Local hyperthermia therapy induces browning of white fat and treats obesity.

Beige fat plays key roles in the regulation of systemic energy homeostasis; however, detailed mechanisms and safe strategy for its activation remain elusive. In this study, we discovered that local hyperthermia therapy (LHT) targeting beige fat promoted its activation in humans and mice. LHT achieved using a hydrogel-based photothermal therapy activated beige fat, preventing and treating obesity in mice without adverse effects. HSF1 is required for the effects since HSF1 deficiency blunted the metabolic benefits of LHT. HSF1 regulates Hnrnpa2b1 (A2b1) transcription, leading to increased mRNA stability of key metabolic genes. Importantly, analysis of human association studies followed by functional analysis revealed that the HSF1 gain-of-function variant p.P365T is associated with improved metabolic performance in humans and increased A2b1 transcription in mice and cells. Overall, we demonstrate that LHT offers a promising strategy against obesity by inducing beige fat activation via HSF1-A2B1 transcriptional axis.

Ring domains are essential for GATOR2-dependent mTORC1 activation.

The GATOR2-GATOR1 signaling axis is essential for amino-acid-dependent mTORC1 activation. However, the molecular function of the GATOR2 complex remains unknown. Here, we report that disruption of the Ring domains of Mios, WDR24, or WDR59 completely impedes amino-acid-mediated mTORC1 activation. Mechanistically, via interacting with Ring domains of WDR59 and WDR24, the Ring domain of Mios acts as a hub to maintain GATOR2 integrity, disruption of which leads to self-ubiquitination of WDR24. Physiologically, leucine stimulation dissociates Sestrin2 from the Ring domain of WDR24 and confers its availability to UBE2D3 and subsequent ubiquitination of NPRL2, contributing to GATOR2-mediated GATOR1 inactivation. As such, WDR24 ablation or Ring deletion prevents mTORC1 activation, leading to severe growth defects and embryonic lethality at E10.5 in mice. Hence, our findings demonstrate that Ring domains are essential for GATOR2 to transmit amino acid availability to mTORC1 and further reveal the essentiality of nutrient sensing during embryonic development.

Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL.

Recently, chimeric antigen receptor (CAR)-T cell therapy has shown great promise in treating haematological malignancies1-7. However, CAR-T cell therapy currently has several limitations8-12. Here we successfully developed a two-in-one approach to generate non-viral, gene-specific targeted CAR-T cells through CRISPR-Cas9. Using the optimized protocol, we demonstrated feasibility in a preclinical study by inserting an anti-CD19 CAR cassette into the AAVS1 safe-harbour locus. Furthermore, an innovative type of anti-CD19 CAR-T cell with PD1 integration was developed and showed superior ability to eradicate tumour cells in xenograft models. In adoptive therapy for relapsed/refractory aggressive B cell non-Hodgkin lymphoma (ClinicalTrials.gov, NCT04213469 ), we observed a high rate (87.5%) of complete remission and durable responses without serious adverse events in eight patients. Notably, these enhanced CAR-T cells were effective even at a low infusion dose and with a low percentage of CAR+ cells. Single-cell analysis showed that the electroporation method resulted in a high percentage of memory T cells in infusion products, and PD1 interference enhanced anti-tumour immune functions, further validating the advantages of non-viral, PD1-integrated CAR-T cells. Collectively, our results demonstrate the high safety and efficacy of non-viral, gene-specific integrated CAR-T cells, thus providing an innovative technology for CAR-T cell therapy.

SENP1-Sirt3 Signaling Controls Mitochondrial Protein Acetylation and Metabolism.

Sirt3, as a major mitochondrial nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, is required for mitochondrial metabolic adaption to various stresses. However, how to regulate Sirt3 activity responding to metabolic stress remains largely unknown. Here, we report Sirt3 as a SUMOylated protein in mitochondria. SUMOylation suppresses Sirt3 catalytic activity. SUMOylation-deficient Sirt3 shows elevated deacetylation on mitochondrial proteins and increased fatty acid oxidation. During fasting, SUMO-specific protease SENP1 is accumulated in mitochondria and quickly de-SUMOylates and activates Sirt3. SENP1 deficiency results in hyper-SUMOylation of Sirt3 and hyper-acetylation of mitochondrial proteins, which reduces mitochondrial metabolic adaption responding to fasting. Furthermore, we find that fasting induces SENP1 translocation into mitochondria to activate Sirt3. The studies on mice show that Sirt3 SUMOylation mutation reduces fat mass and antagonizes high-fat diet (HFD)-induced obesity via increasing oxidative phosphorylation and energy expenditure. Our results reveal that SENP1-Sirt3 signaling modulates Sirt3 activation and mitochondrial metabolism during metabolic stress.

