脉络丛 LAT2 和 SNAT3 作为 CSF 氨基酸稳态维持的伴侣。
Choroid plexus LAT2 and SNAT3 as partners in CSF amino acid homeostasis maintenance.
机构信息
Institute of Physiology, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
Genes, Disease and Therapy Program, Molecular Genetics Laboratory-IDIBELL, Barcelona, Spain.
出版信息
Fluids Barriers CNS. 2020 Feb 11;17(1):17. doi: 10.1186/s12987-020-0178-x.
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.
背景
脑脊液（CSF）主要由位于脑室内的脉络丛（CP）产生。尽管 CSF 源自血浆，但它几乎不含蛋白质（~比血浆低 250 倍），并且游离氨基酸含量也低 2-20 倍，除了谷氨酰胺（Gln），其含量几乎相等。本研究的目的是确定在小鼠 CP 上皮细胞中表达哪些氨基酸转运体，以便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 评估分离的脉络丛（CPs）中氨基酸转运体的表达，然后使用特异性抗体进行免疫荧光定位研究。通过测定 LAT2（Slc7a8）反向转运体缺失对 CSF 氨基酸的影响来确定其作用。
结果
使用特定标志物（特别是转甲状腺素蛋白（Ttr））在 mRNA 水平上测试分离的脉络丛的纯度，Ttr 在 CP 中的富集度比脑组织高 330 倍。在第一轮实验中，通过 qPCR 对 32 个 Slc 氨基酸转运体中的 14 个进行了测试，选择了其中 5 个进一步研究。在这 5 个中，有 3 个被认为高度表达，即 SNAT1（Slc38a1）、SNAT3（Slc38a3）和 LAT2（Slc7a8）。通过免疫荧光观察到其中 3 个：SNAT1（Slc38a1）是一种中性氨基酸-Na 同向转运体，位于 CP 上皮细胞的基底外侧膜的血液侧，而 SNAT3（Slc38a3）是一种氨基酸-Na 同向转运体和 H 反向转运体，以及 LAT2（Slc7a8）是一种中性氨基酸反向转运体，定位于 CSF 面向的腔膜。在 LAT2 敲除小鼠模型中，CSF 谷氨酰胺没有变化，而其他氨基酸通常比血浆低 2-20 倍，增加了，特别是 LAT2 摄取的底物亮氨酸（Leu）、缬氨酸（Val）和色氨酸（Trp）以及其他一些氨基酸，如谷氨酸（Glu）、甘氨酸（Gly）和脯氨酸（Pro）。
结论
这些结果表明，Gln 通过 SNAT1 从血液主动转运到 CP 上皮细胞，然后通过 SNAT3 和 LAT2 从腔面释放到 CSF 中。它通过 LAT2 的外排可能会促使该反向转运体的必需氨基酸底物从 CSF 中再摄取，从而有助于维持血浆和 CSF 之间的氨基酸梯度。
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