Cohesin regulation of genome organization in mature granule neurons in the mouse cerebellum

Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, we have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1. Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABAα6R characteristic of mature granule neurons, formed "T-shaped" axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage.

OPINION article Front. Cell. Neurosci., 26 June 2013Sec. Cellular Neurophysiology https://doi.org/10.3389/fncel.2013.00096

Secreted Frizzled-Related Protein 3 Regulates Activity-Dependent Adult Hippocampal Neurogenesis

Novel connection between newborn granule neurons and the hippocampal CA2 field

Cerebellar granule cell survival and maturation induced by K + and NMDA correlate with c- fos proto-oncogene expression

Cerebellar granule neurons isolated from 7-day-old rats and cultured in normal medium undergo apoptosis, but remain healthy under depolarizing conditions with elevated K(+) (>==25 mM) or in the presence of brain-derived neurotrophic factor. Northern blot analysis showed that cyclin D1 mRNA was up-regulated in this apoptotic process. Both granule neurons and microglia were immunostained with anti-cyclin D1 antibodies, which is consistent with our previous finding that microglia become activated in response to neuronal cell death under these conditions. Only granule neurons, however, showed an enhanced expression of both mRNA and protein levels of cyclin D1 in the presence of aphidicolin that completely eliminated non-neuronal cells. The entire cell body of granule neurons became immunostained prior to cell shrinkage or nuclear condensation. Moreover, cell cycle blockers and an inhibitor of cyclin-dependent kinases suppressed both increased immunoreactivity and cell death, further substantiating the involvement of an abortive cell cycle in this process. In contrast, both levels of cyclin D1 remained unaltered in mature granule neurons undergoing apoptosis following combined serum withdrawal and low K(+) shift, suggesting developmental stage dependence of granule neuron apoptosis in vitro. This culture system is suitable for further analysis of the role of cyclin D1 in cell death.

During the development of the central nervous system, pools of progenitor cells undergo proliferation and subsequent differentiation into mature neurons, establishing a functional neuronal network. However, an imbalance in these processes, characterized by excessive proliferation and loss of differentiation, can lead to tumor formation in pediatric patients. In the developing cerebellum, medulloblastomas of the Sonic Hedgehog (SHH) group arise from excessive proliferation of granule neuron progenitor cells (GNPs) driven by SHH signaling, accompanied by hindered differentiation into mature granule neurons. Genetic predisposition accounts for nearly 40% of all pediatric SHH-medulloblastomas, but the development of humanized models to study these predisposition genes has been lacking. To address this gap, we created an ATOH1-driven EGFP reporter iPSC line to generate reliable cerebellar organoids focused on the development of the granule cell lineage. Our cerebellar organoid protocol yielded homogeneous organoids with robust activation of the ATOH1-EGFP reporter around day 30. Through bulk transcriptomics analysis at day 30, we observed pronounced expression of GNP markers, followed by increased expression of markers associated with mature granule neurons by day 60. Furthermore, by aligning the single-nuclei RNA sequencing (snRNAseq) data of day 60 cerebellar organoids with our human cerebellar development snRNAseq atlas, we identified various cerebellar cell types, including GNPs, mature granule neurons, Purkinje cells, interneurons, as well as glutamatergic and GABAergic neurons. Using this protocol, we examined the development of cerebellar organoids in models of hereditary predisposition to childhood cancer. Using a Gorlin syndrome model, we found that PTCH1HET organoids are larger in size than their isogenic control counterparts, while a model for ELP1HET hereditary predisposition showed no change in size compared with control organoids. These organoid models will allow us to uncover novel molecular mechanisms of hereditary predisposition using human cells.

