Engineering a CRISPRoff Platform to Modulate Expression of Myeloid Cell Leukemia (MCL-1) in Committed Oligodendrocyte Neural Precursor Cells.

In vitro differentiation of human pluripotent stem cell (hPSC) model systems has furthered our understanding of human development. Techniques used to elucidate gene function during early development have encountered technical challenges, especially when targeting embryonic lethal genes. The introduction of CRISPRoff by Nuñez and collaborators provides an opportunity to heritably silence genes during long-term differentiation. We modified CRISPRoff and sgRNA Sleeping Beauty transposon vectors that depend on tetracycline-controlled transcriptional activation to silence the expression of embryonic lethal genes at different stages of differentiation in a stable manner. We provide instructions on how to generate sgRNA transposon vectors that can be used in combination with our CRISPRoff transposon vector and a stable hPSC line. We validate the use of this tool by silencing MCL-1, an anti-apoptotic protein, which results in pre-implantation embryonic lethality in mice; this protein is necessary for oligodendrocyte and hematopoietic stem cell development and is required for the in vitro survival of hPSCs. In this protocol, we use an adapted version of the differentiation protocol published by Douvaras and Fossati (2015) to generate oligodendrocyte lineage cells from human embryonic stem cells (hESCs). After introduction of the CRISPRoff and sgRNAs transposon vectors in hESCs, we silence MCL-1 in committed oligodendrocyte neural precursor cells and describe methods to measure its expression. With the methods described here, users can design sgRNA transposon vectors targeting MCL-1 or other essential genes of interest to study human oligodendrocyte development or other differentiation protocols that use hPSC model systems. Key features • Generation of an inducible CRISPRoff Sleeping Beauty transposon system. • Experiments performed in vitro for generation of inducible CRISPRoff pluripotent stem cell line amenable to oligodendrocyte differentiation. • Strategy to downregulate an essential gene at different stages of oligodendrocyte development.

Leptomeningeal Neural Organoid (LMNO) Fusions as Models to Study Meninges-Brain Signaling.

Neural organoids derived from human induced pluripotent stem cells (iPSCs) provide a model to study the earliest stages of human brain development, including neurogenesis, neural differentiation, and synaptogenesis. However, neural organoids lack supportive tissues and some non-neural cell types that are key regulators of brain development. Neural organoids have instead been co-cultured with non-neural structures and cell types to promote their maturation and model interactions with neuronal cells. One structure that does not form de novo with neural organoids is the meninges, a tri-layered structure that surrounds the CNS and secretes key signaling molecules required for mammalian brain development. Most studies of meninges-brain signaling have been performed in mice or using two-dimensional (2D) cultures of human cells, the latter not recapitulating the architecture and cellular diversity of the tissue. To overcome this, we developed a co-culture system of neural organoids generated from human iPSCs fused with fetal leptomeninges from mice with fluorescently labeled meninges (Col1a1-GFP). These proof-of-concept studies test the stability of the different cell types in the leptomeninges (fibroblast and macrophage) and the fused brain organoid (progenitor and neuron), as well as the interface between the organoid and meningeal tissue. We test the longevity of the fusion pieces after 30 days and 60 days in culture, describe best practices for preparing the meninges sample prior to fusion, and examine the feasibility of single or multiple meninges pieces fused to a single organoid. We discuss potential uses of the current version of the LMNO fusion model and opportunities to improve the system.

Functional Overlap of Inborn Errors of Immunity and Metabolism Genes Define T Cell Immunometabolic Vulnerabilities.

Inborn Errors of Metabolism (IEM) and Immunity (IEI) are Mendelian diseases in which complex phenotypes and patient rarity can limit clinical annotations. Few genes are assigned to both IEM and IEI, but immunometabolic demands suggest functional overlap is underestimated. We applied CRISPR screens to test IEM genes for immunologic roles and IEI genes for metabolic effects and found considerable crossover. Analysis of IEM showed N-linked glycosylation and the de novo hexosamine synthesis enzyme, Gfpt1 , are critical for T cell expansion and function. Interestingly, Gfpt1 -deficient T H 1 cells were more affected than T H 17 cells, which had increased Nagk for salvage UDP-GlcNAc synthesis. Screening IEI genes showed the transcription factor Bcl11b promotes CD4 + T cell mitochondrial activity and Mcl1 expression necessary to prevent metabolic stress. These data illustrate a high degree of functional overlap of IEM and IEI genes and point to potential immunometabolic mechanisms for a previously unappreciated set of these disorders. HIGHLIGHTS: Inborn errors of immunity and metabolism have greater overlap than previously known Gfpt1 deficiency causes an IEM but also selectively regulates T cell subset fate Loss of Bcl11b causes a T cell deficiency IEI but also harms mitochondrial function Many IEM may have immune defects and IEI may be driven by metabolic mechanisms.

