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Keywords:

  • dopamine;
  • ectodomain shedding;
  • EGF;
  • EGFR;
  • neurotrophic factor;
  • striatum

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

J. Neurochem. (2011) 10.1111/j.1471-4159.2011.07295.x

Abstract

Epidermal growth factor (EGF) and structurally related peptides promote neuronal survival and the development of midbrain dopaminergic neurons; however, the regulation of their production has not been fully elucidated. In this study, we found that the treatment of striatal cells with dopamine agonists enhances EGF release both in vivo and in vitro. We prepared neuron-enriched and non-neuronal cell-enriched cultures from the striatum of rat embryos and challenged those with various neurotransmitters or dopamine receptor agonists. Dopamine and a dopamine D1-like receptor agonist (SKF38393) triggered EGF release from neuron-enriched cultures in a dose-dependent manner. A D2-like agonist (quinpirole) increased EGF release only from non-neuronal cell-enriched cultures. The EGF release from striatal neurons and non-neuronal cells was concomitant with ErbB1 phosphorylation and/or with the activation of a disintegrin and metalloproteinase and matrix metalloproteinase. The EGF release from neurons was attenuated by an a disintegrin and metalloproteinase/matrix metalloproteinase inhibitor, GM6001, and a calcium ion chelator, BAPTA/AM. Transfection of cultured striatal neurons with alkaline phosphatase-tagged EGF precursor cDNA confirmed that dopamine D1-like receptor stimulation promoted both ectodomain shedding of the precursor and EGF release. Therefore, the activation of striatal dopamine receptors induces shedding and release of EGF to provide a retrograde neurotrophic signal to midbrain dopaminergic neurons.

Abbreviations used
ADAM

a disintegrin and metalloproteinase

AP

alkaline phosphatase

BoNT/A

botulinum neurotoxin type A

E

embryonic day

EGF

epidermal growth factor

GFAP

glial fibrillary acidic protein

GPCR

G-protein coupled receptors

HB-EGF

heparin-binding EGF-like growth factor

MMP

matrix metalloproteinase

PMA

phorbol 12-myristate 13-acetate

PND

postnatal day

TGFα

transforming growth factor alpha

The epidermal growth factor (EGF) family includes EGF, transforming growth factor-alpha (TGFα), and heparin-binding EGF-like growth factor (HB-EGF) (Schneider and Wolf 2009). Most of these factors are expressed in the CNS together with their receptor, ErbB1 (Wong and Guillaud 2004; Piao et al. 2005) and regulate neural stem cell proliferation, neuronal survival, or differentiation (Seroogy et al. 1995; Cameron et al. 1998; Abe et al. 2009). Members of the EGF family are initially synthesized as large, membrane-anchored precursor proteins (Schneider and Wolf 2009) and released through proteolytic cleavage at their extracellular domain (i.e. shedding) by matrix metalloproteinases (MMPs), such as a disintegrin and metalloproteinases (ADAMs) (Le Gall et al. 2003; Horiuchi et al. 2007; Higashiyama et al. 2008). The stimulation of G-protein coupled receptors (GPCRs) often activates these metalloproteinases and promotes ectodomain shedding, which leads to ErbB1 activation (Daub et al. 1996; Prenzel et al. 1999). However, the biological implication of ectodomain shedding in neural plasticity and neurotrophic signaling is not fully understood (Ozaki et al. 2004; Shishido et al. 2006).

Dopaminergic neurons in the substantia nigra receive neurotrophic signals from their main target region – the striatum (Unsicker 1994; Olson 1997). Surgical lesions of the nigrostriatal pathway result in the degeneration of dopaminergic neurons, but the administration of neurotrophic factors rescues them from cell death or enhances their regeneration (Beck et al. 1995; Volpe et al. 1998; Iwakura et al. 2005). EGF and other ErbB1 ligands appear to be the neurotrophic factors for midbrain dopaminergic neurons, which express ErbB1 (Seroogy et al. 1995; Abe et al. 2009; Iwakura et al. 2011). In vivo and in vitro studies both indicate that the ErbB1 ligands including EGF, HB-EGF and TGFα support cell survival and the growth of midbrain dopaminergic neurons (Alexi and Hefti 1993; Casper et al. 1994; Ho and Blum 1997; Futamura et al. 2003; Iwakura et al. 2005). We found that EGF exhibits distinct neurotrophic activity on the subset of nigra dopaminergic neurons both in vivo and in vitro (Iwakura et al. 2011). Indeed, both mice lacking an ErbB1 ligand and ErbB1-deficient midbrain cultures exhibit abnormalities in the development of dopaminergic neurons (Blum 1998; Iwakura et al. 2011). In spite of these extensive studies on the neurotrophic actions of ErbB1 ligands, the regulation of their production and release in the target brain regions remains to be characterized.

