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Keywords:

  • apoptosis;
  • dopamine;
  • folic acid;
  • 1-methyl- 4-phenyl-1;
  • 2;
  • 3;
  • 6-tetra-hydropyridine;
  • rotenone;
  • substantia nigra.

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Although the cause of Parkinson's disease (PD) is unknown, data suggest roles for environmental factors that may sensitize dopaminergic neurons to age-related dysfunction and death. Based upon epidemiological data suggesting roles for dietary factors in PD and other age-related neurodegenerative disorders, we tested the hypothesis that dietary folate can modify vulnerability of dopaminergic neurons to dysfunction and death in a mouse model of PD. We report that dietary folate deficiency sensitizes mice to MPTP-induced PD-like pathology and motor dysfunction. Mice on a folate-deficient diet exhibit elevated levels of plasma homocysteine. When infused directly into either the substantia nigra or striatum, homocysteine exacerbates MPTP-induced dopamine depletion, neuronal degeneration and motor dysfunction. Homocysteine exacerbates oxidative stress, mitochondrial dysfunction and apoptosis in human dopaminergic cells exposed to the pesticide rotenone or the pro-oxidant Fe2+. The adverse effects of homocysteine on dopaminergic cells is ameliorated by administration of the antioxidant uric acid and by an inhibitor of poly (ADP-ribose) polymerase. The ability of folate deficiency and elevated homocysteine levels to sensitize dopaminergic neurons to environmental toxins suggests a mechanism whereby dietary folate may influence risk for PD.

Abbreviations
used

5-HIAA, 5-hydroxyindoleacetic acid

5-HT

serotonin

DA

dopamine

DMEM

Dulbecco's minimum essential medium

DOPAC

3,4-dihydroxyphenylacetic acid

HVA

homovanillic acid

MPTP

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

PD

Parkinson's disease

SDS–PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SN

substantia nigra

TH

tyrosine hydroxylase.

The risk of coronary artery disease (Refsum et al. 1998) and stroke (Elkind and Sacco 1998) is increased in individuals with a low dietary folate intake and an elevated plasma homocysteine concentration. Homocysteine is a metabolite of methionine, an amino acid critical for the generation of methyl groups required for synthesis of DNA. Normally homocysteine levels are maintained low by remethylation to methionine by enzymes that require folate or cobalamin (vitamin B12), and by catabolism to cysteine by the pyridoxine (vitamin B6)-dependent enzyme cystathionine β-synthase (Finkelstein 1990; Scott and Weir 1998). Variability in levels of homocysteine among individuals can result from genetic or environmental factors, with dietary folate levels having a major impact such that there is generally an inverse relationship between plasma folate and homocysteine levels (Giles et al. 1995). While it has been know for decades that folate deficiency can have major adverse effects on the developing human nervous system (Greenblatt et al. 1994), it is not known whether homocysteine plays a role in such developmental defects. However, patients with genetic defects that result in hyperhomocysteinemia do exhibit neurological abnormalities such as mental retardation, cerebral atrophy and seizures (Watkins and Rosenblatt 1989; van den Berg et al. 1995), and exposure of cultured cortical and hippocampal neurons to homocysteine increases their vulnerability to excitotoxicity (Lipton et al. 1997; Kruman et al. 2000).

Parkinson's disease (PD) is characterized by dysfunction and degeneration of dopaminergic neurons in the substantia nigra (SN) resulting in progressive akinesia, tremor and rigidity (Marsden 1994). At the cellular level, the pathogenic process in PD likely involves increased levels of oxidative stress, mitochondrial dysfunction and a biochemical cell death cascade called apoptosis (Jenner and Olanow 1998; Tatton 2000). Data suggest important roles for both genetic and environmental factors in the pathogenesis of PD (Langston 1998; de Silva et al. 2000). Although specific gene defects have been linked to a very small percentage of cases of PD, increasing evidence points to environmental factors such as exposure to toxins (Betarbet et al. 2000), head trauma (Taylor et al. 1999), high calorie intake (Logroscino et al. 1996; Duan and Mattson 1999) and low antioxidant intake (de Rijk et al. 1997) as being important risk factors for the common sporadic forms of PD. Recent findings suggest that homocysteine levels are increased in PD patients (Allain et al. 1995; Kuhn et al. 1998; Yasui et al. 2000), but it is not known whether this alteration precedes disease onset. In addition, the clinical data are mainly from studies of patients treated with levodopa, a drug that may itself affect homocysteine levels (Miller et al. 1997; Muller et al. 2001). It therefore remains unclear whether folate deficiency and/or elevated homocysteine levels play a critical role in the pathogenesis of PD. Systemic administration of␣the␣toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces PD-like pathology and behavioral symptoms in mice and monkeys, and is widely used as a model of sporadic PD (Schneider et al. 1987; Moratalla et al. 1992; Bezard et al. 1997). In the present study we employed this animal model and a human dopaminergic cell culture model to test the hypothesis that, by increasing homocysteine levels, folate deficiency endangers dopaminergic neurons thereby increasing the risk of PD.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Animals and experimental treatments

