Conditional Knockout Mouse Model Research Articles

Deletion of IFT20 exclusively in the RPE ablates primary cilia and leads to retinal degeneration.

Vision impairment places a serious burden on the aging society, affecting the lives of millions of people. Many retinal diseases are of genetic origin, of which over 50% are due to mutations in cilia-associated genes. Most research on retinal degeneration has focused on the ciliated photoreceptor cells of the retina. However, the contribution of primary cilia in other ocular cell types has largely been ignored. The retinal pigment epithelium (RPE) is a monolayer epithelium at the back of the eye intricately associated with photoreceptors and essential for visual function. It is already known that primary cilia in the RPE are critical for its development and maturation; however, it remains unclear whether this affects RPE function and retinal tissue homeostasis. We generated a conditional knockout mouse model, in which IFT20 is exclusively deleted in the RPE, ablating primary cilia. This leads to defective RPE function, followed by photoreceptor degeneration and, ultimately, vision impairment. Transcriptomic analysis offers insights into mechanisms underlying pathogenic changes, which include transcripts related to epithelial homeostasis, the visual cycle, and phagocytosis. Due to the loss of cilia exclusively in the RPE, this mouse model enables us to tease out the functional role of RPE cilia and their contribution to retinal degeneration, providing a powerful tool for basic and translational research in syndromic and non-syndromic retinal degeneration. Non-ciliary mechanisms of IFT20 in the RPE may also contribute to pathogenesis and cannot be excluded, especially considering the increasing evidence of non-ciliary functions of ciliary proteins.

Regulation of tau‐phosphorylation and other Alzheimer’s disease‐associated phenotypes in REST conditional knockout mice

AbstractBackgroundThe RE1‐silencing transcription factor (REST) has been strongly implicated in Alzheimer’s disease (AD), healthy ageing and longevity, and there is a clear need to understand better the exact mechanisms that mediate that role. Here we study the role of REST in a series of AD‐related phenotypes in the mouse brain.MethodWe have previously generated a conditional knockout (cKO) mouse model, lacking Rest in the postnatal forebrain. Immunohistochemistry and Western blot were used to evaluate the effect of REST on neuronal, synaptic and astrocytic markers and key proteins involved in tau phosphorylation. Furthermore, RNA‐Seq (including gene ontology, gene set enrichment and protein‐protein interaction analyses) was used to study the effect of REST inactivation at the transcriptome.ResultQuantification of labelled neuronal nuclei on brain sections revealed no major neurodegeneration in Rest cKO mice. However, mild phenotypes of dendritic degeneration and as astrocytic activation were suggested. The levels of an important postsynaptic protein, PSD‐95, appeared reduced in the brain of Rest cKO mice. Interestingly, levels of proteins involved in the phosphorylation of tau, such as glycogen synthase kinase‐3beta (GSK‐3β) and p35/p25 (activators of CDK5), as well as the levels of phosphorylated tau, were found increased in the brain of Rest cKO mice. RNA‐seq results suggest that postnatal inactivation of REST down‐regulates synaptic activity and neuronal pathways, while triggering an immune response.ConclusionOur results suggest dysregulation of one of the biochemical hallmarks of Alzheimer’s disease, tau phosphorylation, and of synaptic and neuronal pathways, as well as an immune response, when REST is inactivated in the adult mouse brain.

The effect of microglia activation on cerebral injury induced by conditional Tpk knockout in brain neurons of adult mice and the mechanism exploration

