Transgenic Mice Overexpressing Tyrosine-to-Cysteine Mutant Human α-Synuclein

Abnormal aggregation of human α-synuclein in Lewy bodies and Lewy neurites is a pathological hallmark of Parkinson disease and dementia with Lewy bodies. Studies have shown that oxidation and nitration of α-synuclein lead to the formation of stable dimers and oligomers through dityrosine cross-linking. Previously we have reported that tyrosine-to-cysteine mutations, particularly at the tyrosine 39 residue (Y39C), significantly enhanced α-synuclein fibril formation and neurotoxicity. In the current study, we have generated transgenic mice expressing the Y39C mutant human α-synuclein gene controlled by the mouse Thy1 promoter. Mutant human α-synuclein was widely expressed in transgenic mouse brain, resulting in 150% overexpression relative to endogenous mouse α-synuclein. At age 9–12 months, transgenic mice began to display motor dysfunction in rotarod testing. Older animals aged 15–18 months showed progressive accumulation of human α-synuclein oligomers, associated with worse motor function and cognitive impairment in the Morris water maze. By age 21–24 months, α-synuclein aggregates were further increased, accompanied by severe behavioral deficits. At this age, transgenic mice developed neuropathology, such as Lewy body-like α-synuclein and ubiquitin-positive inclusions, phosphorylation at Ser129 of human α-synuclein, and increased apoptotic cell death. In summary, Y39C human α-synuclein transgenic mice show age-dependent, progressive neuronal degeneration with motor and cognitive deficits similar to diffuse Lewy body disease. The time course of α-synuclein oligomer accumulation coincided with behavioral and pathological changes, indicating that these oligomers may initiate protein aggregation, disrupt cellular function, and eventually lead to neuronal death.

␣-Synuclein is a 140-amino acid protein mainly located at presynaptic terminals (1,2). ␣-Synuclein may have a role in regulating vesicle function and in neurotransmitter release by binding to synaptic vesicles and stabilizing lipid and protein fine structure (3)(4)(5). Recently, ␣-synuclein has been shown to regulate vesicular trafficking from endoplasmic reticulum to Golgi (6,7). Point mutations and genomic duplication in the ␣-synuclein gene have been linked to autosomal-dominant forms of Parkinson disease (8 -11). Accumulation of ␣-synuclein fibrils in Lewy bodies (LBs) 2 and Lewy neurites are the pathological hallmark of Parkinson disease, dementia with Lewy bodies, the Lewy body variant of Alzheimer disease, and neurodegeneration with brain iron accumulation type 1, collectively referred as ␣-synucleinopathies (12)(13)(14)(15)(16)(17). Thus, the process of ␣-synuclein aggregation plays a critical role in the pathogenesis of many neurodegenerative diseases.
The accumulation of ␣-synuclein causes neuronal death when the protein aggregates into protofibrils and fibrils. However, the cellular mechanisms by which the soluble ␣-synuclein monomer forms oligomers and fibrils remain unclear. In vitro studies have shown that human ␣-synuclein proteins are prone to aggregate by increasing concentration, temperature, oxidative stress, and heavy metals (18 -21). Mutations such as A53T, A30P, and E46K can accelerate protein aggregation (22)(23)(24)(25).
A growing body of evidence suggests oxidative stress plays a critical role in initiating ␣-synuclein aggregation and toxicity (19, 26 -29). In vitro, exposure of human recombinant ␣-synuclein protein to nitrating agents leads to the formation of stable dimers and oligomers through o-oЈ-dityrosine crosslinking (30). Oxidative dimer formation appears to be the critical rate-limiting step for protein aggregation and fibrillogenesis (31). Wild type ␣-synuclein has four tyrosine residues at positions 39, 125, 133, and 136 and lacks cysteine. Because cysteine can be easily oxidized to form stable cross-linked disulfide dimers, we have systemically substituted cysteine for tyrosine and analyzed the effect on tyrosine-to-cysteine mutant protein aggregation and neurotoxicity (32). We have found that cysteine substitution in the Y39C and Y125C positions but not the Y133C and Y136C positions significantly enhanced ␣-synuclein aggregation and toxicity (32).
