Over-expression of N-type calcium channels in cortical neurons from a mouse model of Amyotrophic Lateral Sclerosis

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Abstract

Voltage-gated Ca2 + channels (VGCCs) mediate calcium entry into neuronal cells in response to membrane depolarisation and play an essential role in a variety of physiological processes. In Amyotrophic Lateral Sclerosis (ALS), a fatal neurodegenerative disease caused by motor neuron degeneration in the brain and spinal cord, intracellular calcium dysregulation has been shown, while no studies have been carried out on VGCCs. Here we show that the subtype N-type Ca2 + channels are over expressed in G93A cultured cortical neurons and in motor cortex of G93A mice compared to Controls. In fact, by western blotting, immunocytochemical and electrophysiological experiments, we observe higher membrane expression of N-type Ca2 + channels in G93A neurons compared to Controls. G93A cortical neurons filled with calcium-sensitive dye Fura-2, show a net calcium entry during membrane depolarization that is significantly higher compared to Control. Analysis of neuronal vitality following the exposure of neurons to a high K+ concentration (25 mM, 5 h), shows a significant reduction of G93A cellular survival compared to Controls. N-type channels are involved in the G93A higher mortality because ω-conotoxin GVIA (1 μM), which selectively blocks these channels, is able to abolish the higher G93A mortality when added to the external medium.

These data provide robust evidence for an excess of N-type Ca2 + expression in G93A cortical neurons which induces a higher mortality following membrane depolarization. These results may be central to the understanding of pathogenic pathways in ALS and provide novel molecular targets for the design of rational therapies for the ALS disorder.

Highlights

▸ Voltage-dependent Ca2 + current density is increased in G93A neurons. ▸ N-type Ca2 + channels are over expressed in G93A cortical cultures and tissues. ▸ The net Ca2 + entry during membrane depolarization is higher in G93A neurons. ▸ Increased N-type Ca2 + channel density induces higher mortality in G93A cultures.

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