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Keywords:

  • cell death;
  • differentiation;
  • MPP+;
  • Parkinson's disease;
  • α-synuclein

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

The pre-synaptic protein, α-synuclein, has been associated with the pathogenesis of Parkinson's disease. The present study indicates that α-synuclein, but not its mutants (A53T, A30P), can protect CNS dopaminergic cells from the parkinsonism-inducing drug 1-methyl-4-phenylpyridinium (MPP+), whereas it cannot protect from the dopaminergic toxin, 6-hydroxydopamine, hydrogen-peroxide, or the β-amyloid peptide, A-β. Protection from MPP+ was directly correlated with the preservation of mitochondrial function. Specifically, α-synuclein rescued cells from MPP+ mediated decreases in mitochondrial dehydrogenase activity and loss of ATP levels by utilizing ketosis. It also prevented toxin-induced activation of the creatine kinase/creatine phosphate system. Similarly, α-synuclein protected cells from the complex I inhibitor rotenone and 3-nitroproprionic acid, a complex II inhibitor. Wild-type α-synuclein-mediated neuroprotection and subsequent alterations in energy were not found in dbcAMP-differentiated cells. These results suggest that the normal physiological role for α-synuclein may change during development.

Abbreviations used
BHB

d-β-hydroxybutyrate

BSA

bovine serum albumin

CPK

creatine phosphokinase

HA

hemagglutinin

MPP+

1-methyl-4-phenylpyridinium

NGF

nerve growth factor

3-NPA

3-nitropropionic acid

6-OHDA

6-hydroxydopamine

PBS

phosphate-buffered saline

PD

Parkinson's disease

RIPA

radioimmuno precipitation assay

SDS–PAGE

sodium dodecyl sulfate – polyacrylamide gel electrophoresis

The pre-synaptic protein, alpha (α)-synuclein, has been associated with Parkinson's disease (PD) based on genetic linkage studies (Polymeropoulos et al. 1997; Krüger et al. 1998) as well as immunohistochemical evidence showing α-synuclein to be the principle component of Lewy bodies, a hallmark of PD (Spillantini et al. 1997).

Two independent α-synuclein mutations have been described, A53T (Polymeropoulos et al. 1997) and A30P (Krüger et al. 1998). The proteins encoded by these mutations appear to aggregate more rapidly than wild type (Conway et al. 1998; Giasson et al. 1999; Narhi et al. 1999; Wood et al. 1999; Conway et al. 2000). Structurally, α-synuclein consists of an N-terminal region containing six 11-mer repeats and an acidic C-terminal domain. Because the repeated region shares homology with the lipid binding A2 apolipoproteins, a role in lipid binding has been proposed. Consistent with this notion, α-synuclein has been shown to reversibly associate with various membranous structures (McLean et al. 2000; Jo et al. 2000; Perrin et al. 2000; Jo et al. 2002) including synaptic vesicles (Jensen et al. 1998). Studies examining developmental expression of α-synuclein suggest that it might have a role in synapse formation and/or maintenance and stabilization (Hsu et al. 1998; Bayer et al. 1999; Petersen et al. 1999; Galvin et al. 2001). For example, down regulation or loss of α-synuclein expression appears to decrease the size of the distal vesicular pool in hippocampal neurons (Murphy et al. 2000; Cabin et al. 2002), furthering the idea that α-synuclein plays a role in vesicle generation and/or maintenance.

Wild type α-synuclein or its mutants have been overexpressed in a variety of heterologous cell types with variable outcomes. For example, α-synuclein protected TSM1 cells from apoptosis induced by staurosporine (Da Costa et al. 2000, 2002), whereas in SK-N-MC neuroblastoma cells, α-synuclein accelerated cell death in response to this drug (Lee et al. 2001). Conversely, α-synuclein attenuated cell death in the SK-N-MC cells mediated by serum deprivation or hydrogen peroxide (H2O2; Lee et al. 2001), but it neither protected against H2O2 nor exacerbated apoptotic injuries in the neuroblastoma cell line, SH-SY5Y (Kanda et al. 2000). In yet a third neuroblastoma cell line, BE-M17, α-synuclein both accelerated cell death and led to cellular inclusions following treatments inducing apoptosis (Ostrerova-Golts et al. 2000).

Attempts to delineate the mechanisms by which α-synuclein either protects or induces cell death have proven equally complex. For example, previous studies investigating neuronal cell death in response to nerve growth factor (NGF) withdrawal demonstrated that survival pathways were suppressed (ERK/MAPK) whereas JNK and p38 pathways were activated (Xia et al. 1995). Consistent with this paradigm, overexpression of either α-synuclein, A53T, or A30P mutants in N2a cells led to decreased phosphorylation of ERK1/2 and accelerated cell death after serum deprivation (Iwata et al. 2001). Surprisingly, however, p38 and JNK were also markedly decreased which, in theory, should have rescued cells from α-synuclein's adverse actions (Iwata et al. 2001). Indeed, Hashimoto et al. (2002) have demonstrated that overexpression of α-synuclein does decrease JNK expression, thereby affording protection against H2O2 in the hypothalamic cell line GT1-17. Thus, the specific role of α-synuclein may depend upon the cellular context in which it is expressed.

From the studies described above, it would appear that α-synuclein has multiple physiological roles and/or functional responses, depending upon the environmental context or state of activation of other signaling pathways. In as much as many previous studies have been done in non-dopaminergic backgrounds and/or cells without an entire complement of proteins involved in dopamine biosynthesis, release and re-uptake, the goal of these studies was to analyze α-synuclein function in the context of a well-characterized CNS-derived model system. For example, MN9D cells, which are derived from murine mesencephalic neurons, synthesize, release, and take up dopamine (Choi et al. 1991; Tang et al. 1994) as well as exhibit many features of bona fide dopamine neurons such as sensitivity to 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+; Choi et al. 1991; Oh et al. 1995). Thus, this cell line is a highly applicable system for studying the role of α-synuclein. Moreover, MN9D cells were immortalized just after their dopaminergic characteristics became apparent (Choi et al. 1991), and as such they may represent an earlier developmental time period than other cell lines. Therefore, naive MN9D cells may reflect a unique environment, one in which α-synuclein may exhibit developmentally specific properties. Here, we report that overexpression of wild-type but not mutant forms of α-synuclein suppresses cell death mediated by MPP+ in naive but not dbcAMP-treated dopaminergic cells.

Cloning and establishment of α-synuclein stable cell lines

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

A full-length human α-synuclein clone was obtained from Genome Systems. This clone, Syn/WT, served as a template to produce both mutant forms of α-synuclein, Syn/A53T and Syn/A30P, using a PCR-based methodology. All three cDNA molecules were tagged at the amino terminus with the hemagglutinin (HA) epitope. Each resulting construct was subcloned into the pcDNA3 expression vector (Invitrogen, Carlsbad, CA, USA). All clones were subsequently sequenced to verify their integrity. Syn/WT, Syn/A53T, and Syn/A30P, as well as a vector only (pcDNANeo3) construct were transfected into MN9D cells as described previously (Tang et al. 1994). Stable cell lines were purified by repetitive rounds of serial dilution in the above media with the added antibiotic G418 (0.5 mg/mL).

Cell culture and treatments

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

MN9D cells (Choi et al. 1991) were cultured in Iscove's IMDM media (Invitrogen) supplemented with 10% fetal calf serum at 37°C, 10% CO2, and, unless otherwise stated, were plated in 24-well culture dishes and allowed to reach 60% confluency. Cells were subsequently switched to serum-free Iscove's/Hams supplemented with B27 (Invitrogen) 30 min prior to drug addition. MPP+ was dissolved in media, 6-OHDA and H2O2 were resuspended in boiled water, rotenone was dissolved in chloroform and 3-nitroproprionic acid was diluted in ethanol (all from Sigma, St Louis, MO, USA). Both rotenone and 3-NPA were initially prepared as stock solutions prior to dilution in media, such that the final concentration of either chloroform or ethanol was less than 0.01%. β-Amyloid peptide (A-β) (25–35 or 1–40; Bachem, Torrance, CA, USA) was diluted to 1 mg/mL in water and then aged by incubating at 37°C for 3 days prior to addition to the cells. Control cultures were treated with the vehicle used to dissolve that drug.

Measurement of dopaminergic characteristics

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

For dopamine uptake and content measurements, cells were plated at 2 × 104 cells per well of a 24-well plate and allowed to grow for 2 days. Dopamine uptake was performed as described (Oh et al. 1996). Dopamine content was assayed exactly as described (Lotharius and O'Malley 2000). TH activity was measured by coupled non-enzymatic decarboxylation as described in O'Hara et al. (1996). All assays were normalized to relative protein concentrations using the Bradford assay (Bio-Rad, Hercules, CA, USA). TH activity results were further normalized to relative TH protein levels.

Immunocytochemistry

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

For localization of α-synuclein 2 × 104 cells were grown on Laboratory-Tek (Naperville, IL, USA) chamber slides, incubated with a mouse monoclonal HA (1 : 2,000, Babco, Richmond, CA, USA) or a α-synuclein antibody (1 : 300, Transduction Laboratories, Lexington, KY, USA) and processed as previously described (Oh et al. 1995). For western blot analysis, stable cell lines were washed in ice cold phosphate-buffered saline (PBS) and lysed in RIPA buffer (150 mm NaCl, 1% NP-40, 0.5% NaDoc, 0.1% SDS, 50 mm Tris, pH 8.0, and protease inhibitor cocktail, Roche, Mannheim, Germany). Protein concentrations were determined using the Bradford assay (Bio-Rad). Equal amounts of protein were loaded on 15% sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) gels. Resulting blots were blocked and stained in 1% bovine serum albumin (BSA), 0.1% milk, and 0.01% Triton-X-100, in PBS and then incubated with an antibody directed against one or more of the following: α-synuclein (1 : 300, Transduction Laboratories), HA (1 : 1,000, Babco), TH (1 : 5,000, DiaSorin, Stillwater, MN, USA), PARP which recognizes both cleaved and uncleaved PARP (1 : 300, BioMol, Plymouth Meeting, PA, USA), activated caspase 3 (1 : 1,000, Cell Signaling Tech., Beverly, MA, USA), synapsin 1 (1 : 1,000, Transduction Laboratories), SV2 (a gift from Kathleen M. Buckley, Harvard Medical School), and VMAT2 (Chemicon, Temecula, CA, USA). For chemifluorescent detection, blots were incubated with the appropriate horseradish peroxidase labeled secondary antibodies followed by ECL plus (Amersham, Piscataway, NJ, USA) substrate detection. Protein density was quantified by computerized scanning densitometry using Storm software (Molecular Dynamics, Sunnyvale, CA, USA).

Cell viability assays

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

Cell viability was assessed using propidium iodide (1 mg/mL; Molecular Probes, Eugene, OR, USA) exactly as described (Wiegele et al. 1998). Cell counts were conducted by a person blinded to the experimental conditions by scoring photomicrographs taken from four consecutive fields (20 × objective) across a culture well. At least four wells from each individual culture condition were analyzed. The percentage of cell survival was calculated as the number of surviving cells per total cell count. DNA cleavage was detected by terminal deoxynucleotidyl transferase mediated biotinylated UTP nick end labeling (TUNEL) using the manufacturer's recommended procedures (Roche).

Metabolism assays

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

General mitochondrial activity was assayed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma) exactly as described (Oh et al. 1995). Intracellular ATP was measured using a luciferase-based ATP assay kit per the manufacturer's instructions (Calbiochem, San Diego, CA, USA). An ATP standard curve was established by plotting optical readings from known concentrations of ATP. Values were normalized to μg of protein per μL of sample used. The rate of glucose uptake was determined in cells treated with and without MPP+ exactly as described by Oh et al. (1995). Creatine phosphokinase (CPK) activity was quantified using a spectrophotometric assay (Sigma) that followed the reduction over time of NADPH. β-Hydroxybutyrate was assayed by using a spectrophotometric assay as described by the manufacturers (Standbio Laboratory, Boerne, TX, USA). All absorbance values were normalized to μg of protein per μL of sample used.

MN9D cell dbcAMP treatment

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

Cells were plated at a density of 60% with varying cells/mL depending on the size of the well plated and incubated for 24 h to allow for cell attachment and recovery. To induce dbcAMP treatment, cells were grown for 7 days in serum-free Iscove's/Hams supplemented with B27 and G418 with the addition of 1 mm N6, 2′-O-dibutyryladensoine 3′,5′-cyclic monophosphate sodium salt (dbcAMP, Sigma).

