Uncoupling protein-2 promotes nigrostriatal dopamine
neuronal function
Zane B. Andrews,1Alicia Rivera,2John D. Elsworth,3Robert H. Roth,3Luigi Agnati,4 Belen Gago,2Alfonso Abizaid,1 Michael Schwartz,5Kjell Fuxe6andTamas L. Horvath1,5,7
1
Department of Obstetrics, Gynecology & Reproductive Services, Yale University School of Medicine, New Haven, USA 2
Department of Cell Biology, School of Science, University of Ma´laga, Campus Teatinos, Ma´laga, Spain 3Department of Psychiatry, Yale University School of Medicine, New Haven, USA
4
Department of Biomedical Science, Section of Physiology, University of Modena, Modena, Italy 5Department of Neurobiology, Yale University School of Medicine, New Haven, USA
6
Department of Neuroscience, Division of Cellular and Molecular Neurochemistry, Karolinska Institute, Stockholm, Sweden 7
Section of Comparative Medicine, Yale University School of Medicine, 375 Congress Ave, LSOG 117, New Haven, CT 06510, USA
Keywords: locomotor behaviour, mitochondria, mouse, reactive oxygen species, striatum, tyrosine hydroxylase
Abstract
Uncoupling protein 2 (UCP2) is known to promote neuroprotection in many forms of neurological pathologies including Parkinson’s disease. Here, we examined the hypothesis that UCP2 also mediates aspects of normal nigrostriatal dopamine (DA) function. Mice lacking UCP2 exhibited reduced dopamine turnover in the striatum as measured by the 3,4-dihydoxyphenylacetic acid⁄dopamine (DOPAC⁄DA) ratio, reduced tyrosine hydroxylase immunoreactivity (TH IR) in the substantia nigra pars compacta (SNc) and reticulata, striatum and nucleus accumbens. UCP2-knockout (KO) mice also had reduced dopamine transporter immunoreactivity (DAT IR) in the SNc but not other brain regions examined. In order to determine if these biochemical deficits are transcribed into behavioural deficits, we examined locomotor function in UCP2-KO mice compared to wild-type (WT) controls. UCP2-KO mice exhibited significantly reduced total movement distance, movement velocity and increased rest time compared to wild-type controls. These results suggest that UCP2 is an important mitochondrial protein that helps to maintain normal nigrostriatal dopamine neuronal function and a reduction in UCP2 levels may predispose individuals to environmental causes of Parkinson’s disease.
Introduction
Neuronal mitochondria are essential organelles as they provide neurons with the energy required to mediate diverse functions such as synaptic plasticity (Liet al., 2004), neuronal survival (Nicholls & Budd, 2000), neurogenesis and proliferation (Limoli et al., 2004), calcium regulation (Leo et al., 2005), synaptic neurotransmission (Hollenbeck, 2005) and volume transmission (Richter, 1988; Fuxe
et al., 2005; Rivera et al., 2006). Thus, it is not surprising that mitochondrial dysfunction lies at the heart of many neurological pathologies and the ageing process (Beal, 2005). In order to serve their essential role in energy generation, mitochondria rely on a host of both nuclear and mitochondrial genes. The recently identified mitochond-rial protein, uncoupling protein 2 (UCP2), encoded from nuclear DNA, has received a great deal of research attention in the CNS due to its potentially important role in promoting neuronal function and prevent neurological disease (Diano et al., 2003; Mattiasson et al., 2003; Sullivanet al., 2003; Andrews et al., 2005a; Andrewset al., 2005b; Contiet al., 2005). Activation of UCP2 allows protons to enter the mitochondrial matrix without entering the ATP synthase and as such uncouples mitochondrial respiration from ATP production. This uncoupling activity reduces mitochondrial reactive oxygen species
(ROS) production and promotes ATP production through increased mitochondrial biogenesis (Dianoet al., 2003; Andrewset al., 2005a). Through these mechanisms, UCP2 may contribute to important neuronal functions such as synaptic plasticity and neurotransmission. Indeed, perturbed striatal dopamine (DA) neurotransmission and nigral DA loss cell is a hallmark of Parkinson’s disease (Dauer & Przedborski, 2003). In particular, UCP2 prevents DA cell loss after 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) treatment indi-cating UCP2 helps to restrict the pathogenesis of Parkinson’s disease (Andrewset al., 2005b; Contiet al., 2005). Although striatal DA levels are not decreased in unchallenged UCP2 knockout (KO) mice, these mice are more susceptible to striatal DA loss after MPTP injection (Andrews et al., 2005b). This suggests that UCP2 is important to maintain normal striatal DA neurotransmission. Further, UCP2-KO mice possess fewer mitochondria in nigral DA neurons compared to wild-type (WT) controls (Andrews et al., 2005b), highlighting a potential for nigrostriatal DA dysfunction. Our current understanding suggests that the pathogenesis of Parkinson’s disease is 90% sporadic and 10% familial (Dauer & Przedborski, 2003). Thus, genetic⁄allelic variation of UCP2 in humans may underlie an inherent predisposition to nigrostriatal DAergic dysfunction and lower the threshold of cell loss after exposure to deleterious environmental conditions. The purpose of these studies was to establish aspects of normal nigrostriatal DA function in UCP2-KO mice compared to wild-type controls, in order to determine if UCP2 affects this predisposition.
Correspondence: Dr Tamas L. Horvath,7
Section of Comparative Medicine, as above. E-mail: [email protected]
Materials and methods
All male mice used in the following experiments were aged between 12 and 15 weeks at the time of killing. All procedures were approved by the Institutional Animal Care and Use Committee of Yale University. All mice were maintained under standard laboratory conditions with water and food freely available; lights were maintained on a 12-h light : 12-h dark cycle. UCP2 KO and human UCP2 expressing transgenic lines were generated as described previously (Andrewset al., 2005b).
Striatal dopamine turnover
At the time of killing each dorsal striatal sample was dissected, transferred to 400lL ice-cold perchloric acid, sonicated and centri-fuged for 10 min at 10 000g The pellet was saved for protein determination according to the spectrophotometric method of Lowry. 3,4-dihydoxyphenylacetic acid (DOPAC) and DA were assayed as previously reported (Andrewset al., 2005b).
Immunohistochemistry
Animals were killed under ether anaesthesia and perfused transcar-dially with 0.1mphosphate-buffered saline, pH 7.4 (PBS) followed by 4% paraformaldehyde (w⁄v) in 0.1m phosphate buffer, pH 7.4 (PB). Tyrosine hydroxylase (TH) was detected using a mouse monoclonal antibody (DiaSorin, Stillwater, MN, USA) diluted at 1 : 10 000 in PBS containing 0.2% Triton X-100 (PBS-TX) and 0.1% sodium azide. Dopamine transporter (DAT) was detected using a rabbit polyclonal antibody (Chemicom International, Temecula, CA, USA) diluted at 1 : 5000. Details of the immunohistochemical processing procedure are published elsewhere (Rivera et al., 2006). Semiquantitative analysis of optical density (OD) of TH immunore-activity (IR) and DAT IR was performed using the image analysing system NIH image (http://rsb.info.nih.gov/nih-image/). The intensity of TH and DAT IR was expressed as the OD and the value was corrected with the OD from an immunonegative area (Agnatiet al., 1984). Two to six sample areas (200lm2) from six sections of each mouse (WTn¼6; KO,n¼6) were measured.
Behavioural studies
Spontaneous locomotor activity was examined in the same UCP2-KO (n¼5) and WT (n¼5) mice at 2 and 5 months of age. Activity measures were assessed in a 16 inch, square plexiglass arena with photobeams and sensors spaced at intervals sufficient to provide a spatial resolution of animal movement of 1.27 cm in the X–Y dimension (Coulbourn Instruments TruScan, Bilaney Consultants Ltd, Sevenoaks, UK). The arena sensors were computer coupled and sampled every 100 ms using Coulbourn Instruments Truscan 99 software. Activity measures were recorded over a 15-min period and statistical comparisons between KO and WT groups on measures of total distance traveled, average movement velocity and the amount of rest time (time spent not moving) spent during a 15-min session were made using Student’st-tests.
Potential alterations in gross motor coordination and motor learning were assessed using an accelerating Rota Rod. Five-month-old UCP2-KO and WT mice were placed on a rotating cylinder (AccuScan Instruments, Columbus, OH, USA) and the amount of time they were able to walk on the cylinder without falling was recorded for each of six sessions spaced 5 min apart on two consecutive days. The rotation
speed of the cylinder accelerated from 0 to 40 r.p.m. over a 200-s period and the speed remained constant upon reaching 200 r.p.m.
Statistical analysis
All data are present as mean ± SEM. Statistical comparisons were made using Student’st-test or one-wayanovafollowed by Newman– Kuelsposthoctest.P< 0.05 was considered significant.
Results
UCP2-KO mice exhibit reduced striatal DA turnover
The DOPAC⁄DA ratio was used to estimate DA turnover as it measures the coupled neurochemical relationship between the syn-thesis of DA and the levels of its primary metabolite, DOPAC, which is considered to be proportional to the amount of released DA in terminal axon fields. We found that UCP2-KO mice had significantly reduced DA turnover in comparison to WT controls (Fig. 1, DOPAC⁄DA ratio 0.065 ± 0.001 vs. 0.099 ± 0.008, respectively,
P< 0.002, Student’st-test). No differences in striatal DA turnover in UCP2 overexpressing transgenic mice were found (data not shown).
