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The ubiquitin proteasome system in synaptic and axonal degeneration

The ubiquitin proteasome system in synaptic and axonal degeneration JCB Mini-Review The ubiquitin proteasome system in synaptic and axonal degeneration: a new twist to an old cycle 1,2 1,2 Laura Korhonen and Dan Lindholm Department of Neuroscience, Unit of Neurobiology, Biomedical Centre, Uppsala University, S-75123 Uppsala, Sweden Mclean Hospital, Harvard Medical School, Belmont, MA 02478 The ubiquitin proteasome system (UPS) contributes to the that are then degraded by the UPS to small peptides. At the pathophysiology of neurodegenerative diseases, and it is heart of the UPS is the 26S proteasome that consists of a also a major determinant of synaptic protein degradation 20S core particle and two 19S regulatory particles (Fig. 1 A). and activity. Recent studies in rodents and in the fruit fly Apart from ubiquitination, there are deubiquitination en- Drosophila have shown that the activity of the UPS is zymes that replenish the cellular pool of ubiquitin and are involved in axonal degeneration. Increased knowledge of important for the proper function of the proteasome. Modi- the UPS in synaptic and axonal reactions may provide fications of protein substrates through attachment of a novel drug targets for treatments of neuronal injuries and monoubiquitin or polyubiquitin chain are important in neurodegenerative disorders. many cellular processes ranging form cell cycle control, DNA repair, transcription, cell signaling, and regulation of protein trafficking (Hicke, 2001; Weissman, 2001; Adams, Neurodegenerative diseases are characterized by the selective 2003). In the nervous system, ubiquitination plays a role, loss of neurons due to aggregation of different intra- or among others, in neuronal signaling, synapse formation and extracellular proteins (Bence et al., 2001). Apart from the function, and in different diseases (Hegde and DiAntonio, deleterious effects of the protein deposits on the nerve cell 2002; Ciechanover and Brundin, 2003). soma, the axons and dendrites also degenerate in these diseases. It is becoming increasingly evident that altered activities The functional changes observed early in neurodegenerative of the UPS are crucially involved in the pathophysiology of disorders are reflected by alterations in synaptic dysfunctions Parkinson’s disease (PD), Huntington’s disease (HD), and and loss of connectivity. Recent evidence indicates that the in spinocerebellar ataxia (Bence et al., 2001; Lindsten et al., degeneration of axons and synapses plays an important role 2002; Ciechanover and Brundin, 2003). Mutations in parkin, both in chronic diseases and after injuries. The molecular encoding an ubiquitin-E3 ligase result in juvenile recessive mechanisms governing axonal degeneration and synaptic PD (Dawson and Dawson, 2003). -Synuclein, which is maintenance are not fully understood, but a crucial role has mutated in some familiar forms of PD, is highly enriched been ascribed to the activity of the UPS. in presynaptic terminals and Lewy-bodies (Kaplan et al., 2003). Recently, the higher than normal level of wild-type UPS and neurological disorders -synuclein was found in a family with early onset PD, and Ubiquitin is a 76 aa–long protein that becomes attached to a contributing mechanism could be an insufficient clearance other proteins through a multi-enzyme system (Weissman, by the UPS (Eriksen et al., 2003; Singleton et al., 2003). 2001; Adams, 2003). The first step is the activation of Apart from PD, other neurodegenerative disorders also ubiquitin by an enzyme, E1, followed by transfer to an display accumulation of mutated proteins in inclusion bod- ubiquitin conjugating enzyme, E2, and to the ubiquitin ligase, ies and aggregates in conjunction with ubiquitin (Lindsten E3 that covalently attaches the ubiquitin moiety to a lysine et al., 2002; Ciechanover and Brundin, 2003). The presence residue on target proteins (Fig. 1 A). There are several E2 of protein deposits has been amply demonstrated in neu- and many E3 enzymes that are specific for different protein ronal cell bodies, but less is known about their occurrence in substrates that increase the diversity of this system. Reiteration axons and synapses. Recent studies on HD and other PolyQ of the cycle produces polyubiquitin chains on target proteins diseases show that the respective mutated proteins can inter- fere with axonal transport (Gunawardena et al., 2003; Szebenyi et al., 2003). The Huntingtin protein involved in Address correspondence to Dan Lindholm, Dept. of Neuroscience, Unit HD also interacts with synaptic vesicles and proteins involved of Neurobiology, Biomedical Centre, Box 587, Uppsala University, S-75123 Uppsala, Sweden. Tel.: 46-18-471-4435. Fax: 46-18-559-017. email: [email protected] Abbreviations used in this paper: HD, Huntington’s disease; PD, Parkinson’s Key words: neurodegeneration; synapse function; Wallerian degeneration; polyubiquitination; monoubiquitination