ABSTRACT
Polyglutamine (polyQ)-encoding CAG repeat expansions represent a common disease-causing mutation responsible for several dominant spinocerebellar ataxias (SCAs). PolyQ-expanded SCA proteins are toxic for cerebellar neurons, with Purkinje cells (PCs) being the most vulnerable. RNA interference (RNAi) reagents targeting transcripts with expanded CAG reduce the level of various mutant SCA proteins in an allele-selective manner in vitro and represent promising universal tools for treating multiple CAG/polyQ SCAs. However, it remains unclear whether the therapeutic targeting of CAG expansion can be achieved in vivo and if it can ameliorate cerebellar functions. Here, using a mouse model of SCA7 expressing a mutant Atxn7 allele with 140 CAGs, we examined the efficacy of short hairpin RNAs (shRNAs) targeting CAG repeats expressed from PHP.eB adeno-associated virus vectors (AAVs), which were introduced into the brain via intravascular injection. We demonstrated that shRNAs carrying various mismatches with the CAG target sequence reduced the level of polyQ-expanded ATXN7 in the cerebellum, albeit with varying degrees of allele selectivity and safety profile. An shRNA named A4 potently reduced the level of polyQ-expanded ATXN7, with no effect on normal ATXN7 levels and no adverse side effects. Furthermore, A4 shRNA treatment improved a range of motor and behavioral parameters 23 weeks after AAV injection and attenuated the disease burden of PCs by preventing the downregulation of several PC-type-specific genes. Our results show the feasibility of the selective targeting of CAG expansion in the cerebellum using a blood-brain barrier-permeable vector to attenuate the disease phenotype in an SCA mouse model. Our study represents a significant advancement in developing CAG-targeting strategies as a potential therapy for SCA7 and possibly other CAG/polyQ SCAs.
Subject(s)
Ataxin-7 , Dependovirus , Disease Models, Animal , Peptides , Phenotype , RNA, Small Interfering , Spinocerebellar Ataxias , Trinucleotide Repeat Expansion , Animals , Spinocerebellar Ataxias/genetics , Spinocerebellar Ataxias/therapy , Spinocerebellar Ataxias/metabolism , Peptides/genetics , Dependovirus/genetics , Mice , Ataxin-7/genetics , Ataxin-7/metabolism , Trinucleotide Repeat Expansion/genetics , RNA, Small Interfering/genetics , Genetic Vectors/genetics , Genetic Vectors/administration & dosage , Purkinje Cells/metabolism , Purkinje Cells/pathology , Mice, Transgenic , Cerebellum/metabolism , Cerebellum/pathology , Humans , Genetic Therapy/methods , AllelesABSTRACT
Loss of arterial smooth muscle cells (SMCs) and abnormal accumulation of the extracellular domain of the NOTCH3 receptor (Notch3ECD) are the 2 core features of CADASIL, a common cerebral small vessel disease caused by highly stereotyped dominant mutations in NOTCH3. Yet the relationship between NOTCH3 receptor activity, Notch3ECD accumulation, and arterial SMC loss has remained elusive, hampering the development of disease-modifying therapies. Using dedicated histopathological and multiscale imaging modalities, we could detect and quantify previously undetectable CADASIL-driven arterial SMC loss in the CNS of mice expressing the archetypal Arg169Cys mutation. We found that arterial pathology was more severe and Notch3ECD accumulation greater in transgenic mice overexpressing the mutation on a wild-type Notch3 background (TgNotch3R169C) than in knockin Notch3R170C/R170C mice expressing this mutation without a wild-type Notch3 copy. Notably, expression of Notch3-regulated genes was essentially unchanged in TgNotch3R169C arteries. We further showed that wild-type Notch3ECD coaggregated with mutant Notch3ECD and that elimination of 1 copy of wild-type Notch3 in TgNotch3R169C was sufficient to attenuate Notch3ECD accumulation and arterial pathology. These findings suggest that Notch3ECD accumulation, involving mutant and wild-type NOTCH3, is a major driver of arterial SMC loss in CADASIL, paving the way for NOTCH3-lowering therapeutic strategies.
