Sodium channel cleavage is associated with aberrant neuronal activity and cognitive deficits in a mouse model of Alzheimer's disease.

BACE1 is the rate-limiting enzyme that cleaves amyloid precursor protein (APP) to produce the amyloid ß peptides that accumulate in Alzheimer's disease (AD). BACE1, which is elevated in AD patients and APP transgenic mice, also cleaves the ß2-subunit of voltage-gated sodium channels (Navß2). Although increased BACE1 levels are associated with Navß2 cleavage in AD patients, whether Navß2 cleavage occurs in APP mice had not yet been examined. Such a finding would be of interest because of its potential impact on neuronal activity: previous studies demonstrated that BACE1-overexpressing mice exhibit excessive cleavage of Navß2 and reduced sodium current density, but the phenotype associated with loss of function mutations in either Navß-subunits or pore-forming α-subunits is epilepsy. Because mounting evidence suggests that epileptiform activity may play an important role in the development of AD-related cognitive deficits, we examined whether enhanced cleavage of Navß2 occurs in APP transgenic mice, and whether it is associated with aberrant neuronal activity and cognitive deficits. We found increased levels of BACE1 expression and Navß2 cleavage fragments in cortical lysates from APP transgenic mice, as well as associated alterations in Nav1.1α expression and localization. Both pyramidal neurons and inhibitory interneurons exhibited evidence of increased Navß2 cleavage. Moreover, the magnitude of alterations in sodium channel subunits was associated with aberrant EEG activity and impairments in the Morris water maze. Together, these results suggest that altered processing of voltage-gated sodium channels may contribute to aberrant neuronal activity and cognitive deficits in AD.

Neuroprotective profile of novel SRC kinase inhibitors in rodent models of cerebral ischemia.

Src kinase signaling has been implicated in multiple mechanisms of ischemic injury, including vascular endothelial growth factor (VEGF)-mediated vascular permeability that leads to vasogenic edema, a major clinical complication in stroke and brain trauma. Here we report the effects of two novel Src kinase inhibitors, 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methyl-1-piperazinyl)propoxy]-3-quinolinecarbonitrile (SKI-606) and 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[4-(4-methypiperazin-1-yl)but-1-ynyl]-3-quinolinecarbonitrile (SKS-927), on ischemia-induced brain infarction and short- and long-term neurological deficits. Two well established transient [transient middle cerebral artery occlusion (tMCAO)] and permanent [permanent middle cerebral artery occlusion (pMCAO)] focal ischemia models in the rat were used with drug treatments initiated up to 6 h after onset of stroke to mimic the clinical scenario. Brain penetration of Src inhibitors, their effect on blood-brain barrier integrity and VEGF signaling in human endothelial cells were also evaluated. Our results demonstrate that both agents potently block VEGF-mediated signaling in human endothelial cells, penetrate rat brain upon systemic administration, and inhibit postischemic Src activation and vascular leakage. Treatment with SKI-606 or SKS-927 (at the doses of 3-30 mg/kg i.v.) resulted in a dose-dependent reduction in infarct volume and robust protection from neurological impairments even when the therapy was initiated up to 4- to 6-h after tMCAO. Src blockade after pMCAO resulted in accelerated improvement in recovery from motor, sensory, and reflex deficits during a long-term (3 weeks) testing period poststroke. These data demonstrate that the novel Src kinase inhibitors provide effective treatment against ischemic conditions within a clinically relevant therapeutic window and may constitute a viable therapy for acute stroke.

Pancortin-2 interacts with WAVE1 and Bcl-xL in a mitochondria-associated protein complex that mediates ischemic neuronal death.

