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From: Current Clinical Neurology: Atypical Parkinsonian Disorders Edited by: I. Litvan © Humana Press Inc., Totowa, NJ
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Neurodegenerative F-Synucleinopathies
Michel Goedert and Maria Grazia Spillantini
INTRODUCTION
Parkinson’s disease (PD) is the most common movement disorder (1). Neuropathologically, it is defined by nerve cell loss in the substantia nigra and the presence there of Lewy bodies and Lewy neurites. Nerve cell loss and Lewy body pathology are also found in a number of other brain regions, such as the dorsal motor nucleus of the vagus, the nucleus basalis of Meynert, and some autonomic ganglia. Lewy bodies and Lewy neurites also constitute the defining neuropathological characteris- tics of dementia with Lewy bodies (DLB), a common late-life dementia that exists in a pure form or overlaps with the neuropathological characteristics of Alzheimer’s disease (AD) (2). Unlike PD, DLB is characterized by large numbers of Lewy bodies in cortical brain areas. Ultrastructurally, Lewy bodies and Lewy neurites consist of abnormal filamentous material. Despite the fact that the Lewy body was first described in 1912 (3), its molecular composition remained unknown until 1997.
The two developments that imparted a new direction to research on the aetiology and pathogenesis of PD and DLB were the twin discoveries that a missense mutation in the F-synuclein gene is a rare genetic cause of PD (4) and that F-synuclein is the main component of Lewy bodies and Lewy neurites in idiopathic PD, DLB, and several other diseases (5). Subsequently, the filamentous glial and neu- ronal inclusions of multiple system atrophy (MSA) were also found to be made of F-synuclein, reveal- ing an unexpected molecular link with Lewy body diseases (6–8). These findings have placed the dysfunction of F-synuclein at the center of PD and several atypical parkinsonian disorders (Table 1).
THE SYNUCLEIN FAMILY
Synucleins are abundant brain proteins whose physiological functions are only poorly understood.
The human synuclein family consists of three members—F-synuclein, G-synuclein, and L-synuclein—
which range from 127 to 140 amino acids in length and are 55–62% identical in sequence, with a similar domain organization (Fig. 1) (9–11). The amino-terminal half of each protein is taken up by imperfect 11-amino acid repeats that bear the consensus sequence KTKEGV. Individual repeats are separated by an interrepeat region of five to eight amino acids. The repeats are followed by a hydro- phobic intermediate region and a negatively charged carboxy-terminal domain; F- and G-synuclein have identical carboxy-termini. By immunohistochemistry, F- and G-synuclein are concentrated in nerve terminals, with little staining of somata and dendrites. Ultrastructurally, they are found in close proximity to synaptic vesicles (12). In contrast, L-synuclein is present throughout nerve cells in many brain regions. In rat, F-synuclein is most abundant throughout telencephalon and diencephalon, with lower levels in more caudal regions (13). G-Synuclein is distributed fairly evenly throughout the central nervous system, whereas L-synuclein is most abundant in midbrain, pons, and spinal cord,
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Table 1
F-Synuclein diseases Lewy body diseases
Idiopathic Parkinson’s disease Dementia with Lewy bodies Pure autonomic failure Lewy body dysphagia Inherited Lewy body diseases Multiple system atrophy
Olivopontocerebellar atrophy Striatonigral degeneration Shy–Drager syndrome
with much lower levels in forebrain areas. At the level of neurotransmitter systems, F-synuclein is particularly abundant in central catecholaminergic regions, G-synuclein in somatic cholinergic neu- rons, whereas L-synuclein is present in both catecholaminergic and cholinergic systems.
Synucleins are natively unfolded proteins with little ordered secondary structure that have only been identified in vertebrates (14). Experimental studies have shown that F-synuclein can bind to lipid membranes through its amino-terminal repeats, indicating that it may be a lipid-binding protein (15,16). It adopts structures rich in F-helical character upon binding to synthetic lipid membranes containing acidic phospholipids. This conformation is taken up by amino acids 1–98 and consists of two F-helical regions (residues 1–42 and 45–98) that are interrupted by a break of two amino acids (residues 43 and 44) (17,18). Residues 99–140 are unstructured. In cell lines and primary neu- rons treated with high fatty acid concentrations, F-synuclein was found to accumulate on phospholipid monolayers surrounding triglyceride-rich droplets (19). G-Synuclein bound in a similar way, but L- synuclein failed to bind to lipid droplets and remained cytosolic. Accordingly, F-synuclein has been shown to bind fatty acids in vitro, albeit with a lower affinity than physiological fatty acid-binding proteins (20,21). Both F- and G-synucleins have been shown to inhibit phospholipase D2 (22). This Fig. 1. Sequence comparison of human F-synuclein (F Syn), G-synuclein (G Syn), and L-synuclein (L Syn).
Amino acid identities between at least two of the three sequences are indicated by black bars. As a result of a common polymorphism, residue 110 of L-synuclein is either E or G.
isoform of phospholipase D localizes to the plasma membrane, where it might be involved in signal- induced cytoskeletal regulation and endocytosis. It is therefore possible that F- and G-synucleins regulate vesicular transport processes.
Little is known about posttranslational modifications of synucleins in the brain. In transfected cells,F-synuclein is constitutively phosphorylated at residues 87 and 129, with residue 129 being the predominant site (23). However, in normal brain, only a small fraction of F-synuclein is phosphory- lated at S129 (24). Casein kinase-1 and casein kinase-2 phosphorylate S129 of F-synuclein in vitro, as do several G protein-coupled receptor kinases (23–25). Phosphorylation at S129 has been reported to result in a reduced ability of F-synuclein to interact with phospholipids and phospholipase D2 in one study, whereas another study found no effect of phosphorylation on lipid binding (25,26).F-Synuclein contains four tyrosine residues, three of which are located in the carboxy-terminal region. Tyrosine kinases of the Src family phosphorylate Y125 in vitro and in transfected cells (27,28). The same site also becomes phosphorylated following exposure of cells to osmotic stress (29), suggesting that tyrosine phosphorylation of F-synuclein may be regulated. However, it remains to be seen whether synucleins are phosphorylated on tyrosines in brain. Like some other natively unfolded proteins, F-synuclein can be degraded by the proteasome in the absence of polyubiquitination (30).
Inactivation of the F-synuclein gene by homologous recombination does not lead to a neurologi- cal phenotype, with the mice being largely normal (31–35). So, a loss of function of F-synuclein is unlikely to account for its role in neurodegeneration. Analysis of mice lacking L-synuclein has simi- larly failed to reveal any gross abnormalities (34). Mice lacking G-synuclein have not yet been re- ported. Ultimately, mice lacking all three synucleins may be needed for an understanding of the physiological functions of these abundant brain proteins. One study has reported that targeted disrup- tion of the F-synuclein gene confers specific resistance of dopamine neurons to the toxic effects of 1- methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (35). The resistance to neurodegeneration appeared to be mediated through the inability of MPTP to inhibit the complex I activity of the mito- chondrial respiratory chain. A second study on two additional lines of mice without F-synuclein has reported partial resistance to MPTP in one line and normal sensitivity to the toxin in the other (36). It thus appears that F-synuclein is not obligatorily coupled to sensitivity to MPTP, but that it can influ- ence MPTP toxicity on some genetic backgrounds.
LEWY BODY DISEASES The PARK1 Locus
In 1990, Golbe, Duvoisin, and (contursi kindred) colleagues described an autosomal-dominantly inherited form of PD in an Italian-American family (37). It was the first familial form of disease in which Lewy bodies had been shown to be present. Subsequently, several more families with autopsy- confirmed Lewy body disease were identified. In 1996, Polymeropoulos and colleagues mapped the genetic defect responsible for disease in the Contursi kindred to chromosome 4q21-23 (PARK1) (38). One year later, they reported a missense mutation (A53T) in the F-synuclein gene as the cause of familial PD in this kindred and several PD families of Greek origin (Fig. 2) (4). A founder effect probably accounts for the relatively frequent occurrence of the A53T mutation in southern Italy and Greece. In 1998, a second mutation (A30P) in the F-synuclein gene was identified in a German pedigree with early-onset PD (Fig. 2) (39). In 2003, the genetic defect responsible for a familial form of PD dementia in a large family (the Iowa kindred; see ref. 40) was shown to be a triplication of a 1.6–2.0 Mb region on the long arm of chromosome 4 (41). One of an estimated 17 genes located in this region is the F-synuclein gene. These findings thus suggest that that the simple overproduction of wild-type F-synuclein may be sufficient to cause PD dementia. Moreover, polymorphic varia- tions in the 5' noncoding region of the F-synuclein gene have been found to be associated with idiopathic PD in some, but not all, studies (42–45). They probably influence the level of F-synuclein expression (46).
