QSAR studies for the pharmacological inhibition of glycogen synthase kinase-3

The enzyme GSK-3 plays a central role in cells during the phosphorylation of various key regulatory proteins, and consequently pharmacological inhibitors of this enzyme potentially allow the treatment of diseases that include neurodegenerative and bipolar affective disorders, diabetes, and diseases caused by unicellular parasites. Today there is a huge number of reported empirical structure-activity relationships (SAR) that may guide a rational design of more potent and selective inhibitors. However, only a few studies based on Quantitative Structure-Activity Relationships (QSAR) are available for predicting the inhibitor potency against this specific kinase, and they involve mainly molecular modeling and 3D-QSAR. The present review deals with the recent search for a quantitative analysis of GSK-3 inhibition.     

Thapsigargin-induced apoptosis was prevented by glycogen synthase kinase-3 inhibitors in PC12 cells

Uncontrolled calcium stress has been linked causally to a variety of neurodegenerative diseases, including ischemia, excitotoxicity and Alzheimer's disease. Thapsigargin, which increases [Ca²⁺]_i, induces apoptotic cell death (chromatin condensation and DNA fragmentation) accompanied by caspase-3 activation in PC12 cells. We examined whether GSK-3 is involved in thapsigargin-induced cell death by using GSK-3 inhibitors in PC12 cells. Cells treated with 0.1 μM thapsigargin for 24 h shrank. The injured cells underwent chromatin condensation and nuclear fragmentation, indicating apoptotic cell death. We assayed the effects of selective GSK-3 inhibitors, SB216763, azakenpaullone and alsteropaullone on thapsigargin-induced apoptosis. These inhibitors completely protected cells from thapsigargin-induced apoptosis. Alsterpaullone did not reduce the GRP78 protein expression induced by thapsigargin, suggesting that GSK-3 activation is not involved in induction of GRP78. In addition, GSK-3 inhibitors inhibited caspase-3 activation accompanied by thapsigargin-induced apoptosis. We showed in this report that thapsigargin-induced apoptosis is prevented by GSK-3 inhibitors, suggesting that thapsigargin induces caspase-dependent apoptosis mediated through GSK-3 activation in PC12 cells.     

Chronic inhibition of glycogen synthase kinase-3 protects against rotenone-induced cell death in human neuron-like cells by increasing BDNF secretion

Mitochondrial dysfunction is a common feature of many neurodegenerative disorders. Likewise, activation of glycogen synthase kinase-3 (GSK-3) has been proposed to play an important role in neurodegeneration. This multifunctional protein kinase is involved in a number of cellular functions and we previously showed that chronic inhibition of GSK-3 protects neuronal cells against mitochondrial dysfunction-elicited cell death, through a mechanism involving increased glucose metabolism and the translocation of hexokinase II (HKII) to mitochondria. Here, we sought to gain deeper insight into the molecular basis of this neuroprotection. We found that chronic inhibition of GSK-3, either genetically or pharmacologically, elicited a marked increase in brain-derived neurotrophic factor (BDNF) secretion, which in turn conferred resistance to mitochondrial dysfunction through subcellular re-distribution of HKII. These results define a molecular pathway through which chronic inhibition of GSK-3 may protect neuronal cells from death. Moreover, they highlight the potential benefits of enhanced neurotrophic factor secretion as a therapeutic approach to treat neurodegenerative diseases.     

The derivation of mesenchymal stem cells from human embryonic stem cells

Human embryonic stem cells (hESCs) hold promise for tissue regeneration therapies by providing a potentially unlimited source of cells capable of undergoing differentiation into specified cell types. Several preclinical studies and a few clinical studies use human bone marrow stromal cells (hBMSCs) to treat skeletal diseases and repair damaged tissue. However, hBMSCs have limited proliferation and differentiation capacity, suggesting that an alternate cell source is desirable, and hESCs may serve this purpose. Here we describe a protocol for the reproducible derivation of mesenchymal stem cells from hESCs (hES-MSCs). The hES-MSCs have a similar immunophenotype to hBMSCs, specifically they are CD73+, STRO-1+ and CD45-, and are karyotypically stable. The derived hES-MSCs are also capable of differentiating into osteoblasts and adipocytes. When the hES-MSCs were genetically modified with the lineage-specific Col2.3-GFP lentivirus and cultured in osteogenic medium, increased GFP expression was detected over time, indicating the hES-MSCs have the capacity to differentiate down the osteogenic lineage and had progressed toward a mature osteoblast phenotype.     

