Publications by authors named "Matthew F Starost"

63 Publications

SARS-CoV-2 infection induces protective immunity and limits transmission in Syrian hamsters.

Life Sci Alliance 2021 04 11;4(4). Epub 2021 Feb 11.

Division of Viral Products, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA

A critical question in understanding the immunity to SARS-COV-2 is whether recovered patients are protected against re-challenge and transmission upon second exposure. We developed a Syrian hamster model in which intranasal inoculation of just 100 TCID virus caused viral pneumonia. Aged hamsters developed more severe disease and even succumbed to SARS-CoV-2 infection, representing the first lethal model using genetically unmodified laboratory animals. After initial viral clearance, the hamsters were re-challenged with 10 TCID SARS-CoV-2 and displayed more than 4 log reduction in median viral loads in both nasal washes and lungs in comparison to primary infections. Most importantly, re-challenged hamsters were unable to transmit virus to naïve hamsters, and this was accompanied by the presence of neutralizing antibodies. Altogether, these results show that SARS-CoV-2 infection induces protective immunity that not only prevents re-exposure but also limits transmission in hamsters. These findings may help guide public health policies and vaccine development and aid evaluation of effective vaccines against SARS-CoV-2.
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http://dx.doi.org/10.26508/lsa.202000886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893819PMC
April 2021

Pde8b haploinsufficiency in mice is associated with modest adrenal defects, impaired steroidogenesis, and male infertility, unaltered by concurrent PKA or Wnt activation.

Mol Cell Endocrinol 2021 02 15;522:111117. Epub 2020 Dec 15.

Section on Endocrinology and Genetics, Program on Developmental Endocrinology & Genetics (PDEGEN), Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health, Bethesda, MD, 20892, USA. Electronic address:

PDE8B, PRKAR1A and the Wnt/β-catenin signaling are involved in endocrine disorders. However, how PDEB8B interacts with both Wnt and protein kinase A (PKA) signaling in vivo remains unknown. We created a novel Pde8b knockout mouse line (Pde8b); Pde8b haploinsufficient (Pde8b) mice were then crossed with mice harboring: (1) constitutive beta-catenin activation (Pde8b;ΔCat) and (2) Prkar1a haploinsufficieny (Pde8b;Prkar1a). Adrenals and testes from mice (3-12-mo) were evaluated in addition to plasma corticosterone, aldosterone and Dkk3 concentrations, and the examination of expression of steroidogenesis-, Wnt- and cAMP/PKA-related genes. Pde8b male mice were infertile with down-regulation of the Wnt/β-catenin pathway which did not change significantly in the Pde8b;ΔCat mice. Prkar1a haploinsufficiency also did not change the phenotype significantly. In vitro studies showed that PDE8B knockdown upregulated the Wnt pathway and increased proliferation in CTNNB1-mutant cells, whereas it downregulated the Wnt pathway in PRKAR1A-mutant cells. These data support an overall weak, if any, role for PDE8B in adrenocortical tumorigenesis, even when co-altered with Wnt signaling or PKA upregulation; on the other hand, PDE8B appears to play a significant role in male fertility.
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http://dx.doi.org/10.1016/j.mce.2020.111117DOI Listing
February 2021

Ovarian teratoma in a woodchuck (Marmota monax) with hepatocellular carcinoma: radiologic and pathologic features.

BMC Vet Res 2020 Nov 23;16(1):451. Epub 2020 Nov 23.

Center for Interventional Oncology, Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, 10 Center Drive, Room 3N320, MSC 1182, Bethesda, MD, 20892, USA.

Background: Teratomas are germ cell neoplasms composed of a wide variety of tissues. In the woodchuck, only one testicular teratoma has been described in the literature. The objective of this report was to describe the radiologic and pathologic findings in a female woodchuck (Marmota monax) with an ovarian teratoma consisting of mature tissues originating from all three germ layers.

Case Presentation: A 2-year-old female woodchuck that had been infected at birth with woodchuck hepatitis virus and subsequently developed hepatocellular carcinoma was incidentally discovered to have a mobile 6.6 × 4.8 × 4.7 cm abdominal mass on computed tomography (CT) imaging. The tumor was predominantly solid and heterogenous on CT with soft tissue, fat, and areas of dense calcification. The teratoma did not enhance with intravenous contrast administration. On ultrasound, the tumor was solid with heterogeneous echogenicity, reflecting the fat content and areas of calcification. Sonolucent areas were present that may have represented cysts. There was heterogeneously increased signal on T1-weighted magnetic resonance imaging (MRI) and heterogeneous hyperintensity in T2-weighted imaging. Fat was evident within the tumor. At necropsy, the tumor was attached to the distal end of the right uterine horn. Histopathology showed mature tissue types representing all three germ layers.

Conclusions: Ovarian teratoma should be considered in the differential diagnosis of ovarian or abdominal masses in woodchucks. The tumor displayed mature tissue derived from all three germ layers. CT, ultrasound, and MRI findings were presented in detail and matched the typical imaging appearance of teratomas.
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http://dx.doi.org/10.1186/s12917-020-02658-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685576PMC
November 2020

Reducing Fatty Acid Oxidation Improves Cancer-free Survival in a Mouse Model of Li-Fraumeni Syndrome.

Cancer Prev Res (Phila) 2020 Sep 21. Epub 2020 Sep 21.

Cardiovascular Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland.

Germline mutations of , which cause the cancer predisposition disorder Li-Fraumeni syndrome (LFS), can increase mitochondrial activity as well as fatty acid β-oxidation (FAO) in mice. Increased fatty acid metabolism can promote cancer malignancy, but its specific contribution to tumorigenesis in LFS remains unclear. To investigate this, we crossed LFS mice carrying the R172H knock-in mutation ( , homolog of the human R175H LFS mutation) with myoglobin-knockout ( ) mice known to have decreased FAO. double-mutant mice also showed mildly reduced FAO in thymus, a common site of T lymphoma development in LFS mice, in association with an approximately 40% improvement in cancer-free survival time. RNA sequencing profiling revealed that the R172H mutation promotes mitochondrial metabolism and ribosome biogenesis, both of which are suppressed by the disruption of . The activation of ribosomal protein S6, involved in protein translation and implicated in cancer promotion, was also inhibited in the absence of . To further confirm the role of FAO in lymphomagenesis, mitochondrial FAO enzyme, carnitine palmitoyltransferase 2 (CPT2), was specifically disrupted in T cells of mice using a Cre-P-mediated strategy. The heterozygous knockout of resulted in thymus FAO haploinsufficiency and an approximately 30% improvement in survival time, paralleling the antiproliferative signaling observed with disruption. Thus, this study demonstrates that moderating FAO in LFS can suppress tumorigenesis and improve cancer-free survival with potential implications for cancer prevention. PREVENTION RELEVANCE: Mildly inhibiting the increased fatty acid oxidation observed in a mouse model of Li-Fraumeni syndrome, a cancer predisposition disorder caused by inherited mutations of , dampens aberrant pro-tumorigenic cell signaling and improves the survival time of these mice, thereby revealing a potential strategy for cancer prevention in patients.
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http://dx.doi.org/10.1158/1940-6207.CAPR-20-0368DOI Listing
September 2020

Prkar1a haploinsufficiency ameliorates the growth hormone excess phenotype in Aip-deficient mice.