Subject(s)
CADASIL , Mice , Animals , Receptor, Notch3/genetics , CADASIL/genetics , CADASIL/metabolism , CADASIL/pathology , Protein Aggregates , Receptors, Notch/genetics , Receptors, Notch/metabolism , Arteries/pathology , Mice, Transgenic , MutationABSTRACT
Immune checkpoint inhibitors (ICI) represent the cornerstone for the treatment of patients with metastatic clear cell renal cell carcinoma (ccRCC). Despite a favorable response for a subset of patients, others experience primary progressive disease, highlighting the need to precisely understand the plasticity of cancer cells and their cross-talk with the microenvironment to better predict therapeutic response and personalize treatment. Single-cell RNA sequencing of ccRCC at different disease stages and normal adjacent tissue (NAT) from patients identified 46 cell populations, including 5 tumor subpopulations, characterized by distinct transcriptional signatures representing an epithelial-to-mesenchymal transition gradient and a novel inflamed state. Deconvolution of the tumor and microenvironment signatures in public data sets and data from the BIONIKK clinical trial (NCT02960906) revealed a strong correlation between mesenchymal-like ccRCC cells and myofibroblastic cancer-associated fibroblasts (myCAF), which are both enriched in metastases and correlate with poor patient survival. Spatial transcriptomics and multiplex immune staining uncovered the spatial proximity of mesenchymal-like ccRCC cells and myCAFs at the tumor-NAT interface. Moreover, enrichment in myCAFs was associated with primary resistance to ICI therapy in the BIONIKK clinical trial. These data highlight the epithelial-mesenchymal plasticity of ccRCC cancer cells and their relationship with myCAFs, a critical component of the microenvironment associated with poor outcome and ICI resistance. SIGNIFICANCE: Single-cell and spatial transcriptomics reveal the proximity of mesenchymal tumor cells to myofibroblastic cancer-associated fibroblasts and their association with disease outcome and immune checkpoint inhibitor response in clear cell renal cell carcinoma.
Subject(s)
Cancer-Associated Fibroblasts , Carcinoma, Renal Cell , Kidney Neoplasms , Humans , Cancer-Associated Fibroblasts/pathology , Carcinoma, Renal Cell/drug therapy , Carcinoma, Renal Cell/genetics , Gene Expression Profiling , Immunotherapy , Kidney Neoplasms/drug therapy , Kidney Neoplasms/genetics , Prognosis , Tumor Microenvironment , Clinical Trials as TopicABSTRACT
Microglial cells ensure essential roles in brain homeostasis. In pathological condition, microglia adopt a common signature, called disease-associated microglial (DAM) signature, characterized by the loss of homeostatic genes and the induction of disease-associated genes. In X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disease, microglial defect has been shown to precede myelin degradation and may actively contribute to the neurodegenerative process. We previously established BV-2 microglial cell models bearing mutations in peroxisomal genes that recapitulate some of the hallmarks of the peroxisomal ß-oxidation defects such as very long-chain fatty acid (VLCFA) accumulation. In these cell lines, we used RNA-sequencing and identified large-scale reprogramming for genes involved in lipid metabolism, immune response, cell signaling, lysosome and autophagy, as well as a DAM-like signature. We highlighted cholesterol accumulation in plasma membranes and observed autophagy patterns in the cell mutants. We confirmed the upregulation or downregulation at the protein level for a few selected genes that mostly corroborated our observations and clearly demonstrated increased expression and secretion of DAM proteins in the BV-2 mutant cells. In conclusion, the peroxisomal defects in microglial cells not only impact on VLCFA metabolism but also force microglial cells to adopt a pathological phenotype likely representing a key contributor to the pathogenesis of peroxisomal disorders.
ABSTRACT
Androgen deprivation therapy (ADT) is a cornerstone of prostate cancer (PCa) management. Although tumors initially regress, many progress to a hormone-independent state termed castration-resistant PCa (CRPC), for which treatment options are limited. We here report that the major luminal cell population in tumors of Pten(i)pe-/- mice, generated by luminal epithelial cell-specific deletion of the tumor suppressor PTEN after puberty, is castration-resistant and that the expression of inflammation and stemness markers is enhanced in persistent luminal cells. In addition, hypoxia-inducible factor 1 (HIF1) signaling, which we have previously demonstrated to be induced in luminal cells of Pten(i)pe-/- mice and to promote malignant progression, is further activated. Importantly, we show that genetic and pharmacological inhibition of HIF1A sensitizes Pten-deficient prostatic tumors to castration and provides durable therapeutic responses. Furthermore, HIF1A inhibition induces apoptotic signaling in human CRPC cell lines. Therefore, our data demonstrate that HIF1A in prostatic tumor cells is a critical factor that enables their survival after ADT, and identify it as a target for CRPC management.
