Genetic Mouse Research Articles

To unwind the biological knots: The DNA/RNA G-quadruplex resolvase RHAU (DHX36) in development and disease.

The G-quadruplex (G4) sequences are short fragments of 4-interval triple guanine (G) with frequent and ubiquitous distribution in the genome and RNA transcripts. The G4 sequences are usually folded into secondary "knot" structure via Hoogsteen hydrogen bond to exert negative regulation on a variety of biological processes, including DNA replication and transcription, mRNA translation, and telomere maintenance. Recent structural biological and mouse genetics studies have demonstrated that RHAU (DHX36) can bind and unwind the G4 "knots" to modulate embryonic development and postnatal organ function. Deficiency of RHAU gives rise to embryonic lethality, impaired organogenesis, and organ dysfunction. These studies uncovered the pivotal G4 resolvase function of RHAU to release the G4 barrier, which plays fundamental roles in development and physiological homeostasis. This review discusses the latest advancements and findings in deciphering RHAU functions using animal models.

Klf5 defines alveolar epithelial type 1 cell lineage commitment during lung development and regeneration.

Alveolar epithelial cell fate decisions drive lung development and regeneration. Using transcriptomic and epigenetic profiling coupled with genetic mouse and organoid models, we identified the transcription factor Klf5 as an essential determinant of alveolar epithelial cell fate across the lifespan. We show that although dispensable for both adult alveolar epithelial type 1 (AT1) and alveolar epithelial type 2 (AT2) cell homeostasis, Klf5 enforces AT1 cell lineage fidelity during development. Using infectious and non-infectious models of acute respiratory distress syndrome, we demonstrate that Klf5 represses AT2 cell proliferation and enhances AT2-AT1 cell differentiation in a spatially restricted manner during lung regeneration. Moreover, exvivo organoid assays identify that Klf5 reduces AT2 cell sensitivity to inflammatory signaling to drive AT2-AT1 cell differentiation. These data define the roll of a major transcriptional regulator of AT1 cell lineage commitment and of the AT2 cell response to inflammatory crosstalk during lung regeneration.

S109: ONCOGENIC DEUBIQUITINATION CONTROLS TYROSINE KINASE SIGNALING AND THERAPY RESPONSE IN ACUTE LYMPHOBLASTIC LEUKEMIA

Background: Dysregulation of kinase signaling pathways via mutations favors tumor cell survival and resistance to therapy and it is common in cancer. Our data unveil how dysregulated deubiquitination controls signaling pathways, leading to cancer cell survival and drug non-response, and suggest novel therapeutic combinations towards targeting T-cell acute lymphoblastic leukemia (T-ALL). Here, we reveal a novel mechanism of post-translational regulation of kinase signaling and nuclear receptor activity via deubiquitination in acute leukemia. Aims: This study aims at 1) characterizing the function of an oncogenic complex composed by two deubiquitinating enzymes in in vitro and in vivo leukemia systems and 2) testing the association of deubiquitinase activity with resistance to therapy in acute lymphoblastic leukemia. Methods: We use genetic mouse and human:mouse xenograft models of T-cell leukemia, biochemical studies (quantitative global proteomics, phosphoproteomics and ubiquitination analysis) and high-throughput molecular biology (chromatin conformation capture (HiC), chromatin accessibility (ATAC-Seq) and gene expression (RNA-Seq)) analyses. Results: We observed that the ubiquitin specific protease 11 (USP11) is highly expressed in lymphoblastic leukemia and associates with poor prognosis in this disease. USP11 ablation inhibits leukemia growth in vitro and in vivo, sparing normal hematopoiesis and thymus development, suggesting that USP11 could be a therapeutic target in leukemia. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK). Deubiquitination of LCK controls its activity, thereby altering T cell receptor signaling. Impairment of LCK activity leads to increased expression of the glucocorticoid receptor transcript, culminating into transcriptional activation of pro-apoptotic target genes, and sensitizes cells to glucocorticoids in T cell leukemia patient samples. The transcriptional activation of pro-apoptotic target genes, such as BCL2L11, is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Pharmacological inhibition of USP7 or genetic knockout of USP7 in combination treatment of glucocorticoid displayed improved anti-T-ALL efficacy in vivo. Image:Summary/Conclusion: Our data unveil how dysregulated deubiquitination controls signaling pathways, leading to cancer cell survival and drug non-response, and suggest novel therapeutic combinations towards targeting T-cell leukemia.

