Cerebellar Granule Cell Precursors Research Articles

Intracerebellar administration of the chemokine Cxcl3 reduces the volume of medulloblastoma lesions at an advanced stage by promoting the migration and differentiation of preneoplastic precursor cells

AbstractThe prognosis for many pediatric brain tumors, including cerebellar medulloblastoma (MB), remains dismal but there is promise in new therapies. We have previously generated a mouse model developing spontaneous MB at high frequency, Ptch1+/−/Tis21−/−. In this model, reproducing human tumorigenesis, we identified the decline of the Cxcl3 chemokine in cerebellar granule cell precursors (GCPs) as responsible for a migration defect, which causes GCPs to stay longer in the proliferative area rather than differentiate and migrate internally, making them targets of transforming insults. We demonstrated that 4‐week Cxcl3 infusion in cerebella of 1‐month‐old mice, at the initial stage of MB formation, forces preneoplastic GCPs (pGCPs) to leave lesions and differentiate, with a complete suppression of MB development. In this study, we sought to verify the effect of 4‐week Cxcl3 treatment in 3‐month‐old Ptch1+/−/Tis21−/− mice, when MB lesions are at an advanced, irreversible stage. We found that Cxcl3 treatment reduces tumor volumes by sevenfold and stimulates the migration and differentiation of pGCPs from the lesion to the internal cerebellar layers. We also tested whether the pro‐migratory action of Cxcl3 favors metastases formation, by xenografting DAOY human MB cells in the cerebellum of immunosuppressed mice. We showed that DAOY cells express the Cxcl3 receptor, Cxcr2, and that Cxcl3 triggers their migration. However, Cxcl3 did not significantly affect the frequency of metastases or the growth of DAOY‐generated MBs. Finally, we mapped the expression of the Cxcr2 receptor in human MBs, by evaluating a well‐characterized series of 52 human MBs belonging to different MB molecular subgroups. We found that Cxcr2 was variably expressed in all MB subgroups, suggesting that Cxcl3 could be used for therapy of different MBs.

Syndecan-3 regulates the time of transition from cell cycle exit to initial differentiation stage in mouse cerebellar granule cell precursors

To analyze the role of syndecan-3 (SDC3), a heparan sulfate proteoglycan, in cerebellum development, we examined the effect of SDC3 on the transition from cell cycle exit to the initial differentiation stage of cerebellar granule cell precursors (CGCPs). First, we examined SDC3 localization in the developing cerebellum. SDC3 was mainly localized to the inner external granule layer where the transition from the cell cycle exit to the initial differentiation of CGCPs occurs. To examine how SDC3 regulates the cell cycle exit of CGCPs, we performed SDC3-knockdown (SDC3-KD) and -overexpression (Myc-SDC3) assays using primary CGCPs. SDC3-KD significantly increased the ratio of p27Kip1+ cells to total cells at day 3 in vitro (DIV3) and 4, but Myc-SDC3 reduced that at DIV3. Regarding the cell cycle exit efficiency using 24 h-labelled bromodeoxyuridine (BrdU) and a marker of cell cycling, Ki67, SDC3-KD significantly increased cell cycle exit efficiency (Ki67-; BrdU+ cells/BrdU+ cells) in primary CGCP at DIV4 and 5, but Myc-SDC3 reduced that at DIV4 and 5. However, SDC3-KD and Myc-SDC3 did not affect the efficiency of the final differentiation from CGCPs to granule cells at DIV3–5. Furthermore, the ratio of CGCPs in the cell cycle exiting stage to total cells, identified by initial differentiation markers TAG1 and Ki67 (TAG1+; Ki67+ cells), was considerably decreased by SDC3-KD at DIV4, but increased by Myc-SDC3 at DIV4 and 5. Altogether, these results indicate that SDC3 regulates the timing of the transition from the cell cycle exit stage to the initial differentiation stage of CGCP.

β-arrestin1-E2F1-ac axis regulates physiological apoptosis and cell cycle exit in cellular models of early postnatal cerebellum.