disease; PSD, postsynaptic density; UPS, ubiquitin proteasome system. © The Rockefeller University Press, 0021-9525/2004/04/27/4 $8.00 The Journal of Cell Biology, Volume 165, Number 1, April 12, 2004 27–30 http://www.jcb.org/cgi/doi/10.1083/jcb.200311091 27 The Journal of Cell Biology 28 The Journal of Cell Biology | Volume 165, Number 1, 2004 the activity of the ubiquitin ligase, Ube3A, is crucial for long-term potentiation in mouse hippocampus, and the gene is mutated in Angelman’s syndrome, a human disorder causing mental retardation (Miura et al., 2002). Together, these studies show that the UPS and protein modifications via ubiquitination play an important role in regulation of synaptic maintenance and function in different organisms. However, the time window and specific proteins regulated by the UPS vary between different studies. In the work on the PSD proteins, a time scale of 24–48 h was used. In this time, the UPS acts on different targets, including ubiquiti- nation of proteins controlling transcription or translation that may indirectly influence protein abundance in the syn- apse. In their study of the neuromuscular system in Drosoph- ila, Speese et al. (2003) noted a relatively short time window for the degradation of synaptic proteins by the UPS. Using drugs to inhibit the activity, in addition to genetic manipu- lation of the proteasome, DUNC-13 was identified as a se- lective target for UPS in the presynaptic terminal. Electro- physiological recordings showed that proteasome inhibition also enhanced synaptic efficacy and presynaptic transmitter release. This data demonstrates that the activity of the UPS locally regulates the levels of DUNC-13 and influences pre- synaptic efficacy in Drosophila. The correlative changes ob- Figure 1. The activity of the UPS and neuronal compartments. served in synaptic function and regulation of the DUNC-13 (A) UPS, ubiquitin proteasome system. U, ubiquitin; E1, ubiquitin by the UPS also suggest that the levels of DUNC-13 may be activating enzyme; E2, ubiquitin conjugating enzyme; E3, ubiquitin ligase; 20S, catalytic core; 19S, regulator particle; PS. Protein substrate. the crucial mediator for increased neurotransmitter release. (B) Neurons consist of three major subcellular compartments However, this functional link has so far not been directly functionally linked to each other: the cell body, axon, and nerve shown neither are other protein excluded as targets for the terminals. In neurodegenerative diseases, there is an accumulation UPS in this context. of mutant or misfolded proteins (circles) due to insufficient clearance Members of the UNC protein family are found in differ- or the relative dysfunction of the UPS. The protein aggregates can ent species, and are involved in synaptic vesicle priming and further disrupt the UPS and affect the axonal transport and the synapses. Molecular insights into axonal reactions show an involve- regulation of neurotransmitter release. It would be impor- ment of the UPS in axonal degeneration. In nerve endings, the UPS tant to study whether the mouse homologue MUNC-1, is crucial for protein turnover and synapse maintenance and function. shown to be important for proper function of glutamatergic The exact role of the UPS in synaptic dysfunction is not known, but synaptic vesicles (Augustin et al., 1999), also undergoes disturbances in its activity may seriously affect protein trafficking UPS-mediated down-regulation. Mice carrying a gene dele- and neuronal connectivity. Protein components of the UPS, including specific E2 and E3 molecules, and their corresponding protein targets tion for Munc-18-1, exhibit loss of neurotransmitter secre- may differ between compartments. This opens up the possibility for tion during development, without affecting the initial as- specific and local interference with the UPS activity under different sembly of the synapse (Verhage et al., 2000). However, after conditions and in neurodegenerative diseases. birth there is a widespread neurodegeneration with the loss of synapses. The contribution of the UPS to this phenotype in neurotransmission (Song et al., 2003). In spinocerebellar has yet not been studied. ataxia 1, there is an altered trafficking of glutamate receptor Protein modification by ubiquitin at nerve endings may subunits and PKC in Purkinje cells (Skinner et al., 2001). go beyond effects on protein degradation. Polyubiquitina- Given the role of the UPS in disease pathophysiology, it is tion targets proteins for degradation, but monoubiquitina- important to study the key proteins in the axons and in the tion regulates protein trafficking, involving endosomes, as synapses that may be altered in the different disorders. well as other functions (Hicke, 2001). Receptor tyrosine ki- nases, such as the EGF receptor are monoubiquitinated UPS and the function of synapses upon ligand binding (Haglund et al., 2003; Mosesson et al., Synapses undergo large structural changes during matura- 2003). This leads to receptor tyrosine kinase internalization tion