scholarly journals CRISPR Knockdown of Kcnq3 Attenuates the M-current in NPY/AgRP Neurons

2020 ◽  
Author(s):  
Todd L. Stincic ◽  
Martha A. Bosch ◽  
Avery C. Hunker ◽  
Barbara Juarez ◽  
Ashley M. Connors ◽  
...  
Keyword(s):  
Arcuate Nucleus ◽  
Energy State ◽  
Input Resistance ◽  
Field Testing ◽  
Ingestive Behavior ◽  
Loss Of Function ◽  
Guide Rna ◽  
M Current ◽  
Subunit Mrna ◽  
Channel Expression

AbstractArcuate nucleus Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons drive ingestive behavior in response to the internal and external environment of an organism. NPY/AgRP neurons are adjacent to the median eminence, a circumventricular organ, and circulating metabolic factors and hormones communicate the energy state of the animal via these neurons by altering the excitability of NPY/AgRP neurons, which produces an appropriate change in behavior to maintain homeostasis. One example of this plasticity is seen in the M-current, a subthreshold, non-inactivating K+ current that acts to modulate excitability. Fasting decreases while estradiol increases the M-current through regulation of subunit mRNA expression of Kcnq 2, 3, & 5. KCNQ2/3 heteromers are thought to mediate the majority of the M-current. Here we used a recently developed single adeno-associated viral (AAV) vector containing a recombinase-dependent Staphylococcus aureus Cas9 (SaCas9) and a single guide RNA against Kcnq3 to selectively delete Kcnq3 in NPY/AgRP neurons to produce a loss of function in the M-current. We found that this virus was effective at knocking down Kcnq3 but not Kcnq2 expression. With the reduced KCNQ3 channel expression NPY/AgRP neurons were more depolarized, exhibited a higher input resistance, and the rheobase current needed to induce firing was significantly reduced, indicative of increased excitability. Although the resulting decrease in the M-current did not overtly alter ingestive behavior, it did significantly reduce the locomotor activity as measured in open field testing. Therefore, the SaCas9-sgKcnq3 is efficient to knock down Kcnq3 expression thereby reducing the M-current and increasing the excitability of NPY/AgRP neurons.

Abstract TMP25: Long Non-Coding RNA Mediates Stroke-Induced Neurogenesis

Stroke ◽  
2020 ◽  
Vol 51 (Suppl_1) ◽  
Author(s):  
Baoyan Fan ◽  
Wanlong Pan ◽  
Xinli Wang ◽  
Michael Chopp ◽  
Zheng Gang Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Regulation Of Gene Expression ◽  
Artery Occlusion ◽  
Loss Of Function ◽  
Bioinformatics Analyses ◽  
Guide Rna ◽  
Non Coding Rna ◽  
Cerebral Artery Occlusion ◽  
Long Non Coding Rna

Background and Purpose: Adult neurogenesis contributes to functional recovery after stroke. Long non-coding RNAs (lncRNAs) regulate stem cell self-renewal and differentiation. However, the role of lncRNAs in stroke-induced neurogenesis remains unknown. Methods and Results: Using lncRNA array and in situ hybridization, we analyzed lncRNA profiles of adult neural stem cells (NSCs) isolated from the subventricular zone neurogenic region in rats subjected to middle cerebral artery occlusion. We found that H19 was the most highly upregulated lncRNA (19 fold) in ischemic NSCs compared with non-ischemic NSCs. Reduction of endogenous H19 in NSCs by CRISPR-Cas9 genome editing significantly decreased the proliferation and increased the apoptosis of ischemic NSCs, as assayed by the number of BrdU + cells (56±5% vs 22±3%, p<0.01, n=3) and Caspase-3/7 activity compared to NSCs transfected with scrambled small guide RNA (sgRNA). Knockdown of H19 significantly decreased the number of Tuj1 + neuroblasts (8±2% vs 5±0.4%, p<0.01, n=3) and NG 2 + oliogodendrocyte progenitor cells (10±1% vs 5±0.3%, p<0.01, n=3), suggesting that deletion of H19 suppresses the proliferation and survival and blocks the differentiation of NSCs into neurons and oligodendrocytes. Additional RNA-sequencing and bioinformatics analyses revealed that genes deregulated by H19 knockdown were involved in transcription, apoptosis, proliferation, cell cycle and response to hypoxia. Western blot analysis validated that loss-of-function and gain-of-function of H19 significantly increased and reduced, respectively, the transcription of cell cycle-related genes including p27. Using ChIRP assay, we found that upregulated H19 in NSCs was physically associated with EZH2 which catalyzes the repressive H3K27me3 histone marker. Knockdown of H19 significantly reduced the enrichment of H3K27me3 at the promoter of p27, leading to the upregulation of p27 expression and consequently inhibition of NSC proliferation. Conclusions: H19 mediates stroke-induced neurogenesis by regulating genes involved in cell cycle and survival through the interaction with chromatin remodeling proteins. Our data provide novel insights into epigenetic regulation of gene expression by lncRNA in neurogenesis.

