[show abstract][hide abstract] ABSTRACT: Glioblastoma (GBM) is the most common malignant adult brain tumor and carries a poor prognosis due to primary and acquired resistance. While many cellular features of GBM have been documented, it is unclear if cells within these tumors extend a primary cilium, an organelle whose associated signaling pathways may regulate proliferation, migration, and survival of neural precursor and tumor cells. Using immunohistochemical and electron microscopy (EM) techniques, we screened human GBM tumor biopsies and primary cell lines for cilia. Immunocytochemical staining of five primary GBM cell lines revealed that between 8 and 25 % of the cells in each line possessed gamma tubulin-positive basal bodies from which extended acetylated, alpha-tubulin-positive axonemes. EM analyses confirmed the presence of cilia at the cell surface and revealed that their axonemes contained organized networks of microtubules, a structural feature consistent with our detection of IFT88 and Arl13b, two trafficked cilia proteins, along the lengths of the axonemes. Notably, cilia were detected in each of 23 tumor biopsies (22 primary and 1 recurrent) examined. These cilia were distributed across the tumor landscape including regions proximal to the vasculature and within necrotic areas. Moreover, ciliated cells within these tumors co-stained with Ki67, a marker for actively dividing cells, and ZEB1, a transcription factor that is upregulated in GBM and linked to tumor initiation, invasion, and chemoresistance. Collectively, our data show that subpopulations of cells within human GBM tumors are ciliated. In view of mounting evidence supporting roles of primary cilia in tumor initiation and propagation, it is likely that further study of the effects of cilia on GBM tumor cell function will improve our understanding of GBM pathogenesis and may provide new directions for GBM treatment strategies.
Journal of Neuro-Oncology 02/2014; · 3.12 Impact Factor
[show abstract][hide abstract] ABSTRACT: Glioblastoma remains one of the most lethal types of cancer, and is the most common brain tumour in adults. In particular, tumour recurrence after surgical resection and radiation invariably occurs regardless of aggressive chemotherapy. Here, we provide evidence that the transcription factor ZEB1 (zinc finger E-box binding homeobox 1) exerts simultaneous influence over invasion, chemoresistance and tumourigenesis in glioblastoma. ZEB1 is preferentially expressed in invasive glioblastoma cells, where the ZEB1-miR-200 feedback loop interconnects these processes through the downstream effectors ROBO1, c-MYB and MGMT. Moreover, ZEB1 expression in glioblastoma patients is predictive of shorter survival and poor Temozolomide response. Our findings indicate that this regulator of epithelial-mesenchymal transition orchestrates key features of cancer stem cells in malignant glioma and identify ROBO1, OLIG2, CD133 and MGMT as novel targets of the ZEB1 pathway. Thus, ZEB1 is an important candidate molecule for glioblastoma recurrence, a marker of invasive tumour cells and a potential therapeutic target, along with its downstream effectors.
EMBO Molecular Medicine 08/2013; 5(8):1196-1212. · 7.80 Impact Factor
[show abstract][hide abstract] ABSTRACT: The formation of primary cilia is a highly choreographed process that can be disrupted in developing neurons by overexpressing neuromodulatory G-protein-coupled receptors GPCRs or by blocking intraflagellar transport. Here, we examined the effects of overexpressing the ciliary GPCRs, 5HT6 and SSTR3, on cilia structure and the differentiation of neocortical neurons. Neuronal overexpression of 5HT6 and SSTR3 was achieved by electroporating mouse embryo cortex in utero with vectors encoding these receptors. We found that overexpression of ciliary GPCRs in cortical neurons, especially 5HT6, induced the formation of long (>30 μm) and often forked cilia. These changes were associated with increased levels of intraflagellar transport proteins and accelerated ciliogenesis in neonatal neocortex, the induction of which required Kif3a, an anterograde motor critical for cilia protein trafficking and growth. GPCR overexpression also altered the complement of signaling molecules within the cilia. We found that SSTR3 and type III adenylyl cyclase (ACIII), proteins normally enriched in neuronal cilia, were rarely detected in 5HT6-elongated cilia. Intriguingly, the changes in cilia structure were accompanied by changes in neuronal morphology. Specifically, disruption of normal ciliogenesis in developing neocortical neurons, either by overexpressing cilia GPCRs or a dominant-negative form of Kif3a, significantly impaired dendrite outgrowth. Remarkably, coexpression of ACIII with 5HT6 restored ACIII to cilia, normalized cilia structure, and restored dendrite outgrowth, effects that were not observed in neurons coexpressing ACIII and dominant-negative form of Kif3a. Collectively, our data suggest the formation of neuronal dendrites in developing neocortex requires structurally normal cilia enriched with ACIII.