Subject(s)
Prostatic Neoplasms, Castration-Resistant , Male , Humans , Mice , Animals , Prostatic Neoplasms, Castration-Resistant/metabolism , Androgen Antagonists/therapeutic use , Receptors, Androgen/metabolism , Castration , Hypoxia , Cell Line, TumorABSTRACT
BACKGROUND & AIMS: Enteroendocrine cells (EECs) and their hormones are essential regulators of whole-body energy homeostasis. EECs sense luminal nutrients and microbial metabolites and subsequently secrete various hormones acting locally or at a distance. Impaired development of EECs during embryogenesis is life-threatening in newborn mice and humans due to compromised nutrient absorption. However, the physiological importance of the EEC system in adult mice has yet to be directedly studied. Herein, we aimed to determine the long-term consequences of a total loss of EECs in healthy adults on energy metabolism, intestinal transcriptome, and microbiota. METHODS: We depleted intestinal EECs by tamoxifen treatment of adult Neurog3fl/fl; Villin-CreERT2 male mice. We studied intestinal cell differentiation, food efficiency, lipid absorption, microbiota composition, fecal metabolites, and transcriptomic responses in the proximal and distal small intestines of mice lacking EECs. We also determined the high-fat diet-induced transcriptomic changes in sorted Neurog3eYFP/+ EECs. RESULTS: Induction of EEC deficiency in adults is not life-threatening unless fed with a high-fat diet. Under a standard chow diet, mice lose 10% of weight due to impaired food efficiency. Blood concentrations of cholesterol, triglycerides, and free fatty acids are reduced, and lipid absorption is impaired and delayed in the distal small intestine. Genes controlling lipogenesis, carbohydrate metabolism, and neoglucogenesis are upregulated. Microbiota composition is rapidly altered after EECs depletion and is characterized by decreased α-diversity. Bacteroides and Lactobacillus were progressively enriched, whereas Lachnospiraceae declined without impacting fecal short-chain fatty acid concentrations. CONCLUSIONS: EECs are dispensable for survival in adult male mice under a standard chow diet. The absence of EECs impairs intestinal lipid absorption, leading to transcriptomic and metabolic adaptations and remodeling of the gut microbiota.
Subject(s)
Gastrointestinal Microbiome , Humans , Male , Mice , Animals , Intestines , Enteroendocrine Cells/metabolism , Hormones/metabolism , Cholesterol/metabolismABSTRACT
Friedreich ataxia is an autosomal recessive multisystem disorder with prominent neurological manifestations and cardiac involvement. The disease is caused by large GAA expansions in the first intron of the FXN gene, encoding the mitochondrial protein frataxin, resulting in downregulation of gene expression and reduced synthesis of frataxin. The selective loss of proprioceptive neurons is a hallmark of Friedreich ataxia, but the cause of the specific vulnerability of these cells is still unknown. We herein perform an in vitro characterization of human induced pluripotent stem cell-derived sensory neuronal cultures highly enriched for primary proprioceptive neurons. We employ neurons differentiated from healthy donors, Friedreich ataxia patients and Friedreich ataxia sibling isogenic control lines. The analysis of the transcriptomic and proteomic profile suggests an impairment of cytoskeleton organization at the growth cone, neurite extension and, at later stages of maturation, synaptic plasticity. Alterations in the spiking profile of tonic neurons are also observed at the electrophysiological analysis of mature neurons. Despite the reversal of the repressive epigenetic state at the FXN locus and the restoration of FXN expression, isogenic control neurons retain many features of Friedreich ataxia neurons. Our study suggests the existence of abnormalities affecting proprioceptors in Friedreich ataxia, particularly their ability to extend towards their targets and transmit proper synaptic signals. It also highlights the need for further investigations to better understand the mechanistic link between FXN silencing and proprioceptive degeneration in Friedreich ataxia.
