A Link between Aβ and the Ubiquitin-Proteasome System
The paper by Song et al. is a surprising study on the connection between amyloid-β and proteasome inhibition through E2-25K/Hip2 (a ubiquitin-conjugating enzyme) and frameshift ubiquitin (Van Leeuwen et al., 1998). Of course, this study raises a number of questions. For instance, how does Aβ1-42 induce E2-25K/Hip2 activation and how specific is the effect when scrambled controls are used? Furthermore, it will be of interest to study the influence of E2-25K/Hip2 on ubiquitination of Lys29 in UBB+1protein (Lindsten et al., 2002). In my opinion, the translation of the results towards neuropathology in Alzheimer’s disease could have been better. Data from controls (“normal”), whose age is not mentioned, would be much more informative if young and aged (without and with neuropathology, respectively) nondemented controls had been included. Furthermore, the resolution of these...  Read more

A Link between Aβ and the Ubiquitin-Proteasome System
The paper by Song et al. is a surprising study on the connection between amyloid-β and proteasome inhibition through E2-25K/Hip2 (a ubiquitin-conjugating enzyme) and frameshift ubiquitin (Van Leeuwen et al., 1998). Of course, this study raises a number of questions. For instance, how does Aβ1-42 induce E2-25K/Hip2 activation and how specific is the effect when scrambled controls are used? Furthermore, it will be of interest to study the influence of E2-25K/Hip2 on ubiquitination of Lys29 in UBB+1protein (Lindsten et al., 2002). In my opinion, the translation of the results towards neuropathology in Alzheimer’s disease could have been better. Data from controls (“normal”), whose age is not mentioned, would be much more informative if young and aged (without and with neuropathology, respectively) nondemented controls had been included. Furthermore, the resolution of these photographs is such that the localization of anti-E2-25K/Hip2 is unclear: It may be present in dystrophic neurites. If it is co-localized with Aβ in the plaque, one might wonder what is the interpretation of its co-localization with UBB+1 in cerebral cortical cells (of which area?). The latter is important, as Aβ and UBB+1 protein are present in different cellular compartments. Moreover, it is unclear why the neurofibrillary tangle, a major hallmark for Alzheimer’s disease, is not discussed in the paper.

Nevertheless, this is an original contribution which will help to understand conformational diseases such as Alzheimer’s. Congratulations are, therefore, in order for the authors and further contributions from this group are awaited eagerly.

View all comments by Fred van Leeuwen

Ubiquitin and Synaptic Dysfunction: Ataxic Mice Highlight New Common Themes in Neurologic Disease

The gene responsible for the neurologic symptoms in ataxia mice has been identified and shown to encode a ubiquitin-specific protease. This new study reveals new linkages among ubiquitination, synapse function, and neurologic disease.

A final commonality in most neurodegenerative diseases including Alzheimer's disease (AD) is loss of neurons in critical brain areas. However, symptoms of illness often precede detectable neuronal loss, and many neurologic syndromes proceed without appreciable cell death or loss of nervous tissue. In such cases, the underlying deficit is believed to be in how the nerve cells themselves function and communicate at synapses. Indeed, aspects of cognitive impairment in both AD and age-related memory decline have been attributed to synaptic dysfunction.^1-10 Yet, uncovering the links among disease genes or risk factors, synapse dysfunction, and pathologic features of neurologic disease remains a formidable challenge. Now, a...  Read more

Ubiquitin and Synaptic Dysfunction: Ataxic Mice Highlight New Common Themes in Neurologic Disease

The gene responsible for the neurologic symptoms in ataxia mice has been identified and shown to encode a ubiquitin-specific protease. This new study reveals new linkages among ubiquitination, synapse function, and neurologic disease.

A final commonality in most neurodegenerative diseases including Alzheimer's disease (AD) is loss of neurons in critical brain areas. However, symptoms of illness often precede detectable neuronal loss, and many neurologic syndromes proceed without appreciable cell death or loss of nervous tissue. In such cases, the underlying deficit is believed to be in how the nerve cells themselves function and communicate at synapses. Indeed, aspects of cognitive impairment in both AD and age-related memory decline have been attributed to synaptic dysfunction.^1-10 Yet, uncovering the links among disease genes or risk factors, synapse dysfunction, and pathologic features of neurologic disease remains a formidable challenge. Now, a recent paper in Nature Genetics from the laboratory of Nancy Jenkins reports that the neurologic phenotype of ataxia mutant mice is due to loss of a ubiquitin-specific protease which in turn produces profound synaptic dysfunction.¹¹

Jenkins and colleagues set their sights on identifying the genetic lesion responsible for the severe tremors and hindlimb paralysis in ataxia mutant mice, a recessive mutation that arose spontaneously and was first described almost 40 years ago.¹² Through the laborious process of positional cloning, they mapped the mutation within mouse chromosome 18 to a location near Usp14, a gene which encodes ubiquitin-specific protease 14. Consistent with a loss-of-function mutation, expression of Usp14 mRNA was reduced and altered, and Usp14 protein was completely absent from ataxia mutant mice, due to a large insertion in the Usp14 gene. Interestingly, despite a predominantly neurological phenotype, Usp 14 is expressed in many tissues including brain, raising the possibility that these mice have additional, yet unapparent or subtle, defects in other tissues.

So what exactly is Usp14? Usp14 exists as a member of a large family of enzymes dedicated to clipping off ubiquitin (Ub) from proteins to which this small 76 amino acid polypeptide has been attached.¹³ Covalent modification of proteins by Ub is important in controlling myriad cellular processes.¹⁴ Addition of Ub in growing chains (termed poly-Ub) serves as a targeting signal for marked proteins to be destroyed by the proteasome, a protein-eating machine which acts as the garbage disposal of cells. On the other hand, addition of a single Ub (termed mono-ubiquitination) regulates protein trafficking and protein activity,¹⁵ much like other post-translational protein modifications, including phosphorylation.

