Biology subjects

Crossman, D.

Publications and source records attributed to Crossman, D..

7 recordsLinked to original sources

bioRxiv2021

miR-486 is an epigenetic modulator of Duchenne muscular dystrophy pathologies

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle disorder resulting in muscle weakness and cardiomyopathy. MicroRNAs have been shown to play essential roles in muscle development, metabolism, and disease pathologies. We demonstrated that miR-486 expression is reduced in DMD muscles and its expression levels correlate with dystrophic disease severity. MicroRNA-486 knockout mice developed disrupted myofiber architecture, decreased myofiber size, decreased locomotor activity, increased cardiac fibrosis, and metabolic defects that were exacerbated on the dystrophic mdx5cv background. We integrated RNA-sequencing and chimeric eCLIP-sequencing data to identify direct in vivo targets of miR-486 and associated dysregulated gene signatures in skeletal muscle. In comparison to our DMD mouse muscle transcriptomes, we identified several of these miR-486 muscle targets including known modulators of dystrophinopathy disease symptoms. Together, our studies identify miR-486 as a driver of muscle remodeling in DMD, a useful biomarker for dystrophic disease progression, and highlight chimeric eCLIP-sequencing as a useful tool to identify direct in vivo microRNA target transcripts. O_FIG O_LINKSMALLFIG WIDTH=122 HEIGHT=200 SRC="FIGDIR/small/448387v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@13eeaf5org.highwire.dtl.DTLVardef@1b3b2dborg.highwire.dtl.DTLVardef@a36782org.highwire.dtl.DTLVardef@1e0b95f_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2021

Mitochondrial Fission Regulates Transcription of Ribosomal Protein Genes in Embryonic Hearts

Mitochondrial dysfunction causes severe congenital heart diseases and prenatal/neonatal lethality. The lack of sufficient knowledge regarding how mitochondrial abnormalities affect cardiogenesis poses a major barrier for the development of clinical applications that target inborn heart defects due to mitochondrial deficiency. Mitochondrial morphology, which is regulated by fission and fusion, plays key roles in determining mitochondrial activity. Drp1 encodes a dynamin-related GTPase required for mitochondrial fission. To investigate the role of mitochondrial fission on cardiogenesis during the embryonic metabolic shift period, we specifically inactivated Drp1 in second heart field derived structures. Deletion of Drp1 in embryonic cardiomyocytes led to severe defects in mitochondrial morphology, ultrastructure, and activity. These defects caused increased cell death, decreased cell survival, disorganized cardiomyocytes, and embryonic lethality. Through characterizing this model, we reveal a novel AMPK-SIRT7-GABPB axis that relays the mitochondrial fission anomaly to reduced transcription of ribosomal protein genes in mutant cardiomyocytes. We therefore provide the first mouse genetic evidence to show that mitochondrial fission is essential for embryonic heart development. Furthermore, we uncovered a novel signaling cascade that mediates the crosstalk between mitochondrial dysfunction and protein synthesis. Our research provides further mechanistic insight regarding how mitochondrial dysfunction causes pathological molecular and cellular alterations during cardiogenesis.

developmental biology↗

bioRxiv2020

Spatial heterogeneity of glioblastoma cells reveals sensitivity to NAD+ depletion at tumor edge

Even after total resection of glioblastoma core lesions by surgery and aggressive post-surgical treatments, life-threatening tumors inevitably recur. A characteristic obstacle in effective treatment is high intratumoral heterogeneity, both longitudinally and spatially. Recurrence occurs predominantly at the brain parenchyma-tumor core interface, a region termed tumor edge. Given the difficulty of accessing it surgically, the composition of the tumor edge, harboring both cancerous and non-cancerous cells, remains largely unknown. Here, to identify phenotypic diversity among heterogeneous glioblastoma core and edge lesions, we uncovered the existence of three phenotypically-distinct clonal subpopulations within individual tumors from glioblastoma patients. Clones from the tumor core shared the same phenotype, exclusively generating tumor-core cells. In contrast, two distinct clonal subtypes were identified at the tumor edge: one generated only edge-lesion cells and the other expanded more broadly to establish both edge- and core-lesions. Using multiple xenograft experimental models in mouse brains, tumor edge development was found to require that both somatic and tumor cells express the NADase CD38, combinedly elevating glioblastoma malignancy. In vitro data suggested that intracellular NADase activity at the edge was provoked through intercellular communication between edge clones and normal astrocytes. Systemic treatment of tumor-bearing mice with 78c, a small-molecule CD38 inhibitor, attenuated the formation of glioblastoma edge lesions, suggesting its clinical potential to pharmacologically eliminate tumor-edge lesions. Collectively, these findings provide novel phenotypic and mechanistic insights into clonal heterogeneity within glioblastoma, particularly in the surgically unresectable, currently understudied tumor edge.

