scholarly journals Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

2018 ◽  
Author(s):  
Laura E. Schultz-Rogers ◽  
Maira P. Almeida ◽  
Wesley A. Wierson ◽  
Marcel Kool ◽  
Maura McGrail
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Cell Death ◽  
Neural Stem Cells ◽  
Progenitor Cell ◽  
Adaptor Protein ◽  
Genome Integrity ◽  
Neural Precursors ◽  
Chromatin Remodelers

AbstractRetinoblastoma-binding protein 4 (Rbbp4) is a WDR adaptor protein for multiple chromatin remodelers implicated in human oncogenesis. Here we show Rbbp4 is overexpressed in zebrafish rb1-embryonal brain tumors and is upregulated across the spectrum of human embryonal and glial brain cancers. We demonstrate in vivo Rbbp4 is essential for zebrafish neurogenesis and has distinct roles in neural stem and progenitor cells. rbbp4 mutant neural stem cells show delayed cell cycle progression and become hypertrophic. In contrast, rbbp4 mutant neural precursors accumulate extensive DNA damage and undergo programmed cell death that is dependent on Tp53 signaling. Loss of Rbbp4 and disruption of genome integrity correlates with failure of neural precursors to initiate quiescence and transition to differentiation. rbbp4; rb1 double mutants show that survival of neural precursors after disruption of Rb1 is dependent on Rbbp4. Elevated Rbbp4 in Rb1-deficient brain tumors might drive proliferation and circumvent DNA damage and Tp53-dependent apoptosis, lending support to current interest in Rbbp4 as a potential druggable target.Author SummaryExamining the developmental mechanisms controlling neural stem and progenitor cell behavior is critical to our understanding of the processes driving brain tumor oncogenesis. Chromatin remodelers and their associated adaptor proteins are thought to be key drivers of brain development and disease through epigenetic regulation of gene expression and maintenance of genome integrity, but knowledge of their in vivo roles in vertebrate neurogenesis is limited. The chromatin remodeler adaptor protein Rbbp4 has recently been shown to function in a mouse model of neuroblastoma and in glioblastoma multiforme cell resistance to the chemotherapeutic temozolomide. However, an in vivo requirement for Rbbp4 in neurogenesis has only just been shown by isolation of a recessive lethal mutation in zebrafish rbbp4. Here we provide conclusive genetic evidence that zebrafish rbbp4 is essential in neural stem and progenitor cell function during development. Our data reveal for the first time in vivo that Rbbp4 prevents DNA damage and activation of Tp53 signaling pathway that leads to programmed cell death. Importantly, neural progenitors that are mutant for the tumor suppressor Rb1 also depend on Rbbp4 for survival. Finally, we show that neural stem cells that have lost Rbbp4 cease dividing, and may enter a senescent like state. Together, these observations provide novel evidence that elevated expression of Rbbp4 in rb1-mutant tumors may contribute to cancer cell survival by blocking senescence and/or DNA damage-induced cell death.

CBIO-13. THOC1 DRIVES GBM AGGRESSION THROUGH MODULATION OF R-LOOPS AND GENOMIC STABILITY

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi29-vi30
Author(s):  
Shreya Budhiraja ◽  
Shivani Baisiwala ◽  
Khizar Nandoliya ◽  
Li Chen ◽  
Crismita Dmello ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Neural Stem Cells ◽  
Histone Deacetylase ◽  
Genomic Stability ◽  
Loop Formation ◽  
Driver Genes ◽  
A Genome