scholarly journals Improved analysis of CRISPR fitness screens and reduced off-target effects with the BAGEL2 gene essentiality classifier

2020 ◽  
Author(s):  
Eiru Kim ◽  
Traver Hart
Keyword(s):  
Dynamic Range ◽  
Quality Data ◽  
Loss Of Function ◽  
Genetic Screens ◽  
Gene Essentiality ◽  
Guide Rna ◽  
Genome Wide ◽  
Improved Model ◽  
Target Effects

AbstractIdentifying essential genes in genome-wide loss of function screens is a critical step in functional genomics and cancer target finding. We previously described the Bayesian Analysis of Gene Essentiality (BAGEL) algorithm for accurate classification of gene essentiality from short hairpin RNA and CRISPR/Cas9 genome wide genetic screens. Here, we introduce an updated version, BAGEL2, which employs an improved model that offers greater dynamic range of Bayes Factors, enabling detection of tumor suppressor genes, and a multi-target correction that reduces false positives from off-target CRISPR guide RNA. We also suggest a metric for screen quality at the replicate level and demonstrate how different algorithms handle lower-quality data in substantially different ways. BAGEL2 is written in Python 3 and source code, along with all supporting files, are available on github (https://github.com/hart-lab/bagel).

scholarly journals The Peripubertal Decline in Makorin Ring Finger Protein 3 Expression is Independent of Leptin Action

2020 ◽  
Vol 4 (7) ◽  
Author(s):  
Stephanie A Roberts ◽  
Ana Paula Abreu ◽  
Victor M Navarro ◽  
Joy N Liang ◽  
Caroline A Maguire ◽  
...  
Keyword(s):  
Arcuate Nucleus ◽  
Pubertal Development ◽  
Ring Finger ◽  
Central Precocious Puberty ◽  
Mrna Levels ◽  
Ring Finger Protein ◽  
Loss Of Function ◽  
Wild Type ◽  
Finger Protein ◽  
Significant Difference

Abstract A critical body weight is necessary for pubertal development, an effect mediated in part by leptin. The potential regulation by leptin of Makorin Ring Finger Protein 3 (MKRN3), in which loss-of-function mutations are the most common genetic cause of central precocious puberty, has not been previously explored. In mice, expression of Mkrn3 in the hypothalamic arcuate nucleus is high early in life and declines before the onset of puberty. Therefore, we aimed to explore if leptin contributes to the decrease in hypothalamic Mkrn3 mRNA levels observed in mice during pubertal development. We first used a leptin-deficient (ob/ob) mouse model. Mkrn3 mRNA levels in the mediobasal hypothalamus (MBH), which includes the arcuate nucleus, and in the preoptic area (POA), both showed a significant decrease with age from postnatal day (PND) 12 to PND30 in ob/ob mice in both males and females, similar to that observed in wild-type mice. To further explore the effects of leptin on Mkrn3 expression, we exposed prepubertal wild-type mice to high levels of leptin from age PND9-12, which did not result in any significant difference in Mkrn3 expression levels in either the MBH or POA. In summary, regulation of Mkrn3 expression by leptin was not observed in either the MBH or the POA, 2 hypothalamic sites important for pubertal maturation. These data suggest that the decline in Mkrn3 at the onset of puberty may occur independently of leptin and support our hypothesis that MKRN3 is a bona fide controller of puberty initiation.

scholarly journals Hypertension resistance polymorphisms in ROMK (Kir1.1) alter channel function by different mechanisms

2010 ◽  
Vol 299 (6) ◽  
pp. F1359-F1364 ◽  
Author(s):  
Liang Fang ◽  
Dimin Li ◽  
Paul A. Welling
Keyword(s):  
Blood Pressure ◽  
Critical Role ◽  
Pressure Control ◽  
Surface Expression ◽  
Protein Product ◽  
Sequence Variants ◽  
Channel Activity ◽  
Loss Of Function ◽  
Channel Function ◽  
Channel Expression