and in response to neuronal activity. As shown by and degradation in lysosomes, preventing recycling to the Ehlers (2003), the UPS regulates the degradation of mole- plasma membrane. Monoubiquitination of receptor proteins cules, belonging to the postsynaptic densities (PSDs) that can thus exert an important control step in the action of contain various receptors and scaffolding proteins. Synaptic trophic factors. Although not yet studied in brain, this may activity influences the ubiquitination and turnover of a sub- also occur for neurotransmitter receptors, and other mole- set of PSD proteins, important for the control of synapse cules at the synapse. It is crucial to study whether alterations function and maintenance (Ehlers, 2003). Results with Aply- in monoubiquitination per se can affect protein trafficking sia have shown that protein ubiquitination is important for and synaptic connectivity. Wilson et al. (2002) reported re- synaptic plasticity (Hegde and DiAntonio, 2002). Likewise, cently that mutation in Usp14, encoding an ubiquitin-spe- Protein ubiquitination in nerve degeneration | Korhonen and Lindholm 29 cific protease, causes synaptic dysfunction leading to ataxia velopment of particular projecting neurons of the Drosophila in mice. mushroom bodies it was shown that overexpression of an In recent years, novel proteins have been discovered, such ubiquitin protease or a mutant form of the Drosophila E1 as the ubiquitin-like proteins and those carrying ubiquitin ubiquitin-activating enzyme, inhibited pruning (Watts et interacting domains that influence the efficacy of the UPS al., 2003). Likewise, mutations in two of the subunits in the (Weissman, 2001; Adams, 2003). So far little is known 19S regulatory particle (Fig. 1 A) also impaired pruning. about these proteins in the nervous system or locally in the This genetic evidence strongly suggests that local degenera- function of the synapse. tion of protein via the UPS activity is a necessary require- ment for axon pruning during development of the mush- Axonal degeneration and the UPS room bodies. The relevance of these findings for Wallerian After transection, the distal segment of the nerve normally degeneration and axonal reactions in neurodegenerative dis- undergoes a degeneration process with typical morpho- eases is not straightforward. However, there are similarities logical signs, called Wallerian degeneration (Coleman and between these processes both with regard to molecular Perry, 2002; Raff et al., 2002). This type of degeneration oc- mechanisms and the cellular reactions involved. First, study- curs also in many neuropathies and neurodegenerative disor- ing different fly mutants, it was shown that axon pruning is ders and is distinct from the death of the nerve cell body. independent of the activity of grim, hid, and rpr that regulate Studies of the mouse mutant, Wld that displays a signifi- cell death in Drosophila. This is in keeping with results ob- cantly slower Wallerian reaction, demonstrate that axonal tained in rodents in the Wld mice (Finn et al., 2000; Raff et degeneration is an active process (Coleman et al., 1998; al., 2002), and shows that the programs governing axonal Mack et al., 2001). In this mouse, gene rearrangements have degeneration and death of the nerve cell body via apoptosis resulted in the production of an 85-kD chimeric protein are inherently different. Second, local UPS activity seems in- consisting of nicotinamide mononucleotide adenylyl trans- strumental for both pruning and Wallerian degeneration to ferase and the amino-terminal portion of the ubiquitination occur. Third, looking at the cellular changes, the disruption factor E4b (Mack et al., 2001). Overexpression of the fusion of the microtubuli was identified as an early step in both protein, using the -actin promoter or viral vectors, can de- pruning and Wallerian degeneration. Thus, it is tempting to lay axonal degeneration (Coleman and Perry, 2002). The suggest that the two processes may be governed by similar data with the Wld mice suggests an involvement of the UPS mechanisms involving local regulation of axonal proteins via in Wallerian degeneration, but there are some caveats. Thus, the UPS. the cellular targets of the Wld chimeric gene in axonal de- The results on inhibition of the UPS in axons, also suggest generation are not known. It has also to be shown that the therapeutic targets to preserve axons after injury and in de- fusion protein with the truncated region of the E4 enzyme generation. The caveat is that a general inhibition of protea- can function in polyubiquitination and proteasome-medi- some activity may be harmful to the cell. To arrive at a spe- ated degradation of proteins. cific inhibition of the UPS, the exact nature of the different Supporting evidence for the involvement of the UPS in molecules involved in ubiquitination and degradation of axonal degeneration comes from studies of the Gracile ax- protein targets in the axons needs to be studied in more de- onal dystrophy (Gad) mutant mice. In