UCP2-KO mice exhibit a diminution of TH IR
We analysed TH and DAT IR in the main nigrostriatal and mesolimbic DAergic projection fields in both UCP2-KO and UCP2 overexpress-ing transgenic mice. TH and DAT IR were also measured in the DAergic nerve cell groups of substantia nigra pars compacta (SNc) and reticulata (SNr). We observed a significant decrease of TH IR in the caudate putamen by 13%, nucleus accumbens by 33%, SNc by 31% and SNr by 39% of UCP2-KO mice as compared with the WT (Fig. 2A–E). No differences were observed in the same areas in the UCP2 overexpressing transgenic mice (data no shown). DAT IR was significantly reduced by 27% in the SNc but no other statistically significant differences were observed in the caudate putamen, nucleus accumbens or SNr (Fig. 1F).
UCP2-KO mice display locomotor impairments
In order to determine if the observed decline in striatal DA turnover and TH IR results in functional deficits, we examined behavioural
Fig. 1. UCP2-KO (n¼6) mice have reduced striatal dopamine turnover
relative to WT controls (n¼6). Dopamine turnover was estimated as the ratio between DOPAC⁄dopamine. *Significant with respect to WT controls, P< 0.002 Student’st-test.
parameters in UCP2-KO compared to WT mice. Comparison of open field activity between UCP2-KO and WT littermates revealed significant differences on all three measures sampled. At both 2 and 5 months of age the UCP2-KO mice had significantly reduced total movement, reduced movement velocity and spent significantly more time resting (Table 1).
In contrast to the significant differences described aboved, UCP2-KO mice showed no significant gross motor abnormalities when compared to WT littermates on the accelerating Rota Rod task at 5 months of age. Both groups also showed session to session increases in the time spent on the cylinder indicating no impairment of simple motor learning for this task.
Discussion
In this study, we examined biochemical and behavioural indices of nigrostriatal DAergic function in untreated UCP2-KO mice
com-pared to WT controls. The purpose was to establish whether UCP2 plays an important role in maintaining normal DAergic cell function. We discovered that UCP2-KO mice have reduced dorsal striatal DA turnover, reduced striatal and nigral TH immunoreac-tivity, and reduced SNc DAT immunoreactivity. These deficits culminate in reduced indices of locomotor activity and thereby further implicate UCP2 in preventing the pathogenesis of Parkin-son’s disease.
The loss of striatal DA as seen after nigrostriatal DA neurodegen-eration leads to rigidity, tremor at rest, slowness or absence of voluntary movement, postural instability and freezing (Dauer & Przedborski, 2003), broadly similar with the observed locomotor deficits such as decreased movement velocity, total movement distance and increased rest time in this study. In Parkinson’s disease, physical motor symptoms supposedly only manifest once striatal DA concen-tration and nigral DA cell number reaches 20% and 40%, respectively, of original levels. Previously we reported UCP2-KO mice have similar striatal DA and nigral cell number to WT mice in untreated conditions (Andrewset al., 2005b), leading to the question; why do UCP2-KO exhibit locomotor deficits? The answer probably relates to a functional rather than an anatomical loss of DA transmission. Rotenone, a mitochondrial complex I inhibitor that recapitulates features of Parkinson’s disease (Betarbetet al., 2000), causes parkinsonian motor deficits but produces only minimal damage to the nigrostriatal DA system (Hoglingeret al., 2003; Fleminget al., 2004). In an attempt to reconcile these discrepancies, Bao et al. (2005) showed that partial mitochondrial inhibition by rotenone caused a functional suppression of DA release via H2O2and subsequent activation of ATP-sensitive
potassium channels (KATPchannels; Avshalumovet al., 2005) leading
to hyperpolarization without DA or ATP depletion. While we previously observed no difference in striatal DA in UCP2-KO
Table1. Activity measures for WT and UCP2-KO mice
Age and group
Total distance (inches)
Movement velocity
(cm⁄15 min) Rest time (s)
2 months
WT 328.7 ± 26.6 331.1 ± 26.5 383.0 ± 15.6 KO 178.0 ± 20.9** 180.7 ± 20.7** 550.4 ± 37.7**
5 months
WT 382.4 ± 62.1 384.3 ± 62.1 380.7 ± 63.3 KO 197.0 ± 29.9** 145.6 ± 56.5** 603.8 ± 60.3**
Data are presented as means ± SEM. **P <0.01 when compared to WT at the same age.
