Title: Oxidative Stress in Transgenic Mice with Oligodendroglial α-Synuclein Overexpression Replicates the Characteristic Neuropathology of Multiple System Atrophy
Abstract: Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by parkinsonism unresponsive to dopaminergic therapy, cerebellar ataxia, and dysautonomia. Neuropathology shows a characteristic neuronal multisystem degeneration that is associated with widespread oligodendroglial α-synuclein (α-SYN) inclusions. Presently no animal model completely replicates the specific neuropathology of MSA. Here we investigated the behavioral and pathological features resulting from oligodendroglial α-SYN overexpression in transgenic mice exposed to mitochondrial inhibition by 3-nitropropionic acid. In transgenic mice 3-nitropropionic acid induced or augmented motor deficits that were associated with MSA-like pathology including striatonigral degeneration and olivopontocerebellar atrophy. Widespread astrogliosis and microglial activation were also observed in the presence of α-SYN in oligodendrocytes. Our results indicate that combined mitochondrial inhibition and overexpression of oligodendroglial α-SYN generates a novel model of MSA that may be useful for evaluating both pathogenesis and treatment strategies. Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by parkinsonism unresponsive to dopaminergic therapy, cerebellar ataxia, and dysautonomia. Neuropathology shows a characteristic neuronal multisystem degeneration that is associated with widespread oligodendroglial α-synuclein (α-SYN) inclusions. Presently no animal model completely replicates the specific neuropathology of MSA. Here we investigated the behavioral and pathological features resulting from oligodendroglial α-SYN overexpression in transgenic mice exposed to mitochondrial inhibition by 3-nitropropionic acid. In transgenic mice 3-nitropropionic acid induced or augmented motor deficits that were associated with MSA-like pathology including striatonigral degeneration and olivopontocerebellar atrophy. Widespread astrogliosis and microglial activation were also observed in the presence of α-SYN in oligodendrocytes. Our results indicate that combined mitochondrial inhibition and overexpression of oligodendroglial α-SYN generates a novel model of MSA that may be useful for evaluating both pathogenesis and treatment strategies. Multiple system atrophy (MSA) is a sporadic neurodegenerative disorder that presents with levodopa-resistant parkinsonism, cerebellar ataxia, and dysautonomia in any combination.1Gilman S Low PA Quinn N Albanese A Ben-Shlomo Y Fowler CJ Kaufmann H Klockgether T Lang AE Lantos PL Litvan I Mathias CJ Oliver E Robertson D Schatz I Wenning GK Consensus statement on the diagnosis of multiple system atrophy.J Neurol Sci. 1999; 163: 94-98Abstract Full Text Full Text PDF PubMed Scopus (966) Google Scholar, 2Wenning GK Colosimo C Geser F Poewe W Multiple system atrophy.Lancet Neurol. 2004; 3: 93-103Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar Neuropathologically there is multisystem neuronal loss affecting striatum and substantia nigra pars compacta (striatonigral degeneration, SND), cerebellum, pons, inferior olives (olivopontocerebellar atrophy), and the intermediolateral column of the spinal cord.3Daniel S Autonomic failure.in: Bannister R Mathias CJ The Neuropathology and Neurochemistry of Multiple System Atrophy. University Press, Oxford1999: 321-328Google Scholar Microglial activation,4Ishizawa K Komori T Sasaki S Arai N Mizutani T Hirose T Microglial activation parallels system degeneration in multiple system atrophy.J Neuropathol Exp Neurol. 2004; 63: 43-52Crossref PubMed Scopus (128) Google Scholar astrogliosis,5Wenning GK Seppi K Tison F Jellinger K A novel grading scale for striatonigral degeneration (multiple system atrophy).J Neural Transm. 2002; 109: 307-320Crossref PubMed Scopus (70) Google Scholar and demyelination6Matsuo A Akiguchi I Lee GC McGeer EG McGeer PL Kimura J Myelin degeneration in multiple system atrophy detected by unique antibodies.Am J Pathol. 