Functional expression of carnitine/organic cation transporter OCTN1 in mouse brain neurons: Possible involvement in neuronal differentiation |
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Authors: | Noritaka Nakamichi Takayuki Taguchi Hiroshi Hosotani Tomohiko Wakayama Takuya Shimizu Tomoko Sugiura Shoichi Iseki Yukio Kato |
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Institution: | 1. Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan;2. Department of Histology and Embryology, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan |
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Abstract: | The aim of the present study is to clarify the functional expression and physiological role in brain neurons of carnitine/organic cation transporter OCTN1/SLC22A4, which accepts the naturally occurring antioxidant ergothioneine (ERGO) as a substrate in vivo. After intracerebroventricular administration, the distribution of 3H]ERGO in several brain regions of octn1−/− mice was much lower than that in wild-type mice, whereas extracellular marker 14C]mannitol exhibited similar distribution in the two strains. The 3H]ERGO distribution in wild-type mice was well correlated with the amount of ERGO derived from food intake and the OCTN1 mRNA level in each brain region. Immunohistochemical analysis revealed colocalization of OCTN1 with neuronal cell markers microtubule-associated protein 2 (MAP2) and βIII-tubulin in mouse brain and primary cultured cortical neurons, respectively. Moreover, cultured cortical neurons exhibited time-dependent and saturable uptake of 3H]ERGO. These results demonstrate that OCTN1 is functionally expressed in brain neurons. The addition of ERGO simultaneously with serum to culture medium of cortical neurons attenuated mRNA and protein expressions of MAP2, βIII-tubulin and synapse formation marker synapsin I, and induced those of sex determining region Y-box 2 (Sox2), which is required to maintain the properties of undifferentiated neural stem cells. In neuronal model Neuro2a cells, knockdown of OCTN1 by siRNA reduced the uptake of 3H]ERGO with concomitant up-regulation of oxidative stress marker HO-1 and Sox2, and down-regulation of neurite outgrowth marker GAP43. Interestingly, the siRNA knockdown decreased the number of differentiated Neuro2a cells showing long neurites, but increased the total number of cells. Thus, OCTN1 is involved in cellular differentiation, but inhibits their proliferation, possibly via the regulation of cellular oxidative stress. This is the first evidence that OCTN1 plays a role in neuronal differentiation and proliferation, which are required for brain development. |
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Keywords: | ATRA all-trans retinoic acid CL cerebellum CX cerebral cortex DIV days in vitro DMEM Dulbecco&rsquo s modified Eagle&rsquo s medium ERGO ergothioneine FBS fetal bovine serum GAP43 growth-associated protein 43 HC hippocampus HO-1 heme oxygenase-1 HPLC high-performance liquid chromatography HT hypothalamus i c v intracerebroventricular MAP2 microtubule-associated protein 2 MB midbrain MP medulla and pons Nrf2 nuclear factor-erythroid 2 p45-related factor-2 OCTN1 carnitine/organic cation transporter 1 PA paraformaldehyde PBS phosphate-buffered saline ROS reactive oxygen species siOCTN1 siRNA targeting the mouse OCTN1 gene siRNA small interfering RNA SLC solute carrier Sox2 sex determining region Y-box 2 ST striatum Syn1 synapsin I xCT cystine glutamate exchanger subunit Ucp2 mitochondrial uncoupling protein 2 36B4 acidic ribosomal phosphoprotein P0 |
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