Knockout Research Articles

Arylacetamide deacetylase regulates hepatic iron homeostasis to protect against carbon tetrachloride-induced ferroptosis.

Arylacetamide deacetylase (AADAC) catalyzes the hydrolysis of small molecules containing ester and amide bonds. Recently, it has been reported that AADAC can suppress reactive oxygen species production in cancer cells. This study aimed to elucidate the possibility that AADAC protects against drug-induced liver injury accompanied by oxidative stress and to explore its molecular mechanisms. Intraperitoneal administration of carbon tetrachloride induced significantly more severe liver injury in Aadac knockout (KO) mice (plasma alanine aminotransferase level: 19,381 ± 10,578 U/L) than in wild-type (WT) mice (7219 ± 4729 U/L). More severe liver injury in Aadac KO mice was accompanied by higher hepatic malondialdehyde and antioxidant gene mRNA levels than those in WT mice. The increase in plasma alanine aminotransferase levels in Aadac KO mice was substantially suppressed by pretreatment with the ferroptosis inhibitors deferoxamine or ferrostatin-1, suggesting that Aadac deficiency increases susceptibility to ferroptosis. Immunoprecipitation followed by proteomic analysis revealed that AADAC interacts with ceruloplasmin (CP), which oxidizes ferrous iron to ferric iron. Hepatic CP activity was significantly lower in Aadac KO mice than that in WT mice, resulting in elevated hepatic ferrous iron levels in Aadac KO mice. Overexpression of human AADAC in Huh-7 cells significantly attenuated carbon tetrachloride-induced cytotoxicity by suppressing ferrous iron accumulation, suggesting that AADAC interacts with CP to suppress hepatic ferrous iron accumulation. The hepatoprotective role of Aadac in ferroptosis was also observed in mice with acetaminophen-induced liver injury. This study demonstrates a novel function of AADAC in protecting against ferroptosis induced by hepatotoxicants, carbon tetrachlorideand acetaminophen.

12604 Use Of A Novel Program "WordMiner" To Identify Cellular Proliferation Related Genes Induced In FSTL3 KO Mouse Testis

Abstract Disclosure: N. Mukherjee: None. R. Ballesteros: None. A. Mukherjee: None. TGFβ family ligands play crucial roles in promoting cellular proliferation. Moreover, activin and TGFβ are linked with cancer. Deletion of FSTL3, an inhibitor of a subset of TGFβ ligands, in mice leads to increased proliferation in all tissues investigated including the testis. We also find increased SMAD2/3 activation in FSTL3 knockout (KO) compared to wildtype (WT) in these tissues. Therefore, the FSTL3 KO mouse is a model of increased activity of endogenous TGFβ ligands that are inhibited by FSTL3, such as activin. It is likely that increased signalling by these ligands leads to the observed increased cellular proliferation in FSTL3 KO mice. To discover genes that might be involved in activin-dependent mitosis, we studied gene expression datasets linked to cellular proliferation. We hypothesised that genes important in cellular proliferation will be downregulated in senescent cells and upregulated in models of increased cellular proliferation and activin action such as the FSTL3 KO mice. We queried GEO datasets to identify studies related to senescence and selected Hutchinson-Gilford progeria syndrome (HGPS): fibroblast (HG-U133A), GSE3860. We found 4048 transcripts showing significantly (p<0.05) altered expression in HGPS fibroblasts compared to WT. We developed a Python-based program, “WordMiner”, that inputs a keyword and a list of terms (e.g. genes) and then searches a specified database sequentially for publications including the keyword and each term. The output is a table of search result frequencies and hyperlinks to the search result pages, compiled in an auxiliary file, produced with greater speed and efficiency than manual search methods. The greater the search result frequency, the stronger the known link between the keyword and the term. We used WordMiner to query the association of the search term “mitosis” with the 4048 genes above using PubMed as the database and identified 1235 transcripts. We refined this list further using the keyword “activin” to identify 420 transcripts linked to activin and mitosis. Importantly, by comparing against our RNAseq data, we find 20 of these genes upregulated in the FSTL3 KO postnatal day 3 transcriptome. This finding supports our hypothesis, and also demonstrates WordMiner as an efficient and versatile cross-referencing query tool. Among the 20 genes identified are transcripts known to be important in proliferation, such as Akap9 and Wnt2b. These and the remainder in the list of genes highlighted are therefore likely to be involved in pathways induced by activin, promoting cellular proliferation in FSTL3 KO testis. Currently, siRNA transfection experiments are underway to address whether knockdown of these genes in a Sertoli cell line (TM4) limits proliferation. This will help in the elucidation of the molecular pathways involved in postnatal Sertoli cell proliferation. Presentation: 6/3/2024