The actin-modulating protein Wiskott-Aldrich syndrome protein verprolin homologous-1 (WAVE1) and a novel CNS-specific protein, pancortin, are highly enriched in adult cerebral cortex, but their functions are unknown. Here we show that WAVE1 and pancortin-2 interact in a novel cell death cascade in adult, but not embryonic, cerebral cortical neurons. Focal ischemic stroke induces the formation of a protein complex that includes pancortin-2, WAVE1, and the anti-apoptotic protein Bcl-xL. The three-protein complex is associated with mitochondria resulting in increased association of Bax with mitochondria, cytochrome c release, and neuronal apoptosis. In pancortin null mice generated using a Cre-loxP system, ischemia-induced WAVE1-Bcl-xL interaction is diminished, and cortical neurons in these mice are protected against ischemic injury. Thus, pancortin-2 is a mediator of ischemia-induced apoptosis of neurons in the adult cerebral cortex and functions in a novel mitochondrial/actin-associated protein complex that sequesters Bcl-xL.

Angiotensin IV elevates oxytocin levels in the rat amygdala and produces anxiolytic-like activity through subsequent oxytocin receptor activation.

INTRODUCTION: The effects of angiotensin (Ang) IV result from binding to a constitutively active metallopeptidase known as the AT(4) receptor (or oxytocinase/insulin-regulated membrane aminopeptidase). While in vitro evidence indicates that Ang IV inhibits the peptidase activity of AT(4) receptors, leading to increases in the concentrations of several peptides, including oxytocin, the consequence of inhibiting AT(4) peptidase activity in vivo remains unresolved. DISCUSSION: Microdialysis coupled to immunoassay techniques revealed that systemic and intra-amygdala injection of Nle-Ang IV, a metabolically stable derivative of Ang IV, significantly elevated extracellular levels of oxytocin in the rat amygdala. Based on earlier reports describing the anxiolytic-like effects of oxytocin, we investigated whether disrupting AT(4) peptidase activity would yield similar responses. In the mouse four-plate test, acute treatment with either Nle-Ang IV or LVV-hemorphin-7, a related AT(4) receptor ligand, elicited significant increases in the number of punished crossings. These behavioral responses were comparable to the anxiolytic-like effects of oxytocin and to the standard anxiolytic agent, chlordiazepoxide. Cotreatment with either the AT(4) receptor antagonist, divalinal, or the selective oxytocin receptor antagonist, WAY-162720, reversed the anxiolytic-like effects of Nle-Ang IV, while combining ineffective doses of Nle-Ang IV and oxytocin increased the number of punished crossings in this assay. Conversely, Nle-Ang IV and LVV-hemorphin-7 were inactive in the mouse tail suspension test of antidepressant activity. These findings represent the first in vivo demonstration of the peptidase activity of AT(4) receptors, confirm the anxiolytic-like properties of Ang IV, and reveal a unique and previously uncharacterized relationship between AT(4) and oxytocin receptor systems.

Ganglioside inhibition of neurite outgrowth requires Nogo receptor function: identification of interaction sites and development of novel antagonists.

Gangliosides are key players in neuronal inhibition, with antibody-mediated clustering of gangliosides blocking neurite outgrowth in cultures and axonal regeneration post injury. In this study we show that the ganglioside GT1b can form a complex with the Nogo-66 receptor NgR1. The interaction is shown by analytical ultracentrifugation sedimentation and is mediated by the sialic acid moiety on GT1b, with mutations in FRG motifs on NgR1 attenuating the interaction. One FRG motif was developed into a cyclic peptide (N-AcCLQKFRGSSC-NH(2)) antagonist of GT1b, reversing the GT1b antibody inhibition of cerebellar granule cell neurite outgrowth. Interestingly, the peptide also antagonizes neurite outgrowth inhibition mediated by soluble forms of the myelin-associated glycoprotein (MAG). Structure function analysis of the peptide point to the conserved FRG triplet being the minimal functional motif, and mutations within this motif inhibit NgR1 binding to both GT1b and MAG. Finally, using gene ablation, we show that the cerebellar neuron response to GT1b antibodies and soluble MAG is indeed dependent on NgR1 function. The results suggest that gangliosides inhibit neurite outgrowth by interacting with FRG motifs in the NgR1 and that this interaction can also facilitate the binding of MAG to the NgR1. Furthermore, the results point to a rational strategy for developing novel ganglioside antagonists.

The structure of the Lingo-1 ectodomain, a module implicated in central nervous system repair inhibition.

Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200 A2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane.