SEARCH

SEARCH BY CITATION

Keywords:

  • dopamine;
  • GPR37;
  • neurodegeneration;
  • neurotoxin;
  • Pael-R;
  • Parkinson's disease

Abstract

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

G protein-coupled receptor 37 (GPR37) is suggested to be implicated in the pathogenesis of Parkinson's disease and is accumulating in Lewy bodies within afflicted brain regions. Over-expressed GPR37 is prone to misfolding and aggregation, causing cell death via endoplasmic reticulum stress. Although the cytotoxicity of misfolded GPR37 is well established, effects of the functional receptor on cell viability are still unknown. An N2a cell line stably expressing green fluorescent protein (GFP)-tagged human GPR37 was created to study its trafficking and effects on cell viability upon challenge with the toxins 1-methyl-4-phenylpyridinium (MPP+), rotenone and 6-hydroxydopamine (6-OHDA). Neuronal-like differentiation into a tyrosine hydroxylase expressing phenotype, using dibutyryl-cAMP, induced trafficking of GPR37 to the plasma membrane. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability and lactate dehydrogenase (LDH) cell death assays revealed that GPR37 was protective against all three toxins in differentiated cells. In undifferentiated cells, the majority of GPR37 was cytoplasmic and the protective effects were more variable: GPR37 expression protected against rotenone and MPP+ but not against 6-OHDA in MTT assays, while it protected against 6-OHDA but not against MPP+ or rotenone in lactate dehydrogenase (LDH) assays. These results suggest that GPR37 functionally trafficked to the plasma membrane protects against toxicity.

Abbreviations used
AR-JP

autosomal recessive juvenile Parkinson's disease

BSA

bovine serum albumin

DAT

dopamine transporter

dbcAMP

dibuturyl-cyclic

DMEM

Dulbecco's modified essential medium

GPCR

G protein-coupled receptor

GPR37

G protein-coupled receptor 37

LB

Lewy bodies

LDH

lactate dehydrogenase

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NET

norepinephrine transporter

PD

Parkinson's disease

PFA

paraformaldehyde

ROS

reactive oxygen species

SERT

serotonin transporter

TH

tyrosine hydroxylase

Parkinson's disease (PD) is a common and progressive neurodegenerative disorder, in which the loss of nigrostriatal dopaminergic neurons causes motor abnormalities, autonomic dysfunctions and neuropsychiatric symptoms (Dauer and Przedborski 2003). At the subcellular level, a neuropathological feature of PD is the accumulation of heavily ubiquitinated protein aggregates, so-called Lewy bodies (LB). In autosomal recessive juvenile PD (AR-JP), mutations in the gene encoding parkin lead to impaired parkin-mediated protein ubiquitination (Kitada et al. 1998; Imai et al. 2000; Shimura et al. 2000; Zhang et al. 2000). This results in impaired protein degradation and subsequently in neuronal death (reviewed by Wang and Takahashi 2007).

The orphan G protein-coupled receptor (GPCR) GPR37 is a substrate of parkin (Imai et al. 2001). Aggregated GPR37 has been found at the core of LBs in post-mortem brains of PD patients, as well as in the substantia nigra of AR-JP patients (Imai et al. 2001; Murakami et al. 2004). Further, the receptor misfolds, forms aggregates and induces cell death via ER stress when transiently over-expressed in vitro (Imai et al. 2001) and when virally over-expressed in mouse substantia nigra (Kitao et al. 2007; Dusonchet et al. 2009). It has therefore been suggested that over-expression and aggregation of GPR37 may play an important role in the progression of PD pathology, particularly in AR-JP.