Development of the cerebellum is characterized by rapid proliferation of cerebellar granule cell precursors (GCPs) induced by paracrine stimulation of Sonic hedgehog (Shh) signaling from Purkinje cells, in the external granular layer (EGL). Then, granule cell precursors differentiate and migrate into the inner granular layer (IGL) of the cerebellum to form a terminally differentiated cell compartment. Aberrant activation of Sonic hedgehog signaling leads to granule cell precursors hyperproliferation and the onset of Sonic hedgehog medulloblastoma (MB), the most common embryonal brain tumor. β-arrestin1 (ARRB1) protein plays an important role downstream of Smoothened, a component of the Sonic hedgehog pathway. In the medulloblastoma context, β-arrestin1 is involved in a regulatory axis in association with the acetyltransferase P300, leading to the acetylated form of the transcription factor E2F1 (E2F1-ac) and redirecting its activity toward pro-apoptotic gene targets. This axis in the granule cell precursors physiological context has not been investigated yet. In this study, we demonstrate that β-arrestin1 has antiproliferative and pro-apoptotic functions in cerebellar development. β-arrestin1 silencing increases proliferation of Sonic hedgehog treated-cerebellar precursor cells while decreases the transcription of E2F1-ac pro-apoptotic targets genes, thus impairing apoptosis. Indeed, chromatin immunoprecipitation experiments show a direct interaction between β-arrestin1 and the promoter regions of the pro-apoptotic E2F1 target gene and P27, indicating the double role of β-arrestin1 in controlling apoptosis and cell cycle exit in a physiological context. Our data elucidate the role of β-arrestin1 in the early postnatal stages of cerebellar development, in those cell compartments that give rise to medulloblastoma. This series of experiments suggests that the physiological function of β-arrestin1 in neuronal progenitors is to directly control, cooperating with E2F1 acetylated form, transcription of pro-apoptotic genes.

MEDB-11. MYC overexpression and SMARCA4 loss in cerebellar granule cell precursors cooperate to drive medulloblastoma formation in mice

Group 3 medulloblastoma is one of the most aggressive types of childhood brain tumors. Roughly 30 % of cases carry genetic alterations in MYC, SMARCA4 or both genes combined. While overexpression of MYC has previously been shown to drive medulloblastoma formation in mice, the functional significance of SMARCA4 mutations and their suitability as a therapeutic target remain largely unclear. To address this issue, we combined an overexpression of MYC with a loss of SMARCA4 in cerebellar granule cell precursors. Cells were isolated from 7-day-old Math1-creERT2::Smarca4fl/fl pups after tamoxifen-induced loss of SMARCA4. Subsequently, MYC overexpression was achieved by lentiviral transduction, and transduced cells were transplanted into immunodeficient CD1nu/nu recipient mice. Preliminary results show tumor formation in 5/19 transplanted mice (26 %) after 6 months. SMARCA4 loss in all tumor cells was confirmed both immunohistochemically and on a genetic level and suggests a dependency of tumor growth on SMARCA4 loss. In a next step, additional cohorts will clarify if tumor development is accelerated by or even dependent on the loss of SMARCA4 in our model. Additionally, the neoplastic potential of tumor cells will be verified with the aid of secondary recipient mice. To evaluate to what extent the generated tumors are comparable to human Group 3 medulloblastomas, tumors will be extensively analyzed on a morphological, transcriptional, and epigenetic level. Altogether, we hope to establish a suitable mouse model for SMARCA4 mutated Group 3 medulloblastoma that will help to elucidate the role of SMARCA4 in tumor development and to identify new therapeutic targets.

Syndecan-3 Regulates the Time of Transition from Cell Cycle Exit to Initial Differentiation Stage in Mouse Cerebellar Granule Cell Precursors

Syndecan-3 Regulates the Time of Transition from Cell Cycle Exit to Initial Differentiation Stage in Mouse Cerebellar Granule Cell Precursors

Tumor Growth in the High Frequency Medulloblastoma Mouse Model Ptch1+/-/Tis21KO Has a Specific Activation Signature of the PI3K/AKT/mTOR Pathway and Is Counteracted by the PI3K Inhibitor MEN1611.

