Glutamate transporters around the tripartite synapse
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AbstractGlutamate is the major excitatory neurotransmitter in the mammalian nervous system. It is inactivated by cellular uptake catalyzed by a family of glutamate transporter proteins (GLT1, GLAST, EAAC1, EAAT4 and EAAT5). The main aim of the present thesis was to determine the contributions of the individual glutamate transporter subtypes to the total glutamate uptake around hippocampal synapses focusing on the EAAC1 subtype and on the mysterious transporter responsible for nerve terminal uptake of glutamate. The first step on this endeavor was to make antibodies to EAAC1. As outlined in Paper I, it turned out to be more difficult to make good antibodies to EAAC1 than to the other glutamate transporters. Specificity testing using tissue from EAAC1 knockout mice as negative controls revealed highly specific interactions with unrelated proteins. Paper II summarizes of the lessons learnt about immunocytochemical specificity testing, and Paper III illustrates how the antigen pre-adsorption test can be misleading. After having overcome methodological problems, we were in position to address the original question. In Paper IV a new procedure for immunoisolation of EAAC1 was developed, and known amounts of pure EAAC1 protein was used as standard to quantify EAAC1 concentrations in brain tissue extracts. EAAC1 was found to be present at 13 μg per gram hippocampal protein. This is 100 times less than GLT1 and argues against a significant contribution of EAAC1 to rapid transmitter activation. EAAC1is selectively expressed in neuronal somata and dendrites throughout the brain, and thereby in a total surface area similar to that of astrocytes. In Paper V we show that nerve terminal glutamate uptake fully depends on GLT1, and that about 10% of hippocampal GLT1 protein is expressed terminals. This also explains why high levels of GLT1 mRNA is present in CA3 pyramidal cells. In Paper VI we describe antibodies to GLT1 splice variants and show that GLT1a represents about 90 % of total hippocampal GLT1, while GLT1b and GLT1c represented 6 and 1 %, respectively. We also provide the first data on the distribution of the GLT1b and show that this variant does not contribute to nerve terminal uptake.
List of papers. Papers I, II, V and VI are removed from the thesis due to copyright restrictions.
Paper I: Holmseth S, Dehnes Y, Bjørnsen LP, Boulland JL, Furness DN, Bergles D, Danbolt NC. Specificity of antibodies: unexpected cross-reactivity of antibodies directed against the excitatory amino acid transporter 3 (EAAT3). Neuroscience. 2005;136(3):649-60. doi:10.1016/j.neuroscience.2005.07.022
Paper II: Holmseth S, Lehre KP, Danbolt NC. Specificity controls for immunocytochemistry. Anat Embryol (Berl). 2006 Aug;211(4):257-66. doi:10.1007/s00429-005-0077-6
Paper III: Holmseth S, Zhou Y, Follin-Arbelet V, Lehre KP, Bergles D, Danbolt NC. Specificity controls for immunocytochemistry: the antigen pre-adsorption test can lead to inaccurate assessment of antibody specificity. Submitted version. Published in: J Histochem Cytochem. 2012 Mar;60(3):174-87. doi:10.1369/0022155411434828
Paper IV: Holmseth S, Dehnes Y, Huang YH, Follin-Arbelet V, Grutle NJ, Mylonakou NM, Plachez C, Bergles D, Zhou Y, Furness DN, Danbolt NC and Lehre KP, (2011). Low density of EAAC1 (EAAT3; slc1a1) glutamate transporters suggests involvement in neuronal metabolism rather than in rapid control of synaptically released glutamate. Manuscript, published as: The Density of EAAC1 (EAAT3) Glutamate Transporters Expressed by Neurons in the Mammalian CNS. J Neurosci. 2012 Apr 25;32. (17):6000-13. doi:10.1523/JNEUROSCI.5347-11.2012 Distributed under the Creative Commons Attribution‐NonCommercial‐ShareAlike 3.0 Unported license
Paper V: Furness DN, Dehnes Y, Akhtar AQ, Rossi DJ, Hamann M, Grutle NJ, Gundersen V, Holmseth S, Lehre KP, Ullensvang K, Wojewodzic M, Zhou Y, Attwell D, Danbolt NC. A quantitative assessment of glutamate uptake into hippocampal synaptic terminals and astrocytes: New insights into a neuronal role for excitatory amino acid transporter 2 (GLT1). Neuroscience. 2008 Nov 11;157(1):80-94. doi:10.1016/j.neuroscience.2008.08.043
Paper VI: Holmseth S, Scott HA, Real K, Lehre KP, Leergaard TB, Bjaalie JG, Danbolt NC. The concentrations and distributions of three C-terminal variants of the GLT1 (GLT1; slc1a2) glutamate transporter protein in rat brain tissue suggests differential regulation. Neuroscience. 2009 Sep 15;162(4):1055-71. doi:10.1016/j.neuroscience.2009.03.048