Abstract Glioblastoma (GBM) is the most aggressive and common type of adult malignant brain tumor, with a median survival of only 21 months. To identify which genes drive its highly aggressive phenotype, we performed a genome-wide CRISPR-Cas9 knockout screen. Results showed substantial enrichment of ~160 novel essential oncogenic driver genes and pathways, including a previously unstudied gene THOC1—involved in RNA processing—that showed significant elevations in expression at RNA and protein levels (p< 0.05) in GBM, as well as a significant survival benefit in patient datasets when downregulated (p< 0.05). Knocking out THOC1 resulted in cell death in multiple GBM patient-derived xenograft (PDX) lines and extended survival compared to the controls (p< 0.01) in vivo. Overexpression of THOC1 in neural stem cells resulted in transformation to a cancerous phenotype, as evidenced by sphere formation in a soft agar assay (p< 0.01) and in vivo tumor engraftment assays. Further investigation of THOC1 through immunoprecipitation in neural stem cells and multiple GBM lines showed significant interaction in GBM with histone deacetylase complex SIN3A, involved in recruiting major histone deacetylases in order to close the DNA and prevent the accumulation of R-loops, RNA:DNA hybrids that pose a threat to genomic stability. Additional investigation revealed that THOC1-knockdowns in vitro induced R-loop formation and DNA damage, while THOC1-overexpression in vitro resulted in an untenable decrease in R-loops and DNA damage, suggesting that the THOC1-SIN3A axis is elevated in GBM in order to prevent the accumulation of genotoxic R-loops. Additionally, histone deacetylase activity was shown to be elevated in THOC1-overexpression conditions and reduced in THOC1-knockdown conditions, confirming that the THOC1-SIN3A axis functions to prevent R-loop accumulation through the epigenetic regulation. In summary, our whole-genome CRISPR-Cas9 knockout screen has identified a promising therapeutic target for GBM—a disease desperately in need of therapeutic innovations.

Growth Factor Independence 1 (Gfi1) Is An Essential Factor for the Development of Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 297-297
Author(s):  
Cyrus Khandanpour ◽  
Ehssan Sharif- Askari ◽  
Paul Jolicoeur ◽  
Ulrich Duehrsen ◽  
Tarik Moroy
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Cell Death ◽  
T Cell ◽  
Cell Lymphoma ◽  
Latency Period ◽  
T Cell Lymphoma ◽  
Hematopoietic Stem

Abstract Hematopoietic differentiation is controlled to a large extent by a network of transcription factors and chromatin modifiers and disruption of this system can lead to leukemia or lymphoma. One of the transcription factor genes, which is aberrantly expressed in human T-cell lymphoma is Growth Factor Independence 1 (Gfi1). Since over expression of Gfi1 can accelerate experimentally induced T-cell tumors in mice, it is likely that Gfi1 plays a crucial role in establishing or maintaining lymphoid neoplasms. To test this hypothesis we have used, N-ethyl-N-nitrosourea (ENU) to induce T-cell tumors in WT mice (Gfi1+/+), Gfi1-deficient mice (Gfi1−/−) or mice transgenically over expressing Gfi1 under the control of the pan-hematopoietic vav-promoter (vav-Gfi1). As expected, most of Gfi1+/+ mice (25/27) developed T-cell tumors and acute myeloid leukemia within 118 days. Similarly, vav-Gfi1 mice (10/10) developed T-cell lymphoma, but within a shorter latency period (88 days). In contrast, only 3/14 Gfi1−/− mice developed hematopoietic neoplasia with a prolonged median latency period of 126 days. Other approaches using infection of newborn mice with Moloney Murine leukemia virus (MoMuLV) to induce T-cell lymphoma or co expression of an Eμ-myc transgene to induce B-cell lymphoma showed a similar dependency of tumor formation on the presence and expression of Gfi1. Closer analysis of tumors forming in Gfi1−/− mice demonstrated that Gfi1 deficiency correlated with a smaller size of the tumors and a noticeably increased rate of cell death within the tumor samples. This pointed to a potential role of Gfi1 in the regulation of apoptosis. To explore this hypothesis, we exposed both thymocytes and hematopoietic stem cells (Lin-, Sca1+, c-kit+, LSK) to ENU or gamma-irradiation in vitro. We could observe that Gfi1−/− thymocytes and stem cells (LSK cells) have a higher rate of cell death following exposure to these DNA damage inducing agents in vitro than the WT controls. To validate these results, we recapitulated these experiments in vivo. Gfi1−/− mice exhibited severe bone marrow failure and a more pronounced loss of hematopoietic stem cells (LSK) than Gfi1+/+ mice after ENU treatment or gamma irradiation in vivo. To explore this mechanism on the molecular basis we evaluated expression of the different pro and antiapoptotic components in Gfi1+/+ and Gfi1−/− thymocytes after irradiation. Strikingly, Gfi1−/− thymocytes expressed higher levels of the pro-apoptotic proteins such as Bax and Noxa and lower levels of the CDK inhibitor p21WAF than WT thymocytes following induction of DNA damage. Our model would be that Gfi1 represents a new regulator in the cellular response to DNA damage in the hematopoietic system by inhibiting different proapoptotic factors. We propose that Gfi1 is essential for the development of lymphoid and potentially myeloid neoplasms by inhibiting apoptosis. We suggest that Gfi1 could represent a possible new target structure for therapeutic intervention.

