Neuroprotective Effect Of Translocator Protein 18 KDa (TSPO) And Its Putative Mechanism

PartⅠ Over-expression of TSPO in the hippocampal CA1 area alleviates cognitive dysfunction caused by lipopolysaccharide in miceObjective Nowdays, research on neuroprotection is one of the hot spots. The translocator protein 18 kDa (TSPO) is primarily localized in the outer mitochondrial membrane and involved in the rate-limiting step in neurosteroids production, i.e. translocation of cholesterol from the outer to the inner mitochondrial membrane. TSPO in the nervous system is predominantly expressed in glial cells and is closely related with regulation of immune/inflammatory response. TSPO has been reported to be up-regulated greatly during various brain injuries, however, the role that the increased TSPO played is not clear. We used the lipopolysaccharide (LPS)-induced mice model to confirm if TSPO over-expression in hippocampal CA1 area was protective for cognitive dysfunction and whether the neuroprotective effects were related to steroideogenesis.Methods C57BL/6J mice were randomly divided into eight groups:(i) NC group; (ii) TSPOoe group; (iii) NC+LPS group; (iv) TSPOoe+LPS group; (v) NC+LPS+FN group; (vi) TSPOoe+LPS+FN group; (vii) NC+FN group; (viii) TSPOoe+FN group. GV287 lentiviral vectors mediating TSPO over-expression (TSPOoe) or those contained the non-targeting negative control sequence (NC) were injected into bilateral hippocampal CA1 areas of mice by stereotaxic apparatus on day 0. Finasteride (FN,5 mg/kg), LPS (250 μg/kg) or vehicle was administered (i.p.) once a day from day 15 to day 26. Morris Water Maze (MWM) training or testing was conducted 22 days after the microinfusions, precisely 1h after finasteride treatment and 30 min after LPS administration. Punched tissues of hippocampal CA1 were harvested to detect TSPO expression and neurosteroids levels.Results (1) During the acquisition phase of MWM, the animals’ performance improved over time, and the latency to locate platform seemed to be affected by time and LPS (Ftime =157.42,P=0.0001; Ftreament=10.53,P=0.0017). Post hoc analysis showed that, to locate the platform, mice in NC+LPS group needed longer time than those in the NC group on Day 2 of training. On Day 4, the latency to locate the platform in NC group was not significantly different from other groups, which meant that all mice had learned the task during 4 days of training. (2) In the probe trial, mice in NC+LPS group tended to spend less time in the target quadrant than those in NC group. Mice in TSPOoe+LPS group tended to spend more time in the target quadrant than those in NC+LPS group. Moreover, finasteride treatment decreased TSPOoe+LPS-induced long duration. (3) Levels of TSPO were evaluated by Western-blotting, the results suggest that TSPOoe and LPS significantly increased TSPO expression. (4) Mice treated with TSPOoe displayed statistically significant increases in progesterone and allopregnanolone compared to those injected with NC Lentivirus in the injected sites. Finasteride administration attenuated TSPOoe-induced increase of allopregnanolone level while having no influence on progesterone level.Conclusion TSPO over-expression in hippocampal CA1 area significantly mitigated the cognitive dysfunction caused by systemic administration of LPS. TSPOoe microinjections increased progesterone and allopregnanolone synthesis. Blockade of allopregnanolone production using finasteride partially reversed the behavioural effects of TSPO over-expression. These data suggest that up-regulation of TSPO level during brain injury may be an adaptive response mechanism which is supposed to be related with the organism’s need for neurosteroids. We conclude that TSPO may be an attractive therapeutic target for brain injury in the future.PartⅡ Neuroprotective effect of TSPO over-expression in BV-2 cells for primary cultured hippocampal neurons and its putative mechanismObjective To investigate the effect of TSPO over-expression in BV-2 cells on primary cultured hippocampal neurons.Methods BV-2 cells were divided into four groups according to transfected lentivirus types (NC or TSPOoe) and whether LPS(100 ng/ml) was given:NC group, TSPOoe group, NC+LPS group, and TSPOoe+LPS group. TNF-α and IL-6 levels were detected using ELISA assay. The conditioned media (CM) from BV-2 cell cultures was collected and then used to stimulate the primary hippocampal neurons. The hippocampal neurons were also divided into four groups according to the CM sources:NC-CM group, TSPOoe-CM group, NC+LPS-CM group, and TSPOoe+LPS-CM group. CCK-8 assay was used to evaluate the viability of the neurons.Results (1) TNF-α and IL-6 levels significantly increased in the medium from the BV-2 cells in NC+LPS group compared to NC group. Levels of the two cytokines decreased significantly in TSPOoe+LPS group compared to NC+LPS group. (2) The viability of the neurons was significantly lower in NC+LPS-CM group than NC-CM group as well as TSPOoe+LPS-CM group.Conclusion Over-expression of TSPO suppressed LPS-induced TNF-α and IL-6 increases in BV-2 cells, demonstrating its anti-inflammatory effect. The CM from LPS-treated BV-2 cell cultures has neurotoxic effect on primary neurons, and TSPO over-expression in BV-2 cells could attenuate this effect.