Aggregates in neurodegenerative disease: crowds and power?

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Aggregates in neurodegenerative disease: crowds and power? In his famous sociological study ‘Crowds and Power’1, Elias Canetti discussed the relationship between the individual and the crowd. He argued persuasively that different facets of a personality become transmuted on becoming part of a crowd. The crowd can be seen as an enabling device for an individual’s ‘Will to Power’, so that he can act in a fashion that would not normally be deemed possible or at least appropriate. Crowds might often be vehicles of destruction, but they can also sometimes act as safe havens that protect groups of individuals from a hostile environment or that segregate undesirable members of society from the general population. Echoes of Canetti’s work can currently be found in the intense debate concerning the role of protein aggregates or ‘inclusions’ as potential causative agents in neurodegenerative disease. It is a remarkable fact that large molecular aggregates of different types have been observed in a wide variety of such diseases, some of which are listed in Table 1. However, although the occurrence of these aggregates is not in doubt, their significance is unclear but potentially of great importance. Are aggregates the actual entities that cause neurons to die, presumably through mechanical disruption of the cell, or are they an attempt by the cell to sequester potentially toxic molecules in a sort of cellular concentration camp? Several new papers in three separate areas of research have increased our understanding of what is occurring significantly.

Familial amyotrophic lateral sclerosis

Phuong B. Tran and Richard J. Miller Dept of Pharmacological and Physiological Sciences, The University of Chicago, Chicago, IL 60637, USA.

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Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that is characterized by selective degeneration of the motoneurons in the brainstem and spinal cord. Inevitably, this disorder leads to paralysis and death within three to five years. Approximately 10% of ALS cases are inherited [familial ALS (FALS)] and of these cases, 15–20% arise from missense mutations in the gene encoding Cu2+–Zn2+ superoxide dismutase (SOD1)2. The evidence available to date strongly suggests that FALS is caused by a toxic ‘gain of function’ of these mutated forms of SOD1. Transgenic mice that express genes that encode mutant FALS-related SOD1 develop a motoneuron disease that dramatically recapitulates many of the symptoms of human FALS (Ref. 3). What is the nature of the gain of function of the mutated SOD1? Several reasonable and TINS Vol. 22, No. 5, 1999

interesting suggestions have been made. In addition to dismutation of superoxide radicals, SOD1 is also capable of catalyzing several other ‘side reactions’, which include the use of peroxynitrite for the nitrosylation of proteins and the use of its product, hydrogen peroxide, for the generation of highly toxic hydroxyl free radicals. It has been suggested that perhaps one of these reactions is upregulated in animals that have the mutated form of SOD1. Bruijn et al.4 have now tested this hypothesis. Mice were generated that expressed widely differing background levels of the gene encoding SOD1, including homozygous knockouts (0% SOD1), heterozygous knockouts (50% SOD1) and wild-type mice (100% SOD1), as well as mice that expressed an additional 600% of the gene encoding human SOD1 in addition to their normal mouse background. Transgenic mice were then generated with the FALS transgene, SOD/G85R, added to these diverse backgrounds. Would the differing amounts of wild-type SOD1 alter the time course or extent of the ALS-like symptoms produced by SOD/G85R in the different types of mice? The answer was clearly no. The authors argued that if aberrant enzyme activity of the mutant SOD1 had been the cause of the symptoms then the amount of

wild-type SOD1 in the background should indeed have made a difference. They, therefore, concluded that something else must be responsible for the toxic gain of function; but what? One good possibility is that it could be due to the existence of large SOD1-containing aggregates that were originally shown to exist in the neurons and astrocytes of the mutant SOD1 transgenic mice3. Durham et al.5 also observed these aggregates using a very different paradigm. cDNAs that contained mutant Sod1 were microinjected into cultured mouse motoneurons and it was shown that the expression of the mutant, but not wild-type, Sod1 killed motoneurons by glutamate-receptor- and Ca2+-induced apoptosis6. The detection of large SOD1 immunoreactive aggregates in the cytoplasm of many motoneurons, a phenomenon never observed in control cells or those overproducing wild-type SOD1, was correlated with motoneuron death. In their most recent study, Bruijn et al.4 have extended their observations by demonstrating aggregates in the cytoplasm of affected motoneurons from mice carrying the SOD/G85R transgene, as well as from transgenic mice carrying other mutated forms of SOD1. Similar aggregates have been detected in motoneurons from

