Increased Zinc and Manganese in Parallel with Neurodegeneration,Synaptic Protein Changes and Activation of Akt/GSK3 Signaling in Ovine CLN6 Neuronal Ceroid Lipofuscinosis

Mutations in the CLN6 gene cause a variant late infantile form of neuronal ceroid lipofuscinosis (NCL; Batten disease). CLN6 loss leads to disease clinically characterized by vision impairment, motor and cognitive dysfunction, and seizures. Accumulating evidence suggests that alterations in metal homeostasis and cellular signaling pathways are implicated in several neurodegenerative and developmental disorders, yet little is known about their role in the NCLs. To explore the disease mechanisms of CLN6 NCL, metal concentrations and expression of proteins implicated in cellular signaling pathways were assessed in brain tissue from South Hampshire and Merino CLN6 sheep. Analyses revealed increased zinc and manganese concentrations in affected sheep brain in those regions where neuroinflammation and neurodegeneration first occur. Synaptic proteins, the metal-binding protein metallothionein, and the Akt/GSK3 and ERK/MAPK cellular signaling pathways were also altered. These results demonstrate that altered metal concentrations, synaptic protein changes, and aberrant modulation of cellular signaling pathways are characteristic features in the CLN6 ovine form of NCL.     

WNT unrelated activities in commercially available preparations of recombinant WNT3a

WNT signaling pathways play an important role in both development and disease. By analyzing the signaling capabilities of commercially available WNT3a preparations towards the PI3K/AKT/GSK3 signaling pathway, we discovered unexpected inconsistencies from lot to lot of recombinant WNT3a. We provide evidence that: (1) The ability to trigger AKT/GSK3 signaling varies dramatically between different lots of WNT3a, without any variation in their ability to activate the canonical WNT/β‐catenin signaling. (2) sFRP1, a WNT signaling inhibitor, is unable to interfere with the activation of AKT/GSK3 signaling induced by some of the WNT3a lots. (3) Pharmacological inhibition of AKT/GSK3 phosphorylation by PI3K inhibitors fails to affect the stabilization of β‐catenin, the central effector of the canonical WNT/β‐catenin signaling pathway. In summary, while all tested lots of recombinant WNT3a activated WNT/β‐catenin pathway, our results suggest that individual lots of recombinant WNT3a activate the PI3K/AKT/GSK3 pathway in a WNT‐independent manner, hampering thus the analysis of regulation of PI3K/AKT/GSK3 by WNT ligand. J. Cell. Biochem. 111: 1077–1079, 2010. © 2010 Wiley‐Liss, Inc.     

Glycogen synthase kinase-3 signaling in Alzheimer's disease

Alzheimer's disease (AD) is the most common form of neurodegenerative disorder with dementia, accounting for approximately 70% of the all cases. Currently, 5.8 million people in the U.S. are living with AD and by 2050 this number is expected to double resulting in a significant socio-economic burden. Despite intensive research, the exact mechanisms that trigger AD are still not known and at the present there is no cure for it. In recent years, many signaling pathways associated with AD neuropathology have been explored as possible candidate targets for the treatment of this condition including glycogen synthase kinase-3β (GSK3-β). GSK3-β is considered a key player in AD pathophysiology since dysregulation of this kinase influences all the major hallmarks of the disease including: tau phosphorylation, amyloid-β production, memory, neurogenesis and synaptic function. The present review summarizes the current understanding of the GSK3-β neurobiology with particular emphasis on its effects on specific signaling pathways associated with AD pathophysiology. Moreover, it discusses the feasibility of targeting GSK3-β for AD treatment and provides a summary of the current research effort to develop GSK3-β inhibitors in preclinical and clinical studies.     

Two Sides of the Same Coin: Wnt Signaling in Neurodegeneration and Neuro-Oncology

Wnts function through the activation of at least three intracellular signal transduction pathways, of which the canonical β-catenin mediated pathway is the best understood. Aberrant canonical Wnt signaling has been involved in both neurodegeneration and cancer. An impairment of Wnt signals appears to be associated with aspects of neurodegenerative pathologies while overactivation of Wnt signaling is a common theme in several types of human tumors. Therefore, although therapeutic approaches aimed at modulating Wnt signaling in neurodegenerative and hyperproliferative diseases might impinge on the same molecular mechanisms, different pharmacological outcomes are required. Here we review recent developments on the understanding of the role of Wnt signaling in Alzheimer’s disease and CNS tumors, and identify possible avenues for therapeutic intervention within a complex and multi-faceted signaling pathway.     

Synaptotoxicity in Alzheimer's disease: the Wnt signaling pathway as a molecular target

Recent evidence supports a role of the Wnt pathway in neurodegenerative disorders such as Alzheimer's disease (AD). A relationship between amyloid-beta-peptide (Abeta)-induced neurotoxicity and a decrease in the cytoplasmatic levels of beta-catenin has been proposed. Also, the inhibition of glycogen synthase kinase (GSK-3beta), a central modulator of the pathway, protects rat hippocampal neurons from Abeta-induced damage. Interestingly, during the progression of AD, it has been described that active GSK-3beta is found in neuronal cell bodies and neurites, co-localizing with pre-neurofibrillary tangles observed in disease brains. Since Abeta oligomers are associated with the post-synaptic region and we have found that the non-canonical Wnt signaling modulates PSD-95 and glutamate receptors, we propose that the synaptic target for Abeta oligomers in AD is the postsynaptic region and at the molecular level is the non-canonical Wnt signaling pathway. Altogether, our evidence suggests that a sustained loss of Wnt signaling function may be involved in the Abeta-dependent neurodegeneration observed in AD brains and that the activation of this signaling pathway could be of therapeutic interest in AD.     

A TNF receptor 2 selective agonist rescues human neurons from oxidative stress-induced cell death

Tumor necrosis factor (TNF) plays a dual role in neurodegenerative diseases. Whereas TNF receptor (TNFR) 1 is predominantly associated with neurodegeneration, TNFR2 is involved in tissue regeneration and neuroprotection. Accordingly, the availability of TNFR2-selective agonists could allow the development of new therapeutic treatments of neurodegenerative diseases. We constructed a soluble, human TNFR2 agonist (TNC-scTNF(R2)) by genetic fusion of the trimerization domain of tenascin C to a TNFR2-selective single-chain TNF molecule, which is comprised of three TNF domains connected by short peptide linkers. TNC-scTNF(R2) specifically activated TNFR2 and possessed membrane-TNF mimetic activity, resulting in TNFR2 signaling complex formation and activation of downstream signaling pathways. Protection from neurodegeneration was assessed using the human dopaminergic neuronal cell line LUHMES. First we show that TNC-scTNF(R2) interfered with cell death pathways subsequent to H(2)O(2) exposure. Protection from cell death was dependent on TNFR2 activation of the PI3K-PKB/Akt pathway, evident from restoration of H(2)O(2) sensitivity in the presence of PI3K inhibitor LY294002. Second, in an in vitro model of Parkinson disease, TNC-scTNF(R2) rescues neurons after induction of cell death by 6-OHDA. Since TNFR2 is not only promoting anti-apoptotic responses but also plays an important role in tissue regeneration, activation of TNFR2 signaling by TNC-scTNF(R2) appears a promising strategy to ameliorate neurodegenerative processes.     

Augmentation of therapeutic angiogenesis using genetically modified human endothelial progenitor cells with altered glycogen synthase kinase-3beta activity

Previously we reported that inhibition of glycogen synthase kinase-3beta (GSK3beta), a key regulator in many intracellular signaling pathways, enhances the survival and migration of vascular endothelial cells. Here we investigated the effect of inhibition of GSK3beta activity on the angiogenic function of endothelial progenitor cell (EPC) and demonstrated a new therapeutic angiogenesis strategy using genetically modified EPC. As we previously reported, two biologically distinct types of EPC, spindle-shaped "early EPC" and cobblestone-shaped "late EPC" could be cultivated from human peripheral blood. Catalytically inactive GSK3beta gene was transduced into both EPC. Inhibition of GSK3beta signaling pathway led to increased nuclear translocation of beta-catenin and increased secretion of angiogenic cytokines (vascular endothelial growth factor and interleukin-8). It enhanced the survival and proliferation of early EPC, whereas it promoted the survival and differentiation of late EPC. Transplantation of either of these genetically modified EPC into the ischemic hind limb model of athymic nude mouse significantly improved blood flow, limb salvage, and tissue capillary density compared with nontransduced EPC. Inhibition of GSK3beta signaling of either of these genetically modified EPC augmented the in vitro and in vivo angiogenic potency of these cell populations. These data provide evidence that GSK3beta has a key role in the angiogenic properties of EPC. Furthermore, the genetic modification of EPC to alter this signaling step can improve the efficacy of cell-based therapeutic vasculogenesis.     