these mice, there is tail. The first attempt toward this was made by Watts et al. an inactivation of the ubiquitin carboxy-terminal hydrolase, (2003) in Drosophila. However, of the over a dozen of E2 UCH-L1, causing, among others, degeneration of the gracile and E3 enzymes studied, none was found important for tract of the spinal cord (Saigoh et al., 1999). UCH-1 dis- axon pruning. This does not come as a surprise in view of plays dual activities as a deubiquitination enzyme and as an the fact that in Drosophila, as in mammalian cells the diver- ubiquitin ligase (Liu et al., 2002). The exact roles of UCH-1 sity of these enzymes and their corresponding protein sub- and the corresponding protein substrates in the Gad mice strates is large. However, the use of modern large-scale tech- are so far not known. niques for proteome analysis may provide new insights into Recent studies in rats using transection of cultured neurons these important questions. or crush lesions of the optic nerve in vivo showed that inhibi- Concluding remarks tion of the UPS by drugs targeting the proteasome can retard the onset of Wallerian degeneration (Zhai et al., 2003). Simi- Synaptic dysfunction and decreased connectivity herald lar results were obtained by viral expression of an ubiquitin many of the neurodegenerative diseases. The UPS play an protease that can reverse ubiquitination of protein substrates. important role in synaptic function and may contribute to Using specific antibodies for axonal components, the frag- the disease-induced changes. The activity of the UPS is in- mentation of microtubuli was identified as an early sign of volved in local axonal degeneration after nerve injury. The the axonal degeneration that was also sensitive to proteasome mechanisms controlling axonal degeneration are different inhibition (Zhai et al., 2003). These results lend credence to from those regulating death of the nerve cell body. One the view that the local activity of UPS, acting on selective could then envision a two-stage approach in neurodegenera- protein targets, is important during axonal degeneration. tion with treatments of the axon and the soma separately. Further evidence comes from studies in Drosophila, show- However, with regard to such therapies and the effects of re- ing that the degeneration of axons occurring during pruning ducing UPS activity, there is a Scylla and Charybis situation requires the UPS (Watts et al., 2003). Axon pruning is an for these two compartments. Inhibition of local UPS activity important process for the refinement of the neuronal con- is beneficial for retarding axonal degeneration. However, a nections in both vertebrates and invertebrates. Studying de- drawback is that there is already a relative inhibition of the 30 The Journal of Cell Biology | Volume 165, Number 1, 2004 Nat. Rev. Neu- Hegde, A.N., and A. DiAntonio. 2002. Ubiquitin and the synapse. UPS in the neurological disorders, caused by protein aggre- rosci. 3:854–861. gates and toxic products in the cell (Fig. 1 B). A further inhi- Nat. Rev. Mol. Cell Biol. Hicke, L. 2001. Protein regulation by monoubiquitin. bition of the UPS may thus aggravate the underlying disease. 2:195–201. Although one could foresee a local delivery of UPS inhibi- Kaplan, B., V. Ratner, and E. Haas. 2003. Alpha-synuclein: its biological function tors, this may prove cumbersome. Many of the compounds and role in neurodegenerative diseases. J. Mol. Neurosci. 20:83–92. Lindsten, K., F.M. de Vrij, L.G. Verhoef, D.F. Fischer, F. W. van Leeuwen, E.M. used to date are also rather toxic and show unspecific effects. Hol, M.G. Masucci, and N.P. Dantuma. 2002. Mutant ubiquitin found in To circumvent these problems, we need to know more neurodegenerative disorders is a ubiquitin fusion degradation substrate that about the occurrence and the nature of molecules, including blocks proteasomal degradation. J. Cell Biol. 157:417–427. specific E2 and E3 enzymes and the corresponding protein Liu, Y., L. Fallon, H.A. Lashuel, Z. Liu, and P.T. Lansbury, Jr. 2002. The UCH- L1 gene encodes two opposing enzymatic activities that affect -synuclein substrates in the neuron. Such information may allow de- Cell. 111:209–218. degradation and Parkinson’s disease susceptibility. signing novel drug targets to influence the UPS separately in Mack, T.G., M. Reiner, B. Beirowski, W. Mi, M. Emanuelli, D. Wagner, D. the axon and the nerve cell body in different diseases. Given Thomson, T. Gillingwater, F. Court, L. Conforti, et al. 2001. Wallerian de- the current interest in the UPS, increased knowledge on generation of injured axons and synapses is delayed by a Ube4b/Nmnat chi- these matters is likely to occur rapidly. The door to new dis- Nat. Neurosci. 4:1199–1206. meric gene. Mosesson, Y., K. Shtiegman, M. Katz, Y. Zwang, G. Vereb, J. Szollosi, and Y. coveries in this field is now more than ajar. Yarden. 