Mouse Cerebellum Research Articles

Spatially Aware Domain Adaptation Enables Cell Type Deconvolution from Multi-Modal Spatially Resolved Transcriptomics.

Spatially Resolved Transcriptomics (SRT) offers unprecedented opportunities to elucidate the cellular arrangements within tissues. Nevertheless, the absence of deconvolution methods that simultaneously model multi-modal features has impeded progress in understanding cellular heterogeneity in spatial contexts. To address this issue, SpaDA is developed, a novel spatially aware domain adaptation method that integrates multi-modal data (i.e., transcriptomics, histological images, and spatial locations) from SRT to accurately estimate the spatial distribution of cell types. SpaDA utilizes a self-expressive variational autoencoder, coupled with deep spatial distribution alignment, to learn and align spatial and graph representations from spatial multi-modal SRT data and single-cell RNA sequencing (scRNA-seq) data. This strategy facilitates the transfer of cell type annotation information across these two similarity graphs, thereby enhancing the prediction accuracy of cell type composition. The results demonstrate that SpaDA surpasses existing methods in cell type deconvolution and the identification of cell types and spatial domains across diverse platforms. Moreover, SpaDA excels in identifying spatially colocalized cell types and key marker genes in regions of low-quality measurements, exemplified by high-resolution mouse cerebellum SRT data. In conclusion, SpaDA offers a powerful and flexible framework for the analysis of multi-modal SRT datasets, advancing the understanding of complex biological systems.

The ataxia-telangiectasia disease protein ATM controls vesicular protein secretion via CHGA and microtubule dynamics via CRMP5

The autosomal recessive disease ataxia-telangiectasia (A-T) presents with cerebellar degeneration, immunodeficiency, radiosensitivity, capillary dilatations, and pulmonary infections. Most symptoms outside the nervous system can be explained by failures of the disease protein ATM as a Ser/Thr-kinase to coordinate DNA damage repair. However, ATM in adult neurons has cytoplasmic localization and vesicle association, where its roles remain unclear. Here, we defined novel ATM protein targets in human neuroblastoma cells, and filtered initial pathogenesis events in ATM-null mouse cerebellum. Profiles of global proteome and phosphoproteomics - both direct ATM/ATR substrates and overall phosphorylation changes - confirmed previous findings for NBN, MRE11, MDC1, CHEK1, EIF4EBP1, AP3B2, PPP2R5C, SYN1 and SLC2A1. Even stronger downregulation of ATM/ATR substrate phosphopeptides after ATM-depletion was documented for CHGA, EXPH5, NBEAL2 and CHMP6 as key factors of protein secretion and endosome dynamics, as well as for CRMP5, DISP2, PHACTR1, PLXNC1, INA and TPX2 as neurite extension factors. Prominent effects on semaphorin-CRMP5-microtubule signals and ATM association with CRMP5 were validated. As a functional consequence, microtubules were stabilized, and neurite retraction ensued. The impact of ATM on secretory granules confirms previous ATM-null cerebellar transcriptome findings. This study provides the first link of A-T neural atrophy to growth cone collapse and aberrant microtubule dynamics.

N-Acetylcysteine Counteracts Immune Dysfunction and Autism-Related Behaviors in the Shank3b Mouse Model of Autism Spectrum Disorder.

Autism spectrum disorder (ASD) includes a range of neurodevelopmental disabilities characterized by social interaction deficits, communication impairments, and repetitive behaviors. Previous studies have shown that pro-inflammatory conditions play a key role in ASD. Despite this, how oxidative stress and inflammation may contribute to ASD-related behaviors is still poorly understood. Here, we reported that increased levels of molecules related to inflammation are present in the cerebellum and peripheral blood (PB) of mice lacking Shank3b, an established model of syndromic ASD. In parallel, immune dysfunction was documented in the bone marrow (BM) and spleens of mutant mice. N-acetylcysteine (NAC) treatment rescued inflammation in the cerebellum and PB and impaired the production of pro-inflammatory molecules in the BM and spleen. In addition, social impairment was counteracted in NAC-treated Shank3b-/- animals. Taken together, our results provide clear evidence of the key role of cerebellar oxidative stress and inflammation in the establishment of ASD-related behaviors. Furthermore, our findings underscore the importance of considering ASD as a systemic disorder.