Our initial finding that, among various neurotransmitters, dopamine specifically induces EGF release from cultured striatal cells prompted us to characterize this molecular process. We took a pharmacologic approach to identify the dopamine receptor subtypes responsible for EGF release. We also tested the ectodomain shedding and vesicular exocytosis of EGF to investigate the molecular mechanism underlying the EGF release by transfecting the tagged EGF precursor gene as well as by measuring the enzyme activity of metalloproteinases. Finally, we discuss the contribution of EGF release as a retrograde neurotrophic signal to dopaminergic development and pathophysiology.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Animals

Sprague–Dawley (SD) rat pups (Japan SLC, Inc., Shizuoka, Japan) were maintained with their dams in the animal care facility of Niigata University Brain Research Institute. All animals were housed with free access to food and water in a temperature-controlled room under a 12-h light/12-h dark cycle. All animal experiments described were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) and approved by the Animal Use and Care Committee guidelines of Niigata University. All efforts were made to minimize animal discomfort and the number of animals used.

Apomorphin (1 mg/kg; Sigma, St Louis, MO, USA) or vehicle (0.2% ascorbic acid in saline) was subcutaneously administered into postnatal day (PND) 7 rats. Some rats subcutaneously received SCH23390 (1 mg/kg; Sigma) 30 min prior to the dopamine agonist challenge as well. After 30 min, rats were anesthetized with isoflurane and killed by decapitation and striatum were removed and frozen.

Neuonal and non-neuronal cultures

Striatal tissues were dissected from embryonic day (E)18-19 rat embryos and dissociated with 1 mg/mL papain solution. Cells were plated on poly-d-lysin-coated dish at a density of 1.0 × 106 cells/mL with Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. After 1-h incubation, the medium was changed to serum-free medium and cultures were grown for 7 days as described previously (Iwakura et al. 2001). On 7 days in vitro, cultures were exposed to phorbol 12-myristate 13-acetate (PMA, 100 ng/mL; Calbiochem, San Diego, CA, USA), SKF38393 (100 μM; Sigma), quinpirole (30 μM; Sigma), or ionomycin (10 μM; Calbiochem). Some cultures were pre-incubated with the exocytosis inhibitor botulinum neurotoxin type A (BoNT/A, 100 nM; a gift from Dr Kozaki, Osaka Prefecture University), a metalloproteinase inhibitor, GM 6001 (galardin, 100 nM; Calbiochem), calphostin C (1 μM; Sigma) or BAPTA/AM (10 μM; Calbiochem) prior to PMA or SKF38393 challenge. Alternatively, mixed non-neuronal cell cultures were prepared from the same striatal tissue. Striatal tissues were dissociated with 0.1% trypsin and cells were plated to plain dishes with Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. Cells were plated at a density of 1.0 × 106 cells/mL and allowed to grow to 80–90% confluence after one passage. After over night incubation in serum-free conditions, mixed non-neuronal cell cultures were similarly challenged with the above dopamine agonists.

Immunocytochemistry

Striatal cultures were fixed in 4% paraformaldehyde in phosphate-buffered saline. Fixed cells were immunolabeled with the anti-human placental alkaline phosphatase antibody (AP, 1 : 50; DAKO, Glostrup, Denmark) and anti-dopamine D1-like receptor antibody (1 : 100; Chemicon, Temecula, CA, USA). Immunoreactivity was visualized by fluorescent secondary antibodies; streptoavidin-Alexa 488 (Vector Laboratories, Burlingame, CA, USA), Alexa488-conjugated anti-mouse IgG, (Vector Laboratories), Alexa546-conjugated anti-mouse IgG (Vector Laboratories), Alexa546-conjugated anti-rabbit IgG antibodies (Vector Laboratories).

Enzyme immunoassay

EGF, TGFα and HB-EGF levels were measured as described previously (Iwakura et al. 2005). In brief, brain tissues and cultured cells were homogenized in 10 volumes of homogenization buffer. The homogenates were centrifuged at 14 000 g for 30 min at 4°C. Culture supernatants (i.e. conditioned medium) of striatal neurons and non-neuronal cells were similarly centrifuged and stored at −80°C until use. Supernatants of tissue homogenates, culture medium and growth factor standards were loaded into wells of ELISA plates that have been coated with anti-EGF (Santa cruz, Santa cruz, CA, USA), anti-TGFα, (Sigma) and anti-HB-EGF (R&D systems, Minneapolis, MN, USA) antibodies. Biotinylated anti-TGFα and anti-HB-EGF (R&D systems) antibodies were allowed to bind to TGFα and HB-EGF. The biotinylated secondary antibodies were detected using streptoavidin β-galactosidase (1 : 10 000; Sigma). Alternatively, the secondary antibodies for EGF (Santa Cruz) were detected using anti-rabbit IgG β-galactosidase (1 : 1000; Sigma). The fluorescence of the enzyme products from 4-methylumbelliferyl-β-d-galactoside (Sigma) was determined as described above. The cross-reactivity of the EGF immunoassay with TGFα and HB-EGF was less than 0.1% (data not shown).