DRP1 mutations associated with EMPF1 encephalopathy alter mitochondrial membrane potential and metabolic programs.

Mitochondria and peroxisomes are dynamic signaling organelles that constantly undergo fission, driven by the large GTPase dynamin-related protein 1 (DRP1; encoded by DNM1L). Patients with de novo heterozygous missense mutations in DNM1L present with encephalopathy due to defective mitochondrial and peroxisomal fission (EMPF1) - a devastating neurodevelopmental disease with no effective treatment. To interrogate the mechanisms by which DRP1 mutations cause cellular dysfunction, we used human-derived fibroblasts from patients who present with EMPF1. In addition to elongated mitochondrial morphology and lack of fission, patient cells display lower coupling efficiency, increased proton leak and upregulation of glycolysis. Mitochondrial hyperfusion also results in aberrant cristae structure and hyperpolarized mitochondrial membrane potential. Peroxisomes show a severely elongated morphology in patient cells, which is associated with reduced respiration when cells are reliant on fatty acid oxidation. Metabolomic analyses revealed impaired methionine cycle and synthesis of pyrimidine nucleotides. Our study provides insight into the role of mitochondrial dynamics in cristae maintenance and the metabolic capacity of the cell, as well as the disease mechanism underlying EMPF1.

Emerging mitochondrial-mediated mechanisms involved in oligodendrocyte development.

Oligodendrocytes are the myelinating glia of the central nervous system and are generated after oligodendrocyte progenitor cells (OPCs) transition into pre-oligodendrocytes and then into myelinating oligodendrocytes. Myelin is essential for proper signal transmission within the nervous system and axonal metabolic support. Although the intrinsic and extrinsic factors that support the differentiation, survival, integration, and subsequent myelination of appropriate axons have been well investigated, little is known about how mitochondria-related pathways such as mitochondrial dynamics, bioenergetics, and apoptosis finely tune these developmental events. Previous findings suggest that changes to mitochondrial morphology act as an upstream regulatory mechanism of neural stem cell (NSC) fate decisions. Whether a similar mechanism is engaged during OPC differentiation has yet to be elucidated. Maintenance of mitochondrial dynamics is vital for regulating cellular bioenergetics, functional mitochondrial networks, and the ability of cells to distribute mitochondria to subcellular locations, such as the growing processes of oligodendrocytes. Myelination is an energy-consuming event, thus, understanding the interplay between mitochondrial dynamics, metabolism, and apoptosis will provide further insight into mechanisms that mediate oligodendrocyte development in healthy and disease states. Here we will provide a concise overview of oligodendrocyte development and discuss the potential contribution of mitochondrial mitochondrial-mediated mechanisms to oligodendrocyte bioenergetics and development.

Leptomeningeal Neural Organoid (LMNO) Fusions as Models to Study Meninges-Brain Signaling.

Neural organoids derived from human induced pluripotent stem cells (iPSCs) provide a model to study the earliest stages of human brain development, including neurogenesis, neural differentiation, and synaptogenesis. However, neural organoids lack supportive tissues and some non-neural cell types that are key regulators of brain development. Neural organoids have instead been co-cultured with non-neural structures and cell types to promote their maturation and model interactions with neuronal cells. One structure that does not form de novo with neural organoids is the meninges, a tri-layered structure that surrounds the CNS and secretes key signaling molecules required for mammalian brain development. Most studies of meninges-brain signaling have been performed in mice or using two-dimensional (2D) cultures of human cells, the latter not recapitulating the architecture and cellular diversity of the tissue. To overcome this, we developed a co-culture system of neural organoids generated from human iPSCs fused with fetal leptomeninges from mice with fluorescently labeled meninges (Col1a1-GFP). These proof-of-concept studies test the stability of the different cell types in the leptomeninges (fibroblast and macrophage) and the fused brain organoid (progenitor and neuron), as well as the interface between the organoid and meningeal tissue. We test the longevity of the fusion pieces after 30 days and 60 days in culture, describe best practices for preparing the meninges sample prior to fusion, and examine the feasibility of single or multiple meninges pieces fused to a single organoid. We discuss potential uses of the current version of the LMNO fusion model and opportunities to improve the system.

