The I2020T Leucine-rich repeat kinase 2 transgenic mouse exhibits impaired locomotive ability accompanied by dopaminergic neuron abnormalities
© Maekawa et al.; licensee BioMed Central Ltd 2012
Received: 5 October 2011
Accepted: 16 April 2012
Published: 25 April 2012
Leucine-rich repeat kinase 2 (LRRK2) is the gene responsible for autosomal-dominant Parkinson’s disease (PD), PARK8, but the mechanism by which LRRK2 mutations cause neuronal dysfunction remains unknown. In the present study, we investigated for the first time a transgenic (TG) mouse strain expressing human LRRK2 with an I2020T mutation in the kinase domain, which had been detected in the patients of the original PARK8 family.
The TG mouse expressed I2020T LRRK2 in dopaminergic (DA) neurons of the substantia nigra, ventral tegmental area, and olfactory bulb. In both the beam test and rotarod test, the TG mice exhibited impaired locomotive ability in comparison with their non-transgenic (NTG) littermates. Although there was no obvious loss of DA neurons in either the substantia nigra or striatum, the TG brain showed several neurological abnormalities such as a reduced striatal dopamine content, fragmentation of the Golgi apparatus in DA neurons, and an increased degree of microtubule polymerization. Furthermore, the tyrosine hydroxylase-positive primary neurons derived from the TG mouse showed an increased frequency of apoptosis and had neurites with fewer branches and decreased outgrowth in comparison with those derived from the NTG controls.
The I2020T LRRK2 TG mouse exhibited impaired locomotive ability accompanied by several dopaminergic neuron abnormalities. The TG mouse should provide valuable clues to the etiology of PD caused by the LRRK2 mutation.
Leucine-rich repeat kinase 2 (LRRK2) is the gene responsible for autosomal-dominant Parkinson’s disease (PD), PARK8, which originally has been defined by linkage analysis of a Japanese family (Sagamihara family) [1–4]. LRRK2 is a complex kinase consisting of LRR, ROC, COR, kinase, and WD40 domains . The Sagamihara family patients have the I2020T mutation in the kinase domain . Accumulated evidence suggests that LRRK2 may play a key role in axonal extension, autophagy, proliferation, and survival of neurons through its kinase activity [6–9]. In spite of the proposed mechanisms for neurodegeneration in vitro, the mechanism by which LRRK2 mutations affect DA neurons in patients and model animals in vivo is still far from conclusive.
As a mammalian PD model, transgenic (TG) mice expressing the R1441G mutation at the LRRK2 ROC domain reportedly show reduction of locomotive ability and diminished dopamine release . The R1441C knock-in (KI) mouse, on the other hand, appears normal in steady-state, although a reduction of amphetamine-induced locomotor activity and impaired D2 receptor function have been observed . Four different TG mouse lines expressing the G2019S mutation in the LRRK2 kinase domain, have been reported [12–15]. Two of them displayed increased ambulatory activity but the others did not. In terms of pathology, only one of them showed degeneration of DA neurons accompanied by abnormal autophagy, whereas two others showed increased tau-phosphorylation or promotion of tubulin polymerization associated with Golgi fragmentation. Temporal overexpression of G2019S LRRK2 in rat reportedly impairs dopamine reuptake, leading to enhanced locomotive activity .
In contrast to the extensive analysis of R1441G, R1441C, and G2019S TG mice, no LRRK2 TG rodent model with the I2020T mutation has ever been reported. G2019S and I2020T, despite being mutations affecting neighboring residues, have been known to have distinctive effects on the LRRK2 molecule, as reflected in kinase activity and susceptibility to post-translational degradation [17, 18]. In Drosophila, TG flies expressing the I2020T LRRK2, or its homologue I1915T LRRK, have been reported to show either DA neuron loss leading to unusual locomotive activity or a decrease of neuromuscular junction boutons [19–21]. In the present study, we investigated a TG mouse strain expressing I2020T LRRK2 in DA neurons. The TG mouse exhibits impaired locomotive ability, a reduced striatum dopamine content, fragmented Golgi apparatus, and an elevated degree of tubulin polymerization. Furthermore, the tyrosine hydroxylase-positive (TH+) primary neurons of the TG mouse show increased vulnerability and shortened neurites.