Tissue levels of phosphorylated ErbB1 were measured using an ELISA kit (BioSource International Inc., Camarillo, CA, USA) as described by the manufacturer. The peroxidase activity retained in each well was measured by incubation with tetramethylbenzidine. The level of chromogenic product was monitored at 450 nm using a microplate reader (Benchmark, Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Western blotting analysis

Protein levels of phospho-ErbB1, ErbB1 and SNAP-25 were determined by immunoblot analysis as described previously (Iwakura et al. 2008). In brief, culture cell lysates or tissue extracts were denatured with 8%β-mercaptoethanol plus 2% sodium dodecyl sulfate, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride membrane (ImmobilonTM, Millipore, Billerica, MA, USA). The membrane was probed with the anti-phospho-ErbB1 antibody (1 : 500; Santa Cruz), anti-ErbB1 antibody (1 : 1000; Santa Cruz), anti-SNAP-25 antibody (1 : 500, gifted from Dr Takahashi, Kitazato University), anti-dopamine D1 receptor antibody (1 : 500; Chemicon), anti-dopamine D2 receptor antibody (1 : 1000; Chemicon), anti-glial fibrillary acidic protein (GFAP) antibody (1 : 2000; DAKO) and anti-neuron-specific enolase antibody (1 : 2000; Chemicon). After extensive washing, immunoreactivity on the membrane was detected with the anti-rabbit immunoglobulin conjugated to horseradish peroxidase, followed by a chemiluminescence reaction (ECL kit; Amersham Biosciences, Piscataway, NJ, USA).

Alkaline phosphatase assay for pro-EGF processing

Eukaryotic expression vector containing the human EGF precursor cDNA tagged with human placental AP was constructed by modifying the HB-EGF precursor cDNA expression vector (Shishido et al. 2006). A human EGF cDNA fragment encoding the signal sequence and prodomain was inserted into the multiple cloning site (NotI-XbaI) of the pRc/CMV vector (Invitrogen, Carlsbad, CA, USA). AP cDNA was attached to the N-terminus of the core EGF domain and fused to the above cDNA construct for the signal sequence and prodomain. Striatal cultures were prepared as described above and grown in 12-well plates at a density of 4 × 105 cells/well. Striatal neurons were transfected with the expression vector carrying AP-tagged EGF precursor cDNA (pRc-CMV/EGF-AP) by the calcium phosphate method (CalPhos™ Mammalian Transfection Kit; Clontech Laboratory Inc., Mountain View, CA, USA). After 48 h, cultured neurons were exposed to PMA (100 ng/mL) and SKF38393 (100 μM). Some cultures were pre-incubated with BoNT/A or GM 6001 prior to PMA or SKF38393 treatment.

An aliquot (0.1 mL) of culture supernatants was harvested and heated for 30 min at 60°C to inactivate endogenous AP activity. Then culture supernatants were mixed with an equal volume of p-nitrophenyl phosphate solution (pH 9.5, 1 M diethanolamine, 0.01% MgCl2, and 1 mg/mL p-nitrophenyl phosphate) and incubated for 30 min at 37°C. The optical absorbance at 405 nm was measured using a microplate reader (Benchmark, Bio-Rad Laboratories).

Enzyme assay for MMPs and ADAMs

Striatal tissue of postnatal rats (PND7) and cultured cells were homogenized in lysis buffer (20 mM Tris, pH8.0, 0.06% Brij-35, 0.1% Triton X-100). The lysates were incubated with a fluorescent substrate for tumor necrosis factor-alpha converting enzyme (BIOMOL Research Laboratories Inc., Plymouth Meeting, PA, USA) or MMP (SensoLyte 520 MMP Substrate Sampler Kit; AnaSpec, San Jose, CA, USA) for 1 h at 37°C in the presence of 20 mM Tris, pH 8.0, and 0.06% Brij-35. The MMP substrate is, at least, cleaved by MMP-1, -2,- 3, -7, -8, -9, -12, and -13 whereas the tumor necrosis factor-alpha converting enzyme substrate is by ADAM-9, -10, and -17 (Roghani et al. 1999; Moss and Rasmussen 2007). The amount of fluorescence product was measured using a microplate reader (CORONA electric) with excitation at 490 nm and emission at 530 nm.

