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Whole human genome sequencing of individuals is becoming rapid and inexpensive, enabling new strategies for using personal genome information to help diagnose, treat, and even prevent human disorders for which genetic variations are causative or are known to be risk factors. Many of the exploding number of newly discovered genetic variations alter the structure, function, dynamics, stability, and/or interactions of specific proteins and RNA molecules. Accordingly, there are a host of opportunities for biochemists and biophysicists to participate in (1) developing tools to allow accurate and sometimes medically actionable assessment of the potential pathogenicity of individual variations and (2) establishing the mechanistic linkage between pathogenic variations and their physiological consequences, providing a rational basis for treatment or preventive care. In this review, we provide an overview of these opportunities and their associated challenges in light of the current status of genomic science and personalized medicine, the latter often termed precision medicine.
Broad-spectrum muscarinic receptor antagonists have represented the first available treatment for different movement disorders such as dystonia. However, the specificity of these drugs and their mechanism of action is not entirely clear. We performed a systematic analysis of the effects of anticholinergic drugs on short- and long-term plasticity recorded from striatal medium spiny neurons from DYT1 dystonia knock-in (Tor1a(+/Δgag) ) mice heterozygous for ΔE-torsinA and their controls (Tor1a(+/+) mice). Antagonists were chosen that had previously been proposed to be selective for muscarinic receptor subtypes and included pirenzepine, trihexyphenydil, biperiden, orphenadrine, and a novel selective M1 antagonist, VU0255035. Tor1a(+/Δgag) mice exhibited a significant impairment of corticostriatal synaptic plasticity. Anticholinergics had no significant effects on intrinsic membrane properties and on short-term plasticity of striatal neurons. However, they exhibited a differential ability to restore the corticostriatal plasticity deficits. A complete rescue of both long-term depression (LTD) and synaptic depotentiation (SD) was obtained by applying the M1 -preferring antagonists pirenzepine and trihexyphenidyl as well as VU0255035. Conversely, the nonselective antagonist orphenadrine produced only a partial rescue of synaptic plasticity, whereas biperiden and ethopropazine failed to restore plasticity. The selectivity for M1 receptors was further demonstrated by their ability to counteract the M1 -dependent potentiation of N-methyl-d-aspartate (NMDA) current recorded from striatal neurons. Our study demonstrates that selective M1 muscarinic receptor antagonism offsets synaptic plasticity deficits in the striatum of mice with the DYT1 dystonia mutation, providing a potential mechanistic rationale for the development of improved antimuscarinic therapies for this movement disorder.
© 2014 International Parkinson and Movement Disorder Society.
The channel pore-forming α subunit Kv4.2 is a major constituent of A-type (I(A)) potassium currents and a key regulator of neuronal membrane excitability. Multiple mechanisms regulate the properties, subcellular targeting, and cell-surface expression of Kv4.2-encoded channels. In the present study, shotgun proteomic analyses of immunoprecipitated mouse brain Kv4.2 channel complexes unexpectedly identified the voltage-gated Na⁺ channel accessory subunit Navβ1. Voltage-clamp and current-clamp recordings revealed that knockdown of Navβ1 decreases I(A) densities in isolated cortical neurons and that action potential waveforms are prolonged and repetitive firing is increased in Scn1b-null cortical pyramidal neurons lacking Navβ1. Biochemical and voltage-clamp experiments further demonstrated that Navβ1 interacts with and increases the stability of the heterologously expressed Kv4.2 protein, resulting in greater total and cell-surface Kv4.2 protein expression and in larger Kv4.2-encoded current densities. Together, the results presented here identify Navβ1 as a component of native neuronal Kv4.2-encoded I(A) channel complexes and a novel regulator of I(A) channel densities and neuronal excitability.
