Conditional Deletion Research Articles

Kölliker's Organ Functions as a Developmental Hub in Mouse Cochlea regulating spiral limbus and tectorial membrane development.

Kölliker's organ is a transient developmental structure in the mouse cochlea that undergoes significant remodeling postnatally. Utilizing an epithelial-specific conditional deletion mouse model of Prdm16 (marker and regulator of Kölliker's organ), we show that Prdm16 is required for interdental cell development, and thereby the development of the limbal domain of the tectorial membrane and its medial anchorage to the spiral limbus. Additionally, we show that Kölliker's organ is involved in normal tectorial membrane collagen fibril development and maturation. Interestingly, mesenchymal cells of the spiral limbus underneath Prdm16-deficient Kölliker's organ failed to produce interstitial matrix proteins, resulting in a hypoplastic and truncated spiral limbus, indicating a non-cell autonomous role of Prdm16 in regulating spiral mesenchymal matrix development. Single cell RNA sequencing identified differentially expressed genes in Prdm16-deficient Kölliker's organ suggesting a role for connective tissue growth factor (CTGF) downstream Prdm16 in epithelial-mesenchymal signaling involved in spiral limbus matrix deposition. Prdm16-deficient mice showed a hearing deficit, as indicated by elevated auditory brain stem response thresholds at most frequencies, consistent with the cochlear structural defects. Both sexes were studied. This work establishes Prdm16 as a deafness gene in mice through its role in regulating Kölliker's organ development. Such understanding recognizes Kölliker's organ as a developmental hub regulating multiple surrounding cochlear structures.Significance Statement In this study, we show that the Kölliker's organ functions as a developmental hub that orchestrates the development of tectorial membrane and spiral limbus during cochlear development. Utilizing a mouse model of conditional deletion of Prdm16 (marker and regulator of Kölliker's organ), we establish Prdm16's necessity for hearing in mice through its many roles during cochlear development including permitting interdental cell development and thereby the formation of the tectorial membrane limbal domain, secreting collagens essential for tectorial membrane matrix development, and signaling to the underlying mesenchyme to secrete extracellular matrix and develop the spiral limbus.

RUNX2 promotes fibrosis via an alveolar-to-pathological fibroblast transition.

A hallmark of pulmonary fibrosis is the aberrant activation of lung fibroblasts into pathological fibroblasts that produce excessive extracellular matrix1-3. Thus, the identification of key regulators that promote the generation of pathological fibroblasts can inform the development of effective countermeasures against disease progression. Here we use two mouse models of pulmonary fibrosis to show that LEPR+ fibroblasts that arise during alveologenesis include SCUBE2+ alveolar fibroblasts as a major constituent. These alveolar fibroblasts in turn contribute substantially to CTHRC1+POSTN+ pathological fibroblasts. Genetic ablation of POSTN+ pathological fibroblasts attenuates fibrosis. Comprehensive analyses of scRNA-seq and scATAC-seq data reveal that RUNX2 is a key regulator of the expression of fibrotic genes. Consistently, conditional deletion of Runx2 with LeprcreERT2 or Scube2creERT2 reduces the generation of pathological fibroblasts, extracellular matrix deposition and pulmonary fibrosis. Therefore, LEPR+ cells that include SCUBE2+ alveolar fibroblasts are a key source of pathological fibroblasts, and targeting Runx2 provides a potential treatment option for pulmonary fibrosis.

Neuronal cell type specific roles for Nprl2 in neurodevelopmental disorder-relevant behaviors.

