Papers by Maria Papanikolaou
Noradrenergic Signaling and Astroglia
Abstract White matter (WM) tracts contain bundles of myelinated axons and the glia that support t... more Abstract White matter (WM) tracts contain bundles of myelinated axons and the glia that support them, namely oligodendrocytes that form the myelin and astrocytes, which provide structural and homeostatic support, together with small populations of microglia and oligodendrocyte precursor cells. Neurotransmitter signaling is prominent in WM, and there is a significant role for adrenergic signaling, together with glutamatergic and purinergic mechanisms. The site of axoglial signaling in myelinated axons is the node of Ranvier, which is the site of action potential propagation and displays many properties of synapses. Neurotransmitters released during axonal electrical activity activate receptors on perinodal astrocytes, to stimulate their release of gliotransmitters that integrate function within axon–glial–vascular networks. One of the key functions of astroglial adrenergic signaling in WM is to maintain energy supply to axons and oligodendrocytes, ensuring efficient and rapid information transfer throughout the central nervous system and maintaining WM integrity in times of energy stress.
Glia express a variety of ion channels, but the precise subtypes expressed by astrocytes and olig... more Glia express a variety of ion channels, but the precise subtypes expressed by astrocytes and oligodendrocytes has not been fully elucidated. The K ir 7.1 subtype of inwardly rectifying potassium channels (K ir) is highly expressed in retinal pigment epithelium and has been demonstrated in Purkinje neurons of the adult rat cerebellum and pyramidal neurons of the hippocampus, but it has not previously been identified in glia. Using quantitative real time PCR, an ion channel profile for the developing mouse optic nerve was constructed and K ir 7.1 was identified as one of the major ion channels present. Immunostaining revealed widespread expression of K ir 7.1 in glia and neurons in the mouse brain with the highest expression found in optic nerve oligodendrocytes. A major function of K ir is to maintain the membrane potential of glia in the face of large ionic shifts associated with normal neuronal function and pathology. Oligodendrocytes are particularly susceptible to ischemia so the role of K ir 7.1 in maintaining oligodendrocyte integrity during oxygen and glucose deprivation (ODG) in the isolated intact mouse optic nerve was examined, using the K ir 7.1 channel blocker VU590. Blockade of K ir 7.1 resulted in increased cell death of optic nerve oligodendrocytes in normoxic conditions by activating caspase-dependent apoptotic pathways and significantly augmented cell death induced by OGD. Moreover, intracellular calcium fluctuations dependent on store operated calcium entry in optic nerve glia were identified as a potential mechanism for the cellular stress induced by K ir 7.1 inhibition. The results presented within this thesis demonstrate functional expression of K ir 7.1 in glial cells, and indicate they are important in maintaining oligodendrocytic integrity in both physiological and pathological conditions.
Brain Structure and Function, 2017
com the 'calcium toolkit' that underpins SOCE and the sustainability of calcium signalling in whi... more com the 'calcium toolkit' that underpins SOCE and the sustainability of calcium signalling in white matter glia. Keywords Store-operated calcium channel • CRAC • TRP channel • Glia • Astrocyte • Oligodendrocyte • White matter • Calcium signalling Abstract 'Calcium signalling' is the ubiquitous response of glial cells to multiple extracellular stimuli. The primary mechanism of glial calcium signalling is by release of calcium from intracellular stores of the endoplasmic reticulum (ER). Replenishment of ER Ca 2+ stores relies on store-operated calcium entry (SOCE). However, despite the importance of calcium signalling in glial cells, little is known about their mechanisms of SOCE. Here, we investigated SOCE in glia of the mouse optic nerve, a typical CNS white matter tract that comprises bundles of myelinated axons and the oligodendrocytes and astrocytes that support them. Using quantitative RT-PCR, we identified Orai1 channels, both Stim1 and Stim2, and the transient receptor potential M3 channel (TRPM3) as the primary channels for SOCE in the optic nerve, and their expression in both astrocytes and oligodendrocytes was demonstrated by immunolabelling of optic nerve sections and cultures. The functional importance of SOCE was demonstrated by fluo-4 calcium imaging on isolated intact optic nerves and optic nerve cultures. Removal of extracellular calcium ([Ca 2+ ] o) resulted in a marked depletion of glial cytosolic calcium ([Ca 2+ ] i), which recovered rapidly on restoration of [Ca 2+ ] o via SOCE. 2-aminoethoxydiphenylborane (2APB) significantly decreased SOCE and severely attenuated ATPmediated calcium signalling. The results provide evidence that Orai/Stim and TRPM3 are important components of A. M. Butt and A. Lewis contributed equally to this work.
