Functional Polarity of Microvascular Brain Endothelial Cells Supported by Neurovascular Unit Computational Model of Large Neutral Amino Acid Homeostasis

The homeostatic regulation of large neutral amino acid (LNAA) concentration in the brain interstitial fluid (ISF) is essential for proper brain function. LNAA passage into the brain is primarily mediated by the complex and dynamic interactions between various solute carrier (SLC) transporters expressed in the neurovascular unit (NVU), among which SLC7A5/LAT1 is considered to be the major contributor in microvascular brain endothelial cells (MBEC). The LAT1-mediated trans-endothelial transport of LNAAs, however, could not be characterized precisely by available in vitro and in vivo standard methods so far. To circumvent these limitations, we have incorporated published in vivo data of rat brain into a robust computational model of NVU-LNAA homeostasis, allowing us to evaluate hypotheses concerning LAT1-mediated trans-endothelial transport of LNAAs across the blood brain barrier (BBB). We show that accounting for functional polarity of MBECs with either asymmetric LAT1 distribution between membranes and/or intrinsic LAT1 asymmetry with low intraendothelial binding affinity is required to reproduce the experimentally measured brain ISF response to intraperitoneal (IP) L-tyrosine and L-phenylalanine injection. On the basis of these findings, we have also investigated the effect of IP administrated L-tyrosine and L-phenylalanine on the dynamics of LNAAs in MBECs, astrocytes and neurons. Finally, the computational model was shown to explain the trans-stimulation of LNAA uptake across the BBB observed upon ISF perfusion with a competitive LAT1 inhibitor.


Evaluation of brain ISF post-stimuli responses under the assumption of LAT1 as the dominant LNAA transporter in astrocytes
As mentioned in the Introduction section, several studies have shown that LAT2 is the dominant LNAA transporter in primary astrocyte cells (Yudkoff et al., 1996a;Kim et al., 2004;Braun et al., 2011). However, Zhang Y, et al. (Zhang et al., 2014) showed that freshly isolated astrocytes specifically express higher levels of LAT1 mRNA compared LAT2 mRNA (Zhang et al., 2014).

Neu
The bi-directional and expression constant of MBEC LAT1 LAT1 in MBECs. To this end, we assumed LAT1 to be the dominant astrocytic LNAA transporter, replacing LAT2 in the astrocyte. In this situation, given that the intra-compartmental fluxes depend on the choice of the dominant transporters, we simply substituted the parameters of Eq. (7) (Methods section) with those of LAT1 for astrocytes (reported in Suppl. Table 3) and similarly calculated the baseline (pre-stimulus) state of the NVU system as reported in Suppl. Table 3. We then calculated the post-stimulus response of LNAA concentrations (Suppl. Fig.1) upon perturbation of plasma Ltyrosine and L-phenylalanine concentrations ( Figs. 2A and 2C). All model parameters except for kinetic parameters specific to LAT1 in the astrocyte (Suppl. Table 3) remain the same as in the nominal model (Table 1). The bi-directional kinetic constant for astrocyte LAT1 is considered equal to the corresponding value in the MBEC LAT1 ( RK LAT1 ). The time evolution of the plasma concentration of L-tyrosine and L-phenylalanine and of competing LNAAs is plotted as percentage of the baseline values in Suppl. Fig. 1. The error bars given for the baseline concentrations were calculated based on sensitivity studies as described in the Sensitivity analysis section. We compared the model predictions for the ISF response to IP L-tyrosine and L-phenylalanine injection with results of in vivo measurements (Bongiovanni et al., 2003;Bongiovanni et al., 2010), in the range from 1 (symmetric case) and 1300 (highly asymmetric bi-directional kinetics (Meier et al., 2002)) (Suppl. Fig. 1A,C). We found a close agreement between our model calculations and experimental measurements assuming asymmetric MBEC LAT1 kinetics (best with RK LAT1 = 220 and 45, respectively, for L-tyrosine and L-phenylalanine IP injection cases), while the model failed to reproduce the experimental data when the bi-directional kinetics of LAT1 were assumed to be symmetric in MBECs (Suppl. Fig. 1A, C).
To evaluate the hypothesis of asymmetric distribution of LAT1 at the BBB, we varied the abluminal to luminal expression ratio of LAT1, RE LAT1 , between 0.01 to 10 (representing highly asymmetric abluminal to luminal expression ratio) while we assumed the bi-directional kinetics of LAT1 to be symmetric ( RK LAT1 = 1). The numerical results obtained with asymmetric transporter expression agreed well with in vivo experimental data, best for an expression kinetic constant of RE LAT1 =0.12.
Taken together, our results show that assuming LAT1 rather than LAT2 as the dominant astrocytic AAT does not affect our conclusion on functional polarity of MBECs with either strong asymmetric kinetics of LAT1 and/or its expression at the BBB. Further exploration of the correlation between astrocyte mRNA is required to characterize the function of LAT1 in astrocytes in vivo.

RE LAT1
a In this column, TL and CL represent L-tyrosine and L-tyrosine competing LNAAs, respectively. b In this column, TL and CL represent Lphenylalanine and L-phenylalanine competing LNAAs, respectively. c The kinetic parameters for the mixture of L-tyrosine and L-phenylalanine competing LNAAs are calculated based on Eq.10 (Suppl.    . Table 3), respectively. Each experimental data point represents the mean ± SD for three (plasma) and four to eight (ISF) animals (Bongiovanni et al., 2003). The error bar associated with model calculations indicate the standard deviation determined based on the sensitivity analysis results. Panel (C) and (D) show the experimental data for L-phenylalanine (Phe) concentration in the brain ISF (prefrontal cortex (PFC)) (Bongiovanni et al., 2010) in response to IP administration of 200 mg/kg L-phenylalanine (as plotted in Fig. 2D), versus the model calculations (model input is shown in Fig.2D) for various ratios of the bi-directional kinetic constant of LAT1 in MBEC and astrocytes (RK LAT1 ) as well as different abluminal to luminal expression ratios of LAT1 (RE LAT1 ). In panels (C) and (D), the ISF baseline value for L-phenylalanine is 0.4 µM as reported in Suppl. Table 3 Table 4).