A novel "weak toxin" (WTX) from Naja kaouthia snake venom competes with [125 I]␣-bungarotoxin for binding to the membrane-bound Torpedo californica acetylcholine receptor (AChR), with an IC 50 of ϳ2.2 M. In this respect, it is ϳ300 times less potent than neurotoxin II from Naja oxiana and ␣-cobratoxin from N. kaouthia, representing short-type and long-type ␣-neurotoxins, respectively. WTX and ␣-cobratoxin displaced [125 I]␣-bungarotoxin from the Escherichia coli-expressed fusion protein containing the rat ␣7 AChR N-terminal domain 1-208 preceded by glutathione S-transferase with IC 50 values of 4.3 and 9.1 M, respectively, whereas for neurotoxin II the IC 50 value was >100 M. Micromolar concentrations of WTX inhibited acetylcholine-activated currents in Xenopus oocyte-expressed rat muscle AChR and human and rat ␣7 AChRs, inhibiting the latter most efficiently (IC 50 of ϳ8.3 M). Thus, a virtually nontoxic "three-fingered" protein WTX, although differing from ␣-neurotoxins by an additional disulfide in the N-terminal loop, can be classified as a weak ␣-neurotoxin. It differs from the short chain ␣-neurotoxins, which potently block the muscle-type but not the ␣7 AChRs, and is closer to the long ␣-neurotoxins, which have comparable potency against the above-mentioned AChR types.

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The characterisation of interspecies differences in gene regulation is crucial to understanding the molecular basis of phenotypic diversity and evolution. The atonal homologue Atoh7 participates in the ontogenesis of the vertebrate retina. Our study reveals how evolutionarily conserved, non-coding DNA sequences mediate both the conserved and the species-specific transcriptional features of the Atoh7 gene. In the mouse and chick retina, species-related variations in the chromatin-binding profiles of bHLH transcription factors correlate with distinct features of the Atoh7 promoters and underlie variations in the transcriptional rates of the Atoh7 genes. The different expression kinetics of the Atoh7 genes generate differences in the expression patterns of a set of genes that are regulated by Atoh7 in a dose-dependent manner, including those involved in neurite outgrowth and growth cone migration. In summary, we show how highly conserved regulatory elements are put to use in mediating non-conserved functions and creating interspecies neuronal diversity.

J. Neurochem. (2010) 113, 1307–1318. Abstract Retinal excitotoxicity is associated with retinal ischemia, and with glaucomatous and traumatic optic neuropathy. The present study investigates the role of c‐Jun N‐terminal kinase (JNK) activation in NMDA‐mediated retinal excitotoxicity and determines whether neuroprotection can be obtained with the JNK pathway inhibitor, d‐form of JNK‐inhibitor 1 (d‐JNKI‐1). Young adult rats received intravitreal injections of 20 nmol NMDA, which caused extensive neuronal death in the inner nuclear and ganglion cell layers. This excitotoxicity was associated with strong activation of calpain, as revealed by fodrin cleavage, and of JNK. The cell‐permeable peptide d‐JNKI‐1 was used to inhibit JNK. Within 40 min of its intravitreal injection, FITC‐labeled d‐JNKI‐1 spread through the retinal ganglion cell layer into the inner nuclear layer and interfered with the NMDA‐induced phosphorylation of JNK. Injections of unlabeled d‐JNKI‐1 gave unprecedentedly strong neuroprotection against cell death in both layers, lasting for at least 10 days. The NMDA‐induced calpain‐specific fodrin cleavage was likewise strongly inhibited by d‐JNKI‐1. Moreover the electroretinogram was partially preserved by d‐JNKI‐1. Thus, the JNK pathway is involved in NMDA‐mediated retinal excitotoxicity and JNK inhibition by d‐JNKI‐1 provides strong neuroprotection as shown morphologically, biochemically and physiologically.

The HES proteins are known Notch effectors and have long been recognized as important in inhibiting neuronal differentiation. However, the roles that they play in the specification of neuronal fate remain largely unknown. Here, we show that in the differentiating retinal epithelium, the proneural protein ATOH7 (ATH5) is required for the activation of the transcription of the Hes5.3 gene before the penultimate mitosis of progenitor cells. We further show that the HES5.3 protein slows down the cell-cycle progression of Atoh7-expressing cells, thereby establishing conditions for Atoh7 to reach a high level of expression in S phase and induce neuronal differentiation prior to the ultimate mitosis. Our study uncovers how a proneural protein recruits a protein known to be a component of the Notch signaling pathway in order to regulate the transition between an initial phase of selection among uncommitted progenitors and a later phase committing the selected progenitors to neuronal differentiation.

The atonal homolog 5 (ATH5) protein is central to the transcriptional network regulating the specification of retinal ganglion cells, and its expression comes under the spatiotemporal control of several basic helix-loop-helix (bHLH) proteins in the course of retina development. Monitoring the in vivo occupancy of the ATH5 promoter by the ATH5, Ngn2, and NeuroM proteins and analyzing the DNA motifs they bind, we show that three evolutionarily conserved E-boxes are required for the bHLH proteins to control the different phases of ATH5 expression. E-box 4 mediates the activity of Ngn2, ATH5, and NeuroM along the pathway leading to the conversion of progenitors into newborn neurons. E-box 1, by mediating the antagonistic effects of Ngn2 and HES1 in proliferating progenitors, controls the expansion of the ATH5 expression domain in early retina. E-box 2 is required for the positive feedback by ATH5 that underlies the up-regulation of ATH5 expression when progenitors are going through their last cell cycle. The combinatorial nature of the regulation of the ATH5 promoter suggests that the bHLH proteins involved have no assigned E-boxes but use a common set at which they either cooperate or compete to finely tune ATH5 expression as development proceeds.Retina development in vertebrates relies on regulatory proteins, most of which are widely expressed in the developing nervous system. However, the expression of the basic helixloop-helix protein atonal homolog 5 (ATH5) 3 appears to be restricted to retina ontogenesis. ATH5 activates neurogenesis and is required for the production of retinal ganglion cells (RGCs) (1-4). It is transiently expressed during the period of development when RGCs are produced and underlies the pathway leading to the conversion of proliferating progenitors into newborn RGCs (5, 6). Retinotopic differences in the timing of RGC production may reflect the wave-like expression of ATH5 (5, 7). In zebrafish, a signal from the optic stalk appears to induce the first patch of ATH5-expressing cells, and the continued spread of ATH5 expression beyond that patch may require a cascade of cell-to-cell interactions (7,8). It has been suggested that Sonic hedgehog derived from newborn RGCs drives a self-propagating wave of ATH5 expression and RGC production across the zebrafish retina (9, 10). In contrast, related experiments in zebrafish and chick highlight the importance of intrinsic factors for triggering ATH5 expression and neurogenesis (11-13).The presence of consensus E-box binding sites in the highly conserved upstream sequence of the ATH5 gene suggests that bHLH transcription factors are directly involved in the regulation of ATH5. This idea is supported by experiments showing the selective binding of neuronal bHLH proteins to the upstream sequence of ATH5 and the simultaneous changes in the expression level of ATH5 in response to several of these proteins (5, 6, 14 -16). These findings indicate a requirement for different combinations of bHLH proteins in regulating the different phases of ATH5 expression...

Adverse effects observed during atracurium administration may be attributed, at least partly, to an interaction with neuronal nicotinic receptors.

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