Gene-targeted Mice Research Articles

Claustrum and dorsal endopiriform cortex complex cell-identity is determined by Nurr1 and regulates hallucinogenic-like states in mice

The Claustrum/dorsal endopiriform cortex complex (CLA) is an enigmatic brain region with extensive glutamatergic projections to multiple cortical areas. The transcription factor Nurr1 is highly expressed in the CLA, but its role in this region is not understood. By using conditional gene-targeted mice, we show that Nurr1 is a crucial regulator of CLA neuron identity. Although CLA neurons remain intact in the absence of Nurr1, the distinctive gene expression pattern in the CLA is abolished. CLA has been hypothesized to control hallucinations, but little is known of how the CLA responds to hallucinogens. After the deletion of Nurr1 in the CLA, both hallucinogen receptor expression and signaling are lost. Furthermore, functional ultrasound and Neuropixel electrophysiological recordings revealed that the hallucinogenic-receptor agonists’ effects on functional connectivity between prefrontal and sensorimotor cortices are altered in Nurr1-ablated mice. Our findings suggest that Nurr1-targeted strategies provide additional avenues for functional studies of the CLA.

Epigenetic mechanisms differentially regulate blood pressure and renal dysfunction in male and female Npr1 haplotype mice.

We determined the epigenetic mechanisms regulating mean arterial pressure (MAP) and renal dysfunction in guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene-targeted mice. The Npr1 (encoding NPRA) gene-targeted mice were treated with class 1 specific histone deacetylase inhibitor (HDACi) mocetinostat (MGCD) to determine the epigenetic changes in a sex-specific manner. Adult male and female Npr1 haplotype (1-copy; Npr1+/-), wild-type (2-copy; Npr1+/+), and gene-duplicated heterozygous (3-copy; Npr1++/+) mice were intraperitoneally injected with MGCD (2 mg/kg) for 14 days. BP, renal function, histopathology, and epigenetic changes were measured. One-copy male mice showed significantly increased MAP, renal dysfunction, and fibrosis than 2-copy and 3-copy mice. Furthermore, HDAC1/2, collagen1alpha-2 (Col1α-2), and alpha smooth muscle actin (α-SMA) were significantly increased in 1-copy mice compared with 2-copy controls. The expression of antifibrotic microRNA-133a was attenuated in 1-copy mice but to a greater extent in males than females. NF-κB was localized at significantly lower levels in cytoplasm than in the nucleus with stronger DNA binding activity in 1-copy mice. MGCD significantly lowered BP, improved creatinine clearance, and repaired renal histopathology. The inhibition of class I HDACs led to a sex-dependent distinctive stimulation of acetylated positive histone marks and inhibition of methylated repressive histone marks in Npr1 1-copy mice; however, it epigenetically lowered MAP, repaired renal fibrosis, and proteinuria and suppressed NF-kB differentially in males versus females. Our results suggest a role for epigenetic targets affecting hypertension and renal dysfunction in a sex-specific manner.

Propagation of distinct CWD prion strains during peripheral and intracerebral challenges of gene-targeted mice

Since prion diseases result from infection and neurodegeneration of the central nervous system (CNS), experimental characterizations of prion strain properties customarily rely on the outcomes of intracerebral challenges. However, natural transmission of certain prions, including those causing chronic wasting disease (CWD) in elk and deer, depends on propagation in peripheral host compartments prior to CNS infection. Using gene-targeted GtE and GtQ mice, which accurately control cellular elk or deer PrP expression, we assessed the impact that peripheral or intracerebral exposures play on CWD prion strain propagation and resulting CNS abnormalities. Whereas oral and intraperitoneal transmissions produced identical neuropathological outcomes in GtE and GtQ mice and preserved the naturally convergent conformations of elk and deer CWD prions, intracerebral transmissions generated CNS prion strains with divergent biochemical properties in GtE and GtQ mice that were changed compared to their native counterparts. While CWD replication kinetics remained constant during iterative peripheral transmissions and brain titers reflected those found in native hosts, serial intracerebral transmissions produced 10-fold higher prion titers and accelerated incubation times. Our demonstration that peripherally and intracerebrally challenged Gt mice develop dissimilar CNS diseases which result from the propagation of distinct CWD prion strains points to the involvement of tissue-specific cofactors during strain selection in different host compartments. Since peripheral transmissions preserved the natural features of elk and deer prions, whereas intracerebral propagation produced divergent strains, our findings illustrate the importance of experimental characterizations using hosts that not only abrogate species barriers but also accurately recapitulate natural transmission routes of native strains.

