Journal: Molecular Psychiatry

Article Title: Schizophrenia is defined by cell-specific neuropathology and multiple neurodevelopmental mechanisms in patient-derived cerebral organoids

doi: 10.1038/s41380-021-01316-6

Figure Lengend Snippet: a Schematic of proteome mapping pipeline using tandem mass tag (TMT) chemistry and liquid chromatography-mass spectrometry (LC-MS). Briefly, whole organoids were subjected to tryptic digestion to isolate proteins from each sample. Proteins were next isobarically barcoded with TMT reagents, and then condensed into a single multiplexed suspension. This allowed samples to be run concurrently, eliminating batch-specific technical variance, and a low-noise assessment of both organoid variability (using a 4x2 design) and the identification of translated disease factors. Multiplexed suspensions were subsequently subjected to quantitative TMT-LC-MS for peptide detection, deconvolution, and quantification, and then computationally analyzed. b Scz organoids recapitulate the developmental proteome. Given numerous cellular signatures of Scz that separated cases from Ctrls (Fig. ), we sought to determine the proteome diversity of Ctrl and Scz 3D organoids. The Scz organoid proteome was 99.95% identical in pooled protein diversity relative to Ctrls. Thus, Scz proteome differences are likely to be explained not in the induction of differential factors at the posttranslational level, but rather their total molecular quantity. c Individual proteins exhibit low variability in expression between groups. Given the high degree of conservation in peptide diversity between both Ctrl and Scz organoids, we next examined dispersion of individual proteins by generating their coefficient of variation (CVs) for each group. In both Ctrl and Scz samples, over 3000 detectable peptides for each group exhibited low variability (<20% CVs; blue dots). Only 3 and 10 proteins exhibited “high” variability in Ctrls and Scz organoids, respectively (red dots). In these panels, the black lines/box segregate proteins with median expression differences within groups. This reflects that Ctrl and Scz organoids exhibited similar patterns of proteome reproducibility in this particular analysis. d Subtle proteome differences in Scz patient-derived organoids. A heat-map of differentially expressed proteins shows differences in LC-MS expression intensities in Ctrl versus Scz organoids. Specifically, Scz organoids are defined by the differential expression of just 222 peptides, or ~5.9% of the detected proteome (heat-map). This heat-map thus provides a visualization of the individual peptide factors identified via LC-MS, and the consistency of expression differences at the single-protein level. Thus, consistent with our hypothesis, Scz organoid samples principally differed in their quantity, rather than diversity, of developmental factors. e Disease factors are detected and differentially expressed in Scz organoids. A heat-map of selected differentially expressed proteins in Scz organoids (Log2 key from panel d still applies). This included POU-domain peptide fragments that putatively mapped to the forebrain neuronal-development factor POU3F2 (BRN2), and disease factors with known and/or likely involvement in disease pathophysiology (e.g., COMT/PLCL1). In addition, factors with putative genetic risk but otherwise unestablished disease biology (e.g., PTN) were also differentially expressed in Scz organoids. f Schematic of pipeline for live single-cell RNA sequencing of organoids and clustering of 26,335 transcriptomes recapitulates fetal brain cellular identities. Briefly, organoids from 4 Ctrl and 3 Scz lines were generated concurrently, pooled by-line, and dissociated to a single-cell suspension. We rapidly conducted survival-based high-throughput FACS to purify samples to 2000 live cells/ µm per line. For robustness, post-FACS live cell viability was also cross-confirmed using Countess-II. Live cell suspensions were next rapidly loaded into 10x chromium microfluidic devices to produce barcoded single-cell nanodroplet emulsions. This emulsion was later broken, barcoded samples were amplified, libraries prepared, subjected to Illumina sequencing, and then parsed through a suite of unbiased computational analyses. Clustering of marker genes for cell-type specific clusters was conducted via pairwise comparisons of the normalized expression values for cells of a given cluster vs. the cells of all other clusters. Thus yielded unbiased gene sets, which were defined by the top 10 gene markers for each cluster that met a high-pass FDR threshold of 1% and >15,000 total read counts. Many of these prototypic markers defined cell-types consistent with human fetal tissue (see below). Here we present UMAP coordinates for 26,335 transcriptomes split by Ctrl and Scz cases, presenting the cell-type clusters identified in our unbiased clustering analysis. Cell-type labels were determined via a variety of approaches including marker gene-expression, automated annotation, and, namely, comparison with human fetal samples (see Methods for analysis pipeline). Bar chart (right) depicts cell-type proportions, illustrating that ~93% of Ctrl scRNA-Seq transcriptomes were identified as neural progenitors, proliferating cells, or terminal cortical cell-types (e.g., neurons and glia). Compared to this, only ~75% of Scz cell-types exhibited a similar conservation of identity. Compared to remaining cell types in Ctrl organoids (~7% cells), the remaining ~25% of Scz scRNA-Seq transcriptomes reflected enrichment for brain-related cell-types including putative neuroendothelial cells, structural markers, developing vasculature, retinal, and choroid plexus markers in Scz organoids. This analysis therefore revealed the cell-types produced at the expense of neurons in Scz organoids alluded to in pulse-chase experimentation depicted in Fig. . Of note, all cell-types exhibited reproducible proportions across individual iPSC donors within respective groups (i.e., all Scz organoids exhibited similarly reproducible alterations in cell-type diversity, which was defined by an overarching loss of neurons). g Confirmation of progenitor and neuronal depletion in Scz organoids. Scz organoids exhibited a striking depletion of progenitors ( SOX2+ and PAX6 +) and pan-neuronal markers ( MAP2 +, DCX +, and STMN2 +). These expression differences reflect both abundance and magnitude. Thus, replicating our prior results (see Fig. ), scRNA-Seq analysis confirmed that progenitors and neurons are depleted in Scz organoids. Representative UMAPs for SOX2 and MAP2 are provided given that these are the same markers shown for progenitor and neuronal depletion in Fig. . For Fig. 2f–j, total n = 26, 335 transcriptomes, n = 20,844 genes from 7 iPSC lines; Ctrl n = 15,089 transcriptomes from 4 Ctrl iPSC lines, and Scz n = 11,246 transcriptomes from 3 Scz iPSC lines. Ctrl: Control, Scz: Schizophrenia.

