Adult Sox2+ stem cell exhaustion in mice results in cellular senescence and premature aging

Aging is characterized by a gradual functional decline of tissues with age. Adult stem and progenitor cells are responsible for tissue maintenance, repair, and regeneration, but during aging, this population of cells is decreased or its activity is reduced, compromising tissue integrity and causing pathologies that increase vulnerability, and ultimately lead to death. The causes of stem cell exhaustion during aging are not clear, and whether a reduction in stem cell function is a cause or a consequence of aging remains unresolved. Here, we took advantage of a mouse model of induced adult Sox2+ stem cell depletion to address whether accelerated stem cell depletion can promote premature aging. After a short period of partial repetitive depletion of this adult stem cell population in mice, we observed increased kyphosis and hair graying, and reduced fat mass, all of them signs of premature aging. It is interesting that cellular senescence was identified in kidney after this partial repetitive Sox2+ cell depletion. To confirm these observations, we performed a prolonged protocol of partial repetitive depletion of Sox2+ cells, forcing regeneration from the remaining Sox2+ cells, thereby causing their exhaustion. Senescence specific staining and the analysis of the expression of genetic markers clearly corroborated that adult stem cell exhaustion can lead to cellular senescence induction and premature aging.     

A possible role of Drosophila CTCF in mitotic bookmarking and maintaining chromatin domains during the cell cycle

Background

The CCCTC-binding factor (CTCF) is a highly conserved insulator protein that plays various roles in many cellular processes. CTCF is one of the main architecture proteins in higher eukaryotes, and in combination with other architecture proteins and regulators, also shapes the three-dimensional organization of a genome. Experiments show CTCF partially remains associated with chromatin during mitosis. However, the role of CTCF in the maintenance and propagation of genome architectures throughout the cell cycle remains elusive.

Results

We performed a comprehensive bioinformatics analysis on public datasets of Drosophila CTCF (dCTCF). We characterized dCTCF-binding sites according to their occupancy status during the cell cycle, and identified three classes: interphase-mitosis-common (IM), interphase-only (IO) and mitosis-only (MO) sites. Integrated function analysis showed dCTCF-binding sites of different classes might be involved in different biological processes, and IM sites were more conserved and more intensely bound. dCTCF-binding sites of the same class preferentially localized closer to each other, and were highly enriched at chromatin syntenic and topologically associating domains boundaries.

Conclusions

Our results revealed different functions of dCTCF during the cell cycle and suggested that dCTCF might contribute to the establishment of the three-dimensional architecture of the Drosophila genome by maintaining local chromatin compartments throughout the whole cell cycle.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0019-6) contains supplementary material, which is available to authorized users.     

Screening the expression characteristics of several miRNAs in G93A‐SOD1 transgenic mouse: altered expression of miRNA‐124 is associated with astrocyte differentiation by targeting Sox2 and Sox9

MicroRNA s (miRNA s) are suspected to be a contributing factor in amyotrophic lateral sclerosis (ALS ). Here, we assess the altered expression of miRNA s and the effects of miR‐124 in astrocytic differentiation in neural stem cells of ALS transgenic mice. Differentially expressed miRNA ‐positive cells (including miR‐124, miR‐181a, miR‐22, miR‐26b, miR‐34a, miR‐146a, miR‐219, miR‐21, miR‐200a, and miR‐320) were detected by in situ hybridization and qRT ‐PCR in the spinal cord and the brainstem. Our results demonstrated that miR‐124 was down‐regulated in the spinal cord and brainstem. In vitro , miR‐124 was down‐regulated in neural stem cells and up‐regulated in differentiated neural stem cells in G93A‐ superoxide dismutase 1 (SOD 1 ) mice compared with WT mice by qRT ‐PCR . Meanwhile, Sox2 and Sox9 protein levels showed converse change with miR‐124 in vivo and vitro . After over‐expression or knockdown of miR‐124 in motor neuron‐like hybrid (NSC 34) cells of mouse, Sox2 and Sox9 proteins were noticeably down‐regulated or up‐regulated, whereas Sox2 and Sox9 mRNA s remained virtually unchanged. Moreover, immunofluorescence results indicated that the number of double‐positive cells of Sox2/glial fibrillary acidic protein (GFAP) and Sox9/glial fibrillary acidic protein (GFAP) was higher in G93A‐SOD 1 mice compared with WT mice. We also found that many Sox2‐ and Sox9‐positive cells were nestin positive in G93A‐SOD 1 mice, but not in WT mice. Furthermore, differentiated neural stem cells from G93A‐SOD 1 mice generated a greater proportion of astrocytes and lower proportion of neurons than those from WT mice. MiR‐124 may play an important role in astrocytic differentiation by targeting Sox2 and Sox9 in ALS transgenic mice.

Cover Image for this issue: doi: 10.1111/jnc.14171 .

