Measurement of locomotor activity

The test is to place one mouse in a black plexiglass box (30 x 30 x 30 cm) under a light condition. The field is continuously monitored by a computer-operated Etho Vision video tracking system (Noldus, Netherlands), which consists of eight equidistant photoreceptor beams on each side of the box, dividing the field into 64 equally sized squares.The total moving distance and average velocity of each mouse were measured during a 10 min period.

2.8. Rotarod test

The rotarod test evaluates general motor coordination and balance. The test consists of rotating drum (Ugo Basile, Comerio, Italy). The mice were trained for 5 min on two separate days with a fixed speed at 5 rpm. Testing was conducted the following week with a speed at 15 rpm. Mice were subjected to three trials, each with a maximum duration of 180 sec. The latency and the rotation speed at which the mouse falls off the drum were recorded. Mouse performance typically improves with repetition. To control for this improvement, each mouse should undergo four trials with a 30 min interval between trials.

2.9. Immunohostochemistry

Immediately after the rotarod test, mice were anesthetized (0.016 ml/g BW, 2.5%

avertin, Sigma, Germany) and transcardiacally perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). Brains were removed and post-fixed with 4%

paraformaldehyde overnight and then placed in 30% sucrose in PBS for 2 days.

Brains were serially sectioned 30 µm on a cryostat.

For immunohistochemistry, free-floating sections were immunostained. In brief, sections were rinsed in 0.1 M PBS three times (10 min/wash). Endogenous peroxidase activity was blocked by incubation with 3% H2O2 for 30 min. Sections were then washed in PBS for three times (10 min/wash). Nonspecific epitopes were then blocked by incubation in 5% normal goat serum and 0.1% Triton X-100 in PBS for 2 hrs. Sections were incubated overnight at room temperature with primary antibodies, Calbindin (Sigma, 1:1000) and caspase 3 (Chemicon, 1:40). Secondary antibodies were then applied to the sections by a linking reagent (DAKO, CA) for 1 hr. Immunostaining was highlighted using substrate-chromogen solution and diaminobenzidine oxidation. All sections were mounted on coated slides and cover-slipped for light microscopy observation.

2.10. Mitochondria purification

The mitochondrial fractions of tissues from the different regions of mouse brain (cortex, brainstem, hippocampus and cerebellum) were isolated by using a mitochondrial extraction kit (Active Motif, Carlsbad, CA. USA) according to the manufacturer protocol. In brief, tissue samples were gently homogenized with a glass–teflon homogenizer. Homogenates were centrifuged at 800 g for 10 min at

min at 4°C to pellet the mitochondria. The supernatant thus obtained was the cytosolic fraction. The mitochondrial pellet was resuspended in 100 μl of isolation medium. The purity of the mitochondrial fraction was verified by the selective expression of the mitochondrial inner membrane-specific protein prohibitin. Total protein in the mitochondrial or cytosolic extracts was quantified by the BCA Protein Assay (Pierce, USA).

2.11. Western blot analysis

Western blot analysis was conducted on proteins extracted from the frontal cortex, hippocampus and cerebellum with antibodies for Bβ2, Flag, Bcl-2, cytochrome c, prohibitin, and β-actin. The antibodies used were rabbit polyclonal anti-cytochrome c (1:1000; Santa Cruz, CA, USA); mouse monoclonal anti-Bβ2 (1:2000; a gift from Dr. Strack, Department of Pharmacology, University of Iowa Carver College of Medicine), Flag (1:1000; Sigma, USA), Bcl-2 (1:1000; Sigma, USA), prohibitin (1:1000; Labvision/NeoMarkers, Fremont, CA, USA), β-actin (1:5000; Chemicon, Temecula, CA, USA). The secondary antibody of anti-rabbit IgG HRP-linked antibody (Cell signaling, USA) was used for cytochrome c. The second antibody of anti-mouse IgG HRP-linked antibody (Cell signaling, USA) was used for Bβ2, Bcl-2, prohibitin, and β-actin. Specific antibody–antigen complex was detected by an enhanced chemiluminescence Western blot detection system (Amersham Pharmacia Biotech, USA). The intensity of Western analysis was quantified by the Fuji LAS-3000 imaging system (Fuji, Japan), and was expressed as a ratio relative to β-actin protein (for analysis of total protein or proteins in cytosolic fraction) or prohibitin (for analysis of proteins in mitochondrial fraction).