Extraneural infection route restricts prion conformational variability and attenuates the impact of quaternary structure on infectivity.

Prions can exist as different strains that consist of conformational variants of the misfolded, pathogenic prion protein isoform PrPSc. Defined by stably transmissible biological and biochemical properties, strains have been identified in a spectrum of prion diseases, including chronic wasting disease (CWD) of wild and farmed cervids. CWD is highly contagious and spreads via direct and indirect transmission involving extraneural sites of infection, peripheral replication and neuroinvasion of prions. Here, we investigated the impact of infection route on CWD prion conformational selection and propagation. We used gene-targeted mouse models expressing deer PrP for intracerebral or intraperitoneal inoculation with fractionated or unfractionated brain homogenates from white-tailed deer, harboring CWD strains Wisc-1 or 116AG. Upon intracerebral inoculation, Wisc-1 and 116AG-inoculated mice differed in conformational stability of PrPSc. In brains of mice infected intraperitoneally with either inoculum, PrPSc propagated with identical conformational stability and fewer PrPSc deposits in most brain regions than intracerebrally inoculated animals. For either inoculum, PrPSc conformational stability in brain and spinal cord was similar upon intracerebral infection but significantly higher in spinal cords of intraperitoneally infected animals. Inoculation with fractionated brain homogenates resulted in lower variance of survival times upon intraperitoneal compared to intracerebral infection. In summary, we demonstrate that extraneural infection mitigates the impact of PrPSc quaternary structure on infection and reduces conformational variability of PrPSc propagated in the brain. These findings provide new insights into the evolution of stable CWD strains in natural, extraneural transmissions.

Norwegian moose CWD induces clinical disease and neuroinvasion in gene-targeted mice expressing cervid S138N prion protein.

Chronic wasting disease (CWD) is a prion disease affecting deer, elk and moose in North America and reindeer, moose and red deer in Northern Europe. Pathogenesis is driven by the accumulation of PrPSc, a pathological form of the host's cellular prion protein (PrPC), in the brain. CWD is contagious among North American cervids and Norwegian reindeer, with prions commonly found in lymphatic tissue. In Nordic moose and red deer CWD appears exclusively in older animals, and prions are confined to the CNS and undetectable in lymphatic tissues, indicating a sporadic origin. We aimed to determine transmissibility, neuroinvasion and lymphotropism of Nordic CWD isolates using gene-targeted mice expressing either wild-type (138SS/226QQ) or S138N (138NN/226QQ) deer PrP. When challenged with North American CWD strains, mice expressing S138N PrP did not develop clinical disease but harbored prion seeding activity in brain and spleen. Here, we infected these models intracerebrally or intraperitoneally with Norwegian moose, red deer and reindeer CWD isolates. The moose isolate was the first CWD type to cause full-blown disease in the 138NN/226QQ model in the first passage, with 100% attack rate and shortened survival times upon second passage. Furthermore, we detected prion seeding activity or PrPSc in brains and spinal cords, but not spleens, of 138NN/226QQ mice inoculated intraperitoneally with the moose isolate, providing evidence of prion neuroinvasion. We also demonstrate, for the first time, that transmissibility of the red deer CWD isolate was restricted to transgenic mice overexpressing elk PrPC (138SS/226EE), identical to the PrP primary structure of the inoculum. Our findings highlight that susceptibility to clinical disease is determined by the conformational compatibility between prion inoculum and host PrP primary structure. Our study indicates that neuroinvasion of Norwegian moose prions can occur without, or only very limited, replication in the spleen, an unprecedented finding for CWD.

Regulation of placental amino acid transport in health and disease.

