Lewy Body-like Inclusions in Human Midbrain Organoids Carrying Glucocerebrosidase and α-Synuclein Mutations.

OBJECTIVE: We utilized human midbrain-like organoids (hMLOs) generated from human pluripotent stem cells carrying glucocerebrosidase gene (GBA1) and α-synuclein (α-syn; SNCA) perturbations to investigate genotype-to-phenotype relationships in Parkinson disease, with the particular aim of recapitulating α-syn- and Lewy body-related pathologies and the process of neurodegeneration in the hMLO model. METHODS: We generated and characterized hMLOs from GBA1-/- and SNCA overexpressing isogenic embryonic stem cells and also generated Lewy body-like inclusions in GBA1/SNCA dual perturbation hMLOs and conduritol-b-epoxide-treated SNCA triplication hMLOs. RESULTS: We identified for the first time that the loss of glucocerebrosidase, coupled with wild-type α-syn overexpression, results in a substantial accumulation of detergent-resistant, ß-sheet-rich α-syn aggregates and Lewy body-like inclusions in hMLOs. These Lewy body-like inclusions exhibit a spherically symmetric morphology with an eosinophilic core, containing α-syn with ubiquitin, and can also be formed in Parkinson disease patient-derived hMLOs. We also demonstrate that impaired glucocerebrosidase function promotes the formation of Lewy body-like inclusions in hMLOs derived from patients carrying the SNCA triplication. INTERPRETATION: Taken together, the data indicate that our hMLOs harboring 2 major risk factors (glucocerebrosidase deficiency and wild-type α-syn overproduction) of Parkinson disease provide a tractable model to further elucidate the underlying mechanisms for progressive Lewy body formation. ANN NEUROL 2021;90:490-505.

Human Pluripotent Stem Cell-Derived Organoids as Models of Liver Disease.

BACKGROUND & AIMS: There are few in vitro models for studying the 3-dimensional interactions among different liver cell types during organogenesis or disease development. We aimed to generate hepatic organoids that comprise different parenchymal liver cell types and have structural features of the liver, using human pluripotent stem cells. METHODS: We cultured H1 human embryonic stem cells (WA-01, passage 27-40) and induced pluripotent stem cells (GM23338) with a series of chemically defined and serum-free media to induce formation of posterior foregut cells, which were differentiated in 3 dimensions into hepatic endoderm spheroids and stepwise into hepatoblast spheroids. Hepatoblast spheroids were reseeded in a high-throughput format and induced to form hepatic organoids; development of functional bile canaliculi was imaged live. Levels of albumin and apolipoprotein B were measured in cell culture supernatants using an enzyme-linked immunosorbent assay. Levels of gamma glutamyl transferase and alkaline phosphatase were measured in cholangiocytes. Organoids were incubated with troglitazone for varying periods and bile transport and accumulation were visualized by live-imaging microscopy. Organoids were incubated with oleic and palmitic acid, and formation of lipid droplets was visualized by staining. We compared gene expression profiles of organoids incubated with free fatty acids or without. We also compared gene expression profiles between liver tissue samples from patients with nonalcoholic steatohepatitis (NASH) versus without. We quantified hepatocyte and cholangiocyte populations in organoids using immunostaining and flow cytometry; cholangiocyte proliferation of cholangiocytes was measured. We compared the bile canaliculi network in the organoids incubated with versus without free fatty acids by live imaging. RESULTS: Cells in organoids differentiated into hepatocytes and cholangiocytes, based on the expression of albumin and cytokeratin 7. Hepatocytes were functional, based on secretion of albumin and apolipoprotein B and cytochrome P450 activity; cholangiocytes were functional, based on gamma glutamyl transferase and alkaline phosphatase activity and proliferative responses to secretin. The organoids organized a functional bile canaliculi system, which was disrupted by cholestasis-inducing drugs such as troglitazone. Organoids incubated with free fatty acids had gene expression signatures similar to those of liver tissues from patients with NASH. Incubation of organoids with free fatty acid-enriched media resulted in structural changes associated with nonalcoholic fatty liver disease, such as decay of bile canaliculi network and ductular reactions. CONCLUSIONS: We developed a hepatic organoid platform with human cells that can be used to model complex liver diseases, including NASH.

