Targeted disruption of the neuronal nitric oxide synthase gene

doi:10.1016/0092-8674(93)90615-W

Cell

Volume 75, Issue 7, 31 December 1993, Pages 1273-1286

https://doi.org/10.1016/0092-8674(93)90615-W Get rights and content

Abstract

By homologous recombination, we have generated mice that lack the neuronal nitric oxide synthase (NOS) gene. Neuronal NOS expression and NADPH-diaphorase (NDP) staining are absent in the mutant mice. Very low level residual catalytic activity suggests that other enzymes in the brain may generate nitric oxide. The neurons normally expressing NOS appear intact, and the mutant NOS mice are viable, fertile, and without evident histopathological abnormalities in the central nervous system. The most evident effect of disrupting the neuronal NOS gene is the development of grossly enlarged stomachs, with hypertrophy of the pyloric sphincter and the circular muscle layer. This phenotype resembles the human disorder infantile pyloric stenosis, in which gastric outlet obstruction is associated with the lack of NDP neurons in the pylorus.

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The pivotal role of neuronal nitric oxide synthase in the release of 6-nitrodopamine from mouse isolated vas deferens
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6-Nitrodopamine (6-ND) is released from rat and human vas deferens and is considered a major mediator of both tissues contractility. The contractions induced by 6-ND are selectively blocked by both tricyclic antidepressants and α₁-adrenoceptor antagonists. Endothelial nitric oxide synthase (eNOS) is the major isoform responsible for 6-ND release in mouse isolated heart, however the origin of 6-ND in the vas deferens is unknown. Here it was investigated by LC-MS/MS the basal release of 6-ND from isolated vas deferens obtained from control, eNOS^−/−, nNOS^−/−, and iNOS^−/− mice. In addition, it was evaluated in vitro vas deferens contractility following electric field stimulation (EFS).
Basal release of 6-ND was significantly reduced in nNOS^−/− mice compared to control mice, but not decreased when the vas deferens were obtained from either eNOS^−/− or iNOS^−/− mice. Pre-incubation of the vas deferens with tetrodotoxin (1 μM) significantly reduced the basal release of 6-ND from control, eNOS^−/−, and iNOS^−/− mice but had no effect on the basal release of 6-ND from nNOS^−/− mice. EFS-induced frequency-dependent contractions of the vas deferens, which were significantly reduced when the tissues obtained from control, eNOS^−/− and iNOS^−/− mice, were pre-incubated with l-NAME, but unaltered when the vas deferens was obtained from nNOS^−/− mice. In addition, the EFS-induced contractions were significantly smaller when the vas deferens were obtained from nNOS^−/− mice.
The results clearly demonstrate that nNOS is the main NO isoform responsible for 6-ND release in mouse vas deferens and reinforces the concept of 6-ND as a major modulator of vas deferens contractility.
Hypoxia-Inducible Factor 1α Stabilization Restores Epigenetic Control of Nitric Oxide Synthase 1 Expression and Reverses Gastroparesis in Female Diabetic Mice
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Although depletion of neuronal nitric oxide synthase (NOS1)-expressing neurons contributes to gastroparesis, stimulating nitrergic signaling is not an effective therapy. We investigated whether hypoxia-inducible factor 1α (HIF1A), which is activated by high O₂ consumption in central neurons, is a Nos1 transcription factor in enteric neurons and whether stabilizing HIF1A reverses gastroparesis.
Mice with streptozotocin-induced diabetes, human and mouse tissues, NOS1⁺ mouse neuroblastoma cells, and isolated nitrergic neurons were studied. Gastric emptying of solids and volumes were determined by breath test and single-photon emission computed tomography, respectively. Gene expression was analyzed by RNA-sequencing, microarrays, immunoblotting, and immunofluorescence. Epigenetic assays included chromatin immunoprecipitation sequencing (13 targets), chromosome conformation capture sequencing, and reporter assays. Mechanistic studies used Cre-mediated recombination, RNA interference, and clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9)–mediated epigenome editing.
