Cypin regulates synaptic content via K63-linked polyubiquitination

Cypin (cytosolic PSD-95 interactor) is the most highly expressed guanine deaminase in the brain. Cypin regulates neuronal development by increasing dendritogenesis, modifying dendritic spine morphology, and protecting neurons from injury. To identify binding partners to cypin that may act to mediate these functions, we performed a yeast two-hybrid screen of a rat brain cDNA library with cypin as bait. One of the interactors identified is the beta 7 subunit of the proteasome. Using ubiquitin-independent and ubiquitin-dependent sensors in cultured cells, we found that cypin acts via both pathways to regulate proteasome activity. Proteomic studies revealed that cypin overexpression alters the protein ubiquitination pathway and promotes synaptogenesis in cultured hippocampal neurons. Further analysis of proteome changes at the synapses identified that cypin plays a major role in pre- and postsynaptic function, with postsynaptic functions being predominant. In fact, cypin overexpression resulted in elevated K63-linked polyubiquitination of PSD-95, a major scaffolding protein at the postsynaptic density responsible for the clustering and signaling of glutamate receptors at synapses. Based on our in vitro data, we overexpressed cypin bilaterally in the dorsal hippocampus and performed biochemical assays. Cypin overexpression led to increased levels of synaptic proteins. We then knocked down cypin in vivo, and we observed a decrease in K63-linked polyubiquitinated proteins. Taken together, our data demonstrate a novel role for cypin in the regulation of proteasome activity and proteasome-independent post-translational modifications on synaptic proteins, resulting in changes to synaptic content.