Loss of function in the subunits of the GTPase-activating protein (GAP) activity toward Rags-1 (GATOR1) complex, an amino-acid sensitive negative regulator of the mechanistic target of rapamycin complex 1 (mTORC1), is implicated in both genetic familial epilepsies and Neurodevelopmental Disorders (NDDs) (Baldassari et al., 2018). Previous studies have found seizure phenotypes and increased activity resulting from conditional deletion of GATOR1 function from forebrain excitatory neurons (Yuskaitis et al., 2018; Dentel et al., 2022); however, studies focused on understanding mechanisms contributing to NDD-relevant behaviors are lacking, especially studies understanding the contributions of GATOR1's critical GAP catalytic subunit, nitrogen permease regulator like-2 (Nprl2). Given the clinical phenotypes observed in patients with Nprl2 mutations, in this study, we sought to investigate the neuronal cell type contributions of Nprl2 to NDD behaviors. We conditionally deleted Nprl2 broadly in most neurons (Synapsin1cre), in inhibitory neurons only (Vgatcre), and in Purkinje cells within the cerebellum (L7cre). Broad neuronal deletion of Nprl2 resulted in seizures, social and learning deficits, and hyperactivity. In contrast, deleting Nprl2 from inhibitory neurons led to increased motor learning, hyperactive behavior, in addition to social and learning deficits. Lastly, Purkinje cell (PC) loss of Nprl2 also led to learning and social deficits but did not affect locomotor activity. These phenotypes enhance understanding of the spectrum of disease found in human populations with GATOR1 loss of function and highlight the significance of distinct cellular populations to NDD-related behaviors. SIGNIFICANCE STATEMENT: We aim to elucidate the neuronal-specific contributions of nitrogen permease regulator like-2 (Nprl2) to its neurodevelopmental disorder (NDD)-relevant phenotypes. We conditionally deleted Nprl2 broadly in neurons (Syn1cre), in inhibitory neurons (Vgatcre), and in cerebellar Purkinje cells (L7cre). We identify seizures only in the Syn1cre conditional mutant (cKO); hyperactivity, learning difficulties, social deficits, and impulsivity in the Syn1cre and Vgatcre cKOs; and social deficits, and fear learning deficits in L7cre cKOs. To our knowledge, we are the first to describe the behavioral contributions of Nprl2's function across multiple cell types. Our findings highlight both critical roles for Nprl2 in learning and behavior and also distinct contributions of select neuronal populations to these NDD-relevant behaviors.

KLF4 enhances transplantation-induced hematopoiesis by inhibiting TLRs and non-canonical NFκB signaling at a steady state.

The transcription factor Krüppel-like factor 4 (KLF4) acts as a transcriptional activator and repressor. KLF4 plays a role in various cellular processes, including the dedifferentiation of somatic cells into induced pluripotent stem cells. Although it has been shown to enhance self-renewal in embryonic and leukemia stem cells, its role in adult hematopoietic stem cells (HSC) remains underexplored. We demonstrate that conditional deletion of the Klf4 gene in hematopoietic cells led to an increased frequency of immunophenotypic hematopoietic stem cells (HSCs) in the bone marrow, along with a normal distribution of lymphoid and myeloid progenitor cells. Non-competitive bone marrow transplants showed normal engraftment and multi-lineage reconstitution, except for monocytes and T cells. However, the loss of KLF4 hindered hematological reconstitution in competitive serial bone marrow transplants, highlighting a critical role for KLF4 in stress-induced hematopoiesis. Transcriptome analysis revealed an upregulation of NFκB2 and toll-like receptors (e.g., TLR4) in Klf4-null HSCs during homeostasis. Flow cytometry and immunoblot analysis confirmed the increased cell surface expression of TLR4 and the activation of NFκB2 in HSCs under homeostatic conditions, whereas NFκB2 expression drops after radiation compared to steady-state levels. Our findings suggest that the constitutive activation of the TLR4-NFκB2 pathway inhibits the ability of HSCs to regenerate blood after transplantation in cytoablated bone marrow.

Remote Ischemic Conditioning Attenuates Transneuronal Degeneration and Promotes Stroke Recovery via CD36-Mediated Efferocytosis.

