External Granule Layer Research Articles

Stem/progenitor cells in the developing human cerebellum: an immunohistochemical study.

The aim of this study was to analyze, by immunohistochemistry, the occurrence of stem/progenitor cells localized in the different niches of the developing human cerebellum. To this end, cerebellar samples were obtained from 3 fetuses and 3 newborns ranging, respectively, from 11 to 24 and from 30 to 38 weeks of gestation. Specimens were 10% formalin-fixed, routinely processed and paraffin-embedded; 3 μm-tick sections were immunostained with anti-SOX2 and PAX6 antibodies. Our study evidenced SOX2 and PAX6 immunoreactivity in precursors cells in all six developing human cerebella. SOX2 was expressed in precursors of different neural cell types, including Purkinje neurons, stellate cells, basket cells and Golgi cells. In the cerebellar cortex, SOX2 expression changed during gestation, being highly expressed from the 20th up to the 24th week, whereas at the 30th and at the 34th week SOX2 immunoreactivity was restricted to the Purkinje cell layer and the inner zone. Cerebellar human cortex was negative at the 38th week of gestation. PAX6 immunoreactivity was restricted to granule cell precursors in the external granule layer (EGL), being detected at all gestational ages. Our study indicates SOX2 and PAX6 as two useful markers of stem/progenitor cells that highlight the different germinative zones in the developing human cerebellum.

Adsorption of chlorophenoxy pesticides on activated carbon with gradually removed external particle layers

Abstract In the work the role of porous structure of carbon adsorbent is considered taking into account adsorption equilibrium and kinetics of selected chlorophenoxy herbicides. In the study three fractions of activated carbon obtained in the process of gradual removing the external granule layers by abrasion (33 and 66%wt) are used. Porosity of the obtained samples was characterized by low-temperature nitrogen adsorption. The adsorption isotherms of 4-chlorophenoxyacetic acid and 2-(4-chlorophenoxy)-2-methylpropionic acid from aqueous solutions were measured by using a static method. The kinetics of adsorption process was studied by applying UV–vis spectrophotometer with a flow cell. The concentration vs. time and the adsorption vs. time profiles were calculated from the obtained spectra. The adsorption data were analyzed to discuss the influence of carbon properties and adsorbate properties on adsorption effectiveness and rate. Effect of carbon pore structure on adsorption rate was observed. The usefulness of various kinetic models and equations for analysis of the experimental data was studied. In terms of fitting quality and lack of systematic deviations the semi-empirical multi-exponential (m-exp) equation was found to be the best with the fractal-like mixed order equation (f-MOE) and McKay’s pore diffusion models (PDMs) as second-best alternatives with approximately doubled deviations and some systematic deviations. All other models and equations investigated including second order kinetics (PSOE) and intraparticle diffusion model (IDM) showed much higher deviations of systematic character.

Proneurotrophin-3 promotes cell cycle withdrawal of developing cerebellar granule cell progenitors via the p75 neurotrophin receptor.

Cerebellar granule cell progenitors (GCP) proliferate extensively in the external granule layer (EGL) of the developing cerebellum prior to differentiating and migrating. Mechanisms that regulate the appropriate timing of cell cycle withdrawal of these neuronal progenitors during brain development are not well defined. The p75 neurotrophin receptor (p75(NTR)) is highly expressed in the proliferating GCPs, but is downregulated once the cells leave the cell cycle. This receptor has primarily been characterized as a death receptor for its ability to induce neuronal apoptosis following injury. Here we demonstrate a novel function for p75(NTR) in regulating proper cell cycle exit of neuronal progenitors in the developing rat and mouse EGL, which is stimulated by proNT3. In the absence of p75(NTR), GCPs continue to proliferate beyond their normal period, resulting in a larger cerebellum that persists into adulthood, with consequent motor deficits.

Regulation of Tlx3 by Pax6 is required for the restricted expression of Chrnα3 in Cerebellar Granule Neuron progenitors during development.