Uropathogenic Escherichia coli subverts mitochondrial metabolism to enable intracellular bacterial pathogenesis in urinary tract infection.

Urinary tract infections are among the most common human bacterial infections and place a significant burden on healthcare systems due to associated morbidity, cost and antibiotic use. Despite being a facultative anaerobe, uropathogenic Escherichia coli, the primary cause of urinary tract infections, requires aerobic respiration to establish infection in the bladder. Here, by combining bacterial genetics with cell culture and murine models of infection, we demonstrate that the widely conserved respiratory quinol oxidase cytochrome bd is required for intracellular infection of urothelial cells. Through a series of genetic, biochemical and functional assays, we show that intracellular oxygen scavenging by cytochrome bd alters mitochondrial physiology by reducing the efficiency of mitochondrial respiration, stabilizing the hypoxia-inducible transcription factor HIF-1 and promoting a shift towards aerobic glycolysis. This bacterially induced rewiring of host metabolism antagonizes apoptosis, thereby protecting intracellular bacteria from urothelial cell exfoliation and preserving their replicative niche. These results reveal the metabolic basis for intracellular bacterial pathogenesis during urinary tract infection and identify subversion of mitochondrial metabolism as a bacterial strategy to facilitate persistence within the urinary tract.

Human iPSC-derived cerebral organoids model features of Leigh syndrome and reveal abnormal corticogenesis.

Leigh syndrome (LS) is a rare, inherited neurometabolic disorder that presents with bilateral brain lesions caused by defects in the mitochondrial respiratory chain and associated nuclear-encoded proteins. We generated human induced pluripotent stem cells (iPSCs) from three LS patient-derived fibroblast lines. Using whole-exome and mitochondrial sequencing, we identified unreported mutations in pyruvate dehydrogenase (GM0372, PDH; GM13411, MT-ATP6/PDH) and dihydrolipoyl dehydrogenase (GM01503, DLD). These LS patient-derived iPSC lines were viable and capable of differentiating into progenitor populations, but we identified several abnormalities in three-dimensional differentiation models of brain development. LS patient-derived cerebral organoids showed defects in neural epithelial bud generation, size and cortical architecture at 100 days. The double mutant MT-ATP6/PDH line produced organoid neural precursor cells with abnormal mitochondrial morphology, characterized by fragmentation and disorganization, and showed an increased generation of astrocytes. These studies aim to provide a comprehensive phenotypic characterization of available patient-derived cell lines that can be used to study Leigh syndrome.

Revealing the Impact of Mitochondrial Fitness During Early Neural Development Using Human Brain Organoids.

Mitochondrial homeostasis -including function, morphology, and inter-organelle communication- provides guidance to the intrinsic developmental programs of corticogenesis, while also being responsive to environmental and intercellular signals. Two- and three-dimensional platforms have become useful tools to interrogate the capacity of cells to generate neuronal and glia progeny in a background of metabolic dysregulation, but the mechanistic underpinnings underlying the role of mitochondria during human neurogenesis remain unexplored. Here we provide a concise overview of cortical development and the use of pluripotent stem cell models that have contributed to our understanding of mitochondrial and metabolic regulation of early human brain development. We finally discuss the effects of mitochondrial fitness dysregulation seen under stress conditions such as metabolic dysregulation, absence of developmental apoptosis, and hypoxia; and the avenues of research that can be explored with the use of brain organoids.

A taste of the early steps in BAX activation with FLAMBE.

The activation of BAX through intricate intramolecular changes is critical for apoptosis. In this issue of Cell Reports Methods, Gelles et al. report engineering FLAMBE, an elegant fluorescence polarization ligand assay for monitoring the early activation of monomeric BAX via real-time release of a peptide probe, expanding the repertoire of BAX activation assays to the single-molecule level.

Stem cell conversion to the cardiac lineage requires nucleotide signalling from apoptosing cells.