Remote ischemic conditioning (RIC) has been implicated in cross-organ protection in cerebrovascular disease, including stroke. However, the lack of a consensus protocol and controversy over the clinical therapeutic outcomes of RIC suggest an inadequate mechanistic understanding of RIC. The current study identifies RIC-induced molecular and cellular events in the blood, which enhance long-term functional recovery in experimental cerebral ischemia. Naive mice or mice subjected to transient ischemic stroke were randomly selected to receive sham conditioning or RIC in the hindlimb at 2 hours post-stroke. At 3 days post-stroke, monocyte composition in the blood was analyzed, and brain tissue was examined for monocyte-derived macrophage (Mφ), levels of efferocytosis, and CD36 expression. Mouse with a specific deletion of CD36 in monocytes/Mφs was used to establish the role of CD36 in RIC-mediated modulation of efferocytosis, transneuronal degeneration, and recovery following stroke. RIC applied 2 hours after stroke increased the entry of monocytes into the injured brain. In the postischemic brain, Mφ had increased levels of CD36 expression and efferocytosis. These changes in brain Mφ were derived from RIC-induced changes in circulating monocytes. In the blood, RIC increased CD36 expression in circulating monocytes and shifted monocytes to a proinflammatory LY6CHigh state. Conditional deletion of CD36 in Mφ abrogated the RIC-induced monocyte shift in the blood and efferocytosis in the brain. During the recovery phase of stroke, RIC rescued the loss of the volume and of tyrosine hydroxylase+ neurons in substantia nigra and behavioral deficits in wild-type mice but not in mice with a specific deletion of CD36 in monocytes/Mφs. RIC induces a shift in monocytes to a proinflammatory state with elevated CD36 levels, and this is associated with CD36-dependent efferocytosis in Mφs that rescues delayed transneuronal degeneration in the postischemic brain and promotes stroke recovery. Together, these findings provide novel insight into our mechanistic understanding of how RIC improves poststroke recovery.

P0039 Deletion of the autophagy-related gene 16 like 1 (Atg16l1) in the myeloid lineage induces systemic inflammation

Abstract Background The autophagy-related gene 16 like 1 (ATG16L1) is essential for the formation of the autophagosome and deletion of this gene leads, among other things, to the disruption of autophagy.1,2 In 2007, a Genome Wide Association Study identified ATG16L1 as a risk gene for Crohn’s Disease.3 This study aims to understand the mechanisms causing the hyperinflammatory systemic phenotype observed in our mouse model carrying a conditional myeloid deletion of Atg16l1 (Atg16l1LysM). Methods The immune phenotype of Atg16l1LysM and Atg16l1fl/fl mice was characterized using histology staining and flow cytometry of lymphatic organs. In addition, single cell RNA sequencing of splenocytes and determination of cytokine levels in the serum were performed to investigate the inflammatory status of the animals. LPS-stimulated bone marrow-derived macrophages (BMDMs) of Atg16l1LysM and Atg16l1fl/fl mice were used as an in-vitro model to study signaling pathways that might be responsible for the inflammatory phenotype. Results Atg16l1LysM mice display a spontaneous hyperinflammatory phenotype that is accompanied by hepatosplenomegaly and increased cytokine levels in serum. Histology staining revealed a disrupted spleen architecture with an increase of red pulp tissue compared to floxed littermates. Flow cytometry and single cell transcriptome data of splenocytes revealed alterations in the cellular composition of Atg16l1LysM spleens confirming an increase of activated myeloid cells and a simultaneous reduction of lymphoid cells, especially T cells. Additionally, the proportion of myeloid-derived suppressor cells (MDSC) in Atg16l1LysM spleen was found to be increased. Moreover, a significant overexpression of type-I interferon signaling genes was discovered in monocytic cells derived from the Atg16l1LysM spleen. Bulk RNA sequencing data of LPS-stimulated Atg16l1 knockout BMDMs confirmed the overactivation of type-I IFN-related genes compared to wild type BMDMs, pointing towards a role for the Stimulator of IFN genes (STING) and Rig-I-like (RLR) pathways in the Atg16l1-deficient scenario. However, neither inhibiting the STING pathway in vitro nor creating Atg16l1STINGLysM double knockout mice lead to a significant reduction of IFN-β production. Conclusion The increased type-I IFN response observed in Atg16l1LysM mice does not seem to be influenced by STING signaling. Future studies are needed to uncover whether RIG-I-like receptors are major contributors of the increased IFN-β response in absence of Atg16l1 and to determine the internal triggers of this reaction. The results also prompt further investigations on aberrant cell activation in autophagy-deficient myeloid cells.

Nicotinic α7 receptors on cholinergic neurons in the striatum mediate cocaine-reinforcement, but not food reward.

