GTPBP8 modulates mitochondrial fission through a Drp1-dependent process.

Mitochondrial fission is a tightly regulated process involving multiple proteins and cell signaling. Despite extensive studies on mitochondrial fission factors, our understanding of the regulatory mechanisms remains limited. This study shows the critical role of a mitochondrial GTPase, GTPBP8, in orchestrating mitochondrial fission in mammalian cells. Depletion of GTPBP8 resulted in drastic elongation and interconnectedness of mitochondria. Conversely, overexpression of GTPBP8 shifted mitochondrial morphology from tubular to fragmented. Notably, the induced mitochondrial fragmentation from GTPBP8 overexpression was inhibited in cells either depleted of the mitochondrial fission protein Drp1 (also known as DNM1L) or carrying mutated forms of Drp1. Importantly, downregulation of GTPBP8 caused an increase in oxidative stress, modulating cell signaling involved in the increased phosphorylation of Drp1 at Ser637. This phosphorylation hindered the recruitment of Drp1 to mitochondria, leading to mitochondrial fission defects. By contrast, GTPBP8 overexpression triggered enhanced recruitment and assembly of Drp1 at mitochondria. In summary, our study illuminates the cellular function of GTPBP8 as a pivotal modulator of the mitochondrial division apparatus, inherently reliant on its influence on Drp1.

Deletion of IL-27p28 induces CD8 T cell immunity against colorectal tumorigenesis.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide, characterized by molecular and clinical heterogeneity. Interleukin (IL)-27, a heterodimeric cytokine composed of p28 and EBI3 subunits, has been reported to exert potent antitumor activity in several cancer models. However, the precise role of IL-27 in the pathogenesis of CRC remains unclear. Here, we show that during the azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CRC development, IL-27p28 levels are dramatically increased in peripheral blood and tumor tissues, and the cytokine is mainly produced by tumor-infiltrating myeloid cells. IL-27p28 deficient mice display tumor resistances in both inflammation-associated CRC model and syngeneic MC38 colon cancer model. Administration with IL-27p28 neutralizing antibody also reduces the tumor formation in AOM/DSS-treated mice. Mechanically, CD8+ T cells in IL-27p28-/- mice exhibit enhanced tumor infiltration and cytotoxicity, which can be largely attributed to activation of the Akt/mTOR signaling pathway. Furthermore, selective depletion of CD8+ T cells in IL-27p28-/- mice markedly accelerate tumor growth and almost abrogate the protective effects of IL-27p28 deficiency. Most interestingly, the expression of IL-27p28 is also upregulated in tumor tissues of CRC patients and those with high expression of IL-27p28 tend to have a poorer overall survival. Our results suggest that loss of IL-27p28 suppresses colorectal tumorigenesis by augmenting CD8+ T cell-mediated anti-tumor immunity. Targeting IL-27p28 could be developed as a novel strategy for the treatment of colorectal cancers.

Anti-aging mechanism of different age donor-matched adipose-derived stem cells.

BACKGROUND: Adipose-derived stem cells (ASCs) have anti-aging and anti-obesity effects in aged animals, but the underlying molecular mechanism remains unknown. METHODS: In the present study, we evaluated the in vivo transplantation effects of different age donor-matched ASCs on natural aging and leptin knockout mice (ob-/ob- mice). The multi-omics expression profiles of young and aged mouse donor-derived ASCs were also analyzed. RESULTS: The results revealed that ASCs from young donors induced weight and abdominal fat loss for older recipients but not for young or ob-/ob-mice. The young and aged mouse donor ASCs displayed significant phenotypic differences, contributing to the distinguished weight loss and anti-aging effects in aged mice. CONCLUSIONS: Our data suggest an underlying molecular mechanism by which young-donor ASCs reduce immune cells and inflammation in aged mice via secreted immune factors. These findings point to a general anti-aging mechanism of stem cells, which may provide new insights into age-related disturbances of stem cell plasticity in healthy aging and age-related diseases.

Improvements in Gut Microbiota Dysbiosis in Aged Mice Transplanted with Adipose-Derived Stem Cells.