Abstract 1669: Transient IGF1 exposure stably reprograms EWS-FLI1 immortalized embryonal limb-derived mesenchymal stem cell-like cells to full transformation

Abstract Ewing sarcoma (EwS), an aggressive bone and soft tissue cancer predominantly occurring during adolescence, is driven by the chimeric EWS-FLI1 oncogene. It is assumed that a permissive cellular context or potentially additional complementing mutations are necessary for the EWS-FLI1 fusion to exert its full oncogenic effect. Prior efforts to generate genetic EwS mouse models failed because of high EWS-FLI1 induced apoptosis when expressed in various candidate progenitor cell types. In our study, we restricted EWS-FLI1 expression to mesenchymal stem cells during endochondral bone formation by crossing a mouse containing EWS-FLI1 knocked into the Rosa26 locus with a mouse expressing Prx1-driven Cre recombinase. However, the resulting EFprx1 progeny died after birth as functional consequence of severe skeletal malformations resulting from differentiation arrest of early chondrocytes. In vitro, these embryonic EWS-FLI1 transgenic chondrocyte-like mesenchymal stem cells (EFprx1 MSCLC) are immortal, but not fully transformed. However, we observed elevated levels of cleaved PARP-1, a surrogate marker of apoptosis. This apparent increased cellular turnover was paralleled by down-regulation of anti-apoptotic Bcl2a family genes as identified by RNA-seq analysis of EFprx1 MSCLC. The peak incidence of human EwS occurs during puberty, a developmental period in which several growth promoting hormones are highly enriched in the bone microenvironment. Among them is IGF1, which is known for its anti-apoptotic activity through upregulation of Bcl2 family genes. As previous studies implied IGF1 signaling essential in EwS pathogenesis, we speculated that IGF1 treatment may aid in the transformation process of EFprx1 MSCLC. In fact, a significant increase in soft-agar colony formation was observed upon IGF1 treatment for 4 weeks. To test whether IGF1 had irreversibly reprogrammed EFPrx1 MSCLC, we picked colonies grown in presence of IGF1, expanded and re-assayed them after 5 weeks in absence of the hormone. Strikingly, EFprx1 MSCLC retained their transformed phenotype not only in the soft-agar assay but also upon xenotransplantation in mice. Gene set enrichment analysis of upregulated genes from these stably hormone-reprogramed fully transformed EFprx1 MSCLC showed a significant enrichment for the EWS-FLI1 specific transcriptional signature and cell cycle-related pathways, while apoptotic gene sets were downregulated. Our data suggest that transient IGF1 exposure in the bone microenvironment may be required for EWS-FLI1 driven transformation. We propose that EwS formation could be influenced by hormonal changes during puberty. Based on these findings, we postulate that correct developmental timing of EWS-FLI1 expression may play a pivotal role for the generation of an EwS mouse model. Citation Format: Rahil Noorizadeh, Florian Halbritter, Barbara Sax, Wolfgang Mikulits, Richard Moriggl, Heinrich Kovar. Transient IGF1 exposure stably reprograms EWS-FLI1 immortalized embryonal limb-derived mesenchymal stem cell-like cells to full transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1669.

Abstract 1372: Macrophages impair antigen specific CD8+ T cell response against HCC in NAFLD mice