Ub itself is attached to proteins through an isopeptide linkage involving the C-terminal carboxylate of Ub and the epsilon-NH₂ group of a lysine side chain of the substrate protein.¹⁴ At least three distinct sets of enzymatic activities are involved in transferring Ub to proteins. These include the Ub-activating enzyme E1, Ub-conjugating enzymes (E2) and Ub ligases (E3). In addition, a large number of enzymes (including Usp14) control ubiquitin-dependent events by removing Ub.^13,16 These deubiquitinating enzymes (DUBs) release Ub from proteins about to be degraded by the proteasome, recycle monomeric Ub from polymeric Ub chains, edit inappropriately ubiquitinated proteins, and reverse regulatory mono-ubiquitination.^13,16 In the case of Usp14, this cleavage is specific for release of Ub from mono-ubiquitinated proteins in vitro.¹⁷

How does loss of Usp14 lead to an ataxia neurologic phenotype? Although the molecular mechanisms are still unknown, Wilson et al. provide an important clue by showing that synaptic transmission is abnormal in the ataxia mutant mice.¹¹ Such a "microscopic" deficit was suggested by the fact that ataxia mutant mice have only subtle anatomical changes in the central nervous system with no detectable pathological lesions of regions of cell loss.^12,18 Specifically, Wilson et al. found marked alterations in synaptic transmission at both the neuromuscular junction-which makes good sense given the profound muscle wasting and motor deficits in these animals-and, interestingly, at CA3-CA1 synapses in the hippocampus, a brain region involved in learning and memory. The types of synaptic deficits observed, namely decreased quantal content at the neuromuscular junction and decreased paired-pulse facilitation (PPF) and post-tetanic potentiation (PTP) in CA1 hippocampus suggest that the deficit resides on the presynaptic side of the synapse in the machinery that regulates release of neurotransmitter. This finding is quite interesting in light of recent studies demonstrating crucial roles for presynaptic ubiquitination and deubiquitination in axon guidance and synapse development in insects and nonmammalian vertebrates.¹⁹ Together with these previous studies, the results of Wilson et al. highlight a largely unappreciated role for Ub-dependent protein modification in synapse function.

Although the neurologic phenotype of ataxia mutant mice is not associated with significant neurodegeneration, the findings of Wilson et al. emphasize at least two new and emerging themes of relevance to neurodegenerative diseases, including AD. The first theme is the "sub-pathological" or microscopic dysfunction at the level of neuronal synapses. Just as Wilson et al. find subtle, yet physiologically significant synaptic defects in ataxia mutant mice, recent electrophysiological and behavioral studies support a role for synaptic dysfunction in the early stages of AD.² For example, mutant mice overexpressing wild-type and mutant APP display disrupted synaptic morphology, altered hippocampal synaptic transmission or plasticity, and impairment of spatial learning, often before amyloid plaque deposition.^3-7 In addition, exposure of hippocampal neurons to low concentrations of Aβ peptide fragments in brain slices or in vivo results in marked alterations in long-term potentiation (LTP) and long-term depression (LTD) of synaptic strength at CA1 synapses.^1,20-22 More recently, examination of mice expressing presenilin-1 mutations linked to familial Alzheimer's disease (FAD) has demonstrated abnormally sensitized hippocampal LTP.^23,24 Such synaptic dysfunction is not only involved in clinical syndromes but also in "normal" age-related memory decline. In fact, altered hippocampal synaptic function provides one of the primary electrophysiological markers for memory deficits during aging.^8-10

A second important theme emerging from the studies of Wilson et al. and others¹⁹ is the important role of ubiquitin-dependent mechanisms in synaptic function. The accumulation of ubiquitin conjugates and inclusion bodies containing ubiquitin has long been acknowledged in the pathologic lesions associated with a broad array of chronic neurodegenerative diseases, such as the neurofibrillary tangles of AD, Lewy bodies in both Parkinson's disease and Lewy body dementia, Bunina bodies in amyotrophic lateral sclerosis, and nuclear inclusions in CAG repeat expansion disorders such as Huntington's disease, spinocerebellar ataxias, and spinobulbar muscular atrophy.^14,25 More recently, mutations in ubiquitin-modifying enzymes and aberrant ubiquitin-proteasome function have been implicated in Parkinson's, Huntington's, and Alzheimer's disease,^25,26 as well as other neurologic syndromes.^27,28 Intriguingly, many of the mutated ubiquitin-modifying enzymes (e.g., parkin in certain hereditary forms of Parkinson's disease) or potential targets of ubiquitination are present at synapses and may impact synaptic function.^29,30

In the case of AD, a direct relationship between the ubiquitin system and disease pathogenesis has been strongly suggested by the discovery of a frameshift mutation in the ubiquitin which extends the molecule by 20 amino acids [Ub(+1)] in the brains of AD patients, including those with late-onset nonfamilial disease.³¹ Polyubiquitin chains formed from Ub(+1) are resistant to deubiquitination, and thus potently inhibit the degradation of polyubiquitinated proteins.³² Such inhibition could, in principle, lead to toxic buildup of ubiquitinated proteins, or perhaps alter ubiquitin-dependent modifications necessary for normal synaptic transmission, thereby producing both late and early aspects of disease symptoms, respectively. Additional distinct ubiquitin-dependent mechanisms in AD may occur via the presenilins (PS1 and PS2), proteins involved in processing amyloid precursor protein (APP) which are mutated in various familial versions of AD. Indeed, both PS1 and PS2 are targeted by the ubiquitin-proteasome system.^33,34 Whether these ubiquitin-dependent mechanisms are in turn linked mechanistically to synaptic deficits or cell death associated with AD remains to be clarified, but the above discussed findings along with the new results from Wilson et al. further strengthen the link among aberrant ubiquitin-proteasome function, synaptic defects, and neurodegenerative disease.