Statistical analysis

All values are presented as mean ± SE. Pharmacological effects were analyzed using the Student’s t-test (data from two groups) or Fisher’s LSD test (data from multiple groups). The p-values less than 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Dopamine-triggered release of EGF from cultured striatal neurons

The activation of GPCRs is known to induce proteolytic processing and release of membrane-anchored precursors for EGF or its structural homologues, leading to the transactivation of ErbB1 (Le Gall et al. 2003; Horiuchi et al. 2007; Higashiyama et al. 2008). To examine whether ErbB1 ligands can be liberated from striatal neurons following neurotransmitter challenge, we prepared primary neuron-enriched cultures from embryonic striatal tissue. Prior to this examination, we determined the cell populations in this neuronal culture by immunocytochemistry (Table 1 and Figure S1). Eighty percent of the total cells were positive for MAP2 or NeuN. However, GFAP-positive cells constituted a small percentage of the total cells. In addition, we found the expression of dopamine D1 receptors and D2 receptors in cultured striatal neurons (Figure S2). Thus, striatal neurons represented a major cell population in the present neuron-enriched cultures.

Table 1.   Cell populations of neuron-enriched cell culture
  1. Neuron-enriched cell cultures were prepared from striatal tissues of rat embryos (E18-19) and maintained in the serum-free condition (see Materials and methods section). Cells in culture were immunostained with anti-MAP2, anti-NeuN, anti-GFAP and anti-nestin antibodies. Data represent mean ± SE. = 4 sister cultures. Representative pictures are shown in Figure S1.

 MAP2NeuNGFAPNestin
% of total cell number81.2 ± 1.586.2 ± 2.17.8 ± 0.712.5 ± 2.0

We challenged the above striatal cultures with receptor saturating concentrations of neurotransmitters of dopamine, serotonin (5-HT), acetylcholine, glutamate, and phorbol 12-myristate 13-acetate (PMA, a positive control agent), and measured the amounts of EGF, TGFα and HB-EGF released to culture supernatants by ELISA (Fig. 1). Among the various neurotransmitters, only dopamine increased EGF concentrations in culture supernatants. In contrast, there were no changes in TGFα and HB-EGF concentrations following stimulation of any neurotransmitters. PMA, an inducer for ectodomain shedding, elevated the concentrations of all ErbB1 ligands in culture supernatants, verifying the authenticity of this assay (Pandiella and Massague 1991; Gechtman et al. 1999; Le Gall et al. 2003).

image

Figure 1.  Effects of neurotransmitters on EGF release from neuron-enriched striatal cultures. Striatal cultures were treated with control vehicle (0.2% ascorbic acid), dopamine (30 μM, 20 min), 5-hydroxytryptamine (5-HT, 100 μM, 30 min), acetylcholine (100 μM, 30 min), glutamate (100 μM, 30 min), or PMA (100 ng/mL, 30 min) on DIV7. Concentrations of EGF, TGFα, and HB-EGF in culture supernatants were measured by ELISA (= 4 sister cultures). Data represent mean ± SE. *< 0.05.

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Dopamine receptor stimulation triggers EGF release

To identify the dopamine receptor subtype(s) triggering EGF release, we treated neuron-enriched striatal cultures with a dopamine D1-like receptor agonist (SKF38393) and a D2-like receptor agonist (quinpirole) (Fig. 2a). Treatment with SKF38393 triggered an increase in EGF release to culture supernatants, while quinpirole showed no effects. We also determined the dose dependency of EGF release on dopamine and SKF38393 (Fig. 2b and c). The maximum release of EGF was observed with 100 μM dopamine as well as with 100 μM SKF38393. Phosphorylation of ErbB1 receptors following SKF38393 administration paralleled the EGF release but was inhibited by pre-treatment with the ErbB tyrosine kinase inhibitor PD153035 (Fig. 2d).

image

Figure 2.  Effects of dopamine agonists on EGF release from cultured striatal neurons. (a) Neuron-enriched striatal cultures were treated with control vehicle (0.2% ascorbic acid), SKF38393 (100 μM), quinpirole (30 μM), or PMA (100 ng/mL) at 37°C for 15 min (for dopamine agonists) or 30 min (for PMA, positive control) on DIV7. Levels of EGF in culture supernatants were measured by ELISA (= 4 sister cultures). Neuronal cultures were treated with 0, 10, 30, 100 or 300 μM dopamine (b) and SKF38393 (c), and EGF levels in culture supernatants were measured by ELISA (= 4 sister cultures). The concentration of 300 μM dopamine and SKF38393 failed to further increase the EGF levels (data not shown). (d) Cell lysates were prepared from the above cultures and subjected to western blotting with anti-phospho-ErbB1, anti-ErbB1, and anti-NSE antibodies. Typical immunoblots are shown. Data represent mean ± SE. *< 0.05, **< 0.01, compared with vehicle-treated cultures (control).

