Inducible Knockout Research Articles

MST1/2 regulates fibro/adipogenic progenitor fate decisions in skeletal muscle regeneration

Defective skeletal muscle regeneration is often accompanied by fibrosis. Fibroblast/adipose progenitors (FAPs) are important in these processes, however, the regulation of FAP fate decisions is unclear. Here, using inducible conditional knockout mice, we show that blocking mammalian Ste20-like kinases 1/2 (MST1/2) of FAPs prevented apoptosis and reduced interleukin-6 secretion invivo and invitro, which impaired myoblast proliferation and differentiation, as well as impaired muscle regeneration. Deletion of Mst1/2 increased co-localization of Yes-associated protein (YAP) with Smad2/3 in nuclei and promoted differentiation of FAPs toward myofibroblasts, resulting in excessive collagen deposition and skeletal muscle fibrosis. Meanwhile, inhibition of MST1/2 increased YAP/Transcriptional co-activator with PDZ-binding motif activation, which promoted activation of the WNT/β-catenin pathway and impaired the differentiation of FAPs toward adipocytes. These results reveal a new mechanism for MST1/2 action in disrupted skeletal muscle regeneration and fibrosis via regulation of FAP apoptosis and differentiation. MST1/2 is a potential therapeutic target for the treatment of some myopathies.

Abstract 5336: Establishment and characterization of a novel inducible Apoc2 knockout mouse model to study the roles of Apoc2 in normal hematopoiesis

Abstract Apolipoprotein C2 (Apoc2) is upregulated in acute myeloid leukemia and presents a viable therapeutic target. Whether this gene plays a role in normal hematopoiesis remains unknown. To investigate the impact of targeting Apoc2 on normal hematopoiesis, we established and characterized an inducible Apoc2 knockout mouse model. Homozygous floxed Apoc2 mice (Apoc2fl/fl) were obtained from the NIH (Dr. Remaley’s lab) and were crossed with homozygous R26-CreERT2 mice. After three generations, Gt(ROSA)26SorCre/Cre, Apoc2fl/fl mice were generated and confirmed by genotyping. Male (M) and female (F) mice were injected with tamoxifen (TAM: 20mg/mL dissolved in corn oil) intraperitoneally (75mg/kg per day for 5 days) to induce Apoc2 knockout (Apoc2-KO). The control wild type (WT) group included Gt(ROSA)26Sorwt/wt, Apoc2fl/fl mice, and CD45.2+ WT mice injected with TAM, or Gt(ROSA)26SorCre/Cre, Apoc2fl/fl mice injected with corn oil.No obvious abnormal phenotypes were observed in Apoc2-KO mice. The average increase of body weight 3 weeks post TAM or corn oil injections of 5-week-old Gt(ROSA)26SorCre/Cre, Apoc2fl/fl siblings was not statistically significantly different (Apoc2-KO (n = 2M + 2F) vs WT (n = 2M + 2F) = 2.450g vs 2.125g, P = 0.63). Analyses of Apoc2-KO (n = 5M + 3F) and WT (n = 4M + 2F showed no significant difference in livers and spleens weight when normalized to mice age. The percentage of knockdown in liver (99.96%), spleen (77.32%), bone marrow (BM, 83.56%) and blood (82.02%) was confirmed by qPCR. In-vitro cell expansion was performed in the presence of thrombopoietin and stem cell factor for BM cells and IL-2 and PHA for T cells. Apoc2-KO BM cells exhibited similar in vitro expansion to that of control mice (Apoc2-KO vs WT: Day 10/Day 6: 1.22 fold vs 1.57 fold; Day 10/Day 1: 57.26% vs 72.34%; P > 0.05; and Cell Trace assay: mean fluorescence intensity (MFI) of Day 9 normalized to Day1: Apoc2-KO vs WT = 0.37 vs 0.39; P > 0.05). Also, the colony forming ability was not significantly different between Apoc2-KO and WT BM cells (Apoc2-KO vs WT = 79.09 units vs 81.47 units, P = 0.62). In-vitro T cell expansions of splenocytes were not significantly different between Apoc2-KO and WT mice (Apoc2-KO vs WT: Day 10/Day 4: 1.73 fold vs 1.83 fold; Cell Trace: mean MFI of Day 9 normalized to Day1: Apoc2-KO vs WT = 0.6208 vs 0.7088; P > 0.05). Hematology analysis assessing white blood cell count, red blood cell count, hemoglobin count, neutrophil, lymphocytes, showed no significant difference between the groups.Here we report the first inducible Apoc2 knockout mouse model and demonstrated that Apoc2 knockout has limited impact on normal hematopoietic cells in mice. On-going research using this model will assess the impact of Apoc2 knockout on ageing related phenotypes and leukemia progression. Citation Format: Xiaochen Zhang, Ritu Chandwani, Mateusz Pospiech, Yiting Meng, Kanaka Dhuri, Bhakti Thakker, Atham Ali, Shephali M. Kadam, Yu-Chun Wei, Houda Alachkar. Establishment and characterization of a novel inducible Apoc2 knockout mouse model to study the roles of Apoc2 in normal hematopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5336.