We have previously generated a mouse model (Ptch1+/−/Tis21KO), which displays high frequency spontaneous medulloblastoma, a pediatric tumor of the cerebellum. Early postnatal cerebellar granule cell precursors (GCPs) of this model show, in consequence of the deletion of Tis21, a defect of the Cxcl3-dependent migration. We asked whether this migration defect, which forces GCPs to remain in the proliferative area at the cerebellar surface, would be the only inducer of their high frequency transformation. In this report we show, by further bioinformatic analysis of our microarray data of Ptch1+/−/Tis21KO GCPs, that, in addition to the migration defect, they show activation of the PI3K/AKT/mTOR pathway, as the mRNA levels of several activators of this pathway (e.g., Lars, Rraga, Dgkq, Pdgfd) are up-regulated, while some inhibitors (e.g. Smg1) are down-regulated. No such change is observed in the Ptch1+/− or Tis21KO background alone, indicating a peculiar synergy between these two genotypes. Thus we investigated, by mRNA and protein analysis, the role of PI3K/AKT/mTOR signaling in MBs and in nodules from primary Ptch1+/−/Tis21KO MB allografted in the flanks of immunosuppressed mice. Activation of the PI3K/AKT/mTOR pathway is seen in full-blown Ptch1+/−/Tis21KO MBs, relative to Ptch1+/−/Tis21WT MBs. In Ptch1+/−/Tis21KO MBs we observe that the proliferation of neoplastic GCPs increases while apoptosis decreases, in parallel with hyper-phosphorylation of the mTOR target S6, and, to a lower extent, of AKT. In nodules derived from primary Ptch1+/−/Tis21KO MBs, treatment with MEN1611, a novel PI3K inhibitor, causes a dramatic reduction of tumor growth, inhibiting proliferation and, conversely, increasing apoptosis, also of tumor CD15+ stem cells, responsible for long-term relapses. Additionally, the phosphorylation of AKT, S6 and 4EBP1 was significantly inhibited, indicating inactivation of the PI3K/AKT/mTOR pathway. Thus, PI3K/AKT/mTOR pathway activation contributes to Ptch1+/−/Tis21KO MB development and to high frequency tumorigenesis, observed when the Tis21 gene is down-regulated. MEN1611 could provide a promising therapy for MB, especially for patient with down-regulation of Btg2 (human ortholog of the murine Tis21 gene), which is frequently deregulated in Shh-type MBs.

Multifaceted Functions of Rab23 on Primary Cilium-Mediated and Hedgehog Signaling-Mediated Cerebellar Granule Cell Proliferation.

Sonic hedgehog (Shh) signaling from the primary cilium drives cerebellar granule cell precursor (GCP) proliferation. Mutations of hedgehog (Hh) pathway repressors commonly cause medulloblastoma, the most prevalent and malignant childhood brain tumor that arises from aberrant GCP proliferation. We demonstrate that Nestin Cre-driven conditional knock-out (CKO) of a Shh pathway repressor-Rab23 in the mouse brain of both genders caused mis-patterning of cerebellar folia and elevated GCP proliferation during early development, but with no prevalent occurrence of medulloblastoma at adult stage. Strikingly, Rab23-depleted GCPs exhibited upregulated basal level of Shh pathway activities despite showing an abnormal ciliogenesis of primary cilia. In line with the compromised ciliation, Rab23-depleted GCPs were desensitized against Hh pathway activity stimulations by Shh ligand and Smoothened (Smo) agonist-SAG, and exhibited attenuated stimulation of Smo-localization on the primary cilium in response to SAG. These results implicate multidimensional actions of Rab23 on Hh signaling cascade. Rab23 represses the basal level of Shh signaling, while facilitating primary cilium-dependent extrinsic Shh signaling activation. Collectively, our findings unravel instrumental roles of Rab23 in GCP proliferation and ciliogenesis. Furthermore, Rab23's potentiation of Shh signaling pathway through the primary cilium and Smo suggests a potential new therapeutic strategy for Smo/primary cilium-driven medulloblastoma.SIGNIFICANCE STATEMENT Primary cilium and Sonic hedgehog (Shh) signaling are known to regulate granule cell precursor (GCP) proliferation. Aberrant overactivation of Shh signaling pathway ectopically increases GCP proliferation and causes malignant childhood tumor called medulloblastoma. However, the genetic and molecular regulatory cascade of GCP tumorigenesis remains incompletely understood. Our finding uncovers Rab23 as a novel regulator of hedgehog (Hh) signaling pathway activity and cell proliferation in GCP. Intriguingly, we demonstrated that Rab23 confers dual functions in regulating Shh signaling; it potentiates primary cilium and Shh/Smoothened (Smo)-dependent signaling activation, while antagonizes basal level Hh activity. Our data present a previously underappreciated aspect of Rab23 in mediating extrinsic Shh signaling upstream of Smo. This study sheds new light on the mechanistic insights underpinning Shh signaling-mediated GCP proliferation and tumorigenesis.

