Background Exposure to anesthetics during synaptogenesis results in apoptosis and subsequent cognitive dysfunction in adulthood. Probrain derived neurotrophic factor (proBDNF) is involved in synaptogenesis and can induce neuronal apoptosis via p75 neurotrophic receptors (p75NTR). proBDNF is cleaved into mature BDNF (mBDNF) by plasmin, a protease converted from plasminogen by tPA that is released with neuronal activity; mBDNF supports survival and stabilizes synapses through TrkB receptors. We hypothesized that anesthetics suppresses tPA release from neurons, enhances p75NTR signaling, reduces synapses resulting in apoptosis. Methods Primary neurons (DIV5) and postnatal day 5-7 (PND5-7) mice were exposed to isoflurane (1.4%, 4 h) in 5% CO2, 95% air. Apoptosis was assessed by cleaved caspase-3 (Cl-Csp3) immunoblot and immunofluorescence microscopy. Dendritic spine changes were evaluated with the neuronal spine marker, drebrin. Changes in synapses in PND5-7 mouse hippocampi were assessed by electron microscopy. Primary neurons were exposed to tPA, plasmin, or pharmacologic inhibitors of p75NTR (Fc-p75NTR or TAT-Pep5) 15 min prior to isoflurane. TAT-Pep5 was administered by intraperitoneal injection to PND5-7 mice 15 min prior to isoflurane. Results Exposure of neurons in vitro (DIV5) to isoflurane decreased tPA in the culture medium, reduced drebrin expression (marker of dendritic filopodial spines) and enhanced Cl-Csp3. tPA, plasmin or TAT-Pep5 stabilized dendritic filopodial spines and decreased Cl-Csp3 in neurons. TAT-Pep5 blocked isoflurane-mediated increase in Cl-Csp3 and reduced synapses in PND5-7 mouse hippocampi. Conclusion tPA, plasmin, or p75NTR inhibition blocked isoflurane-mediated reduction in dendritic filopodial spines and neuronal apoptosis in vitro. Isoflurane reduced synapses and enhanced Cl-Csp3 in the hippocampus of PND5-7 mice, the latter effect being mitigated by p75NTR inhibition in vivo. These data support the hypothesis that isoflurane neurotoxicity in the developing rodent brain is mediated by reduced synaptic tPA release and enhanced proBDNF/p75NTR-mediated apoptosis.

N-Methyl-D-aspartate (NMDA) receptor (NMDAR) activation and downstream signaling are important for neuronal function. Activation of prosurvival Src family kinases and extracellular signal-regulated kinase (ERK) 1/2 is initiated by NMDAR activation, but the cellular organization of these kinases in relation to NMDARs is not entirely clear. We hypothesized that caveolin-1 scaffolds and coordinates protein complexes involved in NMDAR signaling and that this organization is necessary for neuronal preconditioning, whereby NMDAR activation protects neurons from subsequent ischemic cell death. We found that sublethal ischemia (SLI) or preconditioning via NMDA treatment of primary cortical neurons from neonatal rats or mice increases expression of phosphorylated (P) caveolin-1, P-Src, and P-ERK1/2. The NMDAR antagonist, MK801, or the Src inhibitor, PP2, attenuated SLI-induced preconditioning. NMDAR2B distributed to buoyant fractions and heavy fractions, partially colocalized with caveolin-1 and the membrane raft marker, cholera toxin B. Cultures of primary neurons treated with caveolin-1 small interfering RNA or from caveolin-1(-/-) mice lacked the NMDA-mediated increase in P-Src and P-ERK, as well as SLI- and NMDA-induced preconditioning. Adenovirally mediated expression of caveolin-1 in neurons from caveolin-1(-/-) mice restored NMDA-mediated enhancement of P-Src and P-ERK1/2, redistributed NMDAR2B to buoyant fractions, and enhanced NMDAR2B localization to membrane rafts. We conclude that caveolin-1, perhaps via its ability to scaffold key signaling components, is essential for NMDAR localization to neuronal membrane rafts, NMDAR/Src tyrosine kinase family/ERK signaling, and protection of neurons from ischemic injury and cell death.

