Francisco Wandosell

Estradiol inhibits GSK3 and regulates interaction of estrogen receptors, GSK3, and beta-catenin in the hippocampus

Estrogens regulate a wide set of neuronal functions such as gene expression, survival and differentiation in a manner not very different from that exerted by neurotrophins or by growth factors. The best-studied hormonal action is the transcriptional activation mediated by estrogen receptors. However, the direct effects of estrogen on growth factor signaling have not been well clarified. The present data show that estradiol, in vivo, induces a transient activation of GSK3 in the adult female rat hippocampus, followed by a more sustained inhibition, as inferred from phosphorylation levels of Tau. Similar data was obtained from cultured hippocampal neurons when treated with the hormone. The transient activation was confirmed by direct measure of GSK3 kinase activity. In addition, our results show a novel complex of estrogen receptor alpha, GSK3, and beta-catenin. The presence of the hormone removes beta-catenin from this complex. There is a second complex, also affected by estradiol, in which Tau is associated with GSK3, beta-catenin, and elements of the PI3 kinase complex. Considering the role of GSK3 in neurodegeneration, our data suggest that part of the neuroprotective effects of estrogen may be due to the control of GSK3.

The Neurite Retraction Induced by Lysophosphatidic Acid Increases Alzheimer's Disease-like Tau Phosphorylation

The bioactive phospholipid lysophosphatidic acid (LPA) causes growth cone collapse and neurite retraction in neuronal cells. These changes are brought about by the action of a cell surface receptor coupled to specific G proteins that control morphology and motility through the action of a group of small GTPases, the Rho family of proteins. Many studies have focused on actin reorganization modulated by Rho-GTPases, but almost no information has been obtained concerning microtubular network reorganization after LPA-induced neurite retraction. In the present study, we demonstrate an increase in site-specific Alzheimers disease-like Tau phosphorylation during LPA-induced neurite retraction in differentiated SY-SH5Y human neuroblastoma cells. The phosphorylation state of Tau was inferred from its immunoreactivity with antibodies that recognize phosphorylation-sensitive epitopes. The effects of specific kinase inhibitors indicate that this phosphorylation is mediated by glycogen synthase kinase-3 (GSK-3). In support of this idea, we observed an increase of GSK-3 activity upon growth cone collapse. Our results are consistent with the hypothesis that activation of GSK-3 occurs in the Rho pathway and may represent an important link between microtubules and microfilaments dynamics during neuritogenesis and in pathological situations such as Alzheimers disease.

Chapter 31: CNS glial scar tissue: a source of molecules which inhibit central neurite outgrowth

Publisher Summary The chapter describes the interactions between purified gliotic membranes and embryonic central nervous system (CNS) explants. Gliotic membranes have different effects on neurite outgrowth, depending on whether the lesion is isomorphic or anisomorphic. The partial characterization of a proteoglycan associated with isomorphic gliotic membranes, which appears to inhibit central neurite outgrowth is also described. The major component of the glial scar is reactive glia. Reactive glia formation is the most general and stereotyped reaction of the CNS to any type of insult. From a neuropathological point of view, gliosis can be classified as anisomorphic or isomorphic, based on whether the eliciting stimulus is an open injury. Plasma membranes isolated from fresh tissue maintain the characteristics of the living cell surface; therefore, plasma membranes are isolated from glial scar tissue to study directly the interaction of growing axons with the scar cellular components, predominantly reactive astrocytes and microglial cells.

Glycosaminoglycans and β-amyloid, prion and tau peptides in neurodegenerative diseases

Protein aggregation into dense filamentous inclusions is a characteristic feature of many etiologically diverse neurodegenerative disorders including Alzheimers disease (AD), spongiform encephalopathies, and tauopathies. Thus, beta-amyloid peptide (Abeta) accumulates within senile amyloid plaques in AD, protease-resistant prion protein constitutes the amyloid deposits in spongiform encephalopathies and tau protein gives rise to neurofibrillary tangles (NFT) both in AD and in tauopathies. Curiously, these abnormal protein inclusions contain, in addition to their major peptide components, some associated sulfated glycosaminoglycans (sGAG). Here we discuss the proposal that the binding of sGAG to aggregate-forming peptides may modify the pathogenic process depending on their subcellular localization.

Characterization of a neurite outgrowth inhibitor expressed after CNS injury.

Reactive gliosis, a general response to injury in the central nervous system grey and white matter, represents a serious obstacle to axonal regeneration in mammals. In culture, myelin‐free plasma membranes from normal rat brain tissue promoted neurite outgrowth, whereas myelin‐free membranes purified from injured tissue were inhibitory. The inhibitory activity could be solubilized by detergent, was sensible to glycosaminoglycan lyase digestion and eluted with an apparent molecular weight of 160 – 220 kDa in gel filtration chromatography. When presented as a surface‐bound molecule, the inhibitor prevented neurite initiation; when added in a soluble form to growing neurites, it induced their retraction. These results provide cellular and molecular evidence supporting the classical view that, in the mammalian central nervous system, damage‐evoked gliosis correlates with the expression of molecules capable of preventing neurite outgrowth.

