Essential role for DNA-PK-mediated phosphorylation of NR4A nuclear orphan receptors in DNA double-strand break repair.

DNA-dependent protein kinase (DNA-PK) is a central regulator of DNA double-strand break (DSB) repair; however, the identity of relevant DNA-PK substrates has remained elusive. NR4A nuclear orphan receptors function as sequence-specific DNA-binding transcription factors that participate in adaptive and stress-related cell responses. We show here that NR4A proteins interact with the DNA-PK catalytic subunit and, upon exposure to DNA damage, translocate to DSB foci by a mechanism requiring the activity of poly(ADP-ribose) polymerase-1 (PARP-1). At DNA repair foci, NR4A is phosphorylated by DNA-PK and promotes DSB repair. Notably, NR4A transcriptional activity is entirely dispensable in this function, and core components of the DNA repair machinery are not transcriptionally regulated by NR4A. Instead, NR4A functions directly at DNA repair sites by a process that requires phosphorylation by DNA-PK. Furthermore, a severe combined immunodeficiency (SCID)-causing mutation in the human gene encoding the DNA-PK catalytic subunit impairs the interaction and phosphorylation of NR4A at DSBs. Thus, NR4As represent an entirely novel component of DNA damage response and are substrates of DNA-PK in the process of DSB repair.

Transcription factor-induced lineage programming of noradrenaline and motor neurons from embryonic stem cells.

An important goal in stem cell biology is to develop methods for efficient generation of clinically interesting cell types from relevant stem cell populations. This is particularly challenging for different types of neurons of the central nervous system where hundreds of distinct neuronal cell types are generated during embryonic development. We previously used a strategy based on forced transcription factor expression in embryonic stem cell-derived neural progenitors to generate specific types of neurons, including dopamine and serotonin neurons. Here, we extend these studies and show that noradrenergic neurons can also be generated from pluripotent embryonic stem cells by forced expression of the homeobox transcription factor Phox2b under the signaling influence of fibroblast growth factor 8 (FGF8) and bone morphogenetic proteins. In neural progenitors exposed to FGF8 and sonic hedgehog both Phox2b and the related Phox2a instead promoted the generation of neurons with the characteristics of mid- and hindbrain motor neurons. The efficient generation of these neuron types enabled a comprehensive genome-wide gene expression analysis that provided further validation of the identity of generated cells. Moreover, we also demonstrate that the generated cell types are amenable to drug testing in vitro and we show that variants of the differentiation protocols can be applied to cultures of human pluripotent stem cells for the generation of human noradrenergic and visceral motor neurons. Thus, these studies provide a basis for characterization of yet an additional highly clinically relevant neuronal cell type.

Spatial RNA Sequencing Identifies Robust Markers of Vulnerable and Resistant Human Midbrain Dopamine Neurons and Their Expression in Parkinson's Disease.

Defining transcriptional profiles of substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) dopamine neurons is critical to understanding their differential vulnerability in Parkinson's Disease (PD). Here, we determine transcriptomes of human SNc and VTA dopamine neurons using LCM-seq on a large sample cohort. We apply a bootstrapping strategy as sample input to DESeq2 and identify 33 stably differentially expressed genes (DEGs) between these two subpopulations. We also compute a minimal sample size for identification of stable DEGs, which highlights why previous reported profiles from small sample sizes display extensive variability. Network analysis reveal gene interactions unique to each subpopulation and highlight differences in regulation of mitochondrial stability, apoptosis, neuronal survival, cytoskeleton regulation, extracellular matrix modulation as well as synapse integrity, which could explain the relative resilience of VTA dopamine neurons. Analysis of PD tissues showed that while identified stable DEGs can distinguish the subpopulations also in disease, the SNc markers SLIT1 and ATP2A3 were down-regulated and thus appears to be biomarkers of disease. In summary, our study identifies human SNc and VTA marker profiles, which will be instrumental for studies aiming to modulate dopamine neuron resilience and to validate cell identity of stem cell-derived dopamine neurons.

CYCLIN-B1/2 and -D1 act in opposition to coordinate cortical progenitor self-renewal and lineage commitment.

The sequential generation of layer-specific cortical neurons requires radial glia cells (RGCs) to precisely balance self-renewal and lineage commitment. While specific cell-cycle phases have been associated with these decisions, the mechanisms linking the cell-cycle machinery to cell-fate commitment remain obscure. Using single-cell RNA-sequencing, we find that the strongest transcriptional signature defining multipotent RGCs is that of G2/M-phase, and particularly CYCLIN-B1/2, while lineage-committed progenitors are enriched in G1/S-phase genes, including CYCLIN-D1. These data also reveal cell-surface markers that allow us to isolate RGCs and lineage-committed progenitors, and functionally confirm the relationship between cell-cycle phase enrichment and cell fate competence. Finally, we use cortical electroporation to demonstrate that CYCLIN-B1/2 cooperate with CDK1 to maintain uncommitted RGCs by activating the NOTCH pathway, and that CYCLIN-D1 promotes differentiation. Thus, this work establishes that cell-cycle phase-specific regulators act in opposition to coordinate the self-renewal and lineage commitment of RGCs via core stem cell regulatory pathways.