The development of the dentate gyrus is characterized by distinct phases establishing a durable stem-cell pool required for postnatal and adult neurogenesis. Here, we report that Bcl11b/Ctip2, a zinc finger transcription factor expressed in postmitotic neurons, plays a critical role during postnatal development of the dentate gyrus. Forebrain-specific ablation of Bcl11b uncovers dual phase-specific functions of Bcl11b demonstrated by feedback control of the progenitor cell compartment as well as regulation of granule cell differentiation, leading to impaired spatial learning and memory in mutants. Surprisingly, we identified Desmoplakin as a direct transcriptional target of Bcl11b. Similarly to Bcl11b, postnatal neurogenesis and granule cell differentiation are impaired in Desmoplakin mutants. Re-expression of Desmoplakin in Bcl11b mutants rescues impaired neurogenesis, suggesting Desmoplakin to be an essential downstream effector of Bcl11b in hippocampal development. Together, our data define an important novel regulatory pathway in hippocampal development, by linking transcriptional functions of Bcl11b to Desmoplakin, a molecule known to act on cell adhesion.

Brain-derived neurotrophic factor (bdnf) can prevent apoptosis of rat cerebellar granule neurons in culture

Objective Chlorpyrifos (CPF) is a neurotoxic organophosphorus (OP) insecticide. Itsmechanism of action includes oxidative stress, excitotoxicity, and inhibition of the acetylcholinesterase enzyme (AChE). The aim of the present study is to investigate CPF toxicityin mature and immature cerebellar granule neurons (CGNs), as well as its effect on glutamate induced excitotoxicity.Materials and Methods This study was an in vitro experimental study performed on micecultured CGNs. Immature and mature neurons were exposed to different concentrationsof CPF (1-1000 µM) and glutamate (10-600 µM) for 48 hours after which we used theMTT assay to measure cytotoxicity. Immature neurons had exposure to CPF for 5 daysin order to evaluate the cytotoxic effect on developing neurons. Mature neurons receivedsub-lethal concentrations of CPF (10, 100 µM) combined with different concentrations ofglutamate. AChE activity and reactive oxygen species (ROS) generation were assessedafter treatments.Results Immature CGNs had increased sensitivity to CPF toxicity compared to matureneurons. We observed significantly greater ROS production in immature compared tomature neurons, however AChE activity was more inhibited in mature neurons. AlthoughCPF toxicity was not well correlated with AChE inhibition, it correlated well with ROS production. Glutamate toxicity was potentiated by sub-lethal concentration of CPF, howeverglutamate induced ROS production was not affected. The results suggested that CPFpotentiated glutamate toxicity by mechanisms other than oxidative stress.ConclusionCPF toxicity differed in mature and immature neurons. Potentiated glutamate toxicity by CPF implied that CPF exposure might be a risk factor for neurodegenerative disease.

The glutamate receptor gene GluR-4 is proposed to generate two spliced isoforms (Sommer et al., 1990). Screening a rat cerebellar cDNA library, we have now identified a third type of transcript derived from GluR-4 gene by differential RNA processing. This transcript encodes a protein with a "flop" module between transmembrane regions 3 and 4, but with a C-terminus segment of 36 amino acids different from the previously described GluR-4 flip/flop cDNAs. This subunit was therefore designated as GluR-4c flop. Transcripts synthesized in vitro from GluR-4c cDNA form kainate/AMPA-activated channels when expressed in Xenopus oocytes. The current-voltage relationship for kainate-evoked responses in oocytes injected with GluR-4c showed strong inward rectification. The different transcripts derived from the GluR-4 gene were studied on Northern blots hybridized with either a cDNA probe or oligonucleotides specific for the GluR-4 flip/flop and C-terminal domains. Three transcripts of 6.2, 4.2, and 3.0 kilobases (kb) derived from the GluR-4 gene were identified on Northern blots containing total RNA prepared from different brain regions, using a cDNA probe or an oligonucleotide corresponding to the N-terminal region common to all transcripts. These transcripts were much more abundant in the cerebellum than in other brain areas, and their levels increased during cerebellar development. The maximal increase was observed between postnatal days 1 and 20, an age corresponding to the division and maturation of granule neurons. The flip/flop and the C-terminal oligonucleotides hybridized to the two higher molecular weight transcripts but did not hybridize to the small RNA. Interestingly, using cerebellar cells that were cultured for up to 12 d, we observed that the three transcripts are present in granule neurons, but that astrocytes only express the 6.2 and the 4.2 kb transcripts. The 3.0 kb transcript accumulates in cerebellar granule cells during development in vitro. Furthermore, in situ hybridization histochemistry revealed that the GluR-4c transcripts are preferentially expressed in cerebellar granule cells and Bergmann glial cells, whereas the expression of GluR-4 flip mRNAs is restricted to Bergmann glial cells. Interestingly, we also show that granule cells already express GluR-4c in the premigratory zone of the external granular layer, indicating that intrinsic or highly localized cues induce GluR-4c expression before these cells reach their final position.

