Removal of KCNQ2 from Parvalbumin-expressing Interneurons Improves Anti-Seizure Efficacy of Retigabine

AbstractMost anti-seizure drugs (ASDs) achieve their effects by suppressing neuronal excitability through various drug targets. However, these drug targets are widely expressed in both excitatory and inhibitory neurons. Here, we investigate whether the efficacy of the ASD retigabine (RTG) is altered after removal of the potassium channel subunit KCNQ2, one of its drug targets, from parvalbumin-expressing interneurons (PV-INs). Parvalbumin-Cre (PV-Cre) mice were crossed with Kcnq2-floxed (Kcnq2fl/fl) mice to conditionally delete Kcnq2, the gene encoding KCNQ2, from PV-INs. The efficacy of RTG (10 mg/kg, i.p.) in preventing seizures induced by picrotoxin (PTX, 10 mg/kg, i.p.) and kainic acid (KA, 30mg/kg, i.p.) in conditional knockout mice (cKO, PV-Kcnq2fl/fl) was tested. Immunostaining for KCNQ2 and KCNQ3 and in vitro pharmacological studies with whole-cell recordings were also performed. The cKO mice had no significant change in appearance, body mass, balance, heat sensitivity, depressive behavior, mortality, or EEG spectral power. RTG significantly delayed the onset of PTX- and KA-induced convulsive seizures in cKO mice, but not in wild-type littermates (WT). The expression of both KCNQ2 and KCNQ3 subunits was specifically enriched at the distal axon initial segments (AISs) of hippocampal CA1 PV-INs. In cKO mice, this specific expression and the potassium currents mediated by these subunits were greatly reduced in PV-INs, while their expression in CA1 pyramidal cells (CA1-PCs) remained unchanged. Accordingly, while the ability of RTG to suppress CA1-PC spike activity was unchanged in cKO mice, its suppressive effect on high-frequency spike activity of CA1 PV-INs (elicited by >540pA depolarizing currents) was significantly reduced compared with WT mice. In addition, the RTG-induced suppressive effect on intrinsic membrane excitability of PV-INs in WT mice became absent or decreased in cKO mice. These findings suggest that reducing the suppression of PV-INs by RTG improves its anticonvulsant effect.Key Points(3-5 bullets, no longer than 85 characters each)RTG was effective for seizures only after Kcnq2 was removed from PV-INs.KCNQ2/KCNQ3 was enriched at PV-IN AISs, sites of AP initiation.Kcnq2 removal greatly reduced KCNQ2/KCNQ3 expression and function in CA1 PV-INs.The suppressive effect of RTG on hippocampal PV-INs was blunted in cKO mice.Therefore, the efficacy of RTG may improve with partial sparing of interneurons.

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Genome-wide identification of genetic requirements ofPseudomonas aeruginosaPAO1 for rat cardiomyocyte (H9C2) infection by insertion sequencing

10.1101/2021.03.03.433694 ◽

2021 ◽

Author(s):

Jothi Ranjani ◽

Ramamoorthy Sivakumar ◽

Paramasamy Gunasekaran ◽

Jeyaprakash Rajendhran

Keyword(s):

