Cerebellar Granule Neuron Precursors Research Articles

BIOL-23. LOSS OF MICRORNAS COOPERATES WITH TUMOR SUPPRESSORS TO INITIATE MYC-DEPENDENT METASTATIC MEDULLOBLASTOMA

Abstract To determine the role of microRNA regulation in brain tumor development, we incorporated a conditional allele of the microRNA processing enzyme Dicer to a glioma mouse model based on inactivation of the tumor suppressors Nf1, Trp53, and Pten using the Nestin-creERT2 transgene. Loss of Dicer and tumor suppressors at adult ages led to glioma development; however, mutant mice tamoxifen induced at early postnatal ages developed medulloblastoma (MB) instead of glioma. The switch in tumor spectrum occurred with 100% penetrance and the cerebellar tumors were histologically indistinguishable from human MB. The minimum genetic mutations required for MB formation were Dicer1 and Trp53, while additional loss of Pten produced more invasive tumors with leptomeningeal metastasis. Analysis of tumor transcriptome and MB subtype-specific markers show that Dicer tumors most resemble human Sonic Hedgehog (SHH) MB. The presence of leptomeningeal metastasis, Trp53 loss and upregulation of Mycn and Gli2 in the setting of activated Shh signaling suggests that this model phenocopies extremely high risk p53-mutated SHH MB, which carries poor prognosis and is associated with the majority of treatment failures in SHH MB. Analysis of pre-symptomatic mutant mice showed proliferative defects and retained cells in the external granule cell layer (EGL), suggesting that tumors may arise from cerebellar granule neuron precursors (CGNPs). However, targeting CGNPs using Math1-creERT2 did not lead to MB development, suggesting that an earlier EGL precursor may be required for tumorigenesis. Dicer tumors exhibit reduced microRNAs and RNA-induced silencing complexes consistent with loss of Dicer. One of these downregulated microRNAs, miR-101, is a regulator of Mycn and its overexpression leads to Mycn downregulation and inhibition of MB growth. Likewise, genetic and pharmacologic inhibition of Myc leads to inhibition of MB growth. These studies demonstrate a role for microRNAs in Myc-dependent MB development and show an alternative pathway for SHH MB tumorigenesis.

Targeting H3K27me3 demethylase to inhibit Shh signaling and cholesterol metabolism in medulloblastoma growth.

Previously we uncovered the epigenetic regulation of medulloblastoma that low levels of H3K27me3 are required for Shh target gene expression and medulloblastoma growth. Since Jmjd3, an H3K27me3 demethylase, is responsible for maintaining low H3K27me3 at Shh target genes, targeting Jmjd3 could be an efficient way to inhibit Shh signaling and medulloblastoma growth. Here we show that the small molecule GSK-J4, an inhibitor of Jmjd3, significantly inhibited the expression of Shh target genes in Shh responsive cell models and primary cerebellar granule neuron precursors. GSK-J4 also significantly reduced the growth of primary Shh medulloblastoma cultures. Treating human medulloblastoma cell line DaoY by GSK-J4 led to cell cycle arrest at G0/G1 phase with decreased cells in S-phase. Tumor cell proliferation was significantly inhibited by GSK-J4 treatment. Gene expression analyses showed that GSK-J4 additionally constrained the expression of key genes in cholesterol biosynthesis. Our results highlight the possibility that targeting H3K27me3 demethylase Jmjd3 with GSK-J4 to inhibit Shh signaling and cholesterol metabolism is a potential application to treat Shh medulloblastoma.

