Betaine-homocysteine methyltransferase (BHMT) is an enzyme involved in one-carbon metabolism and plays a crucial role in maintaining liver health. In this study, we investigated the impact of liver-specific deletion of BHMT on liver dysfunction using a mouse model. We generated BHMT floxed mice and bred them with albumin Cre to generate liver-specific BHMT knockout (BHMT LKO) mice. Liver tissues harvested from six-month-old chow-fed BHMT floxed and LKO mice were characterized through histological, biochemical, and molecular analyses. BHMT LKO mice displayed a complete loss of hepatic expression of BHMT mRNA, protein and enzyme activity. Histopathological analysis revealed the development of hepatic steatosis in BHMT LKO mice compared to the floxed mice. These morphological changes were supported by biochemical analysis showing elevated levels of hepatic triglycerides in conjunction with a profound decrease in the methylation potential (i.e., reduced S-adenosylmethionine (SAM): S-adenosylhomocysteine (SAH) ratio), which was mainly driven by a six- to sevenfold increase in SAH levels. BHMT LKO mice also exhibited increased lipid peroxidation and lysosomal dysfunction compared to floxed mice. Early signs of inflammation were seen in the livers of BHMT LKO mice of both sexes, as evident from significant increase in CD68-positive cells and interleukin 1β levels. Additionally, there was a moderate increase in fibrosis, as evidenced by the upregulated expression of α-smooth muscle actin and collagen II levels and the histological assessment of picrosirius red-stained liver sections of BHMT LKO mice of both sexes compared to their respective counterparts. These findings demonstrate that hepatic BHMT deficiency promotes lipid accumulation, lysosomal/proteasomal dysfunction, and early inflammatory and fibrotic changes in the liver by reducing the methylation potential. Collectively, our results underscore BHMT as a critical regulator of liver homeostasis and a potential therapeutic target in liver-related disorders.

Abstract The regulation of nociceptor identity and function is essential, as disruptions can significantly influence pain sensation, yet our understanding of the molecular mechanisms involved remains incomplete. In this study, we identified ribonuclease 4 (RNase4) as selectively expressed in the unmyelinated nociceptor lineage. Analysis of RNase4-deficient mice and single-cell transcriptomic data revealed a cell-autonomous role for RNase4 in regulating nociceptor function. Moreover, in a neuropathic pain model, RNase4 expression was upregulated in nociceptors during the pain and recovery phases, and its deletion altered mechanical sensation. Additionally, RNase4 exerted non-cell- autonomous effects on the myelin structural organization of adjacent myelinated axons. Together, these findings implicate RNase4 as a dual regulator of nociceptor biology and myelin integrity, revealing a molecular pathway for pain regulation and nerve repair.

Buprenorphine has long been recognized as a mu opioid agonist with a distinctive and intricate pharmacological profile. It is a partial agonist at the mu opioid receptor, an antagonist at the kappa and delta opioid receptors, and an agonist at the nociception opioid receptor. Similar to other mu agonists such as morphine and fentanyl, buprenorphine can produce side effects, including tolerance, physical dependence, respiratory depression, and addiction. The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, generating an array of splice variants or isoforms, which are conserved from rodents to humans. These splice variants can be categorized into 2 main types, exon 1 (E1)-associated variants and exon 11 (E11)-associated variants. E1-associated variants primarily consist of full-length, 7-transmembrane C-terminal variants, whereas E11-associated variants are typically truncated 6-transmembrane variants. Previous studies established that buprenorphine analgesia in mice is dependent on both E1- and E11-associated variants. However, the role of these variants in buprenorphine analgesia and respiratory depression in rats remains unclear. In this study, we used CRISPR/Cas9 technology to develop 2 rat Oprm1 gene-targeting models in which E1- and E11-associated variants were selectively disrupted, aiming to investigate their roles in buprenorphine and morphine's actions. The results showed that both E1- and E11-associated variants are essential for buprenorphine's analgesic and respiratory depressional effects in rats, whereas morphine's effects are solely attributed to the E1-associated variants. These findings provide new and important insights into the distinct contributions of the E1- and E11-associated variants to the pharmacological actions of buprenorphine and morphine. SIGNIFICANCE STATEMENT: Differential dependences of buprenorphine and morphine analgesia and respiratory depression on Oprm1 exon 1- and exon 11-associated variants revealed in rat gene-targeting models provide new and important insights into unique contributions of these variants to buprenorphine and morphine actions.

