Protective Effect Of TSPO Ligand PK11195 On Neuroinflammation And Its Mechanism

Translocator protein 18 kDa (TSPO) is a protein primarily localized in the outer membrane of mitochondria in peripheral organs and cerebral glial cells. Translocation of cholesterol from the cytoplasm into the mitochondria for steroidogenesis is one of the important functions of TSPO. TSPO also plays an important role in modulating inflammation. TSPO expression is markedly increased not only in acute brain injury but also in many kinds of neurodegenerative and neurological diseases, including Alzheimer’s disease. However, the role of TSPO upregulation is yet to be determined. Additionally, some TSPO ligands have therapeutic potential in neuroprotection, neuroregeneration and anxiety. Lots of in vivo and in vitro studies have demonstrated that classical ligands of TSPO, PK11195 and Ro5-4864, could reduce microglia activation and have protective effects against neuroinflammation. Studies investigating the effect of TSPO ligands on cognition and amyloidogenesis were rare. In this study, we adopted an animal model of neuroinflammation caused by chronic systemic administration of LPS. This model has been used to mimic pathophysiology of AD characterized by neuroinflammation, gliosis, β-amyloid proteins (AP) accumulation, cognitive dysfunction. Here, we sought to evaluate the effect of PK11195 on LPS-induced chronic neuroinflammation and its subsequent pathology through in vivo and in vitro studies.Part 1 Protective effect of TSPO ligand PK11195 on LPS-induced neuroinflammation and its mechanismObjective:To investigate the effects of TSPO ligand PK11195 on LPS-induced neuroinflammation and mechanisms.Methods:Male C57BL/6J mice were randomly assigned to four groups:Control (n=12), LPS (n=12), LPS+PK11195 (n=12), PK11195 (n=12). PK11195 (3 mg/kg, i.p.) was administered consecutively in group LPS+PK11195 and PK11195 once per day. Meanwhile, an equivalent volume of vehicle was given in group Control and LPS. Three days after PK11195 administration, the first injection of LPS (the dose was based on our preliminary study.) was given 30min after PK11195 administration in group LPS and LPS + PK 11195 while an equivalent volume of sterile saline was given to mice in group Control and PK11195. LPS was also administered once per day. Seven days after LPS administration, cognitive function was assessed by Morris water maze (MWM), i.e. four consecutive days of training followed by one day of probe test. The drugs were not discontinued until the mice were sacrificed. Immunoblotting analyses of hippocampal COX-2> BACE-1、IDE, BDNF were performed. Hippocampal progesterone and allopregnanolone concentrations were assayed by ELISA.Results:1. The results of our preliminary experiment showed that 500 μg/Kg LPS was fit to cause cognitve dysfunction in C57BL/6J mice.2. In the training phase of MWM, to locate the platform, mice in LPS group needed significantly longer time than those in the Control group on Day 1 of training and swam longer distance on Day 4 of training. PK11195 pretreatment protected against the learning disability induced by LPS. Data of the probe trial illustrated that mice in LPS group spent less percentage of time and distance in target quadrant while PK11195 protected memory deficits caused by LPS.3. Inflammatory proteins, TSPO and COX-2, were upregulated by LPS, PK11195 alleviated these effects.4. Hippocampal Aβx-42 contents were elevated by LPS, accompanied by increases of BACE-1 and IDE, PK11195 pretreatment alleviated these effects.5. PK11195 increased brain levels of progesterone, allopregnanolone in LPS-injected mice.6. BDNF content is significantly decreased in LPS-treated mice, which was not affected by PK11195 pretreatment.Conclusion:Our data demonstrated that PK11195 could protect cognitive deficits induced by chronic LPS administration. The underling mechanism may involve alleviated neuroinflammation, increased synthesis of neurosteroid and decreased Aβ accumulation accompanied by down-regulation of BACE-1.Part 2 Protective effect of TSPO ligand PK11195 on Aβ1-42 oligomer-induced inflammatory BV-2 cell model and its mechanismObjective:To determine the cytoprotective effect of PK11195 on Aβ1-42 oligomer-induced damages in BV-2 cells and possible mechanisms.Methods:Aβ1-42 oligomer-induced BV-2 cell model was established. Then the optimal dose of PK11195 on protection of Aβ1-42 oligomer-induced BV-2 damage was determined based on cell viability assessed by the CCK8 assay. Immunoblotting analyses of TSPO、COX-2 and BACE-1 were performed to determine the effects of Aβ1-42 oligomer and PK11195 on BV-2 cells.Results:1. The CCK8 assay showed that 10μM Aβ1-42 exposure for 48h was fit to establish inflammatory BV-2 cell model.2. Pretreatment with PK11195 (0.01 μM 0.1 μM) reduced Aβ1-42 oligomer-induced death of BV-2 cells.3. Immunoblotting analyses demonstrated that TSPO, COX-2 and BACE-1 contents were upregulated by Aβ1-42 oligomer (10μM), PKl 1195 alleviated these effects.Conclusion:Our results demonstrated that microglial activation played an important role in AP induced neuroinflammation. BV-2 cells were activated by Aβ1-42 oligomer, inflammatory proteins, TSPO and COX-2, were upregulated as well as BACE-1. BACE-1 promoted mamyloidogenesis to form a vicious cycle, lastly resulting decreased cell viability. PK11195, a TSPO ligand, could alleviate BV-2 cell activation, prevent inflammatory protein and BACE-1 increases and enhance BV-2 viability. Considering that microglial activation, neuroinflammation, amyloidogenesis were closely correlated with neurodegenerative diseases, especially AD, our data provide new insights into the neuroprotective effect of TSPO in neurodegerative diseases.