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Characterization of Centrally Expressed Solute Carriers: Histological and Functional Studies with Transgenic Mice
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
: His (English)
Abstract [en]

The Solute Carrier (SLC) superfamily is the largest group of membrane-bound transporters, currently with 456 transporters in 52 families. Much remains unknown about the tissue distribution and function of many of these transporters. The aim of this thesis was to characterize select SLCs with emphasis on tissue distribution, cellular localization, and function.       In paper I, we studied the leucine transporter B0AT2 (Slc6a15). Localization of B0AT2 and Slc6a15 in mouse brain was determined using in situ hybridization (ISH) and immunohistochemistry (IHC), localizing it to neurons, epithelial cells, and astrocytes. Furthermore, we observed a lower reduction of food intake in Slc6a15 knockout mice (KO) upon intraperitoneal injections with leucine, suggesting B0AT2 is involved in mediating the anorexigenic effects of leucine.     In paper II, we studied the postnatal, forebrain-specific deletion of Slcz1, belonging to the SLC18 family, in conditional KO mice (cKO). We observed a decreased response to diazepam and a higher neuronal activity in cortex and hippocampus of cKO mice, as well as an impairment in short-term recognition memory. Intracellular expression was found in neurons but not astrocytes with IHC, indicating SLCZ1 is implicated in neuronal regulation of locomotion and memory.    In paper III, we performed the first detailed histological analysis of PAT4, a transporter belonging to the SLC36 family, involved in the activation of mTOR complex 1 on lysosomes. We found abundant Slc36a4 mRNA and PAT4 expression in mouse brain, using ISH and IHC. We used IHC to localize PAT4 to both inhibitory and excitatory neurons and epithelial cells. We also found both intracellular- and plasmalemmal expression and partial colocalization of PAT4 with lysosomal markers.    Lastly, in paper IV, we provided the first tissue mapping of orphan transporter MCT14 (SLC16A14). Using qPCR, we detected moderate to high Slc16a14 mRNA in the central nervous system and kidney. We found widespread Slc16a14 and MCT14 in mouse brain using ISH and IHC. We also found MCT14 to have intracellular and plasmalemmal expression in mainly excitatory but also inhibitory neurons, as well as epithelial cells. We found MCT14 to be most closely related to MCT8, MCT2 and MCT9, suggesting a similar role for this transporter.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 62 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1215
Keyword [en]
SLC, Solute carriers, Amino acid transporter, PAT, B0AT2, mTORC1
National Category
Neurosciences
Research subject
Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-282956ISBN: 978-91-554-9555-8 (print)OAI: oai:DiVA.org:uu-282956DiVA: diva2:918151
Public defence
2016-06-03, B:21, Husargatan. 75124 Uppsala, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2016-05-12 Created: 2016-04-08 Last updated: 2016-06-01
List of papers
1. B(0)AT2 (SLC6A15) is localized to neurons and astrocytes, and is involved in mediating the effect of leucine in the brain
Open this publication in new window or tab >>B(0)AT2 (SLC6A15) is localized to neurons and astrocytes, and is involved in mediating the effect of leucine in the brain
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 3, e58651- p.Article in journal (Refereed) Published
Abstract [en]

The B(0)AT2 protein is a product of the SLC6A15 gene belonging to the SLC6 subfamily and has been shown to be a transporter of essential branched-chain amino acids. We aimed to further characterize the B(0)AT2 transporter in CNS, and to use Slc6a15 knock out (KO) mice to investigate whether B(0)AT2 is important for mediating the anorexigenic effect of leucine. We used the Slc6a15 KO mice to investigate the role of B(0)AT2 in brain in response to leucine and in particular the effect on food intake. Slc6a15 KO mice show lower reduction of food intake as well as lower neuronal activation in the ventromedial hypothalamic nucleus (VMH) in response to leucine injections compared to wild type mice. We also used RT-PCR on rat tissues, in situ hybridization and immunohistochemistry on mouse CNS tissues to document in detail the distribution of SLC6A15 on gene and protein levels. We showed that B(0)AT2 immunoreactivity is mainly neuronal, including localization in many GABAergic neurons and spinal cord motor neurons. B(0)AT2 immunoreactivity was also found in astrocytes close to ventricles, and co-localized with cytokeratin and diazepam binding inhibitor (DBI) in epithelial cells of the choroid plexus. The data suggest that B(0)AT2 play a role in leucine homeostasis in the brain.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-199769 (URN)10.1371/journal.pone.0058651 (DOI)000318334500110 ()23505546 (PubMedID)
Available from: 2013-05-13 Created: 2013-05-13 Last updated: 2017-12-06Bibliographically approved
2. Postnatal forebrain-specific deletion of neuronal transporter Slcz1
Open this publication in new window or tab >>Postnatal forebrain-specific deletion of neuronal transporter Slcz1
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(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-282955 (URN)
Available from: 2016-04-10 Created: 2016-04-08 Last updated: 2016-06-01
3. PAT4 is abundantly expressed in excitatory and inhibitory neurons as well as epithelial cells
Open this publication in new window or tab >>PAT4 is abundantly expressed in excitatory and inhibitory neurons as well as epithelial cells
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2014 (English)In: Brain Research, ISSN 0006-8993, E-ISSN 1872-6240, Vol. 1557, 12-25 p.Article in journal (Refereed) Published
Abstract [en]

