Choroid plexus LAT2 and SNAT3 as partners in CSF amino acid homeostasis maintenance

Elena Dolgodilina,Brigitte Herzog,Simone M Camargo,Francois Verrey,Eva Roth,Manuel Palacín,Virginia Nunes

doi:10.1186/s12987-020-0178-x

Abstract

BackgroundCerebrospinal 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.MethodsExpression 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.ResultsThe 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).ConclusionThese 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.

Highlights

Cerebrospinal fluid (CSF) is mainly produced by the choroid plexus (CP) located in brain ventricles
Amino acid transporters expressed in choroid plexus To study the expression of specific amino acid transporters in CP, we tested first their mRNA levels, they are known not to correlate with protein expression
We tested initially the purity of the CPs isolated from the four ventricles of each individual animal by measuring the mRNA level of four cell specific markers by qPCR, transthyretin (Ttr) as choroidal marker, glial fibrillary acidic protein (Gfap) for astrocytes, platelet endothelial cell adhesion molecule-1 (Pecam 1 or Cd31) for brain endothelial cells and synaptophysin (Syp) for neurons (Fig. 1a)

Summary

Introduction

Cerebrospinal fluid (CSF) is mainly produced by the choroid plexus (CP) located in brain ventricles. Strong concentration gradients between plasma and CSF were reported for proteins (~ 250 fold) and proteinogenic amino acids (AAs) [2–20-fold, with the exception of glutamine (Gln)] in independent studies [3,4,5]. In the case of ALS data about changes in the level of the major excitatory neurotransmitter glutamate (Glu) remains controversial, while neutral non-essential amino acids as Gln and alanine (Ala) have been reported to be elevated in two independent studies [6,7,8]. CSF Glu concentration was shown to raise slightly during the brain disorder essential tremor, while the levels of other neurotransmitters (aspartate (Asp), GABA) and some amino acids [serine (Ser), threonine (Thr), Gln, glycine (Gly) and ornithine (Orn)] declined [9]. These numerous observations confirm that knowledge about the regulatory mechanisms underlying the maintenance of CSF AA homeostasis is important and relevant for clinical practice

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