Kinase signalling in excitatory neurons regulates sleep quantity and depth.

Progress has been made in the elucidation of sleep and wakefulness regulation at the neurocircuit level1,2. However, the intracellular signalling pathways that regulate sleep and the neuron groups in which these intracellular mechanisms work remain largely unknown. Here, using a forward genetics approach in mice, we identify histone deacetylase 4 (HDAC4) as a sleep-regulating molecule. Haploinsufficiency of Hdac4, a substrate of salt-inducible kinase 3 (SIK3)3, increased sleep. By contrast, mice that lacked SIK3 or its upstream kinase LKB1 in neurons or with a Hdac4S245A mutation that confers resistance to phosphorylation by SIK3 showed decreased sleep. These findings indicate that LKB1-SIK3-HDAC4 constitute a signalling cascade that regulates sleep and wakefulness. We also performed targeted manipulation of SIK3 and HDAC4 in specific neurons and brain regions. This showed that SIK3 signalling in excitatory neurons located in the cerebral cortex and the hypothalamus positively regulates EEG delta power during non-rapid eye movement sleep (NREMS) and NREMS amount, respectively. A subset of transcripts biased towards synaptic functions was commonly regulated in cortical glutamatergic neurons through the expression of a gain-of-function allele of Sik3 and through sleep deprivation. These findings suggest that NREMS quantity and depth are regulated by distinct groups of excitatory neurons through common intracellular signals. This study provides a basis for linking intracellular events and circuit-level mechanisms that control NREMS.

Landscape of driver mutations and their clinical effects on Down syndrome-related myeloid neoplasms.

ABSTRACT: Transient abnormal myelopoiesis (TAM) is a common complication in newborns with Down syndrome (DS). It commonly progresses to myeloid leukemia (ML-DS) after spontaneous regression. In contrast to the favorable prognosis of primary ML-DS, patients with refractory/relapsed ML-DS have poor outcomes. However, the molecular basis for refractoriness and relapse and the full spectrum of driver mutations in ML-DS remain largely unknown. We conducted a genomic profiling study of 143 TAM, 204 ML-DS, and 34 non-DS acute megakaryoblastic leukemia cases, including 39 ML-DS cases analyzed by exome sequencing. Sixteen novel mutational targets were identified in ML-DS samples. Of these, inactivations of IRX1 (16.2%) and ZBTB7A (13.2%) were commonly implicated in the upregulation of the MYC pathway and were potential targets for ML-DS treatment with bromodomain-containing protein 4 inhibitors. Partial tandem duplications of RUNX1 on chromosome 21 were also found, specifically in ML-DS samples (13.7%), presenting its essential role in DS leukemia progression. Finally, in 177 patients with ML-DS treated following the same ML-DS protocol (the Japanese Pediatric Leukemia and Lymphoma Study Group acute myeloid leukemia -D05/D11), CDKN2A, TP53, ZBTB7A, and JAK2 alterations were associated with a poor prognosis. Patients with CDKN2A deletions (n = 7) or TP53 mutations (n = 4) had substantially lower 3-year event-free survival (28.6% vs 90.5%; P < .001; 25.0% vs 89.5%; P < .001) than those without these mutations. These findings considerably change the mutational landscape of ML-DS, provide new insights into the mechanisms of progression from TAM to ML-DS, and help identify new therapeutic targets and strategies for ML-DS.

B Cells of Early-life Origin Defined by RAG2-based Lymphoid Cell Tracking under Native Hematopoietic Conditions.