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It is possible that glial cells or neural progenitor cells also contributed to the EGF release in the above neuron-enriched culture, we prepared mixed non-neuronal cell cultures from rat embryonic striatal tissue and similarly examined EGF release from these cells. First, we characterized cell populations in this culture preparation by immunocytochemistry for oligodendrocytes, astrocytes, neural progenitor cells, and neuronal cells (Table 2 and Figure S3). The major cell populations were GFAP-positive cells and nestin-positive cells, suggesting that the non-neuronal cell cultures were mainly composed of astrocytes and/or neural progenitor cells. We found that cultured non-neuronal cells from embryonic striatum also express significant levels of dopamine D1 and D2 receptors (Figure S2) (O’Keeffe et al. 2009; Winner et al. 2009). However, these cells released EGF in response to quinpirole but not SKF38393 (Fig. 3). There was concomitant phosphorylation of ErbB1 in cultured non-neuronal cells. However, the absolute concentrations of EGF released by quinpirole were lower, compared with those released from neuronal cell cultures (Fig. 2). Thus, D1-like receptors-dependent EGF release occurs mainly from striatal neurons whereas D2-like receptors-dependent EGF release is from striatal non-neuronal cells in the present experimental paradigm.

Table 2.   Cell populations of striatal non-neuronal cell culture
  1. Non-neuronal cell-enriched cultures were prepared from striatal tissues of rat embryos (E18-19) and grown in the serum-containing condition (see Materials and methods section). After one cell-culture passage with trypsinization, cells in the secondary culture were immunostained with anti-A2B5, anti-GFAP, anti-MAP2 and anti-nestin antibodies. Data represent mean ± SE. = 4 sister cultures. Representative pictures are shown in Figure S3.

 A2B5GFAPMAP2Nestin
% of total cell number38.2 ± 6.665.0 ± 7.05.5 ± 2.984.7 ± 4.5
image

Figure 3.  Effects of dopamine receptor agonists on the release of EGF from mixed non-neuronal cell cultures. Mixed non-neuronal cells were prepared from the striatal regions of rat embryos (E18) (see Figure S3). (a) Culture supernatants were collected without stimulation from mixed non-neuronal cell cultures as well as from neuron-enriched cultures. Basal concentrations of EGF in culture supernatants were measured by ELISA (= 4 sister cultures). (b) Mixed non-neuronal cell cultures were treated with control vehicle, SKF38393, quinpirole (Quin) or PMA as described in Fig. 2a. EGF concentrations in culture supernatants were measured by ELISA (= 4 sister cultures). (c) Cell lysates were prepared from the above non-neuronal cell cultures and subjected to immunoblotting with anti-phospho-ErbB1, anti-ErbB1, and anti-GFAP antibodies. Typical immunoblots are shown (= 2 sister cultures). Data represent mean ± SE. *< 0.05.

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EGF release from cultured striatal neurons depends on ectodomain shedding or vesicular exocytosis

The release of soluble EGF requires the ectodomain shedding of its precursor proteins that is caused by matrix metalloproteinases, such as MMPs or ADAMs (Le Gall et al. 2003; Sahin et al. 2004). Ectodomain shedding takes place not only at the cell surface, but also in intracellular vesicles (Yokozeki et al. 2007). We tested the involvement of ectodomain shedding in cultured striatal neurons (Fig. 4). We monitored EGF release from cultured striatal neurons in the presence of a broad MMP inhibitor, GM6001, or an exocytosis blocker, BoNT/A (Blasi et al. 1993). Pre-treatment with GM6001 markedly suppressed SKF38393- or PMA-triggered EGF release by 70–90%, whereas pre-treatment with BoNT/A attenuated EGF release by only 20–30%. The results indicate that the release of soluble EGF requires the activation of membrane-anchored metalloproteinases. In contrast, the effects of BoNT/A were less marked compared with the effects of GM6001, even though the proteolytic cleavage of SNAP25 confirmed the action of BoNT/A on exocytosis blockade.

image

Figure 4.  Effects of a matrix metalloproteinase inhibitor and an exocytosis blocker on EGF release. (a) Neuron-enriched striatal cultures were pre-treated with or without GM6001 (100 nM, 1 h) or botulinum neurotoxin type A (BoNT/A, 100 nM, 8 h) and then challenged with control vehicle, SKF38393, or PMA on DIV7 as described in Fig. 2a. Concentrations of EGF released to culture supernatants were measured by ELISA (= 5 sister cultures). Data represent mean ± SE. *< 0.05, ***< 0.001, compared with GM6001- or BoNT/A-untreated culture. ###< 0.001, compared with vehicle-treated control culture. (b) Cell lysates from BoNT/A-treated or BoNT/A-untreated cultures were subjected to western blotting for SNAP25. The authenticity of exocytosis blockade was controlled by the cleavage of this essential component of the exocytosis machinery.

