Abstract
Oxidative stress is linked to dopaminergic (DA) neurodegeneration in Parkinson’s disease. Our laboratory reported slowly progressive DA neurodegeneration in the zitter (zi) rat, which is Attractin (Atrn) deficient. However, little is known about the function of Atrn in the central nervous system (CNS). Thus, we investigated whether DA neurodegeneration in the zi rat was induced by oxidative stress, and how Atrn affects oxidative stress. First, we summarize our previous in vivo data, which revealed suppression of DA neurodegeneration using antioxidants (vitamin E and melatonin) in zi rats. Second, our current ex vivo and in vitro studies are introduced. Using primary neuronal cultures of zi mesencephalon as a model of Atrn-deficient neurons or Atrn-GFP-overexpressing HEK293 cells, accumulation of reactive oxygen species (ROS) in mitochondria and cell viability was examined under oxidative stress. Atrn-deficient neurons accumulated excess ROS in mitochondria, resulting in neurodegeneration, whereas Atrn-overexpressing cells showed suppression of ROS accumulation under oxidative stress. These results showed that Atrn plays a suppressive role against ROS and that the loss of Atrn function induced excess ROS accumulation and led to DA neurodegeneration. This is the first report to show that Atrn directly modulates mitochondrial ROS accumulation in the CNS.
Keywords
- dopaminergic neurodegeneration
- Attractin
- reactive oxygen species
- Parkinson’s disease
- oxidative stress
1. Introduction
Oxidative stress is considered to be the cause of several neurological diseases. In particular, dopaminergic (DA) neurons are vulnerable to oxidative stress, which is normally generated by dopamine metabolism [1, 2]. The progressive DA neurodegeneration that occurs with age in Parkinson’s disease (PD) is caused by increased oxidative stress. The neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and6-hydroxydopamine induce free radicals (H2O2, O2–,and OH) that destroy DA neurons [3, 4] and are used to produce acute animal models of PD. Recently, we reported slowly progressive DA neurodegeneration, which is similar to the symptoms of humans with PD, in a mutant rat, the
The
Our laboratory first reported DA neurodegeneration that occurs with age in
![](http://cdnintech.com/media/chapter/50827/1512345123/media/fig1.png)
Figure 1.
Progressive dopaminergic neurodegeneration with age in
Previous studies have indicated that
2. Materials and methods
2.1. Reagents
Materials for cultures were B27 supplement, B27 supplement minus antioxidants (AO (–)), neurobasal medium, Opti-MEM, and penicillin/streptomycin from Gibco (Life Technologies; Carlsbad, CA); MitoTracker Red CM-H2Xros, MitoTracker green FM, and Lipofectamine Plus Reagent from Invitrogen (Life Technologies); cell proliferation reagent WST-1 from Roche Diagnostics (Basel, Switzerland); cytosine-arabinofuranoside, l-glutamine, and poly-l-lysine from Sigma-Aldrich (St. Louis, MO); pAcGFP1-N1 vector from Clontech Laboratories Inc. (Mountain View, CA). Reagents for the immunocytochemistry were purchased from the following sources: biotinylated horse anti-mouse IgG, normal goat serum, and vectastain ABC kit from Vector (Burlingame, CA); anti-tyrosine hydroxylase (TH) antibody from Incstar (Stillwater, MN); Alexa Fluor 488 goat anti-mouse IgG from Molecular Probes (Life Technologies).
2.2. In vivo study
Male
2.3. Ex vivo study
Ventral mesencephalic neurons were prepared from fetal
The cell viability assay was performed by using the cell proliferation reagent WST-1 according to the manufacturer’s protocol. Cell viability was expressed as a percent of the values of SD-Cont.
ROS accumulation was monitored with a plate reader using MitoTracker Red CM-H2Xros (MTR). Neurons were co-stained with 0.5 μM MTR and 0.2 μM MitoTracker green (MTG: mitochondria marker) in Opti-MEM for 30 min at 37°C and then assessed (MTR, excitation 535 nm, barrier filter 595 nm; MTG, excitation 485 nm, barrier filter 535 nm). The MTR/MTG ratio of each group was normalized to that of SD-Cont, which was used as an index of ROS content.
Immunofluorescence procedure was described previously [16].
2.4. In vitro study
3. Results
3.1. The effect of antioxidants on DA neurodegeneration in zi rats
We treated
Melatonin, which is a synthetic product of the pineal gland, is a direct free radical scavenger and an indirect antioxidant. Melatonin readily crosses the blood-brain barrier and plays a role in protecting against ROS in the brain. Chronic melatonin administration suppressed DA neuron death in the SNc of
![](http://cdnintech.com/media/chapter/50827/1512345123/media/fig2.png)
Figure 2.
Antioxidants suppressed dopaminergic neurodegeneration in
Antioxidants were effective for preventing DA neurodegeneration in
3.2. Accumulation of ROS in zi mesencephalic neurons ex vivo
To investigate whether ROS accumulation in mesencephalic neurons is caused by a genetic factor of the
Cell viability was shown in Figure 3A. Two-way analysis of variance (ANOVA) revealed significant effects of the supplement (
Next, ROS accumulation in mitochondria was monitored using MTR, which produces intense fluorescence depending on ROS accumulation in mitochondria (Figure 3B). Two-way ANOVA revealed significant effects of genotype (
Because the neurons from
![](http://cdnintech.com/media/chapter/50827/1512345123/media/fig3.png)
Figure 3.
Effects of genotype and supplement factors in primary neuronal cultures from the ventral mesencephalon. Neurons from
3.3. Suppression of ROS accumulation in Atrn -overexpressing cells in vitro
To investigate the function of
ROS accumulation in
![](http://cdnintech.com/media/chapter/50827/1512345123/media/fig4.png)
Figure 4.
ROS accumulation in
Under the microscope, the time of addition of H2O2 as a form of oxidative stress was set at 0 min, and live images were scanned at 0 and 60 min (Figure 5).
![](http://cdnintech.com/media/chapter/50827/1512345123/media/fig5.png)
Figure 5.
4. Discussion
This study revealed that the loss of
Z
Acknowledgments
This work was supported by research grant from Dokkyo Medical University (Investigator-Initiated Research Grant No.2014-01).
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