Journal of Neuroscience 02/2013; 33(6):2626-38. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Neuronal primary cilia are not generally recognized, but they are considered to extend from most, if not all, neurons in the neocortex. However, when and how cilia develop in neurons are not known. This study used immunohistochemistry for adenylyl cyclase III (ACIII), a marker of primary cilia, and electron microscopic analysis to describe the development and maturation of cilia in mouse neocortical neurons. Our results indicate that ciliogenesis is initiated in late fetal stages after neuroblast migration, when the mother centriole docks with the plasma membrane, becomes a basal body, and grows a cilia bud that we call a procilium. This procilium consists of a membranous protrusion extending from the basal body but lacking axonemal structure and remains undifferentiated until development of the axoneme and cilia elongation starts at about postnatal day 4. Neuronal cilia elongation and final cilia length depend on layer position, and the process extends for a long time, lasting 8-12 weeks. We show that, in addition to pyramidal neurons, inhibitory interneurons also grow cilia of comparable length, suggesting that cilia are indeed present in all neocortical neuron subtypes. Furthermore, the study of mice with defective ciliogenesis suggested that failed elongation of cilia is not essential for proper neuronal migration and laminar organization or establishment of neuronal polarity. Thus, the function of this organelle in neocortical neurons remains elusive.
The Journal of Comparative Neurology 03/2012; 520(4):848-73. · 3.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: To better understand the short and long-term effects of stress on the developing cerebral cortex, it is necessary to understand how early stress response genes protect or permanently alter cells. One family of highly conserved, stress response genes is the growth arrest and DNA damage-45 (Gadd45) genes. The expression of these genes is induced by a host of genotoxic, drug, and environmental stressors. Here we examined the impact of altering the expression of Gadd45alpha (Gadd45a), a member of the Gadd45 protein family that is expressed throughout the developing cortices of mice and humans. To manipulate levels of Gadd45a protein in developing mouse cortex, we electroporated cDNA plasmids encoding either Gadd45a or Gadd45a shRNA to either overexpress or knockdown Gadd45a levels in the developing cortices of mice, respectively. The effects of these manipulations were assessed by examining the fates and morphologies of the labeled neurons. Gadd45a overexpression both in vitro and in vivo significantly impaired the morphology of neurons, decreasing neurite complexity, inducing soma hypertrophy and increasing cell death. Knockdown of Gadd45a partially inhibited neuronal migration and reduced neurite complexity, an effect that was reversed in the presence of an shRNA-resistant Gadd45a. Finally, we found that shRNA against MEKK4, a direct target of Gadd45a, also stunted neurite outgrowth. Our findings suggest that the expression of Gadd45a in normal, developing brain is tightly regulated and that treatments or environmental stimuli that alter its expression could produce significant changes in neuronal circuitry development.
PLoS ONE 01/2012; 7(9):e44207. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The dyslexia-associated gene DCDC2 is a member of the DCX family of genes known to play roles in neurogenesis, neuronal migration, and differentiation. Here we report the first phenotypic analysis of a Dcdc2 knockout mouse. Comparisons between Dcdc2 knockout mice and wild-type (wt) littermates revealed no significant differences in neuronal migration, neocortical lamination, neuronal cilliogenesis or dendritic differentiation. Considering previous studies showing genetic interactions and potential functional redundancy among members of the DCX family, we tested whether decreasing Dcx expression by RNAi would differentially impair neurodevelopment in Dcdc2 knockouts and wild-type mice. Consistent with this hypothesis, we found that deficits in neuronal migration, and dendritic growth caused by RNAi of Dcx were more severe in Dcdc2 knockouts than in wild-type mice with the same transfection. These results indicate that Dcdc2 is not required for neurogenesis, neuronal migration or differentiation in mice, but may have partial functional redundancy with Dcx.
[show abstract][hide abstract] ABSTRACT: Most, if not all, cortical neurons possess a single primary cilium; however, little is known about the mechanisms that control neuronal ciliogenesis. The Citron kinase-deficient (Citron-K(fh/fh)) rat, a model in which failed cytokinesis during development produces cortical neurons containing multiple cellular organelles, provides a unique system in which to examine the relationship between centriole inheritance and neuronal ciliogenesis. In this study, we analyzed the cerebral cortex of these animals using immunohistochemistry, serial confocal, and electron microscopy to determine if the multinucleated neurons present in the cortex of these animals also possess multiple centrioles and cilia. We found that neurons containing multiple nuclei possessed multiple centrioles and cilia whose lengths varied across cortical regions. Despite the presence of multiple cilia, we found that perinatal expression of adenylyl cyclase III, a cilia-specific marker, and somatostatin receptor 3, a receptor enriched in cilia, were preserved in developing Citron-K(fh/fh) brain. Together, these results show that multinucleated neurons arising from defective cytokinesis can extend multiple cilia.