TABLE 1. Types of aggregates associated with some of the more-common neurodegenerative diseases Disease

Aggregate type

Some aggregate components

Alzheimer’s disease

Extracellular amyloid plaques

b-amyloid NAC Ubiquitin

Parkinson’s disease

Intracytoplasmic Lewy bodies

a-synuclein Ubiquitin UCH-LI Neurofilaments

Huntington’s disease and other CAG-repeat disorders

Intranuclear aggregates

Expanded glutaminerepeat proteins

Ubiquitin Molecular chaperones 2OS proteasome protein Nuclear matrix proteins

ALS and FALS

Cytoplasmic aggregates in neurons and astrocytes

SODI (FALS) Ubiquitin a-synuclein (ALS)

Prion diseases

Extracellular amyloid plaques

Prion protein Ubiquitin

Abbreviations: ALS, amyotrophic lateral sclerosis; FALS, familial amyotrophic lateral sclerosis; NAC, non-amyloid component (fragment of a-synuclein); SODI, superoxide dismutase 1; UCH-L1, ubiquitin carboxyterminal hydrolase L1.

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human FALS patients7. Interestingly, aggregates never occurred in mice that only expressed the gene encoding wild-type SOD1, even when the gene was expressed at increased levels, and they also never occurred in neurons outside the affected region (which mainly contained motoneurons). In agreement with the data on the time course of the disease in these animals, aggregates occurred with similar frequency irrespective of the level of Sod1 in the background. Immunohistochemical analysis showed that these aggregates normally contained both wild-type and mutated SOD1. Nevertheless, the presence of the wild-type SOD1 was not essential for aggregate formation, as aggregates could be detected in homozygous SOD1 knockout mice that carried the SOD/G85R transgene. It is not clear exactly what causes the aggregates to form, which is obviously an important issue. Mutant, but not wild-type SOD1, forms aggregates when transfected into COS cells, but does not cause cell death in this case8. It should also be noted that the wild-type SOD1 molecules that are damaged by oxidation can be shown to aggregate spontaneously in vitro9. Interestingly, overproduction of the HSP70 chaperone in motoneurons decreases mutant-SOD1-aggregate formation and increases neuronal survival10. The authors of the studies discussed above4,5 have suggested that aggregate formation could be a contributing factor in FALS. Understanding the precise causal relationship between SOD1 aggregation and the death of motoneurons will require further investigation. However, it is clear that current experimental paradigms should soon reveal more details about this relationship. It is also interesting to note that the existence of SOD1 aggregates in astrocytes as described by Bruijn et al.3 provides a potential link to one theory concerning the etiology of idiopathic ALS that suggests excitotoxic motoneuronal death occurs as a result of compromised glutamate uptake into astrocytes11.

CAG-repeat disorders Huntington’s disease (HD) is a progressive neurodegenerative condition that is typically associated with neuronal loss in the striatum and cortex. The disease, which exhibits an autosomal-dominant form of inheritance, is associated with involuntary dance-like, writhing movements, together with adverse psychological symptoms, cognitive decline and eventually death. Inheritance of the disease is also associated with ‘anticipation’, that is, worsening of the symptoms and earlier times of onset with succeeding generations. The gene responsible for HD has been shown to code for a large novel 350 kDa protein named huntingtin. This

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TABLE 2. Proteins involved in polyglutamine-repeat disorders Disease

