Cerebellar Granule Neuron Progenitors Research Articles

TMOD-19. GERMLINEELP1 DEFICIENCY SENSITIZES CEREBELLAR GRANULE NEURON PROGENITORS TO SHH MEDULLOBLASTOMA

Abstract Germline loss-of-function (LOF) variants in Elongator complex protein 1 (ELP1) are the most prevalent predisposing genetic events observed in medulloblastoma (MB), accounting for 30% of the Sonic Hedgehog 3 subtype (SHH-3). Molecularly, ELP1-associated SHH-MBs acquire somatic PTCH1 mutations in >80% of cases, and universal loss-of-heterozygosity of the wild-type ELP1 and PTCH1 alleles through loss of chromosome-arm 9q, resulting in their biallelic inactivation. Notably, germline ELP1 LOF occurs mutually exclusive with somatic/germline TP53 mutations in the SHH-3 subtype, suggesting that genetic perturbation of either ELP1 or TP53 may promote similar downstream oncogenic consequences in cerebellar granule neuron progenitors (GNPs), the accepted cellular origin of SHH-MB. Despite these findings, the molecular, biochemical, and cellular mechanism(s) by which ELP1-deficiency provokes malignant pathogenesis remain unknown. In this study, we sought to close this knowledge gap and functionally determine why children harboring pathogenic ELP1 germline variants are at an increased risk of developing SHH-MB. Mice were genetically engineered to mimic heterozygous Elp1 LOF (Elp1HET). We studied the effect of loss of ELP1 in GNPs using a combination of molecular profiling, immunophenotyping, and cellular assays. GNPs from Elp1HET exhibited a spectrum of molecular and biochemical hallmarks of malignant transformation including increased DNA replication stress, DNA damage, accelerated cell cycle progression, and stalled differentiation. Orthotopic transplantation of Elp1HET GNPs engineered to harbor somatic Ptch1 inactivation yielded SHH-MB-like tumors with compromised p53 signaling, providing an explanation for the specificity of ELP1-associated tumors in SHH-3, and their exclusivity with TP53-mutant tumors. Treatment of ELP1-mutant patient-derived xenografts with an FDA-approved MDM2 inhibitor reactivated p53-dependent apoptosis and extended survival. Collectively, our findings functionally substantiate the role of ELP1 deficiency in predisposition to SHH-3 MB and nominate therapeutic strategies to overcome p53 inhibition as a rational treatment option.

Fibrinogen inhibits sonic hedgehog signaling and impairs neonatal cerebellar development after blood–brain barrier disruption

Cerebellar injury in preterm infants with central nervous system (CNS) hemorrhage results in lasting neurological deficits and an increased risk of autism. The impact of blood-induced pathways on cerebellar development remains largely unknown, so no specific treatments have been developed to counteract the harmful effects of blood after neurovascular damage in preterm infants. Here, we show that fibrinogen, a blood-clotting protein, plays a central role in impairing neonatal cerebellar development. Longitudinal MRI of preterm infants revealed that cerebellar bleeds were the most critical factor associated with poor cerebellar growth. Using inflammatory and hemorrhagic mouse models of neonatal cerebellar injury, we found that fibrinogen increased innate immune activation and impeded neurogenesis in the developing cerebellum. Fibrinogen inhibited sonic hedgehog (SHH) signaling, the main mitogenic pathway in cerebellar granule neuron progenitors (CGNPs), and was sufficient to disrupt cerebellar growth. Genetic fibrinogen depletion attenuated neuroinflammation, promoted CGNP proliferation, and preserved normal cerebellar development after neurovascular damage. Our findings suggest that fibrinogen alters the balance of SHH signaling in the neurovascular niche and may serve as a therapeutic target to mitigate developmental brain injury after CNS hemorrhage.

Cerebellar granular neuron progenitors exit their germinative niche via BarH-like1 activity mediated partly by inhibition of T-cell factor.