The neurotransmitter acetylcholine has since long been implicated in reward learning and drug addiction. However, the role of specific cholinergic receptor subtypes on different neuronal populations remain elusive. Here, we studied the function of nicotinic acetylcholinergic alpha 7 receptors (α7 nAChRs) in cocaine and food-enforced behaviors. We found that global deletion of α7 nAChRs in mice attenuates cocaine seeking in a Pavlovian conditioned place preference paradigm and decreases operant responding to cocaine in a runway task and in self-administration, without influencing responding to palatable food. This effect can be attributed to alpha 7 receptor signaling in the striatum, as selective deletion of striatal α7 nAChRs using a viral vector approach resulted in a similar decrease in cocaine-preference as that of global deletion. To investigate which type of striatal neurons are responsible for this effect, we selectively targeted Cholinergic (ChAT-expressing) neurons and dopamine D1-receptor (D1R) expressing neurons. Mice with conditional deletion of α7 nAChRs in ChAT-neurons (α7 nAChR-ChATCre) exhibited decreased cocaine place preference and intact place preference for food, while α7 nAChR-D1RCre mice had no changes in reward learning to neither food nor cocaine. Cocaine induction of striatal immediate early gene expression of cFos, FosB, Arc and EGR2 was blocked in α7 nAChR-ChATCre mice, demonstrating the importance of α7 nAChRs on cholinergic neurons for striatal neuronal activity changes. Collectively, our findings show that α7 nAChRs on cholinergic interneurons in the striatum are pivotal for learning processes related to cocaine, but not food reward.

TRIM32 promotes neuronal ferroptosis by enhancing K63-linked ubiquitination and subsequent p62-selective autophagic degradation of GPX4.

Ferroptosis, characterized by iron-dependent phospholipid peroxidation, is recognized as one of the cell death pathways activated following spinal cord injury (SCI). However, the precise regulatory mechanisms governing this process remain poorly understood. Here, this study identified TRIM32, an E3 ubiquitin ligase, as a key enhancer of neuronal ferroptosis. TRIM32 promoted neuronal ferroptosis by accelerating the degradation of GPX4, which is an essential inhibitor of ferroptosis. Conditional deletion of Trim32 in neurons markedly inhibited neuronal ferroptosis and promoted neuronal survival, eventually improving mouse locomotor functional recovery after SCI. However, overexpression of Trim32 showed aggravated neuronal loss and poor behavioral function, which could be attenuated by ferroptosis inhibitor Liproxstatin-1. Mechanistically, TRIM32 interacted with GPX4, promoted K63-linked ubiquitination modification of GPX4 at K107, thus enhanced p62-dependent autophagic degradation of GPX4. Moreover, ROS-ATM-Chk2 signaling pathway phosphorylates TRIM32 at S55, further contributing to GPX4 ubiquitination and degradation and subsequent neuronal ferroptosis after SCI, suggesting a positive feedback loop between ROS and TRIM32. Clinically, lipid peroxidation was significantly promoted in patients with SCI. These findings reveal that TRIM32 functions as a neuronal ferroptosis enhancer which is detrimental to neuronal survival and locomotor functional recovery in mice after SCI by promoting K63-linked ubiquitination and subsequent p62-dependent autophagic degradation of GPX4, suggesting a promising therapeutic target for SCI.

The FBXO45-GEF-H1 axis controls germinal center formation and B-cell lymphomagenesis.

The role of ubiquitin-mediated degradation mechanisms in the pathogenesis of diffuse large B cell (DLBCL) and follicular lymphoma (FL) is not completely understood. We show that conditional deletion of the E3 ubiquitin ligase Fbxo45 in germinal center B-cells results in B-cell lymphomagenesis in homozygous (100%) and heterozygous (48%) mice. Mechanistically, FBXO45 targets the RHO guanine exchange factor ARHGEF2/GEF-H1 for ubiquitin-mediated degradation. Double genetic ablation of Fbxo45 and Arhgef2 ameliorated lymphoma formation. Transgenic knock-in mice harboring a GEF-H1 mutant unable to bind FBXO45 develop B-cell lymphomas with ~50% penetrance. Genome sequencing in human lymphomas identified mutually-exclusive FBXO45 copy number losses and ARHGEF2 gains, with combined frequencies ranging from 26.32% in FL to 45.12% in DLBCL. Notably, FBXO45 silencing enhances sensitivity to MEK1/2 inhibition. These results identify FBXO45 and ARHGEF2 as a novel tumor-suppressor and oncogene pair involved in the pathogenesis of B-cell lymphomas with significant implications for targeted therapies.

