Oestrogen as a neuroprotective hormone

In addition to its role as a sex hormone, oestrogen affects the structure and function of the nervous system. Oestrogen receptors are expressed in brain regions that are involved in sex differentiation and maturation. But in addition to its well-known effects, oestrogen also has important neuroprotective actions that are both dependent and independent of a nuclear oestrogen-receptor activity. Furthermore, oestrogen can interact with neuroprotective intracellular signalling pathways and is itself a neuroprotective antioxidant. Understanding the mechanisms of oestrogen action will be crucial to determine its potential as a therapeutic agent, particularly in the elderly.     

Rat cerebral cortex corticotropin-releasing hormone receptors: evidence for receptor coupling to multiple G-proteins

The wide distribution of corticotrophin-releasing hormone (CRH) receptors in brain and periphery appear to be important in integrating the responses of the brain, endocrine and immune systems to physiological, psychological and immunological stimuli. The type 1 receptors are highly expressed throughout the cerebral cortex, a region involved in cognitive function and modulation of stress responses, where they are coupled to the adenylyl cyclase system. Using techniques that analyse receptor-mediated guanine-nucleotide binding protein (G-proteins) activation, we recently demonstrated that expressed type 1alpha CRH receptors are capable of activating multiple G-proteins, which suggests that CRH can regulate multiple signalling pathways. In an effort to characterize the intracellular signals generated by CRH in the rat cerebral cortex we sought to identify G-proteins activated by CRH in a physiological membrane environment. Rat cerebral cortical membrane suspensions were analysed for the ability of CRH to stimulate incorporation of [alpha-32P]-GTP-gamma-azidoanilide to various G-protein alpha-chains. Our results show that CRH receptors are coupled to and activate at least five different G-proteins (Gs, Gi, Gq/11, Go and Gz) with subsequent stimulation of at least two intracellular signalling cascades. In addition, the photoaffinity experiments indicated that the CRH receptors preferentially activate the 45 kDa form of the Gs alpha-protein. This data may help elucidate the intracellular signalling pathways mediating the multiple actions of CRH especially under different physiological conditions.     

Acetylcholinesterase in cell adhesion, neurite growth and network formation

The expression of acetylcholinesterase is not restricted to cholinergically innervated tissues and relates to both neurotransmission and multiple biological aspects, including neural development, stress response and neurodegenerative diseases. Therefore, the classical function of acetylcholinesterase has to be distinguished from its non-classical, e.g. enzymatic from non-enzymatic, functions. Here, the roles of acetylcholinesterase in cell adhesion, promoting neurite outgrowth and neural network formation are reviewed briefly, together with potential mechanisms to support these functions. Part of these functions may depend on the structural properties of acetylcholinesterase, for example, protein-protein interactions. Recent findings have revealed that laminin-1 is an interaction partner for acetylcholinesterase. The binding of acetylcholinesterase to this extracellular matrix component may allow cell-to-cell recognition, and also cell signalling via membrane receptors. Studies using monolayer and 3D spheroid retinal cultures, as well as the acetylcholinesterase-knockout mouse, have been instrumental in elaborating the non-classical functions of acetylcholinesterase.     

Anomalous insulin-binding activity in the bovine neural retina: a possible mechanism for regulation of receptor binding specificity

