Interaction of amisulpride with GLUT1 at the blood-brain barrier. Relevance to Alzheimer’s disease

Blood-brain barrier (BBB) dysfunction may be involved in the increased sensitivity of Alzheimer's disease (AD) patients to antipsychotics, including amisulpride. Studies indicate that antipsychotics interact with facilitated glucose transporters (GLUT), including GLUT1, and that GLUT1 BBB expression decreases in AD. We tested the hypotheses that amisulpride (charge: +1) interacts with GLUT1, and that BBB transport of amisulpride is compromised in AD. GLUT1 substrates, GLUT1 inhibitors and GLUT-interacting antipsychotics were identified by literature review and their physicochemical characteristics summarised. Interactions between amisulpride and GLUT1 were studied using in silico approaches and the human cerebral endothelial cell line, hCMEC/D3. Brain distribution of [3H]amisulpride was determined using in situ perfusion in wild type (WT) and 5xFamilial AD (5xFAD) mice. With transmission electron microscopy (TEM) we investigated brain capillary degeneration in WT mice, 5xFAD mice and human samples. Western blots determined BBB transporter expression in mouse and human. Literature review revealed that, although D-glucose has no charge, charged molecules can interact with GLUT1. GLUT1 substrates are smaller (184.95±6.45g/mol) than inhibitors (325.50±14.40g/mol) and GLUT-interacting antipsychotics (369.38±16.04). Molecular docking showed beta-D-glucose (free energy binding: -15.39kcal/mol) and amisulpride (-29.04kcal/mol) interact with GLUT1. Amisulpride did not affect [14C]D-glucose hCMEC/D3 accumulation. [3H]amisulpride uptake into the brain (except supernatant) of 5xFAD mice compared to WT remained unchanged. TEM revealed brain capillary degeneration in human AD. There was no difference in GLUT1 or P-glycoprotein BBB expression between WT and 5xFAD mice. In contrast, caudate P-glycoprotein, but not GLUT1, expression was decreased in human AD capillaries versus controls. This study provides new details about the BBB transport of amisulpride, evidence that amisulpride interacts with GLUT1 and that BBB transporter expression is altered in AD. This suggests that antipsychotics could potentially exacerbate the cerebral hypometabolism in AD. Further research into the mechanism of amisulpride transport by GLUT1 is important for improving antipsychotics safety

Optogenetic cleavage of the Miro GTPase reveals the direct consequences of real-time loss of function in Drosophila.

Miro GTPases control mitochondrial morphology, calcium homeostasis, and regulate mitochondrial distribution by mediating their attachment to the kinesin and dynein motor complex. It is not clear, however, how Miro proteins spatially and temporally integrate their function as acute disruption of protein function has not been performed. To address this issue, we have developed an optogenetic loss of function "Split-Miro" allele for precise control of Miro-dependent mitochondrial functions in Drosophila. Rapid optogenetic cleavage of Split-Miro leads to a striking rearrangement of the mitochondrial network, which is mediated by mitochondrial interaction with the microtubules. Unexpectedly, this treatment did not impact the ability of mitochondria to buffer calcium or their association with the endoplasmic reticulum. While Split-Miro overexpression is sufficient to augment mitochondrial motility, sustained photocleavage shows that Split-Miro is surprisingly dispensable to maintain elevated mitochondrial processivity. In adult fly neurons in vivo, Split-Miro photocleavage affects both mitochondrial trafficking and neuronal activity. Furthermore, functional replacement of endogenous Miro with Split-Miro identifies its essential role in the regulation of locomotor activity in adult flies, demonstrating the feasibility of tuning animal behaviour by real-time loss of protein function

Split-Miro photocleavage does not affect [Ca²⁺]_m abundance.