糖元合酶激酶-3过度激活对神经细丝磷酸化的影响

目的：在细胞水平研究糖元合酶激酶((3SK-3)过度激活对神经细丝磷酸化的影响。方法：采用磷酯酰肌醇三磷酸激酶(P13K)的特异性抑制剂渥曼青霉素(wortmannin,WT)处理野生型小鼠成神经瘤细胞株(N2a／wt),免疫印迹技术及酶活性测定检测GSK-3活性,免疫荧光技术检测神经细丝(NF)的磷酸化状态。进一步采用(3SK-3特异性的抑制剂LiCl处理上述细胞,检测上述相同指标。结果：WT处理N2a细胞1h后,GSK3酶活性显著增加,并使抗体SMI31显色增强,SMI32显色减弱,提示NF被过度磷酸化。而采用WT和LiCl联合处理N2a／wt细胞,GSK-3活性显著降低,与此同时,NF的磷酸化程度明显减少。结论：GSK-3过度激活可引起细胞骨架蛋白NF的异常过度磷酸化,而过度磷酸化的NF可能参与了AD的病理过程。     

Methods for derivation of human embryonic stem cells

The expanded blastocysts, developed from 2PN-stage embryos, are generally divided into three categories: a good blastocyst containing a large and distinguishable inner cell mass (ICM), a blastocyst with a small and distinct ICM, and a blastocyst with a poorly defined ICM. In this study, we introduce methods for the derivation of human embryonic stem cells (hESCs) depending on the quality of the blastocysts. An immunosurgical method was used for the good expanded blastocysts. This method, however, raises the probability of ICM loss in cases of hESC derivation from blastocysts with smaller or indistinct ICMs. Furthermore, this method is also associated with a risk of the contamination of the hESCs with animal pathogens. To overcome these shortcomings, the partial- or whole-embryo culture method was used. For blastocysts with no visible ICM, the whole-embryo culture method was used to establish hESCs via the seeding of the entire blastocyst without its zona pellucida directly on a STO feeder layer. However, trophectodermal overgrowth tends to hinder the expansion of the ICM during the initial steps of hESC derivation. Therefore, the partial-embryo culture method was developed to establish hESCs from blastocysts with smaller ICMs. The surgical isolation of the region containing the ICM with an ultra-fine glass pipette alleviates trophectoderm overgrowth. This method is also applicable to blastocysts with large and distinct ICMs, and the efficiency of this method is comparable to that of the immunosurgical method.     

Unfolded protein response activates glycogen synthase kinase-3 via selective lysosomal degradation

The unfolded protein response (UPR) is a stress response that is activated upon disturbed homeostasis in the endoplasmic reticulum. In Alzheimer's disease, as well as in other tauopathies, the UPR is activated in neurons that contain early tau pathology. A recent genome-wide association study identified genetic variation in a UPR transducer as a risk factor for tauopathy, supporting a functional connection between UPR activation and tau pathology. Here we show that UPR activation increases the activity of the major tau kinase glycogen synthase kinase (GSK)-3 in vitro via a selective removal of inactive GSK-3 phosphorylated at Ser^21/9. We demonstrate that this is mediated by the autophagy/lysosomal pathway. In brain tissue from patients with different tauopathies, lysosomal accumulations of pSer^21/9 GSK-3 are found in neurons with markers for UPR activation. Our data indicate that UPR activation increases the activity of GSK-3 by a novel mechanism, the lysosomal degradation of the inactive pSer^21/9 GSK-3. This may provide a functional explanation for the close association between UPR activation and early tau pathology in neurodegenerative diseases.     

Lipopolysaccharide-induced interleukin-6 production is controlled by glycogen synthase kinase-3 and STAT3 in the brain

Background

Complement represents a crucial mediator of neuroinflammation and neurodegeneration after traumatic brain injury. The role of the terminal complement activation pathway, leading to generation of the membrane attack complex (MAC), has not been thoroughly investigated. CD59 is the major regulator of MAC formation and represents an essential protector from homologous cell injury after complement activation in the injured brain.

