External Granule Layer Research Articles

Essential roles of leucine-rich glioma inactivated 1 in the development of embryonic and postnatal cerebellum.

Leucine-rich glioma inactivated 1 (LGI1) is a secreted protein that interacts with ADAM transmembrane proteins, and its mutations are linked to human epilepsy. The function of LGI1 in CNS development remains undefined. Here, we report novel functions of LGI1 in the generation of cerebellar granule precursors (CGPs) and differentiation of radial glial cells (RGCs) in the cerebellum. A reduction in external granule layer thickness and defects in foliation were seen in embryonic and new-born LGI1 knockout (KO) mice. BrdU staining showed an inhibited proliferation of CGPs in KO embryos, which might be explained by the reduced Sonic hedgehog in embryos. In addition, the differentiation of RGCs into Bergmann glias was suppressed in KO mice. Enhanced Jagged1-Notch1 signaling in KO mice via reduced β-secretase proteolysis suggests that altered phenotype of RGCs is due to abnormal Notch1 signaling. Together, our results demonstrate that LGI1 is an essential player in the cerebellar development.

The apical complex protein Pals1 is required to maintain cerebellar progenitor cells in a proliferative state.

Through their biased localization and function within the cell, polarity complex proteins are necessary to establish the cellular asymmetry required for tissue organization. Well-characterized germinal zones, mitogenic signals and cell types make the cerebellum an excellent model for addressing the crucial function of polarity complex proteins in the generation and organization of neural tissues. Deletion of the apical polarity complex protein Pals1 in the developing cerebellum results in a remarkably undersized cerebellum with disrupted layers in poorly formed folia and strikingly reduced granule cell production. We demonstrate that Pals1 is not only essential for cerebellum organogenesis, but also for preventing premature differentiation and thus maintaining progenitor pools in cerebellar germinal zones, including cerebellar granule neuron precursors in the external granule layer. In the Pals1 mouse mutants, the expression of genes that regulate the cell cycle was diminished, correlating with the loss of the proliferating cell population of germinal zones. Furthermore, enhanced Shh signaling through activated Smo cannot overcome impaired cerebellar cell generation, arguing for an epistatic role of Pals1 in proliferation capacity. Our study identifies Pals1 as a novel intrinsic factor that regulates the generation of cerebellar cells and Pals1 deficiency as a potential inhibitor of overactive mitogenic signaling.

ERBB3-mediated regulation of Bergmann glia proliferation in cerebellar lamination.

Cortical lamination is crucial for the assembly of cerebellar circuitry. In this process, granule neurons (GNs) migrate along Bergmann glia (BG), which are specialized astroglial cells, from the external granule layer to the internal granule layer. However, the molecular mechanisms underlying BG development are not well understood. Here, we show that GFAP::Cre;Erbb3(F/F) mice, which lack Erbb3 in both radial glia and neurons, exhibit impairments in balance and motor coordination. Cerebellar lamination is aberrant, with misplaced Purkinje neurons and GN clusters. These phenotypes were not observed in Math1::CreER(T2);Erbb3(F/F) mice, where the Erbb3 gene was deleted in GNs, suggesting involvement of non-neuronal Erbb3 in cerebellar lamination. Mechanistic studies indicate that ERBB3 is crucial for the proliferation of BG, which are required for GN migration. These observations identify a crucial role for ERBB3 in cerebellar lamination and reveal a novel mechanism that regulates BG development.

Generation and characterization of mice harboring a conditional CXCL12 allele.