Homeobox gene Tlx3 is known to promote glutamatergic differentiation and is expressed in post-mitotic neurons of CNS. Contrary to this here, we discovered that Tlx3 is expressed in the proliferating progenitors of the external granule layer in the cerebellum, and examined factors that regulate this expression. Using Pax6−/−Sey mouse model and molecular interaction studies we demonstrate Pax6 is a key activator of Tlx3 specifically in cerebellum, and induces its expression starting at embryonic day (E)15. By Postnatal day (PN)7, Tlx3 is expressed in a highly restricted manner in the cerebellar granule neurons of the posterior cerebellar lobes, where it is required for the restricted expression of nicotinic cholinergic receptor-α3 subunit (Chrnα3) and other genes involved in formation of synaptic connections and neuronal migration. These results demonstrate a novel role for Tlx3 and indicate that Pax6-Tlx3 expression and interaction is part of a region specific regulatory network in cerebellum and its deregulation during development could possibly lead to Autistic spectral disorders (ASD).

LXR agonist rescued the deficit in the proliferation of the cerebellar granule cells induced by dexamethasone

Dexamethasone (DEX) exposure during early postnatal life produces permanent neuromotor and intellectual deficits and stunts cerebellar growth. The liver X receptor (LXR) plays important roles in CNS development. However, the effects of LXR on the DEX-mediated impairment of cerebellar development remain undetermined. Thus, mice were pretreated with LXR agonist TO901317 (TO) and were later exposed to DEX to evaluate its protective effects on DEX-mediated deficit during cerebellar development. The results showed that an acute exposure of DEX on postnatal day 7 resulted in a significant impairment in cerebellar development and decreased the proliferation of granule neuron precursors in the external granule layer of cerebellum. This effect was attenuated by pretreatment with TO. We further found that the decrease in the proliferation caused by DEX occurred via up-regulation of glucocorticoid receptor and p27kip1, which could be partially prevented by LXR agonist pretreatment. Overall, our results suggest that LXR agonist pretreatment could protect against DEX-induced deficits in cerebellar development in postnatal mice and may thus be perspective recruited to counteract such GC side effects.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

A Mathematical Model of Granule Cell Generation During Mouse Cerebellum Development.

Determining the cellular basis of brain growth is an important problem in developmental neurobiology. In the mammalian brain, the cerebellum is particularly amenable to studies of growth because it contains only a few cell types, including the granule cells, which are the most numerous neuronal subtype. Furthermore, in the mouse cerebellum granule cells are generated from granule cell precursors (gcps) in the external granule layer (EGL), from 1day before birth until about 2weeks of age. The complexity of the underlying cellular processes (multiple cell behaviors, three spatial dimensions, time-dependent changes) requires a quantitative framework to be fully understood. In this paper, a differential equation-based model is presented, which can be used to estimate temporal changes in granule cell numbers in the EGL. The model includes the proliferation of gcps and their differentiation into granule cells, as well as the process by which granule cells leave the EGL. Parameters describing these biological processes were derived from fitting the model to histological data. This mathematical model should be useful for understanding altered gcp and granule cell behaviors in mouse mutants with abnormal cerebellar development and cerebellar cancers.

WIP1 modulates responsiveness to Sonic Hedgehog signaling in neuronal precursor cells and medulloblastoma.

High-level amplification of the protein phosphatase PPM1D (WIP1) is present in a subset of medulloblastomas (MBs) that have an expression profile consistent with active Sonic Hedgehog (SHH) signaling. We found that WIP1 overexpression increased expression of Shh target genes and cell proliferation in response to Shh stimulation in NIH3T3 and cerebellar granule neuron precursor (cGNP) cells in a p53-independent manner. Thus, we developed a mouse in which WIP1 is expressed in the developing brain under control of the Neurod2 promoter (ND2:WIP1). The external granule layer in early post-natal ND2:WIP1 mice exhibited increased proliferation and expression of Shh downstream targets. MB incidence increased and survival decreased when ND2:WIP1 mice were crossed with a Shh-activated MB mouse model. Conversely, Wip1 knock out significantly suppressed MB formation in two independent mouse models of Shh-activated MB. Furthermore, Wip1 knock-down or treatment with a WIP1 inhibitor suppressed the effects of Shh stimulation and potentiated the growth inhibitory effects of SHH pathway-inhibiting drugs in Shh-activated MB cells in vitro. This suggests an important cross-talk between SHH and WIP1 pathways that accelerates tumorigenesis and supports WIP1 inhibition as a potential treatment strategy for MB.