More study is clearly needed. In particular, very little knowledge exists regarding the ubiquitin-related enzymatic machinery at synapses or the synaptic proteins targeted by ubiquitination and deubiquitination. Paramount will be understanding the regulation of these important modifications during normal physiology and disease states. Also important will be deciphering the relationship between ubiquitin-dependent protein turnover and plastic or degenerative change at synapses. In this regard, uncovering the links among neurodegenerative disease genes, the ubiquitin-proteasome system, and synaptic signaling complexes promises to illuminate the mechanisms underlying synapse dysfunction in neurologic disease. By identifying a new role for ubiquitin-dependent mechanisms in the synaptic deficits of ataxic mice, Jenkins and colleagues have taken a steady step forward toward this goal.

References
1. Walsh, D. M., Klyubin, I., Fadeeva, J. V., Cullen, W. K., Anwyl, R., Wolfe, M. S., Rowan, M. J., and Selkoe, D. J. (2002). Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo, Nature 416, 535-9. Abstract

2. Mesulam, M. M. (1999). Neuroplasticity failure in Alzheimer's disease: bridging the gap between plaques and tangles, Neuron 24, 521-9.

3. Nalbantoglu, J., Tirado-Santiago, G., Lahsaini, A., Poirier, J., Goncalves, O., Verge, G., Momoli, F., Welner, S. A., Massicotte, G., Julien, J. P., and Shapiro, M. L. (1997). Impaired learning and LTP in mice expressing the carboxy terminus of the Alzheimer amyloid precursor protein, Nature 387, 500-5. Abstract

4. Chapman, P. F., White, G. L., Jones, M. W., Cooper-Blacketer, D., Marshall, V. J., Irizarry, M., Younkin, L., Good, M. A., Bliss, T. V., Hyman, B. T., et al. (1999). Impaired synaptic plasticity and learning in aged amyloid precursor protein transgenic mice, Nat Neurosci 2, 271-6.

5. Hsia, A. Y., Masliah, E., McConlogue, L., Yu, G. Q., Tatsuno, G., Hu, K., Kholodenko, D., Malenka, R. C., Nicoll, R. A., and Mucke, L. (1999). Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models, Proc Natl Acad Sci U S A 96, 3228-33. Abstract

6. Mucke, L., Masliah, E., Yu, G. Q., Mallory, M., Rockenstein, E. M., Tatsuno, G., Hu, K., Kholodenko, D., Johnson-Wood, K., and McConlogue, L. (2000). High-level neuronal expression of abeta 1-42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation, J Neurosci 20, 4050-8. Abstract

7. Fitzjohn, S. M., Morton, R. A., Kuenzi, F., Rosahl, T. W., Shearman, M., Lewis, H., Smith, D., Reynolds, D. S., Davies, C. H., Collingridge, G. L., and Seabrook, G. R. (2001). Age-related impairment of synaptic transmission but normal long-term potentiation in transgenic mice that overexpress the human APP695SWE mutant form of amyloid precursor protein, J Neurosci 21, 4691-8. Abstract

8. Barnes, C. A. (1994). Normal aging: regionally specific changes in hippocampal synaptic transmission, Trends Neurosci 17, 13-8.

9. Foster, T. C., and Norris, C. M. (1997). Age-associated changes in Ca(2+)-dependent processes: relation to hippocampal synaptic plasticity, Hippocampus 7, 602-12.

10. Foster, T. C. (1999). Involvement of hippocampal synaptic plasticity in age-related memory decline, Brain Res Brain Res Rev 30, 236-49.

11. Wilson, S. M., Bhattacharyya, B., Rachel, R. A., Coppola, V., Tessarollo, L., Householder, D. B., Fletcher, C. F., Miller, R. J., Copeland, N. G., and Jenkins, N. A. (2002). Synaptic defects in ataxia mice result from a mutation in Usp14, encoding a ubiquitin-specific protease, Nat Genet 7, 7.

12. D'Amato, C. J., and Hicks, S. P. (1965). Neuropathologic alterations in the ataxia (paralytic) mouse, Arch Pathol 80, 604-612.

13. Chung, C. H., and Baek, S. H. (1999). Deubiquitinating enzymes: their diversity and emerging roles, Biochem Biophys Res Commun 266, 633-40.

14. Glickman, M. H., and Ciechanover, A. (2002). The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction, Physiol Rev 82, 373-428. Abstract

15. Hicke, L. (2001). Protein regulation by monoubiquitin, Nat Rev Mol Cell Biol 2, 195-201.

16. Wilkinson, K. D. (1997). Regulation of ubiquitin-dependent processes by deubiquitinating enzymes, Faseb J 11, 1245-56.

17. Yin, L., Krantz, B., Russell, N. S., Deshpande, S., and Wilkinson, K. D. (2000). Nonhydrolyzable diubiquitin analogues are inhibitors of ubiquitin conjugation and deconjugation, Biochemistry 39, 10001-10.

18. Burt, A. M. (1980). Morphologic abnormalities in the postnatal differentiation of CA1 pyramidal cells and granule cells in the hippocampal formation of the ataxic mouse, Anat Rec 196, 61-9.

19. Murphey, R., and Godenschwege, T. (2002). New roles for ubiquitin in the assembly and function of neuronal circuits, Neuron 36, 5.