NUBP2 deficiency disrupts the centrosome-check point in the brain and causes primary microcephaly.

Microcephaly affects 1 in 2,500 babies per year. Primary microcephaly results from aberrant neurogenesis leading to a small brain at birth. This is due to altered patterns of proliferation and/or early differentiation of neurons. Premature differentiation of neurons is associated with defects in the centrosome and/or primary cilia. In this study, we report on the first patients identified with NUBP2 -deficiency and utilize a conditional mouse model to ascertain the molecular mechanisms associated with NUBP2 -deficient primary microcephaly. We identified homozygous NUBP2 variants in these patients who displayed profound primary microcephaly in addition to intrauterine growth restriction, cervical kyphosis, severe contractures of joints, and facial dysmorphia. We then generated a mouse model using Emx1-Cre to ablate Nubp2 from the forebrain. The mice presented with severe microcephaly starting at E18.5. Neurospheres generated from the forebrain of Emx1-Cre; Nubp2 flox/flox conditional deletion mice were used to support the pathogenicity of the patient variants. We show that loss of Nubp2 increases both canonical and non-canonical cell death, but that loss of p53 fails to rescue microcephaly in the mouse model. Examination of neurogenesis in Emx1-Cre; Nubp2 flox/flox mice revealed distinct alterations in proliferation and cellular migration accompanied by supernumerary centrosomes and cilia. We therefore propose that NUBP2 is a novel primary microcephaly-related gene and that the role of Nubp2 in centrosome and cilia regulation is crucial for proper neurogenesis.

PKCδ germline variants and genetic deletion in mice augment antitumor immunity through regulation of myeloid cells.

Based on the notion that hypomorphic germline genetic variants are linked to autoimmune diseases, we reasoned that novel targets for cancer immunotherapy might be identified through germline variants associated with greater T-cell infiltration into tumors. Here, we report that while investigating germline polymorphisms associated with a tumor immune gene signature, we identified PKCδ as a candidate. Genetic deletion of PKCδ in mice resulted in improved endogenous antitumor immunity and increased efficacy of anti-PD-L1. Single-cell RNA sequencing revealed myeloid cell expression of Prkcd, and PKCδ deletion caused a shift in macrophage gene expression from an M2-like to an M1-like phenotype. Conditional deletion of PKCδ in myeloid cells recapitulated improved tumor control that was augmented further with anti-PD-L1. Analysis of clinical samples confirmed an association between PRKCD variants and M1/M2 phenotype, as well as between a PKCδ KO-like gene signature and clinical benefit from anti-PD-1. Our results identify PKCδ as a candidate therapeutic target that modulates myeloid cell states.

Tgfβ signaling stimulates glycolysis to promote the genesis of synovial joint interzone in developing mouse embryonic limbs.

The initial interzone cells for synovial joints originate from chondrocytes, but such critical transition is minimally understood. With single-cell RNA sequencing (scRNA-seq) of murine embryonic knee joint primordia, we discovered that heightened expression of glycolysis genes characterized developing interzone cells when compared to flanking chondrocytes. Conditional deletion of the glucose transporters Glut1 and/or Glut3, in either the incipient pre-skeletal mesenchyme with Prx1Cre or in chondrocytes with Col2Cre, disrupted interzone formation dose-dependently. In contrast, deletion of Glut1/3 in established interzone cells with Gdf5Cre did not have similar severe disruption of joint development. scRNA-seq revealed that Glut1/3 deletion by Prx1Cre impeded Tgfβ signaling in the developing interzone cells. Direct elimination of Tgfβ signaling with Prx1Cre partially phenocopied the deletion of Glut1/3 in impairing interzone formation. Tgfβ stimulated glycolysis in chondrocytes via activation of mTOR and Hif1α in vitro. The data support that the essential conversion of chondrocytes to interzone cells requires a transient elevation of glycolysis partly dependent on Tgfβ signaling.