Abnormal fetal growth, i.e., intrauterine growth restriction (IUGR) or fetal growth restriction (FGR) and fetal overgrowth, is associated with increased perinatal morbidity and mortality and is strongly linked to the development of metabolic and cardiovascular disease in childhood and later in life. Emerging evidence suggests that changes in placental amino acid transport may contribute to abnormal fetal growth. This review is focused on amino acid transport in the human placenta, however, relevant animal models will be discussed to add mechanistic insights. At least 25 distinct amino acid transporters with different characteristics and substrate preferences have been identified in the human placenta. Of these, System A, transporting neutral nonessential amino acids, and System L, mediating the transport of essential amino acids, have been studied in some detail. Importantly, decreased placental Systems A and L transporter activity is strongly associated with IUGR and increased placental activity of these two amino acid transporters has been linked to fetal overgrowth in human pregnancy. An array of factors in the maternal circulation, including insulin, IGF-1, and adiponectin, and placental signaling pathways such as mTOR, have been identified as key regulators of placental Systems A and L. Studies using trophoblast-specific gene targeting in mice have provided compelling evidence that changes in placental Systems A and L are mechanistically linked to altered fetal growth. It is possible that targeting specific placental amino acid transporters or their upstream regulators represents a novel intervention to alleviate the short- and long-term consequences of abnormal fetal growth in the future.

Post-transcriptional regulation and subcellular localization of G-protein γ7 subunit: implications for striatal function and behavioral responses to cocaine.

The striatal D1 dopamine receptor (D1R) and A2a adenosine receptor (A2aR) signaling pathways play important roles in drug-related behaviors. These receptors activate the Golf protein comprised of a specific combination of αolfβ2γ7 subunits. During assembly, the γ7 subunit sets the cellular level of the Golf protein. In turn, the amount of Golf protein determines the collective output from both D1R and A2aR signaling pathways. This study shows the Gng7 gene encodes multiple γ7 transcripts differing only in their non-coding regions. In striatum, Transcript 1 is the predominant isoform. Preferentially expressed in the neuropil, Transcript 1 is localized in dendrites where it undergoes post-transcriptional regulation mediated by regulatory elements in its 3' untranslated region that contribute to translational suppression of the γ7 protein. Earlier studies on gene-targeted mice demonstrated loss of γ7 protein disrupts assembly of the Golf protein. In the current study, morphological analysis reveals the loss of the Golf protein is associated with altered dendritic morphology of medium spiny neurons. Finally, behavioral analysis of conditional knockout mice with cell-specific deletion of the γ7 protein in distinct populations of medium spiny neurons reveals differential roles of the Golf protein in mediating behavioral responses to cocaine. Altogether, these findings provide a better understanding of the regulation of γ7 protein expression, its impact on Golf function, and point to a new potential target and mechanisms for treating addiction and related disorders.

Batf3+ DCs and the 4-1BB/4-1BBL axis are required at the effector phase in the tumor microenvironment for PD-1/PD-L1 blockade efficacy

The cellular source of positive signals that reinvigorate Tcells within the tumor microenvironment (TME) for the therapeutic efficacy of programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade has not been clearly defined. We now show that Batf3-lineage dendritic cells (DCs) are essential in this process. Flow cytometric analysis, gene-targeted mice, and blocking antibody studies revealed that 4-1BBL is a major positive co-stimulatory signal provided by these DCs within the TME that translates to CD8+ Tcell functional reinvigoration and tumor regression. Immunofluorescence and spatial transcriptomics on human tumor samples revealed clustering of Batf3+ DCs and CD8+ Tcells, which correlates with anti-PD-1 efficacy. In addition, proximity to Batf3+ DCs within the TME is associated with CD8+ Tcell transcriptional states linked to anti-PD-1 response. Our results demonstrate that Batf3+ DCs within the TME are critical for PD-1/PD-L1 blockade efficacy and indicate a major role for the 4-1BB/4-1BB ligand (4-1BBL) axis during this process.