Although several studies report on the cytotoxicity of over-expressed and misfolded GPR37, the effects of functional GPR37 on cell viability are less known. We therefore created a mouse neuroblastoma cell line stably expressing GFP-tagged human GPR37 to study its trafficking and the consequences for cell viability and cell death, specifically in response to rotenone, MPP+ or 6-OHDA. These toxins are selective for catecholaminergic/dopaminergic cells and induce changes that mimic certain cellular aspects of PD, while acting via different mechanisms. Rotenone and MPP+ are potent inhibitors of complex I and thereby induce mitochondrial dysfunction (reviewed by Dauer and Przedborski 2003). 6-OHDA, on the other hand, accumulates in the cytoplasm of monoaminergic neurons and auto-oxidizes or is degraded by monoamine oxidases into reactive oxygen species (ROS) (reviewed by Blum et al. 2001; Dauer and Przedborski 2003). Further, LB-like formations have been observed upon chronic rotenone treatment (Betarbet et al. 2000) and 6-OHDA activates the unfolded protein response (Elkon et al. 2001; Holtz and O'Malley 2003). Cell death induced by these three toxins in N2a cells with or without GPR37 was assessed. Our results demonstrate a neuroprotective role of GPR37 that correlates with increased receptor levels at the plasma membrane.

Materials and methods

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

Materials

N2a cells were a gift from Dr Patrick Allen, Rockefeller University. Reagents for cell culture and transfection were from Invitrogen, Lidingö, Sweden. Drugs and chemicals including MPP+, rotenone, 6-OHDA, dibutyryl-cAMP (dbcAMP), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), paraformaldehyde (PFA), bovine serum albumin (BSA), HCl and isopropanol were from Sigma-Aldrich, Munich, Germany.

Generation of a stable cell line

N2a mouse neuroblastoma cells (Klebe and Ruddle 1969) were seeded at 1 × 104 cells/mL in culture medium [Dulbecco's modified essential medium (DMEM) containing 4.5 g/L d-glucose, non-essential amino acids, 10% foetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin] in 100 mm petri dishes. Transfections were performed by adding 25 μg of pCMV6-hGPR37-GFP (OriGene, Rockville, MD, USA) and 5 μL lipofectamine in 5 mL OPTI-MEM to each dish. Transfected clones were selected using Geneticin (500 μg/mL) and stable expression was confirmed by fluorescence microscopy (Carl Zeiss, Jena, Germany).

Cell culture

Cells were maintained in culture medium containing 50 μg/mL Geneticin at 37°C, 5% CO2 and split 1 : 5 every 3–4 days. For experiments, cells were seeded in OPTI-MEM supplemented with 1% foetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. For differentiation, 1 mM dbcAMP was added to the culture medium at seeding and experiments were performed after 3–4 days.

Quantification of cell differentiation and hGPR37-GFP expression in live cells

N2a is a neuroblastoma cell line (Klebe and Ruddle 1969) known to express the neuronal βIII tubulin (Luduena et al. 1988; Minana et al. 1990). Cells were exposed to low-serum medium and dbcAMP to induce a neuronal-like differentiation and TH expression, using a protocol adapted from Tremblay et al. (2010). Cells were seeded at 1 × 104 cells/mL in LabTek eight-chambered cover glasses (Nalgene Nunc; Thermo Scientific, Waltham, MA, USA). Confocal images were acquired with an LSM510 ConfoCor3 instrument (Carl Zeiss, Jena, Germany) modified to enable imaging of cells expressing low levels of hGPR37-GFP using silicone avalanche photodiodes (SPCM-AQR-1X; PerkinElmer, Waltham, MA, USA). The C-Apochromat 40×, NA = 1.2, water immersion UV-VIS-IR objective was used for all images. GFP fluorescence was excited using the 488 nm line of the Ar/ArKr laser. Emitted light was collected using the HFT 488/543/633 main dichroic beam splitter and the band-pass filter BP505-530. Three visual fields per well in two different wells from three separate seedings yielded a minimum of 1700 cells per differentiation state and cell type for analysis. All cells and all primary processes of a given cell that exceeded its diameter in length were counted. For each image, the number of processes was divided by the number of cells. The lengths of all counted processes were also determined. One representative image of hGPR37-GFP cells from each well and each seeding was selected for quantification of fluorescence intensity at the membrane and in the cytoplasm respectively (minimum 180 cells per differentiation state). The membrane was identified in overlay images of the transmission channel and the GFP channel. Membrane staining using DiD lipid dye (Invitrogen) was used to confirm correct alignment of the fluorescence and transmission images. Image analysis was performed using the Carl Zeiss LSM image browser software (Carl Zeiss). The average fluorescence intensities (I) in defined regions of interest (ROI) at the membrane and in the cytoplasm were determined for each cell. The average of all Imembrane values in a given image was divided by the average of all Icytoplasm values in the same image, and the ratios of averages for all images were plotted.