Abstract 5456: Identifying the role of tissue factor in osteosarcoma metastasis

Abstract Patients with osteosarcoma who develop metastasis face very poor prognoses. Even when treated with the most effective medical and surgical interventions available, about forty percent of osteosarcoma patients will eventually succumb to lung metastasis. Despite the relevance of metastasis to clinical outcomes, we understand little about the mechanisms that drive these tumor cells into the lung and facilitate their metastatic growth. Consequently, there are no approved antimetastatic agents for osteosarcoma and outcomes have not improved in more than 40 years. Tissue factor is a tightly regulated physiologic initiator of coagulation with functions in inflammation, cytokinesis, angiogenesis, apoptosis, and growth factor secretion. These functions are divided between two separate pathways: initiation of the coagulation cascade via activation of factors VII and X, and propagation of cell signaling pathways via PAR activation. It is aberrantly expressed in many cancers and has been associated with metastatic behavior and inferior outcomes by affecting metastatic vascularity, growth, and migration. Osteosarcoma has been shown to also aberrantly express tissue factor, with descriptive studies suggesting similar effects on metastatic behavior. We hypothesized that tissue factor expressed on the surface of disseminated osteosarcoma cells activates procoagulant pathways and PAR signaling, and that both facilitate pulmonary metastasis. To test this, we first established and quantified expression of tissue factor across six osteosarcoma cell lines using Western blot, flow cytometry, and confocal microscopy in both monoculture and in coculture with human bronchial epithelial cells. PAR1 and PAR2 were also discovered to be expressed in osteosarcoma cells. RNA sequencing performed suggests that both tissue factor and PAR1 expression in osteosarcoma increase over the metastatic period, as does FVII expression by alveolar macrophages; this implies that tissue factor activation during metastasis is dynamic and dependent on host interactions. Ongoing work includes functional assays of osteosarcoma tissue factor and PARs activity as well as preclinical testing of tissue factor inhibition in a murine xenograft model of osteosarcoma. To define how tissue factor propagates metastasis, we are separately inhibiting its coagulant and cell signaling pathways after murine tail vein injection using apixaban or pathway-specific antibodies. We will also use an inducible F3 knockout to alter tumor expression of tissue factor. If we find that manipulation of tissue factor expression changes metastatic behavior across cell lines, this will suggest that tissue factor plays a crucial, mechanistic role in osteosarcoma metastasis. Varying effectiveness of coagulation or signaling pathway inhibition on metastasis will implicate tissue factor’s mechanism of metastatic propagation. Citation Format: Charles C. Treinen, Amy Gross, Maren Cam, Bryce Kerlin, Ryan Roberts. Identifying the role of tissue factor in osteosarcoma metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5456.

Abstract 1973: Discovery of the first-in-class SHP1 covalent inhibitor for cancer immunotherapy

