Fig 1.
The percentage distribution and chemical structures of the two amisulpride microspecies found at physiological pH.
Microspecies A has a single positive charge and Microspecies B has no charge, according to MarvinSketch 22.9.0.
Fig 2.
Physicochemical characteristics of GLUT1 substrates and inhibitors.
Evaluation of the PubMed database identified 9 substrates and 33 inhibitors of GLUT1, and 11 antipsychotics which inhibited cell entry of GLUT substrates. A) Comparison of the molecular weight (g/mol) of GLUT1 substrates, GLUT1 inhibitors and GLUT interacting antipsychotics. Each square, dot and triangle represents a compound. Comparison of the predicted gross charge of GLUT1 substrates, GLUT1 inhibitors and GLUT interacting antipsychotics at pH = 7.4, according to MarvinSketch 22.9.0. Data was analysed using One-way ANOVA, GraphPad Prism 9 followed by Tukey’s multiple comparisons test ****p<0.0001. B) The pie charts show the molecular weight of the GLUT1 substrates, GLUT1 inhibitors and G:UT-interacting antipsychotics which inhibit cell entry of GLUT substrates. Data was analysed using One-way ANOVA, GraphPad Prism 9 followed by Tukey’s multiple comparisons test **p<0.01, ****p<0.0001. C) The pie charts show the charge of the most prevalent microspecies of the GLUT1 substrates, GLUT1 inhibitors, and antipsychotics interacting with GLUT at physiological pH according to MarvinSketch 22.9.0.
Table 1.
Summary of the MW range, Mean±SEM, median (g/mol) and gross charge at physiological pH of GLUT1 substrates, GLUT1 inhibitors and GLUT interacting antipsychotics.
Fig 3.
Molecular level interactions of amisulpride, alpha-D-glucose, beta-D-glucose and sucrose with the binding sites of GLUT1.
2D (left) and 3D (right) representations. In the 2D representations, green dotted lines are used to show hydrogen bonds, pink dotted lines are used to depict hydrophobic interactions. A) Stick representation is used for amisulpride and line representation for the amino acid residues from GLUT1. B) Stick representation is used for alpha-D-glucose and line representation for the amino acid residues from GLUT1. C) Stick representation is used for beta-D-glucose and line representation for the amino acid residues from GLUT1. D) Stick representation is used for sucrose and line representation for the amino acid residues from GLUT1.
Fig 4.
Expression and function of glucose transporters in hCMEC/D3 cells–self inhibition.
A) GLUT1 expression in hCMEC/D3 cells. Three passages of hCMEC/D3 cells (P30, P31 and P33) (30 μg of protein per well) were tested for GLUT1 (40–60 kDa) expression. The figure is an example membrane of three technical repeats. Caco-2 cell lysate was used as a positive control; HEK-293 cell lysate was used as a negative control. GAPDH (37 kDa) was used as a loading control. Antibodies used: anti-GLUT1 antibody– 1:100 000, #ab115730; anti-GAPDH antibody– 1:2500, #ab9485, Abcam; secondary anti-rabbit IgG, HRP-linked antibody– 1:2000, #7074, Cell Signalling Technology. B) Vd of [3H]mannitol was not significantly different between the control and the experimental conditions. C) Non-labelled glucose significantly decreased the accumulation of [14C]D-glucose ([3H]mannitol corrected) in hCMEC/D3 cells after 1 h of incubation. Results are expressed as mean ± SEM, n = 3–4 plates, passages (P33, P34 and P35) with five well replicates per treatment in each plate. Data were analysed with an unpaired one-tailed Student’s t-test, using GraphPad Prism 9, each point represents a plate *p<0.05.
Fig 5.
Interaction of amisulpride with glucose transporters in hCMEC/D3 cells.