Background The mechanisms by which isoflurane injured the developing brain are not clear. Recent work has demonstrated that it is mediated in part by activation of p75 neurotrophin receptor (p75NTR). p75NTR activates RhoA, a small GTPase that can depolymerize actin. It is therefore conceivable that inhibition of RhoA or prevention of cytoskeletal depolymerization might attenuate isoflurane neurotoxicity. This study was conducted to test these hypotheses using primary cultured neurons and hippocampal slice cultures from neonatal mouse pups. Methods Primary neuron cultures (days in vitro, DIV4-7) and hippocampal slice cultures from postnatal day 4-7 mice were exposed to 1.4% isoflurane (4 h). Neurons were pretreated either with TAT-Pep5, an intracellular inhibitor of p75NTR, the cytoskeletal stabilizer Jasplakinolide or their corresponding vehicles. Hippocampal slice cultures were pretreated with TATPep5 prior to isoflurane exposure. RhoA activation was evaluated by immunoblot. Cytoskeletal depolymerization and apoptosis were evaluated with immunofluorescence microscopy using drebrin and cleaved caspase-3 (cl-Csp3) staining respectively. Results RhoA activation was increased following 30 min and 120 min of isoflurane exposure in neurons; TAT-Pep5 (10 μM) decreased isoflurane - mediated RhoA activation at both time intervals. isoflurane decreased drebrin immunofluorescence and enhanced cl-Csp3 in neurons, effects that were attenuated by pretreatment with either Jasplakinolide (1 μM) or TAT-Pep5. TAT-ßPep5 attenuated the isoflurane-mediated decrease in phalloidin immunofluorescence. TAT-Pep5 significantly attenuated isoflurane-mediated loss of drebrin immunofluorescence in hippocampal slices. Conclusion Isoflurane results in RhoA activation, cytoskeletal depolymerization, and apoptosis. Inhibition of RhoA activation or prevention of downstream actin depolymerization significantly attenuated isoflurane-mediated neurotoxicity in developing neurons.

Background Propofol exposure to neurons during synaptogenesis results in apoptosis leading to cognitive dysfunction in adulthood. Previous work from our laboratory showed that isoflurane neurotoxicity occurs through p75 neurotrophin receptor (p75NTR) and subsequent cytoskeleton depolymerization. Given that isoflurane and propofol both suppress neuronal activity, we hypothesized that propofol also induces apoptosis in developing neurons through p75NTR. Methods DIV5-7 neurons were exposed to propofol (3 µM) for 6 hr and apoptosis was assessed by cleaved caspase-3 (Cl-Csp3) immunoblot and immunofluorescence microscopy. Primary neurons from p75NTR−/− mice or wild-type neurons were treated with propofol, with or without pretreatment with TAT-Pep5 (10 µM, 15 min), a specific p75NTR inhibitor. P75NTR−/− neurons were transfected for 72 h with a lentiviral vector containing the synapsin driven p75NTR gene (Syn-p75NTR) or control vector (Syn-GFP) prior to propofol. To confirm our in vitro findings, wild type mice and p75NTR−/− mice (PND5) were pre-treated with either TAT-Pep5 or TAT-ctrl followed by propofol for 6 h. Results Neurons exposed to propofol showed a significant increase in Cl-Csp3, an effect attenuated by TAT-Pep5 and hydroxyfasudil. Apoptosis was significantly attenuated in p75NTR−/− neurons. In p75NTR−/− neurons transfected with Syn-p75NTR, propofol significantly increased Cl-Csp3 in comparison to Syn-GFP transfected p75NTR−/− neurons. Wild type mice exposed to propofol exhibited increased Cl-Csp3 in the hippocampus, an effect attenuated by TAT-Pep5. By contrast, propofol did not induce apoptosis in p75NTR−/− mice. Conclusion These results demonstrate that propofol induces apoptosis in developing neurons in vivo and in vitro and implicate a role for p75NTR and the downstream effector ROCK.