1 1 3 21 7 Open image in new window Methylphenidate is the drug of choice for the treatment of ADHD and its use has increased significantly over the last few years. Several studies have shown beneficial effects of MPH in ADHD [], but nevertheless, its use among children is non-consensual []. In addition, there is currently a concern regarding MPH misuse for cognitive enhancement. Thus, it crucial to better clarify the central effects of MPH. In the present study, we investigated whether MPH affects human brain ECs, the main component of the BBB, using a representative therapeutic dose range of MPH []. We demonstrate, for the first time, that MPH increases brain EC permeability by stimulating vesicular transport and unveil the intracellular signaling pathway responsible for this effect (Fig.).

It was previously demonstrated that MPH augments the number of vesicles in ECs [14] and that methamphetamine, a powerful psychostimulant drug of abuse, increases both paracellular and transcellular transports in ECs [18, 19]. Paralleling these findings, we showed that MPH increased transendothelial flux without altering monolayer electrical resistance or the expression/distribution of intercellular junctional proteins in human brain ECs. Instead, MPH promoted vesicular transport across ECs via a caveolae-dependent mechanism. We also revealed that this psychostimulant increased Cav1 phosphorylation. Induction of Cav1 expression has been identified in a number of brain disorders, such as Alzheimer’s disease [8] and intracerebral hemorrhage [7]. Moreover, caveolae-dependent processes are involved in the transport of macromolecules [37], virus [42], and fungal pathogens across ECs [32]. Caveolae have also been implicated in the internalization of TJ transmembrane proteins leading to brain endothelial barrier disruption during CNS inflammation [27], although recycling of TJs also allows for faster re-establishment of barrier function without new protein synthesis [43]. In addition, Cav1 reduction was associated with the deletion of TJ-associated proteins and a consequent increase of barrier permeability [44, 45]. In the present study, we identified neither alterations in the expression/organization of inter-endothelial junctions nor in the expression of Cav1 protein. Nevertheless, several studies have suggested that Cav1 phosphorylation plays a crucial role in the regulation of caveolae formation and transcytosis in pulmonary endothelial cells [6, 28, 37]. Very recently, it was shown that endocytosis and trafficking of caveolae are associated with a Cav1 Tyr14 phosphorylation-dependent conformational change in rat lung microvascular endothelial cells [11]. Accordingly, in the present study, we demonstrated for the first time that shRNA-induced depletion of Cav1 or the use of Cav1Y14F mutant prevented increase in brain endothelial transcytosis induced by MPH. On the contrary, overexpression of Cav1Y14D by itself enhanced vesicular transport, supporting the hypothesis that phosphorylation of Cav1 at Tyr14 promotes caveolae formation upon MPH stimulation. These data were further corroborated by immunoelectron microscopy, wherein numerous caveolae-like invaginations on the plasma membrane and in the cytosol of ECs were observed after exposure to MPH.

We also investigated the mechanism by which MPH increased caveolae vesicular transport. It has been shown that young rats treated with MPH exhibit oxidative damage as evidenced by an increase in both lipid peroxidation and protein carbonyl adducts in the brain [36]. In addition, MPH seems to interfere with important brain antioxidant defenses [46]. Whereas ROS generation by ECs at low levels signal important physiological activities, such as cell growth and differentiation [38], excessive generation of ROS overwhelms the intracellular antioxidant defense systems leading to an imbalance in redox homeostasis, oxidative stress, and endothelial dysfunction [38]. In fact, we found that intracellular ROS generation in human ECs, namely superoxide anion, increased upon exposure to MPH. Likewise, acute MPH administration in young rats increased the amount of brain superoxide [35]. We also identified NOX as the source of ROS production triggered by MPH. Our findings are consistent with the previous studies showing a crucial role for NOX-generated ROS in BBB disruption [38, 47]. Collectively, these observations led us to hypothesize that an antioxidant strategy could have a beneficial effect on ECs exposed to MPH. Indeed, the antioxidant VitC was able to prevent both MPH-induced ROS generation and transcytosis. Accordingly, others have reported that VitC could prevent microvascular endothelial barrier dysfunction during septic insult by blocking NOX-dependent ROS generation [48].