(A) Representative image of an S2R+ cell showing mitochondrial targeting of mito-GCaMP6f (cyan) in the perinuclear region (white arrows) and in single mitochondria (arrowheads). Yellow is mCherry-tagged wt-Miro (wt-Miro); magenta is MitoTracker DeepRed (MTDR). Scale bar: 5 μm. (B) Cells transfected with mito-GCaMP6f and either mCherry-wt-Miro (wt-Miro) or mCherry-Split-Miro (Split-Miro) were stained with MTDR; and the ratio of mito-GCaMP6f/MTDR signal intensity was analysed at the beginning (first minute) and at the end (seventh minute) of the time-lapse imaging under blue light. Number of cells: wt-Miro = 12, Split-Miro = 16; from 3 independent experiments. Data are shown as mean ± SEM. Repeated measures one-way ANOVA followed by Tukey’s post hoc test did not show any significant difference between groups, indicating that the basal [Ca2+]m does not significantly change after Split-Miro photocleavage. (C) Expression of mCherry-Split-Miro (Split-Miro) or EBFP-SNPH (SNPH) does not significantly alter [Ca2+]m uptake in cells challenged with ionomycin, compared to control conditions. Traces indicate the average mito-GCaMP6f fluorescence intensity values (circles) at individual time point before and after cell exposure to ionomycin (arrow). N = number of cells, from 5 independent experiments. (D, E) Normalised response peak and time to reach the peak, respectively, relative to the data shown in (C). Circles, number of cells. Kruskal–Wallis test followed by Dunn’s multiple comparisons showed no difference between conditions. (F) wt-Miro or Split-Miro (magenta) were coexpressed with the ER-mito::SPLICS probe (cyan) in S2R+ cells. The SPLICS probe displays a typical punctuated stain in these cells, as previously observed in mammalian and Drosophila cells [34,36]. (G, H) Quantification of the SPLICS puncta and statistical analysis by Wilcoxon test showed no significant difference between the beginning (first minute) and the end (seventh minute) of the time-lapse imaging under blue light in wild-type and Split-Miro–transfected cells. Data are presented as % change of SPLICS puncta at seventh minute compared to first minute. Number of contacts analysed are in brackets from 5 (wt-Miro) and 9 (Split-Miro) cells from 2 independent experiments. The data underlying the graphs shown in the figures can be found in S1 Data.</p

Split-Miro–dependent control of mitochondrial motility in vivo.

(A, B) Stills from movies of GFP-labelled mitochondria in wing neuronal axons expressing UAS-wt-Miro (A) or UAS-Split-Miro (B) in miroSd32/+ background during the first minute (top panels) and fifth minute (bottom panels) of blue light exposure. Traces of transported mitochondria in corresponding movies are overlayed onto the images. (C, D) Number of motile mitochondria captured in a 50-μm axonal tract in wing neurons expressing UAS-wt-Miro (C) or UAS-Split-Miro (D). Bar charts show the average mitochondrial content at each time point. Filled circles represent the number of mitochondria within each axonal bundle at minute 1 and 5. Data were analysed by paired Student t test. Number of wings analysed: UAS-wt-Miro = 8, UAS-Split-Miro = 8, from 2 independent experiments. (E, F) Anterograde velocity (E) and run length (F) of axonal mitochondria in wing neurons expressing UAS-wt-Miro or UAS-Split-Miro, during the first and fifth minute of blue light exposure, relative to (C, D). Due to the overall lower number of bidirectional and retrograde-moving mitochondria, a meaningful statistical analysis of their velocity and run length is not possible. Circles represent tracked mitochondria. Data mean ± SEM, Mann–Whitney test. *** p S1 Data.</p

Split-Miro–dependent changes of mitochondrial morphology and connectivity are rescued by SNPH expression.

