Human Cerebellum Research Articles

Cerebellar GABA Change during Visuomotor Adaptation Relates to Adaptation Performance and Cerebellar Network Connectivity: A Magnetic Resonance Spectroscopic Imaging Study.

Motor adaptation is crucial for performing accurate movements in a changing environment and relies on the cerebellum. Although cerebellar involvement has been well characterized, the neurochemical changes in the cerebellum underpinning human motor adaptation remain unknown. We used a novel magnetic resonance spectroscopic imaging (MRSI) technique to measure changes in the inhibitory neurotransmitter GABA in the human cerebellum during visuomotor adaptation. Participants (n = 17, six female) used their right hand to adapt to a rotated cursor in the scanner, compared with a control task requiring no adaptation. We spatially resolved adaptation-driven GABA changes at the cerebellar nuclei and cerebellar cortex in the left and the right cerebellar hemisphere independently and found that simple right-hand movements increase GABA in the right cerebellar nuclei and decreases GABA in the left. When isolating adaptation-driven GABA changes, we found that GABA in the left cerebellar nuclei and the right cerebellar nuclei diverged, although GABA change from baseline at the right cerebellar nuclei was not different from zero at the group level. Early adaptation-driven GABA fluctuations in the right cerebellar nuclei correlated with adaptation performance. Participants showing greater GABA decrease adapted better, suggesting early GABA change is behaviorally relevant. Early GABA change also correlated with functional connectivity change in a cerebellar network. Participants showing greater decreases in GABA showed greater strength increases in cerebellar network connectivity. Results were specific to GABA, to adaptation, and to the cerebellar network. This study provides first evidence for plastic changes in cerebellar neurochemistry during motor adaptation. Characterizing these naturally occurring neurochemical changes may provide a basis for developing therapeutic interventions to facilitate human motor adaptation.SIGNIFICANCE STATEMENT Despite motor adaptation being fundamental to maintaining accurate movements, its neurochemical basis remains poorly understood, perhaps because measuring neurochemicals in the human cerebellum is technically challenging. Using a novel magnetic resonance spectroscopic imaging method, this study provides evidence for GABA changes in the left compared with the right cerebellar nuclei driven by both simple movement and motor adaptation. Although right cerebellar GABA changes were not significantly different from zero at the group level, the adaptation-driven GABA fluctuations in the right cerebellar nuclei correlated with adaptation performance and with functional connectivity change in a cerebellar network. These results show the first evidence for plastic changes in cerebellar neurochemistry during a cerebellar learning task. This provides the basis for developing therapeutic interventions that facilitate these naturally occurring changes to amplify cerebellar-dependent learning.

Three-Dimensional Modeling and Augmented Reality and Virtual Reality Simulation of Fiber Dissection of the Cerebellum and Brainstem.

Surgeons must understand the complex anatomy of the cerebellum and brainstem and their 3-dimensional (3D) relationships with each other for surgery to be successful. To the best of our knowledge, there have been no fiber dissection studies combined with 3D models, augmented reality (AR), and virtual reality (VR) of the structure of the cerebellum and brainstem. In this study, we created freely accessible AR and VR simulations and 3D models of the cerebellum and brainstem. To create 3D models and AR and VR simulations of cadaveric dissections of the human cerebellum and brainstem and to examine the 3D relationships of these structures. Ten cadaveric cerebellum and brainstem specimens were prepared in accordance with the Klingler's method. The cerebellum and brainstem were dissected under the operating microscope, and 2-dimensional and 3D images were captured at every stage. With a photogrammetry tool (Qlone, EyeCue Vision Technologies, Ltd.), AR and VR simulations and 3D models were created by combining several 2-dimensional pictures. For the first time reported in the literature, high-resolution, easily accessible, free 3D models and AR and VR simulations of cerebellum and brainstem dissections were created. Fiber dissection of the cerebellum-brainstem complex and 3D models with AR and VR simulations are a useful addition to the goal of training neurosurgeons worldwide.

