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Characterization of Centrally Expressed Solute Carriers

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Characterization of Centrally Expressed Solute Carriers Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1215 Characterization of Centrally Expressed Solute Carriers Histological and Functional Studies with Transgenic Mice SAHAR ROSHANBIN ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-554-9555-8 UPPSALA urn:nbn:se:uu:diva-282956 2016 Dissertation presented at Uppsala University to be publicly examined in B:21, Husargatan. 75124 Uppsala, Uppsala, Friday, 3 June 2016 at 13:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Biträdande professor David Engblom (Institutionen för klinisk och experimentell medicin, Cellbiologi, Linköpings Universitet). Abstract Roshanbin, S. 2016. Characterization of Centrally Expressed Solute Carriers. Histological and Functional Studies with Transgenic Mice. (. His). Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1215. 62 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9555-8. 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. Keywords: SLC, Solute carriers, Amino acid transporter, PAT, B0AT2, mTORC1 Sahar Roshanbin, Department of Neuroscience, Functional Pharmacology, Box 593, Uppsala University, SE-75124 Uppsala, Sweden. © Sahar Roshanbin 2016 ISSN 1651-6206 ISBN 978-91-554-9555-8 urn:nbn:se:uu:diva-282956 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-282956) To my driving school instructor who told me to use my brain List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Hägglund, MG., Roshanbin, S., Löfqvist, E., Hellsten, SV., Nilsson, VC., Todkar, A., Zhu, Y., Stephansson, O., Drgonova, J., Uhl, GR., Schiöth, HB., Fredriksson, R. (2013) B0 AT2 (SLC6A15) Is Localized to Neurons and Astrocytes, and Is Involved in Mediating the Effect of Leucine in the Brain. PLOS one. 8(3): e58651 II Roshanbin, S., Westerfors, E., Sreedharan, S., Schiöth, HB., Fredriksson, R. :(2016) Postnatal forebrain-specific deletion of neuronal transporter Slcz1. Manuscript III Roshanbin, S., Hellsten, SV., Tafreshiha, A., Zhu, Y., Raine, A., Fredriksson. R. (2014) PAT4 is abundantly expressed in excitatory and inhibitory neurons as well as epithelial cells. Brain research. 1557: 12-25 IV Roshanbin, S., Lindberg, FA., Lekholm, E., Eriksson, MA., Perland, E., Raine, A., Åhlund, J., Fredriksson, R. (2016) His- tological characterization of orphan transporter MCT14 (SLC16A14) shows abundant expression in mouse CNS and kidney. Submitted manuscript Reprints were made with permission from the respective publishers. Additional Publications V Schiöth HB, Roshanbin S, Hägglund MG, Fredriksson R: Evolutionary origin of amino acid transporter families SLC32, SLC36 and SLC38 and physiological, pathological and therapeutic aspects. Mol Aspects Med 2013, 34(2-3):571-585. VI Roshanbin S, Classical Mouse Genetics. In: Stanley Maloy and Kelly Hughes, editors. Brenner's Encyclopedia of Genet- ics, 2nd edition, Vol 2. San Diego: Academic Press; 2013. p. 680-681 VII Caruso V, Hägglund MG, Badiali L, Bagchi S, Roshanbin S, Ahmad T, Schiöth HB, Fredriksson R: The G protein-coupled receptor GPR162 is widely distributed in the CNS and highly expressed in the hypothalamus and in hedonic feeding areas. Gene 2014, 553(1):1-6. VIII Rottkamp DM, Rudenko IA, Maier MT, Roshanbin S, Yulyaningsih E, Perez L, Valdearcos M, Chua S, Koliwad SK, Xu AW: Leptin potentiates astrogenesis in the developing hy- pothalamus. Mol Metab 2015, 4(11):881-889. Contents Introduction ................................................................................................... 11 Solute Carriers .......................................................................................... 12 Nomenclature and classification .............................................................. 13 Structure ................................................................................................... 15 SLC substrates .......................................................................................... 16 Localization of SLCs ................................................................................ 17 SLCs in drug transport ............................................................................. 17 SLCs as drug targets ................................................................................. 18 SLC-mediated transport in vesicles, peroxisomes and lysosomes ........... 20 SLCs in vesicles ................................................................................... 20 SLCs in peroxisomes and lysosomes ................................................... 21 SLCs and amino acid sensing: the mTOR and GCN pathways ............... 23 The mTOR pathway ............................................................................ 23 The GCN2 pathway ............................................................................. 26 The monocarboxylate transporter family ................................................. 27 Transport of monocarboxylates ........................................................... 29 Transport of aromatic amino acids ...................................................... 30 Transport of thyroid hormones ............................................................ 30 Orphan transporters ............................................................................. 31 Aims .............................................................................................................. 32 Paper I ...................................................................................................... 32 Paper II ..................................................................................................... 32 Paper III .................................................................................................... 33 Paper IV ................................................................................................... 33 Methodological considerations: mouse genetics ........................................... 34 Results and discussion .................................................................................. 38 Paper I ...................................................................................................... 38 Paper II ..................................................................................................... 39 Paper III .................................................................................................... 40 Paper IV ................................................................................................... 41 Closing remarks: conclusions and perspectives ............................................ 42 Paper I ...................................................................................................... 42 Paper II ..................................................................................................... 42 Paper III .................................................................................................... 43 Paper IV ................................................................................................... 44 Acknowledgments......................................................................................... 45 References
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