20. Freir, D. B., Holscher, C., and Herron, C. E. (2001). Blockade of long-term potentiation by beta-amyloid peptides in the CA1 region of the rat hippocampus in vivo, J Neurophysiol 85, 708-13. Abstract

21. Kim, J. H., Anwyl, R., Suh, Y. H., Djamgoz, M. B., and Rowan, M. J. (2001). Use-dependent effects of amyloidogenic fragments of (beta)-amyloid precursor protein on synaptic plasticity in rat hippocampus in vivo, J Neurosci 21, 1327-33. Abstract

22. Stephan, A., Laroche, S., and Davis, S. (2001). Generation of aggregated beta-amyloid in the rat hippocampus impairs synaptic transmission and plasticity and causes memory deficits, J Neurosci 21, 5703-14. Abstract

23. Parent, A., Linden, D. J., Sisodia, S. S., and Borchelt, D. R. (1999). Synaptic transmission and hippocampal long-term potentiation in transgenic mice expressing FAD-linked presenilin 1, Neurobiol Dis 6, 56-62. Abstract

24. Zaman, S. H., Parent, A., Laskey, A., Lee, M. K., Borchelt, D. R., Sisodia, S. S., and Malinow, R. (2000). Enhanced synaptic potentiation in transgenic mice expressing presenilin 1 familial Alzheimer's disease mutation is normalized with a benzodiazepine, Neurobiol Dis 7, 54-63.

25. Alves-Rodrigues, A., Gregori, L., and Figueiredo-Pereira, M. E. (1998). Ubiquitin, cellular inclusions and their role in neurodegeneration, Trends Neurosci 21, 516-20. Abstract

26. McNaught, K. S., Olanow, C. W., Halliwell, B., Isacson, O., and Jenner, P. (2001). Failure of the ubiquitin-proteasome system in Parkinson's disease, Nat Rev Neurosci 2, 589-94.

27. Jiang, Y. H., Armstrong, D., Albrecht, U., Atkins, C. M., Noebels, J. L., Eichele, G., Sweatt, J. D., and Beaudet, A. L. (1998). Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation, Neuron 21, 799-811.

28. Moss, A., Blackburn-Munro, G., Garry, E. M., Blakemore, J. A., Dickinson, T., Rosie, R., Mitchell, R., and Fleetwood-Walker, S. M. (2002). A role of the ubiquitin-proteasome system in neuropathic pain, J Neurosci 22, 1363-72.

29. Fallon, L., Moreau, F., Croft, B. G., Labib, N., Gu, W. J., and Fon, E. A. (2002). Parkin and CASK/LIN-2 associate via a PDZ-mediated interaction and are co-localized in lipid rafts and postsynaptic densities in brain, J Biol Chem 277, 486-91.

30. Zhang, Y., Gao, J., Chung, K. K., Huang, H., Dawson, V. L., and Dawson, T. M. (2000). Parkin functions as an E2-dependent ubiquitin- protein ligase and promotes the degradation of the synaptic vesicle-associated protein, CDCrel-1, Proc Natl Acad Sci U S A.

31. van Leeuwen, F. W., de Kleijn, D. P., van den Hurk, H. H., Neubauer, A., Sonnemans, M. A., Sluijs, J. A., Koycu, S., Ramdjielal, R. D., Salehi, A., Martens, G. J., et al. (1998). Frameshift mutants of beta amyloid precursor protein and ubiquitin-B in Alzheimer's and Down patients, Science 279, 242-7.

32. Lam, Y. A., Pickart, C. M., Alban, A., Landon, M., Jamieson, C., Ramage, R., Mayer, R. J., and Layfield, R. (2000). Inhibition of the ubiquitin-proteasome system in Alzheimer's disease, Proc Natl Acad Sci U S A 97, 9902-6.

33. Kim, T. W., Pettingell, W. H., Hallmark, O. G., Moir, R. D., Wasco, W., and Tanzi, R. E. (1997). Endoproteolytic cleavage and proteasomal degradation of presenilin 2 in transfected cells, J Biol Chem 272, 11006-10. Abstract

34. Steiner, H., Capell, A., Pesold, B., Citron, M., Kloetzel, P. M., Selkoe, D. J., Romig, H., Mendla, K., and Haass, C. (1998). Expression of Alzheimer's disease-associated presenilin-1 is controlled by proteolytic degradation and complex formation, J Biol Chem 273, 32322-31. Abstract

View all comments by Michael Ehlers

The USP’s belong to the family of deubiquitinating enzymes (DUB’s) that are essential for subsequent proteasomal activity. The other family members are ubiquitin C-terminal hydrolases (UCH) that have been associated with Parkinson’s disease and gracile axonal dystrophy in the mouse. Ubiquitin is usually associated with proteasomal degradation but is also involved in a variety of other biological functions (e.g. cell cycle progression, signal transduction, antigen presentation and DNA repair). A new role is the assembly and function of neuronal circuits ( see review by Murphy and Godenschwege, Neuron 36,5-8,2002).The paper by Wilson et al is a highly interesting contribution in this regard as it shows in this mutant (ax/ax) that USP-14 is important for regulating synaptic activity (as indicated by a loss of paired-pulse frequency). The unknown target of USP-14 may be an interesting protein. It is possibly involved in neurodegenerative diseases where synapse...  Read more

The USP’s belong to the family of deubiquitinating enzymes (DUB’s) that are essential for subsequent proteasomal activity. The other family members are ubiquitin C-terminal hydrolases (UCH) that have been associated with Parkinson’s disease and gracile axonal dystrophy in the mouse. Ubiquitin is usually associated with proteasomal degradation but is also involved in a variety of other biological functions (e.g. cell cycle progression, signal transduction, antigen presentation and DNA repair). A new role is the assembly and function of neuronal circuits ( see review by Murphy and Godenschwege, Neuron 36,5-8,2002).The paper by Wilson et al is a highly interesting contribution in this regard as it shows in this mutant (ax/ax) that USP-14 is important for regulating synaptic activity (as indicated by a loss of paired-pulse frequency). The unknown target of USP-14 may be an interesting protein. It is possibly involved in neurodegenerative diseases where synapse maintenance is essential (Terry and Katzman 2001).