The properties of depolarization-evoked calcium transients are known to change during the maturation of dissociated cerebellar granule neuron cultures. Here, we assessed the role of the calcium-induced calcium release (CICR) mechanism in granule neuron maturation. Both depletion of intracellular calcium stores and the pharmacological blockade of CICR significantly reduced depolarization stimulated calcium transients in young but not older (>/=1 week) cultures. This functional decrease in the CICR signaling component was associated with the reduction of ryanodine receptor (RyR) immunoreactivity during granule neuron maturation both in culture and in the intact cerebellum. These observations are consistent with the idea that changes in RyR expression result in functional changes in calcium signaling transients during normal neuronal development in the intact mammalian cerebellum as well as in reduced neuronal cultures. Pharmacological disruption of CICR during neuron differentiation in vitro resulted in dose-dependent changes in survival, GAP-43 expression, and the acquisition of the glutamatergic neurotransmitter phenotype. Together, these results indicate that CICR function plays a physiologically relevant role in regulating early granule neuron differentiation in vitro and is likely to play a role in cerebellar maturation.

The properties of depolarization-evoked calcium transients are known to change during the maturation of dissociated cerebellar granule neuron cultures. Here, we assessed the role of the calcium-induced calcium release (CICR) mechanism in granule neuron maturation. Both depletion of intracellular calcium stores and the pharmacological blockade of CICR significantly reduced depolarization stimulated calcium transients in young but not older (≥1 week) cultures. This functional decrease in the CICR signaling component was associated with the reduction of ryanodine receptor (RyR) immunoreactivity during granule neuron maturation both in culture and in the intact cerebellum. These observations are consistent with the idea that changes in RyR expression result in functional changes in calcium signaling transients during normal neuronal development in the intact mammalian cerebellum as well as in reduced neuronal cultures. Pharmacological disruption of CICR during neuron differentiation in vitro resulted in dose-dependent changes in survival, GAP-43 expression, and the acquisition of the glutamatergic neurotransmitter phenotype. Together, these results indicate that CICR function plays a physiologically relevant role in regulating early granule neuron differentiation in vitro and is likely to play a role in cerebellar maturation. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 134–147, 2000

The cerebellum plays an important role in motor control, motor skill acquisition, memory and learning among other brain functions. In rodents, cerebellar development continues after birth, characterized by the maturation of granule neurons. Cerebellar granule neurons (CGNs) are the most abundant neuronal type in the central nervous system, and they provide an excellent model for investigating molecular, -cellular, and physiological mechanisms underlying neuronal development as well as neural circuitry linked to behavior. Here we describe a procedure to isolate and culture CGNs from postnatal day 6 mice. These cultures can be used to examine numerous aspects of CGN differentiation, electrophysiology, and function.

In the developing cerebellum, young granule neurons in the external germinal layer respond preferentially to BDNF, while mature neurons within the inner portion of the cerebellum respond preferentially to NT3. Here we show that this anatomic distinction reflects a developmentally regulated switch at the level of neurotrophin receptor gene expression. The salient feature of the developmental switch is a change in the ration of mRNA transcripts encoding functional BDNF and NT3 receptor tyrosine kinases. The ratio of the BDNF receptor trkB to the NT3 receptor trkC reverses from 5:1 in neonatal cerebellum to 1:3 in adult cerebellum. TrkB and TrkC are closely related transmembrane tyrosine protein kinases. However, activation of BDNF and NT3 receptors in cerebellar granule neurons do not give equivalent biological responses. In aggregate cell culture and single cell assays, BDNF enhances axonal outgrowth of early granule cells by influencing neurite elongation. In contrast, NT3 alters the morphology of outgrowth. Collectively, these findings suggest that regulation of neurotrophin receptors during cerebellar development is important for the timing and morphology of axonal growth.