Faculty Bibliography

Tau fibrillization is a potential therapeutic target for Alzheimer's and other neurodegenerative diseases. Several small-molecule inhibitors of tau aggregation have been developed for this purpose. One of them, 3,3'-bis(beta-hydroxyethyl)-9-ethyl-5,5'-dimethoxythiacarbocyanine iodide (N744), is a cationic thiacarbocyanine dye that inhibits recombinant tau filament formation when present at submicromolar concentrations. To prepare dosing regimens for testing N744 activity in biological models, its full concentration-effect relationship in the range 0.01-60muM was examined in vitro by electron microscopy and laser light scattering methods. Results revealed that N744 concentration dependence was biphasic, with fibrillization inhibitory activity appearing at submicromolar concentration, but with relief of inhibition and increases in fibrillization apparent above 10muM. Therefore, fibrillization was inhibited 50% only over a narrow concentration range, which was further reduced by filament stabilizing modifications such as tau pseudophosphorylation. N744 inhibitory activity also was paralleled by changes in its aggregation state, with dimer predominating at inhibitory concentrations and large dye aggregates appearing at high concentrations. Ligand dimerization was promoted by the presence of tau protein, which lowered the equilibrium dissociation constant for dimerization more than an order of magnitude relative to controls. The results suggest that ligand aggregation may play an important role in both inhibitory and disinhibitory phases of the concentration-effect curve, and may lead to complex dose-response relationships in model systems.

OBJECTIVE: The objective of this study is the development and evaluation of efficient neurophysiological signal statistics, which may assess the driver's alertness level and serve as potential indicators of sleepiness in the design of an on-board countermeasure system. METHODS: Multichannel EEG, EOG, EMG, and ECG were recorded from sleep-deprived subjects exposed to real field driving conditions. A number of severe driving errors occurred during the experiments. The analysis was performed in two main dimensions: the macroscopic analysis that estimates the on-going temporal evolution of physiological measurements during the driving task, and the microscopic event analysis that focuses on the physiological measurements' alterations just before, during, and after the driving errors. Two independent neurophysiologists visually interpreted the measurements. The EEG data were analyzed by using both linear and non-linear analysis tools. RESULTS: We observed the occurrence of brief paroxysmal bursts of alpha activity and an increased synchrony among EEG channels before the driving errors. The alpha relative band ratio (RBR) significantly increased, and the Cross Approximate Entropy that quantifies the synchrony among channels also significantly decreased before the driving errors. Quantitative EEG analysis revealed significant variations of RBR by driving time in the frequency bands of delta, alpha, beta, and gamma. Most of the estimated EEG statistics, such as the Shannon Entropy, Kullback-Leibler Entropy, Coherence, and Cross-Approximate Entropy, were significantly affected by driving time. We also observed an alteration of eyes blinking duration by increased driving time and a significant increase of eye blinks' number and duration before driving errors. CONCLUSIONS: EEG and EOG are promising neurophysiological indicators of driver sleepiness and have the potential of monitoring sleepiness in occupational settings incorporated in a sleepiness countermeasure device. SIGNIFICANCE: The occurrence of brief paroxysmal bursts of alpha activity before severe driving errors is described in detail for the first time. Clear evidence is presented that eye-blinking statistics are sensitive to the driver's sleepiness and should be considered in the design of an efficient and driver-friendly sleepiness detection countermeasure device.

The enzyme beta-galactosidase, encoded by the bacterial gene lac-Z, is commonly used as a histochemical reporter to track transplanted cells in vivo or to analyze temporospatial gene expression patterns by coupling expression of specific target genes to beta-galactosidase activity. Previously, endogenous beta-galactosidase activity has been recognized as a confounding factor in the study of different soft tissues, but there is no description of the typical background on bone marrow sections when using the chromogenic substrate 5-Bromo-4-chloro-3-indolyl beta-D-Galactoside (X-Gal). In this report, we show that osteoclasts in bone marrow sections specifically and robustly stain blue with X-Gal. This leads to a typical background when bone marrow is examined that is present from the first day post partum throughout the adult life of experimental mice and can be confused with transgenic, bacterial beta-galactosidase expressing hematopoietic or stromal cells. Experimental variations in the X-Gal staining procedure, such as pH and time of exposure to substrate, were not sufficient to avoid this background. Therefore, these data demonstrate the need for strenuous controls when evaluating beta-galactosidase positive bone marrow cells. Verifiable bacterial beta-galactosidase positive bone marrow cells should be further identified using immunohistological or other approaches. Specifically, beta-galactosidase positive hematopoietic or stromal cells should be proven specifically not to be osteoclasts by co-staining or staining adjacent sections for specific markers of hematopoietic and stromal cells