EPCT-17. DEVELOPING EYA PHOSPHATASE INHIBITORS WITH ON-TARGET EFFECTS IN SHH-MEDULLOBLASTOMA

Medulloblastoma, one of the most frequent malignant pediatric brain tumors, encompasses four molecularly and clinically distinct cancers. Sonic hedgehog (SHH)-subtype medulloblastoma constitutes about 30% of medulloblastomas, and therapies targeting the SHH pathway can lead to new highly selective treatment. The haloacid dehalogenase (HAD) phosphatase Eyes Absent 1 (EYA1) is critically involved in the development and progression of SHH-medulloblastoma: Eya1 is highly expressed in SHH-medulloblastomas, and single cell sequencing indicates that Eya1 is a consistent feature that can be detected in every individual cancer cell. Inhibition of EYA1 interrupts SHH pathway signaling. During normal development, EYA1 promotes symmetric division of cerebellar granule cell precursors (GCPs), the cells of origin for SHH-subtype medulloblastoma, and reduced levels of EYA1 decrease medulloblastoma mortality rates in mouse models. Therefore, targeting EYA1 may be a novel therapeutic avenue for these pediatric cancers. Benzarone derivatives have been suggested as allosteric EYA-inhibitors, and benzarone provides a promising platform for chemical derivatives. Here, we develop 60 novel benzarone derivatives and assess their efficacy in inhibiting SHH-medulloblastoma growth through the inhibition of EYA1. Several of the new compounds inhibit EYA1 phosphotyrosine phosphatase activity in a cell-based assay, interrupt SHH pathway, and prevent SHH-medulloblastoma growth in vitro. Our results show that these novel benzarone derivatives are a new promising avenue for developing therapeutics for pediatric SHH-medulloblastoma via inhibition of EYA phosphatases.

The Eya1 Phosphatase Mediates Shh-Driven Symmetric Cell Division of Cerebellar Granule Cell Precursors

During neural development, stem and precursor cells can divide either symmetrically or asymmetrically. The transition between symmetric and asymmetric cell divisions is a major determinant of precursor cell expansion and neural differentiation, but the underlying mechanisms that regulate this transition are not well understood. Here, we identify the Sonic hedgehog (Shh) pathway as a critical determinant regulating the mode of division of cerebellar granule cell precursors (GCPs). Using partial gain and loss of function mutations within the Shh pathway, we show that pathway activation determines spindle orientation of GCPs, and that mitotic spindle orientation correlates with the mode of division. Mechanistically, we show that the phosphatase Eya1 is essential for implementing Shh-dependent GCP spindle orientation. We identify atypical protein kinase C (aPKC) as a direct target of Eya1 activity and show that Eya1 dephosphorylates a critical threonine (T410) in the activation loop. Thus, Eya1 inactivates aPKC, resulting in reduced phosphorylation of Numb and other components that regulate the mode of division. This Eya1-dependent cascade is critical in linking spindle orientation, cell cycle exit and terminal differentiation. Together these findings demonstrate that a Shh-Eya1 regulatory axis selectively promotes symmetric cell divisions during cerebellar development by coordinating spindle orientation and cell fate determinants.