Adipose-derived stem cells (ASCs), as a cell therapy with considerable therapeutic potential, have received increasing attention in tissue repair, endocrine regulation, immune regulation, and aging and obesity research. Gut microbiota are present in all organisms and play important roles in the development of aging and obesity. Dysbiosis activates inflammatory pathways that may contribute to the development of aging and obesity. We used C57BL/6 J mice of different ages to carry out the experiment. Young mice were used as donors for ASC. Feces from the three groups were collected for 16sRNA sequencing to analyze the species composition of intestinal microorganisms, and then, predicted metabolic pathways by PICRUSt2 using 16s rRNA gene sequences. Immune cell levels in abdominal adipose tissue were assessed by flow cytometry. The content of IL-6, IL-1ß, TNF-α, and lipopolysaccharides in serum was measured by ELISA kit. Our 16sRNA sequencing data showed restoration of gut microbiota diversity and an increase in beneficial flora (Akkermansia, Lactobacillus, Prevotella) 7 days after ASC transplantation. In addition, the inflammatory environment improved in older transplanted mice.

Nr1d1 Mediated Cell Senescence in Mouse Heart-Derived Sca-1+CD31- Cells.

AIM: Sca-1+CD31- cells are resident cardiac progenitor cells, found in many mammalian tissues including the heart, and able to differentiate into cardiomyocytes in vitro and in vivo. Our previous work indicated that heart-derived Sca-1+CD31- cells increased the Nr1d1 mRNA level of Nr1d1 with aging. However, how Nr1d1 affects the senescence of Sca-1+CD31- cells. METHODS: Overexpression and knockdown of Nr1d1 in Sca-1+CD31- cells and mouse cardiac myocyte (MCM) cell lines were performed by lentiviral transduction. The effects of Nr1d1 abundance on cell differentiation, proliferation, apoptosis, cell cycle, and transcriptomics were evaluated. Moreover, binding of Nr1d1 to the promoter region of Nr4a3 and Serpina3 was examined by a luciferase reporter assay. RESULTS AND CONCLUSIONS: Upregulation Nr1d1 in young Sca-1+CD31- cells inhibited cell proliferation and promoted apoptosis. However, depletion of Nr1d1 in aged Sca-1+CD31- cells promoted cell proliferation and inhibited apoptosis. Furthermore, Nr1d1 was negatively associated with cell proliferation, promoting apoptosis and senescence-associated beta-galactosidase production in MCMs. Our findings show that Nr1d1 stimulates Serpina3 expression through its interaction with Nr4a3. Nr1d1 may therefore act as a potent anti-aging receptor that can be a therapeutic target for aging-related diseases.

Mitochondrial protein dysfunction in pathogenesis of neurological diseases.

Mitochondria are essential organelles for neuronal function and cell survival. Besides the well-known bioenergetics, additional mitochondrial roles in calcium signaling, lipid biogenesis, regulation of reactive oxygen species, and apoptosis are pivotal in diverse cellular processes. The mitochondrial proteome encompasses about 1,500 proteins encoded by both the nuclear DNA and the maternally inherited mitochondrial DNA. Mutations in the nuclear or mitochondrial genome, or combinations of both, can result in mitochondrial protein deficiencies and mitochondrial malfunction. Therefore, mitochondrial quality control by proteins involved in various surveillance mechanisms is critical for neuronal integrity and viability. Abnormal proteins involved in mitochondrial bioenergetics, dynamics, mitophagy, import machinery, ion channels, and mitochondrial DNA maintenance have been linked to the pathogenesis of a number of neurological diseases. The goal of this review is to give an overview of these pathways and to summarize the interconnections between mitochondrial protein dysfunction and neurological diseases.

Imperfect guide-RNA (igRNA) enables CRISPR single-base editing with ABE and CBE.

CRISPR base editing techniques tend to edit multiple bases in the targeted region, which is a limitation for precisely reverting disease-associated single-nucleotide polymorphisms (SNPs). We designed an imperfect gRNA (igRNA) editing methodology, which utilized a gRNA with one or more bases that were not complementary to the target locus to direct base editing toward the generation of a single-base edited product. Base editing experiments illustrated that igRNA editing with CBEs greatly increased the single-base editing fraction relative to normal gRNA editing with increased editing efficiencies. Similar results were obtained with an adenine base editor (ABE). At loci such as DNMT3B, NSD1, PSMB2, VIATA hs267 and ANO5, near-perfect single-base editing was achieved. Normally an igRNA with good single-base editing efficiency could be selected from a set of a few igRNAs, with a simple protocol. As a proof-of-concept, igRNAs were used in the research to construct cell lines of disease-associated SNP causing primary hyperoxaluria construction research. This work provides a simple strategy to achieve single-base base editing with both ABEs and CBEs and overcomes a key obstacle that limits the use of base editors in treating SNP-associated diseases or creating disease-associated SNP-harboring cell lines and animal models.

Functional characterization of CD49f+ hepatic stem/progenitor cells in adult mice liver.