Several genes guide inner ear development, and mutations in these genes can cause malformations that result in congenital hearing loss. However, the contribution of noncoding regulatory elements remains largely unclear. This study investigates the function of distal enhancer elements in the transcriptional regulation of GDF6 , a gene implicated in cochlear development. Using mouse models with targeted deletions, human inner ear organoids, and CRISPR interference (CRISPRi), we identified a downstream regulatory interval harboring a developmental enhancer required to maintain GDF6 expression during otic epithelial maturation and cochlear morphogenesis. Deletion of this regulatory region or targeting of CRISPRi-based repressors to these regions resulted in decreased GDF6 expression, failure of otic-epithelium development, and prevention of hair cell-like differentiation, reflecting cochlear aplasia observed in patients with corresponding genomic deletions. These findings highlight the contribution of long-range regulatory elements to auditory development and illustrate how their disruption contributes to human deafness.

Abstract ID 130415Poster Board 340The single-copy gene (OPRM1) encoding the mu opioid receptor (MOR) undergoes extensive alternative splicing, generating multiple splice variants. One set of OPRM1 variants, exon 7-associated full-length 7 transmembrane (TM) C-terminal splice variants (E7 variants), contain a unique intracellular C-terminal tail with 30 amino acids encoded by E7 that are highly conserved from rodents, primates to humans. E7 variants are abundantly expressed in the central nervous system with distinct distributions among brain regions. Cumulative evidence has indicated that these E7 variants play a crucial role in mediating various adverse effects associated with clinically used mu opioids, such as tolerance, reward, and respiratory depression. For example, truncating E7-enocded C-terminal tails in mice (mE7M-B6) attenuated morphine tolerance and reward, while not affecting analgesia. The current studies further establish the role of E7 variants in mediating mu opioid-induced tolerance, reward, and respiratory depression in naïve mice and mE7M-B6 by using antisense oligos (ASOs) and a newly developed rabbit monoclonal antibody (RabmAb) that target E7 sequences. Intracerebroventricular administration of either the ASO or RabmAb attenuated morphine tolerance measured by radiant-heat tail-flick assay and reward measured by conditioned place preference (CPP) in mice. Additionally, we generated a new mouse model (mMOR-1O-KI) in which only a single E7 variant, mMOR-1O, is expressed to investigate the in vivo functions of mMOR-1O. The results showed that mMOR-1O-KI mice had enhanced morphine tolerance and reward (CPP), complementing those the results from mE7M-B6 mice and further supporting the role of E7 variants in mediating these effects. Furthermore, fentanyl-induced tolerance, reward and respiratory depression measured by whole body plethysmography were significantly reduced in mE7M-B6 mice. Together, these studies indicate that targeting E7 variants presents a promising approach to mitigate tolerance, reward, and respiratory depression associated with clinically used mu opioids, while preserving their analgesic properties mediated by other Oprm1 7TM variants.Supported by grants from NIH (DA042888, DA046714, DA007242 and CA08748), the Mayday Foundation, Peter F. McManus Charitable Trust, Rutgers New Jersey Medical School and Brain Health Institute.

Synaptotagmin-9 (Syt9) is a Ca2+ sensor mediating fast synaptic release expressed in various parts of the brain. The presence and role of Syt9 in retina is unknown. We found evidence for Syt9 expression throughout the retina and created mice to conditionally eliminate Syt9 in a cre-dependent manner. We crossed Syt9fl/fl mice with Rho-iCre, HRGP-Cre, and CMV-Cre mice to generate mice in which Syt9 was eliminated from rods (rodSyt9CKO), cones (coneSyt9CKO), or whole animals (CMVSyt9). CMVSyt9 mice showed an increase in scotopic electroretinogram (ERG) b-waves evoked by bright flashes with no change in a-waves. Cone-driven photopic ERG b-waves were not significantly different in CMVSyt9 knockout mice and selective elimination of Syt9 from cones had no effect on ERGs. However, selective elimination from rods decreased scotopic and photopic b-waves as well as oscillatory potentials. These changes occurred only with bright flashes where cone responses contribute. Synaptic release was measured in individual rods by recording anion currents activated by glutamate binding to presynaptic glutamate transporters. Loss of Syt9 from rods had no effect on spontaneous or depolarization-evoked release. Our data show that Syt9 acts at multiple sites in the retina and suggest that it may play a role in regulating transmission of cone signals by rods.

BACKGROUND:Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called "pronuclear injection-based targeted transgenesis" (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT (i-PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre-mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre-LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad.RESULTS:To enable the i-PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice.CONCLUSIONS:Simultaneous use of PhiC31 and FLP in i-PITT approach allows insertion of donor plasmids containing Cre-loxP-based conditional expression cassettes.