Abstract Cancer immunotherapy refers to the approach leveraging patients’ immune system against tumor, which has gained tremendous progress hallmarked by immune checkpoint blockade and adoptive cell therapies. However, contemporary strategies suffer from limited responses, adverse side effects, and low tissue penetration due to utilization of macromolecules, calling for more efficacious and safer alternative strategies. SHP1 is a protein tyrosine phosphatase (PTP) primarily expressed in hematopoietic cells and has been shown to negatively regulate immune responses in T cells and natural killer (NK) cells, and SHP1 deletion in these cells has shown to promote their anti-tumor functions. Recent studies also demonstrated that inducible SHP1 knockout inhibited tumor growth in vivo through immune activation. Although presented as an attractive target, no high-quality small molecule inhibitors have been reported for SHP1 due to its undruggable nature. Through high-throughput screening and extensive medicinal chemistry, we have acquired the first-in-class SHP1 covalent inhibitor M029, which shows >25-fold selectivity against SHP2, its closest analogues and >60-fold selectivity against other PTPs and cysteine-based proteins. Further proteomics studies reveal that M029 is superiorly selective for SHP1 in cellulo. Additionally, M029 is stable in 100-fold excessive glutathione with a half-life of >35 hours and non-toxic up to 100 µM to healthy cells, such high stability was also reflected by its oral bioavailability with a F% of 10%. M029 treatment significantly activated T cell receptor signaling in T cells and NK cells killing effects in vitro. Furthermore, oral dosage of M029 significantly delayed tumor progression in mice bearing MC38 tumors through enhanced T cell and NK cell infiltration and activation and prolonging of T cell exhaustion. To validate that the compound acted through immune activation, we dosed the mice with anti-CD8+ or anti-NK1.1 during M029 treatment, both of which abolished the compound efficacy, corroborating with its mechanism of action. Meanwhile, to assess the compound safety, we dosed the mice up to 100 mg/kg for 8 days, where no significant body weight change or cytokine storm were observed. Collectively, we have developed the first SHP1 covalent inhibitor with high selectivity and strong anti-tumor efficacy. This study is the first characterization of pharmacological SHP1 inhibition as cancer immunotherapy and solidified its potential as an immunotherapeutic target. The development of M029 will also enlighten drug discovery strategies against SHP1 or other undruggable PTPs. Citation Format: Zihan Qu, Frederick Nguele Meke, Zheng Zhang, Yunpeng Bai, Aaron D. Krabill, Christine S. Muli, Brenson A. Jassim, Jiajun Dong, Jinyue Li, Nguyen Yuyen, Andy W. Tao, Darci T. Trader, Zhong-Yin Zhang. Discovery of the first-in-class SHP1 covalent inhibitor for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1973.

Abstract 1632: Dissecting AMBRA1-cyclin D axis deregulation in embryonal tumors of the central nervous system

Abstract Dissecting AMBRA1-cyclin D axis deregulation in embryonal tumors of the centralnervous systemEmbryonal tumors of the central nervous system (CNS) are malignant growths originating from embryonic neural stem cells, primarily affecting pediatric patients. These tumors pose a significant threat to children's health, leading to high morbidity and mortality rates. To improve survival rates and reduce morbidity, there is a critical need for advanced molecular diagnostics and innovative therapies. Among these tumors are medulloblastomas (MB) and other embryonal tumors, previously classified as primitive neuroectodermal tumors (CNS-PNET). These malignancies are characterized by uncontrolled cell proliferation, primarily driven by abnormalities in cell cycle proteins. Consequently, targeting cell cycle regulators has emerged as a promising approach for their treatment. Two key signaling pathways, namely the MYC and the cyclin D-CDK-RB pathway, govern the decision-making process related to genome replication. Mutations in both MYC and the CDK4/6/cyclin D pathway, leading to the functional inactivation of RB, have been identified as relevant factors in the development of CNS-PNET and medulloblastomas. Our seminal research has highlighted the crucial role of AMBRA1, an upstream master regulator orchestrating the transition from G1 to S phase (Maiani & Milletti et al., 2021). Specifically, AMBRA1 modulates the abundance of D-type cyclins and MYC by facilitating their proteasomal degradation, thereby ensuring genomic stability during DNA replication.Based on these discoveries, we propose that AMBRA1 functions as a multifaceted tumor suppressor across various cancer types. An unbiased Sleeping Beauty analysis identified AMBRA1 as a genetic driver of CNS-PNET, providing a strong foundation for investigating its role further (Beckmann et al., 2019). We hypothesize that the simultaneous deletion of AMBRA1 and p53 in an inducible knockout mouse model can lead to the spontaneous development of CNS-PNET or medulloblastoma, contingent on the specific targeted cell lineage. Additionally, our research demonstrates that AMBRA1 deficiency confers resistance to CDK4/6 inhibitors while creating a synthetic lethality with CHK1 inhibitors. Consequently, we propose that a comparative analysis of AMBRA1, D-type cyclins, and Myc family expression levels could serve as a diagnostic marker. This marker could facilitate patient-based stratification for CHK1 inhibitor treatment efficacy, enabling the differentiation of non- responsive cases to CDK4/6 chemotherapy. Citation Format: Giacomo Milletti, Giulia Cadeddu, Alba Adelantado Rubio, Caterina Ferraina, Apolinar Maya-Mendoza, Francesco Cecconi. Dissecting AMBRA1-cyclin D axis deregulation in embryonal tumors of the central nervous system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1632.

