Perturbations in transporter function and expression have been reported in all neurological diseases, and these perturbations appear to be part of the pathogenetic processes ultimately leading to disabilities.
About the group
To study these mechanisms, the Neurotransporter group has acquired expertise in the construction of transgenic animals, membrane protein purification and reconstitution in artificial cell membranes, neuropharmacology, confocal imaging, electron microscopy, 3D-reconstruction, computer simulations, antibody production, robotics and advanced databasing.
The research impact of our research group
The total number of publications from the neurotransporter group is 110 plus 7 book chapters. The total number of citations (excluding self-citations) to Danbolt's papers is >13800 citations in > 8400 publications according to Clarivate Analytics (Dec 2019; Danbolt N* 1984-2019; all databases). There are 11 publications with more than 300 citations and another 25 in the range 100-299 citations (the latter includes some self-citations).
The "Weighted Relative Citation Ratio" (https://icite.od.nih.gov/analysis) is high: 422.06. Thirty-one of these 98 papers are above the 90-percentile indicating the high quality of the work.
Scientific findings from our research group
1. Danbolt was the first, in collaboration with Baruch I. Kanner (Hebrew University, Israel) , to purify a glutamate transporter protein using reconstitution of transport activity to monitor the purification process (Danbolt et al., 1990). Antibodies to the purified protein was use to localize (Danbolt et al., 1992) and clone a glutamate transporter, which turned out to define a new gene family (Nature 360:464 1992).
2. The group was first to discover that neurons modulate glutamate transporter expression in astrocytes (Levy et al., 1995). The transporters are also regulated by several other mechanisms (J. Biol. Chem. 268:27313 1992; J. Biol. Chem. 271:5976 1996; J.Neurochem. 69:2612 1997; TIPS 19:3281998; Nat Neurosci. 2:427 1999). A disease related functionally impaired mutant was identified (J. Biol. Chem. 276: 576).
3. The group has studied the localization of neurotransmitter transporters using light and electron microscopic immunocytochemistry (e.g. Lehre et al., 1995; Dehnes et al., 1998). Together with Ann Massie (Brussel) we discovered that many of the antibodies used to localize the xCT cystine-glutamate exchanger recognized unrelated proteins (Van Liefferinge et al., 2016 ) and show that xCT is selectively expressed in astroglia (Ottestad-Hansen et al., 2018). In combination with stereological analysis and Western blotting, data on the densities of the number of transporter molecules per square micrometer plasma membranes has been obtained (Lehre and Danbolt, 1998; Dehnes et al., 1998; Holmseth et al., 2012; Otterstad-Hanson et al., 2018; for review see: Danbolt, 2001). The EAAT2-subtype represents about 1 % of total adult hippocampus protein, while EAAT3 (Holmseth et al., 2012) and the cystine-glutamate exchanger (xCT; Slc7a11: Ottestad-Hansen et al., 2018) are expressed at levels about 100 times lower. The levels of C-terminal EAAT2 variant were also determined (Holmseth et al., 2009). The EAAT2-subtype accounts for 93 % of the forebrain glutamate uptake activity and the EAAT-type of transporters form homo-oligomers consisting of non-covalently attached subunits (Haugeto et al., 1996).
4. The identity of the glutamate transporters in glutamatergic nerve terminals has been an unresolved question for a long time. Using antibodies to glutaraldehyde-fixed transporter substrates, we showed by immuno-gold electron microscopy that there is likely uptake of glutamate into axon terminals. We were able to demonstrate that the uptake was solely mediated by EAAT2 and as fast as the glutamate uptake by astrocytes (Furness et al., 2008) despite higher number of transporter molecules in the astrocytes. The follow-up work by the combined use of proteoliposomes and computer modeling (Zhou et al., 2014a) in collaboration with H. Peter Larsson (University of Miami), we showed that this mismatch between glutamate transporter protein densities and transport activity was not due to a higher rate of heteroexchange than of net transport. To resolve the discrepancy in uptake efficiency between neurons and astrocytes, we created a conditional EAAT2 knockout mouse (Zhou et al., 2014b). This enabled us to conclude, in collaboration with Paul Rosenberg, that astrocytic EAAT2 protects against fatal epilepsy while neuronal EAAT2 contributes significantly to glutamate uptake into synaptosomes, at least in the hippocampus (Petr et al., 2015). We subsequently showed axon-terminal expressing EAAT2 are found in most brain regions and that neuronal EAAT2 plays a detectable role in the overall glutamate metabolism in vivo (Zhou et al., 2019a; For review see: Danbolt et al., 2016).