ABSTRACT
Individuals with mutations in CHD8 present with gastrointestinal complaints, yet the underlying mechanisms are understudied. Here, using a stable constitutive chd8 mutant zebrafish model, we found that the loss of chd8 leads to a reduced number of vagal neural crest cells (NCCs), enteric neural and glial progenitors, emigrating from the neural tube, and that their early migration capability was altered. At later stages, although the intestinal colonization by NCCs was complete, we found the decreased numbers of both serotonin-producing enterochromaffin cells and NCC-derived serotonergic neurons, suggesting an intestinal hyposerotonemia in the absence of chd8 Furthermore, transcriptomic analyses revealed an altered expression of key receptors and enzymes in serotonin and acetylcholine signaling pathways. The tissue examination of chd8 mutants revealed a thinner intestinal epithelium accompanied by an accumulation of neutrophils and the decreased numbers of goblet cells and eosinophils. Last, single-cell sequencing of whole intestines showed a global disruption of the immune balance with a perturbed expression of inflammatory interleukins and changes in immune cell clusters. Our findings propose a causal developmental link between chd8, NCC development, intestinal homeostasis, and autism-associated gastrointestinal complaints.
Subject(s)
Autistic Disorder , Neural Crest , Animals , Neural Crest/metabolism , Zebrafish , Serotonin/metabolism , Cell Movement/genetics , Intestines , HomeostasisABSTRACT
Microglia are crucial for brain homeostasis, and dysfunction of these cells is a key driver in most neurodegenerative diseases, including peroxisomal leukodystrophies. In X-linked adrenoleukodystrophy (X-ALD), a neuroinflammatory disorder, very long-chain fatty acid (VLCFA) accumulation due to impaired degradation within peroxisomes results in microglial defects, but the underlying mechanisms remain unclear. Using CRISPR/Cas9 gene editing of key genes in peroxisomal VLCFA breakdown (Abcd1, Abcd2, and Acox1), we recently established easily accessible microglial BV-2 cell models to study the impact of dysfunctional peroxisomal ß-oxidation and revealed a disease-associated microglial-like signature in these cell lines. Transcriptomic analysis suggested consequences on the immune response. To clarify how impaired lipid degradation impacts the immune function of microglia, we here used RNA-sequencing and functional assays related to the immune response to compare wild-type and mutant BV-2 cell lines under basal conditions and upon pro-inflammatory lipopolysaccharide (LPS) activation. A majority of genes encoding proinflammatory cytokines, as well as genes involved in phagocytosis, antigen presentation, and co-stimulation of T lymphocytes, were found differentially overexpressed. The transcriptomic alterations were reflected by altered phagocytic capacity, inflammasome activation, increased release of inflammatory cytokines, including TNF, and upregulated response of T lymphocytes primed by mutant BV-2 cells presenting peptides. Together, the present study shows that peroxisomal ß-oxidation defects resulting in lipid alterations, including VLCFA accumulation, directly reprogram the main cellular functions of microglia. The elucidation of this link between lipid metabolism and the immune response of microglia will help to better understand the pathogenesis of peroxisomal leukodystrophies.
ABSTRACT
BACKGROUND: Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder that primarily affects the cerebellum and retina. SCA7 is caused by a polyglutamine expansion in the ATXN7 protein, a subunit of the transcriptional coactivator SAGA that acetylates histone H3 to deposit narrow H3K9ac mark at DNA regulatory elements of active genes. Defective histone acetylation has been presented as a possible cause for gene deregulation in SCA7 mouse models. However, the topography of acetylation defects at the whole genome level and its relationship to changes in gene expression remain to be determined. METHODS: We performed deep RNA-sequencing and chromatin immunoprecipitation coupled to high-throughput sequencing to examine the genome-wide correlation between gene deregulation and alteration of the active transcription marks, e.g. SAGA-related H3K9ac, CBP-related H3K27ac and RNA polymerase II (RNAPII), in a SCA7 mouse retinopathy model. RESULTS: Our analyses revealed that active transcription marks are reduced at most gene promoters in SCA7 retina, while a limited number of genes show changes in expression. We found that SCA7 retinopathy is caused by preferential downregulation of hundreds of highly expressed genes that define morphological and physiological identities of mature photoreceptors. We further uncovered that these photoreceptor genes harbor unusually broad H3K9ac profiles spanning the entire gene bodies and have a low RNAPII pausing. This broad H3K9ac signature co-occurs with other features that delineate superenhancers, including broad H3K27ac, binding sites for photoreceptor specific transcription factors and expression of enhancer-related non-coding RNAs (eRNAs). In SCA7 retina, downregulated photoreceptor genes show decreased H3K9 and H3K27 acetylation and eRNA expression as well as increased RNAPII pausing, suggesting that superenhancer-related features are altered. CONCLUSIONS: Our study thus provides evidence that distinctive epigenetic configurations underlying high expression of cell-type specific genes are preferentially impaired in SCA7, resulting in a defect in the maintenance of identity features of mature photoreceptors. Our results also suggest that continuous SAGA-driven acetylation plays a role in preserving post-mitotic neuronal identity.
