Homer is concentrated at the postsynaptic density and does not redistribute after acute synaptic stimulation
Introduction
Homer is a scaffold protein at the postsynaptic density (PSD) with isoforms belonging to three gene families, Homer 1, 2, and 3, each containing long and short forms (reviewed in Ehrengruber et al., 2004, Shiraishi-Yamaguchi and Furuichi, 2007, Foa and Gasperini, 2009). The long forms are localized at the PSD and are constitutively expressed, while the short forms are cytosolic with low expression under basal conditions, and are transcriptionally induced upon neuronal stimulation. Homer binds to type I-metabotropic glutamate receptors (mGluR1 and 5) (Soloviev et al., 2000), inositol 1,4,5-trisphosphate receptors (IP3R) (Tu et al., 1998) and Shank (Hayashi et al., 2009), and is thought to be involved in glutamate receptor trafficking (Xiao et al., 2000, Shiraishi-Yamaguchi and Furuichi, 2007) and calcium signaling (Worley et al., 2007). Homer is also implicated in neurological disorders (Szumlinski et al., 2006, Luo et al., 2012).
Homer 1, 2, and 3 are differentially localized to different regions of the brain as demonstrated by in situ hybridization and Western blotting (Xiao et al., 1998), and by immunolabeling at the light microscopy level (Shiraishi et al., 2004). However, studies of Homer at the EM level have been scarce (Xiao et al., 1998, Tu et al., 1999, Petralia et al., 2001, Petralia et al., 2005), and none of these studies specified the activity state of the synapses. Here, we label different parts of the mouse brain by immunogold under validated resting conditions (Tao-Cheng et al., 2007), and compare immunolocalization of different isoforms of Homer. The detailed distribution of Homer is also compared with the known patterns of distribution of its binding partners.
The PSD is a dynamic complex that undergoes activity-dependent structural changes (Tao-Cheng, 2012). Many PSD proteins such as calcium/calmodulin-dependent kinase II (CaMKII; Dosemeci et al., 2001, Dosemeci et al., 2002), Shank (a PSD scaffold protein which binds to Homer; Tao-Cheng et al., 2010) and CYLD (a cylindromatosis tumor suppressor that functions as a deubiquitinase; Dosemeci et al., 2013) rapidly redistribute and accumulate at the PSD upon acute (2 min) stimulation. Furthermore, AMPA receptors (GluR2) become more concentrated at the PSD upon depolarization (Tao-Cheng et al., 2011), while SynGAP (a Ras GTPase activating protein; Yang et al., 2011, Yang et al., 2013) moves out of the PSD core upon excitation. To determine whether Homer distribution similarly is affected under these acute synaptic stimulation protocols, its distribution was compared under resting conditions, high K+ depolarization, acute N-methyl-d-asparic acid (NMDA) treatment, and calcium-free conditions. Dissociated hippocampal cultures were used to allow easy manipulation of the stimulation conditions (Tao-Cheng, 2012).
Section snippets
Materials and antibodies
Mouse monoclonal antibody against Homer 1 (pan Homer 1, clone 2G8), rabbit polyclonal antibodies against Homer 1b/c (raised against aa 152–354 of human Homer 1b), Homer 2 (raised against aa 1–176 of rat Homer 2), Homer 3 (raised against aa 1–177 of rat Homer 3), pan Homer 1/2/3 (raised against N-terminal parts of Homer 1, 2 and 3) were from Synaptic Systems (Göttingen, Germany); goat polyclonal antibody against Homer 1a (raised against M-13, a peptide at the C-terminus) was from Santa Cruz
Homer concentrates at the PSD
Western analysis was carried out to verify the various antibodies against different Homer isoforms (Fig. 1). Consistent with previous biochemical data (Sun et al., 1998, Xiao et al., 1998, Shiraishi et al., 1999), Homer 1, 2, and 3 were enriched at PSDs (Fig. 1 top row) isolated from rat brains. Antibodies against the long and short forms of the Homer 1 demonstrate that the long form (Homer 1b/c) was enriched at the PSD (Fig. 1, bottom row, left), in contrast to the short form (Homer 1a) (Fig. 1
Discussion
The present study uses pre-embedding immunogold to localize Homer in whole mouse brain and in disassociated hippocampal cultures. Our observations yield new insights about the actual distribution of Homer with respect to that of its predicted binding partners. The present study also addresses the question whether Homer, like many other PSD proteins (Tao-Cheng, 2012), translocates into or away from the PSD upon acute synaptic activity. Our finding that Homer does not redistribute relative to
Acknowledgements
We thank Christine A. Winters for hippocampal neuronal cultures, Virginia Crocker and Rita Azzam for expert EM technical support, Dr. Ayse Dosemeci for helpful discussions and critical reading of the manuscript. Supported by the Intramural Research Program of the NIH, NINDS.
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