Patient-Derived Air-Liquid Interface Forebrain Organoids Reveal Functional Synaptic Deficits in Schizophrenia

Schizophrenia (SCZ) is a severe and debilitating neurodevelopmental disorder with lifelong impact on everyday life. Disruptions in synapse functions play a key role in its complex and poorly understood etiological and pathological mechanisms. Here, we investigated both the molecular composition and the spontaneous and stimulated functional properties of synapses in neural organoids from SCZ individuals. Air-liquid interface forebrain organoids (ALI-FOs) were generated from induced pluripotent stem cells (iPSCs) derived from three individuals with SCZ and three healthy controls. At day 170 synaptosomes were enriched and analyzed by data-independent acquisition mass spectrometry to profile the proteome, alongside with TMT-labeled phosphoproteomics both before and after acute KCl-induced depolarization. In parallel, we characterized the PTMome of the surrounding cellular environment, comprising phosphorylation, peptides with free and reversibly modified cysteines, and sialylated N-linked glycopeptides. Functional glutamatergic and GABAergic activity was assessed using calcium imaging to capture spontaneous neuronal signaling. Both conditions exhibited mature synaptic structures, while growth cones were observed only in SCZ-derived ALI-FOs, indicative of ongoing or delayed synaptogenesis. Proteomic analysis of synaptosome preparations revealed 358 differentially regulated proteins between SCZ and controls and 125 phophoproteins with altered phosphorylation, which clustered into three major categories: (1) synaptogenesis and synapse signaling; (2) cytoskeleton and cell junctions; (3) growth cone dynamics and neurite outgrowth. Analysis of the PTMs in the surrounding cellular environment revealed regulation of key regulatory mechanisms in 526 proteins, supporting the synaptic alterations observed. Notably, components of the Wnt signaling pathway were consistently dysregulated across both the synaptosome preparation and the PTMome in SCZ-derived ALIFOs as compared to controls. Depolarization-induced phospho-signaling revealed SCZ-specific response enriched in synaptic vesicle trafficking pathways. Together, these findings provide new insights into early synaptic alterations in SCZ, highlighting changes not only in protein composition, but more in protein regulatory mechanisms underlying synaptic signaling.