During the perinatal period, the immune system sets the threshold to select either response or tolerance to environmental Ags, which leads to the potential to provide a lifetime of protection and health. B-1a B cells have been demonstrated to develop during this perinatal time window, showing a unique and restricted BCR repertoire, and these cells play a major role in natural Ab secretion and immune regulation. In the current study, we developed a highly efficient temporally controllable RAG2-based lymphoid lineage cell labeling and tracking system and applied this system to understand the biological properties and contribution of B-1a cells generated at distinct developmental periods to the adult B-1a compartments. This approach revealed that B-1a cells with a history of RAG2 expression during the embryonic and neonatal periods dominate the adult B-1a compartment, including those in the bone marrow (BM), peritoneal cavity, and spleen. Moreover, the BCR repertoire of B-1a cells with a history of RAG2 expression during the embryonic period was restricted, becoming gradually more diverse during the neonatal period, and then heterogeneous at the adult stage. Furthermore, more than half of plasmablasts/plasma cells in the adult BM had embryonic and neonatal RAG2 expression histories. Moreover, BCR analysis revealed a high relatedness between BM plasmablasts/plasma cells and B-1a cells derived from embryonic and neonatal periods, suggesting that these cell types have a common origin. Taken together, these findings define, under native hematopoietic conditions, the importance in adulthood of B-1a cells generated during the perinatal period.

Activity of estrogen receptor ß expressing neurons in the medial amygdala regulates preference toward receptive females in male mice.

The processing of information regarding the sex and reproductive state of conspecific individuals is critical for successful reproduction and survival in males. Generally, male mice exhibit a preference toward the odor of sexually receptive (RF) over nonreceptive females (XF) or gonadally intact males (IM). Previous studies suggested the involvement of estrogen receptor beta (ERß) expressed in the medial amygdala (MeA) in male preference toward RF. To further delineate the role played by ERß in the MeA in the neuronal network regulating male preference, we developed a new ERß-iCre mouse line using the CRISPR-Cas9 system. Fiber photometry Ca2+ imaging revealed that ERß-expressing neurons in the postero-dorsal part of the MeA (MeApd-ERß+ neurons) were more active during social investigation toward RF compared to copresented XF or IM mice in a preference test. Chemogenetic inhibition of MeApd-ERß+ neuronal activity abolished a preference to RF in "RF vs. XF," but not "RF vs. IM," tests. Analysis with cre-dependent retrograde tracing viral vectors identified the principal part of the bed nucleus of stria terminalis (BNSTp) as a primary projection site of MeApd-ERß+ neurons. Fiber photometry recording in the BNSTp during a preference test revealed that chemogenetic inhibition of MeApd-ERß+ neurons abolished differential neuronal activity of BNSTp cells as well as a preference to RF against XF but not against IM mice. Collectively, these findings demonstrate for the first time that MeApd-ERß+ neuronal activity is required for expression of receptivity-based preference (i.e., RF vs. XF) but not sex-based preference (i.e., RF vs. IM) in male mice.

SIK3-HDAC4 in the suprachiasmatic nucleus regulates the timing of arousal at the dark onset and circadian period in mice.

Mammals exhibit circadian cycles of sleep and wakefulness under the control of the suprachiasmatic nucleus (SCN), such as the strong arousal phase-locked to the beginning of the dark phase in laboratory mice. Here, we demonstrate that salt-inducible kinase 3 (SIK3) deficiency in gamma-aminobutyric acid (GABA)-ergic neurons or neuromedin S (NMS)-producing neurons delayed the arousal peak phase and lengthened the behavioral circadian cycle under both 12-h light:12-h dark condition (LD) and constant dark condition (DD) without changing daily sleep amounts. In contrast, the induction of a gain-of-function mutant allele of Sik3 in GABAergic neurons exhibited advanced activity onset and a shorter circadian period. Loss of SIK3 in arginine vasopressin (AVP)-producing neurons lengthened the circadian cycle, but the arousal peak phase was similar to that in control mice. Heterozygous deficiency of histone deacetylase (HDAC) 4, a SIK3 substrate, shortened the circadian cycle, whereas mice with HDAC4 S245A, which is resistant to phosphorylation by SIK3, delayed the arousal peak phase. Phase-delayed core clock gene expressions were detected in the liver of mice lacking SIK3 in GABAergic neurons. These results suggest that the SIK3-HDAC4 pathway regulates the circadian period length and the timing of arousal through NMS-positive neurons in the SCN.

Impaired Glycosylation of Gastric Mucins Drives Gastric Tumorigenesis and Serves as a Novel Therapeutic Target.