Abstract ID 86969Poster Board 495Fentanyl, a potent synthetic analgesic acting primarily through the mu opioid receptor (MOR), has been implicated in two-thirds of recent US opioid overdose deaths, mainly due to respiratory depression. Understanding the mechanisms underpinning this is essential towards prevention and the development of new effective interventions. The MOR gene, Oprm1, undergoes extensive alternative splicing, generating numerous variants; some of which can classified into the following category: Exon 1-associated full-length 7 transmembrane carboxyl terminal variants (E1-variants). While the role of E1-variants in mu opioid pharmacology is well-established in mouse models studying analgesia, tolerance, dependence, and reward, their contribution to fentanyl-induced respiratory depression (FIRD) in rats remains unclear. Using rat Oprm1 E1-conditional knockout models (E1f/f), in which the coding exon and adjacent introns were floxed with loxPs, we observed dose-dependent FIRD in these rats, as measured by whole-body plethysmography. Conversely, global E1 knockout rats (E1-/-) were resistant to FIRD, highlighting the mediation of E1-variants in this process. Additionally, this study focuses on elucidating the role of E1-variants in brain regions implicated in FIRD: the parabrachial nucleus (PBN) and nucleus tractus solitarii (NTS). Employing tandem mass tags-based phosphorproteomics/proteomics (TMT PP/P-omics) and single-nucleus RNA-sequencing in E1f/f and E1-/- rat models, we identified differential expression of phosphoproteins and transcripts in response to fentanyl. Among several candidate genes, protein kinase C (PKC) emerged as a key regulator in FIRD, influencing critical signaling pathways. Administration of tamoxifen, a non-selective PKC inhibitor, via the intracerebroventricular route significantly reduced FIRD. This study sheds light on the molecular mechanisms underlying FIRD via E1-variants in the PBN and NTS, providing insights into potential therapeutic targets for mitigating opioid-induced respiratory depression.NIDA (R56 DA059061); Department of Anesthesiology, New Jersey Medical School; Brain Health Institute, Rutgers; The Mayday Foundation.

Objective: To elucidate the molecular mechanisms by which CD73 variants contribute to inflammation and atherosclerosis in humans. Approach and Results: To explore this knowledge gap, we conducted phenotype-wide association analysis (PheWAS) in a cohort of 25,000 patients to identify ASCVD-associated variants in the NT5E (CD73) gene in a non-biased manner. PheWAS analysis of coding variants identified that the rs200648774 single nucleotide polymorphisms in ecto-5’-nucleotidase (NT5E; CD73) that was associated with atherosclerosis (ICD-9; 440.x codes) in this cohort. The SNP rs200648774 (C-->T; MAF 0.000188) results in an Arginine (R) to Cysteine (C) substitution at amino acid 354 within the active site of CD73 (OR: 10.18, SE: 1.1; P=0.035). This Arginine residue is evolutionarily conserved across species. The variant protein is expressed but displays a loss of function (LOF; CD73 Cys354 variant). To examine the impact of the CD73 Cys354 variant , we generated a murine model using E fficient a dditions with s sDNA i nserts-CRISPR (Easi-CRISPR) gene editing. CD73-Arg354/Arg354 (cd73 wt/wt ) and CD73-Cys354/Cys354 (cd73 Δ/Δ ) mice in an Ldlr -/- background were placed on HFD for 12 weeks then sacrificed for quantification of aortic root atherosclerosis. Representative images of oil red O staining of aortic valve from CD73-Arg354/Arg354- Ldlr -/- (cd73 wt/wt , Ldlr -/- ) mice (Fig. A; Bar= 100 um) and CD73-Cys354/Cys354 Ldlr -/- (cd73 Δ/Δ , Ldlr -/- ) mice (Fig. B; Bar= 100 um). Quantified area of atherosclerosis in CD73-Cys354/Cys354 (cd73 Δ/Δ ) aortic roots were significantly higher in CD73-Cys354/Cys354 (cd73 Δ/Δ ) than in CD73-Arg354/Arg354 (cd73 wt/wt ) (Fig. C; p=0.0096). The Percentage of Oil Red O staining in aortic root atherosclerotic lesions was also significantly higher in CD73-Cys354/Cys354 (cd73 Δ/Δ ) aortic roots compared to CD73-Arg354/Arg354 (cd73 wt/wt ) aortic roots. Together these data demonstrate that the rs200648774 single nucleotide polymorphisms resulting in cysteine substitution for arginine in the active site of CD73 results in a loss of CD73 function that also associates with increased atherosclerosis in genetically susceptible mice . Conclusion: The LOF rs200648774 single nucleotide polymorphisms associates with atherosclerosis in humans and generation of the CD73-Cys354/Cys354 polymorphism in Ldlr -/- mice results in increased HFD induced atherosclerosis