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Fig. 2. Mutations in the F-synuclein gene in familial Parkinson’s disease. (A) Schematic diagram of human F-synuclein. The seven repeats with the consensus sequence KTKEGV are shown as black bars. The two known missense mutations are indicated. Triplication of a region on chromosome 4 that comprises the F-synuclein gene causes familial PD dementia. Therefore, it appears likely that the overexpression of wild-type F-synuclein can also cause disease (B) Repeats in human F-synuclein. Residues 7–87 of the 140-residue protein are shown. Amino acid identities between at least five of the seven repeats are indicated by black bars. The A to P mutation at residue 30 between repeats two and three and the A to T mutation at residue 53 between repeats four and five are shown.
F-Synuclein and Sporadic Lewy Body Diseases
Shortly after the identification of the genetic defect responsible for PD in the Contursi kindred, Lewy bodies and Lewy neurites in the substantia nigra from patients with sporadic PD were shown to be strongly immunoreactive for F-synuclein (Fig. 3) (5). Subsequently, Lewy bodies and isolated Lewy body filaments from PD brain were found to be decorated by antibodies directed against F-synuclein (47,48). In addition to PD, Lewy bodies and Lewy neurites also constitute the defin- ing neuropathological characteristics of DLB. Unlike PD, DLB is characterized by large numbers of Lewy bodies and Lewy neurites in cortical brain areas. But like PD, DLB is also characterized by Lewy body pathology in the substantia nigra, and the Lewy bodies and neurites associated with DLB are strongly immunoreactive for F-synuclein (Fig. 4) (5). Filaments from Lewy bodies in DLB are decorated by F-synuclein antibodies, and their morphology closely resembles that of filaments extracted from the substantia nigra of PD brains (49,50).
TheF-synuclein filaments associated with PD and DLB are unbranched, with a length of 200–
600 nm and a width of 5–10 nm. Full-length F-synuclein is present, with its amino- and carboxy- termini being exposed on the filament surface. Protease digestion and site-directed spin labeling studies have shown that the core of the F-synuclein filaments extends over a stretch of about 70 amino acids, from residues 34–101 (51,52). This sequence overlaps almost entirely with the lipid- binding region of F-synuclein. Biochemically, the presence of F-synuclein inclusions has been found to correlate with the accumulation of F-synuclein. Of the three human synucleins, only F-synuclein is associated with the filamentous inclusions of Lewy body diseases. Using specific antibodies, G- and L-synucleins are absent from these inclusions (5,50).
Fig. 3. Substantia nigra from patients with Parkinson’s disease immunostained for F-synuclein. (A) Two pigmented nerve cells, each containing an F-synuclein-positive Lewy body. Lewy neurites (small arrows) are also immunopositive. Scale bar, 20 μm. (B) Pigmented nerve cell with two F-synuclein-positive Lewy bodies.
Scale bar 8 μm. (C)F-Synuclein-positive extracellular Lewy body. Scale bar, 4 μm.
Prior to this work, ubiquitin staining had been the preferred immunohistochemical marker of Lewy body pathology (53). By double-labeling immunohistochemistry, the number of F-synuclein-posi- tive structures was greater than that stained for ubiquitin, suggesting that the ubiquitination of F-synuclein occurs after its assembly into filaments (50,54,55). In DLB brain, F-synuclein is mostly mono- or diubiquitinated (54–56). Phosphorylation and nitration are two additional posttranslational modifica- tions of filamentous F-synuclein. Phosphorylation at S129 has been documented in Lewy bodies and Lewy neurites by mass spectrometry and phosphorylation-dependent antibodies (24). It has been suggested that phosphorylation of F-synuclein at S129 may trigger filament assembly. However, it remains to be determined whether it occurs before or after filament assembly in human brain. Nitra- tion of tyrosine residues in proteins is the result of the action of oxygen, nitric oxide, and their prod- ucts, such as peroxynitrite. Nitration of filamentous F-synuclein was found using antibodies specific for nitrated tyrosine residues (57). As for ubiquitin, staining for nitrotyrosine was less extensive than staining for F-synuclein, suggesting that nitration of F-synuclein occurs after its assembly into filaments.
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Fig. 4. Brain tissue from patients with dementia with Lewy bodies immunostained for F-synuclein.
(A,B) F-Synuclein-positive Lewy bodies and Lewy neurites in substantia nigra stained with antibodies recognizing the amino-terminal (A) or the carboxy-terminal (B) region of F-synuclein. Scale bar, 100 μm (in B, for A,B). (C,D)F-Synuclein-positive Lewy neurites in serial sections of hippocampus stained with antibodies recognizing the amino-terminal (C) or the carboxy-terminal (D) region of F-synuclein. Scale bar, 80 μm (in D, for C,D). (E),F-Synuclein-positive intraneuritic Lewy body in a Lewy neurite in substantia nigra. Scale bar, 40 μm.
Lewy body pathology is also the defining feature of several other, rarer diseases. Cases in which F-synuclein immunoreactivity has been examined have shown its presence. Thus, pure autonomic failure is characterized by F-synuclein-positive Lewy body pathology in sympathetic ganglia (58,59).
Incidental Lewy body disease describes the presence of small numbers of Lewy bodies and Lewy neurites in the absence of clinical symptoms. It is observed in 5–10% of the general population over the age of 60, and is believed to represent a preclinical form of Lewy body disease (60,61). In cases with incidental Lewy body disease, the first pathological F-synuclein-immunoreactive structures do not form in dopaminergic nerve cells of the substantia nigra, but in non-catecholaminergic neurons of the dorsal glossopharyngeus-vagus complex, projection neurons of the reticular zone, and in the locus coeruleus, raphe nuclei, and the olfactory system (62). Incidental Lewy body disease may be at one end of the spectrum of Lewy body diseases, with DLB at the other end and PD somewhere in between. The Lewy body pathology that is sometimes associated with other neurodegenerative dis- eases, such as Alzheimer’s disease, the parkinsonism-dementia complex of Guam, and neurodegeneration with brain iron accumulation type I has also been shown to be immunoreactive for F-synuclein (7,63,64). However, it is not an invariant feature of these diseases. Nevertheless, under- standing why F-synuclein pathology develops in a proportion of cases in these apparently unrelated conditions may shed light on the mechanisms operating in the diseases defined by the presence of Lewy body pathology.
F-Synuclein and Inherited Lewy Body Diseases
The discovery that F-synuclein is the major component of Lewy bodies and Lewy neurites in idiopathic PD raised the question of the neuropathological picture in cases of familial PD with mutations
in the F-synuclein gene. Widespread Lewy bodies and Lewy neurites immunoreactive for F-synuclein were described in the brainstem pigmented nuclei, hippocampus, and temporal cortex in two indi- viduals from an Australian family of Greek origin with the A53T mutation (65). Lewy neurites ac- counted for much of the pathology, underscoring their relevance for the neurodegenerative process.
As in other Lewy body diseases, the F-synuclein pathology was neuronal. However, it was much more widespread than in idiopathic PD. Similarly extensive deposits of F-synuclein have been found in a case of the Contursi kindred (66). In addition, substantial tau pathology was reported in this case, suggesting that a missense mutation in the F-synuclein gene can somehow also lead to tau deposition.
This is in line with recent studies that have reported that tau staining is frequently associated with Lewy bodies and that F-synuclein assemblies can induce tau filament formation in vitro (67,68).
Work so far has been on cases with the A53T mutation. Nothing is known about the neuropathology in cases of familial PD with the A30P mutation in F-synuclein.