Cerebellar granule neuron progenitors (GNPs) originate from the upper rhombic lip (URL), a germinative niche in which developmental defects produce human diseases. T-cell factor (TCF) responsiveness and Notch dependence are hallmarks of self-renewal in neural stem cells. TCF activity, together with transcripts encoding proneural gene repressors hairy and enhancer of split (Hes/Hey), are detected in the URL; however, their functions and regulatory modes are undeciphered. Here, we established amphibian as a pertinent model for studying vertebrate URL development. The amphibian long-lived URL is TCF active, whereas the external granular layer (EGL) is non-proliferative and expresses hes4 and hes5 genes. Using functional and transcriptomic approaches, we show that TCF activity is necessary for URL emergence and maintenance. We establish that the transcription factor Barhl1 controls GNP exit from the URL, acting partly through direct TCF inhibition. Identification of Barhl1 target genes suggests that, besides TCF, Barhl1 inhibits transcription of hes5 genes independently of Notch signaling. Observations in amniotes suggest a conserved role for Barhl in maintenance of the URL and/or EGL via co-regulation of TCF, Hes and Hey genes.

MDB-13. GERMLINEELP1 DEFICIENCY SENSITIZES CEREBELLAR GRANULE NEURON PROGENITORS TO SHH MEDULLOBLASTOMA

Abstract BACKGROUND Germline loss-of-function (LOF) variants in Elongator complex protein 1 (ELP1) are the most prevalent predisposing genetic events observed in medulloblastoma (MB), accounting for 30% of the Sonic Hedgehog 3 subtype (SHH-3). Molecularly, ELP1-associated SHH-MBs acquire somatic PTCH1 mutations in >80% of cases, and universal loss-of-heterozygosity of the wild-type ELP1 and PTCH1 alleles through loss of chromosome-arm 9q, resulting in their biallelic inactivation. Notably, germline ELP1 LOF occurs mutually exclusive with somatic/germline TP53 mutations in the SHH-3 subtype, suggesting that genetic perturbation of either ELP1 or TP53 may promote similar tumorigenic consequences in cerebellar granule neuron progenitors (GNPs), the accepted cellular origin of SHH-MB. Despite these findings, the molecular, biochemical, and cellular mechanism(s) by which ELP1-deficiency provokes malignant pathogenesis remain unknown. In this study, we sought to close this knowledge gap and functionally determine why children harboring pathogenic ELP1 germline variants are at an increased risk of developing SHH-MB. METHODS Mice were genetically engineered to mimic heterozygous Elp1 LOF (Elp1HET). We studied the effect of Elp1 LOF in GNPs using a combination of molecular profiling, immunophenotyping, and cellular assays. RESULTS GNPs from Elp1HET mice exhibited a spectrum of molecular and biochemical hallmarks of malignant transformation including increased DNA replication stress, DNA damage, accelerated cell cycle progression, and stalled differentiation. Orthotopic transplantation of Elp1HET GNPs engineered to harbor somatic Ptch1 inactivation yielded SHH-MB-like tumors with compromised p53 signaling, providing an explanation for the specificity of ELP1-associated tumors in SHH-3, and their exclusivity with TP53-mutant tumors. Treatment of ELP1-mutant patient-derived xenografts with an FDA-approved MDM2 inhibitor reactivated p53-dependent apoptosis and extended survival. CONCLUSIONS Collectively, our findings functionally substantiate the role of ELP1 deficiency in predisposition to SHH-3 MB and nominate therapeutic strategies to overcome p53 inhibition as a rational treatment option.

Dual and opposing roles for the kinesin-2 motor, KIF17, in Hedgehog-dependent cerebellar development.

While the kinesin-2 motors KIF3A and KIF3B have essential roles in ciliogenesis and Hedgehog (HH) signal transduction, potential role(s) for another kinesin-2 motor, KIF17, in HH signaling have yet to be explored. Here, we investigated the contribution of KIF17 to HH-dependent cerebellar development, where Kif17 is expressed in both HH-producing Purkinje cells and HH-responding cerebellar granule neuron progenitors (CGNPs). Germline Kif17 deletion in mice results in cerebellar hypoplasia due to reduced CGNP proliferation, a consequence of decreased HH pathway activity mediated through decreased Sonic HH (SHH) protein. Notably, Purkinje cell-specific Kif17 deletion partially phenocopies Kif17 germline mutants. Unexpectedly, CGNP-specific Kif17 deletion results in the opposite phenotype-increased CGNP proliferation and HH target gene expression due to altered GLI transcription factor processing. Together, these data identify KIF17 as a key regulator of HH-dependent cerebellar development, with dual and opposing roles in HH-producing Purkinje cells and HH-responding CGNPs.