Stereological study of the effects of maternal diabetes on cerebellar cortex development in rat

Diabetes during pregnancy is associated with the deficits in balance and motor coordination and altered social behaviors in offspring. In the present study, we have investigated the effect of maternal diabetes and insulin treatment on the cerebellar volume and morphogenesis of the cerebellar cortex of rat neonates during the first two postnatal weeks. Sprague Dawley female rats were maintained diabetic from a week before pregnancy through parturition. At the end of pregnancy, the male offspring euthanized on postnatal days (P) 0, 7, and 14. Cavalieri's principle and fractionator methods were used to estimate the cerebellar volume, the thickness and the number of cells in the different layers of the cerebellar cortex. In spite of P0, there was a significant reduction in the cerebellar volume and the thickness of the external granule, molecular, and internal granule layers between the diabetic and the control animals. In diabetic group, the granular and purkinje cell densities were increased at P0. Moreover, the number of granular and purkinje cells in the cerebellum of diabetic neonates was reduced in comparison with the control group at P7 and P14. There were no significant differences in either the volume and thickness or the number of cells in the different layers of the cerebellar cortex between the insulin-treated diabetic group and controls. Our data indicate that diabetes in pregnancy disrupts the morphogenesis of cerebellar cortex. This dysmorphogenesis may be part of the cascade of events through which diabetes during pregnancy affects motor coordination and social behaviors in offspring.

<em>Ex Vivo</em> Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors.

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Abstract A46: Transcriptome analysis on tumor and its normal cerebellar counterpart from mouse models reveals molecular mechanisms underlying evolution of SHH and Group3 medulloblastoma

Abstract Human medulloblastoma (MB) consists of four molecular subgroups. In spite of enormous effort majority of current MB mouse models are SHH subgroup, represented by Patched heterozygous mice (designated as SHH-A). GTML MB is the only published spontaneous Group3 MB mouse model (designated as G3), which was induced by expressing MycN under the regulation of GLT1 promoter. By using Sleeping Beauty transposon system we have created and published two SHH mouse models: Math1SB11/T2Onc/Ptch+/- (designated as SHH-B) and Math1SB11/T2Onc/P53mut (designated as SHH-C). To dissect the molecular mechanism of SHH and Group3 MB during tumor evolution (initiation, maintenance and maturation), we profiled transcriptomes on SHH-A, SHH-B, SHH-C and G3 MBs along with normal mouse cerebellum (CB) of different developmental stage (E15, E18, P0, P60, P95 and P122). Fetal and/or adult human CBs are routinely used as normal controls for human pediatric MBs. One major advantage of profiling mouse tumor samples over human tumor samples is the availability of age-matched normal mouse CB counterparts. This is particularly true in case of developmental diseases such as pediatric MBs. Indeed, our study showed that transcriptomes of normal mouse CB from E15, E18 and P0 (designated as EdCB, representing early developmental CB), are similar to each other but distinct from mouse CB at P60, P95 and P122 (designated as LdCB, representing late developmental CB). Importantly P60 to P120 are the ages at which most of spontaneous MBs are harvested from a tumor-bearing mouse. We therefore analyzed global gene expression level of MBs and EdCB in comparison with LdCB, the mouse MB's normal counterparts. As a developmental disease, MB is presumably initiated from a single cell or a single cell clone that is transformed at an early developmental stage of a normal CB. In fact, it is well known that a SHH MB is initiated from transformation of granule cell precursors at EGL, the external granule layer. However, it is unclear about the initiation of Group3 MB. In this study we showed that compared to a normal LdCB, a particular MB transriptome harbors four major types of abnormally expressed genes: 1. Genes not only specific to one subgroup but also shared with EdCB; representing tumor initiation events. 2. Genes specific to a subgroup only; representing subgroup-specific tumor promotion events. 3. Genes shared not only by both subgroups but also by EdCB, representing non-subgroup specific tumor maintenance events; 4. Genes shared by both subgroups only; representing non-subgroup specific late tumor maturation events. We confirmed that SHH MBs from SHH-A, B, and C are all initiated at Math1/Atoh1 expressing cells, which are further transformed by up-regulation of Olig3 and MycN among other genes. Rxrg-expressing cells are identified as Group3 MB initiating cells, which are further transformed by over-expression of Crx (Otx3) along with other genes such as Otx2, and down-regulation of cerebellum-specific Zic1 and others such as Lgi1. Both subgroups of MBs may be maintained and mature as genes associated with cell cycle, mitosis and chromosome integrity, such as KIFs, CDK1, PLK1/4, AURKA/B, CENP family and MCMs are abnormally overexpressed. Our study reveals distinct molecular mechanisms underlying tumor evolution of SHH and Group3 MBs. We propose a MB evolution model, illustrating how a normal cell at specific cell of origin can be transformed to a fully developed subgroup specific MB. Furthermore, our results demonstrate that despite the fact that they are initiated from distinct cell of origin, different subgroup of MBs do share common abnormal molecular events for tumor maintenance and maturation, suggesting that subgroup specific target therapies alone may be not as effective as their combination with therapies targeting abnormalities common to different subgroup of MBs. Citation Format: Xiaochong Wu, Adrian Dubuc, William A. Weiss, Robert J. Wechsler-Reya, Michael D. Taylor. Transcriptome analysis on tumor and its normal cerebellar counterpart from mouse models reveals molecular mechanisms underlying evolution of SHH and Group3 medulloblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A46.

