Isoflurane Increases Neuronal Cell Death Vulnerability by Downregulating miR-214

Since accumulating evidence suggests the application of anesthetics may increase the risk of Alzheimer’s disease (AD), we investigated the cytotoxicity of inhaled general anesthesia in neurons and its underlying mechanism. Using primary cultured rat hippocampal neurons as the study model, here we show that isoflurane increases vulnerability to intracellular or extracellular amyloid b with or without serum deprivation. This isoflurane-induced effect is mediated by the downregulation of miR-214 level that lead to an elevated expression of Bax, a prominent target for miR-214. We conclude that isoflurane increases cell death in the presence of amyloid b by increasing Bax level through downregulating miR-214. Our data provide a new insight for inhaled anesthetics toxicity and indicate a possible mechanistic link between anesthetic application and neurodegenration in AD

Prenatal Cocaine Exposure Upregulates BDNF-TrkB Signaling

Prenatal cocaine exposure causes profound changes in neurobehavior as well as synaptic function and structure with compromised glutamatergic transmission. Since synaptic health and glutamatergic activity are tightly regulated by brain-derived neurotrophic factor (BDNF) signaling through its cognate tyrosine receptor kinase B (TrkB), we hypothesized that prenatal cocaine exposure alters BDNF-TrkB signaling during brain development. Here we show prenatal cocaine exposure enhances BDNF-TrkB signaling in hippocampus and prefrontal cortex (PFCX) of 21-day-old rats without affecting the expression levels of TrkB, P75NTR, signaling molecules, NMDA receptor—NR1 subunit as well as proBDNF and BDNF. Prenatal cocaine exposure reduces activity-dependent proBDNF and BDNF release and elevates BDNF affinity for TrkB leading to increased tyrosine-phosphorylated TrkB, heightened Phospholipase C-γ1 and N-Shc/Shc recruitment and higher downstream PI3K and ERK activation in response to ex vivo BDNF. The augmented BDNF-TrkB signaling is accompanied by increases in association between activated TrkB and NMDARs. These data suggest that cocaine exposure during gestation upregulates BDNF-TrkB signaling and its interaction with NMDARs by increasing BDNF affinity, perhaps in an attempt to restore the diminished excitatory neurotransmission

Prenatal Cocaine Exposure Uncouples mGluR1 from Homer1 and Gq Proteins

Cocaine exposure during gestation causes protracted neurobehavioral changes consistent with a compromised glutamatergic system. Although cocaine profoundly disrupts glutamatergic neurotransmission and in utero cocaine exposure negatively affects metabotropic glutamate receptor-type 1 (mGluR1) activity, the effect of prenatal cocaine exposure on mGluR1 signaling and the underlying mechanism responsible for the prenatal cocaine effect remain elusive. Using brains of the 21-day-old (P21) prenatal cocaine-exposed rats, we show that prenatal cocaine exposure uncouples mGluR1s from their associated synaptic anchoring protein, Homer1 and signal transducer, Gq/11 proteins leading to markedly reduced mGluR1-mediated phosphoinositide hydrolysis in frontal cortex (FCX) and hippocampus. This prenatal cocaine-induced effect is the result of a sustained protein kinase C (PKC)-mediated phosphorylation of mGluR1 on the serine residues. In support, phosphatase treatment of prenatal cocaine-exposed tissues restores whereas PKC-mediated phosphorylation of saline-treated synaptic membrane attenuates mGluR1 coupling to both Gq/11 and Homer1. Expression of mGluR1, Homer1 or Gα proteins was not altered by prenatal cocaine exposure. Collectively, these data indicate that prenatal cocaine exposure triggers PKC-mediated hyper-phosphorylation of the mGluR1 leading to uncoupling of mGluR1 from its signaling components. Hence, blockade of excessive PKC activation may alleviate abnormalities in mGluR1 signaling and restores mGluR1-regulated brain functions in prenatal cocaine-exposed brains

Mechanism of Action for rTMS: A Working Hypothesis Based on Animal Studies

Experiments in rodents have elucidated some of the molecular mechanisms underlying repetitive transcranial magnetic stimulation (rTMS). These studies may be useful in a translational perspective so that future TMS studies in rodents can closely match human TMS protocols designed for therapeutic purposes. In the present work we will review the effects of rTMS on glutamate neurotransmission which in turn induce persistent changes in synaptic activity. In particular, we will focus on the role of NMDA and non-NMDA transmission and on the permissive role of BDNF-TrKB interaction in the rTMS induced after-effects

Naloxone's Pentapeptide Binding Site on Filamin A Blocks Mu Opioid Receptor–Gs Coupling and CREB Activation of Acute Morphine

Chronic morphine causes the mu opioid receptor (MOR) to switch its coupling from Gi/o to Gs, resulting in excitatory signaling via both Gαs and its Gβγ dimer. Ultra-low-dose naloxone (NLX) prevents this switch and attenuates opioid tolerance and dependence. This protective effect is mediated via a high-affinity interaction of NLX to a pentapeptide region in c-terminal filamin A (FLNA), a scaffolding protein interacting with MOR. In organotypic striatal slice cultures, we now show that acute morphine induces a dose-dependent Go-to-Gs coupling switch at 5 and 15 min that resolves by 1 hr. The acute Gs coupling induced by 100 µM morphine was completely prevented by co-treatment with 100 pM NLX, (+)NLX, or naltrexone (NTX), or their pentapeptide binding site (FLNA2561–2565), which we show can act as a decoy for MOR or bind to FLNA itself. All of these co-treatments presumably prevent the MOR–FLNA interaction. Since ultra-low-dose NTX also attenuates the addictive properties of opioids, we assessed striatal cAMP production and CREB phosphorylation at S133. Correlating with the Gs coupling, acute morphine induced elevated cAMP levels and a several-fold increase in pS133CREB that were also completely blocked by NLX, NTX or the FLNA pentapeptide. We propose that acute, robust stimulation of MOR causes an interaction with FLNA that allows an initially transient MOR–Gs coupling, which recovers with receptor recycling but persists when MOR stimulation is repeated or prolonged. The complete prevention of this acute, morphine-induced MOR–Gs coupling by 100 pM NLX/NTX or 10 µM pentapeptide segment of FLNA further elucidates both MOR signaling and the mechanism of action of ultra-low-dose NLX or NTX in attenuating opioid tolerance, dependence and addictive potential

