Homer is a postsynaptic density (PSD) scaffold protein that is involved in synaptic plasticity, calcium signaling and neurological disorders. Here, we use pre-embedding immunogold electron microscopy to illustrate the differential localization of three Homer gene products (Homer 1, 2, and 3) in different regions of the mouse brain. In cross-sectioned PSDs, Homer occupies a layer ~30–100 nm from the postsynaptic membrane lying just beyond the dense material that defines the PSD core (~30 nm thick). Homer is evenly distributed within the PSD area along the lateral axis, but not at the peri-PSD locations within 60 nm from the edge of the PSD, where mGluR1 and 5 are concentrated. This distribution of Homer matches that of Shank, another major PSD scaffold protein, but differs from those of other two major binding partners of Homer, type I mGluR and IP3 receptors. Many PSD proteins rapidly redistribute upon acute (2 min) stimulation. To determine whether Homer distribution is affected by acute stimulation, we examined its distribution in dissociated hippocampal cultures under different conditions. Both the pattern and density of label for Homer 1, the isoform that is ubiquitous in hippocampus, remained unchanged under high K+ depolarization (90 mM for 2–5 min), NMDA treatment (50 μM for 2 min), and calcium-free conditions (EGTA at 1 mM for 2 min). In contrast, Shank and CaMKII accumulate at the PSD upon NMDA treatment, and CaMKII is excluded from the PSD complex under low calcium conditions.

Neuroinflammation is an important contributor to neuronal pathology and death in neurodegenerative diseases and neuronal injury. Therapeutic interventions blocking the activity of the inflammatory kinase IKKβ, a key regulator of neuroinflammatory pathways, is protective in several animal models of neurodegenerative disease and neuronal injury. In Huntington’s disease (HD), however, significant questions exist as to the impact of blocking or diminishing the activity of IKKβ on HD pathology given its potential role in Huntingtin (HTT) degradation. In cell culture, IKKβ phosphorylates HTT serine (S) 13 and activates HTT degradation, a process that becomes impaired with polyQ expansion. To investigate the in vivo relationship of IKKβ to HTT S13 phosphorylation and HD progression, we crossed conditional tamoxifen-inducible IKKβ knockout mice with R6/1 HD mice. Behavioral assays in these mice showed a significant worsening of HD pathological phenotypes. The increased behavioral pathology correlated with reduced levels of endogenous mouse full-length phospho-S13 HTT, supporting the importance of IKKβ in the phosphorylation of HTT S13 in vivo. Notably, many striatal autophagy genes were up-regulated in HD vs. control mice; however, IKKβ knockout partially reduced this up-regulation in HD, increased striatal neurodegeneration, and enhanced an activated microglial response. We propose that IKKβ is protective in striatal neurons early in HD progression via phosphorylation of HTT S13. As IKKβ is also required for up-regulation of some autophagy genes and HTT is a scaffold for selective autophagy, IKKβ may influence autophagy through multiple mechanisms to maintain healthy striatal function, thereby reducing neuronal degeneration to slow HD onset.

Polyubiquitin chains on proteins flag them for distinct fates depending on the type of polyubiquitin linkage. While lysine48-linked polyubiquitination directs proteins to proteosomal degradation, lysine63-linked polyubiquitination promotes different protein trafficking and is involved in autophagy. Here we show that postsynaptic density (PSD) fractions from adult rat brain contain deubiquitinase activity that targets both lysine48 and lysine63-linked polyubiquitins. Comparison of PSD fractions with parent subcellular fractions by Western immunoblotting reveals that CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, is highly enriched in the PSD fraction. Electron microscopic examination of hippocampal neurons in culture under basal conditions shows immunogold label for CYLD at the PSD complex in approximately one in four synapses. Following depolarization by exposure to high K+, the proportion of CYLD-labeled PSDs as well as the labeling intensity of CYLD at the PSD increased by more than eighty percent, indicating that neuronal activity promotes accumulation of CYLD at the PSD. An increase in postsynaptic CYLD following activity, would promote removal of lysine63-polyubiquitins from PSD proteins and thus could regulate their trafficking and prevent their autophagic degradation.