A Drosophila Model of ALS: Human ALS-Associated Mutation in VAP33A Suggests a Dominant Negative Mechanism

ALS8 is caused by a dominant mutation in an evolutionarily conserved protein, VAPB (vesicle-associated membrane protein (VAMP)-associated membrane protein B)/ALS8). We have established a fly model of ALS8 using the corresponding mutation in Drosophila VAPB (dVAP33A) and examined the effects of this mutation on VAP function using genetic and morphological analyses. By simultaneously assessing the effects of VAPwt and VAPP58S on synaptic morphology and structure, we demonstrate that the phenotypes produced by neuronal expression of VAPP58S resemble VAP loss of function mutants and are opposite those of VAP overexpression, suggesting that VAPP58S may function as a dominant negative. This is brought about by aggregation of VAPP58S and recruitment of wild type VAP into these aggregates. Importantly, we also demonstrate that the ALS8 mutation in dVAP33A interferes with BMP signaling pathways at the neuromuscular junction, identifying a new mechanism underlying pathogenesis of ALS8. Furthermore, we show that mutant dVAP33A can serve as a powerful tool to identify genetic modifiers of VAPB. This new fly model of ALS, with its robust pathological phenotypes, should for the first time allow the power of unbiased screens in Drosophila to be applied to study of motor neuron diseases

Effective Stimuli for Constructing Reliable Neuron Models

The rich dynamical nature of neurons poses major conceptual and technical challenges for unraveling their nonlinear membrane properties. Traditionally, various current waveforms have been injected at the soma to probe neuron dynamics, but the rationale for selecting specific stimuli has never been rigorously justified. The present experimental and theoretical study proposes a novel framework, inspired by learning theory, for objectively selecting the stimuli that best unravel the neuron's dynamics. The efficacy of stimuli is assessed in terms of their ability to constrain the parameter space of biophysically detailed conductance-based models that faithfully replicate the neuron's dynamics as attested by their ability to generalize well to the neuron's response to novel experimental stimuli. We used this framework to evaluate a variety of stimuli in different types of cortical neurons, ages and animals. Despite their simplicity, a set of stimuli consisting of step and ramp current pulses outperforms synaptic-like noisy stimuli in revealing the dynamics of these neurons. The general framework that we propose paves a new way for defining, evaluating and standardizing effective electrical probing of neurons and will thus lay the foundation for a much deeper understanding of the electrical nature of these highly sophisticated and non-linear devices and of the neuronal networks that they compose

In Vitro Pharmacological Characterization of RXFP3 Allosterism: An Example of Probe Dependency

Recent findings suggest that the relaxin-3 neural network may represent a new ascending arousal pathway able to modulate a range of neural circuits including those affecting circadian rhythm and sleep/wake states, spatial and emotional memory, motivation and reward, the response to stress, and feeding and metabolism. Therefore, the relaxin-3 receptor (RXFP3) is a potential therapeutic target for the treatment of various CNS diseases. Here we describe a novel selective RXFP3 receptor positive allosteric modulator (PAM), 3-[3,5-Bis(trifluoromethyl)phenyl]-1-(3,4-dichlorobenzyl)-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]urea (135PAM1). Calcium mobilization and cAMP accumulation assays in cell lines expressing the cloned human RXFP3 receptor show the compound does not directly activate RXFP3 receptor but increases functional responses to amidated relaxin-3 or R3/I5, a chimera of the INSL5 A chain and the Relaxin-3 B chain. 135PAM1 increases calcium mobilization in the presence of relaxin-3NH2 and R3/I5NH2 with pEC50 values of 6.54 (6.46 to 6.64) and 6.07 (5.94 to 6.20), respectively. In the cAMP accumulation assay, 135PAM1 inhibits the CRE response to forskolin with a pIC50 of 6.12 (5.98 to 6.27) in the presence of a probe (10 nM) concentration of relaxin-3NH2. 135PAM1 does not compete for binding with the orthosteric radioligand, [125I] R3I5 (amide), in membranes prepared from cells expressing the cloned human RXFP3 receptor. 135PAM1 is selective for RXFP3 over RXFP4, which also responds to relaxin-3. However, when using the free acid (native) form of relaxin-3 or R3/I5, 135PAM1 doesn't activate RXFP3 indicating that the compound's effect is probe dependent. Thus one can exchange the entire A-chain of the probe peptide while retaining PAM activity, but the state of the probe's c-terminus is crucial to allosteric activity of the PAM. These data demonstrate the existence of an allosteric site for modulation of this GPCR as well as the subtlety of changes in probe molecules that can affect allosteric modulation of RXFP3

dTip60 HAT Activity Controls Synaptic Bouton Expansion at the Drosophila Neuromuscular Junction

