Seizures, behavioral deficits, and adverse drug responses in two new genetic mouse models of HCN1 epileptic encephalopathy

De novo mutations in voltage- and ligand-gated channels have been associated with an increasing number of cases of developmental and epileptic encephalopathies, which often fail to respond to classic antiseizure medications. Here, we examine two knock-in mouse models replicating de novo sequence variations in the human HCN1 voltage-gated channel gene, p.G391D and p.M153I (Hcn1(G380D/+) and Hcn1(M142I/+) in mouse), associated with severe drug-resistant neonatal- and childhood-onset epilepsy, respectively. Heterozygous mice from both lines displayed spontaneous generalized tonic-clonic seizures. Animals replicating the p.G391D variant had an overall more severe phenotype, with pronounced alterations in the levels and distribution of HCN1 protein, including disrupted targeting to the axon terminals of basket cell interneurons. In line with clinical reports from patients with pathogenic HCN1 sequence variations, administration of the antiepileptic Na+ channel antagonists lamotrigine and phenytoin resulted in the paradoxical induction of seizures in both mouse lines, consistent with an impairment in inhibitory neuron function. We also show that these variants can render HCN1 channels unresponsive to classic antagonists, indicating the need to screen mutated channels to identify novel compounds with diverse mechanism of action. Our results underscore the necessity of tailoring effective therapies for specific channel gene variants, and how strongly validated animal models may provide an invaluable tool toward reaching this objective

Functional roles of Cav1.3, Cav3.1 and HCN channels in automaticity of mouse atrioventricular cells: Insights into the atrioventricular pacemaker mechanism

The atrioventricular node controls cardiac impulse conduction and generates pacemaker activity in case of failure of the sino-atrial node. Understanding the mechanisms of atrioventricular automaticity is important for managing human pathologies of heart rate and conduction. However, the physiology of atrioventricular automaticity is still poorly understood. We have investigated the role of three key ion channel-mediated pacemaker mechanisms namely, Cav1.3, Cav3.1 and HCN channels in automaticity of atrioventricular node cells (AVNCs). We studied atrioventricular conduction and pacemaking of AVNCs in wild-type mice and mice lacking Cav3.1 (Cav3.1−/−), Cav1.3 (Cav1.3−/−), channels or both (Cav1.3−/−/Cav3.1−/−). The role of HCN channels in the modulation of atrioventricular cells pacemaking was studied by conditional expression of dominant-negative HCN4 channels lacking cAMP sensitivity. Inactivation of Cav3.1 channels impaired AVNCs pacemaker activity by favoring sporadic block of automaticity leading to cellular arrhythmia. Furthermore, Cav3.1 channels were critical for AVNCs to reach high pacemaking rates under isoproterenol. Unexpectedly, Cav1.3 channels were required for spontaneous automaticity, because Cav1.3−/− and Cav1.3−/−/Cav3.1−/− AVNCs were completely silent under physiological conditions. Abolition of the cAMP sensitivity of HCN channels reduced automaticity under basal conditions, but maximal rates of AVNCs could be restored to that of control mice by isoproterenol. In conclusion, while Cav1.3 channels are required for automaticity, Cav3.1 channels are important for maximal pacing rates of mouse AVNCs. HCN channels are important for basal AVNCs automaticity but do not appear to be determinant for β-adrenergic regulation

Measles Vaccines Designed for Enhanced CD8+ T Cell Activation

Priming and activation of CD8+ T cell responses is crucial to achieve anti-viral and anti-tumor immunity. Live attenuated measles vaccine strains have been used successfully for immunization for decades and are currently investigated in trials of oncolytic virotherapy. The available reverse genetics systems allow for insertion of additional genes, including heterologous antigens. Here, we designed recombinant measles vaccine vectors for priming and activation of antigen-specific CD8+ T cells. For proof-of-concept, we used cytotoxic T lymphocyte (CTL) lines specific for the melanoma-associated differentiation antigen tyrosinase-related protein-2 (TRP-2), or the model antigen chicken ovalbumin (OVA), respectively. We generated recombinant measles vaccine vectors with TRP-2 and OVA epitope cassette variants for expression of the full-length antigen or the respective immunodominant CD8+ epitope, with additional variants mediating secretion or proteasomal degradation of the epitope. We show that these recombinant measles virus vectors mediate varying levels of MHC class I (MHC-I)-restricted epitope presentation, leading to activation of cognate CTLs, as indicated by secretion of interferon-gamma (IFNγ) in vitro. Importantly, the recombinant OVA vaccines also mediate priming of naïve OT-I CD8+ T cells by dendritic cells. While all vaccine variants can prime and activate cognate T cells, IFNγ release was enhanced using a secreted epitope variant and a variant with epitope strings targeted to the proteasome. The principles presented in this study will facilitate the design of recombinant vaccines to elicit CD8+ responses against pathogens and tumor antigens