In the Iowa kindred with a triplication of the region of chromosome 4 that encompasses the F-synuclein gene, abundant and widespread F-synuclein-immunoreactive Lewy bodies and Lewy neurites have been described (69). The pathological picture resembled that of DLB. In addition, F-synuclein-positive glial inclusions were also present, suggesting that overproduction of wild-type F-synuclein may be a mechanism leading to the formation of glial cytoplasmic inclusions.
A genetic locus (PARK3) that is linked to autosomal-dominantly inherited PD has been mapped to chromosome 2p13 (70). Although the underlying genetic defect is not yet known, neuropathological analysis has shown the presence of F-synuclein-immunoreactive Lewy bodies and Lewy neurites, with a distribution resembling PD (71). It appears likely that the defective PARK3 gene product functions in the pathway that leads from soluble to filamentous F-synuclein.
Subsequent to the identification of the A53T mutation in the F-synuclein gene, a missense muta- tion (I93M) in the ubiquitin carboxy-terminal hydrolase L1 (UCHL1) gene was reported in a patient with familial PD (PARK5) (72). UCHL1 is an abundant neuron-specific deubiquitinating enzyme.
This mutation resulted in a partial loss of the catalytic activity of the thiol protease, which could, in principle, lead to protein aggregation. However, no information is available about neuropathology in this family, and the presence of Lewy body pathology with the accumulation of F-synuclein remains a possibility. In 1998, mutations in the gene parkin (PARK2) were identified as the genetic cause of a form of autosomal-recessive juvenile parkinsonism (AR-JP) (73). These mutations constitute a common cause of parkinsonism, especially when the age of onset is relatively early in life. Parkin functions as a ubiquitin ligase, and the known mutations are believed to lead to a loss of its function (74). It is possible that one or more proteins accumulate as a result, and that this causes nerve cell degeneration. Parkin has been reported to be present in Lewy bodies and to ubiquitinate a minor, O-glycosylated form of F-synuclein (75–77). However, with one exception, mutations in parkin have, so far, not been found to lead to the development of Lewy body pathology (78–80). Further- more, a more rigorous study using well-characterized anti-parkin antibodies failed to confirm colocalization with F-synuclein (81). In 2002, mutations in the gene DJ-1 were found to cause another form of AR-JP (PARK7), presumably through a loss of function mechanism (21). The physi- ological function of DJ-1 remains to be identified. To date, there is no information about the neuro- pathological features of cases with mutations in DJ-1. DJ-1-immunoreactivity does not colocalize with Lewy bodies or Lewy neurites (83). It will be interesting to see whether there are links between the mechanisms that lead to Lewy body diseases and AR-JP, or whether they constitute distinct disease entities.
MULTIPLE SYSTEM ATROPHY
After the discovery of F-synuclein in the filamentous lesions of Lewy body diseases, multiple system atrophy (MSA) was also shown to be characterized by filamentous F-synuclein deposits (Fig. 5) (6–8). Lewy body pathology is not generally observed in MSA. Instead, glial cytoplasmic inclusions,
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Fig. 5. Brain tissue from patients with multiple system atrophy immunostained for F-synuclein. (A–D) F-Synuclein-immunoreactive oligodendrocytes and nerve cells in white matter of pons (A,B,D) and cer- ebellum (C) identified with antibodies recognizing the amino-terminal (A,C) or the carboxy-terminal (B,D) region ofF-synuclein. (E,F) F-Synuclein-immunoreactive oligodendrocytes and nerve cells in gray matter of pons (E) and frontal cortex (F) identified with antibodies recognizing the amino-terminal (E) or the carboxy-terminal (F) region of F-synuclein. Arrows identify representative examples of each of the characteristic lesions stained for F-synuclein: cytoplasmic oligodendroglial inclusions (A,F), cytoplasmic nerve cell inclusions (B), nuclear oli- godendroglial inclusion (C), neuropil threads (D), and nuclear nerve cell inclusion (E). Scale bars, 33 μm (in E);
50μm (in F, for A–D,F).
which consist of filamentous aggregates, are the defining neuropathological feature of MSA (84).
They are found mostly in the cytoplasm and, to a lesser extent, in the nucleus of oligodendrocytes.
Inclusions are also observed in the cytoplasm and nucleus of some nerve cells, and in neuropil threads.
The principal brain regions affected are the substantia nigra, striatum, locus coeruleus, pontine nu- clei, inferior olives, cerebellum, and spinal cord. Typically, nerve cell loss and gliosis are observed.
The formation of glial cytoplasmic inclusions might be the primary lesion that will eventually com- promise nerve cell function and viability. Nerve cell loss and clinical symptoms of MSA in the pres- ence of only glial cytoplasmic inclusions have been described (85).
Glial cytoplasmic inclusions are strongly immunoreactive for F-synuclein, and filaments isolated from the brains of patients with MSA are labeled by F-synuclein antibodies (6–8). No staining is obtained with antibodies specific for G- or L-synuclein. As in DLB, assembled F-synuclein is nitrated and phosphorylated at S129, and the number of F-synuclein-positive structures exceeds that stained by anti-ubiquitin antibodies, indicating again that the accumulation of F-synuclein precedes ubiquitination. The filament morphologies and their staining characteristics were found to be similar to those of filaments extracted from the brains of patients with PD and DLB (8). Two distinct mor- phologies were observed. Some filaments showed a distinctly twisted appearance, alternating in width between 5 and 18 nm, with a period of 70–90 nm. The other class of filaments had a more uniform width of approx 10 nm. The formation of inclusions was shown to correlate with decreased solubility ofF-synuclein. However, the reduction in solubility was less marked than in PD and DLB (86).
This work has revealed an unexpected molecular link between MSA and Lewy body diseases. The main difference is that in MSA most of the F-synuclein pathology is found in glial cells, whereas in Lewy body diseases most of the pathology is present in nerve cells.
SYNTHETIC F-SYNUCLEIN FILAMENTS
Recombinant F-synuclein assembles into filaments that share many of the morphological and ultrastructural characteristics of filaments present in humans (Fig. 6) (87–89). Assembly is a nucle- ation-dependent process and occurs through the repeats in the amino-terminal half (90,91). The carboxy-terminal region, in contrast, inhibits assembly (87,92,93). It has been reported that the struc- tural transformation involves a partially folded intermediate (94). The A53T mutation increases the rate of filament assembly, indicating that this might be its primary effect (88,89,92,95). The effect of the A30P mutation on the assembly of F-synuclein is less clear. One study reported an increase in filament assembly (95), another study found no change (92), and a third found an inhibitory effect on assembly (96). The third study showed that the A30P monomer was consumed at a similar rate to wild-typeF-synuclein, leading to the suggestion that oligomeric, nonfibrillar F-synuclein species might be detrimental to nerve cells. The A30P mutation has also been shown to produce reduced binding of F-synuclein to rat brain vesicles in vitro (16,97), indicating that this might lead to its accumulation over time, resulting in filament assembly. Consistent with this, it has been shown that theF-helical conformation that is induced and stabilized by the interaction between F-synuclein and acidic phospholipids prevents its assembly into filaments (98). Thus, lipid binding and self-assembly ofF-synuclein appear to be mutually exclusive.
The assembly of F-synuclein is accompanied by the transition from random coil to a G-pleated sheet (92,99). By electron diffraction, F-synuclein filaments have been found to show a conforma- tion characteristic of amyloid fibers. Under the conditions of these experiments, G- and L-synuclein failed to assemble into filaments, and remained in a random coil conformation (92,100). This behav- ior is consistent with their absence from the filamentous lesions of the F-synuclein diseases. When incubated together with F-synuclein, G- and L-synuclein markedly inhibit the fibrillation of F-synuclein, suggesting that they could indirectly influence the pathogenesis of Lewy body diseases and MSA (101–103).
The sequence requirements for the assembly of F-synuclein are only incompletely understood.
One difference between F- and G-synuclein is the presence of a stretch of 11 amino acids (residues 73–83) in F-synuclein that is missing from G-synuclein. It has been suggested that this sequence is both necessary and sufficient for the assembly ofF-synuclein and that its absence explains why G-synuclein does not form filaments (101,104). A similar sequence is present in L-synuclein, but it differs at four positions from residues 73–83 of F-synuclein. Other studies have suggested that residues 71–84 (105), 68–76 (106), or 66–74 (107) of F-synuclein are essential for its ability to form filaments.