BACKGROUND & AIMS: Gastric cancer is often accompanied by a loss of mucin 6 (MUC6), but its pathogenic role in gastric carcinogenesis remains unclear. METHODS: Muc6 knockout (Muc6-/-) mice and Muc6-dsRED mice were newly generated. Tff1Cre, Golph3-/-, R26-Golgi-mCherry, Hes1flox/flox, Cosmcflox/flox, and A4gnt-/- mice were also used. Histology, DNA and RNA, proteins, and sugar chains were analyzed by whole-exon DNA sequence, RNA sequence, immunohistochemistry, lectin-binding assays, and liquid chromatography-mass spectrometry analysis. Gastric organoids and cell lines were used for in vitro assays and xenograft experiments. RESULTS: Deletion of Muc6 in mice spontaneously causes pan-gastritis and invasive gastric cancers. Muc6-deficient tumor growth was dependent on mitogen-activated protein kinase activation, mediated by Golgi stress-induced up-regulation of Golgi phosphoprotein 3. Glycomic profiling revealed aberrant expression of mannose-rich N-linked glycans in gastric tumors, detected with banana lectin in association with lack of MUC6 expression. We identified a precursor of clusterin as a binding partner of mannose glycans. Mitogen-activated protein kinase activation, Golgi stress responses, and aberrant mannose expression are found in separate Cosmc- and A4gnt-deficient mouse models that lack normal O-glycosylation. Banana lectin-drug conjugates proved an effective treatment for mannose-rich murine and human gastric cancer. CONCLUSIONS: We propose that Golgi stress responses and aberrant glycans are important drivers of and promising new therapeutic targets for gastric cancer.

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Structure and Function of Neuronal Circuits Linking Ventrolateral Preoptic Nucleus and Lateral Hypothalamic Area.

To understand how sleep-wakefulness cycles are regulated, it is essential to disentangle structural and functional relationships between the preoptic area (POA) and lateral hypothalamic area (LHA), since these regions play important yet opposing roles in the sleep-wakefulness regulation. GABA- and galanin (GAL)-producing neurons in the ventrolateral preoptic nucleus (VLPO) of the POA (VLPOGABA and VLPOGAL neurons) are responsible for the maintenance of sleep, while the LHA contains orexin-producing neurons (orexin neurons) that are crucial for maintenance of wakefulness. Through the use of rabies virus-mediated neural tracing combined with in situ hybridization (ISH) in male and female orexin-iCre mice, we revealed that the vesicular GABA transporter (Vgat, Slc32a1)- and galanin (Gal)-expressing neurons in the VLPO directly synapse with orexin neurons in the LHA. A majority (56.3 ± 8.1%) of all VLPO input neurons connecting to orexin neurons were double-positive for Vgat and Gal Using projection-specific rabies virus-mediated tracing in male and female Vgat-ires-Cre and Gal-Cre mice, we discovered that VLPOGABA and VLPOGAL neurons that send projections to the LHA received innervations from similarly distributed input neurons in many brain regions, with the POA and LHA being among the main upstream areas. Additionally, we found that acute optogenetic excitation of axons of VLPOGABA neurons, but not VLPOGAL neurons, in the LHA of male Vgat-ires-Cre mice induced wakefulness. This study deciphers the connectivity between the VLPO and LHA, provides a large-scale map of upstream neuronal populations of VLPO→LHA neurons, and reveals a previously uncovered function of the VLPOGABA→LHA pathway in the regulation of sleep and wakefulness.SIGNIFICANCE STATEMENT We identified neurons in the ventrolateral preoptic nucleus (VLPO) that are positive for vesicular GABA transporter (Vgat) and/or galanin (Gal) and serve as presynaptic partners of orexin-producing neurons in the lateral hypothalamic area (LHA). We depicted monosynaptic input neurons of GABA- and galanin-producing neurons in the VLPO that send projections to the LHA throughout the entire brain. Their input neurons largely overlap, suggesting that they comprise a common neuronal population. However, acute excitatory optogenetic manipulation of the VLPOGABA→LHA pathway, but not the VLPOGAL→LHA pathway, evoked wakefulness. This study shows the connectivity of major components of the sleep/wake circuitry in the hypothalamus and unveils a previously unrecognized function of the VLPOGABA→LHA pathway in sleep-wakefulness regulation. Furthermore, we suggest the existence of subpopulations of VLPOGABA neurons that innervate LHA.