Hum Mol Genet 2020 Oct;29(17):2951-2961

Section on Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD 20892, USA.

Mutations of the regulatory subunit (PRKAR1A) of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), leading to activation of the PKA pathway, are the genetic cause of Carney complex which is frequently accompanied by somatotroph tumors. Aryl hydrocarbon receptor-interacting protein (AIP) mutations lead to somatotroph tumorigenesis in mice and humans. The mechanisms of AIP-dependent pituitary tumorigenesis are still under investigation and evidence points to a connection between the AIP and PKA pathways. In this study, we explore the combined effects of Aip and Prkar1a deficiency on mouse phenotype and, specifically, pituitary histopathology. Aip+/- mice were compared with double heterozygous Aip+/-, Prkar1a+/- mice. The phenotype (including histopathology and serological studies) was recorded at 3, 6, 9 and 12 months of age. Detailed pituitary histological and immunohistochemical studies were performed at 12 months. Twelve-month old Aip+/- mice demonstrated phenotypic and biochemical evidence of GH excess including significantly elevated insulin-like growth factor 1 levels, larger weight and body length, higher hemoglobin and cholesterol levels and a higher frequency of growth plate thickening in comparison to Aip+/, Prkar1a+/- mice. Pituitary histopathology did not uncover any pituitary adenomas or somatotroph hyperplasia in either group. These results demonstrate a slow progression from elevated GH release to the formation of overt somatotropinomas in Aip+/- mice; the acromegalic phenotype of these mice is surprisingly ameliorated in Aip+/-, Prkar1a+/- mice. This highlights the complexities of interaction between the AIP and PKA pathway. Specifically targeting GH secretion rather than somatotroph proliferation may be an advantage in the medical treatment of AIP-dependent human acromegaly.
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http://dx.doi.org/10.1093/hmg/ddaa178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566352PMC
October 2020

Gene therapy using a novel G6PC-S298C variant enhances the long-term efficacy for treating glycogen storage disease type Ia.

Biochem Biophys Res Commun 2020 06 16;527(3):824-830. Epub 2020 May 16.

Section on Cellular Differentiation, Division of Translational Medicine, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

The current phase I/II clinical trial for human glycogen storage disease type-Ia (GSD-Ia) (NCT03517085) uses a recombinant adeno-associated virus (rAAV) vector expressing a codon-optimized human glucose-6-phosphatase-α (G6Pase-α or G6PC). DNA sequence changes introduced by codon-optimization can negatively impact gene expression. We therefore generated a novel variant in which a single amino acid change, S298C, is introduced into the native human G6PC sequence. Short term gene transfer study in G6pc-/- mice showed that the rAAV-G6PC-S298C vector is 3-fold more efficacious than the native rAAV-G6PC vector. We have shown previously that restoring 3% of normal hepatic G6Pase-α activity in G6pc-/- mice prevents hepatocellular adenoma/carcinoma (HCA/HCC) development and that mice harboring <3% of normal hepatic G6Pase-α activity are at risk of tumor development. We have also shown that G6Pase-α deficiency leads to hepatic autophagy impairment that can contribute to hepatocarcinogenesis. We now undertake a long-term (66-week) preclinical characterization of the rAAV-G6PC-S298C vector in GSD-Ia gene therapy. We show that the increased efficacy of rAAV-G6PC-S298C has enabled the G6pc-/- mice treated with a lower dose of this vector to survive long-term. We further show that mice expressing ≥3% of normal hepatic G6Pase-α activity do not develop hepatic tumors or autophagy impairment but mice expressing <3% of normal hepatic G6Pase-α activity display impaired hepatic autophagy with one developing HCA/HCC nodules. Our study shows that the rAAV-G6PC-S298C vector provides equal or greater efficacy to the codon optimization approach, offering a valuable alternative vector for clinical translation in human GSD-Ia.
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http://dx.doi.org/10.1016/j.bbrc.2020.04.124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309276PMC
June 2020

Noncoding Microdeletion in Mouse Disrupts Neural Crest Migration into the Stria Vascularis, Reduces the Endocochlear Potential, and Suggests the Neuropathology for Human Nonsyndromic Deafness DFNB39.

J Neurosci 2020 04 9;40(15):2976-2992. Epub 2020 Mar 9.

Auditory Development and Restoration Program,

Hepatocyte growth factor (HGF) is a multifunctional protein that signals through the MET receptor. HGF stimulates cell proliferation, cell dispersion, neuronal survival, and wound healing. In the inner ear, levels of HGF must be fine-tuned for normal hearing. In mice, a deficiency of HGF expression limited to the auditory system, or an overexpression of HGF, causes neurosensory deafness. In humans, noncoding variants in are associated with nonsyndromic deafness However, the mechanism by which these noncoding variants causes deafness was unknown. Here, we reveal the cause of this deafness using a mouse model engineered with a noncoding intronic 10 bp deletion (del10) in Male and female mice homozygous for del10 exhibit moderate-to-profound hearing loss at 4 weeks of age as measured by tone burst auditory brainstem responses. The wild type (WT) 80 mV endocochlear potential was significantly reduced in homozygous del10 mice compared with WT littermates. In normal cochlea, endocochlear potentials are dependent on ion homeostasis mediated by the stria vascularis (SV). Previous studies showed that developmental incorporation of neural crest cells into the SV depends on signaling from HGF/MET. We show by immunohistochemistry that, in del10 homozygotes, neural crest cells fail to infiltrate the developing SV intermediate layer. Phenotyping and RNAseq analyses reveal no other significant abnormalities in other tissues. We conclude that, in the inner ear, the noncoding del10 mutation in leads to developmental defects of the SV and consequently dysfunctional ion homeostasis and a reduction in the EP, recapitulating human DFNB39 nonsyndromic deafness. Hereditary deafness is a common, clinically and genetically heterogeneous neurosensory disorder. Previously, we reported that human deafness DFNB39 is associated with noncoding variants in the 3'UTR of a short isoform of encoding hepatocyte growth factor. For normal hearing, HGF levels must be fine-tuned as an excess or deficiency of HGF cause deafness in mouse. Using a mutant mouse with a small 10 bp deletion recapitulating a human noncoding variant, we demonstrate that neural crest cells fail to migrate into the stria vascularis intermediate layer, resulting in a significantly reduced endocochlear potential, the driving force for sound transduction by inner ear hair cells. HGF-associated deafness is a neurocristopathy but, unlike many other neurocristopathies, it is not syndromic.
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http://dx.doi.org/10.1523/JNEUROSCI.2278-19.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141880PMC
April 2020

Transarterial Chemoembolization in a Woodchuck Model of Hepatocellular Carcinoma.