Channels
Coronary Artery Disease (CAD) typically kills more people globally each year than any other singl... more Coronary Artery Disease (CAD) typically kills more people globally each year than any other single cause of death. A better understanding of genetic predisposition to CAD and the underlying mechanisms will help to identify those most at risk and contribute to improved therapeutic approaches. KCNE2 is a functionally versatile, ubiquitously expressed potassium channel β subunit associated with CAD and cardiac arrhythmia susceptibility in humans and mice. Here, to identify novel KCNE2 interaction partners, we employed yeast two-hybrid screening of adult and fetal human heart libraries using the KCNE2 intracellular C-terminal domain as bait. Testin (encoded by TES), an endothelial cell-expressed, CAD-associated, focal adhesion protein, was identified as a high-confidence interaction partner for KCNE2. We confirmed physical association between KCNE2 and Testin in vitro by co-immunoprecipitation. Whole-cell patch clamp electrophysiology revealed that KCNE2 negative-shifts the voltage dependence and increases the rate of activation of the endothelial cell and cardiomyocyte-expressed Kv channel α subunit, Kv1.5 in CHO cells, whereas Testin did not alter Kv1.5 function. However, Testin nullified KCNE2 effects on Kv1.5 voltage dependence and gating kinetics. In contrast, Testin did not prevent KCNE2 regulation of KCNQ1 gating. The data identify a novel role for Testin as a tertiary ion channel regulatory protein. Future studies will address the potential role for KCNE2-Testin interactions in arterial and myocyte physiology and CAD.
Channels, 2021
Coronary Artery Disease (CAD) typically kills more people globally each year than any other singl... more Coronary Artery Disease (CAD) typically kills more people globally each year than any other single cause of death. A better understanding of genetic predisposition to CAD and the underlying mechanisms will help to identify those most at risk and contribute to improved therapeutic approaches. KCNE2 is a functionally versatile, ubiquitously expressed potassium channel β subunit associated with CAD and cardiac arrhythmia susceptibility in humans and mice. Here, to identify novel KCNE2 interaction partners, we employed yeast two-hybrid screening of adult and fetal human heart libraries using the KCNE2 intracellular C-terminal domain as bait. Testin (encoded by TES), an endothelial cell-expressed, CAD-associated, focal adhesion protein, was identified as a high-confidence interaction partner for KCNE2. We confirmed physical association between KCNE2 and Testin in vitro by co-immunoprecipitation. Whole-cell patch clamp electrophysiology revealed that KCNE2 negative-shifts the voltage depe...
Gastrointestinal (GI) motility disorders such as irritable bowel syndrome (IBS) can occur when co... more Gastrointestinal (GI) motility disorders such as irritable bowel syndrome (IBS) can occur when coordinated smooth muscle contractile activity is disrupted, and little is known about the molecular i...
The objective of the present study was the quantitative assessment of the previously documented i... more The objective of the present study was the quantitative assessment of the previously documented inverse relationship between Alzheimer's Disease (AD) and cancer (CA) by conducting a meta-analysis and evaluating systematic differentiations of the aforementioned relationship based on cancer localization. For the purpose of the study all available empirical data of the last decade, which met specific criteria, were included in the analysis by querying PubMed, Web of Science and Cochrane Library databases. Seven studies were included in the analysis, with a total sample of 18,887 (10,859 AD patients, 8,028 non-demented controls) participants to calculate cancer risk among AD patients, and 11 studies, with a total of 5,607,076 (1,853,318 cancer patients, 3,753,758 healthy controls) participants, were assessed to evaluate AD risk among cancer patients. The analysis revealed that AD patients appear to have a reduced risk of cancer, by 40% (RR 0.60, 95% CI 0.45 - 0.79), while cancer his...
Scientific Reports
Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K+ ion di... more Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K+ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These f...