MDB-23. ELP1 GERMLINE DEFICIENCY SENSITIZES THE GRANULE NEURON LINEAGE TO SHH MEDULLOBLASTOMA AND EXPOSES NOVEL THERAPEUTIC VULNERABILITIES

Abstract Germline loss-of-function (LOF) variants in Elongator complex protein 1 (ELP1) are the most prevalent predisposing genetic events in childhood medulloblastoma (MB). ELP1 germline carriers develop SHH-MBs that exhibit coincident somatic PTCH1 mutations and universal loss-of-heterozygosity of the remaining ELP1 allele through chromosome 9q deletion. The molecular, biochemical, and cellular mechanisms by which germline ELP1/Elongator deficiency contribute to SHH-MB tumorigenesis remain largely unknown. Herein, we report that mice engineered to mimic germline Elp1 LOF (i.e., Elp1HET) seen in SHH-MB patients exhibit hallmark features of premalignancy events in cycling cerebellar granule neuron progenitors (GNPs), the lineage-of-origin for SHH-MB. Compared to wild-type counterparts, Elp1HET GNPs exhibit increased replication stress-associated DNA damage, homologous recombination-associated genomic instability, accelerated cell cycle kinetics, reduced p53-dependent apoptosis in response to genotoxic stress, and slowed differentiation. Orthotopic transplantation of Elp1HET GNPs harboring somatic Ptch1 inactivation into the cerebella of immunocompromised mice promotes onset of SHH-MB tumors with incomplete penetrance that exhibit reduced p53 transcriptional activity through a currently unknown mechanism(s). Concomitant Elp1 and Ptch1 gene targeting in p53-null GNPs reproduces highly penetrant cerebellar tumors recapitulating the molecular and phenotypic features of ELP1-associated SHH-MB. Finally, reactivation of the p53 pathway through preclinical treatment with an MDM2 inhibitor promotes cell death and prolongs the survival of patient-derived xenograft tumor (PDX) models harboring deleterious ELP1 mutations. Together, our findings reveal that germline Elp1 LOF heightens genomic instability and malignant transformation in cycling GNPs, providing a mechanistic model for the subgroup-restricted pattern of predisposition associated with pathogenic ELP1 germline carriers. These results provide essential mechanistic insight into the molecular and cellular basis of SHH-MB predisposition driven by ELP1 LOF and nominate therapies that overcome p53 pathway inhibition as a rational treatment option for affected children.

Trisomy 21 induces pericentrosomal crowding delaying primary ciliogenesis and mouse cerebellar development.

Trisomy 21, the genetic cause of Down syndrome, disrupts primary cilia formation and function, in part through elevated Pericentrin, a centrosome protein encoded on chromosome 21. Yet how trisomy 21 and elevated Pericentrin disrupt cilia-related molecules and pathways, and the in vivo phenotypic relevance remain unclear. Utilizing ciliogenesis time course experiments combined with light microscopy and electron tomography, we reveal that chromosome 21 polyploidy elevates Pericentrin and microtubules away from the centrosome that corral MyosinVA and EHD1, delaying ciliary membrane delivery and mother centriole uncapping essential for ciliogenesis. If given enough time, trisomy 21 cells eventually ciliate, but these ciliated cells demonstrate persistent trafficking defects that reduce transition zone protein localization and decrease sonic hedgehog signaling in direct anticorrelation with Pericentrin levels. Consistent with cultured trisomy 21 cells, a mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers at P4. Our work reveals that elevated Pericentrin from trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding mechanism results in signaling deficiencies consistent with the neurological phenotypes found in individuals with Down syndrome.

