During the early development of the cerebellum, a burst of granule cell progenitor (GCP) proliferation occurs in the outer external granule layer (EGL), which is sustained mainly by Purkinje cell-derived Sonic Hedgehog (Shh). Shh response is interrupted once GCPs move into the inner EGL, where granule progenitors withdraw proliferation and start differentiating and migrating toward the internal granule layer (IGL). Failure to interrupt Shh signals results in uncoordinated proliferation and differentiation of GCPs and eventually leads to malignancy (i.e., medulloblastoma). The Shh inhibitory mechanisms that are responsible for GCP growth arrest and differentiation remain unclear. Here we report that REN, a putative tumor suppressor frequently deleted in human medulloblastoma, is expressed to a higher extent in nonproliferating inner EGL and IGL granule cells than in highly proliferating outer EGL cells. Accordingly, upregulated REN expression occurs along GCP differentiation in vitro, and, in turn, REN overexpression promotes growth arrest and increases the proportion of p27/Kip1 ϩ GCPs. REN also impairs both Gli2-dependent gene transcription and Shh-enhanced expression of the target Gli1 mRNA, thus antagonizing the Shh-induced effects on the proliferation and differentiation of cultured GCPs. Conversely, REN functional knock-down impairs Hedgehog antagonism and differentiation and sustains the proliferation of GCPs. Finally, REN enhances caspase-3 activation and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling apoptotic GCP numbers; therefore, the pattern of REN expression, its activity, and its antagonism on the Hedgehog pathway suggest that this gene may represent a restraint of Shh signaling at the outer to inner EGL GCP transitions. Medulloblastoma-associated REN loss of function might withdraw such a limiting signal for immature cell expansion, thus favoring tumorigenesis.
The Niemann-Pick type C1 (NPC1) disease is a neurodegenerative lysosomal storage disorder due to mutations in the NPC1 gene, encoding a transmembrane protein related to the Sonic hedgehog (Shh) receptor, Patched, and involved in intracellular trafficking of cholesterol. We have recently found that the proliferation of cerebellar granule neuron precursors is significantly reduced in Npc1-/- mice due to the downregulation of Shh expression. This finding prompted us to analyze the formation of the primary cilium, a non-motile organelle that is specialized for Shh signal transduction and responsible, when defective, for several human genetic disorders. In this study, we show that the expression and subcellular localization of Shh effectors and ciliary proteins are severely disturbed in Npc1-deficient mice. The dysregulation of Shh signaling is associated with a shortening of the primary cilium length and with a reduction of the fraction of ciliated cells in Npc1-deficient mouse brains and the human fibroblasts of NPC1 patients. These defects are prevented by treatment with 2-hydroxypropyl-β-cyclodextrin, a promising therapy currently under clinical investigation. Our findings indicate that defective Shh signaling is responsible for abnormal morphogenesis of the cerebellum of Npc1-deficient mice and show, for the first time, that the formation of the primary cilium is altered in NPC1 disease.
In fertilized mouse eggs, de novo transcription of embryonic genes is first observed during the S phase of the one-cell stage. This transcription, however, is mostly limited to the male pronucleus and possibly uncoupled from translation, making the functional meaning obscure. We found that one-cell mouse embryos respond to the osmotic shock of in vitro isolation with migration of HSF1, the canonical stress activator of mammalian heat shock genes, to pronuclei and by transient transcription of the hsp70.1, but not hsp70.3 and hsp90, heat shock genes. Isolated growing dictyate oocytes also display a nuclear HSF1 localization, but, in contrast with embryos, they transcribe both hsp70.1 and hsp70.3 genes only after heat shock. Intranuclear injection of double-stranded oligodeoxyribonucleotides containing HSE, GAGA box or GC box consensus sequences, and antibodies raised to transcription factors HSF1, HSF2, Drosophila melanogaster GAGA factor, or Sp1 demonstrated that hsp70.1 transcription depends on HSF1 in both oocytes and embryos and that Sp1 is dispensable in oocytes and inhibitory in the embryos. Hsp70.1 thus represents the first endogenous gene so far identified to be physiologically activated and tightly regulated after fertilization in mammals.
Niemann-Pick type C1 (NPC1) disease is a lysosomal storage disorder caused by defective intracellular trafficking of exogenous cholesterol. Purkinje cell (PC) degeneration is the main sign of cerebellar dysfunction in both NPC1 patients and animal models. It has been recently shown that a significant decrease in Sonic hedgehog (Shh) expression reduces the proliferative potential of granule neuron precursors in the developing cerebellum of Npc1−/− mice. Pursuing the hypothesis that this developmental defect translates into functional impairments, we have assayed Npc1-deficient pups belonging to the milder mutant mouse strain Npc1nmf164 for sensorimotor development from postnatal day (PN) 3 to PN21. Npc1nmf164/ Npc1nmf164 pups displayed a 2.5-day delay in the acquisition of complex motor abilities compared to wild-type (wt) littermates, in agreement with the significant disorganization of cerebellar cortex cytoarchitecture observed between PN11 and PN15. Compared to wt, Npc1nmf164 homozygous mice exhibited a poorer morphological differentiation of Bergmann glia (BG), as indicated by thicker radial shafts and less elaborate reticular pattern of lateral processes. Also BG functional development was defective, as indicated by the significant reduction in GLAST and Glutamine synthetase expression. A reduced VGluT2 and GAD65 expression also indicated an overall derangement of the glutamatergic/GABAergic stimulation that PCs receive by climbing/parallel fibers and basket/stellate cells, respectively. Lastly, Npc1-deficiency also affected oligodendrocyte differentiation as indicated by the strong reduction of myelin basic protein. Two sequential 2-hydroxypropyl-β-cyclodextrin administrations at PN4 and PN7 counteract these defects, partially preventing functional impairment of BG and fully restoring the normal patterns of glutamatergic/GABAergic stimulation to PCs.These findings indicate that in Npc1nmf164 homozygous mice the derangement of synaptic connectivity and dysmyelination during cerebellar morphogenesis largely anticipate motor deficits that are typically observed during adulthood.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0370-z) contains supplementary material, which is available to authorized users.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.