SMARCA4 encodes one of two mutually exclusive ATPase subunits in the BRG/BRM associated factor (BAF) complex that is recruited by transcription factors (TFs) to drive chromatin accessibility and transcriptional activation. SMARCA4 is among the most recurrently mutated genes in human cancer, including ∼30% of germinal center (GC)-derived Burkitt lymphomas. In mice, GC-specific Smarca4 haploinsufficiency cooperated with MYC over-expression to drive lymphomagenesis. Furthermore, monoallelic Smarca4 deletion drove GC hyperplasia with centroblast polarization via significantly increased rates of centrocyte recycling to the dark zone. Mechanistically, Smarca4 loss reduced the activity of TFs that are activated in centrocytes to drive GC-exit, including SPI1 (PU.1), IRF family, and NF-κB. Loss of activity for these factors phenocopied aberrant BCL6 activity within murine centrocytes and human Burkitt lymphoma cells. SMARCA4 therefore facilitates chromatin accessibility for TFs that shape centrocyte trajectories, and loss of fine-control of these programs biases toward centroblast cell-fate, GC hyperplasia and lymphoma.

Custom oligonucleotides (oligos) are widely used reagents in biomedical research. Some common applications of oligos include polymerase chain reaction (PCR), sequencing, hybridization, microarray, and library construction. The reliability of oligos in such applications depends on their purity and specificity. Here, we report that commercially available oligos are frequently contaminated with nonspecific sequences (i.e. other unrelated oligonucleotides). Most of the oligos that we designed to amplify clustered regularly interspersed palindromic repeats (CRISPR) guide sequences contained nonspecific CRISPR guides. These contaminants were detected in research-grade oligos procured from eight commercial oligo-suppliers located in three different geographic regions of the world. Deep sequencing of some of the oligos revealed a variety of contaminants. Given the wide range of applications of oligos, the impact of oligo cross-contamination varies greatly depending on the field and the experimental method. Incorporating appropriate control experiments in research design can help ensure that the quality of oligo reagents meets the intended purpose. This can also minimize risk depending on the purposes for which the oligos are used.

Supplementary Figure 1 from The Human Orthologue of <i>Drosophila</i> Ecdysoneless Protein Interacts with p53 and Regulates Its Function

XLSX file - 43K, Number of metastatic deposits for Ecd Scr and KD cell injected mice

Myocarditis is a predominant cause of congestive heart failure and sudden death in children and young adolescents that can lead to dilated cardiomyopathy. Lymphocytic myocarditis mediated by T cells can result from the recognition of cardiac antigens that may involve CD4 or CD8 T cells or both. In this report, we describe the generation of T cell receptor (TCR) transgenic mice on a C57BL/6 genetic background specific to cardiac myosin heavy chain (Myhc)-α 334–352 and make the following observations: First, we verified that Myhc-α 334–352 was immunogenic in wild-type C57BL/6 mice and induced antigen-specific CD4 T cell responses despite being a poor binder of IAb; however, the immunized animals developed only mild myocarditis. Second, TCRs specific to Myhc-α 334–352 in transgenic mice were expressed in both CD4 and CD8 T cells, suggesting that the expression of epitope-specific TCR is common to both cell types. Third, although T cells from naïve transgenic mice did not respond to Myhc-α 334–352, both CD4 and CD8 T cells from animals immunized with Myhc-α 334–352 responded to the peptide, indicating that antigen priming is necessary to break tolerance. Fourth, although the transgenic T cells could produce significant amounts of interferon-γ and interleukin-17, the immunized animals developed only mild disease, indicating that other soluble factors might be necessary for developing severe myocarditis. Alternatively, the C57BL/6 genetic background might be a major contributing factor for resistance to the development of myocarditis. Taken together, our model permits the determination of the roles of both CD4 and CD8 T cells to understand the disease-resistance mechanisms of myocarditis in a single transgenic system antigen-specifically.