Eset partners with Oct4 to restrict extraembryonic trophoblast lineage potential in embryonic stem cells.

The histone H3 Lys 9 (H3K9) methyltransferase Eset is an epigenetic regulator critical for the development of the inner cell mass (ICM). Although ICM-derived embryonic stem (ES) cells are normally unable to contribute to the trophectoderm (TE) in blastocysts, we find that depletion of Eset by shRNAs leads to differentiation with the formation of trophoblast-like cells and induction of trophoblast-associated gene expression. Using chromatin immmunoprecipitation (ChIP) and sequencing (ChIP-seq) analyses, we identified Eset target genes with Eset-dependent H3K9 trimethylation. We confirmed that genes that are preferentially expressed in the TE (Tcfap2a and Cdx2) are bound and repressed by Eset. Single-cell PCR analysis shows that the expression of Cdx2 and Tcfap2a is also induced in Eset-depleted morula cells. Importantly, Eset-depleted cells can incorporate into the TE of a blastocyst and, subsequently, placental tissues. Coimmunoprecipitation and ChIP assays further demonstrate that Eset interacts with Oct4, which in turn recruits Eset to silence these trophoblast-associated genes. Our results suggest that Eset restricts the extraembryonic trophoblast lineage potential of pluripotent cells and links an epigenetic regulator to key cell fate decision through a pluripotency factor.

PRL1 and PRL3 promote macropinocytosis via its lipid phosphatase activity.

PRL1 and PRL3, members of the protein tyrosine phosphatase family, have been associated with cancer metastasis and poor prognosis. Despite extensive research on their protein phosphatase activity, their potential role as lipid phosphatases remains elusive. Methods: We conducted comprehensive investigations to elucidate the lipid phosphatase activity of PRL1 and PRL3 using a combination of cellular assays, biochemical analyses, and protein interactome profiling. Functional studies were performed to delineate the impact of PRL1/3 on macropinocytosis and its implications in cancer biology. Results: Our study has identified PRL1 and PRL3 as lipid phosphatases that interact with phosphoinositide (PIP) lipids, converting PI(3,4)P2 and PI(3,5)P2 into PI(3)P on the cellular membranes. These enzymatic activities of PRLs promote the formation of membrane ruffles, membrane blebbing and subsequent macropinocytosis, facilitating nutrient extraction, cell migration, and invasion, thereby contributing to tumor development. These enzymatic activities of PRLs promote the formation of membrane ruffles, membrane blebbing and subsequent macropinocytosis. Additionally, we found a correlation between PRL1/3 expression and glioma development, suggesting their involvement in glioma progression. Conclusions: Combining with the knowledge that PRLs have been identified to be involved in mTOR, EGFR and autophagy, here we concluded the physiological role of PRL1/3 in orchestrating the nutrient sensing, absorbing and recycling via regulating macropinocytosis through its lipid phosphatase activity. This mechanism could be exploited by tumor cells facing a nutrient-depleted microenvironment, highlighting the potential therapeutic significance of targeting PRL1/3-mediated macropinocytosis in cancer treatment.

Drosophila octamer elements and Pdm-1 dictate the coordinated transcription of core histone genes.

We reveal a set of divergent octamer elements in Drosophila melanogaster (dm) core histone gene promoters. These elements recruit transcription factor POU-domain protein in D. melanogaster 1 (Pdm-1), which along with co-activator dmOct-1 coactivator in S-phase (dmOCA-S), activates transcription from at least the Drosophila histone 2B (dmH2B) and 4 (dmH4) promoters in a fashion similar to the transcription of mammalian histone 2B (H2B) gene activated by octamer binding transcription factor 1 (Oct-1) and Oct-1 coactivator in S-phase (OCA-S). The expression of core histone genes in both kingdoms is coordinated; however, although the expression of mammalian histone genes involves subtype-specific transcription factors and/or co-activator(s), the expression of Drosophila core histone genes is regulated by a common module (Pdm-1/dmOCA-S) in a directly coordinated manner. Finally, dmOCA-S is recruited to the Drosophila histone locus bodies in the S-phase, marking S-phase-specific transcription activation of core histone genes.

