Floor plate-derived neuropilin-2 functions as a secreted semaphorin sink to facilitate commissural axon midline crossing.

Commissural axon guidance depends on a myriad of cues expressed by intermediate targets. Secreted semaphorins signal through neuropilin-2/plexin-A1 receptor complexes on post-crossing commissural axons to mediate floor plate repulsion in the mouse spinal cord. Here, we show that neuropilin-2/plexin-A1 are also coexpressed on commissural axons prior to midline crossing and can mediate precrossing semaphorin-induced repulsion in vitro. How premature semaphorin-induced repulsion of precrossing axons is suppressed in vivo is not known. We discovered that a novel source of floor plate-derived, but not axon-derived, neuropilin-2 is required for precrossing axon pathfinding. Floor plate-specific deletion of neuropilin-2 significantly reduces the presence of precrossing axons in the ventral spinal cord, which can be rescued by inhibiting plexin-A1 signaling in vivo. Our results show that floor plate-derived neuropilin-2 is developmentally regulated, functioning as a molecular sink to sequester semaphorins, preventing premature repulsion of precrossing axons prior to subsequent down-regulation, and allowing for semaphorin-mediated repulsion of post-crossing axons.

Global ablation of the mouse Rab11a gene impairs early embryogenesis and matrix metalloproteinase secretion.

Rab11a has been conceived as a prominent regulatory component of the recycling endosome, which acts as a nexus in the endo- and exocytotic networks. The precise in vivo role of Rab11a in mouse embryonic development is unknown. We globally ablated Rab11a and examined the phenotypic and molecular outcomes in Rab11a(null) blastocysts and mouse embryonic fibroblasts. Using multiple trafficking assays and complementation analyses, we determined, among multiple important membrane-associated and soluble cargos, the critical contribution of Rab11a vesicular traffic to the secretion of multiple soluble MMPs. Rab11a(null) embryos were able to properly form normal blastocysts but died at peri-implantation stages. Our data suggest that Rab11a critically controls mouse blastocyst development and soluble matrix metalloproteinase secretion.

Molecular mechanisms underlying the apoptotic effect of KCNB1 K+ channel oxidation.

Potassium (K(+)) channels are targets of reactive oxygen species in the aging nervous system. KCNB1 (formerly Kv2.1), a voltage-gated K(+) channel abundantly expressed in the cortex and hippocampus, is oxidized in the brains of aging mice and of the triple transgenic 3xTg-AD mouse model of Alzheimer's disease. KCNB1 oxidation acts to enhance apoptosis in mammalian cell lines, whereas a KCNB1 variant resistant to oxidative modification, C73A-KCNB1, is cytoprotective. Here we investigated the molecular mechanisms through which oxidized KCNB1 channels promote apoptosis. Biochemical evidence showed that oxidized KCNB1 channels, which form oligomers held together by disulfide bridges involving Cys-73, accumulated in the plasma membrane as a result of defective endocytosis. In contrast, C73A-mutant channels, which do not oligomerize, were normally internalized. KCNB1 channels localize in lipid rafts, and their internalization was dynamin 2-dependent. Accordingly, cholesterol supplementation reduced apoptosis promoted by oxidation of KCNB1. In contrast, cholesterol depletion exacerbated apoptotic death in a KCNB1-independent fashion. Inhibition of raft-associating c-Src tyrosine kinase and downstream JNK kinase by pharmacological and molecular means suppressed the pro-apoptotic effect of KCNB1 oxidation. Together, these data suggest that the accumulation of KCNB1 oligomers in the membrane disrupts planar lipid raft integrity and causes apoptosis via activating the c-Src/JNK signaling pathway.

An evolutionarily conserved mode of modulation of Shaw-like K⁺ channels.