J Vasc Interv Radiol 2020 May 24;31(5):812-819.e1. Epub 2020 Feb 24.

Center for Interventional Oncology, Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, 10 Center Dr., Room 3N320B, MSC 1182, Bethesda, MD 20892; National Institute of Biomedical Imaging and Bioengineering and National Cancer Institute Center for Cancer Research, National Institutes of Health, 10 Center Dr., Room 3N320B, MSC 1182, Bethesda, MD 20892.

Purpose: To assess the feasibility of transarterial chemoembolization with drug-eluting embolic (DEE) microspheres in a woodchuck model of hepatocellular carcinoma (HCC).

Materials And Methods: Nine woodchucks were studied: 4 normal animals and 5 animals infected with woodchuck hepatitis virus in which HCC had developed. Three animals with HCC underwent multidetector CT. A 3-F sheath was introduced into the femoral artery, and the hepatic arteries were selectively catheterized with 2.0-2.4-F microcatheters. Normal animals underwent diagnostic angiography and bland embolization. Animals with HCC underwent DEE transarterial chemoembolization with 70-150-μm radiopaque microspheres loaded with 37.5 mg doxorubicin per milliliter. Cone-beam CT and multidetector CT were performed. Following euthanasia, explanted livers underwent micro-CT, histopathologic examination, and fluorescence imaging of doxorubicin.

Results: The tumors were hypervascular and supplied by large-caliber tortuous vessels, with arteriovenous shunts present in 2 animals. There was heterogeneous enhancement on multidetector CT with areas of necrosis. Six tumors were identified. The most common location was the right medial lobe (n = 3). Mean tumor volume was 30.7 cm ± 12.3. DEE chemoembolization of tumors was achieved. Excluding the 2 animals with arteriovenous shunts, the mean volume of DEE microspheres injected was 0.49 mL ± 0.17. Fluorescence imaging showed diffusion of doxorubicin from the DEE microspheres into the tumor.

Conclusions: Woodchuck HCC shares imaging appearances and biologic characteristics with human HCC. Selective catheterization and DEE chemoembolization may similarly be performed. Woodchucks may be used to model interventional therapies and possibly characterize radiologic-pathologic correlations.
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http://dx.doi.org/10.1016/j.jvir.2019.08.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198364PMC
May 2020

A Mouse Homolog of a Human TP53 Germline Mutation Reveals a Lipolytic Activity of p53.

Cell Rep 2020 01;30(3):783-792.e5

Cardiovascular Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA. Electronic address:

The physiological effects of the many germline mutations of TP53, encoding the tumor suppressor protein p53, are poorly understood. Here we report generating a p53 R178C knockin mouse modeling the human TP53 R181C mutation, which is notable for its prevalence and prior molecular characterization. Consistent with its weak cancer penetrance in humans, homozygous p53 mice show a modest increase in tumorigenesis but, surprisingly, are lean with decreased body fat content. They display evidence of increased lipolysis and upregulation of fatty acid metabolism in their inguinal white adipose tissue (iWAT). Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses show that the mutant p53 bound and transactivated Beta-3-Adrenergic Receptor (ADRB3), a gene that is known to promote lipolysis and is associated with obesity. This study reveals that a germline mutation of p53 can affect fat metabolism, which has been implicated in cancer development.
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http://dx.doi.org/10.1016/j.celrep.2019.12.074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021448PMC
January 2020

NPC1 Deficiency in Mice is Associated with Fetal Growth Restriction, Neonatal Lethality and Abnormal Lung Pathology.

J Clin Med 2019 Dec 19;9(1). Epub 2019 Dec 19.

Genomics, Development and Disease Section, Genetic Disease Research Branch, NHGRI, NIH, Bethesda, MD, 20892, USA.

The rare lysosomal storage disorder Niemann-Pick disease type C1 (NPC1) arises from mutation of , which encodes a lysosomal transmembrane protein essential for normal transport and trafficking of cholesterol and sphingolipids. NPC1 is highly heterogeneous in both clinical phenotypes and age of onset. Previous studies have reported sub-Mendelian survival rates for mice homozygous for various mutant alleles but have not studied the potential mechanisms underlying this phenotype. We performed the first developmental analysis of a mouse model, , and discovered significant fetal growth restriction in homozygous mutants beginning at E16.5. mice also exhibited cyanosis, increased respiratory effort, and over 50% lethality at birth. Analysis of neonatal lung tissues revealed lipid accumulation, notable abnormalities in surfactant, and enlarged alveolar macrophages, suggesting that lung abnormalities may be associated with neonatal lethality in mice. The phenotypic severity of the model facilitated this first analysis of perinatal lethality and lung pathology in an NPC1 model organism, and this model may serve as a useful resource for developing treatments for respiratory complications seen in NPC1 patients.
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http://dx.doi.org/10.3390/jcm9010012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019814PMC
December 2019

Gene therapy prevents hepatic tumor initiation in murine glycogen storage disease type Ia at the tumor-developing stage.

J Inherit Metab Dis 2019 05 6;42(3):459-469. Epub 2019 Mar 6.

Section on Cellular Differentiation, Division of Translational Medicine, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland.

Hepatocellular adenoma/carcinoma (HCA/HCC) is a long-term complication of glycogen storage disease type-Ia (GSD-Ia), which is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC), a key enzyme in gluconeogenesis. Currently, there is no therapy to address HCA/HCC in GSD-Ia. We have previously shown that a recombinant adeno-associated virus (rAAV) vector-mediated G6PC gene transfer to 2-week-old G6pc-/- mice prevents HCA development. However, it remains unclear whether G6PC gene transfer at the tumor developing stage of GSD-Ia can prevent tumor initiation or abrogate the pre-existing tumors. Using liver-specific G6pc-knockout (L-G6pc-/-) mice that develop HCA/HCC, we now show that treating the mice at the tumor-developing stage with rAAV-G6PC restores hepatic G6Pase-α expression, normalizes glucose homeostasis, and prevents de novo HCA/HCC development. The rAAV-G6PC treatment also normalizes defective hepatic autophagy and corrects metabolic abnormalities in the nontumor liver tissues of both tumor-free and tumor-bearing mice. However, gene therapy cannot restore G6Pase-α expression in the HCA/HCC lesions and fails to abrogate any pre-existing tumors. We show that the expression of 11 β-hydroxysteroid dehydrogenase type-1 that mediates local glucocorticoid activation is downregulated in HCA/HCC lesions, leading to impairment in glucocorticoid signaling critical for gluconeogenesis activation. This suggests that local glucocorticoid action downregulation in the HCA/HCC lesions may suppress gene therapy mediated G6Pase-α restoration. Collectively, our data show that rAAV-mediated gene therapy can prevent de novo HCA/HCC development in L-G6pc-/- mice at the tumor developing stage, but it cannot reduce any pre-existing tumor burden.
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http://dx.doi.org/10.1002/jimd.12056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6483852PMC
May 2019

The phenotypic landscape of a Tbc1d24 mutant mouse includes convulsive seizures resembling human early infantile epileptic encephalopathy.