CD4 T Cell-Derived IL-21 Is Critical for Sustaining Plasmodium Infection-Induced Germinal Center Responses and Promoting the Selection of Memory B Cells with Recall Potential.

Development of Plasmodium-specific humoral immunity is critically dependent on CD4 Th cell responses and germinal center (GC) reactions during blood-stage Plasmodium infection. IL-21, a cytokine primarily produced by CD4 T cells, is an essential regulator of affinity maturation, isotype class-switching, B cell differentiation, and maintenance of GC reactions in response to many infection and immunization models. In models of experimental malaria, mice deficient in IL-21 or its receptor IL-21R fail to develop memory B cell populations and are not protected against secondary infection. However, whether sustained IL-21 signaling in ongoing GCs is required for maintaining GC magnitude, organization, and output is unclear. In this study, we report that CD4+ Th cells maintain IL-21 expression after resolution of primary Plasmodium yoelii infection. We generated an inducible knockout mouse model that enabled cell type-specific and timed deletion of IL-21 in peripheral, mature CD4 T cells. We found that persistence of IL-21 signaling in active GCs had no impact on the magnitude of GC reactions or their capacity to produce memory B cell populations. However, the memory B cells generated in the absence of IL-21 exhibited reduced recall function upon challenge. Our data support that IL-21 prevents premature cellular dissolution within the GC and promotes stringency of selective pressures during B cell fate determination required to produce high-quality Plasmodium-specific memory B cells. These data are additionally consistent with a temporal requirement for IL-21 in fine-tuning humoral immune memory responses during experimental malaria.

Astrocyte focal adhesion kinase reduces passive stress coping by inhibiting ciliary neurotrophic factor only in female mice

Astrocytes have been implicated in stress responses and produce ciliary neurotrophic factor (CNTF), which we have shown in the mouse medial amygdala (MeA) to promote passive stress coping response only in females. Pharmacological inhibition of focal adhesion kinase (FAK) upregulates CNTF expression. Here, we found that inducible knockout of FAK in astrocytes or systemic treatment with an FAK inhibitor increased passive coping behavior, i.e., immobility, in an acute forced swim stress test in female, but not male, mice. Strikingly, four weeks of chronic unpredictable stress (CUS) did not further increase passive coping in female astrocytic FAK knockout mice, whereas it exacerbated it in female wildtype mice and male mice of both genotypes. These data suggest that astrocyte FAK inhibition is required for chronic stress-induced passive coping in females. Indeed, CUS reduced phospho-FAK and increased CNTF in the female MeA. Progesterone treatment after ovariectomy activated amygdala FAK and alleviated ovariectomy-induced passive coping in wildtype, but not astrocytic FAK knockout females. This suggests that progesterone-mediated activation of FAK in astrocytes reduces female stress responses. Finally, astrocytic FAK knockout or FAK inhibitor treatment increased CNTF expression in the MeA of both sexes, although not in the hippocampus. As mentioned, MeA CNTF promotes stress responses only in females, which may explain the female-specific role of astrocytic FAK inhibition. Together, this study reveals a novel female-specific progesterone-astrocytic FAK pathway that counteracts CNTF-mediated stress responses and points to opportunities for developing treatments for stress-related disorders in women.

TEAD1 is crucial for developmental myelination, Remak bundles, and functional regeneration of peripheral nerves.

Previously we showed that the hippo pathway transcriptional effectors, YAP and TAZ, are essential for Schwann cells (SCs) to develop, maintain and regenerate myelin . Although TEAD1 has been implicated as a partner transcription factor, the mechanisms by which it mediates YAP/TAZ regulation of SC myelination are unclear. Here, using conditional and inducible knockout mice, we show that TEAD1 is crucial for SCs to develop and regenerate myelin. It promotes myelination by both positively and negatively regulating SC proliferation, enabling Krox20/Egr2 to upregulate myelin proteins, and upregulating the cholesterol biosynthetic enzymes FDPS and IDI1. We also show stage-dependent redundancy of TEAD1 and that non-myelinating SCs have a unique requirement for TEAD1 to enwrap nociceptive axons in Remak bundles. Our findings establish TEAD1 as a major partner of YAP/TAZ in developmental myelination and functional nerve regeneration and as a novel transcription factor regulating Remak bundle integrity.