Dual Antiplatelet Treatment up to 72 Hours after Ischemic Stroke.

BACKGROUND: Dual antiplatelet treatment has been shown to lower the risk of recurrent stroke as compared with aspirin alone when treatment is initiated early (≤24 hours) after an acute mild stroke. The effect of clopidogrel plus aspirin as compared with aspirin alone administered within 72 hours after the onset of acute cerebral ischemia from atherosclerosis has not been well studied. METHODS: In 222 hospitals in China, we conducted a double-blind, randomized, placebo-controlled, two-by-two factorial trial involving patients with mild ischemic stroke or high-risk transient ischemic attack (TIA) of presumed atherosclerotic cause who had not undergone thrombolysis or thrombectomy. Patients were randomly assigned, in a 1:1 ratio, within 72 hours after symptom onset to receive clopidogrel (300 mg on day 1 and 75 mg daily on days 2 to 90) plus aspirin (100 to 300 mg on day 1 and 100 mg daily on days 2 to 21) or matching clopidogrel placebo plus aspirin (100 to 300 mg on day 1 and 100 mg daily on days 2 to 90). There was no interaction between this component of the factorial trial design and a second part that compared immediate with delayed statin treatment (not reported here). The primary efficacy outcome was new stroke, and the primary safety outcome was moderate-to-severe bleeding - both assessed within 90 days. RESULTS: A total of 6100 patients were enrolled, with 3050 assigned to each trial group. TIA was the qualifying event for enrollment in 13.1% of the patients. A total of 12.8% of the patients were assigned to a treatment group no more than 24 hours after stroke onset, and 87.2% were assigned after 24 hours and no more than 72 hours after stroke onset. A new stroke occurred in 222 patients (7.3%) in the clopidogrel-aspirin group and in 279 (9.2%) in the aspirin group (hazard ratio, 0.79; 95% confidence interval [CI], 0.66 to 0.94; P = 0.008). Moderate-to-severe bleeding occurred in 27 patients (0.9%) in the clopidogrel-aspirin group and in 13 (0.4%) in the aspirin group (hazard ratio, 2.08; 95% CI, 1.07 to 4.04; P = 0.03). CONCLUSIONS: Among patients with mild ischemic stroke or high-risk TIA of presumed atherosclerotic cause, combined clopidogrel-aspirin therapy initiated within 72 hours after stroke onset led to a lower risk of new stroke at 90 days than aspirin therapy alone but was associated with a low but higher risk of moderate-to-severe bleeding. (Funded by the National Natural Science Foundation of China and others; INSPIRES ClinicalTrials.gov number, NCT03635749.).

Engineering APOBEC3A deaminase for highly accurate and efficient base editing.

Cytosine base editors (CBEs) are effective tools for introducing C-to-T base conversions, but their clinical applications are limited by off-target and bystander effects. Through structure-guided engineering of human APOBEC3A (A3A) deaminase, we developed highly accurate A3A-CBE (haA3A-CBE) variants that efficiently generate C-to-T conversion with a narrow editing window and near-background level of DNA and RNA off-target activity, irrespective of methylation status and sequence context. The engineered deaminase domains are compatible with PAM-relaxed SpCas9-NG variant, enabling accurate correction of pathogenic mutations in homopolymeric cytosine sites through flexible positioning of the single-guide RNAs. Dual adeno-associated virus delivery of one haA3A-CBE variant to a mouse model of tyrosinemia induced up to 58.1% editing in liver tissues with minimal bystander editing, which was further reduced through single dose of lipid nanoparticle-based messenger RNA delivery of haA3A-CBEs. These results highlight the tremendous promise of haA3A-CBEs for precise genome editing to treat human diseases.