Hum Mol Genet 2019 05;28(9):1530-1547

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, Porter Neuroscience Research Center, National Institutes of Health, Bethesda, MD, USA.

Epilepsy, deafness, onychodystrophy, osteodystrophy and intellectual disability are associated with a spectrum of mutations of human TBC1D24. The mechanisms underlying TBC1D24-associated disorders and the functions of TBC1D24 are not well understood. Using CRISPR-Cas9 genome editing, we engineered a mouse with a premature translation stop codon equivalent to human S324Tfs*3, a recessive mutation of TBC1D24 associated with early infantile epileptic encephalopathy (EIEE). Homozygous S324Tfs*3 mice have normal auditory and vestibular functions but show an abrupt onset of spontaneous seizures at postnatal day 15 recapitulating human EIEE. The S324Tfs*3 variant is located in an alternatively spliced micro-exon encoding six perfectly conserved amino acids incorporated postnatally into TBC1D24 protein due to a micro-exon utilization switch. During embryonic and early postnatal development, S324Tfs*3 homozygotes produce predominantly the shorter wild-type TBC1D24 protein isoform that omits the micro-exon. S324Tfs*3 homozygotes show an abrupt onset of seizures at P15 that correlates with a developmental switch to utilization of the micro-exon. A mouse deficient for alternative splice factor SRRM3 impairs incorporation of the Tbc1d24 micro-exon. Wild-type Tbc1d24 mRNA is abundantly expressed in the hippocampus using RNAscope in situ hybridization. Immunogold electron microscopy using a TBC1D24-specific antibody revealed that TBC1D24 is associated with clathrin-coated vesicles and synapses of hippocampal neurons, suggesting a crucial role of TBC1D24 in vesicle trafficking important for neuronal signal transmission. This is the first characterization of a mouse model of human TBC1D24-associated EIEE that can now be used to screen for antiepileptogenic drugs ameliorating TBCID24 seizure disorders.
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http://dx.doi.org/10.1093/hmg/ddy445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466106PMC
May 2019

Suppression of Wnt/β-catenin signaling by EGF receptor is required for hair follicle development.

Mol Biol Cell 2018 11 6;29(22):2784-2799. Epub 2018 Sep 6.

Division of Biotechnology Review and Research IV, Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993.

Mice that lack the epidermal growth factor receptor (EGFR) fail to develop a hair coat, but the mechanism responsible for this deficit is not completely understood. Here, we show that EGFR plays a critical role to attenuate wingless-type MMTV integration site family member (Wnt)/β-catenin signaling during postnatal hair follicle development. Genetic ablation of EGFR in mice resulted in increased mitotic activity in matrix cells, apoptosis in hair follicles, and impaired differentiation of epithelial lineages that form hair. EGFR is activated in wild-type hair follicle stem cells marked with SOX9 or NFATc1 and is essential to restrain proliferation and support stem cell numbers and their quiescence. We observed elevated levels of Wnt4, 6, 7b, 10a, 10b, and 16 transcripts and hyperactivation of the β-catenin pathway in EGFR knockout follicles. Using primary keratinocytes, we linked ligand-induced EGFR activation to suppression of nascent mRNA synthesis of Wnt genes. Overexpression of the Wnt antagonist sFRP1 in mice lacking EGFR demonstrated that elevated Wnts are a major cause for the hair follicle defects. Colocalization of transforming growth factor α and Wnts regulated by EGFR in stem cells and progeny indicates that EGFR autocrine loops control Wnts. Our findings define a novel mechanism that integrates EGFR and Wnt/β-catenin pathways to coordinate the delicate balance between proliferation and differentiation during development.
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http://dx.doi.org/10.1091/mbc.E18-08-0488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249831PMC
November 2018

Mouse Homolog of the Human R337H Mutation Reveals Its Role in Tumorigenesis.

Cancer Res 2018 09 24;78(18):5375-5383. Epub 2018 Jul 24.

Cardiovascular Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland.

Inheritance of germline mutations in the tumor suppressor gene causes Li-Fraumeni syndrome (LFS), a cancer predisposition disorder. The arginine to histidine substitution at amino acid position 337 of p53 (R337H) is a founder mutation highly prevalent in southern and southeastern Brazil and is considered an LFS mutation. Although this mutation is of significant clinical interest, its role in tumorigenesis using animal models has not been described. Here, we generate a knockin mouse model containing the homologous R337H mutation (mouse R334H). tumorigenesis was not significantly increased in either heterozygous ( ) or homozygous ( ) p53 R334H knockin mice compared with wild-type mice. However, susceptibility to diethylnitrosamine (DEN)-induced liver carcinogenesis was increased in a mutant allele dose-dependent manner. In parallel, mice exposed to DEN exhibited increased DNA damage but decreased cell-cycle regulation in the liver. Oligomerization of p53, which is required for transactivation of target genes, was reduced in R334H liver, consistent with its decreased nuclear activity compared with wild-type. By modeling a mutation in mice that has relatively weak cancer penetrance, this study provides evidence that the human R337H mutation can compromise p53 activity and promote tumorigenesis. A germline mutation in the oligomerization domain of p53 decreases its transactivation potential and renders mice susceptible to carcinogen-induced liver tumorigenesis. .
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http://dx.doi.org/10.1158/0008-5472.CAN-18-0016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6139041PMC
September 2018

CDC14A phosphatase is essential for hearing and male fertility in mouse and human.

Hum Mol Genet 2018 03;27(5):780-798

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, MD 20892, USA.

The Cell Division-Cycle-14 gene encodes a dual-specificity phosphatase necessary in yeast for exit from mitosis. Numerous disparate roles of vertebrate Cell Division-Cycle-14 (CDC14A) have been proposed largely based on studies of cultured cancer cells in vitro. The in vivo functions of vertebrate CDC14A are largely unknown. We generated and analyzed mutations of zebrafish and mouse CDC14A, developed a computational structural model of human CDC14A protein and report four novel truncating and three missense alleles of CDC14A in human families segregating progressive, moderate-to-profound deafness. In five of these families segregating pathogenic variants of CDC14A, deaf males are infertile, while deaf females are fertile. Several recessive mutations of mouse Cdc14a, including a CRISPR/Cas9-edited phosphatase-dead p.C278S substitution, result in substantial perinatal lethality, but survivors recapitulate the human phenotype of deafness and male infertility. CDC14A protein localizes to inner ear hair cell kinocilia, basal bodies and sound-transducing stereocilia. Auditory hair cells of postnatal Cdc14a mutants develop normally, but subsequently degenerate causing deafness. Kinocilia of germ-line mutants of mouse and zebrafish have normal lengths, which does not recapitulate the published cdc14aa knockdown morphant phenotype of short kinocilia. In mutant male mice, degeneration of seminiferous tubules and spermiation defects result in low sperm count, and abnormal sperm motility and morphology. These findings for the first time define a new monogenic syndrome of deafness and male infertility revealing an absolute requirement in vivo of vertebrate CDC14A phosphatase activity for hearing and male fertility.
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http://dx.doi.org/10.1093/hmg/ddx440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6059191PMC
March 2018

Liver-directed gene therapy for murine glycogen storage disease type Ib.