AbstractBackgroundWe previously designed and constructed the neuronal Tpk conditional knockout mouse model, which can simulate various pathophysiological changes of Alzheimer’s disease (AD). The objective of this study was to explore the effect and mechanism of microglia activation induced by the conditioned neuronal Tpk knockout on neuronal injury.MethodWe selected the 3‐month‐old CaMKII‐CreERT2/+ ; Tpkfl/fl (Tpk‐cKO) and negative control mice Tpkfl/f (Control) from the same nest. The mice were given CSF1R inhibitor (PLX3397)respectively during the whole stage (the 2nd∼10th weeks after induction), the late stage (the 6th∼10th weeks after induction), and the early stage (the 2nd∼6th weeks after induction) to eliminate microglia during whole/early/late stage. Besides, the single‐cell RNA sequencing (scRNA‐seq) analysis was performed at different time courses.ResultAccording to scRNA‐seq analysis, Microglia can be divided into different subgroups, which have different functions and proportions, and their pseudotime states were quite distinct. Among them, microglia subgroup 5 was highly distributed in the early pseudotime state playing the role of immune regulation and neuroprotection, while subgroup 6 was mainly distributed in the late pseudotime state contributing to phagocytosis damage. Therefore, when microglia were eliminated for the whole period, the protective effect of microglia was completely lost leading to more cerebral injury. Eliminating microglia at the early stage, the cerebral injury was alleviated because the repopulated microglia was still in the early pseudotime state performing immunomodulatory and neuroprotective functions. Eliminating microglia at late stage can also mitigate the cerebral injury, mainly because it not only has received the immunomodulatory and neuroprotective effects in the early stage, but also reduce the phagocytic damage in the late stage.ConclusionThe overall effect and mechanism of microglia activation induced by neuronal Tpk conditional knockout on cerebral injury are distinct in different stages, which is associated with the function and proportion of microglia clusters. The major effect of microglia at the early stage was immunoregulation and neuroprotection induced by cluster 5, and the general effect of the late stage was phagocytic damage mainly provided by cluster 6.

Reduction in Lymphoid-Supporting Factors By Mesenchymal Stromal Cells Drives Myeloid-Biased Hematopoiesis at Middle Age

The bone marrow “niche” for hematopoietic stem cells (HSCs) encompasses various cell types, extracellular matrices and secreted factors that maintain the specialized functions of HSCs such as quiescence, self-renewal, and production of multipotent progenitor cells. In old age, changes in this niche contribute to dysfunction of HSCs and the hematopoietic system including increased inflammation and myeloid-biased molecular programs. We have observed that HSC and hematopoietic aging phenotypes become apparent by mid-age (Young et al. Cell Stem Cell 2021, Young et al. J Exp Med 2016), leading to the hypothesis that evaluating alterations in the niche at this stage would reveal the earliest changes initiating HSC aging. To test this hypothesis, we examined both the hematopoietic and non-hematopoietic compartments in individual middle-aged (12-14 months of age, n = 9) and compared them to young C57BL/6 mice (2-4 months of age, n = 5). Immunostaining of femurs showed an increase in sinusoid volume, vessel dysmorphia, and an increase in megakaryocyte abundance in the bone marrow of middle-aged mice. No alterations were observed in arterioles nor adipocyte volume. This data provides supporting evidence that a subset of HSC niche remodeling phenotypes observed in old age are present by mid-age. We then performed single-cell RNA-seq to comprehensively assess changes in abundance and transcriptomes of hematopoietic and non-hematopoietic cells at mid-age. Analysis of this data revealed significant expansion of HSCs in middle-aged mice. In the expanded HSC population, a reduction in B lymphoid differentiation signatures was observed without alterations in inflammation or myeloid differentiation signatures. Computational predictions suggested that the reduced B lymphopoiesis program in middle-aged HSCs resulted from decreased interaction with soluble factors produced by subsets of mesenchymal stromal cells (MSCs), rather than endothelial cells or other hematopoietic cell compartments. Specifically, reduced MSC-to-HSC interactions were linked to a decline in signaling mediated by adiponectin (ADIPOQ; Adipoq), stem cell factor (SCF; Kitl), midkine (MDK; Mdk), and insulin-like growth factor 1 (IGF1; Igf1). Among these, reductions in Kitl and Igf1 in MSCs were correlated with the loss of B lymphopoiesis programs in HSCs comparing individual middle-aged mice. To test the functional impact of the decline in Igf1 in MSCs on lymphopoiesis, we created three MSC-selective Igf1 conditional knockout mouse models using distinct Cre recombinases (Lepr-Cre, nestin-CreER and Prx1-CreER). In all three of these models, MSC-selective knockout of Igf1 reduced B lymphopoiesis resulting in myeloid-biased hematopoiesis, replicating hematopoietic aging phenotypes observed at middle age. These findings indicate that the decline in B lymphopoiesis from HSCs during aging is initiated by a reduction in lymphoid-supporting factors produced by MSCs in the bone marrow microenvironment, and this occurs independently of inflammation or activation of myeloid-promoting transcriptional programs in HSCs.

Mustn1 ablation in skeletal muscle results in functional alterations.

