Seipin Deficiency as a Model of Severe Adipocyte Dysfunction: Lessons from Rodent Models and Teaching for Human Disease

Obesity prevalence is increasing worldwide, leading to cardiometabolic morbidities. Adipocyte dysfunction, impairing white adipose tissue (WAT) expandability and metabolic flexibility, is central in the development of obesity-related metabolic complications. Rare syndromes of lipodystrophy characterized by an extreme paucity of functional adipose tissue should be considered as primary adipocyte dysfunction diseases. Berardinelli-Seip congenital lipodystrophy (BSCL) is the most severe form with a near absence of WAT associated with cardiometabolic complications such as insulin resistance, liver steatosis, dyslipidemia, and cardiomyopathy. Twenty years ago, mutations in the BSCL2 gene have been identified as the cause of BSCL in human. BSCL2 encodes seipin, an endoplasmic reticulum (ER) anchored protein whose function was unknown back then. Studies of seipin knockout mice or rats demonstrated how seipin deficiency leads to severe lipodystrophy and to cardiometabolic complications. At the cellular levels, seipin is organized in multimers that are particularly enriched at ER/lipid droplet and ER/mitochondria contact sites. Seipin deficiency impairs both adipocyte differentiation and mature adipocyte maintenance. Experiments using adipose tissue transplantation in seipin knockout mice and tissue-specific deletion of seipin have provided a large body of evidence that liver steatosis, cardiomyopathy, and renal injury, classical diabetic complications, are all consequences of lipodystrophy. Rare adipocyte dysfunctions such as in BSCL are the key paradigm to unravel the pathways that control adipocyte homeostasis. The knowledge gathered through the study of these pathologies may bring new strategies to maintain and improve adipose tissue expandability.

Lyz2-Cre-Mediated Genetic Deletion of Septin7 Reveals a Role of Septins in Macrophage Cytokinesis and Kras-Driven Tumorigenesis

By crossing septin7-floxed mice with Lyz2-Cre mice carrying the Cre recombinase inserted in the Lysozyme-M (Lyz2) gene locus we aimed the specific deletion of septin7 in myeloid cells, such as monocytes, macrophages and granulocytes. Septin7flox/flox:Lyz2-Cre mice show no alterations in the myeloid compartment. Septin7-deleted macrophages (BMDMs) were isolated and analyzed. The lack of Septin7 expression was confirmed and a constitutive double-nucleation was detected in Septin7-deficient BMDMs indicating a defect in macrophage cytokinesis. However, phagocytic function of macrophages as judged by uptake of labelled E. coli particles and LPS-stimulated macrophage activation as judged by induction of TNF mRNA expression and TNF secretion were not compromised. In addition to myeloid cells, Lyz2-Cre is also active in type II pneumocytes (AT2 cells). We monitored lung adenocarcinoma formation in these mice by crossing them with the conditional knock-in Kras-LSL-G12D allele. Interestingly, we found that control mice without septin7 depletion die after 3–5 weeks, while the Septin7-deficient animals survived 11 weeks or even longer. Control mice sacrificed in the age of 4 weeks display a bronchiolo-alveolar hyperplasia with multiple adenomas, whereas the Septin7-deficient animals of the same age are normal or show only a weak multifocal brochiolo-alveolar hyperplasia. Our findings indicate an essential role of Septin7 in macrophage cytokinesis but not in macrophage function. Furthermore, septin7 seems absolutely essential for oncogenic Kras-driven lung tumorigenesis making it a potential target for anti-tumor interventions.

Altered Epithelial-mesenchymal Plasticity as a Result of Ovol2 Deletion Minimally Impacts the Self-renewal of Adult Mammary Basal Epithelial Cells

Stem-cell containing mammary basal epithelial cells exist in a quasi-mesenchymal transcriptional state characterized by simultaneous expression of typical epithelial genes and typical mesenchymal genes. Whether robust maintenance of such a transcriptional state is required for adult basal stem cells to fuel self-renewal and regeneration remains unclear. In this work, we utilized SMA-CreER to direct efficient basal cell-specific deletion of Ovol2, which encodes a transcription factor that inhibits epithelial-to-mesenchymal transition (EMT), in adult mammary gland. We identified a basal cell-intrinsic role of Ovol2 in promoting epithelial, and suppressing mesenchymal, molecular traits. Interestingly, Ovol2-deficient basal cells display minimal perturbations in their ability to support tissue homeostasis, colony formation, and transplant outgrowth. These findings underscore the ability of adult mammary basal cells to tolerate molecular perturbations associated with altered epithelia-mesenchymal plasticity without drastically compromising their self-renewal potential.

