Brain, Nervous System, Tissue, Continued

Biological Applications:

Parkinson's disease: Investigating the selective vulnerability of nigrostriatal neurons in mouse brain slices to complex I inhibition by rotenone

Mitochondrial Inhibitors and Parkinson's Disease

The cause of Parkinson's disease (PD) is unknown, but epidemiological studies suggest an association with pesticides and other environmental toxins (Gorell et al. 1998). In particular, the toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the pesticide rotenone have been found to produce, in experimental animals, a Parkinsonian syndrome characterized by highly selective nigrostriatal dopaminergic degeneration just as in idiopathic PD. Biochemical studies have implicated mitochondrial complex I dysfunction in both the pathogenesis of Parkinson's disease and in the neurotoxicity of MPTP and rotenone, suggesting that the nigrostriatal system, selectively affected in idiopathic PD as well as by MPTP and rotenone, may be particularly vulnerable to an impairment of mitochondrial energy metabolism.

The MPP+-Model of Parkinson's Disease

The eurotoxic effects of MPTP reproduce neurochemical and pathological features of human parkinsonism such as the reduction of striatal dopamine (DA) and the loss of dopaminergic cell bodies in the substantia nigra (Javitch et al. 1985). In fact, after the pro-toxin MPTP was reported to produce in humans an acute parkinsonian syndrome that is virtually indistinguishable from idiopathic PD, its metabolite, 1-methyl-4-pyridinium (MPP+), was found to be a mitochondrial poison that inhibits respiration at complex I of the electron transport chain (Betarbet et al. 2000). Similar toxic events may play a role in neurodegeneration in PD since a decrease in complex I activity has been measured post mortem in both the substantia nigra and the striatum of PD patients as compared to control subjects (Dawson, 2000). However, unlike in idiopathic PD, MPTP does not cause a systemic complex I defect. Instead, MPTP is first converted into its active metabolite MPP+, which is then selectively imported into dopaminergic neurons by the plasma membrane dopamine (DA) transporter (Javitch et al. 1985). Once it has been taken up into the cell, MPP+ accumulates in the mitochondria where i acts to inhibit complex I. Therefore, since only cells possessing the DA transporter take up MPP+, the inhibition is not systemic but instead is highly selective for dopaminergic neurons. This selective inhibition is likely to account for the parkinsonian-like pattern of degeneration caused by MPTP/MPP+ administration. Therefore, MPTP animal models do not provide good approximations for mitochondrial dysfunction in human PD.

The Rotenone Model of Parkinson's Disease

In contrast, systemic complex I inhibition by the chronic administration of the pesticide rotenone has recently been shown to reproduce the same progressive, selective nigrostriatal dopaminergic degeneration seen in PD (Betarbet et al. 2000). Rotenone, widely believed to be a safe, natural alternative to synthetic pesticides, is most commonly used as an insecticide in vegetable gardens and to kill or sample fish populations in lakes and reservoirs. It is a well characterized, high-affinity, specific inhibitor of mitochondrial complex I that exerts its inhibitory action by blocking electron transfer in NADH-Q reductase, thus preventing the utilization of NADH as a substrate in oxidative phosphorylation (Stryer, 1999). Furthermore, in contrast to MPP+, rotenone is extremely hydrophobic and crosses biological membranes easily. Therefore, it does not depend on the dopamine transporter for access to the cytoplasm and is thus likely to affect all cells (Betarbet et al. 2000). Hence rotenone, as opposed to MPTP, is well suited to produce a systemic inhibition of complex I that better approximates complex I dysfunction evident in idiopathic PD. However, the question remains as to why rotenone, which inhibits complex I throughout the brain, should preferentially lead to the degeneration of dopaminergic neurons.

A limited number of studies have evaluated the effects of rotenone on dopaminergic neurons both in vitro and in vivo. Treatment of mesencephalic cultures and striatal synaptosomes with rotenone has been shown to cause neurotoxicity as measured by a decreased uptake of neurotransmitters (Marey-Semper et al. 1993). Interestingly, DA uptake was significantly more affected than the uptake of serotonin, GABA or noradrenaline, thus supporting the hypothesis of a selective vulnerability of dopaminergic cells. In an in vivo study, a substantial depletion of striatal DA and its metabolites was reported after the stereotaxic injection of rotenone into the median forebrain bundle of rats (Heikkila et al. 1985). In contrast, systemic acute treatment of mice with relatively high doses of rotenone was found to have no affect on DA concentration, but instead caused a significant increase in DA metabolites (Thiffault et al. 2000). Studies in which rats were chronically treated with rotenone via continuous intravenous infusion provide the best histological (but not biochemical) evidence of selective neuronal damage. Ferrante et al. (1997) have provided evidence showing selective degeneration in the striatum and globus pallidus, but not the substantia nigra, of rats treated with high doses of rotenone for a period of 7-9 days (Ferrante et al. 1997). Similarly, Betarbet et al. (2000) have most recently shown histological evidence of selective degeneration of the nigrostriatal dopaminergic system, as well as the development of alpha-synuclein protein inclusions in degenerating neurons (a hallmark of idiopathic PD), following the continuous intravenous infusion of low doses of rotenone for relatively longer periods of time. However, the possibility that nigrostriatal injury may be induced by rotenone under less severe experimental conditions and different paradigms of administration remains to be further evaluated.

Questions that we are investigating:

(a) Why does administration of rotenone, which inhibits complex I throughout the brain, preferentially affect dopaminergic neurons in the midbrain? Is there a selective vulnerability of these nigral neurons to complex I inhibition?

(b) Can we develop two-photon redox-fluorescence microscopy into an optical imaging assay of complex I activity?

References:

Betarbet, R., T. Sherer, G. MacKenzie, M. Garcia-Osuna, A.V. Panov and J.T. Greenamyre, 2000. Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Nature Neuroscience. 3: 1301-1306

Dawson, T.M. 2000. New Animal Models for Parkinson's Disease. Cell. 101:115-118

Ferrante, R.J., J.B. Schulz, N.W. Kowall, and M.F. Beal., 1997. Systemic administration of rotenone
produces selective damage in the striatum and globus pallidus, but not in the substantia nigra. Brain Res. 753: 157-162.

Gorell, J.M., C.C. Johnson, B.A. Rybicki, E.L. Peterson, and R.J. Richardson, 1998. The risk of
Parkinson's disease with exposure to pesticides, farming, well water and rural living. Neurology. 50:1346-1350.

Javitch, J.A., R.J. D'amato, S.M. Strittmatter, and S.H. Snyder, 1985. Parkinsonism-inducing neurotoxin,
N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine: uptake of the metabolite N-methyl-4-phenylpyridine by dopamine neurons explains selective toxicity. Proc. Natl. Acad. Sci. USA. 82: 2173-2177.

Marey-Semper, I., M. Gelman, and M. Levi-Strauss, 1993. The high sensitivity to rotenone of striatal
dopamine uptake suggests the existence of a constitutive metabolic deficiency in dopaminergic neurons from the substantia nigra. Eur. J. Neurosci. 5: 1029-1034.

Stryer L., 1999. Biochemistry. 4th Ed. W.H. Freeman and Company. New York, NY. p.544

Thiffault, C., J.W. Langston, and D.A. Di Monte, 2000. Increased striatal dopamine turnover following
acute administration of rotenone to mice. Brain Research. 885: 283-288.