PRC2 disruption in cerebellar progenitors produces cerebellar hypoplasia and aberrant myoid differentiation without blocking medulloblastoma growth

We show that Polycomb Repressive Complex-2 (PRC2) components EED and EZH2 maintain neural identity in cerebellar granule neuron progenitors (CGNPs) and SHH-driven medulloblastoma, a cancer of CGNPs. Proliferating CGNPs and medulloblastoma cells inherit neural fate commitment through epigenetic mechanisms. The PRC2 is an epigenetic regulator that has been proposed as a therapeutic target in medulloblastoma. To define PRC2 function in cerebellar development and medulloblastoma, we conditionally deleted PRC2 components Eed or Ezh2 in CGNPs and analyzed medulloblastomas induced in Eed-deleted and Ezh2-deleted CGNPs by expressing SmoM2, an oncogenic allele of Smo. Eed deletion destabilized the PRC2, depleting EED and EZH2 proteins, while Ezh2 deletion did not deplete EED. Eed-deleted cerebella were hypoplastic, with reduced proliferation, increased apoptosis, and inappropriate muscle-like differentiation. Ezh2-deleted cerebella showed similar, milder phenotypes, with fewer muscle-like cells and without reduced growth. Eed-deleted and Ezh2-deleted medulloblastomas both demonstrated myoid differentiation and progressed more rapidly than PRC2-intact controls. The PRC2 thus maintains neural commitment in CGNPs and medulloblastoma, but is not required for SHH medulloblastoma progression. Our data define a role for the PRC2 in preventing inappropriate, non-neural fates during postnatal neurogenesis, and caution that targeting the PRC2 in SHH medulloblastoma may not produce durable therapeutic effects.

Conventional and Spectral Karyotyping of Murine Cerebellar Granule Neuron Progenitors.

Karyotyping remains an invaluable tool to researchers exploring the cause and consequence of genomic instability in biologic systems. It allows investigators to survey the entire chromosome complement in individual cells and in a single experiment, visualize, and measure different forms or features of instability such as aneuploidy, ongoing chromosomal instability, DNA damage/mis-repair, telomere erosion, chromosome mis-segregation, or defects in cell cycle progression. This chapter describes the combined use of conventional (DAPI-banding) and spectral karyotyping (SKY) to characterize genomic instability in murine cerebellar granule neuron progenitors (CGNPs), using CGNPs with conditional deletion of Atr as a positive control for chromosomal rearrangements. Protocols for preparing slides (metaphase spreads) from fixed cell suspension, DAPI-banding, and spectral karyotyping (SKY) are included. Pertinent aspects of image acquisition and analysis are detailed. These protocols can likely be adapted to other tissue types (murine or human).

Maintaining Cerebellar Granule Neuron Progenitors in Cell Culture.

In vitro studies allow the manipulation and sampling of the cellular environment. Using freshly explanted cerebellar granule neuron progenitors (CGNPs) for in vitro studies of neural progenitors avoids the potential confounding effect of culturing cell lines that have adapted to the in vitro environment. CGNPs can be cultured in vitro for up to 72h, and during this period, they will demonstrate SHH-driven proliferation that wanes over time and differentiation that increases over time, approximating their typical developmental trajectory. CGPNs in culture thus provide an ideal system for studying neural progenitor biology with the range of manipulations and analyses that are possible in vitro.

Dissociation of Cerebellar Granule Neuron Progenitors for Culture, FACS, Transcriptomics, and Molecular Biology.

Brain growth reflects the proliferation dynamics of neural progenitors, and understanding brain growth requires molecular, genetic, and functional studies of these specific cells. Cerebellar granule neuron progenitors (CGNPs) proliferate in the early postnatal period in both mice and humans, to generate the largest population of neurons in the central nervous system. CGNPs present a large, spatially segregated source of neural progenitors with a consistent, well-characterized temporal pattern of proliferation and differentiation that facilitates analysis. Dissociating of CGNPs with the methods below will generate a suspension of primary neural progenitors harvested from the postnatal brain that may be used for diverse experimental analyses including cell culture, protein extraction, flow cytometry, metabolomic analysis, and transcriptomic analysis with single-cell resolution (scRNA-seq).

Proliferation Analysis of Cerebellar Granule Neuron Progenitors for Microcephaly Research, Using Immunofluorescent Staining and Flow Cytometry.