cancer biology↗

bioRxiv2020

Brain aging-dependent glioma traits reversible by NAD+/BDNF-mediated neuronal reactivation

The rise in aging population worldwide is increasing death from cancer, including glioblastoma. Here, we explore the impact of brain aging on glioma tumorigenesis. We find that glioblastoma in older patients and older mice displayed reduced neuronal signaling, including a decline of NTRK-like family member 6 (SLITRK6), a receptor for neurotrophic factor BDNF. This reduction was linked to the systemic decline of nicotinamide adenine dinucleotide (NAD+) with aging, as old mice exposed to young blood via parabiosis or supplemented with the NAD+ precursor NMN (nicotinamide mononucleotide) reverted phenotypically to young-brain responses to glioma, with reactivated neuronal signaling and reduced death from tumor burden. Interestingly, the phenotypic reversal by NMN was largely absent in old mice undergoing parabiosis with BDNF+/- young mice and in BDNF+/- mice undergoing tumor challenge, supporting the notion that the lower NAD+-BDNF signaling in the aging brain aggravated glioma tumorigenesis. We propose that the aging-associated decline in brain NAD+ worsens glioma outcomes at least in part by decreasing neuronal/synaptic activity and increasing neuroinflammation.

cancer biology↗

bioRxiv2020

Profiling of the reprogramome and quantification of fibroblast reprogramming to pluripotency

Pluripotent state can be established via reprogramming of somatic nuclei by factors within an oocyte or by ectopic expression of a few transgenes. Considered as being extensive and intensive, the full complement of genes to be reprogrammed, however, has never been defined, nor has the degree of reprogramming been determined quantitatively. Here, we propose a new concept of reprogramome, which is defined as the full complement of genes that need to be reprogrammed to the expression levels found in pluripotent stem cells (PSCs). This concept in combination with RNA-seq enables us to precisely profile reprogramome and sub-reprogramomes, and study the reprogramming process with the help of other available tools such as GO analyses. With reprogramming of human fibroblasts into PSCs as an example, we have defined the full complement of the human fibroblast-to-PSC reprogramome. Furthermore, our analyses of the reprogramome revealed that WNT pathways and genes with roles in cellular morphogenesis have to be extensively and intensely reprogrammed for the establishment of pluripotency. We further developed the first mathematical model to quantitate the overall reprogramming, as well as reprogramming in a specific cellular feature such as WNT signaling pathways and genes regulating cellular morphogenesis. We anticipate that our concept and mathematical model may be applied to study and quantitate other reprogramming (pluripotency reprogramming from other somatic cells, and lineage reprogramming), as well as transcriptional and epigenetic differences between any two types of cells including cancer cells and their normal counterparts.

cell biology↗

bioRxiv2019

Impaired Regulation of Redox Transcriptome during the Differentiation of iPSCs into Induced Cardiomyocytes (iCMs)