The methyltransferase METTL3 negatively regulates nonalcoholic steatohepatitis (NASH) progression

Nonalcoholic steatohepatitis (NASH) is a key step in the progression of nonalcoholic fatty liver (NAFL) to cirrhosis. However, the molecular mechanisms of the NAFL-to-NASH transition are largely unknown. Here, we identify methyltransferase like 3 (METTL3) as a key negative regulator of NASH pathogenesis. Hepatocyte-specific deletion of Mettl3 drives NAFL-to-NASH progression by increasing CD36-mediated hepatic free fatty acid uptake and CCL2-induced inflammation, which is due to increased chromatin accessibility in the promoter region of Cd36 and Ccl2. Antibody blockade of CD36 and CCL2 ameliorates NASH progression in hepatic Mettl3 knockout mice. Hepatic overexpression of Mettl3 protects against NASH progression by inhibiting the expression of CD36 and CCL2. Mechanistically, METTL3 directly binds to the promoters of the Cd36 and Ccl2 genes and recruits HDAC1/2 to induce deacetylation of H3K9 and H3K27 in their promoters, thus suppressing Cd36 and Ccl2 transcription. Furthermore, METTL3 is translocated from the nucleus to the cytosol in NASH, which is associated with CDK9-mediated phosphorylation of METTL3. Our data reveal a mechanism by which METTL3 negatively regulates hepatic Cd36 and Ccl2 gene transcription via a histone modification pathway for protection against NASH progression.

RNA Binding Protein SRSF3 confers an essential role in megakaryocyte maturation and platelet production

RNA processing is increasingly recognised as a critical control point in the regulation of different haematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that Serine-arginine rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterised by megakaryocyte maturation arrest, dramatically reduced platelet counts and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.

Dopamine neuron morphology and output are differentially controlled by mTORC1 and mTORC2

The mTOR pathway is an essential regulator of cell growth and metabolism. Midbrain dopamine neurons are particularly sensitive to mTOR signaling status as activation or inhibition of mTOR alters their morphology and physiology. mTOR exists in two distinct multiprotein complexes termed mTORC1 and mTORC2. How each of these complexes affect dopamine neuron properties and whether they act together or independently is unknown. Here we investigated this in mice with dopamine neuron-specific deletion of Rptor or Rictor, which encode obligatory components of mTORC1 or mTORC2, respectively. We find that inhibition of mTORC1 strongly and broadly impacts dopamine neuron structure and function causing somatodendritic and axonal hypotrophy, increased intrinsic excitability, decreased dopamine production, and impaired dopamine release. In contrast, inhibition of mTORC2 has more subtle effects, with selective alterations to the output of ventral tegmental area dopamine neurons. As mTOR is involved in several brain disorders caused by dopaminergic dysregulation including Parkinson's disease and addiction, our results have implications for understanding the pathophysiology and potential therapeutic strategies for these diseases.

Development of Notch1 Positive T-Lineage Lymphomas or Splenic Marginal Zone Lymphomas with Pan-Hematopoietic or Pro-B Cell Specific Deletion of Trp53 with Distinct Differentially Dysregulated Pathways

Deletion of the tumor suppressor gene TP53 (Trp53 in mice) has been associated with the development of numerous human malignancies. TP53 acts as a central coordinator of the DNA damage response. In mice, pan-Trp53 deletion leads predominantly to the development of T-cell lymphomas, followed by B-cell lymphomas, sarcomas and teratomas. In order to dissect the role of Trp53 in the hematopoietic system, we created two different loss of function mouse models: Pan-hematopoietic Trp53 deletion using Vav1-Cre based deletion; and a B-cell-specific deletion was created using CD19-Cre. Vav1-p53CKO mice developed hematolymphoid malignancies with 100% penetrance by 12 months. Most malignancies observed were CD3e+ T-lineage lymphomas involving the thymus or spleen (37/45). Beyond 200 days, these mice predominantly developed mixed myeloid malignancies. The shift away from T-lineage malignancies in older mice may reflect aging-related decline of pre-malignant lymphoid progenitors and skewing to myeloid progenitors. Flow cytometric characterization of the T-lineage lymphomas identified a mix of tumors, including double-negative (CD4-CD8-), double-positive (CD4+CD8+), or single positive (CD4/CD8). In pre-malignant mice, Vav1-p53CKO thymocytes showed accelerated maturation with most of the cells in the DN4 stage, suggesting a bypass of the p53-dependent DN3 β-selection checkpoint. All T-lineage lymphomas showed overexpression of surface Notch1 as well as overexpression of Notch1 targets Hes1 and p21 at the transcript level. Consistent with prior data, normal murine thymocyte subsets showed high levels of expression of Notch1 target genes at the DN3 stage of development, which appears dysregulated in these T-lineage lymphomas. This Notch1 activation was found to be multifactorial with increased Mdm2 and decreased Numb levels seen in tumors. Overall, we demonstrate Notch1 activation and subsequent acceleration through the T-cell developmental stages in this model of pan-hematopoietic Trp53 deletion. The B-cell specific Trp53 knockout mice (CD19-P53CKO) (n=54) were followed up for up to 2 years. The majority (47/54) developed splenomegaly in an age-dependent manner. Histologic examination showed marginal zone expansion (6/54), frank low-grade marginal zone lymphoma (16/54) or diffuse splenic lymphoma (25/54). The disease was confined to the spleen in the case of lower-grade histology while higher grades correlated with liver and kidney involvement. Flow cytometric analysis of tumors showed B220+ CD19+ IgM+ cells. Interestingly, these tumors demonstrated low levels of Notch2 expression, which normally is highly expressed in marginal zone B-cells. In order to characterize pathogenesis, we sorted follicular and marginal zone B-cells from floxed P53 and pre-malignant CD19-P53CKO mice. RNA was isolated from all these fractions and the spleens of 5 CD19-P53KO mice with diffuse lymphoma and subjected to RNA-Seq. A comparison of the floxed p53 with the CD19-P53CKO fractions (follicular and marginal zone) revealed a highly similar transcriptome. On the other hand, p53-deficient lymphomas showed >10,000 genes significantly differentially expressed demonstrating the unique transcriptome which developed during malignant transformation. Pathway analysis of these genes using Gene Set Enrichment Analysis (GSEA) identified enrichment of PI3K, Rap1 and MAPK signaling pathways, which are associated with cellular proliferation. Overexpression of the PI3K pathway genes Ccne1, Sgk1, Mapk13 and Pik3cb were validated by qPCR in 10 independent tumor samples when compared to the splenic marginal zone fractions. In the B-cell lineage, Trp53 deficiency leads to the dysregulation of multiple genes involved in key cellular signaling pathways, including the PI3K/MAPK pathway. In summary, pan hematopoietic deletion of Trp53 led to T-lineage lymphoma in young mice and myeloid tumors in older mice; with activation of Notch1 signaling in the former. B-cell specific deletion of Trp53 led to splenic marginal zone and diffuse B-cell lymphoma with transcriptional dysregulation of key signaling molecules. Hence, tumorigenesis by Trp53 deletion is tightly linked to lineage and appears to dysregulate key signaling pathways that are operant in those lineages, potentially identifying novel strategies for therapeutic interventions in P53 dependent human hematolymphoid malignancies. Disclosures No relevant conflicts of interest to declare.

Tet1 Regulates Astrocyte Development and Cognition of Mice Through Modulating GluA1

Tet (Ten eleven translocation) family proteins-mediated 5-hydroxymethylcytosine (5hmC) is highly enriched in the neuronal system, and is involved in diverse biological processes and diseases. However, the function of 5hmC in astrocyte remains completely unknown. In the present study, we show that Tet1 deficiency alters astrocyte morphology and impairs neuronal function. Specific deletion of Tet1 in astrocyte impairs learning and memory ability of mice. Using 5hmC high-throughput DNA sequencing and RNA sequencing, we present the distribution of 5hmC among genomic features in astrocyte and show that Tet1 deficiency induces differentially hydroxymethylated regions (DhMRs) and alters gene expression. Mechanistically, we found that Tet1 deficiency leads to the abnormal Ca2+ signaling by regulating the expression of GluA1, which can be rescued by ectopic GluA1. Collectively, our findings suggest that Tet1 plays important function in astrocyte physiology by regulating Ca2+ signaling.