Immunocytochemistry

Cells were seeded on poly-l-lysine-coated cover glasses at 5 × 104 cells/cm2 and immunocytochemistry was performed according to standard protocols 3 days after seeding. Briefly, cells were fixed for 30 min in 4% ice-cold formaldehyde (freshly prepared from PFA) followed by blocking for 1 h in PBS containing 3% BSA and 0.3% Triton X-100. Cover glasses were incubated in primary antibody against tyrosine hydroxylase (TH) (1 : 500, #22941, Immunostar, Hudson, WI, USA) overnight at 4°C, followed by incubation with anti-mouse Alexa 568 secondary antibody (1 : 2000, #A11031; Invitrogen, Lidingö, Sweden) for 1 h. Cover glasses were dried and mounted on poly-lysine-coated glass slides (Histolab, Stockholm, Sweden) using FluorSave mounting medium (Calbiochem, San Diego, CA, USA). Confocal images were collected with the LSM510 ConfoCor3 instrument (Carl Zeiss) described above using the C-Apochromat 40×, NA = 1.2, water immersion UV-VIS-IR objective. Alexa 568 was excited using the 543 He/Ne laser. Emitted light was collected using the HFT 488/543/633 main dichroic beam splitter and the long-pass filter LP580. Brightfield images were used to determine the fraction of positively stained cells.

MTT and LDH assays

Cells were seeded in 96-well plates at a density of 1 × 104 cells per well and incubated at 37°C, 5% CO2 for 3 days before addition of MPP+, rotenone or 6-OHDA. Cytotoxicity was assayed 24 h later. For MTT assays, 0.5 mg/mL MTT was added to the medium and cells were incubated for 1 h at 37°C, 5% CO2. The medium was removed and cells were lysed in isopropanol containing 0.04 M HCl. Absorbance was measured at 550 nm using an Anthos 2020 microplate reader (Biochrom, Berlin, Germany). LDH assays were performed using a kit (#TOX7; Sigma-Aldrich) according to the manufacturer's instructions. Briefly, cell lysis solution was added to half of the wells within each treatment group and cells were incubated for 45 min at 37°C, 5% CO2. Plates were centrifuged to pellet detached cells and debris, and 1/10 of the medium was assayed. Samples were incubated with the assay mixture for 10 min and absorbance was read at 490 nm using a THERMOmax ELISA plate reader (Molecular Devices, Sunnyvale, CA, USA). Absorbance at 650 nm was used as reference. In LDH assays, the lowest concentration of each toxin was used, as the largest cell type effects were seen at these concentrations in MTT assays. For the assessment of cell death, LDH released into the medium was expressed as a fraction of total LDH. Total LDH activity was used as a measure of total number of cells (viable and dead).

For MTT assays, data were pooled from four separate experiments. For LDH assays, data were pooled from three separate experiments. Data were normalized within each plate, containing one control group and several treatment groups. Untreated controls within each cell type were set to 100%. Absorbance in treatment groups was expressed as a percentage of control within cell type.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). Unpaired, two-tailed Student's t-test was used for datasets with two groups. Data sets with more than two groups and two grouping variables were analysed by two-way anova. Where a significant interaction was found, post-hoc analysis was performed by one-way anova for multiple comparisons or by Student's t-test for pairwise comparisons. Where no significant interaction but a significant variable effect was found, post-hoc analysis was performed using the Bonferroni test. Values are reported as mean ± SEM and effects were considered significant at p < 0.05.

