JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2008, 59, 2, 205216 www.jpp.krakow.pl
M. PLAAS a,b,c, A. KARIS c, J. INNOS a, E. REBANE a, V. BAEKELANDT d, A. VAARMANN e, H. LUUK a, E. VASAR a, S. KOKS a,f,*
ALPHA-SYNUCLEIN A30P POINT-MUTATION GENERATES AGE-DEPENDENT NIGROSTRIATAL DEFICIENCY IN MICE Department of Physiology, Center of Molecular and Clinical Medicine, University of Tartu, Tartu, Estonia; b) Institute of Technology, University of Tartu, Tartu, Estonia; c) Department of Integrative Zoology, University of Tartu, Tartu, Estonia; d) Laboratory for Neurobiology and Gene Therapy, Katholieke Universiteit Leuven, Leuven, Belgium; e) Department of Pharmacology, Center of Molecular and Clinical Medicine, University of Tartu, Tartu, Estonia; f) Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, Tartu, Estonia a)
Lewy bodies are mainly composed of α-synuclein (SNCA) and specific mutations in SNCA gene are related to familial forms of Parkinson's disease (PD). The purpose of our study was to generate a mouse line with A30P knock-in point mutation in SNCA gene and to test if a single point-mutation is able to turn otherwise normal SNCA into a toxic form. The behavioral profile of SNCA A30P mice was followed for 16 months. Generally, these mice are healthy and viable without any obvious abnormalities. Starting from the age of 13 months mice developed a significant deficit in motor performance tests related to nigrostriatal function (ink-test and beam walk). In other tests (motility boxes, rotarod) mice continuously performed normally. Moreover, SNCA A30P mice expressed the altered sensitivity to VMAT2 inhibitor reserpine, possibly reflecting a functional deficiency of dopamine. Indeed, mice at 15 months of age had significantly reduced levels of dopamine and its major metabolite DOPAC in the striatum, and reduced levels of dopamine in the mesolimbic system. The present study confirms that SNCA plays an important role in the development of PD and an insertion of a single point mutation is sufficient to generate age-related decline in specific motor performance. The generated mouse line has a potential to become a model for PD with comparable time course and phenotype. K e y w o r d s : alpha-synuclein, SNCA, Parkinson disease, transgenic mice, knockout mice, disease models
* - author for correspondence
206 INTRODUCTION
A-synuclein (SNCA) belongs to a family of structurally related proteins that are prominently expressed in the central nervous system. SNCA was the first gene linked with Parkinson's disease (PD). Aggregated SNCA proteins form intracellular deposits that are hallmarks of degenerative synucleinopathies and constitute the major components of Lewy bodies - characteristic histological features of PD (1). The accumulation of SNCA in cultured human dopaminergic cells results in apoptosis that requires endogenous dopamine production and is mediated by reactive oxygen species (2). Point-mutations (A53T and A30P) in SNCA gene are responsible for the familial forms of PD (3, 4). The identification of a third mutation (E46K) and genomic multiplications of a locus containing SNCA indicate that even subtle defects in the SNCA system are sufficient for the development of the disease and even small changes in the expression level could become a risk factor (5, 6). The exact function of SNCA is not known yet, but it is a 140-amino acid presynaptic protein and possibly is related to vesicle handling and neurotransmitter release (7, 8). Mice lacking SNCA are healthy and fertile, but display functional deficiency in the dopaminergic system (9). Namely, dopamine release is increased in response to paired stimuli and response to amphetamine is attenuated (9). On the other hand, overexpression of wild-type or mutant SNCA in PC12 cells inhibited the release of dopamine and the accumulation of "docked" vesicles (10). In similar experiments, significantly increased cytosolic dopamine concentration was found (11). And finally, gain-offunction haplotypes of vmat2 gene confer protective effect for the development of Parkinson's disease (12). These findings indicate that SNCA has a significant role in the vesicular storage and release of dopamine and in the regulation of nigrostriatal physiology. Taking into account the role of SNCA in the pathogenesis of PD, this protein has been in the focus of research and several transgenic animals have been developed in order to model the pathogenesis of PD (13-16). All these models have some limitations, mostly due to the technology used. These mice have been generated by adding at least one wild-type or mutant copy of SNCA gene into the mouse genome. This approach (microinjection of DNA) is random (uncontrolled integration site and number of transgene copies) and results in genetically different mouse lines. Moreover, an extra copy of SNCA is added, which is pathogenetic in its own right, irrespective of whether it is mutant or wild-type (5). All these mouse lines express very different phenotypes and in some cases degeneration is wide and unspecific leading to very severe motor deficits that are not specific to the nigrostriatal system (17). Moreover, even in case endogenous SNCA was removed, transgenic SNCA induced severe neuronopathy and motor neuron damage in the ventral roots (13). Also, histopathological findings do not necessarily correlate with behavioral phenotypes. For instance, human A30P SNCA transgenic mice with
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Fig. 1. Targeting construct and recombination to achieve insertion of SNCA A30P point-mutation Vector contains floxed Neo-TK expression cassette that was removed by in vitro expression of Crerecombinase. SNCA A30P mutation inserted SmaI restriction site what was used to genotype the mutant mice after PCR amplification.