Atf7ip and Setdb1 interaction orchestrates the hematopoietic stem and progenitor cell state with diverse lineage differentiation.

Hematopoietic stem and progenitor cells (HSPCs) are a heterogeneous group of cells with expansion, differentiation, and repopulation capacities. How HSPCs orchestrate the stemness state with diverse lineage differentiation at steady condition or acute stress remains largely unknown. Here, we show that zebrafish mutants that are deficient in an epigenetic regulator Atf7ip or Setdb1 methyltransferase undergo excessive myeloid differentiation with impaired HSPC expansion, manifesting a decline in T cells and erythroid lineage. We find that Atf7ip regulates hematopoiesis through Setdb1-mediated H3K9me3 modification and chromatin remodeling. During hematopoiesis, the interaction of Atf7ip and Setdb1 triggers H3K9me3 depositions in hematopoietic regulatory genes including cebpß and cdkn1a, preventing HSPCs from loss of expansion and premature differentiation into myeloid lineage. Concomitantly, loss of Atf7ip or Setdb1 derepresses retrotransposons that instigate the viral sensor Mda5/Rig-I like receptor (RLR) signaling, leading to stress-driven myelopoiesis and inflammation. We find that ATF7IP or SETDB1 depletion represses human leukemic cell growth and induces myeloid differentiation with retrotransposon-triggered inflammation. These findings establish that Atf7ip/Setdb1-mediated H3K9me3 deposition constitutes a genome-wide checkpoint that impedes the myeloid potential and maintains HSPC stemness for diverse blood cell production, providing unique insights into potential intervention in hematological malignancy.

Allele-Specific Suppression of Variant MHC With High-Precision RNA Nuclease CRISPR-Cas13d Prevents Hypertrophic Cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy has severe clinical complications of heart failure, arrhythmia, and sudden cardiac death. Heterozygous single nucleotide variants (SNVs) of sarcomere genes such as MYH7 are the leading cause of this type of disease. CRISPR-Cas13 (clustered regularly interspaced short palindromic repeats and their associated protein 13) is an emerging gene therapy approach for treating genetic disorders, but its therapeutic potential in genetic cardiomyopathy remains unexplored. METHODS: We developed a sensitive allelic point mutation reporter system to screen the mutagenic variants of Cas13d. On the basis of Cas13d homology structure, we rationally designed a series of Cas13d variants and obtained a high-precision Cas13d variant (hpCas13d) that specifically cleaves the MYH7 variant RNAs containing 1 allelic SNV. We validated the high precision and low collateral cleavage activity of hpCas13d through various in vitro assays. We generated 2 HCM mouse models bearing distinct MYH7 SNVs and used adenovirus-associated virus serotype 9 to deliver hpCas13d specifically to the cardiomyocytes. We performed a large-scale library screening to assess the potency of hpCas13d in resolving 45 human MYH7 allelic pathogenic SNVs. RESULTS: Wild-type Cas13d cannot distinguish and specifically cleave the heterozygous MYH7 allele with SNV. hpCas13d, with 3 amino acid substitutions, had minimized collateral RNase activity and was able to resolve various human MYH7 pathological sequence variations that cause hypertrophic cardiomyopathy. In vivo application of hpCas13d to 2 hypertrophic cardiomyopathy models caused by distinct human MYH7 analogous sequence variations specifically suppressed the altered allele and prevented cardiac hypertrophy. CONCLUSIONS: Our study unveils the great potential of CRISPR-Cas nucleases with high precision in treating inheritable cardiomyopathy and opens a new avenue for therapeutic management of inherited cardiac diseases.

Therapeutic Effects of Hematopoietic Stem Cell Derived From Gene-Edited Mice on ß654-Thalassemia.