Radiation Triggers a Dynamic Sequence of Transient Microglial Alterations in Juvenile Brain.

Cranial irradiation (IR), an effective tool to treat malignant brain tumors, triggers a chronic pro-inflammatory microglial response, at least in the adult brain. Using single-cell and bulk RNA sequencing, combined with histology, we show that the microglial response in the juvenile mouse hippocampus is rapid but returns toward normal within 1 week. The response is characterized by a series of temporally distinct homeostasis-, sensome-, and inflammation-related molecular signatures. We find that a single microglial cell simultaneously upregulates transcripts associated with pro- and anti-inflammatory microglial phenotypes. Finally, we show that juvenile and adult irradiated microglia are already transcriptionally distinct in the early phase after IR. Our results indicate that microglia are involved in the initial stages but may not be responsible for driving long-term inflammation in the juvenile brain.

Neogenin and RGMa control neural tube closure and neuroepithelial morphology by regulating cell polarity.

In humans, neural tube closure defects occur in 1:1000 pregnancies. The design of new strategies for the prevention of such common defects would benefit from an improved understanding of the molecular events underlying neurulation. Neural fold elevation is a key morphological process that acts during neurulation to drive neural tube closure. However, to date, the molecular pathways underpinning neural fold elevation have not been elucidated. Here, we use morpholino knock-down technology to demonstrate that Repulsive Guidance Molecule (RGMa)-Neogenin interactions are essential for effective neural fold elevation during Xenopus neurulation and that loss of these molecules results in disrupted neural tube closure. We demonstrate that Neogenin and RGMa are required for establishing the morphology of deep layer cells in the neural plate throughout neurulation. We also show that loss of Neogenin severely disrupts the microtubule network within the deep layer cells suggesting that Neogenin-dependent microtubule organization within the deep cells is essential for radial intercalation with the overlying superficial cell layer, thereby driving neural fold elevation. In addition, we show that sustained Neogenin activity is also necessary for the establishment of the apicobasally polarized pseudostratified neuroepithelium of the neural tube. Therefore, our study identifies a novel signaling pathway essential for radial intercalation and epithelialization during neural fold elevation and neural tube morphogenesis.

Axon-Seq Decodes the Motor Axon Transcriptome and Its Modulation in Response to ALS.

Spinal motor axons traverse large distances to innervate target muscles, thus requiring local control of cellular events for proper functioning. To interrogate axon-specific processes we developed Axon-seq, a refined method incorporating microfluidics, RNA sequencing (RNA-seq), and bioinformatic quality control. We show that the axonal transcriptome is distinct from that of somas and contains fewer genes. We identified 3,500-5,000 transcripts in mouse and human stem cell-derived spinal motor axons, most of which are required for oxidative energy production and ribogenesis. Axons contained transcription factor mRNAs, e.g., Ybx1, with implications for local functions. As motor axons degenerate in amyotrophic lateral sclerosis (ALS), we investigated their response to the SOD1G93A mutation, identifying 121 ALS-dysregulated transcripts. Several of these are implicated in axonal function, including Nrp1, Dbn1, and Nek1, a known ALS-causing gene. In conclusion, Axon-seq provides an improved method for RNA-seq of axons, increasing our understanding of peripheral axon biology and identifying therapeutic targets in motor neuron disease.

The Wnt receptor Ryk is required for Wnt5a-mediated axon guidance on the contralateral side of the corpus callosum.

Ryk (receptor related to tyrosine kinase) has been shown to be a novel Wnt receptor in both Caenorhabditis elegans and Drosophila melanogaster. Recently, Ryk-Wnt interactions were shown to guide corticospinal axons down the embryonic mouse spinal cord. Here we show that, in Ryk-deficient mice, cortical axons project aberrantly across the major forebrain commissure, the corpus callosum. Many mouse mutants have been described in which loss-of-function mutations result in the inability of callosal axons to cross the midline, thereby forming Probst bundles on the ipsilateral side. In contrast, loss of Ryk does not interfere with the ability of callosal axons to cross the midline but impedes their escape from the midline into the contralateral side. Therefore, Ryk(-/-) mice display a novel callosal guidance phenotype. We also show that Wnt5a acts as a chemorepulsive ligand for Ryk, driving callosal axons toward the contralateral hemisphere after crossing the midline. In addition, whereas callosal axons do cross the midline in Ryk(-/-) embryos, they are defasciculated on the ipsilateral side, indicating that Ryk also promotes fasciculation of axons before midline crossing. In summary, this study expands the emerging role for Wnts in axon guidance and identifies Ryk as a key guidance receptor in the establishment of the corpus callosum. Our analysis of Ryk function further advances our understanding of the molecular mechanisms underlying the formation of this important commissure.