Methods

Mice deleted in the Cd59a gene (CD59a^-/-) and wild-type littermates (n = 60) were subjected to focal closed head injury. Sham-operated (n = 60) and normal untreated mice (n = 14) served as negative controls. The posttraumatic neurological impairment was assessed for up to one week after trauma, using a standardized Neurological Severity Score (NSS). The extent of neuronal cell death was determined by serum levels of neuron-specific enolase (NSE) and by staining of brain tissue sections in TUNEL technique. The expression profiles of pro-apoptotic (Fas, FasL, Bax) and anti-apoptotic (Bcl-2) mediators were determined at the gene and protein level by real-time RT-PCR and Western blot, respectively.

Results

Clinically, the brain-injured CD59a^-/- mice showed a significantly impaired neurological outcome within 7 days, as determined by a higher NSS, compared to wild-type controls. The NSE serum levels, an indirect marker of neuronal cell death, were significantly elevated in CD59a^-/- mice at 4 h and 24 h after trauma, compared to wild-type littermates. At the tissue level, increased neuronal cell death and brain tissue destruction was detected by TUNEL histochemistry in CD59a^-/- mice within 24 hours to 7 days after head trauma. The analysis of brain homogenates for potential mediators and regulators of cell death other than the complement MAC (Fas, FasL, Bax, Bcl-2) revealed no difference in gene expression and protein levels between CD59a^-/- and wild-type mice.

Conclusion

These data emphasize an important role of CD59 in mediating protection from secondary neuronal cell death and further underscore the key role of the terminal complement pathway in the pathophysiology of traumatic brain injury. The exact mechanisms of complement MAC-induced secondary neuronal cell death after head injury require further investigation.     

Association analysis of the glycogen synthase kinase-3beta gene in bipolar disorder

Glycogen synthase kinase-3beta (GSK3beta) is a target of lithium as well as sodium valproate, both of which are effective mood stabilizing prophylatics/treatments for bipolar disorder, a highly heritable psychiatric disorder. Though it is not clear whether the mood stabilizing effects of these drugs act directly through GSK3beta, it is a good candidate for mediating at least part of lithium's action, and is an aetiological candidate gene for the disease itself. Recently, a potential locus for bipolar disorder was reported on chromosome 3q, close to 3q13.37 where GSK3beta maps. We conducted an association study to test the hypothesis that polymorphism of GSK3beta is involved in susceptibility to bipolar disorder by examining association between GSK3beta-gene polymorphisms and bipolar disorder. Of the five polymorphisms we examined, three were very rare in the study population and were not examined further. Neither of the remaining two polymorphisms we examined showed association with bipolar disorder. Thus, it is unlikely that the GSK3beta-gene is a risk factor for bipolar disorder in our sample, but we cannot exclude the gene completely as other unknown polymorphisms in the gene may increase susceptibility.     

An efficient method for the derivation of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) represent a unique tool for many researchers; however, the process of ESC derivation is often very inefficient and requires high specialization, training, and expertise. To circumvent these limitations, we aimed to develop a simple and efficient protocol based on the use of commercially available products. Here, we present an optimized protocol that we successfully applied to derive ESCs from several knockout mouse strains (Wnt-1, Wnt-5a, Lrp6, and parkin) with 50%-75% efficiency. The methodology is based on the use of mouse embryonic fibroblast feeders, knockout serum replacement (SR), and minimal handling of the blastocyst. In this protocol, all centrifugation steps (as well as the use of trypsin inhibitor) were avoided and replaced by an ESC medium containing fetal calf serum (FCS) after the trypsinizations. We define the potential advantages and disadvantages of using SR and FCS in individual steps of the protocol. We also characterize the ESCs for the expression of ESC markers by immunohistochemistry, Western blot, and a stem cell focused microarray. In summary, we provide a simplified and improved protocol to derive mESCs that can be useful for laboratories aiming to isolate transgenic mESCs for the first time.     

Recent advances in the derivation of germ cells from the embryonic stem cells

In recent years, considerable progress has been made in the establishment and differentiation of human embryonic stem (ES) cell lines. The primordial germ cells (PGCs) and embryonic germ (EG) cells derived from them share many of their properties with ES cells. ES cell lines have now been derived from different stages of germ cell development and they have differentiated into gametes and shown embryonic development in mice, including the production of live pups. Conversely, germ cells can also be derived from ES cells. It has been demonstrated that murine (m) ES cells can differentiate into PGCs and subsequently into early gametes (oocytes and sperms) and blastocysts. Recently, immature sperm cells derived from mES cells in culture have produced live offspring. Preliminary research has indicated that human (h) ES cells probably have the potential to differentiate into germ cells. Adult stem cells have been reported to differentiate into mature germ cells in vitro. Therefore, stem cells may offer a valuable in vitro model for the investigation of germ cell development and the early stages of human gametogenesis, including epigenetic modifications of the germ line. This review discusses recent developments in the derivation and specification of mammalian germ cells from ES cells and describes some of the mechanisms of germ cell development.     