Myeloid deficiency of heparan sulfate 6-O-endosulfatases impairs bone marrow hematopoiesis

The heparan sulfate (HS) 6-O-endosulfatases or the Sulfs (Sulf1 and Sulf2) are the only known enzymes that can modify HS sulfation status extracellularly and have been shown to regulate diverse biological processes. The role of the Sulfs in bone marrow (BM) hematopoiesis is not known. In this study, we generated a novel mouse line with myeloid-specific deletion of the Sulfs by crossing Sulf1/2 double floxed mice with the LysM-cre line. The LysM-Sulf knockout (KO) male mice exhibited age-dependent expansion of hematopoietic stem cells and the granulocyte-monocyte lineages in the BM, whereas common lymphoid progenitors and B lymphocyte populations were significantly reduced. Although megakaryocytic and erythroid progenitors were not reduced in the BM, the LysM-Sulf KO males suffered age-dependent reduction of red blood cells (RBCs) and platelets in the peripheral blood, suggesting that the production of RBCs and platelets was arrested at later stages. In addition, LysM-Sulf KO males displayed progressive splenomegaly with extramedullary hematopoiesis. Compared to males, LysM-Sulf KO females exhibited a much-reduced phenotype, and ovariectomy had little effect. Mechanistically, reduced TGF-β/Smad2 but enhanced p53/p21 signaling were observed in male but not female LysM-Sulf KO mice. Finally, HS disaccharide analysis via LC-MS/MS revealed increased HS 6-O-sulfation in the BM from both male and female LysM-Sulf KO mice, however, the distribution of 6-O-sulfated motifs were different between the sexes with compensatory increase in Sulf1 expression observed only in LysM-Sulf KO females. In conclusion, our study reveals that myeloid deficiency of the Sulfs leads to multilineage abnormalities in BM hematopoiesis in an age- and sex-dependent manner.

Tissue nonspecific alkaline phosphatase deficiency impairs Purkinje cell development and survival in a mouse model of infantile hypophosphatasia

Loss-of-function mutations in the tissue-nonspecific alkaline phosphatase (TNAP) gene can result in hypophosphatasia (HPP), an inherited multi-systemic metabolic disorder that is well-known for skeletal and dental hypomineralization. However, emerging evidence shows that both adult and pediatric patients with HPP suffer from cognitive deficits, higher measures of depression and anxiety, and impaired sensorimotor skills. The cerebellum plays an important role in sensorimotor coordination, cognition, and emotion. To date, the impact of TNAP mutation on the cerebellar circuitry development and function remains poorly understood. The main objective of this study was to investigate the roles of TNAP in cerebellar development and function, with a particular focus on Purkinje cells, in a mouse model of infantile HPP. Male and female wild type (WT) and TNAP knockout (KO) mice underwent behavioral testing on postnatal day 13–14 and were euthanized after completion of behavioral tests. Cerebellar tissues were harvested for gene expression and immunohistochemistry analyses. We found that TNAP mutation resulted in significantly reduced body weight, shorter body length, and impaired sensorimotor functions in both male and female KO mice. These developmental and behavioral deficits were accompanied by abnormal Purkinje cell morphology and dysregulation of genes that regulates synaptic transmission, cellular growth, proliferation, and death. In conclusion, inactivation of TNAP via gene deletion causes developmental delays, sensorimotor impairment, and Purkinje cell maldevelopment. These results shed light on a new perspective of cerebellar dysfunction in HPP.

Rapid increase of C/EBPα p42 induces growth arrest of acute myeloid leukemia (AML) cells by Cop1 deletion in Trib1-expressing AML.