Cell cycle progression is a vital aspect of neural development. Repeated cell division in neural progenitor populations amplifies the numbers of specific cell types and is required to prevent growth failure that manifests as microcephaly. Regulated cycling is also required for cell fate specification. Analysis of cell cycle states is a valuable tool to understand the mechanisms underlying brain growth. Here we describe the preparation of cells for immunofluorescent-stained samples and flow cytometry and how to analyze cell cycle progression and cell cycle exit in progenitors. We describe methods as applied to analysis of cerebellar granule neuron progenitors (CGNPs), but similar methods in brain sections can also be applied to other brain neural progenitor populations, such as the hippocampus and subventricular zone.

TMET-01. DISRUPTION OF THE SERINE-PRODUCING PATHWAYS SLOWS PROGRESSION OF THE SONIC HEDGEHOG-DRIVEN MEDULLOBLASTOMA

Abstract We have found that medulloblastoma metabolism is specialized to promote growth in the CNS and can be targeted for anti-tumor therapy. Prior studies show that serine is scarce in the CNS and that metastatic tumors require serine-producing enzymes to grow in the brain. We analyzed whether medulloblastoma, a primary brain tumor, showed similar serine dependency. We found that Sonic Hedgehog signaling, which induces proliferation in cerebellar granule neuron progenitors in normal development and medulloblastoma formation when hyperactivated, also induces PHGDH and SHMT1, which each catalyze key steps in different serine-production pathways. To define the functional role of PHGDH and SHMT1 in medulloblastomas, we bred mice genetically engineered to develop SHH medulloblastomas with mice carrying deletions of each of these genes. We found that mice with Phgdh-deleted medulloblastomas showed slower progression and longer survival times, compared to mice with Phgdh-intact medulloblastomas. Medulloblastomas with combined deletion of Phgdh and Shmt1 show even slower tumor progression. Stable-isotope flux analysis suggests that different serine-producing pathways compensate for the loss of individual serine-producing enzymes, providing a rationale for the greater anti-tumor effect of disrupting multiple serine-producing enzymes simultaneously. Our data show that serine-producing enzymes can be targeted as a novel approach to medulloblastoma therapy, and underscore the importance of targeting multiple serine-production pathways in complex metabolic interventions.

MEDB-42. GermlineElp1 deficiency promotes genomic instability and survival of granule neuron progenitors primed for SHH medulloblastoma pathogenesis

Germline loss-of-function (LOF) mutations in Elongator complex protein 1 (ELP1) are found in 15-20% of childhood SHH medulloblastoma (MB) and are exceedingly rare in non-SHH-MB or other cancers. ELP1 germline carriers that develop SHH-MB harbor frequent somatic PTCH1 mutations and universally sustain loss-of-heterozygosity of the remaining ELP1 allele through chromosome 9q deletion. ELP1 functions as a scaffolding subunit of the Elongator complex that is required for posttranscriptional modification of tRNAs and maintenance of efficient translational elongation and protein homeostasis. However, the molecular, biochemical, and cellular mechanisms by which ELP1/Elongator LOF contribute to SHH-MB tumorigenesis remain largely unknown. Herein, we report that mice harboring germline Elp1 monoallelic loss (i.e., Elp1+/-) exhibit hallmark features of malignant predisposition in developing cerebellar granule neuron progenitors (GNPs), the lineage-of-origin for SHH-MB. Elp1+/- GNPs are characterized by increased replication stress-induced DNA damage, upregulation of the homologous recombination repair pathway, aberrant cell cycle, and attenuation of p53-dependent apoptosis. CRISPR/Cas9-mediated Elp1 and Ptch1 gene targeting in mouse GNPs reproduces highly penetrant SHH-MB tumors recapitulating the molecular and phenotypic features of patient tumors. Reactivation of the p53 pathway through MDM2 and PAK4 inhibitors promotes selective cell death in patient-derived xenograft tumors (PDX) harboring deleterious ELP1 mutations. Together, our findings reveal that germline Elp1 deficiency heightens genomic instability and survival in GNPs, providing a mechanistic model for the subgroup-restricted pattern of predisposition and malignancy associated with pathogenic ELP1 germline carriers. These results provide rationale for further preclinical studies evaluating drugs that overcome p53 pathway inhibition in ELP1-associated SHH-MB and a renewed outlook for improving treatment options for affected children and their families.*, # Contributed equally

The developmental stage of the medulloblastoma cell-of-origin restricts Sonic hedgehog pathway usage and drug sensitivity.