The chemokine CXCL12 has important functions in immune and central nervous systems. Moreover, a global disruption of CXCL12 in mice results in perinatal lethality. To circumvent this impediment and provide a tool for analyzing CXCL12 functions in specific organ systems, we have generated a mouse line harboring a loxP-site flanked exon 2 of CXCL12. A germ line deleter, β-actin::cre was used to remove a CXCL12 exon 2 and subsequently systemic CXCL12 exon 2 deficient embryos were generated. These mutant embryos showed a marked depletion of CXCL12 transcript. As expected from the global mutant phenotype, our mutants were also characterized by highly irregular cerebellar cytoarchitecture of the external granule layer as well as altered radial migration of midbrain dopaminergic neurons. Importantly, migration of the pontine grey nucleus (PGN) was derailed and remarkably resembled the global mutant phenotype of the CXCL12 receptor - CXCR4 in this system. Despite the fact that CXCL12 signaling can be mediated through receptors other than CXCR4, our results indicate a monogamous relationship between the CXCL12 ligand and CXCR4 receptor in controlling PGN migration. Our findings further expand on the understanding of CXCL12 function in PGN development. Moreover, phenotypic similarities between our mutants and mice harboring a global CXCL12 disruption support the validity of our line. Importantly, these results strongly suggest that our conditional CXCL12 line can be used as a powerful tool to manipulate CXCL12 signaling and function in vivo.

Mice lacking the transcription factor SHOX2 display impaired cerebellar development and deficits in motor coordination

Purkinje cells of the developing cerebellum secrete the morphogen sonic hedgehog (SHH), which is required to maintain the proliferative state of granule cell precursors (GCPs) prior to their differentiation and migration to form the internal granule layer (IGL). Despite a wealth of knowledge regarding the function of SHH during cerebellar development, the upstream regulators of Shh expression during this process remain largely unknown. Here we report that the murine short stature homeobox 2 (Shox2) gene is required for normal Shh expression in dorsal-residing Purkinje cells. Using two different Cre drivers, we show that elimination of Shox2 in the brain results in developmental defects in the inferior colliculus and cerebellum. Specifically, loss of Shox2 in the cerebellum results in precocious differentiation and migration of GCPs from the external granule layer (EGL) to the IGL. This correlates with premature bone morphogenetic protein 4 (Bmp4) expression in granule cells of the dorsal cerebellum. The size of the neonatal cerebellum is reduced in Shox2-mutant animals, which is consistent with a reduction in the number of GCPs present in the EGL, and could account for the smaller vermis and thinner IGL present in adult Shox2mutants. Shox2-mutant mice also display reduced exploratory activity, altered gait and impaired motor coordination. Our findings are the first to show a role for Shox2 in brain development. We provide evidence that Shox2 plays an important role during cerebellar development, perhaps to maintain the proper balance of Shh and Bmp expression levels in the dorsal vermis, and demonstrate that in the absence of Shox2, mice display both cerebellar impairments and deficits in motor coordination, ultimately highlighting the importance of Shox2 in the cerebellum.

LGI1 is involved in the development of mouse brain.

Mutations in leucine-rich glioma inactivated 1 (LGI1) are linked to human autosomal dominant lateral temporal lobe epilepsy. It has been shown that LGI1 prevents the inactivation of voltage-gated potassium channels, mediates postnatal maturation of glutamatergic synapses, and regulates excitatory neurotransmission. However, other functions of LGI1 in the central nervous system have not been elucidated. We found that LGI1 is involved in the development of the cerebellum and cortex. The thickness of external granule layer was reduced, and foliation was affected in the cerebellum of LGI1 knockout mice. Double staining with Pax6 and BrdU showed a significant inhibition of proliferation of granule cell precursors of knockout embryos. The differentiation of radial glia cells was also suppressed in knockout mice, as shown by increased radial glial cells and decreased Bergmann glias in the areas of the cerebellum and cortex. Thus, our data demonstrate that LGI1 may be an essential player in the development of the brain.