Crude membrane from the bovine neural retina contains one IGF-I and two insulin binding sites. Although both insulin binding sites have a high affinity for insulin (IC50 = 0.1 and 7.0 nM), only one exhibits "classical" specificity and binds insulin with higher affinity than IGF-I. The second insulin binding site is "non-classical" in that it has an equal affinity for IGF-I and insulin. Retinal IGF-I binding exceeds insulin binding by a factor of 10-20. Despite this high level of IGF-I binding it is unlikely that non-classical insulin binding represents insulin binding to an IGF-I receptor because 1) anomalous binding is 30 times greater than that predicted from cross-specificity, 2) low concentrations of unlabeled IGF-I increase IGF-I binding to the IGF-I binding site but do not increase IGF-I binding to the non-classical insulin binding site and 3) the IGF-I receptor's affinity for insulin (and IGF-I) increases greatly during receptor purification. In contrast, the insulin affinity of the non-classical insulin binding site is largely unaffected by this process. Although receptor solubilization and purification had no effect on the insulin receptor's affinity for insulin, it did markedly increase this site's affinity for IGF-I. Thus, the major proportion of purified retinal "insulin receptors" have a higher affinity for IGF-I than insulin. The evidence presented here is consistent with the view that the bovine retina contains one IGF-I and two insulin binding sites and that a detergent-sensitive factor regulates IGF-I affinity of both classes of binding sites.     

Growth factor receptor signalling: location, location, location

Ligand binding to plasma membrane receptors initiates a series of events culminating in a variety of changes in cellular phenotypes. Although numerous publications have documented the activation/inactivation of signalling molecules following receptor binding, relatively few investigations have focused on the cellular compartment responsible for either initiating or selecting the particular pathway that mediates the response. Specifically, does receptor signalling occur only at the plasma membrane; is signalling dependent upon the location of defined endosome populations; or are components of both plasma membrane and endosomal activity operative depending upon the particular signalling pathway or cell type? This review addresses aspects of these questions by discussing the evidence supporting or contrasting the interplay between the endocytic and signalling systems for a subset of tyrosine kinase, serine/threonine kinase and G-protein-coupled receptors.     

Non-genomic oestrogen receptor signal in B lymphocytes: An approach towards therapeutic interventions for infection,autoimmunity and cancer

The non-genomic membrane bound oestrogen receptor (mER) regulates intracellular signals through receptor-ligand interactions. The mER, along with G-protein coupled oestrogen receptor GPR 30 (GPER), induces diverse cell signalling pathways in murine lymphocytes. The mER isoform ER-alpha46 has recently been demonstrated in human B and T lymphocytes as an analogue receptor for chemokine CCL18, the signalling events of which are not clearly understood. Ligand-induced mER and GPER signalling events are shared with BCR, CD19 mediated intracellular signalling through phospholipase C, PIP2/IP3/PI3 mediated activation of Akt, MAP kinase, and mTOR. Oestrogen has the ability to induce CD40-mediated activation of B cells. The complete signalling pathways of mER, GPR30 and their interaction with other signals are targeted areas for novel drug development in B cells during infection, autoimmunity and cancer. Therefore, an in depth investigation is critical for determining shared signal outputs during B cell activation. Here, we focus on the mode of action of membrane bound ER in B cells as therapeutic checkpoints.     

An oestrogen membrane receptor participates in estradiol actions for the prevention of amyloid-beta peptide1-40-induced toxicity in septal-derived cholinergic SN56 cells

Although oestrogen [17 beta-estradiol (E2)]-related neuroprotection has been demonstrated in different models, the involvement of non-classical oestrogen receptors (ERs) remains unexplored. Using the SN56 cholinergic cell line, we present evidence indicating that an ER associated with the plasma membrane participates in oestrogen-dependent inhibition of cell death induced by amyloid-beta peptide (A beta) toxicity. Similarly to E2 alone, a 15-min exposure to estradiol-horseradish peroxidase (E-HRP) significantly reduced A beta-induced cell death. This effect was decreased by the ER antagonist ICI 182,780 as well as by MC-20 antibody directed to a region neighbouring the ligand-binding domain of ER alpha. Using confocal microscopy on unpermeabilized SN56 cells exposed to MC-20 antibody, we identified a protein at the plasma membrane level. Western blot analysis of purified SN56 cell membrane fractions using MC-20 antibody revealed the presence of one band with the same electrophoretic mobility as intracellular ER alpha. Using conjugated forms of the steroid, E-HRP and E2 conjugated to bovine serum albumin-FITC, we demonstrated by confocal microscopy that SN56 cells contain surface binding sites for E2. Binding of both conjugates was blocked by pre-incubation with E2 and decreased by either ICI 182,780 or MC-20 antibody in a concentration-dependent manner. Thus, a membrane-related ER that shares some structural homologies with ER alpha may participate in oestrogen-mediated neuroprotection.     

