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. While Split-Miro overexpression is sufficient to augment mitochondrial motility, sustained photocleavage shows Split-Miro is surprisingly dispensable to maintain elevated mitochondrial processivity. 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.

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 and 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 and 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 accumulation in hCMEC/D3. 5xFAD mice showed increased brain [3H]amisulpride uptake, and no cerebrovascular space changes compared to WT. TEM revealed brain capillary degeneration in human AD. There was no significant effect of AD on mouse GLUT1 and P-gp BBB expression, and in human GLUT1 expression. In contrast, caudate P-glycoprotein 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 exacerbate the cerebral hypometabolism in AD. Further research into the mechanism of amisulpride transport by GLUT1 is important for improving antipsychotics safety.