The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.DOI: http://dx.doi.org/10.7554/eLife.14371.001

Oxidative modification of low-density lipoprotein (LDL) particles is a key event in the development of atherosclerosis. Oxidized LDL induces oxidative stress and modifies gene expression in endothelial cells. Berries constitute a rich dietary source of phenolic antioxidants. We found that the endemic Chilean berry Aristotelia chilensis (ach) has higher phenol content and scores better for total radical-trapping potential and total antioxidant reactivity in in vitro antioxidant capacity tests, when compared to different commercial berries. The juice of ach is also effective in inhibiting copper-induced LDL oxidation. In human endothelial cell cultures, the addition of ach juice significantly protects from hydrogen peroxide-induced intracellular oxidative stress and is dose-dependent. The aqueous, anthocyanin-rich fraction of ach juice accounts for most of ach's antioxidant properties. These results show that ach is a rich source of phenolics with high antioxidant capacity and suggest that it may have antiatherogenic properties.

The structural organization of excitatory inputs supporting spike-timing-dependent plasticity (STDP) remains unknown. We performed a spine STDP protocol using two-photon (2P) glutamate uncaging (pre) paired with postsynaptic spikes (post) in layer 5 pyramidal neurons from juvenile mice. Here we report that pre-post pairings that trigger timing-dependent LTP (t-LTP) produce shrinkage of the activated spine neck and increase in synaptic strength; and post-pre pairings that trigger timing-dependent LTD (t-LTD) decrease synaptic strength without affecting spine shape. Furthermore, the induction of t-LTP with 2P glutamate uncaging in clustered spines (<5 μm apart) enhances LTP through a NMDA receptormediated spine calcium accumulation and actin polymerization-dependent neck shrinkage, whereas t-LTD was dependent on NMDA receptors and disrupted by the activation of clustered spines but recovered when separated by >40 μm. These results indicate that synaptic cooperativity disrupts t-LTD and extends the temporal window for the induction of t-LTP, leading to STDP only encompassing LTP.

The frequency sensitivity of auditory hair cells in the inner ear varies with their longitudinal position in the sensory epithelium. Among the factors that determine the differential cellular response to sound is the resonance of a hair cell's transmembrane electrical potential, whose frequency correlates with the kinetic properties of the high-conductance Ca 2؉ -activated K ؉ (BK) channels encoded by a Slo (kcnma1) gene. It has been proposed that the inclusion of specific alternative axons in the Slo transcripts along the cochlea underlies the gradient of BK-channel kinetics. By analyzing the complete sequences of chicken Slo gene (cSlo) cDNAs from the chicken's cochlea, we show that most transcripts lack alternative exons. Transcripts with more than one alternative exon constitute only 10% of the total. Although the fraction of transcripts containing alternative exons increases from the cochlear base to the apex, the combination of alternative exons is not regulated. There is also a clear increase in the expression of BK transcripts with long carboxyl termini toward the apex. When long and short BK transcripts are expressed in HEK-293 cells, the kinetics of single-channel currents differ only slightly, but they are substantially slowed when the channels are coexpressed with the auxiliary ␤ subunit that occurs more widely at the apex. These results argue that the tonotopic gradient is not established by the selective inclusion of highly specific cSlo exons. Instead, a gradient in the expression of ␤ subunits slows BK channels toward the low-frequency apex of the cochlea.The auditory system maps continuous sensory variables, such as the frequency of a sound or the spatial location of its source, onto cellular detector arrays whose individual elements are narrowly tuned. How sensory maps of any kind are established and maintained is an open question. In only a single case, the tonotopic frequency map in the inner ear, is the physiological mechanism responsible for the individual cell's differential tuning understood at the molecular level.In the process of electrical resonance, a combination of voltage-and ion-dependent conductances operates in conjunction with the passive electrical properties of a hair cell's membrane to accentuate cellular responsiveness to a particular range of frequencies while attenuating that to frequencies outside this range. A dynamic interaction between two types of ion channels mediates this band-pass behavior. Voltage-sensitive Ca 2ϩ channels activated by the current through mechanotransduction channels depolarize the hair cell's membrane and increase the intracellular concentration of Ca 2ϩ . With some delay, these ions activate the Ca 2ϩ -sensitive high-conductance Ca 2ϩ -activated K ϩ (BK) channels that repolarize the membrane, thereby closing the Ca 2ϩ channels, decreasing the intracellular concentration of Ca 2ϩ , and deactivating the K ϩ channels. How rapidly this dynamic system progresses through the cycle depends on several factors, most notably on the kinetic properties of th...

support-information-section).