Mustn1, a gene expressed exclusively in the musculoskeletal system, was shown in previous invitro studies to be a key regulator of myogenic differentiation and myofusion. Other studies also showed Mustn1 expression associated with skeletal muscle development and hypertrophy. However, its specific role in skeletal muscle function remains unclear. This study sought to investigate the effects of Mustn1 in a conditional knockout (KO) mouse model in Pax7 positive skeletal muscle satellite cells. Specifically, we investigated the potential effects of Mustn1 on myogenic gene expression, grip strength, alterations in gait, exvivo investigations of isolated skeletal muscle isometric contractions, and potential changes in the composition of muscle fiber types. Results indicate that Mustn1 KO mice did not present any substantial phenotypic changes or significant variations in genes related to myogenic differentiation and fusion. However, an approximately 10% decrease in overall grip strength was observed in the 2-month-old KO mice in comparison to the control wild type (WT), but this decrease was not significant when normalized by weight. KO mice also generated approximately 8% higher vertical force than WT at 4 months in the hindlimb. Exvivo experiments revealed decreases in about 20 to 50% in skeletal muscle contractions and about 10%-20% fatigue in soleus of both 2- and 4-month-old KO mice, respectively. Lastly, immunofluorescent analyses showed a persistent increase of Type IIb fibers up to 15-fold in the KO mice while Type I fibers decreased about 20% and 30% at both 2 and 4 months, respectively. These findings suggest a potential adaptive or compensatory mechanism following Mustn1 loss, as well as hinting at an association between Mustn1 and muscle fiber typing. Collectively, Mustn1's complex roles in skeletal muscle physiology requires further research, particularly in terms of understanding the potential role of Mustn1 in muscle repair and regeneration, as well as with influence of exercise. Collectively, these will offer valuable insights into Mustn1's key biological functions and regulatory pathways.

Polycomb Group Protein Yin Yang 1 Controls DNA Methylation in Murine T Lymphocyte Development

Transcription factors and epigenetic regulators play key functions in T lymphocyte lineage commitment and development. Mammalian PcG proteins are major negative regulators of gene expression that typically bind as large complexes (such as Polycomb Repressive Complex 1 (PRC1) and PRC2) to gene regulatory regions selectively targeted by an as yet unknown mechanism. While nearly all mammalian PcG proteins are lack of specific DNA binding capacities, transcription factor Yin Yang 1 (YY1) can bind at the DNA sites specifically and recruit other PcG proteins to specific DNA sequences. A small 25 amino acid YY1 domain (the REPO domain) is necessary and sufficient for the recruitment of PcG proteins, PcG-mediated stable transcriptional repression and histone modification. While YY1ΔREPO is competent for DNA binding, transcriptional activation, transient transcriptional repression and interaction with transcriptional coregulators such as HDACs, YY1ΔREPO is defective in all YY1 PcG functions. Our prior study demonstrated that deletion of YY1 in the hematopoietic system leads to T cell development blockage at the double-negative 3 (DN3) stage. Interestingly, ectopic expression of YY1∆REPO in YY1 null background by retroviral bone marrow transplantation could not rescue YY1 deficiency mediated T cell survival defect. This suggests that the YY1 REPO domain/PcG function plays a vital role in T cell development. Herein, we generated the YY1 REPO domain conditional knockout mouse model ( Yy1 f/∆REPO Vav-Cre) by CRISPR/Cas9 to further dissect the YY1 REPO domain/PcG function in T cell development. Yy1 f/∆REPO Vav-Cre ( Yy1 -/∆REPO) mice have reduced thymus weight and cellularity with medulla aplasia. Compared with wild-type (WT) and YY1 heterozygous ( Yy1 +/-) mice, Yy1 -/∆REPO mice have an increased percentage of DN3 cells with decreased DN4 percentage supporting a developmental blockage at the DN3 stage. Yy1 -/∆REPO DN T cells fail to go through TCRβ rearrangement with increased apoptosis and reduction of TCRβ + cells in the thymus compared with WT and Yy1 +/-cells. Interestingly, RNA-seq analysis in sorted primary DN3 cells revealed the genetic network governing DNA methylation, histone methylation, chromosome maintenance, and translation initiation complex formation are deregulated in Yy1 -/∆REPO mice compared to WT and Yy1 +/-mice. DN T cell development requires precise and dynamic regulation of histone and DNA methylation and demethylation. Interestingly, the DNA demethylase enzymes ten-eleven translocation (TET) family proteins Tet1 and Tet2 are downregulated in Yy1 -/∆REPO DN3 thymocytes compared with WT and Yy1 +/- DN3 cells. Our data show that the YY1 REPO domain/PcG function is required for DN3-DN4 transition via regulating TCRβ rearrangement and the YY1 REPO domain/PcG function plays a critical role in proper DNA methylation status in early T cell development. As PcG proteins modify histone via trimethylation of histone H3 on lysine 27, we will further assess the impact of YY1 REPO domain/PcG function on histone methylation in T cell development. By utilizing a powerful mouse model for identifying YY1 functions as PcG domain-dependent or PcG domain-independent, our study has generated fundamental new insights into the epigenetic landscape in T cell development.