Role of inter-alpha-trypsin inhibitor heavy chain 4 and its citrullinated form in experimental arthritis murine models.

Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) is a major protein in serum and reported to be upregulated at the onset of rheumatoid arthritis (RA). Its citrullinated form, cit-ITIH4, is specifically found in the serum and synovial fluid of patients with RA. However, the detailed function of ITIH4 in arthritis remains unknown. The aim of this study was to clarify the role of ITIH4 and cit-ITIH4 using experimental arthritis models. ITIH4 and cit-ITIH4 expression was examined in steady-state mice and two different arthritis models, and their pathological effects were examined in Itih4-deficient mice. In naïve C57BL/6 (WT) mice, ITIH4 was expressed as mRNA in the liver and the lung and was expressed as protein in serum and hepatocytes. In K/BxN serum transferred arthritis (K/BxN-STA) and collagen-induced arthritis (CIA), ITIH4 and cit-ITIH4 in sera were increased before the onset of arthritis, and cit-ITIH4 was further increased at the peak of arthritis. In Itih4-deficient mice, citrullinated proteins in serum and joints, especially 120 kDa protein, were clearly diminished; however, there was no significant difference in arthritis severity between WT and itih-/- mice either in the K/BxN-STA or CIA model. CIA mice also exhibited pulmonary lesions and itih4-/- mice tended to show enhanced inflammatory cell aggregation compared to WT mice. Neutrophils in the lungs of itih4-/- mice were significantly increased compared to WT mice. In summary, ITIH4 itself did not alter the severity of arthritis but may inhibit autoimmune inflammation via suppression of neutrophil recruitment.

Clonal germinal center B cells function as a niche for T-cell lymphoma.

Angioimmunoblastic T-cell lymphoma (AITL) is proposed to be initiated by age-related clonal hematopoiesis (ACH) with TET2 mutations, whereas the G17V RHOA mutation in immature cells with TET2 mutations promotes the development of T follicular helper (TFH)-like tumor cells. Here, we investigated the mechanism by which TET2-mutant immune cells enable AITL development using mouse models and human samples. Among the 2 mouse models, mice lacking Tet2 in all the blood cells (Mx-Cre × Tet2flox/flox × G17V RHOA transgenic mice) spontaneously developed AITL for approximately up to a year, while mice lacking Tet2 only in the T cells (Cd4-Cre × Tet2flox/flox × G17V RHOA transgenic mice) did not. Therefore, Tet2-deficient immune cells function as a niche for AITL development. Single-cell RNA-sequencing (scRNA-seq) of >50 000 cells from mouse and human AITL samples revealed significant expansion of aberrant B cells, exhibiting properties of activating light zone (LZ)-like and proliferative dark zone (DZ)-like germinal center B (GCB) cells. The GCB cells in AITL clonally evolved with recurrent mutations in genes related to core histones. In silico network analysis using scRNA-seq data identified Cd40-Cd40lg as a possible mediator of GCB and tumor cell cluster interactions. Treatment of AITL model mice with anti-Cd40lg inhibitory antibody prolonged survival. The genes expressed in aberrantly expanded GCB cells in murine tumors were also broadly expressed in the B-lineage cells of TET2-mutant human AITL. Therefore, ACH-derived GCB cells could undergo independent clonal evolution and support the tumorigenesis in AITL via the CD40-CD40LG axis.

A rare olive compound oleacein functions as a TrkB agonist and mitigates neuroinflammation both in vitro and in vivo.