1998; 153: 735-744Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar appear to be associated with the degeneration process. (Oligodendro)glial cytoplasmic inclusions (GCIs) represent the neuropathological hallmark lesion of MSA.7Papp MI Kahn JE Lantos PL Glial cytoplasmic inclusions in the CNS of patients with multiple system atrophy (striatonigral degeneration, olivopontocerebellar atrophy and Shy-Drager syndrome).J Neurol Sci. 1989; 94: 79-100Abstract Full Text PDF PubMed Scopus (843) Google Scholar The importance of GCIs for the pathogenesis of MSA is generally acknowledged, however, the relation of GCI formation to glial and neuronal cell death remains unclear. 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Reactive oxygen species derived from dysfunctional mitochondria in the presence of environmental toxins may contribute to the pathogenesis of MSA.11Burn DJ Jaros E Multiple system atrophy: cellular and molecular pathology.Mol Pathol. 2001; 54: 419-426PubMed Google Scholar, 12Hanna PA Jankovic J Kirkpatrick JB Multiple system atrophy: the putative causative role of environmental toxins.Arch Neurol. 1999; 56: 90-94Crossref PubMed Scopus (65) Google Scholar Indeed, oxidative damage of α-SYN was detected in GCIs.13Giasson BI Duda JE Murray IV Chen Q Souza JM Hurtig HI Ischiropoulos H Trojanowski JQ Lee VM Oxidative damage linked to neurodegeneration by α-synuclein nitration in synucleinopathy lesions.Science. 2000; 290: 985-989Crossref PubMed Scopus (1396) Google Scholar MSA carries a serious prognosis because of rapid progression of motor disabilities.2Wenning GK Colosimo C Geser F Poewe W Multiple system atrophy.Lancet Neurol. 2004; 3: 93-103Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar In addition, effective therapy for MSA patients is lacking. Therefore, there is a strong need for experimental models as preclinical test beds for novel treatment strategies in MSA. We have developed several animal models of SND, the neuropathological substrate of MSA-associated parkinsonism, based on neurotoxic lesions of striatum and substantia nigra pars compacta (SNc) by either direct unilateral delivery in rats14Wenning GK Granata R Laboyrie PM Quinn NP Jenner P Marsden CD Reversal of behavioural abnormalities by fetal allografts in a novel rat model of striatonigral degeneration.Mov Disord. 1996; 11: 522-532Crossref PubMed Scopus (67) Google Scholar, 15Puschban Z Scherfler C Granata R Laboyrie P Quinn NP Jenner P Poewe W Wenning GK Autoradiographic study of striatal dopamine re-uptake sites and dopamine D1 and D2 receptors in a 6-hydroxydopamine and quinolinic acid double-lesion rat model of striatonigral degeneration (multiple system atrophy) and effects of embryonic ventral mesencephalic, striatal or co-grafts.Neuroscience. 2000; 95: 377-388Crossref PubMed Scopus (35) Google Scholar, 16Puschban Z Waldner R Seppi K Stefanova N Humpel C Scherfler C Levivier M Poewe W Wenning GK Failure of neuroprotection by embryonic striatal grafts in a double lesion rat model of striatonigral degeneration (multiple system atrophy).Exp Neurol. 2000; 164: 166-175Crossref PubMed Scopus (16) Google Scholar, 17Scherfler C Puschban Z Ghorayeb I Goebel GP Tison F Jellinger K Poewe W Wenning GK Complex motor disturbances in a sequential double lesion rat model of striatonigral degeneration (multiple system atrophy).Neuroscience. 2000; 99: 43-54Crossref PubMed Scopus (51) Google Scholar, 18Ghorayeb I Puschban Z Fernagut PO Scherfler C Rouland R Wenning GK Tison F Simultaneous intrastriatal 6-hydroxydopamine and quinolinic acid injection: a model of early-stage striatonigral degeneration.Exp Neurol. 2001; 167: 133-147Crossref PubMed Scopus (50) Google Scholar, 19Stefanova N Lundblad M Tison F Poewe W Cenci MA Wenning GK Effects of pulsatile L-DOPA treatment in the double lesion rat model of striatonigral degeneration (multiple system atrophy).Neurobiol Dis. 2004; 15: 630-639Crossref PubMed Scopus (26) Google Scholar or systemic exposure in mice and primates.20Ghorayeb I Fernagut PO Aubert I Bezard E Poewe W Wenning GK Tison F Toward a primate model of L-dopa-unresponsive parkinsonism mimicking striatonigral degeneration.Mov Disord. 