Cop1 encodes a ubiquitin E3 ligase that has been well preserved during evolution in both plants and metazoans. In metazoans, the C/EBP family transcription factors are targets for degradation by Cop1, and this process is regulated by the Tribbles pseudokinase family. Over-expression of Tribbles homolog 1 (Trib1) induces acute myeloid leukemia (AML) via Cop1-dependent degradation of the C/EBPα p42 isoform. Here, we induced rapid growth arrest and granulocytic differentiation of Trib1-expressing AML cells using a Cop1 conditional knockout (KO), which is associated with a transient increase in the C/EBPα p42 isoform. The growth-suppressive effect of Cop1 KO was canceled by silencing of Cebpa and reinforced by exogenous expression of the p42 isoform. Moreover, Cop1 KO improved the survival of recipients transplanted with Trib1-expressing AML cells. We further identified a marked increase in Trib1 protein expression in Cop1 KO, indicating that Trib1 is self-degraded by the Cop1 degradosome. COP1 downregulation also inhibits the proliferation of human AML cells in a TRIB1-dependent manner. Taken together, our results provide new insights into the role of Trib1/Cop1 machinery in the C/EBPα p42-dependent leukemogenic activity, and a novel idea to develop new therapeutics.

Loss of Cell-Cell Contact Inhibits Cellular Differentiation of α-Catenin Knock Out P19 Embryonal Carcinoma Cells and Their Colonization into the Developing Mouse Embryos.

Cadherin-catenin cell-cell adhesion complexes, composed of cadherin, β-catenin or plakoglobin, and α-catenin (α-cat) molecules, are crucial for maintaining cell-cell contact and are commonly referred to as "adherens junctions (AJs)." Inactivating this system leads to loss of cell-cell contact and developmental arrest in early embryos. However, it remains unclear whether the loss of cell-cell contact affects the differentiation of embryonic cells. In this study, we explored the use of a murine embryonal carcinoma cell line, P19, as an in vitro model for early embryogenesis. P19 cells easily form embryoid bodies (EBs) and are susceptible to cellular differentiation in response to retinoic acid (RA) and teratoma formation. Using CRISPR/Cas9 technology to disrupt the endogenous α-cat gene in P19 cells, we generated α-cat knockout (KO) cells that exhibited a loss of cell-cell contact. When cultivated on non-coated dishes, these α-cat KO cells formed EBs, but their structures were labile. In the RA-containing medium, the α-cat KO EBs failed to produce differentiated cells on their outer layer and continued to express SSEA-1, an antigen specific to pluripotent cells. Teratoma formation assays revealed an absence of overt differentiated cells in tumors derived from α-cat KO P19 cells. Aggregation assays revealed the inability of the KO cells to colonize into the zona pellucida-denuded 8-cell embryos. These findings suggest that the AJs are essential for promoting the early stages of cellular differentiation and for the colonization of early-developing embryos.

EZH2 specifically regulates ISL1 during embryonic urinary tract formation

Isl1 has been described as an embryonic master control gene expressed in the pericloacal mesenchyme. Deletion of Isl1 from the genital mesenchyme in mice leads to an ectopic urethral opening and epispadias-like phenotype. Using genome wide association methods, we identified ISL1 as the key susceptibility gene for classic bladder exstrophy (CBE), comprising epispadias and exstrophy of the urinary bladder. The most significant marker (rs6874700) identified in our recent GWAS meta-analysis achieved a p value of 1.48 × 10− 24 within the ISL1 region. In silico analysis of rs6874700 and all other genome-wide significant markers in Linkage Disequilibrium (LD) with rs6874700 (D’ = 1.0; R2 > 0.90) revealed marker rs2303751 (p value 8.12 × 10− 20) as the marker with the highest regulatory effect predicted. Here, we describe a novel 1.2 kb intragenic promoter residing between 6.2 and 7.4 kb downstream of the ISL1 transcription starting site, which is located in the reverse DNA strand and harbors a binding site for EZH2 at the exact region of marker rs2303751. We show, that EZH2 silencing in HEK cells reduces ISL1 expression. We show that ezh2−/− knockout (KO) zebrafish larvae display tissues specificity of ISL1 regulation with reduced expression of Isl1 in the pronephric region of zebrafish larvae. In addition, a shorter and malformed nephric duct is observed in ezh2−/− ko zebrafish Tg(wt1ß:eGFP) reporter lines. Our study shows, that Ezh2 is a key regulator of Isl1 during urinary tract formation and suggests tissue specific ISL1 dysregulation as an underlying mechanism for CBE formation.