TMIC-81. INCREASED LEVEL OF IGFBP2 PROMOTES TUMOR METASTASIS IN SHH MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is the most common pediatric brain malignancy. MB comprises 5 major subgroups known as WNT, SHH p53wt, SHH p53mut, Group 3 and Group 4. Among the four MB subgroups SHH group is the most dominant molecular subgroup in infants and adults. These tumors are proposed to arise from cerebellar granule neuron precursors (CGNPs), whose developmental expansion requires SHH signaling from the neighboring Purkinje neurons. Previous reports suggest that SHH group features a unique tumor microenvironment compared with other MB groups. To better understand how SHH MB cells interact with Tumor Microenvironment, we performed cytokine array analysis of culture media from SHH group Patient Tumor cells, spontaneous SHH MB mouse tumor cells and SHH MB cell lines. Further, confirmed these results using ELISA, Western blot, and immunofluorescence from human SHH MB cell lines, Smo/A1 mouse tumor primary cells and PZp53Med cell lines. In continuation to the observation of IGFBP2 expression in various cell types in single cell analysis, we analyzed the presence of IGFBP2 in astrocytes using Smo/A1 mouse tumor Immunohistochemistry. Our data showed increased levels of IGFBP2 produced by SHH MB cell lines compared to others. We analyzed the role of IGFBP2 in SHH MB tumor growth and metastasis. IGFBP2 knock-down stable cell lines showed phenotypic changes including reduced cell proliferation, cell migration and EMT. Further western blot analysis of IGFBP2 KD cells showed reduced expression of EMT markers also reduced the activation of STAT3. Our preliminary in vitro data suggest IGFBP2 exerts it metastasis-promoting role in SHH MB by regulating the expression of EMT marker proteins and matrix remodeling proteins. Further functional studies suggest that in SHH MB, IGFBP2 may regulate a STAT3-mediated EMT program to metastasize. These findings provide a strong rationale for further pursuing how IGFBP2 promotes medulloblastoma tumor cell growth and migration in vivo.

Impaired SNF2L Chromatin Remodeling Prolongs Accessibility at Promoters Enriched for Fos/Jun Binding Sites and Delays Granule Neuron Differentiation

Chromatin remodeling proteins utilize the energy from ATP hydrolysis to mobilize nucleosomes often creating accessibility for transcription factors within gene regulatory elements. Aberrant chromatin remodeling has diverse effects on neuroprogenitor homeostasis altering progenitor competence, proliferation, survival, or cell fate. Previous work has shown that inactivation of the ISWI genes, Smarca5 (encoding Snf2h) and Smarca1 (encoding Snf2l) have dramatic effects on brain development. Smarca5 conditional knockout mice have reduced progenitor expansion and severe forebrain hypoplasia, with a similar effect on the postnatal growth of the cerebellum. In contrast, Smarca1 mutants exhibited enlarged forebrains with delayed progenitor differentiation and increased neuronal output. Here, we utilized cerebellar granule neuron precursor (GNP) cultures from Smarca1 mutant mice (Ex6DEL) to explore the requirement for Snf2l on progenitor homeostasis. The Ex6DEL GNPs showed delayed differentiation upon plating that was not attributed to changes in the Sonic Hedgehog pathway but was associated with overexpression of numerous positive effectors of proliferation, including targets of Wnt activation. Transcriptome analysis identified increased expression of Fosb and Fosl2 while ATACseq experiments identified a large increase in chromatin accessibility at promoters many enriched for Fos/Jun binding sites. Nonetheless, the elevated proliferation index was transient and the Ex6DEL cultures initiated differentiation with a high concordance in gene expression changes to the wild type cultures. Genes specific to Ex6DEL differentiation were associated with an increased activation of the ERK signaling pathway. Taken together, this data provides the first indication of how Smarca1 mutations alter progenitor cell homeostasis and contribute to changes in brain size.