A) Amisulpride at three different concentrations (20, 50 or 100 μM) did not have a significant effect on the accumulation of [3H]mannitol in hCMEC/D3 cells after 1 h of incubation. B) Amisulpride at three different concentrations (20, 50 or 100 μM) did not have a significant effect on the accumulation of [14C]glucose ([3H]mannitol corrected) in hCMEC/D3 cells after 1 h of incubation. Results are expressed as mean ± SEM, n = 3–4 plates, passages (P33, P34 and P35) with five well replicates per treatment in each plate. Data were analysed with a One-way ANOVA, using GraphPad Prism 9, each point represents a plate.
Fig 6.
TEM ultrastructural study in WT and 5xFAD mice.
TEM image of the frontal cortex of A) WT mouse, which is free of Aβ plaques and B) 5xFAD mouse, with Aβ plaques. Mouse age– 4.5–6 months. Magnification– 1200x, scale bar– 10 μm, n = 2 mice per group.
Fig 7.
[3H]Amisulpride ([14C]sucrose corrected) and [14C]sucrose uptake into the brain of WT and 5xFAD mice.
WT and 5xFAD mice were perfused with [3H]amisulpride and [14C]sucrose. Perfusion time—10 minutes, fluid flow rate– 5.5 ml/min. Mouse age: 12–15 months. WT n = 6, 5xFAD n = 4, apart for the hypothalamus where WT n = 5, 5xFAD n = 4; the capillary pellet where WT n = 3, 5xFAD n = 3, and the homogenate where WT n = 6, 5xFAD n = 3. No significant differences in paracellular permeability and membrane integrity were observed in any region. Each dot represents data from one mouse. All data are expressed as mean ± SEM. A: The effect of genotype in the different brain regions was analysed by mixed-effects analysis with Holm-Sidak post hoc test. B: The effect of genotype on the capillary depletion samples which included the whole brain homogenate, brain parenchyma containing supernatant and endothelial cell enriched pellet samples were analysed by unpaired two-tailed Student’s t-test. No significant effect was observed except between the supernatant samples of 5xFAD mice compared to WT mice (*p<0.05). Statistical analysis was performed using GraphPad Prism 9.
Fig 8.
TEM image of capillaries in the frontal cortex (A) putamen (B) and caudate (C) of a human AD case. Frontal cortex and caudate magnification– 3000x scale bar– 2 μm. Putamen magnification– 2000x, scale bar– 5 μm. n = 1 case, two sections per brain area were stained and imaged, 5 to 10 images were examined per section. Case number: BBN002.32856; Sex: F; Age: 74; PM delay: 19 h; Alzheimer’s disease, BNE stage VI.
Fig 9.
TEM image of the A) frontal cortex (A and C), putamen (B) and caudate (D) of human AD case. A) TEM image of the frontal cortex of human AD case. B) TEM image of the putamen of human AD case. A and B showing electron dense and lucid portions of lipofuscin granules. Magnification– 1500x, scale bar– 5 μm. C) TEM image of degenerating neurites and degenerating myelinated axon in the frontal cortex of a human AD case. Magnification– 6000x, scale bar– 2 μm. D) TEM image of myelin and axon degeneration in the caudate of a human AD case. Magnification– 2500x, scale bar– 5 μm. Case, n = 1, two sections per brain area were stained and imaged, 5 to 10 images were examined per section. Case number: BBN002.32856; Sex: F; Age: 74; PM delay: 19 h; Alzheimer’s disease, BNE stage VI.
Fig 10.
TfR1, GLUT1 and P-gp expression in human frontal cortex and caudate brain capillary lysates from human control and AD age-matched cases.
The expression of the different transporters was not necessarily assessed in the same samples. The samples were not randomized. Intensity ratio was calculated using the intensity of the band of TfR1, GLUT1 or P-gp and controlling it for the intensity of the loading control GAPDH. Results are presented as mean±SEM, each dot represents a case, data was analysed using unpaired two-tailed Student’s t-test. *P<0.05. All analysis was performed using Image J, Excel and GraphPad Prism 9.