2000; 15: 531-536Crossref PubMed Scopus (41) Google Scholar, 21Ghorayeb I Fernagut PO Stefanova N Wenning GK Bioulac B Tison F Dystonia is predictive of subsequent altered dopaminergic responsiveness in a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine+3-nitropropionic acid model of striatonigral degeneration in monkeys.Neurosci Lett. 2002; 335: 34-38Crossref PubMed Scopus (27) Google Scholar, 22Fernagut PO Diguet E Stefanova N Biran M Wenning GK Canioni P Bioulac B Tison F Subacute systemic 3-nitropropionic acid intoxication induces a distinct motor disorder in adult C57Bl/6 mice: behavioural and histopathological characterisation.Neuroscience. 2002; 114: 1005-1017Crossref PubMed Scopus (130) Google Scholar, 23Stefanova N Puschban Z Fernagut PO Brouillet E Tison F Reindl M Jellinger KA Poewe W Wenning GK Neuropathological and behavioral changes induced by various treatment paradigms with MPTP and 3-nitropropionic acid in mice: towards a model of striatonigral degeneration (multiple system atrophy).Acta Neuropathol (Berl). 2003; 106: 157-166Crossref PubMed Scopus (46) Google Scholar Distinct behavioral and morphological abnormalities have been characterized in these models. However, oligodendroglial α-SYN pathology was not present in any of them. Thus, transgenic (Tg) mouse models were generated in which human α-SYN was specifically expressed in oligodendrocytes.24Kahle PJ Neumann M Ozmen L Muller V Jacobsen H Spooren W Fuss B Mallon B Macklin WB Fujiwara H Hasegawa M Iwatsubo T Kretzschmar HA Haass C Hyperphosphorylation and insolubility of alpha-synuclein in transgenic mouse oligodendrocytes.EMBO Rep. 2002; 3: 583-588Crossref PubMed Scopus (267) Google Scholar In Tg mice overexpressing human wild-type α-SYN under control of the proteolipid protein (PLP) promotor, both GCI-like aggregates and biochemical markers of MSA were observed, including detergent insolubility25Tu PH Galvin JE Baba M Giasson B Tomita T Leight S Nakajo S Iwatsubo T Trojanowski JQ Lee VM Glial cytoplasmic inclusions in white matter oligodendrocytes of multiple system atrophy brains contain insoluble alpha-synuclein.Ann Neurol. 1998; 44: 415-422Crossref PubMed Scopus (596) Google Scholar, 26Dickson DW Lin W Liu WK Yen SH Multiple system atrophy: a sporadic synucleinopathy.Brain Pathol. 1999; 9: 721-732Crossref PubMed Scopus (173) Google Scholar, 27Campbell BC McLean CA Culvenor JG Gai WP Blumbergs PC Jakala P Beyreuther K Masters CL Li QX 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: 87-96Crossref PubMed Scopus (162) Google Scholar and phosphorylation at serine-12928Fujiwara H Hasegawa M Dohmae N Kawashima A Masliah E Goldberg MS Shen J Takio K Iwatsubo T Alpha-synuclein is phosphorylated in synucleinopathy lesions.Nat Cell Biol. 2002; 4: 160-164Crossref PubMed Scopus (161) Google Scholar of the transgenic α-SYN. In the present work we aimed to establish whether oligodendroglial α-SYN overexpression in (PLP)-α-SYN mice24Kahle PJ Neumann M Ozmen L Muller V Jacobsen H Spooren W Fuss B Mallon B Macklin WB Fujiwara H Hasegawa M Iwatsubo T Kretzschmar HA Haass C Hyperphosphorylation and insolubility of alpha-synuclein in transgenic mouse oligodendrocytes.EMBO Rep. 2002; 3: 583-588Crossref PubMed Scopus (267) Google Scholar combined with mitochondrial inhibition by 3-nitropropionic acid (3-NP)22Fernagut PO Diguet E Stefanova N Biran M Wenning GK Canioni P Bioulac B Tison F Subacute systemic 3-nitropropionic acid intoxication induces a distinct motor disorder in adult C57Bl/6 mice: behavioural and histopathological characterisation.Neuroscience. 