Multiomic modelling of schizophrenia using patient-derived brain organoids

Schizophrenia (SCZ) is a highly heritable mental disorder characterized by disabling psychotic and cognitive symptoms, making a significant contribution to the global disease burden. The first symptoms typically appear in late adolescence and typically progress in a relapsing or chronic pattern. Existing treatments are only partially effective in restoring function, with many patients suffering from side effects and a lifelong disability. The growing number of identified genetic and environmental risk factors implicate deviation in early neurodevelopmental processes but despite this, the pathophysiology remains largely elusive. This is primarily due to the unavailability of diseased or "at-risk" brain tissue, as well as the absence of suitable experimental models to elucidate the mechanisms by which these risk factors affect the brain.

However, recent studies have linked the genetic risk for SCZ to excessive microglia-mediated elimination of synapses, utilizing patient-derived cellular models based on induced pluripotent stem cells (iPSCs) from individuals with SCZ. Brain postmortem studies also confirm a loss of synapses in SCZ, and imaging studies suggest that this occurs already during the first psychotic episode.

The main aim of this thesis was to investigate how risk factors for SCZ, genetic as well as environmental, influence early brain development and contribute to pathophysiology and clinical manifestations taking place much later in early adulthood. To do this, we used a multi- pronged approach including patient-derived cellular modeling, with high-resolution sequencing techniques across molecular layers applied to human brain organoids, as well as clinical studies based on cerebrospinal fluid (CSF) collected from patients that experienced their first psychotic episode.

In paper I, we exposed whole brain organoids with innately developing microglia to SARS- CoV-2, a virus linked to a neurodevelopmental spectrum diagnosis in young children and SCZ in adults. We discovered that SARS-CoV-2, despite a limited neurotropism, causes a loss of synaptic termini, similar to genetic risk factors for SCZ. Utilizing high-resolution imaging, we observed an increased uptake of synaptic material in microglia, while single cell transcriptomic profiling revealed an interferon-responsive state of microglia, promoting synapse elimination.

Clinical studies of postmortem brain tissue and CSF obtained from patients with SCZ have revealed elevated levels of kynurenic acid, an endogenous metabolite of tryptophan with antagonistic properties on the N-methyl-D-aspartate (NMDA) and a7 nicotinic acetylcholine (a7nAch) receptors. In paper II, we demonstrated that extrinsic kynurenic acid decreased neuronal activity while leading to a loss of synapses in co-cultures of glutamatergic neurons and microglia-like cells from patients. Further using patient-derived forebrain organoids with innately developing microglia we show that pharmacological inhibition of kynurenic acid production caused a decreased internalization of synaptic structures in microglia. Our transcriptomic analyses also explored gene expression networks centered on the kynurenic acid-producing enzymes that enrich for common genetic SCZ risk variants and genes governing synaptic activity.

In paper III, we focused on another complement factor, C1Q, that initiates the classical pathway used by microglia. The most strongly associated risk locus for SCZ can largely be explained by genomic elements that increase complement component 4A (C4A) expression and C4A proteins levels are elevated in patients first-episode SCZ. Experimental studies then show that C4A can bind synaptic structures and facilitate internalization of synaptic elements. In CSF obtained from patients with first-episode psychosis, we observed lower C1q levels while postmortem brain tissue gene expression data indicated lower C1Q mRNA expression.

In paper IV, we utilized multi-lineage forebrain organoids derived from monozygotic twins discordant for SCZ and undertook an unbiased approach with combined single-cell transcriptomics and epigenomics. Our findings revealed early developmental disruptions in a cell type-specific manner across both modalities as well as an increased uptake of synaptic material in microglia within the patient-derived models.

The work presented in this thesis suggest that different risk factors for SCZ can converge overlapping neurodevelopmental processes with the potential to influence pathophysiology observed in first-episode patients. Further, they strengthen the role of integrated multi- disciplinary approaches to study the causes of SCZ.

List of scientific papers

I. Samudyata*, Ana O. Oliveira*, Susmita Malwade*, Nuno Rufino de Sousa, Sravan K. Goparaju, Jessica Gracias, Funda Orhan, Laura Steponaviciute, Martin Schalling, Steven D. Sheridan, Roy H. Perlis, Antonio G. Rothfuchs & Carl M. Sellgren. SARS-CoV-2 promotes microglial synapse elimination in human brain organoids. Molecular Psychiatry. 27, 3939-3950 (2022). https://doi.org/10.1038/s41380-022-01786-2

II. Funda Orhan, Susmita Malwade, Neda Khanlarkhani, Asimenia Gkoga, Oscar Jungholm, Marja Koskuvi, Šárka Lehtonen, Lilly Schwieler, Kent Jardemark, Jari Tiihonen, Jari Koistinaho, Sophie Erhardt, Göran Engberg, Samudyata, Carl M. Sellgren. Kynurenic acid promoted activity-dependent synaptic pruning in schizophrenia American Journal of Psychiatry. [Accepted]

III. Marja Koskuvi*, Susmita Malwade*, Jessica Gracias Lekander, Elin Hörbeck, Sanna Bruno, Jessica Holmen Larsson, Aurimantas Pelanis, Anniella Isgren, Anneli Goulding, Helena Fatouros-Bergman, Samudyata, Martin Schalling, Fredrik Piehl, Sophie Erhardt, Mikael Landen, Simon Cervenka, Funda Orhan & Carl M. Sellgren. Lower complement C1q levels in first-episode psychosis and in schizophrenia. Brain Behaviour and Immunity. 117, 313-319 (2024). https://doi.org/10.1016/j.bbi.2024.01.219

IV. Susmita Malwade, Samudyata, Jessica Gracias Lekander, Angelika Langeder, Katharina Weinberger, Marja Koskuvi, Luis Enrique Arroyo-García, Jari Koistinaho, Jari Tiihonen, Carl M. Sellgren. Developmental multi-omics of monozygotic twins discordant for schizophrenia. 2024. [Manuscript]

*equal contribution; co-first authors