Generation of I2020T LRRK2 TG mice
I2020T LRRK2 TG mice exhibit impaired locomotive ability
Golgi apparatus fragmentation in DA neurons of I2020T LRRK2 TG mice
Tubulin of the I2020T TG brain shows greater polymerization
Reduced striatal dopamine level in I2020T LRRK2 TG mice
Primary TH+neurons of I2020T LRRK2 TG mice are vulnerable to apoptosis and have defective neurites
In the present study, we investigated for the first time a TG mouse strain expressing human LRRK2 with the I2020T mutation, which affects the kinase domain in the patients of the original PARK8 family (Sagamihara family). The I2020T TG mice exhibited several neurological abnormalities, such as impaired locomotive ability, DA neurons with a fragmented Golgi apparatus, a decreased striatal dopamine content, and an elevated degree of tubulin polymerization. In addition, the primary TH+ neurons derived from the TG brain showed high vulnerability and had neurites with defective characteristics. Our study provides the first in vivo evidence that expression of I2020T LRRK2 in mouse brain causes impaired locomotive ability and neurophysiological abnormality.
Importantly, the I2020T TG mice exhibited motor dysfunction in the beam test and rotarod test under physiological conditions, along with the decreased striatal dopamine content. On the other hand, it has been reported that animals showing striatal dopamine loss do not necessarily exhibit motor deficits in behavioral tests [23–25]. Although the beam test (narrow beam-traverse test) has been employed as a useful behavioral test for model animals with genetic and drug-induced PD [26–28], in the present study we further refined the experimental conditions of the test (width, form, and height of beam, etc.) to detect the abnormality present in the I2020T TG mouse. In addition, the improved rotarod test (high-position rotarod and continuous day trial) used in this study has been reported to have high sensitivity for detecting presymptomatic or early-stage PD, i.e., Parkin-KO mice showing no abnormality in the typical rotarod test reportedly exhibited an obvious motor deficit in the improved rotarod test , and model rats with presymptomatic PD induced by intracranial injection of 6-hydroxydopamine also exhibited a motor deficit in the improved rotarod test . Thus, these sensitive behavioral tests appear to be capable of detecting the motor deficits in I2020T TG mice. On the other hand, neither the beam test nor the rotarod test detected a significant difference between aged TG and NTG mice. Further investigations including methodological refinements will be necessary before it can be proved that locomotive ability is restored with aging. In the cylinder test, the I2020T TG mice showed an increased frequency of rearing. This is in contrast to R1441G TG mice, which reportedly exhibit decreased rearing . The TG mice also showed a non-significant tendency to exhibit a higher frequency of rearing and grooming in the open-field test.
We did not detect any obvious DA neuron loss in the I2020T TG mouse brain like that reported in most other LRRK2 TG mice, except for one G2019S TG line . The I2020T TG brain did not form α-synuclein- or tau-positive aggregated materials. These neuropathological features may not be discordant from those of the Sagamihara patients harboring the I2020T mutation, whose postmortem brains have revealed only mild loss of DA neurons and no detectable Lewy bodies or neurofibrillary tangles [31, 32]. On the other hand, the TG mouse expressing I2020T LRRK2 in the olfactory bulb had a sense of smell similar to that of control mice, whereas the Sagamihara patients show a degree of olfactory dysfunction ranging from slight impairment to anosmia .