Two-month-old male C57Bl/6 mice weighing 21–23 g (obtained from the National Cancer Institute) were maintained under temperature- and light-controlled conditions (20–23°C, 12-h light/12-h dark cycle). The control diet was a standard mouse diet (Dyets, Inc.; diet #518754) which contained 2 mg folate/kg of food. The experimental diet lacked folate, but was otherwise identical to the control diet. All mice were given water and diet ad libitum. The protocol for MPTP administration was similar to that described previously (Duan et al. 1999). Two dosing regimens were employed. In the first study, mice that had been maintained on the control or folate-deficient diet for 3 months (5-months-old at the time of MPTP administration) were given a subtoxic dose of MPTP that resulted in no detectable pathology or symptomology (two i.p. injections of 20 mg MPTP/kg body weight, separated by 4 h). In the second study 2-month-old mice were given 4 i.p. injections of 20 mg/kg MPTP with a 2-h interval between injections, a regimen that caused considerable pathology and motor dysfunction. Homocysteine was administered by stereotaxic infusion into either the striatum or substantia nigra (4.3 ng in 1.0 µL) using methods similar to those described previously (Gibb et al. 1988). Twenty-four hours later mice were killed and brains were rapidly removed; the striata were removed and stored at −80°C until used for measurements of monoamines and metabolites. Additional mice were perfused transcardially with saline followed by cold 4% paraformaldehyde in PBS. Coronal brain sections were cut on a freezing microtome and immunostained with TH antibody as described previously (Duan et al. 1999).

Behavioral testing

Behavioral assessments on each mouse were made 1 day prior to, and 7 days after, MPTP administration. Motor performance was assessed with a rotary rod apparatus using a protocol similar to that described previously (Duan and Mattson 1999; Hengemihle et al. 1999). For the rotarod tests the rotadrum was filled with water to a level just below the bottom of the rod. The mice were placed on the rotating rod and the time until they fell off was recorded. This was repeated (with a rest period that increased by 5 s with each fall) until the total time on the rod for the control group was 5 min. Both the total time spent on the rotating rod and the total number of falls for each mouse were recorded.

Immunoblot and immunohistochemical methods

These methods were similar to those described previously (Duan et al. 1999). Briefly, for immunoblot analysis, 50 µg solubilized proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE; 10% gel) and transferred to a nitrocellulose membrane. The membrane was incubated overnight at 4°C in the presence of 5% non fat milk, and then incubated for 2 h with tyrosine hydroxylase (TH) antibody (1 : 2000; Chemicon, Temecula, CA, USA). The membrane was then exposed for 1 h to HRP-conjugated secondary antibody (1 : 3000; Jackson Immuno-Research, West Grove, PA, USA) and immunoreactive protein was visulized using a chemiluminescence-based detection kit according to the manufacturer's protocol (ECL kit; Amersham Pharmacia Biotech, Piscataway, NJ, USA). Equal loading of lanes was confirmed by probing of blots with an antibody against beta-actin. For immunostaining, mice were perfused through the ascending aorta with physiological saline followed by 4% paraformadehyde in PBS (pH 7.4). Fixed brains were cryoprotected in a 30% sucrose solution, coronal brain sections (30 µm) were cut on a freezing microtome, and sections were immunostained with TH antibody (1 : 400 dilution; Chemicon), which was detected using a biotinylated secondary antibody, peroxidase-labeled avidin, and diaminobenzadine. The numbers of TH-immunoreactive neurons in the SN of the mice were quantified as described previously (Duan et al. 1999). Briefly, numbers of TH-positive cells in the zona-compacta were counted in both the left and right SN; four sections were counted per mouse by an investigator (WD) blinded as to the experimental treatment history of the mice.

Quantification of levels of monoamines and their metabolites and homocysteine

Levels of dopamine, DOPAC (dihydroxyphenylacetic acid), HVA (homovanillic acid), 5-HT (5-hydroxytryptamine) and 5-HIAA (5-hydroxyindoleacetic acid) were measured by HPLC-based analyses using methods described previously (Andrews and Murphy 1993; Tella et al. 1996). Briefly, mice were killed, the brain removed and placed on ice. Striata were removed and weighed, then flash frozen and stored at −80°C until extraction. Striatal tissue was sonicated in a solution of 10% perchloric acid containing 10 ng/mg tissue of the internal standard dihydroxybenzilamine and centrifuged at 20 000 g for 5 min. Concentrations of dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in striatal tissue extracts were measured by HPLC with electrochemical detection. The analytical column was Bondapak C-18 (5 µm, 4.6 × 250 mm; Waters Corp., Milford, MA, USA); the mobile phase consisted of 0.08 m sodium acetate, 1 mm sodium EDTA, 5 mm heptane sulfonic acid, 4.0% acetonitrile, pH 4.0 at flow rate of 1.5 mL/min and a temperature of 25°C. The system consisted of Waters 717 Plus automated injection system, Waters piston pump, Waters manometric module and Bioanalytical System LC-4C amperometric detector. The glassy carbon electrode was used at a potential of 0.75 V. Peak areas and sample concentrations were calculated with a Hewlett Packard integrator. Contents of DA, DOPAC, HVA, 5-HT, 5-HIAA were calculated as pg/mg of tissue weight.

Homocysteine levels in serum samples were quantified using an $ {\rm IM}^\reg _{\rm X} ^{}$ immunoassay analyzer (Abbott Laboratories) according to the protocol provided by the manufacturer. Trunk blood was collected from mice at the time of euthanasia, serum was isolated from whole blood, and a 50-µL aliquot was used for analysis.