Results and discussion

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

Wild-type and GPR37-expressing N2a cells differentiate into a catecholaminergic, neuronal-like phenotype upon dbcAMP treatment

Wild-type N2a and GPR37-expressing N2a cells have similar morphology and differentiation patterns (Fig. 1). The degree of differentiation was determined by the number of primary processes (defined as primary extensions longer than the soma diameter) and the average process length. Upon differentiation, the average number of primary processes per cell increased from 0.14 ± 0.02 to 0.52 ± 0.04 in GPR37-expressing cells and from 0.13 ± 0.03 to 0.51 ± 0.08 in wild-type cells (Fig. 1a, left). GPR37-expression had no effect on average process length (Fig. 1a, right) or overall morphology (Fig. 1a and b).

image

Figure 1. Wild-type N2a and G protein-coupled receptor 37 (GPR37)-expressing cells have similar morphology and differentiation patterns. (a) Quantification of primary processes per cell and process length in undifferentiated and differentiated wild-type versus GPR37-expressing cells. Processes per cell: (treatment: F(1,20) = 57.75, p < 0.01; cell type: F(1,20) = 0.10; interaction: F(1,20) = 0.003). Process length: (treatment: F(1,20) = 3.02; cell type: F(1,20) = 0.71; interaction: F(1,20) = 0.004). (b) Representative images showing the morphology of live undifferentiated and differentiated cells, and immunocytochemistry for tyrosine hydroxylase (TH). TH expression was increased in all differentiated versus undifferentiated cells in both cell types (wild-type and GPR37-expressing). Differentiation and membrane expression data were analysed by two-way anova followed by Student's t-test for pairwise comparisons; ***< 0.001 versus undifferentiated cells.

Download figure to PowerPoint

Expression of TH as a catecholaminergic marker was analysed by immunocytochemistry. Strong staining was detected in all differentiated wild-type and GPR37-expressing cells (Fig. 1b), whereas only weak TH staining was detected in undifferentiated cells. The neuronal-like, catecholaminergic phenotype induced by differentiation was used in subsequent studies of GPR37 effects on cell viability in response to different catecholaminergic toxins.

Plasma membrane localization of GPR37 upon differentiation

Differentiation not only increased TH expression and process formation but also triggered substantial trafficking of GPR37 to the plasma membrane (Fig. 2). The average fluorescence intensity (I) in a region is proportional to the density of hGPR37-GFP in that same region. Thus, the relative density of GPR37 at the plasma membrane was determined by calculation of average Imembrane/ average Icytoplasm. This ratio increased more than twofold in differentiated versus undifferentiated cells, reflecting substantial trafficking of GPR37 to the plasma membrane upon differentiation (Fig. 2a).

image

Figure 2. G protein-coupled receptor 37 (GPR37) is trafficked to the membrane upon differentiation. (a) Quantification of GFP fluorescence intensity at the plasma membrane versus the cytoplasm. Data were analysed by Student's t-test (n = 6 images per differentiation state; ***p < 0.001). (b) Representative images showing hGPR37-GFP (green) expression patterns in undifferentiated and differentiated cells respectively.

Download figure to PowerPoint

Fluorescence intensity distribution in both undifferentiated and differentiated cells suggests limited receptor aggregation (Fig. 2b) and the majority of intracellular GPR37 in our N2a cell line appears functionally trafficked.

GPR37 protects against MPP+-, rotenone- and 6-OHDA-induced toxicity in differentiated cells

To assess the impact of GPR37 expression on cytotoxicity, cells were treated with MPP+, rotenone or 6-OHDA, and the toxic effects were evaluated by MTT and LDH assays (Figs 3 and 4). MTT reduction into formazan by mitochondrial complex II was used as a measure of cell viability. Released LDH was used as a measure of cell death, while total LDH activity was used as a measure of total number of cells (viable and dead). GPR37 expression did not affect viability in the absence of toxin exposure (data not shown). However, in differentiated cells, expression of GPR37 protected against all three toxins as assessed with the MTT and the LDH assays (Figs 3b, d, f, 4b, d and f).