2003. Endocytosis of receptor tyrosine kinases is driven by monou- biquitylation, not polyubiquitylation. J. Biol. Chem. 278:21323–21326. L. Korhonen received a grant from Swedish Brain Foundation. D. Lindholm Miura, K., T. Kishino, E. Li, H. Webber, P. Dikkes, G.L. Holmes, and J. Wag- was supported by Uppsala University and Swedish Cancer Foundation. staff. 2002. Neurobehavioral and electroencephalographic abnormalities in Neurobiol. Dis. 9:149–159. Ube3a maternal-deficient mice. Submitted: 18 November 2003 Raff, M.C., A.V. Whitmore, and J.T. Finn. 2002. Axonal self-destruction and neu- Accepted: 26 February 2004 Science. 296:868–871. rodegeneration. Saigoh, K., Y.L. Wang, J.G. Suh, T. Yamanishi, Y. Sakai, H. Kiyosawa, T. Harada, N. Ichihara, S. Wakana, T. Kikuchi, and K. Wada. 1999. Intragenic dele- References tion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice. Adams, J. 2003. The proteasome: structure, function, and role in the cell. Cancer Nat. 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The ubiquitin proteasome system in synaptic and axonal degeneration

Korhonen, Laura; Lindholm, Dan
The Journal of Cell Biology , Volume 165 (1) – Apr 12, 2004
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Copyright © 2004, The Rockefeller University Press
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Abstract

JCB Mini-Review The ubiquitin proteasome system in synaptic and axonal degeneration: a new twist to an old cycle 1,2 1,2 Laura Korhonen and Dan Lindholm Department of Neuroscience, Unit of Neurobiology, Biomedical Centre, Uppsala University, S-75123 Uppsala, Sweden Mclean Hospital, Harvard Medical School, Belmont, MA 02478 The ubiquitin proteasome system (UPS) contributes to the that are then degraded by the UPS to small peptides. At the pathophysiology of neurodegenerative diseases, and it is heart of the UPS is the 26S proteasome that consists of a also a major determinant of synaptic protein degradation 20S core particle and two 19S regulatory particles (Fig. 1 A). and activity. Recent studies in rodents and in the fruit fly Apart from ubiquitination, there are deubiquitination en- Drosophila have shown that the activity of the UPS is zymes that replenish the cellular pool of ubiquitin and are involved in axonal degeneration. Increased knowledge of important for the proper function of the proteasome. Modi- the UPS in synaptic and axonal reactions may provide fications of protein substrates through attachment of a novel drug targets for treatments of neuronal injuries and monoubiquitin or polyubiquitin chain are important in neurodegenerative disorders. many cellular processes ranging form cell cycle control, DNA repair, transcription, cell signaling, and regulation of protein trafficking (Hicke, 2001; Weissman, 2001; Adams, Neurodegenerative diseases are characterized by the selective 2003). In the nervous system, ubiquitination plays a role, loss of neurons due to aggregation of different intra- or among others, in neuronal signaling, synapse formation and extracellular proteins (Bence et al., 2001). Apart from the function, and in different diseases (Hegde and DiAntonio, deleterious effects of the protein deposits on the nerve cell 2002; Ciechanover and Brundin, 2003). soma, the axons and dendrites also degenerate in these diseases. It is becoming increasingly evident that altered activities The functional changes observed early in neurodegenerative of the UPS are crucially involved in the pathophysiology of disorders are reflected by alterations in synaptic dysfunctions Parkinson’s disease (PD), Huntington’s disease (HD), and and loss of connectivity. Recent evidence indicates that the in spinocerebellar ataxia (Bence et al., 2001; Lindsten et al., degeneration of axons and synapses plays an important role 2002; Ciechanover and Brundin, 2003). Mutations in parkin, both in chronic diseases and after injuries. The molecular encoding an ubiquitin-E3 ligase result in juvenile recessive mechanisms governing axonal degeneration and synaptic PD (Dawson and Dawson, 2003). -Synuclein, which is maintenance are not fully understood, but a crucial role has mutated in some familiar forms of PD, is highly enriched been ascribed to the activity of the UPS. in presynaptic terminals and Lewy-bodies (Kaplan et al., 2003). Recently, the higher than normal level of wild-type UPS and neurological disorders -synuclein was found in a family with early onset PD, and Ubiquitin is a 76 aa–long protein that becomes attached to a contributing mechanism could be an insufficient clearance other proteins through a multi-enzyme system (Weissman, by the UPS (Eriksen et al., 2003; Singleton et al., 2003). 