Pluripotent stem cells can be driven by manipulation of Wnt signalling through a series of states similar to those that occur during early embryonic development, transitioning from an epithelial phenotype into the cardiogenic-mesoderm lineage and ultimately into functional cardiomyocytes. Strikingly, we observed that initiation of differentiation in induced pluripotent stem cells (iPSCs) and embryonic stem cells triggers widespread apoptosis, followed by a synchronous epithelial-mesenchymal transition (EMT). Apoptosis is caused by the absence of bFGF in the differentiation medium. EMT requires induction of the transcription factors SNAI1 and SNAI2 downstream of MESP1 expression, and double knockout of SNAI1 and SNAI2 or loss of MESP1 in iPSCs blocks EMT and prevents cardiac differentiation. Remarkably, blockade of early apoptosis, either chemically or by ablation of pro-apoptotic genes, also completely prevents EMT, suppressing even the earliest events in mesoderm conversion, including T/BRA, TBX6 and MESP1 induction. Conditioned medium from WNT-activated wild-type iPSCs overcomes the block to EMT by cells incapable of apoptosis, suggesting involvement of soluble factors from apoptotic cells in mesoderm conversion. Knockout of the PANX1 channel blocked EMT, whereas treatment with a purinergic P2-receptor inhibitor or addition of apyrase demonstrated a requirement for nucleotide triphosphate signalling. ATP and/or UTP was sufficient to induce a partial EMT in apoptosis-incapable cells treated with WNT activator. Notably, knockout of the ATP/UTP-specific P2Y2 receptor blocked EMT and mesoderm induction. We conclude that in addition to acting as chemo-attractants for clearance of apoptotic cells, nucleotides can function as essential paracrine signals that, with WNT signalling, create a logical AND gate for mesoderm specification.

Oligodendrocytes depend on MCL-1 to prevent spontaneous apoptosis and white matter degeneration.

Neurologic disorders often disproportionately affect specific brain regions, and different apoptotic mechanisms may contribute to white matter pathology in leukodystrophies or gray matter pathology in poliodystrophies. We previously showed that neural progenitors that generate cerebellar gray matter depend on the anti-apoptotic protein BCL-xL. Conditional deletion of Bcl-xL in these progenitors produces spontaneous apoptosis and cerebellar hypoplasia, while similar conditional deletion of Mcl-1 produces no phenotype. Here we show that, in contrast, postnatal oligodendrocytes depend on MCL-1. We found that brain-wide Mcl-1 deletion caused apoptosis specifically in mature oligodendrocytes while sparing astrocytes and oligodendrocyte precursors, resulting in impaired myelination and progressive white matter degeneration. Disabling apoptosis through co-deletion of Bax or Bak rescued white matter degeneration, implicating the intrinsic apoptotic pathway in Mcl-1-dependence. Bax and Bak co-deletions rescued different aspects of the Mcl-1-deleted phenotype, demonstrating their discrete roles in white matter stability. MCL-1 protein abundance was reduced in eif2b5-mutant mouse model of the leukodystrophy vanishing white matter disease (VWMD), suggesting the potential for MCL-1 deficiency to contribute to clinical neurologic disease. Our data show that oligodendrocytes require MCL-1 to suppress apoptosis, implicate MCL-1 deficiency in white matter pathology, and suggest apoptosis inhibition as a leukodystrophy therapy.

Amphibians and reptiles of the Atlantic Forest in Recôncavo Baiano, east Brazil: Cruz das Almas municipality.

A list of amphibian and reptile species that occur in open and forested areas of the Atlantic Forest in the municipality of Cruz das Almas, in the Recôncavo Baiano, eastern Brazil is presented. Field sampling occurred between January 2015 to March 2019, totalling 117 samples distributed in three areas: Parque Florestal Mata de Cazuzinha, Mata da Cascalheira, and Riacho do Machado. A total of 1,848 individuals of 69 species (31 anurans, 14 lizards, 19 snakes, two amphisbaenians, and three testudines) was recorded. Additionally, one individual of Ophiodesstriatus was found in Mata da Cascalheira after the end of sampling, totalling 15 lizard species and 70 herpetofaunal species. The prevalence of open-area species and the presence of Phyllopezuslutzae, Diploglossuslessonae, and Dryadosauranordestina in interior forest patches are discussed. Additionally, a new record of the invasive terrapin Trachemysdorbigni in the State of Bahia is reported.

