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

鉴于癫痫或抑郁症等主要脑部疾病对常用治疗方法的耐药性，以及P-糖蛋白(Pgp)等外排转运体在耐药机制中的潜在作用，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措施是非常重要的。以往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microRNAs对Pgp mRNA翻译的调控)、已经表达的Pgp从细胞内的隔室到顶膜的Pgp转运机制，以及Pgp调节的信号转导。我们观察到从高Pgp表达的细胞到低Pgp表达的细胞之间的Pgp转移，这开启了一种全新的方式来看待Pgp在血脑屏障中的调节。这种Pgp在脑内皮细胞中的转移是通过细胞-细胞直接接触和/或外体介导的，我们在其中发现了Pgp。我们研究的另一个原创性和令人惊讶的发现是，除了在血脑屏障内皮细胞顶端质膜表达的PGP已知的药物外排功能外，PGP还可以将潜在有毒的外源性物质隔离在内皮细胞的溶酶体中。细胞内的溶酶体小泡以一种未知的机制运输到细胞膜上，离开细胞积聚在顶端细胞膜上，形成腺泡状泡团(“障碍体”)，被中性粒细胞吞噬，从而有效地处置隔离的异物，有效地保护脑实质。通过新的拨款申请，我们现在计划进一步表征药物诱导的不同物种(人、猪、鼠)血脑屏障中屏障小体的形成，并更详细地破译中性粒细胞与血脑屏障内皮细胞之间的相互作用及其药理调节。此外，我们还将研究屏障小体的形成对中枢神经系统活性药物(如抗癫痫和抗抑郁药物)和化疗药物产生耐药性的影响。接下来，我们将尝试将体外发现转化为啮齿动物模型中血脑屏障的体内功能。我们期望，表征PGP在血脑屏障中的新调节机制将允许开发新的创新策略来与血脑屏障介导的脑部疾病抵抗机制进行药理相互作用。

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.