Integrin β1–mediated mast cell immune-surveillance of blood vessel content

BackgroundImmunoglobulin E, IgE)-mediated degranulation of mast cells, MCs) provides rapid protection against environmental hazards, including animal venoms. A fraction of tissue-resident MCs intimately associates with blood vessels. These perivascular MCs were reported to extend projections into the vessel lumen and to be the first MCs to acquire intravenously injected IgE, suggesting that IgE loading of MCs depends on their vascular association. ObjectiveWe sought to elucidate the molecular basis of the MC-blood vessel interaction and to determine its relevance for IgE-mediated immune responses. MethodsWe selectively inactivated the Itgb1 gene, encoding the β1 chain of integrin adhesion molecules, ITGB1; in MCs by conditional gene targeting in mice. We analyzed skin MCs for blood vessel association, surface IgE density, capability to bind circulating antibody specific for MC surface molecules, as well as in vivo responses to antigen administered via different routes. ResultsLack of ITGB1 expression severely compromised MC blood vessel association. ITGB1-deficient MCs showed normal densities of surface IgE but reduced binding of intravenously injected antibodies. While their capacity to degranulate in response to IgE ligation in vivo was unimpaired, anaphylactic responses to antigen circulating in the vasculature were largely abolished. ConclusionITGB1-mediated association of MCs with blood vessels is key for MC immune-surveillance of blood vessel content, but is dispensable for slow steady-state loading of endogenous IgE onto tissue-resident MCs.

Demyelination and Na+ Channel Redistribution Underlie Auditory and Vestibular Dysfunction in PMP22-Null Mice.

Altered expression of peripheral myelin protein 22 (PMP22) results in demyelinating peripheral neuropathy. PMP22 exhibits a highly restricted tissue distribution with marked expression in the myelinating Schwann cells of peripheral nerves. Auditory and vestibular Schwann cells and the afferent neurons also express PMP22, suggesting a unique role in hearing and balancing. Indeed, neuropathic patients diagnosed with PMP22-linked hereditary neuropathies often present with auditory and balance deficits, an understudied clinical complication. To investigate the mechanism by which abnormal expression of PMP22 may cause auditory and vestibular deficits, we studied gene-targeted PMP22-null mice. PMP22-null mice exhibit an unsteady gait, have difficulty maintaining balance, and live for only ∼3-5 weeks relative to unaffected littermates. Histological analysis of the inner ear revealed reduced auditory and vestibular afferent nerve myelination and profound Na+ channel redistribution without PMP22. Yet, Na+ current density was unaltered, in stark contrast to increased K+ current density. Atypical postsynaptic densities and a range of neuronal abnormalities in the organ of Corti were also identified. Analyses of auditory brainstem responses (ABRs) and vestibular sensory-evoked potential (VsEP) revealed that PMP22-null mice had auditory and vestibular hypofunction. These results demonstrate that PMP22 is required for hearing and balance, and the protein is indispensable for the formation and maintenance of myelin in the peripheral arm of the eighth nerve. Our findings indicate that myelin abnormalities and altered signal propagation in the peripheral arm of the auditory nerve are likely causes of auditory deficits in patients with PMP22-linked neuropathies.

Microglial Rac1 is essential for experience-dependent brain plasticity and cognitive performance

Microglia, the largest population of brain immune cells, continuously interact with synapses to maintain brain homeostasis. In this study, we use conditional cell-specific gene targeting in mice with multi-omics approaches and demonstrate that the RhoGTPase Rac1 is an essential requirement for microglia to sense and interpret the brain microenvironment. This is crucial for microglia-synapse crosstalk that drives experience-dependent plasticity, a fundamental brain property impaired in several neuropsychiatric disorders. Phosphoproteomics profiling detects a large modulation of RhoGTPase signaling, predominantly of Rac1, in microglia of mice exposed to an environmental enrichment protocol known to induce experience-dependent brain plasticity and cognitive performance. Ablation of microglial Rac1 affects pathways involved in microglia-synapse communication, disrupts experience-dependent synaptic remodeling, and blocks the gains in learning, memory, and sociability induced by environmental enrichment. Our results reveal microglial Rac1 as a central regulator of pathways involved in the microglia-synapse crosstalk required for experience-dependent synaptic plasticity and cognitive performance.

Embryonic Stem Cell Gene Targeting and Chimera Production in Mice.