Protein

Normal repeat range

Expanded repeat range

HD DRPLA SCA-1 SCA-2 SCA-3 SCA-6 SCA-7 SBMA Schizophreniaa

Huntingtin Atrophin 1 Ataxin 1 Ataxin 2 Ataxin 3 P- or Q-type Ca2+ channel Ataxin 7 Androgen receptor Ca2+-activated K+ channel

6–35 3–36 6–39 14–32 12–40 4–18 7–17 9–36 <19

36–121 49–88 40–83 33–77 55–86 21–30 38–130 38–62 >19

a

Linkage is tentative at this point. Abbreviations: DRPLA, dentatorubral pallido-luysian atrophy; HD, Huntington’s disease; SBMA, spinobulbar muscular atrophy; SCA, spinocerebellar ataxia.

protein is very widely distributed both within and outside the CNS. Its normal function is unknown, although it has been shown, using yeast two-hybrid analysis, that it binds to several cellular partners (for example, glyceraldehyde 3-phosphate dehydrogenase), which could yield clues in this regard12. The molecular basis for the transmission of HD is associated with instability in the length of a CAG repeat, which codes for a string of glutamine residues, near the N terminus of the protein13. Normal individuals exhibit a polymorphic variation in this region of between 6 and 34 glutamines. However, in individuals affected with the disease, the length of the repeat runs from 36 to approximately 120 residues. Furthermore, the expansion of the glutamine repeat is associated with increased severity of the disorder and also with anticipation in succeeding generations. Huntington’s disease is one member of a family of eight currently known CAG-repeat disorders (Table 2) and it has also been suggested that certain other conditions might be due to this CAG-repeat mechanism14. In addition, a recently cloned K+ channel, which might be linked to schizophrenia, shows a CAG polymorphism15. In each of the eight CAG-repeat disorders, the protein responsible for the disease exhibits an unstable CAG repeat that produces the disease once it expands outside a ‘normal’ range. As with HD, the normal functions of the proteins associated with each CAGrepeat disorder are unknown with the exception of spinobulbar muscular atrophy, where the protein is the androgen receptor, and spinocerebellar ataxia 6 (SCA-6), where the protein is the P- or Q-type voltagesensitive Ca2+ channel13. It is also interesting to note that although these proteins are often distributed ubiquitously, the neuropathological profile differs in each case and involves unique populations of neurons16. How do genes that contain CAG repeats produce neurodegeneration? It is

clear that the polyglutamine sequence is the major factor involved, as the severity of the disease correlates with its length. In a fascinating experiment carried out by Ordway et al.17, transgenic mice were generated that carried the gene for hypoxanthine phosphoribosyltransferase (a protein not known to be linked to any CAG-repeat disorder) into which they had introduced a 146-unit CAG repeat. Amazingly, these mice developed a neurodegenerative disease with a unique profile, which clearly indicated the key role of polyglutamine. What exactly does the polyglutamine do? From the point of view of the present discussion, it has been shown that in several of the CAG-repeat disorders aggregates of the involved protein can be observed in neurons from the affected area of the nervous system16,18. This is true in tissue from human patients19, from transgenic mice20, from the rhabdomeres of Drosophila eyes or Caenorhabditis elegans neurons that express the genes encoding these expanded CAG repeats21,22, and in some cultured cell lines transfected with these genes23. Aggregates are not seen in normal individuals or in mice or flies expressing genes that carry only a normal number of CAG repeats. In HD patients, huntingtin aggregates can also be seen in dystrophic neurites19. However, it is the striking occurrence of nuclear inclusions that has been shown to be common among CAG-repeat disorders. It has been suggested that, as in the case of FALS (see above), these aggregates could be in some way responsible for the toxic gain of function that produces the disease. How do the aggregates form? Two major mechanisms have been suggested: (1) Expanded glutamine repeats can act as hydrogen-bonded polar zippers, forming pleated sheets of b-strands that are held together by hydrogen bonds between their amides24. Accordingly, it is interesting to note that huntingtin proteins with strings of glutamines in the pathogenic TINS Vol. 22, No. 5, 1999

