Sodium imaging courtesy of Martin A.
Smith, University of California, Irvine,
Irvine, CA

Why would a molecule that arranges the connections between motor neurons and muscle cells be expressed throughout the brain? Hilgenberg et al. propose that agrin regulates synaptic activity, demonstrating a new receptor and mechanism for agrin action in the brain, in a recent study in Cell.

Agrin is secreted by motor neurons and clusters acetylcholine receptors on muscle cells in preparation for neuromuscular innervation. In neurons, agrin increases intracellular Ca²⁺ and induces the expression of the immediate early gene c-fos. However, MuSK, the known agrin receptor, is not expressed in the brain.

The authors searched for a brain-specific receptor for agrin using a 20 kDa carboxyl-terminus fragment of agrin that induces c-fos expression in cultured neurons. The authors chemically crosslinked the agrin agonist to cultured neurons and glia. A 130 kDa band representing agrin and its crosslinked binding partner was found in immunoblots of neuronal, but not glial cell lysates. Mass spectrometry analysis of the crosslinked products identified agrin and the 3 subunit of Na+/K+-ATPase, which in brain is only expressed in neurons. Immunoprecipitation and western blots with an 3Na⁺/K⁺-ATPase antibody confirmed the association between agrin and 3Na⁺/K⁺-ATPase. Agrin binding sites colocalized with 3Na⁺/K⁺-ATPase in cultured mouse cortical neurons, and 3Na⁺/K⁺-ATPase mainly localized to synapses.

The Na⁺/K⁺-ATPase pumps Na⁺ ions out of the cell. The authors loaded cultured mouse cortical neurons with a fluorescent, Na⁺-sensitive dye and antagonists to block action potentials and synaptic transmission. Agrin increased intracellular Na⁺ concentration. A small inhibitory fragment of agrin that displaced bound agrin prevented the agrin-induced increase in intracellular Na⁺ concentration. Therefore, agrin blocks the Na⁺ pump. In whole-cell, current-clamp experiments, agrin increased the cell's membrane potential. The inhibitory agrin fragment not only blocked agrin-induced increases in membrane potential, but also reduced the basal membrane potential. These data suggest that endogenous agrin regulates the electrical gradient of the cell. Agrin raised intracellular Na⁺ concentration and membrane potential only in cells expressing 3Na⁺/K⁺-ATPase, suggesting this receptor is sufficient to mediate agrin's electrophysiological effects.

To examine agrin's effects on neuronal activity, the authors did current-clamp experiments without the action potential and synaptic transmission inhibitors. In dispersed cortical neurons and in cortical slice preparations, agrin increased the frequency of spontaneous action potentials. The inhibitory agrin fragment blocked kainate-induced increases in action potential frequency, suggesting that endogenous agrin regulates neuronal excitability.

The authors showed previously that excitotoxic injury is reduced in neurons from agrin knockout mice. What is the mechanism for the protective effects of agrin gene deletion? The authors suggest that reduction in agrin expression enhances the ability of the 3Na⁺/K⁺-ATPase to maintain Na⁺ ion homeostasis, minimizing the influx of Ca²⁺ through voltage-gated channels, and supporting the activity of the Na⁺/Ca²⁺ exchanger. As a result, the cell is protected from potentially damaging levels of intracellular Ca²⁺. Agrin might therefore be an important target for drugs to prevent or reduce the impact of excitotoxic insults to the brain.

1. Hilgenberg, L. G. W. , Su, H. , Gu, H. , O'Dowd, D. K. & Smith M. A. α3Na⁺/K⁺-ATPase is a neuronal receptor for agrin. Cell. 125, 359– 369 (2006)