Host Cell ◽

Drug Targets ◽

Infectious Agent ◽

Cell Lysis ◽

Growth Media ◽

List Type ◽

H9c2 Cells ◽

Secretion Systems ◽

Chronic Infections ◽

Adhesion And Invasion

AbstractPseudomonas aeruginosais the major infectious agent among Gram-negative bacteria which causes both acute and chronic infections without any tissue specificity. Infections due toP. aeruginosaare hard to treat, as it entails various strategies like virulence factors synthesis, drug efflux systems & resistance and protein secretion systems during pathogenesis. Despite extensive research inPseudomonaspathogenesis, novel drug targets and potential therapeutic strategies are inevitable. In this study, we investigated the genetic requirements ofP.aeruginosaPAO1 for rat cardiomyocyte (H9C2) infection by insertion sequencing (INSeq). A mutant library comprising ~70,000 mutants of PAO1 was generated and the differentiated form of H9C2 cells (d-H9C2) was infected with the library. The infected d-H9C2 cells were maintained with antibiotic-protection and without any antibiotics in the growth media for 24 h. Subsequently, DNA library for INSeq was prepared, sequenced and fitness analysis was performed. A-One hundred and thirteen mutants were negatively selected in the infection condition with antibiotic-protection, whereas 143 mutants were negatively selected in antibiotic-free condition. Surprisingly, a higher number of mutants showed enriched fitness than the mutants of reduced fitness during the infection. We demonstrated that the genes associated with flagella and T3SS are important for adhesion and invasion of cardiomyocytes, while pili and proteases are conditionally essential during host cell lysis.Take away✓Fitness ofP.aeruginosamutants were analyzed during cardiomyocyte infection✓Genes involve amino acid transport & metabolism and signal transduction are important during intracellular lifestyle✓OMVs play a crucial role during infection and pathogenesis✓Flagella and T3SS are conditionally essential for adhesion and invasion, whereas pili and proteases are conditionally essential during host cell lysis

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2007 Russell Ross Memorial Lectureship in Vascular Biology—Epigenetic Control of Vascular Smooth Muscle Differentiation in Development and Disease

Circulation ◽

10.1161/circ.116.suppl_16.ii_a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Gary K. Owens

Keyword(s):

Smooth Muscle ◽

Vascular Injury ◽

Serum Response Factor ◽

Critical Role ◽

Conditional Knockout ◽

Phenotypic Switching ◽

Marker Genes ◽

List Type ◽

Response Factor ◽

Serum Response

There is clear evidence that alterations in the differentiated state of the smooth muscle cell (SMC) play a key role in the pathogenesis of a number of major human diseases, including atherosclerosis and postan-gioplasty restenosis. This process is referred to as “phenotypic switching” and likely evolved to promote repair of vascular injury. However, the mechanisms controlling phenotypic switching as well as normal differentiation of SMCs in vivo are poorly understood. This talk will provide an overview of molecular mechanisms that control differentiation of SMCs during vascular development. A particular focus will be to consider the role of CArG elements found within the promoters of many SMC differentiation marker genes, as well as regulation of their activity by serum response factor and the potent SMC-selective serum response factor coactivator myocardin. In addition, I will summarize recent work in our laboratory showing that SMC- and gene-locus–selective changes in chromatin structure play a critical role both in normal control of SMC differentiation and in phenotypic switching in response to vascular injury. Finally, I will present evidence based on conditional knockout experiments in mice showing that krupple-like factor 4 is induced in SMCs after vascular injury and regulates SMC phenotypic switching and growth through: binding to G/C repressor elements located in close proximity of CArG elements within the promoters of many SMC marker genes, suppressing expression of myocardin, and inducing epigenetic modifications of SMC marker gene loci associated with chromatin condensation and transcriptional silencing. Supported by NIH grants P01 HL19242, R37 HL57353, and R01 HL 38854.

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Dentate EEG spikes and associated interneuronal population bursts in the hippocampal hilar region of the rat

Journal of Neurophysiology ◽

10.1152/jn.1995.73.4.1691 ◽

1995 ◽

Vol 73 (4) ◽

pp. 1691-1705 ◽

Cited By ~ 120

Author(s):

A. Bragin ◽

G. Jando ◽

Z. Nadasdy ◽

M. van Landeghem ◽

G. Buzsaki

Keyword(s):