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Calcium ions, protein kinase C, and divalent metal ions such as zinc are the molecular regulators for the shedding enzyme of ADAMs (Izumi et al. 1998; Merlos-Suarez et al. 2001; Le Gall et al. 2003). We examined the contribution of calcium ion and protein kinase C to the shedding process. SKF38393 application triggered EGF release, but the release was fully inhibited by pre-treatment with a protein kinase C inhibitor, calphostin C (Fig. 5a). A similar inhibitory effect of calphostin C was observed when striatal cultures were challenged with PMA. A calcium ionophore, ionomycin, mimicked SKF38393: The application of ionomycin to cultured striatal neurons triggered EGF release (Fig. 5b). Conversely, pre-treatment with BAPTA/AM, a chelating agent for calcium ions, blocked the effect of SKF38393. These data suggest that calcium signaling plays a major role in the regulation of EGF release from striatal neurons.

image

Figure 5.  Effects of a protein kinase C inhibitor and calcium chelator/ionophore on EGF release from striatal neurons. (a) Striatal neuron-enriched cultures were pre-treated with or without 1 μM calphostin C (calC) and then challenged with control vehicle, SKF38393, or PMA on DIV7 as described in Fig. 2a. (b) In the presence or absence of BAPTA/AM (10 μM, 30 min), neuronal cultures were challenged with control vehicle, SKF38393, or ionomycin (10 μM). Concentrations of EGF released to culture supernatants were measured by ELISA (= 5 sister cultures, each). Data represent mean ± SE. *< 0.05.

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To confirm these findings, we constructed an expression vector carrying EGF precursor cDNA in which the EGF domain was tagged with alkaline phosphatase (AP) (Fig. 6a). We transfected this expression vector into cultured striatal neurons and quantified AP activity in culture supernatants, at cell surface, and in intracellular components following SKF38393 or PMA treatment. SKF38393 stimulation resulted in a significant increase in AP activity in culture supernatants (Fig. 6b). Pre-treatment with GM6001 attenuated the release of AP activity into culture supernatants, whereas the BoNT/A pre-treatment exhibited modest or negligible effects. PMA mimicked the SKF38393 effects by markedly elevating AP activity in culture supernatants, verifying the authenticity of this assay for ectodomain shedding. This process was inhibited by pre-treatment with GM6001 but not with BoNT/A. The opposite trend in AP activity was observed at the cell surface (Fig. 6c). SKF38393 or PMA alone markedly decreased AP activity at the cell surface, whereas culture pre-treatment with GM6001 or BoNT/A attenuated the decrease in AP activity. BoNT/A alone showed a significant basal effect of decreased AP activity at the cell surface in control conditions. We also calculated AP activity in intracellular components by subtracting the activity at the cell surface from the total activity in the whole cell lysates (Fig. 6d), assuming that protein degradation of AP-tagged EGF by MMPs was negligible. This estimation suggests that the level of AP activity in intracellular components was not affected by SKF38393 or PMA, nor by GM6001 or BoNT/A.

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Figure 6.  Effects of GM6001 and botulinum neurotoxin on AP-tag release from the surface of striatal neurons. (a) A schematic diagram of the expression vector for pRc-CMV/EGF-AP. cDNA regions for human placental alkaline phosphatase (AP) tag (blue), EGF domain (red) and transmembrane region (green) are shown. AP activity was monitored in culture supernatants (b), at the cell surface (c), or in intracellular components (d) of cultured striatal neurons expressing AP-tagged EGF precursors. After transfection, neuronal cultures were pre-treated with or without GM6001 (100 nM, 1 h) or BoNT/A (100 nM, 8 h), then challenged with control vehicle, SKF38393 or PMA as described in Fig. 2a (= 5 sister cultures). Data represent mean ± SE. **< 0.01, ***< 0.001 compared with GM6001- or BoNT/A-untreated culture. ###< 0.001, compared with vehicle-treated control culture. (e, f) Immunoreactivity of AP tag was visualized at the cell surface together with immunoreactivity for dopamine D1-like receptors. Live striatal neurons expressing AP-tagged EGF precursors was challenged with control vehicle (e; 0.2% ascorbic acid, 15 min) or SKF38393 (f; 100 μM, 15 min) and then treated with the anti-AP antibody to visualize EGF precursors on cell surface. After fixation, cells were incubated with the anti-dopamine D1-like receptor antibody followed by the fluorescent secondary antibodies (red for AP and green for dopamine D1-like receptor). Scale bar, 100 μm.

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Application of the anti-AP antibody to live striatal cultures marked cells carrying AP-tag at the cell surface, verifying the surface expression of the precursors (Fig. 6e). SKF38393 treatment indeed reduced the immunoreactivity for AP at the cell surface, supporting our argument that the ectodomain shedding of EGF precursors occurs at the cell surface and regulates EGF release from cultured striatal neurons.