Many studies have identified factors that influence the rate and/or extent of F-synuclein assembly in vitro. Methionine-oxidized and nitrated F-synucleins were found to be poor at assembling into
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filaments compared with the unmodified proteins (108,109). For the nitrated protein, stable oligo- meric intermediates formed that were reminiscent of protofibrils. A similar inhibition of the protofibril to fibril conversion of F-synuclein has been reported for a number of catecholamines, including oxidized dopamine (110). This has led to the suggestion that nonvesicular, cytoplasmic dopamine may be harmful by sustaining the accumulation of potentially toxic protofibrils. Factors that have been reported to accelerate the fibrillation of F-synuclein include cytochrome C (111), some pesti- cides (112), several di- and trivalent metal ions (113), sulphated glycosaminoglycans (114), polyamines (115), and oxidized glutathione (116).
The aforementioned substances were used largely because they had previously been implicated in the pathogenesis of PD. These findings in vitro are consistent with many of these hypotheses. How- ever, the true relevance of these factors for the transition from soluble to filamentous F-synuclein in nerve cells in the brain remains to be established.
ANIMAL MODELS Transgenic Mice
Experimental animal models of F-synucleinopathies are being produced by a number of laborato- ries (Table 2). They are essential for studying disease pathogenesis and for identifying ways to inter- fere with the disease process. Several transgenic mouse lines that express wild-type or mutant human Fig. 6. Filaments extracted from the brains of patients with dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) or assembled from bacterially expressed human F-synuclein (SYN) were decorated by an anti-F-synuclein antibody. The gold particles conjugated to the second antibody appear as black dots. Scale bar, 100 nm.
F-synuclein in nerve cells have been described. One study has reported on the effects of F-synuclein overexpression in glial cells. In all published studies, mice developed numerous F-synuclein-immu- noreactive cell bodies and processes.
The first study to be published described the expression of wild-type human F-synuclein driven by the human platelet-derived growth factor-G promoter (117). The mice developed cytoplasmic and nuclear intraneuronal inclusions in neocortex, hippocampus, olfactory bulb, and substantia nigra.
These inclusions were F-synuclein-immunoreactive, with some being ubiquitin-positive as well. By electron microscopy, they consisted of amorphous, nonfilamentous material. The mice showed a reduction in dopaminergic nerve terminals in the striatum and signs of impaired motor function, but they failed to exhibit nerve cell loss in the substantia nigra. Two studies have reported the expression of wild-type and A53T or A30P mutant human F-synuclein driven by the murine Thy-1 promoter. In one study (118), mice expressing wild-type or A53T mutant F-synuclein developed an early-onset motor impairment that was associated with axonal degeneration in the ventral roots and signs of muscle atrophy. Some F-synuclein inclusions were argyrophilic and ubiquitin-immunoreactive, but they lacked the filaments characteristic of the human diseases. In the second study (119,120), mice expressing A30P mutant F-synuclein developed a neurodegenerative phenotype consisting of the accumulation of protease-resistant human F-synuclein phosphorylated at S129. Occasional inclu- sions were also ubiquitin-positive. By electron microscopy, filamentous structures were observed, although they were not shown to be made of F-synuclein. The transgenic mice showed a progressive deterioration of motor function. It remains to be seen whether the accumulation of mutant F-synuclein was accompanied by nerve cell loss.
Three studies have described the expression of wild-type and A53T or A30P mutant human F-synuclein under the control of the murine prion protein promoter (121–123). A severe movement disorder was observed that was accompanied by the accumulation of F-synuclein in nerve cells and their processes. One study documented the presence of abundant F-synuclein filaments in brain and spinal cord of mice transgenic for A53T F-synuclein (121). The formation of filamentous inclusions closely correlated with the appearance of clinical symptoms, suggesting a possible cause-and-effect relationship. A minority of inclusions was ubiquitin-immunoreactive. Signs of Wallerian degenera- tion were much in evidence in ventral roots, but nerve cell numbers in the ventral horn of the spinal cord were unchanged. A major difference with PD was the absence of significant pathology in dopam-
Table 2
Animal Models of F-Synucleinopathies
Toxin Model Genetic Models
Rotenone Worm Fly Mouse Rat Marmoset
Dopaminergic + + + – + +
Nerve cell loss
Filamentous + – + + u u
F-Synuclein inclusions
Motor deficits + – + + u u
Complex I + u u u u u
Inhibition
The rotenone model is in the rat. The genetic models in worm, fly, and mouse are based on the transgenic expression of human F-synuclein (wild-type and mutant), whereas the rat and marmoset models are based on viral vector-mediated transfer of human F-synuclein (wild-type and mutant). u = unknown.
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inergic nerve cells of the substantia nigra. In mice, these neurons appear to be relatively resistant to the effects of F-synuclein expression. This is further supported by reports showing that the expres- sion of wild-type and mutant human F-synuclein under the control of the tyrosine hydroxylase pro- moter did not lead to the formation of inclusions or neurodegeneration (124,125).
Mouse lines transgenic for wild-type human F-synuclein under the control of a proteolipid protein promoter were generated to give high levels of expression in oligodendroglia (126). Expression of humanF-synuclein phosphorylated at S129 was obtained, but there was no sign of argyrophilic glial cytoplasmic inclusions or abnormal filaments. Behavioral changes were also not observed.
Transgenic Flies and Worms
One of the first reports describing the overexpression of F-synuclein made use of Drosophila melanogaster, an organism without synucleins (127). Expression of wild-type and A30P or A53T mutant human F-synuclein in nerve cells of D. melanogaster resulted in the formation of filamentous Lewy body–like inclusions and an age-dependent loss of some dopaminergic nerve cells (Table 2).
The inclusions were F-synuclein- and ubiquitin-immunoreactive. An age-dependent locomotor de- fect was observed that could be reversed by the administration of L-DOPA or several dopamine agonists, underscoring the validity of this model for PD (128). Overexpression of wild-type and A53T mutant human F-synuclein in nerve cells of Caenorhabditis elegans resulted in a loss of dopam- inergic nerve cells and motor deficits, in the apparent absence of filamentous F-synuclein inclusions (Table 2) (129).
Disease models in D. melanogaster and C. elegans offer some advantages over mouse models, in particular with regard to the speed and relative ease with which genetic modifiers of disease pheno- type can be discovered and pharmacological modifiers can be screened. Coexpression of human heat- shock protein 70 alleviated the toxicity of F-synuclein in transgenic flies (130). Conversely, a reduction in the fly chaperone system exacerbated nerve cell loss. Increasing chaperone activity through the administration of geldanamycin delayed neurodegeneration in transgenic flies (131). It thus appears that chaperones can modulate the neurotoxicity resulting from the overexpression of humanF-synuclein.
Viral Vector-Mediated Gene Transfer
Viral vector-mediated gene transfer differs from standard transgenic approaches by being tar- geted to a defined region of the central nervous system and by being inducible at any point during the life of the animal. Recombinant adeno-associated virus and recombinant lentivirus vector sys- tems have been used to express wild-type and mutant F-synuclein in the substantia nigra of rat and marmoset (Table 2).
In the rat, expression of F-synuclein was maximal 2–3 wk after virus injection (132–134). At 8–10 wk, numerous swollen, dystrophic axons and dendrites were observed in conjunction with F-synuclein inclu- sions. Nerve cell bodies contained Lewy body–like inclusions and substantial nerve cell loss (30–
80%) was present in the substantia nigra. The inclusions were F-synuclein-positive and ubiquitin-negative, but it remains to be determined whether they were also filamentous. Behavior- ally, about a quarter of animals were impaired in spontaneous and drug-induced motor behaviors.
Similar findings were reported for wild-type and A30P or A53T mutant human F-synuclein. One study has reported that degeneration of transduced nigral cells was seen upon expression of human, but not rat, F-synuclein (134). In the marmoset, dopaminergic nerve cells of the substantia nigra were transduced with high efficiency and human F-synuclein was expressed (135). Similar to the rat, F-synuclein-positive inclusions were observed, together with a loss of 40–75% of tyrosine hydroxylase-positive neurons. Overexpression of disease-causing gene products by using recom- binant viral vectors constitutes a promising way forward for modeling human neurodegenerative diseases in a number of species, including primates.
Rotenone Neurotoxicity
A model of F-synuclein pathology has been developed in the rat by using the chronic administra- tion of the pesticide rotenone, a high-affinity inhibitor of complex I, one of the five enzyme com- plexes of the inner mitochondrial membrane involved in oxidative phosphorylation (136,137). The rats developed a progressive degeneration of nigrostriatal neurons and Lewy body-like inclusions that were immunoreactive for F-synuclein and ubiquitin (Table 2). Behaviorally, they showed bradykinesia, postural instability, and some evidence of resting tremor. Using this regime of rotenone administration, the inhibition of complex I was only partial, indicating that a bioenergetic defect with ATP (adenosine 5'-triphosphate) depletion was probably not involved. Instead, oxidative damage might have contributed to this condition, as partial inhibition of complex I by rotenone is known to stimulate the production of reactive oxygen species. It would therefore seem that oxidative stress can lead to the assembly of F-synuclein into filaments. Although it remains to be seen how robust a model rotenone administration is, it appears clear that it can lead to the degeneration of nigrostriatal dopaminergic nerve cells in association with F-synuclein-positive inclusions. This has so far not been achieved following the administration of either 6-hydroxydopamine or MPTP, the two most widely used toxin models of PD. It has been reported that the systemic administration of rotenone leads to the specific degeneration of dopaminergic nerve cells in the substantia nigra. However, a subsequent study has described additional nerve cell loss in the striatum (138), raising the question of how valid a model rotenone intoxication is for PD.
CONCLUSION
The relevance of F-synuclein for the neurodegenerative process in Lewy body diseases and MSA is now well established. The development of experimental models of F-synucleinopathies has opened the way to the identification of the detailed mechanisms by which the formation of inclusions causes disease. These model systems have also made it possible to identify disease modifiers (enhancers and suppressors) that may well lead to the development of the first mechanism-based therapies for these diseases. At a conceptual level, it will be important to understand whether F-synuclein has a role to play in disorders, such as the autosomal-recessive juvenile forms of parkinsonism caused by muta- tions in the parkin and DJ-1 genes, or whether there are entirely separate mechanisms by which the dopaminergic nerve cells of the substantia nigra degenerate in PD and in inherited disorders with parkinsonism.
BOX 1: MAJOR RESEARCH ISSUES
The new work has established that a neurodegenerative pathway leading from soluble to insoluble, filamentous F-synuclein is central to Lewy body diseases and multiple system atro- phy. The study of familial forms of diseases with filamentous F-synuclein inclusions has re- vealed that missense mutations in the F-synuclein gene or overexpression of wild-type protein are sufficient to cause neurodegeneration. It appears likely that these genetic defects induce the assembly of F-synuclein into filaments. What causes assembly of F-synuclein in the much more common cases of sporadic disease remains to be discovered. In normal brain, the bio- physically driven propensity of F-synuclein to undergo ordered self-assembly is probably coun- terbalanced by a number of factors, including the ability of cells to dispose of the protein prior to assembly. Other factors may include the binding of F-synuclein to lipid membranes and its interactions with G- and L-synucleins. Identification of the detailed mechanisms at work con- stitutes an important area for future research. In particular, we need to know more about the factors that regulate the production of F-synuclein and the mechanisms that ensure its degrada- tion. We need to understand what the toxic F-synuclein species are and how they exert their effects. It appears probable that all familial forms of Lewy body disease are caused by genetic defects that act at the level of the neurodegenerative F-synuclein pathway. It remains to be seen
90 Goedert and Spillantini
whether they are mechanistically linked to familial forms of parkinsonism that lead to nerve cell loss in the substantia nigra, without formation of Lewy body pathology. The availability of ever better animal models of the F-synucleinopathies opens the way to the identification of genetic and pharmacological modifiers (enhancers and suppressors) of the disease process.
This will be essential for a better understanding of the pathogenesis of Lewy body diseases and multiple system atrophy and for the discovery of novel therapeutic avenues.
REFERENCES
1. Jellinger KA, Mizuno Y. Parkinson’s disease. In: Dickson D, ed. Neurodegeneration: The Molecular Pathology of Dementia and Movement Disorders. Basel: ISN Neuropath Press, 2003: 159–187.
2. Ince PG, McKeith IG. Dementia with Lewy bodies. In: Dickson D, ed. Neurodegeneration: The Molecular Pathology of Dementia and Movement Disorders. Basel: ISN Neuropath Press, 2003:188–199.
3. Lewy F. Paralysis agitans. In: Lewandowski M, Abelsdorff G, eds. Handbuch der Neurologie. Berlin: Springer Verlag, 1912: 920–933.
4. Polymeropoulos MH, Lavedan C, Leroy E, et al. Mutation in the F-synuclein gene identified in families with Parkinson’s disease. Science 1997;276:2045–2047.
5. Spillantini MG, Schmidt ML, Lee VMY, Trojanowski JQ, Jakes R, Goedert M. F-Synuclein in Lewy bodies. Nature 1997;388:839–840.
6. Wakabayashi K, Yoshimoto M, Tsuji S, Takahashi H. F-Synuclein immunoreactivity in glial cytoplasmic inclusions in multiple system atrophy. Neurosci Lett 1998;249:180–182.
7. Tu PH, Galvin JE, Baba M, et al. Glial cytoplasmic inclusions in white matter oligodendrocytes of multiple system atrophy brains contain insoluble F-synuclein. Ann Neurol 1998;44:415–422.
8. Spillantini MG, Crowther RA, Jakes R, Cairns NJ, Lantos PL, Goedert M. Filamentous F-synuclein inclusions link multiple system atrophy with Parkinson’s disease and dementia with Lewy bodies. Neurosci Lett 1998;251:205–208.
9. Ueda K, Fukushima H, Masliah E, et al. Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc Natl Acad Sci USA 1993;90:11282–11286.
10. Jakes R, Spillantini MG, Goedert M. Identification of two distinct synucleins from human brain. FEBS Lett 1994;345:27–32.
11. Ji H, Liu YE, Jia T, et al. Identification of a breast cancer-specific gene, BCSG1, by direct differential cDNA sequenc- ing. Cancer Res 1997;57:759–764.
12. Clayton DF, George JM. Synucleins in synaptic plasticity and neurodegenerative disorders. J Neurosci Res 1999;58:120–129.
13. Li JY, Jensen PH, Dahlström A. Differential localization of F-, G- and L-synucleins in the rat CNS. Neuroscience 2002;113:463–478.
14. Weinreb PH, Zhen W, Poon AW, Conway KA, Lansbury PT. NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded. Biochemistry 1996;35:13709–13715.
15. Davidson WS, Jonas A, Clayton DF, George, JM. Stabilization of F-synuclein secondary structure upon binding to synthetic membranes. J Biol Chem 1998;273:9443–9449.
16. Jensen PH, Nielsen MH, Jakes R, Dotti CG, Goedert M. Binding of F-synuclein to rat brain vesicles is abolished by familial Parkinson’s disease mutation. J Biol Chem 1998;273:26292–26294.
17. Chandra S, Cheng X, Rizo J, Jahn R, Südhof TC. A broken F-helix in folded F-synuclein. J Biol Chem 2003;278:
15313–15318.
18. Bussell R, Eliezer D. A structural and functional role for 11-mer repeats in F-synuclein and other exchangeable lipid binding proteins. J Mol Biol 2003;329:763–778.
19. Cole NB, Murphy DD, Grider T, Rueter S, Brasaemle D, Nussbaum RL. Lipid droplet binding and oligomerization properties of the Parkinson’s disease protein F-synuclein. J Biol Chem 2002;277:6344–6352.
20. Sharon R, Goldberg MS, Bar-Joseph I, Betensky RA, Shen J, Selkoe DJ. F-Synuclein occurs in lipid-rich high molecu- lar weight complexes, binds fatty acids, and shows homology to the fatty acid-binding proteins. Proc Natl Acad Sci USA 2002;98:9110–9115.