In this study, we have created transgenic (Tg) mice expressing human ␣-synuclein with Y39C mutations under control of the mouse Thy1 promoter (Tg-Y39C). We report here that the Tg-Y39C mice developed ␣-synuclein pathology and progressive motor and cognitive deficits.

EXPERIMENTAL PROCEDURES
Generation of Y39C Human ␣-Synuclein Tg Mice-We have generated Tg mice expressing Y39C mutant human ␣-synuclein under the murine Thy1 promoter (33) (kindly provided by Dr. Herman van der Putten). The Y39C human ␣-synuclein fragment was isolated from pcDNA3.1-Y39C-hsyn by the restriction enzyme XhoI (32) and ligated into the XhoI site of the mThy1 cassette (Fig. 1A). The constructs were linearized by NotI and gel-purified. Microinjection of DNA into one-cell embryos (FVB/N) was done by the University of Colorado Transgenic Core according to standard procedures. Founder mice were identified by PCR using tail genomic DNA. The PCR was performed with the P1 (5Ј-TCTGAGTGGCAAAG-GACCTTAG-3Ј) and P2 (5Ј-CCCTTCCTCAGAAGGCATT-TCAT-3Ј) primers and 30 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, resulting in a 440-bp fragment. The founder mice were confirmed by Southern blotting following standard procedures, using a 360-bp DNA probe corresponding to human ␣-synuclein cDNA sequence (Fig. 1A). A transgenic line with the highest expression of human ␣-synuclein was selected for subsequent analysis. For all experiments, heterozygous human ␣-synuclein Tg mice were crossed with wild type FVB/N mice to generate Tg and nontransgenic (nTg) littermates. Mice were genotyped by PCR using primers P1 and P2.
Terminal Deoxynucleotidyltransferase-mediated Biotinylated UTP Nick End Labeling (TUNEL) Assay-The apoptotic cell nuclei in brain sections were analyzed by TUNEL (In Situ Cell Death Detection Kit; Roche Applied Science). Sections were treated for 10 min with proteinase K (5 mg/ml) at 37°C and then stained according to the kit (35,36).
Open Field Activity Test-Mice were tested in 50 ϫ 50-cm square boxes with clear Plexiglas walls and floors, evenly illuminated by white overhead fluorescent lighting. Mice were FIGURE 1. Generation of Y39C human ␣-synuclein Tg mice. A, the DNA structure of the mThy1 promoter and mThy1-Y39C human ␣-synuclein targeting vector. Exons 1-4 are labeled. N, NotI; X, XhoI; P1 and P2, PCR primers. DNA probe location is also shown. B, four founders were identified by PCR using primers P1 and P2, which resulted in a 400 bp band. WT, wild type as control. C, Southern blotting using a human ␣-synucleinspecific probe verified that two lines, B5365 and B5366, had the expected 6 kb band. D, Western blotting confirmed that lines B5365 and B5366 expressed human ␣-synuclein (antibody LB509). The blot was reprobed with synuclein-1 antibody, which has affinity for both mouse and human ␣-synuclein, and results were normalized to ␤-actin. The total ␣-synuclein levels (mouse ϩ human) as determined by synuclein-1 were quantified from three independent Western blotting experiments. E, the Tg and nTg mouse survival rates were analyzed from 40 animals each over 24 months. Tg mice had significantly lower survival at 24 months than did nTg littermates (*, p Ͻ 0.05).
individually placed in the center of the open field and left to freely explore for 30 min. Activity was measured by a computerassisted Photobeam Activity System with Flexfield (San Diego Instruments). The total distance traveled and the total movement times were recorded in 5-min intervals.