BACKGROUND: Neuroinflammation is widely acknowledged as a characteristic feature of almost all neurological disorders and specifically in depression- and anxiety-like disorders. In recent years, there has been significant attention on natural compounds with potent anti-inflammatory effects due to their potential in mitigating neuroinflammation and neuroplasticity. METHODS: In the present study, we aimed to evaluate the neuroprotective effects of oleacein (OC), a rare secoiridoid derivative found in extra virgin olive oil. Our goal was to explore the BDNF/TrkB neurotrophic activity of OC and subsequently assess its potential for modulating neuroinflammatory response using human neuroblastoma cells (SH-SY5Y cells) and an in vivo model of depression induced by lipopolysaccharide (LPS)-mediated inflammation. RESULTS: In SH-SY5Y cells, OC exhibited a significant dose-dependent increase in BDNF expression. This enhancement was absent when cells were co-treated with inhibitors of BDNF's receptor TrkB, as well as downstream molecules PI3K and MEK. Whole-transcriptomics analysis revealed that OC upregulated cell cycle-related genes under normal conditions, while downregulating inflammation-associated genes in LPS-induced conditions. Furthermore, surface plasmon resonance (SPR) assays demonstrated that OC exhibited a stronger and more stable binding affinity to TrkB compared to the positive control, 7,8-dihydroxyflavone. Importantly, bioluminescence imaging revealed that a single oral dose of OC significantly increased BDNF expression in the brains of Bdnf-IRES-AkaLuc mice. Furthermore, oral administration of OC at a dosage of 10 mg/kg body weight for 10 days significantly reduced immobility time in the tail suspension test compared to the LPS-treated group. RT-qPCR analysis revealed that OC significantly decreased the expression of pro-inflammatory cytokines Tnfα, Il6, and Il1ß, while simultaneously enhancing Bdnf expression, as well as both pro and mature BDNF protein levels in mice hippocampus. These changes were comparable to those induced by the positive control antidepressant drug fluoxetine. Additionally, microarray analysis of mouse brains confirmed that OC could counteract LPS-induced inflammatory biological events. CONCLUSION: Altogether, our study represents the first report on the potential antineuroinflammatory and antidepressant properties of OC via modulation of BDNF/TrkB neurotrophic activity. This finding underscores the potential of OC as a natural therapeutic agent for depression- and anxiety-related disorders.

Birth of mice from meiotically arrested spermatocytes following biparental meiosis in halved oocytes.

Microinjection of spermatozoa or spermatids into oocytes is a major choice for infertility treatment. However, the use of premeiotic spermatocytes has never been considered because of its technical problems. Here, we show that the efficiency of spermatocyte injection in mice can be improved greatly by reducing the size of the recipient oocytes. Live imaging showed that the underlying mechanism involves reduced premature separation of the spermatocyte's meiotic chromosomes, which produced much greater (19% vs. 1%) birth rates in smaller oocytes. Application of this technique to spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans, resulted in the production of live offspring. Thus, the microinjection of primary spermatocytes into oocytes may be a potential treatment for overcoming a form of nonobstructive azoospermia caused by meiotic failure.

FGF-23 from erythroblasts promotes hematopoietic progenitor mobilization.

Fibroblast growth factor 23 (FGF-23) hormone is produced by bone-embedded osteocytes and regulates phosphate homeostasis in kidneys. We found that administration of granulocyte colony-stimulating factor (G-CSF) to mice induced a rapid, substantial increase in FGF-23 messenger RNA in bone marrow (BM) cells. This increase originated mainly from CD45-Ter119+CD71+ erythroblasts. FGF-23 protein in BM extracellular fluid was markedly increased during G-CSF-induced hematopoietic progenitor cell (HPC) mobilization, but remained stable in the blood, with no change in the phosphate level. Consistent with the BM hypoxia induced by G-CSF, low oxygen concentration induced FGF-23 release from human erythroblast HUDEP-2 cells in vitro. The efficient mobilization induced by G-CSF decreased drastically in both FGF-23-/- and chimeric mice with FGF-23 deficiency, only in hematopoietic cells, but increased in osteocyte-specific FGF-23-/- mice. This finding suggests that erythroblast-derived, but not bone-derived, FGF-23 is needed to release HPCs from BM into the circulation. Mechanistically, FGF-23 did not influence CXCL-12 binding to CXCR-4 on progenitors but interfered with their transwell migration toward CXCL-12, which was canceled by FGF receptor inhibitors. These results suggest that BM erythroblasts facilitate G-CSF-induced HPC mobilization via FGF-23 production as an intrinsic suppressor of chemoattraction.