Activation of matrix metalloproteinases following dopamine receptor stimulation

The effects of GM6001 on shedding of EGF precursors suggest the recruitment of metalloproteinases (Prenzel et al. 1999; Yan et al. 2002). Previous studies confirm the expression of MMPs and ADAMs in the striatum (Karkkainen et al. 2000; Kim et al. 2003). Therefore, we examined whether the above dopamine agonists activate ADAMs or MMPs in cultured striatal neurons, monitoring the cleavage of their synthetic substrates in vitro (Fig. 7). SKF38393 and PMA treatment both markedly enhanced the cleavage of the ADAM substrate, whereas quinpirole had no effect. We also assessed the effects on MMPs. Again, treatment with SKF38393 and PMA, but not quinpirole, elevated the enzyme activity, although the magnitude of the increase in MMP activity appeared to be less marked than that of ADAM activity.

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Figure 7.  Dopamine receptor agonists increased matrix metalloproteinase activity in cultured striatal neurons. Neuron-enriched striatal cultures were treated with control vehicle, SKF38393, quinpirole or PMA as described in Fig 2a. Cell lysates were prepared from these cultures and incubated with synthetic substrates for ADAMs (a) or MMPs (b) in vitro. Cleavage rates of these substrates were measured as their enzyme activity (see the Materials and methods section) (= 5 sister cultures). Data represent mean ± SE. **< 0.01.

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Dopamine D1-like receptor-dependent EGF release and ErbB1 phosphorylation in the stratum in vivo

Finally, we examined whether dopamine receptor stimulation in vivo triggers EGF release and activates local ErbB1 in the striatum. We administered a broad dopamine receptor agonist, apomorphine, to postnatal rats (PND7) and quantified protein levels of EGF in the tissue supernatant fraction and phosphorylated ErbB1 in the membrane fraction by ELISA (Fig. 8). Prior to the apomorphine challenge, some rats were pre-treated with the dopamine D1 receptor antagonist SCH23390. Injection of apomorphine alone elevated tissue content of free soluble EGF in the striatum, whereas the combination of apomorphine and SCH23390 or SCH23390 pre-treatment alone had no effect (Fig. 8a). The increase in free EGF content was concomitant with the activation of local ErbB1 (Fig. 8b). Apomorphine increased ErbB1 phosphorylation levels in the striatum, while the co-administration of SCH23390 blocked the increase in ErbB1 phosphorylation. In parallel, we monitored the enzyme activity of ADAMs in striatal tissue homogenates (Fig. 8c). Apomorphine alone increased the enzyme activity whereas the combination of apomorphine and SCH23390 attenuated the increase. These results suggest that EGF shedding and release in vivo also depends on the activation of dopamine D1-like receptors at the early postnatal stage.

image

Figure 8.  EGF release and ErbB1 phosphorylation following dopamine D1-like receptor activation in vivo. Postnatal rats (PND7) were pre-treated with SCH23390 (SCH; 1 mg/kg, sc) or saline for 30 min, and then challenged with apomorphine (Apo; 1 mg/kg, sc) or vehicle (0.2% ascorbic acid). Thirty minutes after apomorphine challenge, striatum was dissected and homogenized. A part of tissue homogenates were centrifuged to separate the membrane components including membrane-anchored EGF receptors. Levels of soluble-free EGF in the supernatants (a) and ErbB1 phosphorylation (b) in the membrane fraction were determined by ELISA (= 5 rats for each group). (c) Alternatively, crude tissue homogenates were incubated with synthetic substrates for ADAMs. Cleavage rates of ADAM substrates were measured in vitro as described in Fig. 7 (= 5 rats for each group). Data represent mean ± SE. **< 0.01, compared with saline- and vehicle-treated control.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In this study, we characterized the dopamine-dependent EGF release from target cell populations of midbrain dopaminergic neurons. To consider the neurotrophic contribution of EGF, we used striatal neurons at the late gestation stage for our assay and obtained the following results: (i) among various neurotransmitters examined, only dopamine evoked EGF release from neuron-enriched striatal cultures; (ii) the dopamine D1-like receptor agonist enhanced the release of mature EGF and ErbB1 phosphorylation; (iii) the release from non-neuronal cells was triggered by the D2-like receptor agonist; (iv) transfection with AP-tagged EGF precursor cDNA verified the ectodomain shedding of EGF precursors on the surface of striatal neurons; (v) D1-like receptor stimulation activated MMPs and ADAMs along with EGF release from striatal neurons; (vi) the activation of the D1-like receptors in postnatal rats similarly elevated striatal content of free EGF and ErbB1 phosphorylation. These observations suggest that the activation of D1-like receptors induces ectodomain shedding of EGF precursors at the cell surface of striatal neurons as well as in secretory vesicles whereas the activation of D2-like receptors triggers EGF release from non-neuronal cells (Fig. 9).

image

Figure 9.  Neurotrophic interactions between dopaminergic afferents and striatal cells. Dopamine released from nigrostriatal dopaminergic terminals reacts with the dopamine D1-like receptor in striatal neurons or D2-like receptors in non-neuronal cells and activates the ADAM enzyme (anterograde signals) via protein kinase C (PKC). Then, ADAM cleaves the ectodomain of EGF precursor and liberates mature EGF into secretory vesicles or intercellular spaces. Free EGF reacts with ErbB1 in dopaminergic terminals, potentially promoting dopaminergic development and/or maturation (retrograde trophic signals). Alternatively, released EGF also acts on ErbB1 in the striatal neuron or glia itself. This picture illustrates neuron–target interactions mediated by dopamine and the neurotrophic factor EGF.