Cell cultures, experimental treatments, and quantification of cell death

Previous studies have characterized human neuroblastoma SK-N-MC cells (Seaton et al. 1998; Duan and Mattson 1999). Cells were maintained in T-flasks containing DMEM (Dulbecco's minimum essential medium) supplemented 10% with heat-inactivated fetal bovine serum, 26 mm sodium bicarbonate (pH 7.2). For experiments, cells were plated in 35-mm diameter plastic or glass-bottom dishes on a gelatin substrate in 0.8 mL of culture maintenance medium. Immediately prior to experimental treatment the culture medium was replaced with serum-free DMEM. Homocysteine, 3-aminobenzamide, FeSO4 and uric acid were prepared as 200 × stocks in sterile water. Rotenone was prepared as a 500 × stock in dimethylsulfoxide. To quantify apoptosis, cells were fixed in 4% paraformaldehyde and stained with the fluorescent DNA-binding dye Hoechst 33342 as described previously (Kruman et al. 1997). Hoechst-stained cells were visualized and photographed under epifluorescence illumination (340 nm excitation and 510 nm barrier filter) using a 40 × oil immersion objective (200 cells/culture were counted, and counts were made in at least six separate cultures/treatment condition). Analyses were performed without knowledge of the treatment history of the cultures. The percentage of ‘apoptotic’ cells (cells with condensed and fragmented nuclear chromatin were considered apoptotic) in each culture was determined.

Measurements of mitochondrial transmembrane potential and oxyradical levels

The dye rhodamine 123 (Molecular Probes, Eugene, OR, USA) was employed as a measure of mitochondrial membrane potential using methods described previously (Guo et al. 1999). Briefly, cells were incubated for 30 min in the presence of 10 µm of the dye, washed three times in fresh culture medium, and confocal images of cellular rhodamine 123 fluorescence were acquired using a Zeiss 510 CLSM (488 nm excitation and 510 nm emission; Zeiss, Thornwood, NY, USA). The average pixel intensity in individual cell bodies was determined using the software supplied by the manufacturer (Zeiss); all images were coded and analysed without knowledge of experimental treatment history of the cultures. The dye dihydrorhodamine (DHR) was used to quantify relative levels of mitochondrial reactive oxygen species using methods similar to those described previously (Guo et al. 1999). DHR localizes to mitochondria and fluoresces when oxidized by hydroxyl radical and peroxynitrite to the positively charged rhodamine 123 derivative. Briefly, cells were incubated for 30 min in the presence of 5 µm DHR, washed three times with fresh medium, and confocal images of cellular fluorescence were acquired and analyzed as described for rhodamine 123 fluorescence. All images were coded and analysed without knowledge of experimental treatment history of the cultures.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Dietary folate deficiency induces hyperhomocysteinemia and sensitizes dopaminergic neurons to dysfunction and death in a mouse model of PD

Beginning at 2 months of age, mice were maintained for 3 months on either a folate-deficient diet or a control diet containing folate. Mice on the folate-deficient exhibited an eight-fold increase in the plasma concentration of homocysteine (Fig. 1a), demonstrating that dietary folate is a major regulator of homocysteine metabolism in mice. In order to determine whether folate deficiency would sensitize the brain to environmental toxins that can cause a PD syndrome in humans, the mice on folate-deficient and control diets were administered MPTP at a subtoxic dose (two i.p. injections of 20 mg/kg; 4 h between injections), and one week later their motor function was assessed using a rotarod apparatus. In contrast to mice on the control diet that were resistant to the subtoxic dose of MPTP, mice that had been maintained on the folate-deficient diet exhibited profound motor dysfunction as indicated by a decrease in the time period they could maintain themselves on the rotarod and by an increased numbers of falls (Figs 1b and c). Folate deficiency alone did not impair performance on the rotarod tests. In order to determine the extent of MPTP-induced damage to dopaminergic neurons, we measured levels of tyrosine hydroxylase (TH; an enzyme required for dopamine synthesis) in the striatum and counted TH immunoreactive neurons in the SN of each mouse. Whereas MPTP caused little or no decrease in TH levels in the striatum of mice on the control diet, it caused a marked decrease in TH levels in folate-deficient mice (Fig. 1d). Quantification of TH-positive neurons in the SN revealed no loss of dopaminergic neurons in MPTP-treated mice on the control diet, but a marked 50–60% loss of dopaminergic neurons in folate-deficient mice (Figs 1e and f). Folate deficiency alone did not affect striatal TH levels nor numbers of TH-positive neurons in the SN (Figs 1d and f).

image

Figure 1. Dietary folate deficiency sensitizes mice to dopaminergic dysfunction and degeneration in the MPTP model of PD. Mice were maintained for 3 months on either a folate-deficient diet or a control diet containing 2 mg/kg folate. (a) Blood samples were drawn and levels of homocysteine in plasma were quantified. Values are the mean and SE of determinations made in 20 mice/group (*p < 0.001; paired t-test). (b) and (c) Mice were given saline (Control) or MPTP and 7 days later were tested on a rotarod apparatus. Summary data for measurements of running times (b) and numbers of falls per 5 min (c) are shown. Values are the mean and SE of determinations made in 10 mice/group (*p < 0.01 compared with each of the other values; anova with Scheffe post hoc tests). (d) Mice were fed control or folate-deficient diets for 3 months and were given either saline (Control) or MPTP; 7 days later mice were killed and striatal tissue samples were removed and levels of TH were determined by immunoblot analysis (50 µg total protein/lane). (e) Representative micrographs showing TH immunoreactivity in the substantia nigra (SN) of mice subjected to the indicated treatments. (f) Numbers of TH-positive neurons in the SN were quantified. Values are the mean and SE of counts made in 10 mice per group (*p < 0.01 compared with each of the other values (anova with Scheffe post hoc tests).