DS-01 * GLUCOCORTICOIDS POTENTLY INDUCE APOPTOSIS IN THE CEREBELLAR EXTERNAL GRANULE LAYER: IMPLICATIONS FOR MEDULLOBLASTOMA TREATMENT

Premature infants suffering from respiratory dysfunction are often administered glucocorticoids (GCs) to mature the lungs. Unfortunately, neonates exposed to GCs can exhibit neuromotor deficits and selective cerebellar stunting. In order to test the safety of GCs, we administered the synthetic GC dexamethasone to neonatal mouse pups and found it potently increased apoptosis in the external granule layer (EGL) of the cerebellum. The EGL is responsible for producing over 90% of the neurons in the cerebellum, which represents over half the neurons in the entire brain. We subsequently discovered that endogenous GC release, receptors, and enzymatic metabolism is carefully orchestrated to selectively increase stimulation in the EGL as it is naturally eliminated. This suggests the GC system may signal the permanent disappearance of the EGL during development. Unfortunately, EGL neural progenitor cells (NPCs) can transform into medulloblastomas (MBs), the most common malignant brain tumor in children. As a result, the GC system may play a critical role in MB development. Up to 30% of human medulloblastomas are thought to be driven by excessive Hedgehog signaling (a potent EGL mitogen). Consistent with this idea, mice with mutations causing constitutive Hedgehog signaling are also vulnerable to MB formation. Interestingly, excessive Hedgehog signaling is also thought to decrease GC stimulation in the EGL due to selective GC metabolism. Therefore, we used MB prone (Patched) mice to examine if this vulnerability still exists during preneoplastic tumor transformation. We found GC stimulation potently increased apoptotic cell death in preneoplasms. This suggests GCs may both play a role in MB development and have potential as a therapeutic treatment.

DS-03 * SONIC HEDGEHOG ANTAGONISTS POTENTLY INDUCE APOPTOSIS IN THE CEREBELLAR EXTERNAL GRANULE LAYER: IMPLICATIONS FOR MEDULLOBLASTOMA TREATMENT

There is a great interest in Hedgehog signaling both for its role in cerebellar development and medulloblastoma (MB) treatment. The cerebellum maintains its own proliferative layer called the external granule layer (EGL) that produces over 90% of its neurons. During development, the established dogma views Hedgehog signaling as a robust mitogenic stimulator of EGL proliferation. However, in other regions of the body, Hedgehog stimulation acts as a survival signal by potently inducing NPC apoptosis when signaling is lost. In this manner, the sonic hedgehog ligand's concentration gradient determines NPC survival or death thereby morphologically sculpting the developing nervous system. Therefore, we tested whether Hedgehog signaling also acts as a survival signal in the EGL by administering several Hedgehog antagonists (vismodegib, cyclopamine, and jervine). Remarkably, we found all Hedgehog antagonists (HAs) potently induced EGL apoptosis within a few hours of administration. This suggests a large portion of the HAs' anti-proliferative effects are due to the apoptotic loss of a large number of EGL NPCs. This research may also have important implications for MB formation and treatment. There is convincing evidence that EGL neural progenitor cells (NPCs) can be the tumor initiating cells for MBs (the most common malignant brain tumor in children). Therefore, we examined if HAs can also produce apoptosis in Patched mice which exhibit constitutive Hedgehog stimulation and are prone to MB formation. We found HA administration also potently increased apoptosis in both EGL NPCs and preneoplasms. This may have important implications for the treatment of MBs with HAs. For example, apoptosis involves signaling mechanisms distinct from proliferation that may need to be disabled for malignant transformation. In addition, the requirement for Hedgehog signaling may prevent metastasis by killing tumor cells as they spread to regions where such signaling is absent.