BST2 facilitates activation of hematopoietic stem cells through ERK signaling

The proinflammatory cytokine interferon gamma (IFNγ) is upregulated in a variety of infections and contributes to bone marrow failure through hematopoietic stem cell (HSC) activation and subsequent exhaustion. The cell-surface protein, bone marrow stromal antigen 2 (BST2), is a key mediator of this process, because it is induced upon IFN stimulation and required for IFN-dependent HSC activation. To identify the mechanism by which BST2 promotes IFN-dependent HSC activation, we evaluated its role in niche localization, immune cell function, lipid raft formation, and intracellular signaling. Our studies indicated that knockout (KO) of BST2 in a murine model does not disrupt immune cell responses to IFN-inducing mycobacterial infection. Furthermore, intravital imaging studies indicate that BST2 KO does not disrupt localization of HSCs relative to endothelial or osteoblastic niches in the bone marrow. However, using imaging-based flow cytometry, we found that IFNγ treatment shifts the lipid raft polarity of wild-type (WT) but not Bst2-/- hematopoietic stem and progenitor cells (HSPCs). Furthermore, RNAseq analysis, reverse-phase protein array and western blot analysis of HSPCs indicate that BST2 promotes ERK1/2 phosphorylation during IFNγ-mediated stress. Overall, we find that BST2 facilitates HSC division by promoting cell polarization and ERK activation, thus elucidating a key mechanism of IFN-dependent HSPC activation. These findings inform future approaches in the treatment of cancer and bone marrow failure.

Interplay between the SAFE and the sphingolipid pathway for cardioprotection

AimActivation of both the Survivor Activating Factor Enhancement (SAFE) pathway (including Tumor Necrosis Factor-alpha (TNF-α) and Signal Transducer and Activator of Transcription-3 (STAT-3)) and the sphingolipid signalling pathway (including sphingosine kinase-1 (SK1) and sphingosine-1 phosphate (S1P)) play a key role in promoting cardioprotection against ischemia-reperfusion injury (IRI). We investigated whether the activation of the SAFE pathway by exogenous S1P is dependent on the activation of SK1 for cardioprotection. Materials and methodsIsolated cardiomyocytes from TNF-α knockout (KO) mice, cardiomyocyte-specific STAT-3 KO mice and their wild-type (WT) littermates were exposed to simulated ischemia in the presence of a trigger of the SAFE pathway (S1P) and SK1 inhibitor (SK1-I). Similarly, isolated perfused hearts from adult TNF-α KO, STAT-3 KO and WT mice were subjected to IRI with S1P and/or SK1-I. Cell viability, infarct size (IS) and SK1 activity were assessed. Key findingsIn isolated cardiomyocytes and in isolated hearts subjected to simulated ischemia/IRI, S1P pretreatment decreased cell death in WT mice, an effect that was abrogated in the presence of SK1-I. S1P failed to reduce cell death after simulated ischemia/IRI in both cardiomyocytes or hearts isolated from TNF-α KO and STAT-3 KO mice. Interestingly, S1P pretreatment increased SK1 activity in WT and STAT-3 KO mice, with no changes in TNF-α KO mice. SignificanceOur data strongly suggest SK1 as a key component to activate STAT-3 downstream of TNF-α in the SAFE pathway, paving the way for the development of novel cardioprotective strategies that may target SK1 to modulate the SAFE pathway and increase cell survival following IRI.

Gut microbiota of miR-30a-5p-deleted mice aggravate high-fat diet-induced hepatic steatosis by regulating arachidonic acid metabolic pathway.

