Abeta(1-42) reduces synapse number and inhibits neurite outgrowth in primary cortical and hippocampal neurons: a quantitative analysis.

Synaptic loss represents one of the earliest signs of neuronal damage and is observed within both Alzheimer's disease patients and transgenic mouse models of the disease. We have developed a novel in vitro assay using high content screening technology to measure changes in a number of cell physiological parameters simultaneously within a neuronal population. Using Hoechst-33342 to label nuclei, betaIII-tubulin as a neuron-specific marker, and synapsin-I as an indicator of pre-synaptic sites, we have designed software to interrogate triple-labelled images, counting only those synaptic puncta associated with tubulin-positive structures. Here we demonstrate that addition of amyloid beta peptide (Abeta(1-42)), to either primary hippocampal or cortical neurons for 4 days in vitro has deleterious effects upon synapse formation, neurite outgrowth and arborisation in a concentration-dependent manner. Control reverse peptide showed no effect over the same concentration range. The effects of Abeta(1-42) were inhibited by D-KLVFFA, which contains residues 16-20 of Abeta that function as a self-recognition element during Abeta assembly and bind to the homologous region of Abeta and block its oligomerisation. These effects of Abeta(1-42) on synapse number and neurite outgrowth are similar to those described within AD patient pathology and transgenic mouse models.

Hippocalcin functions as a calcium sensor in hippocampal LTD.

It is not fully understood how NMDAR-dependent LTD causes Ca(2+)-dependent endocytosis of AMPARs. Here we show that the neuronal Ca(2+) sensor hippocalcin binds the beta2-adaptin subunit of the AP2 adaptor complex and that along with GluR2 these coimmunoprecipitate in a Ca(2+)-sensitive manner. Infusion of a truncated mutant of hippocalcin (HIP(2-72)) that lacks the Ca(2+) binding domains prevents synaptically evoked LTD but has no effect on LTP. These data indicate that the AP2-hippocalcin complex acts as a Ca(2+) sensor that couples NMDAR-dependent activation to regulated endocytosis of AMPARs during LTD.

Identification of neuroprotective properties of anti-MAG antibody: a novel approach for the treatment of stroke?

The inhibitory activity of myelin-associated glycoprotein (MAG) on neurons is thought to contribute to the lack of regenerative capacity of the CNS after injury. The interaction of MAG and its neuronal receptors mediates bidirectional signaling between neurons and oligodendrocytes. The novel finding that an anti-MAG monoclonal antibody not only possesses the ability to neutralise the inhibitory effect of MAG on neurons but also directly protects oligodendrocytes from glutamate-mediated oxidative stress-induced cell death is reported here. Furthermore, administration of anti-MAG antibody (centrally and systemically) starting 1 hour after middle cerebral artery occlusion in the rat significantly reduced lesion volume at 7 days. This neuroprotection was associated with a robust improvement in motor function compared with animals receiving control IgG1. Together, these data highlight the potential for the use of anti-MAG antibodies as therapeutic agents for the treatment of stroke.

KCNQ/M currents in sensory neurons: significance for pain therapy.

Neuronal hyperexcitability is a feature of epilepsy and both inflammatory and neuropathic pain. M currents [IK(M)] play a key role in regulating neuronal excitability, and mutations in neuronal KCNQ2/3 subunits, the molecular correlates of IK(M), have previously been linked to benign familial neonatal epilepsy. Here, we demonstrate that KCNQ/M channels are also present in nociceptive sensory systems. IK(M) was identified, on the basis of biophysical and pharmacological properties, in cultured neurons isolated from dorsal root ganglia (DRGs) from 17-d-old rats. Currents were inhibited by the M-channel blockers linopirdine (IC50, 2.1 microm) and XE991 (IC50, 0.26 microm) and enhanced by retigabine (10 microm). The expression of neuronal KCNQ subunits in DRG neurons was confirmed using reverse transcription-PCR and single-cell PCR analysis and by immunofluorescence. Retigabine, applied to the dorsal spinal cord, inhibited C and Adelta fiber-mediated responses of dorsal horn neurons evoked by natural or electrical afferent stimulation and the progressive "windup" discharge with repetitive stimulation in normal rats and in rats subjected to spinal nerve ligation. Retigabine also inhibited responses to intrapaw application of carrageenan in a rat model of chronic pain; this was reversed by XE991. It is suggested that IK(M) plays a key role in controlling the excitability of nociceptors and may represent a novel analgesic target.

Lipid rafts mediate the interaction between myelin-associated glycoprotein (MAG) on myelin and MAG-receptors on neurons.

The interaction between myelin-associated glycoprotein (MAG), expressed at the periaxonal membrane of myelin, and receptors on neurons initiates a bidirectional signalling system that results in inhibition of neurite outgrowth and maintenance of myelin integrity. We show that this involves a lipid-raft to lipid-raft interaction on opposing cell membranes. MAG is exclusively located in low buoyancy Lubrol WX-insoluble membrane fractions isolated from whole brain, primary oligodendrocytes, or MAG-expressing CHO cells. Localisation within these domains is dependent on cellular cholesterol and occurs following terminal glycosylation in the trans-Golgi network, characteristics of association with lipid rafts. Furthermore, a recombinant form of MAG interacts specifically with lipid-raft fractions from whole brain and cultured cerebellar granule cells, containing functional MAG receptors GT1b and Nogo-66 receptor and molecules required for transduction of signal from MAG into neurons. The localisation of both MAG and MAG receptors within lipid rafts on the surface of opposing cells may create discrete areas of high avidity multivalent interaction, known to be critical for signalling into both cell types. Localisation within lipid rafts may provide a molecular environment that facilitates the interaction between MAG and multiple receptors and also between MAG ligands and molecules involved in signal transduction.

Molecular cloning, distribution and functional analysis of the NA(V)1.6. Voltage-gated sodium channel from human brain.

We have cloned and expressed the full-length human Na(V)1.6 sodium channel cDNA. Northern analysis showed that the hNa(V)1.6 gene, like its rodent orthologues, is abundantly expressed in adult brain but not other tissues including heart and skeletal muscle. Within the adult brain, hNa(V)1.6 mRNA is widely expressed with particularly high levels in the cerebellum, occipital pole and frontal lobe. When stably expressed in human embryonic kidney cells (HEK293), the hNa(V)1.6 channel was found to be very similar in its biophysical properties to human Na(V)1.2 and Na(V)1.3 channels [Eur. J. Neurosci. 12 (2000) 4281-4289; Pflügers Arch. 441 (2001) 425-433]. Only relatively subtle differences were observed, for example, in the voltage dependence of gating. Like hNa(V)1.3 channels, hNa(V)1.6 produced sodium currents with a prominent persistent component when expressed in HEK293 cells. These persistent currents were similar to those reported for the rat Na(V)1.2 channel [Neuron 19 (1997) 443-452], although they were not dependent on over-expression of G protein betagamma subunits. These data are consistent with the proposal that Na(V)1.6 channels may generate the persistent currents observed in cerebellar Purkinje neurons [J. Neurosci. 17 (1997) 4157-4536]. However, in our hNa(V)1.6 cell line we have been unable to detect the resurgent currents that have also been described in Purkinje cells. Although Na(V)1.6 channels have been implicated in producing these resurgent currents [Neuron 19 (1997) 881-891], our data suggest that this may require modification of the Na(V)1.6 alpha subunit by additional factors found in Purkinje neurons but not in HEK293 cells.