Abstract Background: Liver cancer is a leading cause of cancer-related deaths. Non-alcoholic fatty liver disease (NAFLD) has become a burgeoning etiology of liver cancer. Emerging data shows that NAFLD alters adaptive T cell immunity and has a profound influence on both liver cancer development and efficacy of immunotherapy. Tumor antigen-specific T cells recognize tumor and are crucial for controlling malignancy. However, their populations are often low, and detection requires special tools. This study utilized a novel mouse model of HCC to investigate how the tumor antigen-specific T cell response is affected in NAFLD. Methods: We generated a doxycycline-inducible MHC-I OVA257-264 and MHC-II OVA323-339 antigen-expressing HCC cell line which allowed us to investigate tumor antigen-specific T cell responses. The cell line was seeded orthotopically into the livers of diet-induced (methionine choline-deficient [MCD]) and genetic (Ob/Ob [Ob]) NAFLD mouse models. OVA257-264 specific CD8+ T cells were detected by flow cytometry using H-2Kb/OVA257-264 tetramer. Pharmacological depletion experiments were used to identify additional effector cells. Results: The creation of an inducible MHC-I and -II OVA antigen-expressing HCC cell line was validated in both in-vitro and in-vivo settings. Tumor expression of highly immunogenic OVA antigens caused a strong tumor suppression which was mostly mediated by CD8+ T cells in mice fed with normal diet. The inverse correlation between percentage of OVA257-264 specific CD8+ T cells with tumor size further supported their critical role in tumor control. In contrast, in NAFLD mice fed MCD diet as well as Ob/Ob mice, tumor OVA antigens induction failed to reduce liver tumor growth. The presence of OVA257-264 specific CD8+ T cells in NAFLD mice suggested that NAFLD did not inhibit the generation of tumor antigen-specific T cells. This was supported by the finding that OVA257-264 specific CD8+ T cells from NAFLD mice had similar cytotoxic potential, activation, degranulation, PD-1 expression, and effector memory phenotype compared to controls, suggesting an indirect mechanism for the impaired anti-tumor immunity. Immunoprofiling revealed a drastic increase of macrophages in tumor-bearing NAFLD livers. Depletion of macrophages with clodronate reversed the NAFLD caused resistance to tumor-antigen dependent tumor suppression. It was observed that there were minimal changes to the amount of TAS CD8+ T cells in doxycycline-treated mice irrespective of clodronate depletion. There was, however, higher markers of activation such as CD69 and TNFα in clodronate-depleted mice compared to controls. Conclusions: NAFLD impairs antigen-specific CD8+ T cell immunity against liver tumor. The effect was not due to reduced generation or intrinsic functional changes of tumor antigen-specific CD8+ T cells but caused by accumulated macrophages in the liver environment. Citation Format: John C. McVey, Benjamin L. Green, Benjamin Ruf, Tim F. Greten, Chi Ma. Macrophages impair antigen specific CD8+ T cell response against HCC in NAFLD mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1372.

Abstract 3420: Tgfbm3b is a natural germline genetic variant that modifies anti-tumor responses to anti-PD-1 immunotherapy through its action on the tumor microenvironment

Abstract Our goal for this study is to use a mouse genetics approach to identify host germline genetic variants that determine therapeutic responses to cancer immunotherapy (IMT) and to validate these findings in cancer patients treated with α-PD-1. The information gathered may lead to drug targets that potentiate IMT responses and genetic markers may contribute to risk scores for response to IMTs. Immune Checkpoint blockade (ICB) therapy leads to remarkable clinical responses, including highly durable complete responses in a fraction of patients. Consequently, α-PD-1/PDL-1 is now first-line therapy for treating melanoma and NSCLC (non-small cell lung carcinoma). However, most patients do not respond to α-PD-1 therapy, and few show systemic adverse effects. There is therefore an unmet need to develop biomarkers and drug targets that will potentiate therapeutic responses to ICBs. Firstly, we found that blockade of active TGFβ using therapeutic antibodies synergizes with α-PD-1 therapy to potentiate tumor rejection, thus, we hypothesized that variant genetic modifiers of TGFβ1 may influence outcomes of α-PD-1 therapy. By utilizing Tgfbm3b-C57BL/6 (Tgfbm3b-C57) mice carrying a genetic variation of TGFβ1 to study the anti-tumor response to IMT, we confirmed that Tgfbm3b-C57 potentiated anti-tumor responses to α-PD-1 and to combinatorial α-PD-1/α-TGFβ IMT with higher induction of CD8+ T cell-mediated anti-tumor cytotoxicity. The C57 allele contains gene Adam17 that encodes an amino acid variant, L613V, and a charge change at amino acid 113 (D113N). Protein ADAM17 is a sheddase that involves in TNFα biosynthesis and sheds the TGFβ type I receptor (TβRI) to attenuate TGFβ signaling. To understand the molecular function of the variants, the vectors encoding wildtype (NIH) ADAM17 that carries D113, L613 versus the variant (C57) ADAM17 that has N113, V613, are being transfected and tested for the action on a panel of ADAM17 substrates including TNFα and TβRI on NIH3T3 cell line. We are also investigating whether genetic variation within the syntenic human TGFBM3 locus confers differential survival benefit to patients after α-PD-1 therapy. We will confirm the genetic association of ADAM17 SNPs and screen the TGFBM3b locus from LPIN1 to HPCAL1, and YWHAQ to DDEF2. The dataset includes genome-wide association data from progression-free survival analysis of 2000 NSCLC patients following α-PD-1 therapy. The preliminary data from our initial cohort (n=862) of NSCLC patients on PD-1 inhibitors are currently being evaluated. By investigating the molecular and cellular mechanisms by which Tgfbm loci influence ICB outcomes and addressing the relevance to human cancer patients, this project not only addresses an important basic science question but may ultimately lead to novel biomarkers or drug targets that can improve IMT outcomes and reduce adverse effects. Citation Format: Szu-Ying Chen, Ons Mamai, Rosemary Akhurst. Tgfbm3b is a natural germline genetic variant that modifies anti-tumor responses to anti-PD-1 immunotherapy through its action on the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3420.

