Identification of neuron-glia signaling feedback in human schizophrenia using patient-derived, mix-and-match forebrain assembloids

Background

Schizophrenia is a challenging disease to study since numerous environmental and genetic factors contribute to its development¹. To better understand the mechanisms involved in schizophrenia, it is necessary to assess the impacts of the disease on the brain at different stages of development in the same individual, which isn’t a trivial task². In vitro models, such as patients’ induced pluripotent stem cells (iPSCs), have been used to model schizophrenia while conserving the individual’s personalized genetic information. However, iPSCs do not capture the complexity of the neural connections in the human brain.

The revolutionary advent of brain organoids derived from patient iPSCs has made it possible to model the complex connections between neurons in disease^3,4. Though 3D brain organoids are an improvement on 2D in vitro models, organoids still present some limitations (like any model). Indeed, brain organoids lack cellular diversity and essential information on how different brain regions interact^2,4.

In this preprint², the authors propose a novel model to study schizophrenia development. Their forebrain assembloid model involves reconstituting an organoid with glial cells derived from either healthy or schizophrenia patients’ iPSCs. The authors reveal novel structural and genetic features across multiple cell types that are likely to contribute to disease progression. These findings come from an extensive study of the assembloids at various stages of development and subsequent recapitulation in a chemically-induced mouse model of schizophrenia.

Key Findings

TP53 and NFATC4 drive neurodevelopmental defects in schizophrenia organoids

Using transcriptomic profiling of schizophrenia patient-derived assembloids and post-mortem brain tissue, the authors found increased expression of tumour protein p53 (TP53) and nuclear factor of activated T-cells 4 (NFATC4) during early stages of forebrain development. These proteins act as master transcription regulators, which epigenetically reprogram the transcriptomic landscape in schizophrenia brains. Increased expression of these master regulators leads to reduced proliferation of neural progenitor cells (NPCs), premature neuronal differentiation, and a marked reduction in cortical layer thickness. However, genetic ablation of TP53 and NAFTC4 rescued these defects, as these organoids exhibited enhanced proliferation of NPCs, an increase in SOX2-positive progenitors, and improved cortical thickness; forebrain organoids, specifically, displayed proper laminar organization similar to healthy controls.

SCZ patient-derived astrocytes and microglia cells exacerbate synapse deficits in assembloid models of schizophrenia

To begin their investigation of glial cell involvement in schizophrenia neuropathology, the authors characterized synapse density and function in the assembloids using immunohistochemistry, electrophysiology and calcium imaging. The authors observed that schizophrenia organoids reconstituted with schizophrenia or healthy glial cells displayed comparable synaptic deficits. The authors reasoned this is due to transcriptome-altering signalling between diseased neurons and diseased or healthy glial cells. The authors also observed that schizophrenia organoids reconstituted with schizophrenia glial cells displayed worse synaptic deficits than schizophrenia organoids lacking glial cells. These results show that schizophrenia glial cells exacerbate synapse deficits in schizophrenia assembloids.

Up-regulation of the UCN-WNT11 and PTPRF-THBS4 gene axes between neurons and glial cells contributes to synaptic deficits in schizophrenia assembloids

To identify reciprocally-regulating genes between neurons/astrocytes and neurons/microglia in assembloids, the authors performed RNA sequencing of the transcriptomes of neurons, astrocytes and microglia derived from SCZ iPSCs or isolated from assembloids. To validate the physiological relevance of their search, the authors made a consensus with parallel analyses of single-cell RNA sequencing data from post-mortem SCZ brain tissue. Through their efforts, the authors identified an up-regulation of the UCN-WNT11 signalling axis in neurons/astrocytes and the PTPRF-THBS4 signalling axis in neurons/microglia. The authors then performed CRISPR on schizophreniapatient-derived iPSCs to generate organoids with knocked out UCN and PTPR genes (dKO organoids), astrocytes with knocked out WNT11 and microglia with knocked out THBS4. The authors observed that dKO schizophrenia organoids reconstituted with non-KO schizophrenia glial cells, or non-dKO schizophrenia organoids reconstituted with KO schizophrenia glial cells exhibited improved synaptic function compared to schizophrenia assembloids. These results suggest that the up-regulation of the UCN-WNT11 and PTPRF-THBS4 gene axes contributed to the synapse deficits observed in schizophrenia assembloids.

