Constitutive Notch2 signaling in neural stem cells promotes tumorigenic features and astroglial lineage entry

Recent studies identified a highly tumorigenic subpopulation of glioma stem cells (GSCs) within malignant gliomas. GSCs are proposed to originate from transformed neural stem cells (NSCs). Several pathways active in NSCs, including the Notch pathway, were shown to promote proliferation and tumorigenesis in GSCs. Notch2 is highly expressed in glioblastoma multiforme (GBM), a highly malignant astrocytoma. It is therefore conceivable that increased Notch2 signaling in NSCs contributes to the formation of GBM. Here, we demonstrate that mice constitutively expressing the activated intracellular domain of Notch2 in NSCs display a hyperplasia of the neurogenic niche and reduced neuronal lineage entry. Neurospheres derived from these mice show increased proliferation, survival and resistance to apoptosis. Moreover, they preferentially differentiate into astrocytes, which are the characteristic cellular population of astrocytoma. Likewise, we show that Notch2 signaling increases proliferation and resistance to apoptosis in human GBM cell lines. Gene expression profiling of GBM patient tumor samples reveals a positive correlation of Notch2 transcripts with gene transcripts controlling anti-apoptotic processes, stemness and astrocyte fate, and a negative correlation with gene transcripts controlling proapoptotic processes and oligodendrocyte fate. Our data show that Notch2 signaling in NSCs produces features of GSCs and induces astrocytic lineage entry, consistent with a possible role in astrocytoma formation

TDP-43 induces p53-mediated cell death of cortical progenitors and immature neurons

TAR DNA-binding protein 43 (TDP-43) is a key player in neurodegenerative diseases including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Accumulation of TDP-43 is associated with neuronal death in the brain. How increased and disease-causing mutant forms of TDP-43 induce cell death remains unclear. Here we addressed the role of TDP-43 during neural development and show that reduced TDP-43 causes defects in neural stem/progenitor cell proliferation but not cell death. However, overexpression of wild type and TDP-43A315T proteins induce p53-dependent apoptosis of neural stem/progenitors and human induced pluripotent cell (iPS)-derived immature cortical neurons. We show that TDP-43 induces expression of the proapoptotic BH3-only genes Bbc3 and Bax, and that p53 inhibition rescues TDP-43 induced cell death of embryonic mouse, and human cortical neurons, including those derived from TDP-43G298S ALS patient iPS cells. Hence, an increase in wild type and mutant TDP-43 induces p53-dependent cell death in neural progenitors developing neurons and this can be rescued. These findings may have important implications for accumulated or mutant TDP-43 induced neurodegenerative diseases

The RSPO–LGR4/5–ZNRF3/RNF43 module controls liver zonation and size

LGR4/5 receptors and their cognate RSPO ligands potentiate Wnt/β-catenin signalling and promote proliferation and tissue homeostasis in epithelial stem cell compartments. In the liver, metabolic zonation requires a Wnt/β-catenin signalling gradient, but the instructive mechanism controlling its spatiotemporal regulation is not known. We have now identified the RSPO-LGR4/5-ZNRF3/RNF43 module as a master regulator of Wnt/β-catenin-mediated metabolic liver zonation. Liver-specific LGR4/5 loss of function (LOF) or RSPO blockade disrupted hepatic Wnt/β-catenin signalling and zonation. Conversely, pathway activation in ZNRF3/RNF43 LOF mice or with recombinant RSPO1 protein expanded the hepatic Wnt/β-catenin signalling gradient in a reversible and LGR4/5-dependent manner. Recombinant RSPO1 protein increased liver size and improved liver regeneration, whereas LGR4/5 LOF caused the opposite effects, resulting in hypoplastic livers. Furthermore, we show that LGR4(+) hepatocytes throughout the lobule contribute to liver homeostasis without zonal dominance. Taken together, our results indicate that the RSPO-LGR4/5-ZNRF3/RNF43 module controls metabolic liver zonation and is a hepatic growth/size rheostat during development, homeostasis and regeneration

A Cre-conditional MYCN-driven neuroblastoma mouse model as an improved tool for preclinical studies

