Neurogranin enhances spontaneous activity and neuronal survival of hippocampal neurons

Abstract Neurogranin (Ng) is a postsynaptic protein highly enriched in forebrain neurons and implicated in synaptic plasticity through its ability to bind calmodulin. However, its impact on neuronal development, network dynamics, and cellular homeostasis remains incompletely understood. In this study, we examined the effects of manipulating Ng expression in primary hippocampal neurons using viral gene delivery, with emphasis on structural, functional, and molecular outcomes. Restoring Ng expression to adult physiological levels enhanced dendritic growth, increased synaptic number, and induced a proximal shift of the axon initial segment, consistent with adaptive responses to increased connectivity. Functionally, Ng markedly increased spontaneous neuronal activity and network synchronization, even under culture conditions that normally show minimal baseline activity. Electrophysiological recordings revealed enhanced burst firing and spike synchrony, indicating strengthened functional coupling rather than increased membrane excitability. Ng-dependent activity required action potential firing and glutamatergic transmission. At the molecular level, Ng increased total calmodulin levels in a binding-dependent manner, reduced overall calcium/calmodulin-dependent protein kinase II abundance while enhancing its relative autophosphorylation, and selectively decreased both total and surface levels of ionotropic glutamate receptors. These changes are consistent with a coordinated homeostatic reorganization of calcium-dependent signaling. Despite robust increases in activity, Ng expression improved neuronal viability, reduced cellular stress markers, and increased expression of the anti-apoptotic protein Bcl-2. Active caspase-3 was selectively elevated without triggering apoptosis, suggesting a non-apoptotic role in activity-dependent structural remodeling. Together, these findings identify Ng as a homeostatic regulator that promotes coordinated network activity, adaptive synaptic remodeling, and neuronal survival. Competing Interest Statement The authors have declared no competing interest. Abbreviations - 2-APB - (2-aminoethoxydiphenyl borate), - AAV - (adeno-associated virus), - AD - (Alzheimer’s disease), - AIS - (axon initial segment), - AnkG - (Ankyrin G), - AraC - (1-beta-arabino-furanosylcytosine), - BSA - (bovine serum albumin), - Ca2+ - (calcium ion), - CaM - (calmodulin), - CaMKII - (calcium/calmodulin-dependent protein kinase II), - CSF - (cerebrospinal fluid), - DIV - (days in vitro), - DMEM - (Dulbecco’s modified Eagle’s medium), - E19 - (embryonic day 19), - ECL - (enhanced chemiluminescence), - FBS - (fetal bovine serum), - HBSS - (Hank’s balanced salt solution), - LDH - (lactate dehydrogenase), - LTD - (long-term depression), - LTP - (long-term potentiation), - LVs - (lentiviral particles), - MCI - (mild cognitive impairment), - mEPSC - (miniature excitatory postsynaptic current), - MPEP - (2-methyl-6-(phenylethynyl)pyridine), - MPMS - (1-methoxyphenazine methosulfate), - MTT - (thiazolyl blue tetrazolium bromide), - NA - (numerical aperture), - NAD - (β-nicotinamide adenine dinucleotide), - NB - (neurobasal culture medium), - Ng - (Neurogranin), - NMDA - (N-methyl-D-Aspartate), - PA - (phosphatidic acid), - PFA - (paraformaldehyde), - PI - (propidium iodide), - PKC - (protein kinase C), - PLL - (poly-L-lysine), - ROI - (region of interest), - RT - (room temperature), - TTX - (tetrodotoxin)