Vitronectin promotes the progress of the initial differentiation stage in cerebellar granule cells.

Vitronectin (VN), which is an extracellular matrix protein, is known to be involved in the proliferation and differentiation of primary cultured cerebellar granule cell precursors (CGCPs); however, the effect of VN is not fully understood. In this study, we analyzed the effects of VN loss on the proliferation and differentiation of CGCPs in VN knockout (VNKO) mice in vivo. First, immunohistochemistry showed that VN was distributed in the region from the inner external granule layer (iEGL) through the internal granule layer (IGL) in wild-type (WT) mice. Next, we observed the formation of the cerebellar cortex using sagittal sections of VNKO mice at postnatal days (P) 5, 8 and 11. Loss of VN suppressed the ratio of NeuN, a neuronal differentiation marker, to positive cerebellar granule cells (CGCs) in the external granule layer (EGL) and the ratio of CGCs in the IGL at P8, indicating that the loss of VN suppresses the differentiation into CGCs. However, the loss of VN did not significantly affect the proliferation of CGCPs. Next, the effect of VN loss on the initial differentiation stage of CGCPs was examined. The loss of VN increased the expression levels of Transient axonal glycoprotein 1 (TAG1), a marker of neurons in the initial differentiation stage, in the cerebella of VNKO mice at P5 and 8 and increased the ratio of TAG1-positive cells in the primary culture of VNKO-derived CGCPs, indicating that the loss of VN accumulates the CGCPs in the initial differentiation stage. Taken together, these results demonstrate that VN promotes the progress of the initial differentiation stage of CGCPs.

Altered cerebellum development and impaired motor coordination in mice lacking the Btg1 gene: Involvement of cyclin D1

Cerebellar granule neurons develop postnatally from cerebellar granule precursors (GCPs), which are located in the external granule layer (EGL) where they massively proliferate. Thereafter, GCPs become postmitotic, migrate inward to form the internal granule layer (IGL), further differentiate and form synapses with Purkinje cell dendrites.We previously showed that the Btg family gene, Tis21/Btg2, is required for normal GCP migration. Here we investigated the role in cerebellar development of the related gene, Btg1, which regulates stem cell quiescence in adult neurogenic niches, and is expressed in the cerebellum.Knockout of Btg1 in mice caused a major increase of the proliferation of the GCPs in the EGL, whose thickness increased, remaining hyperplastic even after postnatal day 14, when the EGL is normally reduced to a few GCP layers. This was accompanied by a slight decrease of differentiation and migration of the GCPs and increase of apoptosis. The GCPs of double Btg1/Tis21-null mice presented combined major defects of proliferation and migration outside the EGL, indicating that each gene plays unique and crucial roles in cerebellar development. Remarkably, these developmental defects lead to a permanent increase of the adult cerebellar volume in Btg1-null and double mutant mice, and to impairment in all mutants, including Tis21-null, of the cerebellum-dependent motor coordination. Gain- and loss-of-function strategies in a GCP cell line revealed that Btg1 regulates the proliferation of GCPs selectively through cyclin D1. Thus, Btg1 plays a critical role for cerebellar maturation and function.