scholarly journals Roles of Retinoids and Retinoic Acid Receptors in the Regulation of Hematopoietic Stem Cell Self-Renewal and Differentiation

PPAR Research ◽  
2007 ◽  
Vol 2007 ◽  
pp. 1-7 ◽  
Author(s):  
Louise E. Purton
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Progenitor Cell ◽  
Retinoic Acid Receptors ◽  
Key Factors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Complex Processes ◽  
Major Interest

Multipotent hematopoietic stem cells (HSCs) sustain blood cell production throughout an individual's lifespan through complex processes ultimately leading to fates of self-renewal, differentiation or cell death decisions. A fine balance between these decisions in vivo allows for the size of the HSC pool to be maintained. While many key factors involved in regulating HSC/progenitor cell differentiation and cell death are known, the critical regulators of HSC self-renewal are largely unknown. In recent years, however, a number of studies describing methods of increasing or decreasing the numbers of HSCs in a given population have emerged. Of major interest here are the emerging roles of retinoids in the regulation of HSCs.

scholarly journals Altered HSC Metabolism in Response to Stress Leads to De Novo dna Damage and Cellular Attrition

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 255-255
Author(s):  
Anja Geiselhart ◽  
Dagmar Walter ◽  
Amelie Lier ◽  
Frederic B. Thalheimer ◽  
Sina Huntscha ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
Cell Death ◽  
De Novo ◽  
Bone Marrow Failure ◽  
Fold Increase ◽  
Quiescent State ◽  
Response To Stress