Hepatic Stem/progenitor cells (HSPCs) have gained a large amount of interest for treating acute liver disease. However, the isolation and identification of HSPCs are unclear due to the lack of cell-specific surface markers. To isolate adult HSPCs, we used cell surface-marking antibodies, including CD49f and Sca-1. Two subsets of putative HSPCs, Lin-CD45-Sca-1-CD49f+ (CD49f+) and Lin-CD45-Sca-1+CD49f- (Sca-1+) cells, were isolated from adult mice liver by flow cytometry. Robust proliferative activity and clonogenic activity were found in both CD49f+ and Sca-1+ cells through colony-forming tests and cell cycle analyses. Immunofluorescence staining revealed that CD49f+ cells expressed ALB and CK-19 while Sca-1+ cells expressed only ALB, indicating that CD49f+ cells were bipotential and capable of differentiating into hepatocyte and cholangiocyte. Consequently, PAS stain showed that differentiated CD49f+ and Sca-1+ cells synthesised glycogen, indicating they could differentiate into functional hepatocytes. mRNA expression profile indicated that both CD49f+ and Sca-1+ cells showed differential expression of genes that are associated with liver progenitor function such as Sox9 and EpCam. Moreover, two subsets of putative HSPCs were activated by DDC and we found that their abundance and proliferation increased with age. In summary, we hypothesized that CD49f+ cells were a type of potential HSPCs and may be utilised for clinical stem cell therapy.

Identification and functional characterization of CD133+GFAP+CD117+Sca1+ neural stem cells.

Neural stem cells (NSCs) are responsible for maintaining the nervous system and repairing damages. Utility of NSCs could provide a novel solution to treat neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. However, we have no idea the exact phenotypic and functional characteristics of NSCs and their precise role in geriatric neurological and aging-related diseases. In this study, C57BL/6 mice were used to isolate and identify CD133+GFAP+CD117+Sca1+ cells in the hippocampal dentate gyrus region of the mouse brain as a novel neural stem cell population, in terms of cell phenotype, self-renewal capacity, and differentiation capability. With increasing in aging, the function, total cell number, and self-renewal capacity of CD133+GFAP+CD117+Sca1+ cells decreased, and the activity of differentiated cells also decreased. Meanwhile, we investigated differentially expressed genes in order to further classify their gene signature and pathways associated with their functional changes. Taken together, these findings demonstrate the existence of a rare population of NSCs in the hippocampal dentate gyrus region. Identification of specific NSCs offers ample opportunities for alleviating neural diseases.

Molecular Mechanism of the Cytosine CRISPR Base Editing Process and the Roles of Translesion DNA Polymerases.

CRISPR-mediated base editing causes damage to DNA, mainly uracil, apurinic/apyrimidinic (AP) sites, and nicks, which require various DNA repair mechanisms to complete the base conversion process. Currently, there are only hypotheses explaining the base editing process, but the molecular mechanism and roles of the repair systems in the process are relatively unknown. To explore the mechanism of base editing repair, a base editor, nCas9-PmCDA1, was applied in the model eukaryote, Saccharomyces cerevisiae, either with the wild type or its derivatives with genes encoding translesion DNA synthesis (TLS) polymerases knocked out. We found that C-to-G and C-to-A conversions resulted mainly from the repair of AP sites created by Ung and required Polζ as an extender. Rev1 is the main TLS polymerase for specifically incorporating Cs on the opposite position of AP sites to cause the dominant C-to-G conversion, while Polδ incorporates Ts or As on the opposite of AP sites, resulting in C-to-A and C-to-T conversions. Polη is not involved in the repair of AP sites caused by the base editor. Furthermore, our data suggested that the indels of base editing are mainly caused by the breakage of AP sites. Different from the current hypothesis model of the base editing mechanism, this work first elucidates the key roles of TLS polymerases in the cytosine base editing process. This work also suggests a new direction for the development of genomic and base editing techniques by employing, manipulating, and engineering TLS polymerases.

Comparison of Antiobesity Effects of Adipose-Derived Stromal/Stem Cells from Different Sources in a Natural Aging Model.

PURPOSE: Our previous study found that white adipose stem cells (W-ASCs) derived from abdominal and femoral sulcus white adipose stem cells (ASCs) have antiaging and age-related obesity effects. Whether interscapular brown adipose stem cells (B-ASCs) have the same effect has not been reported. The study objective was to compare the effects of ASCs from different tissues on aging and aging-related obesity. PATIENTS AND METHODS: C57BL/6J mice at 22 months of age were transplanted with either B-ASCs or W-ASCs from young mice at 2 months of age. Changes in body weight, biochemistry, cytokines, hormone secretion, cell senescence, lipid metabolism, and ASC function were assessed after transplanted 1 month. RESULTS: W-ASCs were superior to B-ASCs as aging and age-related obesity indicators, based on change in body weight, organ weight, antioxidant and anti-inflammatory activity, lipid metabolism, and liver and kidney function. CONCLUSION: Difference in the tissue source was reflected by the heterogeneity of antiaging and age-related obesity effects of transplanted ASCs. Based on the study results, we recommend W-ASCs over B-ASCs in aging and age-related obesity applications.