Rotarod Test-Mice were tested for their ability to maintain balance on a rotating rod (rotarod) at a selected speed ranging from 3 to 33 rpm. The protocol consisted of two phases: habituation (day 1) and rotarod training/testing (days 2-7). On day 1, the mice were trained to remain on a 3-cm diameter rod at 3 rpm. During training and testing, mice had to run on the rod as it rotated at a constant speed of 7, 10, 14, 20, 26, or 33 rpm from day 2 to day 7, respectively. Each mouse received three 1-min trials, with a 5-min interval between trials. The time the mice spent on the rod without falling was recorded for each trial.
Morris Water Maze Testing-Spatial learning was assessed using the Morris water maze (37). The maze included a circular tank (120 cm in diameter) filled to 5 cm below the edge of the tank with 27°C water that was made opaque by the addition of nontoxic black ink. A circular escape platform (10 cm in diameter) was located 1 cm below the surface of the water in a constant location in the northwest quadrant of the tank. On day 1, mice were first acclimated to the maze during a three-trial habituation session. On day 2, mice were tested for their ability to find the visible platform, which was located 4 cm above the water levels. Each mouse had four trials with a 5-min interval between trials. From day 3 to day 6, mice were trained to find the hidden platform for four consecutive days. Each mouse received four trials per day, with a 5-min interval between trials. The mice were allowed to swim for up to 60 s before being returned to the home cage. The times required to find the platform (latency) were recorded for each trial.
Statistics-Data were analyzed using multivariate analysis of variance and the Fisher least significant difference post hoc test. Significance was set at p Ͻ 0.05. Values are shown as mean Ϯ S.E.

Characterization of Y39C Mutant Human ␣-Synuclein Tg
Mice-Transgenic mice were generated by standard pronuclear injection procedures, and four founders were identified by PCR (Fig. 1B). Southern blotting using a human ␣-synucleinspecific probe showed two lines (B5365 and B5366) with a strong band at 6 kb as expected (Fig. 1C); two other lines (B5464 and B5368) had very low expression of the desired gene fragment (data not shown). Western blotting using the human ␣-synuclein-specific antibody LB509 confirmed that human O, quantification of TUNEL staining from 12-, 18-, and 24-month-old Tg and nTg control mice. Three mice from each age group with two representative sections from each mouse (n ϭ 6) were analyzed for the rate of apoptosis by TUNEL staining. The number of TUNEL-positive cells was counted in each section. The data were normalized to 12-month-old nTg control mice. There was a significant increase in the apoptotic rate in 24-month-old Tg mice compared with their nTg littermates (**, p Ͻ 0.01). Scale bars, 20 m in B applied to A-E; 5 m in H applied to F-L; 10 m in M applied to M and N.
␣-synuclein was expressed in both B5365 and B5366 lines (Fig.  1D). The Western blot was stripped and reprobed with synuclein-1 antibody, which detects both mouse and human ␣-synuclein. Results showed a 150 and 115% overexpression of human ␣-synuclein relative to endogenous mouse ␣-synuclein in line B5365 and B5366, respectively. Because line B5365 had higher human ␣-synuclein levels than line B5366, we used line B5365 for the subsequent experiments. The Tg mice were healthy and fertile. The body weights of Tg and nTg littermates were similar (data not shown). The Tg mice showed significantly lower survival rates beginning at age 24 months compared with nTg littermates (*, p Ͻ 0.05; Fig. 1E).