Somatic Genetics Analysis of Sleep in Adult Mice.

Classical forward and reverse mouse genetics require germline mutations and, thus, are unwieldy to study sleep functions of essential genes or redundant pathways. It is also time-consuming to conduct EEG/EMG-based mouse sleep screening because of labor-intensive surgeries and genetic crosses. Here, we describe a highly accurate SleepV (video) system and adeno-associated virus (AAV)-based adult brain chimeric (ABC)-expression/KO platform for somatic genetics analysis of sleep in adult male or female mice. A pilot ABC screen identifies CREB and CRTC1, of which constitutive or inducible expression significantly reduces quantity and/or quality of non-rapid eye movement sleep. Whereas ABC-KO of exon 13 of Sik3 by AAV-Cre injection in Sik3-E13flox/flox adult mice phenocopies Sleepy (Sik3Slp/+) mice, ABC-CRISPR of Slp/Sik3 reverses hypersomnia of Sleepy mice, indicating a direct role of SLP/SIK3 kinase in sleep regulation. Multiplex ABC-CRISPR of both orexin/hypocretin receptors causes narcolepsy episodes, enabling one-step analysis of redundant genes in adult mice. Therefore, this somatic genetics approach should facilitate high-throughput analysis of sleep regulatory genes, especially for essential or redundant genes, in adult mice by skipping mouse development and minimizing genetic crosses.SIGNIFICANCE STATEMENT The molecular mechanisms of mammalian sleep regulation remain unclear. Classical germline mouse genetics are unwieldy to study sleep functions of essential genes or redundant pathways. The EEG/EMG-based mouse sleep screening is time-consuming because of labor-intensive surgeries and lengthy genetic crosses. To overcome these “bottlenecks,” we developed a highly accurate video-based sleep analysis system and adeno-associated virus-mediated ABC-expression/KO platform for somatic genetics analysis of sleep in adult mice. These methodologies facilitate rapid identification of sleep regulatory genes, but also efficient mechanistic studies of the molecular pathways of sleep regulation in mice.

Sustained hyperarousal induced by acute stress in tryptophan-hydroxylase-2 genetic deficient male mice

Sustained hyperarousal induced by acute stress in tryptophan-hydroxylase-2 genetic deficient male mice

Aberrant methylmalonylation underlies methylmalonic acidemia and is attenuated by an engineered sirtuin.

Organic acidemias such as methylmalonic acidemia (MMA) are a group of inborn errors of metabolism that typically arise from defects in the catabolism of amino and fatty acids. Accretion of acyl-CoA species is postulated to underlie disease pathophysiology, but the mechanism(s) remain unknown. Here, we surveyed hepatic explants from patients with MMA and unaffected donors, in parallel with samples from various mouse models of methylmalonyl-CoA mutase deficiency. We found a widespread posttranslational modification, methylmalonylation, that inhibited enzymes in the urea cycle and glycine cleavage pathway in MMA. Biochemical studies and mouse genetics established that sirtuin 5 (SIRT5) controlled the metabolism of MMA-related posttranslational modifications. SIRT5 was engineered to resist acylation-driven inhibition via lysine to arginine mutagenesis. The modified SIRT5 was used to create an adeno-associated viral 8 (AAV8) vector and systemically delivered to mutant and control mice. Gene therapy ameliorated hyperammonemia and reduced global methylmalonylation in the MMA mice.

Further Evidence that Inhibition of Neuronal Voltage-Gated Calcium Channels Contributes to the Hypnotic Effect of Neurosteroid Analogue, 3β-OH.