PDIp is a major intracellular oestrogen-storage protein that modulates tissue levels of oestrogen in the pancreas

E2 (17β-oestradiol), a female sex hormone, has important biological functions in a woman's body. The pancreas, often considered a non-classical E2-targeting organ, is known to be functionally regulated by E2, but little is known about how oestrogen actions are regulated in this organ. In the present study we report that PDIp (pancreas-specific protein disulfide isomerase), a protein-folding catalyst, can act as a major intracellular E2 storage protein in a rat model to modulate the pancreatic tissue level, metabolism and action of E2. The purified endogenous PDIp from both rat and human pancreatic tissues can bind E2 with a Kd value of approximately 150?nM. The endogenous PDIp-bound E2 accounts for over 80% of the total protein-bound E2 present in rat and human pancreatic tissues, and this binding protects E2 from metabolic disposition and prolongs its duration of action. Importantly, we showed in ovariectomized female rats that the E2 level in the pancreas reaches its highest level (9-fold increase over its basal level) at 24-48?h after a single injection of E2, and even at 96?h its level is still approximately 5-fold higher. In contrast, the E2 level in the uterus quickly returns to its basal level at 48?h after reaching its maximal level (approximately 2-fold increase) at 24?h. Taken together, these results show for the first time that PDIp is a predominant intracellular oestrogen storage protein in the pancreas, which offers novel mechanistic insights into the accumulation and action of oestrogen inside pancreatic cells.     

Modulation of synaptic signalling complexes by Homer proteins

The number of neurotransmitter receptors in the postsynaptic membrane and their functional coupling to intracellular signalling cascades are important determinants of synaptic strength--and hence potential targets for plasticity related modulation. In this context, Homer/Vesl proteins have gained particular interest for three main reasons: (i) they constitute part of the molecular scaffold at postsynaptic densities of excitatory synapses in the mammalian brain; (ii) they physically link type-I metabotropic glutamate receptors to the postsynaptic density and to inositol 1,4,5-triphosphate receptors in the subsynaptic endoplasmic reticulum; and (iii) Homer-1a, which has been categorized as an immediate early gene isoform, exerts dominant-negative activity, suggesting that it is involved in activity dependent rearrangements at synaptic junctions. Although these fundamental aspects have been reviewed previously by Xiao et al., this review will address primarily more recent studies on the regulation of Homer 1a expression and on the role of Homer/Vesl proteins in spine morphogenesis and receptor targeting and signalling.     

Neurotransmitters and Integration in Neuronal-Astroglial Networks

Two major neural cell types, glia, astrocytes in particular, and neurones can release chemical transmitters that act as soluble signalling compounds for intercellular communication. Exocytosis, a process which depends on an increase in cytosolic Ca²⁺ levels, represents a common denominator for release of neurotransmitters, stored in secretory vesicles, from these neural cells. While neurones rely predominately on the immediate entry of Ca²⁺ from the extracellular space to the cytosol in this process, astrocytes support their cytosolic Ca²⁺ increases by appropriating this ion from the intracellular endoplasmic reticulum store and extracellular space. Additionally, astrocytes can release neurotransmitters using a variety of non-vesicular pathways which are mediated by an assortment of plasmalemmal channels and transporters. Once a neuronal and/or astrocytic neurotransmitter is released into the extracellular space, it can activate plasma membrane neurotransmitter receptors on neural cells, causing autocrine and/or paracrine signalling. Moreover, chemical transmission is essential not only for homocellular, but also for heterocellular bi-directional communication in the brain. Further detailed understanding of chemical transmission will aid our comprehension of the brain (dys)function in heath and disease.     