Abstract B16: Dissecting MDM2 function in medulloblastoma pathogenesis

Abstract Treatment for medulloblastoma (MB), a tumor of the cerebellum, involves an aggressive regimen of surgical resection, radiation and adjuvant chemotherapy. Although the 5-year cure rate is over 50%, most children retain life-long side effects associated with treatment, including reduced intellect. There is an urgent need to develop multi-targeted therapeutic strategies that enhance the killing of MB tumor cells while sparing normal cells. As a critical component of the cellular response to stress, p53 function is required for the cytotoxic effects of many chemotherapeutics. As p53 mutations are relatively rare in MB, adjuvant chemotherapeutics that enhance p53 function may have a profound effect in MB. Indeed, we previously reported that pharmacological disruption of the interaction of p53 with its negative regulator, MDM2, potentiates cell killing in human medulloblastoma cells. In order to gain further insight into the role of MDM2 in MB pathogenesis, here we have used a combination of wild-type, null, and hypomorphic alleles of MDM2 to perturb the MDM2-p53 pathway in Ptch1+/- mice, a model of human Sonic hedgehog (Shh)-induced MB. Our studies demonstrate that a 70% reduction in the level of MDM2 abrogates the formation of preneoplastic lesions (PNLs) in the cerebellum of Ptch1+/- mice, thereby supporting a requirement for MDM2 in MB initiation. Significantly, even a modest ~50% reduction in the level of MDM2 was sufficient to decrease PNL formation in the Ptch1+/- cerebellum in two-fold. Although reduced in number, detailed histological analyses of PNLs at P21 revealed no difference in the size, proliferation or apoptosis of PNLs between Ptch1+/- mice expressing a wild-type or 50% reduced level of MDM2. Next, we examined granule neuron precursors (GNPs) in the external granule layer (EGL) at P7, the pool of cells from which PNLs are presumed to originate. Consistent with the partial deregulation of Shh signaling in Ptch1+/- mice, the Ptch1+/- EGL was more highly proliferative than that of wild-type mice. Importantly, we found that the enhanced proliferation of EGL cells afforded by loss of one Ptch1 allele was restored back to wild-type levels when the level of MDM2 is reduced. Additionally, a 50% in the level of MDM2 potentiates apoptosis and enhances the steady state level of p53SER15 in the EGL in Ptch1+/- mice as compared to control mice. Dysregulation of the MDM2-p53 and Sonic hedgehog (Shh)-Gli pathways are both implicated in the pathogenesis of human MB; however, no approach to date has yielded a detailed understanding of how crosstalk between these two important pathways contributes to the proliferation, survival, and differentiation of GNPs, the presumed cell of origin for some MB subtypes. Our prior studies placed MDM2 at a critical node between the growth inhibiting the p53 pathway and the growth-promoting Shh-Gli pathway in normal cerebellar development. New work presented here further suggests that MDM2 is required for MB tumorigenesis to prevent p53 activation, which may in turn function to counteract deregulated Shh-mediated proliferation in the tumor initiating cell population. The coordinate regulation of these two important pathways may provide a mechanism to sustain tumor-promoting levels of Shh-Gli signaling without activating the tumor-suppressing functions of p53. By nature of its requirement in MB tumorigenesis, MDM2 may provide an important therapeutic target for boosting the efficacy of current MB treatments. Citation Format: Susan M. Mendrysa, Reem Malek. Dissecting MDM2 function in medulloblastoma pathogenesis. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B16.

Deficits in cerebellar granule cell development and social interactions in CD47 knockout mice.

CD47 is involved in neurite differentiation in cultured neurons, but the function of CD47 in brain development is largely unknown. We determined that CD47 mRNA was robustly expressed in the developing cerebellum, especially in granule cells. CD47 protein was mainly expressed in the inner layer of the external granule layer (EGL), molecular layer, and internal granule layer (IGL), where granule cells individually become postmitotic and migrate, leading to neurite fasciculation. At postnatal day 8 (P8), CD47 knockout mice exhibited an increased number of proliferating granule cells in the EGL, whereas the CD47 agonist peptide 4N1K increased the number of postmitotic cells in primary granule cells. Knocking out the CD47 gene and anti-CD47 antibody impaired the radial migration of granule cells from the EGL to the IGL individually in mice and slice cultures. In primary granule cells, knocking out CD47 reduced the number of axonal collaterals and dendritic branches; by contrast, overexpressing CD47 or 4N1K treatment increased the axonal length and numbers of axonal collaterals and dendritic branches. Furthermore, the length of the fissure between Lobules VI and VII was decreased in CD47 knockout mice at P21 and at 14 wk after birth. Lastly, CD47 knockout mice exhibited increased social interaction at P21 and depressive-like behaviors at 10 wk after birth. Our study revealed that the cell adhesion molecule CD47 participates in multiple phases of granule cell development, including proliferation, migration, and neurite differentiation implying that aberrations of CD47 are risk factors that cause abnormalities in cerebellar development and atypical behaviors.