Erk5 in Bone Marrow Mesenchymal Stem Cells Regulates Bone Homeostasis by Preventing Osteogenesis in Adulthood.

Extracellular signal-regulated kinase 5 (Erk5) belongs to the mitogen-activated protein kinase (MAPK) family. Previously, we demonstrated that Erk5 directly phosphorylates Smad-specific E3 ubiquitin protein ligase 2 (Smurf2) at Thr249 (Smurf2Thr249) to activate its E3 ubiquitin ligase activity. Although we have clarified the importance of Erk5 in embryonic mesenchymal stem cells (MSCs) on skeletogenesis, its role in adult bone marrow (BM)-MSCs on bone homeostasis remains unknown. Leptin receptor-positive (LepR+) BM-MSCs represent a major source of bone in adult bone marrow and are critical regulators of postnatal bone homeostasis. Here, we identified Erk5 in BM-MSCs as an important regulator of bone homeostasis in adulthood. Bone marrow tissue was progressively osteosclerotic in mice lacking Erk5 in LepR+ BM-MSCs with age, accompanied by increased bone formation and normal bone resorption in vivo. Erk5 deficiency increased the osteogenic differentiation of BM-MSCs along with a higher expression of Runx2 and Osterix, essential transcription factors for osteogenic differentiation, without affecting their stemness in vitro. Erk5 deficiency decreased Smurf2Thr249 phosphorylation and subsequently increased Smad1/5/8-dependent signaling in BM-MSCs. The genetic introduction of the Smurf2T249E mutant (a phosphomimetic mutant) suppressed the osteosclerotic phenotype in Erk5-deficient mice. These findings suggest that the Erk5-Smurf2Thr249 axis in BM-MSCs plays a critical role in the maintenance of proper bone homeostasis by preventing excessive osteogenesis in adult bone marrow.

Generation and mutational analysis of a transgenic murine model of the human MAF mutation.

Aymé-Gripp syndrome is an autosomal dominant multisystem disorder. The major clinical features of this syndrome include congenital cataracts, sensorineural hearing loss, intellectual disability, and a distinctive flat facial appearance. MAF has been identified as a causative gene of the syndrome, and heterozygous variants owing to impairment in glycogen synthase kinase 3 (GSK3)-mediated MAF phosphorylation shows related disorders. However, the underlying mechanisms of these types of disorders in affected individuals remain poorly understood. To explore the underlying mechanisms and discover new phenotypes, a murine model with a Maf mutation on a GSK3 phosphorylation motif, p.Thr58Ile, was generated using CRISPR-Cas9 gene editing. This is a homologous mutation to that in human patients. Our murine model exhibited similar phenotypes to those in humans, such as lens abnormalities, short stature, growth retardation, and abnormal skull morphology. The murine model showed decreased brain volume and malocclusion. Considering the sequencing and genotyping data, our models were successfully generated for the first time (to the best of our knowledge). Therefore, this study offers new and unique functional insights into human and murine MAF and novel clinical values of MAF pathogenic variants associated with changes in the functions of several organs based on a viable murine model.

Ribosome binding protein GCN1 regulates the cell cycle and cell proliferation and is essential for the embryonic development of mice.