Developmentally Arrested Basket/Stellate Cells in Postnatal Human Brain as Potential Tumor Cells of Origin for Cerebellar Hemangioblastoma in von Hippel-Lindau Patients.

von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary cancer disorder caused by a germline mutation in the VHL tumor suppressor gene. Loss of the wild-type allele results in VHL deficiency and the potential formation of cerebellar hemangioblastomas, which resemble embryonic hemangioblast proliferation and differentiation processes. Multiple, microscopic, VHL-deficient precursors, termed developmentally arrested structural elements (DASEs), consistently involve the cerebellar molecular layer in VHL patients, indicating the tumor site of origin. Unlike hemangioblastomas, however, cerebellar DASEs do not express brachyury, a mesodermal marker for hemangioblasts. In this study, neuronal progenitors occupying the molecular layer were investigated as tumor cells of origin. By immunohistochemistry, cerebellar DASEs and hemangioblastomas lacked immunoreactivity with antibody ZIC1 (Zic family member 1), a granule cell progenitor marker with concordance from oligonucleotide RNA expression array analyses. Rather, cerebellar DASEs and hemangioblastomas were immunoreactive with antibody PAX2 (paired box 2), a marker of basket/stellate cell progenitors. VHL cerebellar cortices also revealed PAX2-positive cells in Purkinje and molecular layers, resembling the histological and molecular development of basket/stellate cells in postnatal non-VHL mouse and human cerebella. These data suggest that VHL deficiency can result in the developmental arrest of basket/stellate cells in the human cerebellum and that these PAX2-positive, initiated cells await another insult or signal to form DASEs and eventually, tumors.

A multifaceted gradient in human cerebellum of structural and functional development

The organization of the basic tissue and functional properties of the cerebellum across development is unknown. Combining several large datasets, we demonstrate in the human cerebellum a static tissue gradient in adults that mirrors a similar growth-rate gradient across development. Quantitative tissue metrics corroborate unique densities of certain lipids and proteins among lobules, and cerebellar structural development closely follows cerebellar functional properties through childhood.

Phenotypic, metabolic, and biogenesis properties of human stem cell-derived cerebellar spheroids

Human cerebellum consists of high density and complexity of neurons. Thus, it is challenging to differentiate cerebellar-like organoids with similar cellular markers and function to the human brain. Our previous study showed that the combination of retinoic acid (RA), Wingless/integrated (Wnt) activator, and Sonic Hedgehog (SHH) activator promotes cerebellar differentiation from human induced pluripotent stem cells (hiPSCs). This study examined phenotypic, metabolic, and biogenesis in early cerebellar development. Cerebellum spheroids were differentiated from human iPSK3 cells. During day 7–14, RA and Wnt activator CHIR99021 were used and SHH activator purmorphamine (PMR) was added later to promote ventralization. Gene expression for early cerebellar layer markers, metabolism, and extracellular vesicle (EV) biogenesis were characterized. Zinc-induced neurotoxicity was investigated as a proof-of-concept of neurotoxicity study. Flow cytometry results showed that there was no significant difference in NEPH3, PTF1A, OLIG2, and MATH1 protein expression between RCP (RA-CHIR-PMR) versus the control condition. However, the expression of cerebellar genes for the molecular layer (BHLE22), the granule cell layer (GABRB2, PAX6, TMEM266, KCNIP4), the Bergmann glial cells (QK1, DAO), and the Purkinje cell layer (ARHGEF33, KIT, MX1, MYH10, PPP1R17, SCGN) was significantly higher in the RCP condition than the control. The shift in metabolic pathways toward glycolysis was observed for RCP condition. The EV biogenesis marker expression was retained. Mild zinc-induced neurotoxicity may exist when zinc exposure exceeds 1.0 µM. RCP treatment can promote specific cerebellar-like differentiation from hiPSCs indicated by gene expression of early cerebellar markers and regionally enriched genes. The higher cerebellar marker expression is accompanied by the elevated glycolysis with the retained EV biogenesis. This study should advance the understanding of biomarkers during early cerebellar development for cerebellum organoid engineering and neurotoxicity study.

The Discovery of Anti-Yo (Anti-PCA1) Antibody in Patients with Paraneoplastic Cerebellar Degeneration: Opening a Window into Autoimmune Neurological Disease.