5. Loss of glutamine synthetase (Glul; GS) in the human epileptogenic hippocampus might be a mechanism behind mesial temporal lobe epilepsy (Eid et al., 2004). To uncover the molecular mechanism from GS-deficiency to epilepsy, we created in collaboration with Tore Eid (Yale School of Medicine) and Siu-Pok Yee (University of Connecticut) a conditional GS knockout mouse line. We overcame the early lethality by limiting the deletion of the gene to the cerebral cortex (Zhou et al., 2019b ). The initial investigation on GS conditional knockouts revealed that deficiency in glutamine synthetase during the development induces a chain of events culminating in epilepsy and neurodegeneration (including glial dysfunction and vascular impairment) rather than a direct excitotoxic mechanism.
6. This work has shown that the transporters play complex roles in the normal functioning of the nervous system as well as in neurological disorders (for review see: Danbolt 2001). The main interest in recent years has been to obtain quantitative data in order to perform computer simulations. What are all of these transporters doing together? Precise quantitative data, however, requires rigorous specificity controls and access to transgenic animals (Holmseth 2005, 2006, Holmseth et al., 2012a and 2012b; Danbolt et al., 2016 )
7. The betaine-GABA transporter (BGT1) line was developed to test the hypothesis that BGT1 plays a role in seizure control. Surprisingly, BGT1 does not a play a role in the brain and only plays a minor role in the kidney. The main function of BGT1 is in the liver (Lehre et al., 2011; Zhou et al., 2012a) illustrating the importance of using transgenic animals. This opens new areas of multidiscipline research involving molecular biology, physiology and nutrition (Kempson et al., 2014; Zhou et al., manuscript in preparation).
8. Deletion of the gene encoding the GABA transporter 2 (GAT2, slc6a13) in mice revealed that this transporter is mostly expressed in the liver where it unexpectedly serves as a taurine transporter (Zhou et al., 2012b ). It is also expressed in the kidneys. In the brain, it is found in some cells along blood vessels and in the leptomeninges at the brain surface. Thus, neither GAT2 nor BGT1 are likely to be important for controlling the action of transmitter GABA in the brain.
9. The logistics of this research activity has become complex, and that lead to an interest in databasing to improve the efficiency of the research process. Due to increasing numbers of methods, reagents, electronic files and higher mobility of both people and materials it is hard to keep track. The traditional notebooks are inadequate, and the group started developing an electronic notebook system based on novel principles of database design patented by Knut Petter Lehre. The software is further developed and commercialized by Science Linker AS (Oslo Norway), and it is now used to manage the animal laboratory facilities at our institute and at Oslo University Hospital.
- Zhou Y, Dhaher R, Parent M, Hu QX, Hassel B, Yee SP, Hyder F, Gruenbaum SE, Eid T, Danbolt NC (2019) Selective deletion of glutamine synthetase in the mouse cerebral cortex induces glial dysfunction and vascular impairment that precede epilepsy and neurodegeneration. Neurochem Int. 123:22-33.
- Zhou Y, Hassel B, Eid T, Danbolt NC (2019) Axon-terminals expressing EAAT2 (GLT-1; Slc1a2) are common in the forebrain and not limited to the hippocampus. Neurochem Int. 123:101-113.
- Ottestad-Hansen S, Hu QX, Follin-Arbelet VV, Bentea E, Sato H, Massie A, Zhou Y, Danbolt NC (2018) The cysteine-glutamate exchanger (xCT, Slc7a11) is expressed in significant concentrations in a subpopulation of astrocytes in the mouse brain. Glia 66(5):951-970.
- Danbolt NC, Zhou Y, Furness DN, Holmseth S (2016) Strategies for immunohistochemical protein localization using antibodies: what did we learn from neurotransmitter transporters in glial cells and neurons. Glia 64:2045-2064.
- Danbolt NC, Furness DN, Zhou Y (2016) Neuronal vs glial glutamate uptake: resolving the conundrum. Neurochem Int 98:29-45.
- Zhou Y, Waanders LF, Holmseth S, Guo C, Berger UV, Li Y, Lehre A-C, Lehre KP, Danbolt NC (2014a) Proteome analysis and conditional deletion of the EAAT2 glutamate transporter provide evidence against a role of EAAT2 in pancreatic insulin secretion in mice. J Biol Chem 289:1329-1344.
- Zhou Y, Wang XY, Tzingounis AV, Danbolt NC, Larsson HP (2014b) EAAT2 (GLT-1,, slc1a2) glutamate transporters reconstituted in liposomes argues against heteroexchange being substantially faster than net uptake. J Neurosci 34:13472–13485.
