Faculty Bibliography

AMPK is an important sensor of cellular energy levels. The aim of these studies was to investigate whether cardiac K(ATP) channels, which couple cellular energy metabolism to membrane excitability, are regulated by AMPK activity. We investigated effects of AMPK on rat ventricular K(ATP) channels using electrophysiological and biochemical approaches. Whole-cell K(ATP) channel current was activated by metabolic inhibition; this occurred more rapidly in the presence of AICAR (an AMPK activator). AICAR had no effects on K(ATP) channel activity recorded in the inside-out patch clamp configuration, but ZMP (the intracellular intermediate of AICAR) strongly activated K(ATP) channels. An AMPK-mediated effect is demonstrated by the finding that ZMP had no effect on K(ATP) channels in the presence of Compound C (an AMPK inhibitor). Recombinant AMPK activated Kir6.2/SUR2A channels in a manner that was dependent on the AMP concentration, whereas heat-inactivated AMPK was without effect. Using mass-spectrometry and co-immunoprecipitation approaches, we demonstrate that the AMPK alpha-subunit physically associates with K(ATP) channel subunits. Our data demonstrate that the cardiac K(ATP) channel function is directly regulated by AMPK activation. During metabolic stress, a small change in cellular AMP that activates AMPK can be a potential trigger for K(ATP) channel opening. This article is part of a Special Issue entitled 'Local Signaling in Myocytes'

Purpose:To investigate technical feasibility, test-retest reproducibility, and the ability to differentiate healthy subjects from subjects with osteoarthritis (OA) with diffusion-tensor (DT) imaging parameters and T2 relaxation time.Materials and Methods:This study was approved by the institutional review board and was HIPAA compliant. All subjects provided written informed consent. DT imaging parameters and T2 (resolution = 0.6 x 0.6 x 2 mm) of patellar cartilage were measured at 7.0 T in 16 healthy volunteers and 10 patients with OA with subtle inhomogeneous signal intensity but no signs of cartilage erosion at clinical magnetic resonance (MR) imaging. Ten volunteers were imaged twice to determine test-retest reproducibility. After cartilage segmentation, maps of mean apparent diffusion coefficient (ADC), fractional anisotropy (FA), and T2 relaxation time were calculated. Differences for ADC, FA, and T2 between the healthy and OA populations were assessed with nonparametric tests. The ability of each MR imaging parameter to help discriminate healthy subjects from subjects with OA was assessed by using receiver operating characteristic curve analysis.Results:Test-retest reproducibility was better than 10% for mean ADC (8.1%), FA (9.7%), and T2 (5.9%). Mean ADC and FA differed significantly (P < .01) between the OA and healthy populations, but T2 did not. For ADC, the optimal threshold to differentiate both populations was 1.2 x 10(-3) mm(2)/sec, achieving specificity of 1.0 (16 of 16) and sensitivity of 0.80 (eight of 10). For FA, the optimal threshold was 0.25, yielding specificity of 0.88 (14 of 16) and sensitivity of 0.80 (eight of 10). T2 showed poor differentiation between groups (optimal threshold = 22.9 msec, specificity = 0.69 [11 of 16], sensitivity = 0.60 [six of 10]).Conclusion:In vivo DT imaging of patellar cartilage is feasible, has good test-retest reproducibility, and may be accurate in discriminating healthy subjects from subjects with OA. ADC and FA are two promising biomarkers for early OA.(c) RSNA, 2011

The hTau mouse model of tauopathy was utilized to assess gene expression changes in vulnerable hippocampal CA1 neurons. CA1 pyramidal neurons were microaspirated via laser capture microdissection followed by RNA amplification in combination with custom-designed microarray analysis and qPCR validation in hTau mice and nontransgenic (ntg) littermates aged 11-14months. Statistical analysis revealed ~8% of all the genes on the array platform were dysregulated, with notable downregulation of several synaptic-related markers including synaptophysin (Syp), synaptojanin, and synaptobrevin, among others. Downregulation was also observed for select glutamate receptors (GluRs), Psd-95, TrkB, and several protein phosphatase subunits. In contrast, upregulation of tau isoforms and a calpain subunit were found. Microarray assessment of synaptic-related markers in a separate cohort of hTau mice at 7-8months of age indicated only a few alterations compared to the 11-14month cohort, suggesting progressive synaptic dysfunction occurs as tau accumulates in CA1 pyramidal neurons. An assessment of SYP and PSD-95 expression was performed in the hippocampal CA1 sector of hTau and ntg mice via confocal laser scanning microscopy along with hippocampal immunoblot analysis for protein-based validation of selected microarray observations. Results indicate significant decreases in SYP-immunoreactive and PSD-95-immunoreactive puncta as well as downregulation of SYP-immunoreactive and PSD-95-immunoreactive band intensity in hTau mice compared to age-matched ntg littermates. In summary, the high prevalence of downregulation of synaptic-related genes indicates that the moderately aged hTau mouse may be a model of tau-induced synaptodegeneration, and has profound effects on how we perceive progressive tau pathology affecting synaptic transmission in AD

