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Protection by the NDI1 gene against neurodegeneration in a rotenone rat model of Parkinson's disease.

Marella M, Seo BB, Nakamaru-Ogiso E, Greenamyre JT, Matsuno-Yagi A, Yagi T - PLoS ONE (2008)

Bottom Line: It is widely recognized that mitochondrial dysfunction, most notably defects in the NADH-quinone oxidoreductase (complex I), is closely related to the etiology of sporadic Parkinson's disease (PD).A single, unilateral injection of recombinant adeno-associated virus carrying the NDI1 gene into the vicinity of the substantia nigra resulted in expression of the Ndi1 protein in the entire substantia nigra of that side.The present study shows, for the first time, the powerful neuroprotective effect offered by the Ndi1 enzyme in a rotenone rat model of PD.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, United States of America.

ABSTRACT
It is widely recognized that mitochondrial dysfunction, most notably defects in the NADH-quinone oxidoreductase (complex I), is closely related to the etiology of sporadic Parkinson's disease (PD). In fact, rotenone, a complex I inhibitor, has been used for establishing PD models both in vitro and in vivo. A rat model with chronic rotenone exposure seems to reproduce pathophysiological conditions of PD more closely than acute mouse models as manifested by neuronal cell death in the substantia nigra and Lewy body-like cytosolic aggregations. Using the rotenone rat model, we investigated the protective effects of alternative NADH dehydrogenase (Ndi1) which we previously demonstrated to act as a replacement for complex I both in vitro and in vivo. A single, unilateral injection of recombinant adeno-associated virus carrying the NDI1 gene into the vicinity of the substantia nigra resulted in expression of the Ndi1 protein in the entire substantia nigra of that side. It was clear that the introduction of the Ndi1 protein in the substantia nigra rendered resistance to the deleterious effects caused by rotenone exposure as assessed by the levels of tyrosine hydroxylase and dopamine. The presence of the Ndi1 protein also prevented cell death and oxidative damage to DNA in dopaminergic neurons observed in rotenone-treated rats. Unilateral protection also led to uni-directional rotation of the rotenone-exposed rats in the behavioral test. The present study shows, for the first time, the powerful neuroprotective effect offered by the Ndi1 enzyme in a rotenone rat model of PD.

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Characterization of the rotenone rat model.Rats received a subcutaneous injection of microspheres encapsulating rotenone. A: Concentration of rotenone in the plasma was determined on an HPLC system from 200 µl of blood samples drawn from the tail vein from 4 rats every week for 60 days. Values are given with standard deviation (SD). B: Rat brain coronal sections at the level of SN were subjected to immunohistochemical staining with antibody against α-synuclein or ubiquitin. Left panel, Lewy body-like cytoplasmic inclusions in SN neurons (arrows). Scale bar is 10 µm. Right panels, images at a higher magnification. C: The rat SN sections were stained with antibody specific for 8-oxo-dG. Oxidatively modified DNA appeared in green. Nuclei were stained in blue with DAPI. The image shows the damage to both mitochondrial DNA (left arrow) and nuclear DNA (right arrow) in SN neurons. Scale bar is 15 µm.
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pone-0001433-g001: Characterization of the rotenone rat model.Rats received a subcutaneous injection of microspheres encapsulating rotenone. A: Concentration of rotenone in the plasma was determined on an HPLC system from 200 µl of blood samples drawn from the tail vein from 4 rats every week for 60 days. Values are given with standard deviation (SD). B: Rat brain coronal sections at the level of SN were subjected to immunohistochemical staining with antibody against α-synuclein or ubiquitin. Left panel, Lewy body-like cytoplasmic inclusions in SN neurons (arrows). Scale bar is 10 µm. Right panels, images at a higher magnification. C: The rat SN sections were stained with antibody specific for 8-oxo-dG. Oxidatively modified DNA appeared in green. Nuclei were stained in blue with DAPI. The image shows the damage to both mitochondrial DNA (left arrow) and nuclear DNA (right arrow) in SN neurons. Scale bar is 15 µm.