Cardiac arrhythmia induced by genetic silencing of 'funny' (f) channels is rescued by GIRK4 inactivation

The mechanisms underlying cardiac automaticity are still incompletely understood and controversial. Here we report the complete conditional and time-controlled silencing of the 'funny' current (If) by expression of a dominant-negative, non-conductive HCN4-channel subunit (hHCN4-AYA). Heart-specific I-f silencing caused altered [Ca2+](i) release and Ca2+ handling in the sinoatrial node, impaired pacemaker activity and symptoms reminiscent of severe human disease of pacemaking. The effects of I-f silencing critically depended on the activity of the autonomic nervous system. We were able to rescue the failure of impulse generation and conduction by additional genetic deletion of cardiac muscarinic G-protein-activated (GIRK4) channels in I-f-deficient mice without impairing heartbeat regulation. Our study establishes the role of f-channels in cardiac automaticity and indicates that arrhythmia related to HCN loss-of-function may be managed by pharmacological or genetic inhibition of GIRK4 channels, thus offering a new therapeutic strategy for the treatment of heart rhythm diseases

Pacemaker activity and ionic currents in mouse atrioventricular node cells

It is well established that pacemaker activity of the sino-atrial node (SAN) initiates the heartbeat. However, the atrioventricular node (AVN) can generate viable pacemaker activity in case of SAN failure, but we have limited knowledge of the ionic bases of AVN automaticity. We characterized pacemaker activity and ionic currents in automatic myocytes of the mouse AVN. Pacemaking of AVN cells (AVNCs) was lower than that of SAN pacemaker cells (SANCs), both in control conditions and upon perfusion of isoproterenol (ISO). Block of INa by tetrodotoxin (TTX) or of ICa,L by isradipine abolished AVNCs pacemaker activity. TTX-resistant (INar) and TTX-sensitive (INas) Na+ currents were recorded in mouse AVNCs, as well as T-(ICa,T) and L-type (ICa,L) Ca2+ currents. ICa,L density was lower than in SANCs (51%). The density of the hyperpolarization-activated current, (If) and that of the fast component of the delayed rectifier current (IKr) were, respectively, lower (52%) and higher (53%) in AVNCs than in SANCs. Pharmacological inhibition of If by 3 µM ZD-7228 reduced pacemaker activity by 16%, suggesting a relevant role for If in AVNCs automaticity. Some AVNCs expressed also moderate densities of the transient outward K+ current (Ito). In contrast, no detectable slow component of the delayed rectifier current (IKs) could be recorded in AVNCs. The lower densities of If and ICa,L, as well as higher expression of IKr in AVNCs than in SANCs may contribute to the intrinsically slower AVNCs pacemaking than that of SANCs

Effects of Kv1.2 Intracellular Regions on Activation of Kv2.1 Channels

Depolarizing voltage steps activate voltage-dependent K(+) (Kv) channels by moving the voltage sensor, which triggers a coupling reaction leading to the opening of the pore. We constructed chimeric channels in which intracellular regions of slowly activating Kv2.1 channels were replaced by respective regions of rapidly activating Kv1.2 channels. Substitution of either the N-terminus, S4–S5 linker, or C-terminus generated chimeric Kv2.1/1.2 channels with a paradoxically slow and approximately exponential activation time course consisting of a fast and a slow component. Using combined chimeras, each of these Kv1.2 regions further slowed activation at the voltage of 0 mV, irrespective of the nature of the other two regions, whereas at the voltage of 40 mV both slowing and accelerating effects were observed. These results suggest voltage-dependent interactions of the three intracellular regions. This observation was quantified by double-mutant cycle analysis. It is concluded that interactions between N-terminus, S4–S5 linker, and/or C-terminus modulate the activation time course of Kv2.1 channels and that part of these interactions is voltage dependent

Guidelines for the use of flow cytometry and cell sorting in immunological studies

International audienceThe classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127(-) and CD127(+) early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127(-) and CD127(+) ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127(-) ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127(+) ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis