Does dopamine contribute to striatal damage caused by impaired mitochondrial function?
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ABSTRACT: Exposure of PC12 cells to metamphetamine (MA) induces the formation of multilamellar structures (whorls) resembling autophagic granules that subsequently develop as intracellular inclusions. These inclusions stain for a variety of antigens belonging to the ubiquitin proteasome pathway. Since MA-induced intracellular bodies require the presence of dopamine in the present study we analyzed the role of dopamine (DA) receptors in producing neuronal inclusions. Moreover, we investigated potential signaling pathways which could lead to ubiquitination in the presence of MA. Based on recent reports that ubiquitination of beta-adrenergic receptors is promoted by beta-arrestin which shuttles proteins from the plasma membrane to the ubiquitin proteasome system we investigated whether beta-arrestin is involved in MA-induced inclusion formation. Our experiments document that (i) beta-arrestin was associated with MA-induced intracellular bodies; (ii) MA induced a rapid and reversible ubiquitination of beta-arrestin; (iii) dopamine antagonists reduced both MA-induced beta-arrestin ubiquitination and intracellular whorls formation; (iv) the number of MA-induced intracellular bodies was reduced in cells transfected with the beta-arrestin dominant negative mutant, betaarrV53D and was increased by the persistently ubiquitinated beta-arrestin-ubiquitin fusion protein. In conclusion, the present study demonstrates the involvement of beta-arrestin in MA-induced intracellular bodies and the participation of dopamine receptors in this process.Journal of Neurochemistry 07/2008; 105(5):1939-47. · 4.24 Impact Factor
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ABSTRACT: The transcription factor Nrf2 controls inducible expression of multiple antioxidant/detoxification genes. We previously found that Nrf2-/- mice have increased sensitivity to in vivo mitochondrial stress and ischemia. Although Nrf2 regulated these forms of neuronal toxicity, it was unclear which injury-triggered signal(s) led to Nrf2 activation in vivo. In this study, we use primary cultures to test the hypothesis that excessive dopamine release can act as an endogenous Nrf2-inducing signal. We cultured two cell types that show increased Nrf2 activity during ischemia in vivo, astrocytes and meningeal cells. Cultures were infected with an adenovirus reporter of Nrf2 transcriptional activity. Dopamine-induced Nrf2 activity in both cell types by generating oxidative stressors, H2O2 and dopamine-quinones. Nrf2 activation in meningeal cells was significantly higher than astrocytes. The effect of dopamine was blocked by antioxidants, and by over-expression of either dominant-negative Nrf2 or Keap1. Nrf2 induction was specific to oxidative stress caused by catecholaminergic neurotransmitters as epinephrine also induced Nrf2, but the monoamine serotonin had no significant effect. These in vitro results suggest Nrf2 activity in astrocytes and meningeal cells link the neurotoxic actions of dopamine to neuroprotective pathways that may potentially modulate ischemic injury and neurodegeneration.Journal of Neurochemistry 04/2007; 101(1):109-19. · 4.24 Impact Factor
Does Dopamine Contribute to Striatal Damage
Caused by Impaired Mitochondrial Function?
WILLIAM F. MARAGOS,a,b,e REBEKAH J. JAKEL,a M. DATHAN CHESNUT,a
JAMES W. GEDDES,c AND LINDA P. DWOSKINd
aDepartment of Neurology, bDepartment of Anatomy and Neurobiology, cSanders Brown
Center on Aging, and dSchool of Pharmacy, University of Kentucky Medical Center,
Lexington, Kentucky 40536-0284, USA
Huntington’s disease (HD) is a heredofamilial disorder characterized clinically by
involuntary and often incapacitating “choreiform” movements, dementia, and psy-
chiatric disturbances.1 Neuropathological changes include neuronal loss and inclu-
sions, gliosis, and atrophy. Although several brain regions including the cerebral
cortex, hypothalamus, and cerebellum show these changes, the basal ganglia are pre-
There is increasing evidence that diminished energy metabolism is a factor in the
pathophysiology of HD. In rodents, administration of 3 nitropropionic acid (3NP)
and intrastriatal injections of malonate (both inhibitors of the mitochondrial enzyme
succinate dehydrogenase) replicate many of the neuropathological abnormalities
seen in HD and have thus been used to model this disorder.2,3 The mechanism of ac-
tion of these compounds has been postulated to occur via the indirect activation of
excitatory amino acid (EAA) receptors and induction of “cell death” pathways. De-
spite the insights gained from studies investigating 3NP and malonate toxicity, the
preferential vulnerability of the striatum is unclear. We hypothesize that one factor
contributing to the preferential striatal damage is the presence of the neurotransmit-
ter dopamine (DA). DA is present in high concentrations in the striatum and under
certain circumstances may be neurotoxic. In this study, we have investigated the ef-
fects of (1) dopamine deafferentation and (2) MAO inhibition on the striatal damage
produced by 3NP and malonate. We also evaluated whether mitochondrial inhibition
altered dopamine uptake into synaptosomes.