Abstract Hematopoietic stem cells (HSCs) reside in a quiescent state, which is thought to preserve their genomic stability during aging. HSCs are forced to exit this so-called dormant state and enter into cycle in response to stress stimuli such as infections or severe bleeding. This situation may provoke high levels of proliferative stress in HSCs and a subsequent decline in stem cell function. We recently found that de novo DNA damage can be precipitated in HSCs in vivo by enforcing cell cycle progression using agonists that mimic physiologic stress, such as interferons, G-CSF, TPO or serial bleeding, (Walter et al., Blood, 122, 21:799). The Fanconi anemia (FA) DNA repair pathway is an important route via which this replication damage is resolved in HSCs in vivo. In FA deficient mice, DNA damage repair was impaired, provoking HSC depletion and severe aplastic anemia (p<0.01) upon serial treatment with the synthetic double-stranded RNA mimetic polyI:polyC (pI:pC). Here, we sought to identify the mechanistic basis of the stress-induced DNA damage acquisition and concomitant HSC attrition in vivo. Activated HSCs exhibited elevated mitochondrial membrane potential, indicative of increased energy production via oxidative phosphorylation (>2-fold increase, p<0.01). Next, to determine whether there was an associated increase in intracellular reactive oxygen species (ROS) production, we made use of genetically encoded fluorescent biosensors to detect the status of specific redox couples within different HSC compartments in vivo. Activated HSCs demonstrated increased levels of oxidized mitochondrial glutathione (2.3-fold increase, p<0.01) and cytoplasmic hydrogen peroxide (1.6-fold increase, p<0.05) compared to dormant HSC controls. These enhanced ROS levels directly correlated with elevated 8-Oxo-dG lesions on the DNA of HSCs that had been activated into cycle in vivo(>1.3-fold increase, p<0.05). Finally, retroviral over-expression of ROS-detoxifying enzymes completely rescued gH2AX foci formation in cycling HSCs, demonstrating a direct functional link between stress-induced DNA damage and altered redox biology. We next performed live cell video imaging on individual WT and Fanca-/- LT-HSCs in vitro in order to track cell fate decisions upon exit from quiescence. In the first division upon exit from quiescence, Fanca-/- HSCs were frequently observed to undergo abnormal mitoses while this was not evident in WT HSCs. At this time point, we observed elevated DNA damage in Fanca-/- HSCs as measured by gH2AX, 53BP1 and RAD51 foci, as well as increased ROS-induced 8-Oxo-dG lesions (>5-fold increase, p<0.01). HSCs from Fanca-/- mice demonstrated a significantly higher rate of replication-dependent cell death following the first division (24% vs. 6%, p<0.05%) suggesting that apoptosis is the major route via which HSCs are lost in response to stress-induced DNA damage. Taken together, these data strongly implicate stress-induced exit from dormancy as a cause of physiologic DNA damage in HSCs in vivo. Under stress conditions, the increased energy demand of cycling stem cells leads to elevated levels of ROS in mitochondria and cytoplasm, which is a direct source of DNA damage. If unresolved by the FA-dependent DNA damage response, this DNA damage accumulates in the cell and provokes apoptotic cell death. This recapitulates the highly penetrant bone marrow failure syndrome in FA patients and suggests that their HSCs are lost due to an aberrant response to HSC activation, most likely as a consequence of infection or other physiologic stress. These data provide a novel link between stress hematopoiesis, ROS, DNA damage and HSC loss and may have important clinical implications in the study of age-related hematopoietic defects in both FA and non-FA patients. Moreover, these data provide the first evidence that FA knockout mouse models can be utilized to accurately recapitulate the etiology of bone marrow failure through the progressive application of stress-induced alterations in HSC function that mimic usual physiologic stressors such as infection. Disclosures No relevant conflicts of interest to declare.

scholarly journals Loss of mitochondrial transcription factor A in neural stem cells leads to immature brain development and triggers the activation of the integral stress response in vivo

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0255355
Author(s):  
Rintaro Kuroda ◽  
Kaoru Tominaga ◽  
Katsumi Kasashima ◽  
Kenji Kuroiwa ◽  
Eiji Sakashita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Transcription Factor ◽  
Stress Response ◽  
Neural Stem Cells ◽  
Mitochondrial Dysfunction ◽  
Mitochondrial Transcription ◽  
Mitochondrial Transcription Factor ◽  
Mitochondrial Transcription Factor A

Mitochondrial dysfunction is significantly associated with neurological deficits and age-related neurological diseases. While mitochondria are dynamically regulated and properly maintained during neurogenesis, the manner in which mitochondrial activities are controlled and contribute to these processes is not fully understood. Mitochondrial transcription factor A (TFAM) contributes to mitochondrial function by maintaining mitochondrial DNA (mtDNA). To clarify how mitochondrial dysfunction affects neurogenesis, we induced mitochondrial dysfunction specifically in murine neural stem cells (NSCs) by inactivating Tfam. Tfam inactivation in NSCs resulted in mitochondrial dysfunction by reducing respiratory chain activities and causing a severe deficit in neural differentiation and maturation both in vivo and in vitro. Brain tissue from Tfam-deficient mice exhibited neuronal cell death primarily at layer V and microglia were activated prior to cell death. Cultured Tfam-deficient NSCs showed a reduction in reactive oxygen species produced by the mitochondria. Tfam inactivation during neurogenesis resulted in the accumulation of ATF4 and activation of target gene expression. Therefore, we propose that the integrated stress response (ISR) induced by mitochondrial dysfunction in neurogenesis is activated to protect the progression of neurodegenerative diseases.