Hedgehog regulates cerebellar progenitor cell and medulloblastoma apoptosis

The external granule layer (EGL) is a proliferative region that produces over 90% of the neurons in the cerebellum but can also malignantly transform into a cerebellar tumor called the medulloblastoma (the most common malignant brain tumor in children). Current dogma considers Hedgehog stimulation a potent proliferative signal for EGL neural progenitor cells (NPCs) and medulloblastomas. However, the Hedgehog pathway also acts as a survival signal in the neural tube where it regulates dorsoventral patterning by controlling NPC apoptosis. Here we show that Hedgehog stimulation is also a potent survival signal in the EGL and medulloblastomas that produces a massive apoptotic response within hours of signal loss in mice. This toxicity can be produced by numerous Hedgehog antagonists (vismodegib, cyclopamine, and jervine) and is Bax/Bak dependent but p53 independent. Finally, since glucocorticoids can also induce EGL and medulloblastoma apoptosis, we show that Hedgehog's effects on apoptosis can occur independent of glucocorticoid stimulation. This effect may play a major role in cerebellar development by directing where EGL proliferation occurs thereby morphologically sculpting growth. It may also be a previously unknown major therapeutic effect of Hedgehog antagonists during medulloblastoma therapy. Results are discussed in terms of their implications for both cerebellar development and medulloblastoma treatment.

NrCAM modulates sonic hedgehog signalling by controlling smoothened translocation in the cilium

Objective: Cerebellar development involves a spurt of proliferation in external granule layer (EGL) in response to shh, causing granule neuron precursors (GNPs) to proliferate. These cells subsequently differentiate into granule neurons in the inner granule layer (IGL). F3, a CNTN family molecule, can interact with NrCAM to switch GNPs from proliferation to differentiation. We aim to identify the role of NrCAM in the sonic hedgehog response in GNPs. Methods: GNPs were extracted from wildtype and NrCAM mutant P5 cerebella using Percoll gradient centrifugation. Proliferation response to shh was measured using EdU in presence/absence of F3-Fc. GNPs treated with shh/SAG were stained with antibodies against Arl13b and smo to look for differences in cilia size and smo occupancy after different treatment times. Results: NrCAM-/- and wildtype GNPs both proliferated equally in response to shh. F3 was found to block the proliferation response in wildtype but not in NrCAM-/- GNPs. F3 also failed to affect proliferation in SmoA1 GNPs with a constitutively active smo suggesting that the F3-NrCAM mediated block lay upstream of Smo. NrCAM was detected in wildtype cilia and Smo localization was affected in NrCAM-/- GNPs. No differences in cilia length were observed. Conclusion: Our results suggest that NrCAM affects shh-mediated proliferation by controlling smo movement into the cilium.

Asymmetric cell division of granule neuron progenitors in the external granule layer of the mouse cerebellum.

ABSTRACTThe plane of division of granule neuron progenitors (GNPs) was analysed with respect to the pial surface in P0 to P14 cerebellum and the results showed that there was a significant bias towards the plane of cell division being parallel to pial surface across this developmental window. In addition, the distribution of β-Catenin in anaphase cells was analysed, which showed that there was a significant asymmetry in the distribution of β-Catenin in dividing GNPs. Further, inhibition of Sonic Hedgehog (Shh) signalling had an effect on plane of cell division. Asymmetric distribution of β-Catenin was shown to occur towards the source of a localized extracellular cue.