The renal outer medullary K+ (ROMK) channel plays a critical role in renal sodium handling. Recent genome sequencing efforts in the Framingham Heart Study offspring cohort (Ji W, Foo JN, O'Roak BJ, Zhao H, Larson MG, Simon DB, Newton-Cheh C, State MW, Levy D, and Lifton RP. Nat Genet 40: 592–599, 2008) recently revealed an association between suspected loss-of-function polymorphisms in the ROMK channel and resistance to hypertension, suggesting that ROMK activity may also be a determinant of blood pressure control in the general population. Here we examine whether these sequence variants do, in fact, alter ROMK channel function and explore the mechanisms. As assessed by two-microelectrode voltage clamp in Xenopus oocytes, 3/5 of the variants (R193P, H251Y, and T313FS) displayed an almost complete attenuation of whole cell ROMK channel activity. Surface antibody binding measurements of external epitope-tagged channels and analysis of glycosylation-state maturation revealed that these variants prevent channel expression at the plasmalemma, likely as a consequence of retention in the endoplasmic reticulum. The other variants (P166S, R169H) had no obvious effects on the basal channel activity or surface expression but, instead, conferred a gain in regulated-inhibitory gating. As assessed in giant excised patch-clamp studies, apparent phosphotidylinositol 4,5-bisphosphate (PIP2) binding affinity of the variants was reduced, causing channels to be more susceptible to inhibition upon PIP2 depletion. Unlike the protein product of the major ROMK allele, these two variants are sensitive to the inhibitory affects of a G protein-coupled receptor, which stimulates PIP2 hydrolysis. In summary, we have found that hypertension resistance sequence variants inhibit ROMK channel function by different mechanisms, providing new insights into the role of the channel in the maintenance of blood pressure.

scholarly journals Serotonin 5-HT2C Receptor Agonist Promotes Hypophagia via Downstream Activation of Melanocortin 4 Receptors

Endocrinology ◽  
2007 ◽  
Vol 149 (3) ◽  
pp. 1323-1328 ◽  
Author(s):  
Daniel D. Lam ◽  
Magdalena J. Przydzial ◽  
Simon H. Ridley ◽  
Giles S. H. Yeo ◽  
Justin J. Rochford ◽  
...  
Keyword(s):  
Energy Balance ◽  
Food Intake ◽  
Arcuate Nucleus ◽  
Weight Percent ◽  
Ingestive Behavior ◽  
The Novel ◽  
Functional Importance ◽  
Serotonin 2C Receptor ◽  
Pomc Mrna ◽  
Melanocortin 4 Receptor

The neurotransmitter serotonin (5-hydroxytryptamine) is a well-established modulator of energy balance. Both pharmacological and genetic evidence implicate the serotonin 2C receptor (5-HT2CR) as a critical receptor mediator of serotonin’s effects on ingestive behavior. Here we characterized the effect of the novel and selective 5-HT2CR agonist BVT.X on energy balance in obese and lean mice and report that BVT.X significantly reduces acute food intake without altering locomotor activity or oxygen consumption. In an effort to elucidate the mechanism of this effect, we examined the chemical phenotype of 5-HT2CR-expressing neurons in a critical brain region affecting feeding behavior, the arcuate nucleus of the hypothalamus. We show that 5-HT2CRs are coexpressed with neurons containing proopiomelanocortin, known to potently affect appetite, in the arcuate nucleus of the hypothalamus of the mouse. We then demonstrate that prolonged infusion with BVT.X in obese mice significantly increases Pomc mRNA and reduces body weight, percent body fat, and initial food intake. To evaluate the functional importance of melanocortin circuitry in the effect of BVT.X on ingestive behavior, we assessed mice with disrupted melanocortin pathways. We report that mice lacking the melanocortin 4 receptor are not responsive to BVT.X-induced hypophagia, demonstrating that melanocortins acting on melanocortin 4 receptor are a requisite downstream pathway for 5-HT2CR agonists to exert effects on food intake. The data presented here not only indicate that the novel 5-HT2CR agonist BVT.X warrants further investigation as a treatment for obesity but also elucidate specific neuronal pathways potently affecting energy balance through which 5-HT2CR agonists regulate ingestive behavior.

scholarly journals Nicotine excites hypothalamic arcuate anorexigenic proopiomelanocortin neurons and orexigenic neuropeptide Y neurons: similarities and differences