Single-Cell Analysis Reveals a Close Relationship between Differentiating Dopamine and Subthalamic Nucleus Neuronal Lineages.

Stem cell engineering and grafting of mesencephalic dopamine (mesDA) neurons is a promising strategy for brain repair in Parkinson's disease (PD). Refinement of differentiation protocols to optimize this approach will require deeper understanding of mesDA neuron development. Here, we studied this process using transcriptome-wide single-cell RNA sequencing of mouse neural progenitors expressing the mesDA neuron determinant Lmx1a. This approach resolved the differentiation of mesDA and neighboring neuronal lineages and revealed a remarkably close relationship between developing mesDA and subthalamic nucleus (STN) neurons, while also highlighting a distinct transcription factor set that can distinguish between them. While previous hESC mesDA differentiation protocols have relied on markers that are shared between the two lineages, we found that application of these highlighted markers can help to refine current stem cell engineering protocols, increasing the proportion of appropriately patterned mesDA progenitors. Our results, therefore, have important implications for cell replacement therapy in PD.

Predictive Markers Guide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson's Disease.

Stem cell treatments for neurodegenerative diseases are expected to reach clinical trials soon. Most of the approaches currently under development involve transplantation of immature progenitors that subsequently undergo phenotypic and functional maturation in vivo, and predicting the long-term graft outcome already at the progenitor stage remains a challenge. Here, we took an unbiased approach to identify predictive markers expressed in dopamine neuron progenitors that correlate with graft outcome in an animal model of Parkinson's disease through gene expression analysis of >30 batches of grafted human embryonic stem cell (hESC)-derived progenitors. We found that many of the commonly used markers did not accurately predict in vivo subtype-specific maturation. Instead, we identified a specific set of markers associated with the caudal midbrain that correlate with high dopaminergic yield after transplantation in vivo. Using these markers, we developed a good manufacturing practice (GMP) differentiation protocol for highly efficient and reproducible production of transplantable dopamine progenitors from hESCs.

Netrin-1 is required for efficient neural tube closure.

During neural tube formation, neural plate cells migrate from the lateral aspects of the dorsal surface towards the midline. Elevation of the lateral regions of the neural plate produces the neural folds which then migrate to the midline where they fuse at their dorsal tips, generating a closed neural tube comprising an apicobasally polarized neuroepithelium. Our previous study identified a novel role for the axon guidance receptor neogenin in Xenopus neural tube formation. We demonstrated that loss of neogenin impeded neural fold apposition and neural tube closure. This study also revealed that neogenin, via its interaction with its ligand, RGMa, promoted cell-cell adhesion between neural plate cells as the neural folds elevated and between neuroepithelial cells within the neural tube. The second neogenin ligand, netrin-1, has been implicated in cell migration and epithelial morphogenesis. Therefore, we hypothesized that netrin-1 may also act as a ligand for neogenin during neurulation. Here we demonstrate that morpholino knockdown of Xenopus netrin-1 results in delayed neural fold apposition and neural tube closure. We further show that netrin-1 functions in the same pathway as neogenin and RGMa during neurulation. However, contrary to the role of neogenin-RGMa interactions, neogenin-netrin-1 interactions are not required for neural fold elevation or adhesion between neuroepithelial cells. Instead, our data suggest that netrin-1 contributes to the migration of the neural folds towards the midline. We conclude that both neogenin ligands work synergistically to ensure neural tube closure.

Transcription factor-induced lineage selection of stem-cell-derived neural progenitor cells.

The generation of specific types of neurons from stem cells offers important opportunities in regenerative medicine. However, future applications and proper verification of cell identities will require stringent ways to generate homogeneous neuronal cultures. Here we show that transcription factors like Lmx1a, Phox2b, Nkx2.2, and Olig2 can induce desired neuronal lineages from most expressing neural progenitor cells by a mechanism resembling developmental binary cell-fate switching. Such efficient selection of cell fate resulted in remarkable cellular enrichment that enabled global gene-expression validation of generated neurons and identification of previously unrecognized features in the studied cell lineages. Several sources of stem cells have a limited competence to differentiate into specific neuronal cell types; e.g., dopamine neurons. However, we show that the combination of factors that normally promote either regional or dedicated neuronal specification can overcome limitations in cellular competence and also promote efficient reprogramming in more remote neural contexts, including human neural progenitor cells.