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P.B. Tran and R.J. Miller – Aggregates and neuronal death

range can form aggregates of amyloid-like fibrils in vitro25. (2) Aggregates of glutamine-repeat proteins could be stabilized by transglutaminase catalysed cross-linking26. The aggregates found in HD and the other disorders might also contain elements in addition to the proteins that contain glutamine repeats and these could be significant for aggregate formation. Ubiquitination of the proteins in aggregates has been observed in the case of each disorder. Immunohistochemical analysis of aggregates in patients with HD showed that they did not contain b-amyloid or neurofilament proteins, which distinguishes them from Lewy bodies in Parkinson’s disease (see below), or a variety of other proteins. However, ataxin-1 aggregates in SCA-1 have been shown to contain a nuclear matrix protein and LANP (leucinerich acidic nuclear protein) as well as the 20S proteasome protein and a molecular chaperone27,28. Much of the huntingtin found in the aggregates in HD patients appears to be the truncated N-terminal region of the molecule that contains the polyglutamine tract19. It is therefore of great interest to note that huntingtin can be cleaved by caspase 3, an enzyme that is clearly important in the final steps of apoptotic cell death and which is probably one of the enzymes that produces truncated huntingtin in vivo. Other proteins that contain glutamine repeats can also be cleaved by caspase 3 as well as a number of other caspases29. This might be of particular significance as neuronal death produced by glutamine-repeat proteins is probably due to apoptosis or something similar30. The discovery that much of the huntingtin found in nuclear aggregates is in the form of cleavage products could help to explain how it is able to access the nucleus, as there is a limit on the molecular weights of proteins that can freely diffuse into the nucleus from the cytoplasm. This could also be true for some of the other glutamine-repeat proteins, although some of these contain specific nuclear-localization motifs or possibly enter the nucleus by using other nucleus-bound proteins. Are the nuclear aggregates observed responsible for neurodegeneration? Three recent papers have addressed this question directly and the conclusions have been most informative. Using different types of caspase inhibitors, Kim et al. dissociated neuronal death from aggregate formation31. Klement et al.32 observed that a particular mutant of ataxin 1 that carried a string of 77 glutamines failed to form aggregates either in vitro or in the Purkinje cells of transgenic mice, even though it was clear that the protein did become localized to the nucleus. However, the transgenic mice did eventually develop a neurological disease 196

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and lesions that closely resembled those obtained with a wild-type ataxin 1 (which contained 82 glutamines and also produced nuclear inclusions). Using a model of aggregate formation and apoptosis in transfected cultured striatal neurons, Saudou et al.33 provided further evidence to suggest that nuclear import of glutamine-expanded huntingtin was vital for its deleterious effects and they also tackled the issue of the importance of aggregate formation. Using mice with a dominant negative mutation of the gene encoding the ubiquitinconjugating enzyme, they were able to reduce the overall level of protein ubiquitination. Overproduction of glutamineexpanded huntingtin under these conditions failed to produce nuclear aggregates, but toxicity was still observed (and actually increased). It should be noted that it is still possible that the proteins in these experiments were in the form of microaggregates that would not have been detected. Nevertheless, the observations strongly suggest that nuclear inclusions in CAGrepeat disorders could be more of a cellular protective device than agents of destruction. It is of particular interest to note the effect of interfering with ubiquitination in the light of recent findings with Parkinson’s disease (PD).