Intracranial recording is an important diagnostic method routinely used in a number of neurological monitoring scenarios. In recent years, advancements in such recordings have been extended to include unit activity of an ensemble of neurons. However, a detailed functional characterization of excitatory and inhibitory cells has not been attempted in human neocortex, particularly during the sleep state. Here, we report that such feature discrimination is possible from high-density recordings in the neocortex by using 2D multielectrode arrays. Successful separation of regular-spiking neurons (or bursting cells) from fast-spiking cells resulted in well-defined clusters that each showed unique intrinsic firing properties. The high density of the array, which allowed recording from a large number of cells (up to 90), helped us to identify apparent monosynaptic connections, confirming the excitatory and inhibitory nature of regular-spiking and fast-spiking cells, thus categorized as putative pyramidal cells and interneurons, respectively. Finally, we investigated the dynamics of correlations within each class. A marked exponential decay with distance was observed in the case of excitatory but not for inhibitory cells. Although the amplitude of that decline depended on the timescale at which the correlations were computed, the spatial constant did not. Furthermore, this spatial constant is compatible with the typical size of human columnar organization. These findings provide a detailed characterization of neuronal activity, functional connectivity at the microcircuit level, and the interplay of excitation and inhibition in the human neocortex.

Most neurons fire in bursts, imposing episodic energy demands, but how these demands are coordinated with oxidative phosphorylation is still unknown. Here, using fluorescence imaging techniques on presynaptic termini of Drosophila motor neurons (MNs), we show that mitochondrial matrix pH (pH(m)), inner membrane potential (Deltapsi(m)), and NAD(P)H levels ([NAD(P)H](m)) increase within seconds of nerve stimulation. The elevations of pH(m), Deltapsi(m), and [NAD(P)H](m) indicate an increased capacity for ATP production. Elevations in pH(m) were blocked by manipulations that blocked mitochondrial Ca(2+) uptake, including replacement of extracellular Ca(2+) with Sr(2+) and application of either tetraphenylphosphonium chloride or KB-R7943, indicating that it is Ca(2+) that stimulates presynaptic mitochondrial energy metabolism. To place this phenomenon within the context of endogenous neuronal activity, the firing rates of a number of individually identified MNs were determined during fictive locomotion. Surprisingly, although endogenous firing rates are significantly different, there was little difference in presynaptic cytosolic Ca(2+) levels ([Ca(2+)](c)) between MNs when each fires at its endogenous rate. The average [Ca(2+)](c) level (329 +/- 11 nm) was slightly above the average Ca(2+) affinity of the mitochondria (281 +/- 13 nm). In summary, we show that when MNs fire at endogenous rates, [Ca(2+)](c) is driven into a range where mitochondria rapidly acquire Ca(2+). As we also show that Ca(2+) stimulates presynaptic mitochondrial energy metabolism, we conclude that [Ca(2+)](c) levels play an integral role in coordinating mitochondrial energy metabolism with presynaptic activity in Drosophila MNs

Regulation of the hypothalamic-pituitary-adrenal (HPA) axis is critical for adaptation to environmental changes. The principle regulator of the HPA axis is corticotrophin-releasing hormone (CRH), which is made in the parventricular nucleus and is an important target of negative feedback by glucocorticoids. However, the molecular mechanisms that regulate CRH are not fully understood. Disruption of normal HPA axis activity is a major risk factor of neuropsychiatric disorders in which decreased expression of the glucocorticoid receptor (GR) has been documented. To investigate the role of the GR in CRH neurons, we have targeted the deletion of the GR, specifically in the parventricular nucleus. Impairment of GR function in the parventricular nucleus resulted in an enhancement of CRH expression and an up-regulation of hypothalamic levels of BDNF and disinhibition of the HPA axis. BDNF is a stress and activity-dependent factor involved in many activities modulated by the HPA axis. Significantly, ectopic expression of BDNF in vivo increased CRH, whereas reduced expression of BDNF, or its receptor TrkB, decreased CRH expression and normal HPA functions. We find the differential regulation of CRH relies upon the cAMP response-element binding protein coactivator CRTC2, which serves as a switch for BDNF and glucocorticoids to direct the expression of CRH.