2011 ◽  
Vol 106 (3) ◽  
pp. 1191-1202 ◽  
Author(s):  
Hao Huang ◽  
Youfen Xu ◽  
Anthony N. van den Pol
Keyword(s):  
Weight Loss ◽  
Neuropeptide Y ◽  
Arcuate Nucleus ◽  
Input Resistance ◽  
Cell Types ◽  
Underlying Mechanism ◽  
Synaptic Activity ◽  
Cognitive Arousal ◽  
Hypothalamic Arcuate Nucleus ◽  
Prevalence Of Obesity

Two of the biggest health problems facing us today are addiction to nicotine and the increased prevalence of obesity. Interestingly, nicotine attenuates obesity, but the underlying mechanism is not clear. Here we address the hypothesis that if weight-reducing actions of nicotine are mediated by anorexigenic proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus, nicotine should excite these cells. Nicotine at concentrations similar to those found in smokers, 100–1,000 nM, excited POMC cells by mechanisms based on increased spike frequency, depolarization of membrane potential, and opening of ion channels. This was mediated by activation of both α7 and α4β2 nicotinic receptors; by itself, this nicotine-mediated excitation could explain weight loss caused by nicotine. However, in control experiments nicotine also excited the orexigenic arcuate nucleus neuropeptide Y (NPY) cells. Nicotine exerted similar actions on POMC and NPY cells, with a slightly greater depolarizing action on POMC cells. Immunocytochemistry revealed cholinergic axons terminating on both cell types. Nicotine actions were direct in both cell types, with nicotine depolarizing the membrane potentials and reducing input resistance. We found no differences in the relative desensitization to nicotine between POMC and NPY neurons. Nicotine inhibited excitatory synaptic activity recorded in NPY, but not POMC, cells. Nicotine also excited hypocretin/orexin neurons that enhance cognitive arousal, but the responses were smaller than in NPY or POMC cells. Together, these results indicate that nicotine has a number of similar actions, but also a few different actions, on POMC and NPY neurons that could contribute to the weight loss associated with smoking.

Gene regulation of ENaC subunits by serum- and glucocorticoid-inducible kinase-1

2005 ◽  
Vol 288 (3) ◽  
pp. F505-F512 ◽  
Author(s):  
Cary Boyd ◽  
Anikó Náray-Fejes-Tóth
Keyword(s):  
Gene Expression ◽  
Maximum Level ◽  
Dominant Negative ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Delayed Effect ◽  
Subunit Mrna ◽  
Fold Induction ◽  
Free Media ◽  
First Time

Aldosterone is a key regulator of epithelial Na+ channels (ENaC) in renal cortical collecting ducts (CCD). The goal of this study was to examine whether serum- and glucocorticoid-inducible kinase-1 (SGK1), an aldosterone-induced gene, is vital to the delayed effect of aldosterone by increasing the gene expression of ENaC subunits. To test this hypothesis, we compared the levels of ENaC mRNA in mouse CCD cells that stably express either full-length (FL)-SGK1 or a kinase-dead dominant negative (K127M)-SGK1. Our results revealed that SGK1 regulates gene expression of ENaC, whether cells are maintained in steroid-free media or in the presence of corticosteroids (CS) and/or other growth factors. Under all conditions, the loss of function of SGK1 caused a significant decrease in the expression of α- and β-ENaC, but not γ-ENaC. Compared with cells expressing FL-SGK1, K127M-SGK1 decreased the expression of α- and β-subunit mRNA by ∼45 and ∼90%, respectively. Next, to determine whether SGK1 is one of the proteins mediating the induction of α-ENaC mRNA by CS, we compared steroid induction of α-ENaC in cells expressing K127M-SGK1 vs. FL-SGK1. The maximum level of α-ENaC mRNA levels following CS was significantly (∼45%) higher in FL-SGK1- vs. K127M-SGK1-expressing cells, although the fold-induction by CS was similar in both FL-SGK1- and K127M-SGK1-expressing cells. In summary, we report for the first time that SGK1 regulates transcription of ENaC subunits. We propose that the effect of SGK1 on ENaC transcription is mediated by the activation of unidentified transcription factors.

scholarly journals GUIDES: sgRNA design for loss-of-function screens

2017 ◽  
Author(s):  
Joshua A. Meier ◽  
Feng Zhang ◽  
Neville E. Sanjana
Keyword(s):  
Graphical User Interface ◽  
Large Scale ◽  
Protein Annotation ◽  
Loss Of Function ◽  
Sequencing Data ◽  
Web Based ◽  
Guide Rna ◽  
Sgrna Design ◽  
Dna Editing ◽  
Human And Mouse