Vitronectin regulates the axon specification of mouse cerebellar granule cell precursors via αvβ5 integrin in the differentiation stage

Vitronectin, an extracellular matrix protein, controls the differentiation of cerebellar granule cell precursors (CGCPs) via αvβ5 integrin, particularly in the initial stage of differentiation to granule cells. In this study, we determined whether vitronectin regulates axon specification in this initial differentiation stage of CGCPs. First, we analyzed whether vitronectin deficiency, β5 integrin knockdown (KD), and β5 integrin overexpression affect axon specification of primary cultured CGCPs. Vitronectin deficiency and β5 integrin KD inhibited axon formation, while vitronectin administrated- and β5 integrin overexpressed-neurons formed multiple axons. Moreover, KD of β5 integrin suppressed vitronectin-induced multiple axon formation. These findings indicate that vitronectin contributes to regulating axon specification via αvβ5 integrin in CGCPs. Next, we determined the signaling pathway involved in regulating vitronectin-induced axon specification. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited vitronectin-induced multiple axon specification, and lithium chloride, an inhibitor of glyocogen synthase kinase 3 beta (GSK3β), attenuated the inhibitory effect of vitronectin-KO and β5 integrin KD on the specification of CGCPs. In addition, vitronectin induced the phosphorylation of protein kinase B (Akt) and GSK3β in neuroblastoma Neuro2a cells. Taken together, our results indicate that vitronectin plays an important factor in axon formation process in CGCPs via a β5 integrin/PI3K/GSK3β pathway.

The role of SCFSkp2 and SCFβ-TrCP1/2 in the cerebellar granule cell precursors.

A proper balance between proliferation and differentiation of cerebellar granule cell precursors (GCPs) is required for appropriate cerebellar morphogenesis. The Skp1-Cullin1-F-box (SCF) complex, an E3 ubiquitin ligase complex, is involved in polyubiquitination and subsequent degradation of various cell cycle regulators and transcription factors. However, it remains unknown how the SCF complex affects proliferation and differentiation of GCPs. In this study, we found that the scaffold protein Cullin1, and F-box proteins Skp2, β-TrCP1 and β-TrCP2 are expressed in the external granule layer (EGL). Knockdown of these molecules in the EGL showed that Cullin1, Skp2 and β-TrCP2 enhanced differentiation of GCPs. We also observed accumulation of cyclin-dependent kinase inhibitor p27 in GCPs when treated with a Cullin1 inhibitor or proteasome inhibitor. Furthermore, knockdown of p27 rescued enhancement of differentiation by Cullin1 knockdown. These results suggest that the SCF complex is involved in the maintenance of the proliferative state of GCPs through p27 degradation. In addition, inhibition of Cullin1 activity also prevented cell proliferation and enhanced accumulation of p27 in Daoy cells, a cell line derived from the sonic hedgehog subtype of medulloblastoma. This suggested that excess degradation of p27 through the SCF complex causes overproliferation of medulloblastoma cells.

Deletion of Btg1 Induces Prmt1-Dependent Apoptosis and Increased Stemness in Shh-Type Medulloblastoma Cells Without Affecting Tumor Frequency.

About 30% of medulloblastomas (MBs), a tumor of the cerebellum, arise from cerebellar granule cell precursors (GCPs) undergoing transformation following activation of the Sonic hedgehog (Shh) pathway. To study this process, we generated a new MB model by crossing Patched1 heterozygous (Ptch1+/−) mice, which develop spontaneous Shh-type MBs, with mice lacking B-cell translocation gene 1 (Btg1), a regulator of cerebellar development. In MBs developing in Ptch1+/− mice, deletion of Btg1 does not alter tumor and lesion frequencies, nor affect the proliferation of neoplastic precursor cells. However, in both tumors and lesions arising in Ptch1+/− mice, ablation of Btg1 increases by about 25% the apoptotic neoplastic precursor cells, as judged by positivity to activated caspase-3. Moreover, although Btg1 ablation in early postnatal GCPs, developing in the external granule cell layer, leads to a significant increase of proliferation, and decrease of differentiation, relative to wild-type, no synergy occurs with the Ptch1+/− mutation. However, Btg1 deletion greatly increases apoptosis in postnatal GCPs, with strong synergy between Btg1-null and Ptch1+/− mutations. That pronounced increase of apoptosis observed in Ptch1+/−/Btg1 knockout young or neoplastic GCPs may be responsible for the lack of effect of Btg1 ablation on tumorigenesis. This increased apoptosis may be a consequence of increased expression of protein arginine methyltransferase 1 (Prmt1) protein that we observe in Btg1 knockout/Ptch1+/− MBs. In fact, apoptotic genes, such as BAD, are targets of Prmt1. Moreover, in Btg1-null MBs, we observed a two-fold increase of cells positive to CD15, which labels tumor stem cells, raising the possibility of activation of quiescent tumor cells, known for their role in long-term resistance to treatment and relapses. Thus, Btg1 appears to play a role in cerebellar tumorigenesis by regulating the balance between apoptosis and proliferation during MB development, also influencing the number of tumor stem cells.