Hum Mol Genet 2017 11;26(22):4395-4405

Section on Cellular Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development.

Glycogen storage disease type-Ib (GSD-Ib), deficient in the glucose-6-phosphate transporter (G6PT), is characterized by impaired glucose homeostasis, myeloid dysfunction, and long-term risk of hepatocellular adenoma (HCA). We examined the efficacy of G6PT gene therapy in G6pt-/- mice using recombinant adeno-associated virus (rAAV) vectors, directed by either the G6PC or the G6PT promoter/enhancer. Both vectors corrected hepatic G6PT deficiency in murine GSD-Ib but the G6PC promoter/enhancer was more efficacious. Over a 78-week study, using dose titration of the rAAV vectors, we showed that G6pt-/- mice expressing 3-62% of normal hepatic G6PT activity exhibited a normalized liver phenotype. Two of the 12 mice expressing < 6% of normal hepatic G6PT activity developed HCA. All treated mice were leaner and more sensitive to insulin than wild-type mice. Mice expressing 3-22% of normal hepatic G6PT activity exhibited higher insulin sensitivity than mice expressing 44-62%. The levels of insulin sensitivity correlated with the magnitudes of hepatic carbohydrate response element binding protein signaling activation. In summary, we established the threshold of hepatic G6PT activity required to prevent tumor formation and showed that mice expressing 3-62% of normal hepatic G6PT activity maintained glucose homeostasis and were protected against age-related obesity and insulin resistance.
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http://dx.doi.org/10.1093/hmg/ddx325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886224PMC
November 2017

Glycogen storage disease type Ia mice with less than 2% of normal hepatic glucose-6-phosphatase-α activity restored are at risk of developing hepatic tumors.

Mol Genet Metab 2017 03 10;120(3):229-234. Epub 2017 Jan 10.

Section on Cellular Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, United States. Electronic address:

Glycogen storage disease type Ia (GSD-Ia), characterized by impaired glucose homeostasis and chronic risk of hepatocellular adenoma (HCA) and carcinoma (HCC), is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC). We have previously shown that G6pc-/- mice receiving gene transfer mediated by rAAV-G6PC, a recombinant adeno-associated virus (rAAV) vector expressing G6Pase-α, and expressing 3-63% of normal hepatic G6Pase-α activity maintain glucose homeostasis and do not develop HCA/HCC. However, the threshold of hepatic G6Pase-α activity required to prevent tumor formation remained unknown. In this study, we constructed rAAV-co-G6PC, a rAAV vector expressing a codon-optimized (co) G6Pase-α and showed that rAAV-co-G6PC was more efficacious than rAAV-G6PC in directing hepatic G6Pase-α expression. Over an 88-week study, we showed that both rAAV-G6PC- and rAAV-co-G6PC-treated G6pc-/- mice expressing 3-33% of normal hepatic G6Pase-α activity (AAV mice) maintained glucose homeostasis, lacked HCA/HCC, and were protected against age-related obesity and insulin resistance. Of the eleven rAAV-G6PC/rAAV-co-G6PC-treated G6pc-/- mice harboring 0.9-2.4% of normal hepatic G6Pase-α activity (AAV-low mice), 3 expressing 0.9-1.3% of normal hepatic G6Pase-α activity developed HCA/HCC, while 8 did not (AAV-low-NT). Finally, we showed that the AAV-low-NT mice exhibited a phenotype indistinguishable from that of AAV mice expressing ≥3% of normal hepatic G6Pase-α activity. The results establish the threshold of hepatic G6Pase-α activity required to prevent HCA/HCC and show that GSD-Ia mice harboring <2% of normal hepatic G6Pase-α activity are at risk of tumor development.
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http://dx.doi.org/10.1016/j.ymgme.2017.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5346453PMC
March 2017

Inhibiting mitochondrial respiration prevents cancer in a mouse model of Li-Fraumeni syndrome.

J Clin Invest 2017 01 21;127(1):132-136. Epub 2016 Nov 21.

Li-Fraumeni syndrome (LFS) is a cancer predisposition disorder caused by germline mutations in TP53 that can lead to increased mitochondrial metabolism in patients. However, the implications of altered mitochondrial function for tumorigenesis in LFS are unclear. Here, we have reported that genetic or pharmacologic disruption of mitochondrial respiration improves cancer-free survival in a mouse model of LFS that expresses mutant p53. Mechanistically, inhibition of mitochondrial function increased autophagy and decreased the aberrant proliferation signaling caused by mutant p53. In a pilot study, LFS patients treated with metformin exhibited decreases in mitochondrial activity concomitant with activation of antiproliferation signaling, thus reproducing the effects of disrupting mitochondrial function observed in LFS mice. These observations indicate that a commonly prescribed diabetic medicine can restrain mitochondrial metabolism and tumorigenesis in an LFS model, supporting its further consideration for cancer prevention in LFS patients.
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http://dx.doi.org/10.1172/JCI88668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5199691PMC
January 2017

Celecoxib treatment of fibrous dysplasia (FD) in a human FD cell line and FD-like lesions in mice with protein kinase A (PKA) defects.

Mol Cell Endocrinol 2017 01 4;439:165-174. Epub 2016 Aug 4.

Section on Endocrinology and Genetics (SEGEN), Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD 20892, USA. Electronic address:

Osteochondromyxomas (OMX) in the context of Carney complex (CNC) and fibrous dysplasia (FD)-like lesions (FDLL) in mice, as well as isolated myxomas in humans may be caused by inactivation of PRKAR1A, the gene coding for the type 1a regulatory subunit (R1α) of cAMP-dependent protein kinase (PKA). OMXs and FDLL in mice lacking Prkar1a grow from abnormal proliferation of adult bone stromal cells (aBSCs). Prkar1a and Prkaca (coding for Cα) haploinsufficiency leads to COX2 activation and prostaglandin E2 (PGE2) production that, in turn, activates proliferation of aBSCs. Celecoxib is a cyclooxygenase-2 (COX2) inhibitor. We hypothesized that COX-2 inhibition may have an effect in FD and FDLL. In vitro treatment of a human cell line prepared from a FD patient with Celecoxib resulted in decreased PGE2 and cell proliferation. Treatment of mice haploinsufficient for R1α and Cα with 1500 mg/kg Celecoxib led to decreased PGE2 and proliferation and increased apoptosis, with a corresponding gene expression profile, resulting in dramatic reduction of tumor growth. Furthermore, the treatment improved the organization of cortical bone that was adjacent to the tumor. We conclude that, in vitro and in vivo, Celecoxib had an inhibitory effect on FD cell proliferation and in mouse FDLL structure, respectively. We speculate that COX-2 inhibitors offer an attractive alternative to current treatments for benign tumors such as OMX and FD that, apart from tumor suppression, may mechanically stabilize affected bones.
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http://dx.doi.org/10.1016/j.mce.2016.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123938PMC
January 2017

Studies of mice with cyclic AMP-dependent protein kinase (PKA) defects reveal the critical role of PKA's catalytic subunits in anxiety.