GUIDES (Graphical User Interface for DNA Editing Screens) is a web-based tool for the design of custom, large-scale CRISPR libraries for loss-of-function screens in human and mouse. GUIDES combines multi-tissue RNA-sequencing data to target expressed exons, protein annotation to target functional domains, sophisticated on-target and off-target guide RNA scoring and other optimizations to create CRISPR libraries directly from a list of genes without requiring any programming expertise.

scholarly journals Dravet Variant SCN1AA1783V Impairs Interneuron Firing Predominantly by Altered Channel Activation

2021 ◽  
Vol 15 ◽  
Author(s):  
Nikolas Layer ◽  
Lukas Sonnenberg ◽  
Emilio Pardo González ◽  
Jan Benda ◽  
Ulrike B. S. Hedrich ◽  
...  
Keyword(s):  
Current Density ◽  
Input Resistance ◽  
Epileptic Encephalopathy ◽  
Dravet Syndrome ◽  
Slow Inactivation ◽  
Loss Of Function ◽  
Peak Current Density ◽  
Channel Activation ◽  
Peak Current

Dravet syndrome (DS) is a developmental epileptic encephalopathy mainly caused by functional NaV1.1 haploinsufficiency in inhibitory interneurons. Recently, a new conditional mouse model expressing the recurrent human p.(Ala1783Val) missense variant has become available. In this study, we provided an electrophysiological characterization of this variant in tsA201 cells, revealing both altered voltage-dependence of activation and slow inactivation without reduced sodium peak current density. Based on these data, simulated interneuron (IN) firing properties in a conductance-based single-compartment model suggested surprisingly similar firing deficits for NaV1.1A1783V and full haploinsufficiency as caused by heterozygous truncation variants. Impaired NaV1.1A1783V channel activation was predicted to have a significantly larger impact on channel function than altered slow inactivation and is therefore proposed as the main mechanism underlying IN dysfunction. The computational model was validated in cortical organotypic slice cultures derived from conditional Scn1aA1783V mice. Pan-neuronal activation of the p.Ala1783V in vitro confirmed a predicted IN firing deficit and revealed an accompanying reduction of interneuronal input resistance while demonstrating normal excitability of pyramidal neurons. Altered input resistance was fed back into the model for further refinement. Taken together these data demonstrate that primary loss of function (LOF) gating properties accompanied by altered membrane characteristics may match effects of full haploinsufficiency on the neuronal level despite maintaining physiological peak current density, thereby causing DS.

Smooth muscle ATP-sensitive potassium channels mediate migraine-relevant hypersensitivity in mouse models

Cephalalgia ◽  
2021 ◽  
pp. 033310242110535
Author(s):  
Sarah L Christensen ◽  
Rikke H Rasmussen ◽  
Sanne La Cour ◽  
Charlotte Ernstsen ◽  
Thomas F Hansen ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Mouse Models ◽  
Ex Vivo ◽  
Target Tissue ◽  
Loss Of Function ◽  
Dilatory Response ◽  
Channel Opening ◽  
Nociceptive Responses ◽  
Channel Expression ◽  
Mrna And Protein Expression

Background Opening of KATP channels by systemic levcromakalim treatment triggers attacks in migraine patients and hypersensitivity to von Frey stimulation in a mouse model. Blocking of these channels is effective in several preclinical migraine models. It is unknown in what tissue and cell type KATP-induced migraine attacks are initiated and which KATP channel subtype is targeted. Methods In mouse models, we administered levcromakalim intracerebroventricularly, intraperitoneally and intraplantarily and compared the nociceptive responses by von Frey and hotplate tests. Mice with a conditional loss-of-function mutation in the smooth muscle KATP channel subunit Kir6.1 were given levcromakalim and GTN and examined with von Frey filaments. Arteries were tested for their ability to dilate ex vivo. mRNA expression, western blotting and immunohistochemical stainings were made to identify relevant target tissue for migraine induced by KATP channel opening. Results Systemic administration of levcromakalim induced hypersensitivity but central and local administration provided antinociception respectively no effect. The Kir6.1 smooth muscle knockout mouse was protected from both GTN and levcromakalim induced hypersensitivity, and their arteries had impaired dilatory response to the latter. mRNA and protein expression studies showed that trigeminal ganglia did not have significant KATP channel expression of any subtype, whereas brain arteries and dura mater primarily expressed the Kir6.1 + SUR2B subtype. Conclusion Hypersensitivity provoked by GTN and levcromakalim in mice is dependent on functional smooth muscle KATP channels of extracerebral origin. These results suggest a vascular contribution to hypersensitivity induced by migraine triggers.

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