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Dynamic control of protein degradation via the ubiquitin proteasome system (UPS) is thought to play a crucial role in neuronal function and synaptic plasticity. The proteasome subunit Rpt6, an AAA ATPase subunit of the 19S regulatory particle (RP), has emerged as an important site for regulation of 26S proteasome function in neurons. Phosphorylation of Rpt6 on serine 120 (S120) can stimulate the catalytic rate of substrate degradation by the 26S proteasome and this site is targeted by the plasticity-related kinase Ca2+/calmodulin-dependent kinase II (CaMKII), making it an attractive candidate for regulation of proteasome function in neurons. Several in vitro studies have shown that altered Rpt6 S120 phosphorylation can affect the structure and function of synapses. To evaluate the importance of Rpt6 S120 phosphorylation in vivo, we created two mouse models which feature mutations at S120 that block or mimic phosphorylation at this site. We find that peptidase and ATPase activities are upregulated in the phospho-mimetic mutant and downregulated in the phospho-dead mutant [S120 mutated to aspartic acid (S120D) or alanine (S120A), respectively]. Surprisingly, these mutations had no effect on basal synaptic transmission, long-term potentiation (LTP), and dendritic spine dynamics and density in the hippocampus. Furthermore, these mutants displayed no deficits in cued and contextual fear memory. Thus, in a mouse model that blocks or mimics phosphorylation at this site, either compensatory mechanisms negate these effects, or small variations in proteasome activity are not enough to induce significant changes in synaptic structure, plasticity, or behavior.

Sanfilippo syndrome, MPS IIIA-D, results from deficits in lysosomal enzymes that specifically degrade heparan sulfate, a sulfated glycosaminoglycan. The accumulation of heparan sulfate results in neurological symptoms, culminating in extensive neurodegeneration and early death. To study the impact of storage in postnatal neurodevelopment, we examined murine models of MPS IIIA, which lack the enzyme sulfamidase. We show that changes occur in excitatory postsynaptic structure and function in the somatosensory cortex prior to signs of neurodegeneration. These changes coincide with accumulation of heparan sulfate with characteristic non-reducing ends, which is present at birth in the mutant mice. Accumulation of heparan sulfate was also detected in primary cultures of cortical neural cells, especially astrocytes. Accumulation of heparan sulfate in cultured astrocytes corresponded with augmented extracellular heparan sulfate and glypican 4 levels. Heparan sulfate from the cerebral cortex of MPS IIIA mice showed enhanced ability to increase glutamate AMPA receptor subunits at the cell surface of wild type neurons. These data support the idea that abnormalities in heparan sulfate content and distribution contribute to alterations in postsynaptic function. Our findings identify a disease-induced developmental phenotype that temporally overlaps with the onset of behavioral changes in a mouse model of MPS IIIA.

The trafficking of AMPA receptors (AMPARs) to and from synapses is crucial for synaptic plasticity. Previous work has demonstrated that AMPARs undergo activity-dependent ubiquitination by the E3 ubiquitin ligase Nedd4-1, which promotes their internalization and degradation in lysosomes. Here, we define the molecular mechanisms involved in ubiquitination and deubiquitination of AMPARs. We report that Nedd4-1 is rapidly redistributed to dendritic spines in response to AMPAR activation and not in response to NMDA receptor (NMDAR) activation in cultured rat neurons. In contrast, NMDAR activation directly antagonizes Nedd4-1 function by promoting the deubiquitination of AMPARs. We show that NMDAR activation causes the rapid dephosphorylation and activation of the deubiquitinating enzyme (DUB) USP8. Surface AMPAR levels and synaptic strength are inversely regulated by Nedd4-1 and USP8. Strikingly, we show that homeostatic downscaling of synaptic strength is accompanied by an increase and decrease in Nedd4-1 and USP8 protein levels, respectively. Furthermore, we show that Nedd4-1 is required for homeostatic loss of surface AMPARs and downscaling of synaptic strength. This study provides the first mechanistic evidence for rapid and opposing activity-dependent control of a ubiquitin ligase and DUB at mammalian CNS synapses. We propose that the dynamic regulation of these opposing forces is critical in maintaining synapses and scaling them during homeostatic plasticity. [ABSTRACT FROM AUTHOR]