Behav Brain Res 2016 07 16;307:1-10. Epub 2016 Mar 16.

Section on Endocrinology and Genetics, Program in Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD 20892, United States.

Cyclic adenosine mono-phosphate-dependent protein kinase (PKA) is critically involved in the regulation of behavioral responses. Previous studies showed that PKA's main regulatory subunit, R1α, is involved in anxiety-like behaviors. The purpose of this study was to determine how the catalytic subunit, Cα, might affect R1α's function and determine its effects on anxiety-related behaviors. The marble bury (MB) and elevated plus maze (EPM) tests were used to assess anxiety-like behavior and the hotplate test to assess nociception in wild type (WT) mouse, a Prkar1a heterozygote (Prkar1a(+/-)) mouse with haploinsufficiency for the regulatory subunit (R1α), a Prkaca heterozygote (Prkaca(+/-)) mouse with haploinsufficiency for the catalytic subunit (Cα), and a double heterozygote mouse (Prkar1a(+/-)/Prkaca(+/-)) with haploinsufficiency for both R1α and Cα. We then examined specific brain nuclei involved in anxiety. Results of MB test showed a genotype effect, with increased anxiety-like behavior in Prkar1a(+/-) and Prkar1a(+/-)/Prkaca(+/-) compared to WT mice. In the EPM, Prkar1a(+/-) spent significantly less time in the open arms, while Prkaca(+/-) and Prkar1a(+/-)/Prkaca(+/-) mice displayed less exploratory behavior compared to WT mice. The loss of one Prkar1a allele was associated with a significant increase in PKA activity in the basolateral (BLA) and central (CeA) amygdala and ventromedial hypothalamus (VMH) in both Prkar1a(+/-) and Prkar1a(+/-)/Prkaca(+/-) mice. Alterations of PKA activity induced by haploinsufficiency of its main regulatory or most important catalytic subunits result in anxiety-like behaviors. The BLA, CeA, and VMH are implicated in mediating these PKA effects in brain.
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http://dx.doi.org/10.1016/j.bbr.2016.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4853257PMC
July 2016

Hematopoietic neoplasms in Prkar2a-deficient mice.

J Exp Clin Cancer Res 2015 Nov 25;34:143. Epub 2015 Nov 25.

Section on Endocrinology and Genetics, Program on Developmental Endocrinology & Genetics (PDEGEN) & Pediatric Endocrinology Inter-institute Training Program, Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD, 20892, USA.

Background: Protein kinase A (PKA) is a holoenzyme that consists of a dimer of regulatory subunits and two inactive catalytic subunits that bind to the regulatory subunit dimer. Four regulatory subunits (RIα, RIβ, RIIα, RIIβ) and four catalytic subunits (Cα, Cβ, Cγ, Prkx) have been described in the human and mouse genomes. Previous studies showed that complete inactivation of the Prkar1a subunit (coding for RIα) in the germline leads to embryonic lethality, while Prkar1a-deficient mice are viable and develop schwannomas, thyroid, and bone neoplasms, and rarely lymphomas and sarcomas. Mice with inactivation of the Prkar2a and Prkar2b genes (coding for RIIα and RIIβ, respectively) are also viable but have not been studied for their susceptibility to any tumors.

Methods: Cohorts of Prkar1a (+/-) , Prkar2a (+/-) , Prkar2a (-/-) , Prkar2b (+/-) and wild type (WT) mice have been observed between 5 and 25 months of age for the development of hematologic malignancies. Tissues were studied by immunohistochemistry; tumor-specific markers were also used as indicated. Cell sorting and protein studies were also performed.

Results: Both Prkar2a (-/-) and Prkar2a (+/-) mice frequently developed hematopoietic neoplasms dominated by histiocytic sarcomas (HS) with rare diffuse large B cell lymphomas (DLBCL). Southern blot analysis confirmed that the tumors diagnosed histologically as DLBCL were clonal B cell neoplasms. Mice with other genotypes did not develop a significant number of similar neoplasms.

Conclusions: Prkar2a deficiency predisposes to hematopoietic malignancies in vivo. RIIα's likely association with HS and DLBCL was hitherto unrecognized and may lead to better understanding of these rare neoplasms.
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http://dx.doi.org/10.1186/s13046-015-0257-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4660639PMC
November 2015

Celecoxib reduces glucocorticoids in vitro and in a mouse model with adrenocortical hyperplasia.

Endocr Relat Cancer 2016 Jan 5;23(1):15-25. Epub 2015 Oct 5.

Section on Endocrinology and Genetics (SEGEN)Program on Developmental Endocrinology and Genetics (PDEGEN), Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health, 10 Center Drive, Building 10-Clinical Research Center, Room 1-3330, Bethesda, Maryland 20892, USAOffice of Research Services (ORS)Division of Veterinary Resources (DVR), Office of the Director (OD), National Institutes of Health, Bethesda, Maryland 20892, USACNRS UMR6247Génétique Reproduction et Développement, Clermont Université, Aubière, France.

Primary pigmented nodular adrenocortical disease (PPNAD), whether in the context of Carney complex (CNC) or isolated, leads to ACTH-independent Cushing's syndrome (CS). CNC and PPNAD are caused typically by inactivating mutations of PRKAR1A, a gene coding for the type 1a regulatory subunit (R1α) of cAMP-dependent protein kinase (PKA). Mice lacking Prkar1a, specifically in the adrenal cortex (AdKO) developed CS caused by bilateral adrenal hyperplasia (BAH), which is formed from the abnormal proliferation of fetal-like adrenocortical cells. Celecoxib is a cyclooxygenase 2 (COX2) inhibitor. In bone, Prkar1a inhibition is associated with COX2 activation and prostaglandin E2 (PGE2) production that, in turn, activates proliferation of bone stromal cells. We hypothesized that COX2 inhibition may have an effect in PPNAD. In vitro treatment of human cell lines, including one from a patient with PPNAD, with celecoxib resulted in decreased cell viability. We then treated AdKO and control mice with 1500 mg/kg celecoxib or vehicle. Celecoxib treatment led to decreased PGE2 and corticosterone levels, reduced proliferation and increased apoptosis of adrenocortical cells, and decreased steroidogenic gene expression. We conclude that, in vitro and in vivo, celecoxib led to decreased steroidogenesis. In a mouse model of PPNAD, celecoxib caused histological changes that, at least in part, reversed BAH and this was associated with a reduction of corticosterone levels.
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http://dx.doi.org/10.1530/ERC-15-0472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4659722PMC
January 2016

Haploinsufficiency for either one of the type-II regulatory subunits of protein kinase A improves the bone phenotype of Prkar1a+/- mice.