Cholinergic nucleus basalis (NB) neurons provide the major cholinergic innervation to the cortical mantle, are selectively vulnerable in late stage Alzheimer's disease (AD) and require the neurotrophin, nerve growth factor (NGF) and its receptors (TrkA and p75(NTR)), for their survival. The molecular events underlying the demise of these neurons in AD were investigated using tissue harvested from participants in a longitudinal clinical pathological study of aging and AD who agreed to an annual clinical evaluation providing a categorization of no cognitive impairment (NCI), mild cognitive impairment (MCI) or AD and postmortem brain donation. Although the number of choline acetyltransferase (ChAT)-positive neurons was unchanged, TrkA and p75(NTR) receptor-containing neurons, which co-localize with ChAT, were significantly reduced in the NB of subjects with MCI and AD compared to those with NCI. These observations indicate a phenotypic down-regulation rather than frank NB neuronal degeneration in MCI. Expression profiling of single cholinergic NB neurons revealed TrkA but not p75(NTR) mRNA is reduced in MCI, suggesting that decreased neurotrophin responsiveness may be an early biomarker for AD. The NGF precursor molecule, proNGF, is increased in the cortex in MCI and AD. Since proNGF accumulates in the presence of reduced cortical TrkA and sustained levels of p75(NTR), a shift in the balance between cell survival and death molecules may occur in prodromal AD. Coincident with these phenomena, brain derived neurotrophic factor (BDNF) and its precursor molecule, proBDNF, are reduced in the MCI cortex, potentially depriving CBF neurons of additional trophic factor support. Moreover, there is a shift in the ratio of 3 repeat tau to 4 repeat tau gene expression, whereas total tau message is stable in NB neurons during the disease process. These data suggest there is a shift in cholinotrophic molecular events in MCI and early AD which may lead to cell dysfunction and eventual cell death over the course of the disease. These findings support the concept that from a neurotrophic pathobiologic perspective, MCI is already early AD.

BACKGROUND: Attention deficit hyperactivity disorder (ADHD) is a prevalent neuropsychiatric disorder in childhood with established problems in cognitive control and associated fronto-striatal circuitry. More recently, fronto-cerebellar circuits have been implicated in this disorder. Both of these circuits are important in predicting the occurrence and timing of behaviorally relevant events and in detecting violations of these predictions. Therefore, we hypothesized that the ability to predict the occurrence of frequent events would be compromised in ADHD, as well as the ability to adapt behavior when expectancy was violated. METHODS: We used rapid, mixed-trial, event-related functional magnetic resonance imaging (fMRI) to examine cognitive and neural processes in two independent samples of children and adolescents with ADHD and matched controls. Subjects performed a variation of a go-no/go task where the predictability of stimulus identity (what) and timing (when) was manipulated. RESULTS: Behaviorally, children and adolescents with ADHD had increased variability in reaction times, and decreased benefit in reaction time when events were predictable. Differences in accuracy between groups were most reliable for temporally unpredictable trials. Functional imaging results from both samples showed that relative to the control children and adolescents, individuals with ADHD had diminished cerebellar activity to violations of stimulus timing and diminished ventral prefrontal and anterior cingulate activity to violations in stimulus timing and identity. CONCLUSIONS: These findings are consistent with the view that disruptive behaviors in inappropriate contexts, a major criterion in diagnosing ADHD, may be related to an impaired ability to predict temporal and contextual cues in the environment, thus hindering the ability to alter behavior when they change. This ability requires intact fronto-cerebellar, as well as fronto-striatal circuitry.

Potential advantages of magnetic resonance (MR) imaging at 3 T include higher signal-to-noise ratios, better image contrast, particularly in gadolinium-enhanced applications, and better spectral separation for spectroscopic applications. In terms of clinical imaging, these advantages can mean higher-spatial-resolution images, faster imaging, and improved MR spectroscopy. However, achieving superior imaging and spectroscopic quality at 3 T can be challenging. This review discusses many of the problems encountered in body and cardiovascular MR imaging at 3 T, such as increased susceptibility, B(1) field inhomogeneity, and increased specific absorption rate. The article also considers solutions that are being pursued, such as parallel imaging, variable-rate selective excitation, and variable flip angle sequences. A review of the most commonly used pulse sequences provides practical tips on how these can be optimized for 3-T imaging. In the coming few years, substantial improvements in 3-T technology for clinical imaging and spectroscopy will undoubtedly be seen. An understanding of the basic principles on which these developments are based will help radiologists translate the advances into better imaging studies and, ultimately, better patient care. (c) RSNA, 2007