Prior to 1982, ovarian and certain other cancers were known to have a rare complication of progressive cerebellar ataxia, a disorder characterized pathologically by severe-often total-obliteration of cerebellar Purkinje cells. However, the cause of cerebellar injury in these patients was unknown. In that year, we began studies in which sera from individuals with this disorder were reacted with frozen sections of human cerebellum. These studies revealed that patients with ovarian cancer and cerebellar degeneration had high titers of antibodies directed against cytoplasmic antigens of Purkinje cells and deep cerebellar nuclei-a previously undescribed pattern of antibody response which was subsequently found not to be present in ovarian cancer patients who remained neurologically normal. This antibody, now known as "anti-Yo" or "anti-PCA1" provides a marker for rapidly progressive cerebellar ataxia and is heavily associated with gynecological and breast malignancies. Although the role of anti-Yo antibody in cerebellar injury has not been established in living animals, in vitro studies have demonstrated that anti-Yo antibody causes Purkinje cell death in the absence of T lymphocytes. In this commentary, we describe our studies leading to initial discovery of anti-Yo antibody, discuss the relationship of this discovery to current knowledge of paraneoplastic neurological disease, and outline some important questions which remain to be resolved before we fully understand the pathogenesis and optimal treatment of this disorder.

The Cerebellar Gene Database: a Collective Database of Genes Critical for Cerebellar Development

This report presents the first comprehensive database that specifically compiles genes critical for cerebellar development and function. The Cerebellar Gene Database details genes that, when perturbed in mouse models, result in a cerebellar phenotype according to available data from both Mouse Genome Informatics and PubMed, as well as references to the corresponding studies for further examination. This database also offers a compilation of human genetic disorders with a cerebellar phenotype and their associated gene information from the Online Mendelian Inheritance in Man (OMIM) database. By comparing and contrasting the mouse and human datasets, we observe that only a small proportion of human mutant genes with a cerebellar phenotype have been studied in mouse knockout models. Given the highly conserved nature between mouse and human genomes, this surprising finding highlights how mouse genetic models can be more frequently employed to elucidate human disease etiology. On the other hand, many mouse genes identified in the present study that are known to lead to a cerebellar phenotype when perturbed have not yet been found to be pathogenic in the cerebellum of humans. This database furthers our understanding of human cerebellar disorders with yet-to-be-identified genetic causes. It is our hope that this gene database will serve as an invaluable tool for gathering background information, generating hypotheses, and facilitating translational research endeavors. Moreover, we encourage continual inputs from the research community in making this compilation a living database, one that remains up-to-date with the advances in cerebellar research.

ExoPLOT: Representation of alternative splicing in human tissues and developmental stages with transposed element (TE) involvement

Advances in RNA high-throughput sequencing and large-scale functional assays yield new insights into the multifaceted activities of transposed elements (TE) and many other previously undiscovered sequence elements. Currently, no tool for easy access, analysis, quantification, and visualization of alternatively spliced exons across multiple tissues or developmental stages is available. Also, analysis pipelines demand computational skills or hardware requirements, which often are hard to meet by wet-lab scientists. We developed ExoPLOT to enable simplified access to massive RNA high throughput sequencing datasets to facilitate the analysis of alternative splicing across many biological samples. To demonstrate the functonality of ExoPLOT, we analyzed the contributon of exonized TEs to human coding sequences (CDS). mRNA splice variants containing the TE-derived exon were quantified and compared to expression levels of TE-free splice variants. For analysis, we utilized 313 human cerebrum, cerebellum, heart, kidney, liver, ovary, and testis transcriptomes, representing various pre- and postnatal developmental stages. ExoPLOT visualizes the relative expression levels of alternative transcripts, e.g., caused by the insertion of new TE-derived exons, across different developmental stages of and among multiple tissues. This tool also provides a unique link between evolution and function during exonization (gain of a new exon) and exaptation (recruitment/co-optation) of a new exon. As input for analysis, we derived a database of 1151 repeat-masked, exonized TEs, representing all prominent families of transposons in the human genome and the collection of human consensus coding sequences (CCDS). ExoPLOT screened preprocessed RNA high-throughput sequencing datasets from seven human tissues to quantify and visualize the dynamics in RNA splicing for these 1151 TE-derived exons during the entire human organ development. In addition, we successfully mapped and analyzed 993 recently described exonized sequences from the human frontal cortex onto these 313 transcriptome libraries. ExoPLOT's approach to preprocessing RNA deep sequencing datasets facilitates alternative splicing analysis and significantly reduces processing times. In addition, ExoPLOT's design allows studying alternative RNA isoforms other than TE-derived in a customized – coordinate-based manner and is available at http://retrogenomics3.uni-muenster.de:3838/exz-plot-d/.

