The Neuroprotective Effect Of Tissure-type Plasminogen Activator And Neuroserpin

Objective:TPA is an important serine protease in the central nervous system. It plays a critical role, along with its inhibitor neuroserpin, in neuronal survival under physiological and pathological conditions. Since cotical and hippocampal neurons are highly vulnerable to hypoxia-induced cell death, and the concentrations of tPA and neuroserpin in these regions of the brain are higher, we investigated the roles of tPA and neuroserpin in neuronal cell-survival after a hypoxic insult. The roles of exogenous as well as endogenous tPA and neuroserpin were examined.Methods:We assessed the activity of tPA released from cortical or hippocampal neurons using Elisa. We also looked into tPA-confered neuronal survival in wild- type and tPA deficient mice from OGD induced cell death in vitro model using MTT assay. In vivo, the effects of endogenous tPA and neuroserpin in cortical or hippocampal neurons on neuronal cell death were assessed after the induction of ischemia using BCCAO and MCAO models, which result in the ischemia of the forebrain and focal brain respectively. Furthermore, the neuroprotective mechanisms and effects of tPA and neuroserpin were elucidated by treating with exsogenous tPA and neuroserpin at different time points as a method of preconditioning.Results:Using an in situ zymography assay, we found that proteolytic activity of tPA is absent in the CA1 layer under normal oxygen concentrations. The activity of tPA in the cortical and CA1 layers increased immediately after exposure to hypoxia. The increased levels of endogenous tPA induced by brief hypoxia,resulted in the protection of hippocampal but not cortical neurons from subsequent lethal hypoxia in wild-type mice. In contrast, the protective effect of ischemic preconditioning was absent in tPA and plasminogen deficient mice. Our results indicate that treatment with 500 nM recombinant tPA simultaneously with OGD increases hippocampal neuronal survival from 55.71%±1.9% to 73.6%±6.4%, whereas inactive tPA increased hippocampal neuronal survival to 80.02±1.55%. Moreover, We found that not only did?2-antiplasmin, MK-801 or wortmannin not abrogate the protective effects of tPA, but also incubation with plasmin alone failed to induce neuronal survival. Furthermore,the LRP-extracellular domain binding protein, RAP, abrogated the protective effects of tPA in the early preconditioning model showing that tPA does not need proteolytic activity to confer this protection.In a delayed preconditioning model,however,we found that incubation with 60 nM of tPA or plasmin 24 hours before exposure to lethal OGD increased neuronal survival from 62±2.88% to 88.28%±2.72% and 86.44%±1.64%, respectively, but treatment with inactive tPA or plasminogen alone did not have a protective effect. The protective effect of tPA was inhibited by coincubation with a2-antiplasmin, aprotinin, EACA, MK-801 or wortmannin,showing that it is proteolysis dependent. Moreover in contrast to our observations with early preconditioning, we found that incubation with tPA 24 hours before the excitotoxic injury with kainic acid had a neuroprotective effect.Immunofluorescence analysis showed that there is low expression of neuroserpin in the hippocampus and cortex under normoxia. The activity of neuroserpin in cortical and hippocampal CA1 layer increases immediately after exposure to hypoxic insult. In contrast to lethal ischemia, a sublethal ischemic injury induces a significant increase in the expression of neuroserpin in the hippocampus and cortex. Treatment with exogenous neuroserpin during OGD increases the survival of hippocampal neurons in wild-type and tPA deficient mice to 83.3±3.8% and 92.0±1.6% respectively and cortical neurons to 79.8±2.2% and 82.3±2.4%, respectively. Compared to untreated cells, plasmin decreases neuronal survival to 74.2±1.7%, and that this effect is significantly inhibited by co-incubation with either MK-801 (88.8±1.1%) or neuroserpin (94±1.3%). Incubation with kainic acid alone reduces neuronal survival to 78.2±1.8%, and that early/immediate preconditioning with neuroserpin increases cell survival to 98.3±2.3%. Despite a decrease in the volume of the ischemic lesion associated with genetic deficiency of tPA, treatment with neuroserpin caused a further reduction of 48.3% and 32.7% in the volume of the ischemic lesion in wild-type and tPA deficient mice.Conclusions:Our data indicate that treatment of hippocampal neurons with tPA prior to lethal hypoxia confers neuro-protection. This protective effect is independent of plasminogen/plasmin and instead requires the engagement of a member of the LDL receptor family. Additionally, treatment with tPA also induces tolerance against a lethal insult applied 24 hours later. This delayed protective effect of tPA requires tPA's proteolytic activity and is mediated by plasmin via NMDAR-dependent Akt phosphorylation. Neuroserpin induces ischemic tolerance in wild-type and tPA deficient neurons and mice. Neuroprotective effect of neuroserpin is due, largely, to the inhibition of plasmin-mediated excitotoxin-induced cell death and is independent of neuroserpin's ability to inhibit tPA activity.