View all comments by Fred van Leeuwen

Ubiquitin and Synaptic Dysfunction: Ataxic Mice Highlight New Common Themes in Neurologic Disease
The gene responsible for the neurologic symptoms in ataxia mice has been identified and shown to encode a ubiquitin-specific protease. This new study reveals new linkages among ubiquitination, synapse function, and neurologic disease.

A final commonality in most neurodegenerative diseases including Alzheimer’s disease (AD) is loss of neurons in critical brain areas. However, symptoms of illness often precede detectable neuronal loss, and many neurologic syndromes proceed without appreciable cell death or loss of nervous tissue. In such cases, the underlying deficit is believed to be in how the nerve cells themselves function and communicate at synapses. Indeed, aspects of cognitive impairment in both AD and age-related memory decline have been attributed to synaptic dysfunction.^1-10 Yet, uncovering the links among disease genes or risk factors, synapse dysfunction, and pathologic features of neurologic disease remains a formidable challenge. Now, a recent paper in...  Read more

Ubiquitin and Synaptic Dysfunction: Ataxic Mice Highlight New Common Themes in Neurologic Disease
The gene responsible for the neurologic symptoms in ataxia mice has been identified and shown to encode a ubiquitin-specific protease. This new study reveals new linkages among ubiquitination, synapse function, and neurologic disease.

A final commonality in most neurodegenerative diseases including Alzheimer’s disease (AD) is loss of neurons in critical brain areas. However, symptoms of illness often precede detectable neuronal loss, and many neurologic syndromes proceed without appreciable cell death or loss of nervous tissue. In such cases, the underlying deficit is believed to be in how the nerve cells themselves function and communicate at synapses. Indeed, aspects of cognitive impairment in both AD and age-related memory decline have been attributed to synaptic dysfunction.^1-10 Yet, uncovering the links among disease genes or risk factors, synapse dysfunction, and pathologic features of neurologic disease remains a formidable challenge. Now, a recent paper in Nature Genetics from the laboratory of Nancy Jenkins reports that the neurologic phenotype of ataxia mutant mice is due to loss of a ubiquitin-specific protease which in turn produces profound synaptic dysfunction.¹¹

Jenkins and colleagues set their sights on identifying the genetic lesion responsible for the severe tremors and hindlimb paralysis in ataxia mutant mice, a recessive mutation that arose spontaneously and was first described almost 40 years ago.¹² Through the laborious process of positional cloning, they mapped the mutation within mouse chromosome 18 to a location near Usp14, a gene which encodes ubiquitin-specific protease 14. Consistent with a loss-of-function mutation, expression of Usp14 mRNA was reduced and altered, and Usp14 protein was completely absent from ataxia mutant mice, due to a large insertion in the Usp14 gene. Interestingly, despite a predominantly neurological phenotype, Usp 14 is expressed in many tissues including brain, raising the possibility that these mice have additional, yet unapparent or subtle, defects in other tissues.

So what exactly is Usp14? Usp14 exists as a member of a large family of enzymes dedicated to clipping off ubiquitin (Ub) from proteins to which this small 76 amino acid polypeptide has been attached.¹³ Covalent modification of proteins by Ub is important in controlling myriad cellular processes.¹⁴ Addition of Ub in growing chains (termed poly-Ub) serves as a targeting signal for marked proteins to be destroyed by the proteasome, a protein-eating machine which acts as the garbage disposal of cells. On the other hand, addition of a single Ub (termed mono-ubiquitination) regulates protein trafficking and protein activity,¹⁵ much like other post-translational protein modifications, including phosphorylation.

Ub itself is attached to proteins through an isopeptide linkage involving the C-terminal carboxylate of Ub and the epsilon-NH₂ group of a lysine side chain of the substrate protein.¹⁴ At least three distinct sets of enzymatic activities are involved in transferring Ub to proteins. These include the Ub-activating enzyme E1, Ub-conjugating enzymes (E2) and Ub ligases (E3). In addition, a large number of enzymes (including Usp14) control ubiquitin-dependent events by removing Ub.^13,16 These deubiquitinating enzymes (DUBs) release Ub from proteins about to be degraded by the proteasome, recycle monomeric Ub from polymeric Ub chains, edit inappropriately ubiquitinated proteins, and reverse regulatory mono-ubiquitination.^13,16 In the case of Usp14, this cleavage is specific for release of Ub from mono-ubiquitinated proteins in vitro.¹⁷

How does loss of Usp14 lead to an ataxia neurologic phenotype? Although the molecular mechanisms are still unknown, Wilson et al. provide an important clue by showing that synaptic transmission is abnormal in the ataxia mutant mice.¹¹ Such a "microscopic" deficit was suggested by the fact that ataxia mutant mice have only subtle anatomical changes in the central nervous system with no detectable pathological lesions of regions of cell loss.^12,18 Specifically, Wilson et al. found marked alterations in synaptic transmission at both the neuromuscular junction—which makes good sense given the profound muscle wasting and motor deficits in these animals—and, interestingly, at CA3-CA1 synapses in the hippocampus, a brain region involved in learning and memory. The types of synaptic deficits observed, namely decreased quantal content at the neuromuscular junction and decreased paired-pulse facilitation (PPF) and post-tetanic potentiation (PTP) in CA1 hippocampus suggest that the deficit resides on the presynaptic side of the synapse in the machinery that regulates release of neurotransmitter. This finding is quite interesting in light of recent studies demonstrating crucial roles for presynaptic ubiquitination and deubiquitination in axon guidance and synapse development in insects and nonmammalian vertebrates.¹⁹ Together with these previous studies, the results of Wilson et al. highlight a largely unappreciated role for Ub-dependent protein modification in synapse function.

