Nonmotor symptoms in Parkinson's disease: Investigating early-phase onset of behavioral dysfunction in the 6-hydroxydopamine-lesioned rat model

To investigate the psychiatric symptoms accompanying the early phases of Parkinson's disease (PD), we injected adult rats with 10.5 μg 6-hydroxydopamine (6-OHDA) bilaterally into the dorsal striatum. The resulting neurodegeneration led, 12 weeks after injection, to a mild (36%) reduction of striatal dopamine. We tested the behavioral response of sham and 6-OHDA-lesioned animals at different time points after injection to evaluate the onset and progression of behavioral abnormalities. The results showed that such a mild reduction of dopamine levels was associated with a decrease in anxiety-like behavior, an increase in "depression"-like behavior, and a marked change in social behavior. Learning and memory abilities were not affected. Overall, the PD rat model used here displays behavioral alterations having face validity with psychiatric symptoms of the pathology and thus appears to be a valuable tool for investigating the neural bases of the early phases of PD. © 2008 Wiley-Liss, Inc

Sensitivity to 3-NP of striatal cells expressing hMTH1 and wild-type or mutant murine <i>htt</i>.

<p>Striatal cells derived from wild-type Hdh<i>^Q7/Q7</i> and mutant <i>Hdh^Q111/Q111</i> mice were transfected with hMTH1. (A) Proteins were separated and probed with an antibody against hMTH1. (B) Intracellular localization of hMTH1 (green fluorescence) in <i>Hdh^Q111/Q111</i> and <i>Hdh^Q111/Q111</i>+hMTH1. Nuclei were counterstained by DAPI. (C) LDH release. LDH release from striatal cells into culture medium was measured 24 hr after continuous exposure to 20 mM 3-NP. Hdh<i>^Q7/Q7</i> (grey bar) and Hdh<i>^Q7/Q7</i>+hMTH1 (dashed bar); <i>Hdh^Q111/Q111</i> (black bars) and <i>Hdh^Q111/Q111</i>+hMTH1 (open bar). Mean±SE, n = 4. (D) Clonal survival. Cloning efficiency was determined at 33°C after 24 hr continuous exposure to the indicated 3-NP concentrations. Mean±SD, n = 2. (E) Coulter counter assay. Survival of non-proliferating cells measured in a Coulter Counter. Mean±SD, n = 3. The asterisks indicate significant differences by Student's <i>t</i>-test (p values = 0.02) between <i>Hdh^Q111/Q111</i> and <i>Hdh^Q111/Q111</i>+hMTH1.</p

Oxidative DNA damage in the brain.

<p>Brains from wild-type mice that had been treated twice per day for 5 days with 60 mg/kg 3-NP were examined for DNA 8-oxodG by immunohistochemistry. Nuclei of striatum, parietal and frontal cortex were counterstained by DAPI.</p

3-NP-induced toxicity in wild-type and hMTH1-Tg^+/− and hMTH1-Tg^+/+ transgenic mice.

<p>Groups of wild-type (n = 20), <i>hMTH1-Tg^+/−</i> (n = 16) and <i>hMTH1-Tg^+/+</i> (n = 16) mice were injected i.p. twice daily for 5 days with 60 mg/kg 3-NP. Wild-type (black bars), <i>hMTH1-Tg^+/−</i> (grey bars) and <i>hMTH1-Tg^+/+</i> (white bars). (A) Weight loss. Body weight, measured immediately before the first injection on the indicated days, is expressed as a percentage of the pretreated body weight. (B) Motor impairment. Mice were monitored twice a day for dystonia and/or gait abnormalities. Neurological score was as follows: intermittent dystonia of one hindlimb: 1; intermittent dystonia of two hindlimbs: 2; permanent dystonia of hindimbs: 3; uncoordinated and wobbling gait or recumbency: 3; near death recumbency: 4. For each animal, the highest neurological score reached at any time of the observation period was considered. Values are mean±standard errors. (C) Cumulative mortality. The non-surviving fraction at the end of 5-day treatment is expressed as a percentage of starting total. (D) Striatal lesion formation. The percentage of animals with detectable post-mortem striatal lesions is shown. (E) Size of striatal lesions. Postmortem measurements of striatal lesions along the rostrocaudal axis. The asterisks indicate a P<0.05 <i>vs</i> wild-type according to One-way Anova and Tukey multiple comparison post-hoc test for panels A, B and E and to χ² test for panels C and D.</p

hMTH1 protein in <i>hMTH1-Tg^+/+</i> MEFs and protection against oxidative stress.