Mucin4 (MUC4) appears early during pancreatic intraepithelial neoplasia-1 (PanIN1), coinciding with the expression of epidermal growth factor receptor-1 (EGFR). The EGFR signaling is required for the onset of Kras-driven pancreatic ductal adenocarcinoma (PDAC); however, the players and mechanisms involved in sustained EGFR signaling in early PanIN lesions remain elusive. We generated a unique Esai -CRISPR-based Muc4 conditional knockout murine model to evaluate its effect on PDAC pathology. The Muc4 depletion in the autochthonous murine model carrying K-ras and p53 mutations (K-ras G12D ; TP53 R172H ; Pdx-1cre, KPC) to generate the KPCM4 −/− murine model showed a significant delay in the PanIN lesion formation with a significant reduction ( p < 0.01) in EGFR (Y1068) and ERK1/2 (T202/Y204) phosphorylation. Further, a significant decrease ( p < 0.01) in Sox9 expression in PanIN lesions of KPCM4 −/− mice suggested the impairment of acinar-to-ductal metaplasia in Muc4-depleted cells. The biochemical analyses demonstrated that MUC4, through its juxtamembrane EGF-like domains, interacts with the EGFR ectodomain, and its cytoplasmic tail prevents EGFR ubiquitination and subsequent proteasomal degradation upon ligand stimulation, leading to sustained downstream oncogenic signaling. Targeting the MUC4 and EGFR interacting interface provides a promising strategy to improve the efficacy of EGFR-targeted therapies in PDAC and other MUC4-expressing malignancies.

mRNAs produced in a cell are almost always translated within the same cell. Some mRNAs are transported to other cells of the organism through processes involving membrane nanotubes or extracellular vesicles. A recent report describes a surprising new phenomenon of encapsulating mRNAs inside virus-like particles (VLPs) to deliver them to other cells in a process that was named SEND (Selective Endogenous eNcapsidation for cellular Delivery). Although the seminal work demonstrates the SEND process in cultured cells, it is unknown whether this phenomenon occurs in vivo . Here, we demonstrate the SEND process in living organisms using specially designed genetically engineered mouse models. Our proof of principle study lays a foundation for the SEND-VLP system to potentially be used as a gene therapy tool to deliver therapeutically important mRNAs to tissues.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology has made it possible to produce genome-edited (GE) animals more easily and rapidly than before. In most cases, GE mice are produced by microinjection (MI) or by in vitro electroporation (EP) of CRISPR reagents into fertilized eggs (zygotes). Both of these approaches require ex vivo handling of isolated embryos and their subsequent transfer into another set of mice (called recipient or pseudopregnant mice). Such experiments are performed by highly skilled technicians (especially for MI). We recently developed a novel genome editing method, called "GONAD (Genome-editing via Oviductal Nucleic Acids Delivery)," which can completely eliminate the ex vivo handling of embryos. We also made improvements to the GONAD method, termed "improved-GONAD (i-GONAD)." The i-GONAD method involves injection of CRISPR reagents into the oviduct of an anesthetized pregnant female using a mouthpiece-controlled glass micropipette under a dissecting microscope, followed by EP of the entire oviduct allowing the CRISPR reagents to enter into the zygotes present inside the oviduct, in situ. After the i-GONAD procedure, the mouse recovered from anesthesia is allowed to continue the pregnancy to full term to deliver its pups. The i-GONAD method does not require pseudopregnant female animals for embryo transfer, unlike the methods relying on ex vivo handling of zygotes. Therefore, the i-GONAD method can reduce the number of animals used, compared to the traditional methods. In this chapter, we describe some newer technical tips about the i-GONAD method. Additionally, even though the detailed protocols of GONAD and i-GONAD have been published elsewhere (Gurumurthy et al., Curr Protoc Hum Genet 88:15.8.1-15.8.12, 2016 Nat Protoc 14:2452-2482, 2019), we provide all the protocol steps of i-GONAD in this chapter so that the reader can find most of the information, needed for performing i-GONAD experiments, in one place.

Patients with mutations in the TMPRSS3 gene suffer from recessive deafness DFNB8/DFNB10 for whom cochlear implantation is the only treatment option. Poor cochlear implantation outcomes are seen in some patients. To develop biological treatment for TMPRSS3 patients, we generated a knock-in mouse model with a frequent human DFNB8 TMPRSS3 mutation. The Tmprss3A306T/A306T homozygous mice display delayed onset progressive hearing loss similar to human DFNB8 patients. Using AAV2 as a vector to carry a human TMPRSS3 gene, AAV2-h TMPRSS3 injection in the adult knock-in mouse inner ears results in TMPRSS3 expression in the hair cells and the spiral ganglion neurons. A single AAV2-h TMPRSS3 injection in aged Tmprss3A306T/A306T mice leads to sustained rescue of the auditory function, to a level similar to the wildtype mice. AAV2-h TMPRSS3 delivery rescues the hair cells and the spiral ganglions. This is the first study to demonstrate successful gene therapy in an aged mouse model of human genetic deafness. This study lays the foundation to develop AAV2-h TMPRSS3 gene therapy to treat DFNB8 patients, as a standalone therapy or in combination with cochlear implantation.