EXTH-49. DEVELOPMENTAL ANTIGENS CAN TARGET EMBRYONAL PEDIATRIC BRAIN CANCERS IN ADOPTIVE CELLULAR THERAPY

Abstract Mutations in tumor neoantigens have shown promise in immunotherapies for many cancers, yet pediatric brain tumors, which typically have a lower mutational burden, offer fewer opportunities to capitalize on these targeted therapies. Studies indicate that medulloblastoma (MB) and brain stem gliomas (BSG) originate from abnormal reactivation of post-natal developmental processes. This led us to investigate whether proteins expressed during the early post-natal development of the mouse cerebellum and brainstem could be powerful antigens to fight MB and BSG, respectively. We performed both in vitro and in vivo studies in mice with the RNA of developmental antigens from the brain stem and cerebellum in an adoptive cellular therapy (ACT) platform. We evaluated the reactivity and therapeutic efficacy of these treatments as sustainable antigenic targets against BSG and MBs. We demonstrate that T cells activated towards these non-mutated, tissue-specific developmental antigens can recognize distinct subtypes of MB and BSG, and activate specifically without cross-reacting to the normal brain. This conferred a survival benefit in established orthotopic models of these pediatric brain tumor types. Our research exhibits the value developmental antigens can offer when serving as tumor rejection antigens in MB and BSG.

Purkinje cell-specific deficiency in SEL1L-hrd1 endoplasmic reticulum-associated degradation causes progressive cerebellar ataxia in mice.

Recent studies have identified multiple genetic variants of SEL1L-HRD1 endoplasmic reticulum-associated degradation (ERAD) in humans with neurodevelopmental disorders and locomotor dysfunctions, including ataxia. However, the relevance and importance of SEL1L-HRD1 ERAD in the pathogenesis of ataxia remain unexplored. Here, we showed that SEL1L deficiency in Purkinje cells leads to early-onset progressive cerebellar ataxia with progressive loss of Purkinje cells with age. Mice with Purkinje cell-specific deletion of SEL1L (Sel1LPcp2Cre) exhibited motor dysfunction beginning around 9 weeks of age. Transmission electron microscopy analysis revealed dilated ER and fragmented nuclei in Purkinje cells of adult Sel1LPcp2Cre mice, indicative of altered ER homeostasis and cell death. Finally, loss of Purkinje cells was associated with a secondary neurodegeneration of granular cells, as well as robust activation of astrocytes and proliferation of microglia, in the cerebellums of Sel1LPcp2Cre mice. These data demonstrate the pathophysiological importance of SEL1L-HRD1 ERAD in Purkinje cells in the pathogenesis of cerebellar ataxia.

Characterization of Single-Cell Cis-regulatory Elements Informs Implications for Cell Differentiation.

Cis-regulatory elements govern the specific patterns and dynamics of gene expression in cells during development, which are the fundamental mechanisms behind cell differentiation. However, the genomic characteristics of single-cell cis-regulatory elements closely linked to cell differentiation during development remain unclear. To explore this, we systematically analyzed ∼250,000 putative single-cell cis-regulatory elements obtained from snATAC-seq analysis of the developing mouse cerebellum. We found that over 80% of these single-cell cis-regulatory elements show pleiotropic effects, being active in 2 or more cell types. The pleiotropic degrees of proximal and distal single-cell cis-regulatory elements are positively correlated with the density and diversity of transcription factor binding motifs and GC content. There is a negative correlation between the pleiotropic degrees of single-cell cis-regulatory elements and their distances to the nearest transcription start sites, and proximal single-cell cis-regulatory elements display higher relevance strengths than distal ones. Furthermore, both proximal and distal single-cell cis-regulatory elements related to cell differentiation exhibit enhanced sequence-level evolutionary conservation, increased density and diversity of transcription factor binding motifs, elevated GC content, and greater distances from their nearest genes. Together, our findings reveal the general genomic characteristics of putative single-cell cis-regulatory elements and provide insights into the genomic and evolutionary mechanisms by which single-cell cis-regulatory elements regulate cell differentiation during development.