TMOD-06. LOSS OF DICER COOPERATES WITH TUMOR SUPPRESSORS TO INITIATE METASTATIC MEDULLOBLASTOMA

To determine the role of microRNA regulation in brain tumor development, we incorporated a conditional allele of the microRNA processing enzyme Dicer to a previously characterized glioma mouse model based on inactivation of the tumor suppressors Nf1, Trp53, and Pten using the Nestin-creERT2 transgene. Loss of Dicer and tumor suppressors at adult ages led to glioma development; however, mutant mice tamoxifen induced at early postnatal ages developed medulloblastoma instead of glioma. The switch in tumor spectrum occurred with 100% penetrance and tumors were histologically indistinguishable from human medulloblastoma (MB). The minimum genetic mutations required for MB formation were Dicer and Trp53. Nf1 was dispensable, while additional loss of Pten produced more invasive tumors and leptomeningeal metastases. The time window for initiation of tumorigenesis was until the 2nd postnatal week, coinciding with the disappearance of the external granule layer (EGL), where cerebellar granule neuron precursors (CGNPs) undergo proliferation. Analysis of pre-symptomatic mutant mice showed proliferative defects and retained cells in the EGL, suggesting that the tumors may arise from CGNPs. However, targeting a subset of CGNPs using Math1-creERT2 did not lead to MB development, suggesting that an earlier EGL precursor may be required for tumorigenesis. Analysis of tumor transcriptome and MB subtype-specific genes and markers show that Dicer tumors most resemble extremely high risk p53-mutated SHH MB. Small RNA and mRNA sequencing analyses showed downregulation of microRNAs and dysregulation of its targets such as N-Myc. These studies demonstrate a role for microRNAs in MB development and show a fully penetrant genetic mouse model of highly metastatic MB.

BIOL-08. IGFBP2 PROMOTES TUMOR METASTASIS IN SHH MEDULLOBLASTOMA

Medulloblastoma (MB) is the most common pediatric brain malignancy. MB comprises 5 major subgroups known as WNT, SHH p53wt, SHH p53mut, Group 3 and Group 4. Among the four MB subgroups SHH group is the most dominant molecular subgroup in infants and adults. These tumors are proposed to arise from cerebellar granule neuron precursors (CGNPs), whose developmental expansion requires SHH signaling from the neighboring Purkinje neurons. Previous reports suggest that SHH group features a unique tumor microenvironment compared with other MB groups. Recently, we performed cytokine array analysis of culture media from different MB cell lines. Interestingly, our data showed increased levels of IGFBP2 produced by SHH MB cell lines compared to others. We confirmed these results using ELISA and Western blotting from 3 human SHH MB cell lines, and Smo/A1 mouse tumor cells. IGFBP2 is a member of IGFBP super family of proteins; it plays important roles in tumor cell proliferation, metastasis and drug resistance. We analyzed the role of IGFBP2 in SHH group medulloblastoma tumor growth and metastasis. IGFBP2 knock-down stable cell lines showed phenotypic changes including reduced cell proliferation, cell migration and colony size. Our preliminary in vitro data suggest IGFBP2 exerts it metastasis-promoting role in SHH MB by regulating the expression of EMT marker proteins such as N cadherin, slug etc. and matrix remodeling proteins like MMPs and TIMPs. We are currently performing functional studies in organotypic tumor slice cultures to validate these findings and establish IGFBP2 as a novel regulator of aggressive tumor growth and spread in SHH MB.

Simultaneous Brg1 Knockout and MYCN Overexpression in Cerebellar Granule Neuron Precursors Is Insufficient to Drive Tumor Formation but Temporarily Enhances their Proliferation and Delays their Migration

Medulloblastoma (MB) is the most common malignant brain tumor in childhood. According to the World Health Organization (WHO) classification of central nervous system (CNS) tumors, this embryonal tumor is divided into a wingless (WNT)-activated, Sonic hedgehog (SHH)-activated, and non-WNT/non-SHH entity. The latter is poorly defined but frequently carries mutations in Brahma-related gene 1 (BRG1) or amplifications of MYCN. Here, we investigated whether a combination of a Brg1 knockout and an overexpression of MYCN in cerebellar granule neuron precursors or multipotent neural stem cells is sufficient to drive brain tumor formation in mice. To this end, we generated Math1-creERT2::Brg1fl/fl::lslMYCN and hGFAP-cre::Brg1fl/fl::lslMYCN mice, respectively. We did not observe brain tumor formation in any of these models. hGFAP-cre::Brg1fl/fl::lslMYCN mice revealed severe CNS abnormalities with short survival, similar to the situation with a sole loss of Brg1, as we previously described. Investigation of Math1-creERT2::Brg1fl/fl::lslMYCN mice with a tamoxifen induction at postnatal day 3 revealed a regular survival but significant increase in cerebellar granule neuron precursor proliferation, followed by a delayed inward migration of these cells. This is in stark contrast to the hypoplastic cerebellum that we previously observed after embryonic deletion of Brg1 in Math1 positive cerebellar granule neurons. Our results indicate a time-specific function of Brg1 in cerebellar granule neuron precursors. Yet, the exact temporal and spatial origin of non-WNT/non-SHH MB remains unclear.