Patients with non-alcoholic fatty liver disease (NAFLD) often exhibit hepatic steatosis and dyslipidemia. Studies have shown that intestinal microorganisms are closely related to the occurrence of NAFLD and atherosclerosis. Our previous study has underscored the protective role of microRNA-30a-5p (miR-30a-5p) against atherosclerosis. In the present study, we aimed to elucidate the effect and underlying mechanism of the intestinal microorganisms of miR-30a-5p knockout (KO) mice on NAFLD. Our findings demonstrated that KO exacerbated high-fat diet (HFD)-induced hepatic steatosis and disrupted liver function, as evidenced by elevated levels of total cholesterol, low-density lipoprotein, alanine aminotransferase, aspartate transaminase, and total bile acids in serum. Fecal microbiota from HFD-fed KO mice induced hepatic steatosis, dyslipidemia, and higher levels of enzymes indicative of liver damage in wild-type mice. Remarkably, KO mice significantly intensified the above effects. 16s rDNA sequencing and metabolomics of the intestinal microbiota in the HFD-treated KO and WT mice showed that the loss of miR-30a-5p resulted in intestinal microbiota imbalance and was highly related to the arachidonic acid metabolic pathway. Targeted metabolomic in the liver tissues unveiled upregulation of COX-related (PGF2a, 8-iso-PGF2a and PGF2) and LOX-related (LTB4, LTD4, 12S-HETE and 15S-HETE) factors in HFD-treated KO mice. Immunohistochemistry and transcriptional analyses showed that miR-30a-5p affected arachidonic acid metabolism through the LOX/COX pathways. Besides, COX/LOX pathways and hepatic steatosis were reversed after reintroducing miR-30a-5p in HFD-treated KO mice. This study reveals the pivotal mechanism by which miR-30a-5p and intestinal microbes regulate hepatic steatosis and abnormal lipid metabolism, offering promising avenues for NAFLD and atherosclerosis therapeutics. MiR-30a-5p deletion aggravated hepatic steatosis and lipid disorder induced by an HFD in mice. Gut microbiota participated in the regulation of hepatic steatosis in the context of miR-30a-5p. Gut microbiota metabolism-related arachidonic acid metabolic pathway contributed to miR-30a-5p-regulated hepatic steatosis and lipid disorder. Reintroducing miR-30a-5p reversed hepatic steatosis and arachidonic acid metabolism disorder caused by HFD and miR-30a-5p deletion.

ZnR/GPR39 regulates hepatic insulin signaling, tunes liver bioenergetics and ROS production, and mitigates liver fibrosis and injury

Adequate supply of zinc is essential for hepatic function and its deficiency is associated with acute liver injury (ALI) and chronic nonalcoholic fatty liver disease (NAFLD). However, how zinc controls hepatic function is unknown. We found that the zinc sensitive ZnR/GPR39, a mediator of zinc signaling, enhances hepatic phosphorylation of ERK1/2, which is reduced in ZnR/GPR39 deficient livers. Surprisingly, livers from ZnR/GPR39 knockout (KO) mice exhibited elevated insulin receptor expression and downstream AKT activation. Moreover, ZnR/GPR39 KO mice had higher blood fasting glucose level, pronounced hepatic lipid accumulation, increased hepatocyte oxygen consumption rate (OCR) and reactive oxygen species (ROS) levels. These data suggest that ZnR/GPR39 modulates insulin receptor signaling, a major pathway in hepatic metabolism. Associated with the impaired signaling, ZnR/GPR39 KO livers exhibited increased tissue fibrosis, manifested by marked elevation of collagen expression, compared to wildtype (WT). Additionally, we found alteration of hepatocyte junctional proteins that was accompanied by increased macrophage infiltration and higher liver inflammation in ZnR/GPR39 KO mice. To determine the role of ZnR/GPR39 in ALI, we applied a mild LPS challenge that induced profound decrease in hepatic OCR, also leading to higher ROS generation in ZnR/GPR39 KO hepatocytes, but not in WT. We further found increased serum IL-2 and AST/ALT ratio only in ZnR/GPR39 KO mice. Our findings reveal a role of ZnR/GPR39 in controlling hepatic insulin receptor signaling and mitigating liver fibrosis and inflammation, thus underscoring the important role of ZnR/GPR39 in liver signaling and function.