SMO-M2 mutation does not support cell-autonomous Hedgehog activity in cerebellar granule cell precursors

Growth and patterning of the cerebellum is compromised if granule cell precursors do not properly expand and migrate. During embryonic and postnatal cerebellar development, the Hedgehog pathway tightly regulates granule cell progenitors to coordinate appropriate foliation and lobule formation. Indeed, granule cells impairment or defects in the Hedgehog signaling are associated with developmental, neurodegenerative and neoplastic disorders. So far, scant and inefficient cellular models have been available to study granule cell progenitors, in vitro. Here, we validated a new culture method to grow postnatal granule cell progenitors as hedgehog-dependent neurospheres with prolonged self-renewal and ability to differentiate into granule cells, under appropriate conditions. Taking advantage of this cellular model, we provide evidence that Ptch1-KO, but not the SMO-M2 mutation, supports constitutive and cell-autonomous activity of the hedgehog pathway.

TMOD-03. SOMATIC MUTATIONS OF CHROMATIN REGULATOR Kmt2d IN CEREBELLAR PRECURSORS INFLUENCES SHH-MEDULLOBLASTOMA TUMORIGENESIS

Abstract Medulloblastoma (MB), the most common malignant pediatric brain tumor, is a classic example of dysregulation of developmental pathways leading to tumorogenesis. Despite advancements in multi-modal therapies, most patients suffer from long-term neurocognitive and neuroendocrine disabilities. The Sonic Hedgehog subgroup of MB (SHH-MB) accounts for ~30% of all cases and originates from ATOH1+ cerebellar granule cell precursors (GCPs). Experimental data in mice has shown that activating mutations in the SHH pathway induce tumors only in rare GCPs, suggesting that additional mutations and epigenetic changes are required to influence tumor progression. The KMT2D gene, encoding the histone-lysine N-methyltransferase 2D, is amongst the ten most frequently mutated genes in MB, with somatic mutations seen in ~15% of all SHH-MB patients. We developed sporadic mouse models of SHH-MB with a low penetrance to enable studies of secondary mutations (Tan, PNAS, 2018). Immunofluorescence staining for KMT2D on early-stage SHH-MB lesions, mid-stage and late-stage tumors revealed that a subset of lesions/tumors (16/98) do not express KMT2D and are negative for H3K4me3. Interestingly, P53 and KMT2D expression showed a positive correlation in ~94% of tumors/lesions and NeuN and KMT2D showed a positive correlation in ~92% of tumors/lesions. In order to determine the roles for KMT2D in GCP proliferation and differentiation, and uncover whether and how KMT2D promotes SHH-MB tumorigenesis, we are using genetic mouse-models whereby Kmt2d is heterozygously or homozygously deleted alone, or in conjunction with activation of the SHH pathway. Mice with SHH-MB tumors expressing SmoM2 and a loss of Kmt2d develop aggressive tumors at high penetrance, with metastatic leptomeningeal spread in the brain stem and spinal cord. Thus, loss of Kmt2d increases SHH-MB tumor progression and leads to malignancy. Ongoing studies are determining how the chromatin landscape and gene expression are changed when Kmt2d is deleted in GCPs.