The DA neurons of I2020T LRRK2 TG mice showed increased fragmentation of the Golgi apparatus. The structure of the Golgi apparatus is maintained by an appropriate microtubule polymerization status . Microtubule-polymerizing reagents such as taxol and microtubule-depolymerizing reagent such as vinblastine reportedly disrupt the Golgi apparatus . Our finding that brain tubulin in I2020T TG mice had a tendency to be excessively polymerized suggests that one of the mechanisms responsible for Golgi fragmentation could be impaired tubulin stability. These features of this TG mouse strain are very similar to those of one of the reported G2019S LRRK2 TG mouse strains showing a fragmented Golgi apparatus and an increased insoluble tubulin fraction . Although we did not detect any alteration of tau phosphorylation in the LRRK2 TG brain, other microtubule-associated proteins such as CRMP-2, a known substrate for LRRK2 , could influence the polymerization of tubulin in the TG brain. Alternatively, the mutant LRRK2 might have a directly deleterious effect on the Golgi apparatus, because we found that I2020T LRRK2 is located there, in accord with other studies of mouse neurons and C. elegans (in this case, a LRRK2-homolog LRK-1) [35, 36]. In any event, the Golgi fragmentation appears to have resulted in some sort of defective characteristic of DA neurons in I2020T TG mice, as has been reported in brain tissues of patients with various neurodegenerative diseases, those of animal PD models, and even in neurons at the pre-apoptotic stage [37–40].
In accordance with their impaired locomotive ability, I2020T TG mice exhibited a reduced striatal dopamine content. Analysis of the TH enzymatic activity and dopamine metabolites revealed that this low dopamine content might not be ascribable to either reduced synthesis or increased metabolism. It has been proposed that LRRK2 plays a key role in the trafficking of pre-synaptic vesicles by regulating membrane dynamics [41–43]. In the DA neurons of I2020T TG mice, fragmentation of the Golgi apparatus might hamper the maturation of some vesicle proteins, and hyper-polymerization of tubulin might disrupt their proper organization into membranes and vesicles [43, 44]. Although the exact mechanism responsible for the reduced dopamine content of the I2020T TG striatum is unknown, a possible distortion of membrane/vesicle dynamics might affect the consumption and recycling of synaptic dopamine.
Although no significant neuronal loss was obvious in the adult brain up to 18 months old, the primary TH+ neurons derived from the I2020T TG midbrain showed a higher degree of apoptosis than those from the NTG controls, indicating that the I2020T mutation might confer intrinsic vulnerability on TH+ neurons. This finding is consistent with reports demonstrating that overexpression of I2020T LRRK2 in primary neurons induces neurotoxicity . Also, we and other groups have demonstrated that neuroblastoma cell lines expressing I2020T LRRK2 are more susceptible to oxidative stress than those expressing wild-type LRRK2, although no consensus has been established regarding the molecular mechanism involved [19, 46, 47]. The neurites of primary TH+ neurons in the TG mouse also exhibited abnormal features, i.e., few branches and decreased outgrowth, consistent with one of the reported G2019S TG mouse strains whose cultured TH+ neurons also showed a reduction of neurite complexity . These intrinsic defects detectable in primary TH+ neurons may play some role in the neural dysfunction observed in adult I2020T TG mice.
We have established a TG mouse strain expressing the I2020T mutant human LRRK2. The TG mouse exhibits impaired locomotive ability and several neurological abnormalities. This strain should provide valuable clues to the etiology of PD caused by the LRRK2 mutation, as well as data relevant to the future development of therapeutic approaches.
Mice were housed in a light- and temperature-controlled room with water and food available ad libitum. For sacrifice, mice were euthanatized by cervical dislocation or exsanguination. All procedures had been approved by the Animal Experimentation and Ethics Committee of Kitasato University.
Generation of I2020T LRRK2 transgenic mice
The V5-tagged human LRRK2 cDNA with the I2020T mutation and SNPs of the Sagamihara family patient has been described previously . The 8,958-bp DNA fragment containing the CMV promoter and the LRRK2 cDNA including the tag was cut with Ahd I and Fsp I from the plasmid and microinjected into fertilized eggs of C57BL/6 J x C3H F1 female mice. The eggs were then transferred to the oviducts of pseudo-pregnant foster mothers of random-bred ICR. The founder TG mouse was back-crossed with the C57BL/6 J mouse more than 9 times, and their offspring were genotyped by polymerase chain reaction (PCR). The TG mice were backcrossed at least 9 times before being used for experiments. Methods for Southern blotting and RT-PCR are described in Additional file 2.