Antiobesity Effects of Adipose-Derived Stromal/Stem Cells in a Naturally Aged Mouse Model.

OBJECTIVE: Adipose-derived stromal/stem cells (ASCs) have multilineage differentiation potential and functional properties, as well as applications for cell-based therapies in tissue repair and regeneration. However, there is a lack of evidence regarding the efficacy of ASCs as an antiobesity agent in aged organisms. This study aimed to clarify the effectiveness of ASCs at treating obesity using a naturally aged mouse model. METHODS: Old (22 months) C57BL/6J mice with transplanted young-mice (2 months) donor ASCs were measured for weight change, biochemistry, cytokines, hormone secretion, cell senescence, lipid metabolism, and functional changes of ASCs. RESULTS: The results indicated that old mice treated with ASCs showed antiaging and antiobesity effects such as significant loss of body and organ weight, improved stem cell plasticity, increased antioxidant capacity (superoxide dismutase and catalase), improved liver and kidney function, improved lipid metabolism, and increased hormone secretion (sex hormone-binding globulin, thyrotropin, and leptin). Treatment with ASCs decreased cell senescence and suppressed secretion of inflammatory agents (interleukin-6 and tumor necrosis factor alpha). CONCLUSIONS: Traditional drugs used in the treatment of obesity have limitations and are unsuitable for the elderly. Based on the results, the future use of ASCs as primary antiaging and antiobesity agents is suggested because of their positive effects on aged animals.

Mitochondrial Membrane Remodeling.

Mitochondria are key regulators of many important cellular processes and their dysfunction has been implicated in a large number of human disorders. Importantly, mitochondrial function is tightly linked to their ultrastructure, which possesses an intricate membrane architecture defining specific submitochondrial compartments. In particular, the mitochondrial inner membrane is highly folded into membrane invaginations that are essential for oxidative phosphorylation. Furthermore, mitochondrial membranes are highly dynamic and undergo constant membrane remodeling during mitochondrial fusion and fission. It has remained enigmatic how these membrane curvatures are generated and maintained, and specific factors involved in these processes are largely unknown. This review focuses on the current understanding of the molecular mechanism of mitochondrial membrane architectural organization and factors critical for mitochondrial morphogenesis, as well as their functional link to human diseases.

Impact of Aging on the Characterization of Brown and White Adipose Tissue-Derived Stem Cells in Mice.

Adipose tissue enriched with adipose tissue-derived stem cells (ASCs) is often used for stem cell-based therapies. However, the characteristics of ASCs from different types of adipose tissue have varying biochemical and functional properties. We aimed to investigate how age affected the biological and functional characteristics of ASCs from brown (BAT) and white adipose tissue (WAT). ASCs were obtained and cultured from mouse BAT and WAT at different ages: young (2 months of age) and older mice (22 months of age). Mesenchymal markers were characterized by flow cytometry, and cell proliferation, apoptosis, differentiation potential, senescence, and metabolism were then determined. The percentage of WAT was higher in elderly mice, and the percentage of BAT was higher in young mice. All ASC sample phenotypes were characterized as CD29+/CD44+/CD105+/CD45-; the proliferation rate was not statistically different among all age groups. However, the number of senescent cells and the percentage of apoptosis in elderly mouse ASCs were significantly increased, and the ability of osteogenic and lipogenic differentiation was decreased in these same animals. In addition, ASCs from young mice were more inclined to undergo osteogenic differentiation, especially BAT-ASCs, whose gene expression of fat-consuming components was also significantly higher than of WAT-ASCs. The results indicated that ASCs derived from both WAT and BAT possessed different characteristics of fat metabolism and cell differentiation relative to the osteo- and adipolineages. In particular, because BAT-ASCs from young mice contributed to fat consumption, if used for cell grafting, they may potentially be attractive vehicles for treating obesity.

Latexin deficiency in mice up-regulates inflammation and aggravates colitis through HECTD1/Rps3/NF-κB pathway.