We recently reported that a neurosteroid analogue with T-channel-blocking properties (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH), induced hypnosis in rat pups without triggering neuronal apoptosis. Furthermore, we found that the inhibition of the CaV3.1 isoform of T-channels contributes to the hypnotic properties of 3β-OH in adult mice. However, the specific mechanisms underlying the role of other subtypes of voltage-gated calcium channels in thalamocortical excitability and oscillations in vivo during 3β-OH-induced hypnosis are largely unknown. Here, we used patch-clamp recordings from acute brain slices, in vivo electroencephalogram (EEG) recordings, and mouse genetics with wild-type (WT) and CaV2.3 knock-out (KO) mice to further investigate the molecular mechanisms of neurosteroid-induced hypnosis. Our voltage-clamp recordings showed that 3β-OH inhibited recombinant CaV2.3 currents. In subsequent current-clamp recordings in thalamic slices ex vivo, we found that selective CaV2.3 channel blocker (SNX-482) inhibited stimulated tonic firing and increased the threshold for rebound burst firing in WT animals. Additionally, in thalamic slices we found that 3β-OH inhibited spike-firing more profoundly in WT than in mutant mice. Furthermore, 3β-OH reduced bursting frequencies in WT but not mutant animals. In ensuing in vivo experiments, we found that intra-peritoneal injections of 3β-OH were less effective in inducing LORR in the mutant mice than in the WT mice, with expected sex differences. Furthermore, the reduction in total α, β, and low γ EEG power was more profound in WT than in CaV2.3 KO females over time, while at 60 min after injections of 3β-OH, the increase in relative β power was higher in mutant females. In addition, 3β-OH depressed EEG power more strongly in the male WT than in the mutant mice and significantly increased the relative δ power oscillations in WT male mice in comparison to the mutant male animals. Our results demonstrate for the first time the importance of the CaV2.3 subtype of voltage-gated calcium channels in thalamocortical excitability and the oscillations that underlie neurosteroid-induced hypnosis.

The role of intraspinal sensory neurons in the control of quadrupedal locomotion

From swimming to walking and flying, animals have evolved specific locomotor strategies to thrive in different habitats. All types of locomotion depend on the integration of motor commands and sensory information to generate precisely coordinated movements. Cerebrospinal-fluid-contacting neurons (CSF-cN) constitute a vertebrate sensory system that monitors CSF composition and flow. In fish, CSF-cN modulate swimming activity in response to changes in pH and bending of the spinal cord; however, their role in mammals remains unknown. We used mouse genetics to study their function in quadrupedal locomotion. We found that CSF-cN are directly integrated into spinal motor circuits. The perturbation of CSF-cN function does not affect general motor activity nor the generation of locomotor rhythm and pattern but results in specific defects in skilled movements. These results identify a role for mouse CSF-cN in adaptive motor control and indicate that this sensory system evolved a novel function to accommodate the biomechanical requirements of limb-based locomotion.

Leveraging mouse genetics to generate heavy-chain only antibodies for therapeutic application

Abstract Conventional antibodies (Abs) consist of two heavy and two light immunoglobulin chains, while heavy-chain only antibodies (HCAbs), like those found in camelid and shark species, are unique because they lack a light chain binding partner. The unique structure of these HCAbs impart advantageous properties to these smaller Abs, including greater accessibility to their target antigens, higher hydrophilicity, and greater conformational stability under environmental stresses. We have generated a mouse model, the IgG3dCH1 strain, that can produce HCAbs from the endogenous murine heavy chain locus. We have shown that these mice undergo efficient B cell development in the bone marrow, and generate a diverse repertoire of B cells that populate all peripheral immune compartments. While single-cell sequencing analysis of B cells revealed some biases in VH and JH usage, it also highlighted the tremendous diversity of rearrangements that give rise to B cells bearing these single chain antibodies. We’ve been able to show that upon immunization, IgG3dCH1 animals are protected from lethal bacterial challenge with a pathogenic strain of Enterobacter hormaechei and generate pathogen specific IgG. Further, immunization with SARS-CoV2 stabilized prefusion spike protein elicited HCAbs with a KD in the nM range, measured via bio-layer interferometry. These data support this mouse being utilized as a platform for the generation of pathogen-specific HCAbs that can be further engineered for testing and application for translational outcomes. Supported by grants from NIH (T32 AI007180, R43 AI136141).