(A) Representative images of mitochondria in Drosophila S2R+ cells at the beginning (0 minutes) and after 3 and 7 minutes of exposure to blue light, which leads to Split-Miro, but not wt-Miro, photocleavage. Control cells (top panels) were cotransfected with mCherry-tagged wt-Miro (magenta) and EGFP targeted to the mitochondria via the Zdk1-MiroC anchor (EGFP-mito, grey). Split-Miro (bottom panels) was tagged with both EGFP and mCherry to independently follow the C-terminal and N-terminal half, respectively. The mCherry is shown at the beginning and end of the imaging period to confirm retention on and release from the mitochondria in wt-Miro and Split-Miro transfected cells, respectively, under blue light. Scale bar: 10 μm. (B) Quantification of mitochondrial aspect ratio (AR) and (C) of the number of mitochondrial branches within the network, relative to A. (D) Quantifications of the mitochondrial collapse phenotype after 7 minutes of time-lapse imaging with 488-nm blue light in S2R+ cells overexpressing wt-Miro and Split-Miro with or without a Miro dsRNA construct (MiroRNAi). Number of cells: wt-Miro = 15, Split-Miro = 15, Split-Miro + MiroRNAi = 10, Fisher’s exact test. (E) Representative images of cells expressing mCherry-Split-Miro (Split-Miro, yellow) with either an empty vector (top panels) or with EGFP-SNPH (SNPH, bottom panels, grey) and stained with MitoTracker DeepRed (MTDR, magenta). White arrows show examples of mitochondria that have retracted after Split-Miro photocleavage. Diffuse cytoplasmic yellow signal indicates release of Split-Miro N-terminus from the mitochondria. Scale bar: 10 μm. (E’) Magnified inset shows examples of stable SNPH-positive mitochondria (white arrowheads) and dynamic mitochondrial membranes devoid of SNPH (magenta arrowheads). Scale bar: 2 μm. Not shown, Split-Miro. (F) Quantification of mitochondrial AR and (G) number of mitochondrial branches at the time points indicated, relative to E. Circles represent the average AR calculated from single mitochondria within the same cell (B, F) and the average number of branches per cell normalised to the average group value (Split-Miro, Split-Miro + SNPH) at time point 0 (C, G). Comparison across time points was performed by repeated measures one-way ANOVA followed by Tukey’s post hoc test (B, C, F) and Friedman test followed by Dunn’s post hoc test (G), from 3 independent experiments. Data are reported as mean ± SEM. * p p p S1 Data.</p

The role of actin in the Split-Miro–dependent retraction of the mitochondrial network in S2R+ cells.

(A) S2R+ cell transfected with mCherry/EGFP-tagged Split-Miro and not treated with cytochalasin D (to maintain an intact actin network) were imaged by time-lapse with a 488-nm laser for 7 minutes. The presence of an intact actin network does not prevent mitochondrial network collapse. Scale bar: 10 μm. Cartoon depicts the reconstitution state of Split-Miro at 0 and 7 minutes (min) under blue light. (B) Representative images of S2R+ cells treated with or without cytochalasin D (cytoD) for 4 hours before imaging. Before imaging, cells were stained with MitoTracker Green (MitoTracker) and ActinTracker DeepRed (ActinTracker) to visualise the mitochondria and the actin network, respectively. Note the depolymerisation of the actin network after cytoD treatment, which is required for process extension in S2R+ cells. Scale bar: 5 μm. (TIF)</p

Extended characterisation of the effects of Split-Miro photocleavage and SNPH overexpression in S2R+ cells.