Although the neurologic phenotype of ataxia mutant mice is not associated with significant neurodegeneration, the findings of Wilson et al. emphasize at least two new and emerging themes of relevance to neurodegenerative diseases, including AD. The first theme is the "sub-pathological" or microscopic dysfunction at the level of neuronal synapses. Just as Wilson et al. find subtle, yet physiologically significant synaptic defects in ataxia mutant mice, recent electrophysiological and behavioral studies support a role for synaptic dysfunction in the early stages of AD.² For example, mutant mice overexpressing wild-type and mutant APP display disrupted synaptic morphology, altered hippocampal synaptic transmission or plasticity, and impairment of spatial learning, often before amyloid plaque deposition.^3-7 In addition, exposure of hippocampal neurons to low concentrations of Aβ peptide fragments in brain slices or in vivo results in marked alterations in long-term potentiation (LTP) and long-term depression (LTD) of synaptic strength at CA1 synapses.^1,20-22 More recently, examination of mice expressing presenilin-1 mutations linked to familial Alzheimer’s disease (FAD) has demonstrated abnormally sensitized hippocampal LTP.^23,24 Such synaptic dysfunction is not only involved in clinical syndromes but also in "normal" age-related memory decline. In fact, altered hippocampal synaptic function provides one of the primary electrophysiological markers for memory deficits during aging.^8-10

A second important theme emerging from the studies of Wilson et al. and others¹⁹ is the important role of ubiquitin-dependent mechanisms in synaptic function. The accumulation of ubiquitin conjugates and inclusion bodies containing ubiquitin has long been acknowledged in the pathologic lesions associated with a broad array of chronic neurodegenerative diseases, such as the neurofibrillary tangles of AD, Lewy bodies in both Parkinson’s disease and Lewy body dementia, Bunina bodies in amyotrophic lateral sclerosis, and nuclear inclusions in CAG repeat expansion disorders such as Huntington’s disease, spinocerebellar ataxias, and spinobulbar muscular atrophy.^14,25 More recently, mutations in ubiquitin-modifying enzymes and aberrant ubiquitin-proteasome function have been implicated in Parkinson’s, Huntington’s, and Alzheimer’s disease,^25,26 as well as other neurologic syndromes.^27,28 Intriguingly, many of the mutated ubiquitin-modifying enzymes (e.g., parkin in certain hereditary forms of Parkinson’s disease) or potential targets of ubiquitination are present at synapses and may impact synaptic function.^29,30

In the case of AD, a direct relationship between the ubiquitin system and disease pathogenesis has been strongly suggested by the discovery of a frameshift mutation in the ubiquitin which extends the molecule by 20 amino acids [Ub(+1)] in the brains of AD patients, including those with late-onset nonfamilial disease.³¹ Polyubiquitin chains formed from Ub(+1) are resistant to deubiquitination, and thus potently inhibit the degradation of polyubiquitinated proteins.³² Such inhibition could, in principle, lead to toxic buildup of ubiquitinated proteins, or perhaps alter ubiquitin-dependent modifications necessary for normal synaptic transmission, thereby producing both late and early aspects of disease symptoms, respectively. Additional distinct ubiquitin-dependent mechanisms in AD may occur via the presenilins (PS1 and PS2), proteins involved in processing amyloid precursor protein (APP) which are mutated in various familial versions of AD. Indeed, both PS1 and PS2 are targeted by the ubiquitin-proteasome system.^33,34 Whether these ubiquitin-dependent mechanisms are in turn linked mechanistically to synaptic deficits or cell death associated with AD remains to be clarified, but the above discussed findings along with the new results from Wilson et al. further strengthen the link among aberrant ubiquitin-proteasome function, synaptic defects, and neurodegenerative disease.

More study is clearly needed. In particular, very little knowledge exists regarding the ubiquitin-related enzymatic machinery at synapses or the synaptic proteins targeted by ubiquitination and deubiquitination. Paramount will be understanding the regulation of these important modifications during normal physiology and disease states. Also important will be deciphering the relationship between ubiquitin-dependent protein turnover and plastic or degenerative change at synapses. In this regard, uncovering the links among neurodegenerative disease genes, the ubiquitin-proteasome system, and synaptic signaling complexes promises to illuminate the mechanisms underlying synapse dysfunction in neurologic disease. By identifying a new role for ubiquitin-dependent mechanisms in the synaptic deficits of ataxic mice, Jenkins and colleagues have taken a steady step forward toward this goal.