ABSTRACTSonic hedgehog (SHH) medulloblastoma originates from the cerebellar granule neuron progenitor (CGNP) lineage, which depends on Hedgehog signaling for its perinatal expansion. Whereas SHH tumors exhibit overall deregulation of this pathway, they also show patient age-specific aberrations. To investigate whether the developmental stage of the CGNP can account for these age-specific lesions, we analyzed developing murine CGNP transcriptomes and observed highly dynamic gene expression as a function of age. Cross-species comparison with human SHH medulloblastoma showed partial maintenance of these expression patterns, and highlighted low primary cilium expression as hallmark of infant medulloblastoma and early embryonic CGNPs. This coincided with reduced responsiveness to upstream SHH pathway component Smoothened, whereas sensitivity to downstream components SUFU and GLI family proteins was retained. Together, these findings can explain the preference for SUFU mutations in infant medulloblastoma and suggest that drugs targeting the downstream SHH pathway will be most appropriate for infant patients.

Neuronal Polarity Pathways as Central Integrators of Cell-Extrinsic Information During Interactions of Neural Progenitors With Germinal Niches.

Germinal niche interactions and their effect on developing neurons have become the subject of intense investigation. Dissecting the complex interplay of cell-extrinsic and cell-intrinsic factors at the heart of these interactions reveals the critical basic mechanisms of neural development and how it goes awry in pediatric neurologic disorders. A full accounting of how developing neurons navigate their niches to mature and integrate into a developing neural circuit requires a combination of genetic characterization of and physical access to neurons and their supporting cell types plus transformative imaging to determine the cell biological and gene-regulatory responses to niche cues. The mouse cerebellar cortex is a prototypical experimental system meeting all of these criteria. The lessons learned therein have been scaled to other model systems and brain regions to stimulate discoveries of how developing neurons make many developmental decisions. This review focuses on how mouse cerebellar granule neuron progenitors interact with signals in their germinal niche and how that affects the neuronal differentiation and cell polarization programs that underpin lamination of the developing cerebellum. We show how modeling of these mechanisms in other systems has added to the growing evidence of how defective neuronal polarity contributes to developmental disease.

Stem cell differentiation with consistent lineage commitment induced by a flash of ultrafast-laser activationin vitroand in vivo.

Recent technological advancements on stem cell differentiation induction have been making great progress in stem cell research, regenerative medicine, and therapeutic applications. However, the risk of off-target differentiation limits the wide application of stem cell therapy strategies. Here, we report a non-invasive all-optical strategy to induce stem cell differentiation invitro and invivo that activates individual target stem cells in situ by delivering a transient 100-ms irradiation of a tightly focused femtosecond laser to a submicron cytoplasmic region of primary adipose-derived stem cells (ADSCs). The ADSCs differentiate to osteoblasts with stable lineage commitment that cannot further transdifferentiate because of simultaneous initiation of multiple signaling pathways through specific Ca2+ kinetic patterns. This method can work invivo to direct mouse cerebellar granule neuron progenitors to granule neurons in intact mouse cerebellums through the skull. Hence, this optical method without any genetic manipulations or exogenous biomaterials holds promising potential in biomedical research and cell-based therapies.

Deletion of CHD8 in cerebellar granule neuron progenitors leads to severe cerebellar hypoplasia, ataxia, and psychiatric behavior in mice

CHD8 is a candidate gene for autism spectrum disorders and neurological development delay. It has been reported to be essential for neurogenesis in the cerebral cortex, but the function of CHD8 in cerebellum has not been comprehensively investigated. The potential relationship of cerebellum dysplasia with psychiatric disorders in patients with CHD8 mutations is still not clear. In this study, we establish different conditional knockout mouse models to investigate the roles of CHD8 in cerebellar development. Mice with neural stem cell-specific Chd8 deletion exhibit significant reduction of cerebellum volume and no layering structure is detected. Genetic deletion of Chd8 in cerebellar granule neuron progenitors (GNPs) leads to cerebellar hypoplasia, absent of proliferation layer and ectopic of Purkinje neuron. However, no substantial cerebellar dysplasia is detected in mice with Purkinje neuron- or oligodendrocyte-specific Chd8 ablation. Single-cell RNA sequencing indicates that ribosome-related genes and pathways are most significantly disrupted in GNPs, indicating the potential mechanism. Importantly, in addition to the ataxia phenotype, mice with GNP-specific Chd8 ablation present a neuropsychiatric phenotype in three-chamber and light/dark tests. Taken together, our results provide insights not only into the function of CHD8 in cerebellar development, but also the pathogenesis of neuropsychiatric disorders in patients with CHD8 mutations.