image

Figure 3. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays reveal G protein-coupled receptor 37 (GPR37)-mediated protection against MPP+-, rotenone- and 6-OHDA-induced toxicity in N2a cells. Cell viability was quantified by MTT assay. (a) Undifferentiated cells treated with MPP+ versus vehicle (toxin: F(4,291) = 4.18, < 0.01; cell type: F(1,291) = 63.84, < 0.01; interaction: F(4,291) = 4.59, < 0.01). (b) Differentiated cells treated with MPP+ versus vehicle (toxin: F(4, 308) = 249.8, < 0.01; cell type: F(1,308) = 27.32, < 0.01; interaction: F(4,308) = 5.88, < 0.01). (c) Undifferentiated cells treated with rotenone versus vehicle (toxin: F(2,186) = 24.70, < 0.01; cell type: F(1,186) = 45.64, < 0.01; interaction: F(2,186) = 12.44, < 0.01). (d) Differentiated cells treated with rotenone versus vehicle (toxin: F(2,186) = 1481, < 0.01; cell type: F(1,186) = 16.96, < 0.01; interaction: F(2,186) = 4.81, < 0.01). (e) Undifferentiated cells treated with 6-OHDA versus vehicle (toxin: F(2,234) = 160.6 < 0.01; cell type: F(1,234) = 3.27; interaction: F(2,234) = 1.11). (f) Differentiated cells treated with 6-OHDA versus vehicle (toxin: F(2,186) = 89.03, < 0.01; cell type: F(1,186) = 76.29, < 0.01; interaction: F(2,186) = 20.03, < 0.01). Data were analysed by two-way anova followed by Student's t-test for pairwise comparisons and one-way anova for multiple comparisons; ***< 0.001 versus vehicle within cell type, ##< 0.01, ###< 0.001 versus wild-type at a given concentration.

Download figure to PowerPoint

image

Figure 4. Lactate dehydrogenase (LDH) assays reveal G protein-coupled receptor 37 (GPR37)-mediated protection against MPP+-, rotenone- and 6-OHDA-induced toxicity in N2a cells. (a) Undifferentiated cells treated with MPP+ versus vehicle. Released LDH: (toxin: F(1,52) = 24.47, < 0.01; cell type: F(1,52) = 0.006; interaction: F(1,52) = 0.009), total LDH: (toxin: F(1,52) = 14.94, < 0.01; cell type: F(1,52) = 7.34, < 0.01; interaction: F(1,52) = 7.47, p = 0.01). (b) Differentiated cells treated with MPP+ versus vehicle. Released LDH: (toxin: F(1,51) = 11.62, < 0.01; cell type: F(1,51) = 8.03, < 0.01; interaction: F(1,51) = 7.84, < 0.01), total LDH: (toxin: F(1,50) = 0.11; cell type: F(1,50) = 9.47, < 0.01; interaction: F(1,50) = 9.45, < 0.01). (c) Undifferentiated cells treated with rotenone versus vehicle. Released LDH: (toxin: F(1,56) = 8.37, < 0.01; cell type: F(1,56) = 1.95; interaction: F(1,56) = 1.97), total LDH: (toxin: F(1,54) = 1.38; cell type: F(1,54) = 3.60; interaction: F(1,54) = 3.66). (d) Differentiated cells treated with rotenone versus vehicle. Released LDH: (toxin: F(1,56) = 10.58, < 0.01; cell type: F(1,56) = 10.08, < 0.01; interaction: F(1,56) = 9.77, < 0.01), total LDH: (toxin: F(1,56) = 1.41; cell type: F(1,56) = 13.12, < 0.01; interaction: F(1,56) = 12.93, < 0.01). (e) Undifferentiated cells treated with 6-OHDA versus vehicle. Released LDH: (toxin: F(1,52) = 133.4, < 0.01; cell type: F(1,52) = 9.41, < 0.01; interaction: F(1,52) = 9.50, < 0.01), total LDH: (toxin: F(1,52) = 21.0, < 0.01; cell type: F(1,52) = 9.09, < 0.01; interaction: F(1,52) = 9.17, < 0.01). (f) Differentiated cells treated with 6-OHDA versus vehicle. Released LDH: (toxin: F(1,52) = 57.31, < 0.01; cell type: F(1,52) = 9.09, < 0.01; interaction: F(1,52) = 8.84, < 0.01), total LDH: (toxin: F(1,50) = 16.84, p = 0.01; cell type: F(1,50) = 18.24, < 0.01; interaction: F(1,50) = 18.12, < 0.01) Data were analysed by two-way anova followed by Student's t-test for pairwise comparisons; *< 0.05, **< 0.01, ***< 0.001 versus vehicle within cell type, ##< 0.01, ###< 0.001 versus wild-type within treatment.