[show abstract][hide abstract] ABSTRACT: Neuronal migration is a fundamental component of brain development whose failure is associated with various neurological and psychiatric disorders. Reelin is essential for the stereotypical inside-out sequential lamination of the neocortex, but the molecular mechanisms of its action still remain unclear. Here we show that regulation of Notch activity plays an important part in Reelin-signal-dependent neuronal migration. We found that Reelin-deficient mice have reduced levels of the cleaved form of Notch intracellular domain (Notch ICD) and that loss of Notch signaling in migrating neurons results in migration and morphology defects. Further, overexpression of Notch ICD mitigates the laminar and morphological abnormalities of migrating neurons in Reeler. Finally, our in vitro biochemical studies show that Reelin signaling inhibits Notch ICD degradation via Dab1. Together, our results indicate that neuronal migration in the developing cerebral cortex requires a Reelin-Notch interaction.
[show abstract][hide abstract] ABSTRACT: Primary cilia are present on mammalian neurons and glia, but their function is largely unknown. We generated conditional homozygous mutant mice for a gene we termed Stumpy. Mutants lack cilia and have conspicuous abnormalities in postnatally developing brain regions, including a hypoplasic hippocampus characterized by a primary deficiency in neural stem cells known as astrocyte-like neural precursors (ALNPs). Previous studies suggested that primary cilia mediate sonic hedgehog (Shh) signaling. Here, we find that loss of ALNP cilia leads to abrogated Shh activity, increased cell cycle exit, and morphological abnormalities in ALNPs. Processing of Gli3, a mediator of Shh signaling, is also altered in the absence of cilia. Further, key mediators of the Shh pathway localize to ALNP cilia. Thus, selective targeting of Shh machinery to primary cilia confers to ALNPs the ability to differentially respond to Shh mitogenic signals compared to neighboring cells. Our data suggest these organelles are cellular "antennae" critically required to modulate ALNP behavior.
Proceedings of the National Academy of Sciences 10/2008; 105(35):13127-32. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cilia are present on nearly all cell types in mammals and perform remarkably diverse functions. However, the mechanisms underlying ciliogenesis are unclear. Here, we cloned a previously uncharacterized highly conserved gene, stumpy, located on mouse chromosome 7. Stumpy was ubiquitously expressed, and conditional loss in mouse resulted in complete penetrance of perinatal hydrocephalus (HC) and severe polycystic kidney disease (PKD). We found that cilia in stumpy mutant brain and kidney cells were absent or markedly deformed, resulting in defective flow of cerebrospinal fluid. Stumpy colocalized with ciliary basal bodies, physically interacted with gamma-tubulin, and was present along ciliary axonemes, suggesting that stumpy plays a role in ciliary axoneme extension. Therefore, stumpy is essential for ciliogenesis and may be involved in the pathogenesis of human congenital malformations such as HC and PKD.
Proceedings of the National Academy of Sciences 03/2008; 105(8):2853-8. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Postmitotic cortical neurons that fail to initiate migration can remain near their site of origin and form persistent periventricular nodular heterotopia (PH). In human telencephalon, this malformation is most commonly associated with Filamin-A (FLNa) mutations. The lack of genetic animal models that reliably produce PH has delayed our understanding of the underlying molecular mechanisms. This review examines PH pathogenesis using a new mouse model. Although PH have not been observed in Flna-deficient mice generated thus far, the loss of MEKK4, a regulator of Flna, produces striking PH in mice and offers insight into the mechanisms involved in neuronal migration initiation. Elucidating the basic functions of FLNa and associated molecules is crucial for understanding the causes of PH and for developing prevention for at-risk patients.
Trends in Neurosciences 03/2008; 31(2):54-61. · 13.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: The techniques and concepts of modern molecular biology and experimental neurobiology give new insights into molecular mechanisms involved in neuronal migration within the cerebral cortex. The findings collectively indicate that a diverse family of genes and transcription factors co-operate in orchestrating the multistage, interrelated phenomena that include control of mode of cell proliferation, fate determination, establishment of polarity, detachment from the local substrate and migration to the proper laminar and a real position in the cortex. Herein, we will review some new data from our laboratory on the initiation of migration, nuclear translocation and attainment of final positions. We can now propose working models of the sequence of gene expression, cascade of multiple molecular pathways and cell-cell interactions that are involved in neuronal migration. Disruption or even slowing down of any step in neuronal migration during embryonic development can result in either gross or subtle abnormalities in neuronal positioning that may only later, during postnatal life, affect the formation of synaptic circuits and eventually behaviour.
Novartis Foundation symposium 02/2007; 288:45-53; discussion 53-8, 96-8.
