Mathieu Marella

Protection by the NDI1 Gene against Neurodegeneration in a Rotenone Rat Model of Parkinson's Disease

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 Parkinsons 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.

Successful Amelioration of Mitochondrial Optic Neuropathy Using the Yeast NDI1 Gene in a Rat Animal Model

Background Lebers hereditary optic neuropathy (LHON) is a maternally inherited disorder with point mutations in mitochondrial DNA which result in loss of vision in young adults. The majority of mutations reported to date are within the genes encoding the subunits of the mitochondrial NADH-quinone oxidoreductase, complex I. Establishment of animal models of LHON should help elucidate mechanism of the disease and could be utilized for possible development of therapeutic strategies. Methodology/Principal Findings We established a rat model which involves injection of rotenone-loaded microspheres into the optic layer of the rat superior colliculus. The animals exhibited the most common features of LHON. Visual loss was observed within 2 weeks of rotenone administration with no apparent effect on retinal ganglion cells. Death of retinal ganglion cells occurred at a later stage. Using our rat model, we investigated the effect of the yeast alternative NADH dehydrogenase, Ndi1. We were able to achieve efficient expression of the Ndi1 protein in the mitochondria of all regions of retinal ganglion cells and axons by delivering the NDI1 gene into the optical layer of the superior colliculus. Remarkably, even after the vision of the rats was severely impaired, treatment of the animals with the NDI1 gene led to a complete restoration of the vision to the normal level. Control groups that received either empty vector or the GFP gene had no effects. Conclusions/Significance The present study reports successful manifestation of LHON-like symptoms in rats and demonstrates the potential of the NDI1 gene therapy on mitochondrial optic neuropathies. Our results indicate a window of opportunity for the gene therapy to be applied successfully after the onset of the disease symptoms.

Mechanism of cell death caused by complex I defects in a rat dopaminergic cell line

Defects in the proton-translocating NADH-quinone oxidoreductase (complex I) of mammalian mitochondria are linked to neurodegenerative disorders. The mechanism leading to cell death elicited by complex I deficiency remains elusive. We have shown that expression of a rotenone-insensitive yeast NADH-quinone oxidoreductase (Ndi1) can rescue mammalian cells from complex I dysfunction. By using the Ndi1 enzyme, we have investigated the key events in the process of cell death using a rat dopaminergic cell line, PC12. We found that complex I inhibition provokes the following events: 1) activation of specific kinase pathways; 2) release of mitochondrial proapoptotic factors, apoptosis inducing factor, and endonuclease G. AS601245, a kinase inhibitor, exhibited significant protection against these apoptotic events. The traditional caspase pathway does not seems to be involved because caspase 3 activation was not observed. Our data suggest that overproduction of reactive oxygen species (ROS) caused by complex I inhibition is responsible for triggering the kinase activation, for the release of the proapoptotic factors, and then for cell death. Nearly perfect prevention of apoptotic cell death by Ndi1 agrees with our earlier observation that the presence of Ndi1 diminishes rotenone-induced ROS generation from complex I. In fact, this study demonstrated that Ndi1 keeps the redox potential high even in the presence of rotenone. Under these conditions, ROS formation by complex I is known to be minimal. Possible use of our cellular model is discussed with regard to development of therapeutic strategies for neurodegenerative diseases caused by complex I defects.

The single subunit NADH dehydrogenase reduces generation of reactive oxygen species from complex I

Using rat dopaminergic and human neuroblastoma cell lines transduced with the NDI1 gene encoding the internal NADH dehydrogenase (Ndi1) from Saccharomyces cerevisiae, we investigated reactive oxygen species (ROS) generation caused by complex I inhibition. Incubation of non‐transduced cells with rotenone elicited oxidative damage to mitochondrial DNA as well as lipid peroxidation. In contrast, oxidative stress was significantly decreased when the cells were transduced with NDI1. Furthermore, mitochondria from the NDI1‐transduced cells showed a suppressed rate of ROS formation by the complex I inhibitors. We conclude that the Ndi1 enzyme is able to suppress ROS overproduction from defective complex I.

Parkinson’s disease and mitochondrial complex I: a perspective on the Ndi1 therapy

Mitochondrial impairment has been collecting more and more attention as a contributing factor to the etiology of Parkinson’s disease. Above all, the NADH-quinone oxidoreductase, complex I, of the respiratory chain seems to be most culpable. Complex I dysfunction is translated to an increased production of reactive oxygen species and a decreased energy supply. In the brain, the dopaminergic neurons are one of the most susceptible cells. Their death is directly linked to the disease apparition. Developing an effective gene therapy is challenged by harmful actions of reactive oxygen species. To overcome this problem a therapeutic candidate must be able to restore the NADH-quinone oxidoreductase activity regardless of how complex I is impaired. Here we discuss the potency of the yeast alternative NADH dehydrogenase, the Ndi1 protein, to reinstate the mitochondrial respiratory chain compensating for disabled complex I and the benefit Ndi1 brings toward retardation of Parkinson’s disease.

A phenotypic model recapitulating the neuropathology of Parkinson's disease

This study was undertaken to develop a phenotypic model recapitulating the neuropathology of Parkinsons disease (PD). Such a model would show loss of dopamine in the basal ganglia, appearance of Lewy bodies, and the early stages of motor dysfunction. The model was developed by subcutaneously injecting biodegradable microspheres of rotenone, a complex I inhibitor in 8–9 month old, ovariectomized Long–Evans rats. Animals were observed for changes in body weight and motor activity. At the end of 11–12 weeks animals were euthanized and the brains examined for histopathological changes. Rotenone treated animals gain weight and appear normal and healthy as compared to controls but showed modest hypokinesia around 5–6 weeks posttreatment. Animals showed loss of dopaminergic (DA) neurons and the appearance of putative Lewy bodies in the substantia nigra. Neuroinflammation and oxidative stress were evidenced by the appearance of activated microglia, iron precipitates, and 8‐oxo‐2′‐deoxyguanosine a major product of DNA oxidation. The dorsal striatum, the projection site of midbrain DA neurons, showed a significant reduction in tyrosine hydroxylase immunostaining, together with an increase in reactive astrocytes, an early sign of DA nerve terminal damage. Levels of vesicular monoamine transporter 2 (VMAT2) were significantly reduced in the dorsal striatum; however, there was an unexpected increase in dopamine transporter (DAT) levels. Old, ovariectomized females treated with rotenone microspheres present with normal weight gain and good health but a modest hypokinesia. Accompanying this behavioral phenotype are a constellation of neuropathologies characteristic of PD that include loss of DA neurons, microglia activation, oxidative damage to nuclear DNA, iron deposition, and appearance of putative Lewy bodies. This phenotypic model recapitulating the neuropathology of Parkinsons disease could provide insight into early mechanisms of pathogenesis and could aid in the identification of biomarkers to identify patients in early stage, PD.

No immune responses by the expression of the yeast Ndi1 protein in rats

Background The rotenone-insensitive internal NADH-quinone oxidoreductase from yeast, Ndi1, has been shown to work as a replacement molecule for complex I in the respiratory chain of mammalian mitochondria. In the so-called transkingdom gene therapy, one major concern is the fact that the yeast protein is foreign in mammals. Long term expression of Ndi1 observed in rodents with no apparent damage to the target tissue was indicative of no action by the hosts immune system. Methodology/Principal Findings In the present study, we examined rat skeletal muscles expressing Ndi1 for possible signs of inflammatory or immune response. In parallel, we carried out delivery of the GFP gene using the same viral vector that was used for the NDI1 gene. The tissues were subjected to H&E staining and immunohistochemical analyses using antibodies specific for markers, CD11b, CD3, CD4, and CD8. The data showed no detectable signs of an immune response with the tissues expressing Ndi1. In contrast, mild but distinctive positive reactions were observed in the tissues expressing GFP. This clear difference most likely comes from the difference in the location of the expressed protein. Ndi1 was localized to the mitochondria whereas GFP was in the cytosol. Conclusions/Significance We demonstrated that Ndi1 expression did not trigger any inflammatory or immune response in rats. These results push forward the Ndi1-based molecular therapy and also expand the possibility of using foreign proteins that are directed to subcellular organelle such as mitochondria.

Complex I inhibition in the visual pathway induces disorganization of the node of Ranvier

Mitochondrial defects can have significant consequences on many aspects of neuronal physiology. In particular, deficiencies in the first enzyme complex of the mitochondrial respiratory chain (complex I) are considered to be involved in a number of human neurodegenerative diseases. The current work highlights a tight correlation between the inhibition of complex I and the state of axonal myelination of the optic nerve. Exposing the visual pathway of rats to rotenone, a complex I inhibitor, resulted in disorganization of the node of Ranvier. The structure and function of the node depend on specific cell adhesion molecules, among others, CASPR (contactin associated protein) and contactin. CASPR and contactin are both on the axonal surfaces and need to be associated to be able to anchor their myelin counterpart. Here we show that inhibition of mitochondrial complex I by rotenone in rats induces reactive oxygen species, disrupts the interaction of CASPR and contactin couple, and thus damages the organization and function of the node of Ranvier. Demyelination of the optic nerve occurs as a consequence which is accompanied by a loss of vision. The physiological impairment could be reversed by introducing an alternative NADH dehydrogenase to the mitochondria of the visual system. The restoration of the nodal structure was specifically correlated with visual recovery in the treated animal.

Long-term evaluation of Leber’s hereditary optic neuropathy-like symptoms in rotenone administered rats

Lebers hereditary optic neuropathy (LHON) is an inherited disorder affecting the retinal ganglion cells (RGCs) and their axons that lead to the loss of central vision. This study is aimed at evaluating the LHON symptoms in rats administered with rotenone microspheres into the superior colliculus (SC). Optical coherence tomography (OCT) analysis showed substantial loss of retinal nerve fiber layer (RNFL) thickness in rotenone injected rats. Optokinetic testing in rotenone treated rats showed decrease in head-tracking response. Electrophysiological mapping of the SC surface demonstrated attenuation of visually evoked responses; however, no changes were observed in the ERG data. The progressive pattern of disease manifestation in rotenone administered rats demonstrated several similarities with human disease symptoms. These rats with LHON-like symptoms can serve as a model for future investigators to design and implement reliable tests to assess the beneficial effects of therapeutic interventions for LHON disease.