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Neurotransporters

The Neurotransporter Group focuses on the transporter proteins moving transmitter amino acids (GABA, glutamate and glycine) across cell membranes, and on the roles of these transporters in normal brain physiology and disease.

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.

Achievements

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.

Published Dec. 16, 2011 2:03 PM - Last modified Jan. 18, 2024 3:18 PM

Contact

Dept. of Molecular Medicine
Domus Medica
Gaustad
Sognsvannsveien 9
0372 Oslo
 

Group leader

Participants

Detailed list of participants