Common molecular mechanisms of SLC6A1 variant-mediated neurodevelopmental disorders in astrocytes and neurons.

Solute carrier family 6 member 1 (SLC6A1) is abundantly expressed in the developing brain even before the CNS is formed. Its encoded GABA transporter 1 (GAT-1) is responsible for the reuptake of GABA into presynaptic neurons and glia, thereby modulating neurotransmission. GAT-1 is expressed globally in the brain, in both astrocytes and neurons. The GABA uptake function of GAT-1 in neurons cannot be compensated for by other GABA transporters, while the function in glia can be partially replaced by GABA transporter 3. Recently, many variants in SLC6A1 have been associated with a spectrum of epilepsy syndromes and neurodevelopmental disorders, including myoclonic atonic epilepsy, childhood absence epilepsy, autism, and intellectual disability, but the pathomechanisms associated with these phenotypes remain unclear. The presence of GAT-1 in both neurons and astrocytes further obscures the role of abnormal GAT-1 in the heterogeneous disease phenotype manifestations. Here we examine the impact on transporter trafficking and function of 22 SLC6A1 variants identified in patients with a broad spectrum of phenotypes. We also evaluate changes in protein expression and subcellular localization of the variant GAT-1 in various cell types, including neurons and astrocytes derived from human patient induced pluripotent stem cells. We found that a partial or complete loss-of-function represents a common disease mechanism, although the extent of GABA uptake reduction is variable. The reduced GABA uptake appears to be due to reduced cell surface expression of the variant transporter caused by variant protein misfolding, endoplasmic reticulum retention, and subsequent degradation. Although the extent of reduction of the total protein, surface protein, and the GABA uptake level of the variant transporters is variable, the loss of GABA uptake function and endoplasmic reticulum retention is consistent across induced pluripotent stem cell-derived cell types, including astrocytes and neurons, for the surveyed variants. Interestingly, we did not find a clear correlation of GABA uptake function and the disease phenotypes, such as myoclonic atonic epilepsy versus developmental delay, in this study. Together, our study suggests that impaired transporter protein trafficking and surface expression are the major disease-associated mechanisms associated with pathogenic SLC6A1 variants. Our results resemble findings from pathogenic variants in other genes affecting the GABA pathway, such as GABAA receptors. This study provides critical insight into therapeutic developments for SLC6A1 variant-mediated disorders and implicates that boosting transporter function by either genetic or pharmacological approaches would be beneficial.

A proteomics approach for the identification of cullin-9 (CUL9) related signaling pathways in induced pluripotent stem cell models.

CUL9 is a non-canonical and poorly characterized member of the largest family of E3 ubiquitin ligases known as the Cullin RING ligases (CRLs). Most CRLs play a critical role in developmental processes, however, the role of CUL9 in neuronal development remains elusive. We determined that deletion or depletion of CUL9 protein causes aberrant formation of neural rosettes, an in vitro model of early neuralization. In this study, we applied mass spectrometric approaches in human pluripotent stem cells (hPSCs) and neural progenitor cells (hNPCs) to identify CUL9 related signaling pathways that may contribute to this phenotype. Through LC-MS/MS analysis of immunoprecipitated endogenous CUL9, we identified several subunits of the APC/C, a major cell cycle regulator, as potential CUL9 interacting proteins. Knockdown of the APC/C adapter protein FZR1 resulted in a significant increase in CUL9 protein levels, however, CUL9 does not appear to affect protein abundance of APC/C subunits and adapters or alter cell cycle progression. Quantitative proteomic analysis of CUL9 KO hPSCs and hNPCs identified protein networks related to metabolic, ubiquitin degradation, and transcriptional regulation pathways that are disrupted by CUL9 deletion in both hPSCs. No significant changes in oxygen consumption rates or ATP production were detected in either cell type. The results of our study build on current evidence that CUL9 may have unique functions in different cell types and that compensatory mechanisms may contribute to the difficulty of identifying CUL9 substrates.

Dynamic properties of mitochondria during human corticogenesis.