A miRNA signature of prion induced neurodegeneration

MicroRNAs (miRNAs) are small, non-coding RNA molecules which are emerging as key regulators of numerous cellular processes. Compelling evidence links miRNAs to the control of neuronal development and differentiation, however, little is known about their role in neurodegeneration. We used microarrays and RT-PCR to profile miRNA expression changes in the brains of mice infected with mouse-adapted scrapie. We determined 15 miRNAs were de-regulated during the disease processes; miR-342-3p, miR-320, let-7b, miR-328, miR-128, miR-139-5p and miR-146a were over 2.5 fold up-regulated and miR-338-3p and miR-337-3p over 2.5 fold down-regulated. Only one of these miRNAs, miR-128, has previously been shown to be de-regulated in neurodegenerative disease. De-regulation of a unique subset of miRNAs suggests a conserved, disease-specific pattern of differentially expressed miRNAs is associated with prion-induced neurodegeneration. Computational analysis predicted numerous potential gene targets of these miRNAs, including 119 genes previously determined to be also de-regulated in mouse scrapie. We used a co-ordinated approach to integrate miRNA and mRNA profiling, bioinformatic predictions and biochemical validation to determine miRNA regulated processes and genes potentially involved in disease progression. In particular, a correlation between miRNA expression and putative gene targets involved in intracellular protein-degradation pathways and signaling pathways related to cell death, synapse function and neurogenesis was identified.     

Lithium Fails to Protect Dopaminergic Neurons in the 6-OHDA Model of Parkinson’s Disease

Lithium has been used for the treatment of bipolar mood disorder and is shown to have neuroprotective properties. Since lithium inhibits the activity of glycogen synthase kinase 3 (GSK3) which is implicated in various human diseases, particularly neurodegenerative diseases, the therapeutic potential of lithium receives great attention. Parkinson’s disease (PD) is the second most common neurodegenerative disease, characterized by the pathological loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Intranigral injection of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) causes selective and progressive degeneration of dopaminergic neurons in SNpc, and is a commonly used animal model of PD. The current study was designated to determine whether lithium is effective in alleviating 6-OHDA-induced neurodegeneration in the SNpc of rats. We demonstrated that chronic subcutaneous administration of lithium inhibited GSK3 activity in the SNpc, which was evident by an increase in phosphorylation of GSK3β at serine 9, cyclin D1 expression, and a decrease in tau phosphorylation. 6-OHDA did not affect GSK3 activity in the SNpc. Moreover, lithium was unable to alleviate 6-OHDA-induced degeneration of SNpc dopaminergic neurons. The results suggest that GSK3 is minimally involved in the neurodegeneration in the rat 6-OHDA model of PD.     

The kynurenine pathway modulates neurodegeneration in a Drosophila model of Huntington's disease

Neuroactive metabolites of the kynurenine pathway (KP) of tryptophan degradation have been implicated in the pathophysiology of neurodegenerative disorders, including Huntington's disease (HD) [1]. A central hallmark of HD is neurodegeneration caused by a polyglutamine expansion in the huntingtin (htt) protein [2]. Here we exploit a transgenic Drosophila melanogaster model of HD to interrogate the therapeutic potential of KP manipulation. We observe that genetic and pharmacological inhibition of kynurenine 3-monooxygenase (KMO) increases levels of the neuroprotective metabolite kynurenic acid (KYNA) relative to the neurotoxic metabolite 3-hydroxykynurenine (3-HK) and ameliorates neurodegeneration. We also find that genetic inhibition of tryptophan 2,3-dioxygenase (TDO), the first and rate-limiting step in the pathway, leads to a similar neuroprotective shift toward KYNA synthesis. Importantly, we demonstrate that the feeding of KYNA and 3-HK to HD model flies directly modulates neurodegeneration, underscoring the causative nature of these metabolites. This study provides the first genetic evidence that inhibition of KMO and TDO activity protects against neurodegenerative disease in an animal model, indicating that strategies targeted?at?two key points within the KP may have therapeutic relevance in HD, and possibly other neurodegenerative disorders.     