High-Affinity Naloxone Binding to Filamin A Prevents Mu Opioid Receptor–Gs Coupling Underlying Opioid Tolerance and Dependence

Ultra-low-dose opioid antagonists enhance opioid analgesia and reduce analgesic tolerance and dependence by preventing a G protein coupling switch (Gi/o to Gs) by the mu opioid receptor (MOR), although the binding site of such ultra-low-dose opioid antagonists was previously unknown. Here we show that with approximately 200-fold higher affinity than for the mu opioid receptor, naloxone binds a pentapeptide segment of the scaffolding protein filamin A, known to interact with the mu opioid receptor, to disrupt its chronic opioid-induced Gs coupling. Naloxone binding to filamin A is demonstrated by the absence of [3H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [3H]naloxone binding to its filamin A-transfected subclone A7 or to immunopurified filamin A. Naloxone binding to A7 cells was displaced by naltrexone but not by morphine, indicating a target distinct from opioid receptors and perhaps unique to naloxone and its analogs. The intracellular location of this binding site was confirmed by FITC-NLX binding in intact A7 cells. Overlapping peptide fragments from c-terminal filamin A revealed filamin A2561-2565 as the binding site, and an alanine scan of this pentapeptide revealed an essential mid-point lysine. Finally, in organotypic striatal slice cultures, peptide fragments containing filamin A2561-2565 abolished the prevention by 10 pM naloxone of both the chronic morphine-induced mu opioid receptor–Gs coupling and the downstream cAMP excitatory signal. These results establish filamin A as the target for ultra-low-dose opioid antagonists previously shown to enhance opioid analgesia and to prevent opioid tolerance and dependence

The Post-Synaptic Density of Human Postmortem Brain Tissues: An Experimental Study Paradigm for Neuropsychiatric Illnesses

Recent molecular genetics studies have suggested various trans-synaptic processes for pathophysiologic mechanisms of neuropsychiatric illnesses. Examination of pre- and post-synaptic scaffolds in the brains of patients would greatly aid further investigation, yet such an approach in human postmortem tissue has yet to be tested. We have examined three methods using density gradient based purification of synaptosomes followed by detergent extraction (Method 1) and the pH based differential extraction of synaptic membranes (Methods 2 and 3). All three methods separated fractions from human postmortem brains that were highly enriched in typical PSD proteins, almost to the exclusion of pre-synaptic proteins. We examined these fractions using electron microscopy (EM) and verified the integrity of the synaptic membrane and PSD fractions derived from human postmortem brain tissues. We analyzed protein composition of the PSD fractions using two dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) and observed known PSD proteins by mass spectrometry. Immunoprecipitation and immunoblot studies revealed that expected protein-protein interactions and certain posttranscriptional modulations were maintained in PSD fractions. Our results demonstrate that PSD fractions can be isolated from human postmortem brain tissues with a reasonable degree of integrity. This approach may foster novel postmortem brain research paradigms in which the stoichiometry and protein composition of specific microdomains are examined

Prenatal Cocaine Exposure Increases Synaptic Localization of a Neuronal RasGEF, GRASP-1 via Hyperphosphorylation of AMPAR Anchoring Protein, GRIP

Prenatal cocaine exposure causes sustained phosphorylation of the synaptic anchoring protein, glutamate receptor interacting protein (GRIP1/2), preventing synaptic targeting of the GluR2/3-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs; J. Neurosci. 29: 6308–6319, 2009). Because overexpression of GRIP-associated neuronal rasGEF protein (GRASP-1) specifically reduces the synaptic targeting of AMPARs, we hypothesized that prenatal cocaine exposure enhances GRASP-1 synaptic membrane localization leading to hyper-activation of ras family proteins and heightened actin polymerization. Our results show a markedly increased GRIP1-associated GRASP-1 content with approximately 40% reduction in its rasGEF activity in frontal cortices (FCX) of 21-day-old (P21) prenatal cocaine-exposed rats. This cocaine effect is the result of a persistent protein kinase C (PKC)- and downstream Src tyrosine kinase-mediated GRIP phosphorylation. The hyperactivated PKC also increased membrane-associated GRASP-1 and activated small G-proteins RhoA, cdc42/Rac1 and Rap1 as well as filamentous actin (F-actin) levels without an effect on the phosphorylation state of actin. Since increased F-actin facilitates protein transport, our results suggest that increased GRASP-1 synaptic localization in prenatal cocaine-exposed brains is an adaptive response to restoring the synaptic expression of AMPA-GluR2/3. Our earlier data demonstrated that persistent PKC-mediated GRIP phosphorylation reduces GluR2/3 synaptic targeting in prenatal cocaine-exposed brains, we now show that the increased GRIP-associated GRASP-1 may contribute to the reduction in GluR2/3 synaptic expression and AMPAR signaling defects