2002; 114: 1005-1017Crossref PubMed Scopus (130) Google Scholar replicates the combined glial and neuronal pathology of MSA. The generation and characterization of the (PLP)-α-SYN mice was previously described.24Kahle PJ Neumann M Ozmen L Muller V Jacobsen H Spooren W Fuss B Mallon B Macklin WB Fujiwara H Hasegawa M Iwatsubo T Kretzschmar HA Haass C Hyperphosphorylation and insolubility of alpha-synuclein in transgenic mouse oligodendrocytes.EMBO Rep. 2002; 3: 583-588Crossref PubMed Scopus (267) Google Scholar The following in vivo protocols were approved by the Federal Ministry of Science and Transport of Austria. In the present study we used a total of 41 homozygous (PLP)-α-SYN mice (Tg) and 38 C57BL/6 mice (control) older than 10 months of age. Animals were maintained in a temperature-controlled room under 12-hour light/dark cycle with free access to food and water. Mice of each genotype (Tg or control) were subgrouped into saline-treated, low-dose 3-NP, and high-dose 3-NP groups. The 3-NP intraperitoneal treatment was done according to the following scheme22Fernagut PO Diguet E Stefanova N Biran M Wenning GK Canioni P Bioulac B Tison F Subacute systemic 3-nitropropionic acid intoxication induces a distinct motor disorder in adult C57Bl/6 mice: behavioural and histopathological characterisation.Neuroscience. 2002; 114: 1005-1017Crossref PubMed Scopus (130) Google Scholar: low-dose 3-NP: 4 × 10 mg/kg, 4 × 20 mg/kg, 4 × 40 mg/kg, 3 × 50 mg/kg, total dose 430 mg/kg; high-dose 3-NP: 8 × 40 mg/kg, 3 × 80 mg/kg, total dose 560 mg/kg. 3-NP was dissolved in saline and pH 7.4 was adjusted with 1 mol/L NaOH. The concentration was calculated to keep the injected volume (∼250 μl) stable. Intraperitoneal injections were done every 12 hours during the intoxication period. We used a previously described rating scale22Fernagut PO Diguet E Stefanova N Biran M Wenning GK Canioni P Bioulac B Tison F Subacute systemic 3-nitropropionic acid intoxication induces a distinct motor disorder in adult C57Bl/6 mice: behavioural and histopathological characterisation.Neuroscience. 2002; 114: 1005-1017Crossref PubMed Scopus (130) Google Scholar for evaluation of hindlimb clasping, general locomotor activity, hindlimb dystonia, truncal dystonia, and postural challenge response (0, normal; 1, slightly disturbed; and 2, markedly disabled). The rating was performed every 12 hours before the injection during the intoxication period and every day during the period after intoxication. The total score for each session was determined. The pole test22Fernagut PO Diguet E Stefanova N Biran M Wenning GK Canioni P Bioulac B Tison F Subacute systemic 3-nitropropionic acid intoxication induces a distinct motor disorder in adult C57Bl/6 mice: behavioural and histopathological characterisation.Neuroscience. 2002; 114: 1005-1017Crossref PubMed Scopus (130) Google Scholar, 29Matsuura K Kabuto H Makino H Ogawa N Pole test is a useful method for evaluating the mouse movement disorder caused by striatal dopamine depletion.J Neurosci Methods. 1997; 73: 45-48Crossref PubMed Scopus (282) Google Scholar was performed at baseline and on day 13 after the beginning of intoxication. Each mouse was habituated to the test the day before. A wooden vertical pole with rough surface, 1 cm wide and 50 cm high was applied. The mouse was placed with the head up at the top of the pole and the time for turning downwards (Tturn) as well as the total time for climbing down the pole until the mouse reached the floor with the four paws (Ttotal) was taken in five trials. The best performance of all of the five trials was kept for the statistical analysis.22Fernagut PO Diguet E Stefanova N Biran M Wenning GK Canioni P Bioulac B Tison F Subacute systemic 3-nitropropionic acid intoxication induces a distinct motor disorder in adult C57Bl/6 mice: behavioural and histopathological characterisation.Neuroscience. 2002; 114: 1005-1017Crossref PubMed Scopus (130) Google Scholar If a mouse was unable to perform the test, a default value of 120 seconds was taken into account. To test the locomotor activity of the mice we applied the Flex Field Activity System (San Diego Instruments, San Diego, CA), which allows monitoring and real-time counting of horizontal and vertical locomotor activity by 544 photo-beam channels. Mice were placed in the center of the open field (40.5 × 40.5 × 36.5 cm) and tested for a 15-minute period always at the same time of the day (5:00 p.m.). The tests were performed in a dark room that was completely isolated from external noises and light during the test period. The sum of counts in the horizontal and vertical plane at baseline and on day 13 after the first injection was further analyzed. The stride length22Fernagut PO Diguet E Stefanova N Biran M Wenning GK Canioni P Bioulac B Tison F Subacute systemic 3-nitropropionic acid intoxication induces a distinct motor disorder in adult C57Bl/6 mice: behavioural and histopathological characterisation.Neuroscience. 