Estrogen-induced cell signalling in a cellular model of Alzheimer's disease

Alzheimer's disease (AD) is characterised by deposition of a 4 kDa amyloid-beta peptide (Abeta) into senile plaques of the affected brain. Abeta is a proteolytic product of the membrane protein, amyloid precursor protein (APP). An alternative cleavage pathway involves alpha-secretase activity and results in secretion of a 100 kDa non-amyloidogenic APP (sAPPalpha) and therefore a potential reduction in Abeta secretion. We have shown that estrogen induces alpha-cleavage and therefore results in the secretion of sAPPalpha. This secretion is signalled via MAP-kinase and PI-3 kinase signal-transduction pathways. These pathways also have the potential to inhibit the activation of glycogen synthase kinase 3beta (GSK), a protein involved in cell death. Therefore, the aim of this work was to further elucidate the estrogen-mediated signaling pathways involved in APP processing, with particular emphasis on GSK activity. By stimulating rat hypothalamic neuronal GT1-7 cells with estradiol, we found that estrogen decreases the activation state of GSK via the MAP kinase pathway. Moreover, the inhibition of GSK activity by LiCl causes enhanced sAPPalpha secretion in a pattern similar to that seen in response to estrogen, suggesting a pivotal role for this deactivation in APP processing. Further, inactivation of GSK by estrogen can be confirmed in an in vivo model. Elucidation of the signaling pathways involved in APP processing may help to understand the pathology of AD and may also prove beneficial in developing therapeutic strategies to combat AD.     

Differential regulation of glycogen synthase kinase 3beta by insulin and Wnt signaling

Glycogen synthase kinase 3beta (GSK3beta) is a key component in many biological processes including insulin and Wnt signaling. Since the activation of each signaling pathway results in a decrease in GSK3beta activity, we examined the specificity of their downstream effects in the same cell type. Insulin induces an increased activity of glycogen synthase but has no influence on the protein level of beta-catenin. In contrast, Wnt increases the cytosolic pool of beta-catenin but not glycogen synthase activity. We found that, unlike insulin, neither the phosphorylation status of the serine9 residue of GSK3beta nor the activity of protein kinase B is regulated by Wnt. Although the decrease in GSK3beta activity is required, GSK3beta may not be the limiting component for Wnt signaling in the cells that we examined. Our results suggest that the axin-conductin complexed GSK3beta may be dedicated to Wnt rather than insulin signaling. Insulin and Wnt pathways regulate GSK3beta through different mechanisms, and therefore lead to distinct downstream events.     

Natural products attenuate PI3K/Akt/mTOR signaling pathway: A promising strategy in regulating neurodegeneration

BackgroundAs common, progressive, and chronic causes of disability and death, neurodegenerative diseases (NDDs) significantly threaten human health, while no effective treatment is available. Given the engagement of multiple dysregulated pathways in neurodegeneration, there is an imperative need to target the axis and provide effective/multi-target agents to tackle neurodegeneration. Recent studies have revealed the role of phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) in some diseases and natural products with therapeutic potentials.PurposeThis is the first systematic and comprehensive review on the role of plant-derived secondary metabolites in managing and/or treating various neuronal disorders via the PI3K/Akt/mTOR signaling pathway.Study design and methodsA systematic and comprehensive review was done based on the PubMed, Scopus, Web of Science, and Cochrane electronic databases. Two independent investigators followed the PRISMA guidelines and included papers on PI3K/Akt/mTOR and interconnected pathways/mediators targeted by phytochemicals in NDDs.ResultsNatural products are multi-target agents with diverse pharmacological and biological activities and rich sources for discovering and developing novel therapeutic agents. Accordingly, recent studies have shown increasing phytochemicals in combating Alzheimer's disease, aging, Parkinson's disease, brain/spinal cord damages, depression, and other neuronal-associated dysfunctions. Amongst the emerging targets in neurodegeneration, PI3K/Akt/mTOR is of great importance. Therefore, attenuation of these mediators would be a great step towards neuroprotection in such NDDs.ConclusionThe application of plant-derived secondary metabolites in managing and/or treating various neuronal disorders through the PI3K/Akt/mTOR signaling pathway is a promising strategy towards neuroprotection.     

The inhibition of glycogen synthase kinase 3beta by a metabotropic glutamate receptor 5 mediated pathway confers neuroprotection to Abeta peptides

Activation of glycogen synthase kinase 3beta (Gsk3beta) has been shown to be a key component in signaling pathways that underlie neurodegeneration and neurodegenerative disease. Conversely, inactivation of Gsk3beta by phosphoinositide 3-kinase (PI3K)/Akt is an important neuroprotective mechanism. Previous studies have shown that agonist activation of group I metabotropic glutamate receptors (mGluRs) can increase neuronal survival and prevent apoptosis. However, little is known about the signaling pathways that couple mGluR5 to neuroprotection. In this report, we investigated whether activation of the PI3K/Akt/Gsk3beta pathway, which has been shown to have an important neuroprotective mechanism, is required for mGluR5 activation mediated neuroprotection against beta-amyloid. We found that brief incubations of mouse hippocampal slices with (R,S)-3,5-dihydroxyphenylglycine (DHPG) resulted in increased phosphorylation of Akt and Gsk3beta. The PI3K inhibitors, LY294002 and wortmannin, blocked the DHPG-induced increased phosphorylation of Akt and Gsk3beta. Similar results were observed in rat primary hippocampal cultures. Finally, we found that the PI3K inhibitor LY294002 can block (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) mediated neuroprotection against beta-amyloid. Thus, these findings suggest that mGluR5 can modulate the PI3K/Akt/Gsk3beta pathway in the hippocampus, and that modulation of this signaling pathway can reverse beta-amyloid-induced neuronal toxicity.     