Histone demethylase UTX/KDM6A enhances tumor immune cell recruitment, promotes differentiation and suppresses medulloblastoma.

The deregulation of epigenetic pathways has been implicated as a critical step in tumorigenesis including in childhood brain tumor medulloblastoma. The H3K27me3 demethylase UTX/KDM6A plays important roles in development and is frequently mutated in various types of cancer. However, how UTX regulates tumor development remains largely unclear. Here, we report the generation of a UTX-deleted mouse model of SHH medulloblastoma that demonstrates the tumor suppressor functions of UTX, which could be antagonized by the deletion of another H3K27me3 demethylase JMJD3/KDM6B. Intriguingly, UTX deletion in cancerous cerebellar granule neuron precursors (CGNPs) resulted in the impaired recruitment of host CD8+ T cells to the tumor microenvironment through a non-cell autonomous mechanism. In both mouse medulloblastoma models and in human medulloblastoma cells, we showed that UTX activates Th1-type chemokines, which are responsible for T cell migration. Surprisingly, our results showed that the depletion of cytotoxic CD8+ T cells did not affect mouse medulloblastoma growth. Nevertheless, the UTX/chemokine/T cell recruitment pathway we identified may be applied to many other cancers and may be important for improving cancer immunotherapy. In addition, UTX is required for the expression of NeuroD2 in precancerous progenitors, which encodes a potent proneural transcription factor. Overexpression of NEUROD2 in CGNPs decreased cell proliferation and increased neuron differentiation. We showed that UTX deletion led to impaired neural differentiation, which could coordinate with active SHH signaling to accelerate medulloblastoma development. Thus, UTX regulates both cell-intrinsic oncogenic processes and the tumor microenvironment in medulloblastoma. Our study provides insights into both medulloblastoma development and context dependent functions of UTX in tumorigenesis.

Medulloblastoma cells resemble neuronal progenitors in their differentiation

ABSTRACT Medulloblastoma (MB) often originate from cerebellar granule neuron precursors (GNPs). We recently found that medulloblastoma cells undergo differentiation as GNPs. Differentiated MB cells have permanently lost their proliferative capacity and tumorigenicity. The differentiation of MB cells is driven by the transcription factor NeuroD1 (Neurogenic differentiation 1), and NeuroD1 expression in MB cells is repressed by EZH2-mediated H3K27me3.

Transient Primary Cilia Mediate Robust Hedgehog Pathway-Dependent Cell Cycle Control

The regulation of proliferation is a primary function of Hedgehog (Hh) signaling in development. Hh signal transduction requires the primary cilium for several steps in the pathway [1-5]. Many cells only build a primary cilium upon cell cycle exit, in G0. In those proliferating cells that do make a cilium, it is a transient organelle, being assembled in G1 and disassembled sometime prior to mitosis [6-9]. Thus, the requirement for primary cilia presents a conundrum: how are proliferative signals conveyed through an organelle that is present for only part of the cell cycle? Here, we investigate this question in a mouse medulloblastoma cell line, SMB55, that requires cilium-mediated Hh pathway activity for proliferation [10]. We show that SMB55 cells, and the primary cerebellar granule neuron precursors (GNPs) from which they derive, are often ciliated beyond G1 into S phase, and the presence of the cilium in SMB55 cells determines the periods of Hh pathway activity. Using live imaging over multiple cell cycles, we demonstrate that Hh pathway activity in either G1-S of the previous cell cycle or G1 of the cell cycle in which the decision is made is sufficient for cell cycle entry. We also show that cyclin D1 contributes to the persistent effects of pathway activity over multiple cell cycles. Together, our results reveal that, even though the signaling organelle itself is transient, Hh pathway control of proliferation is remarkably robust. Further, primary cilium transience may have implications for other Hh-mediated events in development.