Parkinson’s disease Parkinson’s disease is the second most common neurodegenerative disorder after Alzheimer’s disease34. The disease is characterized by tremor, bradykinesia, rigidity and postural instability that results primarily from a loss of dopaminergic neurons of the nigro–striatal pathway. In addition to the loss of nigral neurons, PD is also characterized by the widespread distribution of Lewy bodies, which are intracytoplasmic aggregates 5–25 mm in diameter with a dense eosinophilic core and a pale surrounding ‘halo’. As with other types of aggregates discussed here, it has been suggested that Lewy bodies might have a causative role in neurodegeneration. It is worth noting that they are not completely specific for PD and are also found in association with several other types of neurodegenerative disorders, which include some cases of Alzheimer’s disease and dementia with Lewy bodies (DLB)34,35. Patients with PD also exhibit Lewy neurites, which appear bloated with an unidentified proteinaceous material. It has been of great interest to establish the composition of Lewy bodies and neurites. Until recently, Lewy bodies were known to contain ubiquitin, neurofilaments and several other proteins35. However, recent work on inherited forms of PD has added significantly to our understanding of their composition. As in the case of ALS, most cases of PD occur

spontaneously. However, in a small percentage of cases, PD can be inherited in an autosomally dominant fashion34. Polymeropoulos and colleagues36 demonstrated that one form of familial PD was associated with a missense mutation in a protein called a-synuclein. Another mutation in the same protein has also subsequently been linked to familial PD (Ref. 34). a-Synuclein is a 140 kDa protein that is homologous to two other gene products: b- and g-synuclein37. The functions of all of these proteins are unknown. aSynuclein is found in high concentrations in the nervous system, where it is primarily localized to nerve terminals, as well as other tissues. Curiously, one of the mutations in a-synuclein that has been linked to inherited PD (a Thr to Ala substitution at residue 53) occurs normally in certain species, for example, rats and some birds, that show no evidence of developing PD (Ref. 37). It has been suggested that these species do not live long enough to develop the disease, although other explanations are also possible. It was subsequently shown that a-synuclein was a major component of the Lewy bodies and Lewy neurites in PD as well as in DLB (Refs 38,39). Other members of the synuclein family were absent. Significantly, a-synuclein has also been found in aggregates in other types of neurodegenerative disease. For example, a portion of the a-synuclein molecule known as the ‘non-amyloid component’ (NAC) has been detected in the neuritic plaques of patients with Alzheimer’s disease34–36. Interestingly, NAC has been shown to self aggregate in vitro37. In addition, a-synucleincontaining aggregates are found in glial cells in ‘multiple system atrophy’ and even in sporadic ALS (Ref. 35). The latter observation seems of great interest in view of current speculation as to the role of glial dysfunction in ALS (Ref. 11). It has been suggested that the protein mutations observed in familial PD allow a-synuclein to aggregate more easily. In idiopathic PD, however, a-synuclein aggregation could be triggered by damage to the normal protein, through free-radical-mediated oxidation or some other mechanism. The widespread detection of a-synuclein in many types of aggregates in different neurodegenerative diseases has lead to the suggestion that it could be a common ‘seeding’ factor in initiating their formation. Recently, another type of gene mutation that is linked to familial PD has been discovered. Leroy et al.40 have reported that familial PD in a German family is not linked to a mutation in a-synuclein but in the enzyme ubiquitin carboxy-terminal hydrolase L1 (UCH-L1). Immunoreactivity for this protein has been previously associated with Lewy bodies, together with ubiquitin40. The enzyme, which is very

P.B. Tran and R.J. Miller – Aggregates and neuronal death

abundant in the brain, normally hydrolyses the bonds between ubiquitin molecules or between ubiquitin and other molecules such as glutathione. The mutation detected by Leroy et al. leads to a decrease in the enzyme activity of UCH-L1. However, exactly how this produces PD is currently unclear, although a change in the state of ubiquitination of a key protein might be important. These results are analogous to those obtained from experiments on HD by Saudou et al. discussed above. However, it is also possible that the mutated UCH-L1 itself is prone to aggregate. The widespread detection of a-synuclein-related peptides in aggregates and the ability to manipulate aggregate formation via changes in ubiquitination are certainly important. However, the precise role of aggregates in each of the diseases discussed still remains to be defined. Answers will certainly be forthcoming from current studies on the emerging sociology of macromolecules. References 1 Canetti, E. (1998) Crowds and Power, Penguin 2 Deng, H-X. et al. (1993) Science 261, 1047–1051