A Survey of the Metabolic Landscape of the Developing Cerebellum at Single-Cell Resolution

The use of cell-culture models to investigate development and disease of the cerebellum is a recent advance, facilitated by the discovery that patterning of precursors is capable of giving rise to cells with specific neuronal identity. Pluripotent stem cell-derived organoids, which exhibit self-organisational characteristics reminiscent of early cerebellar tissue, present a number of challenges including recapitulation of conditions resembling the mature brain. An understanding of the processes driving fetal and postnatal maturation is required to reproduce these conditions in vitro and advance the capability of the system to model adult-onset disease. A key tool for achieving this is single-cell RNA sequencing, which enables visualisation of key transcriptional features of subpopulations comprising tissues. Here, we explore and compare available single-cell RNA sequencing data derived from the developing human cerebellum and its synthetic, in vitro counterpart (stem cell-derived cerebellar organoids). We focus on performing a qualitative assessment of the expression of key metabolic pathway genes, given recent findings exemplifying tissue-specific metabolic activity, including hypoxia and metabolic shifts associated with neuronal expansion. Signatures indicative of known cell type-specific metabolic differences, such as the astrocyte-neuron lactate shuttle and glutamate-glutamine cycle were evident at a transcriptional level. Cerebellar tissue and cerebellar organoids showed a number of behavioural similarities, including HIF1 signalling, which may serve to drive expansion of granule cell progenitors in both settings. We further highlight numerous differences between cultured organoids and native tissue which may provide clarity on the state of metabolic state following differentiation of organoids, providing the future framework to test and further hypotheses regarding promoting maturation. Overall, this analysis provides insight into understanding the state of in vitro models of the cerebellum, a critical factor required for modelling susceptibility of various cell types to cerebellar disease.

Changes in Phospholipid Composition of the Human Cerebellum and Motor Cortex during Normal Ageing.

(1) Background: Changes in phospholipid (phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, i.e., PC, PE and PS) composition with age in the mitochondrial and microsomal membranes of the human cerebellum and motor cortex were examined and compared to previous analyses of the prefrontal cortex, hippocampus and entorhinal cortex. (2) Methods: Nano-electrospray ionization on a hybrid triple quadrupole–linear ion trap mass spectrometer was used to analyse the brain regions of subjects aged 18–104 years. (3) Results: With age, the cerebellum showed many changes in the major phospholipids (>10% of the phospholipid class). In both membrane types, these included increases in PE 18:0_22:6 and PS 18:0_22:6, decreases in PE 18:0_20:4 and PS 18:0_18:1 and an increase in PC 16:0_16:0 (microsomal membrane only). In addition, twenty-one minor phospholipids also changed. In the motor cortex, only ten minor phospholipids changed with age. With age, the acyl composition of the membranes in the cerebellum increased in docosahexaenoic acid (22:6) and decreased in the arachidonic (20:4) and adrenic (22:4) acids. A comparison of phospholipid changes in the cerebellum, motor cortex and other brain areas is provided. (4) Conclusions: The cerebellum is exceptional in the large number of major phospholipids that undergo changes (with consequential changes in acyl composition) with age, whereas the motor cortex is highly resistant to change.

Abstract 5977: Discovery and preclinical validation of [11C]AZ3391: A first in class blood-brain barrier permeable, subtype selective PARP-1 PET radioligand

Abstract Positron emission tomography (PET) is a molecular imaging technique used to study the distribution of radiolabeled tracer molecules in vivo in a non-invasive fashion. We here report the discovery and preclinical validation of a novel PET tracer for PARP1. Its exquisite selectivity and high contrast in vivo opens up unique opportunities for exploring target engagement and relationships between drug exposure in plasma and PARP1 occupancy during clinical development of PARP1 inhibitors. AZ3391 was identified as a PET radioligand candidate from our medicinal chemistry program investigating novel PARP1 selective inhibitors and radiolabeled with carbon-11 (t1/2=20.4 min) for a series of in vitro and in vivo imaging experiments. Using in vitro autoradiography, dense binding of [11C]AZ3391 was observed in tissues known to be rich in PARP1 (eg tumour xenograft; NHP and human brain cerebellum). [11C]AZ3391 binding was completely abolished by the inclusion of a high concentration of Olaparib or AZD5305 in the incubate, thus demonstrating specific binding to PARP1 in vitro. Following intravenous injection of [11C]AZ3391 in non-human primates, high binding was observed in organs known to express PARP1, eg brain, spleen and bone marrow. The radioactivity in these organs could be blocked in a dose-dependent manner after pre-treatment with increasing doses of AZD9574 (up to 0.05 mg/kg), thus demonstrating specific binding to PARP1 in vivo. This set of preclinical data supports further development of [11C]AZ3391 as a tool to assess drug induced PARP1 engagement in patients. Citation Format: Peter Johnström, Jeffrey Johannes, Andy Pike, Zsolt Cselényi, Magnus Schou. Discovery and preclinical validation of [11C]AZ3391: A first in class blood-brain barrier permeable, subtype selective PARP-1 PET radioligand [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5977.

Abstract 2505: Predicting prognosis in PFA ependymoma and group 3/4 medulloblastoma methylomes using interpretable epigenetic models

Abstract Introduction: Our goal is to develop interpretable, prognostic models for Group 3/4 medulloblastoma (G3/4 MB) and PFA ependymomas (PFA-EP) using brain tumor DNA methylomes. Our models use prior knowledge of genome regulation and may identify cellular processes that help develop molecular therapies. We previously developed a classifier algorithm, netDx, which uses similarity networks to integrate heterogeneous patient data and predict outcome1. netDx demonstrates excellent performance and allows measures to be grouped into biologically meaningful features (e.g. grouping genes into pathway features). Ependymoma Methods: We predicted survival in PFA-EP using 569 methylomes2 (Illumina 450K). Using netDx, we evaluated a model that grouped CpG-level methylation into sets reflecting 25 cell types from the developing human cerebellum, 3 chromatin states, EZH2 binding sites, and brain super enhancers (80:20 train/test split, feature selection >=8/10; 10 splits). This model was compared to a baseline lacking prior knowledge. Preliminary Results: Organizing methylomes by prior knowledge significantly improves prognostic prediction (Table 1, p < 5x10-3, one-sided WMW). Features that predict prognosis are consistent with known dysregulation in PFA-EP2 (Table 1). G3/4 MB Methods: We predicted binarized survival using 285 methylomes3 (Illumina 450K), using identical methods as above. Preliminary results: Tumor methylomes carry predictive signal for survival prediction (Table 1), consistent with previous findings. We are currently evaluating the effect of including prior knowledge. Interim Conclusion: Prior knowledge can improve survival prediction in PFA-EP and identifies features reflecting tumor biology. We are interested in extending interpretable modeling to other tumours.1. Pai et al. (2019) Mol Sys Biol. 15. 2. Pajtler et al. (2018) Acta Neuropathol. 136. 3. Northcott et al. (2017) Nature 547. Table 1. Average model performance and top features. Dataset Predictor design AUPR (mean+/- SD, 10 train/test splits) Predictive features PFA-EP; 252 good/317 poor survivors2 DNAm, no prior knowledge 64.2 +/- 2.3 n/a * Covariates: CXOrf67 mutation status, sex DNAm, prior knowledge* 67.5 +/- 3.5(p < 5x10-3; one-sided WMW test compared to baseline model) CXOrf67mut, methylation in: {H3K9me3 sites; marker genes for ependymal cells of choroid plexus; marker genes for multiple interneuron classes} G3/4 MB: 146 good/139 poor survivors3 DNAm, no prior knowledge 0.61 +/- 6.3 n/a ** Covariates: age, sex DNAm, prior knowledge** In progress Citation Format: Indy Ng, Alexander Fricke, Shraddha Pai. Predicting prognosis in PFA ependymoma and group 3/4 medulloblastoma methylomes using interpretable epigenetic models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2505.

MEDB-88. BAF60C/SMARCD3-mediated novel neurodevelopmental epigenomic program promotes metastatic dissemination in medulloblastoma

Normal brain development relies on precise genetic and epigenetic spatiotemporal regulation of gene expression. How dysregulation of neurodevelopment relates to medulloblastoma, the most common pediatric brain tumor, remains elusive. Here, we uncovered a novel neurodevelopmental epigenomic program that regulates Purkinje cell migration in developing cerebellum is hijacked to induce tumor metastatic dissemination in medulloblastoma. Integrating publicly available datasets with our in-house data, unsupervised analyses revealed that BAF60C/SMARCD3, a subunit of SWI/SNF chromatin remodeling complex, promotes tumor cell migration in vitro and metastasis in vivo. Based on analyzing the single-cell RNAseq data of cerebellum developmental trajectory in mice and humans, aligning with the medulloblastoma patients’ datasets, we found that BAF60C/SMARCD3 regulated DAB1-mediated Reelin signaling is involved in Purkinje cell positioning during cerebellum development and medulloblastoma metastasis by orchestrating the cis-regulatory elements (CREs) at the DAB1 gene locus. Analysis of spatiotemporal gene expression and chromatin architecture in the human and mouse cerebellum demonstrated that transcription activity of the BAF60C/SMARCD3-DAB1 circuit is downregulated in a mature state of cerebellar development, however, is upregulated in metastatic medulloblastoma. We further identified that a core set of transcription factors, enhancer of zeste homolog 2 (EZH2) and nuclear factor I X (NFIX), bi-directionally control BAF60C/SMARCD3 transcriptional regulation by coordinating with the CREs at the BAF60C/SMARCD3 gene locus to form a chromatin hub during developing cerebellar development and medulloblastoma metastatic dissemination. Highly expressed BAF60C/SMARCD3 activates the Reelin/DAB1 signaling pathway downstream Src kinase, which was validated in the pair-wised primary and metastatic tumors from medulloblastoma patients. Preclinical medulloblastoma mouse models revealed that inhibiting Src activity reduces tumor cell migration and metastatic dissemination at a lower and safe dose. Together, these data deepen our understanding of how the developmental program influences disease progression and provide an opportunity for the development of therapeutics for this devastating brain cancer in children.

MEDB-78. Unified rhombic lip origins of Group 3 and Group 4 medulloblastoma

Identification and characterization of lineage-specific beginnings of distinct medulloblastoma (MB) subgroups is a fundamental challenge in the field. Genetically engineered mouse models and cross-species transcriptomics have provided mounting evidence of discrete, subgroup-specific developmental origins. Likewise, murine single-cell transcriptional atlases of cerebellar development have recently provided further clues into MB subgroup origins, particularly for poorly defined Group 3 and Group 4-MB. However, initial studies were underpowered to characterize rare populations and lacked robust validation, resulting in incomplete findings. Herein, we leveraged a large harmonized murine cerebellar atlas, targeted lineage enrichment, and integrative multi-omic strategies to deeply dissect MB origins. Isolation of spatially and temporally discrete developmental trajectories of key glutamatergic lineages born out of the murine upper rhombic lip provided an enhanced reference for mapping MB subgroup origins, especially for Group 3 and Group 4-MB. However, human-specific anatomic and cellular complexity, particularly within the rhombic lip germinal zone complicated murine-derived inferences. Further tumor-normal integrations using a novel single-cell atlas of the human fetal cerebellum, companioned with laser-capture micro-dissected transcriptional and epigenetic datasets, reinforced developmental insights extracted from candidate murine cerebellar lineages. Characterization of compartment-specific transcriptional signatures identified in the human upper rhombic lip implicated convergent cellular correlates of Group 3 and Group 4-MB, suggestive of a common developmental trajectory underlying their ancestry. Systematic imaging review and 3D summarization of a large clinical trial series of patient tumors, coupled with our advanced insights into developmental signatures, substantiated subgroup-specific tumor location patterns observed at diagnosis. Together, our results strongly implicate a common lineage trajectory of the upper rhombic lip as the probable origin of Group 3 and Group 4-MB. These important findings provide unprecedented opportunities to explore context-dependent mechanisms of MB pathogenesis and will foster generation of improved preclinical models that more faithfully recapitulate tumor biology.

TBIO-02. Mapping the origins of pediatric brain tumors to cell type lineages in the developing cerebellum.