Tg Mice Develop ␣-Synuclein Neuropathology in Aging Brain-We characterized human ␣-synuclein distribution in mouse brain using immunohistochemistry. Brain sections from 6-, 12-, 18-, and 24-month-old Tg mice were immunostained with LB509 antibody. We found that human ␣-synuclein was widely expressed in mouse brain, with highest expression in the cortex ( Fig. 2A). Expression of human ␣-synuclein was also seen in hippocampus, striatum, thalamus, and substantia nigra (Fig. 2, B-E) as well as in spinal cord (data not shown). Human ␣-synuclein staining was present in the cytoplasm and extended to the proximal neurites. In aged animals (24 months old), ␣-synuclein inclusions similar to Lewy bodies were found, particularly in cortex (arrow in Fig. 2F). Since phosphorylation at Ser 129 is a pathological hallmark of synucleinopathies, we stained sections with phospho-Ser 129 antibody. The phospho-Ser 129 -positive inclusions were only found in 24-month Tg brain (arrow in Fig. 2H) and not in 12-month Tg brain (Fig. 2G). In age-matched non-Tg controls, there was no phospho-Ser 129 staining (24 months shown in Fig. 2I). We also performed ubiquitin staining, another marker for neurodegeneration. In 24-month Tg mice, ubiquitin-positive aggregations were observed in cell bodies and neurites (arrows and arrowheads in Fig. 2K). There were no ubiquitin inclusions in 12-month Tg mice (Fig. 2J). In age-matched non-Tg controls, the ubiquitin staining was all negative (24 months shown in Fig. 2L). FIGURE 3. Biochemical characterization of Tg mice by Western blotting. Fresh brain tissues from Tg and nTg control mice were dissected according to the following areas: cortex (Ctx), hippocampus (Hip), thalamus (Thal), and spinal cord (S.C.). Then tissue was homogenized and separated into Triton X-100 fractions (Triton-Soluble). Triton X-100-insoluble material was further extracted into SDS-soluble fractions (SDS-Soluble). Equal amounts of protein (50 g) were separated on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membrane. A, representative sample blot from multiple brain regions of 15-month-old Tg and nTg control mice probed with human ␣-synuclein-specific LB509 antibody. Note the 50 -150-kDa oligomers in both Triton-soluble and SDS-soluble fractions from Tg brain. Note also the absence of human ␣-synuclein in nTg control mouse cortex. B, the blot from A was reprobed with synuclein-1 antibody, detecting both human and mouse ␣-synuclein. With this antibody, a single 16-kDa band of mouse ␣-synuclein is seen in nTg control mouse brain. C, representative sample blots from 21-month-old Tg and nTg control mice probed with synuclein-1. Note that 50 -150-kDa oligomers were intensified in both Triton-soluble fractions and SDS-soluble fractions compared with B. Nontransgenic control mice showed no oligomer formation even when the gel was loaded with 100 g of total protein (nTg 2ϫ), indicating that oligomer formation was not caused by protein "crowding" or higher concentrations in Tg mouse samples. D, quantification of ␣-synuclein oligomers relative to monomers. Western blotting was performed from three animals in each age group. Experiments were repeated twice (n ϭ 6). The Western blot images were scanned into the computer and analyzed by NIH Image J software. The data show that the oligomer/monomer ratios progressively increased with age. There were significant increases in oligomer formation at 15-18 months compared with younger ages and further significant increases at 21-24 months in both Triton-soluble and SDSsoluble fractions (15-18 months versus 10 -12 months (**, p Ͻ 0.01); 21-24 months versus 15-18 months (**, p Ͻ 0.01)). To assess whether accelerated cell death occurred in aging Tg mouse brain, TUNEL assays were performed on tissue sections from 12-, 18-, and 24-month-old mice. There was a 100% increase in apoptotic cell death in 24-month-old Tg mouse brain (Fig. 2N) compared with nTg control mice (Fig. 2, M and O; **, p Ͻ 0.01). By contrast, the 18-month-old Tg brain had TUNEL staining similar to nTg brain (Fig.  2O), suggesting that Y39c Tg animals have age-dependent neuronal degeneration. The increased apoptosis rate in aged Tg mice compared with nTg littermates may be the cause of significantly lower survival seen at 24 months (Fig. 1E).