very high SNCA expression level have severe motor dysfunctions, but do not show any loss of dopaminergic cells (18). On the other hand, in several cases, motor dysfunctions are unspecific and not limited to the nigrostriatal system only (13, 19). Taken together, present transgenic models for PD are very useful to model synucleinopathies, to study the role of SNCA in neurodegeneration and to study the toxicity of accumulating SNCA. However, the existing transgenic models do not display specific motor impairment involving mainly nigrostriatal function. Our study aimed to generate a mouse model for PD with a more specific behavioral deficit. We generated a mouse line with inserted point mutation in the SNCA protein in position 30, by replacing alanine with proline (A30P, Fig. 1). As a result we got a mouse line with a similar point mutation as in humans with familial PD forms. This paper describes behavioral and pharmacological validation of this model. Starting from the age of 4 months a set of different behavioral tests was performed to evaluate the motor performance of these mice. We applied the following tests: open field (automated MOTI boxes with infrared beams), beam walk, rotarod and ink-test. To assess the functionality of the dopaminergic system pharmacologically we tested the effect of reserpine and amphetamine and finally measured the content of dopamine in the striatum, mesolimbic system and ventral mesencephalon (includes the ventral tegmental area and substantia nigra).
208 MATERIALS AND METHODS In our experiment we used transgenic mice generated in our lab. As SNCA is related to a degenerative process we performed behavioral analysis repeatedly with mice of different age. All animal procedures and animal care methods were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and with the institutional guidelines.
Generation of mutant mice with A30P SNCA Targeting construct (Fig. 1) was transformed into E14.1 ES (129/OlaHsd) cell line by standard electroporation. With PCR analysis of 200 clones we detected homologous recombination in 5 clones and in these clones recombination was verified by sequencing. After checking the number of chromosomes, temporary in vitro Cre-recombinase expression was performed to get Cre-lox recombination. We analyzed 50 clones with PCR and detected 6 clones with correct recombination (Neo-TK cassette removed, only 1 loxP site left, Fig. 1). Recombination positive clones were injected into C57BL/6 blastocysts. Chimeric mice were back-crossed with C57BL/6 strain to obtain genetically homogenous background. For behavioral studies we used mice from three different litters from different stages of backcrossing (3, 5 and 7 times back-crossed). Genotyping of mice was performed by PCR amplification of exon 2, followed by the digestion of the PCR product with SmaI. Insertion of mutation (P instead of A in 30th position) generates SmaI restriction site in SNCA gene. This site does not exist in the wild-type allele (Fig. 1).
Behavioral testing Behavioral testing started at the age of 4 months and lasted up to 16 month. Wild-type and SNCA A30P mutant groups were generated by heterozygous mating. Wild-type littermates (instead of C57BL6) were used as control animals to avoid possible differences caused by genetic background. In the present study we used at least 10 male mice in each group and behavioral experiments were repeated blind. Preliminary testing revealed the lack of gender specific differences.
Locomotor activity For the study of locomotor activity the animals were placed singly into photoelectric motility boxes (448 mm×448 mm×450 mm) connected to a computer (ActiMot/MoTil, TSE Technical & Scientific Equipment GmbH, Germany). The illumination level of the transparent test boxes was approximately 400 lux. Time in locomotion (s), distance travelled (m), number of rearing and number of corner entries were registered during 30 min.