Lipids are key regulators of brain function and have been increasingly implicated in neurodegenerative disorders including Alzheimer disease (AD). Here, a systems-based approach was employed to determine the lipidome of brain tissues affected by AD. Specifically, we used liquid chromatography-mass spectrometry to profile extracts from the prefrontal cortex, entorhinal cortex, and cerebellum of late-onset AD (LOAD) patients, as well as the forebrain of three transgenic familial AD (FAD) mouse models. Although the cerebellum lacked major alterations in lipid composition, we found an elevation of a signaling pool of diacylglycerol as well as sphingolipids in the prefrontal cortex of AD patients. Furthermore, the diseased entorhinal cortex showed specific enrichment of lysobisphosphatidic acid, sphingomyelin, the ganglioside GM3, and cholesterol esters, all of which suggest common pathogenic mechanisms associated with endolysosomal storage disorders. Importantly, a significant increase in cholesterol esters and GM3 was recapitulated in the transgenic FAD models, suggesting that these mice are relevant tools to study aberrant lipid metabolism of endolysosomal dysfunction associated with AD. Finally, genetic ablation of phospholipase D(2), which rescues the synaptic and behavioral deficits of an FAD mouse model, fully normalizes GM3 levels. These data thus unmask a cross-talk between the metabolism of phosphatidic acid, the product of phospholipase D(2), and gangliosides, and point to a central role of ganglioside anomalies in AD pathogenesis. Overall, our study highlights the hypothesis generating potential of lipidomics and identifies novel region-specific lipid anomalies potentially linked to AD pathogenesis

TBX3 is critical for human development: mutations in TBX3 cause congenital anomalies in patients with ulnar-mammary syndrome. Data from mice and humans suggest multiple roles for Tbx3 in development and function of the cardiac conduction system. The mechanisms underlying the functional development, maturation, and maintenance of the conduction system are not well understood. We tested the requirements for Tbx3 in these processes. We generated a unique series of Tbx3 hypomorphic and conditional mouse mutants with varying levels and locations of Tbx3 activity within the heart, and developed techniques for evaluating in vivo embryonic conduction system function. Disruption of Tbx3 function in different regions of the developing heart causes discrete phenotypes and lethal arrhythmias: sinus pauses and bradycardia indicate sinoatrial node dysfunction, whereas preexcitation and atrioventricular block reveal abnormalities in the atrioventricular junction. Surviving Tbx3 mutants are at increased risk for sudden death. Arrhythmias induced by knockdown of Tbx3 in adults reveal its requirement for conduction system homeostasis. Arrhythmias in Tbx3-deficient embryos are accompanied by disrupted expression of multiple ion channels despite preserved expression of previously described conduction system markers. These findings indicate that Tbx3 is required for the conduction system to establish and maintain its correct molecular identity and functional properties. In conclusion, Tbx3 is required for the functional development, maturation, and homeostasis of the conduction system in a highly dosage-sensitive manner. TBX3 and its regulatory targets merit investigation as candidates for human arrhythmias.

We have investigated the source(s) and targeting of components to PNS nodes of Ranvier. We show adhesion molecules are freely diffusible within the axon membrane and accumulate at forming nodes from local sources, whereas ion channels and cytoskeletal components are largely immobile and require transport to the node. We further characterize targeting of NF186, an adhesion molecule that pioneers node formation. NF186 redistributes to nascent nodes from a mobile, surface pool. Its initial accumulation and clearance from the internode require extracellular interactions, whereas targeting to mature nodes, i.e., those flanked by paranodal junctions, requires intracellular interactions. After incorporation into the node, NF186 is immobile, stable, and promotes node integrity. Thus, nodes assemble from two sources: adhesion molecules, which initiate assembly, accumulate by diffusion trapping via interactions with Schwann cells, whereas ion channels and cytoskeletal components accumulate via subsequent transport. In mature nodes, components turnover slowly and are replenished via transport. VIDEO ABSTRACT: