Alternative Splicing and the Intracellular Domain Mediate TM-agrin's Ability to Differentially Regulate the Density of Excitatory and Inhibitory Synapse-like Specializations in Developing CNS Neurons.

Agrin is a multi-domain protein best known for its essential function during formation of the neuromuscular junction. Alternative mRNA splicing at sites named y and z in the C-terminal part of agrin regulates its interaction with a receptor complex consisting of the agrin-binding low-density lipoprotein receptor-related protein 4 (Lrp4) and the muscle-specific kinase (MuSK). Isoforms with inserts at both splice sites bind to Lrp4, activate MuSK and are synaptogenic at the neuromuscular junction. Agrin is also expressed as a transmembrane protein in the central nervous system (CNS) but its function during interneuronal synapse formation is unclear. Recently we demonstrated that transfection of a full-length cDNA coding for transmembrane agrin (TM-agrin) in cultured embryonic cortical neurons induced an Lrp4-dependent but MuSK-independent increase in dendritic glutamatergic synapses and an Lrp4- and MuSK-independent reduction of inhibitory synapses. Here we show that presynaptic specializations were similarly affected by TM-agrin overexpression. In addition, we mapped the regions within TM-agrin responsible for TM-agrin's effects on dendritic aggregates of synapse-associated proteins. We show that the presence of a four amino acid insert at splice site y is essential for the increase in the density of puncta containing the postsynaptic density protein 95 kDa. This effect was independent of splice site z. The reduction of the gephyrin puncta density was independent of the entire extracellular part of TM-agrin but required a highly conserved serine residue in the intracellular domain of TM-agrin. These results provide further evidence for a function of TM-agrin during CNS synaptogenesis and demonstrate that different domains and alternative splicing of TM-agrin differentially affect excitatory and inhibitory synapse formation in cultured embryonic CNS neurons.

The role of agrin, Lrp4 and MuSK during dendritic arborization and synaptogenesis in cultured embryonic CNS neurons.

The role of agrin, Lrp4 and MuSK, key organizers of neuromuscular synaptogenesis, in the developing CNS is only poorly understood. We investigated the role of these proteins in cultured mouse embryonic cortical neurons from wildtype and from Lrp4- and MuSK-deficient mice. Neurons from Lrp4-deficient mice had fewer but longer primary dendrites and a decreased density of puncta containing excitatory and inhibitory synapse-associated proteins. Neurons from MuSK-deficient mice had an altered dendritic branching pattern but no change in the density of puncta stained by antibodies against synapse-associated proteins. Transfection of TM-agrin compensated the dendritic branching deficits in Lrp4-deficient but not in MuSK-deficient neurons. TM-agrin transfection increased the density of excitatory synaptic puncta in MuSK-deficient but not in Lrp4-deficient mice and reduced the number of inhibitory synaptic puncta irrespective of MuSK and Lrp4 expression. Addition of purified soluble agrin to microisland cultures of cortical neurons revealed an Lrp4-dependent increase in the size and density of glutamatergic synaptic puncta and in mEPSC but not in mIPSC frequency and amplitude. Thus, agrin induced an Lrp4-independent increase in dendritic branch complexity, an Lrp4-dependent increase of excitatory synaptic puncta and an Lrp4- and MuSK-independent decrease in the density of puncta containing inhibitory synapse-associated proteins. These results establish selective roles for agrin, Lrp4 and MuSK during dendritogenesis and synaptogenesis in cultured CNS neurons.