The ubiquitin-proteasome system, which operates in almost every eukaryotic cell, has been implicated in the pathogenesis of numerous neurodegenerative diseases, including Parkinson’s disease (PD) and Alzheimer’s (AD). So does this pathway have a specialized role in neurons?

Earlier this year, Michael Ehlers reported that synaptic activity leads to de-novo ubiquitination of proteins at the postsynaptic density (PSD), a large, specialized complex, which, through remodeling, can contribute to synaptic plasticity (scroll to Ehlers in ARF related news story); also see Ehlers, 2003). Now, in the online PNAS, Pietro De Camilli and colleagues at Yale University and the European Institute of Oncology, Milan, Italy, report data suggesting the reverse—that synaptic activity may lead to de-ubiquitination in presynaptic synaptosomes.

First author Hong Chen and colleagues examined the effect of depolarizing isolated synaptosomes with K+ and Ca2+ ions. This is thought to mimic what happens on the proximal side of a synaptic junction when an action potential reaches axonal termini. When Chen examined these depolarized synaptosomes, he found a general and dramatic reduction in the amount of ubiquitinated protein. This effect was rapid, taking as little as 15 seconds after depolarization, and it was reversible. Many proteins appeared to be de-ubiquitinated, most of them having been poly-ubiquitinated as judged by the "smeary" nature of Western blots. But some of the affected proteins, which appeared as discrete bands, seemed to have been mono-ubiquitinated. One example was Epsin 1, a protein known to be modified by the addition of only one ubiquitin peptide. The K+/Ca2+ combination resulted in a reduction of ubiquitinated Epsin.

To probe the nature of the de-ubiquitination step, Chen next looked at the role of the protein FAM, the mammalian homolog of the Drosophila protein fat facets, which is known to catalyze the de-ubiquitination of Epsin. To do this, Chen switched to HeLa cells, which are susceptible to the inhibitory action of RNAi. In these cells, ionomycin, which renders them permeable to Ca2+, also caused de-ubiquitination of many proteins including exogenous Epsin 1. Blocking expression of FAM by RNAi, however, abolished Epsin de-ubiquitination without interfering with the removal of the ubiquitin peptide from other proteins.

The data suggest that there may be general and specific de-ubiquitination events taking place at synaptosomes. These are most likely unrelated to removal of ubiquitin-tagged proteins by the proteasome, because when Chen added the general proteasome inhibitor YU101 to synaptosomes, Ca2+-induced de-ubiquitination did not change.

How calcium, in combination with potassium, achieves global de-ubiquitination is still a bit of a mystery. The authors rule out general stimulation of enzymes like FAM, instead favoring a scenario where calcium prevents ubiquitination and therefore upsets the ubiquitination/de-ubiquitination balance. Chen also shows that Ca2+-induced depolarization causes dephosphorylation of a variety of synaptosome proteins, suggesting protein phosphatases may play a role.

Reconciling the differences between their data and that of Ehlers, the authors note that pre- and postsynaptic complexes may be affected differently. An experimental difference is that Ehlers used a chronic mode of stimulation, while in the present study it was acute. Time scales may be critical, and indeed, Chen found that the de-ubiquitination is all but reversed in as little as 10 minutes.—Tom Fagan

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  1. This paper demonstrates rapid Ca2+-dependent de-ubiquitination of synaptic proteins. Using a classical biochemical preparation of synaptosomes, which are pinched-off nerve terminals with attached postsynaptic elements capable of neurotransmitter release, De Camilli and colleagues demonstrate that strong depolarization (a stimulus that triggers neurotransmitter release followed by rapid endocytosis of synaptic vesicles) causes a rapid loss of ubiquitinated proteins, likely due to "de-ubiquitination." They demonstrate that two prominent endocytic proteins—Epsin1 and Eps15—are among the proteins subject to depolarization- and Ca2+-dependent de-ubiquitination, and provide strong evidence that the enzyme that clips off ubiquitin from Epsin is the deubiquitinating enzyme fat facets/FAM. The authors perform biochemical experiments to assess what functional effect ubiquitination has on Epsin, and show that ubiquitin-conjugated Epsin is less capable of binding lipid membranes, clathrin, and AP-2—the essential molecular ingredients for synaptic vesicle endocytosis. From these data, the authors propose that Ca2+-dependent de-ubiquitination of endocytic proteins in the presynaptic nerve terminal helps mediate the rapid endocytosis of synaptic vesicles necessary to sustain synaptic transmission.

    The ubiquitin-proteasome system (UPS) has been implicated in numerous neurodegenerative disorders including Alzheimer’s disease (AD). Moreover, synaptic dysfunction has emerged as an important and perhaps initiating element in the early-stage pathogenesis of AD. By demonstrating a further link between the UPS and synaptic transmission in normal physiological states, the study by Chen et al. brings us closer to unifying these two models of AD pathogenesis. It will be of interest to examine the ubiquitinated state of synaptic proteins in animal models of AD and in human neuropathology.

References

News Citations

  1. Synapses Sizzle in Limelight of Symposium Preceding Neuroscience Conference, Orlando: Day I

Paper Citations

  1. . Activity level controls postsynaptic composition and signaling via the ubiquitin-proteasome system. Nat Neurosci. 2003 Mar;6(3):231-42. PubMed.

Further Reading

No Available Further Reading

Primary Papers

  1. . Rapid Ca2+-dependent decrease of protein ubiquitination at synapses. Proc Natl Acad Sci U S A. 2003 Dec 9;100(25):14908-13. PubMed.