In utero gene suppression reverses schizophrenia-associated deficits in a mouse model

Using a chemically induced mouse model of schizophrenia, the authors demonstrated that early and late gene expression patterns, specifically the increase in TP53 and NFATC4 expression at early developmental stages, and in UCN, WNT11, PTPRF, and THBS4 at later stages, mimic the transcriptomic and histological changes observed in schizophrenia patient-derived organoids. This includes reduced NPC proliferation, decreased cortical layer thickness, and behavioural deficits analyzed through open field tests, elevated plus maze test, and sucrose preference tests. In utero suppression of these dysregulated genes reversed both brain and behavioural abnormalities in the mouse model, highlighting the temporally and functionally distinct roles these two gene sets play in schizophrenia pathogenesis.

Why we chose to highlight this preprint

Schizophrenia remains a challenging disease to study due to its polygenic nature and diverse environmental risk factors. These complexities make studying transitional systems, such as animal models and post-mortem human brains, challenging. This preprint eloquently addresses such limitations using forebrain assembloids derived from patient iPSCs. Combining neurons, astrocytes, and microglia from patients and controls, the authors dissected neuron-glia interactions and identified cell-type-specific contributions to schizophrenia pathology. Additionally, the assembloid model enabled the authors to track these interactions across neurodevelopmental stages, offering a dynamic and human-relevant perspective on disease progression. For these reasons, we believe this study provides a novel, yet powerful approach for modelling schizophrenia.

Questions for the authors

1) You reconstituted the SCZ organoids with only astrocytes and microglia. If you were to reconstitute them with oligodendrocytes derived from SCZ iPSCs, would you expect to see reciprocal alterations between neurons and the oligodendrocytes?

2) You focused on signalling axes between neurons and glial cells, however, you omitted analyzing signalling axes between astrocytes and microglia. Since astrocytes and microglia are known to communicate, would you expect to see reciprocal alterations between glial cells?

3) Your findings suggest that over-regulation of the UNC-WNT11 and PTPRF-THBS4 pathways between neurons/astrocytes and neurons/microglia contributes to developing schizophrenia neuropathology. Furthermore, you show that these gene pathways regulate the synthesis of biomolecules and the detection of stimuli in neurons. With that said, which specific biomolecules or stimuli do you think to be most implicated in the developmental pathophysiology of schizophrenia?

References

1. Thompson, P. M., Vidal, C., Giedd, J. N., Gochman, P., Blumenthal, J., Nicolson, R., Toga, A. W., & Rapoport, J. L. (2001). Mapping adolescent brain change reveals a dynamic wave of accelerated gray matter loss in very early-onset schizophrenia. Proceedings of the National Academy of Sciences of the United States of America, 98(20), 11650–11655. https://doi.org/10.1073/pnas.201243998
2. Kim, E., Kim, Y., Hong, S., Kim, I., Lee, J., Lee, K., An, M., Kim, S.-Y., Kim, S. et Shin, K. (2024, 23 décembre). Identification of neuron-glia signaling feedback in human schizophrenia using patient-derived, mix-and-match forebrain assembloids. https://doi.org/10.1101/2024.12.22.629557
3. Centeno, E. G. Z., Cimarosti, H., & Bithell, A. (2018). 2D versus 3D human induced pluripotent stem cell-derived cultures for neurodegenerative disease modelling. Molecular neurodegeneration, 13(1), 27. https://doi.org/10.1186/s13024-018-0258-4
4. Sloan, S.A., Andersen, J., Pașca, A.M. et al. Generation and assembly of human brain region–specific three-dimensional cultures. Nat Protoc 13, 2062–2085 (2018). https://doi.org/10.1038/s41596-018-0032-7

Tags: assembloids, glia, neurons, schizophrenia

doi: https://doi.org/10.1242/prelights.40829

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