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

Summary The sheer complexity of the brain has complicated our ability to understand the cellular and molecular mechanisms underlying its function 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 GSK3β 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. We find that APOE-ε4-expressing astrocytes may promote more neuronal activity as compared to APOE-ε3-expressing astrocytes. This platform expands the toolbox for the unbiased identification of mechanisms of cell-cell interactions in brain health and disease.

(D) Most significant Gene Ontology Biological Processes enriched in the 500 top genes expressed more highly in neurons in iAssembloids vs. 2D monocultured neurons.Adjusted P values were calculated using the EASE Score, a Modified Fisher Exact P-value and Benjamini-Hochberg method for correction for multiple hypothesis testing.
(E) UMAP representation of integrated dataset from astrocyte and neuron 2D co-culture versus iAssembloids.
(G) BFP+ neurons (blue) in monoculture and cultured within iAssembloids were stained for c-FOS (green).Scale bars represent 100 µm.(A) Cells identified as astrocytes from snRNA-seq in iAssembloids (this study) were integrated with data from single cell sequencing of monocultured astrocytes 9 using Seurat's data integration pipeline.A UMAP was generated and split by origin of sample and time point.Cluster names were defined by running defined markers through EnrichR.Clusters include a CDK15+ cluster, cells undergoing DNA replication, an immature astrocyte cluster (TOP2A+), cells expressing genes related to BDNF signaling, axon guidance, glycolysis, synaptic transmission modulation, the unfolded protein response, and a cluster that had no specific markers (miscellaneous).The final cluster (IL1α, TNF, C1q induced) is based on previous studies for cytokine-induced markers 9 .
(B) EnrichR BioPlanet 2019 pathway analysis was used to define clusters.Redundant terms (terms that consist of the same genes) were removed and only the term that had the highestlog 10 (adjusted p-value) is displayed.E.g.PRIM2, POLA2, PRIM1 fall into the category of DNA replication initiation and Leading strand biosynthesis, but only DNA replication is displayed here.Colors represent clusters that fell into pathway/gene ontology categories.P represents Fisher's Exact Test.Multiple correction testing is performed with the Benjamini-Hochberg method.
(C) Heatmap was generated based on markers defined in A and B.
(D) After clustering, the percent of total astrocytes that falls within the "inflammatory response category was plotted (E) Green channel represents astrocytes tagged with GFP sparsely seeded with neurons and non-fluorescent astrocytes.Astrocytes are seed at either a 2 neuron to 1 astroycte or 1 astrocyte to 1 astrocyte ratio.Representative images shown with iAssembloids treated with media (vehicle) and with media plus the triple cytokines (IL1α, TNF, C1q).The number of processes per astrocyte in focus (outlined in white) was manually counted.N=8 cells across 4 iAssembloids for vehicle and 8 cells across 3 iAssembloids for induced.Standard error of the mean is represented.P-value determined by student's t-test.Scale bar represents 100 micron.(B) Scatterplot comparing CRISPRi-based neuronal survival screens using astrocytes from serum-based differentiation protocol 17,53 vs. the serum-free differentiation protocol 11,12 .
(C) Correlation heatmap of CRISPRi-based neuronal survival screens in the context of 2D monocultures 6 vs. iAssembloids (this study).

Figure S5
. Focused validation screens for top hits from the primary screen (related to Fig. 3 and 4) (A) Scatterplot of gene scores from the primary iAssembloid screen hits vs the secondary validation screen.Phenotypes from the secondary screen have a narrower dynamic range but are highly correlated with the primary screen (r = 0.9).
(B) Results from secondary screens comparing iAssembloid culture in various media: normal media (composed of 0.5X astrocyte media and 0.5X microglia base media), normal media plus cytokines (our iAssembloid culture condition), astrocyte media, and astrocytes media plus cytokines, as well as 3D cultures with different cell type compositions: neurons only (N) and neurons plus microglia (N,M).

Figure S2 .
Figure S2.Astrocytes in iAssembloids express genes associated with neuronal support (related to Fig. 2)

Figure S3 .
Figure S3.Microglia within iAssembloids express MHC Class II proteins (related to Fig. 2)(A) Cells identified as microglia from snRNA-seq in iAssembloids were integrated with single-cell sequencing of microglia 8 using Seurat's data integration pipeline.A UMAP was generated and split by origin of sample and the time point.Clusters include those that fall into the interferon, chemokine, metallothioneins, P54, proliferative and SPP1+ cluster.iAssembloid microglia specific clusters (iAssembloid SPP1+, iAssembloid chemokine, and iAssembloid proliferative are also highlighted).Microglia mainly map to existing clusters.However, cells in iAssembloids uniquely express MHC Class II proteins such as HLA-DRA and HLA-DRB1.(B)Heatmap was generated based on markers defined in A.