[show abstract][hide abstract] ABSTRACT: The dentate gyrus is a site of continual neurogenesis in the postnatal mammalian brain. Here we investigated postnatal neurogenesis in the citron kinase (citron-K) null-mutant rat (flathead). The flathead rat has substantial deficits in embryonic neurogenesis that are due to failed cytokinesis and cell death. We report here the loss of citron-K function has an even severer effect on postnatal neurogenesis in the dentate gyrus. Analysis of phosphorylated histone H3 expression in postnatal neurogenic regions of the flathead mutant revealed a complete lack of mitotic cells in the dentate gyrus and a large reduction in the number of dividing cells in the flathead subventricular zone. Examination of 5-bromodeoxyuridine incorporation in the flathead rat revealed that the flathead rat had a 99% reduction in the number of newly generated cells in the dentate gyrus at postnatal day 10. In addition, doublecortin-positive cells were essentially absent from the postnatal flathead dentate gyrus which also lacked the vimentin- and nestin-positive radial glia scaffold that defines the neurogenic niche in the postnatal subgranular zone. Together these results indicate that postnatal neurogenesis in the dentate gyrus is eliminated by loss of citron-K function, and suggests that a citron-K-dependent progenitor lineage forms the postnatal neuronal progenitor population in the dentate gyrus.
[show abstract][hide abstract] ABSTRACT: Periventricular heterotopia (PVH) is a congenital malformation of human cerebral cortex frequently associated with Filamin-A (FLN-A) mutations but the pathogenetic mechanisms remain unclear. Here, we show that the MEKK4 (MAP3K4) pathway is involved in Fln-A regulation and PVH formation. MEKK4(-/-) mice developed PVH associated with breaches in the neuroependymal lining which were largely comprised of neurons that failed to reach the cortical plate. RNA interference (RNAi) targeting MEKK4 also impaired neuronal migration. Expression of Fln was elevated in MEKK4(-/-) forebrain, most notably near sites of failed neuronal migration. Importantly, recombinant MKK4 protein precipitated a complex containing MEKK4 and Fln-A, and MKK4 mediated signaling between MEKK4 and Fln-A, suggesting that MKK4 may bridge these molecules during development. Finally, we showed that wild-type FLN-A overexpression inhibited neuronal migration. Collectively, our results demonstrate a link between MEKK4 and Fln-A that impacts neuronal migration initiation and provides insight into the pathogenesis of human PVH.
[show abstract][hide abstract] ABSTRACT: Neural tube defects (NTDs) are prevalent human birth defects. Mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinase (JNK), are implicated in facilitating neural tube closure, yet upstream regulators remain to be identified. Here, we show that MAP kinase kinase kinase 4 (MEKK4) is strongly expressed in the developing neuroepithelium. Mice deficient in MEKK4 develop highly penetrant NTDs that cannot be rescued by supplementation with folic acid or inositol. Unlike most mouse models of NTDs, MEKK4 mutant embryos display genetically co-segregated exencephaly and spina bifida, recapitulating the phenotypes observed in human patients. To identify downstream targets of MEKK4 during neural tube development, we examined the activity of MAP kinase kinase 4 (MKK4), a signaling intermediate between MAP kinase kinase kinase and JNK/p38. We found a significant reduction in MKK4 activity in MEKK4-deficient neuroepithelium at sites of neural tube closure. MAPK pathways are key regulators of cell apoptosis and proliferation. Analyses of the neuroepithelium in MEKK4-deficient embryos showed massively elevated apoptosis before and during neural tube closure, suggesting an antiapoptotic role for MEKK4 during development. In contrast, proliferation of MEKK4-deficient neuroepithelial cells appeared to be largely unaffected. MEKK4 therefore plays a critical role in regulating MKK4 activity and apoptotic cell death during neural tube development. Disruption of this signaling pathway may be clinically relevant to folate-resistant human NTDs.
Proceedings of the National Academy of Sciences 04/2005; 102(10):3846-51. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cells undergoing apoptosis during development are removed by phagocytes, but the underlying mechanisms of this process are not fully understood. Phagocytes lacking the phosphatidylserine receptor (PSR) were defective in removing apoptotic cells. Consequently, in PSR-deficient mice, dead cells accumulated in the lung and brain, causing abnormal development and leading to neonatal lethality. A fraction of PSR knockout mice manifested a hyperplasic brain phenotype resembling that of mice deficient in the cell death-associated genes encoding Apaf-1, caspase-3, and caspase-9, which suggests that phagocytes may also be involved in promoting apoptosis. These data demonstrate a critical role for PSR in early stages of mammalian organogenesis and suggest that this receptor may be involved in respiratory distress syndromes and congenital brain malformations.