Quercetin attenuates SiO2-induced ZBP-1-mediated PANoptosis in mouse neuronal cells via the ROS/TLR4/NF-κB pathway

With the increasing development of the society, silicon dioxide (SiO2) has been used in various fields, such as agriculture, food industry, etc., and its residues can pose a potential health threat to organisms. Quercetin (Que) is a potent free radical scavenger commonly found in plants. C57BL/6 mice were chosen to established a mouse model of SiO2 exposure and Que antagonism to investigate the mechanism of action of Que in rescuing the toxic damage of SiO2 on mouse cerebellum tissue. The results showed that cytoplasmic vacuolization, and inflammatory cell infiltration caused by SiO2 were alleviated by the addition of Que. and reduced oxidative stress in mouse cerebellum, alleviated the activation of TLR4 pathway induced by SiO2, and substantially reduced the occurrence of ZBP-1-mediated PANoptosis induced by SiO2 exposure in mouse cerebellum. In NS20Y cells, the oxidative stress activator (Elesclomol) and inhibitor N-acetyl cysteine (NAC), and the NF-κB activator 2 (NA2) were added. Elesclomol and NAC confirm the involvement of ROS in regulating the TLR4/NF-κB pathway, the TLR4/NF-κB pathway regulated ZBP-1-mediated PANoptosis in cerebellum and NS20Y cells induced by SiO2 exposure. In conclusion, the present experimental data suggest that Que mitigates the onset of ZBP-1-mediated PANoptosis in neuronal cells induced by SiO2 through the ROS/TLR4/NF-κB pathway. The present experimental findings help to understand the detoxification effect of Que in more tissues and provide an important reference for the rescue of organisms in long-term SiO2 environment.

Comparative Analysis of Six Adeno-Associated Viral Vector Serotypes in Mouse Inferior Colliculus and Cerebellum.

Adeno-associated viral vector (AAV) serotypes vary in how effectively they express genes across different cell types and brain regions. Here we report a systematic comparison of the AAV serotypes 1, 2, 5, 8, 9, and the directed evolution derived AAVrg, in the inferior colliculus (IC) and cerebellum. The AAVs were identical apart from their different serotypes, each having a synapsin promotor and expressing GFP (AAV-hSyn-GFP). Identical titers and volumes were injected into the IC and cerebellum of adult male and female mice, and brains were sectioned and imaged 2 weeks later. Transduction efficacy, anterograde labeling of axonal projections, and retrograde labeling of somata were characterized and compared across serotypes. Cell-type tropism was assessed by analyzing the morphology of the GFP-labeled neurons in the cerebellar cortex. In both the cerebellum and IC, AAV1 expressed GFP in more cells, labeled a larger volume, and produced significantly brighter labeling than all other serotypes, indicating superior transgene expression. AAV1 labeled more Purkinje cells, unipolar brush cells, and molecular layer interneurons than the other serotypes, while AAV2 labeled a greater number of granule cells. These results provide guidelines for the use of AAVs as gene delivery tools in these regions.

Comparative analysis of six adeno-associated viral vector serotypes in mouse inferior colliculus and cerebellum.

Adeno-associated viral vector (AAV) serotypes vary in how effectively they express genes across different cell types and brain regions. Here we report a systematic comparison of the AAV serotypes 1, 2, 5, 8, 9, and the directed evolution derived AAVrg, in the inferior colliculus and cerebellum. The AAVs were identical apart from their different serotypes, each having a synapsin promotor and expressing GFP (AAV-hSyn-GFP). Identical titers and volumes were injected into the inferior colliculus and cerebellum of adult male and female mice and brains were sectioned and imaged 2 weeks later. Transduction efficacy, anterograde labeling of axonal projections, and retrograde labeling of somata, were characterized and compared across serotypes. Cell-type tropism was assessed by analyzing the morphology of the GFP-labeled neurons in the cerebellar cortex. In both the cerebellum and inferior colliculus, AAV1 expressed GFP in more cells, labeled a larger volume, and produced significantly brighter labeling than all other serotypes, indicating superior transgene expression. AAV1 labeled more Purkinje cells, unipolar brush cells, and molecular layer interneurons than the other serotypes, while AAV2 labeled a greater number of granule cells. These results provide guidelines for the use of AAVs as gene delivery tools in these regions.