Mentions: A few methods are available to administer rotenone in rats. For example, intravenous infusion via minipump [20] or intraperitoneal injection [24] may be used. Our preliminary experiments conducted with an intraperitoneal injection of rotenone, emulsified in oil, turned out to exert noxious effects; the rats presented liver necrosis and large weight loss (unpublished data). Subcutaneous delivery of rotenone, on the other hand, was reported to be more efficient in altering selectively the dopaminergic cells [22]. To eliminate toxic effects derived from the materials used for injections such as organic solvents, we carried out subcutaneous injections of biodegradable microspheres containing rotenone, a method originally adopted by Huang et al. [25]. After the injection, we monitored the concentration of rotenone in the plasma. As shown in Figure 1A, there was an initial increase in the rotenone concentration during the first 2 weeks which was followed by a gradual decrease. The rotenone concentration remained above 1 µM throughout the period of our experiments (60 days) presumably due to a slow and constant release of rotenone from the polymer undergoing disintegration in the animal body [26]. The rats lost about 2% of their body weight during the first 15 days after the injection which coincided with an increase in the rotenone concentration in the plasma, but there was no further loss throughout the rest of the period (data not shown). After 30 or 60 days of rotenone exposure, we examined the brain for the extent and nature of the damage to the nigral neurons. One of the features of PD is formation of filamentous amyloid deposits, Lewy bodies. Such cytoplasmic aggregations were observed in rotenone rat models of PD [22], [27]. We performed immunohistochemical staining of brain sections using an antibody against α-synuclein or ubiquitin and detected Lewy-body like inclusions in SN neurons of all the rats that were exposed to rotenone (Figure 1B). Furthermore, Nissl staining of the brain sections revealed a substantial decrease in the number of viable neurons in the SN of rotenone-treated animals (see below). The SN neurons of the rotenone-treated rats also exhibited extensive staining with antibody against 8-oxo-dG, indicating oxidative modification of DNA (Figure 1C). The oxidative damage was observed in both mitochondrial and nuclear DNA.


Protection by the NDI1 gene against neurodegeneration in a rotenone rat model of Parkinson's disease.

Marella M, Seo BB, Nakamaru-Ogiso E, Greenamyre JT, Matsuno-Yagi A, Yagi T - PLoS ONE (2008)

Characterization of the rotenone rat model.Rats received a subcutaneous injection of microspheres encapsulating rotenone. A: Concentration of rotenone in the plasma was determined on an HPLC system from 200 µl of blood samples drawn from the tail vein from 4 rats every week for 60 days. Values are given with standard deviation (SD). B: Rat brain coronal sections at the level of SN were subjected to immunohistochemical staining with antibody against α-synuclein or ubiquitin. Left panel, Lewy body-like cytoplasmic inclusions in SN neurons (arrows). Scale bar is 10 µm. Right panels, images at a higher magnification. C: The rat SN sections were stained with antibody specific for 8-oxo-dG. Oxidatively modified DNA appeared in green. Nuclei were stained in blue with DAPI. The image shows the damage to both mitochondrial DNA (left arrow) and nuclear DNA (right arrow) in SN neurons. Scale bar is 15 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2175531&req=5

pone-0001433-g001: Characterization of the rotenone rat model.Rats received a subcutaneous injection of microspheres encapsulating rotenone. A: Concentration of rotenone in the plasma was determined on an HPLC system from 200 µl of blood samples drawn from the tail vein from 4 rats every week for 60 days. Values are given with standard deviation (SD). B: Rat brain coronal sections at the level of SN were subjected to immunohistochemical staining with antibody against α-synuclein or ubiquitin. Left panel, Lewy body-like cytoplasmic inclusions in SN neurons (arrows). Scale bar is 10 µm. Right panels, images at a higher magnification. C: The rat SN sections were stained with antibody specific for 8-oxo-dG. Oxidatively modified DNA appeared in green. Nuclei were stained in blue with DAPI. The image shows the damage to both mitochondrial DNA (left arrow) and nuclear DNA (right arrow) in SN neurons. Scale bar is 15 µm.
Mentions: A few methods are available to administer rotenone in rats. For example, intravenous infusion via minipump [20] or intraperitoneal injection [24] may be used. Our preliminary experiments conducted with an intraperitoneal injection of rotenone, emulsified in oil, turned out to exert noxious effects; the rats presented liver necrosis and large weight loss (unpublished data). Subcutaneous delivery of rotenone, on the other hand, was reported to be more efficient in altering selectively the dopaminergic cells [22]. To eliminate toxic effects derived from the materials used for injections such as organic solvents, we carried out subcutaneous injections of biodegradable microspheres containing rotenone, a method originally adopted by Huang et al. [25]. After the injection, we monitored the concentration of rotenone in the plasma. As shown in Figure 1A, there was an initial increase in the rotenone concentration during the first 2 weeks which was followed by a gradual decrease. The rotenone concentration remained above 1 µM throughout the period of our experiments (60 days) presumably due to a slow and constant release of rotenone from the polymer undergoing disintegration in the animal body [26]. The rats lost about 2% of their body weight during the first 15 days after the injection which coincided with an increase in the rotenone concentration in the plasma, but there was no further loss throughout the rest of the period (data not shown). After 30 or 60 days of rotenone exposure, we examined the brain for the extent and nature of the damage to the nigral neurons. One of the features of PD is formation of filamentous amyloid deposits, Lewy bodies. Such cytoplasmic aggregations were observed in rotenone rat models of PD [22], [27]. We performed immunohistochemical staining of brain sections using an antibody against α-synuclein or ubiquitin and detected Lewy-body like inclusions in SN neurons of all the rats that were exposed to rotenone (Figure 1B). Furthermore, Nissl staining of the brain sections revealed a substantial decrease in the number of viable neurons in the SN of rotenone-treated animals (see below). The SN neurons of the rotenone-treated rats also exhibited extensive staining with antibody against 8-oxo-dG, indicating oxidative modification of DNA (Figure 1C). The oxidative damage was observed in both mitochondrial and nuclear DNA.

Bottom Line: It is widely recognized that mitochondrial dysfunction, most notably defects in the NADH-quinone oxidoreductase (complex I), is closely related to the etiology of sporadic Parkinson's disease (PD).A single, unilateral injection of recombinant adeno-associated virus carrying the NDI1 gene into the vicinity of the substantia nigra resulted in expression of the Ndi1 protein in the entire substantia nigra of that side.The present study shows, for the first time, the powerful neuroprotective effect offered by the Ndi1 enzyme in a rotenone rat model of PD.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, United States of America.

ABSTRACT
It is widely recognized that mitochondrial dysfunction, most notably defects in the NADH-quinone oxidoreductase (complex I), is closely related to the etiology of sporadic Parkinson's disease (PD). In fact, rotenone, a complex I inhibitor, has been used for establishing PD models both in vitro and in vivo. A rat model with chronic rotenone exposure seems to reproduce pathophysiological conditions of PD more closely than acute mouse models as manifested by neuronal cell death in the substantia nigra and Lewy body-like cytosolic aggregations. Using the rotenone rat model, we investigated the protective effects of alternative NADH dehydrogenase (Ndi1) which we previously demonstrated to act as a replacement for complex I both in vitro and in vivo. A single, unilateral injection of recombinant adeno-associated virus carrying the NDI1 gene into the vicinity of the substantia nigra resulted in expression of the Ndi1 protein in the entire substantia nigra of that side. It was clear that the introduction of the Ndi1 protein in the substantia nigra rendered resistance to the deleterious effects caused by rotenone exposure as assessed by the levels of tyrosine hydroxylase and dopamine. The presence of the Ndi1 protein also prevented cell death and oxidative damage to DNA in dopaminergic neurons observed in rotenone-treated rats. Unilateral protection also led to uni-directional rotation of the rotenone-exposed rats in the behavioral test. The present study shows, for the first time, the powerful neuroprotective effect offered by the Ndi1 enzyme in a rotenone rat model of PD.

Show MeSH
Related in: MedlinePlus