Neuroblastoma, a childhood cancer that originates from neural crest-derived cells, is the most common deadly solid tumor of infancy. Amplification of the MYCN oncogene, which occurs in approximately 20-25% of human neuroblastomas, is the most prominent genetic marker of high-stage disease. The availability of valid preclinical in vivo models is a prerequisite to develop novel targeted therapies. We here report on the generation of transgenic mice with Cre-conditional induction of MYCN in dopamine β-hydroxylase-expressing cells, termed LSL-MYCN;Dbh-iCre. These mice develop neuroblastic tumors with an incidence of >75%, regardless of strain background. Molecular profiling of tumors revealed upregulation of the MYCN-dependent miR-17-92 cluster as well as expression of neuroblastoma marker genes, including tyrosine hydroxylase and the neural cell adhesion molecule 1. Gene set enrichment analyses demonstrated significant correlation with MYC-associated expression patterns. Array comparative genome hybridization showed that chromosomal aberrations in LSL-MYCN;Dbh-iCre tumors were syntenic to those observed in human neuroblastomas. Treatment of a cell line established from a tumor derived from a LSL-MYCN;Dbh-iCre mouse with JQ1 or MLN8237 reduced cell viability and demonstrated oncogene addiction to MYCN. Here we report establishment of the first Cre-conditional human MYCN-driven mouse model for neuroblastoma that closely recapitulates the human disease with respect to tumor localization, histology, marker expression and genomic make up. This mouse model is a valuable tool for further functional studies and to assess the effect of targeted therapies

sox9b Is a Key Regulator of Pancreaticobiliary Ductal System Development

The pancreaticobiliary ductal system connects the liver and pancreas to the intestine. It is composed of the hepatopancreatic ductal (HPD) system as well as the intrahepatic biliary ducts and the intrapancreatic ducts. Despite its physiological importance, the development of the pancreaticobiliary ductal system remains poorly understood. The SRY-related transcription factor SOX9 is expressed in the mammalian pancreaticobiliary ductal system, but the perinatal lethality of Sox9 heterozygous mice makes loss-of-function analyses challenging. We turned to the zebrafish to assess the role of SOX9 in pancreaticobiliary ductal system development. We first show that zebrafish sox9b recapitulates the expression pattern of mouse Sox9 in the pancreaticobiliary ductal system and use a nonsense allele of sox9b, sox9bfh313, to dissect its function in the morphogenesis of this structure. Strikingly, sox9bfh313 homozygous mutants survive to adulthood and exhibit cholestasis associated with hepatic and pancreatic duct proliferation, cyst formation, and fibrosis. Analysis of sox9bfh313 mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure. The intrahepatic biliary cells are specified but fail to assemble into a functional network. Similarly, intrapancreatic duct formation is severely impaired in sox9bfh313 mutants, while the embryonic endocrine and acinar compartments appear unaffected. The defects in the intrahepatic and intrapancreatic ducts of sox9bfh313 mutants worsen during larval and juvenile stages, prompting the adult phenotype. We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells. Together, these data reveal key roles for SOX9 in the morphogenesis of the pancreaticobiliary ductal system, and they cast human Sox9 as a candidate gene for pancreaticobiliary duct malformation-related pathologies

Expression of PIK3CA mutant E545K in the mammary gland induces heterogeneous tumors but is less potent than mutant H1047R

The phosphoinositide 3-kinase (PI3K) signaling cascade is a key mediator of cellular growth, survival and metabolism and is frequently subverted in human cancer. The gene encoding for the alpha catalytic subunit of PI3K (PIK3CA) is mutated and/or amplified in ∼30% of breast cancers. Mutations in either the kinase domain (H1047R) or the helical domain (E545K) are most common and result in a constitutively active enzyme with oncogenic capacity. PIK3CA(H1047R) was previously demonstrated to induce tumors in transgenic mouse models; however, it was not known whether overexpression of PIK3CA(E545K) is sufficient to induce mammary tumors and whether tumor initiation by these two types of mutants differs. Here, we demonstrate that expression of PIK3CA(E545K) in the mouse mammary gland induces heterogenous mammary carcinomas but with a longer latency than PIK3CA(H1047R)-expressing mice. Our results suggest that the helical domain mutant PIK3CA(E545K) is a less potent inducer of mammary tumors due to less efficient activation of downstream Akt signaling

Transneuronal propagation of mutant huntingtin contributes to non-cell autonomous pathology in neurons

In Huntington's disease (HD), whether transneuronal spreading of mutant huntingtin (mHTT) occurs and its contribution to non-cell autonomous damage in brain networks is largely unknown. We found mHTT spreading in three different neural network models: human neurons integrated in the neural network of organotypic brain slices of HD mouse model, an ex vivo corticostriatal slice model and the corticostriatal pathway in vivo. Transneuronal propagation of mHTT was blocked by two different botulinum neurotoxins, each known for specifically inactivating a single critical component of the synaptic vesicle fusion machinery. Moreover, healthy human neurons in HD mouse model brain slices displayed non-cell autonomous changes in morphological integrity that were more pronounced when these neurons bore mHTT aggregates. Altogether, our findings suggest that transneuronal propagation of mHTT might be an important and underestimated contributor to the pathophysiology of HD