2001; Adams, 2003). The first step is the activation of Apart from PD, other neurodegenerative disorders also ubiquitin by an enzyme, E1, followed by transfer to an display accumulation of mutated proteins in inclusion bod- ubiquitin conjugating enzyme, E2, and to the ubiquitin ligase, ies and aggregates in conjunction with ubiquitin (Lindsten E3 that covalently attaches the ubiquitin moiety to a lysine et al., 2002; Ciechanover and Brundin, 2003). The presence residue on target proteins (Fig. 1 A). There are several E2 of protein deposits has been amply demonstrated in neu- and many E3 enzymes that are specific for different protein ronal cell bodies, but less is known about their occurrence in substrates that increase the diversity of this system. Reiteration axons and synapses. Recent studies on HD and other PolyQ of the cycle produces polyubiquitin chains on target proteins diseases show that the respective mutated proteins can inter- fere with axonal transport (Gunawardena et al., 2003; Szebenyi et al., 2003). The Huntingtin protein involved in Address correspondence to Dan Lindholm, Dept. of Neuroscience, Unit HD also interacts with synaptic vesicles and proteins involved of Neurobiology, Biomedical Centre, Box 587, Uppsala University, S-75123 Uppsala, Sweden. Tel.: 46-18-471-4435. Fax: 46-18-559-017. email: [email protected] Abbreviations used in this paper: HD, Huntington’s disease; PD, Parkinson’s Key words: neurodegeneration; synapse function; Wallerian degeneration; polyubiquitination; monoubiquitination disease; PSD, postsynaptic density; UPS, ubiquitin proteasome system. © The Rockefeller University Press, 0021-9525/2004/04/27/4 $8.00 The Journal of Cell Biology, Volume 165, Number 1, April 12, 2004 27–30 http://www.jcb.org/cgi/doi/10.1083/jcb.200311091 27 The Journal of Cell Biology 28 The Journal of Cell Biology | Volume 165, Number 1, 2004 the activity of the ubiquitin ligase, Ube3A, is crucial for long-term potentiation in mouse hippocampus, and the gene is mutated in Angelman’s syndrome, a human disorder causing mental retardation (Miura et al., 2002). Together, these studies show that the UPS and protein modifications via ubiquitination play an important role in regulation of synaptic maintenance and function in different organisms. However, the time window and specific proteins regulated by the UPS vary between different studies. In the work on the PSD proteins, a time scale of 24–48 h was used. In this time, the UPS acts on different targets, including ubiquiti- nation of proteins controlling transcription or translation that may indirectly influence protein abundance in the syn- apse. In their study of the neuromuscular system in Drosoph- ila, Speese et al. (2003) noted a relatively short time window for the degradation of synaptic proteins by the UPS. Using drugs to inhibit the activity, in addition to genetic manipu- lation of the proteasome, DUNC-13 was identified as a se- lective target for UPS in the presynaptic terminal. Electro- physiological recordings showed that proteasome inhibition also enhanced synaptic efficacy and presynaptic transmitter release. This data demonstrates that the activity of the UPS locally regulates the levels of DUNC-13 and influences pre- synaptic efficacy in Drosophila. The correlative changes ob- Figure 1. The activity of the UPS and neuronal compartments. served in synaptic function and regulation of the DUNC-13 (A) UPS, ubiquitin proteasome system. U, ubiquitin; E1, ubiquitin by the UPS also suggest that the levels of DUNC-13 may be activating enzyme; E2, ubiquitin conjugating enzyme; E3, ubiquitin ligase; 20S, catalytic core; 19S, regulator particle; PS. Protein substrate. the crucial mediator for increased neurotransmitter release. (B) Neurons consist of three major subcellular compartments However, this functional link has so far not been directly functionally linked to each other: the cell body, axon, and nerve shown neither are other protein excluded as targets for the terminals. In neurodegenerative diseases, there is an accumulation UPS in this context. of mutant or misfolded proteins (circles) due to insufficient clearance Members of the UNC protein family are found in differ- or the relative dysfunction of the UPS. The protein aggregates can ent species, and are involved in synaptic vesicle priming and further disrupt the UPS and affect the axonal transport and the synapses. Molecular insights into axonal reactions show an involve- regulation of neurotransmitter release. It would be impor- ment of the UPS in axonal degeneration. In nerve endings, the UPS tant to study whether the mouse homologue MUNC-1, is crucial for protein turnover and synapse maintenance and function. shown to be important for proper function of glutamatergic The exact role of the UPS in synaptic dysfunction is not known, but synaptic vesicles (Augustin et al., 1999), also undergoes disturbances in its activity may seriously affect protein trafficking UPS-mediated down-regulation. Mice carrying a gene dele- and neuronal connectivity. Protein components of the UPS, including specific E2 and E3 molecules, and their corresponding protein targets tion for Munc-18-1, exhibit loss of neurotransmitter secre- may differ between compartments. This opens up the possibility for tion during development, without affecting the initial as- specific and local interference with the UPS activity under different sembly of the synapse (Verhage et al., 2000). However, after conditions and in neurodegenerative diseases. birth there is a widespread neurodegeneration with the loss