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1.
Front Cell Neurosci ; 11: 180, 2017.
Article in English | MEDLINE | ID: mdl-28725180

ABSTRACT

Nitric oxide exerts important regulatory functions in various brain processes. Its synthesis in neurons has been most commonly ascribed to the neuronal nitric oxide synthase (nNOS) isoform. However, the endothelial isoform (eNOS), which is significantly associated with caveolae in different cell types, has been implicated in synaptic plasticity and is enriched in the dendrites of CA1 hippocampal neurons. Using high resolution microscopy and co-distribution analysis of eNOS with synaptic and raft proteins, we now show for the first time in primary cortical and hippocampal neuronal cultures, virtually devoid of endothelial cells, that eNOS is present in neurons and is localized in dendritic spines. Moreover, eNOS is present in a postsynaptic density-enriched biochemical fraction isolated from these neuronal cultures. In addition, qPCR analysis reveals that both the nNOS as well as the eNOS transcripts are present in neuronal cultures. Moreover, eNOS inhibition in cortical cells has a negative impact on cell survival after excitotoxic stimulation with N-methyl-D-aspartate (NMDA). Consistent with previous results that indicated nitric oxide production in response to the neurotrophin BDNF, we could detect eNOS in immunoprecipitates of the BDNF receptor TrkB while nNOS could not be detected. Taken together, our results show that eNOS is located at excitatory synapses where it could represent a source for NO production and thus, the contribution of eNOS-derived nitric oxide to the regulation of neuronal survival and function deserves further investigations.

2.
Neurosci Lett ; 520(1): 98-103, 2012 Jun 27.
Article in English | MEDLINE | ID: mdl-22640895

ABSTRACT

Acylpeptide hydrolase (ACPH), a serine protease present in the central nervous system (CNS), is believed to have a function in modulating synaptic plasticity, cleavage of beta amyloid peptide and degradation of aggregated oxidized proteins. In this report, we demonstrate for the first time the presence of ACPH in the synapse and its preferential localization at the pre-synaptic side. We isolated subcellular fractions from the rat telencephalon enriched in pre- versus post-synaptic components by using differential centrifugation steps to evaluate ACPH catalytic activity and expression level. Relative ACPH levels were determined by Western blot techniques while antibodies against synaptophysin and PSD-95 were used as positive pre- and post-synaptic markers, respectively. Our results show that ACPH protein levels are significantly increased at the synapse, which correlates with a 56% increase in ACPH activity. Furthermore, Western blot experiments show that ACPH is preferentially located at the pre-synaptic side and this is consistent with the increase of its enzymatic activity in fractions enriched in pre-synaptic components. These results give new insights regarding the localization and a putative role of ACPH in the CNS.


Subject(s)
Peptide Hydrolases/metabolism , Telencephalon/enzymology , Acetylcholinesterase/metabolism , Animals , Male , Rats , Rats, Sprague-Dawley , Synapses/enzymology
3.
Neuroreport ; 19(11): 1123-6, 2008 Jul 16.
Article in English | MEDLINE | ID: mdl-18596612

ABSTRACT

Odour-mediated signal transduction is a complex process that occurs in the cilia of olfactory sensory neurons. To gain insight in to the molecular organization of the odour transduction machinery, we developed a procedure to purify olfactory cilia membranes by differential centrifugation of rat olfactory epithelium extracts. We tested whether known scaffolding proteins that might participate in the assembly of the complex chemotransduction apparatus are present in the purified membrane fraction. Utilizing immunoblotting and immunohistochemistry, we show that the multidomain scaffolding proteins ProSAP/Shanks and calcium/calmodulin-dependent serine protein kinase CASK are present in the olfactory cilia. Ion channels involved in chemotransduction could be reconstituted into planar lipid bilayers for electrophysiological recordings. Our procedure should allow the identification of further chemotransduction-related proteins.