ΔNp63 regulates MDSC survival and metabolism in triple-negative breast cancer

Triple-negative breast cancer (TNBC) contributes greatly to mortality of breast cancer, demanding new targetable options. We have shown that TNBC patients have high ΔNp63 expression in tumors. However, the function of ΔNp63 in established TNBC is yet to be explored. In current studies, targeting ΔNp63 with inducible CRISPR knockout and Histone deacetylase inhibitor Quisinostat showed that ΔNp63 is important for tumor progression and metastasis in established tumors by promoting myeloid-derived suppressor cell (MDSC) survival through tumor necrosis factor alpha. Decreasing ΔNp63 levels are associated with decreased CD4+ and FOXP3+ T-cells but increased CD8+ T-cells. RNA sequencing analysis indicates that loss of ΔNp63 alters multiple MDSC properties such as lipid metabolism, chemotaxis, migration, and neutrophil degranulation besides survival. We further demonstrated that targeting ΔNp63 sensitizes chemotherapy. Overall, we showed that ΔNp63 reprograms the MDSC-mediated immunosuppressive functions in TNBC, highlighting the benefit of targeting ΔNp63 in chemotherapy-resistant TNBC.

Endothelial TDP-43 controls sprouting angiogenesis and vascular barrier integrity, and its deletion triggers neuroinflammation.

TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA-binding protein that regulates gene expression, and its malfunction in neurons has been causally associated with multiple neurodegenerative disorders. Although progress has been made in understanding the functions of TDP-43 in neurons, little is known about its roles in endothelial cells (ECs), angiogenesis, and vascular function. Using inducible EC-specific TDP-43-KO mice, we showed that TDP-43 is required for sprouting angiogenesis, vascular barrier integrity, and blood vessel stability. Postnatal EC-specific deletion of TDP-43 led to retinal hypovascularization due to defects in vessel sprouting associated with reduced EC proliferation and migration. In mature blood vessels, loss of TDP-43 disrupted the blood-brain barrier and triggered vascular degeneration. These vascular defects were associated with an inflammatory response in the CNS with activation of microglia and astrocytes. Mechanistically, deletion of TDP-43 disrupted the fibronectin matrix around sprouting vessels and reduced β-catenin signaling in ECs. Together, our results indicate that TDP-43 is essential for the formation of a stable and mature vasculature.

Aminic Organoselenium Compounds as Glutathione Peroxidase Mimics and Inhibitors of Ferroptosis.

The synthesis of diarylamine-based organoselenium compounds via the nucleophilic substitution reactions has been described. Symmetrical monoselenides and diselenides were conveniently synthesized by the reduction of their corresponding selenocyanates using sodium borohydride. Selenocyanates were obtained from 2-chloro acetamides by the nucleophilic displacement with potassium selenocyanate. Selenides were synthesized by treating the 2-chloro acetamides with in situ generated sodium butyl selenolate as nucleophile. Further, the newly synthesized organoselenium compounds were evaluated for their glutathione peroxidase (GPx)-like activity in thiophenol assay. This study revealed that the methoxy-substituted organoselenium compounds showed significant effect on the GPx-like activity. The catalytic parameters for the most efficient catalysts were also determined. The anti-ferroptotic activity for all GPx-mimics evaluated in a 4-OH-tamoxifen (TAM) inducible GPx4 knockout cell line using liproxstatin as standard.

CXCR2 inhibition in G-MDSCs enhances CD47 blockade for melanoma tumor cell clearance

The use of colony-stimulating factor-1 receptor (CSF1R) inhibitors has been widely explored as a strategy for cancer immunotherapy due to their robust depletion of tumor-associated macrophages (TAMs). While CSF1R blockade effectively eliminates TAMs from the solid tumor microenvironment, its clinical efficacy is limited. Here, we use an inducible CSF1R knockout model to investigate the persistence of tumor progression in the absence of TAMs. We find increased frequencies of granulocytic myeloid-derived suppressor cells (G-MDSCs) in the bone marrow, throughout circulation, and in the tumor following CSF1R deletion and loss of TAMs. We find that G-MDSCs are capable of suppressing macrophage phagocytosis, and the elimination of G-MDSCs through CXCR2 inhibition increases macrophage capacity for tumor cell clearance. Further, we find that combination therapy of CXCR2 inhibition and CD47 blockade synergize to elicit a significant anti-tumor response. These findings reveal G-MDSCs as key drivers of tumor immunosuppression and demonstrate their inhibition as a potent strategy to increase macrophage phagocytosis and enhance the anti-tumor efficacy of CD47 blockade in B16-F10 melanoma.

