Neurogranin Expression Is Regulated by Synaptic Activity and Promotes Synaptogenesis in Cultured Hippocampal Neurons
Neurogranin (Ng) is a calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC) and is highly enriched in the dendrites and spines of telencephalic neurons. It is proposed to be involved in regulating CaM availability in the post-synaptic environment to modulate the efficiency of excitatory synaptic transmission. There is a close relationship between Ng and cognitive performance; its expression peaks in the forebrain coinciding with maximum synaptogenic activity, and it is reduced in several conditions of impaired cognition. We studied the expression of Ng in cultured hippocampal neurons and found that both protein and mRNA levels were about 10% of that found in the adult hippocampus. Long-term blockade of NMDA receptors substantially decreased Ng expression. On the other hand, treatments that enhanced synaptic activity such as long-term bicuculline treatment or co-culture with glial cells or cholesterol increased Ng expression. Chemical long-term potentiation (cLTP) induced an initial drop of Ng, with a minimum after 15 min followed by a slow recovery during the next 2–4 h. This effect was most evident in the synaptosome-enriched fraction, thus suggesting local synthesis in dendrites. Lentiviral expression of Ng led to increased density of both excitatory and inhibitory synapses in the second and third weeks of culture. These results indicate that Ng expression is regulated by synaptic activity and that Ng promotes the synaptogenesis process. Given its relationship with cognitive function, we propose targeting of Ng expression as a promising strategy to prevent or alleviate the cognitive deficits associated with aging and neuropathological conditions.
KeywordsNeurogranin Synaptic plasticity Glutamate receptors Synaptogenesis Hippocampal neurons
We thank Dr. FG Scholl (IBiS, Sevilla, Spain) and P. Scheifelle (Biozentrum, Basel, Switzerland) for providing the lentivector pLOX-Syn-DsRed-Syn-GFP. We would like to thank the Advanced Light Microscopy Core Facility, from Centro de Biología Molecular Severo Ochoa (CSIC-UAM), for assistance with the imaging studies.
AG-G, RA, AJ-P, PS, DS-F, and EM-B carried out the experiments and analyzed the data. FJD-G conceived the study and wrote the manuscript. All the authors have read and approved the final version of the manuscript submitted.
This work was supported by the Spanish Ministry of Science and Innovation and MINECO (grants BFU2010-18297 and SAF2014- 55686-R). We also thank the “Fundación Ramón Areces” for providing institutional support to CBMSO.
Compliance with Ethical Standards
All procedures were carried out in accordance with the Spanish Royal Decree 1201/2005 for the protection of animals used in scientific research, and the European Union Directive 2010/63/EU regarding the protection of animals used for scientific purposes. The procedures were approved by local Ethical Committees.
Conflict of Interest
The authors declare that they have no conflict of interest.
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