2002; 114: 1005-1017Crossref PubMed Scopus (130) Google Scholar of the forelimbs and hindlimbs of the mice was measured at baseline and before sacrificing them after a habituation to the test for 3 days before its performance. The limbs of each animal were wetted with a nontoxic food color and the mouse was let to run on a strip of paper (42 cm long, 4.5 cm wide) down a bright corridor toward a dark goal box. After three runs, the stride length of the forelimbs and the hindlimbs on each side was measured, excluding the beginning (7 cm) and the end (7 cm) of the run. The mean stride length for each forelimbs and hindlimbs was determined. Animals were perfused under thiopental anesthesia (day 15 after the first 3-NP injection) with 5 ml of phosphate-buffered saline followed by 20 ml of 4% paraformaldehyde (pH 7.4). Brains were quickly removed, postfixed in the same fixative for 1 hour at 4°C, and then kept in 25% sucrose solution until they sank. Brains were slowly frozen with 2-methylbutan and kept at −80°C until processing. Four series of two adjacent 20-μm sections (directly mounted on slides) and six series of 50-μm free-floating sections throughout the whole brain were cut on a freezing microtome (Leica, Nussloch, Germany) and either dried or kept in assorter buffer at 4°C, respectively. The series on slides were used for standard cresyl violet and Klüver-Barrera stainings and the free-floating sections were used for immunohistochemical stainings. A group of mice was decapitated under thiopental anesthesia, brains were quickly removed, fixed in 4% formaldehyde in phosphate-buffered saline, and embedded in paraffin. Sections (4 μm) were stained with hematoxylin and eosin, Gallyas silver, Campbell-Switzer silver, thioflavin S, and Luxol fast blue-periodic acid-Schiff. The proteinase K-digested paraffin-embedded tissue blot was performed as described previously.30Neumann M Kahle PJ Giasson BI Ozmen L Borroni E Spooren W Muller V Odoy S Fujiwara H Hasegawa M Iwatsubo T Trojanowski JQ Kretzschmar HA Haass C Misfolded proteinase K-resistant hyperphosphorylated alpha-synuclein in aged transgenic mice with locomotor deterioration and in human alpha-synucleinopathies.J Clin Invest. 2002; 110: 1429-1439Crossref PubMed Scopus (299) Google Scholar The following antibodies were used in this study: monoclonal anti-DARPP-32 (1:20000; generous gift from Prof. H. Hemmings, New York Presbyterian Hospital, NY), monoclonal anti-TH (1:500; Sigma, St. Louis, MO), monoclonal anti-calbindin D28k (1:1000, Sigma), monoclonal anti-GFAP (1:100; Roche, Vienna, Austria), monoclonal anti-CNP (1:100, Sigma), monoclonal anti-CD68 (1:100, MCA 1957; Serotec, Oxford, UK), monoclonal anti-human-α-synuclein (1:10, 15G731Kahle PJ Neumann M Ozmen L Muller V Jacobsen H Schindzielorz A Okochi M Leimer U van Der Putten H Probst A Kremmer E Kretzschmar HA Haass C Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain.J Neurosci. 2000; 20: 6365-6373Crossref PubMed Google Scholar), monoclonal anti-MBP (1:100, mAb 381; Chemicon, Temecula, CA), monoclonal anti-MOG (1:100, 8.18-C532Brunner C Lassmann H Waehneldt TV Matthieu JM Linington C Differential ultrastructural localization of myelin basic protein, myelin oligodendroglial glycoprotein, and 2′,3′-cyclic nucleotide 3′-phosphodiesterase in the CNS of adult rats.J Neurochem. 