Understanding the cellular origins of cerebellar tumors at single cell resolution may help identify novel therapeutic vulnerabilities to overcome current impediments in pediatric neuro-oncology. One approach to answer this important question involves the mapping of tumor profiles onto normal embryonal central nervous system development. Previous attempts in this regard focused on integrating human tumor data with developing mouse cerebellum atlases, however, these approaches were inherently limited due to species-specific differences. In order to address this critical issue, we use a high resolution developing human cerebellum single cell atlas in order to compare to extended bulk and single cell transcriptomes profiles from pediatric solid tumors. In result, we provide novel and confirmatory findings for the cellular association of medulloblastoma SHH, Group3 and Group4 as well as pilocytic astrocytoma, using comprehensive approaches to decipher the cellular composition and map the origins of childhood brain tumors. We also shed light on the cellular origins of posterior fossa ependymoma and radiation-induced glioma (secondary tumor, occurring after medulloblastoma). As a common feature among the tumor similarity to normal cell types, we identify gradients of differentiation, starting from early progenitor cells to more differentiated cell states, observed in all cerebellar tumor entities investigated at single cell level; thus reflecting possible lineages of origin. Pertaining to clinical application, we identified specific developmental genes shared between cerebellar lineages and associated tumors, and tumor-specific genes absent in all cerebellar lineages. These two categories of genes comprise candidate lineage markers for faithful modeling and, if absent from other organ systems after birth, could become an important source of potential therapeutic targets. Importantly, all analysis results are publicly available via an interactive online user interface (brain-match.org) that serves as an open valuable resource for the scientific community.

MEDB-59. A draft atlas of medulloblastoma cellular evolution under therapy

How standard care shapes the cellular composition of recurrent medulloblastoma (MB), if therapy selects for specific tumor or immune cell types, is unknown. We report the pilot phase of our ongoing effort to profile human longitudinal MB specimens via single-cell transcriptomics and epigenetics. We profiled 11 diagnostic and eight recurrent specimens from 19 subjects via single-nucleus RNA sequencing (snRNA-seq), and four subjects via single-nucleus assay for transposase-accessible chromatin. Specimens from select subjects were also profiled to assess genome-wide enhancer activity via single-nucleus cleavage-under-targets and tagmentation. We found an upregulation of the DNA-damage response, RNA translation, WNT and NOTCH signaling in recurrent specimens. The percentages of stem-like cells increased by over two-fold at recurrence. We found that microglia and oligodendrocyte-lineage cells were the most abundant non-malignant tumor-associated cell types, representing 2%-10% of cells profiled. Microglia abundances were relatively stable across molecular subtypes, and when comparing primary to recurrent tumors. There was a moderate, but statistically significant, increase in oligodendrocyte abundance in SSH and WNT tumors, compared to Group 3/4 tumors. We compared gene expression in tumor cells with public snRNA-seq from developing human cerebella (PCW 9-21). Combined Group-3/4 cell analysis supports a common lineage hierarchy, with an enrichment for unipolar brush-cell and Purkinje-cell phenotypes found in Group-4 tumors. All Group-3/4 cases contained cycling cells expressing markers of PAX2+ interneuron progenitors, most cycling cells had this phenotype. All specimens contained populations of non-cycling granule-cell progenitor-like cells. We performed single-cell co-expression receptor/ligand analysis to infer paracrine signaling between tumor and non-malignant cell types. This identified both tumor cells and microglia as sources of growth factors, pro-inflammatory cytokines, and pro-apoptotic ligands. Non-malignant oligodendrocyte-lineage cells uniquely expressed IL6-family cytokines, pleiotrophin, and class-III semaphorins. These studies shed light on the cellular heterogeneity of MB and the effect of standard therapy in shaping composition at recurrence.

Spatiotemporal multiomic landscape of human medulloblastoma at single cell resolution.

2069 Background: Medulloblastoma is the most common malignant childhood tumor type with distinct molecular subgroups. While advances in the comprehensive treatment have been made, the mortality in the high-risk group is still very high, driven by an incomplete understanding of cellular diversity. Methods: We use single-nucleus RNA expression, chromatin accessibility and spatial transcriptomic profiling to generate an integrative multi-omic map in 40 human medulloblastomas spanning all molecular subgroups and human postnatal cerebella, which is supplemented by the bulk whole genome and RNA sequences across 300 cases. Results: This approach provides spatially resolved insights into the medulloblastoma and cerebellum transcriptome and epigenome with identification of distinct cell-type in the tumor microenvironment. Medulloblastoma exhibited three tumor subpopulations including the quiescent, the differentiated, and a stem-like (proliferating) population unique to cancer, which localized to an immunosuppressive-vascular niche. We identified and validated mechanisms of stem-like to differentiated process among the malignant cells that drive tumor progression. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing stem-like malignant cells as a hub for intercellular communication. Multiple features of potential immunosuppression and angiogenesis were observed, including Treg cells and endothelial cells co-localization in compartmentalized tumor stroma. Conclusions: Our study provides an integrative molecular landscape of human medulloblastoma and represents a reference to advance mechanistic and therapeutic studies of pediatric neuro-oncological disease.

Cerebellar Gray Matter Volume in Tinnitus.

Tinnitus is the perception of sound without an external source. The flocculus (FL) and paraflocculus (PFL), which are small lobules of the cerebellum, have recently been implicated in its pathophysiology. In a previous study, the volume of the (P)FL-complex correlated with tinnitus severity in patients that had undergone cerebellopontine angle (CPA) tumor removal. In this study, the relation between tinnitus and gray matter volume (GMV) of the (P)FL-complex, GMV of the other cerebellar lobules and GMV of the cerebellar nuclei is investigated in otherwise healthy participants. Data was processed using the SUIT toolbox, which is dedicated to analysis of imaging data of the human cerebellum. GMV of all cerebellar lobules and nuclei were similar between tinnitus and non-tinnitus participants. Moreover, no relation was present between tinnitus severity, as measured by the Tinnitus Handicap Inventory, and (P)FL-complex GMV, tonsil GMV, or total cerebellar cortical GMV. These results suggest that in otherwise healthy participants, in contrast to participants after CPA tumor removal, no relation between the GMV of neither the (P)FL-complex nor other cerebellar lobules and tinnitus presence and severity exists. These findings indicate that a relation only exists when the (P)FL-complex is damaged, for instance by a CPA tumor. Alternatively, it is possible that differences in (P)FL-complex GMVs are too small to detect with a voxel-based morphometry study. Therefore, the role of the (P)FL-complex in tinnitus remains to be further studied.

Uncovering the genetic profiles underlying the intrinsic organization of the human cerebellum.

The functional diversity of the human cerebellum is largely believed to be derived more from its extensive connections rather than being limited to its mostly invariant architecture. However, whether and how the determination of cerebellar connections in its intrinsic organization interact with microscale gene expression is still unknown. Here we decode the genetic profiles of the cerebellar functional organization by investigating the genetic substrates simultaneously linking cerebellar functional heterogeneity and its drivers, i.e., the connections. We not only identified 443 network-specific genes but also discovered that their co-expression pattern correlated strongly with intra-cerebellar functional connectivity (FC). Ninety of these genes were also linked to the FC of cortico-cerebellar cognitive-limbic networks. To further discover the biological functions of these genes, we performed a "virtual gene knock-out" by observing the change in the coupling between gene co-expression and FC and divided the genes into two subsets, i.e., a positive gene contribution indicator (GCI+) involved in cerebellar neurodevelopment and a negative gene set (GCI-) related to neurotransmission. A more interesting finding is that GCI- is significantly linked with the cerebellar connectivity-behavior association and many recognized brain diseases that are closely linked with the cerebellar functional abnormalities. Our results could collectively help to rethink the genetic substrates underlying the cerebellar functional organization and offer possible micro-macro interacted mechanistic interpretations of the cerebellum-involved high order functions and dysfunctions in neuropsychiatric disorders.

Exploring the Genomic Patterns in Human and Mouse Cerebellums Via Single-Cell Sequencing and Machine Learning Method.

In mammals, the cerebellum plays an important role in movement control. Cellular research reveals that the cerebellum involves a variety of sub-cell types, including Golgi, granule, interneuron, and unipolar brush cells. The functional characteristics of cerebellar cells exhibit considerable differences among diverse mammalian species, reflecting a potential development and evolution of nervous system. In this study, we aimed to recognize the transcriptional differences between human and mouse cerebellum in four cerebellar sub-cell types by using single-cell sequencing data and machine learning methods. A total of 321,387 single-cell sequencing data were used. The 321,387 cells included 4 cell types, i.e., Golgi (5,048, 1.57%), granule (250,307, 77.88%), interneuron (60,526, 18.83%), and unipolar brush (5,506, 1.72%) cells. Our results showed that by using gene expression profiles as features, the optimal classification model could achieve very high even perfect performance for Golgi, granule, interneuron, and unipolar brush cells, respectively, suggesting a remarkable difference between the genomic profiles of human and mouse. Furthermore, a group of related genes and rules contributing to the classification was identified, which might provide helpful information for deepening the understanding of cerebellar cell heterogeneity and evolution.