Entorhinal Cortex ◽

Slow Wave Sleep ◽

Molecular Layer ◽

Current Source ◽

Pyramidal Cells ◽

Suppressive Effect ◽

Simultaneous Recording ◽

Field Potentials ◽

Electrode Arrays ◽

Hilar Region

1. This paper describes two novel population patterns in the dentate gyrus of the awake rat, termed type 1 and type 2 dentate spikes (DS1, DS2). Their cellular generation and spatial distribution were examined by simultaneous recording of field potentials and unit activity using multiple-site silicon probes and wire electrode arrays. 2. Dentate spikes were large amplitude (2-4 mV), short duration (< 30 ms) field potentials that occurred sparsely during behavioral immobility and slow-wave sleep. Current-source density analysis revealed large sinks in the outer (DS1) and middle (DS2) thirds of the dentate molecular layer, respectively. DS1 and DS2 had similar longitudinal, lateral, and interhemispheric synchrony. 3. Dentate spikes invariably were coupled to synchronous population bursts of putative hilar interneurons. CA3 pyramidal cells, on the other hand were suppressed during dentate spikes. 4. After bilateral removal of the entorhinal cortex, dentate spikes disappeared, whereas sharp wave-associated bursts, reflecting synchronous discharge of the CA3-CA1 network, increased several fold. 5. These physiological characteristics of the dentate spikes suggest that they are triggered by a population burst of layer II stellate cells of the lateral (DS1) and medial (DS2) entorhinal cortex. 6. We suggest that dentate spike-associated synchronized bursts of hilar-region interneurons provide a suppressive effect on the excitability of the CA3-CA1 network in the intact brain.

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A distinct form of calcium release down-regulates membrane excitability in neocortical pyramidal cells

Neuroscience ◽

10.1016/s0306-4522(01)00486-9 ◽

2002 ◽

Vol 109 (4) ◽

pp. 665-676 ◽

Cited By ~ 21

Author(s):

K Yamamoto ◽

K Hashimoto ◽

M Nakano ◽

S Shimohama ◽

N Kato

Keyword(s):

Calcium Release ◽

Pyramidal Cells ◽

Distinct Form ◽

Membrane Excitability

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Topography, Spectral Characteristics, and Extra-to-Intracranial Propagation Pathways of EMG

10.1101/808253 ◽

2019 ◽

Author(s):

J. Lahr ◽

L.D.J. Fiederer ◽

O. Glanz ◽

A. Schulze-Bonhage ◽

T. Ball

Keyword(s):

Spectral Power ◽

Brain Activity ◽

Spectral Characteristics ◽

Signal Propagation ◽

Emg Activity ◽

List Type ◽

Intracranial Volume ◽

Volume Conduction ◽

Temporal Muscle ◽

The Individual

AbstractObjectiveIntracranial EEG (iEEG) plays an increasingly important role in neuroscientific research and can provide informative control signal for brain-machine interfaces (BMI). While it is clear that electromyographic (EMG) activity of extracranial origin reaches intracranial recordings, the topographic and spectral characteristics of intracranial EMG have been scarcely investigated. It is currently unclear how these characteristics compare to those of physiological brain activity. Little is also known about the exact pathways of extra- to intracranial volume conduction, including the role of craniotomy defects.MethodsIn 5 epilepsy patients under invasive pre-neurosurgical EEG monitoring, we examined chewing-related effects (ChREs) as a source of intracranial EMG activity and compared those effects with physiological brain activity of 9 patients during several behavioural tasks. These included speech production, finger movements, and music perception. Further, we analyzed the association of craniotomy defects (burr-holes and saw-lines) and the intracranial EMG-effects based on the individual post-operative images.ResultsChRE presented with a spatially smooth distribution across almost all intracranial electrodes with the maximum below the temporal muscle. In contrast, the responses of neural origin were spatially more focalized. ChREs were broad-banded and had a higher spectral power and affected higher frequencies than event-related neural activity. ChRE were largely independent of the individual configuration of craniotomy defects. However, we found indications that the silicone sheet, in which electrocorticography (ECoG) electrodes are embedded, attenuates EMG influences, when sufficiently large.ConclusionThe present work is the first comprehensive evaluation of topographic and spectral characteristics of EMG effects in iEEG based on a large sample of subjects. It shows that chewing-related EMG can affect iEEG recordings with higher power than typical physiological brain activity, especially in higher spectral frequencies. As the topographic pattern of ChRE is largely independent of the individual position of craniotomy defects, a direct pathway of volume conduction through the intact skull plays an important role for extra- to-intracranial signal propagation. Intracranial EMG activity related to natural behavior should be accounted for in neuroscientific and BMI applications, especially when based on high-frequency iEEG components. A detailed knowledge of EMG properties may help to design both EMG-reducing algorithms and ECoG grids with a high shielding factor.HighlightsFirst comprehensive description of chewing-related EMG artifacts in iEEG recordingsEMG artifacts and brain activity have distinct topographic and spectral iEEG characteristicsChewing EMG reaches the brain with higher spectral power than task-related brain activityChewing-related EMG artifacts are largely independent of the the position of craniotomy defects