Background: Histone acetylation of chromatin plays a key role in promoting the dynamic transcriptional responses in neurons that influence the neuroplasticity linked to cognitive ability, yet the specific histone acetyltransferases (HATs) that create such epigenetic marks remain to be elucidated. Methods and Findings: Here we use the Drosophila neuromuscular junction (NMJ) as a well-characterized synapse model to identify HATs that control synaptic remodeling and structure. We show that the HAT dTip60 is concentrated both pre and post-synaptically within the NMJ. Presynaptic targeted reduction of dTip60 HAT activity causes a significant increase in synaptic bouton number that specifically affects type Is boutons. The excess boutons show a suppression of the active zone synaptic function marker bruchpilot, suggesting defects in neurotransmission function. Analysis of microtubule organization within these excess boutons using immunohistochemical staining to the microtubule associated protein futsch reveals a significant increase in the rearrangement of microtubule loop architecture that is required for bouton division. Moreover, a-tubulin acetylation levels of microtubules specifically extending into the terminal synaptic boutons are reduced in response to dTip60 HAT reduction. Conclusions: Our results are the first to demonstrate a causative role for the HAT dTip60 in the control of synaptic plasticity that is achieved, at least in part, via regulation of the synaptic microtubule cytoskeleton. These findings have implication

Role of Kinesin Heavy Chain in Crumbs Localization along the Rhabdomere Elongation in Drosophila Photoreceptor

BACKGROUND:Crumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the extension of the apical membrane domain, adherens junction (AJ), and rhabdomere along the growing proximal-distal axis during Drosophila photoreceptor morphogenesis. In developing Drosophila photoreceptors, a stabilized microtubule structure was discovered and its presence was linked to polarity protein localization. It was therefore hypothesized that the microtubules may provide trafficking routes for the polarity proteins during photoreceptor morphogenesis. This study has examined whether Kinesin heavy chain (Khc), a subunit of the microtubule-based motor Kinesin-1, is essential in polarity protein localization in developing photoreceptors. METHODOLOGY/PRINCIPAL FINDINGS:Because a genetic interaction was found between crb and khc, Crb localization was examined in the developing photoreceptors of khc mutants. khc was dispensable during early eye differentiation and development. However, khc mutant photoreceptors showed a range of abnormalities in the apical membrane domain depending on the position along the proximal-distal axis in pupal photoreceptors. The khc mutant showed a progressive mislocalization in the apical domain along the distal-proximal axis during rhabdomere elongation. The khc mutation also led to a similar progressive defect in the stabilized microtubule structures, strongly suggesting that Khc is essential for microtubule structure and Crb localization during distal to proximal rhabdomere elongation in pupal morphogenesis. This role of Khc in apical domain control was further supported by khc's gain-of-function phenotype. Khc overexpression in photoreceptors caused disruption of the apical membrane domain and the stabilized microtubules in the developing photoreceptors. CONCLUSIONS/SIGNIFICANCE:In summary, we examined the role of khc in the regulation of the apical Crb domain in developing photoreceptors. Since the rhabdomeres in developing pupal eyes grow along the distal-proximal axis, these phenotypes suggest that Khc is essential for the microtubule structures and apical membrane domains during the distal-proximal elongation of photoreceptors, but is dispensable for early eye development