A marked paucity of granule cells in the developing cerebellum of the Npc1−/− mouse is corrected by a single injection of hydroxypropyl-β-cyclodextrin

In this study we show that postnatal development of cerebellar granule neurons (GNs) is defective in Npc1−/− mice. Compared to age-matched wild-type littermates, there is an accelerated disappearance of the external granule layer (EGL) in these mice. This is due to a premature exit from the cell cycle of GN precursors residing at the level of the EGL. As a consequence, the size of cerebellar lobules of these mice displays a 20%–25% reduction compared to that of age-matched wild-type mice. This size reduction is detectable at post-natal day 28 (PN28), when cerebellar GN development is completed while signs of neuronal atrophy are not yet apparent.Based on the analysis of EGL thickness and the determination of proliferating GN fractions at increasing developmental times (PN8–PN14), we trace the onset of this GN developmental defect during the second postnatal week. We also show that during this developmental time Shh transcripts undergo a significant reduction in Npc1−/− mice compared to age-matched wild-type mice. In light of the mitogenic activity of Shh on GNs, this observation further supports the presence of defective GN proliferation in Npc1−/− mice. A single injection of hydroxypropyl-β-cyclodextrin at PN7 rescues this defect, restoring the normal patterns of granule neuron proliferation and cerebellar lobule size.To our knowledge, these findings identify a novel developmental defect that was underappreciated in previous studies. This defect was probably overlooked because Npc1 loss-of-function does not affect cerebellar foliation and causes the internal granule layer and molecular layer to decrease proportionally, giving rise to a normally appearing, yet harmoniously smaller, cerebellum.

ASC deficiency suppresses proliferation and prevents medulloblastoma incidence.

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is silenced by promoter methylation in many types of tumors, yet ASC's role in most cancers remains unknown. Here, we show that ASC is highly expressed in a model of medulloblastoma, the most common malignant pediatric brain cancer; ASC is also expressed in human medulloblastomas. Importantly, while ASC deficiency did not affect normal cerebellar development, ASC knockout mice on the Smoothened (ND2:SmoA1) transgenic model of medulloblastoma exhibited a profound reduction in medulloblastoma incidence and a delayed tumor onset. A similar decrease in tumorigenesis with ASC deficiency was also seen in the hGFAP-Cre:SmoM2 mouse model of medulloblastoma. Interestingly, hyperproliferation of the external granule layer (EGL) was comparable at P20 in both wild-type and ASC-deficient SmoA1 mice. However, while the apoptosis and differentiation markers remained unchanged at this age, proliferation makers were decreased, and the EGL was reduced in thickness and area by P60. This reduction in proliferation with ASC deficiency was also seen in isolated SmoA1 cerebellar granule precursor cells in vitro, indicating that the effect of ASC deletion on proliferation was cell autonomous. Interestingly, ASC-deficient SmoA1 cerebella exhibited disrupted expression of genes in the transforming growth factor-β pathway and increased level of nuclear Smad3. Taken together, these results demonstrate an unexpected role for ASC in Sonic hedgehog-driven medulloblastoma tumorigenesis, thus identifying ASC as a promising novel target for antitumor therapy.

Glucocorticoid Induced Cerebellar Toxicity in the Developing Neonate: Implications for Glucocorticoid Therapy during Bronchopulmonary Dysplasia

Prematurely born infants commonly suffer respiratory dysfunction due to the immature state of their lungs. As a result, clinicians often administer glucocorticoid (GC) therapy to accelerate lung maturation and reduce inflammation. Unfortunately, several studies have found GC therapy can also produce neuromotor/cognitive deficits and selectively stunt the cerebellum. However, despite its continued use, relatively little is known about how exposure to this hormone might produce neurodevelopmental deficits. In this review, we use rodent and human research to provide evidence that GC therapy may disrupt cerebellar development through the rapid induction of apoptosis in the cerebellar external granule layer (EGL). The EGL is a transient proliferative region responsible for the production of over 90% of the neurons in the cerebellum. During normal development, endogenous GC stimulation is thought to selectively signal the elimination of the EGL once production of new neurons is complete. As a result, GC therapy may precociously eliminate the EGL before it can produce enough neurons for normal cerebellar function. It is hoped that this review may provide information for future clinical research in addition to translational guidance for the safer use of GC therapy.