NeuroD1 Dictates Tumor Cell Differentiation in Medulloblastoma

SUMMARYTumor cells are characterized by unlimited proliferation and perturbed differentiation. Using single-cell RNA sequencing, we demonstrate that tumor cells in medulloblastoma (MB) retain their capacity to differentiate in a similar way as their normal originating cells, cerebellar granule neuron precursors. Once they differentiate, MB cells permanently lose their proliferative capacity and tumorigenic potential. Differentiated MB cells highly express NeuroD1, a helix-loop-helix transcription factor, and forced expression of NeuroD1 promotes the differentiation of MB cells. The expression of NeuroD1 in bulk MB cells is repressed by trimethylation of histone 3 lysine-27 (H3K27me3). Inhibition of the histone lysine methyltransferase EZH2 prevents H3K27 trimethylation, resulting in increased NeuroD1 expression and enhanced differentiation in MB cells, which consequently reduces tumor growth. These studies reveal the mechanisms underlying MB cell differentiation and provide rationales to treat MB (potentially other malignancies) by stimulating tumor cell differentiation.

A Light-Inducible Hedgehog Signaling Activator Modulates Proliferation and Differentiation of Neural Cells

The Hedgehog signaling pathway shapes our body by regulating the proliferation and differentiation of cells. The spatial and temporal distribution pattern of its ligands finely controls the activity of the Hedgehog pathway during development. To model the control of Hedgehog signaling activities in vitro, we developed a light-inducible Hedgehog signaling activator 6-nitroveratryloxy-carbonyl Smoothened agonist (NVOC-SAG). NVOC-SAG controls the proliferation of mouse cerebellar granule neuron precursor cells and ventral and neural differentiation of human iPS cells in a light dependent manner. The compound provides a new method to control Hedgehog signaling activities.

Erbb4 Is Required for Cerebellar Developmentand Malignant Phenotype of Medulloblastoma.

Medulloblastoma is the most common and malignant pediatric brain tumor in childhood. It originates from dysregulation of cerebellar development, due to an excessive proliferation of cerebellar granule neuron precursor cells (CGNPs). The underlying molecular mechanisms, except for the role of SHH and WNT pathways, remain largely unknown. ERBB4 is a tyrosine kinase receptor whose activity in cancer is tissue dependent. In this study, we characterized the role of ERBB4 during cerebellum development and medulloblastoma progression paying particular interests to its role in CGNPs and medulloblastoma stem cells (MBSCs). Our results show that ERBB4 is expressed in the CGNPs during cerebellum development where it plays a critical role in migration, apoptosis and differentiation. Similarly, it is enriched in the population of MBSCs, where also controls those critical processes, as well as self-renewal and tumor initiation for medulloblastoma progression. These results are translated to clinical samples where high levels of ERBB4 correlate with poor outcome in Group 4 and all medulloblastomas groups. Transcriptomic analysis identified critical processes and pathways altered in cells with knock-down of ERBB4. These results highlight the impact and underlying mechanisms of ERBB4 in critical processes during cerebellum development and medulloblastoma.

Pik3ca mutations significantly enhance the growth of SHH medulloblastoma and lead to metastatic tumour growth in a novel mouse model.