Autonomic control of energy balance and glucose homeostasis.

Neurons in the central nervous system (CNS) communicate with peripheral organs largely via the autonomic nervous system (ANS). Through such communications, the sympathetic and parasympathetic efferent divisions of the ANS may affect thermogenesis and blood glucose levels. In contrast, peripheral organs send feedback to the CNS via hormones and autonomic afferent nerves. These humoral and neural feedbacks, as well as neural commands from higher brain centers directly or indirectly shape the metabolic function of autonomic neurons. Notably, recent developments in mouse genetics have enabled more detailed studies of ANS neurons and circuits, which have helped elucidate autonomic control of metabolism. Here, we will summarize the functional organization of the ANS and discuss recent updates on the roles of neural and humoral factors in the regulation of energy balance and glucose homeostasis by the ANS.

Transient receptor potential melastatin 8 is required for nitroglycerin- and calcitonin gene-related peptide-induced migraine-like pain behaviors in mice.

Migraine is a complex neurovascular disorder that is one of the leading causes of disability and a reduced quality of life. Even with such a high societal impact, our understanding of the cellular and molecular mechanisms that contribute to migraine headaches is limited. To address this complex disorder, several groups have performed genome-wide association studies to elucidate migraine susceptibility genes, with many identifying transient receptor potential melastatin 8 (TRPM8), a cold-sensitive cation channel expressed in peripheral afferents innervating the trigeminovascular system, and the principal mediator of cold and cold pain associated with injury and disease. Interestingly, these migraine-associated single-nucleotide polymorphisms reside in noncoding regions of TRPM8, with those correlated with reduced migraine risk exhibiting lower TRPM8 expression and decreased cold sensitivity. Nonetheless, as a role for TRPM8 in migraine has yet to be defined, we sought to address this gap in our knowledge using mouse genetics and TRPM8 antagonism to determine whether TRPM8 channels or neurons are required for migraine-like pain (mechanical allodynia and facial grimace) in inducible migraine models. Our results show that both evoked and spontaneous pain behaviors are dependent on both TRPM8 channels and neurons, as well as required in both acute and chronic migraine models. Moreover, inhibition of TRPM8 channels prevented acute but not established chronic migraine-like pain. These results are consistent with its association with migraine in genetic analyses and establish that TRPM8 channels are a component of the underlying mechanisms of migraine.

In vitro investigation of mammalian peri-implantation embryogenesis†.

The embryos attach and invade the uterus, establishing the connection with their mother in peri-implantation development. During this period, the pluripotent epiblast cells of the embryo undergo symmetry breaking, cell lineage allocation, and morphogenetic remodeling, accompanied by the dramatic changes of transcriptomic, epigenomic, and signaling pathways, and preparing the stage for their differentiation and gastrulation. The progress in mouse genetics and stem cell biology has advanced the knowledge of these transformations, which are still largely hindered by the hard accessibility of natural embryos. To gain insight into mammalian peri-implantation development, much effort has been made in the field. Recently, advances in the prolonged in vitro culture of blastocysts, the derivation of multiple pluripotent stem cells, and the construction of stem cell-based embryo-like models have opened novel avenues to investigate peri-implantation development in mammals, especially humans. Combining with other emerging new technologies, these new models will substantially promote the comprehension of mammalian peri-implantation development, thus accelerating the progress of reproductive and regenerative medicine.

L-cysteine modulates visceral nociception mediated by the CaV2.3 R-type calcium channels

CaV2.3 channels are subthreshold voltage-gated calcium channels that play crucial roles in neurotransmitter release and regulation of membrane excitability, yet modulation of these channels with endogenous molecules and their role in pain processing is not well studied. Here, we hypothesized that an endogenous amino acid l-cysteine could be a modulator of these channels and may affect pain processing in mice. To test this hypothesis, we employed conventional patch-clamp technique in the whole-cell configuration using recombinant CaV2.3 subunit stably expressed in human embryonic kidney (HEK-293) cells. We found in our in vitro experiments that l-cysteine facilitated gating and increased the amplitudes of recombinant CaV2.3 currents likely by chelating trace metals that tonically inhibit the channel. In addition, we took advantage of mouse genetics in vivo using the acetic acid visceral pain model that was performed on wildtype and homozygous Cacna1e knockout male littermates. In ensuing in vivo experiments, we found that l-cysteine administered both subcutaneously and intraperitoneally evoked more prominent pain responses in the wildtype mice, while the effect was completely abolished in knockout mice. Conversely, intrathecal administration of l-cysteine lowered visceral pain response in the wildtype mice, and again the effect was completely abolished in the knockout mice. Our study strongly suggests that l-cysteine-mediated modulation of CaV2.3 channels plays an important role in visceral pain processing. Furthermore, our data are consistent with the contrasting roles of CaV2.3 channels in mediating visceral nociception in the peripheral and central pain pathways.

