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.

Mitochondrial trafficking to synapses in cultured primary cortical neurons.

Functional synapses require mitochondria to supply ATP and regulate local [Ca2+]i for neurotransmission. Mitochondria are thought to be transported to specific cellular regions of increased need such as synapses. However, little is known about how this occurs, including the spatiotemporal distribution of mitochondria relative to presynaptic and postsynaptic sites, whether mitochondria are dynamically recruited to synapses, and how synaptic activity affects these trafficking patterns. We used primary cortical neurons in culture that form synaptic connections and show spontaneous synaptic activity under normal conditions. Neurons were cotransfected with a mitochondrially targeted cyan fluorescent protein and an enhanced yellow fluorescent protein-tagged synaptophysin or postsynaptic density-95 plasmid to label presynaptic or postsynaptic structures, respectively. Fluorescence microscopy revealed longer dendritic mitochondria that occupied a greater fraction of neuronal process length than axonal mitochondria. Mitochondria were significantly more likely to be localized at synaptic sites. Although this localization was unchanged by inhibition of synaptic activity by tetrodotoxin, it increased in dendritic synapses and decreased in axonal synapses during overactivity by veratridine. Mitochondrial movement and recruitment to synapses also differed between axons and dendrites under basal conditions and when synaptic activity was altered. Additionally, we show that movement of dendritic mitochondria can be selectively impaired by glutamate and zinc. We conclude that mitochondrial trafficking to synapses is dynamic in neurons and is modulated by changes in synaptic activity. Furthermore, mitochondrial morphology and distribution may be optimized differentially to best serve the synaptic distributions in axons and dendrites. Last, selective cessation of mitochondrial movement in dendrites suggests early postsynaptic dysfunction in neuronal injury and degeneration.

Zn2+ inhibits mitochondrial movement in neurons by phosphatidylinositol 3-kinase activation.

Mitochondria have been identified as targets of the neurotoxic actions of zinc, possibly through decreased mitochondrial energy production and increased reactive oxygen species accumulation. It has been hypothesized that impairment of mitochondrial trafficking may be a mechanism of neuronal injury. Here, we report that elevated intraneuronal zinc impairs mitochondrial trafficking. At concentrations just sufficient to cause injury, zinc rapidly inhibited mitochondrial movement without altering morphology. Zinc chelation initially restored movement, but the actions of zinc became insensitive to chelator in <10 min. A search for downstream signaling events revealed that inhibitors of phosphatidylinositol (PI) 3-kinase prevented this zinc effect on movement. Moreover, transient inhibition of PI 3-kinase afforded neuroprotection against zinc-mediated toxicity. These data illustrate a novel mechanism that regulates mitochondrial trafficking in neurons and also suggest that mitochondrial trafficking may be closely coupled to neuronal viability.