Sequential stabilization of RNF220 by RLIM and ZC4H2 during cerebellum development and Shh-group medulloblastoma progression.

ABSTRACTSonic hedgehog (Shh) signaling is essential for the proliferation of cerebellar granule neuron progenitors (CGNPs), and its misregulation is linked to various disorders, including cerebellar cancer medulloblastoma (MB). During vertebrate neural development, RNF220, a ubiquitin E3 ligase, is involved in spinal cord patterning by modulating the subcellular location of glioma-associated oncogene homologs (Glis) through ubiquitination. RNF220 is also required for full activation of Shh signaling during cerebellum development in an epigenetic manner through targeting embryonic ectoderm development. ZC4H2 was reported to be involved in spinal cord patterning by acting as an RNF220 stabilizer. Here, we provided evidence to show that ZC4H2 is also required for full activation of Shh signaling in CGNP and MB progression by stabilizing RNF220. In addition, we found that the ubiquitin E3 ligase RING finger LIM domain-binding protein (RLIM) is responsible for ZC4H2 stabilization via direct ubiquitination, through which RNF220 is also thus stabilized. RLIM is a direct target of Shh signaling and is also required for full activation of Shh signaling in CGNP and MB cell proliferation. We further provided clinical evidence to show that the RLIM‒ZC4H2‒RNF220 cascade is involved in Shh-group MB progression. Disease-causative human RLIM and ZC4H2 mutations affect their interaction and regulation. Therefore, our study sheds light on the regulation of Shh signaling during cerebellar development and MB progression and provides insights into neural disorders caused by RLIM or ZC4H2 mutations.

CBIO-23. ANTIAPOPTOTIC Bcl-xL RESTRICTS APOPTOSIS IN SHH MEDULLOBLASTOMA AND PROMOTES PROGRESSION

Abstract Medulloblastomas in most patients are distinctively sensitive to radiation therapy, but the mechanisms that mediate this sensitivity are unclear. Current treatments still fail 20%-60% of patients with SHH medulloblastoma and can leave survivors with long-term neurocognitive and social deficits. Understanding the mechanisms driving the typical radiation-sensitivity may identify less-toxic therapeutic strategies and provide insight into treatment failure. We previously showed that radiation sensitivity depends on the intrinsic apoptotic pathway, mediated by pro-apoptotic BAX. In cerebellar granule neuron progenitors (CGNPs), the cell of origin for SHH medulloblastoma, BAX activity is directly inhibited by anti-apoptotic BCL-xL; Bcl-xL-deleted CGNPs undergo spontaneous apoptosis. To test the therapeutic potential of disrupting BCL-xL in medulloblastoma, we conditionally deleted Bcl-xL in mice genetically engineered to develop SHH medulloblastoma. Here, I show that Bcl-xL deletion slows SHH medulloblastoma growth and prolongs survival of medulloblastoma-bearing mice. Bcl-xL-deleted tumors initially showed increased rates of spontaneous apoptosis, but this effect waned over time, suggesting the emergence of BCL-xL-independent survival mechanisms. We also noted increased microglial infiltration in Bcl-xL-deleted medulloblastomas. We hypothesize that IGF1 produced by microglia in the tumor microenvironment may be contributing to tumor resistance by upregulating translation of MCL-1, an anti-apoptotic BCL-xL homolog. IGF1 is known to upregulate translation through the mTOR pathway, while anti-apoptotic MCL-1 protein abundance is dependent upon translation regulation. Our on-going studies are testing the efficacy of pharmacologically targeting BCL-xL in mice with medulloblastoma, in combination with targeting IGF1 signaling using mTORC1 inhibitors.