Endothelial Nitric Oxide Synthase Is Present in Dendritic Spines of Neurons in Primary Cultures.

Ariel Caviedes,Carlos Lafourcade,Manuel Varas-Godoy,Angela Massmann,Ursula Wyneken,Thilo Kaehne,Soledad Sandoval,Javiera Bravo-Alegria,Jorge P Figueroa,Francisco Nualart

doi:10.3389/fncel.2017.00180

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.

Highlights

Nitric oxide (NO) is a free radical gas with pivotal signaling capacities in the central nervous system (Benarroch, 2011; Hardingham et al, 2013)
Clean and highly reproducible results were obtained with the monoclonal endothelial isoform (eNOS) antibody of Transduction Laboratories, while the phospho-eNOS and neuronal nitric oxide synthase (nNOS) antibodies were proven to be reliable in Western Blots (Supplementary Figure S1)
In RFP-positive neurons transduced with shRNA1, no eNOS staining could be detected at day 12 in vitro, while RFP-positive neurons transfected with the control shRNA revealed a punctate staining pattern

Summary

Introduction

Nitric oxide (NO) is a free radical gas with pivotal signaling capacities in the central nervous system (Benarroch, 2011; Hardingham et al, 2013). It has a recognized role in the regulation of synaptic plasticity, excitability and excitotoxicity (Calabrese et al, 2007; Brunert et al, 2009; Steinert et al, 2010, 2011). ENOS which is known for its prominent role in the regulation of cerebral blood flow, was surprisingly detected in neocortical and hippocampal neurons where it constitutes a primary source of NO necessary for the induction of long-term potentiation (LTP), while membrane targeting is necessary to induce plasticity (Dinerman et al, 1994; O’Dell et al, 1994; Roskams et al, 1994; Haley et al, 1996; Kantor et al, 1996; Haul et al, 1999). We used primary neuronal cultures, a system free of endothelial cells, to assess eNOS localization in hippocampal and cortical neurons by confocal and super-resolution microscopy as well as by Western Blot and qPCR

Methods

Results

Conclusion