of synapses. The contribution of the UPS to this phenotype in neurotransmission (Song et al., 2003). In spinocerebellar has yet not been studied. ataxia 1, there is an altered trafficking of glutamate receptor Protein modification by ubiquitin at nerve endings may subunits and PKC in Purkinje cells (Skinner et al., 2001). go beyond effects on protein degradation. Polyubiquitina- Given the role of the UPS in disease pathophysiology, it is tion targets proteins for degradation, but monoubiquitina- important to study the key proteins in the axons and in the tion regulates protein trafficking, involving endosomes, as synapses that may be altered in the different disorders. well as other functions (Hicke, 2001). Receptor tyrosine ki- nases, such as the EGF receptor are monoubiquitinated UPS and the function of synapses upon ligand binding (Haglund et al., 2003; Mosesson et al., Synapses undergo large structural changes during matura- 2003). This leads to receptor tyrosine kinase internalization tion and in response to neuronal activity. As shown by and degradation in lysosomes, preventing recycling to the Ehlers (2003), the UPS regulates the degradation of mole- plasma membrane. Monoubiquitination of receptor proteins cules, belonging to the postsynaptic densities (PSDs) that can thus exert an important control step in the action of contain various receptors and scaffolding proteins. Synaptic trophic factors. Although not yet studied in brain, this may activity influences the ubiquitination and turnover of a sub- also occur for neurotransmitter receptors, and other mole- set of PSD proteins, important for the control of synapse cules at the synapse. It is crucial to study whether alterations function and maintenance (Ehlers, 2003). Results with Aply- in monoubiquitination per se can affect protein trafficking sia have shown that protein ubiquitination is important for and synaptic connectivity. Wilson et al. (2002) reported re- synaptic plasticity (Hegde and DiAntonio, 2002). Likewise, cently that mutation in Usp14, encoding an ubiquitin-spe- Protein ubiquitination in nerve degeneration | Korhonen and Lindholm 29 cific protease, causes synaptic dysfunction leading to ataxia velopment of particular projecting neurons of the Drosophila in mice. mushroom bodies it was shown that overexpression of an In recent years, novel proteins have been discovered, such ubiquitin protease or a mutant form of the Drosophila E1 as the ubiquitin-like proteins and those carrying ubiquitin ubiquitin-activating enzyme, inhibited pruning (Watts et interacting domains that influence the efficacy of the UPS al., 2003). Likewise, mutations in two of the subunits in the (Weissman, 2001; Adams, 2003). So far little is known 19S regulatory particle (Fig. 1 A) also impaired pruning. about these proteins in the nervous system or locally in the This genetic evidence strongly suggests that local degenera- function of the synapse. tion of protein via the UPS activity is a necessary require- ment for axon pruning during development of the mush- Axonal degeneration and the UPS room bodies. The relevance of these findings for Wallerian After transection, the distal segment of the nerve normally degeneration and axonal reactions in neurodegenerative dis- undergoes a degeneration process with typical morpho- eases is not straightforward. However, there are similarities logical signs, called Wallerian degeneration (Coleman and between these processes both with regard to molecular Perry, 2002; Raff et al., 2002). This type of degeneration oc- mechanisms and the cellular reactions involved. First, study- curs also in many neuropathies and neurodegenerative disor- ing different fly mutants, it was shown that axon pruning is ders and is distinct from the death of the nerve cell body. independent of the activity of grim, hid, and rpr that regulate Studies of the mouse mutant, Wld that displays a signifi- cell death in Drosophila. This is in keeping with results ob- cantly slower Wallerian reaction, demonstrate that axonal tained in rodents in the Wld mice (Finn et al., 2000; Raff et degeneration is an active process (Coleman et al., 1998; al., 2002), and shows that the programs governing axonal Mack et al., 2001). In this mouse, gene rearrangements have degeneration and death of the nerve cell body via apoptosis resulted in the production of an 85-kD chimeric protein are inherently different. Second, local UPS activity seems in- consisting of nicotinamide mononucleotide adenylyl trans- strumental for both pruning and Wallerian degeneration to ferase and the amino-terminal portion of the ubiquitination occur. Third, looking at the cellular changes, the disruption factor E4b (Mack et al., 2001). Overexpression of the fusion of the microtubuli was identified as an early step in both protein, using the -actin promoter or viral vectors, can de- pruning and Wallerian degeneration. Thus, it is tempting to lay axonal degeneration (Coleman and Perry, 2002). The suggest that the two processes may be governed by similar data with the