A highly reproducible rotenone model of Parkinson's disease.

The systemic rotenone model of Parkinson's disease (PD) accurately replicates many aspects of the pathology of human PD and has provided insights into the pathogenesis of PD. The major limitation of the rotenone model has been its variability, both in terms of the percentage of animals that develop a clear-cut nigrostriatal lesion and the extent of that lesion. The goal here was to develop an improved and highly reproducible rotenone model of PD. In these studies, male Lewis rats in three age groups (3, 7 or 12-14 months) were administered rotenone (2.75 or 3.0 mg/kg/day) in a specialized vehicle by daily intraperitoneal injection. All rotenone-treated animals developed bradykinesia, postural instability, and/or rigidity, which were reversed by apomorphine, consistent with a lesion of the nigrostriatal dopamine system. Animals were sacrificed when the PD phenotype became debilitating. Rotenone treatment caused a 45% loss of tyrosine hydroxylase-positive substantia nigra neurons and a commensurate loss of striatal dopamine. Additionally, in rotenone-treated animals, alpha-synuclein and poly-ubiquitin positive aggregates were observed in dopamine neurons of the substantia nigra. In summary, this version of the rotenone model is highly reproducible and may provide an excellent tool to test new neuroprotective strategies.

Peroxidase mechanism of lipid-dependent cross-linking of synuclein with cytochrome C: protection against apoptosis versus delayed oxidative stress in Parkinson disease.

Damage of presynaptic mitochondria could result in release of proapoptotic factors that threaten the integrity of the entire neuron. We discovered that alpha-synuclein (Syn) forms a triple complex with anionic lipids (such as cardiolipin) and cytochrome c, which exerts a peroxidase activity. The latter catalyzes covalent hetero-oligomerization of Syn with cytochrome c into high molecular weight aggregates. Syn is a preferred substrate of this reaction and is oxidized more readily than cardiolipin, dopamine, and other phenolic substrates. Co-localization of Syn with cytochrome c was detected in aggregates formed upon proapoptotic stimulation of SH-SY5Y and HeLa cells and in dopaminergic substantia nigra neurons of rotenone-treated rats. Syn-cardiolipin exerted protection against cytochrome c-induced caspase-3 activation in a cell-free system, particularly in the presence of H(2)O(2). Direct delivery of Syn into mouse embryonic cells conferred resistance to proapoptotic caspase-3 activation. Conversely, small interfering RNA depletion of Syn in HeLa cells made them more sensitive to dopamine-induced apoptosis. In human Parkinson disease substantia nigra neurons, two-thirds of co-localized Syn-cytochrome c complexes occurred in Lewy neurites. Taken together, these results indicate that Syn may prevent execution of apoptosis in neurons through covalent hetero-oligomerization of cytochrome c. This immediate protective function of Syn is associated with the formation of the peroxidase complex representing a source of oxidative stress and postponed damage.

A novel transferrin/TfR2-mediated mitochondrial iron transport system is disrupted in Parkinson's disease.

More than 80 years after iron accumulation was initially described in the substantia nigra (SN) of Parkinson's disease (PD) patients, the mechanisms responsible for this phenomenon are still unknown. Similarly, how iron is delivered to its major recipients in the cell - mitochondria and the respiratory complexes - has yet to be elucidated. Here, we report a novel transferrin/transferrin receptor 2 (Tf/TfR2)-mediated iron transport pathway in mitochondria of SN dopamine neurons. We found that TfR2 has a previously uncharacterized mitochondrial targeting sequence that is sufficient to import the protein into these organelles. Importantly, the Tf/TfR2 pathway can deliver Tf bound iron to mitochondria and to the respiratory complex I as well. The pathway is redox-sensitive and oxidation of Tf thiols to disulfides induces release from Tf of highly reactive ferrous iron, which contributes to free radical production. In the rotenone model of PD, Tf accumulates in dopamine neurons, with much of it accumulating in the mitochondria. This is associated with iron deposition in SN, similar to what occurs in PD. In the human SN, TfR2 is also found in mitochondria of dopamine neurons, and in PD there is a dramatic increase of oxidized Tf in SN. Thus, we have discovered a novel mitochondrial iron transport system that goes awry in PD, and which may provide a new target for therapeutic intervention.

A FRET-based method to study protein thiol oxidation in histological preparations.

Cysteine residues in proteins have important biological roles. For example, disulfide bonds are important structural elements; additionally, reversible oxidation of thiols to disulfides functions as a molecular switch and constitutes an early response to oxidative damage. Because organs are heterogeneous structures composed of diverse cell types, there is a compelling need for a histological approach to investigate thiol oxidation in situ in order to address the role of specific cell types in oxidative imbalance. Here we describe a fluorescence technique-which can be used in association with standard immunological staining procedures-to detect variations in disulfides in histological preparations. Moreover, by monitoring the fluorescence resonance energy transfer (FRET) between a labeled specific primary antibody and the thiol probe described here, this method can detect thiol oxidation in candidate proteins of interest. When applied to an animal model of Parkinson's disease, our technique demonstrated that thiol oxidation occurs selectively in the dopaminergic neurons of the substantia nigra, the same neurons that are lost selectively in the disease. In summary, this technique provides a new, powerful tool for providing further understanding of oxidative imbalance, a phenomenon common to many diseases.

Intersecting pathways to neurodegeneration in Parkinson's disease: effects of the pesticide rotenone on DJ-1, alpha-synuclein, and the ubiquitin-proteasome system.

Sporadic Parkinson's disease (PD) is most likely caused by a combination of environmental exposures and genetic susceptibilities, although there are rare monogenic forms of the disease. Mitochondrial impairment at complex I, oxidative stress, alpha-synuclein aggregation, and dysfunctional protein degradation, have been implicated in PD pathogenesis, but how they are related to each other is unclear. To further evaluated PD pathogenesis here, we used in vivo and in vitro models of chronic low-grade complex I inhibition with the pesticide rotenone. Chronic rotenone exposure in vivo caused oxidative modification of DJ-1, accumulation of alpha-synuclein, and proteasomal impairment. Interestingly, the effects become more regionally restricted such that systemic complex I inhibition eventually results in highly selective degeneration of the nigrostriatal pathway. DJ-1 modifications, alpha-synuclein accumulation, and proteasomal dysfunction were also seen in vitro and these effects could be prevented with alpha-tocopherol. Thus, chronic exposure to a pesticide and mitochondrial toxin brings into play three systems, DJ-1, alpha-synuclein, and the ubiquitin-proteasome system, and implies that mitochondrial dysfunction and oxidative stress link environmental and genetic forms of the disease.