21. Sharon R, Bar-Joseph I, Frosch MP, Walsh DM, Hamilton JA, Selkoe DJ. The formation of highly soluble oligomers of F-synuclein is regulated by fatty acids and enhanced in Parkinson’s disease. Neuron 2003;37:583–595.
22. Jenco RM, Rawlingson A, Daniels B, Morris AJ. Regulation of phospholipase D2: selective inhibition of mammalian phospholipase D isoenzymes by F- and G-synucleins. Biochemistry 1998;37:4901–4909.
23. Okochi M, Walter J, Koyama A, et al. Constitutive phosphorylation of the Parkinson’s disease–associated F-synuclein.
J Biol Chem 2000;275:390–397.
24. Fujiwara H, Hasegawa M, Dohmae N, et al. F-Synuclein is phosphorylated in synucleinopathy lesions. Nature Cell Biol 2002;4:160–164.
25. Pronin AN, Morris AJ, Surguchov A, Benovic JL. Synucleins are a novel class of substrates for G protein–coupled receptor kinases. J Biol Chem 2000;275:26515–26522.
26. Ahn BH, Rhim H, Kim SY, et al. F-Synuclein interacts with phospholipase D isozymes and inhibits pervanadate- induced phospholipase D activation in human embryonic kidney-293 cells. J Biol Chem 2002;277:12334–12342.
27. Nakamura T, Yamashita H, Takahashi T, Nakamura S. Activated Fyn phosphorylates F-synuclein at tyrosine residue 125. Biochem Biophys Res Commun 2001;280:1085–1092.
28. Ellis CE, Schwartzberg PL, Grider TL, Fink DW, Nussbaum RL. F-Synuclein is phosphorylated by members of the Src family of protein tyrosine kinases. J Biol Chem 2001;276:3879–3884.
29. Nakamura T, Yamshita H, Nagano Y, et al. Activation of Pyk2/RAFTK induces tyrosine phosphorylation of F-synuclein via Src-family kinases. FEBS Lett 2002;521:190–194.
30. Tofaris GK, Layfield R, Spillantini MG. F-Synuclein metabolism and aggregation is linked to ubiquitin-independent degradation by the proteasome. FEBS Lett 2001;509:22–26.
31. Abeliovich A, Schmitz Y, Farinas I, et al. Mice lacking F-synuclein display functional deficits in the nigrostriatal dopamine system. Neuron 2000;25:239–252.
32. Cabin DE, Shimazu K, Murphy D, et al. Synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking F-synuclein. J Neurosci 2002;22:8797–8807.
33. Specht CG, Schoepfer R. Deletion of the F-synuclein locus in a subpopulation of C57BL/6J inbred mice. BMC Neurosci 2001;2:11.
34. Ninkina N, Papachroni K, Robertson DC, et al. Neurons expressing the highest levels of L-synuclein are unaffected by targeted inactivation of the gene. Mol Cell Biol 2003;23:8233–8245.
35. Dauer W, Kholodilov N, Vila M, et al. Resistance of F-synuclein null mice to the parkinsonian neurotoxin MPTP. Proc Natl Acad Sci USA 2002;99:14254–14259.
36. Schlüter OM, Fornai F, Alessandri MG, et al. Role of F-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- induced parkinsonism in mice. Neuroscience 2003;118:985–1002.
37. Golbe LI, Di Iorio G, Sanges G, et al. Clinical genetic analysis of Parkinson’s disease in the Contursi kindred. Ann Neurol 1990;27:276–282.
38. Polymeropoulos MH, Higgins JJ, Golbe LI, et al. Mapping of a gene for Parkinson’s disease to chromosome 4q21-q23.
Science 1996;274:1197–1199.
39. Krüger T, Kuhn W, Müller T, et al. Ala30Pro mutation in the gene encoding F-synuclein in Parkinson’s disease. Nature Genet 1998;18:106–108.
40. Muenter MD, Forno LS, Hornykiewicz O, et al. Hereditary form of Parkinsonism-dementia. Ann Neurol 1998;43:768–781.
41. Singleton AB, Farrer M, Johnson J, et al. F-Synuclein locus triplication causes Parkinson’s disease. Science 2003;302:841.
42. Krüger R, Vieira-Saecker AMM, Kuhn W, et al. Increased susceptibility to sporadic Parkinson’s disease by a certain combinedF-synuclein/apolipoprotein E genotype. Ann Neurol 1998;45:611–617.
43. Tan EK, Matsuura T, Nagamitsu S, Khajavi M, Jankovic J, Ashizawa T. Polymorphism of NACP-Rep1 in Parkinson’s disease: an etiologic link with essential tremor? Neurology 2000;54:1195–1198.
44. Khan N, Graham E, Dixon P, et al. Parkinson’s disease is not associated with the combined F-synuclein/apolipoprotein E susceptibility genotype. Ann Neurol 2001;49:665–668.
45. Farrer M, Maraganore DM, Lockhart P, et al. F-Synuclein gene haplotypes are associated with Parkinson’s disease.
Hum Mol Genet 2001;10:1847–1851.
46. Chiba-Falek O, Nussbaum RL. Effect of allelic variation at the NACP-Rep1 repeat upstream of the F-synuclein gene (SNCA) on transcription in a cell culture luciferase reporter system. Hum Mol Genet 2001;10:3101–3109.
47. Arima K, Ueda K, Sunohara N, et al. Immunoelectron microscopic demonstration of NACP/F-synuclein epitopes on the filamentous component of Lewy bodies in Parkinson’s disease and in dementia with Lewy bodies. Brain Res 1998;808:93–100.
48. Crowther RA, Daniel SE, Goedert M. Characterisation of isolated F-synuclein filaments from substantia nigra of Parkinson’s disease brain. Neurosci Lett 2000;292:128–130.
49. Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M. F-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies. Proc Natl Acad Sci USA 1998;95:6469–6473.
50. Baba M, Nakajo S, Tu PS, et al. Aggregation of F-synuclein in Lewy bodies of sporadic Parkinson’s disease and dementia with Lewy bodies. Am J Pathol 1998;152:879–884.
51. Miake H, Mizusawa H, Iwatsubo T, Hasegawa M. Biochemical characterization of the core structure of F-synuclein filaments. J Biol Chem 2002;277:19213–19219.
52. Der-Sarkissian A, Jao CC, Chen J, Langen R. Structural organization of F-synuclein fibrils studied by site-directed spin labeling. J Biol Chem 2003;278:37530–37535.
53. Kuzuhara S, Mori H, Izumiyama N, Yoshimura M, Ihara Y. Lewy bodies are ubiquitinated: a light and electron micro- scopic immunocytochemical study. Acta Neuropathol 1988;75:345–353.
54. Sampathu DM, Giasson BI, Pawlyk AC, Trojanowski JQ, Lee VMY. Ubiquitination of F-synuclein is not required for formation of pathological inclusions in F-synucleinopathies. Am J Pathol 2003;163:91–100.
92 Goedert and Spillantini
55. Tofaris GK, Razzaq A, Ghetti B, Lilley K, Spillantini MG. Ubiquitination of F-synuclein in Lewy bodies is a patho- logical event not associated with impairment of proteasome function. J Biol Chem 2003;278:44405–44411.
56. Hasegawa M, Fujiwara H, Nonaka T, et al. Phosphorylated F-synuclein is ubiquitinated in F-synucleinopathy lesions.
J Biol Chem 2002;277:49071–49076.
57. Giasson BI, Duda JE, Murray IV, et al. Oxidative damage linked to neurodegeneration by selective F-synuclein nitra- tion in synucleinopathy lesions. Science 2000;290:985–989.
58. Arai K, Kato N, Kashiwado K, Hattori T. Pure autonomic failure in association with human F-synucleinopathy. Neurosci Lett 2000;296:171–173.
59. Kaufmann H, Hague K, Perl D. Accumulation of alpha-synuclein in autonomic nerves in pure autonomic failure. Neu- rology 2001;56: 980–981.