Wld mice suggests an involvement of the UPS mechanisms involving local regulation of axonal proteins via in Wallerian degeneration, but there are some caveats. Thus, the UPS. the cellular targets of the Wld chimeric gene in axonal de- The results on inhibition of the UPS in axons, also suggest generation are not known. It has also to be shown that the therapeutic targets to preserve axons after injury and in de- fusion protein with the truncated region of the E4 enzyme generation. The caveat is that a general inhibition of protea- can function in polyubiquitination and proteasome-medi- some activity may be harmful to the cell. To arrive at a spe- ated degradation of proteins. cific inhibition of the UPS, the exact nature of the different Supporting evidence for the involvement of the UPS in molecules involved in ubiquitination and degradation of axonal degeneration comes from studies of the Gracile ax- protein targets in the axons needs to be studied in more de- onal dystrophy (Gad) mutant mice. In these mice, there is tail. The first attempt toward this was made by Watts et al. an inactivation of the ubiquitin carboxy-terminal hydrolase, (2003) in Drosophila. However, of the over a dozen of E2 UCH-L1, causing, among others, degeneration of the gracile and E3 enzymes studied, none was found important for tract of the spinal cord (Saigoh et al., 1999). UCH-1 dis- axon pruning. This does not come as a surprise in view of plays dual activities as a deubiquitination enzyme and as an the fact that in Drosophila, as in mammalian cells the diver- ubiquitin ligase (Liu et al., 2002). The exact roles of UCH-1 sity of these enzymes and their corresponding protein sub- and the corresponding protein substrates in the Gad mice strates is large. However, the use of modern large-scale tech- are so far not known. niques for proteome analysis may provide new insights into Recent studies in rats using transection of cultured neurons these important questions. or crush lesions of the optic nerve in vivo showed that inhibi- Concluding remarks tion of the UPS by drugs targeting the proteasome can retard the onset of Wallerian degeneration (Zhai et al., 2003). Simi- Synaptic dysfunction and decreased connectivity herald lar results were obtained by viral expression of an ubiquitin many of the neurodegenerative diseases. The UPS play an protease that can reverse ubiquitination of protein substrates. important role in synaptic function and may contribute to Using specific antibodies for axonal components, the frag- the disease-induced changes. The activity of the UPS is in- mentation of microtubuli was identified as an early sign of volved in local axonal degeneration after nerve injury. The the axonal degeneration that was also sensitive to proteasome mechanisms controlling axonal degeneration are different inhibition (Zhai et al., 2003). These results lend credence to from those regulating death of the nerve cell body. One the view that the local activity of UPS, acting on selective could then envision a two-stage approach in neurodegenera- protein targets, is important during axonal degeneration. tion with treatments of the axon and the soma separately. Further evidence comes from studies in Drosophila, show- However, with regard to such therapies and the effects of re- ing that the degeneration of axons occurring during pruning ducing UPS activity, there is a Scylla and Charybis situation requires the UPS (Watts et al., 2003). Axon pruning is an for these two compartments. Inhibition of local UPS activity important process for the refinement of the neuronal con- is beneficial for retarding axonal degeneration. However, a nections in both vertebrates and invertebrates. Studying de- drawback is that there is already a relative inhibition of the 30 The Journal of Cell Biology | Volume 165, Number 1, 2004 Nat. Rev. Neu- Hegde, A.N., and A. DiAntonio. 2002. Ubiquitin and the synapse. UPS in the neurological disorders, caused by protein aggre- rosci. 3:854–861. gates and toxic products in the cell (Fig. 1 B). A further inhi- Nat. Rev. Mol. Cell Biol. Hicke, L. 2001. Protein regulation by monoubiquitin. bition of the UPS may thus aggravate the underlying disease. 2:195–201. Although one could foresee a local delivery of UPS inhibi- Kaplan, B., V. Ratner, and E. Haas. 2003. Alpha-synuclein: its biological function tors, this may prove cumbersome. Many of the compounds and role in neurodegenerative diseases. J. Mol. Neurosci. 20:83–92. Lindsten, K., F.M. de Vrij, L.G. Verhoef, D.F. Fischer, F. W. van Leeuwen, E.M. used to date are also rather toxic and show unspecific effects. Hol, M.G. Masucci, and N.P. Dantuma. 2002. Mutant ubiquitin found in To circumvent these problems, we need to know more neurodegenerative disorders is a ubiquitin fusion degradation substrate that about the occurrence and the nature of molecules, including blocks proteasomal degradation. J. 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Journal

The Journal of Cell Biology – Pubmed Central

Published: Apr 12, 2004

References