The function of Latexin (LXN) in inflammation has attracted attention. However, no data are available regarding its role in colitis. We report that LXN is a suppressor of colitis. LXN deficiency leads to the severity of colitis in DSS-induced mice, and LXN is required for the therapeutic effect of retinoic acid on colitis. Using a proteomics approach, we demonstrate that LXN interacts and forms a functional complex with HECTD1 (an E3 ubiquitin ligase) and ribosomal protein subunit3 (Rps3). IκBα is one of the substrates of HECTD1. Ectopic expression of LXN leads to IκBα accumulation in intestinal epithelial cells, however, LXN knockdown enhances the interaction of HECTD1 and Rps3, contributing to the ubiquitination degradation of IκBα, and subsequently enhances inflammatory response. Thus, our findings provided a novel mechanism underlying LXN modulates colitis via HECTD1/Rps3/NF-κB pathway and significant implications for the development of novel strategies for the treatment of colitis by targeting LXN.

Cited2 regulates proliferation and survival in young and old mouse cardiac stem cells.

BACKGROUND: Cardiac stem cells (CSCs) exhibit age-dependent characteristics. Cited2 has been implicated in the regulation of heart development; however, there is little known about how Cited2 affects CSC aging. RESULTS: Cited2 mRNA and protein level was downregulated in aging heart tissue and CSCs. Old (O)-CSCs showed decreased differentiation and proliferation capacities as compared to Young (Y)-CSCs, the decrease in cell proliferation, increase in apoptosis, and cell cycle arrest in G0/G1 phase in CSCs are mediated by knocdown CITED2expression in (Y)-CSCs. CONCLUSIONS: Cited2 plays an important role in cell cycle progression and in maintaining the balance between CSC proliferation and apoptosis in the process of aging without influencing cell fate decisions. These findings have important implications for cell-based therapies for heart repair.

The mechanistic study behind suppression of GVHD while retaining GVL activities by myeloid-derived suppressor cells.

Graft-versus-host disease (GVHD) is a major barrier to the widespread use of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for treating hematologic malignancies. Myeloid-derived suppressor cells (MDSCs) have been recognized as crucial immunosuppressive cells in various pathologic settings. Here, we investigated whether the unique functional properties of MDSCs could be harnessed to control allo-HSCT-associated GVHD. Using multiple murine GVHD/GVL models including both MHC-mismatched and miHA-mismatched, we demonstrated that treatment with CD115+ MDSCs efficiently suppressed GVHD but did not significantly impair graft-versus-leukemia (GVL) activity, leading to 80 and 67% protection in treated mice in GVHD and GVL models, respectively. The mechanism for this dissociation of GVHD from GVL, specifically the emergence of donor-derived NKG2D+ CD8 T cells with a memory phenotype in MDSC-treated recipient mice, was identified. NKG2D expression on donor T cells was required for eradication of allogeneic lymphoma cells. Furthermore, long-term surviving MDSC recipients that exhibited cytolytic activities against allogeneic leukemia cells had a significantly increased percentage of T regulatory cells and, more importantly, NKG2D+ CD8 T cells. These findings indicate that MDSCs can be used as a novel cell-based therapy to suppress GVHD while maintaining GVL activities through selective induction of NKG2D+ CD8 memory T cells.

Association of HIF-1α and NDRG2 Expression with EMT in Gastric Cancer Tissues.

OBJECTIVE: This study aims to investigate the differences in the expression of hypoxia-inducible factor-1α (HIF-1α), N-myc downstream-regulated gene 2 (NDRG2) and epithelial mesenchymal transition (EMT)-related proteins in normal gastric tissues, gastric cancer tissues and lymph node metastasis. METHODS: Immunohistochemistry was used to detect the expression of HIF-1α, NDRG2, E-cadherin, Snail and Twist in normal gastric tissues, gastric cancer tissues and lymph node metastasis. RESULTS: In normal gastric tissues, HIF-1α was not expressed, NDRG2 was highly expressed. There was a significant between the expression of NDRG2 and Snail, as well as of NDRG2 and Twist. In gastric cancer tissues, there was no statistically difference between the expression of HIF-1α and E-cadherin, NDRG2 and E-cadherin. However, there was a significant difference in expression between the expression of HIF-1α and Snail, HIF-1α and Twist, NDRG2 and Snail, and NDRG2 and Twist. In lymph node metastasis tissues, we show that HIF-1α was highly expressed, while NDRG2 was not, and the difference between the expression of HIF-1α and E-cadherin, HIF-1α and Snail, HIF-1α and Twist was not significant. CONCLUSION: HIF-1α may promote EMT, possibly by inhibiting the expression of NDRG2.

Retraction Note: The Role of Scleraxis in Fate Determination of Mesenchymal Stem Cells for Tenocyte Differentiation.

This paper has been retracted.