Article Snippet: Primary antibodies comprised SOX2 (1:1000; R&D Systems, CAT#: MAB2018-SP), PAX6 (1:1000; Biolegend, CAT#: 901301), β-tubulin III (1:1000; Abcam, CAT#: AB41489), MAP2 (1:1000, Abcam, CAT#: AB11267; 1:1000, Abcam, CAT#: AB32454), BrdU (1:1000, BD Pharminogen, CAT#: 555627), DCX (1:1000, Santa Cruz, sc-271390), Activated/Cleaved Caspase-3 (1:1000; Cell Signaling Biology, CAT#: 9661S and Thermo Fisher Scientific, CAT#: 66470-2-IG), Nestin (1:1000, Abcam, CAT#: AB176571), pH3 (1:1000; Millipore, CAT#: 06-570), Ki67 (1:1000; BD Biosciences, CAT#: 550609), TBR2 (1:300; Abcam, CAT#: AB23345 and EMD Millipore, CAT#: AB15894), GFP (1:1000, Thermofisher, CAT#: A10262), CTIP2 (1:300; Abcam, CAT#: AB18465), and BRN2 (1:750, Santa Cruz, CAT#: SC-393324).

Techniques: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Suspension, Expressing, Dispersion, Derivative Assay, Quantitative Proteomics, RNA Sequencing, Generated, High Throughput Screening Assay, Emulsion, Amplification, Illumina Sequencing, Marker, Gene Expression, Comparison, Produced, Pulse Chase, Control

Journal: Molecular Psychiatry

Article Title: Schizophrenia is defined by cell-specific neuropathology and multiple neurodevelopmental mechanisms in patient-derived cerebral organoids