     

Sox2在骨肉瘤组织中的表达及其临床病理意义

目的研究Sox2在临床骨肉瘤标本中表达,并探讨其表达与肿瘤的生物学特征及临床预后的关系。方法采用免疫组织化学Maxvision检测Sox2蛋白在54例人骨肉瘤标本的表达,12例骨化性肌炎作为正常对照。结果骨肉瘤标本中Sox2阳性表达率为20.69%(12/58),而在骨化性肌炎中Sox2阳性表达率为0%(0/12),Sox2在骨肉瘤标本中的阳性率显著高于对照组骨化性肌炎(P0.01)。Sox2的表达与骨肉瘤临床Enneking分期有关(P0.05),与患者的年龄、性别、部位、组织学类型等其它临床病理因素无关(P0.05)。结论 Sox2可能在骨肉瘤的发生、发展和转移中发挥重要作用,提示Sox2的表达可考虑作为骨肉瘤临床评价生物学行为及判断预后的指标之一。     

Dual role for Sox2 in specification of sensory competence and regulation of Atoh1 function

The formation of inner ear sensory epithelia is believed to occur in two steps, initial specification of sensory competent (prosensory) regions followed by determination of specific cell‐types, such as hair cells (HCs) and supporting cells. However, studies in which the HC determination factor Atoh1 was ectopically expressed in nonprosensory regions indicated that expression of Atoh1 alone is sufficient to induce HC formation suggesting that prosensory formation may not be a prerequisite for HC development. To test this hypothesis, interactions between Sox2 and Atoh1, which are required for prosensory and HC formation respectively, were examined. Forced expression of Atoh1 in nonprosensory cells resulted in transient expression of Sox2 prior to HC formation, suggesting that expression of Sox2 is required for formation of ectopic HCs. Moreover, Atoh1 overexpression failed to induce HC formation in Sox2 mutants, confirming that Sox2 is required for prosensory competence. To determine whether expression of Sox2 alone is sufficient to induce prosensory identity, Sox2 was transiently activated in a manner that mimicked endogenous expression. Following transient Sox2 activation, nonprosensory cells developed as HCs, a result that was never observed in response to persistent expression of Sox2. These results, suggest a dual role for Sox2 in inner ear formation. Initially, Sox2 is required to specify prosensory competence, but subsequent down‐regulation of Sox2 must occur to allow Atoh1 expression, most likely through a direct interaction with the Atoh1 promoter. These results implicate Sox2‐mediated changes in prosensory cells as an essential step in their ability to develop as HCs. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 3–13, 2017     

带有穿膜结构域的转录因子蛋白Oct4和Sox2的融合表达

目的:克隆、表达及纯化带有穿膜结构域的转录因子蛋白Oct4和Sox2。方法:根据GenBank中的Oct4和Sox2基因序列,在其3’端引入穿膜结构域11R,并在其两端引入NdeⅠ和XhoⅠ酶切位点,进行全基因合成;将目的基因克隆至pET41a载体,进行酶切鉴定及测序;将所获阳性重组质粒转化感受态大肠杆菌BL21(DE3),经IPTG诱导表达后,对表达产物进行Western印迹鉴定;最后用Ni-NTA亲和层析柱对所获目的蛋白进行纯化。结果:质粒酶切鉴定结果表明带有目的基因的重组质粒构建成功;SDS-PAGE结果显示有相对分子质量约42×103和38×103的特异性蛋白表达条带,经Western印迹证实为目的蛋白;用Ni-NTA亲和层析柱纯化后,得到均一的Oct4和Sox2目的蛋白。结论:得到带有穿膜结构域的转录因子融合蛋白Oct4和Sox2,为今后安全开展诱导性多能干细胞研究奠定了基础。     

Critical role of anti-apoptotic Bcl-2 protein phosphorylation in mitotic death

Microtubule inhibiting agents (MIAs) characteristically induce phosphorylation of the major anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2 and Bcl-xL, and although this leads to Mcl-1 degradation, the role of Bcl-2/Bcl-xL phosphorylation in mitotic death has remained controversial. This is in part due to variation in MIA sensitivity among cancer cell lines, the dependency of cell fate on drug concentration and uncertainty about the modes of cell death occurring, thus making comparisons of published reports difficult. To circumvent problems associated with MIAs, we used siRNA knockdown of the anaphase-promoting complex activator, Cdc20, as a defined molecular system to investigate the role, specifically in mitotic death, of individual anti-apoptotic Bcl-2 proteins and their phosphorylated forms. We show that Cdc20 knockdown in HeLa cells induces mitotic arrest and subsequent mitotic death. Knockdown of Cdc20 in HeLa cells stably overexpressing untagged wild-type Bcl-2, Bcl-xL or Mcl-1 promoted phosphorylation of the overexpressed proteins in parallel with their endogenous counterparts. Overexpression of Bcl-2 or Bcl-xL blocked mitotic death induced by Cdc20 knockdown; phospho-defective mutants were more protective than wild-type proteins, and phospho-mimic Bcl-xL was unable to block mitotic death. Overexpressed Mcl-1 failed to protect from Cdc20 siRNA-mediated death, as the overexpressed protein was susceptible to degradation similar to endogenous Mcl-1. These results provide compelling evidence that phosphorylation of anti-apoptotic Bcl-2 proteins has a critical role in regulation of mitotic death. These findings make an important contribution toward our understanding of the molecular mechanisms of action of MIAs, which is critical for their rational use clinically.