Y39C Human ␣-Synuclein
Tg Mice Accumulate Human ␣-Synuclein Oligomers in an Agedependent Manner-We analyzed ␣-synuclein protein from different brain regions by Western blotting. The proteins were separated into Triton X-100-soluble and SDS-soluble fractions for comparison. Protein samples from 3-6-month, 10 -12-month, 15-18-month, and 21-24-month Tg and nTg brain were analyzed. Sample blots from 15-month-old Tg and nTg are shown in Fig. 3, A and B, sequentially probed with LB509 (Fig. 3A) and synuclein-1 (Fig. 3B). In the 15-18-month age group, we found that human ␣-synuclein formed ladder-like oligomers with molecular masses of 50 -150 kDa in Tritonsoluble fractions in cortex, hippocampus, thalamus, and spinal cord (Fig. 3A). In SDS-soluble fractions, we found only a single human ␣-synuclein oligomer of 120 kDa in all four brain areas (Fig. 3A). However, when these same SDS-soluble fractions were probed with synuclein-1 antibody, a ladder of oligomers was seen similar to that observed in Triton-soluble samples, suggesting that synuclein-1 antibody may be more sensitive than LB509. Nontransgenic mice showed no oligomers in Triton or SDS fractions. In older Tg animals (21-24 months), oligomer bands were more intense than in younger Tg mice (15-18 months) (Fig. 3C). Quantify- FIGURE 4. Tg mice displayed motor function deficits that were age-dependent and progressive. Tg and age-matched wild-type (WT) mice were tested on the rotarod task (n ϭ 10 for each age group: 3-6, 9 -12, 15-18, and 21-24 months). Testing was done on six consecutive days, with each day tested at a single speed (day 1 to day 6 at 7, 10, 14, 20, 26, and 33 rpm, respectively). The time that mice could remain on the rotating rod was recorded for each day. A, testing at 3-6 months of age showed no significant differences in the running time between Tg and WT animals. B-D, beginning at 9 -12 months and progressing to 21-24 months of age, Tg mice had worse performance on the rotarod compared with age-matched WT mice (*, p Ͻ 0.05; **, p Ͻ 0.01). FIGURE 5. Tg mice showed an age-dependent decline in cognitive function. Tg and age-matched wild-type (WT) mice were tested in a Morris water maze (n ϭ 10 for each age group: 3-6, 9 -12, 15-18, and 21-24 months). The animals were tested on four consecutive days. The latency was recorded and analyzed according to age group for each day. At age 3-6 months (A) and 9 -12 months (B), there were no significant differences in the time to find the platform between Tg and wild-type mice (p Ͼ 0.1). In the 15-18 months group (C), Tg mice did not improve their performance over several days of practice compared with wild-type control (*, p Ͻ 0.05 at day 4). D, the delays in learning and performance were even more obvious in the 21-24-month Tg mice. Tg animals were significantly worse after 3 and 4 days of testing compared with wild-type control (**, p Ͻ 0.01), showing that they failed to learn the task as quickly as nTg control animals. APRIL 11, 2008 • VOLUME 283 • NUMBER 15 ing oligomer to monomer revealed that there were significant age-dependent increases in oligomer aggregation in both Triton and SDS fractions (**, p Ͻ 0.01) (Fig. 3D).

Y39C Human ␣-Synuclein Transgenic Mouse
Tg Mice Develop Progressive Motor Function Deficits-We performed rotarod tests comparing Tg and nTg mice at four different age groups: 3-6, 9 -12, 15-18, and 21-24 months (Fig. 4, A-D). Over the 6-day testing period, most animals learned to stay on the rod. Tg mice started to display motor deficits at age 9 -12 months compared with nTg littermates (*, p Ͻ 0.05; Fig. 4B). Motor performance became progressively worse with age, as shown in Fig. 4, C and D. By 21-24 months, the Tg mice had severe motor impairment, staying on the rotarod only half as long as nTg littermates (**, p Ͻ 0.01; Fig. 4D).
