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Due to an inability to ethically access developing human brain tissue as well as identify prospective cases, early-arising neurodevelopmental and cell-specific signatures of Schizophrenia (Scz) have remained unknown and thus undefined. To overcome these challenges, we utilized patient-derived induced pluripotent stem cells (iPSCs) to generate 3D cerebral organoids to model neuropathology of Scz during this critical period. We discovered that Scz organoids exhibited ventricular neuropathology resulting in altered progenitor survival and disrupted neurogenesis. This ultimately yielded fewer neurons within developing cortical fields of Scz organoids. Single-cell sequencing revealed that Scz progenitors were specifically depleted of neuronal programming factors leading to a remodeling of cell-lineages, altered differentiation trajectories, and distorted cortical cell-type diversity. While Scz organoids were similar in their macromolecular diversity to organoids generated from healthy controls (Ctrls), four GWAS factors (PTN, COMT, PLCL1, and PODXL) and peptide fragments belonging to the POU-domain transcription factor family (e.g., POU3F2/BRN2) were altered. This revealed that Scz organoids principally differed not in their proteomic diversity, but specifically in their total quantity of disease and neurodevelopmental factors at the molecular level. Single-cell sequencing subsequently identified cell-type specific alterations in neuronal programming factors as well as a developmental switch in neurotrophic growth factor expression, indicating that Scz neuropathology can be encoded on a cell-type-by-cell-type basis. Furthermore, single-cell sequencing also specifically replicated the depletion of BRN2 (POU3F2) and PTN in both Scz progenitors and neurons. Subsequently, in two mechanistic rescue experiments we identified that the transcription factor BRN2 and growth factor PTN operate as mechanistic substrates of neurogenesis and cellular survival, respectively, in Scz organoids. Collectively, our work suggests that multiple mechanisms of Scz exist in patient-derived organoids, and that these disparate mechanisms converge upon primordial brain developmental pathways such as neuronal differentiation, survival, and growth factor support, which may amalgamate to elevate intrinsic risk of Scz.

Schizophrenia (Scz) is a brain disorder that has a typical onset in early adulthood but otherwise maintains unknown disease origins. Unfortunately, little progress has been made in understanding the molecular mechanisms underlying neurodevelopment of Scz due to ethical and technical limitations in accessing developing human brain tissue. To overcome this challenge, we have previously utilized patient-derived Induced Pluripotent Stem Cells (iPSCs) to generate self-developing, self-maturating, and self-organizing 3D brain-like tissue known as cerebral organoids. As a continuation of this prior work, here we provide an architectural map of the developing Scz organoid proteome. Utilizing iPSCs from n = 25 human donors (n = 8 healthy Ctrl donors, and n = 17 Scz patients), we generated 3D cerebral organoids, employed 16-plex isobaric sample-barcoding chemistry, and simultaneously subjected samples to comprehensive high-throughput liquid-chromatography/mass-spectrometry (LC/MS) quantitative proteomics. Of 3,705 proteins identified by high-throughput proteomic profiling, we identified that just ~2.62% of the organoid global proteomic landscape was differentially regulated in Scz organoids. In sum, just 43 proteins were up-regulated and 54 were down-regulated in Scz patient-derived organoids. Notably, a range of neuronal factors were depleted in Scz organoids (e.g., MAP2, TUBB3, SV2A, GAP43, CRABP1, NCAM1 etc.). Based on global enrichment analysis, alterations in key pathways that regulate nervous system development (e.g., axonogenesis, axon development, axon guidance, morphogenesis pathways regulating neuronal differentiation, as well as substantia nigra development) were perturbed in Scz patient-derived organoids. We also identified prominent alterations in two novel GWAS factors, Pleiotrophin (PTN) and Podocalyxin (PODXL), in Scz organoids. In sum, this work serves as both a report and a resource that researchers can leverage to compare, contrast, or orthogonally validate Scz factors and pathways identified in observational clinical studies and other model systems.