scholarly journals mRNA-Driven Generation of Transgene-Free Neural Stem Cells from Human Urine-Derived Cells

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 1043 ◽  
Author(s):  
Phil Jun Kang ◽  
Daryeon Son ◽  
Tae Hee Ko ◽  
Wonjun Hong ◽  
Wonjin Yun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Human Urine ◽  
Neurological Disorders ◽  
Therapeutic Potential ◽  
Embryonic Stem ◽  
Global Gene Expression ◽  
Patient Specific ◽  
Human Neural Stem Cells

Human neural stem cells (NSCs) hold enormous promise for neurological disorders, typically requiring their expandable and differentiable properties for regeneration of damaged neural tissues. Despite the therapeutic potential of induced NSCs (iNSCs), a major challenge for clinical feasibility is the presence of integrated transgenes in the host genome, contributing to the risk for undesired genotoxicity and tumorigenesis. Here, we describe the advanced transgene-free generation of iNSCs from human urine-derived cells (HUCs) by combining a cocktail of defined small molecules with self-replicable mRNA delivery. The established iNSCs were completely transgene-free in their cytosol and genome and further resembled human embryonic stem cell-derived NSCs in the morphology, biological characteristics, global gene expression, and potential to differentiate into functional neurons, astrocytes, and oligodendrocytes. Moreover, iNSC colonies were observed within eight days under optimized conditions, and no teratomas formed in vivo, implying the absence of pluripotent cells. This study proposes an approach to generate transplantable iNSCs that can be broadly applied for neurological disorders in a safe, efficient, and patient-specific manner.

scholarly journals Dynamic integration of enteric neural stem cells in ex vivo organotypic colon cultures

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Georgina Navoly ◽  
Conor J. McCann
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Organotypic Culture ◽  
Ex Vivo ◽  
Donor Cell ◽  
Colonic Tissue ◽  
Cell Therapies ◽  
Enteric Ganglia ◽  
Donor Cells

AbstractEnteric neural stem cells (ENSC) have been identified as a possible treatment for enteric neuropathies. After in vivo transplantation, ENSC and their derivatives have been shown to engraft within colonic tissue, migrate and populate endogenous ganglia, and functionally integrate with the enteric nervous system. However, the mechanisms underlying the integration of donor ENSC, in recipient tissues, remain unclear. Therefore, we aimed to examine ENSC integration using an adapted ex vivo organotypic culture system. Donor ENSC were obtained from Wnt1cre/+;R26RYFP/YFP mice allowing specific labelling, selection and fate-mapping of cells. YFP+ neurospheres were transplanted to C57BL6/J (6–8-week-old) colonic tissue and maintained in organotypic culture for up to 21 days. We analysed and quantified donor cell integration within recipient tissues at 7, 14 and 21 days, along with assessing the structural and molecular consequences of ENSC integration. We found that organotypically cultured tissues were well preserved up to 21-days in ex vivo culture, which allowed for assessment of donor cell integration after transplantation. Donor ENSC-derived cells integrated across the colonic wall in a dynamic fashion, across a three-week period. Following transplantation, donor cells displayed two integrative patterns; longitudinal migration and medial invasion which allowed donor cells to populate colonic tissue. Moreover, significant remodelling of the intestinal ECM and musculature occurred upon transplantation, to facilitate donor cell integration within endogenous enteric ganglia. These results provide critical evidence on the timescale and mechanisms, which regulate donor ENSC integration, within recipient gut tissue, which are important considerations in the future clinical translation of stem cell therapies for enteric disease.

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