An increase in reactive oxygen species underlies neonatal cerebellum repair.

The neonatal mouse cerebellum shows remarkable regenerative potential upon injury at birth, wherein a subset of Nestin-expressing progenitors (NEPs) undergoes adaptive reprogramming to replenish granule cell progenitors that die. Here, we investigate how the microenvironment of the injured cerebellum changes upon injury and contributes to the regenerative potential of normally gliogenic-NEPs and their adaptive reprogramming. Single cell transcriptomic and bulk chromatin accessibility analyses of the NEPs from injured neonatal cerebella compared to controls show a temporary increase in cellular processes involved in responding to reactive oxygen species (ROS), a known damage-associated molecular pattern. Analysis of ROS levels in cerebellar tissue confirm a transient increased one day after injury at postanal day 1, overlapping with the peak cell death in the cerebellum. In a transgenic mouse line that ubiquitously overexpresses human mitochondrial catalase (mCAT), ROS is reduced 1 day after injury to the granule cell progenitors, and we demonstrate that several steps in the regenerative process of NEPs are curtailed leading to reduced cerebellar growth. We also provide evidence that microglia are involved in one step of adaptive reprogramming by regulating NEP replenishment of the granule cell precursors. Collectively, our results highlight that changes in the tissue microenvironment regulate multiple steps in adaptative reprogramming of NEPs upon death of cerebellar granule cell progenitors at birth, highlighting the instructive roles of microenvironmental signals during regeneration of the neonatal brain.

Silicon dioxide particles induce DNA oxidative damage activating the AIM2-mediated PANoptosis in mice cerebellum

Silicon dioxide (SiO2) particles are novel materials with wide-ranging applications across various fields, posing potential neurotoxic effects. This study investigates the toxicological mechanisms of SiO2 particles of different sizes on murine cerebellar tissue and cells. Six-week-old C57BL/6 mice were orally administered SiO2 particles of three sizes (1 μm, 300 nm, 50 nm) for 21 days to establish an in vivo model, and mice cerebellar astrocytes (C8-D1A cells) were cultured in vitro. Indicators of oxidative stress, DNA damage, and the PANoptosis pathway were detected using methods such as immunofluorescence staining, comet assay, western blotting, and qRT-PCR. The results show that SiO2 particles induce oxidative stress leading to DNA oxidative damage. The aberrant DNA is recognized by AIM2 (absent in melanoma 2), which activates the assembly of the PANoptosome complex, subsequently triggering PANoptosis. Furthermore, the extent of damage is inversely correlated with the size of SiO2 particles. This study elucidates the toxicological mechanism of SiO2 particles causing cerebellar damage via PANoptosis, extending research on PANoptosis in neurotoxicology, and aiding in the formulation of stricter safety standards and protective measures to reduce the potential toxic risk of SiO2 particles to humans.

Cross-species single-cell spatial transcriptomic atlases of the cerebellar cortex.

The molecular and cellular organization of the primate cerebellum remains poorly characterized. We obtained single-cell spatial transcriptomic atlases of macaque, marmoset, and mouse cerebella and identified primate-specific cell subtypes, including Purkinje cells and molecular-layer interneurons, that show different expression of the glutamate ionotropic receptor Delta type subunit 2 (GRID2) gene. Distinct gene expression profiles were found in anterior, posterior, and vestibular regions in all species, whereas region-selective gene expression was predominantly observed in the granular layer of primates and in the Purkinje layer of mice. Gene expression gradients in the cerebellar cortex matched well with functional connectivity gradients revealed with awake functional magnetic resonance imaging, with more lobule-specific differences between primates and mice than between two primate species. These comprehensive atlases and comparative analyses provide the basis for understanding cerebellar evolution and function.