It is widely accepted that narcotic use during pregnancy and specific environmental factors (e.g., maternal immune activation and chronic stress) may increase risk of neuropsychiatric illness in offspring. However, little progress has been made in defining human-specific in utero neurodevelopmental pathology due to ethical and technical challenges associated with accessing human prenatal brain tissue. Here we utilized human induced pluripotent stem cells (hiPSCs) to generate reproducible organoids that recapitulate dorsal forebrain development including early corticogenesis. We systemically exposed organoid samples to chemically defined “enviromimetic” compounds to examine the developmental effects of various narcotic and neuropsychiatric-related risk factors within tissue of human origin. In tandem experiments conducted in parallel, we modeled exposure to opiates (μ-opioid agonist endomorphin), cannabinoids (WIN 55,212-2), alcohol (ethanol), smoking (nicotine), chronic stress (human cortisol), and maternal immune activation (human Interleukin-17a; IL17a). Human-derived dorsal forebrain organoids were consequently analyzed via an array of unbiased and high-throughput analytical approaches, including state-of-the-art TMT-16plex liquid chromatography/mass-spectrometry (LC/MS) proteomics, hybrid MS metabolomics, and flow cytometry panels to determine cell-cycle dynamics and rates of cell death. This pipeline subsequently revealed both common and unique proteome, reactome, and metabolome alterations as a consequence of enviromimetic modeling of narcotic use and neuropsychiatric-related risk factors in tissue of human origin. However, of our 6 treatment groups, human-derived organoids treated with the cannabinoid agonist WIN 55,212-2 exhibited the least convergence of all groups. Single-cell analysis revealed that WIN 55,212-2 increased DNA fragmentation, an indicator of apoptosis, in human-derived dorsal forebrain organoids. We subsequently confirmed induction of DNA damage and apoptosis by WIN 55,212-2 within 3D human-derived dorsal forebrain organoids. Lastly, in a BrdU pulse-chase neocortical neurogenesis paradigm, we identified that WIN 55,212-2 was the only enviromimetic treatment to disrupt newborn neuron numbers within human-derived dorsal forebrain organoids. Cumulatively this study serves as both a resource and foundation from which human 3D biologics can be used to resolve the non-genomic effects of neuropsychiatric risk factors under controlled laboratory conditions. While synthetic cannabinoids can differ from naturally occurring compounds in their effects, our data nonetheless suggests that exposure to WIN 55,212-2 elicits neurotoxicity within human-derived developing forebrain tissue. These human-derived data therefore support the long-standing belief that maternal use of cannabinoids may require caution so to avoid any potential neurodevelopmental effects upon developing offspring in utero.

Background The T-cell inhibitory molecule PD-L1 (B7-H1, CD274) is expressed on tumor cells of a subset of breast cancer patients. However, the mechanism that regulates PD-L1 expression in this group of patients is still not well-identified. Methods We have used loss and gain of function gene manipulation approach, multi-parametric flow cytometry, large scale gene expression dataset analysis and immunohistochemistry of breast cancer tissue sections. Results Induction of epithelial to mesenchymal transition (EMT) in human mammary epithelial cells upregulated PD-L1 expression, which was dependent mainly on the activation of the PI3K/AKT pathway. Interestingly, gene expression signatures available from large cohort of breast tumors showed a significant correlation between EMT score and the PD-L1 mRNA level (p < 0.001). Strikingly, very strong association (p < 0.0001) was found between PD-L1 expression and claudin-low subset of breast cancer, which is known to have high EMT score. On the protein level, significant correlation was found between PD-L1 expression and standard markers of EMT ( p  = 0.005) in 67 breast cancer patients. Importantly, specific downregulation of PD-L1 in claudin-low breast cancer cells showed signs of EMT reversal as manifested by CD44 and Vimentin downregulation and CD24 upregulation. Conclusions We have demonstrated a bidirectional effect between EMT status and PD-L1 expression especially in claudin-low subtype of breast cancer cells. Our findings highlights the potential dual benefit of anti-PD-L1 particularly in this subset of breast cancer patients that will likely benefit more from anti-PD-L1 targeted therapy as well as in monitoring biological changes upon treatment.