SUMO E3 Ligase ZMIZ1 Drives Leukemogenesis Via Transcriptional Deregulation in Acute Myeloid Leukemia

Transcriptional deregulation is one of the hallmarks of acute myeloid leukemia (AML), which causes the aberrant expression of genes essential for hematopoiesis, proliferation, stemness et, al. Emerging evidence has demonstrated that transcription is regulated by post-translational modification of transcription factors, such as SUMOylation, and the recruitment of co-regulators, such as epigenetic regulators. Here, we conducted a CRISPR-Cas9 screen using sgRNAs targeting epigenetic regulators and a SUMO E3 ligase to explore essential regulators for hematopoiesis and leukemogenesis. Intriguingly, sgRNAs targeting ZMIZ1 was identified to be significantly decreased in proliferated leukemia cells and enriched in differentiated ones. ZMIZ1 (also known as ZIMP10 or RAI17) is a SUMO E3 ligase and transcriptional co-regulator of the Protein Inhibitor of Activated STAT (PIAS)-like family. Moreover, we found ZMIZ1 was highly expressed in AML and related to unfavorable outcomes, suggesting a potential diagnostic and prognostic target for AML. Next, we analyzed the function of ZMIZ1 in AML using both in vivo and in vitro approaches. We knocked down the expression of ZMIZ1 in MOLM-13, THP1, OCI-AML3, HL60, and NB4 cells and found that knockdown of ZMIZ1 suppressed cell viability and induced apoptosis in all five cell lines. Meanwhile, knockdown of ZMIZ1 enhanced the sensitivity to chemotherapy (such as Ara-C). Additionally, knockdown of ZMIZ1 induced cell differentiation in NB4 cells. Moreover, we established conditional knockout mouse models (Zmiz1 flox/floxMx1-Cre), in which Zmiz1 was deleted with the Mx1-Cre transgene. We isolated the bone marrow of these mice and transfected it with the MLL-AF9 fusion gene, followed by transplantation into recipient mice. The conditional knockout was induced after two weeks of transfection. We found that inactivation of Zmiz1 significantly prolonged survival and relieved the leukemic burden. To examine the regulatory function of ZMIZ1 in AML, we conducted ChIP-seq analysis in MOLM-13, THP1, OCI-AML3, HL60, and NB4 cells using antibodies against ZMIZ1. The binding peaks of ZMIZ1 differ in AML cell lines, as fewer than 30% of binding sites were overlapped between different cells. However, the binding motifs for ZMIZ1 showed a similar pattern. Motifs bound by MYB, CENPE, and RUNX1 were enriched in all five cell lines. To test the regulatory effects of ZMIZ1 on these transcription factors, we conducted luciferase reporter assays and found that ZMIZ1 enhanced the transcriptional activity of MYB. Taken together, our study suggests an essential function of ZMIZ1 in leukemogenesis, presumably via SUMOylating essential transcription factors, and sheds new light on AML targeting therapy probably by targeting ZMIZ1.

A promoterless AAV6.2FF-based lung gene editing platform for the correction of surfactant protein B deficiency

Surfactant protein B (SP-B) deficiency is a rare genetic disease that causes fatal respiratory failure within the first year of life. Currently, the only corrective treatment is lung transplantation. Here, we co-transduced the murine lung with adeno-associated virus 6.2FF (AAV6.2FF) vectors encoding a SaCas9-guide RNA nuclease or donor template to mediate insertion of promoterless reporter genes or the (murine) Sftpb gene in frame with the endogenous surfactant protein C (SP-C) gene, without disrupting SP-C expression. Intranasal administration of 3×1011 vg donor template and 1×1011 vg nuclease consistently edited approximately 6% of lung epithelial cells. Frequency of gene insertion increased in a dose-dependent manner, reaching 20%-25% editing efficiency with the highest donor template and nuclease doses tested. We next evaluated whether this promoterless gene editing platform could extend survival in the conditional SP-B knockout mouse model. Administration of 1×1012 vg SP-B-donor template and 5×1011 vg nuclease significantly extended median survival (p= 0.0034) from 5days in the untreated off doxycycline group to 16days in the donor AAV and nuclease group, with one gene-edited mouse living 243days off doxycycline. This AAV6.2FF-based gene editing platform has the potential to correct SP-B deficiency, as well as other disorders of alveolar type II cells.