Microglial morphology aligns with vigilance stage‐specific neuronal oscillations in a brain region‐dependent manner

AbstractMicroglia, the resident immune cells in the brain, dynamically adapt their morphology based on their functional state. This study explored the relationship between microglial morphology and sleep–wake cycles in mice. Using Iba1 immunostaining to identify microglia, we quantified morphological changes in microglia at different timepoints in multiple brain regions (cortex, hippocampus, basal forebrain, hindbrain, and cerebellum) in B6 male mice using semi‐automated 3D structural analysis. Simultaneously, in a separate group, we monitored wake and sleep stage‐specific brain activity using EEG/EMG recordings. During natural sleep–wake cycles, we observed increased microglial complexity (enlarged volume, territorial coverage, and ramification) during wakefulness, characterized by high‐frequency theta (8–12 Hz) and gamma activity (30–80 Hz). Conversely, during NREM sleep, which is dominated by delta activity (0.5–4 Hz), microglia displayed reduced complexity. Notably, this pattern was absent in brain regions lacking direct functional connections to areas generating vigilance stage‐dependent thalamocortical oscillations. We then extended wakefulness to decouple circadian influence from sleep–wake‐specific neuronal activity. This procedure attenuated the decrease in microglial complexity observed during natural sleep, suggesting a crucial role for neuronal activity. Subsequent recovery sleep restored microglial features, independent of the time of day (zeitgeber time). These findings reveal a dynamic interplay between vigilance stage‐specific thalamocortical activity and microglial morphology across various brain regions. This suggests a potential role for microglia in sleep regulation and warrants further investigation to understand the underlying mechanisms.

Adaptive protein synthesis in genetic models of copper deficiency and childhood neurodegeneration.

Copper dysregulation is present in neurodevelopmental, neurodegenerative, and neurological conditions ranging from rare conditions such as Menkes disease and CTR1 deficiency to more common diseases like Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis. However, the precise mechanisms through which copper deficiency leads to pathology and the survival mechanisms by which the brain exhibits resilience remain unknown. We demonstrate that in a human cell line, Drosophila , and the mouse cerebellum, copper-depleted neuronal cells exhibit increased protein synthesis through two distinct signaling pathways: mTORC1 activation and decreased PERK (EIF2AK3) activity. This upregulation of protein synthesis facilitates resilience of neuronal cells to copper deficiency, including partial restoration of dendritic arborization. Our findings offer a new framework for understanding copper deficiency-related pathology in neurological disorders.

Heterogeneity in Slow Synaptic Transmission Diversifies Purkinje Cell Timing.

The cerebellum plays an important role in diverse brain functions, ranging from motor learning to cognition. Recent studies have suggested that molecular and cellular heterogeneity within cerebellar lobules contributes to functional differences across the cerebellum. However, the specific relationship between molecular and cellular heterogeneity and diverse functional outputs of different regions of the cerebellum remains unclear. Here, we describe a previously unappreciated form of synaptic heterogeneity at parallel fiber synapses to Purkinje cells in the mouse cerebellum (both sexes). In contrast to uniform fast synaptic transmission, we found that the properties of slow synaptic transmission varied by up to threefold across different lobules of the mouse cerebellum, resulting in surprising heterogeneity. Depending on the location of a Purkinje cell, the time of peak of slow synaptic currents varied by hundreds of milliseconds. The duration and decay time of these currents also spanned hundreds of milliseconds, based on lobule. We found that, as a consequence of the heterogeneous synaptic dynamics, the same brief input stimulus was transformed into prolonged firing patterns over a range of timescales that depended on Purkinje cell location.

Genome binding properties of Zic transcription factors underlie their changing functions during neuronal maturation

BackgroundThe Zic family of transcription factors (TFs) promote both proliferation and maturation of cerebellar granule neurons (CGNs), raising the question of how a single, constitutively expressed TF family can support distinct developmental processes. Here we use an integrative experimental and bioinformatic approach to discover the regulatory relationship between Zic TF binding and changing programs of gene transcription during postnatal CGN differentiation.ResultsWe first established a bioinformatic pipeline to integrate Zic ChIP-seq data from the developing mouse cerebellum with other genomic datasets from the same tissue. In newborn CGNs, Zic TF binding predominates at active enhancers that are co-bound by developmentally regulated TFs including Atoh1, whereas in mature CGNs, Zic TF binding consolidates toward promoters where it co-localizes with activity-regulated TFs. We then performed CUT&RUN-seq in differentiating CGNs to define both the time course of developmental shifts in Zic TF binding and their relationship to gene expression. Mapping Zic TF binding sites to genes using chromatin looping, we identified the set of Zic target genes that have altered expression in RNA-seq from Zic1 or Zic2 knockdown CGNs.ConclusionsOur data show that Zic TFs are required for both induction and repression of distinct, developmentally regulated target genes through a mechanism that is largely independent of changes in Zic TF binding. We suggest that the differential collaboration of Zic TFs with other TF families underlies the shift in their biological functions across CGN development.