Adaptive immune cells antagonize ILC2 homeostasis via SLAMF3 and SLAMF5.

Type 2 innate lymphoid cells (ILC2s) mainly reside in tissues with few lymphoid cells. How their tissue residency is regulated remains poorly understood. This study explores the inhibitory role of SLAM-family receptors (SFRs) on adaptive immune cells in ILC2 maintenance. We observed an increase in the population of ILC2s in Rag1-deficient mice. Homotypic engagement of SFRs between ILC2s and adaptive immune cells was identified as a potential mechanism. SFR deficiency led to an increase in ILC2s. Conditional deletion of SFRs on T and/or B cells led to an increased ILC2 abundance. Mechanistically, as ILC precursors differentiate into ILC2s, SFRs, primarily SLAMF3 and SLAMF5, are inhibitory, which impair IL-7-induced PI3K activation and enhance apoptosis via SHP-1. These findings reveal a mechanism by which adaptive immune cells negatively regulate the homeostasis of ILC2s and contribute to our understanding of the complex interplay between innate and adaptive immune cells in the regulation of immune responses.

Sarcolemma resilience and skeletal muscle health require O-mannosylation of dystroglycan

BackgroundMaintaining the connection between skeletal muscle fibers and the surrounding basement membrane is essential for muscle function. Dystroglycan (DG) serves as a basement membrane extracellular matrix (ECM) receptor in many cells, and is also expressed in the outward-facing membrane, or sarcolemma, of skeletal muscle fibers. DG is a transmembrane protein comprised of two subunits: alpha-DG (α-DG), which resides in the peripheral membrane, and beta-DG (β-DG), which spans the membrane to intracellular regions. Extensive post-translational processing and O-mannosylation are required for α-DG to bind ECM proteins, which is mediated by a glycan structure known as matriglycan. O-mannose glycan biosynthesis is initiated by the protein O-mannosyltransferase 1 (POMT1) and POMT2 enzyme complex and leads to three subtypes of glycans called core M1, M2, and M3. The lengthy core M3 is capped with matriglycan. Genetic defects in post-translational O-mannosylation of DG interfere with its receptor function and result in muscular dystrophy with central nervous system and skeletal muscle pathophysiology.MethodsTo evaluate how the loss of O-mannosylated DG in skeletal muscle affects the development and progression of myopathology, we generated and characterized mice in which the Pomt1 gene was specifically deleted in skeletal muscle (Pomt1skm) to interfere with POMT1/2 enzyme activity. To investigate whether matriglycan is the primary core M glycan structure that provides the stabilizing link between the sarcolemma and ECM, we generated mice that retained cores M1, M2, and M3, but lacked matriglycan (conditional deletion of like-acetylglucosaminyltransferase 1; Large1skm). Next, we restored Pomt1 using gene transfer via AAV2/9-MCK-mPOMT1 and determined the effect on Pomt1skm pathophysiology.ResultsOur data showed that in Pomt1skm mice O-mannosylated DG is required for sarcolemma resilience, remodeling of muscle fibers and muscle tissue, and neuromuscular function. Notably, we observed similar body size limitations, sarcolemma weakness, and neuromuscular weakness in Large1skm mice that only lacked matriglycan. Furthermore, our data indicate that genetic rescue of Pomt1 in Pomt1skm mice limits contraction-induced sarcolemma damage and skeletal muscle pathology.ConclusionsCollectively, our data indicate that DG modification by Pomt1/2 results in core M3 capped with matriglycan, and that this is required to reinforce the sarcolemma and enable skeletal muscle health and neuromuscular strength.