ClpP/ClpX deficiency impairs mitochondrial functions and mTORC1 signaling during spermatogenesis

Caseinolytic protease proteolytic subunit (ClpP) and caseinolytic protease X (ClpX) are mitochondrial matrix peptidases that activate mitochondrial unfolded protein response to maintain protein homeostasis in the mitochondria. However, the role of ClpP and ClpX in spermatogenesis remains largely unknown. In this study, we demonstrated the importance of ClpP/ClpX for meiosis and spermatogenesis with two conditional knockout (cKO) mouse models. We found that ClpP/ClpX deficiency reduced mitochondrial functions and quantity in spermatocytes, affected energy supply during meiosis and attenuated zygotene-pachytene transformation of the male germ cells. The dysregulated spermatocytes finally underwent apoptosis resulting in decreased testicular size and vacuolar structures within the seminiferous tubules. We found mTORC1 pathway was over-activated after deletion of ClpP/ClpX in spermatocytes. Long-term inhibition of the mTORC1 signaling via rapamycin treatment in vivo partially rescue spermatogenesis. The data reveal the critical roles of ClpP and ClpX in regulating meiosis and spermatogenesis.

OR16-04 Androgen Deprivation Restricts Anti-tumor Immunity In Adrenocortical Carcinoma

Abstract Disclosure: K.M. Warde: None. L. Smith: None. C.J. Stubben: None. P.W. Willet: None. G. Castaneda-Hernandez: None. L. Liu: None. K. Basham: None. Adrenocortical carcinoma (ACC) is a rare cancer that is often diagnosed late and progresses rapidly, resulting in routinely poor outcomes. Through The Cancer Genome Atlas (TCGA) and other genomic studies, homozygous loss of ZNRF3 was newly identified as a frequent genetic alteration in human ACC tumors. ZNRF3 is an E3 ubiquitin ligase that negatively regulates Wnt signaling by degrading Wnt receptors. To examine the role of ZNRF3 in adrenal biology, we generated a Znrf3 conditional knockout (cKO) mouse model. Despite roust early hyperplasia, we found that Znrf3 cKO mice only develop adrenal tumors with advanced aging. Moreover, tumors emerge in a sex dimorphic manner whereby males, who exhibit a high myeloid immune response, predominately get benign tumors and females, who have a dampened myeloid response, develop metastatic ACC. This phenomenon is reflected clinically in patients whereby the incidence of ACC is up to 2.5x higher in women compare to men. In human ACC patients, females exhibit a lower myeloid signature compared to males. Moreover, a low myeloid response signature is associated with shorter progression-free and overall survival. These data suggest a potential relationship between anti-tumor immunity and sex in ACC, yet the underlying mechanisms remain unclear. Sex differences are common in cancer, and although there are many potential factors, gonadal hormones including androgens and estrogens, have been implicated as major biological drivers. Previous studies of the adrenal cortex highlight a specific role for androgens in repressing stem cell recruitment and proliferation. We hypothesized androgens promote anti-tumor immunity to help protect against ACC. To test this hypothesis, we castrated male Znrf3 cKO mice to lower androgen levels. We found that androgen deprivation significantly reduced myeloid cell infiltration, resulting in significantly larger adrenals in castrated as compared to sham controls. RNA-seq analysis revealed a decrease in immune activation pathways and monocyte-derived myeloid cell populations. Additionally, pro-inflammatory cytokines, including TNF, INFG, and IL-6, were significantly inhibited in castrated animals compared to controls. These data support a protective role for androgens in ACC through driving an intra-adrenal myeloid immune response. Consistent with these results, analysis of the TCGA-ACC patient cohort demonstrated that high androgen receptor expression is associated with better overall survival. Taken together, our results reveal an important role for androgens in promoting anti-tumor immunity in a mouse model of ACC, and we highlight the prognostic value of androgen receptor expression in patients. Further understanding of androgen signaling and downstream biological mechanisms that regulate the immune response may ultimately offer novel therapeutics and improve patient outcomes in ACC. Presentation: Friday, June 16, 2023