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Formal model of Parkinson’s disease neurons unveils possible causality links in the pathophysiology of the disease

10.1101/2020.02.06.937664 ◽

2020 ◽

Author(s):

Morgane Nadal ◽

Gabriele S. Kaminski Schierle ◽

Duygu Dikicioglu

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Drug Targets ◽

Iron Homeostasis ◽

Formal Model ◽

Disease Onset ◽

List Type ◽

Ros Production ◽

Sequence Of Events ◽

Neurophysiological Mechanisms

SummaryParkinson’s Disease is the second most common neurodegenerative disease after Alzheimer’s disease. Despite extensive research, the initial cause of the disease is still unknown, although substantial advances were made in understanding of its genetics and the cognate neurophysiological mechanisms. Determining the causality relationships and the chronological steps pertaining to Parkinson’s Disease is essential for the discovery of novel drug targets. We developed a systematic in silico model based on available data, which puts the possible sequence of events occurring in a neuron during disease onset into light. This is the first ever attempt, to our knowledge, to model comprehensively the primary modifications in the molecular pathways that manifest in compromised neurons from the commencement of the disease to the consequences of its progression. We showed that our proposed disease pathway was relevant for unveiling yet incomplete knowledge on calcium homeostasis in mitochondria, ROS production and α-synuclein misfolding.Graphical abstractHighlightsVarying calcium concentration in aging dopaminergic neurons triggers disease onset.ROS production in the mitochondria potentially causes iron accumulation.Iron homeostasis dysregulation is linked to α-synuclein aggregation.

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Spatial drivers and pre-cancer populations collaborate with the microenvironment in untreated and chemo-resistant pancreatic cancer

10.1101/2021.01.13.426413 ◽

2021 ◽

Author(s):

Daniel Cui Zhou ◽

Reyka G. Jayasinghe ◽

John M. Herndon ◽

Erik Storrs ◽

Chia-Kuei Mo ◽

...

Keyword(s):