Benefits and Caveats in the Use of Retinal Pigment Epithelium-Specific Cre Mice.

The retinal pigment epithelium (RPE) is an important monolayer of cells present in the outer retina, forming a major part of the blood-retina barrier (BRB). It performs many tasks essential for the maintenance of retinal integrity and function. With increasing knowledge of the retina, it is becoming clear that both common retinal disorders, like age-related macular degeneration, and rare genetic disorders originate in the RPE. This calls for a better understanding of the functions of various proteins within the RPE. In this regard, mice enabling an RPE-specific gene deletion are a powerful tool to study the role of a particular protein within the RPE cells in their native environment, simultaneously negating any potential influences of systemic changes. Moreover, since RPE cells interact closely with adjacent photoreceptors, these mice also provide an excellent avenue to study the importance of a particular gene function within the RPE to the retina as a whole. In this review, we outline and compare the features of various Cre mice created for this purpose, which allow for inducible or non-inducible RPE-specific knockout of a gene of interest. We summarize the various benefits and caveats involved in the use of such mouse lines, allowing researchers to make a well-informed decision on the choice of Cre mouse to use in relation to their research needs.

MITOL deficiency triggers hematopoietic stem cell apoptosis via ER stress response.

Hematopoietic stem cell (HSC) divisional fate and function are determined by cellular metabolism, yet the contribution of specific cellular organelles and metabolic pathways to blood maintenance and stress-induced responses in the bone marrow remains poorly understood. The outer mitochondrial membrane-localized E3 ubiquitin ligase MITOL/MARCHF5 (encoded by the Mitol gene) is known to regulate mitochondrial and endoplasmic reticulum (ER) interaction and to promote cell survival. Here, we investigated the functional involvement of MITOL in HSC maintenance by generating MX1-cre inducible Mitol knockout mice. MITOL deletion in the bone marrow resulted in HSC exhaustion and impairment of bone marrow reconstitution capability in vivo. Interestingly, MITOL loss did not induce major mitochondrial dysfunction in hematopoietic stem and progenitor cells. In contrast, MITOL deletion induced prolonged ER stress in HSCs, which triggered cellular apoptosis regulated by IRE1α. In line, dampening of ER stress signaling by IRE1α inihibitor KIRA6 partially rescued apoptosis of long-term-reconstituting HSC. In summary, our observations indicate that MITOL is a principal regulator of hematopoietic homeostasis and protects blood stem cells from cell death through its function in ER stress signaling.

Microglia-Derived Insulin-like Growth Factor 1 Is Critical for Neurodevelopment.

Insulin-like growth factor 1 (IGF-1) is a peptide hormone essential for the proper development and growth of the organism, as a complete knockout of Igf1 in mice is lethal, causing microcephaly, growth retardation and the defective development of organs. In the central nervous system, neurons and glia have been reported to express Igf1, but their relative importance for postnatal development has not yet been fully defined. In order to address this, here, we obtained mice with a microglia-specific inducible conditional knockout of Igf1. We show that the deficiency in microglial Igf1, starting in the first postnatal week, leads to body and brain growth retardation, severely impaired myelination, changes in microglia numbers, and behavioral abnormalities. These results emphasize the importance of microglial-derived Igf1 for brain development and function and open new perspectives for the investigation of the role of microglial-Igf1 in neurological diseases.

Ptpn23 Controls Cardiac T-Tubule Patterning by Promoting the Assembly of Dystrophin-Glycoprotein Complex.