THU569 Steroid Receptor Coactivator 3 Is Required To Prevent Early Pregnancy Loss In The Mouse

Abstract Disclosure: V. Maurya: None. L. Hai: None. M.M. Szwarc: None. W. San-Pin: None. F.J. DeMayo: None. J.P. Lydon: None. Members of the Steroid Receptor Coactivator (SRC) family enact distinct transcriptional coregulator functions within a broad spectrum of physiologies and pathologies. We previously demonstrated that endometrial SRC-3 is critical for in vitro human endometrial stromal cell decidualization, a critical cellular process in the establishment of the maternofetal interface. To address SRC-3’s in vivo role in endometrial periimplantation biology, we recently generated a SRC-3 conditional knockout mouse model (SRC-3d/d) in which SRC-3 is selectively ablated in cells that express the progesterone receptor. A six-month breeding trial with wild type proven stud males demonstrated that the SRC-3d/d female fails to produce litters (n>10 per genotype). Unlike the SRC-2d/d female, however, both embryo attachment and implantation into the stromal compartment of the SRC-3d/d endometrium are not impaired at gestation day (GD) 5 or 6. While endometrial stromal decidualization initiates with embryo invasion, pregnant SRC-3d/d mice exhibit early pregnancy loss (EPL) by GD-7.5 (before placentation). At GD-6, the SRC-3d/d decidua is significantly smaller than those of sibling controls. Histological analyses reveal extensive decidual cell death in the SRC-3d/d decidua by GD-7, which results in the sudden demise of the embryo. These histological findings are supported by recent genome-wide RNA profiling studies that show a pro-survival role for SRC-3 in this tissue. Based on these results, we conclude that endometrial SRC-3 is critical for maintaining the functionality and survival of the decidual stromal cell and embryo developmental progression to placentation. Therefore, endometrial SRC-3 (and its downstream signaling networks) are predicted to represent new molecular determinants of non-aneuploid EPL, a poorly understood pregnancy complication for which diagnostic and treatment options are limited. Presentation Date: Thursday, June 15, 2023

Comparative multi-omic analyses of cardiac mitochondrial stress in three mouse models of frataxin deficiency.

Cardiomyopathy is often fatal in Friedreich ataxia (FA). However, FA hearts maintain adequate function until advanced disease stages, suggesting initial adaptation to the loss of frataxin (FXN). Conditional cardiac knockout mouse models of FXN show transcriptional and metabolic profiles of the mitochondrial integrated stress response (ISRmt), which could play an adaptive role. However, the ISRmt has not been investigated in models with disease-relevant, partial decrease in FXN. We characterized the heart transcriptomes and metabolomes of three mouse models with varying degrees of FXN depletion: YG8-800, KIKO-700 and FXNG127V. Few metabolites were changed in YG8-800 mice, which did not provide a signature of cardiomyopathy or ISRmt; several metabolites were altered in FXNG127V and KIKO-700 hearts. Transcriptional changes were found in all models, but differentially expressed genes consistent with cardiomyopathy and ISRmt were only identified in FXNG127V hearts. However, these changes were surprisingly mild even at advanced age (18 months), despite a severe decrease in FXN levels to 1% of those of wild type. These findings indicate that the mouse heart has low reliance on FXN, highlighting the difficulty in modeling genetically relevant FA cardiomyopathy.

Loss of succinyl-CoA synthetase in mouse forebrain results in hypersuccinylation with perturbed neuronal transcription and metabolism

Lysine succinylation is a subtype of protein acylation associated with metabolic regulation of succinyl-CoA in the tricarboxylic acid cycle. Deficiency of succinyl-CoA synthetase (SCS), the tricarboxylic acid cycle enzyme catalyzing the interconversion of succinyl-CoA to succinate, results in mitochondrial encephalomyopathy in humans. This report presents a conditional forebrain-specific knockout (KO) mouse model of Sucla2, the gene encoding the ATP-specific beta isoform of SCS, resulting in postnatal deficiency of the entire SCS complex. Results demonstrate that accumulation of succinyl-CoA in the absence of SCS leads to hypersuccinylation within the murine cerebral cortex. Specifically, increased succinylation is associated with functionally significant reduced activity of respiratory chain complex I and widescale alterations in chromatin landscape and gene expression. Integrative analysis of the transcriptomic data also reveals perturbations in regulatory networks of neuronal transcription in the KO forebrain. Together, these findings provide evidence that protein succinylation plays a significant role in the pathogenesis of SCS deficiency.

