Role of the ubiquitin-proteasome system in nervous system function and disease: using C. elegans as a dissecting tool

Abstract

In addition to its central roles in protein quality control, regulation of cell cycle, intracellular signaling, DNA damage response and transcription regulation, the ubiquitin-proteasome system (UPS) plays specific roles in the nervous system, where it contributes to precise connectivity through development, and later assures functionality by regulating a wide spectrum of neuron-specific cellular processes. Aberrations in this system have been implicated in the etiology of neurodevelopmental and neurodegenerative diseases. In this review, we provide an updated view on the UPS and highlight recent findings concerning its role in normal and diseased nervous systems. We discuss the advantages of the model organism Caenorhabditis elegans as a tool to unravel the major unsolved questions concerning this biochemical pathway and its involvement in nervous system function and dysfunction, and expose the new possibilities, using state-of-the-art techniques, to assess UPS function using this model system.

Fig. 1

Comparison of the UPS between humans and in C. elegans. E1s for both species have already been identified and characterized. C. elegans E2s have been identified based on the criteria of having a UBC motif and the catalytic cysteine residue that accepts activated ubiquitin, and their function has been determined recurring to RNAi studies; human E2s have been identified through sequence homology using E2 protein sequences from C. elegans and Arabidopsis thaliana as an initial set. Predictions for E3s number in humans and C. elegans have been made based in a genome-wide screen based on the presence of specific catalytic domains. One E4 has been described and characterized up to now in each species, although it is possible that many more exist

Fig. 2

Major contributions of C. elegans to the current understanding of the role of the UPS in the synapse. a RPM-1, a RING finger domain protein, regulates pre-synaptic differerentiation by interacting with GLO-4 (Rab GEF), that promotes vesicule-specific membrane proteins trafficking through GLO-1 and AP-3; this pathway is involved in the regulation of axon termination and synaptogenesis. b RPM-1 co-ordinates axon outgrowth by negatively regulating SAX-3 and UNC-5 guidance receptors; this is achieved by controlling vesicular trafficking via the GLO-4 pathway. c RPM-1 contributes to regulate pre-synaptic architecture by forming, with FSN-1 and SKR-1, an SCF complex ubiquitin ligase that leads to DLK-1 ubiquitination and consequent downregulation, thus inhibiting the MAP kinase cascade that includes MKK-4 and PMK-3, known to be involved in synaptic organization. d The transmembrane adhesive molecule SYG-1 determines synaptic sites by preventing SCF^SEL−10 ubiquitin ligase complex assembly and synapse elimination, through its interaction with SKR-1. e RPM-1 and FSN-1, working as an E3, negatively regulate DLK-1, and consequently the p38MAPK pathway thus leading to GLR-1 accumulation in neurites. f The deubiquitylase USP-46 cleaves ubiquitin from GLR-1 receptors, thus promoting GLR-1 surface membrane expression and preventing its degradation in the lysosome. g Post-synaptic ubiquitylation of GLR-1, in a mechanism that requires KEL-8, negatively regulates its levels in the membrane by promoting internalization and posterior degradation by the lysosome. h APC E3 ligase promotes loss of GLR-1 containing synapses possibly through the ubiquitination of a scaffold protein associated with GLR-1. Red arrows represent mechanisms/pathways still not fully elucidated

Fig. 3

Examples of in vivo degradation assays to characterize UPS activity in C. elegans. a In an effort to identify the role of polyQ-containing aggregates in the pathogenesis of neurodegenerative diseases, a construct containing ubiquitin fused with the fluorescent reporter Discosoma Red fluorescent protein (ds-Red) was expressed in C. elegans GABAergic neurons (unc-47 promoter). L1 larvae animals with GABAergic neurons expressing ataxin-3 with 19 glutamine repeats showed no accumulation of aggregates or red fluorescence. Animals expressing ataxin-3 with 127 glutamine repeats and that form aggregates showed red staining corresponding to accumulation of the ubiquitin fusion reporter, indicating UPS impairment. b A ubiquitin fusion degradation substrate was generated by the ligation of GFP to a ubiquitin hidrolase-insensitive (non-cleavable) ubiquitin form (UbV). This construct was stably integrated in the genome of unc-119 (ed4) worms and expressed under the control of the sur-5 promoter in most C. elegans tissues. Mild protein-folding stress using DTT 3 mM or 1% ethanol resulted in UbV-GFP stabilization indicating UPS impairment, whereas acute heat stress—worms grown to day 1 of adulthood and subjected to 37°C for 2 h—enhanced UbV-GFP turnover. RNAi for subunits of the 19S regulatory particles showed different UPS response in different tissues. As an example, rpn-8 subunit depletion resulted in the stabilization of the substrate in the pharynx, vulva, tail, and intestine, whereas rpn-10 depletion resulted in the UPS impairment specifically in the intestine and vulva and not in pharynx or tail. These results show that different types of stress affect the UPS differently and also indicate the tissue-specific requirement of proteasome subunits, besides showing the usefulness of this technique for high-throughput screening approaches. c To understand how the UPS degradation machinery works in different cell types and in a specific time window, a system was developed in which a non-cleavable form of ubiquitin—UbG76V—was fused to the green fluorescent protein Dendra2. Dendra2 can be irreversibly photoconverted from green to red if 405 nm light is used. Making use of specific promoters, this reporter system can be expressed in virtually any cell type and after photoconvertion allows the assessment of UPS activity at the chosen time window