References
1. Walsh, D. M., Klyubin, I., Fadeeva, J. V., Cullen, W. K., Anwyl, R., Wolfe, M. S., Rowan, M. J., and Selkoe, D. J. (2002). Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo, Nature 416, 535-9. Abstract

2. Mesulam, M. M. (1999). Neuroplasticity failure in Alzheimer's disease: bridging the gap between plaques and tangles, Neuron 24, 521-9. Abstract

3. Nalbantoglu, J., Tirado-Santiago, G., Lahsaini, A., Poirier, J., Goncalves, O., Verge, G., Momoli, F., Welner, S. A., Massicotte, G., Julien, J. P., and Shapiro, M. L. (1997). Impaired learning and LTP in mice expressing the carboxy terminus of the Alzheimer amyloid precursor protein, Nature 387, 500-5. Abstract

4. Chapman, P. F., White, G. L., Jones, M. W., Cooper-Blacketer, D., Marshall, V. J., Irizarry, M., Younkin, L., Good, M. A., Bliss, T. V., Hyman, B. T., et al. (1999). Impaired synaptic plasticity and learning in aged amyloid precursor protein transgenic mice, Nat Neurosci 2, 271-6. Abstract

5. Hsia, A. Y., Masliah, E., McConlogue, L., Yu, G. Q., Tatsuno, G., Hu, K., Kholodenko, D., Malenka, R. C., Nicoll, R. A., and Mucke, L. (1999). Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models, Proc Natl Acad Sci U S A 96, 3228-33. Abstract

6. Mucke, L., Masliah, E., Yu, G. Q., Mallory, M., Rockenstein, E. M., Tatsuno, G., Hu, K., Kholodenko, D., Johnson-Wood, K., and McConlogue, L. (2000). High-level neuronal expression of abeta 1-42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation, J Neurosci 20, 4050-8. Abstract

7. Fitzjohn, S. M., Morton, R. A., Kuenzi, F., Rosahl, T. W., Shearman, M., Lewis, H., Smith, D., Reynolds, D. S., Davies, C. H., Collingridge, G. L., and Seabrook, G. R. (2001). Age-related impairment of synaptic transmission but normal long-term potentiation in transgenic mice that overexpress the human APP695SWE mutant form of amyloid precursor protein, J Neurosci 21, 4691-8. Abstract

8. Barnes, C. A. (1994). Normal aging: regionally specific changes in hippocampal synaptic transmission, Trends Neurosci 17, 13-8. Abstract

9. Foster, T. C., and Norris, C. M. (1997). Age-associated changes in Ca(2+)-dependent processes: relation to hippocampal synaptic plasticity, Hippocampus 7, 602-12. Abstract

10. Foster, T. C. (1999). Involvement of hippocampal synaptic plasticity in age-related memory decline, Brain Res Brain Res Rev 30, 236-49. Abstract

11. Wilson, S. M., Bhattacharyya, B., Rachel, R. A., Coppola, V., Tessarollo, L., Householder, D. B., Fletcher, C. F., Miller, R. J., Copeland, N. G., and Jenkins, N. A. (2002). Synaptic defects in ataxia mice result from a mutation in Usp14, encoding a ubiquitin-specific protease, Nat Genet 7, 7. Abstract

12. D'Amato, C. J., and Hicks, S. P. (1965). Neuropathologic alterations in the ataxia (paralytic) mouse, Arch Pathol 80, 604-612.

13. Chung, C. H., and Baek, S. H. (1999). Deubiquitinating enzymes: their diversity and emerging roles, Biochem Biophys Res Commun 266, 633-40. Abstract

14. Glickman, M. H., and Ciechanover, A. (2002). The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction, Physiol Rev 82, 373-428. Abstract

15. Hicke, L. (2001). Protein regulation by monoubiquitin, Nat Rev Mol Cell Biol 2, 195-201. Abstract

16. Wilkinson, K. D. (1997). Regulation of ubiquitin-dependent processes by deubiquitinating enzymes, Faseb J 11, 1245-56. Abstract

17. Yin, L., Krantz, B., Russell, N. S., Deshpande, S., and Wilkinson, K. D. (2000). Nonhydrolyzable diubiquitin analogues are inhibitors of ubiquitin conjugation and deconjugation, Biochemistry 39, 10001-10. Abstract

18. Burt, A. M. (1980). Morphologic abnormalities in the postnatal differentiation of CA1 pyramidal cells and granule cells in the hippocampal formation of the ataxic mouse, Anat Rec 196, 61-9. Abstract

19. Murphey, R., and Godenschwege, T. (2002). New roles for ubiquitin in the assembly and function of neuronal circuits, Neuron 36, 5. Abstract

20. Freir, D. B., Holscher, C., and Herron, C. E. (2001). Blockade of long-term potentiation by beta-amyloid peptides in the CA1 region of the rat hippocampus in vivo, J Neurophysiol 85, 708-13. Abstract

21. Kim, J. H., Anwyl, R., Suh, Y. H., Djamgoz, M. B., and Rowan, M. J. (2001). Use-dependent effects of amyloidogenic fragments of (beta)-amyloid precursor protein on synaptic plasticity in rat hippocampus in vivo, J Neurosci 21, 1327-33. Abstract

22. Stephan, A., Laroche, S., and Davis, S. (2001). Generation of aggregated beta-amyloid in the rat hippocampus impairs synaptic transmission and plasticity and causes memory deficits, J Neurosci 21, 5703-14. Abstract

23. Parent, A., Linden, D. J., Sisodia, S. S., and Borchelt, D. R. (1999). Synaptic transmission and hippocampal long-term potentiation in transgenic mice expressing FAD-linked presenilin 1, Neurobiol Dis 6, 56-62. Abstract

24. Zaman, S. H., Parent, A., Laskey, A., Lee, M. K., Borchelt, D. R., Sisodia, S. S., and Malinow, R. (2000). Enhanced synaptic potentiation in transgenic mice expressing presenilin 1 familial Alzheimer's disease mutation is normalized with a benzodiazepine, Neurobiol Dis 7, 54-63. Abstract

25. Alves-Rodrigues, A., Gregori, L., and Figueiredo-Pereira, M. E. (1998). Ubiquitin, cellular inclusions and their role in neurodegeneration, Trends Neurosci 21, 516-20. Abstract

26. McNaught, K. S., Olanow, C. W., Halliwell, B., Isacson, O., and Jenner, P. (2001). Failure of the ubiquitin-proteasome system in Parkinson's disease, Nat Rev Neurosci 2, 589-94. Abstract