2.12. Mitochondria PP2A activity

PP2A activity was determined using a PP2A immunoprecipitation phosphatase assay (Upstate, USA ) that measures free phosphate with a malachite green dye. To immunoprecipitate PP2A, lysates containing 200 μg of protein were incubated with 4 μg of anti-PP2A-C subunit antibody (clone 1D6) and 40 μl of protein A-agarose slurry for 2 hrs at 4°C with constant rocking. The immunoprecipitates were washed three times in Tris-buffered saline and once with Ser/Thr assay buffer (50 mM Tris-HCl, pH 7.0, 100 μM CaCl2), and resuspended in 20 μl of Ser/Thr assay buffer.

The reaction was initiated by the addition of 60 μl of phosphopeptide substrate (750 μM KRpTIRR). Following incubation for 10 min at 30°C in a shaking incubator, the reaction mixture was centrifuged briefly and the supernatant was transferred to a 96-well microtiter plate. The reaction was terminated by the addition of malachite green phosphate detection solution for 10–15 min at room temperature, and free phosphate was quantified by measuring the absorbance of the mixture at 650 nm using a microplate reader.

2.13. Measurement of mitochondria membrane potential

Mitochondrial membrane potential assay with Isolated mitochondria staining kit (Sigma, USA) is designed for a total volume of 100 μl. First, the isolated mitochondrial sample (up to 10 μl) or valinomycin( a mitochondrial membrane dissipating agent, for control experiments) treated mitochondrial sample equivalent to 5 μg of protein was added to each well. JC-1 Staining Solution 90 μl was then added to the well. If required, bring the total reaction volume to 100 μl with JC-1 Staining Solution. Fluorescence intensity was measured in a spectrofluorometer (Gemini, Molecular Devices Corp., CA, USA) using the 490 nm wavelength for excitation and

2.14. Data analysis

To compare the Bβ2-overexpression effects between the mito-Bβ2 transgenic mice and their wild-type littermate, independent samples Student’s t tests were used in the study. The statistical results were expressed as means ± SEM.

3. Results

3.1. Generation of mito-Bβ2 transgenic mice

Total 6 transgenic founder lines (786, 790, 791, 1433, 1445 and 1461) were generated according to the results of PCR analysis (Fig. 2). However, 786 and 790 founder mice died at the age of 3 weeks. Western blot analysis also confirmed the expression of the transgene encoded protein in transgenic mouse brain (Fig. 3). The results of Western analysis indicates that line 791 and 1433 had the higher expression level among these 4 lines (Fig. 3). Therefore, lines 791 line and 1433 were selected for breeding and the generated offspring were used in the study.

3.2. Mophological phenotype of mito-Bβ2 transgenic mice

Mito-Bβ2 transgenic mice could easily be distinguished from age-matched, wild-type littermates by their abnormal appearance. For example, smaller body size, feeble, thin hair, easily got frightened, and curved spine were all conspicuous features of these transgenic mice (Fig. 4A). The mito-Bβ2 transgenic mice have a substantially reduced lifespan (Fig. 4B). They began to die at 9 weeks of age, and some transgenic mice (lines 786 and 790) even died at 3 weeks old. From the characterization of body weight in every other week, we found that both of the male and female

transgenic mice grow much slowly than those of wild type mice (p < 0.05; Fig. 4C &

D).

3.3. Abnormal neurobehavior of mito-Bβ2 transgenic mice

Clasping, spontaneous seizures and tremor were also identified in some of the transgenic mice. For scoring the paw clasping occurrence, we found that both male and female transgenic mice have higher frequency in showing clasping (p < 0.05; Fig.

5). In addition, transgenic male mice possess more and earlier clasping behavior than transgenic female mice (p < 0.05; Fig. 5B). We also characterized mouse behavior inside the homecage by using the HomeCageScan system (Clever Sys. Reston VA.

USA). We found the number of behavior was slightly fewer in transgenic mice than in wild type littermate (Fig. 6A). However, explaratory, motor, and eating behaviors were significantly decreased in transgenic mice as compared to wild type mice (p <

0.05, Fig. 6B). Furthermore, the resting behavior was significantly prolonged in transgenic mice compared to wild type mice (p < 0.05, Fig. 6B) For the nociception test, we found that there is no difference in the length of latency on the hot plate between trangenic and wild type mice (p > 0.05, Fig.7). In addition, locomotor activity was significantly reduced in the transgenic group in comparison with the wild type group (p < 0.05, Fig. 8A & B). For the rotarod test, we found that the learning impairment of motor task was significant enhanced in transgenic mice as compared to the wild type mice (p < 0.05, Fig 9A). Furthermore, the impairment of motor coordination was also enhanced in the transgenic mice as compared to wild type mice (p<0.05, Fig. 9B).