ß-thalassemia is an inherited blood disease caused by reduced or inadequate ß-globin synthesis due to ß-globin gene mutation. Our previous study developed a gene-edited mice model (ß654-ER mice) by CRISPR/Cas9-mediated genome editing, targeting both the ßIVS2-654 (C > T) mutation site and the 3' splicing acceptor site at 579 and corrected abnormal ß-globin mRNA splicing in the ß654-thalassemia mice. Herein, we further explored the therapeutic effect of the hematopoietic stem cells (HSCs) from ß654-ER mice on ß-thalassemia by consecutive HSC transplantation. The results indicated that HSC transplantation derived from gene-edited mice can significantly improve the survival rate of mice after lethal radiation doses and effectively achieve hematopoietic reconstruction and long-term hematopoiesis. Clinical symptoms, including hematologic parameters and tissue pathology of transplanted recipients, were significantly improved compared to the non-transplanted ß654 mice. The therapeutic effect of gene-edited HSC transplantation demonstrated no significant difference in hematological parameters and tissue pathology compared with wild-type mouse-derived HSCs. Our data revealed that HSC transplantation from gene-edited mice completely recovered the ß-thalassemia phenotype. Our study systematically investigated the therapeutic effect of HSCs derived from ß654-ER mice on ß-thalassemia and further confirmed the efficacy of our gene-editing approach. Altogether, it provided a reference and primary experimental data for the clinical usage of such gene-edited HSCs in the future.

Engineering a precise adenine base editor with minimal bystander editing.

Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications, but their bystander mutations and off-target editing effects raise safety concerns. Through structure-guided engineering, we found ABE8e with an N108Q mutation reduced both adenine and cytosine bystander editing, and introduction of an additional L145T mutation (ABE9), further refined the editing window to 1-2 nucleotides with eliminated cytosine editing. Importantly, ABE9 induced very minimal RNA and undetectable Cas9-independent DNA off-target effects, which mainly installed desired single A-to-G conversion in mouse and rat embryos to efficiently generate disease models. Moreover, ABE9 accurately edited the A5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites (up to 342.5-fold precision over ABE8e) and was further confirmed through a library of guide RNA-target sequence pairs. Owing to the minimized editing window, ABE9 could further broaden the targeting scope for precise correction of pathogenic single-nucleotide variants when fused to Cas9 variants with expanded protospacer adjacent motif compatibility. bpNLS, bipartite nuclear localization signals.

In vivo base editing rescues primary hyperoxaluria type 1 in rats.

Primary hyperoxaluria type 1 (PH1) is a childhood-onset autosomal recessive disease, characterized by nephrocalcinosis, multiple recurrent urinary calcium oxalate stones, and a high risk of progressive kidney damage. PH1 is caused by inherent genetic defects of the alanine glyoxylate aminotransferase (AGXT) gene. The in vivo repair of disease-causing genes was exceedingly inefficient before the invention of base editors which can efficiently introduce precisely targeted base alterations without double-strand DNA breaks. Adenine base editor (ABE) can precisely convert A·T to G·C with the assistance of specific guide RNA. Here, we demonstrated that systemic delivery of dual adeno-associated virus encoding a split-ABE8e could artificially repair 13% of the pathogenic allele in AgxtQ84X rats, a model of PH1, alleviating the disease phenotype. Specifically, ABE treatment partially restored the expression of alanine-glyoxylate-aminotransferase (AGT), reduced endogenous oxalate synthesis and alleviated calcium oxalate crystal deposition. Western blot and immunohistochemistry confirmed that ABE8e treatment restored AGT protein expression in hepatocytes. Moreover, the precise editing efficiency in the liver remained stable six months after treatment. Thus, our findings provided a prospect of in vivo base editing as a personalized and precise medicine for PH1 by directly correcting the mutant Agxt gene.

Epigenetic inactivation of ERF reactivates γ-globin expression in ß-thalassemia.

The fetal-to-adult hemoglobin switch is regulated in a developmental stage-specific manner and reactivation of fetal hemoglobin (HbF) has therapeutic implications for treatment of ß-thalassemia and sickle cell anemia, two major global health problems. Although significant progress has been made in our understanding of the molecular mechanism of the fetal-to-adult hemoglobin switch, the mechanism of epigenetic regulation of HbF silencing remains to be fully defined. Here, we performed whole-genome bisulfite sequencing and RNA sequencing analysis of the bone marrow-derived GYPA+ erythroid cells from ß-thalassemia-affected individuals with widely varying levels of HbF groups (HbF ≥ 95th percentile or HbF ≤ 5th percentile) to screen epigenetic modulators of HbF and phenotypic diversity of ß-thalassemia. We identified an ETS2 repressor factor encoded by ERF, whose promoter hypermethylation and mRNA downregulation are associated with high HbF levels in ß-thalassemia. We further observed that hypermethylation of the ERF promoter mediated by enrichment of DNMT3A leads to demethylation of γ-globin genes and attenuation of binding of ERF on the HBG promoter and eventually re-activation of HbF in ß-thalassemia. We demonstrated that ERF depletion markedly increased HbF production in human CD34+ erythroid progenitor cells, HUDEP-2 cell lines, and transplanted NCG-Kit-V831M mice. ERF represses γ-globin expression by directly binding to two consensus motifs regulating γ-globin gene expression. Importantly, ERF depletion did not affect maturation of erythroid cells. Identification of alterations in DNA methylation of ERF as a modulator of HbF synthesis opens up therapeutic targets for ß-hemoglobinopathies.

Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies.

Editing efficiency is pivotal for the efficacies of CRISPR-based gene therapies. We found that fusing an HMG-D domain to the N terminus of SpCas9 (named efficiency-enhanced Cas9 [eeCas9]) significantly increased editing efficiency by 1.4-fold on average. The HMG-D domain also enhanced the activities of non-NGG PAM Cas9 variants, high-fidelity Cas9 variants, smaller Cas9 orthologs, Cas9-based epigenetic regulators, and base editors in cell lines. Furthermore, we discovered that eeCas9 exhibits comparable off-targeting effects with Cas9, and its specificity could be increased through ribonucleoprotein delivery or using hairpin single-guide RNAs and high-fidelity Cas9s. The entire eeCas9 could be packaged into an adeno-associated virus vector and exhibited a 1.7- to 2.6-fold increase in editing efficiency targeting the Pcsk9 gene in mice, leading to a greater reduction of serum cholesterol levels. Moreover, the efficiency of eeA3A-BE3 also surpasses that of A3A-BE3 in targeting the promoter region of γ-globin genes or BCL11A enhancer in human hematopoietic stem cells to reactivate γ-globin expression for the treatment of ß-hemoglobinopathy. Together, eeCas9 and its derivatives are promising editing tools that exhibit higher activity and therapeutic efficacy for both in vivo and ex vivo therapeutics.

Preparation of magnetic polyacrylamide hydrogel with chitosan for immobilization of glutamate decarboxylase to produce γ-aminobutyric acid.

Gamma-aminobutyric acid (GABA) is an vital neurotransmitter, and the reaction to obtain GABA through biocatalysis requires coenzymes, which are therefore limited in the production of GABA. In this study, polyacrylamide hydrogels doped with chitosan and waste toner were synthesized for glutamate decarboxylase (GAD) and coenzyme co-immobilization to realize the production of GABA and the recovery of coenzymes. Enzymatic properties of immobilized GAD were discussed. The immobilized enzymes have significantly improved pH and temperature tolerance compared to free enzymes. In terms of reusability, after 10 repeated reuses of the immobilized GAD, the residual enzyme activity of immobilized GAD still retains 100% of the initial enzyme activity, and the immobilized coenzyme can also be kept at about 32%, with better stability and reusability. And under the control of no exogenous pH, immobilized GAD showed good performance in producing GABA. Therefore, in many ways, the new composite hydrogel provides another way for the utilization of waste toner and promises the possibility of industrial production of GABA.

Erk and MAPK signaling is essential for intestinal development through Wnt pathway modulation.

Homeostasis of intestinal stem cells (ISCs) is maintained by the orchestration of niche factors and intrinsic signaling networks. Here, we have found that deletion of Erk1 and Erk2 (Erk1/2) in intestinal epithelial cells at embryonic stages resulted in an unexpected increase in cell proliferation and migration, expansion of ISCs, and formation of polyp-like structures, leading to postnatal death. Deficiency of epithelial Erk1/2 results in defects in secretory cell differentiation as well as impaired mesenchymal cell proliferation and maturation. Deletion of Erk1/2 strongly activated Wnt signaling through both cell-autonomous and non-autonomous mechanisms. In epithelial cells, Erk1/2 depletion resulted in loss of feedback regulation, leading to Ras/Raf cascade activation that transactivated Akt activity to stimulate the mTor and Wnt/ß-catenin pathways. Moreover, Erk1/2 deficiency reduced the levels of Indian hedgehog and the expression of downstream pathway components, including mesenchymal Bmp4 - a Wnt suppressor in intestines. Inhibition of mTor signaling by rapamycin partially rescued Erk1/2 depletion-induced intestinal defects and significantly prolonged the lifespan of mutant mice. These data demonstrate that Erk/Mapk signaling functions as a key modulator of Wnt signaling through coordination of epithelial-mesenchymal interactions during intestinal development.