Hum Mol Genet 2015 Nov 5;24(21):6080-92. Epub 2015 Aug 5.

Section on Endocrinology and Genetics (SEGEN), Program on Developmental Endocrinology & Genetics (PDEGEN), Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD),

Carney Complex (CNC), a human genetic syndrome predisposing to multiple neoplasias, is associated with bone lesions such as osteochondromyxomas (OMX). The most frequent cause for CNC is PRKAR1A deficiency; PRKAR1A codes for type-I regulatory subunit of protein kinase A (PKA). Prkar1a(+/-) mice developed OMX, fibrous dysplasia-like lesions (FDL) and other tumors. Tumor tissues in these animals had increased PKA activity due to an unregulated PKA catalytic subunit and increased PKA type II (PKA-II) activity mediated by the PRKAR2A and PRKAR2B subunits. To better understand the effect of altered PKA activity on bone, we studied Prkar2a and Prkar2b knock out (KO) and heterozygous mice; none of these mice developed bone lesions. When Prkar2a(+/-) and Prkar2b(+/-) mice were used to generate Prkar1a(+/-)Prkar2a(+/-) and Prkar1a(+/-)Prkar2b(+/-) animals, bone lesions formed that looked like those of the Prkar1a(+/-) mice. However, better overall bone organization and mineralization and fewer FDL lesions were found in both double heterozygote groups, indicating a partial restoration of the immature bone structure observed in Prkar1a(+/-) mice. Further investigation indicated increased osteogenesis and higher new bone formation rates in both Prkar1a(+/-)Prkar2a(+/-) and Prkar1a(+/-)Prkar2b(+/-) mice with some minor differences between them. The observations were confirmed with a variety of markers and studies. PKA activity measurements showed the expected PKA-II decrease in both double heterozygote groups. Thus, haploinsufficiency for either of PKA-II regulatory subunits improved bone phenotype of mice haploinsufficient for Prkar1a, in support of the hypothesis that the PRKAR2A and PRKAR2B regulatory subunits were in part responsible for the bone phenotype of Prkar1a(+/-) mice.
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http://dx.doi.org/10.1093/hmg/ddv320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599668PMC
November 2015

Galnt1 is required for normal heart valve development and cardiac function.

PLoS One 2015 23;10(1):e0115861. Epub 2015 Jan 23.

Section on Biological Chemistry, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, United States of America.

Congenital heart valve defects in humans occur in approximately 2% of live births and are a major source of compromised cardiac function. In this study we demonstrate that normal heart valve development and cardiac function are dependent upon Galnt1, the gene that encodes a member of the family of glycosyltransferases (GalNAc-Ts) responsible for the initiation of mucin-type O-glycosylation. In the adult mouse, compromised cardiac function that mimics human congenital heart disease, including aortic and pulmonary valve stenosis and regurgitation; altered ejection fraction; and cardiac dilation, was observed in Galnt1 null animals. The underlying phenotype is aberrant valve formation caused by increased cell proliferation within the outflow tract cushion of developing hearts, which is first detected at developmental stage E11.5. Developing valves from Galnt1 deficient animals displayed reduced levels of the proteases ADAMTS1 and ADAMTS5, decreased cleavage of the proteoglycan versican and increased levels of other extracellular matrix proteins. We also observed increased BMP and MAPK signaling. Taken together, the ablation of Galnt1 appears to disrupt the formation/remodeling of the extracellular matrix and alters conserved signaling pathways that regulate cell proliferation. Our study provides insight into the role of this conserved protein modification in cardiac valve development and may represent a new model for idiopathic valve disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115861PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4304789PMC
May 2016

TRIM5α restriction affects clinical outcome and disease progression in simian immunodeficiency virus-infected rhesus macaques.

J Virol 2015 Feb 3;89(4):2233-40. Epub 2014 Dec 3.

Laboratory of Molecular Microbiology, NIAID, NIH, Bethesda, Maryland, USA

Unlabelled: Tripartite motif-containing protein 5α (TRIM5α) is considered to be a potential target for cell-based gene modification therapy against human immunodeficiency virus type 1 (HIV-1) infection. In the present study, we used a relevant rhesus macaque model of infection with simian immunodeficiency virus from sooty mangabey (SIVsm) to evaluate the effect of TRIM5α restriction on clinical outcome. For macaques expressing a restrictive TRIM5 genotype, the disease outcomes of those infected with the wild-type TRIM-sensitive SIVsm strain and those infected with a virus with escape mutations in the capsid were compared. We found that TRIM5α restriction significantly delayed disease progression and improved the survival rate of SIV-infected macaques, supporting the feasibility of exploiting TRIM5α as a target for gene therapy against HIV-1. Furthermore, we also found that preservation of memory CD4 T cells was associated with protection by TRIM5α restriction, suggesting memory CD4 T cells or their progenitor cells as an ideal target for gene modification. Despite the significant effect of TRIM5α restriction on survival, SIV escape from TRIM5α restriction was also observed; therefore, this may not be an effective stand-alone strategy and may require combination with other targets.

Importance: Recent studies suggest that it may be feasible not only to suppress viral replication with antiviral drugs but also potentially to eliminate or "cure" human immunodeficiency virus (HIV) infection. One approach being explored is the use of gene therapy to introduce genes that can restrict HIV replication, including a restrictive version of the host factor TRIM5α. TRIM5 was identified as a factor that restricts HIV replication in macaque cells. The rhesus gene is polymorphic, and some alleles are restrictive for primary SIVsm isolates, although escape mutations arise late in infection. Introduction of these escape mutations into the parental virus conferred resistance to TRIM5 on macaques. The present study evaluated these animals for long-term outcomes and found that TRIM5α restriction significantly delayed disease progression and improved the survival rate of SIV-infected macaques, suggesting that this could be a valid gene therapy approach that could be adapted for HIV.
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http://dx.doi.org/10.1128/JVI.02978-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338887PMC
February 2015

Investigation and identification of etiologies involved in the development of acquired hydronephrosis in aged laboratory mice with the use of high-frequency ultrasound imaging.

Pathobiol Aging Age Relat Dis 2014 1;4. Epub 2014 Aug 1.

Office of Research Services, Division of Veterinary Resources, National Institutes of Health, Bethesda, MD, USA.