Amino acids exert many biological functions, serving as allosteric regulators and neurotransmitters, as constituents in proteins and as nutrients. GCN2-mediated phosphorylation of eukaryotic initiation factor 2 alpha (elF2α) restores homeostasis in response to amino acid starvation (AAS) through the inhibition of the general translation and upregulation of amino acid biosynthetic enzymes and transporters by activating the translation of Gcn4 and ATF4 in yeast and mammals, respectively. GCN1 is a GCN2-binding protein that possesses an RWD binding domain (RWDBD) in its C-terminus. In yeast, Gcn1 is essential for Gcn2 activation by AAS; however, the roles of GCN1 in mammals need to be established. Here, we revealed a novel role of GCN1 that does not depend on AAS by generating two Gcn1 mutant mouse lines: Gcn1-knockout mice (Gcn1 KO mice (Gcn1-/-)) and RWDBD-deleted mutant mice (Gcn1ΔRWDBD mice). Both mutant mice showed growth retardation, which was not observed in the Gcn2 KO mice, such that the Gcn1 KO mice died at the intermediate stage of embryonic development because of severe growth retardation, while the Gcn1ΔRWDBD embryos showed mild growth retardation and died soon after birth, most likely due to respiratory failure. Extension of pregnancy by 24 h through the administration of progesterone to the pregnant mothers rescued the expression of differentiation markers in the lungs and prevented lethality of the Gcn1ΔRWDBD pups, indicating that perinatal lethality of the Gcn1ΔRWDBD embryos was due to simple growth retardation. Similar to the yeast Gcn2/Gcn1 system, AAS- or UV irradiation-induced elF2α phosphorylation was diminished in the Gcn1ΔRWDBD mouse embryonic fibroblasts (MEFs), suggesting that GCN1 RWDBD is responsible for GCN2 activity. In addition, we found reduced cell proliferation and G2/M arrest accompanying a decrease in Cdk1 and Cyclin B1 in the Gcn1ΔRWDBD MEFs. Our results demonstrated, for the first time, that GCN1 is essential for both GCN2-dependent stress response and GCN2-independent cell cycle regulation.

Generation and Characterization of a Cell Type-Specific, Inducible Cre-Driver Line to Study Olfactory Processing.

In sensory systems of the brain, mechanisms exist to extract distinct features from stimuli to generate a variety of behavioral repertoires. These often correspond to different cell types at various stages in sensory processing. In the mammalian olfactory system, complex information processing starts in the olfactory bulb, whose output is conveyed by mitral cells (MCs) and tufted cells (TCs). Despite many differences between them, and despite the crucial position they occupy in the information hierarchy, Cre-driver lines that distinguish them do not yet exist. Here, we sought to identify genes that are differentially expressed between MCs and TCs of the mouse, with an ultimate goal to generate a cell type-specific Cre-driver line, starting from a transcriptome analysis using a large and publicly available single-cell RNA-seq dataset (Zeisel et al., 2018). Many genes were differentially expressed, but only a few showed consistent expressions in MCs and at the specificity required. After further validating these putative markers using ISH, two genes (i.e., Pkib and Lbdh2) remained as promising candidates. Using CRISPR/Cas9-mediated gene editing, we generated Cre-driver lines and analyzed the resulting recombination patterns. This indicated that our new inducible Cre-driver line, Lbhd2-CreERT2, can be used to genetically label MCs in a tamoxifen dose-dependent manner, both in male and female mice, as assessed by soma locations, projection patterns, and sensory-evoked responses in vivo Hence, this is a promising tool for investigating cell type-specific contributions to olfactory processing and demonstrates the power of publicly accessible data in accelerating science.SIGNIFICANCE STATEMENT In the brain, distinct cell types play unique roles. It is therefore important to have tools for studying unique cell types specifically. For the sense of smell in mammals, information is processed first by circuits of the olfactory bulb, where two types of cells, mitral cells and tufted cells, output different information. We generated a transgenic mouse line that enables mitral cells to be specifically labeled or manipulated. This was achieved by looking for genes that are specific to mitral cells using a large and public gene expression dataset, and creating a transgenic mouse using the gene editing technique, CRISPR/Cas9. This will allow scientists to better investigate parallel information processing underlying the sense of smell.

Induction of Mutant Sik3Sleepy Allele in Neurons in Late Infancy Increases Sleep Need.

