How does ubiquitin regulate synapses? Let me count the ways

Ubiquitination is a ‘ubiquitous’ cellular signalling system that can regulate the abundance and/or activity of proteins. Ubiquitin, either as a single molecule or in poly-ubiquitin chains, can be attached to proteins and alter their turnover, cellular localization or function. Perhaps the best known activity of ubiquitination is to regulate the degradation of proteins by the proteasome. Ubiquitination is a dynamic process that is regulated by different families of enzymes, some of which attach ubiquitin to proteins and build ubiquitin chains and others which remove ubiquitin from proteins. The many potential functions of ubiquitination are suggested by the observation that ubiquitin chains can be constructed in a variety of topologies, each of which has unique signalling consequences. One of the early studies linking ubiquitin to synaptic transmission was the cloning of the spontaneously occurring mouse mutation ataxia by Wilson et al. (2002). Ataxia mice exhibit progressive motor co-ordination problems and die a few months after birth. These motor problems are accompanied by reduced neurotransmission at the neuromuscular junction (NMJ). Cloning of the gene responsible for the ataxia phenotype demonstrated that the mouse was a severe hypomorph of the enzyme USP14, a proteasome-associated ubiquitin-specific protease (USP) that is responsible for trimming ubiquitin chains, thereby regulating substrate degradation by the proteasome. Obviously, this raises the question of exactly how these changes in synaptic function might be related to ubiquitin signalling. Some progress in understanding what is going on at the NMJ has already been made by Bhattacharyya et al. (2012) and her colleagues. The NMJ of the ataxia mouse was found to exhibit a decrease in the number of synaptic vesicles in the ‘readily releasable pool’, and also exhibited other abnormal features, such as an increase in the release probability of each synaptic vesicle. As a decrease in the ubiquitin hydrolase activity of USP14 results in a reduction in the size of the free ubiquitin pool, Chen and his colleagues (2011) and his colleagues examined the consequences of overexpressing wild-type USP14 or ubiquitin in the ataxia mouse and found that these procedures re-established normal synaptic transmission at the NMJ. In this issue of The Journal of Physiology, Walters et al. (2014) address the function of ubiquitin signalling at central synapses. The approach they take combines several types of experiments, including mouse genetics, which they have effectively used in the past, together with electron microscopy, electrophysiology and mathematical modelling. When they investigated baseline synaptic transmission in the hippocampus at Schaffer collateral or lateral perforant path synapses of ataxia mice, they observed nothing abnormal. However, when they examined a type of short-term plasticity known as ‘paired pulse facilitation’ (PPF), they found that the magnitude of PPF was decreased. The magnitude of PPF is usually found to be inversely proportional to the release probability; however, that does not seem to be what is going on in this instance. Rather, there was a decrease in the release probability of facilitating pulses that correlated with a nearly 55% reduction in synaptic vesicles at both Schaffer collateral and lateral perforant path synapses. The docked vesicle pool is normally taken as representing the morphological equivalent of the physiologically defined readily releasable vesicle pool. Indications of the mechanism underlying these phenomena were provided by using a variety of genetic models. Mice in which UPS14 was overexpressed in ataxia mice corrected the magnitude of PPF at lateral perforant path synapses; no surprise there. One reasonable hypothesis for explaining the deficits in hippocampal PPF would be that, as in the case of the NMJ, it results from the reduced size of the free ubiquitin pool. However, when Walters et al. (2014) overexpressed ubiquitin, in contrast to previous results at the NMJ, the deficits in PPF were not corrected. Indeed, when they overexpressed a version of USP14 that lacked ubiquitin hydrolase activity altogether, they observed that, exactly as with wild-type USP14, this mutant protein was also able to correct PPF and vesicle number at the lateral perforant path synapses. These data indicate that there is a property of USP14, aside from its known enzymatic activity, which is responsible for regulating the size of the readily releasable pool of transmitter vesicles at hippocampal synapses, which is a very surprising and original result. Furthermore, Walters et al. (2014) demonstrated that the magnitude of PPF in the hippocampus of ataxia mice could be corrected by a drug that inhibited the activity of the proteasome, suggesting that the lack of USP14 leads to the increased degradation of one or more proteins that have an important role in vesicle docking. Biochemical analysis of some of the likely candidate proteins did not reveal any changes in their expression levels in the hippocampus of ataxia mice; therefore, we can assume that any change in protein degradation is not a general feature but is directed specifically to one or more particular proteins, although how such selectivity is produced is not known. Alternatively, a reduction in free ubiquitin pools by proteasome inhibition or overexpression of the USP14 inactive form could block the ubiquitination of a synaptic protein and change its function. Future studies that inactivate USP14 acutely will be required to clarify the role of USP14 in regulating synaptic activity. These results illustrate how ubiquitin signalling can potentially regulate the size of the releasable pool of transmitter vesicles at central synapses. We should not be surprised that ubiquitin signalling is involved in aspects of synaptic function, but the paper by Walters et al. (2014) further reveals the diverse ways in which this can happen. Clearly, the enzyme USP14 has both ubiquitin hydrolase-dependent and -independent functions depending on precisely which synapse and which type of synaptic communication is being examined. The next stage will be to identify exactly which are the protein substrates involved in these interesting regulatory mechanisms.