Yap/Taz are required for establishing the cerebellar radial glia scaffold and proper foliation

Yap/Taz are well-established downstream effectors of the Hippo pathway, known to regulate organ size by directing proliferation and apoptosis. Although the functions of Yap/Taz have been extensively studied, little is known about their role in brain development. Here, through genetic ablation, we show that Yap/Taz are required for cerebellar morphogenesis. Yap/Taz deletion in neural progenitors causes defects in secondary fissure formation, leading to abnormal folia development. Although they seemed very likely to serve an important function in the development of cerebellar granule cell precursors, Yap/Taz are dispensable for their proliferation. Furthermore, Yap/Taz loss does not rescue the medulloblastoma phenotype caused by constitutively active Smoothened. Importantly, Yap/Taz are highly expressed in radial glia and play a crucial role in establishing the radial scaffold and cellular polarity of neural progenitors during embryogenesis. We found that Yap/Taz are necessary to establish and maintain junctional integrity of cerebellar neuroepithelium as prominent junction proteins are not maintained at the apical junction in the absence of Yap/Taz. Our study identifies a novel function of Yap/Taz in cerebellar foliation and finds that they are required to establish the radial glia scaffold and junctional stability.

Expression of transcription factors and signaling molecules in the cerebellar granule cell development

Cerebellar granule cell precursors (GCPs) and granule cells (GCs) constitute a good model system to investigate proliferation of neural precursors and differentiation of neurons. During development, GCPs proliferate in the outer external granule cell layer (outer EGL) and then exit the cell cycle in the inner EGL to become GCs, which inwardly migrate to the inner granule cell layer (IGL). Misregulation of GCP proliferation or GC differentiation leads to maldevelopment of the cerebellum and the formation of a cerebellar tumor, medulloblastoma. Despite many efforts in this field, the mechanisms underlying GC development remain elusive. In this study, we performed detailed immunostaining in the developing cerebellum, with particular focus on GCPs and GCs, looking at several transcription factors, signaling molecules, cell cycle regulators, some of which are known to regulate neural development. Interestingly, we found distinct distribution patterns of certain proteins within the outer and inner EGL, suggesting the existence of subpopulations of GCPs and GCs in those layers. This study provides a basis for future studies on the cerebellar GC development and medulloblastoma.

MEMRI-based imaging pipeline for guiding preclinical studies in mouse models of sporadic medulloblastoma.

Genetically engineered mouse models of sporadic cancers are critical for studying tumor biology and for preclinical testing of therapeutics. We present an MRI-based pipeline designed to produce high resolution, quantitative information about tumor progression and response to novel therapies in mouse models of medulloblastoma (MB). Sporadic MB was modeled in mice by inducing expression of an activated form of the Smoothened gene (aSmo) in a small number of cerebellar granule cell precursors. aSmo mice were imaged and analyzed at defined time-points using a 3D manganese-enhanced MRI-based pipeline optimized for high-throughput. A semi-automated segmentation protocol was established that estimates tumor volume in a time-frame compatible with a high-throughput pipeline. Both an empirical, volume-based classifier and a linear discriminant analysis-based classifier were tested to distinguish progressing from nonprogressing lesions at early stages of tumorigenesis. Tumor centroids measured at early stages revealed that there is a very specific location of the probable origin of the aSmo MB tumors. The efficacy of the manganese-enhanced MRI pipeline was demonstrated with a small-scale experimental drug trial designed to reduce the number of tumor associated macrophages and microglia. Our results revealed a high level of heterogeneity between tumors within and between aSmo MB models, indicating that meaningful studies of sporadic tumor progression and response to therapy could not be conducted without an imaging-based pipeline approach.

A Functional Interaction Between Na,K-ATPase β2-Subunit/AMOG and NF2/Merlin Regulates Growth Factor Signaling in Cerebellar Granule Cells.