Developmental and oncogenic radiation effects on neural stem cells and their differentiating progeny in mouse cerebellum

Neural stem cells are highly susceptible to radiogenic DNA damage, however, little is known about their mechanisms of DNA damage response (DDR) and the long-term consequences of genotoxic exposure. Patched1 heterozygous mice (Ptc1(+/-)) provide a powerful model of medulloblastoma (MB), a frequent pediatric tumor of the cerebellum. Irradiation of newborn Ptc1(+/-) mice dramatically increases the frequency and shortens the latency of MB. In this model, we investigated the mechanisms through which multipotent neural progenitors (NSCs) and fate-restricted progenitor cells (PCs) of the cerebellum respond to DNA damage induced by radiation, and the long-term developmental and oncogenic consequences. These responses were assessed in mice exposed to low (0.25 Gy) or high (3 Gy) radiation doses at embryonic day 13.5 (E13.5), when NSCs giving rise to the cerebellum are specified but the external granule layer (EGL) has not yet formed, or at E16.5, during the expansion of granule PCs to form the EGL. We found crucial differences in DDR and apoptosis between NSCs and fate-restricted PCs, including lack of p21 expression in NSCs. NSCs also appear to be resistant to oncogenesis from low-dose radiation exposure but more vulnerable at higher doses. In addition, the pathway to DNA repair and the pattern of oncogenic alterations were strongly dependent on age at exposure, highlighting a differentiation-stage specificity of DNA repair pathways in NSCs and PCs. These findings shed light on the mechanisms used by NSCs and PCs to maintain genome integrity during neurogenesis and may have important implications for radiation risk assessment and for development of targeted therapies against brain tumors.

Prenatal exposure to bisphenol A interferes with the development of cerebellar granule neurons in mice and chicken

In mice, prenatal exposure to low doses of bisphenol A has been shown to affect neurogenesis and neuronal migration in cortex, resulting in disturbance of both neuronal positioning and the network formation between thalamus and cortex in the offspring brain. In the present study we investigated whether prenatal exposure to bisphenol A disturbs the neurodevelopment of the cerebellum. Two different model systems were used; offspring from two strains of mice from mothers receiving bisphenol A in the drinking water before mating, during gestation and lactation, and chicken embryos exposed to bisphenol A (in the egg) on embryonic day 16 for 24h before preparation of cerebellar granule cell cultures. In the cerebellum, tight regulation of the level of transcription factor Pax6 is critical for correct development of granule neurons. During the development, the Pax6 level in granule neurons is high when these cells are located in the external granule layer and during their migration to the internal granule layer, and it is then reduced. We report that bisphenol A induced an increase in the thickness of the external granule layer and also an increase in the total cerebellar Pax6 level in 11 days old mice offspring. In cultured chicken cerebellar granule neurons from bisphenol A injected eggs the Pax6 level was increased day 6 in vitro. Together, these findings indicate that bisphenol A may affect the granule neurons in the developing cerebellum and thereby may disturb the correct development of the cerebellum.

Critical role of zinc finger protein 521 in the control of growth, clonogenicity and tumorigenic potential of medulloblastoma cells