Cardiac transverse tubules (T-tubules) are anchored to sarcomeric Z-discs by costameres to establish a regular spaced pattern. One of the major components of costameres is the dystrophin-glycoprotein complex (DGC). Nevertheless, how the assembly of the DGC coordinates with the formation and maintenance of T-tubules under physiological and pathological conditions remains unclear. Given the known role of Ptpn23 (protein tyrosine phosphatase, nonreceptor type 23) in regulating membrane deformation, its expression in patients with dilated cardiomyopathy was determined. Taking advantage of Cre/Loxp, CRISPR/Cas9, and adeno-associated virus 9 (AAV9)-mediated in vivo gene editing, we generated cardiomyocyte-specific Ptpn23 and Actn2 (α-actinin-2, a major component of Z-discs) knockout mice. We also perturbed the DGC by using dystrophin global knockout mice (DmdE4*). MM 4-64 and Di-8-ANEPPS staining, Cav3 immunofluorescence, and transmission electron microscopy were performed to determine T-tubule structure in isolated cells and intact hearts. In addition, the assembly of the DGC with Ptpn23 and dystrophin loss of function was determined by glycerol-gradient fractionation and SDS-PAGE analysis. The expression level of Ptpn23 was reduced in failing hearts from dilated cardiomyopathy patients and mice. Genetic deletion of Ptpn23 resulted in disorganized T-tubules with enlarged diameters and progressive dilated cardiomyopathy without affecting sarcomere organization. AAV9-mediated mosaic somatic mutagenesis further indicated a cell-autonomous role of Ptpn23 in regulating T-tubule formation. Genetic and biochemical analyses showed that Ptpn23 was essential for the integrity of costameres, which anchor the T-tubule membrane to Z-discs, through interactions with α-actinin and dystrophin. Deletion of α-actinin altered the subcellular localization of Ptpn23 and DGCs. In addition, genetic inactivation of dystrophin caused similar T-tubule defects to Ptpn23 loss-of-function without affecting Ptpn23 localization at Z-discs. Last, inducible Ptpn23 knockout at 1 month of age showed Ptpn23 is also required for the maintenance of T-tubules in adult cardiomyocytes. Ptpn23 is essential for cardiac T-tubule formation and maintenance along Z-discs. During postnatal heart development, Ptpn23 interacts with sarcomeric α-actinin and coordinates the assembly of the DGC at costameres to sculpt T-tubule spatial patterning and morphology.

A new procedure to induce aortic aneurysms in mice.

Aortic aneurysms (AAs) are a major public health challenge, featured by a progressive impairs in aortic wall integrity that drives to aortic dilation and, in end stage, to its rupture. Despite important advances in the surgical treatment of aortic aneurysms, there is currently no pharmacological intervention that prevents their development, reduces their expansion, or avoids their rupture. In addition to classic risk factors such age or gender, several heritable connective tissue disorders have been associated with AA developing, highlighting the role of extracellular matrix (ECM) genes alterations in the developing of AA. In this sense, we have recently demonstrated that global deletion of the cellular communicating network factor 2 (CCN2), previously known as connective tissue growth factor (CTGF) due to its role in the extracellular matrix formation, predisposes to early and lethal AAs development after Angiotensin II (Ang II) infusion in mice. Here, we detail the protocol to induce and detect AAs generation in inducible global CCN2 knockout mice after Ang II infusion which allow the characterization of CCN role in AA development and may help to the development of pharmacological target for AA treatment.

Characterization of gene expression in the kidney of renal tubular cell-specific NFAT5 knockout mice.

Nuclear factor of activated T cells 5 (NFAT5; also called TonEBP/OREBP) is a transcription factor that is activated by hypertonicity and induces osmoprotective genes to protect cells against hypertonic conditions. In the kidney, renal tubular NFAT5 is known to be involved in the urine concentration mechanism. Previous studies have suggested that NFAT5 modulates the immune system and exerts various effects on organ damage, depending on organ and disease states. Pathophysiological roles of NFAT5 in renal tubular cells, however, still remain obscure. We conducted comprehensive analysis by performing transcription start site (TSS) sequencing on the kidney of inducible and renal tubular cell-specific NFAT5 knockout (KO) mice. Mice were subjected to unilateral ureteral obstruction to examine the relevance of renal tubular NFAT5 in renal fibrosis. TSS sequencing analysis identified 722 downregulated TSSs and 1,360 upregulated TSSs, which were differentially regulated ≤-1.0 and ≥1.0 in log2 fold, respectively. Those TSSs were annotated to 532 downregulated genes and 944 upregulated genes, respectively. Motif analysis showed that sequences that possibly bind to NFAT5 were enriched in TSSs of downregulated genes. Gene Ontology analysis with the upregulated genes suggested disorder of innate and adaptive immune systems in the kidney. Unilateral ureteral obstruction significantly exacerbated renal fibrosis in the renal medulla in KO mice compared with wild-type mice, accompanied by enhanced activation of immune responses. In conclusion, NFAT5 in renal tubules could have pathophysiological roles in renal fibrosis through modulating innate and adaptive immune systems in the kidney.NEW & NOTEWORTHY TSS-Seq analysis of the kidney from renal tubular cell-specific NFAT5 KO mice uncovered novel genes that are possibly regulated by NFAT5 in the kidney under physiological conditions. The study further implied disorders of innate and adaptive immune systems in NFAT5 KO mice, thereby exacerbating renal fibrosis at pathological states. Our results may implicate the involvement of renal tubular NFAT5 in the progression of renal fibrosis. Further studies would be worthwhile for the development of novel therapy to treat chronic kidney disease.