PAT4, the fourth member of the SLC36/proton dependent amino acid transporter (PAT) family, is a high-affinity, low capacity electroneutral transporter of neutral amino acids like proline and tryptophan. It has also been associated with the function of mTORC1, a complex in the mammalian target of rapamycin (mTOR) pathway. We performed in situ hybridization and immunohistological analysis to determine the expression profile of PAT4, as well as an RT-PCR study on tissue from mice exposed to leucine. We performed a phylogenetic analysis to determine the evolutionary origin of PAT4. The in situ hybridization and the immunohistochemistry on mouse brain sections and hypothalamic cells showed abundant PAT4 expression in the mouse brain intracellularly in both inhibitory and excitatory neurons, partially co-localizing with lysosomal markers and epithelial cells lining the ventricles. Its location in epithelial cells around the ventricles indicates a transport of substrates across the blood brain barrier. Phylogenetic analysis showed that PAT4 belongs to an evolutionary old family most likely predating animals, and PAT4 is the oldest member of that family.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-220143 (URN)10.1016/j.brainres.2014.02.014 (DOI)000334081800002 ()24530433 (PubMedID)
Available from: 2014-03-11 Created: 2014-03-11 Last updated: 2017-12-05Bibliographically approved
4. Histological characterization of orphan transporter MCT14 (SLC16A14) shows abundant expression in mouse CNS and kidney
Open this publication in new window or tab >>Histological characterization of orphan transporter MCT14 (SLC16A14) shows abundant expression in mouse CNS and kidney
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2016 (English)In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 17, 43Article in journal (Refereed) Published
Abstract [en]

Background: MCT14 (SLC16A14) is an orphan member of the monocarboxylate transporter (MCT) family, also known as the SLC16 family of secondary active transmembrane transporters. Available expression data for this transporter is limited, and in this paper we aim to characterize MCT14 with respect to tissue distribution and cellular localization in mouse brain. Results: Using qPCR, we found that Slc16a14 mRNA was highly abundant in mouse kidney and moderately in central nervous system, testis, uterus and liver. Using immunohistochemistry and in situ hybridization, we determined that MCT14 was highly expressed in excitatory and inhibitory neurons as well as epithelial cells in the mouse brain. The expression was exclusively localized to the soma of neurons. Furthermore, we showed with our phylogenetic analysis that MCT14 most closely relate to the aromatic amino acid- and thyroid-hormone transporters MCT8 (SLC16A2) and MCT10 (SLC16A10), in addition to the carnitine transporter MCT9 (SLC16A9). Conclusions: We provide here the first histological mapping of MCT14 in the brain and our data are consistent with the hypothesis that MCT14 is a neuronal aromatic-amino-acid transporter.

Keyword
Monocarboxylate; Transporter; Mouse; Amino acid
National Category
Neurology
Identifiers
urn:nbn:se:uu:diva-282951 (URN)10.1186/s12868-016-0274-7 (DOI)000379823100001 ()27364523 (PubMedID)
Funder
Swedish Research CouncilThe Swedish Brain FoundationSwedish Society for Medical Research (SSMF)Novo Nordisk
Available from: 2016-04-10 Created: 2016-04-08 Last updated: 2017-11-30Bibliographically approved

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