P320 VMA21 conditional knockout mice model XMEA with myopathy and dysfunctional autophagy

P320 VMA21 conditional knockout mice model XMEA with myopathy and dysfunctional autophagy

Core fucosylation regulates alveolar epithelial cells senescence through activating of transforming growth factor-β pathway in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF), a fatal disorder associated with aging, has a terrible prognosis. However, the potential causes of IPF remain a riddle. In this study, we designed to explore whether the modification of the core fucosylation (CF) can ameliorate pulmonary fibrosis by targeting alveolar epithelial cells (AECs) senescence. First, we verified that cellular senescence occurs in the bleomycin-induced lung fibrosis mice models and CF modifications accompanying senescent AECs in pulmonary fibrosis. Next, both gain- and loss- of function research on CF were performed to elucidate its role in promoting AECs senescence and triggering pulmonary fibrosis in vitro. Notably, using alveolar epithelial cell-specific FUT8 conditional knockout mouse models, however, inhibition of cellular senescence by deleting the FUT8 gene could attenuate pulmonary fibrosis in vivo. Finally, blocking the CF modification of transforming growth factor -β type I receptor (TGF-βR I) could reduce the activation of downstream transforming growth factor -β (TGF-β) pathways in AECs senescence both in vivo and in vitro. This study reveals that CF is a crucial interventional target for the treatment of pulmonary fibrosis. Blocking CF modification contributes importantly to inhibiting AECs senescence resulting in pulmonary fibrosis lessen.

Physiological and regenerative functions of sterile-α motif protein-14 in hematopoiesis

Sterile α-motif domain-14 (Samd14) protein expression increases the regenerative capacity of the erythroid system. Samd14 is transcriptionally upregulated and promotes cell signaling via the receptor tyrosine kinase Kit in a critical window of acute erythroid regeneration. We generated a hematopoietic-specific conditional Samd14 knockout mouse model (Samd14-CKO) to study the role of Samd14 in hematopoiesis. The Samd14-CKO mouse was viable and exhibited no steady-state hematopoietic phenotype. Samd14-CKO mice were hypersensitive to 5-fluorouracil, resulting in more severe anemia during recovery and impaired erythroid progenitor colony formation. Ex vivo, Samd14-CKO hematopoietic progenitors were defective in their ability to form mast cells. Samd14-CKO mast cells exhibited altered Kit/stem cell factor (SCF), IL-3/IL-3R signaling, and less granularity than Samd14-FL/FL cells. Our findings indicate that Samd14 promotes both erythroid and mast cell functions. The Samd14-CKO mouse phenotype exhibits striking similarities to the KitW/W-v mice, which carry Kit mutations resulting in reduced tyrosine kinase-dependent signaling, causing mast cell and erythroid abnormalities. The Samd14-CKO mouse model is a new tool for studying hematologic pathologies involving Kit signaling.

Germinal center output is sustained by HELLS-dependent DNA-methylation-maintenance in B cells

HELLS/LSH (Helicase, Lymphoid Specific) is a SNF2-like chromatin remodelling protein involved in DNA methylation. Its loss-of-function in humans causes humoral immunodeficiency, called ICF4 syndrome (Immunodeficiency, Centromeric Instability, Facial anomalies). Here we show by our newly generated B-cell-specific Hells conditional knockout mouse model that HELLS plays a pivotal role in T-dependent B-cell responses. HELLS deficiency induces accelerated decay of germinal center (GC) B cells and impairs the generation of high affinity memory B cells and circulating antibodies. Mutant GC B cells undergo dramatic DNA hypomethylation and massive de-repression of evolutionary recent retrotransposons, which surprisingly does not directly affect their survival. Instead, they prematurely upregulate either memory B cell markers or the transcription factor ATF4, which is driving an mTORC1-dependent metabolic program typical of plasma cells. Treatment of wild type mice with a DNMT1-specific inhibitor phenocopies the accelerated kinetics, thus pointing towards DNA-methylation maintenance by HELLS being a crucial mechanism to fine-tune the GC transcriptional program and enable long-lasting humoral immunity.

