The Symmetry of Neural Stem Cell and Progenitor Divisions in the Vertebrate Brain

Robust brain development requires the tight coordination between tissue growth, neuronal differentiation and stem cell maintenance. To achieve this, neural stem cells need to balance symmetric proliferative and terminal divisions with asymmetric divisions. In recent years, the unequal distribution of certain cellular components in mitosis has emerged as a key mechanism to regulate the symmetry of division, and the determination of equal and unequal sister cell fates. Examples of such components include polarity proteins, signaling components, and cellular structures such as endosomes and centrosomes. In several types of neural stem cells, these factors show specific patterns of inheritance that correlate to specific cell fates, albeit the underlying mechanism and the potential causal relationship is not always understood. Here, we review these examples of cellular neural stem and progenitor cell asymmetries and will discuss how they fit into our current understanding of neural stem cell function in neurogenesis in developing and adult brains. We will focus mainly on the vertebrate brain, though we will incorporate relevant examples from invertebrate organisms as well. In particular, we will highlight recent advances in our understanding of the complexities related cellular asymmetries in determining division mode outcomes, and how these mechanisms are spatiotemporally regulated to match the different needs for proliferation and differentiation as the brain forms

Nitric oxide contributes to resistance of the Brown Norway Rat to experimental autoimmune encephalomyelitis

The Brown Norway (BN) rat is reported to be resistant to the induction of experimental autoimmune encephalomyelitis (EAE) and a number of mechanisms have been suggested to explain this resistance. In work reported here we provide evidence that such resistance in the BN rat can be accounted for, at least in part, by their ability to produce higher levels of nitric oxide (NO) than susceptible strains of rats. Spleen cells from the BN rat make significantly more NO following in vitro stimulation than do cells from the Lewis or PVG rat and following in vivo immunization using complete Freund's adjuvant (CFA) the BN rat makes substantially more NO than either susceptible strain. If carbonyl iron is used as adjuvant in vivo there is no increase in NO levels in the BN rat and they are rendered highly susceptible to EAE. Immunizing with CFA simultaneously with neuroantigen and carbonyl iron drives up NO levels and the resistance is restored. EAE produced using carbonyl iron is characterized by extensive macrophage/microglia presence in the central nervous system lesions of the BN rat yet the cytokine profile in the lymph nodes does not differ from that in the EAE Lewis rats

The innate immune response to adjuvants dictates the adaptive immune response to autoantigens

To elucidate the role of innate immunity in susceptibility to the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we induced EAE by immunization with spinal cord homogenate (SCH) plus complete Freund adjuvant or carbonyl iron in 3 inbred rat strains. Lewis are considered "susceptible," PVG/c-Rt7a (PVG) as "semisusceptible," and Brown Norway (BN) as "resistant" to EAE. Immunization with SCH-carbonyl iron resulted in clinical disease in all 3 strains, but the pathologic features of EAE in the resistant BN and the semisusceptible PVG rats differed from those in the Lewis and PVG model of EAE induced with SCH-complete Freund adjuvant. In BN and PVG rats, there were numerous inflammatory lesions with prominent involvement of microglia and, to a lesser extent, perivascular macrophages. These data suggest that different levels of activation of the innate immune system by different adjuvants determine whether EAE will or will not develop. Accordingly, the widely accepted scale of susceptibility to EAE development (Lewis > PVG > BN) should be revised because it does not take into account the important contribution of the composition of the adjuvant to the quality and quantity of the innate immune response and, consequently, to the generation and extent of the pathogenic T-cell-mediated, that is, adaptive, autoimmune disease

A comparative study of nucleostemin family members in zebrafish reveals specific roles in ribosome biogenesis

Nucleostemin (NS) is an essential protein for the growth and viability of developmental stem cells. Its functions are multi-faceted, including important roles in ribosome biogenesis and in the p53-induced apoptosis pathway. While NS has been well studied, the functions of its family members GNL2 and GNL3-like (GNL3L) remain relatively obscure despite a high degree of sequence and domain homology. Here, we use zebrafish lines carrying mutations in the ns family to compare and contrast their functions in vertebrates. We find the loss of zebrafish ns or gnl2 has a major impact on 60S large ribosomal subunit formation and/or function due to cleavage impairments at distinct sites of pre-rRNA transcript. In both cases this leads to a reduction of total protein synthesis. In contrast, gnl3l loss shows relatively minor rRNA processing delays that ultimately have no appreciable effects on ribosome biogenesis or protein synthesis. However, the loss of gnl3l still results in p53 stabilization, apoptosis, and lethality similarly to ns and gnl2 loss. The depletion of p53 in all three of the mutants led to partial rescues of the morphological phenotypes and surprisingly, a rescue of the 60S subunit collapse in the ns mutants. We show that this rescue is due to an unexpected effect of p53 loss that even in wild type embryos results in an increase of 60S subunits. Our study presents an in-depth description of the mechanisms through which ns and gnl2 function in vertebrate ribosome biogenesis and shows that despite the high degree of sequence and domain homology, gnl3l has critical functions in development that are unrelated to the ribosome

YAP Activity Is Necessary and Sufficient for Basal Progenitor Abundance and Proliferation in the Developing Neocortex