Tg Mice Display an Age-dependent Decline in Cognitive Function-We used a Morris water maze to test cognitive function in Tg and nTg mice in four age groups: 3-6, 9 -12, 15-18, and 21-24 months (Fig. 5, A-D). To rule out potential visual impairments in Tg mice, animals were tested for their ability to find the platform positioned above the water level. In this visible platform test, the latencies for each group were as follows: 3-6 months, 24.1 Ϯ 2.9 and 22.5 Ϯ 2.6 s (Tg and control mice, respectively; same order for other groups, p Ͼ 0.1); 9 -12 months, 25.7 Ϯ 2.4 and 23.5 Ϯ 1.8 s (p Ͼ 0.1); 15-18 months, 27.7 Ϯ 2.1 and 26.9 Ϯ 3.2 s (p Ͼ 0.1); 21-24 months, 31.8 Ϯ 2.5 and 29.2 Ϯ 3.6 s (p Ͼ 0.1). The results indicated that transgenic mice had similar visual and locomotor function as age-matched controls. After 4 days of training, nTg control mice were able to quickly locate the hidden platform in the water maze. In 3-6-and 9 -12month groups, there were no significant differences in the latency between Tg and nTg mice (Fig. 5, A and B). However, Tg mice at 15-18 months of age took significantly longer to find the platform compared with nTg littermates (*, p Ͻ 0.05; Fig. 5C). In the oldest group, 21-24 months, the difference in the latency between Tg and nTg mice was even larger; Tg mice took twice as long to find the platform (**, p Ͻ 0.01; Fig.  5D). The data indicate that Tg mice had age-dependent spatial learning and memory deficits.
Tg Mice had Open Field Activity Similar to That of nTg Mice-To demonstrate that the motor and cognitive deficits observed in Tg mice were not caused by muscle weakness or some other restriction on general locomotor ability, we compared the open field exploratory activity in Tg and nTg mice in four age groups. With increasing age, both Tg and nTg animals showed progressive reduction in open field activity. Measurements of total distance traveled (Fig. 6A) and total movement time (Fig. 6B) showed no differences between age-matched Tg and nTg animals (p Ͼ 0.1). The results indicate that Tg mice had open field activity similar to that of nTg mice.
Midbrain Dopamine Neurons Are Not Affected in Tg Mice-Although there was some expression of human ␣-synuclein in midbrain neurons, the protein rarely colocalized with TH-positive dopamine neurons (data not shown). There were no significant losses of dopamine neurons in substantia nigra or dopamine nerve terminals in striatum, even in aged Tg mouse brain (Fig. 7, A-D).
The Time Course of Human ␣-Synuclein Oligomer Accumulation Correlates with Behavioral Deficits and Neuropathology-We summarize the biochemical, behavioral, and pathological data according to age groups in Table 1. At age 9 -12 months, Tg mice had minimal oligomer accumulation (2% of oligomer relative to monomer). Behaviorally, Tg mice showed deficits in rotarod testing but not in water maze performance. As ␣-synuclein oligomers accumulated at 15-18 and 21-24 months of age, Tg mice displayed progressively worse performance in both rotarod and water maze tests. Obvious changes in histopathology were seen in the oldest Tg mice as noted above. From the table, it can be noted that the time course of ␣-synuclein oligomer accumulation was highly correlated with motor and cognitive decline.

DISCUSSION
In our previous work, we have found that tyrosine-to-cysteine substitution, particularly at the tyrosine 39 residue, significantly enhances aggregation of human ␣-synuclein through accelerated formation of oligomers, particularly after oxidative stress (32). In the current study, we have generated and characterized a transgenic mouse overexpressing the Y39C mutant human ␣-synuclein (Y39C) under control of the mouse Thy1 promoter. These transgenic mice have age-dependent, progressive motor and cognitive deficits that correlate with widespread deposition of oligomers in the brain, leading to cellular neuropathology in the older animals. The spontaneous locomotor activity was tested in an open field over 30 min. Total distance traveled and total movement times were recorded in both Tg and age-matched wild-type mice (n ϭ 10 for each age group: 3-6, 9 -12, 15-18, and 21-24 months). Although there was a general trend for all mice to move less with age, there were no significant differences between Tg and wild-type mice in the total distance traveled (A) and in the total movement time (p Ͼ 0.1) (B).