The stem cell-associated transcription co-factor ZNF521 has been implicated in the control of hematopoietic, osteo-adipogenic and neural progenitor cells. ZNF521 is highly expressed in cerebellum and in particular in the neonatal external granule layer that contains candidate medulloblastoma cells-of-origin, and in the majority of human medulloblastomas. Here we have explored its involvement in the control of human and murine medulloblastoma cells. The effect of ZNF521 on growth and tumorigenic potential of human medulloblastoma cell lines as well as primary Ptc1-/+ mouse medulloblastoma cells was investigated in a variety of in vitro and in vivo assays, by modulating its expression using lentiviral vectors carrying the ZNF521 cDNA, or shRNAs that silence its expression. Enforced overexpression of ZNF521 in DAOY medulloblastoma cells significantly increased their proliferation, growth as spheroids and ability to generate clones in single-cell cultures and semisolid media, and enhanced their migratory ability in wound-healing assays. Importantly, ZNF521-expressing cells displayed a greatly enhanced tumorigenic potential in nude mice. All these activities required the ZNF521 N-terminal motif that recruits the nucleosome remodeling and histone deacetylase complex, which might therefore represent an appealing therapeutic target. Conversely, silencing of ZNF521 in human UW228 medulloblastoma cells that display high baseline expression decreased their proliferation, clonogenicity, sphere formation and wound-healing ability. Similarly, Zfp521 silencing in mouse Ptc1-/+ medulloblastoma cells drastically reduced their growth and tumorigenic potential. Our data strongly support the notion that ZNF521, through the recruitment of the NuRD complex, contributes to the clonogenic growth, migration and tumorigenicity of medulloblastoma cells.

The role of Bergmann glial cells in cerebellar development

CorrespondenceA response to Gershon et al. : Hexokinase-2-mediatedaerobic glycolysis is integral to cerebellar neurogen-esis and pathogenesis of meduloblastoma. Cancer andMetabolism 2013, 1(2):17Gershon et al. [1] have investigated the role thatHexokinase-2 (Hk2) plays in the control of aerobic gly-colysis during cerebellar neurogenesis and pathogenesisof medulloblastoma. They conclude from experimentsusing cerebellar granule neurons progenitors (CGNPs)in culture, dissected normal cerebella and medulloblas-toma and cerebellar slices that Hk2 mediates a glycolyticphenotype in proliferating CGNP that is recapitulated inmedulloblastoma, a malignant tumor of CGNP origin.While the role of Hk2 in the aggressive growth of me-dulloblastoma is well documented in their work, itsimportance during the proliferation, migration and dif-ferentiation of cerebellar granule neurons may have beenoverestimated by not taking into account the metabolicactivity and histogenetic role of Bergmann glial cells,a major actor of cerebellar development. Indeed theBergmann glia palisade is already fully developed and oc-cupies most of the cerebellar cortex between pia and thePurkinje cells layer at P7 in the mouse at a time whenGershon et al. claim that CGNPs are mostly responsiblefor an elevated Hk2 activity in the cerebellum. In theirfigure 3, this area where Hk2 is expressed the most at P7that they have labeled as EGL (external granule layer),actually corresponds to the Molecular Layer (ML) inwhich Bergmann glia send their dense, branched pro-cesses. The EGL sensu stricto is made up of a 4 to 8cells-thick layer located at the superficial part of the ML,and CGNPs proliferate very actively only in the outer-most half of that EGL. This is visible in figure 6H of thepaper of Gershon et al., where the proliferating CGNPsare stained in green, while the post-mitotic CGNPs arestained in red. Beyond that, CGNPs cell bodies are onlysparsely present in the ML as they rapidly migratetoward the internal granule layer (IGL) along Bergmannglia processes.Gershon et al. interpretation of the results of their Fig. 5mayalsobediscussedasthehGFAP-cre;Hk2

Expression of transferrin receptor 1, proliferating cell nuclear antigen, p27Kip1 and calbindin in the fetal and neonatal feline cerebellar cortex

Cerebellar cortices from feline fetuses with estimated gestational ages of 40–66days and from kittens aged 2days to 2months, all negative for feline panleukopenia virus (FPV) infection, were analysed for expression of the transferrin receptor 1 (TrFR1), proliferating cell nuclear antigen (PCNA), p27Kip1 and calbindin. TrFR1, the receptor used by FPV to enter target cells, was expressed in capillary endothelial cells in the cerebellum at all fetal stages investigated and in Purkinje cells of a 3-week-old kitten, but not in the neuroblasts in the external granule layer (EGL). PCNA was expressed in cells of the superficial layer of the EGL. The cyclin-dependent kinase inhibitor p27Kip1 was expressed in cells of the deep layer of the EGL. Purkinje cells expressed calbindin from the earliest fetal stage investigated. Co-expression of PCNA and calbindin could not be demonstrated, indicating that feline Purkinje cells are post-mitotic from at least 40days gestation.