HIF1A signaling from physiological intracellular hypoxia was active in mouse and human NOS1⁺ myenteric neurons but reduced in diabetes. Deleting Hif1a in Nos1-expressing neurons reduced NOS1 protein by 50% to 92% and delayed gastric emptying of solids in female but not male mice. Stabilizing HIF1A with roxadustat (FG-4592), which is approved for human use, restored NOS1 and reversed gastroparesis in female diabetic mice. In nitrergic neurons, HIF1A up-regulated Nos1 transcription by binding and activating proximal and distal cis-regulatory elements, including newly discovered super-enhancers, facilitating RNA polymerase loading and pause-release, and by recruiting cohesin to loop anchors to alter chromosome topology.
Pharmacologic HIF1A stabilization is a novel, translatable approach to restoring nitrergic signaling and treating diabetic gastroparesis. The newly recognized effects of HIF1A on chromosome topology may provide insights into physioxia- and ischemia-related organ function.
Stem cell derived therapies to preserve and repair the developing intestine
2023, Seminars in Perinatology
Stem cell research and the use of stem cells in therapy have seen tremendous growth in the last two decades. Neonatal intestinal disorders such as necrotizing enterocolitis, Hirschsprung disease, and gastroschisis have high morbidity and mortality and limited treatment options with varying success rates. Stem cells have been used in several pre-clinical studies to address various neonatal disorders with promising results. Stem cell and patient population selection, timing of therapy, as well as safety and quality control are some of the challenges that must be addressed prior to the widespread clinical application of stem cells. Further research and technological advances such as the use of cell delivery technology can address these challenges and allow for continued progress towards clinical translation.
nNOS-induced tyrosine nitration of TRKB impairs BDNF signaling and restrains neuronal plasticity
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Nitric oxide (NO) has been long recognized as an important modulator of neural plasticity, but characterization of the molecular mechanisms involved - specially the guanylyl cyclase-independent ones - has been challenging. There is evidence that NO could modify BDNF-TRKB signaling, a key mediator of neuronal plasticity. However, the mechanism underlying the interplay of NO and TRKB remains unclear. Here we show that NO induces nitration of the tyrosine 816 in the TRKB receptor in vivo and in vitro, and that post-translational modification inhibits TRKB phosphorylation and binding of phospholipase Cγ1 (PLCγ1) to this same tyrosine residue. Additionally, nitration triggers clathrin-dependent endocytosis of TRKB through the adaptor protein AP-2 and ubiquitination, thereby increasing translocation of TRKB away from the neuronal surface and directing it towards lysosomal degradation. Accordingly, inhibition of nitric oxide increases TRKB phosphorylation and TRKB-dependent neurite branching in neuronal cultures. In vivo, chronic inhibition of neuronal nitric oxide synthase (nNOS) dramatically reduced TRKB nitration and facilitated TRKB signaling in the visual cortex, and promoted a shift in ocular dominance upon monocular deprivation - an indicator of increased plasticity. Altogether, our data describe and characterize a new molecular brake on plasticity, namely nitration of TRKB receptors.
Male minipuberty involves the gonad-independent activation of preoptic nNOS neurons
2023, Free Radical Biology and Medicine
The maturation of the hypothalamic-pituitary-gonadal (HPG) axis is crucial for the establishment of reproductive function. In female mice, neuronal nitric oxide synthase (nNOS) activity appears to be key for the first postnatal activation of the neural network promoting the release of gonadotropin-releasing hormone (GnRH), i.e. minipuberty. However, in males, the profile of minipuberty as well as the role of nNOS-expressing neurons remain unexplored.
nNOS-deficient and wild-type mice were studied during postnatal development. The expression of androgen (AR) and estrogen receptor alpha (ERα) as well as nNOS phosphorylation were evaluated by immunohistochemistry in nNOS neurons in the median preoptic nucleus (MePO), where most GnRH neuronal cell bodies reside, and the hormonal profile of nNOS-deficient male mice was assessed using previously established radioimmunoassay and ELISA methods. Gonadectomy and pharmacological manipulation of ERα were used to elucidate the mechanism of minipubertal nNOS activation and the maturation of the HPG axis.