Prion peptide induces neuronal cell death through a pathway involving glycogen synthase kinase 3.

Prion diseases are characterized by neuronal cell death, glial proliferation and deposition of prion peptide aggregates. An abnormal misfolded isoform of the prion protein (PrP) is considered to be responsible for this neurodegeneration. The PrP 106-126, a synthetic peptide obtained from the amyloidogenic region of the PrP, constitutes a model system to study prion-induced neurodegeneration as it retains the ability to trigger cell death in neuronal cultures. In the present study, we show that the addition of this prion peptide to cultured neurons increases the activity of glycogen synthase kinase 3 (GSK-3), which is accompanied by the enhanced phosphorylation of some microtubule-associated proteins including tau and microtubule-associated protein 2. Prion peptide-treated neurons become progressively atrophic, and die ultimately. Both lithium and insulin, which inhibit GSK-3 activity, significantly decrease prion peptide-induced cell death both in primary neuronal cultures and in neuroblastoma cells. Finally, the overexpression of a dominant-negative mutant of GSK-3 in transfected neuroblastoma cells efficiently prevents prion peptide-induced cell death. These results are consistent with the view that the activation of GSK-3 is a crucial mediator of prion peptide-induced neurodegeneration.

MAP1B Is Required for Netrin 1 Signaling in Neuronal Migration and Axonal Guidance

BACKGROUND The signaling cascades governing neuronal migration and axonal guidance link extracellular signals to cytoskeletal components. MAP1B is a neuron-specific microtubule-associated protein implicated in the crosstalk between microtubules and actin filaments. RESULTS Here we show that Netrin 1 regulates, both in vivo and in vitro, mode I MAP1B phosphorylation, which controls MAP1B activity, in a signaling pathway that depends essentially on the kinases GSK3 and CDK5. We also show that map1B-deficient neurons from the lower rhombic lip and other brain regions have reduced chemoattractive responses to Netrin 1 in vitro. Furthermore, map1B mutant mice have severe abnormalities, similar to those described in netrin 1-deficient mice, in axonal tracts and in the pontine nuclei. CONCLUSIONS These data indicate that MAP1B phosphorylation is controlled by Netrin 1 and that the lack of MAP1B impairs Netrin 1-mediated chemoattraction in vitro and in vivo. Thus, MAP1B may be a downstream effector in the Netrin 1-signaling pathway.

The marine compound spisulosine, an inhibitor of cell proliferation, promotes the disassembly of actin stress fibers.

Spisulosine is a novel antiproliferative (antitumoral) compound of marine origin. In this work the molecular target for this toxic agent has been analyzed. In the presence of spisulosine, cultured cells change their morphology, first acquiring a fusiform morphology, and later becoming rounded without focal adhesions. Analysis of the cytoskeleton of treated cells indicate the absence of actin stress fibers.

Deconstructing GSK-3: The Fine Regulation of Its Activity

Glycogen synthase kinase-3 (GSK-3) unique position in modulating the function of a diverse series of proteins in combination with its association with a wide variety of human disorders has attracted significant attention to the protein both as a therapeutic target and as a means to understand the molecular basis of these disorders. GSK-3 is ubiquitously expressed and, unusually, constitutively active in resting, unstimulated cells. In mammals, GSK-3α and β are each expressed widely at both the RNA and protein levels although some tissues show preferential levels of some of the two proteins. Neither gene appears to be acutely regulated at the transcriptional level, whereas the proteins are controlled posttranslationally, largely through protein-protein interactions or by posttranslational regulation. Control of GSK-3 activity thus occurs by complex mechanisms that are each dependent upon specific signalling pathways. Furthermore, GSK-3 appears to be a cellular nexus, integrating several signalling systems, including several second messengers and a wide selection of cellular stimulants. This paper will focus on the different ways to control GSK-3 activity (phosphorylation, protein complex formation, truncation, subcellular localization, etc.), the main signalling pathways involved in its control, and its pathological deregulation.

Cross-talk between estrogen receptors and insulin-like growth factor-I receptor in the brain: cellular and molecular mechanisms.

Accumulating evidence suggests that insulin-like growth factor-I (IGF-I) and estradiol interact to regulate neural function. In this review, we focus on the cellular and molecular mechanisms involved in this interaction. The expression of estrogen receptors (ERs) and IGF-I receptor is cross-regulated in the central nervous system and many neurons and astrocytes coexpress both receptors. Furthermore, estradiol activates IGF-I receptor and its intracellular signaling. This effect may involve classical ERs since recent findings suggest that ERalpha may affect IGF-I actions in the brain by a direct interaction with some of the components of IGF-I signaling. In turn, IGF-I may regulate ER transcriptional activity in neuronal cells. In conclusion, ERs appear to be part of the signaling mechanism of IGF-I, and IGF-I receptor part of the mechanism of estradiol signaling in the nervous system.