The positive health effects derived from moderate wine consumption are pleiotropic. They appear as improvements in cardiovascular risk factors such as plasma lipids, haemostatic mechanisms, endothelial function and antioxidant defences. The active principles would be ethanol and mainly polyphenols. Results from our and other laboratories support the unifying hypothesis that the improvements in risk factors after red wine consumption are mediated by endothelial nitric oxide synthase (eNOS). Many genes are involved, but the participation of eNOS would be a constant feature. The metabolic syndrome is a cluster of metabolic risk factors associated with high risk of cardiovascular disease (CVD). The National Cholesterol Education Programmmes Adult Treatment Panel III (NCEPATP III) clinical definition of the metabolic syndrome requires the presence of at least three risk factors, from among abdominal obesity, high plasma triacylglycerols, low plasma HDL, high blood pressure and high fasting plasma glucose. The molecular mechanisms responsible for the metabolic syndrome are not known. Since metabolic syndrome apparently affects 10-30% of the population in the world, research on its pathogenesis and control is needed. The recent finding that eNOS knockout mice present a cluster of cardiovascular risk factors comparable to those of the metabolic syndrome suggests that defects in eNOS function may cause human metabolic syndrome. These mice are hypertensive, insulin resistant and dyslipidemic. Further support for a pathogenic role of eNOS comes from the finding in humans that eNOS polymorphisms associate with insulin resistance and diabetes, with hypertension, with inflammatory and oxidative stress markers and with albuminuria. So, the data sustain the hypothesis that eNOS enhancement should reduce metabolic syndrome incidence and its consequences. Therefore red wine, since it enhances eNOS function, should be considered as a potential tool for the control of metabolic syndrome. This hypothesis is supported by epidemiological observations and needs experimental validation in human intervention studies.

Cu(II) mediated low density lipoprotein (LDL) oxidation has been followed by the changes in absorbance at 234 nm and the emitted low level chemiluminescence (CL). The similarity of the time profiles allows us to conclude that the emitted CL is due to the decomposition of a transient product, most likely a hydroperoxide. Red wine, as well as its fractions, afford a noticeable protection when added prior to the start of the LDL oxidation process. On the other hand, when they are added after the onset of the autocatalytic oxidation phase, red wine and its fractions behave as pro-oxidants. This is particularly evidenced by a strong burst of CL (enhancement of the light by a factor approximately 20). This burst is reduced by metal chelators (EDTA and DFO) and can be associated to a sequence of reactions such as XOH + Cu(II) --> X* + H(+) + Cu(I), Cu(I) + LOOH --> chemiluminescence where XOH is a phenolic compound and LOOH is a peroxide-like compound produced in the LDL oxidation.

18The structural organization of excitatory inputs that supports spike-timing-dependent plasticity 19 (STDP) remains unknown. Here we performed a spine STDP protocol using two-photon 20 glutamate uncaging (pre) paired with postsynaptic spikes (post). We found that pre-post pairings 21 that trigger LTP (t-LTP) produce shrinkage of the activated spine neck and increase in synaptic 22 32 Dendritic spines, the main recipient of excitatory information in the brain 1 , are tiny protrusions 33 with a small head (~1 µm in diameter and <1 fL volume) separated from the dendrite by a 34 slender neck. Spines can undergo structural remodeling that is tightly coupled with synaptic 35 function 1, 2, 3, 4 , and are the preferential site for the induction of long-term potentiation (LTP) 4, 5, 36 6, 7 and long-term depression (LTD) 8 , thought to be the underlying mechanisms for learning and 37 memory in the brain 9 . A variation of LTP and LTD has been described in pyramidal neurons 38 that involves the pairing of pre-and postsynaptic action potentials, known as spike-timing 39 dependent plasticity (STDP) 10, 11 . In this process, the timing between pre-and postsynaptic 40 action potentials modulates synaptic strength, triggering LTP or LTD 11 . The sign and magnitude 41 of the change in synaptic strength depends on the relative timing between spikes of two 42 connected neurons (the pre-and postsynaptic neuron 12 ). The STDP learning rules and their 43 dependency on postsynaptic dendritic depolarization 13, 14 , firing rate 13 , and somatic distance of 44 excitatory inputs 14, 15, 16 have been extracted from studies using connected neuronal pairs or by 45 using extracellular stimulating electrodes, but the precise location and structural organization of 46 excitatory inputs capable of supporting STDP at its minimal functional unit -the dendritic spine 47 -are unknown. 48 Activity-dependent spine morphological changes (spine head 4 , neck 2 , or both 17 ) have been 49 correlated with changes in synaptic strength in cortical pyramidal neurons by mechanisms 50 involving biochemical and electrical spine changes 1, 6 . Thus, here we asked what patterns of 51 activity and structural organization of excitatory synaptic inputs support the generation of t-LTP 52 and t-LTD, and which morphological, biophysical and molecular changes observed in dendritic 53 spines can account for the induction of t-LTP and t-LTD? 54 4To induce synapse-specific STDP we performed a protocol whereby two-photon (2P) uncaging 55 of a caged glutamate (MNI-glutamate 3 ) at a single spine -to mimic synaptic release -is 56 preceded or followed in time (STDP timing window 11 ) by a backpropagating action potential 57 (bAP) to trigger t-LTP or t-LTD, respectively. Two-photon uncaging of a caged glutamate at a 58 single spine triggered excitatory postsynaptic potentials (uncaging(u)EPSP) that were recorded in 59 the soma of layer 5 (L5) pyramidal neurons before and after the induction of STDP, while the 60 morphology of the activat...