae, and Lamiaceae families, has been demonstrated, including in inflammation and cancers. However, its pharmacological functions and mechanisms of action in ovarian cancer remain unclear. We investigated the anticancer properties of α,β‐thujone in ES2 and OV90 human ovarian cancer cells and its effect on sensitization to cisplatin. α,β‐thujone inhibited cancer cell proliferation and induced cell death through caspase‐dependent intrinsic apoptotic pathways. Moreover, α,β‐thujone‐mediated endoplasmic reticulum stress was associated with the loss of mitochondrial functions and altered metabolic landscape of ovarian cancer cells. α,β‐Thujone attenuated blood vessel formation in transgenic zebrafish, implying it has significant antiangiogenic potential. In addition, α,β‐thujone sensitized ovarian cancer cells to cisplatin, causing synergistic pharmacological effects. Collectively, our results suggest that α,β‐thujone has therapeutic potential in human ovarian cancer and functions via regulating multiple intracellular stress‐associated metabolic reprogramming and caspase‐dependent apoptotic pathways.