Subject(s)
Retinal Diseases , Spinocerebellar Ataxias , Mice , Animals , Spinocerebellar Ataxias/genetics , Transcription Factors/genetics , Disease Models, Animal , Retinal Diseases/genetics , Gene Expression , Epigenesis, GeneticABSTRACT
BACKGROUND: Medulloblastoma is the most frequent brain malignancy of childhood. The current multimodal treatment comes at the expense of serious and often long-lasting side effects. Drug repurposing is a strategy to fast-track anti-cancer therapy with low toxicity. Here, we showed the ability of ß-blockers to potentiate radiotherapy in medulloblastoma with bad prognosis. METHODS: Medulloblastoma cell lines, patient-derived xenograft cells, 3D spheroids and an innovative cerebellar organotypic model were used to identify synergistic interactions between ß-blockers and ionising radiations. Gene expression profiles of ß-adrenergic receptors were analysed in medulloblastoma samples from 240 patients. Signaling pathways were explored by RT-qPCR, RNA interference, western blotting and RNA sequencing. Medulloblastoma cell bioenergetics were evaluated by measuring the oxygen consumption rate, the extracellular acidification rate and superoxide production. FINDINGS: Low concentrations of ß-blockers significantly potentiated clinically relevant radiation protocols. Although patient biopsies showed detectable expression of ß-adrenergic receptors, the ability of the repurposed drugs to potentiate ionising radiations did not result from the inhibition of the canonical signaling pathway. We highlighted that the efficacy of the combinatorial treatment relied on a metabolic catastrophe that deprives medulloblastoma cells of their adaptive bioenergetics capacities. This led to an overproduction of superoxide radicals and ultimately to an increase in ionising radiations-mediated DNA damages. INTERPRETATION: These data provide the evidence of the efficacy of ß-blockers as potentiators of radiotherapy in medulloblastoma, which may help improve the treatment and quality of life of children with high-risk brain tumours. FUNDING: This study was funded by institutional grants and charities.
Subject(s)
Cerebellar Neoplasms , Medulloblastoma , Child , Energy Metabolism , Humans , Medulloblastoma/drug therapy , Medulloblastoma/genetics , Medulloblastoma/radiotherapy , Quality of Life , Receptors, Adrenergic, beta/metabolism , Receptors, Adrenergic, beta/therapeutic use , SuperoxidesABSTRACT
Prostate cancer (PCa) is a leading cause of cancer-related deaths. The slow evolution of precancerous lesions to malignant tumors provides a broad time frame for preventing PCa. To characterize prostatic intraepithelial neoplasia (PIN) progression, we conducted longitudinal studies on Pten(i)pe-/- mice that recapitulate prostate carcinogenesis in humans. We found that early PINs are hypoxic and that hypoxia-inducible factor 1 alpha (HIF1A) signaling is activated in luminal cells, thus enhancing malignant progression. Luminal HIF1A dampens immune surveillance and drives luminal plasticity, leading to the emergence of cells that overexpress Transglutaminase 2 (TGM2) and have impaired androgen signaling. Elevated TGM2 levels in patients with PCa are associated with shortened progression-free survival after prostatectomy. Last, we show that pharmacologically inhibiting HIF1A impairs cell proliferation and induces apoptosis in PINs. Therefore, our study demonstrates that HIF1A is a target for PCa prevention and that TGM2 is a promising prognostic biomarker of early relapse after prostatectomy.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Prostatic Intraepithelial Neoplasia , Prostatic Neoplasms , Animals , Cell Plasticity , Disease Progression , Humans , Hypoxia , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Male , Mice , Prostatic Intraepithelial Neoplasia/genetics , Prostatic Intraepithelial Neoplasia/metabolism , Prostatic Intraepithelial Neoplasia/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathologyABSTRACT
Despite the fact that the cell cycle is a fundamental process of life, a detailed quantitative understanding of gene regulation dynamics throughout the cell cycle is far from complete. Single-cell RNA-sequencing (scRNA-seq) technology gives access to these dynamics without externally perturbing the cell. Here, by generating scRNA-seq libraries in different cell systems, we observe cycling patterns in the unspliced-spliced RNA space of cell cycle-related genes. Since existing methods to analyze scRNA-seq are not efficient to measure cycling gene dynamics, we propose a deep learning approach (DeepCycle) to fit these patterns and build a high-resolution map of the entire cell cycle transcriptome. Characterizing the cell cycle in embryonic and somatic cells, we identify major waves of transcription during the G1 phase and systematically study the stages of the cell cycle. Our work will facilitate the study of the cell cycle in multiple cellular models and different biological contexts.