Mouse brains were homogenized in TNE buffer [10 mM Tris–HCl buffer (pH 7.6) containing 150 mM NaCl, 1 % Nonidet P-40, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Roche)] and kept gently agitated by slow rotation at 4 °C for 1 hour. The brain lysate was obtained by centrifugation at 13,000 rpm for 15 min at 4 °C, and its protein concentration was determined using BCA protein assay reagents (Thermoscientific). The lysates (40 μg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 5-20 % gradient e-PAGEL (ATTO) or 10 % gels, and blotted onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 2 % skim milk or 2 % ECL Advance Blocking Agent (GE Healthcare) in phosphate-buffered saline (PBS)-0.1%Tween 20 for 60 min at room temperature and probed with the appropriate primary antibodies overnight at 4 °C. After incubation with secondary antibodies for 30 min at room temperature, protein bands were visualized using an ECL- or ECL-Advance Western Blotting Detection Kit (GE Healthcare).
Mice were subjected to flush-perfusion with heparinized saline, followed by perfusion-fixation with 4 % paraformaldehyde. The brains were removed, immersed in 4 % paraformaldehyde overnight, and subsequently in 30 % sucrose for 48 h at 4 °C. Brain sections 30 μm thick were subjected to H2O2-inactivation of endogenous peroxidase activity and treated with 2 % BSA in PBS-0.2 % Triton X-100 for 60 min at room temperature to block non-specific protein binding. For immunofluorescence staining, the tissue sections were incubated with rabbit polyclonal antibody against V5-tag (MBL), rabbit polyclonal antibody against TH (Millipore), mouse monoclonal antibody against β-III tubulin (R&D Systems), mouse monoclonal antibody against GM130 (BD Transduction Laboratories), and mouse monoclonal antibody against TH (Millipore) for 48 h at 4 °C, and subsequently with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated appropriate secondary antibodies for 120 min at room temperature. For TH-immunohistochemical analysis of the substantia nigra and striatum, a biotinylated secondary antibody against rabbit IgG was used together with an ABC kit (Vector Laboratories) for detection. Optical density was analyzed using NIH ImageJ software (http://rsbweb.nih.gov/ij/). Method for measurement of LRRK2 immuno fluorescence intensity in TH+-neurons is described in Additional file 2.
In vitro polymerization of brain tubulin
Brain tissues were homogenized in low-salt reassembly buffer (RAB) [0.1 M Tris–HCl (pH 6.8), 0.5 mM MgSO4, 1 mM EGTA, and 2 mM dithiothreitol] at 4 °C, and centrifuged at 15,000 rpm for 1 h at 4 °C . The supernatants were supplemented with 4 M glycerol and 1 mM GTP, and incubated for 90 min at 37 °C to polymerize the microtubules. After centrifugation at 15,000 rpm for 60 min at 37 °C, the resulting pellets were resuspended in 500μl RAB and incubated on ice for 30 min to depolymerize the microtubules. The polymerization/depolymerization cycle was repeated once, and the resulting microtubule material was resuspended in 250 μl RAB and subjected to Western analysis using the antibody against -III tubulin.
Determination of catecholamines
The dorsal striatum was dissected out and quickly frozen in liquid nitrogen. Samples were homogenized in 500 μl of sample buffer for HPLC [0.2 M perchloric acid, 100 μM EDTA (pH 7.5)] and centrifuged at 15,000 rpm for 5 min at 4 °C. The supernatants were analyzed for dopamine, DOPAC, and HVA using HPLC coupled with electrochemical detection. Levels of DA, DOPAC, and HVA were determined by using standard curves, and normalized by tissue weight.