Experiment 1: Effects of 6 Hydroxydopamine (6OHDA) Lesions
Malonate Lesions. Eight rats received 6OHDA (8 µg/4 µl), and 10 received sterile
saline injections into the right medial forebrain bundle (mfb). Two weeks later, ani-
mals in which apomorphine induced >100 turns were injected into the right striatum
with 1.0 µl of malonate (0.8 µmol/µl). Animals survived 5 days, were sacrificed, and
lesion volumes determined.
eAddress for correspondence: University of Kentucky Medical Center. Department of Neurol-
ogy, Kentucky Clinic, Room L-445, Lexington, Kentucky 40536-0284. Phone: 606-323-2522;
346 ANNALS NEW YORK ACADEMY OF SCIENCES
3NP Lesions. Four animals were injected with 6OHDA (12 µg/2 µl) into the right
substantia nigra (SN) and four with sterile saline. One week later, rats were admin-
istered 20 mg/kg i.p. daily for 4–8 days. Animals were perfused with 4% paraform-
aldehyde, and every 6th striatal section was mounted and analyzed. Tyrosine
hydroxylase immunohistochemistry was performed on sections taken through the
substantia nigra to verify the efficacy of the 6OHDA lesion.
Experiment 2: Effects of MAO Inhibition
Malonate Lesions. Eight animals received i.p. injections of both deprenyl and clo-
rgyline (5 mg/kg each in sterile saline), and eight control animals were treated with
sterile saline for three consecutive days. On the fourth day, all animals were injected
with 1.5 µl of malonate and allowed to survive 3 days, at which time the animals
were euthanized, their brains rapidly removed, frozen, and tissue damage assessed.
3NP Lesions. Four animals were injected on three consecutive days with both de-
prenyl and clorgyline (5 mg/kg each in sterile saline) and 4 animals with sterile sa-
line. On day four, animals began daily i.p. injections of 3NP (20 mg/kg in sterile
saline, pH 7.4) while continuing to receive clorgyline and deprenyl. On day 8 of 3NP
treatment, animals were sacrificed and lesion volumes determined.
Experiment 3: Effects of Mitochondrial Toxins on Dopamine Uptake
The method of Masserano et al.4 was used. Briefly, striatal synaptosomes (20 µg
protein/500 µl sample) were incubated for 10 min with 5 × 105 dpm/ml [3H]-dopam-
ine at 37°C in the presence of varying concentrations of the succinate dehydrogenase
(complex II) inhibitors 3NP and malonate as well as the complex IV inhibitor azide.
Nonspecific binding was determined in the presence of 10 µM nomifensine. Exper-
iments with each compound were replicated in triplicate.
In the control group, the seven animals that survived both sham mfb and striatal
malonate injections had striatal lesions that were histologically similar to those pre-
viously described.2,3 In this group of animals the average lesion volume was 2.3 ±
0.43 mm3. In the group of animals receiving 6OHDA injections into the mfb all but
one animal had apomorphine-induced rotation. The average malonate lesion volume
in these animals was 0.8 ± 0.28 mm3 (p < 0.01; TABLE 1).
In the animals treated with 3NP, tyrosine hydroxylase staining of the substantia
nigra confirmed the efficacy of the prior 6OHDA lesion of dopaminergic neurons
(not shown). In these four animals, the lesion volume induced by 3NP on the non-
6OHDA–lesioned side was 15.5 ± 1.5 mm3 and on the injected side 6.9 ± 1.7mm3
(p < 0.01; TABLE 1).