The functional brain networks that underlie Early Stone Age tool manufacture

After 800,000 years of making simple Oldowan tools, early humans began manufacturing Acheulian handaxes around 1.75 million years ago. This advance is hypothesized to reflect an evolutionary change in hominin cognition and language abilities. We used a neuroarchaeology approach to investigate this hypothesis, recording brain activity using functional near-infrared spectroscopy as modern human participants learned to make Oldowan and Acheulian stone tools in either a verbal or nonverbal training context. Here we show that Acheulian tool production requires the integration of visual, auditory and sensorimotor information in the middle and superior temporal cortex, the guidance of visual working memory representations in the ventral precentral gyrus, and higher-order action planning via the supplementary motor area, activating a brain network that is also involved in modern piano playing. The right analogue to Broca’s area—which has linked tool manufacture and language in prior work1,2—was only engaged during verbal training. Acheulian toolmaking, therefore, may have more evolutionary ties to playing Mozart than quoting Shakespeare

Measurement of the CP asymmetry and branching fraction of B-0 ->rho K-0(0)

We present a measurement of the branching fraction and time-dependent CP asymmetry of B-0 -> POKO. The results are obtained from a data sample of 227 x 10(6) Y(4S) -> BB decays collected with the BABAR detector at the PEP-II asymmetric-energy B factory at Stanford Linear Accelerator Center. From a time-dependent maximum likelihood fit yielding 111 +/- 19 signal events, we find B(B-0 -> rho K-0(0)) = (4.9 +/- 0.8 +/- 0.9) x 10(-6), where the first error is statistical and the second systematic. We report the measurement of the CP parameters S-rho 0KS0 = 0.20 +/- 0.52 +/- 0.24 and C-rho 0KS0 = 0.64 +/- 0.41 +/- 0.20

Observation of CP violation in B ->eta/K-0 decays

We present measurements of the time-dependent CP-violation parameters S and C in B-0 -> eta K-'(0) decays. The data sample corresponds to 384 x 10(6) B (B) over bar pairs produced by e(+)e(-) annihilation at the Upsilon(4S). The results are S = 0.58 +/- 0.10 +/- 0.03 and C = -0.16 +/- 0.07 +/- 0.03. We observe mixing-induced CP violation with a significance of 5.5 standard deviations in this b -> s penguin dominated mode

Measurements of CP-violating asymmetries in B-0 -> a(1)(+/-)(1260)pi(-/+) decays

We present measurements of CP-violating asymmetries in the decay B-0 -> a(1)(+/-)(1260)pi(-/+) with a(1)(+/-)(1260)->pi(-/+)pi(+/-)pi(+/-). The data sample corresponds to 384x10(6) B(b) over bar pairs collected with the BABAR detector at the PEP-II asymmetric B factory at SLAC. We measure the CP-violating asymmetry A(CP)(a1 pi)=-0.07 +/- 0.07 +/- 0.02, the mixing-induced CP violation parameter S-a1 pi=0.37 +/- 0.21 +/- 0.07, the direct CP violation parameter C-a1 pi=-0.10 +/- 0.15 +/- 0.09, and the parameters Delta C-a1 pi=0.26 +/- 0.15 +/- 0.07 and Delta S-a1 pi=-0.14 +/- 0.21 +/- 0.06. From these measured quantities we determine the angle alpha(eff)=78.6 degrees +/- 7.3 degrees

Branching fraction measurements of B+->rho(+)gamma, B-0 ->rho(0)gamma, and B-0 ->omega gamma

We present a study of the decays B+->rho(+)gamma, B-0 ->rho(0)gamma, and B-0 ->omega gamma. The analysis is based on data containing 347x10(6) B (B) over bar events recorded with the BABAR detector at the PEP-II asymmetric B factory. We measure the branching fractions B(B+->rho(+)gamma)=(1.10(-0.33)(+0.37)+/- 0.09)x10(-6) and B(B-0 ->rho(0)gamma)=(0.79(-0.20)(+0.22)+/- 0.06)x10(-6), and set a 90% C.L. upper limit B(B-0 ->omega gamma)(rho/omega)gamma)=(1.25(-0.24)(+0.25)+/- 0.09)x10(-6), from which we determine vertical bar V-td/V-ts vertical bar=0.200(-0.020)(+0.021)+/- 0.015, where the first uncertainty is experimental and the second is theoretical