<p>(A) Expression of hMTH1 in wild-type and homozygous <i>hMTH1-Tg^+/+</i> MEFs. Total cell extracts were separated by 12%-SDS polyacrylamide electrophoresis, blotted and probed with an antibody against hMTH1 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000266#pgen.1000266-Kang1" target="_blank">[23]</a>. The human SHSY5S neuroblastoma and SV40-transformed MRC5 cell lines are shown for comparison (left panel). Western blotting of subcellular fractions of <i>hMTH1-Tg^+/+</i> MEFs with anti-hMTH1 (right panel). Proteins were separated on 18%-SDS polyacrylamide electrophoresis and cytocrome <i>c</i> was used to quantify mitocondrial cell extracts. (B) Steady-state levels of oxidized guanine. DNA and RNA from wild-type (filled bars) and <i>hMTH1-Tg^+/+</i> (open bars) MEFs were digested to nucleosides and 8-oxodG and 8-oxoG were separated and quantified by HPLC-EC. Values are expressed as ratio to DNA and RNA guanine, respectively. Values are the mean±SE of 3 independent determinations. Asterisks indicate statistically significant differences (p-value = 0.005 and 0.015 for DNA and RNA, respectively; <i>t</i>-test). (C) Levels of oxidized guanine following oxidant treatment. 8-oxodG and 8-oxoG were measured by HPLC-EC in DNA and RNA extracted from MEFs exposed to 40 mM KBrO₃ for 30 min (time 0) and after the indicated times of repair incubation in drug-free medium (30, 60, 120 min). Wild type (filled bars) and <i>hMTH1-Tg^+/+</i> (open bars). Values are the mean±SE of 3 independent determinations. Asterisks indicate a p-value≤0.05; <i>t</i>-test.</p

Construction and characterization of a transgenic mouse expressing the <i>hMTH1</i> cDNA.

<p>(A) A BamH1-EcoRV fragment (509 bp) derived from pcDEBΔ <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000266#pgen.1000266-Kang1" target="_blank">[23]</a> encoding the <i>hMTH1</i> cDNA was subcloned into the gWIZ vector under the control of the CMV promoter. This vector transfected into wild-type MEFs expressed the hMTH1 protein (data not shown). The MscI-KpnI fragment (2481 bp) was used in the construction of the transgenic mouse. (B) Determination of transgene copy number. Genomic DNA from mouse tails was analysed by Southern blot analysis (left panel). In the right panel 2, 5, 10, 20, 40 copies of the MscI-KpnI fragment were analysed. Both blots were probed with the same probe. Copy number was determined by comparison. The arrow indicates the DNA from the mouse that was used as a founder for the colony. (C) Analysis by FISH of the number of hMTH1 integrations in hemizygous (<i>hMTH1-Tg^+/−</i>) and homozygous (<i>hMTH1-Tg^+/+</i>) strains. (D) Expression of <i>hMTH1</i> mRNA. RT-PCR was performed using total RNA from the indicated organs and human specific primers. RT-PCR for the <i>GADPH</i> gene is used as an internal control. <i>hMTH1</i> and <i>GAPDH</i> fragments are respectively 200 bp and 330 bp. (E) Western blot analysis of transgene expression. Total proteins (20–40 µg) from a range of tissues were separated by SDS polyacrylamide electrophoresis, blotted and probed with an antibody against hMTH1. β-tubulin was used as a loading control.</p

KBrO₃ sensitivity of <i>hMTH1-Tg^+/+</i> and wild-type MEFs.

<p>Wild-type (closed symbols) and <i>hMTH1-Tg^+/+</i> (open symbols) MEFs were treated with KBrO₃ for 30 min at the indicated concentrations. Viability was measured by MTT assay 48 hr later. The graphs are the mean±SD of 3 independent experiments.</p