Endothelin-1 overexpression leads to further water accumulation and brain edema after middle cerebral artery occlusion via aquaporin 4 expression in astrocytic end-feet.

Stroke patients have increased levels of endothelin-1 (ET-1), a strong vasoconstrictor, in their plasma or cerebrospinal fluid. Previously, we showed high level of ET-1 mRNA expression in astrocytes after hypoxia/ischemia. It is unclear whether the contribution of ET-1 induction in astrocytes is protective or destructive in cerebral ischemia. Here, we generated a transgenic mouse model that overexpress ET-1 in astrocytes (GET-1) using the glial fibrillary acidic protein promoter to examine the role of astrocytic ET-1 in ischemic stroke by challenging these mice with transient middle cerebral artery occlusion (MCAO). Under normal condition, GET-1 mice showed no abnormality in brain morphology, cerebrovasculature, absolute cerebral blood flow, blood-brain barrier (BBB) integrity, and mean arterial blood pressure. Yet, GET-1 mice subjected to transient MCAO showed more severe neurologic deficits and increased infarct, which were partially normalized by administration of ABT-627 (ET(A) antagonist) 5 mins after MCAO. In addition, GET-1 brains exhibited more Evans blue extravasation and showed decreased endothelial occludin expression after MCAO, correlating with higher brain water content and increased cerebral edema. Aquaporin 4 expression was also more pronounced in astrocytic end-feet on blood vessels in GET-1 ipsilateral brains. Our current data suggest that astrocytic ET-1 has deleterious effects on water homeostasis, cerebral edema and BBB integrity, which contribute to more severe ischemic brain injury.

In vivo epigenomic profiling of germ cells reveals germ cell molecular signatures.

The limited number of in vivo germ cells poses an impediment to genome-wide studies. Here, we applied a small-scale chromatin immunoprecipitation sequencing (ChIP-seq) method on purified mouse fetal germ cells to generate genome-wide maps of four histone modifications (H3K4me3, H3K27me3, H3K27ac, and H2BK20ac). Comparison of active chromatin state between somatic, embryonic stem, and germ cells revealed promoters and enhancers needed for stem cell maintenance and germ cell development. We found the nuclear receptor Nr5a2 motif to be enriched at a subset of germ cell cis-regulatory regions, and our results implicate Nr5a2 in germ cell biology. Interestingly, in germ cells, the H3K27me3 histone modification occurs more frequently at regions that are enriched for retrotransposons and MHC genes, indicating that these loci are specifically silenced in germ cells. Together, our study provides genome-wide histone modification maps of in vivo germ cells and reveals the molecular chromatin signatures of germ cells.

Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb.

The dominant effect of transcription factors in imparting expanded potency is best exemplified by the reprogramming of fibroblasts to pluripotent cells using retrovirus-mediated transduction of defined transcription factors. In the murine system, Oct4, Sox2, c-Myc and Klf4 are sufficient to convert fibroblasts to induced pluripotent stem (iPS) cells that have many characteristics of embryonic stem (ES) cells. Here we show that the orphan nuclear receptor Esrrb functions in conjunction with Oct4 and Sox2 to mediate reprogramming of mouse embryonic fibroblasts (MEFs) to iPS cells. Esrrb-reprogrammed cells share similar expression and epigenetic signatures as ES cells. These cells are also pluripotent and can differentiate in vitro and in vivo into the three major embryonic cell lineages. Furthermore, these cells contribute to mouse chimaeras and are germline transmissible. In ES cells, Esrrb targets many genes involved in self-renewal and pluripotency. This suggests that Esrrb may mediate reprogramming through the upregulation of ES-cell-specific genes. Our findings also indicate that it is possible to reprogram MEFs without exogenous Klf transcription factors and link a nuclear receptor to somatic cell reprogramming.