In male mice, minipubertal FSH release occurred at P23, preceding the LH surge at P30, when balanopreputial separation occurs. Progesterone and testosterone remained low during minipuberty, increasing around puberty, whereas estrogen levels were high throughout postnatal development. nNOS neurons showed a sharp increase in Ser¹⁴¹² phosphorylation of nNOS at P23, a phenomenon that occurred even in the absence of the gonads. In male mice, nNOS neurons did not appear to express AR, but abundantly expressed ERα throughout postnatal development. Selective pharmacological blockade of ERα during the infantile period blunted Ser¹⁴¹² phosphorylation of nNOS at P23.
Our results show that the timing of minipuberty differs in male mice when compared to females, but as in the latter, nNOS activity in the preoptic region plays a role in this process. Additionally, akin to male non-human primates, the profile of minipuberty in male mice is shaped by sex-independent mechanisms, and possibly involves extragonadal estrogen sources.
Caveolin 3 suppresses phosphorylation-dependent activation of sarcolemmal nNOS
2022, Biochemical and Biophysical Research Communications
Mutations of the caveolin 3 gene cause autosomal dominant limb-girdle muscular dystrophy (LGMD)1C. In mice, overexpression of mutant caveolin 3 leads to loss of caveolin 3 and results in myofiber hypotrophy in association with activation of neuronal nitric oxide synthase (nNOS) at the sarcolemma. Here, we show that caveolin 3 directly bound to nNOS and suppressed its phosphorylation-dependent activation at a specific residue, Ser1412 in the nicotinamide adenine dinucleotide phosphate (NADPH)−flavin adenine dinucleotide (FAD) module near the C-terminus of the reduction domain, in vitro. Constitutively active nNOS enhanced myoblast fusion, but not myogenesis, in vitro. Phosphorylation-dependent activation of nNOS occurred in muscles from caveolin 3-mutant mice and LGMD1C patients. Mating with nNOS-mutant mice exacerbated myofiber hypotrophy in the caveolin 3-mutant mice. In nNOS-mutant mice, regenerating myofibers after cardiotoxin injury became hypotrophic with reduced myoblast fusion. Administration of NO donor increased myofiber size and the number of myonuclei in the caveolin 3-mutant mice. Exercise also increased myofiber size accompanied by phosphorylation-dependent activation of nNOS in wild-type and caveolin 3-mutant mice. These data indicate that caveolin 3 inhibits phosphorylation-dependent activation of nNOS, which leads to myofiber hypertrophy via enhancing myoblast fusion. Hypertrophic signaling by nNOS phosphorylation could act in a compensatory manner in caveolin 3-deficient muscles.

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Evidence for nitric oxide as mediator of non-adrenergic non-cholinergic relaxations induced by ATP and GABA in the canine gut

Br. J. Pharmacol.

Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum

Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme

Localization of nitric oxide synthase indicating a neural role for nitric oxide

Nature

Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase

Nature

Neural control of adrenal medullary and cortical blood flow during hemorrhage

Am. J. Physiol.

Role of nitric oxide in adrenal medullary vasodilation during catecholamine secretion

Eur. J. Pharmacol.

Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter

Nature

Nitric oxide: a physiologic mediator of penile erection

Science

Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues

A novel neuronal messenger molecule in brain: the free radical, nitric oxide

Ann. Neurol.

Mechanisms of nitric oxide mediated neurotoxicity in primary brain cultures

J. Neurosci.

Involvement of nitric oxide in the reflex relaxation of the stomach to accommodate food or fluid

Nature

Nitric oxide: linking space and time in the brain

Regulation of the cerebral circulation by endothelium

Pharmacol. Ther.

Selective sparing of a class of striatal neurons in Huntington's disease

Science

Article
Targeted disruption of the neuronal nitric oxide synthase gene