Epigenetic gene silencing is seen in several repeat-expansion diseases. In fragile X syndrome, the most common genetic form of mental retardation, a CGG trinucleotide–repeat expansion adjacent to the fragile X mental retardation 1 (FMR1) gene promoter results in its epigenetic silencing. Here, we show that FMR1 silencing is mediated by the FMR1 mRNA. The FMR1 mRNA contains the transcribed CGG-repeat tract as part of the 5′ untranslated region, which hybridizes to the complementary CGG-repeat portion of the FMR1 gene to form an RNA • DNA duplex. Disrupting the interaction of the mRNA with the CGG-repeat portion of the FMR1 gene prevents promoter silencing. Thus, our data link trinucleotide-repeat expansion to a form of RNA-directed gene silencing mediated by direct interactions of the trinucleotide-repeat RNA and DNA.

Synaptic plasticity requires a tight control of mRNA levels in dendrites. RNA translation and degradation pathways have been recently linked to neurodevelopmental and neuropsychiatric diseases, suggesting a role for RNA regulation in synaptic plasticity and cognition. While the local translation of specific mRNAs has been implicated in synaptic plasticity, the tightly controlled mechanisms that regulate local quantity of specific mRNAs remain poorly understood. Despite being the only RNA regulatory pathway that is associated with multiple mental illnesses, the nonsense-mediated mRNA decay (NMD) pathway presents an unexplored regulatory mechanism for synaptic function and plasticity. Here, we show that neuron-specific disruption of UPF2, an NMD component, in adulthood attenuates learning, memory, spine density, synaptic plasticity (L-LTP), and potentiates perseverative/repetitive behavior in mice. We report that the NMD pathway operates within dendrites to regulate Glutamate Receptor 1 (GLUR1) surface levels. Specifically, UPF2 modulates the internalization of GLUR1 and promotes its local synthesis in dendrites. We identified neuronal Prkag3 mRNA as a mechanistic substrate for NMD that contributes to the UPF2-mediated regulation of GLUR1 by limiting total GLUR1 levels. These data establish that UPF2 regulates synaptic plasticity, cognition, and local protein synthesis in dendrites, providing fundamental insight into the neuron-specific function of NMD within the brain.

The liver is a unique organ that has a phenomenal capacity to regenerate after injury. Different surgical procedures, including partial hepatectomy (PH), intraoperative portal vein ligation (PVL), and associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) show clinically distinct recovery patterns and regeneration. The observable clinical differences likely mirror some underlying variations in the patterns of gene activation and regeneration pathways. In this study, we provided a comprehensive comparative transcriptomic analysis of gene regulation in regenerating rat livers temporally spaced at 24 h and 96 h after PH, PVL, and ALPPS. The time-dependent factors appear to be the most important determinant of post-injury alterations of gene expression in liver regeneration. Gene expression profile after ALPPS showed more similar expression pattern to the PH than the PVL at the early phase of the regeneration. Early transcriptomic changes and predicted upstream regulators that were found in all three procedures included cell cycle associated genes ( E2F1, CCND1, FOXM1, TP53 , and RB1 ), transcription factors ( Myc, E2F1, TBX2, FOXM1 ), DNA replication regulators ( CDKN1A, EZH2, RRM2 ), G1/S-transition regulators ( CCNB1, CCND1, RABL6 ), cytokines and growth factors ( CSF2, IL-6, TNF, HGF, VEGF , and EGF ), ATM and p53 signaling pathways. The functional pathway, upstream, and network analyses revealed both unique and overlapping molecular mechanisms and pathways for each surgical procedure. Identification of molecular signatures and regenerative signaling pathways for each surgical procedure further our understanding of key regulators of liver regeneration as well as patient populations that are likely to benefit from each procedure.