BackgroundReprogramming of somatic cells into pluripotent stem cells (iPSC) and subsequent differentiation into iPSC-derived cardiomyocytes (iCM) seems to be a promising strategy for cardiac regenerative therapy. However, recent failure or poor outcomes in cardiac cell therapy warrants further investigation focusing on the infarction/wound environment (site of healing) to improve the cardiac regenerative medicine. Here, using next generation sequencing (NGS), we analyzed the global transcriptome to discover the unidentified genes/pathways that are crucial for cell survival, cytoprotection and mitochondrial dynamics during the differentiation of iPSC into iCM.\n\nMethodsHigh throughput NGS was performed RNA from human iPSCs and iCMs (n=3/group) and analyzed the global changes in the transcriptome during differentiation. Furthermore, Ingenuity Pathway Analysis (IPA) and Gene Ontology (GO) for biological process were performed to understand the transcriptional networks that are involved during iCM differentiation. RNA-seq data were further validated by qRT-PCR analyses.\n\nResultsGlobal transcriptome analysis revealed that ~9,290 genes (log2 FC >2) were significantly altered in human iCMs compared to the parent iPSCs, in which 4,784 transcripts were substantially upregulated and 4,506 transcripts were down-regulated during differentiation. GO enrichment and IPA analyses revealed the top 10 regulatory networks (i.e. hierarchical order) involved in differentiation of iCMs including cardiomyocyte remodeling, integrin-linked kinase signaling, Rho family of GTPases, etc. Surprisingly, none of the top 10 pathways listed the genes liable for redox signaling networks that are crucial for the basal cellular redox homeostasis, Nrf2-dependent antioxidant defense, mitochondrial functions and cell survival. Our deeper and unbiased analysis of this data revealed that the genes involved in above canonical signaling pathways are found in the middle of the inverted vertical cone. Of note, although these pathways are significantly altered during the differentiation (of iPS into cardiomyocytes), a majority of them are ranked low in the hierarchical list (>150). Validation of the randomly selected genes representing various pathways real-time qPCR confirmed the global transcriptome changes observed in NGS.\n\nConclusionWe highlight the significance of Nrf2-redox and mitochondrial transcriptome during differentiation of iPSC into iCMs. Thus, targeting the redox signaling mechanisms in iCMs may enhance their efficiency for cell therapy and improved myocardial repair.

cell biology↗

bioRxiv2018

Basal Role for Nrf2-transgne on transcriptional (mRNA/miRNA) regulation in the mouse myocardium

BackgroundNFE2L2 (nuclear factor, erythroid 2 like 2; Nrf2), is a stress-responsive transcription factor that regulates cellular redox homeostasis. The action mechanism of Nrf2 occurs through sensing oxidative stress in the cellular environment in responses to various stresses including altered metabolism, toxic insult and xenobiotic stresses. However, under the basal condition, the role of excess Nrf2 on global miRNA-mRNA interactions in the myocardium is unknown. Here, we tested a hypothesis that excess Nrf2 (transgene) will promote the transcription of antioxidants, which then might escalate the basal defense mechanisms in the heart. Furthermore, we investigated whether changes in the miRNA profile might have a strong role on the transcriptome in TG hearts.\n\nMethodsNon-transgenic (NTG), and Cardiac specific Nrf2 transgenic (Nrf2-TG) mice in the C57/BL6 background at the age of 6-8 months were used to examine transcriptional and post-transcriptional signatures (mRNA and miRNA expressions) by performing next generation sequencing (NGS) for RNA (RNAseq) and small RNA (sRNAseq) for miRNA expressions in the heart (n=3-6/group). Validation of the NGS data was performed by quantitative real time PCR (qRT-PCR) using specific primers targeting selected miRNAs or mRNAs. Finally, in-silico analyses were performed to determine the miRNA-mRNA interactome networks and the pathways that are potentially regulated by these networks.\n\nResultsNGS analysis for mRNA indicated that there were 5727 differently expressed genes (DEGs) in the Nrf2-TG vs. NTG myocardium. Of which, 3552 were upregulated and 2175 were downregulated significantly. Small RNAseq analysis revealed that 112 miRNAs were significantly altered in Nrf2-TG versus NTG hearts. Among these miRNAs, 68 were upregulated and 44 were downregulated significantly. Validation of key targets for mRNA and miRNA by qPCR confirmed the NGS results. In-silico analysis (IPA) revealed that the majority of the miRNAs and mRNAs that are significantly altered in response to transgene expression are potentially involved in Nrf2 mediated oxidative stress response, ILK Signaling, hypoxia signaling in the cardiovascular system and glutathione-mediated detoxification etc.\n\nConclusionThese high-throughput sequencing data sets from cardiac specific Nrf2-TG mice revealed the transcriptome-wide effects of Nrf2 in the myocardium. Furthermore, our comprehensive analysis indicates that Nrf2 may directly or indirectly regulates these sub-sets of cardiac miRNA-mRNA interactome networks under basal physiological setting.

molecular biology↗