Progesterone and insulin stimulation of CPEB-dependent polyadenylation is regulated by Aurora A and glycogen synthase kinase-3

Progesterone stimulation of Xenopus oocyte maturation requires the cytoplasmic polyadenylation-induced translation of mos and cyclin B mRNAs. One cis element that drives polyadenylation is the CPE, which is bound by the protein CPEB. Polyadenylation is stimulated by Aurora A (Eg2)-catalyzed CPEB serine 174 phosphorylation, which occurs soon after oocytes are exposed to progesterone. Here, we show that insulin also stimulates Aurora A-catalyzed CPEB S174 phosphorylation, cytoplasmic polyadenylation, translation, and oocyte maturation. However, these insulin-induced events are uniquely controlled by PI3 kinase and PKC-zeta, which act upstream of Aurora A. The intersection of the progesterone and insulin signaling pathways occurs at glycogen synthase kinase 3 (GSK-3), which regulates the activity of Aurora A. GSK-3 and Aurora A interact in vivo, and overexpressed GSK-3 inhibits Aurora A-catalyzed CPEB phosphorylation. In vitro, GSK-3 phosphorylates Aurora A on S290/291, the result of which is an autophosphorylation of serine 349. GSK-3 phosphorylated Aurora A, or Aurora A proteins with S290/291D or S349D mutations, have reduced or no capacity to phosphorylate CPEB. Conversely, Aurora A proteins with S290/291A or S349A mutations are constitutively active. These results suggest that the progesterone and insulin stimulate maturation by inhibiting GSK-3, which allows Aurora A activation and CPEB-mediated translation.     

Efficient induction of oligodendrocytes from human embryonic stem cells

Oligodendrocytes form myelin sheaths around axons to support rapid nerve conduction in the central nervous system (CNS). Damage to myelin can cause severe CNS disorders. In this study, we attempted to devise a protocol for the induction of oligodendrocytes from human embryonic stem (ES) cells to treat demyelinated axons. Four days after embryoid body formation, human ES cells were differentiated into neural precursors through selection and expansion procedures. Neural precursors were then grown in the presence of epidermal growth factor and then platelet-derived growth factor to generate oligodendrocyte precursor cells. After withdrawal of the growth factors, the cells were treated with thyroid hormone to induce differentiation into oligodendrocytes. This method resulted in approximately 81%-91% oligodendrocyte precursor cells and approximately 81% oligodendrocytes among total cells. The ability of the oligodendrocyte precursors to myelinate axons has been verified by coculturing with rat hippocampal neurons, confirming their biological functionality.     

Expression of endometrial glycogen synthase kinase-3beta protein throughout the menstrual cycle and its regulation by progesterone

Glycogen synthase kinase-3beta (GSK-3beta) is a serine/threonine kinase that plays a role in glycogen synthesis by inhibiting glycogen synthase (GS) through phosphorylation. We hypothesized that GSK-3beta by virtue of its role in glycogen synthesis through the inhibition of GS will play a role in the preparation of the endometrium for blastocyst implantation. Immunohistochemical (IHC) analysis and Western blot analysis (WBA) detected GSK-3beta in the endometrium, myometrium, Fallopian tube and ovary. WBA showed more than 5-fold higher endometrial expression of the phosphorylated GSK-3beta (pGSK-3beta) isoform (inactive) in the secretory phase as compared with the proliferative phase (P < 0.001), whereas no differences in total GSK-3beta expression were detected. IHC analysis confirmed the WBA and showed marked expression of pGSK-3beta predominantly in glandular epithelial cells in early and mid secretory endometrium with scant expression during the proliferative phase. In in vitro experiments using human endometrial-derived epithelial cell line (HES), progesterone did not alter total GSK mRNA or protein expression. However, progesterone induced a dose-dependent increase in the expression of pGSK-3beta, which could be blocked by RU486. Cyclic expression of GSK-3beta's active and inactive forms in the endometrium suggests that sex hormones regulate the expression of this enzyme. In vitro experiments demonstrate that progesterone through receptor-mediated mechanisms induces phosphorylation of endometrial GSK-3beta.