Subject(s)
Deep Learning , Single-Cell Analysis , Gene Expression Profiling/methods , Genes, cdc , RNA/genetics , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , TranscriptomeABSTRACT
Epidemiological data have linked vitamin D deficiency to the onset and severity of various cancers, including prostate cancer, and although in vitro studies have demonstrated anticancer activities for vitamin D, clinical trials provided conflicting results. To determine the impact of vitamin D signaling on prostatic precancerous lesions, we treated genetically engineered Pten(i)pe-/- mice harboring prostatic intraepithelial neoplasia (PIN) with Gemini-72, a vitamin D analog with reported anticancer activities. We show that this analog induces apoptosis in senescent PINs, normalizes extracellular matrix remodeling by stromal fibroblasts, and reduces the prostatic infiltration of immunosuppressive myeloid-derived suppressor cells. Moreover, single-cell RNA-sequencing analysis demonstrates that while a subset of luminal cells expressing Krt8, Krt4, and Tacstd2 (termed luminal-C cells) is lost by such a treatment, antiapoptotic pathways are induced in persistent luminal-C cells. Therefore, our findings delineate the distinct responses of PINs and the microenvironment to Gemini-72, and shed light on mechanisms that limit treatment's efficacy.
Subject(s)
Precancerous Conditions , Prostatic Intraepithelial Neoplasia , Prostatic Neoplasms , Animals , Humans , Male , Mice , Precancerous Conditions/drug therapy , Prostatic Intraepithelial Neoplasia/drug therapy , Prostatic Intraepithelial Neoplasia/metabolism , Prostatic Intraepithelial Neoplasia/pathology , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Single-Cell Analysis , Tumor Microenvironment , Vitamin D/pharmacology , Vitamin D/therapeutic useABSTRACT
Omics analyses are powerful methods to obtain an integrated view of complex biological processes, disease progression, or therapy efficiency. However, few studies have compared different disease forms and different therapy strategies to define the common molecular signatures representing the most significant implicated pathways. In this study, we used RNA sequencing and mass spectrometry to profile the transcriptomes and proteomes of mouse models for three forms of centronuclear myopathies (CNMs), untreated or treated with either a drug (tamoxifen), antisense oligonucleotides reducing the level of dynamin 2 (DNM2), or following modulation of DNM2 or amphiphysin 2 (BIN1) through genetic crosses. Unsupervised analysis and differential gene and protein expression were performed to retrieve CNM molecular signatures. Longitudinal studies before, at, and after disease onset highlighted potential disease causes and consequences. Main pathways in the common CNM disease signature include muscle contraction, regeneration and inflammation. The common therapy signature revealed novel potential therapeutic targets, including the calcium regulator sarcolipin. We identified several novel biomarkers validated in muscle and/or plasma through RNA quantification, western blotting, and enzyme-linked immunosorbent assay (ELISA) assays, including ANXA2 and IGFBP2. This study validates the concept of using multi-omics approaches to identify molecular signatures common to different disease forms and therapeutic strategies.
