Abstract

An improved understanding of the molecular mechanisms in synapse formation provides insight into both learning and memory and the etiology of neurodegenerative disorders. Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein methyltransferase that negatively regulates synaptic gene expression and inhibits neuronal differentiation. Despite its regulatory function in neurons, little is known about the CARM1 cellular location and its role in dendritic maturation and synapse formation. Here, we examined the effects of CARM1 inhibition on dendritic spine and synapse morphology in the rat hippocampus. CARM1 was localized in hippocampal post-synapses, with immunocytochemistry and electron microscopy revealing co-localization of CARM1 with post-synaptic density (PSD)-95 protein, a post-synaptic marker. Specific siRNA-mediated suppression of CARM1 expression resulted in precocious dendritic maturation, with increased spine width and density at sites along dendrites and induction of mushroom-type spines. These changes were accompanied by a striking increase in the cluster size and number of key synaptic proteins, including N-methyl-d-aspartate receptor subunit 2B (NR2B) and PSD-95. Similarly, pharmacological inhibition of CARM1 activity with the CARM1-specific inhibitor AMI-1 significantly increased spine width and mushroom-type spines and also increased the cluster size and number of NR2B and cluster size of PSD-95. These results suggest that CARM1 is a post-synaptic protein that plays roles in dendritic maturation and synaptic formation and that spatiotemporal regulation of CARM1 activity modulates neuronal connectivity and improves synaptic dysfunction.

Highlights

  • The authors declare that they have no conflicts of interest with the contents of this article

  • Coactivator-associated arginine methyltransferase 1 (CARM1) is a post-synaptic protein that clusters at synapses in hippocampal neurons

  • The relative enrichment of CARM1 in whole hippocampal tissue homogenate, synaptosomes, and postsynaptic density (PSD) fractions treated once with Triton X-100 was tested by immunoblotting analyses and compared with that of N-methyl-D-aspartate receptor subunit 2B (NR2B) and PSD-95 proteins that are known to be associated with the PSD fraction

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Summary

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Spines receive synaptic input from pre-synaptic axon terminals and regulate post-synaptic transmission [7]. Post-translational modification of proteins within signaling cascades, such as phosphorylation and methylation of synaptic proteins, is a fundamental and essential mechanism for regulating axonal elongation and dendrite branching [8, 15]. Activated PKC⑀ modulates morphological changes in dendritic spines and enhances long-term memory by activating structural changes in the synaptic cytoskeleton (18 –20) or by up-regulating HuD-mediated post-transcriptional controls [21, 22]. Coactivator-associated arginine methyltransferase 1 (CARM1), called PRMT4, modulates protein methylation in neurons [28, 29]. CARM1 methylates HuD protein and plays an important role in mRNA processing [22, 30] It inhibits neuronal differentiation in vitro [28]. We examined the effects of genetic and pharmacological inhibition of CARM1 on dendritic complexity, spine number and density, and arborization, and the accumulation of excitatory synaptic proteins at synaptic sites in primary cultures of differentiating rat hippocampal neurons

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