In vitro machine learning-based CAR T immunological synapse quality measurements correlate with patient clinical outcomes.

The human immune system consists of a highly intelligent network of billions of independent, self-organized cells that interact with each other. Machine learning (ML) is an artificial intelligence (AI) tool that automatically processes huge amounts of image data. Immunotherapies have revolutionized the treatment of blood cancer. Specifically, one such therapy involves engineering immune cells to express chimeric antigen receptors (CAR), which combine tumor antigen specificity with immune cell activation in a single receptor. To improve their efficacy and expand their applicability to solid tumors, scientists optimize different CARs with different modifications. However, predicting and ranking the efficacy of different "off-the-shelf" immune products (e.g., CAR or Bispecific T-cell Engager [BiTE]) and selection of clinical responders are challenging in clinical practice. Meanwhile, identifying the optimal CAR construct for a researcher to further develop a potential clinical application is limited by the current, time-consuming, costly, and labor-intensive conventional tools used to evaluate efficacy. Particularly, more than 30 years of immunological synapse (IS) research data demonstrate that T cell efficacy is not only controlled by the specificity and avidity of the tumor antigen and T cell interaction, but also it depends on a collective process, involving multiple adhesion and regulatory molecules, as well as tumor microenvironment, spatially and temporally organized at the IS formed by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The optimal function of cytotoxic lymphocytes (including CTL and NK) depends on IS quality. Recognizing the inadequacy of conventional tools and the importance of IS in immune cell functions, we investigate a new strategy for assessing CAR-T efficacy by quantifying CAR IS quality using the glass-support planar lipid bilayer system combined with ML-based data analysis. Previous studies in our group show that CAR-T IS quality correlates with antitumor activities in vitro and in vivo. However, current manually quantified IS quality data analysis is time-consuming and labor-intensive with low accuracy, reproducibility, and repeatability. In this study, we develop a novel ML-based method to quantify thousands of CAR cell IS images with enhanced accuracy and speed. Specifically, we used artificial neural networks (ANN) to incorporate object detection into segmentation. The proposed ANN model extracts the most useful information to differentiate different IS datasets. The network output is flexible and produces bounding boxes, instance segmentation, contour outlines (borders), intensities of the borders, and segmentations without borders. Based on requirements, one or a combination of this information is used in statistical analysis. The ML-based automated algorithm quantified CAR-T IS data correlates with the clinical responder and non-responder treated with Kappa-CAR-T cells directly from patients. The results suggest that CAR cell IS quality can be used as a potential composite biomarker and correlates with antitumor activities in patients, which is sufficiently discriminative to further test the CAR IS quality as a clinical biomarker to predict response to CAR immunotherapy in cancer. For translational research, the method developed here can also provide guidelines for designing and optimizing numerous CAR constructs for potential clinical development. Trial Registration: ClinicalTrials.gov NCT00881920.

Lipid Receptor G2A-Mediated Signal Pathway Plays a Critical Role in Inflammatory Response by Promoting Classical Macrophage Activation.

Macrophage polarization is a dynamic and integral process in tissue inflammation and remodeling. In this study, we describe that lipoprotein-associated phospholipase A2 (Lp-PLA2) plays an important role in controlling inflammatory macrophage (M1) polarization in rodent experimental autoimmune encephalomyelitis (EAE) and in monocytes from multiple sclerosis (MS) patients. Specific inhibition of Lp-PLA2 led to an ameliorated EAE via markedly decreased inflammatory and demyelinating property of M1. The effects of Lp-PLA2 on M1 function were mediated by lysophosphatidylcholine, a bioactive product of oxidized lipids hydrolyzed by Lp-PLA2 through JAK2-independent activation of STAT5 and upregulation of IRF5. This process was directed by the G2A receptor, which was only found in differentiated M1 or monocytes from MS patients. M1 polarization could be inhibited by a G2A neutralizing Ab, which led to an inhibited disease in rat EAE. In addition, G2A-deficient rats showed an ameliorated EAE and an inhibited autoimmune response. This study has revealed a mechanism by which lipid metabolites control macrophage activation and function, modification of which could lead to a new therapeutic approach for MS and other inflammatory disorders.

The ubiquitination of rag A GTPase by RNF152 negatively regulates mTORC1 activation.

mTORC1 is essential for regulating cell growth and metabolism in response to various environmental stimuli. Heterodimeric Rag GTPases are required for amino-acid-mediated mTORC1 activation at the lysosome. However, the mechanism by which amino acids regulate Rag activation remains not fully understood. Here, we identified the lysosome-anchored E3 ubiquitin ligase RNF152 as an essential negative regulator of the mTORC1 pathway by targeting RagA for K63-linked ubiquitination. RNF152 interacts with and ubiquitinates RagA in an amino-acid-sensitive manner. The mutation of RagA ubiquitination sites abolishes this effect of RNF152 and enhances the RagA-mediated activation of mTORC1. Ubiquitination by RNF152 generates an anchor on RagA to recruit its inhibitor GATOR1, a GAP complex for Rag GTPases. RNF152 knockout results in the hyperactivation of mTORC1 and protects cells from amino-acid-starvation-induced autophagy. Thus, this study reveals a mechanism for regulation of mTORC1 signaling by RNF152-mediated K63-linked polyubiquitination of RagA.

Efficient photoactivatable Dre recombinase for cell type-specific spatiotemporal control of genome engineering in the mouse.

Precise genetic engineering in specific cell types within an intact organism is intriguing yet challenging, especially in a spatiotemporal manner without the interference caused by chemical inducers. Here we engineered a photoactivatable Dre recombinase based on the identification of an optimal split site and demonstrated that it efficiently regulated transgene expression in mouse tissues spatiotemporally upon blue light illumination. Moreover, through a double-floxed inverted open reading frame strategy, we developed a Cre-activated light-inducible Dre (CALID) system. Taking advantage of well-defined cell-type-specific promoters or a well-established Cre transgenic mouse strain, we demonstrated that the CALID system was able to activate endogenous reporter expression for either bulk or sparse labeling of CaMKIIα-positive excitatory neurons and parvalbumin interneurons in the brain. This flexible and tunable system could be a powerful tool for the dissection and modulation of developmental and genetic complexity in a wide range of biological systems.

GATA-binding protein 4 promotes neuroinflammation and cognitive impairment in Aß1-42 fibril-infused rats through small nucleolar RNA host gene 1/miR-361-3p axis.

Aging with dysregulated metabolic and immune homeostasis stimulates pyroptosis, neuroinflammation, and cellular senescence, thus contributing to etiopathogenesis of Alzheimer's disease. GATA-binding protein 4 (GATA4) functions as a transcriptional factor in response to DNA damage, and is associated with neuroinflammation and cellular senescence. The role of GATA4 in Alzheimer's disease was investigated. GATA4 was elevated in hippocampus of Aß1-42 fibril-infused rats. Injection with shRNA targeting GATA4 reduced escape latency with increase of time in target quadrant and number of platform crossings in Aß1-42 fibril-infused rats. Moreover, knockdown of GATA4 ameliorated morphological changes of hippocampus and reduced amyloid plaque deposition in Aß1-42 fibril-infused rats. Silence of GATA4 repressed neuroinflammation and apoptosis in Aß1-42 fibril-infused rats. Loss of GATA4 in Aß1-42 fibril-infused rats reduced the expression of specificity protein 1 (Sp1) to downregulate long noncoding RNA small nucleolar RNA host gene 1 (SNHG1) and upregulated miR-361-3p. Loss of SNHG1 ameliorated learning and memory impairments in Aß1-42 fibril-infused rats. Overexpression of Sp1 attenuated GATA4 silence-induced decrease of escape latency, increase of time in target quadrant, and number of platform crossings in Aß1-42 fibril-infused rats. In conclusion, silence of GATA4 ameliorated cognitive dysfunction and inhibited hippocampal inflammation and cell apoptosis through regulation of Sp1/SNHG1/miR-361-3p.