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Focal administration of homocysteine enhances MPTP-induced motor dysfunction and loss of striatal dopaminergic markers

Because folate deficiency resulted in an increase in homocysteine levels, we determined whether homocysteine might have a direct adverse effect on dopaminergic neurons. For these studies we employed 2-month-old mice and an MPTP dosing regimen that causes considerable loss of dopaminergic neurons and motor dysfunction (four i.p. injections of MPTP at a dose 20 mg/kg, with a 2-h interinjection interval). Two hours after the final MPTP dose, either saline or homocysteine was infused into the striatum or the substantia nigra on the right side of the brain only. One week later the motor performance of the mice was tested. As expected, mice given MPTP exhibited a marked deficit in motor function as indicated by a decrease in the time they were able to maintain themselves on the rotarod and by an increase in the number of falls per test period (Figs 2a and b). Motor performance on the rotarod was not significantly different in saline-treated mice in which homocysteine was infused into either the striatum or substantia nigra compared with control mice. The extent of motor dysfunction caused by MPTP was significantly exacerbated in mice in which homocysteine was infused into either the striatum or the substantia nigra (Figs 2a and b). In light of the exacerbation of motor dysfunction caused by homocysteine, we analyzed striatal tissue from these same mice in order to determine whether homocysteine affected neurochemical markers of dopaminergic function.

image

Figure 2. Homocysteine exacerbates MPTP-induced motor dysfunction when infused directly into the substantia nigra or striatum. Mice were given saline (Control) or MPTP and 2 h later homocysteine was infused into either the striatum (Str) or substantia nigra (SN) on the right side. Seven days later mice were tested on a rotarod apparatus. Summary data for measurements of running times (a) and numbers of falls (b) are shown. Values are the mean and SE of determinations made in 10 mice/group (*p < 0.001 compared with the control value; #p < 0.05 compared with MPTP value; anova with Scheffe post hoc tests).

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Levels of dopamine and two of its metabolites (DOPAC and HVA) were quantified in striatal tissue from mice in each experimental group (Table 1). In control mice the levels of dopamine were between 7000 and 10 000 pg/mg tissue weight. In MPTP-treated mice the levels of dopamine were significantly decreased bilaterally to 2000–3000 pg/mg tissue weight (Table 1). In mice receiving a unilateral intrastriatal infusion of homocysteine only, there was no significant change in dopamine levels in either the right or left striatum, although there was an overall decrease on either side which did not reach statistical significance due to a high interanimal variability. In MPTP-treated mice receiving homocysteine infusions into either the striatum or substantia nigra there was a significant exacerbation of striatal dopamine depletion in the right striatum with levels being near the lower limit of detection in many of the mice. Homocysteine also enhanced dopamine depletion in the left striatum, but to a lesser extent than in the right striatum. Changes in striatal DOPAC and HVA levels among the different experimental groups were, in general, similar to the changes in dopamine levels (Table 1). MPTP-treated mice exhibited a 70–80% decrease in DOPAC levels bilaterally. Homocysteine alone had no significant effect on DOPAC levels, but significantly exacerbated DOPAC depletion in the right striatum when infused into either the striatum or substantia nigra. MPTP caused a 60–70% decrease in HVA levels bilaterally. Homocysteine alone had no significant effect on HVA levels, but significantly exacerbated HVA depletion in the right striatum when infused into either the striatum or substantia nigra. The effects of MPTP and homocysteine were specific for dopaminergic neurons, in that markers of serotonergic neurons (serotonin and its metabolite 5-HIAA) were unaffected by MPTP treatment alone, homocysteine infusion alone, or combined treatment with MPTP and homocysteine (Table 1).

Table 1.  Homocysteine exacerbates MPTP-induced depletion of dopamine and its metabolites
 DopamineDOPACHVA5-HT5-HIAA
LeftRightLeftRightLeftRightLeftRightLeftRight
  1. * p < 0.01 compared with the control value; p < 0.05 compared with MPTP value. anova with Scheffe post hoc tests. Mice were given saline (control) or MPTP and 2 h later homocysteine was infused into either the striatum (Str) or substantial nigra (SN) on the right side. Seven days later mice were killed and striatal tissue samples were removed and levels of dopamine, DOPAC, HVA, 5-HT, 5-HIAA were quantified. Values (pg/mg tissue weight) are the mean ± SE of determinations made in 10 mice/group.

Control8812 ± 11347603 ± 6612 ± 279515 ± 731087 ± 3981001 ± 90617 ± 36491 ± 15347 ± 132257 ± 9
MPTP2664 ± 523*2362 ± 538*130 ± 21*143 ± 28*358 ± 82*364 ± 103*,502 ± 182611 ± 323269 ± 25272 ± 100
Str Homocysteine5890 ± 24184756 ± 1778581 ± 113492 ± 45806 ± 51813 ± 60688 ± 324573 ± 261301 ± 23261 ± 109
Str Hom + MPTP1276 ± 163*183 ± 53*,135 ± 51*13 ± 25*,518 ± 102*203 ± 57*,568 ± 152528 ± 152255 ± 69327 ± 49
SN Homocysteine9074 ± 33978882 ± 3592664 ± 191585 ± 921279 ± 82939 ± 182665 ± 34771 ± 293378 ± 69304 ± 99
SN Hom + MPTP1554 ± 318*383 ± 56*,146 ± 40*26 ± 41*,468 ± 44*174 ± 47*,591 ± 175663 ± 327389 ± 119268 ± 78