Neocortex expansion during mammalian evolution has been linked to an increase in proliferation of basal progenitors in the subventricular zone. Here, we explored a potential role of YAP, the major down-stream effector of the Hippo pathway, in proliferation of basal progenitors. YAP expression and activity are high in ferret and human basal progenitors, which exhibit high proliferative capacity, but low in mouse basal progenitors, which lack such capacity. Conditional expression of a constitutively active YAP in mouse basal progenitors resulted in increased proliferation of basal progenitor and promoted production of upper-layer neurons. Pharmacological and genetic interference with YAP function in ferret and human developing neocortex resulted in decreased abundance of cycling basal progenitors. Together, our data indicate that YAP is necessary and sufficient to promote the proliferation of basal progenitors and suggest that increases in YAP levels and presumably activity contributed to the evolutionary expansion of the neocortex

CD146 increases stemness and aggressiveness in glioblastoma and activates YAP signaling

Glioblastoma (GBM), a highly malignant and lethal brain tumor, is characterized by diffuse invasion into the brain and chemo-radiotherapy resistance resulting in poor prognosis. In this study, we examined the involvement of the cell adhesion molecule CD146/MCAM in regulating GBM aggressiveness. Analyses of GBM transcript expression databases revealed correlations of elevated CD146 levels with higher glioma grades, IDH-wildtype and unmethylated MGMT phenotypes, poor response to chemo-radiotherapy and worse overall survival. In a panel of GBM stem cells (GSCs) variable expression levels of CD146 were detected, which strongly increased upon adherent growth. CD146 was linked with mesenchymal transition since expression increased in TGF-ss-treated U-87MG cells. Ectopic overexpression of CD146/GFP in GG16 cells enhanced the mesenchymal phenotype and resulted in increased cell invasion. Conversely, GSC23-CD146 knockouts had decreased mesenchymal marker expression and reduced cell invasion in transwell and GBM-cortical assembloid assays. Moreover, using GSC23 xenografted zebrafish, we found that CD146 depletion resulted in more compact delineated tumor formation and reduced tumor cell dissemination. Stem cell marker expression and neurosphere formation assays showed that CD146 increased the stem cell potential of GSCs. Furthermore, CD146 mediated radioresistance by stimulating cell survival signaling through suppression of p53 expression and activation of NF-kappa B. Interestingly, CD146 was also identified as an inducer of the oncogenic Yes-associated protein (YAP). In conclusion, CD146 carries out various pro-tumorigenic roles in GBM involving its cell surface receptor function, which include the stimulation of mesenchymal and invasive properties, stemness, and radiotherapy resistance, thus providing an interesting target for therapy

The developmental stage of the medulloblastoma cell-of-origin restricts Hedgehog pathway usage and drug sensitivity

Sonic Hedgehog (SHH) medulloblastoma originates from the cerebellar granule neuron progenitor (CGNP) lineage that depends on Hedgehog signaling for its perinatal expansion. While SHH tumors exhibit overall deregulation of this pathway, they also show patient age-specific aberrations. To investigate if the developmental stage of the CGNP can account for these age-specific lesions, we analyzed developing murine CGNP transcriptomes and observed highly dynamic gene expression as function of age. Cross-species comparison with human SHH medulloblastoma showed partial maintenance of these expression patterns, and highlighted low primary cilium expression as hallmark of infant medulloblastoma and early embryonic CGNPs. This coincided with reduced responsiveness to upstream Shh pathway component Smoothened, while sensitivity to downstream components Sufu and Gli was retained. Together, these findings can explain the preference for SUFU mutations in infant medulloblastoma and suggest that drugs targeting the downstream SHH pathway will be most appropriate for infant patients

Essential role for the d-Asb11 cul5 Box domain for proper notch signaling and neural cell fate decisions in vivo

ECS (Elongin BC-Cul2/Cul5-SOCS-box protein) ubiquitin ligases recruit substrates to E2 ubiquitin-conjugating enzymes through a SOCS-box protein substrate receptor, an Elongin BC adaptor and a cullin (Cul2 or Cul5) scaffold which interacts with the RING protein. In vitro studies have shown that the conserved amino acid sequence of the cullin box in SOCS-box proteins is required for complex formation and function. However, the in vivo importance of cullin boxes has not been addressed. To explore the biological functions of the cullin box domain of ankyrin repeat and SOCS-box containing protein 11 (d-Asb11), a key mediator of canonical Delta-Notch signaling, we isolated a zebrafish mutant lacking the Cul5 box (Asb11(Cul)). We found that homozygous zebrafish mutants for this allele were defective in Notch signaling as indicated by the impaired expression of Notch target genes. Importantly, asb11(Cul) fish were not capable to degrade the Notch ligand DeltaA during embryogenesis, a process essential for the initiation of Notch signaling during neurogenesis. Accordingly, proper cell fate specification within the neurogenic regions of the zebrafish embryo was impaired. In addition, Asb11(Cul) mRNA was defective in the ability to transactivate a her4::gfp reporter DNA when injected in embryos. Thus, our study reporting the generation and the characterization of a metazoan organism mutant in the conserved cullin binding domain of the SOCS-box demonstrates a hitherto unrecognized importance of the SOCS-box domain for the function of this class of cullin-RING ubiquitin ligases and establishes that the d-Asb11 cullin box is required for both canonical Notch signaling and proper neurogenesis.