Medulloblastoma (MB) is the most frequent malignant brain tumour in children with a poor outcome. Divided into four molecular subgroups, MB of the Sonic hedgehog (SHH) subgroup accounts for approximately 25% of the cases and is driven by mutations within components of the SHH pathway, such as its receptors PTCH1 or SMO. A fraction of these cases additionally harbour PIK3CA mutations, the relevance of which is so far unknown. To unravel the role of Pik3ca mutations alone or in combination with a constitutively activated SHH signalling pathway, transgenic mice were used. These mice show mutated variants within Smo, Ptch1 or Pik3ca genes in cerebellar granule neuron precursors, which represent the cellular origin of SHH MB. Our results show that Pik3ca mutations alone are insufficient to cause developmental alterations or to initiate MB. However, they significantly accelerate the growth of Shh MB, induce tumour spread throughout the cerebrospinal fluid, and result in lower survival rates of mice with a double Pik3caH1047R/SmoM2 or Pik3caH1047R/Ptch1 mutation. Therefore, PIK3CA mutations in SHH MB may represent a therapeutic target for first and second line combination treatments.

Reactive Oxygen Species Signaling Promotes Hypoxia-Inducible Factor 1α Stabilization in Sonic Hedgehog-Driven Cerebellar Progenitor Cell Proliferation.

Cerebellar development is a highly regulated process involving numerous factors acting with high specificity, both temporally and by location. Part of this process involves extensive proliferation of cerebellar granule neuron precursors (CGNPs) induced by Sonic Hedgehog (SHH) signaling, but downstream effectors of mitogenic signaling are still being elucidated. Using primary CGNP cultures, a well-established model for SHH-driven proliferation, we show that SHH-treated CGNPs feature high levels of hypoxia-inducible factor 1α (HIF1α), which is known to promote glycolysis, stemness, and angiogenesis. In CGNPs cultured under normoxic conditions, HIF1α is posttranslationally stabilized in a manner dependent upon reactive oxygen species (ROS) and NADPH oxidase (NOX), both of which are also upregulated in these cells. Inhibition of NOX activity resulted in HIF1α destabilization and reduced levels of cyclin D2, a marker of CGNP proliferation. As CGNPs are the putative cells of origin for the SHH subtype of medulloblastoma and aberrant SHH signaling is implicated in other neoplasms, these studies may also have future relevance in the context of cancer. Taken together, our findings suggest that a better understanding of nonhypoxic HIF1α stabilization through NOX-induced ROS generation can provide insights into normal cell proliferation in cerebellar development and SHH-driven cell proliferation in cancers with aberrant SHH signaling.

TCF4 (E2-2) harbors tumor suppressive functions in SHH medulloblastoma.

The TCF4 gene encodes for the basic helix-loop-helix transcription factor 4 (TCF4), which plays an important role in the development of the central nervous system (CNS). Haploinsufficiency of TCF4 was found to cause Pitt-Hopkins syndrome (PTHS), a severe neurodevelopmental disorder. Recently, the screening of a large cohort of medulloblastoma (MB), a highly aggressive embryonal brain tumor, revealed almost 20% of adult patients with MB of the Sonic hedgehog (SHH) subtype carrying somatic TCF4 mutations. Interestingly, many of these mutations have previously been detected as germline mutations in patients with PTHS. We show here that overexpression of wild-type TCF4 in vitro significantly suppresses cell proliferation in MB cells, whereas mutant TCF4 proteins do not to the same extent. Furthermore, RNA sequencing revealed significant upregulation of multiple well-known tumor suppressors upon expression of wild-type TCF4. In vivo, a prenatal knockout of Tcf4 in mice caused a significant increase in apoptosis accompanied by a decreased proliferation and failed migration of cerebellar granule neuron precursor cells (CGNP), which are thought to be the cells of origin for SHH MB. In contrast, postnatal in vitro and in vivo knockouts of Tcf4 with and without an additional constitutive activation of the SHH pathway led to significantly increased proliferation of CGNP or MB cells. Finally, publicly available data from human MB show that relatively low expression levels of TCF4 significantly correlate with a worse clinical outcome. These results not only point to time-specific roles of Tcf4 during cerebellar development but also suggest a functional linkage between TCF4 mutations and the formation of SHH MB, proposing that TCF4 acts as a tumor suppressor during postnatal stages of cerebellar development.

