Regulatory mechanisms of P-glycoprotein in brain endothelial cells of different species and novel in vivo approaches for the translation of in vitro findings

鉴于癫痫或抑郁等主要脑部疾病对常用疗法的耐药性以及外排转运蛋白如P-糖蛋白（Pgp）在耐药机制中的潜在作用，重要的是破译Pgp在血脑屏障（BBB）的调节机制，从而实现创新的治疗干预。以前的研究主要集中在转录调节（转录因子活性和Pgp表达的变化），转录后修饰（Pgp蛋白的变化和microRNA对Pgp mRNA翻译的调节），已表达的Pgp从细胞内室到顶端膜的Pgp运输机制，以及Pgp调节信号转导。我们观察到Pgp从高表达细胞向低表达细胞的细胞间转移，这是一种观察Pgp在血脑屏障中调控的全新方法。这种脑内皮细胞中的Pgp转移是通过直接细胞-细胞接触和/或外来体介导的，其中我们已经鉴定了Pgp。我们研究的另一个原始和令人惊讶的发现是-除了在BBB内皮细胞的顶端质膜中表达的Pgp的已知药物外排功能之外- Pgp可以在内皮细胞的溶酶体中螯合潜在毒性的外源性物质。细胞内溶酶体囊泡通过一种未知的机制运输到细胞膜，并使细胞积聚在顶端细胞膜上，形成被中性粒细胞吞噬的腺泡状囊泡团块（“屏障体”），从而有效处理隔离的异生物质并有效保护脑实质。通过新的拨款申请，我们现在计划进一步表征不同物种（人，猪，大鼠）BBB中药物诱导的屏障体形成，并更详细地破译中性粒细胞和BBB内皮细胞之间的相互作用及其药理学调节。此外，我们将研究屏障体形成对CNS活性药物（例如，抗癫痫和抗抑郁药物）和化疗药物。接下来，我们将尝试将体外发现转化为啮齿动物模型中BBB的体内功能。我们希望，在BBB的Pgp的新的调节机制的特点将允许开发新的创新策略，以避免与BBB介导的脑疾病的耐药机制的相互作用。

In view of the pharmacoresistance of major brain diseases such as epilepsy or depression to common therapies and the potential role of efflux transporters such as P-glycoprotein (Pgp) in the mechanisms underlying drug resistance, it is important to decipher the regulatory mechanisms of Pgp at the blood-brain barrier (BBB), and thereby enable innovative therapeutic interventions. Previous studies focused mainly on transcriptional adjustments (changes in the activity of transcription factors and the expression of Pgp), post-transcriptional modifications (changes in the Pgp protein and regulation of translation of Pgp mRNA by microRNAs), Pgp trafficking mechanisms of already expressed Pgp from intracellular compartments to the apical membrane, as well as Pgp-modulating signal transduction. An entirely new way of looking at Pgp regulation at the BBB is initiated by our observations of cell-to-cell Pgp transfer from cells with high Pgp expression to cells with low Pgp expression. This Pgp-transfer in the brain endothelial cells is mediated via direct cell-cell contact and/or exosomes, in which we have identified Pgp. Another original and surprising finding of our studies is that - in addition to the known drug efflux function of Pgp expressed in the apical plasma membrane of BBB endothelial cells - Pgp can sequester potentially toxic xenobiotics in lysosomes of the endothelial cells. The intracellular lysosomal vesicles traffick by an as yet unknown mechanism to the cell membrane and leave the cell to accumulate on the apical cell membrane and form aciniform vesicle conglomerates (“barrier bodies”) that are phagocytosed by neutrophil granulocytes, thus leading to an effective disposal of the sequestered xenobiotic and an effective protection of brain parenchyma. By the new grant application, we now plan to further characterize the drug-induced formation of barrier bodies in the BBB of different species (human, pig, rat) and decipher the interaction between neutrophils and BBB endothelial cells and its pharmacological modulation in more detail. Furthermore, we will study the impact of barrier body formation for development of drug resistance in response to CNS active drugs (e.g., antiepileptic and antidepressant drugs) and chemotherapeutics. Next, we will try to translate the in vitro findings to the in vivo functioning of the BBB in rodent models. We expect that characterizing the novel regulation mechanisms of Pgp at the BBB will allow developing novel innovative strategies to pharmacologically interact with BBB-mediated resistance mechanisms of brain diseases.