Biophysical Journal, Feb 1, 2018
Hyperpolarization-activated, Cyclic Nucleotide-gated (HCN) channels are major determinants of the... more Hyperpolarization-activated, Cyclic Nucleotide-gated (HCN) channels are major determinants of the firing rate of pacemaker centers in the heart and brain. Direct binding of cAMP to the cyclic nucleotide binding domain (CNBD) activates HCN channels by increasing the maximum open probability and shifting the voltage-dependence of activation to less hyperpolarized potentials. HCN isoforms differ greatly in kinetics, voltage gating, and response to cyclic nucleotides. HCN2 responds strongly to cAMP despite having similar ligand affinity to HCN1, an isoform that responds very poorly to cAMP. To identify the key molecular determinants responsible for cAMP activation, we progressively mutated sites in the C-linker and CNBD of the mHCN2 to HCN1. Our studies identified two clusters of mutations that determine the differences in voltage dependent activation between these two isoforms. One mapped to the C-linker region (M485F, G497D, and S514T), far from the cAMP-binding site. Another was found in proximity to the binding site (V562A/S563G, L565I, and S575T). Concurrent substitutions of five sites (M485F, G497D, S514T, and V562A/ S563G) is sufficient to confer HCN1 phenotype on the mutated HCN2 channels. To understand the role of these residues on various allosteric parameters that determine the gating of these channels, we built an allosteric model consisting of four separate modules: pore gates, four voltage-sensors, ligand binding domain, and C-linker. Our models suggest that these substitutions must alter at least the oligomerization state of the linker and two coupling parameters: one between the linker and the pore and the other between the linker and the voltage sensors. These findings will be discussed in light of the highresolution structures of HCN channels.
Reviews in the Neurosciences
The field of cannabinoid research has been receiving ever-growing interest. Ongoing debates world... more The field of cannabinoid research has been receiving ever-growing interest. Ongoing debates worldwide about the legislation of medical cannabis further motivates research into cannabinoid function within the central nervous system (CNS). To date, two well-characterized cannabinoid receptors exist. While most research has investigated Cb1 receptors (Cb1Rs), Cb2 receptors (Cb2Rs) in the brain have started to attract considerable interest in recent years. With indisputable evidence showing the wide-distribution of Cb2Rs in the brain of different species, they are no longer considered just peripheral receptors. However, in contrast to Cb1Rs, the functionality of central Cb2Rs remains largely unexplored. Here we review recent studies on hippocampal Cb2Rs. While conflicting results about their function have been reported, we have made significant progress in understanding the involvement of Cb2Rs in modulating cellular properties and network excitability. Moreover, Cb2Rs have been shown t...
Glia express a variety of ion channels, but the precise subtypes expressed by astrocytes and olig... more Glia express a variety of ion channels, but the precise subtypes expressed by astrocytes and oligodendrocytes has not been fully elucidated. The Kir7.1 subtype of inwardly rectifying potassium channels (Kir) is highly expressed in retinal pigment epithelium and has been demonstrated in Purkinje neurons of the adult rat cerebellum and pyramidal neurons of the hippocampus, but it has not previously been identified in glia. Using quantitative real time PCR, an ion channel profile for the developing mouse optic nerve was constructed and Kir7.1 was identified as one of the major ion channels present. Immunostaining revealed widespread expression of Kir7.1 in glia and neurons in the mouse brain with the highest expression found in optic nerve oligodendrocytes. A major function of Kir is to maintain the membrane potential of glia in the face of large ionic shifts associated with normal neuronal function and pathology. Oligodendrocytes are particularly susceptible to ischemia so the role of...
Neuroglia in Neurodegenerative Diseases
Brain Structure and Function
Inward rectifying potassium channels (Kir) are a large family of ion channels that play key roles... more Inward rectifying potassium channels (Kir) are a large family of ion channels that play key roles in ion homeostasis in oligodendrocytes, the myelinating cells of the central nervous system (CNS). Prominent expression of Kir4.1 has been indicated in oligodendrocytes, but the extent of expression of other Kir subtypes is unclear. Here, we used qRT-PCR to determine expression of Kir channel transcripts in the mouse optic nerve, a white matter tract comprising myelinated axons and the glia that support them. A novel finding was the high relative expression of Kir7.1, comparable to that of Kir4.1, the main glial Kir channel. Significantly, Kir7.1 immunofluorescence labelling in optic nerve sections and in isolated cells was localised to oligodendrocyte somata. Kir7.1 are known as a K+ transporting channels and, using patch clamp electrophysiology and the Kir7.1 blocker VU590, we demonstrated Kir7.1 channels carry a significant proportion of the whole cell potassium conductance in oligod...
