3.3. Molecular mechanisms involved in the establishment and maintenance of neuronal networks
3.3.2.8 Synaptic modulation: local protein synthesis and degradation
Experience-dependent remodelling of the postsynaptic density (PSD) is critical for synapse formation and plasticity in the mammalian brain. A key mechanism contributing to enduring forms of synaptic plasticity is considered the rapid changes in local protein synthesis at synaptic sites (reviewed in (Sutton and Schuman, 2005). Activity level also regulates the postsynaptic composition and signalling through the ubiquitin-proteasome system (UPS)(Ehlers, 2003).
Local protein synthesis
Durable activity-dependent changes in the synaptic strength (required in memory and long-term synaptic plasticity) depend on the new protein synthesis and the growth or remodelling of excitatory synapses (reviewed by (Bramham and Wells, 2007). The discovery of mRNA, ribosomes and translation factors in dendrites and in the dendritic spines in granule cells of the dentate gyrus (Steward and Levy, 1982), suggested that synapses could be modified directly and individually through regulation of local protein synthesis (Sutton and Schuman, 2006). Dendrites and dendritic spines use their “own” translational machinery for protein synthesis (Job and Eberwine, 2001), which occurs even if the dendrites are isolated from the cell body (Kang and Schuman, 1996). Recent data suggests that the demand for the de novo protein synthesis varies with the level of synaptic activity (Fonseca et al., 2006a) and that long-term synaptic plasticity is the result of a balance of protein synthesis and proteasome-dependent degradation (Fonseca et al., 2006b).
Local protein degradation
Numerous cellular mechanisms regulate the machinery of synaptic transmission including the intracellular levels of calcium and cyclic nucleotides, protein phosphorylation and dephosphorylation, and the localization and translation of synaptic proteins. Hence, the activity, location, and levels of key synaptic proteins are central determinants of synaptic efficacy.
Ubiquitination regulates the amount and subcellular localisation of target proteins, qualifying as a candidate mechanism to regulate synaptic transmission (DiAntonio and Hicke, 2004; Hegde, 2004). Recent work demonstrated that ubiquitination regulates presynaptic neurotransmitter release (Chin et al., 2002; Speese et al., 2003; Wheeler et al., 2002; Wilson et al., 2002) and postsynaptic neurotransmitter receptors (Bedford et al., 2001; Burbea et al., 2002; Buttner et al., 2001; Malinow and Malenka, 2002; Patrick et al., 2003).
When a protein needs to be specifically degraded in the cell, it is marked by covalent attachment of ubiquitin to an #-amino group of lysine residues, forming a polyubiquitin chain by
sequential linkages of monoubiquitins. The polyubiquitinated substrate is then recognized by the proteasome and is degraded to small peptides and amino acids (Ciechanover et al., 1980).
Ubiquitin is disassembled by a class of enzymes called de-ubiquitinating enzymes (DUBs)(D'Andrea and Pellman, 1998) and recycled. This process of degradation of a substrate protein can be divided into two steps (Hegde, 2004):
(1) covalent attachment of the ubiquitin to the substrate (named ubiquitin conjugation or ubiquitination),
(2) degradation of the polyubiquitinated substrate, disassembly of the polyubiquitin chain and recycling of free ubiquitin
The ubiquitin conjugation step is a highly regulated process involving three classes of enzymes or complexes called E1, E2 and E3. E1, the ubiquitin-activating enzyme activates the free ubiquitin in an ATP-dependent reaction. Activated ubiquitin is then transferred to an E2, an ubiquitin-carrier enzyme. Next, E3 ubiquitin ligases, which can work solo or form complexes, ligates the activated ubiquitin to specific substrate targeted and recognized by these proteins (Hershko and Ciechanover, 1998; Hershko et al., 1980)(Figure 11). A detailed description of UPS in C. elegans was made by (Kipreos, 2005).
The first molecular function described for ubiquitin was as a signal to target substrates for degradation by the proteasome. Polyubiquitin chains of at least four ubiquitin linked through Lys48 are the principal signal for targeting substrates to the 26S proteasome (Thrower et al., 2000). Ubiquitin has also regulatory roles in the cell that are proteasome-independent.
Monoubiquitination regulates protein transport between membrane compartments by serving as a sorting signal on protein cargo and by controlling the activity of trafficking machinery (reviewed in (Hicke and Dunn, 2003). Monoubiquitination can regulate protein localization within Figure 11. The Ubiquitin/26S proteasome pathway. The protein ubiquitination begins with the activation of the ubiquitin molecule (Ub) in an ATP-dependent manner. The activated ubiquitin is then transferred to the active site of the ubiquitin-conjugating enzyme (E2). Finally, an ubiquitin-ligase (E3) binds E2 and catalyzes the formation of an isopeptide linkage between the activated ubiquitin and the Lysine residue of the substrate protein. After a chain of multiple ubiquitins is attached to the target protein, it is usually destined to the 26S proteasome, where the target protein is degraded and the ubiquitin monomers are reclaimed by the action of de-ubiquitination enzymes (DUBs)(from Zeng et al., 2006).
the nucleus (Gregory et al., 2003) and also is required for normal transactivation activity of many transcription factors, in which the transcriptional activation domain overlaps with sequences that promote rapid proteasomal degradation (degrons) (Muratani and Tansey, 2003).
Regulation of neuronal function involving UPS encompasses a large number of processes: synapse development, synapse size, vesicle cycling, neurotransmitter release, receptor trafficking, spine size, synaptic plasticity and downstream signalling (Hegde, 2004).
The role of UPS in synaptic regulation seems to be a mechanism conserved in many organisms including worms, flies and mammals, including humans. The most convincing data demonstrating that ubiquitination regulates synaptic transmission come from the analysis of DUNC-13 (a molecule known to control synaptic strength by regulating synaptic vesicle priming) in Drosophila (Speese et al., 2003) and Usp14 in mouse (Wilson et al., 2002). usp14 encodes a protein that cleaves ubiquitin from target proteins, but it can process only short ubiquitin side chains and not polyubiquitin, which regulate the monoubiquitination state, and hence the localization or activity, of target proteins that regulate synaptic transmission. In mammals, two E3 ligases, Staring and Siah, are considered excellent candidates to mediate ubiquitin-dependent regulation of synaptic transmission. Staring is a RING-finger E3 ubiquitin-ligase, which recruits the E2 ubiquitin-conjugating enzyme UbcH8 to syntaxin 1 (a SNARE protein involved in synaptic vesicle fusion with the plasma membrane and thus in neurotransmitter release), promoting its ubiquitination and subsequent degradation by the proteasome (Chin et al., 2002). Similarly, the RING-domain proteins Siah-1A and Siah-2 bind the synaptic vesicle protein synaptophysin and facilitate its ubiquitination and degradation (Wheeler et al., 2002).
The fact that ubiquitin regulates levels of synaptic receptors was shown in:
o Xenopus oocytes, where glycine receptors are ubiquitinated, internalized, and cleaved in the lysosome (Buttner et al., 2001);
o C. elegans, where GLR-1 synaptic levels are regulated by ubiquitin (Burbea et al., 2002)(see glutamate reception in Chapter 3.3.2.6);
o vertebrate hippocampal neurons, where GABAA (Bedford et al., 2001) and AMPA receptors (Patrick et al., 2003) surface number and subunit stability are regulated by ubiquitination.
Unlike phosphorylation, the importance of the ubiquitin pathway in regulating the synapse recently started to be investigated. At the moment, only few components of the ubiquitination machinery at the synapse have been described and a handful of substrates identified (see Table 2).
Protein Function Synaptic action
Ubiquitination machinery
bendless E2 ubiquitin conjugase Axon guidance
Nedd4 E3 ubiquitin ligase Axon guidance
fat facets Deubiquitinating enzyme Synapse development
SKR-1, SEL-10 E3 ubiquitin ligase (SCF complex) Synapse development
RPM-1 E3 ubiquitin ligase Synapse development
Usp14 Deubiquitinating enzyme Synaptic transmission
Staring E3 ubiquitin ligase Synaptic transmission
Siah E3 ubiquitin ligase Synaptic transmission
APC/C E3 ubiquitin ligase Synapse development and transmission
Ap-uch Deubiquitinating enzyme Synaptic plasticity
E6-AP E3 ubiquitin ligase Synaptic plasticity
Ubiquitinated substrates
Commissureless Regulates Robo levels Axon guidance
Unc-13 Synaptic vesicle release Synaptic transmission
Syntaxin Synaptic vesicle release Synaptic transmission
Synaptophysin Synaptic vesicle protein Synaptic transmission Glycine, GABA, &
glutamate receptors
Neurotransmitter receptors Synaptic transmission
PKA-R Regulatory subunit of PKA Synaptic plasticity
Shank, GKAP, & AKAP79 Postsynaptic scaffolding proteins Synaptic plasticity
Table 2. Ubiquitination machinery and substrates regulating the synapse (Modified from (DiAntonio and Hicke, 2004).