Rotarod test In the rotarod test, mice were trained for 1 min on the "easy" rod (O 35 mm, metal rod covered with rough adhesive tape, 9 rotations per min). About 1 h later the mice were re-tested first on the "easy" rod and about 1 h later on the "difficult" rod (O 35 mm, smooth metal surface, 9 rotations per min). Latency to fall down was measured and a cutoff score of 120 s was used.
Beam walk test In the beam walk test three beams with different diameters were used (O 17.5, 14 and 9 mm). Initially mice were trained to walk on a 100 cm wooden round training-beam (O 20 mm) for 10 min for three consecutive days (4 beam crossings per day). On the 4th day test was performed. Before testing, mice were allowed to walk two times on the training-beam. About 1 hour later the mice were tested on Beam 1 (O 17.5 mm), 30 min later on Beam 2 (O 14 mm) and 30 min later on Beam
209 3 (O 9 mm). Traversing time in seconds, the number of steps and the number of slips were measured for each beam (the sum of two consecutive trials for each mouse).
Ink-test Ink-test was used to analyze the effect of the mutation upon the gait of animals. Mice were trained to run through a lighted 50 cm canal into a dark box. The floor of the canal was covered with white paper. Before each trial the forefeet of the animals were marked with red and hind limbs with blue nontoxic paint. We measured the stride width (distance between left and right foot) and the stride length (distance between two ipsilateral prints).
Pharmacological tests We challenged dopaminergic system pharmacologically to test for potential subtle changes. For this purpose reserpine provocation was used - as reserpine inhibits vesicular transporter of dopamine (vmat2) and releases intracellular pool of dopamine, its efficiency generally characterizes activity/affinity of vmat2 and vesicular storage of dopamine. This allows us to test the functionality of presynaptic vesicles. 7.5 mg/kg of reserpine (Sigma-Aldrich Co) was injected intraperitoneally (ip). Body temperature was measured before and 60 min, 120 min and 24 h after reserpine injection. Catalepsy scoring was performed at 120 minutes after reserpine and cut-off time was set to 60 sec. We also measured body weight before and 24 h after reserpine injection. After 24 hours, 6 mg/kg amphetamine (Sigma-Aldrich Co) was administered ip and 15 minutes later motor activity of mice during next 120 min was measured. The control group received two injections of saline. To detect the effect of amphetamine, saline pre-treated mice were injected with dopamine agonist.
HPLC analysis of dopamine content The content of dopamine and DOPAC in the striatum, mesolimbic system and ventral mesencephalon of 15-month-old mice was measured by HPLC with electrochemical detection (ESA CoulArray System 5600, ESA Inc., Chelmsford, MA, USA) essentially as previously described (20). Briefly, the brain tissues were weighted and homogenized with an ultrasonic homogenizer in an ice-cold solution of 0.1 M perchloric acid (20-30 µl per mg of tissue). The homogenate was then centrifuged at 13,000×g for 10 min at 4°C and 20 µl of supernatant was injected into the HPLC system. The chromatographic separation was achieved on an ESA MD-150 reversed-phase C18 (150×3.0 mm ID, 3 µm particle size) column with a Hypersil pre-column (7.5×4.6 mm, 5 µm) by using following mobile phase: 8% (v/v) methanol in 0.1 M monobasic sodium phosphate at pH 3.10 and 0.4 mM octanesulfonic acid. Both the column and electrodes were housed in a thermal chamber maintained at 35°C and the flow rate was 0.5 ml/min. The potentials of array detector were set +25 mV, +150 mV and +400 mV versus palladium reference electrode.