Voltage-gated K(+) channels of the Shaw family (also known as the KCNC or Kv3 family) play pivotal roles in mammalian brains, and genetic or pharmacological disruption of their activities in mice results in a spectrum of behavioral defects. We have used the model system of Caenorhabditis elegans to elucidate conserved molecular mechanisms that regulate these channels. We have now found that the C. elegans Shaw channel KHT-1, and its mammalian homologue, murine Kv3.1b, are both modulated by acid phosphatases. Thus, the C. elegans phosphatase ACP-2 is stably associated with KHT-1, while its mammalian homolog, prostatic acid phosphatase (PAP; also known as ACPP-201) stably associates with murine Kv3.1b K(+) channels in vitro and in vivo. In biochemical experiments both phosphatases were able to reverse phosphorylation of their associated channel. The effect of phosphorylation on both channels is to produce a decrease in current amplitude and electrophysiological analyses demonstrated that dephosphorylation reversed the effects of phosphorylation on the magnitude of the macroscopic currents. ACP-2 and KHT-1 were colocalized in the nervous system of C. elegans and, in the mouse nervous system, PAP and Kv3.1b were colocalized in subsets of neurons, including in the brain stem and the ventricular zone. Taken together, this body of evidence suggests that acid phosphatases are general regulatory partners of Shaw-like K(+) channels.

Toxic role of K+ channel oxidation in mammalian brain.

Potassium (K(+)) channels are essential to neuronal signaling and survival. Here we show that these proteins are targets of reactive oxygen species in mammalian brain and that their oxidation contributes to neuropathy. Thus, the KCNB1 (Kv2.1) channel, which is abundantly expressed in cortex and hippocampus, formed oligomers upon exposure to oxidizing agents. These oligomers were ∼10-fold more abundant in the brain of old than young mice. Oxidant-induced oligomerization of wild-type KCNB1 enhanced apoptosis in neuronal cells subject to oxidative insults. Consequently, a KCNB1 variant resistant to oxidation, obtained by mutating a conserved cysteine to alanine, (C73A), was neuroprotective. The fact that oxidation of KCNB1 is toxic, argues that this mechanism may contribute to neuropathy in conditions characterized by high levels of oxidative stress, such as Alzheimer's disease (AD). Accordingly, oxidation of KCNB1 channels was exacerbated in the brain of a triple transgenic mouse model of AD (3xTg-AD). The C73A variant protected neuronal cells from apoptosis induced by incubation with ß-amyloid peptide (Aß(1-42)). In an invertebrate model (Caenorhabditis elegans) that mimics aspects of AD, a C73A-KCNB1 homolog (C113S-KVS-1) protected specific neurons from apoptotic death induced by ectopic expression of human Aß(1-42). Together, these data underscore a novel mechanism of toxicity in neurodegenerative disease.

Reactive oxygen species are related to ionic fluxes and volume decrease in apoptotic cerebellar granule neurons: role of NOX enzymes.

Reactive oxygen species (ROS) are produced early during apoptosis of cerebellar granule neurons induced by low potassium (K5) and staurosporine (Sts). In addition, K5 and Sts activate NADPH oxidases (NOX). Recently, we described that K5 and Sts induce apoptotic volume decrease (AVD) at a time when ROS generation and NOX activity occur. In the present study, we evaluated the relationship between ROS generation and ionic fluxes during AVD. Here, we showed that K5- and Sts-induced AVD was inhibited by antioxidants and that direct ROS production induced AVD. Moreover, NOX inhibitors eliminated AVD induced by both K5 and Sts. Sts, but not K5, failed to induce AVD in cerebellar granule neurons from NOX2 knockout mice. These findings suggest that K5- and Sts-induced AVD is largely mediated by ROS produced by NOX. On the other hand, we also found that the blockage of ionic fluxes involved in AVD inhibited both ROS generation and NOX activity. These findings suggest that ROS generation and NOX activity are involved in ionic fluxes activation, which in turn could maintain ROS generation by activating NOX, leading to a self-amplifying cycle.