RFC2 may contribute to the pathogenicity of Williams syndrome revealed in a zebrafish model

Williams syndrome (WS) is a rare multisystemic disorder caused by recurrent microdeletions on 7q11.23, characterized by intellectual disability, distinctive craniofacial and dental features, and cardiovascular problems. Previous studies have explored the roles of individual genes within these microdeletions in contributing to WS phenotypes. Here, we report five patients with WS with 1.4 Mb–1.5 Mb microdeletions that include RFC2, as well as one patient with a 167 kb microdeletion involving RFC2 and six patients with intragenic variants within RFC2. To investigate the potential involvement of RFC2 in WS pathogenicity, we generate a rfc2 knockout (KO) zebrafish using CRISPR-Cas9 technology. Additionally, we generate a KO zebrafish of its paralog gene, rfc5, to better understand the functions of these RFC genes in development and disease. Both rfc2 and rfc5 KO zebrafish exhibit similar phenotypes reminiscent of WS, including small head and brain, jaw and dental defects, and vascular problems. RNA-seq analysis reveals that genes associated with neural cell survival and differentiation are specifically affected in rfc2 KO zebrafish. In addition, heterozygous rfc2 KO adult zebrafish demonstrate an anxiety-like behavior with increased social cohesion. These results suggest that RFC2 may contribute to the pathogenicity of Williams syndrome, as evidenced by the zebrafish model.

Autophagy deficiency exacerbated hypoxia-reoxygenation induced inflammation and cell death via a mitochondrial DNA/STING/IRF3 pathway

AimsAutophagy is an important cellular process for maintaining physiological homeostasis and is known to protect against cardiovascular diseases including ischemia reperfusion (I/R) injury. The underlying mechanisms behind its protection require further characterization. Materials and methodsAtg7 knock out (AKO) mice were generated and subjected to I/R injury, complemented by Atg7 KO in a H9c2 cardiomyoblast cellular model ± hypoxia-reoxygenation. Subsequently, in both models, inflammation and cell death were studied. Key findingsWe confirmed that Atg7 KO led to autophagy, including mitophagy, deficiency. Upon H/R, Atg7 KO cells exhibited increased cell death compared to WT cells. Notably, we found that autophagy deficiency increased stress-induced mitochondrial fission, release of mitochondrial DNA, and sterile inflammation, namely activation of a STING/IRF3 axis leading to elevated interferon-α. Following I/R injury, AKO mice showed elevated cell death which correlated with a gene expression profile indicative of decreased anti-inflammatory responses. SignificanceAutophagy deficiency in the cardiomyocyte setting results in detrimental effects during I/R injury in mice or H/R injury in cells, mediated in part via mtDNA/IRF3/STING pathway. As such, modulation of this pathway may yield novel and promising therapeutics to treat or prevent I/R injury.

TPC1 regulates melanoma tumourigenesis via mTORC1 and TFEB

The major cause of death in cancer patients is a combination of metastatic dissemination combined with therapy resistance. Over recent years, intratumour phenotypic heterogeneity arising from the bi-directional interplay between plastic cancer cells and the microenvironment has been identified as key to disease progression. Most notably metastatic outgrowth and resistance to targeted therapies are frequently associated with activity of mTORC1, a key metabolic hub that promotes protein synthesis and proliferation in the presence of nutrients. Yet while the regulation of mTORC1 by amino acids and glucose availability is well characterized, whether other mechanisms are important in controlling mTORC1 and its downstream signalling is less well understood. Here we show, using the murine B16-F0 melanoma cell line as a model, that mTORC1 activity is decreased following the knockout (KO) of TPC1, a cation channel localised to early and recycling endosomes. Consequently, TPC1 KO melanoma cells exhibit reduced proliferation and invasiveness, as well as increased pigmentation associated with nuclear localisation of the MITF-related transcription factor TFEB. Our results demonstrate that the knockout of TPC1 has induced significant tumour-suppressive effects in melanoma, during which the altered activity of mTORC1 and TFEB play the key roles. The results help us further understand the link between mTORC1 and endolysosomal ion channels, and reveal that TPC1 controls melanoma progression and represents a potential therapeutic target.

The critical role of Gαi3 in oral squamous cell carcinoma cell growth.

The identification of novel and effective therapeutic targets for oral squamous cell carcinoma (OSCC) is of paramount importance. This study investigates the expression, potential functions, and mechanistic insights of G protein inhibitory subunit 3 (Gαi3) in OSCC. Gαi3 is found to be upregulated in human OSCC tissues as well as in various primary and established OSCC cells. In different OSCC cells, silencing of Gαi3 through shRNA resulted in inhibited cell proliferation and migration, while also inducing apoptosis. Knockout (KO) of Gαi3 via the CRISPR/Cas9 method produced significant anti-cancer effects in OSCC cells. Conversely, ectopic overexpression of Gαi3 enhanced OSCC cell growth, promoting cell proliferation and migration. Gαi3 plays a crucial role in activating the Akt-mTOR signaling pathway in OSCC cells. Silencing or KO of Gαi3 led to decreased phosphorylation levels of Akt and S6K, whereas overexpression of Gαi3 increased their phosphorylation. Restoration of Akt-mTOR activation through a constitutively active mutant Akt1 mitigated the anti-OSCC effects induced by Gαi3 shRNA. In vivo, Gαi3 silencing significantly suppressed the growth of subcutaneous OSCC xenografts in nude mice, concomitant with inactivation of the Akt-mTOR pathway and induction of apoptosis. Collectively, these findings underscore the critical role of Gαi3 in OSCC cell growth both in vitro and in vivo.

SARS-CoV-2 spike S1 protein induces microglial NLRP3-dependent neuroinflammation and cognitive impairment in mice

Cognitive impairment is often found at the acute stages and sequelae of coronavirus disease 2019 (COVID-19), and the underlying mechanisms remain unclear. The S1 protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might be a cause of cognitive impairment associated with COVID-19. The nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) inflammasome and neuroinflammation play important roles in Alzheimer's disease (AD) with cognitive impairment. However, their roles remain unknown in COVID-19 with cognitive impairment. We stimulated BV2 cells with S1 protein in vitro and injected the hippocampi of wild-type (WT) mice, NLRP3 knockout (KO), and microglia NLRP3 KO mice in vivo with S1 protein to induce cognitive impairment. We assessed exploratory behavior as associative memory using novel object recognition and Morris water maze tests. Neuroinflammation was analyzed using immunofluorescence and western blotting to detect inflammatory markers. Co-localized NLRP3 and S1 proteins were investigated using confocal microscopy. We found that S1 protein injection led to cognitive impairment, neuronal loss, and neuroinflammation by activating NLRP3 inflammation, and this was reduced by global NLRP3 KO and microglia NLRP3 KO. Furthermore, TAK 242, a specific inhibitor of Toll-like receptor-4, resulted in a significant reduction in NLRP3 and pro-IL-1β in BV2 cells with S1 protein stimulation. These results reveal a distinct mechanism through which the SARS-CoV-2 spike S1 protein promotes NLRP3 inflammasome activation and induces excessive inflammatory responses.

NudCL2 is required for cytokinesis by stabilizing RCC2 with Hsp90 at the midbody.

Cytokinesis is required for faithful division of cytoplasmic components and duplicated nuclei into two daughter cells. Midbody, a protein-dense organelle that forms at the intercellular bridge, is indispensable for successful cytokinesis. However, the regulatory mechanism of cytokinesis at the midbody still remains elusive. Here, we unveil a critical role for NudC-like protein 2 (NudCL2), a co-chaperone of heat shock protein 90 (Hsp90), in cytokinesis regulation by stabilizing regulator of chromosome condensation 2 (RCC2) at the midbody in mammalian cells. NudCL2 localizes at the midbody, and its downregulation results in cytokinesis failure, multinucleation, and midbody disorganization. Using iTRAQ-based quantitative proteomic analysis, we find that RCC2 levels are decreased in NudCL2 knockout (KO) cells. Moreover, Hsp90 forms a complex with NudCL2 to stabilize RCC2, which is essential for cytokinesis. RCC2 depletion mirrors phenotypes observed in NudCL2-downregulated cells. Importantly, ectopic expression of RCC2 rescues the cytokinesis defects induced by NudCL2 deletion, but not vice versa. Together, our data reveal the significance of the NudCL2/Hsp90/RCC2 pathway in cytokinesis at the midbody.