Mitochondria are signaling hubs responsible for the generation of energy through oxidative phosphorylation, the production of key metabolites that serve the bioenergetic and biosynthetic needs of the cell, calcium (Ca2+) buffering and the initiation/execution of apoptosis. The ability of mitochondria to coordinate this myriad of functions is achieved through the exquisite regulation of fundamental dynamic properties, including remodeling of the mitochondrial network via fission and fusion, motility and mitophagy. In this Review, we summarize the current understanding of the mechanisms by which these dynamic properties of the mitochondria support mitochondrial function, review their impact on human cortical development and highlight areas in need of further research.

Modeling the function of BAX and BAK in early human brain development using iPSC-derived systems.

Intrinsic apoptosis relies on the ability of the BCL-2 family to induce the formation of pores on the outer mitochondrial membrane. Previous studies have shown that both BAX and BAK are essential during murine embryogenesis, and reports in human cancer cell lines identified non-canonical roles for BAX and BAK in mitochondrial fission during apoptosis. BAX and BAK function in human brain development remains elusive due to the lack of appropriate model systems. Here, we generated BAX/BAK double knockout human-induced pluripotent stem cells (hiPSCs), hiPSC-derived neural progenitor cells (hNPCs), neural rosettes, and cerebral organoids to uncover the effects of BAX and BAK deletion in an in vitro model of early human brain development. We found that BAX and BAK-deficient cells have abnormal mitochondrial morphology and give rise to aberrant cortical structures. We suggest crucial functions for BAX and BAK during human development, including maintenance of homeostatic mitochondrial morphology, which is crucial for proper development of progenitors and neurons of the cortex. Human pluripotent stem cell-derived systems can be useful platforms to reveal novel functions of the apoptotic machinery in neural development.

Inhibition of focal adhesion kinase increases myofibril viscosity in cardiac myocytes.

The coordinated generation of mechanical forces by cardiac myocytes is required for proper heart function. Myofibrils are the functional contractile units of force production within individual cardiac myocytes. At the molecular level, myosin motors form cross-bridges with actin filaments and use ATP to convert chemical energy into mechanical forces. The energetic efficiency of the cross-bridge cycle is influenced by the viscous damping of myofibril contraction. The viscoelastic response of myofibrils is an emergent property of their individual mechanical components. Previous studies have implicated titin-actin interactions, cell-ECM adhesion, and microtubules as regulators of the viscoelastic response of myofibrils. Here we probed the viscoelastic response of myofibrils using laser-assisted dissection. As a proof-of-concept, we found actomyosin contractility was required to endow myofibrils with their viscoelastic response, with blebbistatin treatment resulting in decreased myofibril tension and viscous damping. Focal adhesion kinase (FAK) is a key regulator of cell-ECM adhesion, microtubule stability, and myofibril assembly. We found inhibition of FAK signaling altered the viscoelastic properties of myofibrils. Specifically, inhibition of FAK resulted in increased viscous damping of myofibril retraction following laser ablation. This damping was not associated with acute changes in the electrophysiological properties of cardiac myocytes. These results implicate FAK as a regulator of mechanical properties of myofibrils.

Break on Through: Golgi-Derived Vesicles Aid in Mitochondrial Fission.

Mitochondrial fission is sustained through contact with several organelles, including the endoplasmic reticulum, lysosomes, and the actin cytoskeleton. Nagashima et al. (2020) now demonstrate that PI(4)P-containing Golgi-derived vesicles also modulate mitochondrial fission, driven by Arf1 and PI(4)KIIIß activity, identifying a new organelle contact involved in maintaining mitochondrial homeostasis.

A connection in life and death: The BCL-2 family coordinates mitochondrial network dynamics and stem cell fate.

The B cell CLL/lymphoma-2 (BCL-2) family of proteins control the mitochondrial pathway of apoptosis, also known as intrinsic apoptosis. Direct binding between members of the BCL-2 family regulates mitochondrial outer membrane permeabilization (MOMP) after an apoptotic insult. The ability of the cell to sense stress and translate it into a death signal has been a major theme of research for nearly three decades; however, other mechanisms by which the BCL-2 family coordinates cellular homeostasis beyond its role in initiating apoptosis are emerging. One developing area of research is understanding how the BCL-2 family of proteins regulate development using pluripotent stem cells as a model system. Understanding BCL-2 family-mediated regulation of mitochondrial homeostasis in cell death and beyond would uncover new facets of stem cell maintenance and differentiation potential.