Granule neuron precursor cell proliferation is regulated by NFIX and intersectin 1 during postnatal cerebellar development.

Cerebellar granule neurons are the most numerous neuronal subtype in the central nervous system. Within the developing cerebellum, these neurons are derived from a population of progenitor cells found within the external granule layer of the cerebellar anlage, namely the cerebellar granule neuron precursors (GNPs). The timely proliferation and differentiation of these precursor cells, which, in rodents occurs predominantly in the postnatal period, is tightly controlled to ensure the normal morphogenesis of the cerebellum. Despite this, our understanding of the factors mediating how GNP differentiation is controlled remains limited. Here, we reveal that the transcription factor nuclear factor I X (NFIX) plays an important role in this process. Mice lacking Nfix exhibit reduced numbers of GNPs during early postnatal development, but elevated numbers of these cells at postnatal day 15. Moreover, Nfix-/- GNPs exhibit increased proliferation when cultured in vitro, suggestive of a role for NFIX in promoting GNP differentiation. At a mechanistic level, profiling analyses using both ChIP-seq and RNA-seq identified the actin-associated factor intersectin 1 as a downstream target of NFIX during cerebellar development. In support of this, mice lacking intersectin 1 also displayed delayed GNP differentiation. Collectively, these findings highlight a key role for NFIX and intersectin 1 in the regulation of cerebellar development.

The Transcriptional Regulator SnoN Promotes the Proliferation of Cerebellar Granule Neuron Precursors in the Postnatal Mouse Brain.

Control of neuronal precursor cell proliferation is essential for normal brain development, and deregulation of this fundamental developmental event contributes to brain diseases. Typically, neuronal precursor cell proliferation extends over long periods of time during brain development. However, how neuronal precursor proliferation is regulated in a temporally specific manner remains to be elucidated. Here, we report that conditional KO of the transcriptional regulator SnoN in cerebellar granule neuron precursors robustly inhibits the proliferation of these cells and promotes their cell cycle exit at later stages of cerebellar development in the postnatal male and female mouse brain. In laser capture microdissection followed by RNA-Seq, designed to profile gene expression specifically in the external granule layer of the cerebellum, we find that SnoN promotes the expression of cell proliferation genes and concomitantly represses differentiation genes in granule neuron precursors in vivo Remarkably, bioinformatics analyses reveal that SnoN-regulated genes contain binding sites for the transcription factors N-myc and Pax6, which promote the proliferation and differentiation of granule neuron precursors, respectively. Accordingly, we uncover novel physical interactions of SnoN with N-myc and Pax6 in cells. In behavior analyses, conditional KO of SnoN impairs cerebellar-dependent learning in a delayed eye-blink conditioning paradigm, suggesting that SnoN-regulation of granule neuron precursor proliferation bears functional consequences at the organismal level. Our findings define a novel function and mechanism for the major transcriptional regulator SnoN in the control of granule neuron precursor proliferation in the mammalian brain.SIGNIFICANCE STATEMENT This study reports the discovery that the transcriptional regulator SnoN plays a crucial role in the proliferation of cerebellar granule neuron precursors in the postnatal mouse brain. Conditional KO of SnoN in granule neuron precursors robustly inhibits the proliferation of these cells and promotes their cycle exit specifically at later stages of cerebellar development, with biological consequences of impaired cerebellar-dependent learning. Genomics and bioinformatics analyses reveal that SnoN promotes the expression of cell proliferation genes and concomitantly represses cell differentiation genes in vivo Although SnoN has been implicated in distinct aspects of the development of postmitotic neurons, this study identifies a novel function for SnoN in neuronal precursors in the mammalian brain.