In vulnerable individuals, exposure to stressors can result in chronic disorders such as generalized anxiety disorder (GAD), major depressive disorder (MDD), and post-traumatic stress disorder (PTSD). The extended amygdala is critically implicated in mediating acute and chronic stress responsivity and anxiety-like behaviors. The bed nucleus of the stria terminalis (BNST), a subregion of the extended amygdala, serves as a relay of corticolimbic information to the paraventricular nucleus of the hypothalamus (PVN) to directly influence the stress response. To investigate the influence of the corticosteroid milieu and housing conditions on BNST function, adult C57Bl/6J were either acutely or chronically administered corticosterone (CORT, 25mg/kg in sesame oil) or vehicle (sesame oil) or were group housed or socially isolated for 1 day (acute) or 6-8 weeks (chronic). To ascertain whether these stressors could influence anxiety-like behavior, studies were performed using the novel open-field (NOF) and the elevated zero maze (EZM) tests. To investigate potential associated changes in plasticity, alterations in BNST function were assessed using ex vivo extracellular field potential recordings in the (dorsal-lateral) dlBNST and a high frequency stimulus protocol to induce long-term potentiation (LTP). Our results suggest that chronic CORT injections and chronic social isolation housing conditions lead to an increase in anxiety-like behavior on the EZM and NOF. Chronically stressed mice also displayed a parallel blunting of LTP in the dlBNST. Conversely, acute social isolation housing had no effect on anxiety-like behavior but still resulted in a blunting of LTP in the dlBNST. Collectively, our results suggest acute and chronic stressors can have a distinct profile on plasticity in the BNST that is not uniformly associated with an increase in anxiety-like behavior.
Copyright © 2011 Elsevier Inc. All rights reserved.
GABA(A) receptors are composed predominantly of alphabetagamma receptors, which mediate primarily synaptic inhibition, and alphabetadelta receptors, which mediate primarily extrasynaptic inhibition. At saturating GABA concentrations, the barbiturate pentobarbital substantially increased the amplitude and desensitization of the alpha1beta3delta receptor but not the alpha1beta3gamma2L receptor currents. To explore the structural domains of the delta subunit that are involved in pentobarbital potentiation and increased desensitization of alpha1beta3delta currents, chimeric cDNAs were constructed by progressive replacement of gamma2L subunit sequence with a delta subunit sequence or a delta subunit sequence with a gamma2L subunit sequence, and HEK293T cells were co-transfected with alpha1 and beta3 subunits or alpha1 and beta3 subunits and a gamma2L, delta, or chimeric subunit. Currents evoked by a saturating concentration of GABA or by co-application of GABA and pentobarbital were recorded using the patch clamp technique. By comparing the extent of enhancement and changes in kinetic properties produced by pentobarbital among chimeric and wild type receptors, we concluded that although potentiation of alpha1beta3delta currents by pentobarbital required the delta subunit sequence from the N terminus to proline 241 in the first transmembrane domain (M1), increasing desensitization of alpha1beta3delta currents required a delta subunit sequence from the N terminus to isoleucine 235 in M1. These findings suggest that the delta subunit N terminus and N-terminal portion of the M1 domain are, at least in part, involved in transduction of the allosteric effect of pentobarbital to enhance alpha1beta3delta currents and that this effect involves a distinct but overlapping structural domain from that involved in altering desensitization.
Normal human consciousness may be impaired by two possible routes: direct reduced function in widespread cortical regions or indirect disruption of subcortical activating systems. The route through which temporal lobe limbic seizures impair consciousness is not known. We recently developed an animal model that, like human limbic seizures, exhibits neocortical deactivation including cortical slow waves and reduced cortical cerebral blood flow (CBF). We now find through functional magnetic resonance imaging (fMRI) that electrically stimulated hippocampal seizures in rats cause increased activity in subcortical structures including the septal area and mediodorsal thalamus, along with reduced activity in frontal, cingulate, and retrosplenial cortex. Direct recordings from the hippocampus, septum, and medial thalamus demonstrated fast poly-spike activity associated with increased neuronal firing and CBF, whereas frontal cortex showed slow oscillations with decreased neuronal firing and CBF. Stimulation of septal area, but not hippocampus or medial thalamus, in the absence of a seizure resulted in cortical deactivation with slow oscillations and behavioral arrest, resembling changes seen during limbic seizures. Transecting the fornix, the major route from hippocampus to subcortical structures, abolished the negative cortical and behavioral effects of seizures. Cortical slow oscillations and behavioral arrest could be reconstituted in fornix-lesioned animals by inducing synchronous activity in the hippocampus and septal area, implying involvement of a downstream region converged on by both structures. These findings suggest that limbic seizures may cause neocortical deactivation indirectly, through impaired subcortical function. If confirmed, subcortical networks may represent a target for therapies aimed at preserving consciousness in human temporal lobe seizures.
BACKGROUND - Inherited long-QT syndrome is characterized by prolonged QT interval on the ECG, syncope, and sudden death caused by ventricular arrhythmia. Causative mutations occur mostly in cardiac potassium and sodium channel subunit genes. Confidence in mutation pathogenicity is usually reached through family genotype-phenotype tracking, control population studies, molecular modeling, and phylogenetic alignments; however, biophysical testing offers a higher degree of validating evidence.