Single Cell ◽

Tumor Cells ◽

Drug Targets ◽

Treatment Options ◽

Receptor Expression ◽

Ductal Adenocarcinoma ◽

List Type ◽

Treatment Regimens ◽

Coordinated Expression ◽

Treatment Naïve

SUMMARYPancreatic Ductal Adenocarcinoma (PDAC) is a lethal disease with limited treatment options and poor survival. We studied 73 samples from 21 patients (7 treatment-naïve and 14 treated with neoadjuvant regimens), analyzing distinct spatial units and performing bulk proteogenomics, single cell sequencing, and cellular imaging. Spatial drivers, including mutant KRAS, SMAD4, and GNAQ, were associated with differential phosphosignaling and metabolic responses compared to wild type. Single cell subtyping discovered 12 of 21 tumors with mixed basal and classical features. Trefoil factor family members were upregulated in classical populations, while the basal populations showed enhanced expression of mesenchymal genes, including VIM and IGTB1. Acinar-ductal metaplasia (ADM) populations, present in 95% of patients, with 46% reduction of driver mutation fractions compared to tumor populations, exhibited suppressive and oncogenic features linked to morphologic states. We identified coordinated expression of TIGIT in exhausted and regulatory T cells and Nectin receptor expression in tumor cells. Higher expression of angiogenic and stress response genes in dendritic cells compared to tumor cells suggests they have a pro-tumorigenic role in remodeling the microenvironment. Treated samples contain a three-fold enrichment of inflammatory CAFs when compared to untreated samples, while other CAF subtypes remain similar. A subset of tumor and/or ADM-specific biomarkers showed differential expression between treatment groups, and several known drug targets displayed potential cross-cell type reactivities. This resolution that spatially defined single cell omics provides reveals the diversity of tumor and microenvironment populations in PDAC. Such understanding may lead to more optimal treatment regimens for patients with this devastating disease.HIGHLIGHTSAcinar-ductal metaplasia (ADM) cells represent a genetic and morphologic transition state between acinar and tumor cells.Inflammatory cancer associated fibroblasts (iCAFs) are a major component of the PDAC TME and are significantly higher in treated samplesReceptor-ligand analysis reveals tumor cell-TME interactions through NECTIN4-TIGITTumor and ADM cell proteogenomics differ between treated and untreated samples, with unique and shared potential drug targets

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AP-2γ is Required for Maintenance of Pluripotent Mammary Stem Cells

10.1101/2020.06.10.107078 ◽

2020 ◽

Author(s):

Vivian W. Gu ◽

Edward Cho ◽

Dakota T. Thompson ◽

Victoria C. Cassady ◽

Nicholas Borcherding ◽

...

Keyword(s):

Stem Cells ◽

Mammary Gland ◽

Target Genes ◽

Mammary Development ◽

Conditional Knockout ◽

List Type ◽

Mammary Stem Cells ◽

Regenerative Capacity ◽

Mammary Stem ◽

Deficient Mice

SUMMARYMammary gland ductal morphogenesis depends on the differentiation of mammary stem cells (MaSCs) into basal and luminal lineages. The AP-2γ transcription factor, encoded by Tfap2c, has a central role in mammary gland development but its effect in mammary lineages and specifically MaSCs is largely unknown. Herein, we utilized an inducible, conditional knockout of Tfap2c to elucidate the role of AP-2γ in maintenance and differentiation of MaSCs. Loss of AP-2γ in the basal epithelium profoundly altered the transcriptomes and decreased the number of cells within several clusters of mammary epithelial cells, including adult MaSCs and luminal progenitors.AP-2γ regulated the expression of genes known to be required for mammary development including C/EBPβ, IκBα, and Rspo1. As a result, AP-2γ-deficient mice exhibited repressed mammary gland ductal outgrowth and inhibition of regenerative capacity. The findings demonstrate that AP-2γ is required for maintenance of pluripotent MaSCs and their ability to develop mammary gland structures.HighlightsAP-2γ-deficient mice exhibited repressed ductal outgrowth and regenerative capacityLoss of AP-2γ reduced the number of mammary stem and luminal progenitor cellsAP-2γ target genes, including C/EBPβ, IκBα, and Rspo1, regulate mammary developmentAP-2γ is required for maintenance of pluripotent mammary stem cellseTOC blurbGu, Cho and colleagues utilized a conditional knockout of Tfap2c to examine transcriptional effects of AP-2γ on mammary stem cells. Single cell analysis demonstrated that AP-2γ-deficient mice have decreased numbers of mammary stem cells and alteration of genes required for mammary development including C/EBPβ, IκBα, and Rspo1. They demonstrate that AP-2γ is necessary for maintenance of pluripotent mammary stem cells.

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Neuronal gain modulability is determined by dendritic morphology: a computational optogenetic study

10.1101/096586 ◽

2016 ◽

Author(s):

Sarah Jarvis ◽

Konstantin Nikolic ◽

Simon R Schultz

Keyword(s):

Dendritic Tree ◽

Gain Control ◽

Pyramidal Cells ◽

Dendritic Morphology ◽

Inhibitory Input ◽

List Type ◽

Moderate Degree ◽

Gain Modulation ◽

Input Output

AbstractThe mechanisms by which the gain of the neuronal input-output function may be modulated have been the subject of much investigation. However, little is known of the role of dendrites in neuronal gain control. New optogenetic experimental paradigms based on spatial profiles or patterns of light stimulation offer the prospect of elucidating many aspects of single cell function, including the role of dendrites in gain control. We thus developed a model to investigate how competing excitatory and inhibitory input within the dendritic arbor alters neuronal gain, incorporating kinetic models of opsins into our modeling to ensure it is experimentally testable. To investigate how different topologies of the neuronal dendritic tree affect the neuron’s input-output characteristics we generate branching geometries which replicate morphological features of most common neurons, but keep the number of branches and overall area of dendrites approximately constant. We found a relationship between a neuron’s gain modulability and its dendritic morphology, with neurons with bipolar dendrites with a moderate degree of branching being most receptive to control of the gain of their input-output relationship. The theory was then tested and confirmed on two examples of realistic neurons: 1) layer V pyramidal cells - confirming their role in neural circuits as a regulator of the gain in the circuit in addition to acting as the primary excitatory neurons, and 2) stellate cells. In addition to providing testable predictions and a novel application of dual-opsins, our model suggests that innervation of all dendritic subdomains is required for full gain modulation, revealing the importance of dendritic targeting in the generation of neuronal gain control and the functions that it subserves. Finally, our study also demonstrates that neurophysiological investigations which use direct current injection into the soma and bypass the dendrites may miss some important neuronal functions, such as gain modulation.Author SummaryGain modulability indicated by dendritic morphologyPyramidal cell-like shapes optimally receptive to modulationAll dendritic subdomains required for gain modulation, partial illumination is insufficientComputational optogenetic models improve and refine experimental protocols

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Oncogenic activation of Nrf2 by specific knockout of Nrf1α that acts as a dominant tumor repressor

10.1101/403220 ◽

2018 ◽

Author(s):

Lu Qiu ◽

Meng Wang ◽

Shaofan Hu ◽

Xufang Ru ◽

Yonggang Ren ◽

...

Keyword(s):

Feedback Loop ◽

A Priori ◽

Underlying Mechanism ◽

Suppressive Effect ◽

List Type ◽

Inflammation Marker ◽

Malignant Growth ◽

Pathological Model ◽

Xenograft Mice ◽

Tumor Suppressive Effect

SUMMARYLiver-specific knockout of Nrf1 in mice leads to non-alcoholic steatohepatitis with dyslipidemia, and its deterioration results in spontaneous hepatoma, but the underlying mechanism remains elusive. A similar pathological model is herein reconstructed by using human Nrf1α-specific knockout cell lines. We demonstrated that a marked increase of the inflammation marker COX2 in Nrf1α−/− cells. Loss of Nrf1α leads to hyperactivation of Nrf2, which results from substantial decreases in both Keap1 and PTEN in Nrf1α−/− cells. Further investigation of xenograft mice showed that malignant growth of Nrf1α−/−-derived tumor is almost abolished by silencing Nrf2, while Nrf1α+/+-tumor is markedly repressed by inactive Nrf2−/−ΔTA, but unaffected by a priori constitutive activator of caNrf2ΔN. Mechanistic studies unraveled there exist opposing and unifying inter-regulatory cross-talks between Nrf1 and Nrf2. Collectively, Nrf1α manifests a dominant tumor-suppressive effect by confining Nrf2 oncogenicity, while Nrf2 can directly activate the transcriptional expression of Nrf1 to form a negative feedback loop.HIGHLIGHTSOpposing and unifying inter-regulatory cross-talks between Nrf1α and Nrf2Malignant growth of Nrf1α−/−-derived tumor is prevented by silencing Nrf2Hyper-activation of Nrf2 by Nrf1α−/− results from decreased Keap1 and PTENNrf1α+/+-tumor is repressed by Nrf2−/−ΔTA, but unaltered by its active caNrf2ΔN

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