Overexpression of Parkin Ameliorates Dopaminergic Neurodegeneration Induced by 1- Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine in Mice

<div><p>Mutations in the <em>parkin</em> gene are currently thought to be the most common cause of recessive familial Parkinsonism. Parkin functions as an E3 ligase to regulate protein turnover, and its function in mitochondrial quality control has been reported recently. Overexpression of parkin has been found to prevent neuronal degeneration under various conditions both in vivo and in vitro. Here, we generated a transgenic mouse model in which expression of wild type parkin was driven by neuron-specific enolase (NSE) promoter. We reported that both young and old parkin transgenic mice exhibited less reduction of striatal TH protein and number of TH positive neurons in the substantia nigra induced by 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP), compared to wild type littermates. MPTP-induced mitochondrial impairment in the substantia nigra was improved in young parkin transgenic mice. Decreased striatal α-synuclein was demonstrated in old parkin transgenic mice. These results provide reliable evidence from the transgenic mouse model for parkin that overexpression of parkin may attenuate dopaminergic neurodegeneration induced by MPTP through protection of mitochondria and reduction of α-synuclein in the nigrostriatal pathway.</p> </div

Levels of α-synuclein protein expression in the striatum of wild type and parkin transgenic mice.

<p>Western blot showing α-synuclein protein in the striatum of young (A) and old (C) mice after saline or MPTP treatment. (B, D) Quantification of relative α-synuclein protein expression. Data presented are the means±SE. # p<0.05, significant differences between wild type and transgenic mice; n = 4−6 per group.</p

Less morphological damage of mitochondria in the SNpc neurons of parkin transgenic mice induced by MPTP.

<p>Electron microscopy showing the morphology of mitochondria in the SNpc from young mice at 3 days after MPTP (A, B) or saline treatment (C, D). (A, C) wild type mice; (B, D) parkin transgenic mice. Arrows represent abnormal mitochondria with numerous vacuoles and fragmented cristae; Triangles represent normal mitochondria. (E) Quantitative analyses of the percentage of damaged mitochondria in the SNpc neurons. Scale bar, 1 μm; Data presented are the means±SE. *p <0.05, significant differences between saline and MPTP-treated mice; n = 3 per group.</p

Transcriptional expression of bcl-2, bax, PINK1 and DJ-1 in the substantia nigra.

<p>Samples were from young and old mice treated with MPTP or saline. mRNA expression levels of bcl-2 (A), bax (B), PINK1 (C), DJ-1 (D) were determined by real-time PCR and normalized to GAPDH. Values are means±SE. *p <0.05, significant differences between saline and MPTP-treated mice; #p<0.05, significant differences between wild type and transgenic mice; n = 4−7 per group.</p

No changes of Hsp70 protein expression in the striatum of wild type and parkin transgenic mice.

<p>Western blot showing Hsp70 protein in the striatum of young (A) and old (C) mice after saline or MPTP treatment. Quantification of relative Hsp70 protein expression shown in B, D. Data presented are the means±SE. n = 4−6 per group.</p

MPTP elicited less dopaminergic toxicity in parkin transgenic mice.

<p>Samples were collected at 1 day or 3 days from the striatum (A, B) and the substantia nigra (C, D) after saline or MPTP treatment. Western blot showing striatal TH protein expression in young (A-a) and old (B-a) mice. Quantification of relative TH protein expression was showed in the right panel. Immunohistochemical staining of the striatum showing TH positive nerve fibers of young (A-b) and old (B-b) mice. Scale bar, 0.2 mm. Relative optical density of the staining was showed in the right panel. Immunohistochemical staining showing TH positive cells in the substantia nigra of young (C) and old (D) mice. The sections were from wild type (a, b, c) or parkin transgenic (d, e, f) mice; (a, d) saline; (b, e) 1 day after MPTP treatment; (c, f) 3 days after MPTP treatment. Images to the right are higher magnification of the SN sections. Statistical data of the number of TH positive neurons (left panel) and nissl positive neurons (right panel) were showed in the bottom. Scale bar, 20 µm. Data presented are the means±SE. *p <0.05 and * *p <0.01, significant differences between saline and MPTP-treated mice; #p<0.05 and # #p<0.01, significant differences between wild type and transgenic mice; n = 3−6.</p

Generation and identification of parkin transgenic mice.