ERCC6L facilitates the onset of mammary neoplasia and promotes the high malignance of breast cancer by accelerating the cell cycle

BackgroundBreast cancer (BC) is the leading cause of morbidity and the second leading cause of death among female malignant tumors. Although available drugs have been approved for the corresponding breast cancer subtypes (ER-positive, HER2+) currently, there are still no effective targeted drugs or treatment strategies for metastatic breast cancer or triple-negative breast cancer that lack targets. Therefore, it is urgent to discover new potential targets. ERCC6L is an essential protein involved in chromosome separation during cell mitosis. However, the effect of ERCC6L on the tumorigenesis and progression of breast cancer is unclear.Methods and resultsHere, we found that ERCC6L was highly expressed in breast cancer, especially in TNBC, which was closely related to poor outcomes of patients. An ERCC6L conditional knockout mouse model was first established in this study, and the results confirmed that ERCC6L was required for the development of the mammary gland and the tumorigenesis and progression of mammary gland cancers. In in vitro cell culture, ERCC6L acted as a tumor promoter in the malignant progression of breast cancer cells. Overexpression of ERCC6L promoted cell proliferation, migration and invasion, while knockdown of ERCC6L caused the opposite results. Mechanistically, ERCC6L accelerated the cell cycle by regulating the G2/M checkpoint signalling pathway. Additionally, we demonstrated that there is an interaction between ERCC6L and KIF4A, both of which are closely related factors in mitosis and are involved in the malignant progression of breast cancer.ConclusionsWe first demonstrated that ERCC6L deficiency can significantly inhibit the occurrence and development of mammary gland tumors. ERCC6L was found to accelerate the cell cycle by regulating the p53/p21/CDK1/Cyclin B and PLK/CDC25C/CDK1/Cyclin B signalling pathways, thereby promoting the malignant progression of breast cancer cell lines. There was a direct interaction between KIF4A and ERCC6L, and both are closely associated with mitosis and contribute to growth and metastasis of breast tumor. To sum up, our results suggest that ERCC6L may be used as a promising target for the treatment of BC.

ZNF750 Regulates Skin Barrier Function by Driving Cornified Envelope and Lipid Processing Pathways

The epidermis is a constantly renewing stratified epithelial tissue that provides essential protective barrier functions. The major barrier is located at the outermost layers of the epidermis, formed by terminally differentiated keratinocytes reinforced by proteins of their cornified envelope and sequestered intercellular lipids. Disruptions to epidermal differentiation characterize various skin disorders. ZNF750 is an epithelial transcription factor essential for invitro keratinocyte differentiation, whose truncating mutation in humans causes autosomal dominant psoriasis-like skin disease. In this study, we utilized an epidermal-specific Znf750 conditional knockout mouse model to uncover the role ZNF750 plays in epidermal development. We show that deletion of Znf750 in the developing skin does not block epidermal differentiation completely, suggesting invivo compensatory feedback mechanisms, although it does result in impaired barrier function and perinatal lethality. Molecular dissection revealed ultrastructural defects in the differentiated layers of the epidermis, accompanied by alterations in the expression of ZNF750-dependent genes encoding key cornified envelope precursor proteins and lipid-processing enzymes, including gene subsets known to be mutated in human skin diseases involving impaired barrier function. Together, our findings provide molecular insights into the pathogenesis of human skin disease by linking ZNF750 to a subset of epidermal differentiation genes involved in barrier formation pathways.

PARP inhibitor rescues hearing and hair cell impairment in Cx26‐null mice

AbstractGJB2 (encoding connexin26, Cx26) mutation is the most common genetic cause of hereditary deafness. Cochlear sensory hair cell (HC) death is the core pathologic phenomenon of GJB2‐related deafness. However, mechanism‐based therapy is still obscure. A targeted‐cell conditional Gjb2 knockout mouse model was established in which the Cx26 in Deiters cells and pillar cells were knocked out at birth. We explored the mechanism of HC death caused by deficiency of GJB2 in supporting cells (SCs), which exhibited moderate deafness, early HC death without SCs, and a decrease in distortion product otoacoustic emission. Here, a DNA damage response was observed in HCs during the onset of hearing loss. Apoptosis, necroptosis, and ferroptosis do not contribute to the death of HCs. We identified and demonstrated that parthanatos, a poly‐(ADP‐ribose) polymerase (PARP)‐dependent and caspase‐independent form of cell death, is a mechanism of HC degeneration. Furthermore, we observed that the use of PARP inhibitors effectively alleviated both deafness and HC loss. Our study reveals the specific mechanism of HC death caused by GJB2 deficiency. These findings demonstrate that targeting parthanatos is an HC protective strategy in the prevention of GJB2‐related hereditary deafness.