In other human ␣-synuclein Tg mouse studies, oligomers only appeared in detergent-insoluble fractions (33, 38 -40). By contrast, in our Tg mice, oligomers were recovered in the Triton X-100-soluble fraction as well as in the detergentinsoluble fraction. The difference was probably due to the tyrosine-to-cysteine modification of human ␣-synuclein in our Tg model. We have previously shown that Y39C human ␣-synuclein, but not wild-type or A53T human ␣-synuclein, is able to form dimers and oligomers under very mild oxidative stress in vitro (32). Our Tg mice displayed motor dysfunction at an earlier age than their decline in cognitive function. Because the Thy1 promoter leads to gene expression in all brain neurons, we cannot say which brain structure con-tributed most to the behavioral effects observed. Although Tg animals showed behavioral deficits beginning at age 9 -12 months, ␣-synuclein inclusions and apoptotic cell death first appeared at age 21-24 months. It is possible that oligomer accumulation causes cellular dysfunction. Eventually, oligomer aggregation leads to inclusion formation and finally to cell death. A recent study has demonstrated that ␣-synuclein accumulation can block vesicular trafficking from endoplasmic reticulum to Golgi, resulting in functional impairment and cell death (7). Our Tg mouse model suggests that the ␣-synuclein fibrilization process is linked to neuropathology.
In our Tg mouse model, the panneuronal Thy1 promoter induced human ␣-synuclein expression equivalent to 150% of endogenous ␣-synuclein levels for a total ␣-synuclein content that was 250% of normal. A number of human ␣-synuclein transgenic mouse models have been developed with different promoters (33,38,39,41). These models show varying behavioral phenotypes, neuropathology, and disease duration, depending on the choice of promoters and the magnitude of overexpression (40,(42)(43)(44)(45)(46)(47). In some Thy1 and prion promoter ␣-synuclein Tg mice, the overexpression is about 10-fold higher than normal and is observed throughout the brain, including the cortex, thalamus, and substantia nigra (38 -40). The relatively lower overexpression of human ␣-synuclein in our Tg mouse model may explain the age-dependent, progressive behavioral phenotype. Our animals should make it possible to test novel agents for their ability to prevent progression of the ␣-synucleinopathy and to possibly reverse neuropathologic changes.
Most remarkable about all of the transgenic animals generated to date is the lack of severe pathology in midbrain dopamine neurons (33,42,45,46). Unlike in humans, mouse midbrain dopamine neurons are not selectively damaged by genetic overexpression of mutant ␣-synuclein. Explanations for this phenomenon remain conjectural.
In summary, Y39C human ␣-synuclein transgenic mice develop age-dependent progressive motor and cognitive deficits and ␣-synuclein neuropathology. Motor and cognitive testing indicates that these animals are a useful model of human diffuse Lewy body disease. Further study of these mice should help in understanding the molecular pathogenesis of synucleinopathies and may identify drugs to treat diffuse Lewy body disease in humans. FIGURE 7. Midbrain dopamine neurons were not affected in Tg mice. Brain sections from substantia nigra (A) and striatum (B) were stained with TH antibody. The Tg mice had normal TH staining patterns in both substantia nigra and striatum, without any obvious dopamine cell death. Quantitative data of the total number of TH ϩ cells in substantia nigra and TH ϩ fiber density in striatum from Tg and nTg littermate mice are shown in C and D, respectively. Three mice from each age group (9 -12, 15-18, and 21-24 months) were analyzed for TH ϩ cell number and terminal fibril density. Four sections evenly spanning the substantia nigra and three sections spanning the striatum were stained with TH antibody. The total TH ϩ cell numbers from four sections were counted and averaged for each animal. The image of striatum after TH staining was acquired by SPOT software, and TH ϩ fiber densities were analyzed by Adobe Photoshop. The data show that there was no significant difference in either total TH ϩ cells in substantia nigra or TH ϩ fiber density in striatum between Tg and nTg littermate mice (p Ͼ 0.1).