We next assessed levels of TH in striatal tissue samples from the different groups of mice. Immunoblot analysis showed that TH levels were significantly decreased in both striata of MPTP-treated mice (Fig. 3) Homocysteine alone had no effect on TH levels when infused into either the striatum or substantia nigra. Homocysteine significantly exacerbated the MPTP-induced decrease in striatal TH levels when infused into the substantia nigra, but did not exacerbate striatal TH loss when infused into the striatum (Fig. 3), suggesting an adverse effect of homocysteine on cell bodies of dopaminergic neurons, but not a direct toxic effect on their axons. Analysis of brain sections immunostained with TH antibody revealed loss of TH-positive neurons in the substantia nigra which was significantly exacerbated by homocysteine administration in the substantia nigra (Fig. 4). Homocysteine alone had no effect on the number of TH-positive neurons present in the substantia nigra. The results of these analyses of TH-immunoreactive cells were supported by examination of Nissl-stained sections (data not shown). Interestingly, there appeared to be a dissociation of the effects of intrastriatal versus intranigral homocysteine administration on loss of dopaminergic markers in the striatum (Table 1) in comparison with loss of TH-positive cell bodies in the substantia nigra (Fig. 4). This suggests that a local increase in homocysteine levels in the striatum may promote dopamine depletion without killing the dopaminergic cells. Collectively, the data show that homocysteine exacerbates depletion of striatal dopamine and its metabolites, loss of TH-positive neurons, and motor dysfunction in a mouse model of PD.

image

Figure 3. Homocysteine exacerbates MPTP-induced loss of striatal tyrosine hydroxylase. Mice were given saline (Control) or MPTP and 2 h later homocysteine was infused into either the striatum (Str) or substantia nigra (SN) on the right side. Seven days later mice were killed and striatal tissue samples were removed and levels of TH were determined by immunoblot analysis. (a) shows a representative immunoblot, and (b) and (c) show results of densitometric analysis of blots of samples from 10 different mice/group (mean and SE; *p < 0.01 compared with the control value; #p < 0.01 compared with MPTP value; anova with Scheffe post hoc tests).

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image

Figure 4. Homocysteine exacerbates MPTP-induced loss of substantia nigra dopaminergic neurons. Mice were given saline (Control) or MPTP and 2 h later homocysteine was infused into either the striatum (Str) or substantia nigra (SN) on the right side. Seven days later mice were killed, brain sections were immunostained with an antibody against TH, and numbers of TH-positive neurons in the substantia nigra were quantified. (a) Representative micrographs showing TH immunoreactivity in the SN of mice subjected to the indicated treatments. (b) Values are the mean and SE of counts made in 10 mice/group (*p < 0.01 compared with the control value; #p < 0.05 compared with MPTP value; anova with Scheffe post hoc tests).

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Homocysteine renders human dopaminergic cells vulnerable to death induced by rotenone and iron: involvement of oxidative stress and mitochondrial dysfunction

Environmental toxins, such as the complex I inhibitor rotenone and the pro-oxidant iron, are implicated in the pathogenesis of some cases of PD (Betarbet et al. 2000; Double et al. 2000). In order to determine whether homocysteine directly endangers dopaminergic neurons, and to elucidate the underlying mechanisms, we performed a series of studies in cultures of human dopaminergic cells. These cells express tyrosine hydroxylase and have previously been shown to be vulnerable to apoptosis when exposed to rotenone and ferrous iron (Duan et al. 1999). Based upon the latter study and additional preliminary studies, we employed concentrations of rotenone (1 µm) and iron (5 µm) that induced apoptosis of 20–30% of the cells within a 24-h exposure period. Cells were exposed to rotenone and iron, alone or in combination with homocysteine, and numbers of cells undergoing apoptosis were quantified 24 h later. Whereas homocysteine alone did not induce apoptosis, it significantly enhanced apoptosis induced by rotenone and iron (Fig. 5a). Previous studies have provided evidence that oxidative stress and DNA damage play important roles in the pathogenesis of PD (Marsden 1994; Tatton 2000). We found that the antioxidant uric acid (Yu et al. 1998) and 3-aminobenzamide, an inhibitor of the DNA damage-response enzyme poly (ADP-ribose) polymerase (PARP) (Kruman et al. 2000), completely prevented death of the dopaminergic cells cotreated with homocysteine plus rotenone or iron (Fig. 5a). Mitochondrial alterations including membrane depolarization and oxyradical production play pivotal roles in neuronal apoptosis, including that which occurs in models of PD (Jenner and Olanow 1998; Duan et al. 1999). Assessment of mitochondrial membrane potential revealed that homocysteine exacerbates membrane depolarization in cells exposed to rotenone or iron (Fig. 5b). Uric acid and 3-aminobenzamide each largely prevented membrane depolarization in cells exposed to homocysteine plus rotenone or iron (Fig. 5b). Levels of mitochondrial reactive oxygen species were increased in cells exposed to rotenone and iron, and these increases were exacerbated by cotreatment with homocysteine (Fig. 5c). Uric acid completely suppressed oxidative stress, whereas 3-aminobenzamide afforded only a partial attenuation of the increases. Collectively, the data show that homocysteine sensitizes human dopaminergic neurons to apoptosis under conditions of mitochondrial impairment and oxidative stress.

image

Figure 5. Homocysteine renders human dopaminergic cells vulnerable to death induced by the pesticide rotenone, and by iron: involvement of oxidative stress and PARP activation. (a) Cultures of human dopaminergic cells were exposed for 24 h to the indicated treatments (50 µm homocysteine, 5 µm Fe2+, 1 µm rotenone, 5 mm 3-aminobenzamide, and 100 µm uric acid) and numbers of cells exhibiting apoptotic nuclei were quantified. Values are the mean and SE of determinations made in at least six separate cultures (*p < 0.01 compared with control value; #p < 0.01 compared with value for cultures exposed to rotenone or Fe2+ alone; anova with Scheffe post hoc tests). (b) and (c) Cultures of human dopaminergic cells were exposed for 6 h to the indicated treatments. Measurements of mitochondrial membrane potential were made using the probe rhodamine 123 (b), and levels of reactive oxygen species were determined using the probe dihydrorhodamine (c) (see Methods). Values are the mean and SE of determinations made in at least six separate cultures (*p < 0.01 compared with control value; #p < 0.01 compared with value for cultures exposed to rotenone or Fe2+ alone, and p < 0.01 compared with value for cultures treated with 3AB or uric acid and then exposed to homocysteine plus Fe2+ or rotenone; anova with Scheffe post hoc tests).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