27. Jiang, Y. H., Armstrong, D., Albrecht, U., Atkins, C. M., Noebels, J. L., Eichele, G., Sweatt, J. D., and Beaudet, A. L. (1998). Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation, Neuron 21, 799-811. Abstract

28. Moss, A., Blackburn-Munro, G., Garry, E. M., Blakemore, J. A., Dickinson, T., Rosie, R., Mitchell, R., and Fleetwood-Walker, S. M. (2002). A role of the ubiquitin-proteasome system in neuropathic pain, J Neurosci 22, 1363-72. Abstract

29. Fallon, L., Moreau, F., Croft, B. G., Labib, N., Gu, W. J., and Fon, E. A. (2002). Parkin and CASK/LIN-2 associate via a PDZ-mediated interaction and are co-localized in lipid rafts and postsynaptic densities in brain, J Biol Chem 277, 486-91. Abstract

30. Zhang, Y., Gao, J., Chung, K. K., Huang, H., Dawson, V. L., and Dawson, T. M. (2000). Parkin functions as an E2-dependent ubiquitin- protein ligase and promotes the degradation of the synaptic vesicle-associated protein, CDCrel-1, Proc Natl Acad Sci U S A. Abstract

31. van Leeuwen, F. W., de Kleijn, D. P., van den Hurk, H. H., Neubauer, A., Sonnemans, M. A., Sluijs, J. A., Koycu, S., Ramdjielal, R. D., Salehi, A., Martens, G. J., et al. (1998). Frameshift mutants of beta amyloid precursor protein and ubiquitin-B in Alzheimer's and Down patients, Science 279, 242-7. Abstract

32. Lam, Y. A., Pickart, C. M., Alban, A., Landon, M., Jamieson, C., Ramage, R., Mayer, R. J., and Layfield, R. (2000). Inhibition of the ubiquitin-proteasome system in Alzheimer's disease, Proc Natl Acad Sci U S A 97, 9902-6. Abstract

33. Kim, T. W., Pettingell, W. H., Hallmark, O. G., Moir, R. D., Wasco, W., and Tanzi, R. E. (1997). Endoproteolytic cleavage and proteasomal degradation of presenilin 2 in transfected cells, J Biol Chem 272, 11006-10. Abstract

34. Steiner, H., Capell, A., Pesold, B., Citron, M., Kloetzel, P. M., Selkoe, D. J., Romig, H., Mendla, K., and Haass, C. (1998). Expression of Alzheimer's disease-associated presenilin-1 is controlled by proteolytic degradation and complex formation, J Biol Chem 273, 32322-31. Abstract

View all comments by Michael Ehlers

Ubiquitin Is Implicated Not Only at the End of the Chain of Neuropathological Events
Most investigators think that ubiquitin, as a heat shock protein, is involved in reaction to neuronal damage. The results by Wilson et al. (Nature Genetics 2002), several others (e.g., Hegde and DiAntonio, Nature Reviews Neuroscience 2002;3:854-861) and the report by Ehlers, clearly have taught us that ubiquitin can also be implicated in early stages of neuronal dysfunctioning. If we extrapolate these data to Alzheimer’s disease, this could mean that the synaptic network does not function as efficiently as before (during mild cognitive impairments).

Several years ago we published a paper on frameshift mutations (by dinucleotide deletions in simple repeats) in ubiquitin mRNA (Van Leeuwen et al., 1998). Remarkably, the resulting ubiquitin +1 (UBB⁺¹) is present in the hallmarks of Alzheimer, suggesting an important role in neuropathogenesis.

UBB⁺¹, lacking its very C-terminal amino acid...  Read more

Ubiquitin Is Implicated Not Only at the End of the Chain of Neuropathological Events
Most investigators think that ubiquitin, as a heat shock protein, is involved in reaction to neuronal damage. The results by Wilson et al. (Nature Genetics 2002), several others (e.g., Hegde and DiAntonio, Nature Reviews Neuroscience 2002;3:854-861) and the report by Ehlers, clearly have taught us that ubiquitin can also be implicated in early stages of neuronal dysfunctioning. If we extrapolate these data to Alzheimer’s disease, this could mean that the synaptic network does not function as efficiently as before (during mild cognitive impairments).

Several years ago we published a paper on frameshift mutations (by dinucleotide deletions in simple repeats) in ubiquitin mRNA (Van Leeuwen et al., 1998). Remarkably, the resulting ubiquitin +1 (UBB⁺¹) is present in the hallmarks of Alzheimer, suggesting an important role in neuropathogenesis.

UBB⁺¹, lacking its very C-terminal amino acid (glycine), which is essential for its biological activity, would in fact be a substrate for ubiquitination (Van Leeuwen et al., 2000; Lam et al., 2000; De Vrij et al., 2001). More recently we showed that UBB⁺¹ inhibits the proteasome using a GFP reporter construct (Lindsten et al., 2002). In fact, UBB⁺¹ is recognized as a ubiquitin fusion degradation substrate. Full blockade of the proteosome requires ubiquitination at Lys²⁹ and Lys⁴⁸. Furthermore, UBB⁺¹ overexpression results in neuronal apoptosis (De Vrij et al., ibid.). As UBB⁺¹ expression also occurs in non-neuronal cells, it might contribute to pathogenesis in other diseases, as well (McPhaul et al., 2002). These findings thus suggest that the inhibitory effect of UBB⁺¹ may be an important determinant of neurotoxicity and contribute to an environment that favors the accumulation of misfolded proteins that occur in numerous neurodegenerative diseases.

View all comments by Fred van Leeuwen