<p>(A) Schematic representation of the NSE-globin-parkin transgene construct. The expression of wild type parkin is under the control of NSE promoter. (B) Transcriptional expression of transgene in different regions of parkin transgenic mouse brain. The pair of primers P1 and P2 could identify mRNA expression (∼400 bp). Samples were from the cortex, hippocampus, striatum, substantia nigra, cerebellum and remaining of the brains from transgenic and wild type mice. (C) Transcriptional expression of parkin detected by real-time PCR in the cortex, hippocampus, striatum, substantia nigra, cerebellum and remaining parts of the brain, parkin-specific primers (parkin forward and parkin reverse) were used. (D) Expression of Parkin protein in the cortex, hippocampus, striatum and substantia nigra of wild type and two lines of transgenic mice. Transgenic mice showed increased parkin expression in these regions. Data presented are the means±SE. *p <0.05, **p<0.01, significant differences between wild type and transgenic mice; n = 3−7. (E) Colocalization of parkin and TH in the Substania nigra of parkin transgenic mice. Scale bar, 50 µm.</p

Functional imaging of interleukin 1 beta expression in inflammatory process using bioluminescence imaging in transgenic mice-5

cotreated with vehicle or dexamethasone (3 mg/kg) during the LPS (3 mg/kg) injection. All the mice were imaged at 0 and 5 h after LPS injection. (B) Quantification of luciferase activity (photons/sec) with the LivingImage software. Data presented were mean ± SE. (C) Dexamethasone inhibits hIL-1βP-driven luciferase expression during zymosan induced acute arthritis. Female cHS4I-hIL-1βP-Luc mice (n = 3/group) were cotreated with vehicle or dexamethasone (50 μg/mouse) during the administration of zymosan (300 μg/mouse). All the mice were imaged at 0 and 4 h after zymosan injection. (D) Quantification of luciferase activity (photons/sec) of the articular region of the acute arthritis mice with the LivingImage software. Data presented were mean ± SE. *** indicate significant differences at p < 0.001. p/s/cm/sr: photons/sec/cm/steradian.<p><b>Copyright information:</b></p><p>Taken from "Functional imaging of interleukin 1 beta expression in inflammatory process using bioluminescence imaging in transgenic mice"</p><p>http://www.biomedcentral.com/1471-2172/9/49</p><p>BMC Immunology 2008;9():49-49.</p><p>Published online 19 Aug 2008</p><p>PMCID:PMC2529264.</p><p></p

Functional imaging of interleukin 1 beta expression in inflammatory process using bioluminescence imaging in transgenic mice-3

Nd vehicle (5% glucose in PBS) into the left rear knee joint. The mice were imaged at 0, 1, 3, 4, 6, 8, 24, 30, 48, 72, and 96 h following injection. The color overlay on the images represents the photons/sec/cm/steradian (p/s/cm/sr), as indicated by the color scale next to the images. (B) Quantification of luciferase activity (photons/sec) of the knee region by the LivingImage software. Data presented were mean ± SE.<p><b>Copyright information:</b></p><p>Taken from "Functional imaging of interleukin 1 beta expression in inflammatory process using bioluminescence imaging in transgenic mice"</p><p>http://www.biomedcentral.com/1471-2172/9/49</p><p>BMC Immunology 2008;9():49-49.</p><p>Published online 19 Aug 2008</p><p>PMCID:PMC2529264.</p><p></p

Functional imaging of interleukin 1 beta expression in inflammatory process using bioluminescence imaging in transgenic mice-4

With oxazolone and the left ears were treated with vehicle. Imaging analysis was performed on day – 6, 0, 1, and 30. (B) Quantification of luciferase expression (photons/sec) in all the ears with LivingImage software. Data were presented as mean ± SE. (C) Ear thickness was measured on day – 6, 0, 1, and 30 with a micrometer. Data represent mean ± SE. *** indicate significant differences at p < 0.001. p/s/cm/sr: photons/sec/cm/steradian.<p><b>Copyright information:</b></p><p>Taken from "Functional imaging of interleukin 1 beta expression in inflammatory process using bioluminescence imaging in transgenic mice"</p><p>http://www.biomedcentral.com/1471-2172/9/49</p><p>BMC Immunology 2008;9():49-49.</p><p>Published online 19 Aug 2008</p><p>PMCID:PMC2529264.</p><p></p