doi: 10.1038/s41380-021-01316-6

Figure Lengend Snippet: a Computational analyses reveal altered single-cell lineages in Scz organoids. Diffusion maps of single-cell differentiation trajectories were generated for all 26,335 cells via harmonized principal components. This revealed similar, but subtly altered, differentiation trajectories in Scz organoids ( a ). Heat-maps of slingshot-identified genes most-associated with pseudotime cell lineages of Scz organoids are also presented. Note differences in enrichment for collagen and matrix organization factors in Scz single-cell transcriptomes, whereas Ctrl organoids tended to exhibit up-regulation of neuronal factors (e.g., STMN2 , TUBB2B , CRABP1 ) as expected. Notably, markers such as IFITM3 and POU5F1 (OCT4), as well as cell adhesion and vascularization factors, segregated Scz from Ctrl transcriptomes within pseudotime trajectories. Expression of these markers did not appear to identify a broader pool of undifferentiated stem cells, but rather indicated a series of intrinsic alterations within pseudotime trajectories of Scz cell-types and their differentiation patterns. These pseudotime differentiation trajectory analyses broadly replicated data presented in Figs. – , notably disrupted neuronal differentiation dynamics that contributed to fewer total neurons in Scz organoids. In addition, these computational data support the notion that progenitors are diverted towards altered lineages (notably, neuroendothelial and vascularization-related lineages, see Fig. ) via differentially enriched gene-sets in Scz organoids. The net result of these transcriptional differences is that while Scz progenitors and organoids produce some neurons, vascular-related cell-types tend to be overproduced in Scz organoids. b Scz progenitors exhibit disrupted transcription of axon development factors. SOX2 + progenitor scRNA-Seq transcriptomes were isolated and differentially expressed genes (DEG) examined (5% FDR threshold average logFC Scz/Ctrl ≤2.5, and >3). This revealed down-regulation of a gene-set involved in axon development. Other DEG targets in Scz progenitors by biological process are presented here as a dotplot, and resulted in gene-set enrichment for extracellular organization, adhesion, as well as angiogenesis in Scz progenitors. These differentially enriched gene-sets further support the notion that Scz progenitors exhibit cell-specific transcriptional remodeling, which is consistent with the fact that very early progenitor cell-types retain the ability to putatively differentiate into neuronal or neuroendothelial lineages. c – d , Scz neurons exhibit pathway enrichment for growth factor binding. Similar to progenitors, neuron scRNA-Seq transcriptomes were examined for DEGs (5% FDR threshold average LogFC Scz/Ctrl ≤ 2.5, and >3). Analysis of gene ontologies by molecular function revealed enrichment for “growth factor binding” in Scz neurons ( c ), which was defined by down-regulation of TrkA ( NTRK1 ) neurotrophin receptor gene expression and up-regulation of factors such as FURIN (neurotrophin pro→mature processing), FGFR2 , PDGFA , TGFBR3 , and the interleukin-6 signal transducer IL6ST ( d ). Protein–protein interactions between enriched growth factor binding factors in Scz neurons revealed notable interplay and interaction pathways (e.g., Furin →NGF→NTRK1) that place neuron-specific DEGs such as IL6ST and S100A13 as downstream targets of growth factor binding enrichment ( d ). Alternatively, these downstream factors may be putatively repressed by the inhibitory interaction effect of IGFBP3 on VEGFA ( d ), which are prototypic markers of neurovascular cells which also exhibited increased abundance within Scz organoids (see Fig. ). Other pathway analysis (e.g., KEGG) identified altered Pl3K-Akt signaling in Scz neurons (see Fig. ). e Cell-specific disruption of neurotrophic factors and receptors in Scz organoids. Given unbiased detection of alterations in NT-3 ( NTF3 ) in Scz progenitors and TrkA ( NTRK1 ) receptors in Scz neurons, we next examined progenitor cell-types for specific differences in neurotrophins ( BDNF , BDNF-Antisense / BDNF-AS , NGF , NT-3 , and NT-4 ; left) and their cognate tyrosine kinase receptors ( TrkA , TrkB , TrkC , & p75 NTR/NGFR ; right). SOX2 + progenitors and radial glial cell (RGC) progenitors examined almost identical neurotrophin dysregulation and were defined by the trend for decreased expression of BDNF , BDNF-AS, NT-3 , and TrkB expression. Curiously, NT-4 was up-regulated in Scz progenitor cell-types. Scz neurons were defined only by their near 3-fold reduction of TrkA gene expression. Thus, Scz progenitors and neurons exhibit a developmental “switch” in neurotrophic growth factor pathology within organoids. f – g , Validating gene-expression differences of candidate proteome-derived targets, BRN2 ( POU3F2 ) and PTN , in specific cell-types of Scz organoids. Candidate proteome targets (Fig. ) were cross-examined for differences at the single-cell level (see Fig. for expression of proteome targets in global, progenitor-only and neuron-only scRNA-Seq datasets). Given survival, differentiation, and diminished growth factor support, here we emphasize the neuronal transcription factor BRN2 ( POU3F2 ) and putative growth factor PTN as putative targets for mechanism experiments. Scz progenitors exhibited almost complete depletion of BRN2 relative to Ctrl progenitors, indicating disrupted induction of neuronal differentiation. PTN expression was disrupted in both Scz progenitors and neurons but exhibited a greater difference in progenitors. h Resolving cell-type specificity of Scz neuropathology in 3D cerebral organoids. Schematic summary of cell-specific alterations in Scz progenitors (left) and neurons (right). Scz progenitors exhibited entropy in neurotrophic growth factors, specific reduction of TrkB ( NTRK2 ), and depletion of axon development factors. Scz progenitors also exhibit enrichment for extracellular structure, matrix organization, and angiogenesis factors, which partially explains the intrinsic alteration in cell lineage (Fig. 3a) and differentiation trajectories towards neuroendothelial factors and cell types (Figs. , 3). Total scRNA-Seq dataset comprised n = 26, 335 cells, n = 20,844 genes from 7 iPSC lines; Ctrl n = 15,089 scRNA-Seq transcriptomes from 4 Ctrl iPSC lines, and Scz n = 11, 246 scRNA-seq transcriptomes from 3 Scz iPSC lines. * significant p and FDR values. # = p value significant, with a final FDR score at the cut-off threshold. Ctrl control, Scz schizophrenia.