Fig. 2. (A–D) Darkfield photomicrographs showing TH IR in WT and UCP2-KO mice at bregma levels +1.20 mm (A and B) and)5.30 mm (C and D). KO mice
exhibit lower levels of TH IR than wild-type mice. Scale bar, 500lm (A and B; caudate putamen) and 150 mm (C and D; SNc and SNr). Graphical representation of mean ± SEM percentage of optical density of TH IR (E) and DAT (F) in caudate putamen (CPu), nucleus accumbens (Acb), substantia nigra pars compacta (SNc) and substantia nigra pars reticula (SNr) of wild-type mice (white bars;n¼6) and knockout mice (black bars;n¼6).
compared to WT mice (Andrews et al., 2005b), in this study we witnessed a decrease in DA turnover as estimated by the DOPAC⁄DA ratio. Thus, the absence of UCP2 affects DA release, but not total DA content in the striatum, which in turn may underlie the observed locomotor deficits. Interestingly, H2O2is responsible for
mitochond-rial inhibition that leads to suppression of DA release but not depletion after rotenone exposure (Baoet al., 2005) and the primary function of UCP2 is to limit ROS, including H2O2. Moreover, the ability of UCP2
to buffer ROS in nigral TH cell in vivo provides neuroprotection against MPTP (Andrews et al., 2005b). The same mechanisms are proposed to occur in the remaining DAergic terminals expressing UCP2 after a partial 6-OHDA lesion of DAergic nigrostriatal and mesostriatal pathways (Riveraet al., 2006). UCP2 cannot limit ROS production in KO mice, and as such ROS negatively affects DA release as measured by the decrease in dorsal striatal DA turnover. This collectively culminates in motor deficits similar to those witnessed in Parkinson’s disease.
We observed decreased TH IR in SNc and striatum suggesting UCP2 is important in TH production. This is most likely because UCP2 mediates mitochondrial biogenesis and consequently increases ATP in neuronal tissue (Diano et al., 2003; Andrewset al., 2005a). Further, a reduction in mitochondria and overall cell energy produc-tion due to diminished ATP levels in UCP2-KO mice compromises the ability to release DA at the nerve terminal via activation of KATP
channels. Moreover, the activation of KATPchannels reduces the firing
rate of DA cells in the SNc, further contributing to the reduction of striatal DA release (Liss & Roeper, 2001) and to an unexpected promotion of degeneration of the nigrostriatal DA system in chronic disease (Lisset al., 2005). Indeed, mitochondrial number is decreased in TH cells of the SNc (Andrews et al., 2005b) and VTA (Z. B. Andrews and T. L. Horvath unpublished observations), sup-porting the idea that reduced ATP levels in UCP2-KO mice compromised the cell’s ability to produce TH protein. TH is the rate limiting step in DA synthesis and despite reduced TH IR, striatal DA is not different between UCP2-KO and WT mice (Andrews et al., 2005b). We suggest that although TH IR is reduced, there are sufficient levels to maintain normal striatal DA concentrations. However, after toxic insults mitochondrial dysfunction in the UCP2-KO mice render these neurons more vulnerable to cell death and the reduced TH IR contributes to the further reduction of DA transmission and degeneration in the nigrostriatal dopaminergic system. Indeed, MPTP administration produces greater nigral TH cell loss and striatal dopamine depletion in UCP2-KO mice compared to WT controls (Andrewset al., 2005b).
We also observed a significant decrease in DAT IR in only the SNc, while the reason for this is unknown we suggest a decrease in TH and DA at the cell body results in a down-regulation in DAT, as MPTP is known to reduce DA and TH and a concurrent decrease in DAT binding (D’Astouset al., 2004; Jourdainet al., 2005). Moreover, the selective loss of DAT in the SNc may be the result of greater ROS production and decreased ATP in the cell body due a larger population of mitochondria in the cell body.
In conclusion, UCP2-KO mice exhibited deficits in indices of nigrostriatal DA function including reduced striatal DA turnover, reduced striatal and substantia nigral TH IR and reduced nigral DAT IR. Together, these deficits led to locomotor impairments similar to those in Parkinson’s disease. The present results support the growing body of literature that suggests neuronal UCPs are neuroprotective (Andrews et al., 2005a). More specifically, UCP2 is an important mitochondrial protein that helps to maintain normal nigrostriatal DA neuronal function and a reduction in UCP2 levels may predispose individuals to environmental causes of Parkinson’s disease.
Acknowledgements
This work was supported by NIH grant R01 NS-41725, AG-22880 (TLH), MH14092 and MH57483 (R.H.R. & J.D.E.); and a Swedish Research Council grant K2006-04X-00715-42-2 (K.F.), and grants from the Spanish Research Council (BFI 2002-00587, BFU 2005-06615) to A.R. UCP2 knockout animals were generously provided by Dr. Bradford Lowell.
Abbreviations
DA, dopamine; DAT, dopamine transporter; DOPAC, IR, immunoreactivity; KO, knock out; 3,4-dihydoxyphenylacetic, acid; MPTP, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine; OD, optical density; ROS, reactive oxygen species; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; TH, tyrosine hydroxylase; UCP2, uncoupling protein 2; WT, wild-type.
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