Producing a custom gene mutation in embryonic stem (ES) cells, whether through homologous recombination or CRISPR-Cas gene editing, is the first step along the way to getting the mutation into live mice. However, there are a number of additional steps along the way, each presenting technical challenges. Here, we provide a guide for troubleshooting when the results are not as expected and to distinguish technical problems from possible biological effects of the mutation. From the isolation of clonal lines of targeted ES cells through the production of ES cell chimeras with the targeted ES cell clone, we discuss common technical problems and their most likely causes and solutions. We also provide guidance for situations where the mutation has a phenotype in the form of a dominant effect on ES cells or in chimeras.

Alk1 acts in non-endothelial VE-cadherin+ perineurial cells to maintain nerve branching during hair homeostasis

Vascular endothelial (VE)-cadherin is a well-recognized endothelial cell marker. One of its interacting partners, the TGF-β receptor Alk1, is essential in endothelial cells for adult skin vasculature remodeling during hair homeostasis. Using single-cell transcriptomics, lineage tracing and gene targeting in mice, we characterize the cellular and molecular dynamics of skin VE-cadherin+ cells during hair homeostasis. We describe dynamic changes of VE-cadherin+ endothelial cells specific to blood and lymphatic vessels and uncover an atypical VE-cadherin+ cell population. The latter is not a predicted adult endovascular progenitor, but rather a non-endothelial mesenchymal perineurial cell type, which forms nerve encapsulating tubular structures that undergo remodeling during hair homeostasis. Alk1 acts in the VE-cadherin+ perineurial cells to maintain proper homeostatic nerve branching by enforcing basement membrane and extracellular matrix molecular signatures. Our work implicates the VE-cadherin/Alk1 duo, classically known as endothelial-vascular specific, in perineurial-nerve homeostasis. This has broad implications in vascular and nerve disease.

Amino-terminal proteolytic fragment of the axon growth inhibitor Nogo-A (Rtn4A) is upregulated by injury and promotes axon regeneration

After adult mammalian central nervous system injury, axon regeneration is extremely limited or absent, resulting in persistent neurological deficits. Axon regeneration failure is due in part to the presence of inhibitory proteins, including NogoA (Rtn4A), from which two inhibitory domains have been defined. When these inhibitory domains are deleted, but an amino-terminal domain is still expressed in a gene trap line, mice show axon regeneration and enhanced recovery from injury. In contrast, when there is no amino-terminal Nogo-A fragment in the setting of inhibitory domain deletion, then axon regeneration and recovery are indistinguishable from WT. These data indicated that an amino-terminal Nogo-A fragment derived from the gene trap might promote axon regeneration, but this had not been tested directly and production of this fragment without gene targeting was unclear. Here, we describe posttranslation production of an amino-terminal fragment of Nogo-A from the intact gene product. This fragment is created by proteolysis near amino acid G214-N215 and levels are enhanced by axotomy. Furthermore, this fragment promotes axon regeneration in vitro and acts cell autonomously in neurons, in contrast to the inhibitory extracellular action of other Nogo-A domains.Proteins interacting with the amino-terminal Nogo-A fragment by immunoprecipitation include HSPA8 (HSC70, HSP7C). Suppression of HSPA8 expression by shRNA decreases axon regeneration from cerebral cortical neurons and overexpression increases axon regeneration. Moreover, the amino-terminal Nogo-A fragment increases HSPA8 chaperone activity. These data provide an explanation for varied results in different gene-targeted Nogo-A mice, as well as revealing an axon regeneration promoting domain of Nogo-A.

Abstract 134: Role Of Epigenetic Mechanisms Regulating Renal Pathology And Disorders In Global Npr1 Gene-targeted Mutant Mice In Sex-dependent Manner