Article Snippet: Primary antibodies comprised SOX2 (1:1000; R&D Systems, CAT#: MAB2018-SP), PAX6 (1:1000; Biolegend, CAT#: 901301), β-tubulin III (1:1000; Abcam, CAT#: AB41489), MAP2 (1:1000, Abcam, CAT#: AB11267; 1:1000, Abcam, CAT#: AB32454), BrdU (1:1000, BD Pharminogen, CAT#: 555627), DCX (1:1000, Santa Cruz, sc-271390), Activated/Cleaved Caspase-3 (1:1000; Cell Signaling Biology, CAT#: 9661S and Thermo Fisher Scientific, CAT#: 66470-2-IG), Nestin (1:1000, Abcam, CAT#: AB176571), pH3 (1:1000; Millipore, CAT#: 06-570), Ki67 (1:1000; BD Biosciences, CAT#: 550609), TBR2 (1:300; Abcam, CAT#: AB23345 and EMD Millipore, CAT#: AB15894), GFP (1:1000, Thermofisher, CAT#: A10262), CTIP2 (1:300; Abcam, CAT#: AB18465), and BRN2 (1:750, Santa Cruz, CAT#: SC-393324).

Techniques: Diffusion-based Assay, Cell Differentiation, Generated, Expressing, Isolation, Binding Assay, Gene Expression, Protein-Protein interactions, Disruption, Derivative Assay, Control

Journal: Molecular Psychiatry

Article Title: Schizophrenia is defined by cell-specific neuropathology and multiple neurodevelopmental mechanisms in patient-derived cerebral organoids