Download figure to PowerPoint

In response to 50, 100 and 250 μM MPP+, expression of GPR37 improved cell survival more than 1.5-fold compared with wild-type as measured with the MTT assay (Fig. 3b). At 500 μM, almost all cells died and there was no difference between the cell types. Similarly, expression of GPR37 doubled the cell survival compared with wild-type at both 1 and 10 μM rotenone (Fig. 3d). On treatment with 20 μM 6-OHDA, viability decreased by 51 ± 16% in wild-type cells, whereas GPR37 expressing cells were unaffected (Fig. 3f). Moreover, at the higher concentration (100 μM), expression of GPR37 improved cell survival more than 2.5-fold.

Similarly to MTT assays, LDH assays revealed GPR37-mediated protection against all three toxins in differentiated cells. LDH release was used as a measure of cell death. MPP+ and rotenone increased LDH release in wild-type cells by 34 ± 9% and 53 ± 14%, respectively, whereas GPR37-expressing cells were unaffected (Figs 4b and d). The 6-OHDA-induced increase in LDH release was attenuated by 65 ± 21% in the GPR37-expressing cells compared with wild-type (Fig. 4f). Total LDH activity was measured to assess the total number of cells (viable and dead). MPP+ reduced total LDH activity by 17 ± 8% in wild-type cells, but caused 21 ± 9% increase in GPR37-expressing cells. Rotenone had no effect in wild-type cells. However, in GPR37-expressing cells, rotenone increased total LDH activity by 28 ± 9%. 6-OHDA reduced total LDH activity by 41 ± 5% in wild-type cells. This effect was abolished in GPR37-expressing cells. Taken together, we found that expression of GPR37 protects differentiated N2a cells against all three studied toxins.

GPR37-mediated protective effects are less pronounced in undifferentiated cells

To investigate the effect of GPR37 on toxicity when the receptor is mainly located in the cytoplasm, we also studied the response to MPP+, rotenone and 6-OHDA in undifferentiated cells. GPR37 mediated attenuation of both MPP+ and rotenone-induced toxicity in the MTT assay, but not of that of 6-OHDA (Fig. 3). In wild-type N2a cells, the number of viable cells was reduced by 25 ± 3%, 28 ± 4% and 27 ± 3% at 100, 250 and 500 μM MPP+ respectively. These toxin effects were abolished in GPR37-expressing cells (Fig. 3a). A similar pattern was observed after treatment with 1 and 10 μM rotenone, which decreased the number of viable wild-type cells by 22 ± 4% and 32 ± 4% respectively. The toxic effects of rotenone were abolished in the GPR37-expressing cells (Fig. 3c). On the other hand, expression of GPR37 did not prevent the toxic effects of 6-OHDA (Fig. 3e).

Expression of GPR37 protected against 6-OHDA-induced effects on LDH release, but not against MPP+- or rotenone-induced effects. In cells treated with MPP+ or rotenone, there was no difference between wild-type and GPR37-expressing cells (Fig. 4a and c). After treatment with 6-OHDA, on the other hand, expression of GPR37 attenuated the toxin-induced increase of LDH release by 34 ± 11% (Fig. 4e). We also measured total LDH activity. Here, MPP+ and 6-OHDA caused a significant reduction, and GPR37 had a protective effect in both cases. MPP+ reduced the total LDH activity by 22 ± 3% in wild-type cells. This toxic effect was abolished in GPR37-expressing cells (Fig. 4a). Similarly, 6-OHDA-treatment decreased total LDH activity by 43 ± 2% in wild-type cells, whereas GPR37-expressing cells were unaffected (Fig. 4e). However, expression of GPR37 did not affect total LDH activity after rotenone treatment (Fig. 4c).

Functionally trafficked GPR37 protects N2a cells, while the misfolded receptor is cytotoxic

Taken together, expression of GPR37 was found to robustly protect N2a cells against MPP+-, rotenone- and 6-OHDA-induced toxicity. In contrast, previous reports using transiently transfected cell lines or viral GPR37 over-expression in vivo have reported that GPR37 forms aggregates and increases cell death (Imai et al. 2001; Kitao et al. 2007; Dusonchet et al. 2009). This aggregation also correlates with accumulation of GPR37 in LBs in post-mortem PD brains (Murakami et al. 2004) and with progressive loss of catecholaminergic neurons in parkin-deficient GPR37 over-expressing mice (Wang et al. 2008). Overall, there is strong evidence for GPR37 toxicity being caused by misfolded and aggregated receptors. Moreover, previous observations of resistance to MPTP in GPR37 KO mice and hypersensitivity to 6-OHDA in GPR37 over-expressing transgenic mice (Marazziti et al. 2004; Imai et al. 2007) may be because of lack of GPR37 aggregation in the former and increased GPR37 aggregation in the latter.

In our cell line, GPR37 was expressed at moderate levels (about 6 × 102 to 1.8 × 104 molecules per cell as measured by fluorescence correlation spectroscopy; data not shown). Further, we observed an even receptor distribution in the cytoplasm with a differentiation-induced trafficking of the receptor to the plasma membrane (Fig. 2). These observations suggest that the receptor is functionally trafficked, that is, that loss of receptors because of aggregation is limited. This may explain the lack of basal GPR37-induced cytotoxicity in our system. The receptor trafficking observed in this study is likely to be important for the protective effects on cell viability, and might explain the differences to the previous studies reporting toxic effects of misfolded and aggregated GPR37 both in vitro and in vivo. Although GPR37 is prone to misfolding, and when misfolded may induce cell death, our results show that its functional trafficking in N2a cells is protective against MPP+, rotenone and 6-OHDA. Further, this protection is enhanced upon differentiation into a catecholaminergic neuronal phenotype with increased receptor levels at the plasma membrane. In accordance with our data, the molecular chaperone 4-phenylbutyrate, which reduces ER accumulation of GPR37 and promotes trafficking to the plasma membrane, also reduces cytotoxicity caused by receptor over-expression (Mimori et al. 2012).

Conclusions and perspectives

The pathways determining trafficking of GPR37, are still poorly understood. We show here that differentiation of N2a cells into a catecholaminergic phenotype leads to forward trafficking of stably expressed GPR37 from the cytoplasm to the membrane. In differentiated N2a cells, expression of GPR37 protects against MPP+, rotenone and 6-OHDA-induced toxicity, while the receptor is only partly protective in undifferentiated cells. Further elucidation of the protective mechanisms against these three toxins will be important to understand the potential role of GPR37 in neuroprotection. These findings indicate that the development of GPR37-directed molecular chaperones that not only reduce aggregate formation but also promote plasma membrane localization of GPR37, may be novel neuroprotective agents in PD.

Acknowledgements

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

The work was funded by the Swedish Research Council and Karolinska Institutet. The authors have no conflict of interest to declare.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References
  • 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.
  • Blum D., Torch S., Lambeng N., Nissou M., Benabid A. L., Sadoul R. and Verna J. M. (2001) Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson's disease. Prog. Neurobiol. 65, 135172.
  • Dauer W. and Przedborski S. (2003) Parkinson's disease: mechanisms and models. Neuron 39, 889909.
  • Dusonchet J., Bensadoun J. C., Schneider B. L. and Aebischer P. (2009) Targeted overexpression of the parkin substrate Pael-R in the nigrostriatal system of adult rats to model Parkinson's disease. Neurobiol. Dis. 35, 3241.
  • Elkon H., Melamed E. and Offen D. (2001) 6-Hydroxydopamine increases ubiquitin-conjugates and protein degradation: implications for the pathogenesis of Parkinson's disease. Cell. Mol. Neurobiol. 21, 771781.
  • Holtz W. A. and O'Malley K. L. (2003) Parkinsonian mimetics induce aspects of unfolded protein response in death of dopaminergic neurons. J. Biol. Chem. 278, 1936719377.
  • Imai Y., Soda M. and Takahashi R. (2000) Parkin suppresses unfolded protein stress-induced cell death through its E3 ubiquitin-protein ligase activity. J. Biol. Chem. 275, 3566135664.
  • Imai Y., Soda M., Inoue H., Hattori N., Mizuno Y. and Takahashi R. (2001) An unfolded putative transmembrane polypeptide, which can lead to endoplasmic reticulum stress, is a substrate of Parkin. Cell 105, 891902.
  • Imai Y., Inoue H., Kataoka A. et al. (2007) Pael receptor is involved in dopamine metabolism in the nigrostriatal system. Neurosci. Res. 59, 413425.
  • Kitada T., Asakawa S., Hattori N., Matsumine H., Yamamura Y., Minoshima S., Yokochi M., Mizuno Y. and Shimizu N. (1998) Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392, 605608.
  • Kitao Y., Imai Y., Ozawa K. et al. (2007) Pael receptor induces death of dopaminergic neurons in the substantia nigra via endoplasmic reticulum stress and dopamine toxicity, which is enhanced under condition of parkin inactivation. Hum. Mol. Genet. 16, 5060.
  • Klebe R. J. and Ruddle F. H. (1969) Neuroblastoma: cell culture analysis of a differentiating stem cell system. J. Cell Biol. 43, 69a.
  • Luduena R. F., Zimmermann H. P. and Little M. (1988) Identification of the phosphorylated beta-tubulin isotype in differentiated neuroblastoma cells. FEBS Lett. 230, 142146.
  • Marazziti D., Golini E., Mandillo S., Magrelli A., Witke W., Matteoni R. and Tocchini-Valentini G. P. (2004) Altered dopamine signaling and MPTP resistance in mice lacking the Parkinson's disease-associated GPR37/parkin-associated endothelin-like receptor. Proc. Natl Acad. Sci. USA 101, 1018910194.
  • Mimori S., Okuma Y., Kaneko M., Kawada K., Hosoi T., Ozawa K., Nomura Y. and Hamana H. (2012) Protective effects of 4-phenylbutyrate derivatives on the neuronal cell death and endoplasmic reticulum stress. Biol. Pharm. Bull. 35, 8490.
  • Minana M. D., Felipo V. and Grisolia S. (1990) Inhibition of protein kinase C induces differentiation in Neuro-2a cells. Proc. Natl Acad. Sci. USA 87, 43354339.
  • Murakami T., Shoji M., Imai Y., Inoue H., Kawarabayashi T., Matsubara E., Harigaya Y., Sasaki A., Takahashi R. and Abe K. (2004) Pael-R is accumulated in Lewy bodies of Parkinson's disease. Ann. Neurol. 55, 439442.
  • Shimura H., Hattori N., Kubo S. et al. (2000) Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase. Nat. Genet. 25, 302305.
  • Tremblay R. G., Sikorska M., Sandhu J. K., Lanthier P., Ribecco-Lutkiewicz M. and Bani-Yaghoub M. (2010) Differentiation of mouse Neuro 2A cells into dopamine neurons. J. Neurosci. Methods 186, 6067.
  • Wang H.-Q. and Takahashi R. (2007) Expanding insights on the involvement of endoplasmic reticulum stress in Parkinson's disease. Antioxid. Redox Signal. 9, 553561.
  • Wang H.-Q., Imai Y., Inoue H., Kataoka A., Iita S., Nukina N. and Takahashi R. (2008) Pael-R transgenic mice crossed with parkin deficient mice displayed progressive and selective catecholaminergic neuronal loss. J. Neurochem. 107, 171185.
  • Zhang Y., Gao J., Chung K. K., Huang H., Dawson V. L. and Dawson T. M. (2000) Parkin functions as an E2-dependent ubiquitin- protein ligase and promotes the degradation of the synaptic vesicle-associated protein, CDCrel-1. Proc. Natl Acad. Sci. USA 97, 1335413359.