Laboratory mice develop naturally occurring lesions that affect biomedical research. Hydronephrosis is a recognized pathologic abnormality of the mouse kidney. Acquired hydronephrosis can affect any mouse, as it is caused by any naturally occurring disease that impairs free urine flow. Many etiologies leading to this condition are of particular significance to aging mice. Non-invasive ultrasound imaging detects renal pelvic dilation, renal enlargement, and parenchymal loss for pre-mortem identification of this condition. High-frequency ultrasound transducers produce high-resolution images of small structures, ideal for detecting organ pathology in mice. Using a 40 MHz linear array transducer, we obtained high-resolution images of a diversity of pathologic lesions occurring within the abdomen of seven geriatric mice with acquired hydronephrosis that enabled a determination of the underlying etiology. Etiologies diagnosed from the imaging results include pyelonephritis, neoplasia, urolithiasis, mouse urologic syndrome, and spontaneous hydronephrosis, and were confirmed at necropsy. A retrospective review of abdominal scans from an additional 149 aging mice shows that the most common etiologies associated with acquired hydronephrosis are mouse urologic syndrome and abdominal neoplasia. This report highlights the utility of high-frequency ultrasound for surveying research mice for age-related pathology, and is the first comprehensive report of multiple cases of acquired hydronephrosis in mice.
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http://dx.doi.org/10.3402/pba.v4.24932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4119937PMC
August 2014

Late multiple organ surge in interferon-regulated target genes characterizes staphylococcal enterotoxin B lethality.

PLoS One 2014 13;9(2):e88756. Epub 2014 Feb 13.

Functional Genomics and Proteomics Facility, Critical Care Medicine Department, Clinical Research Center, National Institutes of Health, Bethesda, Maryland, United States of America.

Background: Bacterial superantigens are virulence factors that cause toxic shock syndrome. Here, the genome-wide, temporal response of mice to lethal intranasal staphylococcal enterotoxin B (SEB) challenge was investigated in six tissues.

Results: The earliest responses and largest number of affected genes occurred in peripheral blood mononuclear cells (PBMC), spleen, and lung tissues with the highest content of both T-cells and monocyte/macrophages, the direct cellular targets of SEB. In contrast, the response of liver, kidney, and heart was delayed and involved fewer genes, but revealed a dominant genetic program that was seen in all 6 tissues. Many of the 85 uniquely annotated transcripts participating in this shared genomic response have not been previously linked to SEB. Nine of the 85 genes were subsequently confirmed by RT-PCR in every tissue/organ at 24 h. These 85 transcripts, up-regulated in all tissues, annotated to the interferon (IFN)/antiviral-response and included genes belonging to the DNA/RNA sensing system, DNA damage repair, the immunoproteasome, and the ER/metabolic stress-response and apoptosis pathways. Overall, this shared program was identified as a type I and II interferon (IFN)-response and the promoters of these genes were highly enriched for IFN regulatory matrices. Several genes whose secreted products induce the IFN pathway were up-regulated at early time points in PBMCs, spleen, and/or lung. Furthermore, IFN regulatory factors including Irf1, Irf7 and Irf8, and Zbp1, a DNA sensor/transcription factor that can directly elicit an IFN innate immune response, participated in this host-wide SEB signature.

Conclusion: Global gene-expression changes across multiple organs implicated a host-wide IFN-response in SEB-induced death. Therapies aimed at IFN-associated innate immunity may improve outcome in toxic shock syndromes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0088756PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3923834PMC
October 2014

A null mutation of mouse Kcna10 causes significant vestibular and mild hearing dysfunction.

Hear Res 2013 Jun 22;300:1-9. Epub 2013 Mar 22.

Section on Human Genetics, Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, 5 Research Ct, 2A-19, Rockville, MD 20850, USA.

KCNA10 is a voltage gated potassium channel that is expressed in the inner ear. The localization and function of KCNA10 was studied in a mutant mouse, B6-Kcna10(TM45), in which the single protein coding exon of Kcna10 was replaced with a beta-galactosidase reporter cassette. Under the regulatory control of the endogenous Kcna10 promoter and enhancers, beta-galactosidase was expressed in hair cells of the vestibular organs and the organ of Corti. KCNA10 expression develops in opposite tonotopic gradients in the inner and outer hair cells. Kcna10(TM45) homozygotes display only a mild elevation in pure tone hearing thresholds as measured by auditory brainstem response (ABR), while heterozygotes are normal. However, Kcna10(TM45) homozygotes have absent vestibular evoked potentials (VsEPs) or elevated VsEP thresholds with prolonged peak latencies, indicating significant vestibular dysfunction despite the lack of any overt imbalance behaviors. Our results suggest that Kcna10 is expressed primarily in hair cells of the inner ear, with little evidence of expression in other organs. The Kcna10(TM45) targeted allele may be a model of human nonsyndromic vestibulopathy.
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http://dx.doi.org/10.1016/j.heares.2013.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3684051PMC
June 2013

Development of neurological disease is associated with increased immune activation in simian immunodeficiency virus-infected macaques.

J Virol 2012 Dec 3;86(24):13795-9. Epub 2012 Oct 3.

Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

Simian immunodeficiency virus (SIV) infection of macaques can result in central nervous system disorders, such as meningitis and encephalitis. We studied 10 animals inoculated with brain-derived virus from animals with SIV encephalitis. Over half of the macaques developed SIV-induced neurologic disease. Elevated levels of systemic immune activation were observed to correlate with viral RNA in the cerebral spinal fluid but not with plasma viral load, consistent with a role for SIV in the pathogenesis of neurologic disease.
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http://dx.doi.org/10.1128/JVI.02174-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503031PMC
December 2012

Accumulation of the inner nuclear envelope protein Sun1 is pathogenic in progeric and dystrophic laminopathies.

Cell 2012 Apr;149(3):565-77

National Institute of Allergy and Infectious Diseases, The National Institutes of Health, Bethesda, MD 20892, USA.

Human LMNA gene mutations result in laminopathies that include Emery-Dreifuss muscular dystrophy (AD-EDMD) and Hutchinson-Gilford progeria, the premature aging syndrome (HGPS). The Lmna null (Lmna(-/-)) and progeroid LmnaΔ9 mutant mice are models for AD-EDMD and HGPS, respectively. Both animals develop severe tissue pathologies with abbreviated life spans. Like HGPS cells, Lmna(-/-) and LmnaΔ9 fibroblasts have typically misshapen nuclei. Unexpectedly, Lmna(-/-) or LmnaΔ9 mice that are also deficient for the inner nuclear membrane protein Sun1 show markedly reduced tissue pathologies and enhanced longevity. Concordantly, reduction of SUN1 overaccumulation in LMNA mutant fibroblasts and in cells derived from HGPS patients corrected nuclear defects and cellular senescence. Collectively, these findings implicate Sun1 protein accumulation as a common pathogenic event in Lmna(-/-), LmnaΔ9, and HGPS disorders.
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http://dx.doi.org/10.1016/j.cell.2012.01.059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340584PMC
April 2012