The ability of a folate deficient diet to elevate plasma homocysteine levels and sensitize dopaminergic neurons to MPTP-induced degeneration in mice provides direct evidence that a single dietary nutrient can modify the pathogenic cascade thought to occur in PD. Because focal infusion of homocysteine into the substantia nigra also increased the vulnerability of the dopaminergic neurons to MPTP-induced degeneration, it seems likely that the endangering effect of folate deficiency is due to direct effects of homocysteine on neurons. Indeed, direct application of homocysteine to cultured human dopaminergic cells increased their vulnerability to apoptosis induced by rotenone and iron. Rotenone induces apoptosis by inhibiting mitochondrial complex I, resulting in energy depletion and increased mitochondrial oxyradical production (Barrientos and Moraes 1999). Iron induces apoptosis by promoting hydroxyl radical production and membrane lipid peroxidation (Kruman et al. 1997). Each of the three dopaminergic toxins employed in the present study have been implicated in PD. MPTP was discovered when it was ingested by a group of drug users in California resulting in PD-like symptomology and neuropathology (Langston 1987). Rotenone is a widely used pesticide that causes PD-like pathology in rats (Betarbet et al. 2000) and is implicated as a contributing factor to the increased risk of PD among farmers (Le Couteur et al. 1999). Levels of iron, a well-known inducer of oxyradical production, are elevated in dopaminergic neurons of PD patients (Double et al. 2000). The ability of homocysteine to increase the vulnerability of human dopaminergic cells to more than one insult believed to be relevant to the pathogenesis of PD increases the likely importance of this mechanism in humans.

Homocysteine exacerbated mitochondrial oxyradical production and membrane depolarization in dopaminergic cells exposed to rotenone or iron, suggesting that homocysteine exerts its adverse effect at a step prior to mitochondrial alterations. Humans with inherited forms of hyperhomocysteinemia exhibit a greatly increased risk of several different kinds of cancer, and experimental data suggest that the carcinogenic effect of homocysteine may result from increased oxyradical-mediated DNA damage leading to an increased mutation frequency (Lucock 2000). Homocysteine can also promote DNA damage in cultured embryonic rat hippocampal neurons, resulting in an apoptotic DNA damage response involving activation of PARP and the tumor suppressor protein p53 (Kruman et al. 2000). DNA damage and activation of similar apoptotic cell death cascades occurs in SN dopaminergic neurons in PD patients and in animal models of PD (Marsden 1994). The ability of the antioxidant uric acid and the PARP inhibitor 3-aminobenzamide to protect human dopaminergic cells against the death-promoting effect of homocysteine indicates pivotal roles for oxidative stress and a DNA damage response in the pathogenic action of folate deficiency/hyperhomocysteinemia. In addition, it is possible that the activation of glutamate receptors by homocysteine (Lipton et al. 1997) contributes to its ability to increase the vulnerability of dopaminergic neurons to MPTP-induced cell death.