Cytokine receptors and signal transduction

Cytokines are important regulators of hemopoiesis which exert their actions by binding to specific, high affinity, cell surface receptors. In the past several years, molecular cloning of these receptors has revealed a new superfamily referred to as the hemopoietic growth factor receptors. Members of this family are defined by a 200 amino acid conserved domain; however, it has become increasingly apparent that another characteristic of these receptors is the shared usage of a common signalling subunit among subgroups in this family. The shared signalling component explains the functional redundancy of many cytokines; however, the mechanism by which these receptors transduce a signal across the membrane is not yet clear. Studies into cytokine action have shown that many of the events that occur in response to ligand stimulation are similar to those observed for the better characterized intrinsic tyrosine kinase receptors. Thus, although the cytokine receptors do not possess intrinsic tyrosine kinase activity, these observations have led to a model of cytokine signal transduction adapted from the signalling mechanisms described for the tyrosine kinase receptors.     

Endophilin-1: a multifunctional protein

Endophilin-1, a cytoplasmic Src homology 3 (SH3) domain-containing protein, localises in brain presynaptic nerve termini. Endophilin dimerises through its N-terminus, and participates at multiple stages in clathrin-coated endocytosis, from early membrane invagination to synaptic vesicle uncoating. Both its C-terminal SH3 domain and N-terminus are required for endocytosis. Through its SH3 domain, endophilin bound to proline-rich domains (PRDs) in other endocytic proteins, including synaptojanin and dynamin. The N-terminal region possesses unique functions affecting lipid membrane curvature, through lysophosphatidic acid acyl transferase (LPAAT) activity and direct binding and tubulating activity. In addition to synaptic vesicle formation, endophilin-1 complexes with signalling molecules, including cell surface receptors, metalloprotease disintegrins and germinal centre kinase-like kinase (GLK). Therefore, endophilin-1 may serve to couple vesicle biogenesis with intracellular signalling cascades.     

The interaction between uPAR and vitronectin triggers ligand‐independent adhesion signalling by integrins

The urokinase‐type plasminogen activator receptor (uPAR) is a non‐integrin vitronectin (VN) cell adhesion receptor linked to the plasma membrane by a glycolipid anchor. Through structure–function analyses of uPAR, VN and integrins, we document that uPAR‐mediated cell adhesion to VN triggers a novel type of integrin signalling that is independent of integrin–matrix engagement. The signalling is fully active on VN mutants deficient in integrin binding site and is also efficiently transduced by integrins deficient in ligand binding. Although integrin ligation is dispensable, signalling is crucially dependent upon an active conformation of the integrin and its association with intracellular adaptors such as talin. This non‐canonical integrin signalling is not restricted to uPAR as it poses no structural constraints to the receptor mediating cell attachment. In contrast to canonical integrin signalling, where integrins form direct mechanical links between the ECM and the cytoskeleton, the molecular mechanism enabling the crosstalk between non‐integrin adhesion receptors and integrins is dependent upon membrane tension. This suggests that for this type of signalling, the membrane represents a critical component of the molecular clutch.     

Purinergic signalling in neuron-glia interactions

Activity-dependent release of ATP from synapses, axons and glia activates purinergic membrane receptors that modulate intracellular calcium and cyclic AMP. This enables glia to detect neural activity and communicate among other glial cells by releasing ATP through membrane channels and vesicles. Through purinergic signalling, impulse activity regulates glial proliferation, motility, survival, differentiation and myelination, and facilitates interactions between neurons, and vascular and immune system cells. Interactions among purinergic, growth factor and cytokine signalling regulate synaptic strength, development and responses to injury. We review the involvement of ATP and adenosine receptors in neuron-glia signalling, including the release and hydrolysis of ATP, how the receptors signal, the pharmacological tools used to study them, and their functional significance.