TH activity assay
Striatal TH activity was determined as reported previously . Briefly, the dorsal striatum was homogenized in 10 mM potassium phosphate buffer (pH 7.4), centrifuged at 3,000 rpm for 60 min at 4 °C, and the supernatant was mixed with an equal volume of reaction buffer [100 mM sodium acetic acid buffer (pH 6.0), 10 μg catalase, 1 mM NSD-1015, an inhibitor of aromatic L-amino acid decarboxylase, and 2 mM ferrous ammonium sulphate], and preincubated at 37 °C for 5 min. The reaction was started by adding 200 μM L-tyrosine and 10 mM (6R) tetrahydrobiopterin (BH4). After incubation for 10 min at 37 °C, the reaction was terminated by adding 100 μl of 0.1 M perchloric acid containing 0.4 mM sodium metabisulphite and 0.1 mM disodium EDTA. As the blank control, a similar reaction mixture containing D-tyrosine instead of L-isomer and 100 μM 3-iodo-L-tyrosine was used. The samples were subjected to HPLC and the amount of L-DOPA was measured.
Primary neuron culture
Fetal mice at embryonic day 14.5-15.5 were obtained from the uterus and genotyped for the LRRK2 transgene. Fetal midbrain sections were dissected under a microscope and digested with papain at 37 °C for 20 min. The dispersed cells were suspended in growth medium [F-12 HAM (Sigma), B27 (Gibco), penicillin/streptomycin] and cultured on polyethyleneimine-coated cover slips (Sigma) placed in 24-well plastic tissue culture plates. Three days after plating, Ara C (Sigma) was added to inhibit the growth of glial cells and the medium was changed twice a week. TH+ neurons were identified by immunostaining. The numbers of branches and total outgrowth of neurites were analyzed using NIH ImageJ software (NeuronJ). Apoptotic cells were detected by TUNEL staining using an In Situ Cell Death Detection Kit (Roche).
Beam test. First, mice were trained to walk on a wide beam (100 cm long, 25 mm wide) to motivate walking towards a dark platform. For the experimental test, each mouse was forced to walk along a narrow square beam (5 mm wide and 100 cm long, set at a height of 50 cm) to reach the platform. The time taken and the number of steps required to reach the platform, and the frequency of slips, were recorded.
Rotarod test. Each mouse was placed on a rubber-covered rod (3 cm in diameter) that was rotating at 16 rpm at a height of 50 cm (Shinano Seisakusho Co.). The length of time taken until the mouse fell from the rod was recorded (cut-off time: 180 s). The test was carried out three times per day and continued for 5 days.
Cylinder test. Each mouse was placed individually in an acrylic cylinder (25 cm high, 9.5 cm diameter) and video-recorded for 20 min. The frequency of vertical rearing by placing the forepaws on the wall was counted.
Methods for open-field test and olfactory testaredescribed in Additional file 2.
The numbers and genders of mice used for behavioral tests were summarized in Additional file 3. The other experiments were performed using only male mice.
Statistical analysis. All data are expressed as mean ± SEM. Significance of differences was assessed by Student’s t test.
This study was supported by Kitasato University School of Allied Health Sciences (Grant-in-Aid for Research Project 2009–2011). We thank the following staff of Kitasato University School of Allied Health Sciences for valuable technical advice: Drs. H. Akita and M. Ogata for behavioral tests and perfusion fixation, Dr. Y. Kadoya for immunofluorescence staining, and Dr. H. Maruyama for pathological analysis. We are grateful to Dr. S. Yamamori of Kitasato University School of Medicine for advice regarding the measurement of catecholamines, and Dr. H. Ichinose of Tokyo Institute of Technology, Graduate School of Bioscience and Biotechnology, for advice regarding TH activity measurement. We also thank Miss K. Komiya and Miss N. Komatsu for technical assistance in the behavioral tests.
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