- Zhou Y, Danbolt NC (2013) GABA and Glutamate Transporters in Brain. Front Endocrinol (Lausanne) 4:165.
- Holmseth S, Dehnes Y, Huang YH, Follin-Arbelet VV, Grutle NJ, Mylonakou MN, Plachez C, Zhou Y, Furness DN, Bergles DE, Lehre KP, Danbolt NC (2012) The Density of EAAC1 (EAAT3) Glutamate Transporters Expressed by Neurons in the Mammalian CNS. J Neurosci 32:6000-6013.
- Zhou Y, Holmseth S, Guo C, Hassel B, Hofner G, Huitfeldt HS, Wanner KT, Danbolt NC (2012) Deletion of the gamma-Aminobutyric Acid Transporter 2 (GAT2 and SLC6A13) Gene in Mice Leads to Changes in Liver and Brain Taurine Contents. J Biol Chem 287:35733-35746.
- Zhou Y, Holmseth S, Hua R, Lehre AC, Olofsson AM, Poblete-Naredo I, Kempson SA, Danbolt NC (2012) The betaine-GABA transporter (BGT1, slc6a12) is predominantly expressed in the liver and at lower levels in the kidneys and at the brain surface. Am J Physiol Renal Physiol 302:F316-328.
- Mathiisen TM, Lehre KP, Danbolt NC, Ottersen OP (2010) The perivascular astroglial sheath provides a complete covering of the brain microvessels: an electron microscopic 3D reconstruction. Glia 58:1094-1103.
- Furness D, Dehnes Y, Akhtar A, Rossi D, Hamann M, Grutle N, Gundersen V, Holmseth S, Lehre KP, Ullensvang K, Wojewodzic M, Zhou Y, Attwell D, Danbolt NC (2008) A quantitative assessment of glutamate uptake into hippocampal synaptic terminals and astrocytes: New insights into a neuronal role for excitatory amino acid transporter 2 (EAAT2). Neuroscience 157:80-94.
- Danbolt NC (2001) Glutamate uptake. Progress in Neurobiology 65:1-105.
- Trotti D, Rolfs A, Danbolt NC, Brown RH jr., Hediger MA (1999) SOD1 mutants linked to amyotrophic lateral sclerosis selectively inactivate a glial glutamate transporter. Nature Neuroscience 2:427-433.
- Lehre KP, Danbolt NC (1998) The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain. Journal of Neuroscience 18:8751-8757.
- Levy LM, Lehre KP, Walaas SI, Storm-Mathisen J, Danbolt NC (1995) Down-regulation of glial glutamate transporters after glutamatergic denervation in the rat brain. Eur J Neurosci 7:2036-2041.
- Dehnes Y, Chaudhry FA, Ullensvang K, Lehre KP, Storm-Mathisen J, Danbolt NC (1998) The glutamate transporter EAAT4 in rat cerebellar Purkinje cells: a glutamate- gated chloride channel concentrated near the synapse in parts of the dendritic membrane facing astroglia. Journal of Neuroscience 18:3606-3619.
- Haugeto Ø, Ullensvang K, Levy LM, Chaudhry FA, Honoré T, Nielsen M, Lehre KP, Danbolt NC (1996) Brain glutamate transporter proteins form homomultimers. Journal of Biological Chemistry 271:27715- 27722.
- Lehre KP, Levy LM, Ottersen OP, Storm-Mathisen J, Danbolt NC (1995) Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. Journal of Neuroscience 15:1835- 1853.
- Chaudhry FA, Lehre KP, Campagne MV, Ottersen OP, Danbolt NC, Storm-Mathisen J (1995) Glutamate transporters in glial plasma membranes: highly differentiated localizations revealed by quantitative ultrastructural immunocytochemistry. Neuron 15:711-720.
- Pines G, Danbolt NC, Bjørås M, Zhang Y, Bendahan A, Eide L, Koepsell H, Storm-Mathisen J, Seeberg E, Kanner BI (1992) Cloning and expression of a rat brain L- glutamate transporter. Nature 360:464-467.
- Danbolt NC, Storm-Mathisen J, Kanner BI (1992) An [Na++ K+]coupled L-glutamate transporter purified from rat brain is located in glial cell processes. Neuroscience 51:295-310.
- Danbolt NC, Pines G, Kanner BI (1990) Purification and reconstitution of the sodium- and potassium-coupled glutamate transport glycoprotein from rat brain. Biochemistry US 29:6734-6740.