A newly generated neuronal cell model of CLN7 disease reveals aberrant lysosome motility and impaired cell survival

Mutations in the CLN7/MFSD8 gene encoding the lysosomal membrane protein CLN7 are causative of CLN7 disease, an inherited neurodegenerative disorder that typically affects children. To gain insight into the pathomechanisms of CLN7 disease, we established an immortalized cell line based on cerebellar (Cb) granule neuron precursors isolated from Cln7−/− mice. Here, we demonstrate that Cln7-deficient neuron-derived Cb cells display an abnormal phenotype that includes increased size and defective outward movement of late endosomes and lysosomes as well as impaired lysosomal exocytosis. Whereas Cln7−/− Cb cells appeared to be autophagy-competent, loss of Cln7 resulted in enhanced cell death under prolonged nutrient deprivation. Furthermore, reduced cell survival of Cln7-deficient cells was accompanied by a significantly impaired protein kinase B/Akt phosphorylation at Ser473 during long-term starvation. In summary, our data demonstrate for the first time that the putative lysosomal transporter CLN7 is relevant for lysosome motility and plays an important role for neuronal cell survival under conditions of starvation.

Abstract 4134: HELLS is upregulated in Sonic hedgehog-associated medulloblastoma and proliferating cerebellar progenitors in a YAP-dependent manner

Abstract Aberrant hedgehog signaling has been implicated in many human cancers, including medulloblastoma (MB), the most common malignant pediatric brain tumor. In fact, 30% of MB tumors are driven by aberrant activity of the Sonic hedgehog (SHH) signaling pathway. Our efforts focus on unraveling the downstream components of this pathway to better understand medulloblastoma and better inform therapeutic treatment development and decisions. While some of the signaling alterations in SHH MB are due to gene mutations or amplifications, others may have their roots in epigenetics. Therefore, we examined expression levels of several candidate epigenetic regulators in our systems and identified lymphoid-specific helicase (HELLS) as a gene whose expression is markedly induced by SHH. HELLS is a unique member of the SNF2 family of chromatin remodelers with multiple epigenetic functions in DNA methylation, histone acetylation and methylation, and chromatin remodeling. Additional roles in transcription activation and DNA repair have also been reported. Of interest, HELLS was shown to delay senescence by inhibiting the expression of CDKN2A, the genetic locus of P16INK4A, a key tumor suppressor whose inactivation has recently been reported as critical to MB progression. Confirming our initial finding, we observed considerably higher levels of HELLS in primary cultures of cerebellar granule neuron precursors (CGNPs) treated with SHH and in SmoA1 mouse MB tumor tissue when compared to adjacent normal cerebellum. Our preliminary analysis of a large cohort of MB patients also indicates overexpression of HELLS in human SHH MB. Bioinformatic promoter analysis and experiments with SHH pathway inhibitors suggest regulation of HELLS expression through members of the SHH proliferation program. Downstream effectors of SHH include GLI1/2 and YAP1. Inhibition of GLI1 and GLI2 with Gant61 in both CGNPs and cultured mouse MB cells resulted in a reduction of HELLS, but an increase of apoptosis markers may indicate this reduction is due to cell death rather than specific downregulation of HELLS. In contrast, verteporfin, an inhibitor of the interaction between the transcriptional co-activator YAP1 and the transcription factor TEAD1, resulted in a reduction of HELLS at the mRNA and protein level without a concomitant increase of apoptosis markers. These results suggest that transcriptional upregulation of HELLS in mouse cerebellar progenitors and MB tumors is mediated by the SHH effector YAP1. Experiments to confirm direct involvement of YAP1 in HELLS gene transcription are underway. Future studies to understand the role of HELLS in SHH MB have the potential to provide a better understanding of the disease and new avenues for development of targeted treatments to improve the lives of these patients. Citation Format: M. Hope Robinson, Anna M. Kenney. HELLS is upregulated in Sonic hedgehog-associated medulloblastoma and proliferating cerebellar progenitors in a YAP-dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4134.