Inhibition of coactivator-associated arginine methyltransferase 1 modulates dendritic arborization and spine maturation of cultured hippocampal neurons

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.

Keywords: CARM1; PSD; dendritic maturation; dendritic spine; hippocampal neurons; hippocampus; neuron; signal transduction; synapse; synaptic clustering.

Figure 1.

CARM1 is a post-synaptic protein. A, a total of 20 or 40 μg PSD fraction isolated from rat hippocampi was separated in SDS-PAGE, and gels were stained with Coomassie Brilliant Blue solution. Arrow indicates CARM1 at 68 kDa. B, immunoblotting analysis to detect CARM1 protein levels in protein complexes extracted from PAGE gels in A using anti-CARM1 (lane 1) or anti-serum pre-absorbed with CARM1 antigen (lane 2). C, immunoblotting analysis to detect CARM1 protein levels in whole tissue homogenates (WTH), synaptosomes (Syn), or One-Triton PSD fraction (10 μg/each fraction). Mean ± S.E., three independent experiments. **, p < 0.01; ***, p < 0.001, compared with WTH).

Figure 2.

CARM1 co-localizes with PSD-95 in hippocampal neurons. A and B, cultured hippocampal neurons (14 DIV) were double-stained with anti-CARM1 and PSD-95 (a post-synaptic marker protein) or synapsin (a pre-synaptic marker), and then co-localization from overlay images was analyzed. Scale bar, 10 μm. C, rat brain sections were labeled with antibodies against CARM1 or PSD-95 and gold particle labelings in the CA1 stratum radiatum of the hippocampus were observed using electron microscopy to show post-embedded localization of CARM1 or PSD-95. Scale bar, 500 nm. D, each CARM1 or PSD-95 labeling was counted to show pre- or post-synaptic positions of gold particles and the axodendritic distribution was quantitatively expressed.

Figure 3.

Reduction of CARM1 expression increases the complexity of dendritic arborization of cultured hippocampal neurons. A, cultured hippocampal neurons were untreated or transduced with control scrambled (Cont), CARM1-specific, or PRMT1-specific siRNA at 9 DIV for 5 days. At 14 DIV, neurons were lysed and used for total RNA isolation and RT-qPCR. Relative CARM1 mRNA level was analyzed after normalization with GAPDH mRNA level. B, immunoblotting results with anti-CARM1, anti-PRMT1, and anti-actin antibodies from hippocampal neurons treated as in (A). C, cultured hippocampal neurons treated as in (A) were fixed at 14 DIV and stained with MAP-2, a dendritic marker. Whole cell shape is shown. Scale bar, 30 μm. D and E, quantitative analysis of TDBL (μm) and TDBTN from tracing images of neurons treated as in (C). F and G, Sholl analysis of dendritic complexity in neurons treated as in (C). Mean ± S.E., n = 40 neurons/condition from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with untreated condition.

Figure 4.

Treatment of CARM1 inhibitor promotes dendritic spine maturation in cultured hippocampal neurons. A, cultured hippocampal neurons were transfected with GFP-actin at 7 DIV and untreated or treated at 10 DIV with control media (untreated) or AMI-1 (2.5 or 10 μm) for 4 days. At 14 DIV, neurons were fixed and dendritic spine morphology was visualized by confocal microscopy. Scale bar, 10 μm. B–D, spine length (B), spine head width (C), or spine density (number of spines/10 μm dendrite length) (D) was calculated and statistically analyzed. E, dendritic spine shape was counted and expressed as %. Mean ± S.E., n > 100 spines, from three independent experiments. **, p < 0.01, compared with untreated condition.

Figure 5.

Reduction of CARM1 expression modifies dendritic spine maturation in cultured hippocampal neurons. A, cultured hippocampal neurons were untreated or transfected with GFP-actin at 7 DIV. After 2 days, cells were transduced with control siRNA (Cont) or CARM1-specific siRNA and incubated for another 5 days. At 14 DIV, neurons were fixed and dendritic spine morphology was visualized by confocal microscopy. Scale bar, 10 μm. B–D, spine length (B), spine head width (C), or spine density (D) (number of spines/10-μm dendrite length) was calculated and statistically analyzed. E, dendritic spine shape was counted and expressed as %. Mean ± S.E., n > 100 spines, from three independent experiments. *, p < 0.05, compared with untreated condition).

Figure 6.

CARM1 inhibition increases post-synaptic targeting of NR2B and PSD-95. A, cultured hippocampal neurons at 10 DIV were treated with control media (untreated) or AMI-1 (10 μm) and incubated for 4 days. At 14 DIV, neurons were fixed and used for double-staining with NR2B and PSD-95 antibodies. Scale bar, 10 μm. B and C, synaptic cluster size (μm²) and synaptic clustering (number per 10 μm) were calculated and statistically analyzed. Mean ± S.E., n > 100 spines, from three independent experiments. *, p < 0.05; **, p < 0.01, compared with untreated condition).

Figure 7.

Knockdown of CARM1 protein promotes post-synaptic clustering of NR2B and PSD-95 proteins. A–C, cultured hippocampal neurons were untreated or transduced with control (Cont) or CARM1-specific siRNA at 9 DIV and incubated for 5 days. At 14 DIV, neurons were fixed and used for double-staining with CARM1 and synapsin (A), NR2B (B), or PSD-95 (C) antibodies. Scale bar, 10 μm. D and E, Synaptic cluster size (μm²) and synaptic clustering (number per 10 μm) were calculated and statistically analyzed. Mean ± S.E., three independent experiments. *, p < 0.05, **, p < 0.01, compared with untreated).