Characterization of stable synuclein-expressing MN9D cell lines

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

To investigate the physiological role of alpha (α)-synuclein in dopaminergic cell types, stable cell lines were created by introducing α-synuclein, the A53T and A30P mutations as well as an empty vector control into the mesencephalic, dopaminergic cell line, MN9D. All three constructs of synuclein were tagged at the amino terminus with the hemagglutinin (HA) epitope to help facilitate identification of positive cells. Several cell lines per construct were purified to homogeneity as determined by immunocytochemistry using an antibody directed against HA (Fig. 1a). Studies were subsequently restricted to cell lines exhibiting approximately the same levels of α-synuclein determined by western blotting (Fig. 1b). Vector cells did not express endogenous α-synuclein at either the protein (Fig. 1b) or RNA level (not shown). The dopaminergic biosynthetic enzyme, TH, a protein marker for these cells, was expressed at comparable levels in the vector and over-expressing α-synuclein cell lines (Syn/WT) and at slightly higher levels in the two mutant cell lines (Syn/A53T, Syn/A30P, Fig. 1b).

image

Figure 1. Characterization of Syn/WT, Syn/A53T, and Syn/A30P in MN9D cells. (a) Immunofluorescent staining of indicated stable cell lines using anti-HA as described in the text. Homogenous distribution of synuclein immunoreactivity was seen throughout the cytoplasm and the nucleus. (b) Western blot analysis of equal amounts of protein (25 μg) from each of the indicated cell lines. After protein transfer, blots were cut and probed with either anti-α-synuclein or anti-TH antibodies.

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In addition to dopamine synthesis, parental MN9D cells express a variety of dopaminergic characteristics, including a high-affinity dopamine uptake system (Choi et al. 1991; Tang et al. 1994), the vesicular monoaminergic transporter (VMAT2; O'Malley, unpublished observation) and calcium-induced vesicular release (Choi et al. 1991; Tang et al. 1994). As shown in Table 1, overexpression of Syn/WT, Syn/A53T, and Syn/A30P consistently inhibited dopamine levels as compared with overexpressing vector control cells. In contrast, TH activity was significantly increased in the Syn/A53T and Syn/A30P cell lines whereas, with the exception of the Syn/A53T cells, dopamine uptake was largely unaffected (Table 1). Taken together, overexpressing α-synuclein and its mutants in MN9D cells increased some and decreased other dopaminergic properties such that no general trend was apparent. With the exception of decreased dopamine content, Syn/WT cells exhibited similar dopaminergic properties as the vector expressing cells.

Table 1.  Dopaminergic characteristics of stable cell lines
MeasurementVectorSyn/WTSyn/A53TSyn/A30P
  1. Cell lines were plated, maintained for 2 days and then assayed individually for the different measurements. For dopamine uptake, cells were loaded with [3H]dopamine, harvested and counted as described (Oh et al. 1995). Dopamine content was determined in lysed cells using HPLC coupled with electrochemical detection (Lotharius et al. 2000). TH activity was measured by coupled non-enzymatic decarboxylation as described (O'Hara et al. 1996). Values from quadruplicated wells for uptake and content and triplicate pellets for TH activity were normalized to protein content. +Dopamine uptake is expressed as nmol dopamine/15 min/μg protein. #Dopamine content is expressed as ng dopamine/μg protein. §TH activity is expressed as nmol DOPA/μg protein/h/cell-specific TH protein levels. The latter were determined by western blotting. Data denote the mean ±SEM. Asterisks indicate statistically significant differences between vector control cells with the different α-synuclein stable cell lines (*p < 0.01 Student's t-test).

Dopamine uptake+0.258 ± 0.0110.236 ± 0.0120.138 ± 0.012*0.219 ± 0.023
Dopamine content#105 ± 11.138.2 ± 2.4 *26.2 ± 4.5*60.9 ± 6.1*
TH activity§2.07 ± 0.282.18 ± 0.065.6 ± 1.07*14.7 ± 3.04*

Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

Given that some studies have demonstrated that α-synuclein by itself and/or its mutant forms can be toxic when overexpressed in heterologous cells (Ostrerova et al. 1999; Hsu et al. 2000; Saha et al. 2000; Zhou et al. 2000), each cell line was tested to see if it had equivalent doubling times or exhibited any differences in cell death. No obvious phenotypic alteration was observed with the exception that cells overexpressing the A53T mutation appeared more spindle shaped than oval (Fig. 1a). To ascertain whether Syn/WT, Syn/A53T, or Syn/A30P caused an increase in cell death, various assays were performed, including propidium iodide staining, TUNEL staining, analysis of DNA fragmentation and PARP cleavage as well as activated caspase 3 western blotting. All assays demonstrated that there was no change in normal cell survival by the addition of α-synuclein (Fig. 2) or its mutants (not shown). In the time course of these studies, no evidence of protein aggregation or inclusion body formation was ever observed (not shown). Therefore, overexpression of any form of α-synuclein did not alter these fundamental characteristics of the MN9D cells.

image

Figure 2. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways. (a) Syn/WT or vector-only cells were treated with 15 μm MPP+, 50 μm 6-OHDA, 1 mm H2O2, or 2.5 μm A-β for 24 h and then fixed and processed for synuclein immunoreactivity together with TUNEL detection. (b) Western blot analysis of Syn/WT protein lysates derived from sibling cultures co-treated with indicated toxins. After transfer, blots were cut and probed with an antibody directed against PARP as well as activated caspase 3. Syn/WT cells treated with 6-OHDA or H2O2 exhibited hallmarks of apoptosis, whereas untreated or MPP+-treated cells did not. Results for MN9D/vector, Syn/A53T, or Syn/A30P were similar (not shown).

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Previously, we have shown that the dopaminergic toxins MPP+ and 6-OHDA induce distinct cell-death processes in dopaminergic cells (Oh et al. 1995; Choi et al. 1999a,b; Lotharius et al. 1999; Kim et al. 2001). To determine whether the overexpression of α-synuclein or its mutants altered these cell death pathways, Syn/WT cells were treated with MPP+, 6-OHDA, the oxidative stress inducer hydrogen peroxide (H2O2), and the Alzheimer's disease associated A-β. Consistent with our earlier studies, both 6-OHDA and H2O2 induced TUNEL staining, the appearance of cleaved PARP and activated caspase 3 (Fig. 2) whereas MPP+ and A-β did not induce these hallmarks of apoptosis (Fig. 2). Thus, overexpression of α-synuclein or its mutants (not shown) did not alter specific cellular responses to the selected toxin injury.

Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

To determine whether α-synuclein or its mutants renders dopaminergic cells more vulnerable to environmental stressors, each cell line was treated with various concentrations of different toxins and cell viability was quantified using the propidium iodide assay. Exposure of MN9D/vector cells to the dopaminergic toxins MPP+ or 6-OHDA (Figs 3a and b) led to cell death with a mean toxic dose (TD50) similar to what we have previously reported (Oh et al. 1995; MPP+, TD50 = 0.8 ± 0.02 μm; 6-OHDA, TD50 = 18.75 ±0.03 μm). MN9D/vector cells were also killed by H2O2 (Fig. 3c; TD50 = 21.27 ± 0.07 μm) as well as A-β (Fig. 3d; TD50 = 1.0 ± 0.09 μm). Overexpression of α-synuclein completely rescued cells from MPP+ injury at concentrations of 1 μm and blocked at least 50% of cell death at concentrations up to 50 μm, the highest concentration tested. Although an accurate TD50 could not be estimated from the Syn/WT set of data, it must be greater than 40 μm (Fig. 3a). The mutants were ineffectual, particularly at low concentrations (Syn/A53T, TD50 = 0.75 ± 0.1 μm; Syn/A30P, TD50 = 0.7 ± 0.09 μm). There was a modest but significant attenuation seen with the Syn/A30P construct at higher MPP+ concentrations. No form of α-synuclein protected against 6-OHDA, H2O2, or A-β-mediated cell death. In fact, the Syn/WT, Syn/A53T, and Syn/A30P cell lines were significantly more sensitive to A-β than vector-only expressing cells (TD50 = 0.07 ± 0.018 μm, 0.096 ± 0.032 μm, and 0.09 ± 0.011 μm, respectively). Thus, overexpression of α-synuclein but not its mutants protects dopaminergic cells from one dopaminergic toxin but not another.

image

Figure 3. Overexpression of α-synuclein attenuates cell death induced by MPP+ but not 6-OHDA, H2O2 or A-β. Vector, Syn/WT, Syn/A53T, and Syn/A30P were plated in 24-well plates at 2 × 104 cells/well and maintained for 2 days. Cells were subsequently switched to serum-free B-27 media and treated with the indicated concentrations of MPP+ (a) for 48 h; statistical analysis: Syn/WT, two-way anova, F[5, 10] = 238, p < 10−4; Syn/A30P, two-way anova, F[5, 0.8] = 13.5, p < 3 × 10−4; 6-OHDA (b) for 24 h; statistical analysis: two-way anova, not significant; H2O2 (c), 24 h; statistical analysis: two-way anova, not significant; and A-β (d), 48 h; statistical analysis: Syn/WT, two-way anova, F[5, 75] = 1137, p < 10−4; Syn/A53T, two-way anova, F[5, 5] = 515, p < 10−4; Syn/A30P, two-way anova, F[4, 48] = 878, p < 10−4. Cell viability was measured using propidium iodide. Results represent three to six independent experiments in quadruplicate. Values from each treatment were expressed as a per cent over the non-treated control (100%). Each point represents the mean ± SEM. Bars with less than 2% of SEM are buried within the symbol. *p < 10−5 post hoc comparisons using Student's t-test at single drug concentrations.

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Syn/WT attenuates cell death induced by other inhibitors of electron transport

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

Given that MPP+ can block electron transport by binding to the same site as the complex I inhibitor rotenone (Krueger et al. 1993), we examined whether overexpression of α-synuclein would protect MN9D cells from rotenone or the complex II inhibitor 3-nitropropionic acid (3-NPA). Overexpression of Syn/WT significantly inhibited cell death induced by either rotenone (vector, TD50 = 2.2 ± 0.03 μm; Syn/WT, TD50 = 26.7 ± 0.76 μm) or 3-NPA (vector, TD50 = 1.4 ± 0.22 μm; Syn/WT, TD50 = 15.4 ± 0.3 μm). These data suggest that, within the context of these dopaminergic cells, α-synuclein protects cells from mitochondrial dysfunction.

Overexpression of α-synuclein affects energy levels in dopaminergic cells

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

Mitochondrial inhibitors such as MPP+ or rotenone block ATP production by impairing the electron transport chain. To test whether overexpression of Syn/WT protected cells from MPP+-induced mitochondrial dysfunction, general mitochondrial dehydrogenase activity was assessed using the MTT assay. Mitochondrial function in MN9D/vector cells decreased in a dose-dependent fashion with an IC50 of 15.35 ± 0.12 μm(Fig. 4a). In contrast, mitochondrial function in Syn/WT overexpressing cells was significantly protected at all MPP+ concentrations tested (Fig. 4a; IC50 = 109 ± 8.6 μm; p < 0.006 at all drug concentrations). Interestingly, the A53T and A30P mutants exhibited an intermediate pattern of protection (Syn/A53T, IC50 = 31.4 ± 0.12 μm; p < 0.04 at drug concentrations greater than 5 μm and Syn/A30P, IC50 = 27.08 ± 1.3 μm; p < 0.004). These results are consistent with the hypothesis that overexpression of α-synuclein and, to a lesser extent, its mutant forms, blocks MPP+ toxicity by blocking or overriding MPP+-mediated mitochondrial dysfunction.

image

Figure 4. Overexpression of α-synuclein alters energy levels when cells are challenged with MPP+. Cells were plated in 24-well plates at 2 × 104 cells/well and maintained for 2 days. Cells were then switched to serum-free B-27 media and treated with MPP+. (a) Mitochondrial function was assayed using MTT after 48 h of exposure to the indicated concentration of MPP+. Statistical analysis: Syn/WT, two-way anova, F[5, 17] = 223, p < 10−4; Syn/A53T, two-way anova, F [5, 25] = 39, p < 10−4; Syn/A30P, two-way anova, F [5, 4.9] = 60.5, p < 10−4. (b) Intracellular ATP levels were measured over time for each cell line after exposure to 15 μm of MPP+. All absorbance values were normalized to the protein content of the sample. Statistical analysis: Syn/WT, two-way anova, F[6, 5.4] = 117, p < 10−4; Syn/A53T, two-way anova, F[6, 0.4] = 8.8, p < 10−4; Syn/A30P, two-way anova, F[6, 0.9] = 19, p < 10−4. *Indicates that the point was significant from vector-only cells p < 0.05, **p < 5 × 10−3, ***p < 5 × 10−4 post hoc Student's t-test.

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Decreased ATP levels following MPP+ treatment have been shown in many studies (DiMonte et al. 1986; Singer et al. 1988; Scotcher et al. 1990). To test whether overexpression of α-synuclein prevented MPP+-induced ATP losses, a luciferase-based assay was used to measure ATP levels over time following MPP+ treatment. As predicted, MN9D/vector cells exhibited a time-dependent loss of ATP in response to a mean toxic dose of MPP+, whereas ATP levels in Syn/WT were significantly elevated particularly at early time points (Fig. 4b; p < 0.004 at all time points tested). In contrast, neither mutant was particularly effective at preventing ATP loss although Syn/A30P was significantly different from vector 2 hours after MPP+ treatment (Figs 4b; p < 0.04). Neither mitochondrial function nor ATP levels were protected in Syn/WT cells following 6-OHDA or H2O2 treatments (not shown). Taken together, these data suggest that overexpression of α-synuclein blocks MPP+-mediated ATP losses.

Ketone bodies are increased in Syn/WT cells following MPP+ treatment

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

To determine how Syn/WT cells withstood MPP+-induced mitochondrial dysfunction, we tested several alternative energy sources. For example many in vivo and in vitro studies have shown that the cytotoxic effects of MPP+ exposure are preceded by increased rates of glucose utilization (Reinhard et al. 1990). This is thought to be a compensatory mechanism in response to toxin-induced energy depletion. MN9D cells also show increased rates of glucose uptake following treatment with MPP+ (Oh et al. 1995). Consistent with these earlier experiments (Oh et al. 1995), MPP+ treatment resulted in increased glucose utilization in vector expressing cells as well as in the Syn/WT cell line (Fig. 5a). Thus, overexpression of α-synuclein did not affect this pathway.

image

Figure 5. Alteration of select metabolic parameters after MPP+ treatment. (a) The rate of glucose uptake was measured after incubation with 15 μm MPP+ for the indicated time intervals. (b) Temporal induction of CPK activity levels in vector or Syn/WT cells treated with 15 μm MPP+. Statistical analysis: two-way anova, F[6, 58] = 258, p < 10−4. (c) Temporal induction of β-hydroxybutyrate levels in vector or Syn/WT cells treated with 15 μm MPP+. Statistical analysis: two-way anova, F[6, 58]= 258, p < 10−4. @Indicates that the point is significantly different from the non-treated control (p < 0.01). *indicates that the Syn/WT value was significant from the vector-only value p < 5 × 10−5 post hoc Student's t-test. All assay values were normalized to protein content and expressed as a per cent over the non-treated control (100%). Each point represents the mean ± SEM from three to six independent experiments done in quadruplicate.

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In various tissues, the creatine kinase system acts as an energy buffer capable of regulating intracellular ATP. Accordingly, CPK activity would be expected to increase if ATP levels dropped. In agreement with this model, time-dependent increases in CPK activity were seen in the vector/MN9D cells following MPP+ treatment (Fig. 5b), whereas CPK activity was not increased in Syn/WT cells. Increased levels of ATP in Syn/WT cells following MPP+ treatment (Fig. 4b) might preclude activation of CPK. The mutants A53T and A30P exhibited responses that were intermediate with vector and Syn/WT (not shown). Collectively, these data argue for a direct effect of α-synuclein on mitochondria such that ATP is utilized and/or recruited more effectively.

Recently ketone bodies such as d-beta-hydroxybutyrate (BHB) have been shown to serve as an alternative energy substrate by increasing mitochondrial acetyl CoA and hence the major substrate for electron transport, NADH (Sato et al. 1995; Kashiwaya et al. 1997). To test whether elevation of ketones could account for Syn/WT's preservation/induction of ATP, BHB levels were measured. Notably, BHB rose markedly in Syn/WT cells within 2 hours of MPP+ treatment (Fig. 5c). They remained elevated for at least 4 hours before declining 6 hours after MPP+ addition. Vector-only cells showed a modest increase in ketone bodies at 2 hours, which was not sustained at later time points (Fig. 5c). These data suggest that the Syn/WT cells are able to respond to MPP+ toxicity by elevating levels of ketone bodies as an alternative energy source.

dbcAMP treatment of stable cell lines

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

Because it is thought that α-synuclein plays a role in development, we examined whether dbcAMP treatment affected any of the properties observed in the naive synuclein-expressing stable cell lines. Following 7 days of 1 mm dbcAMP treatment, all of the cell lines exhibited differentiated features. Specifically, cells stopped dividing, appeared flattened, membranes were ruffled, cell bodies were enlarged, and long neuritic processes were apparent (Fig. 6a). dbcAMP treatment did not lead to changes in α-synuclein levels in either the over-expressing cell lines or the vector-only cells (not shown). However, dbcAMP treatment did change markers that are normally up-regulated during differentiation in vivo. For example, the synaptic makers, SV2 and synapsin-1, were both increased after treatment with dbcAMP, as was TH and the vesicular monoamine transporter (VMAT2; Fig. 6b). As shown, increased levels of synaptic markers were essentially the same between Syn/WT and vector-only cells following dbcAMP treatment (Fig. 6b). Examination of dopaminergic characteristics showed that both dopamine content and TH activity were increased in the dbcAMP treated MN9D vector and Syn/WT cells compared with naive cells, whereas dopamine uptake did not change significantly (Table 2). Despite morphological and biochemical differences, there were no significant changes in the pattern of cell death in dbcAMP-treated cells similar to what was observed in naive cells. Thus, 6-OHDA and H2O2-treated dbcAMP-treated cells still died via caspase 3 positive pathways whereas MPP+ and A-β treated cells did not (Fig. 2a; not shown).

image

Figure 6. Characteristics of dbcAMP-treated MN9D vector and Syn/WT cells. (a) Phase-bright photomicrographs of cells before and after 1 mm dbcAMP treatment. (b) Western blot analysis of proteins altered after dbcAMP treatment.

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Table 2.  Dopaminergic characteristics of dbcAMP-treated stable cell lines
MeasurementVector-diffSyn/WT-diff
  1. Cell lines were plated, maintained for 2 days and then 1 mm dbcAMP was added for 7 days prior to being assayed individually for the different measurements. For dopamine uptake, cells were loaded with [3H]dopamine, harvested and counted as described (Oh et al. 1995). Dopamine content was determined in lysed cells using HPLC coupled with electrochemical detection (Lotharius et al. 2000). TH activity was measured by coupled non-enzymatic decarboxylation as described (O'Hara et al. 1996). Values from quadruplicated wells for uptake and content and triplicate pellets for TH activity were normalized to protein content. +Dopamine uptake is expressed as nmol dopamine/15 min/μg protein. #Dopamine content is expressed as ng dopamine/μg protein. §TH activity is expressed as nmol DOPA/μg protein/h/cell specific TH protein levels. The latter were determined by western blotting. Data denote the mean ±SEM.

Dopamine uptake+0.256 ± 0.0110.243 ± 0.013
Dopamine content#605 ± 87720 ± 144
TH activity§1.86 ± 0.181.88 ± 0.14

Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

To determine whether dbcAMP treatment affected the Syn/WT response to MPP+, naive and dbcAMP-treated vector and Syn/WT cells were treated side by side with either 5 or 15 μm MPP+. As previously shown (Fig. 3a), naive Syn/WT cells rescued 71 and 53% cell death induced by 5 and 15 μm MPP+, respectively (Fig. 7). In contrast, no such rescue was observed in dbcAMP-treated cells at either toxin dose (Fig. 7). Interestingly, dbcAMP treatment per se rescued about 55% of cell death at either MPP+ dose in comparison with naive cells. These data suggest that over-expression of α-synuclein mimicked some aspects of dbcAMP treatment, at least in response to MPP+. dbcAMP treatment protected vector and α-synuclein-expressing cells from other toxins as well, albeit to a lesser extent (Table 3). One difference observed was seen with the Syn/A30P cells. The latter consistently exhibited greater cell death in dbcAMP-treated cells than counterparts in response to every toxin (Table 3).

image

Figure 7. Overexpression of α-synuclein attenuates cell death induced by MPP+ in naive but not dbcAMP treated cells. Vector and Syn/WT cells were plated in 24-well plates at 2 × 104 cells/well and maintained for 2 days. Cells were subsequently switched to serum-free B-27 media and half were treated with 1 mm dbcAMP for 7 days prior to treatment with the indicated concentrations of MPP+ for 48 h. Cell viability was measured using propidium iodide. Results represent three independent experiments in quadruplicate. Values from each treatment were expressed as a per cent over the non-treated control (100%). Each point represents the mean ± SEM. *indicates significant from untreated p < 10−5; ^indicates significant from vector-only cells; ∼ indicates significant from naive cells via post hoc comparisons using Student's t-test at single drug concentrations.

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Table 3.  Toxin-induced cell death in dbcAMP-treated cells
TreatmentVector-diffSyn/ WT-diffSyn/ A53T-diffSyn/ A30P-diff
  1. Cell lines were treated with 1 mm dbcAMP as stated in the text and assayed for cell death using propidium iodide exclusion. Data denote percentage of untreated control ±SEM. *Indicate statistically significant differences between vector control cells with the α-synuclein stable cell lines (p < 0.01 Student's t-test).

5 μm MPP+65.5 ± 3.664.8 ± 2.260.0 ± 2.655.0 ± 3.1
15 μm MPP+56.4 ± 2.456.7 ± 2.142.4 ± 2.3*41.9 ± 2.3*
50 μm 6-OHDA49.0 ± 1.550.2 ± 3.047.3 ± 1.628.7 ± 1.5*
100 μm 6-OHDA36.2 ± 1.936.5 ± 2.234.4 ± 2.423.7 ± 1.7*
1.5 mm H2O248.6 ± 2.252.9 ± 3.934.7 ± 2.2*30.6 ± 2.2*
2.5 μm A-β55.4 ± 1.537.8 ± 2.0*28.5 ± 1.0*22.5 ± 1.0*

Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

To determine whether dbcAMP-mediated differentiation altered metabolic responses to MPP+ treatment, dbcAMP treated cultures were analyzed at various time points following addition of 15 μm MPP+(Fig. 8). In contrast with the results noted in naive cultures (Figs 4 and 5), there were no differential responses between Syn/WT and vector-only cells for any of the measured parameters over the time period presented (Fig. 8) or longer (not shown). Specifically, there were no changes in MTT or ATP levels, rate of glucose uptake, CPK activity or BHB levels in response to MPP+. Thus, the dbcAMP-mediated differentiation not only altered vector responses (increased CPK activity and rate of glucose uptake; Fig. 5) but also Syn/WT responses (increased rate of glucose uptake and BHB levels; Fig. 5). Conceivably, the dbcAMP treatment-mediated resistance to MPP+ toxicity is due to alterations in processes leading to enhanced mitochondrial function and/or ATP levels, as neither dropped precipitously across the indicated time periods (Fig. 8). To examine whether dbcAMP treatment altered baseline levels of BHB, ATP, glucose uptake and/or CPK activity, naive and dbcAMP-treated cells were compared (Table 4). Although ATP and CPK activity increased dramatically upon dbcAMP treatment, they did so for both cell types. Taken together, the process of dbcAMP treatment per se leads to fundamental changes within dopaminergic cell types that increase their resistance to MPP+ toxicity. dbcAMP treatment-mediated attenuated responses are similar in magnitude to those seen in naive Syn/WT cells. These data imply that overexpression of α-synuclein mirrors aspects of dbcAMP-mediated differentiation.

image

Figure 8. DbcAMP treatment leads to enhanced metabolic parameters that are not affected by MPP+ treatment. Cells were plated in 24-well plates at 2 × 104 cells/well and maintained for 2 days. Cells were then switched to serum-free B-27 media, differentiated with dbcAMP as stated in the text, and treated with 15 μm MPP+. (a) Mitochondrial function was assayed using MTT after 48 h of exposure to the indicated concentration of MPP+. Intracellular ATP levels (b), the rate of glucose uptake (c), CPK levels (d), and BHB levels (e) were measured over time following exposure to MPP+.

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Table 4.  Effect of dbcAMP treatment on various energy parameters
AssayNaivedbcAMP-treated
VectorSyn/WTVectorSyn/WT
  1. Cell lines were dbcAMP treated with 1 mm dbcAMP as stated in the text and individually assayed for the different metabolic substrates. Data denote ± SEM. *Indicate statistically significant differences between dbcAMP-treated and naive values (p < 0.01 Student's t-test). m/mg protein, #cpm/min/mg protein, §μm/mg protein, ¶μm/mg protein.

ATP+93 ± 0.0893 ± 0.1480 ± 2.5*480 ± 4.3*
Glucose#63.2 ± 2.2862.9 ± 9.565.4 ± 5.158.8 ± 3.48
CPK§0.83 ± 0.160.82 ± 0.124.5 ± 0.51*4.4 ± 0.39*
BHB0.075 ± 0.010.06 ± 0.010.06 ± 0.010.075 ± 0.01

Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

The physiological role of α-synuclein is still unknown despite its widespread abundance in the CNS. The present study highlights a novel function of this protein, its ability to block energy depletion after complex I inhibition by the parkinsonism-inducing toxin MPP+. α-Synuclein-mediated protection is associated with increased ketone bodies, which serve as an alternative energy source. Preservation of mitochondrial function was directly linked to viability as cells were protected from cell death induced by MPP+, rotenone or 3-nitroproprionic acid but not from 6-OHDA, H2O2 or A-beta toxicity. Following dbcAMP treatment, dopaminergic cells with or without α-synuclein were significantly protected from neurotoxin treatment, suggesting that overexpression of α-synuclein mirrors aspects of dbcAMP-mediated differentiation. These data imply that the physiological role(s) of α-synuclein may change over the normal time course of differentiation and maturation.

Context-specific role of α-synuclein in various cell death pathways

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

Given the genetic association of α-synuclein with PD, many investigators have looked for and found a correlation between α-synuclein and cell death both in vivo and in vitro (Kahle et al. 2000). Anomalies abound, however, as, in some cells and with some injurious stimuli, α-synuclein served as a neuroprotectant, but not in others (Da Costa et al. 2000; Ostrerova-Golts et al. 2000; Zhou et al. 2000; Lee et al. 2001). Even in ostensibly similar backgrounds such as various neuroblastoma cell lines, different outcomes have been observed in response to over-expressing α-synuclein (Kanda et al. 2000; Zhou et al. 2000). Despite similarities, however, many of these neuroblastomas have arisen from a variety of peripheral sources at different developmental time periods, so it is not surprising that each would exhibit unique phenotypes and/or that α-synuclein might interact with different proteins and/or signaling pathways in response to external stimuli. Moreover, because increased levels of α-synuclein increase its propensity to aggregate (Hashimoto et al. 1998), differences in the level of expression achieved by introducing α-synuclein into various cell types may also influence cellular fate. For example, cells transduced with viral expression systems promoting high levels of foreign gene expression are more likely than other transfection technologies to exhibit cell death and inclusion body formation in response to over-expressing α-synuclein or its mutants (Kanda et al. 2000; Zhou et al. 2000, 2002). The striking neuropathology seen when α-synuclein was overexpressed using viral approaches in rat substantia nigra (Kirik et al. 2002) further emphasizes that reaching a critical mass and/or overwhelming the cell's capacity to properly process a protein with aggregating capabilities may readily trigger just that consequence. Taken together, the inability to generalize the effects of α-synuclein across all experimental paradigms is probably due to different cellular contexts, expression levels, means of introduction, treatment variables, differentiated states, etc. As knowledge regarding the physiological role of α-synuclein increases, seemingly disparate results may be more explicable.

Role of α-synuclein in CNS-derived dopaminergic cells

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

Previously, we have shown that 6-OHDA and H2O2 induce apoptosis whereas MPP+ and A-β induce a caspase-independent cell death in CNS-derived dopaminergic cells (Oh et al. 1995 and Fig. 2). Neither over-expressing α-synuclein nor its mutants altered 6-OHDA, H2O2 or A-β-mediated cell death, produced inclusions, or significantly changed the cells' normal growth patterns (not shown). Wild-type α-synuclein, however, significantly attenuated cell death induced by MPP+ whereas the mutant A53T was unable to do so (Fig. 3a). The A30P mutant exhibited a modest but significant protective effect in response to MPP+ (Fig. 3a). Thus, wild type α-synuclein selectively protected naive dopaminergic cells from a drug shown to produce PD-like symptoms in both humans and other animal models.

Previous studies have suggested that over-expressing α-synuclein may alter dopaminergic processes (Lee et al. 2001; Perez et al. 2002). For example, in a co-transfected fibroblast cell line, α-synuclein bound to the dopamine transporter, thereby increasing uptake (Lee et al. 2001). Other data have demonstrated that α-synuclein can also bind to TH, decreasing cellular dopamine content via inhibition of TH activity (Perez et al. 2002). Although dopamine content was significantly decreased in the cell lines described here, TH activity was unaltered in the Syn/WT cells and significantly increased in both mutants (Table 1). Moreover, dopamine uptake was unaltered as well. Taken together, these data emphasize the diverse roles α-synuclein may subserve, presumably due to its interaction with cell-type specific effectors that contribute to its overall response.

Several studies have suggested that α-synuclein might function as part of a survival response in dopaminergic cells. For example, Vila et al. (2000) showed that α-synuclein levels were increased in healthy, non-apoptotic neurons in the substantia nigra of mice treated with MPTP. Similarly, levels of α-synuclein mRNA and protein were increased in rat mid-brain dopaminergic neurons following excitotoxic lesioning in the striatum (Kholodilov et al. 1999). Collectively, these results suggest that certain neurotoxic stimuli trigger a protective response by α-synuclein in dopaminergic cells. Presumably the loss of susceptibility to MPP+ toxicity seen in the Syn/WT cells reflects this process. The present findings would suggest that this protective role is severely compromised in the case of the A53T and A30P mutants (Fig. 3a).

Mechanisms of neuroprotection

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

What are the mechanisms associated with this protective response? Previously, we have shown that MPP+ neurotoxicity is a multicomponent process involving both energy depletion due to electron transport inhibition as well as re-distribution of vesicular dopamine and ensuing oxidation (Lotharius et al. 2000). Although differences in dopaminergic properties were apparent in the MN9D cell lines (Table 1), these differences were either not consistent across the cell lines tested (increased TH activity in mutants vs. WT, Table 1) or were in a direction not predicted to affect viability (decreased dopamine content in all cell types, not just wild type, Table 1). Hence, the current studies focused on changes in mitochondrial function. Decreased susceptibility to MPP+-induced mitochondrial dysfunction was directly correlated with decreased cell death in the α-synuclein over-expressing cells (Fig. 4a). Moreover, ATP levels actually rose in response to MPP+ in the Syn/WT cells, whereas the predicted drop in ATP was only seen in the vector-expressing cells (Fig. 4b). For example, 4 hours after MPP+ treatment vector-expressing cells had dropped to 40% of control ATP levels versus Syn/WT, cells were still above their starting levels (Fig. 4b). Interestingly, both mutants appeared to have an intermediate effect in these assays, which was not apparent when only viability was assessed (Figs 4a and b vs. Fig. 3a).

Specific mechanisms have evolved to maintain energy levels at times of maximal activity and/or when energy production has been impaired. In the brain, these primarily include glycolysis, ketosis, or energy transfer via the creatine kinase/creatine phosphate system (Ames 2000). Decreased consumption could offset energy depletion as well (Nicholls and Budd 2000). The speed with which ATP levels were increased in Syn/WT cells (< 30 min; Fig. 5b) suggests that ATP is being utilized/recruited from a different pool rather than being newly synthesized. One such pool would come from glycolysis as the cells switched from aerobic to anaerobic metabolism in response to complex I inhibition via MPP+ (Nicklas et al. 1985; Vyas et al. 1986). In general, a biphasic curve is observed as glucose utilization increases rapidly shortly after MPP+ treatment and then declines. Presumably, the latter represents the inability of glycolysis to keep up with the cell's energy needs. Both Syn/WT and vector-expressing cells exhibited biphasic glucose utilization curves, suggesting that both cell types tapped into this pool for energy compensation (Fig. 5a). CPK activity, however, was not increased following MPP+ treatment of Syn/WT cells, whereas a time-dependent induction in CPK activity was seen in the vector-expressing cells (Fig. 5b). Thus, the increased levels of ATP in Syn/WT cells following MPP+ treatment (Fig. 4b) did not arise from the activation of the creatine kinase/creatine phosphate system.

Normally the brain derives most of its energy from the oxidation of glucose, although ketone bodies can replace glucose during development or starvation (Robinson and Williamson 1980; Edmond et al. 1985). Recently, it has been found that ketone bodies can also serve as an alternative energy source in adult brains particularly during ischemia (Zou et al. 2002; Veech et al. 2001; El-Abhar et al. 2002; Suzuki et al. 2002). Because ketone bodies such as BHB feed into the acetyl CoA pathway, they ensure the continuing function of the tricarboxy acid cycle and, hence a supply of NADH for electron transport (Kashiwaya et al. 1997; Sato et al. 1995). In Syn/WT cells, energy depletion via MPP+-mediated mitochondrial impairment appeared to trigger a process whereby BHB levels were dramatically induced providing a critical alternative resource (Fig. 5c). Cells without α-synuclein or those expressing mutant forms did not appear to utilize this pathway. In support of the present findings, addition of BHB has been shown to protect mesencephalic neurons from MPP+ toxicity (Kashiwaya et al. 2000). The mechanism by which α-synuclein overexpression leads to increased BHB levels is unclear and remains to be elucidated.

dbcAMP-treated cells are more resistant MPP+ toxicity

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References

Molecular programs underlying brain development are spatially and temporally complex. However, recent advances in large-scale gene expression profiling have begun to detail comprehensive genetic programs associated with these processes. For example, in the developing hippocampus, microarray analysis revealed alterations in at least 16 transcriptional clusters related to distinct functional pathways (Mody et al. 2001). Of particular note is the finding that all of the major genes involved in glycolysis were up-regulated in the course of differentiation (Mody et al. 2001). This is in keeping with the maturational shift from ketone bodies to glucose in the brain. The present data point to a similar shift in that, following dbcAMP treatment of MN9D cells, increased ATP levels and CPK activity were observed (Table 4) as well as increased resistance to mitochondrial insults (Figs 7 and 8, Table 3). These findings are consistent with studies showing that dbcAMP treatment of dopaminergic neurons prolongs their survival following transplantation (Branton et al. 1998) and protects them from MPP+-induced degeneration (Hartikka et al. 1992; Mena et al. 1995). Thus, genome-wide expression profiling (Mody et al. 2001), as well as functional studies such as this one, underscores the relationship between energy production and dbcAMP-mediated differentiation.

The present findings highlight a role for α-synuclein as a neuroprotectant expressed in dopaminergic cells. This may reflect a developmental phenomenon, a normal physiological function of this protein, or the unique cellular context of this cell system. In as much as both α-synuclein and MPP+ have been associated with PD, further studies delineating the pathways by which α-synuclein prevents mitochondrial dysfunction may enhance our understanding of this disorder. The Syn/WT MN9D cell line will be a good model system with which to explore the mechanisms associated with α-synuclein protection.

References

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cloning and establishment of α-synuclein stable cell lines
  5. Cell culture and treatments
  6. Measurement of dopaminergic characteristics
  7. Immunocytochemistry
  8. Cell viability assays
  9. Metabolism assays
  10. MN9D cell dbcAMP treatment
  11. Data analysis
  12. Results
  13. Characterization of stable synuclein-expressing MN9D cell lines
  14. Overexpression of α-synuclein does not alter distinct toxin-induced cell death pathways
  15. Overexpression of α-synuclein attenuates MPP+ but not 6-OHDA, H2O2, or A-β-induced cell death
  16. Syn/WT attenuates cell death induced by other inhibitors of electron transport
  17. Overexpression of α-synuclein affects energy levels in dopaminergic cells
  18. Ketone bodies are increased in Syn/WT cells following MPP+ treatment
  19. dbcAMP treatment of stable cell lines
  20. Overexpression of Syn/WT does not rescue dbcAMP treated cells from MPP+ toxicity
  21. Metabolic characteristics are unchanged in response to MPP+ following dbcAMP treatment
  22. Discussion
  23. Context-specific role of α-synuclein in various cell death pathways
  24. Role of α-synuclein in CNS-derived dopaminergic cells
  25. Mechanisms of neuroprotection
  26. dbcAMP-treated cells are more resistant MPP+ toxicity
  27. Acknowledgements
  28. References
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