Background Cerebrospinal fluid (CSF) is mainly produced by the choroid plexus (CP) located in brain ventricles. Although derived from blood plasma, it is nearly protein-free (~ 250-fold less) and contains about 2–20-fold less free amino acids, with the exception of glutamine (Gln) which is nearly equal. The aim of this study was to determine which amino acid transporters are expressed in mouse CP epithelium in order to gain understanding about how this barrier maintains the observed amino acid concentration gradient. Methods Expression of amino acid transporters was assessed in isolated choroid plexuses (CPs) by qRT-PCR followed by localization studies using immunofluorescence with specific antibodies. The impact of LAT2 ( Slc7a8 ) antiporter deletion on CSF amino acids was determined. Results The purity of isolated choroid plexuses was tested on the mRNA level using specific markers, in particular transthyretin (Ttr) that was enriched 330-fold in CP compared to cerebral tissue. In a first experimental round, 14 out of 32 Slc amino acid transporters tested on the mRNA level by qPCR were selected for further investigation. Out of these, five were considered highly expressed, SNAT1 ( Slc38a1 ), SNAT3 ( Slc38a3 ), LAT2 ( Slc7a8 ), ASC1 ( Slc7a10 ) and SIT1 ( Slc6a20b ). Three of them were visualized by immunofluorescence: SNAT1 ( Slc38a1 ), a neutral amino acid-Na + symporter, found at the blood side basolateral membrane of CP epithelium, while SNAT3 ( Slc38a3 ), an amino acid-Na + symporter and H + antiporter, as well as LAT2 ( Slc7a8 ), a neutral amino acid antiporter, were localized at the CSF-facing luminal membrane. In a LAT2 knock-out mouse model, CSF Gln was unchanged, whereas other amino acids normally 2–20-fold lower than in plasma, were increased, in particular the LAT2 uptake substrates leucine (Leu), valine (Val) and tryptophan (Trp) and some other amino acids such as glutamate (Glu), glycine (Gly) and proline (Pro). Conclusion These results suggest that Gln is actively transported by SNAT1 from the blood into CP epithelial cells and then released luminally into CSF via SNAT3 and LAT2. Its efflux via LAT2 may drive the reuptake from the CSF of essential amino acid substrates of this antiporter and thereby participates to maintaining the amino acid gradient between plasma and CSF.

中文翻译：

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脉络丛 LAT2 和 SNAT3 作为 CSF 氨基酸稳态维持的伙伴

背景脑脊液 (CSF) 主要由位于脑室的脉络丛 (CP) 产生。虽然来源于血浆，但它几乎不含蛋白质（约少 250 倍），并且含有约 2-20 倍的游离氨基酸，谷氨酰胺 (Gln) 除外，几乎相等。本研究的目的是确定哪些氨基酸转运蛋白在小鼠 CP 上皮细胞中表达，以了解该屏障如何维持观察到的氨基酸浓度梯度。方法 通过 qRT-PCR 在分离的脉络丛 (CP) 中评估氨基酸转运蛋白的表达，然后使用具有特异性抗体的免疫荧光进行定位研究。确定了 LAT2 (Slc7a8) 逆向转运蛋白缺失对 CSF 氨基酸的影响。结果分离的脉络丛的纯度使用特定标记物在 mRNA 水平上进行测试，特别是与脑组织相比，在 CP 中富含 330 倍的转甲状腺素蛋白 (Ttr)。在第一轮实验中，通过 qPCR 在 mRNA 水平上测试的 32 个 Slc 氨基酸转运蛋白中的 14 个被选择用于进一步研究。其中，有五个被认为是高度表达的，SNAT1 (Slc38a1)、SNAT3 (Slc38a3)、LAT2 (Slc7a8)、ASC1 (Slc7a10) 和 SIT1 (Slc6a20b)。其中三个通过免疫荧光显示：SNAT1 ( Slc38a1 )，一种中性氨基酸-Na + 同向转运蛋白，发现于 CP 上皮的血液侧基底外侧膜，而 SNAT3 ( Slc38a3 )，一种氨基酸-Na + 同向转运蛋白和 H + 反向转运蛋白以及 LAT2 (Slc7a8)，一种中性氨基酸反向转运蛋白，位于面向脑脊液的腔膜上。在 LAT2 敲除小鼠模型中，CSF Gln 没有变化，而其他氨基酸通常比血浆中低 2-20 倍，增加了，尤其是 LAT2 摄取底物亮氨酸 (Leu)、缬氨酸 (Val) 和色氨酸。 Trp) 和其他一些氨基酸，如谷氨酸 (Glu)、甘氨酸 (Gly) 和脯氨酸 (Pro)。结论 这些结果表明 Gln 被 SNAT1 从血液主动转运到 CP 上皮细胞，然后通过 SNAT3 和 LAT2 腔内释放到 CSF 中。它通过 LAT2 的外流可能会驱动脑脊液中这种反向转运蛋白的必需氨基酸底物的再摄取，从而参与维持血浆和脑脊液之间的氨基酸梯度。特别是 LAT2 摄取底物亮氨酸 (Leu)、缬氨酸 (Val) 和色氨酸 (Trp) 以及一些其他氨基酸，如谷氨酸 (Glu)、甘氨酸 (Gly) 和脯氨酸 (Pro)。结论 这些结果表明 Gln 被 SNAT1 从血液主动转运到 CP 上皮细胞，然后通过 SNAT3 和 LAT2 腔内释放到 CSF 中。它通过 LAT2 的外流可能会驱动脑脊液中这种反向转运蛋白的必需氨基酸底物的再摄取，从而参与维持血浆和脑脊液之间的氨基酸梯度。特别是 LAT2 摄取底物亮氨酸 (Leu)、缬氨酸 (Val) 和色氨酸 (Trp) 以及一些其他氨基酸，如谷氨酸 (Glu)、甘氨酸 (Gly) 和脯氨酸 (Pro)。结论 这些结果表明 Gln 被 SNAT1 从血液主动转运到 CP 上皮细胞，然后通过 SNAT3 和 LAT2 腔内释放到 CSF 中。它通过 LAT2 的外流可能会驱动脑脊液中这种反向转运蛋白的必需氨基酸底物的再摄取，从而参与维持血浆和脑脊液之间的氨基酸梯度。