Subject(s)
Cilia/physiology , Lipid Bilayers/metabolism , Olfactory Marker Protein/metabolism , Olfactory Mucosa/metabolism , Olfactory Receptor Neurons/physiology , Adaptor Proteins, Signal Transducing/metabolism , Adenylyl Cyclases/metabolism , Animals , Blotting, Western , Carrier Proteins/metabolism , Cilia/metabolism , Cyclic Nucleotide-Gated Cation Channels/metabolism , Guanylate Kinases/metabolism , Immunohistochemistry , Ion Channels/physiology , Isoenzymes/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins , Olfactory Receptor Neurons/metabolism , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Signal Transduction/physiology
4.
Brain Res ; 1150: 225-38, 2007 May 30.
Article in English | MEDLINE | ID: mdl-17397806

ABSTRACT

Induction of status epilepticus (SE) with kainic acid results in a large reorganization of neuronal brain circuits, a phenomenon that has been studied primarily in the hippocampus. The neurotrophin BDNF, by acting through its receptor TrkB, has been implicated in such reorganization. In the present work we investigated, by Western blot and immunohistochemistry, whether regional changes of TrkB expression within the rat brain cortex are correlated with altered neuronal morphology and/or with apoptotic cell death. We found that the full-length TrkB protein decreased within the cortex when measured 24 h to 1 week after induction of SE. Analysis by immunohistochemistry revealed that TrkB staining diminished within layer V of the retrosplenial granular b (RSGb) and motor cortices, but not within the auditory cortex. In layer II/III, differential changes were also observed: TrkB decreased in the motor cortex, did not change within the RSGb but increased within the auditory cortex. Reduced TrkB was associated with dendritic atrophy and decreased spine density in pyramidal neurons within layer V of the RSGb. No correlation was observed between regional and cellular changes of TrkB protein and apoptosis, measured by the TdT-mediated dUTP nick end labeling (TUNEL) method. The global decrease of TrkB within the neocortex and the associated dendritic atrophy may counteract seizure propagation in the epileptic brain but may also underlie cognitive impairment after seizures.


Subject(s)
Cerebral Cortex/pathology , Dendritic Spines/pathology , Neurons/pathology , Receptor, trkB/metabolism , Status Epilepticus/metabolism , Status Epilepticus/pathology , Animals , Dendritic Spines/ultrastructure , Disease Models, Animal , In Situ Nick-End Labeling/methods , Kainic Acid , Male , Rats , Rats, Sprague-Dawley , Silver Staining/methods , Statistics, Nonparametric , Status Epilepticus/chemically induced , Time Factors
5.
Neuropsychopharmacology ; 31(11): 2415-23, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16554746

ABSTRACT

We have studied the effect of low doses of two widely used antidepressants, fluoxetine (Flx) and reboxetine (Rbx), on excitatory synapses of rat brain cortex and hippocampus. After 15 days of Flx treatment (0.67 mg/kg/day), its plasma level was 20.7+/-5.6 ng/ml. Analysis of postsynaptic densities (PSDs) by immunoblotting revealed no changes in the glutamate receptor subunits GluR1, NR1, NR2A/B, mGluR1alpha nor in the neurotrophin receptor p75(NTR). However, the brain-derived neurotrophic factor (BDNF) receptor TrkB decreased by 42.8+/-6%, and remained decreased after 6 weeks of treatment. The BDNF and TrkB content in homogenates of cortex and hippocampus began to rise at 9 and 15 days, respectively, and remained high for up to 6 weeks. Similar results were obtained following chronic Rbx administration at 0.128 mg/kg/day. We propose that BDNF, whose synthesis is increased by antidepressants, and which is in part released at synaptic sites, binds to TrkB in PSDs, leading to the internalization of the BDNF-TrkB complex and, thus, to a decrease of TrkB in the PSDs. This was paralleled by greater levels of phosphorylated (ie activated) TrkB in the light membrane fraction, that contains signaling endosomes. The retrograde transport of endocyted BDNF/TrkB complexes from spines to cell bodies, where it activates the synthesis of more BDNF, is a protracted process, potentially requiring several cycles of TrkB/BDNF complex endocytosis and transport. This positive feedback mechanism may help explain the time-lag between drug administration and its therapeutic effect, that is, the antidepressant drug paradox.


Subject(s)
Antidepressive Agents/pharmacology , Brain/drug effects , Fluoxetine/pharmacology , Morpholines/pharmacology , Receptor, trkB/metabolism , Synapses/drug effects , Animals , Antidepressive Agents/blood , Blotting, Western/methods , Brain/cytology , Brain-Derived Neurotrophic Factor/metabolism , Dose-Response Relationship, Drug , Fluoxetine/blood , Glutamic Acid/metabolism , Immunoprecipitation/methods , Male , Morpholines/blood , Phosphotyrosine/metabolism , Rats , Rats, Sprague-Dawley , Reboxetine , Subcellular Fractions/drug effects , Time Factors , rab5 GTP-Binding Proteins/metabolism
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