1989; 52: 296-304Crossref PubMed Scopus (336) Google Scholar), monoclonal anti-ubiquitin (1:300, mAb 1510; Chemicon). Secondary antibodies were biotinylated goat anti-mouse IgG or goat anti-rat IgG (Vector, Burlingame, CA). All morphometric analyses were done by a blinded observer on Olympus BX60 microscope (Olympus, Hamburg, Germany) supplied with computerized image analysis system (Sony 3CCD video camera, Image Pro Plus software; Media Cybernetics, Silver Spring, MD, and Neurolucida software, MicroBrightField, Inc., Colchester, VT). Stereological methods33Mayhew TM Gundersen HJ If you assume, you can make an ass out of u and me: a decade of the disector for stereological counting of particles in 3D space.J Anat. 1996; 188: 1-15PubMed Google Scholar were used to count neurons in the striatum, substantia nigra pars compacta, locus ceruleus, pontine nuclei, deep cerebellar nuclei, and inferior olive. The entire cerebellar hemisphere was outlined on serial sections to determine cerebellar volume. Purkinje cells were counted in a region outlined to include only the Purkinje cell layer.34German DC Quintero EM Liang C Xie C Dietschy JM Degeneration of neurons and glia in the Niemann-Pick C mouse is unrelated to the low-density lipoprotein receptor.Neuroscience. 2001; 105: 999-1005Crossref PubMed Scopus (38) Google Scholar The density per mm2 of 2′3′-cyclic nucleotide 3′-phosphodiesterase (CNP)-positive oligodendrocytes in the striatum was determined. The optical density of the dopaminergic terminals in the striatum was measured in serial sections and a mean density score was determined for each mouse. Behavioral data were analyzed with Kruskal-Wallis nonparametric analysis and posthoc Dunn's test. After confirming normality of distribution, the histological morphometric data were subjected to two-way analysis of variance to compare control and Tg mice after intoxication with different doses of 3-NP. Posthoc Bonferroni test was applied where appropriate. P < 0.05 was used to determine statistical significance. Both (PLP)-α-SYN and C57BL/6 control mice developed progressive motor disability after treatment with 3-NP in a dose-dependent manner as established by the Bordeaux motor behavioral scale for assessment of the severity of 3-NP-induced motor disorders in mice.22Fernagut PO Diguet E Stefanova N Biran M Wenning GK Canioni P Bioulac B Tison F Subacute systemic 3-nitropropionic acid intoxication induces a distinct motor disorder in adult C57Bl/6 mice: behavioural and histopathological characterisation.Neuroscience. 2002; 114: 1005-1017Crossref PubMed Scopus (130) Google Scholar In the low-dose treatment group only 43% of the control mice developed motor disability evaluated by this scale, whereas all of the Tg mice in the low-dose group (100%) showed signs of motor impairment. In low-dose 3-NP-treated mice the mean motor behavioral score was significantly greater in Tg compared to control mice at day 15 (Figure 1a). In contrast, motor disability increased in both high-dose 3-NP-treated Tg and control mice reaching a maximum on the last test day (day 15) before sacrifice (Figure 1a). Comparison of overall motor disability scores in Tg and control mice revealed dose dependence (Figure 1b). Forty-six percent of the high-dose 3-NP-treated Tg mice died before the end of the test period, whereas none of the control mice died after the high-dose intoxication. The 3-NP dose-dependent motor impairment was confirmed by open field activity (Figure 1, c and d), pole (Figure 1, e and f) and stride length tests (Figure 1, g and h). The general locomotor activity in an open field arena decreased because of 3-NP intoxication both in the horizontal and vertical plane (Figure 1, c and d). Further, the rearing scores (vertical movements) were significantly more reduced in Tg than in control mice receiving low-dose 3-NP (Figure 1d). The time that mice needed to turn their head and body down (Tturn) and to descend along a pole (Ttotal) significantly increased with escalating dose levels of 3-NP (Figure 1, e and f). Additionally, Tturn and Ttotal were significantly longer in 3-NP-treated Tg compared to control mice indicating a more severe motor deficit with disturbance of balance and coordination. Exposure to 3-NP induced a dose-dependent shortening of the stride length in both control and Tg mice (Figure 1, g and h). In addition, hindlimb stride length was significantly reduced in untreated Tg compared to control mice (Figure 1h). Striatal neuronal cell loss was induced by treatment with 3-NP in both control and Tg mice as shown by cresyl violet staining and DARPP-32 immunohistochemistry. Neuronal loss was most prominent in the dorsolateral striatum with well-circumscribed symmetrical striatal