3 Bruijn, L.I. et al. (1997) Neuron 18, 327–338 4 Bruijn, L.I. et al. (1998) Science 281, 1851–1854 5 Durham, H.D. et al. (1997) J. Neuropath. Exp. Neurol. 56, 523–530 6 Roy, J. et al. (1998) J. Neurosci. 18, 9673–9684 7 Shibata, N. et al. (1996) J. Neuropathol. Exp. Neurol. 55, 481–490 8 Koide, T. et al. (1998) Neurosci. Lett. 257, 29–32 9 Kwon, O.J. et al. (1998) Biochem. Biophys. Acta 1387, 249–256 10 Bruening, W. et al. (1999) J. Neurochem. 72, 693–699 11 Rothstein, J.D., Martin, L.J. and Kuncl, R.W. (1992) New Engl. J. Med. 326, 1464–1468 12 Gusella, J.F. and MacDonald, M.E. (1998) Curr. Opin. Neurobiol. 8, 425–430 13 Reddy, P.S. and Housman, D.E. (1997) Curr. Opin. Cell Biol. 9, 364–372 14 Davies, S.W. et al. (1998) Lancet 351, 131–133 15 Bowen, T. et al. (1998) Mol. Psychiatry 3, 266–269 16 Hackam, A.S., Wellington, C.L. and Haydon, M.R. (1998) Clin. Genet. 53, 233–242 17 Ordway, J.M. et al. (1997) Cell 91, 753–763 18 Ross, C.A. (1997) Neuron 19, 1147–1150 19 DiFiglia, M. et al. (1997) Science 277, 1990–1993 20 Davies, S.W. et al. (1997) Cell 90, 537–548 21 Jackson, G.R. et al. (1998) Neuron 21, 633–642

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Bridging cognition, the brain and molecular genetics: evidence from Williams syndrome Ursula Bellugi, Liz Lichtenberger, Debra Mills, Albert Galaburda and Julie R. Korenberg Williams syndrome (WMS) is a rare sporadic disorder that yields a distinctive profile of medical, cognitive, neurophysiological, neuroanatomical and genetic characteristics. The cognitive hallmark of WMS is a dissociation between language and face processing (relative strengths) and spatial cognition (profound impairment). Individuals with WMS also tend to be overly social, behavior that is opposite to that seen in autism. A genetic hallmark of WMS is a deletion on chromosome band 7q11.23. Williams syndrome is also associated with specific neuromorphological and neurophysiological profiles:proportional sparing of frontal,limbic and neocerebellar structures is seen using MRI; and abnormal functional organization of the neural systems that underlie both language and face processing is revealed through studies using event-related potentials.The non-uniformity in the cognitive, neuromorphological and neurophysiological domains of WMS make it a compelling model for elucidating the relationships between cognition, the brain and, ultimately, the genes. Trends Neurosci. (1999) 22, 197–207

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understanding of the neurobiological bases of WMS. The unusual cognitive profile of WMS, with strengths and weaknesses in cognitive abilities, is currently being mapped out towards achieving this goal1–10. PII: S0166-2236(99)01397-1

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Ursula Bellugi and Liz Lichtenberger are at The Salk Institute for Biological Studies, La Jolla, CA 92037, USA, Debra Mills is at the University of California, San Diego, CA 92093, USA, Albert Galaburda is at the Beth Israel Deaconess Medical Center, Boston, MA 02215, USA, and Julie R. Korenberg is at the CedarsSinai Medical Center, Los Angeles, CA 90048, USA.

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