Journal of Pharmacology and Experimental Therapeutics
Journal of Anatomy
Inward Rectifying Potassium channels (Kir) are a large family of ion channels that play key roles... more Inward Rectifying Potassium channels (Kir) are a large family of ion channels that play key roles in ion homeostasis and neuronal excitability. The most recently described Kir subtype is Kir7.1, which is known as a K + transporting subtype. Earlier studies localised Kir7.1 to subpopulations of neurones in the brain. However, the pattern of Kir7.1 expression across the brain has not previously been examined. Here, we have determined neuronal and glial expression of Kir7.1 in the adult mouse brain, using immunohistochemistry and transgenic mouse lines expressing reporters specific for astrocytes [glial fibrillary acidic protein-enhanced green fluorescent protein (GFAP-EGFP], myelinating oligodendrocytes (PLP-DsRed), oligodendrocyte progenitor cells (OPC, Pdgfra-creER T2 /Rosa26-YFP double-transgenic mice) and all oligodendrocyte lineage cells (SOX10-EGFP). The results demonstrate significant neuronal Kir7.1 immunostaining in the cortex, hippocampus, cerebellum and pons, as well as the striatum and hypothalamus. In addition, astrocytes are shown to be immunopositive for Kir7.1 throughout grey and white matter, with dense immunostaining on cell somata, primary processes and perivascular end-feet. Immunostaining for Kir7.1 was observed in oligodendrocytes, myelin and OPCs throughout the brain, although immunostaining was heterogeneous. Neuronal and glial expression of Kir7.1 is confirmed using neurone-glial cortical cultures and optic nerve glial cultures. Notably, Kir7.1 have been shown to regulate the excitability of thalamic neurones and our results indicate this may be a widespread function of Kir7.1 in neurones throughout the brain. Moreover, based on the function of Kir7.1 in multiple transporting epithelia, Kir7.1 are likely to play an equivalent role in the primary glial function of K + homeostasis. Our results indicate Kir7.1 are far more pervasive in the brain than previously recognised and have potential importance in regulating neuronal and glial function.
Journal of Cellular Physiology
Hellenic journal of nuclear medicine
The objective of the present study was the quantitative assessment of the previously documented i... more The objective of the present study was the quantitative assessment of the previously documented inverse relationship between Alzheimer's Disease (AD) and cancer (CA) by conducting a meta-analysis and evaluating systematic differentiations of the aforementioned relationship based on cancer localization. For the purpose of the study all available empirical data of the last decade, which met specific criteria, were included in the analysis by querying PubMed, Web of Science and Cochrane Library databases. Seven studies were included in the analysis, with a total sample of 18,887 (10,859 AD patients, 8,028 non-demented controls) participants to calculate cancer risk among AD patients, and 11 studies, with a total of 5,607,076 (1,853,318 cancer patients, 3,753,758 healthy controls) participants, were assessed to evaluate AD risk among cancer patients. The analysis revealed that AD patients appear to have a reduced risk of cancer, by 40% (RR 0.60, 95% CI 0.45 - 0.79), while cancer his...
Nature communications, Jan 10, 2018
Voltage-gated potassium channels KCNQ2-5 generate the M-current, which controls neuronal excitabi... more Voltage-gated potassium channels KCNQ2-5 generate the M-current, which controls neuronal excitability. KCNQ2-5 subunits each harbor a high-affinity anticonvulsant drug-binding pocket containing an essential tryptophan (W265 in human KCNQ3) conserved for >500 million years, yet lacking a known physiological function. Here, phylogenetic analysis, electrostatic potential mapping, in silico docking, electrophysiology, and radioligand binding assays reveal that the anticonvulsant binding pocket evolved to accommodate endogenous neurotransmitters including γ-aminobutyric acid (GABA), which directly activates KCNQ5 and KCNQ3 via W265. GABA, and endogenous metabolites β-hydroxybutyric acid (BHB) and γ-amino-β-hydroxybutyric acid (GABOB), competitively and differentially shift the voltage dependence of KCNQ3 activation. Our results uncover a novel paradigm: direct neurotransmitter activation of voltage-gated ion channels, enabling chemosensing of the neurotransmitter/metabolite landscape ...
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Papers by Maria Papanikolaou