Modulation of the development of human monocyte‐derived dendritic cells by lithium chloride

Lithium has been used or explored to treat psychiatric and neurodegenerative diseases that are frequently associated with an abnormal immune status. It is likely that lithium may work through modulation of immune responses in these patients. Because dendritic cells (DC) play a central role in regulating immune responses, this study investigated the influence of lithium chloride (LiCl) on the development and function of DC. Exposure to LiCl during the differentiation of human monocyte‐derived immature DCs (iDC) enhances CD86 and CD83 expression and increases the production of IL‐1β, IL‐6, IL‐8, IL‐10, and TNF‐α. However, the presence of LiCl during LPS‐induced maturation of iDC has the opposite effect. During iDC differentiation, LiCl suppresses the activity of glycogen synthase kinase (GSK)‐3β, and activates PI3K and MEK. In addition, LiCl activates peroxisome proliferator‐activated receptor γ (PPARγ) during iDC differentiation, a pathway not described before. Each of these signaling pathways appears to have distinct impact on the differentiating iDC. The enhanced CD86 expression by LiCl involves the PI3K/AKT and GSK‐3β pathway. LiCl modulates the expression of CD83 in iDC mainly through MEK/ERK, PI3K/AKT, and PPARγ pathways, while the increased production of IL‐1β and TNF‐α mainly involves the MEK/ERK pathway. The effect of LiCl on IL‐6/IL‐8/IL‐10 secretion in iDC is mediated through inhibition of GSK‐3β. We have also demonstrated that PPARγ is downstream of GSK‐3β and is responsible for the LiCl‐mediated modulation of CD86/83 and CD1 expression, but not IL‐6/8/10 secretion. The combined influence of these molecular signaling pathways may account for certain clinical effect of lithium. J. Cell. Physiol. 226: 424–433, 2011. © 2010 Wiley‐Liss, Inc.     

Glycogen synthase kinase 3: a point of convergence for the host inflammatory response

The phosphatidylinositol 3-kinase (PI3K) pathway has been shown to play a central role in regulating the host inflammatory response. Recent studies characterizing the downstream effector molecules within the PI3K pathway have identified that the serine/threonine kinase, glycogen synthase kinase 3 (GSK3), plays a pivotal role in regulating the production of pro- and anti-inflammatory cytokines. In innate immune cells, GSK3 inactivation augments anti-inflammatory cytokine production while concurrently suppressing the production of pro-inflammatory cytokines. The role of GSK3 in T cell biology has also been studied in detail and is involved in regulating multiple downstream signaling processes mediated by the T cell receptor (TCR), the co-stimulatory molecule CD28, and the IL-17 receptor. In vivo studies assessing the therapeutic properties of GSK3 inhibitors have shown that the inactivation of GSK3 can protect the host from immune-mediated pathology and death. This review will highlight the immunological importance GSK3 plays within different signal transduction pathways of the immune system, the cellular mechanisms regulating the activity of GSK3, the role of GSK3 in innate and adaptive immune responses, and the in vivo use of GSK3 inhibitors to treat inflammatory mediated diseases in animals.     

糖原合酶激酶-3，一个信号通路的重要调控因子

糖原合酶激酶-3 (glycogen synthase kinase-3,GSK-3) 是一种多功能的丝氨酸／苏氨酸蛋白激酶,在蛋白质合成、信号传递、细胞增殖、细胞分化、神经功能、肿瘤形成及胚胎发育等众多细胞进程中均扮演重要的角色.GSK-3 能够使多种底物发生磷酸化,并参与胰岛素、Wnt及Hedgehog 等多个信号通路的调控. GSK-3抑制剂在信号通路中能有效地抑制病理情况下GSK-3活性的异常增高,达到治疗的目的.GSK-3的抑制剂将作为一种潜在的药物对治疗糖尿病、阿尔海默茨症、肿瘤等疾病发挥效用.