The objective of this study was to investigate the effect of class I-specific histone deacetylase inhibitor, mocetinostat (MGCD0103; MGCD) in the regulation of epigenetic mechanisms and renal pathology and disorders in Npr1 (coding for natriuretic peptide receptor A; NPRA) gene-disrupted haplotype mice. Male and female Npr1 gene-disrupted (1-copy; +/- ), wild-type (2-copy; +/+ ) and gene-duplicated (3-copy; ++/+) mice were administrated with MGCD (2 mg/kg) by intraperitoneal injection at alternate days for 2-weeks. The renal NPRA protein levels and guanylyl cyclase (GC) activity were significantly lower in 1-copy mice and higher in 3-copy than in 2-copy mice; however, MGCD increased NPRA protein levels and GC activity. The vehicle-treated females showed lower SBP than male mice, whereas both sexes of 1-copy mice had higher SBPs, and 3-copy mice had lower SBPs as compared with 2-copy mice. Treatment with MGCD distinctly reduced SBP in male (1-copy, Δ-21±2.0 mmHg, p<0.001; 2-copy, Δ-5±0.6 mmHg, p<0.01; and 3-copy, Δ-4±0.5 mmHg, p<0.05) and female mice (1-copy, Δ-17±2.1 mmHg, p<0.01; 2-copy, Δ-3±0.5 mmHg, p<0.01; and 3-copy, Δ-3±0.8 mmHg). Hematoxylin and eosin staining showed pronounced renal morphological damages in 1-copy male than female mice as compared to 2-copy and 3-copy mice. One-copy male mice exhibited a greater degree of mesangial matrix expansion and interstitial nephritis as compared with 2-copy and 3-copy mice, which was significantly (p<0.1) decreased with MGCD treatment as compared with vehicle-treated mice. Creatinine clearance was significantly reduced in 1-copy male mice (52%; p<0.05) than female mice (35%; p<0.05) compared with 2-copy mice. The renal HDAC activity and HDAC1 and 2 protein levels in 1-copy male mice were significantly (p<0.05) higher than female mice as compared with 2-copy mice. MGCD markedly increased acetylation of histone marks (H3-K9, H3-K27, and H3-K18) and greatly reduced the renal pathologies as compared with controls. The present results demonstrate that MGCD upregulates NPRA activity and repairs renal pathology epigenetically in 1-copy haplotype mice. These findings will have important implications for treatment of hypertension and renal injury in humans in both sexes.

Conservation of vCJD Strain Properties After Extraction and In Vitro Propagation of PrPSc from Archived Formalin-Fixed Brain and Appendix Tissues Using Highly Sensitive Protein Misfolding Cyclic Amplification.

Three retrospective lymphoreticular tissue studies (Appendix I, II, and III) aimed to estimate the UK prevalence of variant Creutzfeldt-Jakob disease (vCJD), following exposure of the population to the bovine spongiform encephalopathy (BSE) agent, in the late 1980s and 1990s. These studies evaluated the presence of abnormal prion protein aggregates, in archived formalin-fixed paraffin-embedded (FFPE) appendectomy samples, by immunohistochemical detection. Although there was concordance in the estimated prevalence of vCJD from these studies, the identification of positive specimens from pre- and post-BSE-exposure periods in Appendix III study has raised questions regarding the nature and origin of the detected abnormal prion protein. We applied a robust and novel approach in the extraction of disease-associated prion protein (PrPSc) present in frozen and FFPE samples of brain and appendix from a patient with pathologically confirmed vCJD. The extracted material was used to seed the highly sensitive protein misfolding cyclic amplification assay (hsPMCA) to investigate the in vitro and in vivo propagation properties of the extracted abnormal prion protein. We demonstrate that PrPSc can be successfully extracted from FFPE appendix tissue and propagated in vitro. Bioassay in wild-type and gene-targeted mouse models confirmed that the extracted and amplified product is infectious and retains strain properties consistent with vCJD. This provides a highly sensitive and reliable platform for subsequent analysis of the archived FFPE appendix tissue derived from the Appendix II and III surveys, to further evaluate the nature of the abnormal PrP detected in the positive samples.

LYVE-1+ macrophages form a collaborative CCR5-dependent perivascular niche that influences chemotherapy responses in murine breast cancer

Tumor-associated macrophages (TAMs) are a heterogeneous population of cells that facilitate cancer progression. However, our knowledge of the niches of individual TAM subsets and their development and function remain incomplete. Here, we describe a population of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)-expressing TAMs, which form coordinated multi-cellular "nest" structures that are heterogeneously distributed proximal to vasculature in tumors of a spontaneous murine model of breast cancer. We demonstrate that LYVE-1+ TAMs develop in response to IL-6, which induces their expression of the immune-suppressive enzyme heme oxygenase-1 and promotes a CCR5-dependent signaling axis, which guides their nest formation. Blocking the development of LYVE-1+ TAMs or their nest structures, using gene-targeted mice, results in an increase in CD8+ Tcell recruitment to the tumor and enhanced response to chemotherapy. This study highlights an unappreciated collaboration of a TAM subset to form a coordinated niche linked to immune exclusion and resistance to anti-cancer therapy.