Increased Expression of the Neuropeptides PACAP/VIP in the Brain of Mice with CNS Targeted Production of IL-6 Is Mediated in Part by Trans-Signalling.

Inflammation with expression of interleukin 6 (IL-6) in the central nervous system (CNS) occurs in several neurodegenerative/neuroinflammatory conditions and may cause neurochemical changes to endogenous neuroprotective systems. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are two neuropeptides with well-established protective and anti-inflammatory properties. Yet, whether PACAP and VIP levels are altered in mice with CNS-restricted, astrocyte-targeted production of IL-6 (GFAP-IL6) remains unknown. In this study, PACAP/VIP levels were assessed in the brain of GFAP-IL6 mice. In addition, we utilised bi-genic GFAP-IL6 mice carrying the human sgp130-Fc transgene (termed GFAP-IL6/sgp130Fc mice) to determine whether trans-signalling inhibition rescued PACAP/VIP changes in the CNS. Transcripts and protein levels of PACAP and VIP, as well as their receptors PAC1, VPAC1 and VPAC2, were significantly increased in the cerebrum and cerebellum of GFAP-IL6 mice vs. wild type (WT) littermates. These results were paralleled by a robust activation of the JAK/STAT3, NF-κB and ERK1/2MAPK pathways in GFAP-IL6 mice. In contrast, co-expression of sgp130Fc in GFAP-IL6/sgp130Fc mice reduced VIP expression and activation of STAT3 and NF-κB pathways, but it failed to rescue PACAP, PACAP/VIP receptors and Erk1/2MAPK phosphorylation. We conclude that forced expression of IL-6 in astrocytes induces the activation of the PACAP/VIP neuropeptide system in the brain, which is only partly modulated upon IL-6 trans-signalling inhibition. Increased expression of PACAP/VIP neuropeptides and receptors may represent a homeostatic response of the CNS to an uncontrolled IL-6 synthesis and its neuroinflammatory consequences.

Isolation of Stem Cells from Postnatal Mouse Cerebellum and Differentiation into Neural Cells

Isolation of Stem Cells from Postnatal Mouse Cerebellum and Differentiation into Neural Cells

Isolation of Stem Cells from Postnatal Mouse Cerebellum and Differentiation into Neural Cells

Isolation of Stem Cells from Postnatal Mouse Cerebellum and Differentiation into Neural Cells

Pressure Cycling Technology Combined With MicroLC-SWATH Mass Spectrometry for the Analysis of Sex-Related Differences Between Male and Female Cerebella: A Promising Approach to Investigating Proteomics Differences in Psychiatric and Neurodegenerative Diseases.

Pressure cycling technology (PCT) coupled with data-independent sequential window acquisition of all theoretical mass spectra (SWATH-MS) can be a powerful tool for identifying and quantifying biomarkers (e.g., proteins) in complex biological samples. Mouse models are frequently used in brain studies, including those focusing on different neurodevelopmental and psychiatric disorders. More and more pieces of evidence have suggested that sex-related differences in the brain impact the rates, clinical manifestations, and therapy outcomes of these disorders. However, sex-based differences in the proteomic profiles of mouse cerebella have not been widely investigated. In this pilot study, we evaluate the applicability of coupling PCT sample preparation with microLC-SWATH-MS analysis to map and identify differences in the proteomes of two female and two male mice cerebellum samples. We identified and quantified 174 proteins in mice cerebella. A comparison of the proteomic profiles revealed that the levels of 11 proteins in the female and male mice cerebella varied significantly. Although this study utilizes a small sample, our results indicate that the studied male and female mice cerebella possessed differing proteome compositions, mainly with respect to energy metabolism processes.