347 MARAGOS et al.: DOPAMINE AND STRIATAL DAMAGE
In the vehicle-treated animals, the severity of malonate-induced tissue destruc-
tion was homogenous, and the average lesion volume was 26.4 ± 2.2 mm3. In the
group of animals treated with MAO inhibitors, the lesions appeared qualitatively
similar to the control animals but were significantly smaller (15.3 ± 1.5 mm3,
p < 0.001; TABLE 1).
In the control group treated with 3NP, the average lesion volume was 15.5 ± 4.0
mm3, while the average lesion volume of the clorgyline/deprenyl-treated group was
3.8 ± 1.6 mm3 (p < 0.001; TABLE 1).
The addition of mitochondrial toxins to synaptosomes inhibited [3H]dopamine
uptake in a dose-dependent manner (FIG. 1). The rank order of potency was similar
to their toxicity profile in vitro, with azide being the most potent uptake inhibitor and
malonate the least.
In the first experiment, we demonstrated that the removal of striatal dopamine
was neuroprotective against lesions generated by either systemic or direct intrastri-
atal administration of the mitochondrial toxins 3NP and malonate, respectively. Our
data support the observations that dopamine enhanced methyl-malonate toxicity in
striatal neurons in culture7 and support the findings of Reynolds et al., who recently
reported neuroprotection against 3NP toxicity following dopamine depletion.8 As
neuronal death caused by mitochondrial poisons is thought to involve the activation
of EAA receptors,2,3 our findings are consistent with other laboratories that have
shown dopamine depletion to confer neuroprotection against striatal damage in-
duced by the EAA agonists N-methyl-D-aspartate (NMDA)9 and quinolinic acid10
and the non-NMDA agonist kainic acid.9
TABLE 1. Effects of various treatments on malonate- and 3NP-induced striatal lesion
Lesion Volume (mm3)
% Protection ControlExperimental
2.3 ± 0.4
15.5 ± 1.5
0.8 ± 0.3a
6.9 ± 1.7a
26.4 + 2.2
15.5 + 4.0
15.3 + 1.5b
3.8 + 1.6b
ap < 0.01; taken from Ref. 5.bp < 0.001; taken from Ref. 6.
348ANNALS NEW YORK ACADEMY OF SCIENCES
Although the mechanism of dopamine toxicity has not been completely deter-
mined, the production of oxygen-based free radicals have been implicated.11,12 In
the second experiment, inhibition of MAO using the combination of clorgyline and
deprenyl also resulted in significant protection against malonate and 3NP-induced
striatal lesions. MAO is an important enzyme that metabolizes dopamine to dihy-
droxyphenylacetic acid and hydrogen peroxide (H2O2).13 Since H2O2 can be con-
verted to the highly toxic hydoxyl radical, it is possible that the protection we
observed following MAO blockade was due to decreased H2O2 (and ultimately hy-
droxyl radical) production. Treatment of animals with the dopamine precursor L-
DOPA have been shown to result in the increased production of hydroxyl radicals.14
We have shown that mitochondrial toxins inhibit the uptake of [3H]dopamine
(FIG. 1). Although we are uncertain whether this phenomenon is specific to the
dopamine transporter, blockade of dopamine uptake could result in enhanced
dopamine metabolism by MAO (hence, the production of H2O2) and is consistent
with the observation that systemic administration of 3NP increased dopamine turn-
over.2 We concluded from the findings presented herein that dopamine and/or one of
its metabolites contribute(s) to striatal damage caused by impaired energy metabo-
lism and may thus be involved in the striatal damage seen in HD.
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2. BEAL, M.F. et al. 1993. Neurochemical and histological characterization of striatal
excitotoxic lesions produced by the mitochondrial toxin 3-nitropropionic acid. J.
Neurosci. 13: 4181–4192.
FIGURE 1. Dose-dependent inhibition of [3H]dopamine uptake by mitochondrial tox-
ins. The rank order of potency is azide > 3NP > malonate, and the respective IC50s are
7.2 × 10−4, 1.6 × 10−3, and 5.1 × 10−2 M.
349 MARAGOS et al.: DOPAMINE AND STRIATAL DAMAGE
3. GREENE, J.G. et al. 1993. Inhibition of succinate dehydrogenase by malonic acid pro-
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