The Na,K-ATPase, consisting of a catalytic α-subunit and a regulatory β-subunit, is a ubiquitously expressed ion pump that carries out the transport of Na+ and K+ across the plasma membranes of most animal cells. In addition to its pump function, Na,K-ATPase serves as a signaling scaffold and a cell adhesion molecule. Of the three β-subunit isoforms, β1 is found in almost all tissues, while β2 expression is mostly restricted to brain and muscle. In cerebellar granule cells, the β2-subunit, also known as adhesion molecule on glia (AMOG), has been linked to neuron-astrocyte adhesion and granule cell migration, suggesting its role in cerebellar development. Nevertheless, little is known about molecular pathways that link the β2-subunit to its cellular functions. Using cerebellar granule precursor cells, we found that the β2-subunit, but not the β1-subunit, negatively regulates the expression of a key activator of the Hippo/YAP signaling pathway, Merlin/neurofibromin-2 (NF2). The knockdown of the β2-subunit resulted in increased Merlin/NF2 expression and affected downstream targets of Hippo signaling, i.e., increased YAP phosphorylation and decreased expression of N-Ras. Further, the β2-subunit knockdown altered the kinetics of epidermal growth factor receptor (EGFR) signaling in a Merlin-dependent mode and impaired EGF-induced reorganization of the actin cytoskeleton. Therefore, our studies for the first time provide a functional link between the Na,K-ATPase β2-subunit and Merlin/NF2 and suggest a role for the β2-subunit in regulating cytoskeletal dynamics and Hippo/YAP signaling during neuronal differentiation.

Na,K‐ATPase β2‐subunit/AMOG as a regulator of Merlin/NF2 signaling in the cerebellum

The Na,K‐ATPase, consisting of a catalytic α‐subunit and a regulatory β‐subunit, is a ubiquitously expressed ion pump that carries out the transport of Na+ and K+ across the plasma membranes of most animal cells. In addition to its pump function, Na,K‐ATPase serves as a signaling scaffold and a cell adhesion molecule. Of three b‐subunit isoforms, β1 is found almost in all tissues, while β2 expression is mostly restricted to brain and muscle. In cerebellar granule cells, the b2‐subunit, also known as Adhesion Molecule on Glia (AMOG), has been linked to neuron‐astrocyte adhesion and granule cell migration, suggesting its role in cerebellar development. Nevertheless, little is known about molecular pathways that link the b2‐subunit to its cellular functions. Using cerebellar granule precursor cells, we found that the β2‐subunit, but not the b1‐subunit, negatively regulates expression of a key activator of the Hippo/YAP signaling pathway, Merlin/neurofibromin‐2 (NF2). The knockout of the β2‐subunit in mice or its knockdown in cerebellar granule cell precursors resulted in increased Merlin/NF2 expression and affected down‐stream targets of Hippo signaling, i.e. increased YAP phosphorylation and decreased expression of N‐Ras. Further, the β2‐subunit knockdown altered the kinetics of Epidermal Growth Factor Receptor (EGFR) signaling in a Merlin‐dependent mode and impaired EGF‐induced reorganization of the actin cytoskeleton. Therefore, our studies for the first time provide a functional link between the Na,K‐ATPase β2‐subunit and Merlin/NF2 and suggest a role for the β2‐subunit in regulating cytoskeletal dynamics and Hippo/YAP signaling during neuronal differentiation.Support or Funding InformationThis work was supported by funds from NIGMS‐8P20GM103464, the Nemours Foundation and the DO Believe Foundation. The Biomolecular Core Lab is supported by grants NIGMS P30GM114736 (COBRE) and NIGMS‐P20GM103446 (INBRE).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mutation in NADPH oxidase 3 (NOX3) impairs SHH signaling and increases cerebellar neural stem/progenitor cell proliferation

Abnormalities in cerebellar structure and function may cause ataxia, a neurological dysfunction of motor coordination. In the course of the present study, we characterized a mutant mouse lineage with an ataxia-like phenotype. We localized the mutation on chromosome 17 and mapped it to position 1534 of the Nox3 gene, resulting in p.Asn64Tyr change. The primary defect observed in Nox3eqlb mice was increased proliferation of cerebellar granule cell precursors (GCPs). cDNA microarray comparing Nox3eqlb and BALB/c neonatal cerebellum revealed changes in the expression of genes involved in the control of cell proliferation. Nox3eqlb GCPs and NSC produce higher amounts of reactive oxygen species (ROS) and upregulate the expression of SHH target genes, such as Gli1–3 and Ccnd1 (CyclinD1). We hypothesize that this new mutation is responsible for an increase in proliferation via stimulation of the SHH pathway. We suggest this mutant mouse lineage as a new model to investigate the role of ROS in neuronal precursor cell proliferation.