Elevated homocysteine levels can result from folate deficiency or from genetic aberrancies in one or more of the enzymes involved in homocysteine metabolism. The normal range of homocysteine concentrations in plasma is 5–15 µm, and levels of homocysteine in cerebrospinal fluid and brain tissue are reported to range from 0.5 to 10 µm (Welch and Loscalzo 1998). In patients with inherited hyperhomocysteinemia, plasma homocysteine levels reach millimolar concentrations and cerebrospinal fluid levels are in elevated into the mid-micromolar range (Surtees et al. 1997). In the present study, the plasma homocysteine concentration of mice maintained on the control diet were approximately 3 µm, while in mice on a folate deficient diet homocysteine concentrations were between 20 and 30 µm. Homocysteine may be rapidly taken up by neurons via a specific membrane transporter (Grieve et al. 1992), and the resulting high levels of intracellular homocysteine may promote DNA strand breaks by disturbing the DNA methylation cycle (Blount et al. 1997). Levels of plasma homocysteine increase with age (Andersson et al. 1992; Brattstrom et al. 1994), possibly as a result of age-related impairment of renal function or a decline in cystathionine β-synthase activity (Meleady and Graham 1998), and this increase could contribute to the age-specific risk of PD. Recent studies have documented elevated levels of homocysteine in PD patients (Allain et al. 1995; Kuhn et al. 1998), but it is not known whether this abnormality precedes and contributes to the neurodegenerative process. However, a genetic predisposition towards hyperhomocysteinemia does appear to be associated with increased risk of PD (Yasui et al. 2000). Our findings provide the first direct evidence that homocysteine can sensitize dopaminergic neurons to dysfunction and death in models of PD, and suggest a mechanism whereby dietary folate may reduce risk of PD.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  • Allain P., Le Bouil A., Cordillet E., Le Quay L., Bagheri H. and Montastruc J. L. (1995) Sulfate and cysteine levels in the plasma␣of␣patients with Parkinson's disease. Neurotoxicology 16, 527529.
  • Andersson A., Brattstrom L., Israelsson B., Isaksson A., Hamfelt A. and Hultberg B. (1992) Plasma homocysteine before and after methionine loading with regard to age, gender, and menopausal status. Eur. J. Clin. Invest. 22, 7987.
  • Andrews A. M. and Murphy D. L. (1993) Sustained depletion of cortical and hippocampal serotonin and norepinephrine but not striatal dopamine by 1-methyl-4-(2′-aminophenyl)-1,2,3,6-tetrahydropyridine (2′-NH2-MPTP): a comparative study with 2′-CH3-MPTP and MPTP. J. Neurochem. 60, 11671170.
  • Barrientos A. and Moraes C. T. (1999) Titrating the effects of mitochondrial complex I impairment in the cell physiology. J. Biol. Chem. 274, 1618816197.
  • Van Den Berg M., Van Der Knaap M. S., Boers G. H., Stehouwer C. D., Rauwerda J. A. and Valk J. (1995) Hyperhomocysteinaemia; with reference to its neuroradiological aspects. Neuroradiology 37, 403411.DOI: 10.1007/s002340050119
  • Betarbet R., Sherer T. B., MacKenzie G., Garcia-Osuna M., Panov A. V. and Greenamyre J. T. (2000) Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Nat. Neurosci. 3, 13011306.
  • Bezard E., Dovero S., Bioulac B. and Gross C. (1997) Effects of different schedules of MPTP administration on dopaminergic neurodegeneration in mice. Exp. Neurol. 148, 288292.
  • Blount B. C., Mack M. M., Wehr C. M., MacGregor J. T., Hiatt R. A., Wang G., Wickramasinghe S. N., Everson R. B. and Ames B. N. (1997) Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: implication for cancer and neuronal damage. Proc. Natl Acad. Sci. USA 94, 32903295.
  • Brattstrom L., Lindgren A., Israelsson B., Andersson A. and Hultberg B. (1994) Homocysteine and cysteine: determinants of plasma levels␣in middle aged and elderly subjects. J. Intern. Med. 236, 633641.
  • Double K. L., Gerlach M., Youdim M. B. and Riederer P. (2000) Impaired iron homeostasis in Parkinson's disease. J. Neural. Transm. Supplement 60, 3758.
  • Duan W. and Mattson M. P. (1999) Dietary restriction and 2-deoxyglucose administration improve behavioral outcome and reduce degeneration of dopaminergic neurons in models of Parkinson's disease. J.␣Neurosci. Res. 57, 195206.
  • Duan W., Zhang Z., Gash D. M. and Mattson M. P. (1999) Participation of prostate apoptosis response-4 in degeneration of dopaminergic neurons in models of Parkinson's disease. Ann. Neurol. 46, 587597.DOI: 10.1002/1531-8249(199910)46:4<587::AID-ANA6>3.0.CO;2-M
  • Elkind M. S. and Sacco R. L. (1998) Stroke risk factors and stroke prevention. Semin. Neurol. 18, 429440.
  • Finkelstein J. D. (1990) Methionine metabolism in mammals. J. Nutr. Biochem. 1, 228237.
  • Gibb W. R., Costall B., Domeney A. M., Kelly M. E. and Naylor R. J. (1988) The histological effects of intracerebral injection or infusion of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and MPP+ (1-methyl-4-phenylpyridinium) in rat and mouse. Brain. Res. 461, 361366.
  • Giles W. H., Kittner S. J., Anda R. F., Croft J. B. and Casper M. L. (1995) Serum folate and risk for ischemic stroke. First National Health and Nutrition Examination Survey epidemiologic follow-up study. Stroke 26, 11661170.
  • Greenblatt J. M., Huffman L. C. and Reiss A. L. (1994) Folic acid in neurodevelopment and child psychiatry. Prog. Neuropsychopharmacol. Biol. Psychiatry 18, 647660.
  • Grieve A., Butcher S. P. and Griffiths R. (1992) Synaptosomal plasma membrane transport of excitatory sulphur amino acid transmitter candidates: kinetic characterisation and analysis of carrier specificity. J. Neurosci. Res. 32, 6068.
