Introduction: Docosahexaenoic acid (DHA), the predominant omega-3 (ω-3) fatty acid in the brain, which contributes to synaptic membrane organization and plasticity, is commonly recommended for brain health. However, ω-3 formulations commonly combine DHA with eicosapentaenoic acid (EPA) in varying proportions, and the impact of the DHA ratio on postsynaptic scaffold regulation remains unclear. Methods: In this study, differentiated human SH-SY5Y neurons were cultured in the presence of DHA, EPA, or defined DHA ratios (2:1, 1:1, 1:2) at equivalent total concentrations. PSD-95 expression, puncta density, colocalization with the presynaptic marker synaptophysin (SYN), and glutamatergic signaling proteins were quantified by immunofluorescence and Western blot analysis. Metabolic activity was assessed using a colorimetric MTT assay. Results: DHA produced the largest increase in PSD-95 protein abundance by immunoblot and the strongest effects on PSD-95 puncta density and SYN colocalization, accompanied by coordinated upregulation of N-methyl-D-aspartate receptor (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), and calcium/calmodulin-dependent protein kinase II (CaMKII). DHA-enriched compositions preserved this profile, whereas balanced or EPA-dominant compositions showed attenuated responses relative to DHA-dominant conditions. EPA alone failed to enhance postsynaptic scaffold abundance and reduced NMDAR levels relative to control. These effects occurred without a detectable reduction in cellular metabolic activity. Discussion: Collectively, these findings demonstrate that the composition of ω-3 fatty acid has a significant bearing on postsynaptic scaffold organization and glutamatergic signaling in differentiated neurons, with DHA serving as the chief mediator of observed effects. This composition-dependent framework provides an experimental basis for considering DHA ratio when evaluating omega-3 effects on postsynaptic protein organization.

DLG4-related synaptopathy (SHINE syndrome) is a rare neurodevelopmental disorder caused by pathogenic variants in DLG4, which encodes the postsynaptic scaffolding protein (PSD)-95, a central organizer of excitatory synapses. However, the cellular and network-level consequences of DLG4 haploinsufficiency in human neurons remain incompletely understood, and disease-targeted therapeutic strategies are lacking. Here, we used human induced neurons derived from an individual with SHINE syndrome to investigate the effects of PSD-95 deficiency on synaptic protein expression, neuronal morphology, and neuronal network activity. SHINE neurons exhibited reduced PSD-95 levels, impaired neurite morphology, reduced firing rate and burst frequency, prolonged burst duration, and reduced network synchrony. Treatment with the omega-3 (ω-3) polyunsaturated fatty acid docosahexaenoic acid (DHA) significantly increased PSD-95 protein levels and improved multiple parameters of neuronal network activity. DHA exposure was also associated with trends toward increased extracellular signal-regulated kinase and cAMP response element-binding protein phosphorylation. Together, these findings show that patient-derived SHINE neurons exhibit altered postsynaptic organization, neuronal morphology, and network function and provide evidence that DHA may partially restore synaptic and network function in this disorder.

DLG4-related synaptopathy (SHINE syndrome) is a rare neurodevelopmental disorder caused by pathogenic variants in DLG4, which encodes the postsynaptic scaffolding protein PSD-95, a central organizer of excitatory synapses. However, the cellular and network-level consequences of DLG4 haploinsufficiency in human neurons remain incompletely understood, and disease-targeted therapeutic strategies are lacking. Here, we used human induced neurons derived from an individual with SHINE syndrome to investigate the effects of PSD-95 deficiency on synaptic protein expression, neuronal morphology, and neuronal network activity. SHINE neurons exhibited reduced PSD-95 levels, impaired neurite morphology, reduced firing rate and burst frequency, prolonged burst duration, and reduced network synchrony. Treatment with the omega-3 (ω-3) polyunsaturated fatty acid docosahexaenoic acid (DHA) significantly increased PSD-95 protein levels and improved multiple parameters of neuronal network activity. DHA exposure was also associated with trends toward increased ERK and CREB phosphorylation. Together, these findings demonstrate that DLG4 haploinsufficiency disrupts postsynaptic organization, neuronal morphology, and network function in human neurons and provide evidence that DHA may partially restore synaptic and network function in this disorder.