Restoring glucose balance: Conditional HMGB1 knockdown mitigates hyperglycemia in a Streptozotocin induced mouse model

Diabetes mellitus (DM) poses a significant global health burden, with hyperglycemia being a primary contributor to complications and high morbidity associated with this disorder. Existing glucose management strategies have shown suboptimal effectiveness, necessitating alternative approaches. In this study, we explored the role of high mobility group box 1 (HMGB1) in hyperglycemia, a protein implicated in initiating inflammation and strongly correlated with DM onset and progression. We hypothesized that HMGB1 knockdown will mitigate hyperglycemia severity and enhance glucose tolerance. To test this hypothesis, we utilized a novel inducible HMGB1 knockout (iHMGB1 KO) mouse model exhibiting systemic HMGB1 knockdown. Hyperglycemic phenotype was induced using low dose streptozotocin (STZ) injections, followed by longitudinal glucose measurements and oral glucose tolerance tests to evaluate the effect of HMGB1 knockdown on glucose metabolism. Our findings showed a substantial reduction in glucose levels and enhanced glucose tolerance in HMGB1 knockdown mice. Additionally, we performed RNA sequencing analyses, which identified potential alternations in genes and molecular pathways within the liver and skeletal muscle tissue that may account for the in vivo phenotypic changes observed in hyperglycemic mice following HMGB1 knockdown. In conclusion, our present study delivers the first direct evidence of a causal relationship between systemic HMGB1 knockdown and hyperglycemia in vivo, an association that had remained unexamined prior to this research. This discovery positions HMGB1 knockdown as a potentially efficacious therapeutic target for addressing hyperglycemia and, by extension, the DM epidemic. Furthermore, we have revealed potential underlying mechanisms, establishing the essential groundwork for subsequent in-depth mechanistic investigations focused on further elucidating and harnessing the promising therapeutic potential of HMGB1 in DM management.

MicroRNA-19 is regulated by aldosterone in a sex-specific manner to alter kidney sodium transport.

A key regulator of blood pressure homeostasis is the steroid hormone aldosterone, which is released as the final signaling hormone of the renin-angiotensin-aldosterone-signaling (RAAS) system. Aldosterone increases sodium (Na+) reabsorption in the kidney distal nephron to regulate blood volume. Unregulated RAAS signaling can lead to hypertension and cardiovascular disease. The serum and glucocorticoid kinase (SGK1) coordinates much of the Na+ reabsorption in the cortical collecting duct (CCD) tubular epithelial cells. We previously demonstrated that aldosterone alters the expression of microRNAs (miRs) in CCD principal cells. The aldosterone-regulated miRs can modulate Na+ transport and the cellular response to aldosterone signaling. However, the sex-specific regulation of miRs by aldosterone in the kidney distal nephron has not been explored. In this study, we report that miR-19, part of the miR-17-92 cluster, is upregulated in female mouse CCD cells in response to aldosterone activation. Mir-19 binding to the 3'-untranslated region of SGK1 was confirmed using a dual-luciferase reporter assay. Increasing miR-19 expression in CCD cells decreased SGK1 message and protein expression. Removal of this cluster using a nephron-specific, inducible knockout mouse model increased SGK1 expression in female mouse CCD cells. The miR-19-induced decrease in SGK1 protein expression reduced the response to aldosterone stimulation and may account for sex-specific differences in aldosterone signaling. By examining evolution of the miR-17-92 cluster, phylogenetic sequence analysis indicated that this cluster arose at the same time that other Na+-sparing and salt regulatory proteins, specifically SGK1, first emerged, indicating a conserved role for these miRs in kidney function of salt and water homeostasis.NEW & NOTEWORTHY Expression of the microRNA-17-92 cluster is upregulated by aldosterone in mouse cortical collecting duct principal cells, exclusively in female mice. MiR-19 in this cluster targets the serum and glucocorticoid kinase (SGK1) to downregulate both mRNA and protein expression, resulting in a decrease in sodium transport across epithelial cells of the collecting duct. The miR-17-92 cluster is evolutionarily conserved and may act as a novel feedback regulator for aldosterone signaling in females.