Subject(s)
Gene Expression Profiling/methods , Myopathies, Structural, Congenital/drug therapy , Oligonucleotides, Antisense/therapeutic use , Protein Tyrosine Phosphatases, Non-Receptor/genetics , Proteomics/methods , Tamoxifen/therapeutic use , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Animals , Disease Models, Animal , Dynamin II/antagonists & inhibitors , Humans , Longitudinal Studies , Mass Spectrometry , Mice , Myopathies, Structural, Congenital/genetics , Myopathies, Structural, Congenital/metabolism , Nerve Tissue Proteins/antagonists & inhibitors , Sequence Analysis, RNA , Tumor Suppressor Proteins/antagonists & inhibitorsABSTRACT
OBJECTIVE: GPR64/ADGRG2 is an orphan Adhesion G protein-coupled receptor (ADGR) known to be mainly expressed in the parathyroid gland and epididymis. This investigation aimed to delineate the cellular expression of GPR64 throughout the body with focus on the gastrointestinal (GI) tract. METHODS: Transgenic Gpr64mCherry reporter mice were histologically examined throughout the body and reporter protein expression in intestinal tuft cells was confirmed by specific cell ablation. The GPCR repertoire of intestinal Gpr64mCherry-positive tuft cells was analyzed by quantitative RT-PCR analysis and in situ hybridization. The Gpr64mCherry was crossed into the general tuft cell reporter Trpm5GFP to generate small intestinal organoids for time-lapse imaging. Intestinal tuft cells were isolated from small intestine, FACS-purified and transcriptionally compared using RNA-seq analysis. RESULTS: Expression of the Gpr64mCherry reporter was identified in multiple organs and specifically in olfactory microvillous cells, enteric nerves, and importantly in respiratory and GI tuft cells. In the small intestine, cell ablation targeting Gpr64-expressing epithelial cells eliminated tuft cells. Transcriptional analysis of small intestinal Gpr64mCherry -positive tuft cells confirmed expression of Gpr64 and the chemo-sensors Sucnr1, Gprc5c, Drd3, and Gpr41/Ffar3. Time-lapse studies of organoids from Trpm5GFP:Gpr64mCherry mice revealed sequential expression of initially Trpm5GFP and subsequently also Gpr64mCherry in maturing intestinal tuft cells. RNA-seq analysis of small intestinal tuft cells based on these two markers demonstrated a dynamic change in expression of transcription factors and GPCRs from young to mature tuft cells. CONCLUSIONS: GPR64 is expressed in chemosensory epithelial cells across a broad range of tissues; however, in the GI tract, GPR64 is remarkably selectively expressed in mature versus young immunoregulatory tuft cells.
Subject(s)
Chemoreceptor Cells/metabolism , Epithelial Cells/metabolism , Intestine, Small/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Female , Intestine, Small/cytology , Male , Mice , Mice, Transgenic , Receptors, G-Protein-Coupled/analysis , Receptors, G-Protein-Coupled/geneticsABSTRACT
Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease mainly characterized by motor incoordination because of progressive cerebellar degeneration. SCA7 is caused by polyglutamine expansion in ATXN7, a subunit of the transcriptional coactivator SAGA, which harbors histone modification activities. Polyglutamine expansions in specific proteins are also responsible for SCA1-SCA3, SCA6, and SCA17; however, the converging and diverging pathomechanisms remain poorly understood. Using a new SCA7 knock-in mouse, SCA7140Q/5Q, we analyzed gene expression in the cerebellum and assigned gene deregulation to specific cell types using published datasets. Gene deregulation affects all cerebellar cell types, although at variable degree, and correlates with alterations of SAGA-dependent epigenetic marks. Purkinje cells (PCs) are by far the most affected neurons and show reduced expression of 83 cell-type identity genes, including these critical for their spontaneous firing activity and synaptic functions. PC gene downregulation precedes morphologic alterations, pacemaker dysfunction, and motor incoordination. Strikingly, most PC genes downregulated in SCA7 have also decreased expression in SCA1 and SCA2 mice, revealing converging pathomechanisms and a common disease signature involving cGMP-PKG and phosphatidylinositol signaling pathways and LTD. Our study thus points out molecular targets for therapeutic development, which may prove beneficial for several SCAs. Furthermore, we show that SCA7140Q/5Q males and females exhibit the major disease features observed in patients, including cerebellar damage, cerebral atrophy, peripheral nerves pathology, and photoreceptor dystrophy, which account for progressive impairment of behavior, motor, and visual functions. SCA7140Q/5Q mice represent an accurate model for the investigation of different aspects of SCA7 pathogenesis.SIGNIFICANCE STATEMENT Spinocerebellar ataxia 7 (SCA7) is one of the several forms of inherited SCAs characterized by cerebellar degeneration because of polyglutamine expansion in specific proteins. The ATXN7 involved in SCA7 is a subunit of SAGA transcriptional coactivator complex. To understand the pathomechanisms of SCA7, we determined the cell type-specific gene deregulation in SCA7 mouse cerebellum. We found that the Purkinje cells are the most affected cerebellar cell type and show downregulation of a large subset of neuronal identity genes, critical for their spontaneous firing and synaptic functions. Strikingly, the same Purkinje cell genes are downregulated in mouse models of two other SCAs. Thus, our work reveals a disease signature shared among several SCAs and uncovers potential molecular targets for their treatment.