Conditional Deletion of Gremlin-1 in Cathepsin K-expressing Mature Osteoclasts Altered the Skeletal Response to Calcium Depletion in Sex-Dependent Manner

This study assessed the novel concept that osteoclast-derived Grem1 has regulatory functions in the skeletal response to calcium stress using an osteoclastic Grem1 conditional knockout (cKO) mouse model. The calcium stress was initiated by feeding cKO mutants and wildtype (WT) littermates a calcium-deficient diet for 2 weeks. Deletion of Grem1 in mature osteoclasts did not affect developmental bone growth nor basal bone turnover. In response to calcium depletion, male cKO mutants showed greater increases in osteoclastic resorption and trabecular bone loss than male WT littermates, indicating an enhanced skeletal sensitivity to calcium depletion in male mutants. The enhanced sensitivity to calcium depletion was sex-dependent, as female cKO mutants showed lower increases in osteoclastic resorption and bone loss than female WT littermates as well as male cKO mutants. The sex disparity in osteoclastic resorption response to calcium stress was intrinsic to osteoclasts since osteoclasts of male but not female cKO mutants showed greater in vitro bone resorption activity than osteoclasts of WT littermates of respective sex. Male cKO mutants displayed smaller bone formation response to calcium depletion than male WT littermates, while female mutants showed bigger bone formation response than female WT littermates, indicating that cKO mutants also displayed sex disparity in bone formation response. The sex disparity in bone formation response was not caused by intrinsic differences in osteoblasts but might be due to sex-dependent differential osteoclastic release of osteogenic factors. In summary, osteoclast-derived gremlin-1 has complicated and sex-dependent regulatory roles in skeletal response to calcium stress.

Chronic Rapamycin Prevents Electrophysiological and Morphological Alterations Produced by Conditional Pten Deletion in Mouse Cortex.

Abnormalities in the mammalian target of the rapamycin (mTOR) pathway have been implicated in numerous developmental brain disorders. While the molecular and histological abnormalities have been described, less is known about alterations in membrane and synaptic excitability with chronic changes in the mTOR pathway. In the present study, we used a conditional mouse model with a deletion of the phosphatase and tensin homologue (Pten-/-, a negative regulator of mTOR) from cortical pyramidal neurons (CPNs). Whole-cell patch clamp recordings in ex vivo slices examined the intrinsic and synaptic membrane properties of layer II/III CPNs in normal mice treated with rapamycin for four weeks, and Pten-/- mice with and without chronic treatment with rapamycin. Compared with control mice, CPNs from Pten-/- mice demonstrated increased membrane capacitance and time constant in association with increased neuronal somatic size, reduced neuronal firing, and decreased frequency of spontaneous and miniature inhibitory postsynaptic currents, consistent with decreased pre-synaptic GABA release. Rapamycin treatment for four weeks prevented these changes in Pten-/- mice. CPNs from normal mice chronically treated with rapamycin, compared with CPNs from naïve mice, showed reduced capacitance and time constant, increased input resistance, and changes in inhibitory synaptic inputs, consistent with increased pre-synaptic GABA release. These results support the concept that Pten deletion results in significant changes in inhibitory inputs onto CPNs, and these alterations can be prevented with chronic rapamycin treatment. In addition, normal mice treated with rapamycin also display altered membrane and synaptic properties. These findings have potential implications for the treatment of neurological disorders associated with mTOR pathway dysfunction, such as epilepsy and autism.

Deletion of Neuroligins from Astrocytes Does Not Detectably Alter Synapse Numbers or Astrocyte Cytoarchitecture by Maturity.

Astrocytes perform multifarious roles in the formation, regulation, and function of synapses in the brain, but the mechanisms involved are incompletely understood. Interestingly, astrocytes abundantly express neuroligins, postsynaptic adhesion molecules that function as synaptic organizers by binding to presynaptic neurexins. Here we examined the function of neuroligins in astrocytes with a rigorous genetic approach that uses the conditional deletion of all major neuroligins ( Nlgn1-3 ) in astrocytes in vivo and complemented this approach by a genetic deletion of neuroligins in glia cells that are co-cultured with human neurons. Our results show that early postnatal deletion of neuroligins from astrocytes in vivo has no detectable effect on cortical or hippocampal synapses and does not alter the cytoarchitecture of astrocytes when evaluated in young adult mice. Moreover, deletion of astrocytic neuroligins in co-cultures of human neurons produced no detectable consequences for the formation and function of synapses. Thus, astrocytic neuroligins are unlikely to fundamentally shape synapse formation or astrocyte morphogenesis but likely perform other important roles that remain to be discovered.

Ablation of PI3Kγ in neurons protects mice from diet-induced obesity MASLD and insulin resistance.