  • Guo Q., Sebastian L., Sopher B. L., Miller M. W., Glazner G. W., Ware C. B., Martin G. M. and Mattson M. P. (1999) Neurotrophic factors [activity-dependent neurotrophic factor (ADNF) and basic fibroblast growth factor (bFGF)] interrupt excitotoxic neurodegenerative cascades promoted by a PS1 mutation. Proc. Natl Acad. Sci. USA 96, 41254130.
  • Hengemihle J. M., Long J. M., Betkey J., Jucker M. and Ingram D. K. (1999) Age-related psychomotor and spatial learning deficits in 129/SvJ mice. Neurobiol. Aging 20, 918.
  • Jenner P. and Olanow C. W. (1998) Understanding cell death in Parkinson's disease. Ann. Neurol. 44, S72S84.
  • Kruman I., Bruce-Keller A. J., Bredesen D. E., Waeg G. and Mattson M.␣ P. (1997) Evidence that 4-hydroxynonenal mediates oxidative stress-induced neuronal apoptosis. J. Neurosci. 17, 50895100.
  • Kruman I. I., Culmsee C., Chan S. L., Kruman Y., Guo Z., Penix L. and Mattson M. P. (2000) Homocysteine elicits a DNA damage response in neurons that promotes apoptosis and hypersensitivity to excitotoxicity. J. Neurosci. 20, 69206926.
  • Kuhn W., Roebroek R., Blom H., Van Oppenraaij D., Przuntek H., Kretschmer A., Buttner T., Woitalla D. and Muller T. (1998) Elevated plasma levels of homocysteine in Parkinson's disease. Eur. Neurol. 40, 225227.
  • Langston J. W. (1987) Parkinson's disease: current view. Am. Fam. Phys. 35, 201206.
  • Langston J. W. (1998) Epidemiology versus genetics in Parkinson's disease: progress in resolving an age-old debate. Ann. Neurol. 44, S45S52.
  • Le Couteur D. G., McLean A. J., Taylor M. C., Woodham B. L. and Board P. G. (1999) Pesticides and Parkinson's disease. Biomed. Pharmacother. 53, 122130.
  • Lipton S. A., Kim W. K., Choi Y. B., Kumar S., D'Emilia D. M., Rayudu P. V., Arnelle D. R. and Stamler J. S. (1997) Neurotoxicity␣associated with dual actions of homocysteine at the N-methyl-d-aspartate receptor. Proc. Natl Acad. Sci. USA 94, 59235928.
  • Logroscino G., Marder K., Cote L., Tang M. X., Shea S. and Mayeux R. (1996) Dietary lipids and antioxidants in Parkinson's disease:␣a␣population-based, case-control study. Ann. Neurol. 39, 8994.
  • Lucock M. (2000) Folic acid: nutritional biochemistry, molecular biology, and role in disease processes. Mol. Genet. Metab. 71, 121138.
  • Marsden C. D. (1994) Parkinson's disease. J. Neurol. Neurosurg. Psychiatry 57, 672681.
  • Meleady R. A. and Graham I. M. (1998) Homocysteine and vascular disease: nature or nurture? J. Cardiovasc. Risk 5, 233237.
  • Miller J. W., Shukitt-Hale B., Villalobos-Molina R., Nadeau M. R., Selhub J. and Joseph J. A. (1997) Effect of 1-dopa and the catechol-O-methyltransferase inhibitor Ro 41–0960 on sulfur amino acid metabolites in rats. Clin. Neuropharmacol. 20, 5566.
  • Moratalla R., Quinn B., DeLanney L. E., Irwin I., Langston J. W. and Graybiel A. M. (1992) Differential vulnerability of primate caudate-putamen and striosome-matrix dopamine systems to the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Proc. Natl Acad. Sci. USA 89, 38593863.
  • Muller T., Woitalla D., Hauptmann B., Fowler B. and Kuhn W. (2001) Decrease of methionine and S-adnenosylmethionine and increase of homocysteine in treated patients with Parkinson's disease. Neurosci. Lett. 308, 5456.
  • Refsum H., Ueland P. M., Nygard O. and Vollset S. E. (1998) Homocysteine and cardiovascular disease. Annu. Rev. Med. 49, 3162.
  • De Rijk M. C., Breteler M. M., Den Breeijen J. H., Launer L. J., Grobbee D. E., Van Der Meche F. G. and Hofman A. (1997) Dietary antioxidants and Parkinson disease. The Rotterdam Study. Arch. Neurol. 54, 762765.
  • Schneider J. S., Yuwiler A. and Markham C. H. (1987) Selective loss of subpopulations of ventral mesencephalic dopaminergic neurons␣in the monkey following exposure to MPTP. Brain Res. 411, 144150.
  • Scott J. M. and Weir D. G. (1998) Folic acid, homocysteine and one-carbon metabolism: a review of the essential biochemistry. J.␣Cardiovasc. Risk 5, 223227.
  • Seaton T. A., Cooper J. M. and Schapira A. H. (1998) Cyclosporin inhibition of apoptosis induced by mitochondrial complex I toxins. Brain Res. 809, 1217.
  • De Silva H. R., Khan N. L. and Wood N. W. (2000) The genetics of Parkinson's disease. Curr. Opin. Genet. Dev. 10, 292298.
  • Surtees R., Bowron A. and Leonard J. (1997) Cerebrospinal fluid and plasma total homocysteine and related metabolites in children with cystathionine β-synthase deficiency: the effect of treatment. Pediatr. Res. 42, 577582.
  • Tatton N. A. (2000) Increased caspase 3 and bax immunoreactivity accompany nuclear GAPDH translocation and neuronal apoptosis in Parkinson's disease. Exp. Neurol. 166, 2943.
  • Taylor C. A., Saint-Hilaire M. H., Cupples L. A., Thomas C. A., Burchard A. E., Feldman R. G. and Myers R. H. (1999) Environmental, medical, and family history risk factors for Parkinson's disease: a New England-based case control study. Am. J. Med. Genet. 88, 742749.
  • Tella S. R., Ladenheim B., Andrews A. M., Goldberg S. R. and Cadet J.␣ L. (1996) Differential reinforcing effects of cocaine and GBR-12909: biochemical evidence for divergent neuroadaptive changes in the mesolimbic dopaminergic system. J. Neurosci. 16, 74167427.
  • Watkins D. and Rosenblatt D. S. (1989) Functional methionine synthase deficiency (cblE and cblG): clinical and biochemical heterogeneity. Am. J. Med. Genet. 34, 427434.
  • Welch G. N. and Loscalzo J. (1998) Homocysteine and atherothrombosis. N. Engl. J. Med. 338, 10421050.
  • Yasui K., Kowa H., Nakaso K., Takeshima T. and Nakashima K. (2000) Plasma homocysteine and MTHFR C677T genotype in levodopa-treated patients with PD. Neurology 55, 437440.
  • Yu Z. F., Bruce-Keller A. J., Goodman Y. and Mattson M. P. (1998) Uric acid protects neurons against excitotoxic and metabolic insults in cell culture, and against focal ischemic brain injury in vivo. J.␣Neurosci. Res. 53, 613625.