Neuroendocrine mechanism of gastric acid secretion: Historical perspectives and recent developments in physiology and pharmacology.

The physiology of gastric acid secretion is one of the earliest subjects in medical literature and has been continuously studied since 1833. Starting with the notion that neural stimulation alone drives acid secretion, progress in understanding the physiology and pathophysiology of this process has led to the development of therapeutic strategies for patients with acid-related diseases. For instance, understanding the physiology of parietal cells led to the developments of histamine 2 receptor blockers, proton pump inhibitors (PPIs), and recently, potassium-competitive acid blockers. Furthermore, understanding the physiology and pathophysiology of gastrin has led to the development of gastrin/CCK2 receptor (CCK2 R) antagonists. The need for refinement of existing drugs in patients have led to second and third generation drugs with better efficacy at blocking acid secretion. Further understanding of the mechanism of acid secretion by gene targeting in mice has enabled us to dissect the unique role for each regulator to leverage and justify the development of new targeted therapeutics for acid-related disorders. Further research on the mechanism of stimulation of gastric acid secretion and the physiological significances of gastric acidity in gut microbiome is needed in the future.

Heterozygosity for cervid S138N polymorphism results in subclinical CWD in gene-targeted mice and progressive inhibition of prion conversion

Prions are proteinaceous infectious particles that replicate by structural conversion of the host-encoded cellular prion protein (PrPC), causing fatal neurodegenerative diseases in mammals. Species-specific amino acid substitutions (AAS) arising from single nucleotide polymorphisms within the prion protein gene (Prnp) modulate prion disease pathogenesis, and, in several instances, reduce susceptibility of homo- or heterozygous AAS carriers to prion infection. However, a mechanistic understanding of their protective effects against clinical disease is missing. We generated gene-targeted mouse infection models of chronic wasting disease (CWD), a highly contagious prion disease of cervids. These mice express wild-type deer or PrPC harboring the S138N substitution homo- or heterozygously, a polymorphism found exclusively in reindeer (Rangifer tarandus spp.) and fallow deer (Dama dama). The wild-type deer PrP-expressing model recapitulated CWD pathogenesis including fecal shedding. Encoding at least one 138N allele prevented clinical CWD, accumulation of protease-resistant PrP (PrPres) and abnormal PrP deposits in the brain tissue. However, prion seeding activity was detected in spleens, brains, and feces of these mice, suggesting subclinical infection accompanied by prion shedding. 138N-PrPC was less efficiently converted to PrPres in vitro than wild-type deer (138SS) PrPC. Heterozygous coexpression of wild-type deer and 138N-PrPC resulted in dominant-negative inhibition and progressively diminished prion conversion over serial rounds of protein misfolding cyclic amplification. Our study indicates that heterozygosity at a polymorphic Prnp codon can confer the highest protection against clinical CWD and highlights the potential role of subclinical carriers in CWD transmission.

The leukotriene B4 receptors BLT1 and BLT2 as potential therapeutic targets.

Leukotriene B4 (LTB4 ) was recognized as an arachidonate-derived chemotactic factor for inflammatory cells and an important drug target even before the molecular identification of its receptors. We cloned the high- and low-affinity LTB4 receptors, BLT1 and BLT2, respectively, and examined their functions by generating and studying gene-targeted mice. BLT1 is involved in the pathogenesis of various inflammatory and immune diseases, including asthma, psoriasis, contact dermatitis, allergic conjunctivitis, age-related macular degeneration, and immune complex-mediated glomerulonephritis. Meanwhile, BLT2 is a high-affinity receptor for 12-hydroxyheptadecatrienoic acid, which is involved in the maintenance of dermal and intestinal barrier function, and the acceleration of skin and corneal wound healing. Thus, BLT1 antagonists and BLT2 agonists are promising candidates in the treatment of inflammatory diseases.