The cellular mechanisms of autism spectrum disorder (ASD) are poorly understood. Cumulative evidence suggests that abnormal synapse function underlies many features of this disease. Astrocytes regulate several key neuronal processes, including the formation of synapses and the modulation of synaptic plasticity. Astrocyte abnormalities have also been identified in the postmortem brain tissue of ASD individuals. However, it remains unclear whether astrocyte pathology plays a mechanistic role in ASD, as opposed to a compensatory response. To address this, we combined stem cell culturing with transplantation techniques to determine disease-specific properties inherent to ASD astrocytes. We demonstrate that ASD astrocytes induce repetitive behavior as well as impair memory and long-term potentiation when transplanted into the healthy mouse brain. These in vivo phenotypes were accompanied by reduced neuronal network activity and spine density caused by ASD astrocytes in hippocampal neurons in vitro. Transplanted ASD astrocytes also exhibit exaggerated Ca2+ fluctuations in chimeric brains. Genetic modulation of evoked Ca2+ responses in ASD astrocytes modulates behavior and neuronal activity deficits. Thus, this study determines that astrocytes derived from ASD iPSCs are sufficient to induce repetitive behavior as well as cognitive deficit, suggesting a previously unrecognized primary role for astrocytes in ASD.

Schizophrenia (SCZ) is a complex neurodevelopmental disorder characterized by the manifestation of psychiatric symptoms in early adulthood. While many research avenues into the origins of SCZ during brain development have been explored, the contribution of endothelial/vascular dysfunction to the disease remains largely elusive. To model the neuropathology of SCZ during early critical periods of brain development, we utilized patient-derived induced pluripotent stem cells (iPSCs) to generate 3D cerebral organoids and define cell-specific signatures of disease. Single-cell RNA sequencing revealed that while SCZ organoids were similar in their macromolecular diversity to organoids generated from healthy controls (CTRL), SCZ organoids exhibited a higher percentage of endothelial cells when normalized to total cell numbers. Additionally, when compared to CTRL, differential gene expression analysis revealed a significant enrichment in genes that function in vessel formation, vascular regulation, and inflammatory response in SCZ endothelial cells. In line with these findings, data from 23 donors demonstrated that PECAM1 + microvascular vessel-like structures were increased in length and number in SCZ organoids in comparison to CTRL organoids. Furthermore, we report that patient-derived endothelial cells displayed higher paracellular permeability, implicating elevated vascular activity. Collectively, our data identified altered gene expression patterns, vessel-like structural changes, and enhanced permeability of endothelial cells in patient-derived models of SCZ. Hence, brain microvascular cells could play a role in the etiology of SCZ by modulating the permeability of the developing blood brain barrier (BBB).

Decades of research have unequivocally demonstrated that fetal exposure to both recreational and prescription drugs in utero negatively impacts the developing brain. More recently, the application of cutting-edge techniques in neurodevelopmental research has attempted to identify how the fetal brain responds to specific environmental stimuli. Meanwhile, human fetal brain studies still encounter ethical considerations and technical limitations in tissue collection. Human-induced pluripotent stem cell (iPSC)-derived brain organoid technology has emerged as a powerful alternative to examine fetal neurobiology. In fact, human 3D organoid tissues recapitulate cerebral development during the first trimester of pregnancy. In this review, we aim to provide a comprehensive summary of fetal brain metabolic studies related to drug abuse in animal and human models. Additionally, we will discuss the current challenges and prospects of using brain organoids for large-scale metabolomics. Incorporating cutting-edge techniques in human brain organoids may lead to uncovering novel molecular and cellular targets relevant to neurodevelopment, direct novel therapeutic approaches and raise new exciting questions.