60. Perry RH, Irving D, Tomlinson BE. Lewy body prevalence in the aging brain: relationship to neuropsychiatric disor- ders, Alzheimer-type pathology and catecholaminergic nuclei. J Neurol Sci 1990;100:223–233.
61. Saito Y, Kawashima A, Ruberu NN, et al. Accumulation of phosphorylated F-synuclein in aging human brain. J Neuropathol Exp Neurol 2003;62:644–654.
62. Del Tredici K, Rüb U, De Vos RAI, Bohl JRE, Braak H. Where does Parkinson disease pathology begin in the brain?
J Neuropathol Exp Neurol 2002;61:413–426.
63. Lippa CF, Fujiwara H, Mann DM, et al. Lewy bodies contain altered F-synuclein in brains of many familial Alzheimer’s disease patients with mutations in presenilin and amyloid precursor protein genes. Am J Pathol 1998;153:1365–1370.
64. Yamazaki M, Arai Y, Baba M, et al. F-Synuclein inclusions in amygdala in the brains of patients with the parkin- sonism-dementia complex of Guam. J Neuropathol Exp Neurol 2000;59:585–591.
65. Spira PJ, Sharpe DM, Halliday G, Cavanagh J, Nicholson GA. Clinical and pathological features of a parkinsonian syndrome in a family with an Ala53Thr F-synuclein mutation. Ann Neurol 2001;49:313–319.
66. Duda JE, Giasson BI, Mahon ME, et al. Concurrence of F-synuclein and tau brain pathology in the Contursi kindred.
Acta Neuropathol 2002;104:7–11.
67. Ishizawa T, Mattila P, Davies P, Wang D, Dickson DW. Colocalization of tau and alpha-synuclein epitopes in Lewy bodies. J Neuropathol Exp Neurol 2003;62:389–397.
68. Giasson BI, Forman MS, Higuchi M, et al. Initiation and synergistic fibrillization of tau and alpha-synuclein. Science 2003;300:636–640.
69. Gwinn-Hardy K, Mehta ND, Farrer M, et al. Distinctive neuropathology revealed by F-synuclein antibodies in heredi- tary parkinsonism and dementia linked to chromosome 4p. Acta Neuropathol 2000;99:663–672.
70. Gasser T, Müller-Myhsok B, Wszolek ZK, et al. A susceptiibility locus for Parkinson’s disease maps to chromosome 2p13. Nature Genet 1998;18:262–265.
71. Wszolek ZK, Gwinn-Hardy K, Wszolek EK, et al. Neuropathology of two members of a German-American kindred (Family C) with late onset parkinsonism. Acta Neuropathol 2002;103:344–350.
72. Leroy E, Boyer R, Auburger G, et al. The ubiquitin pathway in Parkinson’s disease. Nature 1998;395:451–452.
73. Kitada T, Asakawa S, Hattori N, et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism.
Nature 1998;392:605–608.
74. Shimura H, Hattori N, Kubo S, et al. Familial Parkinson’s disease gene product, parkin, is a ubiquitin-protein ligase.
Nature Genet 2000;25:302–305.
75. Choi P, Golts N, Snyder H, et al. Co-association of parkin and alpha-synuclein. Neuroreport 2001;12:2839–2843.
76. Schlossmacher MG, Frosch MP, Gai WP, et al. Parkin localizes to the Lewy bodies of Parkinson disease and dementia with Lewy bodies. Am J Pathol 2002;160:1655–1667.
77. Shimura H, Schlossmacher MG, Hattori N, et al. Ubiquitination of a new form of alpha-synuclein by parkin from human brain: implications for Parkinson’s disease. Science 2001;293:263–269.
78. Takahashi H, Ohama E, Suzuki S, et al. Familial juvenile parkinsonism: clinical and pathologic study in a family.
Neurology 1994;44:437–441.
79. Mori H, Kondo T, Yokochi M, et al. Pathologic and biochemical studies of juvenile parkinsonism linked to chromo- some 6q. Neurology 1998;51:890–892.
80. Farrer M, Chan P, Chen R, et al. Lewy bodies and parkinsonism in families with parkin mutations. Neurology 2001;50:293–300.
81. Pawlyk AC, Giasson BI, Sampathu DM, et al. Novel monoclonal antibodies demonstrate biochemical variation of brain parkin with age. J Biol Chem 2003;278:48,120–48,128.
82. Bonifati V, Rizzu P, van Baren MJ, et al. Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science 2003;299:256–259.
83. Takao M, Ghetti B, Yoshida H, et al. Early-onset dementia with Lewy bodies. Brain Pathol 2004;14:137–147.
84. Lantos PL, Quinn N. Multiple system atrophy. In: Dickson D, ed. Neurodegeneration: The Molecular Pathology of Dementia and Movement Disorders. Basel: ISN Neuropath Press, 2003: 203–214.
85. Wenning GK, Quinn N, Magalhaes M, Mathias C, Daniel SE. “Minimal change” multiple system atrophy. Mov Disord 1994;9:161–166.
86. Campbell BC, McLean CA, Culvenor JG, et al. The solubility of alpha-synuclein in multiple system atrophy differs from that of dementia with Lewy bodies and Parkinson’s disease. J Neurochem 2001;76:87–96.
87. Crowther RA, Jakes R, Spillantini MG, Goedert M. Synthetic filaments assembled from C-terminally truncated F- synuclein. FEBS Lett 1998;436:309–312.
88. Conway KA, Harper DJ, Lansbury PT. Accelerated in vitro fibril formation by a mutant F-synuclein linked to early- onset Parkinson’s disease. Nature Med 1998;4:1318–1320.
89. El-Agnaf IMA, Jakes R, Curran MD, Wallace A. Effects of the mutations Ala30 to Pro and Ala53 to Thr on the physical and morphological properties of F-synuclein implicated in Parkinson’s disease. FEBS Lett 1998;440:67–70.
90. Giasson BI, Uryu K, Trojanowski JQ, Lee VMY. Mutant and wild-type human F-synuclein assemble into elongated filaments with distinct morphologies in vitro. J Biol Chem 1999;274:7619–7622.
91. Wood SJ, Wypych J, Steavenson S, Louis JC, Citron M, Biere AL. F-Synuclein fibrillogenesis is nucleation-depen- dent. J Biol Chem 1999;274:19509–19512.
92. Serpell LC, Berriman J, Jakes R, Goedert M, Crowther RA. Fiber diffraction of synthetic alpha-synuclein filaments shows amyloid-like cross-beta conformation. Proc Natl Acad Sci USA 2000;97:4897–4902.
93. Murray IVJ, Giasson BI, Quinn SM, et al. Role of F-synuclein carboxy-terminus on fibril formation in vitro. Biochem- istry 2003;42:8530–8540.
94. Uversky VN, Li J, Fink AL. Evidence for a partially folded intermediate in F-synuclein fibril formation. J Biol Chem 2001;276:10737–10744.
95. Narhi L, Wood SJ, Steavenson S, et al. Both familial Parkinson’s disease mutations accelerate F-synuclein aggrega- tion. J Biol Chem 1999;274:9843–9846.
96. Conway KA, Lee SJ, Rochet JC, Ding TT, Williamson RE, Lansbury PT. Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson’s disease: implica- tions for pathogenesis and therapy. Proc Natl Acad Sci USA 2000;97:571–576.
97. Jo E, Fuller N, Rand PR, St George-Hyslop P, Fraser PE. Defective membrane interactions of familial Parkinson’s disease mutant A30P F-synuclein. J Mol Biol 2002;315:799–807.
98. Zhu M, Fink AL. Lipid binding inhibits F-synuclein filament formation. J Biol Chem 2003;278:16873–16877.
99. Conway KA, Harper JD, Lansbury PT. Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson’s disease are typical amyloid. Biochemistry 2000;39:2552–2563.
100. Biere AL, Wood SJ, Wypych J, et al. Parkinson’s disease-associated F-synuclein is more fibrillogenic than G- and L- synuclein and cannot cross-seed its homologs. J Biol Chem 2000;275:34574–34579.
101. Hashimoto M, Rockenstein E, Mante, Mallory M, Masliah E. G-Synuclein inhibits F-synuclein aggregation: a possible role as an antiparkinsonian factor. Neuron 2001;32:213–223.