doi: 10.1038/s41380-021-01316-6

Figure Lengend Snippet: a Schematic of self-regulating BRN2 lentiviral vector and supplementation strategy for BRN2 rescue experiments. Briefly, we modified a previously validated lentiviral construct that transiently induces exogenous BRN2 expression but “switches-off” upon completion of neuronal differentiation and assumption of a post-mitotic neuronal fate. After supplementing neuronal transcriptional programs, exogenous BRN2 -Virus transcripts are decayed via binding of the neuron-specific noncoding RNA, miRNA-124, to recognition units embedded within viral transcripts (see Fig. ). This BRN2 -Virus construct thus allows transient supplementation of BRN2 levels in Scz progenitors undergoing differentiation without sustained overexpression of this target in mature neurons. b – e , BRN2 supplementation increases neuron numbers in Scz organoids. Ctrl and Scz organoids were infected with CTRL-Virus and BRN2 -Virus during organoid neural induction. Images show GFP+ cells within ventricular zones. Viral infection rates appeared similar between Ctrl and Scz organoids, and viral GFP expression was detected within progenitor pools as expected ( b ). Scz organoids infected with CTRL -Virus exhibited fewer neurons than Ctrl organoids. However, when comparing Scz organoids infected with CTRL- Virus versus BRN2- Virus, we observed a significant rescue of BRN2+ neuron number. Scz organoids infected with BRN2 -Virus exhibited substantially increased BRN2+ neuron numbers, which were comparable to Ctrl organoids. Enlarged whole-organoid images are provided in Supplementary Material (see Fig. ), and graphs reflect raw data for complete data transparency of rescue effects ( d , CTRL -Virus Ctrl organoids n = 43 fields, n = 16 organoids, and n = 3 independent Ctrl lines; BRN2 -Virus Ctrl organoids n = 40 fields, n = 18 organoids, and n = 3 Ctrl independent lines; CTRL -Virus Scz organoids n = 65 fields, n = 25 organoids, and n = 5 independent Scz lines; BRN2 -Virus Scz organoids n = 42 fields, n = 14 organoids, and n = 4 independent Scz lines). To determine if this effect was reflected in pan neuronal numbers, we also examined MAP2+ neurons in infected organoids. Consistent with data in Fig. , CTRL -Virus+ Scz organoids exhibited a decreased number of MAP2+ neurons compared to Ctrl organoids. However, MAP2+ neuron numbers were significantly increased in the BRN2 -Virus+ Scz organoids ( e , CTRL -Virus Ctrl organoids n = 73 fields, n = 35 organoids, and n = 5 independent Ctrl lines; BRN2 -Virus Ctrl organoids n = 55 fields, n = 26 organoids, and n = 3 Ctrl independent lines; CTRL -Virus Scz organoids n = 52 fields, n = 25 organoids, and n = 5 independent Scz lines; BRN2 -Virus Scz organoids n = 64 fields, n = 28 organoids, and n = 5 independent Scz lines). Thus, transient BRN2 supplementation resulted in a significant recovery of neurons in 3D Scz patient-derived organoids, which confirms a mechanistic role for BRN2 within Scz organoids. Each data point on graphs reflects raw data (an independent, non-overlapping, cortical field) for complete data transparency, with the average of individual iPSC lines provided in (see Fig. ). f No effect of BRN2 supplementation on progenitor cell death in Scz organoids. Scz organoids are associated with increased rates of cell death of ventricular zone neural progenitors (Fig. ). To determine if BRN2 regulates the survival of progenitors, we assessed the number of CAS3+ in infected organoids. In both CTRL- and BRN2 -Virus infected Scz organoids, there was an increase in progenitor death relative to Ctrl samples ( e , CTRL -Virus Ctrl organoids n = 34 fields, n = 19 organoids, and n = 3 independent Ctrl lines; BRN2 -Virus Ctrl organoids n = 35 fields, n = 19 organoids, and n = 3 independent Ctrl lines; CTRL -Virus Scz organoids n = 35 fields, n = 20 organoids, and n = 4 independent Scz lines; BRN2 -Virus Scz organoids n = 45 fields, n = 23 organoids, and n = 4 independent Scz lines). These data indicate that decreased levels of BRN2 do not contribute to the increased apoptosis of progenitors in Scz organoids. Each data point on graphs reflects raw data (comprising an independent ventricular zone) for complete data transparency (for the average of groups, see Fig. ). In sum, we found that BRN2 has a mechanistic role in promoting neuron production in Scz organoids, but not the survival of neuronal progenitors. This selective rescuing effect of BRN2 highlights that multiple factors and pathways likely combine to produce progenitor and neuronal pathology in developing cortical assemblies of Scz organoids. **** p < 0.0001. Error bars reflect Standard Error of the Mean. Scale bar: b-20x = 60 µm, b-40x = 20 µm, c – f = 60 µm. Ctrl: Control, Scz: Schizophrenia. X in schematic denotes cell death.

Article Snippet: Primary antibodies comprised SOX2 (1:1000; R&D Systems, CAT#: MAB2018-SP), PAX6 (1:1000; Biolegend, CAT#: 901301), β-tubulin III (1:1000; Abcam, CAT#: AB41489), MAP2 (1:1000, Abcam, CAT#: AB11267; 1:1000, Abcam, CAT#: AB32454), BrdU (1:1000, BD Pharminogen, CAT#: 555627), DCX (1:1000, Santa Cruz, sc-271390), Activated/Cleaved Caspase-3 (1:1000; Cell Signaling Biology, CAT#: 9661S and Thermo Fisher Scientific, CAT#: 66470-2-IG), Nestin (1:1000, Abcam, CAT#: AB176571), pH3 (1:1000; Millipore, CAT#: 06-570), Ki67 (1:1000; BD Biosciences, CAT#: 550609), TBR2 (1:300; Abcam, CAT#: AB23345 and EMD Millipore, CAT#: AB15894), GFP (1:1000, Thermofisher, CAT#: A10262), CTIP2 (1:300; Abcam, CAT#: AB18465), and BRN2 (1:750, Santa Cruz, CAT#: SC-393324).

Techniques: Plasmid Preparation, Modification, Construct, Expressing, Virus, Binding Assay, Over Expression, Infection, Derivative Assay, Control