2026 ECNP Congress

PS01-1216

SP4 dosage-dependent disruption of neuronal maturation and excitatory/inhibitory balance in human iPSC-derived forebrain neurons

Debbie Lim Xiu En — S.Y. Yeo, J. Chua Jia En · MSD International GmBH Singapore, Quantitative Biosciences, Singapore, Singapore

2026 ECNP Congress

Schizophrenia (SCZ) is a severe neuropsychiatric disorder characterized by positive symptoms such as hallucinations and delusions, negative symptoms including social withdrawal and affective flattening, and cognitive impairments that disrupt attention, memory, and executive function. Despite decades of research, the cellular and molecular mechanisms underlying SCZ remain incompletely understood. Converging evidence from neuroimaging, postmortem tissue analysis, and genetic studies implicates cortical glutamate/GABA dysregulation as a central feature of the disorder. In particular, abnormalities in excitatory/inhibitory balance and synaptic refinement have been consistently observed, suggesting that disruptions in transcriptional programs governing neuronal maturation may contribute to disease pathophysiology.

Among candidate regulators, Specificity Protein 4 (SP4) has emerged as a critical zinc‑finger transcription factor with high expression in the brain. SP4 belongs to the specificity protein family (Sp1–Sp4), which binds GC‑rich promoter elements to regulate genes essential for neuronal differentiation and synaptic function. Reduced SP4 expression has been reported in cortical tissue from individuals with SCZ, especially in patients exhibiting severe negative symptoms. Genetic studies further support SP4 as a risk gene, with rare truncating mutations conferring elevated odds ratios for SCZ. Functionally, SP4 regulates NMDA receptor subunits and dendritic patterning, processes that are central to excitatory neurotransmission and circuit refinement. Loss of SP4 in animal models impairs dendritic pruning and synaptic maturation, paralleling structural abnormalities observed in SCZ patient brains [1-2]. These findings highlight SP4 as a mechanistically relevant factor linking transcriptional dysregulation to synaptic pathology.

To examine dosage‑dependent effects of SP4 in a human cellular context, we generated CRISPR/Cas9‑engineered SP4‑deficient induced pluripotent stem cell (iPSC)‑derived forebrain neurons containing both excitatory and inhibitory subtypes. Cultures were analyzed at DIV 48 and 64, developmental stages corresponding to synaptic maturation. Immunocytochemistry for Homer1 (postsynaptic) and synaptophysin (presynaptic) colocalization revealed increased synaptic puncta in both SP4+/- and SP4-/- neurons compared to wild‑type controls. SP4+/- neurons additionally displayed excessive dendritic growth and branching, whereas SP4-/- cultures lacked this expansion. Despite this structural overproduction, electrophysiological recordings at D48 demonstrated immature excitatory transmission: both SP4+/- and SP4-/- neurons exhibited elevated sEPSC amplitude but reduced frequency, with no change in sIPSCs (n=4; one‑way ANOVA, Dunnett’s multiple comparison test). This mismatch between synaptic structure and function indicates that SP4 deficiency promotes synaptic overgrowth without activity‑dependent refinement. Intrinsic properties of SP4+/- neurons further included increased burst strength, reduced firing rates, and elevated rheobase and capacitance, consistent with electrophysiological immaturity. Thus, partial SP4 loss drives excitatory/inhibitory imbalance and ineffective glutamatergic signaling, despite dendritic expansion. These findings parallel dendritic pathology observed in postmortem and neuroimaging studies of SCZ patients with prominent negative symptoms, highlighting SP4 as a critical regulator of neuronal maturation and circuit refinement.

Impact

Our study establishes SP4 as a dosage‑sensitive regulator of dendritic growth, synaptic maturation, and excitatory/inhibitory balance in human neurons. By linking transcriptional dysregulation to structural overproduction and immature transmission, these results provide a mechanistic bridge between genetic risk and circuit pathology in SCZ. SP4 emerges as a potential biomarker for negative symptom severity and a candidate target for therapeutic strategies aimed at restoring synaptic refinement and glutamatergic signaling.

References

  1. Sun X, Pinacho R, Saia G, Punko D, Meana JJ, Ramos B, Gill G. Transcription factor Sp4 regulates expression of nervous wreck 2 to control NMDAR1 levels and dendrite patterning. Dev Neurobiol. 2015 Jan;75(1):93-108. doi: 10.1002/dneu.22212. Epub 2014 Jul 22. PMID: 25045015; PMCID: PMC4261064.
  2. Ramos B, Gaudillière B, Bonni A, Gill G. Transcription factor Sp4 regulates dendritic patterning during cerebellar maturation. Proc Natl Acad Sci U S A. 2007 Jun 5;104(23):9882-7. doi: 10.1073/pnas.0701946104. Epub 2007 May 29. PMID: 17535924; PMCID: PMC1887555.