ROS signaling plays an important role in the control of endothelial permeability [38] by interfering with the dynamics of the actin cytoskeleton via Rho GTPases [39]. It is known that Rho-regulated cytoskeletal remodeling is essential for targeting vesicles to their correct location, enabling exocytosis [32, 33]. However, the mechanisms by which Rho GTPases exert their effects on intracellular trafficking are still largely not known. Herein, we observed that MPH had different effects on the activities of the endothelial Rho GTPases Rac1, RhoA, and Cdc42. In particular, while MPH induced the rapid activation of Rac1, it decreased the activity of RhoA and had no effect on Cdc42. Likewise, Rac-mediated ROS production in HeLa cells results in the downregulation of Rho activity, which is required for Rac-induced formation of membrane ruffles and integrin-mediated cell spreading [49]. Interestingly, others have demonstrated the involvement of Rac1 in the recruitment and assembly of the endothelial NOX complex [40]. Here, we showed that MPH activated Rac1 and that shRNA-mediated Rac1 knockdown prevented ROS generation and, consequently, the effect of MPH. Although the exact role of Rac1 in EC barrier function is not fully understood, Rac-dependent generation of ROS is known to cause barrier dysfunction [40]. Chen et al. [40] reported that Rac1 inhibition in human pulmonary artery ECs contributes to barrier protection via the inhibition of NOX and superoxide generation. Similarly, we demonstrated that Rac1 knockdown inhibits MPH-induced caveolae-mediated transcytosis. Furthermore, Rac1 activation was reported to be required for bacterial entry into human ECs [33]. Several pathogens use caveolae as a carrier vacuole to hijack endosomal trafficking in host cells and escape lysosomal degradation [50]. In light of these observations, it is plausible to speculate that MPH-induced caveolae formation and trafficking might ultimately promote the entrance of pathogens into the brain.

RhoA activation is known to be involved in functional changes of TJ proteins, such as claudin-5, and also reorganization of the actin cytoskeleton leading to disruption of endothelial cell–cell contacts and paracellular hyperpermeability [43, 51]. RhoA inhibition and dominant negative RhoA mutant prevented the loss of tight and adherens junctions, the decrease of transendothelial resistance, and stress fiber formation in human umbilical vein endothelial cells [52]. Moreover, RhoA inactivation caused the disassembly of actomyosin stress fibers and reorganized F-actin and phosphotyrosine-containing proteins to β-catenin-containing cell–cell junctions, a process that increased the size-selective permeability of endothelial monolayers [53]. In addition, the reduction of RhoA activity and elevation of Rac1 signaling are important steps in the control of endothelial permeability due to their involvement in the re-annealing of the intercellular junctions [54]. In our study, we did not observe alterations in the intercellular junctions, which may be justified by data indicating the inactivation of RhoA in response to MPH.

The activity of c-Src has been implicated in endothelial hyperpermeability [55], and its inhibition ameliorates vascular leakage and inflammation in rodent brains [56]. Here, we demonstrated that oxidant signaling is a key feature of c-Src activation in response to MPH in brain ECs, since the antioxidant VitC or NOX inhibition abrogated MPH-induced c-Src activation. Intracellular ROS may regulate the activity of c-Src via oxidation of two cysteine residues that control c-Src conformational changes necessary for its activation [41]. The c-Src and Rac1 hierarchy is generally context and stimuli specific in different cells. Our results show that MPH-mediated Rac1 activation is upstream of c-Src in human brain ECs. In response to oxidative stress, Tyr14 on Cav1 is the principal c-Src target [28, 37]. We showed that MPH leads to Cav1 phosphorylation by c-Src, and, consequently, to HRP transcytosis. Direct activation of c-Src via rapamycin increased vesicular transport and promoted the interaction between c-Src and Cav1 at the plasma membrane. In this respect, our results are consistent with other reports showing that Src-Cav1 interaction requires c-Src activity to promote caveolae-mediated pulmonary endothelial hyperpermeability and edema formation [37]. In addition, overexpression of Cav1Y14F in brain ECs abrogated RapR-Src-mediated transcytosis, supporting the premise that c-Src-dependent phosphorylation of Cav1 on Tyr14 is necessary and sufficient to promote caveolae trafficking dynamics and endothelial hyperpermeability. Accordingly, Zimnicka and collaborators [11] have recently demonstrated a key role of Src-dependent Cav1 phosphorylation in promoting caveolae release from the plasma membrane via phosphorylation-dependent destabilization of Cav1 oligomers. Overall, our data indicate that MPH induces Rac1/NOX-dependent ROS generation and subsequent c-Src activation-dependent Cav1 Tyr14 phosphorylation promotes transcellular transport (transcytosis) in human brain ECs.

The present study shows for the first time that MPH has a direct effect on human brain ECs and provides new insights into the mechanism underlying MPH-induced BBB hyperpermeability. In this context, our data raise the question of whether MPH use enhances brain susceptibility to peripheral factors and the risk of neurological disease. However, while the cerebral vasculature provides a crucial protective role in maintaining brain homeostasis, the BBB also represents a substantial obstacle to the delivery of many neurotherapeutic drugs. Thus, our results also suggest that transient and controlled MPH exposure might constitute a potential strategy for enhancing drug delivery into the brain.