Subject(s)
Cerebellum/pathology , Purkinje Cells/pathology , Spinocerebellar Ataxias/pathology , Animals , Down-Regulation , Female , Gene Knock-In Techniques , Male , Mice , TranscriptomeABSTRACT
Amyotrophic lateral sclerosis and frontotemporal dementia are two neurodegenerative diseases with currently no cure. These two diseases share a clinical continuum with overlapping genetic causes. Mutations in the CHMP2B gene are found in patients with ALS, FTD and ALS-FTD. To highlight deregulated mechanisms occurring in ALS-FTD linked to the CHMP2B gene, we performed a whole transcriptomic study on lumbar spinal cord from CHMP2Bintron5 mice, a model that develops progressive motor alterations associated with dementia symptoms reminiscent of both ALS and FTD. To gain insight into the transcriptomic changes taking place during disease progression this study was performed at three stages: asymptomatic, symptomatic and end stage. We showed that before appearance of motor symptoms, the major disrupted mechanisms were linked with the immune system/inflammatory response and lipid metabolism. These processes were progressively replaced by alterations of neuronal electric activity as motor symptoms appeared, alterations that could lead to motor neuron dysfunction. To investigate overlapping alterations in gene expression between two ALS-causing genes, we then compared the transcriptome of symptomatic CHMP2Bintron5 mice with the one of symptomatic SOD1G86R mice and found the same families deregulated providing further insights into common underlying dysfunction of biological pathways, disrupted or disturbed in ALS. Altogether, this study provides a database to explore potential new candidate genes involved in the CHMP2Bintron5-based pathogenesis of ALS, and provides molecular clues to further understand the functional consequences that diseased neurons expressing CHMP2B mutant may have on their neighbor cells.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Disease Models, Animal , Endosomal Sorting Complexes Required for Transport/genetics , Frontotemporal Dementia/genetics , Nerve Tissue Proteins/genetics , Superoxide Dismutase-1/genetics , Transcriptome/genetics , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Endosomal Sorting Complexes Required for Transport/biosynthesis , Frontotemporal Dementia/metabolism , Frontotemporal Dementia/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/biosynthesis , Spinal Cord/metabolism , Spinal Cord/pathologyABSTRACT
BACKGROUND: Pediatric high-grade gliomas (pHGGs) are facing a very dismal prognosis and representative pre-clinical models are needed for new treatment strategies. Here, we examined the relevance of collecting functional, genomic, and metabolomics data to validate patient-derived models in a hypoxic microenvironment. METHODS: From our biobank of pediatric brain tumor-derived models, we selected 11 pHGGs driven by the histone H3.3K28M mutation. We compared the features of four patient tumors to their paired cell lines and mouse xenografts using NGS (next generation sequencing), aCGH (array comparative genomic hybridization), RNA sequencing, WES (whole exome sequencing), immunocytochemistry, and HRMAS (high resolution magic angle spinning) spectroscopy. We developed a multicellular in vitro model of cell migration to mimic the brain hypoxic microenvironment. The live cell technology Incucyte© was used to assess drug responsiveness in variable oxygen conditions. RESULTS: The concurrent 2D and 3D cultures generated from the same tumor sample exhibited divergent but complementary features, recreating the patient intra-tumor complexity. Genomic and metabolomic data described the metabolic changes during pHGG progression and supported hypoxia as an important key to preserve the tumor metabolism in vitro and cell dissemination present in patients. The neurosphere features preserved tumor development and sensitivity to treatment. CONCLUSION: We proposed a novel multistep work for the development and validation of patient-derived models, considering the immature and differentiated content and the tumor microenvironment of pHGGs.