METHODS AND RESULTS - By using in vitro electrophysiological testing of transfected mutant and wild-type long-QT syndrome constructs into Chinese hamster ovary cells, we investigated the biophysical properties of 9 KCNQ1 missense mutations (A46T, T265I, F269S, A302V, G316E, F339S, R360G, H455Y, and S546L) identified in a New Zealand-based long-QT syndrome screening program. We demonstrate through electrophysiology and molecular modeling that 7 of the missense mutations have profound pathological dominant-negative loss-of-function properties, confirming their likely disease-causing nature. This supports the use of these mutations in diagnostic family screening. Two mutations (A46T, T265I) show suggestive evidence of pathogenicity within the experimental limits of biophysical testing, indicating that these variants are disease-causing via delayed- or fast-activation kinetics. Further investigation of the A46T family has revealed an inconsistent cosegregation of the variant with the clinical phenotype.
CONCLUSIONS - Electrophysiological characterization should be used to validate long-QT syndrome pathogenicity of novel missense channelopathies. When such results are inconclusive, great care should be taken with genetic counseling and screening of such families, and alternative disease-causing mechanisms should be considered.
Learning and memory depend on neuronal alterations induced by electrical activity. Most examples of activity-dependent plasticity, as well as adaptive responses to neuronal injury, have been linked explicitly or implicitly to induction by Ca(2+) signals produced by depolarization. Indeed, transient Ca(2+) signals are commonly assumed to be the only effective transducers of depolarization into adaptive neuronal responses. Nevertheless, Ca(2+)-independent depolarization-induced signals might also trigger plastic changes. Establishing the existence of such signals is a challenge because procedures that eliminate Ca(2+) transients also impair neuronal viability and tolerance to cellular stress. We have taken advantage of nociceptive sensory neurons in the marine snail Aplysia, which exhibit unusual tolerance to extreme reduction of extracellular and intracellular free Ca(2+) levels. The axons of these neurons exhibit a depolarization-induced memory-like hyperexcitability that lasts a day or longer and depends on local protein synthesis for induction. Here we show that transient localized depolarization of these axons in an excised nerve-ganglion preparation or in dissociated cell culture can induce short- and intermediate-term axonal hyperexcitability as well as long-term protein synthesis-dependent hyperexcitability under conditions in which Ca(2+) entry is prevented (by bathing in nominally Ca(2+) -free solutions containing EGTA) and detectable Ca(2+) transients are eliminated (by adding BAPTA-AM). Disruption of Ca(2+) release from intracellular stores by pretreatment with thapsigargin also failed to affect induction of axonal hyperexcitability. These findings suggest that unrecognized Ca(2+)-independent signals exist that can transduce intense depolarization into adaptive cellular responses during neuronal injury, prolonged high-frequency activity, or other sustained depolarizing events.
The mechanisms of chain selection and assembly of fibril-associated collagens with interrupted triple helices (FACITs) must differ from that of fibrillar collagens, since they lack the characteristic C-propeptide. We analyzed two carboxyl-terminal noncollagenous domains, NC2 and NC1, of collagen XIX as potential trimerization units and found that NC2 forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. In contrast, the NC1 domain requires formation of an adjacent collagen triple helix to form interchain disulfide bridges. The NC2 domain of collagen XIX and probably of other FACITs is responsible for chain selection and trimerization.
This paper reports on the development and preliminary testing of a three-dimensional implementation of an inverse problem technique for extracting soft-tissue elasticity information via non-rigid model-based image registration. The modality-independent elastography (MIE) algorithm adjusts the elastic properties of a biomechanical model to achieve maximal similarity between images acquired under different states of static loading. A series of simulation experiments with clinical image sets of human breasts were performed to test the ability of the method to identify and characterize a radiographically occult stiff lesion. Because boundary conditions are a critical input to the algorithm, a comparison of three methods for semi-automated surface point correspondence was conducted in the context of systematic and randomized noise processes. The results illustrate that 3D MIE was able to successfully reconstruct elasticity images using data obtained from both magnetic resonance and x-ray computed tomography systems. The lesion was localized correctly in all cases and its relative elasticity found to be reasonably close to the true values (3.5% with the use of spatial priors and 11.6% without). In addition, the inaccuracies of surface registration performed with thin-plate spline interpolation did not exceed empiric thresholds of unacceptable boundary condition error.