102. Uversky VN, Li J, Souillac P, et al. Biophysical properties of the synucleins and their propensities to fibrillate: inhibi- tion of F-synuclein assembly by G- and L-synucleins. J Biol Chem 2002;277:11970–11978.
103. Park JY, Lansbury PT. G-Synuclein inhibits formation of F-synuclein protofibrils: a possible therapeutic strategy against Parkinson’s disease. Biochemistry 2003;42:3696–3700.
104. Kahle PJ, Neumann M, Ozmen L, et al. Selective insolubility of F-synuclein in human Lewy body diseases is recapitu- lated in a transgenic mouse model. Am J Pathol 2001;159:2215–2225.
105. Giasson BI, Murray IVJ, Trojanowski JQ, Lee VMY. A hydrophobic stretch of 12 amino acid residues in the middle of F-synuclein is essential for filament assembly. J Biol Chem 2001;276:2380–2386.
106. Bodles AM, Guthrie DJS, Greer B, Irvine GB. Identification of the region of non-AG component (NAC) of Alzheimer’s disease amyloid responsible for its aggregation and toxicity. J Neurochem 2001;78:384–395.
107. Du HN, Tang L, Luo XY, et al. A peptide motif consisting of glycine, alanine, and valine is required for the fibrillation and cytotoxicity of human F-synuclein. Biochemistry 2003;42:8870–8878.
108. Uversky VN, Yamin G, Souillac PO, Goers J, Glaser CB, Fink AL. Methionine oxidation inhibits fibrillation of human F-synuclein in vitro. FEBS Lett 2002;517:239–244.
109. Yamin G, Uversky VN, Fink AL. Nitration inhibits fibrillation of human F-synuclein in vitro by formation of soluble oligomers. FEBS Lett 2003;542:147–152.
110. Conway KA, Rochet JC, Bieganski RM, Lansbury PT. Kinetic stabilization of the F-synuclein protofibril by a dopam- ine-F-synuclein adduct. Science 2001;294:1346–1349.
111. Hashimoto M, Takeda A, Hsu LJ, Takenouchi T, Masliah E. Role of cytochrome C as a stimulator of F-synuclein aggregation in Lewy body disease. J Biol Chem 1999;274:28849–28852.
112. Uversky VN, Li J, Fink AL. Pesticides directly accelerate the rate of F-synuclein fibril formation: a possible factor in Parkinson’s disease. FEBS Lett 2001;500:105–108.
113. Uversky VN, Li J, Fink AL. Metal-triggered structural transformations, aggregation and fibril formation of human F- synuclein. A possible molecular link between Parkinson’s disease and heravy metal exposure. J Biol Chem 2001;276:44284–44296.
114. Cohlberg JA, Li J, Uversky VN, Fink AL. Heparin and other glycosaminoglycans stimulate the formation of amyloid fibrils from F-synuclein in vitro. J Biol Chem 2002;41:1502–1511.
94 Goedert and Spillantini
115. Antony T, Hoyer W, Cherny D, Heim G, Jovin TM, Subramaniam V. Cellular polyamines promote the aggregation of F-synuclein. J Biol Chem 2003;278:3235–3240.
116. Paik SR, Lee D, Cho HJ, Lee EN, Chang CS. Oxidized glutathione stimulated the amyloid formation of F-synuclein.
FEBS Lett 2003;537:63–67.
117. Masliah E, Rockenstein E, Veinbergs I, et al. Dopaminergic loss and inclusion body formation in F-synuclein mice:
implications for neurodegenerative disorders. Science 2000;287:1265–1269.
118. Van der Putten H, Wiederhold KH, Probst A, et al. Neuropathology in mice expressing human F-synuclein. J Neurosci 2000;20:6021–6029.
119. Kahle PJ, Neumann N, Ozmen L, et al. Subcellular localization of wild-type and Parkinson’s disease–associated mutant F-synuclein in human and transgenic mouse brain. J Neurosci 2000;20:6365–6373.
120. Neumann M, Kahle PJ, Giasson BI, et al. Misfolded proteinase K–resistant hyperphosphorylated F-synuclein in aged transgenic mice with locomotor deterioration and in human F-synucleinopathies. J Clin Invest 2002;110:1429–1439.
121. Giasson BI, Duda JE, Quinn SM, Zhang B, Trojanowski JQ, Lee VMY. Neuronal F-synucleinopathy with severe movement disorder in mice expressing A53T human F-synuclein. Neuron 2002;34:521–533.
122. Lee MK, Stirling W, Xu Yet al. Human F-synuclein-harboring familial Parkinson’s disease–linked Ala53 to Thr muta- tion causes neurodegenerative disease with F-synuclein aggregation in transgenic mice. Proc Natl Acad Sci USA 2002;99:8968–8973
123. Gomez-Isla T, Irizarry MC, Mariash A, et al. Motor dysfunction and gliosis with preserved dopaminergic markers in humanF-synuclein A30P transgenic mice. Neurobiol Aging 2003;24:245–258.
124. Matsuoka Y, Vila M, Lincoln S, et al. Lack of nigral pathology in transgenic mice expressing human F-synuclein driven by the tyrosine hydroxylase promoter. Neurobiol Dis 2001;8:535–539.
125. Richfield EK, Thiruchelvam MJ, Cory-Schlechta DA, et al. Behavioral and neurochemical effects of wild-type and mutated human F-synuclein in transgenic mice. Exp Neurol 2002;175:35–48.
126. Kahle PJ, Neumann M, Ozmen L, et al. Hyperphosphorylation and insolubility of F-synuclein in transgenic mouse oligodendrocytes. EMBO Rep 2002;3:583–588.
127. Feany MB, Bender WW. A Drosophila model of Parkinson’s disease. Nature 2000;404:394–398.
128. Pendleton RG, Parvez F, Sayed M, Hillman R. Effects of pharmacological agents upon a transgenic model of Parkinson’s disease in Drosophila melanogaster. J Pharmacol Exp Ther 2002;300:91–96.
129. Lakso M, Vartiainen S, Moilanen AM, et al. Dopaminergic neuronal loss and motor deficits in Caenorhabditis elegans overexpressing human F-synuclein. J Neurochem 2003;86:165–172.
130. Auluck PK, Chan E, Trojanowski JQ, Lee VMY, Bonini NM. Chaperone suppression of F-synuclein toxicity in a Drosophila model for Parkinson’s disease. Science 2002;295:865–868.
131. Auluck PK, Bonini NM. Pharmacologic prevention of Parkinson’s disease in Drosophila. Nature Med 2002;8:1185–1186.
132. Klein RL, King MA, Hamby ME, Meyer EM. Dopaminergic cell loss induced by human A30P F-synuclein gene trans- fer to the rat substantia nigra. Hum Gene Ther 2002;13:605–612.
133. Kirik D, Rosenblad C, Burger C, et al. Parkinson-like neurodegeneration induced by targeted overexpression of F-synuclein in the nigrostriatal system. J Neurosci 2002;22:2780–2791.
134. Lo Bianco C, Ridet JL, Schneider BL, Déglon N, Aebischer P. F-Synucleinopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson’s disease. Proc Natl Acad Sci USA 2002;99:10813–10818.
135. Kirik D, Annett LE, Burger C, Muzyczka N, Mandel RJ, Björklund A. Nigrostriatal F-synucleinopathy induced by viral vector–mediated overexpression of human F-synuclein: a new primate model of Parkinson’s disease. Proc Natl Acad Sci USA 2003;100:2884–2889.
136. Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT. Chronic systemic pesticide expo- sure reproduces features of Parkinson’s disease. Nature Neurosci 2000;3:1301–1306.
137. Sherer TB, Kim JH, Betarbet R, Greenamyre JT. Subcutaneous rotenone exposure causes highly selective dopaminer- gic degeneration and F-synuclein aggregation. Exp Neurol 2003;179:9–16.
138. Höglinger GU, Féger J, Prigent A, et al. Chronic systemic complex I inhibition induces a hypokinetic multisystem degeneration in rats. J Neurochem 2003;84:491–502.