Beatrice Mintz (1921-2022): an innovator in embryo research and cancer biology

Beatrice Mintz (1921-2022): an innovator in embryo research and cancer biology

GIPR Is Predominantly Localized to Nonadipocyte Cell Types Within White Adipose Tissue.

The incretin hormone glucose-dependent insulinotropic polypeptide (GIP) augments glucose-dependent insulin secretion through its receptor expressed on islet β-cells. GIP also acts on adipose tissue; yet paradoxically, both enhanced and reduced GIP receptor (GIPR) signaling reduce adipose tissue mass and attenuate weight gain in response to nutrient excess. Moreover, the precise cellular localization of GIPR expression within white adipose tissue (WAT) remains uncertain. We used mouse genetics to target Gipr expression within adipocytes. Surprisingly, targeting Cre expression to adipocytes using the adiponectin (Adipoq) promoter did not produce meaningful reduction of WAT Gipr expression in Adipoq-Cre:Giprflx/flx mice. In contrast, adenoviral expression of Cre under the control of the cytomegalovirus promoter, or transgenic expression of Cre using nonadipocyte-selective promoters (Ap2/Fabp4 and Ubc) markedly attenuated WAT Gipr expression. Analysis of single-nucleus RNA-sequencing, adipose tissue data sets localized Gipr/GIPR expression predominantly to pericytes and mesothelial cells rather than to adipocytes. Together, these observations reveal that adipocytes are not the major GIPR+ cell type within WAT-findings with mechanistic implications for understanding how GIP and GIP-based co-agonists control adipose tissue biology.

Thalamic T-Type Calcium Channels as Targets for Hypnotics and General Anesthetics.

General anesthetics mainly act by modulating synaptic inhibition on the one hand (the potentiation of GABA transmission) or synaptic excitation on the other (the inhibition of NMDA receptors), but they can also have effects on numerous other proteins, receptors, and channels. The effects of general anesthetics on ion channels have been the subject of research since the publication of reports of direct actions of these drugs on ion channel proteins. In particular, there is considerable interest in T-type voltage-gated calcium channels that are abundantly expressed in the thalamus, where they control patterns of cellular excitability and thalamocortical oscillations during awake and sleep states. Here, we summarized and discussed our recent studies focused on the CaV3.1 isoform of T-channels in the nonspecific thalamus (intralaminar and midline nuclei), which acts as a key hub through which natural sleep and general anesthesia are initiated. We used mouse genetics and in vivo and ex vivo electrophysiology to study the role of thalamic T-channels in hypnosis induced by a standard general anesthetic, isoflurane, as well as novel neuroactive steroids. From the results of this study, we conclude that CaV3.1 channels contribute to thalamocortical oscillations during anesthetic-induced hypnosis, particularly the slow-frequency range of δ oscillations (0.5–4 Hz), by generating “window current” that contributes to the resting membrane potential. We posit that the role of the thalamic CaV3.1 isoform of T-channels in the effects of various classes of general anesthetics warrants consideration.

What Have We Learned (or Expect to) From Analysis of Murine Genetic Models Related to Substance Use Disorders?

The tremendous public health problem created by substance use disorders (SUDs) presents a major opportunity for mouse genetics. Inbred mouse strains exhibit substantial and heritable differences in their responses to drugs of abuse (DOA) and in many of the behaviors associated with susceptibility to SUD. Therefore, genetic discoveries emerging from analysis of murine genetic models can provide critically needed insight into the neurobiological effects of DOA, and they can reveal how genetic factors affect susceptibility drug addiction. There are already indications, emerging from our prior analyses of murine genetic models of responses related to SUDs that mouse genetic models of SUD can provide actionable information, which can lead to new approaches for alleviating SUDs. Lastly, we consider the features of murine genetic models that enable causative genetic factors to be successfully identified; and the methodologies that facilitate genetic discovery.