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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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Expression of the Ndi1 protein in the rat SN and its effect on the TH levels in the SN and striatum after rotenone exposure.Rats received a unilateral injection of recombinant AAV carrying the NDI1 gene targeted at the right SN. Control rats were injected with PBS. After the expression of the Ndi1 protein has been established, microspheres containing rotenone were injected subcutaneously. For control, microspheres with PBS were used. Thirty or 60 days later, brains were collected for analysis. A: After 30 days of rotenone exposure, brain sections at the level of SN were stained for the NADH dehydrogenase (diaphorase) activity. The right SN that received the NDI1 gene showed high NADH dehydrogenase as revealed by dense staining in both rotenone-treated (b) and PBS-treated (a) group. Non-NDI1 control rats did not exhibit discernible staining (c and d). B: Brain sections from the same groups of rats as A were double-stained for TH (red) and the Ndi1 protein (green). The staining intensities across the right SN (NDI1 injection side) and the left SN are plotted in arbitrary units below each set of the images. Each picture displays a representative section of an animal from each group. C: Rats expressing the Ndi1 protein (NDI1) and non-NDI1 control group (Ctl) were treated with rotenone (Rot) for 60 days and immunohistochemical staining for TH staining were carried out as in B. The fluorescence images of the brain sections were collected and the staining intensity of TH from the left and the right SN was statistically analyzed (student T-test) using ImageJ [39]. Note that the right SN received the NDI1 injection. The number of sections used was; Ctl (n = 6), Ctl+Rot (n = 9), NDI1 (n = 18) and NDI1+Rot (n = 18). Images in each group were collected from evenly spaced sections that cover the entire SN in which the presence of the Ndi1 protein was detected. Error bars represent standard deviation. D: Levels of TH staining in the left and right striatum from the same four groups of rats as in C are compared. The right striatum is ipsilateral to the SN expressing the Ndi1 protein. Representative images of DAB staining of TH are given in Figure S2. The number of sections used for analysis was; Ctl (n = 6), Ctl+Rot (n = 9), NDI1 (n = 9) and NDI1+Rot (n = 18). Error bars represent standard deviation.
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pone-0001433-g002: Expression of the Ndi1 protein in the rat SN and its effect on the TH levels in the SN and striatum after rotenone exposure.Rats received a unilateral injection of recombinant AAV carrying the NDI1 gene targeted at the right SN. Control rats were injected with PBS. After the expression of the Ndi1 protein has been established, microspheres containing rotenone were injected subcutaneously. For control, microspheres with PBS were used. Thirty or 60 days later, brains were collected for analysis. A: After 30 days of rotenone exposure, brain sections at the level of SN were stained for the NADH dehydrogenase (diaphorase) activity. The right SN that received the NDI1 gene showed high NADH dehydrogenase as revealed by dense staining in both rotenone-treated (b) and PBS-treated (a) group. Non-NDI1 control rats did not exhibit discernible staining (c and d). B: Brain sections from the same groups of rats as A were double-stained for TH (red) and the Ndi1 protein (green). The staining intensities across the right SN (NDI1 injection side) and the left SN are plotted in arbitrary units below each set of the images. Each picture displays a representative section of an animal from each group. C: Rats expressing the Ndi1 protein (NDI1) and non-NDI1 control group (Ctl) were treated with rotenone (Rot) for 60 days and immunohistochemical staining for TH staining were carried out as in B. The fluorescence images of the brain sections were collected and the staining intensity of TH from the left and the right SN was statistically analyzed (student T-test) using ImageJ [39]. Note that the right SN received the NDI1 injection. The number of sections used was; Ctl (n = 6), Ctl+Rot (n = 9), NDI1 (n = 18) and NDI1+Rot (n = 18). Images in each group were collected from evenly spaced sections that cover the entire SN in which the presence of the Ndi1 protein was detected. Error bars represent standard deviation. D: Levels of TH staining in the left and right striatum from the same four groups of rats as in C are compared. The right striatum is ipsilateral to the SN expressing the Ndi1 protein. Representative images of DAB staining of TH are given in Figure S2. The number of sections used for analysis was; Ctl (n = 6), Ctl+Rot (n = 9), NDI1 (n = 9) and NDI1+Rot (n = 18). Error bars represent standard deviation.

Mentions: Using the rotenone rat model of PD, we carried out the experiments that allow evaluation of capability and efficiency of the Ndi1 protein as a therapeutic agent. We have previously reported that the Ndi1 protein was functionally expressed in the rat SN following a stereotaxic injection of recombinant AAV carrying the NDI1 gene [16]. However, the level of the Ndi1 protein expression was variable and, therefore, not most suitable for our aims. In an attempt to achieve a consistently high level and a wider area of the Ndi1 protein expression in the SN, we selected AAV serotype 5 [28] which was reported to deliver the transgene in the SN more efficiently than AAV serotype 2 that was used in our previous experiments [16]. The injection point was set at 0.4 mm above the SN in order to avoid any physical damage to the dopaminergic pathway during the surgery. In all animals tested, a single, unilateral injection of the viral particles resulted in the expression of the Ndi1 protein in the entire SN spanning 1.8 mm sagittally. The wide distribution of the Ndi1 protein in the SN dopaminergic neurons was evident in a coronal section of the brain double-stained with antibodies to the Ndi1 protein and TH (Figure S1, A and B). It was also confirmed that the Ndi1 protein was localized to mitochondria (Figure S1, C–E) just as we reported earlier using cultured cells of various origins [14], [15], [29] as well as in rodents [16], [18]. Near perfect colocalization of the Ndi1 protein and mitochondria was most clearly visualized in a 3D image of an SN neuron constructed from a series of confocal images of a brain section double-stained for the Ndi1 protein and the α-subunit of F1-ATPase (Movie S1). The level of TH and the amount of dopamine were not affected by the Ndi1 protein expression. The expressed Ndi1 protein was enzymatically active as seen in dense staining derived from NADH dehydrogenase activity in the SN on the injected side (Figure 2A).


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)

Expression of the Ndi1 protein in the rat SN and its effect on the TH levels in the SN and striatum after rotenone exposure.Rats received a unilateral injection of recombinant AAV carrying the NDI1 gene targeted at the right SN. Control rats were injected with PBS. After the expression of the Ndi1 protein has been established, microspheres containing rotenone were injected subcutaneously. For control, microspheres with PBS were used. Thirty or 60 days later, brains were collected for analysis. A: After 30 days of rotenone exposure, brain sections at the level of SN were stained for the NADH dehydrogenase (diaphorase) activity. The right SN that received the NDI1 gene showed high NADH dehydrogenase as revealed by dense staining in both rotenone-treated (b) and PBS-treated (a) group. Non-NDI1 control rats did not exhibit discernible staining (c and d). B: Brain sections from the same groups of rats as A were double-stained for TH (red) and the Ndi1 protein (green). The staining intensities across the right SN (NDI1 injection side) and the left SN are plotted in arbitrary units below each set of the images. Each picture displays a representative section of an animal from each group. C: Rats expressing the Ndi1 protein (NDI1) and non-NDI1 control group (Ctl) were treated with rotenone (Rot) for 60 days and immunohistochemical staining for TH staining were carried out as in B. The fluorescence images of the brain sections were collected and the staining intensity of TH from the left and the right SN was statistically analyzed (student T-test) using ImageJ [39]. Note that the right SN received the NDI1 injection. The number of sections used was; Ctl (n = 6), Ctl+Rot (n = 9), NDI1 (n = 18) and NDI1+Rot (n = 18). Images in each group were collected from evenly spaced sections that cover the entire SN in which the presence of the Ndi1 protein was detected. Error bars represent standard deviation. D: Levels of TH staining in the left and right striatum from the same four groups of rats as in C are compared. The right striatum is ipsilateral to the SN expressing the Ndi1 protein. Representative images of DAB staining of TH are given in Figure S2. The number of sections used for analysis was; Ctl (n = 6), Ctl+Rot (n = 9), NDI1 (n = 9) and NDI1+Rot (n = 18). Error bars represent standard deviation.
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pone-0001433-g002: Expression of the Ndi1 protein in the rat SN and its effect on the TH levels in the SN and striatum after rotenone exposure.Rats received a unilateral injection of recombinant AAV carrying the NDI1 gene targeted at the right SN. Control rats were injected with PBS. After the expression of the Ndi1 protein has been established, microspheres containing rotenone were injected subcutaneously. For control, microspheres with PBS were used. Thirty or 60 days later, brains were collected for analysis. A: After 30 days of rotenone exposure, brain sections at the level of SN were stained for the NADH dehydrogenase (diaphorase) activity. The right SN that received the NDI1 gene showed high NADH dehydrogenase as revealed by dense staining in both rotenone-treated (b) and PBS-treated (a) group. Non-NDI1 control rats did not exhibit discernible staining (c and d). B: Brain sections from the same groups of rats as A were double-stained for TH (red) and the Ndi1 protein (green). The staining intensities across the right SN (NDI1 injection side) and the left SN are plotted in arbitrary units below each set of the images. Each picture displays a representative section of an animal from each group. C: Rats expressing the Ndi1 protein (NDI1) and non-NDI1 control group (Ctl) were treated with rotenone (Rot) for 60 days and immunohistochemical staining for TH staining were carried out as in B. The fluorescence images of the brain sections were collected and the staining intensity of TH from the left and the right SN was statistically analyzed (student T-test) using ImageJ [39]. Note that the right SN received the NDI1 injection. The number of sections used was; Ctl (n = 6), Ctl+Rot (n = 9), NDI1 (n = 18) and NDI1+Rot (n = 18). Images in each group were collected from evenly spaced sections that cover the entire SN in which the presence of the Ndi1 protein was detected. Error bars represent standard deviation. D: Levels of TH staining in the left and right striatum from the same four groups of rats as in C are compared. The right striatum is ipsilateral to the SN expressing the Ndi1 protein. Representative images of DAB staining of TH are given in Figure S2. The number of sections used for analysis was; Ctl (n = 6), Ctl+Rot (n = 9), NDI1 (n = 9) and NDI1+Rot (n = 18). Error bars represent standard deviation.
Mentions: Using the rotenone rat model of PD, we carried out the experiments that allow evaluation of capability and efficiency of the Ndi1 protein as a therapeutic agent. We have previously reported that the Ndi1 protein was functionally expressed in the rat SN following a stereotaxic injection of recombinant AAV carrying the NDI1 gene [16]. However, the level of the Ndi1 protein expression was variable and, therefore, not most suitable for our aims. In an attempt to achieve a consistently high level and a wider area of the Ndi1 protein expression in the SN, we selected AAV serotype 5 [28] which was reported to deliver the transgene in the SN more efficiently than AAV serotype 2 that was used in our previous experiments [16]. The injection point was set at 0.4 mm above the SN in order to avoid any physical damage to the dopaminergic pathway during the surgery. In all animals tested, a single, unilateral injection of the viral particles resulted in the expression of the Ndi1 protein in the entire SN spanning 1.8 mm sagittally. The wide distribution of the Ndi1 protein in the SN dopaminergic neurons was evident in a coronal section of the brain double-stained with antibodies to the Ndi1 protein and TH (Figure S1, A and B). It was also confirmed that the Ndi1 protein was localized to mitochondria (Figure S1, C–E) just as we reported earlier using cultured cells of various origins [14], [15], [29] as well as in rodents [16], [18]. Near perfect colocalization of the Ndi1 protein and mitochondria was most clearly visualized in a 3D image of an SN neuron constructed from a series of confocal images of a brain section double-stained for the Ndi1 protein and the α-subunit of F1-ATPase (Movie S1). The level of TH and the amount of dopamine were not affected by the Ndi1 protein expression. The expressed Ndi1 protein was enzymatically active as seen in dense staining derived from NADH dehydrogenase activity in the SN on the injected side (Figure 2A).

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