Systems biology approach for enhancing limonene yield by re-engineering Escherichia coli

Engineered microorganisms have emerged as viable alternatives for limonene production. However, issues such as low enzyme abundance or activities, and regulatory feedback/forward inhibition may reduce yields. To understand the underlying metabolism, we adopted a systems biology approach for an engineered limonene-producing Escherichia coli strain K-12 MG1655. Firstly, we generated time-series metabolomics data and, secondly, developed a dynamic model based on enzyme dynamics to track the native metabolic networks and the engineered mevalonate pathway. After several iterations of model fitting with experimental profiles, which also included 13C-tracer studies, we performed in silico knockouts (KOs) of all enzymes to identify bottleneck(s) for optimal limonene yields. The simulations indicated that ALDH/ADH (aldehyde dehydrogenase/alcohol dehydrogenase) and LDH (lactate dehydrogenase) suppression, and HK (hexokinase) enhancement would increase limonene yields. Experimental confirmation was achieved, where ALDH-ADH and LDH KOs, and HK overexpression improved limonene yield by 8- to 11-fold. Our systems biology approach can guide microbial strain re-engineering for optimal target production.

The TMEM132B-GABAA receptor complex controls alcohol actions in the brain.

Alcohol is the most consumed and abused psychoactive drug globally, but the molecular mechanisms driving alcohol action and its associated behaviors in the brain remain enigmatic. Here, we have discovered a transmembrane protein TMEM132B that is a GABAA receptor (GABAAR) auxiliary subunit. Functionally, TMEM132B promotes GABAAR expression at the cell surface, slows receptor deactivation, and enhances the allosteric effects of alcohol on the receptor. In TMEM132B knockout (KO) mice or TMEM132B I499A knockin (KI) mice in which the TMEM132B-GABAAR interaction is specifically abolished, GABAergic transmission is decreased and alcohol-induced potentiation of GABAAR-mediated currents is diminished in hippocampal neurons. Behaviorally, the anxiolytic and sedative/hypnotic effects of alcohol are markedly reduced, and compulsive, binge-like alcohol consumption is significantly increased. Taken together, these data reveal a GABAAR auxiliary subunit, identify the TMEM132B-GABAAR complex as a major alcohol target in the brain, and provide mechanistic insights into alcohol-related behaviors.

Ndufs4 knockout mice with isolated complex I deficiency engage a futile adaptive brain response

Paediatric Leigh syndrome (LS) is an early-onset and fatal neurodegenerative disorder lacking treatment options. LS is frequently caused by mutations in the NDUFS4 gene, encoding an accessory subunit of mitochondrial complex I (CI), the first complex of the oxidative phosphorylation (OXPHOS) system. Whole-body Ndufs4 knockout (KO) mice (WB-KO mice) are widely used to study isolated CI deficiency, LS pathology and interventions. These animals develop a brain-specific phenotype via an incompletely understood pathomechanism. Here we performed a quantitative analysis of the sub-brain proteome in six-weeks old WB-KO mice vs. wildtype mice. Brain regions comprised of a brain slice (BrSl), cerebellum (CB), cerebral cortex (CC), hippocampus (HC), inferior colliculus (IC), and superior colliculus (SC). Proteome analysis demonstrated similarities between CC/HC, and between IC/SC, whereas BrSl and CB differed from these two groups and each other. All brain regions displayed greatly reduced levels of two CI structural subunits (NDUFS4, NDUFA12) and an increased level of the CI assembly factor NDUFAF2. The level of CI-Q module subunits was significantly more reduced in IC/SC than in BrSl/CB/CC/HC, whereas other OXPHOS complex levels were not reduced. Gene ontology and pathway analysis demonstrated specific and common proteome changes between brain regions.Across brain regions, upregulation of cold-shock-associated proteins, mitochondrial fatty acid (FA) oxidation and synthesis (mtFAS) were the most prominent. FA-related pathways were predominantly upregulated in CB and HC. Based upon these results, we argue that stimulation of these pathways is futile and pro-pathological and discuss alternative strategies for therapeutic intervention in LS. SignificanceThe Ndufs4 knockout mouse model is currently the most relevant and most widely used animal model to study the brain-linked pathophysiology of human Leigh Syndrome (LS) and intervention strategies. We demonstrate that the Ndufs4 knockout brain engages futile and pro-pathological responses. These responses explain both negative and positive outcomes of intervention studies in Leigh Syndrome mice and patients, thereby guiding novel intervention opportunities.