MB-36 * WIP1 AUGMENTS SONIC HEDGEHOG (SHH) SIGNALING AND IS A TARGET IN SHH MEDULLOBLASTOMAS

Sonic Hedgehog (SHH) signaling is active in 30% of childhood medulloblastomas. Dysregulation of p53 signaling is especially prognostic for poor survival in SHH medulloblastoma. But, p53 is mutated in <10% of medulloblastomas. Genomic analyses have demonstrated high-level amplification of PPM1D (WIP1) in p53 wild-type SHH medulloblastomas. WIP1 functions as an oncogene, in part by inactivating p53. We hypothesized that, in the absence of p53 mutation, WIP1 overexpression augments SHH-driven medulloblastoma tumorigenesis and is an important target in the treatment of SHH-active medulloblastomas. WIP1 overexpression in murine granule neuron precursor (GNP) cells enhanced expression of Shh target genes, increased the percentage of cells in S phase, and increased cell viability in response to Shh stimulation. Although WIP1 is known to primarily act through p53 pathways, we found that the interaction between WIP1 and Shh was partially p53-independent. To further understand WIP1 mechanisms in vivo, we developed a mouse in which WIP1 is expressed in the developing brain under control of the Neurod2 promoter (ND2:WIP1). The external granule layer in early post-natal ND2:WIP1 mice exhibited increased proliferation and expression of Shh downstream targets. When ND2:WIP1 transgenic mice were crossed with SmoA1/SmoA1 medulloblastoma-prone mice, medulloblastoma incidence increased and double transgenic SmoA1/SmoA1; ND2:WIP1+ mice demonstrated reduced survival. Conversely, Wip1 knock out significantly suppressed medulloblastoma formation in SmoA1/SmoA1 as well as Tamoxifen-treated Math1-creER; Ptc1 fl/fl mice. Treatment with a lentivirus that knocks down Wip1 or with the WIP1 inhibitor CCT007093 inhibits the effects of Shh stimulation and potentiates the effects of the SHH pathway-inhibiting drugs SANT-1, cyclopamine, or the clinically-relevant LDE225 on the growth of either SmoA1/SmoA1 or Math1-creER + ; Ptc1 fl/fl medulloblastoma cells in vitro. This suggests an important cross-talk between SHH and WIP1 that accelerates tumorigenesis. Our findings also suggest an important role for WIP1 inhibition in the treatment of SHH-active medulloblastomas.

PACT/RAX regulates the migration of cerebellar granule neurons in the developing cerebellum.

PACT and its murine ortholog RAX were originally identified as a protein activator for the dsRNA-dependent, interferon-inducible protein kinase PKR. Recent studies indicated that RAX played a role in embryogenesis and neuronal development. In this study, we investigated the expression of RAX during the postnatal development of the mouse cerebellum and its role in the migration of cerebellar granule neurons (CGNs). High expression of RAX was observed in the cerebellum from postnatal day (PD) 4 to PD9, a period when the CGNs migrate from the external granule layer (EGL) to the internal granule layer (IGL). The migration of the EGL progenitor cells in vivo was inhibited by RAX knockdown on PD4. This finding was confirmed by in vitro studies showing that RAX knockdown impaired the migration of CGNs in cerebellar microexplants. PACT/RAX-regulated migration required its third motif and was independent of PKR. PACT/RAX interacted with focal adhesion kinase (FAK) and PACT/RAX knockdown disturbed the FAK phosphorylation in CGNs. These findings demonstrated a novel function of PACT/RAX in the regulation of neuronal migration.

Delineating the factors and cellular mechanisms involved in the survival of cerebellar granule neurons.

Cerebellar granule neurons (CGNs) constitute the most abundant neuronal population in the mammalian brain. Their postnatal generation and the feasibility to induce their apoptotic death in vitro make them an excellent model to study the effect of several neurotransmitters and neurotrophins. Here, we first review which factors are involved in the generation and proliferation of CGNs in the external granule layer (EGL) and in the regulation of their differentiation and migration to internal granule layer (IGL). Special attention was given to the role of several neurotrophins and the NMDA subtype of glutamate receptor. Then, using the paradigm of potassium deprivation in cultured CGNs, we address several extracellular factors that promote the survival of CGNs, with particular emphasis on the cellular mechanisms. The role of specific protein kinases leading to the regulation of transcription factors and recent data involving the small G protein family is also discussed. Finally, the participation of some members of Bcl-2 family and the inhibition of mitochondria-related apoptotic pathway is also considered. Altogether, these studies evidence that CGNs are a key model to understand the development and the survival of neuronal populations.