Sleep is regulated in a homeostatic manner. Sleep deprivation increases sleep need, which is compensated mainly by increased EEG δ power during non-rapid eye movement sleep (NREMS) and, to a lesser extent, by increased sleep amount. Although genetic factors determine the constitutive level of sleep need and sleep amount in mice and humans, the molecular entity behind sleep need remains unknown. Recently, we found that a gain-of-function Sleepy (Slp) mutation in the salt-inducible kinase 3 (Sik3) gene, which produces the mutant SIK3(SLP) protein, leads to an increase in NREMS EEG δ power and sleep amount. Since Sik3Slp mice express SIK3(SLP) in various types of cells in the brain as well as multiple peripheral tissues from the embryonic stage, the cell type and developmental stage responsible for the sleep phenotype in Sik3Slp mice remain to be elucidated. Here, we generated two mouse lines, synapsin1CreERT2 and Sik3ex13flox mice, which enable inducible Cre-mediated, conditional expression of SIK3(SLP) in neurons on tamoxifen administration. Administration of tamoxifen to synapsin1CreERT2 mice during late infancy resulted in higher recombination efficiency than administration during adolescence. SIK3(SLP) expression after late infancy increased NREMS and NREMS δ power in male synapsin1CreERT2; Sik3ex13flox/+ mice. The expression of SIK3(SLP) after adolescence led to a higher NREMS δ power without a significant change in NREMS amounts. Thus, neuron-specific expression of SIK3(SLP) after late infancy is sufficient to increase sleep.SIGNIFICANCE STATEMENT The propensity to accumulate sleep need during wakefulness and to dissipate it during sleep underlies the homeostatic regulation of sleep. However, little is known about the developmental stage and cell types involved in determining the homeostatic regulation of sleep. Here, we show that Sik3Slp allele induction in mature neurons in late infancy is sufficient to increase non-rapid eye movement sleep amount and non-rapid eye movement sleep δ power. SIK3 signaling in neurons constitutes an intracellular mechanism to increase sleep.

Alternative splicing for germ cell-specific Mga transcript can be eliminated without compromising mouse viability or fertility.

The stimulated by retinoic acid gene 8 (STRA8)/MEIOSIN complex and polycomb repressive complex (PRC) 1.6, a PRC1 subtype, are believed to be positive and negative regulators of meiotic onset, respectively. During meiotic initiation, the transcription repressive activity of PRC1.6 must be attenuated so that meiosis-related genes can be effectively activated by the STRA8/MEIOSIN complex. However, the molecular mechanisms that control the impairment of PRC1.6 function remain unclear. We recently demonstrated that the Mga gene, which encodes a scaffolding component of PRC1.6, produces variant mRNA by alternative splicing specifically during meiosis. Furthermore, the anomalous MGA protein encoded by the variant mRNA bears an intrinsic ability to function as a dominant negative regulator against the construction of PRC1.6 and is therefore assumed to be, at least in part, involved in impairment of the complex. Therefore, to unequivocally evaluate the physiological significance of Mga variant mRNA production in gametogenesis, we examined the consequences of a genetic manipulation that renders mice unable to produce Mga variant mRNA. Our data revealed that mutant mice were equivalent to wild-type mice in terms of viability and fertility. Our detailed examination of spermatogenesis also revealed that this genetic alteration is not associated with any apparent abnormalities in testis size, spermatogenic cycle, timing of meiotic onset, or marker gene expression of spermatogonia and spermatocytes. Taken together, these data indicate that the production of germ cell-specific Mga variant mRNA is dispensable not only for viability but also for gametogenesis.

Starvation-induced transcription factor CREBH negatively governs body growth by controlling GH signaling.

cAMP responsive element-binding protein H (CREBH) is a hepatic transcription factor to be activated during fasting. We generated CREBH knock-in flox mice, and then generated liver-specific CREBH transgenic (CREBH L-Tg) mice in an active form. CREBH L-Tg mice showed a delay in growth in the postnatal stage. Plasma growth hormone (GH) levels were significantly increased in CREBH L-Tg mice, but plasma insulin-like growth factor 1 (IGF1) levels were significantly decreased, indicating GH resistance. In addition, CREBH overexpression significantly increased hepatic mRNA and plasma levels of FGF21, which is thought to be as one of the causes of growth delay. However, the additional ablation of FGF21 in CREBH L-Tg mice could not correct GH resistance at all. CREBH L-Tg mice sustained GH receptor (GHR) reduction and the increase of IGF binding protein 1 (IGFBP1) in the liver regardless of FGF21. As GHR is a first step in GH signaling, the reduction of GHR leads to impairment of GH signaling. These data suggest that CREBH negatively regulates growth in the postnatal growth stage via various pathways as an abundant energy response by antagonizing GH signaling.