3.4. PP2A activity and mitochondria membrane potential

In order to find out whether the mitochondria function or PP2A activity altered by the overexpression of Bβ2 in the mitochondria. We first tested the PP2A activity in the mitochondria from different brain regions of transgenic and wild type mice.

The results indicated that the PP2A activity in transgenic mice was significantly higher than that in wild type mice , of all the different brain regions examined, including the cerebellum, brainstem, cortex, and hippocampus (Fig. 10).

The mitochondria membrane potential of transgenic mice was significantly increased in cerebellum, cortex, and hippocampus regions of transgenic mice but decreased in brain stem region as compared to wild type mice (Fig. 11).

3.5. The neuropathology of mito-Bβ2 transgenic mice

There was no obvious difference in gross brain morphology between transgenic and wild type mice. The hippocampus and cerebellum were the most affected brain regions in transgenic mice as evaluated by immunohistochemical analyses (Fig. 12).

We found that the level of the MnSOD and caspase 3 were significantly enhanced in the hippocampus of the transgenic mice as compared to the wild type mice (p < 0.05, Fig. 12.1). In addition, the level of the oxdative stress was significantly enhanced in the cerebellum of the transgenic mice as compared to the wild type mice (p < 0.05, Fig. 12.2). The number of calbindin-positive cerebellar neurons was also decreased in the transgenic mice as compared to wild type mice ( p <0.05, Fig. 12.2).

The result of western blot analysis also showed higher cytochrome c and Bcl-2 in cerebellum, brainstem, cortex, and hippocampus regions of transgenic mice as compared to wild type mice ( p < 0.05, Fig. 13)

4. Discussion

In this study, we found that mitochondria Bβ2 overexpression induces distinctive neurological phenotypes in mice. The progressive neurological phenotype of the early onset Mito-Bβ2 mice are characterized with smaller body size, feeble, thin hair, easily got frightened, seizure, tremor, clasping, curved spine and early death. These variable phenotypes are consistent with previous reports of SCA12 case study (Holmes et al., 1999). One recent evidence also suggests that mitochondria dysfunction increases the rate of lethality (Liu et al., 2007). Consistent with the role in neuronal phenotype in Mito-Bβ2 mice, Bβ2 is implicated in a number of neurological or neurodegenerative disorders (Strack et al., 2004). The observation that the SCA12 mutation is detrimental for neurons suggests that proper regulation of the Bβ gene is critical for neuronal survival (Schmidt et al., 2002). Therefore, the phenotype of the mito-Bβ2 mice provides valuable insight into the pathogenesis of mitochondrial dysfunction or SCA12 disease.

We found that mitochondria Bβ2 overexpression induced the impairment of motor function, but not nociception. A lot of studies suggest that a spectrum of myopathic and neuropathic symptoms in humans have been correlated to mitochondria dysfunction (Manfredi et al., 2005; Shukkur et al., 2006). From the results of calbindin immunohistochemistry, we also found that a significant loss of Purkinje cells was identified in the transgenic mice compared to wild type mice. Furthermore, the oxidative stress of the cerebellum was enhanced in transgenic mice but not wild type mice. Previous studies also suggest that increased oxidative stress and disruption of neuronal calcium homeostasis appear to be interrelated final common pathways that mediate the neurodegenerative process in AD (Appel et al., 2001; Cao et al., 2007;

suggest that the impairment in calcium homeostasis might induce the oxidative stress in Purkinje cells. Finaly, the loss of Purkinje cells induced the impairment in motor coordination. About the impairment in the learning and memory of motor in the animal model, the early degeneration presented outside of the cerebellum as hippocampus region could provide the explanation. Previous study also shows that the signs of both cerebellar and cortical are both involved in the clinical features of SCA12 patients (Holmes et al., 2001).

Our study show that overexpression of Bβ2 in the mitochondria increased PP2A activity, the release of cytochrome c, and the caspase 3 expression in different mouse brain regions. However, membrane potential was reduced in most of the regions examined except for brain stem. On the other hand, the anti-apoptic molecule, Bcl-2 was also found increased in all the different regions examined. Whether some protection responses in the mitochondria induced by the Bβ2 overexpression damage in order to sustain the neuronal survival needs further investigation.

In summary, we have established the animal model and evaluated some of the molecular effect of mitochondria Bβ2 overexpression in these transgenic mice.

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APPENDIX

Figure 1. Construct the pNSE-cox8-Bβ2-flag-EGFP plasmid. (A) The mouse cox-8 presequence was subcloned in front of Bβ2-Flag-EGFP, the plasmid was denominated pNSE-cox8-Bβ2-Flag-EGFP. (B) The mouse cox-8 presequence was PCR amplified with NheI and HindIII cloning sites within the primers