CRISPRi-based screens in iAssembloids to elucidate neuron-glia interactions.

The sheer complexity of the brain has complicated our ability to understand its cellular mechanisms in health and disease. Genome-wide association studies have uncovered genetic variants associated with specific neurological phenotypes and diseases. In addition, single-cell transcriptomics have provided molecular descriptions of specific brain cell types and the changes they undergo during disease. Although these approaches provide a giant leap forward towards understanding how genetic variation can lead to functional changes in the brain, they do not establish molecular mechanisms. To address this need, we developed a 3D co-culture system termed iAssembloids (induced multi-lineage assembloids) that enables the rapid generation of homogenous neuron-glia spheroids. We characterize these iAssembloids with immunohistochemistry and single-cell transcriptomics and combine them with large-scale CRISPRi-based screens. In our first application, we ask how glial and neuronal cells interact to control neuronal death and survival. Our CRISPRi-based screens identified that GSK3ß inhibits the protective NRF2-mediated oxidative stress response in the presence of reactive oxygen species elicited by high neuronal activity, which was not previously found in 2D monoculture neuron screens. We also apply the platform to investigate the role of APOE-ε4, a risk variant for Alzheimer's Disease, in its effect on neuronal survival. This platform expands the toolbox for the unbiased identification of mechanisms of cell-cell interactions in brain health and disease.

A CRISPRi/a platform in human iPSC-derived microglia uncovers regulators of disease states.

Microglia are emerging as key drivers of neurological diseases. However, we lack a systematic understanding of the underlying mechanisms. Here, we present a screening platform to systematically elucidate functional consequences of genetic perturbations in human induced pluripotent stem cell-derived microglia. We developed an efficient 8-day protocol for the generation of microglia-like cells based on the inducible expression of six transcription factors. We established inducible CRISPR interference and activation in this system and conducted three screens targeting the 'druggable genome'. These screens uncovered genes controlling microglia survival, activation and phagocytosis, including neurodegeneration-associated genes. A screen with single-cell RNA sequencing as the readout revealed that these microglia adopt a spectrum of states mirroring those observed in human brains and identified regulators of these states. A disease-associated state characterized by osteopontin (SPP1) expression was selectively depleted by colony-stimulating factor-1 (CSF1R) inhibition. Thus, our platform can systematically uncover regulators of microglial states, enabling their functional characterization and therapeutic targeting.

Flexibility of in vitro cortical circuits influences resilience from microtrauma.

Background: Small clusters comprising hundreds to thousands of neurons are an important level of brain architecture that correlates single neuronal properties to fulfill brain function, but the specific mechanisms through which this scaling occurs are not well understood. In this study, we developed an in vitro experimental platform of small neuronal circuits (islands) to probe the importance of structural properties for their development, physiology, and response to microtrauma. Methods: Primary cortical neurons were plated on a substrate patterned to promote attachment in clusters of hundreds of cells (islands), transduced with GCaMP6f, allowed to mature until 10-13 days in vitro (DIV), and monitored with Ca2+ as a non-invasive proxy for electrical activity. We adjusted two structural factors-island size and cellular density-to evaluate their role in guiding spontaneous activity and network formation in neuronal islands. Results: We found cellular density, but not island size, regulates of circuit activity and network function in this system. Low cellular density islands can achieve many states of activity, while high cellular density biases islands towards a limited regime characterized by low rates of activity and high synchronization, a property we summarized as "flexibility." The injury severity required for an island to lose activity in 50% of its population was significantly higher in low-density, high flexibility islands. Conclusion: Together, these studies demonstrate flexible living cortical circuits are more resilient to microtrauma, providing the first evidence that initial circuit state may be a key factor to consider when evaluating the consequences of trauma to the cortex.