Work in behaving primates indicates that midbrain dopamine neurons encode a prediction error, the difference between an obtained reward and the reward expected. Studies of dopamine action potential timing in the alert and anesthetized rat indicate that dopamine neurons respond in tonic and phasic modes, a distinction that has been less well characterized in the primates. We used spike train models to examine the relationship between the tonic and burst modes of activity in dopamine neurons while monkeys were performing a reinforced visuo-saccadic movement task. We studied spiking activity during four task-related intervals; two of these were intervals during which no task-related events occurred, whereas two were periods marked by task-related phasic activity. We found that dopamine neuron spike trains during the intervals when no events occurred were well described as tonic. Action potentials appeared to be independent, to occur at low frequency, and to be almost equally well described by Gaussian and Poisson-like (gamma) processes. Unlike in the rat, interspike intervals as low as 20 ms were often observed during these presumptively tonic epochs. Having identified these periods of presumptively tonic activity, we were able to quantitatively define phasic modulations (both increases and decreases in activity) during the intervals in which task-related events occurred. This analysis revealed that the phasic modulations of these neurons include both bursting, as has been described previously, and pausing. Together bursts and pauses seemed to provide a continuous, although nonlinear, representation of the theoretically defined reward prediction error of reinforcement learning.

BACKGROUND: The EGFR T790M mutation confers acquired resistance to kinase inhibitors in human EGFR mutant lung adenocarcinoma, is occasionally detected before treatment, and may confer genetic susceptibility to lung cancer. METHODOLOGY/PRINCIPAL FINDINGS: To study further its role in lung tumorigenesis, we developed mice with inducible expression in type II pneumocytes of EGFR(T790M) alone or together with a drug-sensitive L858R mutation. Both transgenic lines develop lung adenocarcinomas that require mutant EGFR for tumor maintenance but are resistant to an EGFR kinase inhibitor. EGFR(L858R+T790M)-driven tumors are transiently targeted by hsp90 inhibition. Notably, EGFR(T790M)-expressing animals develop tumors with longer latency than EGFR(L858R+T790M)-bearing mice and in the absence of additional kinase domain mutations. CONCLUSIONS/SIGNIFICANCE: These new mouse models of mutant EGFR-dependent lung adenocarcinomas provide insight into clinical observations. The models should also be useful for developing improved therapies for patients with lung cancers harboring EGFR(T790M) alone or in conjunction with drug-sensitive EGFR kinase domain mutations.

PURPOSE: To study the influence of actin cytoskeleton reorganization on the subcellular distribution of Ca(v)1.3 L-type Ca2+ channels in salamander retinal third-order neurons. METHODS: Immunocytochemistry with confocal microscopy was used to demonstrate internalization of the Ca(v)1.3 isoform of L-type voltage-gated Ca2+ channels in third-order retinal neurons. A specificity of antibody was confirmed with Western blotting and in control experiments preabsorbing antibody wit its respective peptide. Whole-cell patch clamp technique was applied to record L-type currents from ganglion cells in slice preparations in the presence of N- and P/Q type Ca2+ channel blockers. RESULTS: A high level of Ca(v)1.3 labeling was present in cone photoreceptor terminals in the outer plexiform layer (OPL), as aggregates of puncta. Punctate Ca(v)1.3 labeling was evident throughout the IPL and around the cell bodies in the outer nuclear (ONL), inner nuclear (INL) and on somas and axons of ganglion cells labeled with rhodamine-conjugated dextran. Doubly labeled sections for synaptophysin and Ca(v)1.3 revealed colocalization in the OPL and IPL. Depolymerization of the actin cytoskeleton caused a dynamin-dependent internalization of Ca(v)1.3 but not Ca(v)1.2 subtype of voltage-gated Ca2+ channels in dissociated neurons. In ganglion cells, the inhibition of L-type Ca2+ currents by F-actin disrupters was mediated by Ca2+ channel internalization. Treatment with cytochalasin D protected retinal neurons against kainate-induced excitotoxicity. CONCLUSIONS: Actin cytoskeleton dynamics plays an important role in the regulation of subcellular distribution and function of Ca(v)1.3 L-type Ca2+ channels in salamander retinal neurons. Ca2+-dependent actin depolymerization may serve as a negative feedback mechanism to reduce excessive Ca2+ influx and thereby protect neurons against glutamate-induced excitotoxicity

Axon-glial interactions are critical for the induction of myelination and the domain organization of myelinated fibers. Although molecular complexes that mediate these interactions in the nodal region are known, their counterparts along the internode are poorly defined. We report that neurons and Schwann cells express distinct sets of nectin-like (Necl) proteins: axons highly express Necl-1 and -2, whereas Schwann cells express Necl-4 and lower amounts of Necl-2. These proteins are strikingly localized to the internode, where Necl-1 and -2 on the axon are directly apposed by Necl-4 on the Schwann cell; all three proteins are also enriched at Schmidt-Lanterman incisures. Binding experiments demonstrate that the Necl proteins preferentially mediate heterophilic rather than homophilic interactions. In particular, Necl-1 on axons binds specifically to Necl-4 on Schwann cells. Knockdown of Necl-4 by short hairpin RNA inhibits Schwann cell differentiation and subsequent myelination in cocultures. These results demonstrate a key role for Necl-4 in initiating peripheral nervous system myelination and implicate the Necl proteins as mediators of axo-glial interactions along the internode