Mice with genetic ablation of PI3Kγ are protected from diet-induced obesity. However, the cell type responsible for PI3Kγ action in obesity remains unknown. We generated mice with conditional deletion of PI3Kγ in neurons using the nestin promoter to drive the expression of the Cre recombinase (PI3KγNest mice) and investigated their metabolic phenotype in a model of diet-induced obesity. On a chow diet, lean PI3KγNest mice display reduced linear growth and a normal metabolic phenotype. PI3KγNest mice were largely protected from diet-induced obesity and liver steatosis and showed improved glucose tolerance and insulin sensitivity. This phenotype was associated with increased phosphorylation of hormone-sensitive lipase (HSL) at protein kinase A (PKA) sites in white fat. It is concluded that PI3Kγ action in diet-induced obesity depends on its activity in neurons controlling adipose tissue lipolysis. Future clinical studies on PI3Kγ inhibitors capable of crossing the brain-blood barrier will reveal the relevance of these findings to humans.

Pyrimidine synthesis enzyme CTP synthetase 1 suppresses antiviral interferon induction by deamidating IRF3.

Metabolism is typically contextualized in conjunction with proliferation and growth. The roles of metabolic enzymes beyond metabolism-such as in innate immune responses-are underexplored. Using a focused short hairpin RNA (shRNA)-mediated screen, we identified CTP synthetase 1 (CTPS1), a rate-limiting enzyme of pyrimidine synthesis, as a negative regulator of interferon induction. Mechanistically, CTPS1 interacts with and deamidates interferon regulatory factor 3 (IRF3). Deamidation at N85 impairs IRF3 binding to promoters containing IRF3-responsive elements, thus muting interferon (IFN) induction. Employing CTPS1 conditional deletion and IRF3 deamidated or deamidation-resistant knockin mice, we demonstrated that CTPS1-driven IRF3 deamidation restricts IFN induction in response to viral infection invivo. However, during immune activation, IRF3 deamidation by CTPS1 is inhibited by glycogen synthase kinase 3 beta (GSK3β) to promote IFN induction. This work demonstrates how CTPS1 tames innate immunity independent of its role in pyrimidine synthesis, thus expanding the functional repertoire of metabolic enzymes into immune regulation.

Neutrophil behavior during the metabolic adaptations to short term high fat feeding

Background and Aims: Neutrophils participate to the chronic metabolic consequences of High Fat Diet (HFD). Nevertheless, neutrophils are characterized by short half-life which is determined by a fine tuning in expression and function of CXCR4, which dictates the egress from the bone marrow (BM), and CXCR2, which facilitates their mobilization from BM and the patrolling activity in the periphery. In the quest to study the behavior of the neutrophil with the short-term consequences of HFD feeding, we studied whether the metabolic adaptations affect blood neutrophil count and the membrane expression of their indirect markers of function. Methods: To assess the gluco-metabolic impact of a short-term HFD feeding, indirect calorimetry and plasma glucose dosage were performed on mice previously fed a HFD (60% Kcal from fat) for seven days, followed by immunophenotyping of blood over 24 hours. To test whether changes in circulating neutrophil count during short-term HFD feeding were related to a differential egress from the BM, we repeated the same experimental design in mice harboring a conditional deletion of CXCR4 (CXCR4fl/flMrp8Cre+). Results: Short-term HFD feeding was sufficient to induce a profound metabolic impact (e.g. reduced respiratory exchange ratio, increased energy expenditure, and insulin levels), and to induce an increase of circulating neutrophils (p=0,052), without impacting other leukocytic fractions over 24 hours, compared to chow diet feeding mice (20% Kcal from fat). HFD feeding significantly altered the expression pattern of multiple membrane markers of neutrophil function (CD11b, CD62L, CXCR2) over 24 hours, driving neutrophils toward a phenotype featuring increased migration and activation. Finally, the CXCR4fl/flMrp8Cre+ mice, which present significantly higher circulating neutrophilia, showed lower insulin sensitivity upon HFD compared to WT. Conclusions: We suggest that the metabolic adaptations induced by a short-term exposure to HFD affect neutrophil behavior, surmising it as an appealing target for cardio-metabolic diseases.