lesions in mice treated with high-dose 3-NP (Figure 2, a and b). DARPP-32+ neurons in the striatum were lost dose dependently after 3-NP intoxication (Figure 2c). In addition, Tg mice showed greater striatal neuronal loss than control mice after low-dose 3-NP exposure. Neuronal loss was accompanied by striatal shrinkage as measured by decrease in striatal volume (Figure 2d). Again, striatal volume loss was greater in Tg compared to control mice receiving low-dose 3-NP. In addition to the loss of DARPP-32+ neurons in the striatum, 3-NP intoxication also caused a dose- and genotype-dependent loss of dopaminergic innervation of the striatum, as evidenced by reduced TH immunoreactivity (Figure 2e). A significant reduction of TH-immunoreactive neurons was observed in the SNc of Tg mice at baseline (Figure 2f), suggesting that the presence of α-SYN in oligodendrocytes alone induced dopaminergic neuron loss. 3-NP exposure exacerbated dopaminergic cell loss in both Tg and control mice (Figure 2, g and h) in a dose- and genotype-dependent manner (Figure 2f). In the low-dose and high-dose 3-NP regimens there was no consistent effect of the toxin on cerebellar neurons in control mice. In contrast, Tg mice showed dose-dependent 3-NP-induced cerebellar Purkinje cell loss as measured by cresyl violet staining (Figure 3, a and b) and calbindin D28K immunohistochemistry (data not shown). 3-NP treatment induced dose-dependent loss of the cerebellar volume in Tg mice that was not observed in control mice (Figure 3b). The deep cerebellar nuclei of neither control nor Tg mice were affected by 3-NP intoxication (Figure 3b). Analysis of the neuronal loss in the pons demonstrated TH+ neuron loss in the locus ceruleus (LC) already at baseline in Tg mice (Figure 3, c and d). After 3-NP intoxication, TH+ cell loss was evident in both control and Tg mice, and this was more marked in Tg than in control mice (Figure 3, c and d). Furthermore, neuronal loss in the pontine nuclei and in the inferior olivary nucleus on 3-NP treatment was observed in Tg but not in control mice (Figure 3d). There was no apparent difference between the GFAP staining pattern in untreated control and Tg mice. As a result of the 3-NP intoxication we observed astroglial activation in the striatum of both control and Tg mice forming a scar around the lesion in the dorsolateral striatum (data not shown). In SN the astroglial activation was more prominent in Tg than in control mice treated with 3-NP (Figure 4). Further, 3-NP induced astroglial activation in cerebellum, pons, and inferior olives was only seen in Tg but not in control mice (Figure 4). Prominent microglial activation was observed in the striatum, SNc, and in the white matter throughout the brain of the untreated Tg but not the control mice (Figure 5). Intoxication with 3-NP increased microglial activation in the striatum and substantia nigra of Tg mice and to a lesser degree also induced microglial activation in these areas in control mice (data not shown). In addition the activation of microglia in the white matter of the Tg mice was markedly intensified by 3-NP, whereas in control mice it was absent or just single CD68-positive cells were seen (Figure 5). CD68-positive cells were observed also in the cerebellar cortex and deep cerebellar nuclei, the pontine nuclei, and the inferior olive of 3-NP-treated Tg mice but not in control mice (data not shown). Oligodendrocytes of Tg mice showed human α-SYN-positive profiles (Figure 6, a and b), as previously described.24Kahle PJ Neumann M Ozmen L Muller V Jacobsen H Spooren W Fuss B Mallon B Macklin WB Fujiwara H Hasegawa M Iwatsubo T Kretzschmar HA Haass C Hyperphosphorylation and insolubility of alpha-synuclein in transgenic mouse oligodendrocytes.EMBO Rep. 2002; 3: 583-588Crossref PubMed Scopus (267) Google Scholar Morphometric analysis of the density of oligodendrocytes revealed a significant cell loss because of high-dose, but not low-dose, 3-NP intoxication (Figure 6c). No widespread specific positive inclusions were detectable with ubiquitin antibodies (data not shown). Argyrophilic thioflavin S-positive or proteinase K-resistant