Down Syndrome Cell Adhesion Molecule (DSCAM) Associates with Uncoordinated-5C (UNC5C) in Netrin-1-mediated Growth Cone Collapse

In the developing nervous system, neuronal growth cones explore the extracellular environment for guidance cues, which can guide them along specific trajectories toward their targets. Netrin-1, a bifunctional guidance cue, binds to deleted in colorectal cancer (DCC) and DSCAM mediating axon attraction, and UNC5 mediating axon repulsion. Here, we show that DSCAM interacts with UNC5C and this interaction is stimulated by netrin-1 in primary cortical neurons and postnatal cerebellar granule cells. DSCAM partially co-localized with UNC5C in primary neurons and brain tissues. Netrin-1 induces axon growth cone collapse of mouse cerebellum external granule layer (EGL) cells, and the knockdown of DSCAM or UNC5C by specific shRNAs or blocking their signaling by overexpressing dominant negative mutants suppresses netrin-1-induced growth cone collapse. Similarly, the simultaneous knockdown of DSCAM and UNC5C also blocks netrin-1-induced growth cone collapse in EGL cells. Netrin-1 increases tyrosine phosphorylation of endogenous DSCAM, UNC5C, FAK, Fyn, and PAK1, and promotes complex formation of DSCAM with these signaling molecules in primary postnatal cerebellar neurons. Inhibition of Src family kinases efficiently reduces the interaction of DSCAM with UNC5C, FAK, Fyn, and PAK1 and tyrosine phosphorylation of these proteins as well as growth cone collapse of mouse EGL cells induced by netrin-1. The knockdown of DSCAM inhibits netrin-induced tyrosine phosphorylation of UNC5C and Fyn as well as the interaction of UNC5C with Fyn. The double knockdown of both receptors abolishes the induction of Fyn tyrosine phosphorylation by netrin-1. Our study reveals the first evidence that DSCAM coordinates with UNC5C in netrin-1 repulsion.

Reciprocal actions of ATF5 and Shh in proliferation of cerebellar granule neuron progenitor cells

Precise regulation of neuroprogenitor cell proliferation and differentiation is required for successful brain development, but the factors that contribute to this are only incompletely understood. The transcription factor ATF5 promotes proliferation of cerebral cortical neuroprogenitor cells and its down regulation permits their differentiation. Here, we examine the expression and regulation of ATF5 in cerebellar granule neuron progenitor cells (CGNPs) as well as the role of ATF5 in the transition of CGNPs to postmitotic cerebellar granule neurons (GCNs). We find that ATF5 is expressed by proliferating CGNPs in both the embryonic and postnatal cerebellar external granule layer (EGL) and in the rhombic lip, the embryonic structure from which the EGL arises. In contrast, ATF5 is undetectable in postmitotic GCNs. In highly enriched dissociated cultures of CGNPs and CGNs, ATF5 is expressed only in CGNPs. Constitutive ATF5 expression in CGNPs does not affect their proliferation or exit from the cell cycle. In contrast, in presence of sonic hedgehog (Shh), a mitogen for CGNPs, constitutively expressed ATF5 promotes CGNP proliferation and delays their cell cycle exit and differentiation. Conversely, ATF5 loss-of-function conferred by a dominant-negative form of ATF5 significantly diminishes Shh-stimulated CGNP proliferation and promotes differentiation. In parallel with its stimulation of CGNP proliferation, Shh enhances ATF5 expression by what appeared to be a posttranscriptional mechanism involving protein stabilization. These findings indicate a reciprocal interaction between ATF5 and Shh in which Shh stimulates ATF5 expression and in which ATF5 contributes to Shh-stimulated CGNP expansion.