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、抑郁症等重大脑病对常见药物的耐药性，以及p -糖蛋白（Pgp）等外排转运体在耐药机制中的潜在作用，研究Pgp在血脑屏障（BBB）的调控机制，从而实现创新的治疗干预具有重要意义。以往的研究主要集中在转录调节（转录因子活性的变化和Pgp的表达）、转录后修饰（Pgp蛋白的变化和microrna对Pgp mRNA翻译的调控）、已表达的Pgp从细胞间室到顶膜的转运机制以及Pgp调节的信号转导等方面。我们通过观察Pgp从高表达细胞向低表达细胞的细胞间转移，开创了一种全新的观察血脑屏障中Pgp调控的方法。这种Pgp在脑内皮细胞中的转移是通过直接的细胞间接触和/或外泌体介导的，我们已经在其中鉴定了Pgp。我们研究的另一个原始和令人惊讶的发现是，除了已知的在血脑屏障内皮细胞顶质膜中表达的Pgp的药物外排功能外，Pgp还可以在内皮细胞的溶酶体中隔离潜在的有毒外来生物。细胞内溶酶体囊泡通过一种尚不清楚的机制运输到细胞膜，离开细胞积聚在顶细胞膜上，形成被中性粒细胞吞噬的腺状囊泡聚集体（“屏障体”），从而有效地处理被隔离的外来生物，有效地保护脑实质。通过新的资助申请，我们现在计划进一步表征不同物种（人类，猪，大鼠）血脑屏障中药物诱导的屏障形成，并更详细地破译中性粒细胞和血脑屏障内皮细胞之间的相互作用及其药理调节。此外，我们将研究屏障形成对中枢神经系统活性药物（如抗癫痫药和抗抑郁药）和化疗药物耐药发展的影响。接下来，我们将尝试将体外研究结果转化为啮齿动物血脑屏障的体内功能。我们期望通过描述脑屏障中Pgp的新调控机制，可以开发出新的创新策略，从药理学上与脑屏障介导的脑疾病耐药机制相互作用。

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.