Statistical analysis For statistical analysis Student's t test or two-way ANOVA was used where appropriate, post hoc analysis was performed with Tukey HSD test (Prism 4, GraphPad Software Inc). RESULTS
Motor system and age-dependent changes Generally SNCA A30P mice do not have any gross abnormalities, they breed and grow well. Groups were with almost equal body weight at the age of 16
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Fig. 2. A) General motor activity of SNCA A30P mice (N=20) is not changed compared to wildtype (WT, N=20) littermates. B) Performance of mice in the beam walk test is age-dependent, number of slips per 1 m beam. SNCA A30P mice develop an age-dependent decline in performance; differences became statistically significant from the age of 13 months. * - P < 0.05 (Student's t test) compared to WT mice of the same age, number of mice in each group is 20.
months (37.3 g wild-type mice and 40.1 g SNCA A30P mice). The behavior of mice was similar in all tests up to the age of 13 months. From this time point a significant difference in the beam walk test was evident for the first time - mice with SNCA A30P point mutation slipped more than their wild type littermates on the narrowest beam (9 mm, Fig. 2B). Interestingly, the number of slips on wider beams (14 and 17.5 mm, data not shown) was similar in different genotypes, indicating that more difficult tasks are needed to see subtle motor disability in these mice. Other tests did not reveal any differences at this time point. We continued to follow the performance of motor system in these mice and continuously found more slips in A30P mice compared to wild type littermates in beam walk only on the 9 mm beam (Fig. 2B). This difference was independent of gender of mice. During all the testing period we did not find significant differences in the general motor activity of animals. Fig. 2A illustrates the general locomotor activity of the mice. Also, in the rotarod test the two groups did not display any differences during the testing period (data not shown).
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Fig. 3. A) Mean stride length difference of all four paws. Difference of the longest and shortest stride of from all paws from all mice was calculated. * p < 0.05 (Student's t test), compared to WT mice, number of mice in each group is 20. B) SNCA A30P mice at age of 13 months had a significantly shorter stride length. Stride length for right forepaw (SLFR) data are given as an example. * P Thr mutation causes neurodegenerative disease with alpha-synuclein aggregation in transgenic mice. Proc Natl Acad Sci U S A 2002; 99: 8968-8973. 18. Gomez-Isla T, Irizarry MC, Mariash A, et al. Motor dysfunction and gliosis with preserved dopaminergic markers in human alpha-synuclein A30P transgenic mice. Neurobiol Aging 2003; 24: 245-258. 19. Fernagut PO, Chesselet MF. Alpha-synuclein and transgenic mouse models. Neurobiol Dis 2004; 17: 123-130. 20. Vaarmann A, Kask A, Maeorg U. Novel and sensitive high-performance liquid chromatographic method based on electrochemical coulometric array detection for simultaneous determination of catecholamines, kynurenine and indole derivatives of tryptophan. J Chromatogr B Analyt Technol Biomed Life Sci 2002; 769: 145-153. 21. Eilam R, Peter Y, Elson A, et al. Selective loss of dopaminergic nigro-striatal neurons in brains of Atm-deficient mice. Proc Natl Acad Sci U S A 1998; 95: 12653-12656. 22. Fernagut PO, Diguet E, Labattu B, Tison F. A simple method to measure stride length as an index of nigrostriatal dysfunction in mice. J Neurosci Methods 2002; 113: 123-130. 23. Erickson JD, Eiden LE, Hoffman BJ. Expression cloning of a reserpine-sensitive vesicular monoamine transporter. Proc Natl Acad Sci U S A 1992; 89: 10993-10997. 24. Tillerson JL, Caudle WM, Reveron ME, Miller GW. Detection of behavioral impairments correlated to neurochemical deficits in mice treated with moderate doses of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine. Exp Neurol 2002; 178: 80-90. 25. Fon EA, Pothos EN, Sun BC, Killeen N, Sulzer D, Edwards RH. Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action. Neuron 1997; 19: 1271-1283. 26. Colebrooke RE, Humby T, Lynch PJ, McGowan DP, Xia J, Emson PC. Age-related decline in striatal dopamine content and motor performance occurs in the absence of nigral cell loss in a genetic mouse model of Parkinson's disease. Eur J Neurosci 2006; 24: 2622-2630. 27. Cleren C, Naudin B, Costentin J. Apparent opposite effects of tetrabenazine and reserpine on the toxic effects of 1-methyl-4-phenylpyridinium or 6-hydroxydopamine on nigro-striatal dopaminergic neurons. Brain Res 2003; 989: 187-195. R e c e i v e d : October 23, 2007 A c c e p t e d : April 25, 2008 Authors address: Sulev Koks Department of Physiology University of Tartu 19 Ravila Street 50411 Tartu, Estonia; e-mail:
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