(A) Quantification of the total area covered by the mitochondria within the cell. Each measurement was normalised to the average group value (wt-Miro, Split-Miro) at time point 0. Comparison across time points was performed by repeated measures one-way ANOVA followed by Tukey’s post hoc test. Data are reported as mean ± SEM. Circle, number of cells, from 3 independent experiments. (B) S2 cells are transfected with EGFP-tagged Split-Miro N-terminus (green) and stained with MitoTracker DeepRed (MTDR, cyan). Middle panel: white and magenta stars indicate untransfected and transfected cells, respectively. Scale bar: 10 μm. (C) Duty cycle analysis describes the average time peroxisomes spend on long runs, short runs, or pausing. For each parameter, all peroxisomal values from each cell were averaged and compared between time points. Statistical significance was evaluated by multiple Mann–Whitney tests. (D) Bar chart shows the average peroxisomal content at minute 1 and 7 of time-lapse imaging with blue light. Circles represent the number of peroxisomes within each process. Statistical significance was evaluated by Wilcoxon test. In (C, D), number of processes and cells: wt-Miro = 24, 11, Split-Miro = 39, 16, from 3 independent experiments. There is no significant difference in the motility and number of peroxisomes in each process between timepoints. (E) Representative images showing S2R+ cells transfected with Split-Miro and imaged by time-lapse with blue light for 7 minutes. Exposure to blue light (to induce Split-Miro photocleavage) leads to altered mitochondrial morphology and distribution, without noticeable disorganisation of the microtubule network as detected by the Tubulin Tracker (magenta). Scale bar: 10 μm. Not shown, mCherry-tagged Split-Miro N-terminus. (F) Bar chart shows the average mitochondrial content at minute 1, 3, and 7 of time-lapse imaging with blue light in the processes of S2R+ transfected with either wt-Miro or Split-Miro. Circles represent the number of mitochondria within each process at minute 1 and 7. Data were analysed by Friedman test with Dunn’s multiple comparison test. Number of processes: wt-Miro = 39, Split-Miro = 50 from 11 and 17 cells, respectively, from 3 independent experiments. (G) Length of cellular processes imaged for 7 minutes under time-lapse exposure to 488-nm blue light to achieve Split-Miro photocleavage. Circles, number of the processes. Number of cells: wt-Miro = 11, Split-Miro = 17, from 3 independent experiments. Statistical significance was evaluated by Mann–Whitney test. (H) Representative kymographs from processes of cells transfected with an empty vector (Control) or EGFP-SNPH (SNPH). Mitochondria were stained with MitoTracker Deep Red (MitoTracker). Scale bars: 2 μm (distance) and 10 seconds (time). (I) Percentage of motile mitochondria in cellular processes of control or SNPH-expressing cells. Number of processes: control = 9, SNPH = 9, from 8 cells, from 2 independent experiments. Data are shown as mean ± SEM and were analysed by unpaired Student t test. *p p S1 Data. (TIF)</p

Split-Miro photocleavage does not affect mitochondrial membrane potential in S2R+ cells.

(A) Representative images of cells cotransfected either with wild-type Miro (mCherry-wt-Miro, EGFP-mito) or Split-Miro (mCherry-Split-Miro-EGFP). EGFP signal is used to mark the mitochondria over time; mCherry, not shown. MitoView 405 was used to monitor the mitochondrial membrane potential during the imaging period. Scale bar: 10 μm. (B) Ratio of MitoView and EGFP mitochondrial fluorescence intensity at the time points indicated. Number of cells: wt-Miro = 7, Split-Miro = 7, from 2 independent experiments. Data are shown as mean ± SEM. Each group was analysed by repeated measures one-way ANOVA followed by Dunnett’s post hoc test, with each time point compared to the t = 0 minutes. The data underlying the graphs shown in the figures can be found in S1 Data. (TIF)</p

Primers used in this study.

For NEBuilder HiFi DNA Assembly, bold fonts indicate primer overlap with the amplified gene product, underline indicated overlap with the adjacent insert or plasmid sequence. For site-directed mutagenesis, bold fonts indicate mutated nucleotides. For dsRNA template production, underline indicates T7 promoter sequence which precedes a sequence overlapping with either Miro 3′-UTR (dsRNA) or a nontargeting sequence within the pT2-DsRed-UAS plasmid backbone (Control). (DOCX)</p

Representative time-lapse movie of S2R+ cells transfected with mCherry-Split-MiroN (magenta) and EGFP-tagged Split-MiroC (green).

mCherry-Split-Miro-EGFP is reconstituted at the mitochondria, and, upon irradiation with 488-nm blue light, mCherry-Split-MiroN (but not EGFP-tagged Split-MiroC) is released into the cytoplasm and fully reconstitutes within 3 minutes. Note mCherry-Split-MiroN release is already evident at 2 seconds, followed by complete diffusion throughout the cytoplasm. (AVI)</p