Background: Microglia are the main resident immune cells of the brain, contributing to the brain’s development, protection and homeostasis1. Given the versatility of microglia’s roles, disruptions in microglia functioning have been implicated in the pathophysiology of schizophrenia2. While antipsychotics, the main treatment for schizophrenia, are believed to exert immunomodulatory effects3,4, their precise impact on microglia remains unknown.
Aims: This study aimed to characterize the molecular and functional profiles of human induced pluripotent stem cell (hiPSC)–derived microglia from schizophrenia patients relative to healthy controls, and to assess how acute antipsychotic exposure modulates these phenotypes.
Methods: hiPSCs from schizophrenia patients (n=4) and healthy controls (n=4) were differentiated into microglia-like cells as previously described5. A pilot dose-response study was conducted to aid in the selection of the appropriate antipsychotic treatment dosage, zooming into cellular viability and cytotoxicity. Microglia were exposed to an acute treatment (24 hours) of clozapine, olanzapine and haloperidol (0, 1, 10, 100, 1000nM). Upon dosage selection, at day 14, microglia were treated with clozapine [1uM], haloperidol [50nM], olanzapine [100nM] or vehicle [DMSO] for 24 hours. To investigate differences in motility, hiPSC-derived microglia were labelled, and live imaging (2 hours) was conducted at the Opera Phenix High Content Microscope. To assess differential phagocytic capacity of microglia according to treatment and diagnosis, phagocytosis of zymosan beads was measured through the BD LSRFortessa flow cytometer. The Proteome Profiler Human Cytokine array was used to evaluate changes in cytokine secretion in pooled media samples. Lastly, to investigate differential transcriptomic signatures, bulk RNA-Sequencing was conducted.
Results: Clozapine treatment did not affect cell viability across the distinct doses (H=2.935, p=0.569) and showed no cytotoxic effects (H=0.9461, p=0.918). The same was observed for haloperidol, with no trace of cytotoxicity (H=0.3614, p=0.9855) nor reduced cellular viability (H=4.124, p=0.3895). Exposure to increasing doses of olanzapine did not exhibit significant cytotoxic effects (H=4.050, p=0.3993), however an effect of olanzapine dosage on cell viability was reported (H=16.00, p=0.0030). Posthoc analysis revealed a significant 25% viability decline from baseline relative to 1μM (p=0.0109). Based on the pilot results, microglia were treated for 24 hours with 50nM of haloperidol, 100nM of olanzapine, 1μM of clozapine or vehicle (DMSO) for all subsequent experiments. Antipsychotic treatment and diagnosis did not impact the percentage of phagocytic microglia (DiagnosisxTreatment: F(3,16)=0.0099, p=0.999, Diagnosis F(1,16) = 0.0196, p=0.890, Treatment: F(3,16)=0.0295, p=0.993), nor the amount of phagocytosed particles (DiagnosisxTreatment: F(3,16)=0.0602, p=0.980, Diagnosis: F(1,16)=3.03×10⁻⁵, p=0.996, Treatment: F(3,16)=0.0391, p=0.989). Motility analysis also reported no significant effects of Treatment or Diagnosis. Qualitative assessment of cytokine profiles suggested a heightened SERPINE1 secretion at baseline in the patient population, further enhanced by clozapine and haloperidol treatment. Transcriptomic analysis is currently ongoing.
Conclusion: This study suggests that antipsychotic treatment does not impact functional phenotypes in microglia. The largely absent phenotypic differences between patient-derived samples and controls might indicate that microglia may require interactions with neurons to unmask disease-relevant abnormalities. To test this hypothesis, hiPSC-derived microglia–neuronal co-cultures will be established, employing a matched and mismatch patient-control design, to determine how cellular context shapes microglial behaviour.