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EGF or its homologue reacts with midbrain dopaminergic neurons and promotes their functional development or cell survival (Futamura et al. 2003; Iwakura et al. 2005, 2011; Abe et al. 2009; Namba et al. 2009). The neurotrophic activity of EGF suggests that EGF, which is liberated from striatal cells via dopamine-triggered ectodomain shedding and/or release, functions as a retrograde neurotrophic signal to dopaminergic neurons (Fig. 9). The cellular processes fit the well-known concept of neurotrophic interactions between neurons and their targets (Purves 1986). Once dopamine is released and bound to dopamine D1-like receptors on striatal neurons, the activated ADAM enzyme sheds the membrane–anchored EGF precursors and lets soluble EGF released to dopaminergic afferent terminals; this promotes dopaminergic development or dopamine release (Iwakura et al. 2005; Mizuno et al. 2007). From the juvenile stage, however, glial cell population occupies a significant proportion of striatal cells and may also contribute to the target-derived neurotrophic production in a dopamine D2 receptor-dependent manner (Fig. 9).

Cell population and subcellular components of EGF ectodomain shedding

EGF, TGFα and HB-EGF are all present in the CNS and produced by neurons and glial cells (Wong and Guillaud 2004; Piao et al. 2005). In the late gestation stage of rats, brain contents of these ErbB1 ligands were at nearly equivalent concentrations. The equivalency of their expressions in the prenatal stage contrasts with the remarkably high expression of HB-EGF in the adult stage (Piao et al. 2005). To distinguish the cellular origin of EGF, we prepared two types of striatal cultures, neuron-enriched culture and non-neuronal cell-enriched culture, and compared the magnitude of EGF release and dopamine receptor subtype-dependency between these cultures. The basal EGF release from neuron-enriched cultures was approximately four times larger than that from non-neuronal cell-enriched cultures. The dopamine D1-like receptor stimulation promoted EGF release from neuron-enriched cultures whereas the EGF release from non-neuronal cell-enriched cultures depended on the dopamine D2-like agonist. Recent studies suggest that neural progenitor cells in the adult SVZ respond to a dopamine D2-like agonist by releasing EGF through ectodomain shedding (O’Keeffe et al. 2009; Winner et al. 2009). Therefore, the dopamine receptor subtypes responsible for EGF precursor shedding appear to be different depending on the target cell populations.

Ectodomain shedding occurs not only at the cell surface, but also in intracellular vesicles (Yokozeki et al. 2007). As dopamine analogues and GM6001 failed to alter the amount of AP-tagged EGF precursors in the intracellular fraction, it is likely that the majority of EGF precursors were cleaved at the cell surface. However, we cannot fully rule out the possibility that EGF release from striatal neurons may involve vesicular exocytosis as well because there was a modest but significant inhibitory effect of the exocytosis inhibitor BoNT/A on net EGF release. Alternatively, it is possible that BoNT/A attenuated intracellular trafficking of EGF precursors to the cell surface rather than EGF release. The present results reveal a biological role of ectodomain shedding in EGF release from striatal neurons; a retrograde neurotrophic supply triggered by dopamine from afferent nerves.

Ectodomain shedding is responsible for soluble EGF release from striatal neurons

All dopamine receptors belong to the GPCR superfamily and can activate MMPs and ADAMs, which are responsible for ectodomain shedding (Prenzel et al. 1999; Yan et al. 2002). We found that MMPs and ADAMs were activated following dopamine D1-like receptor agonist stimulation, concomitant with EGF release. The synthetic substrate employed in our ADAM assay is known to be cleaved by ADAM-9, -10, and -17 (Roghani et al. 1999; Moss and Rasmussen 2007). This is consistent with reports showing that ADAM-9, -10, and -17 are the major enzymes responsible for the ectodomain shedding of EGF precursors and expressed in the striatum (Karkkainen et al. 2000; Kim et al. 2003; Le Gall et al. 2003; Horiuchi et al. 2007). In the present study, we were able to trigger the release of HB-EGF and TGFα with PMA but not with neurotransmitters or dopamine receptor agonists. With the given heterogeneity of cultured striatal cells, we speculate that the cells expressing HB-EGF and TGFα precursors were different from those expressing EGF precursors or those carrying the MMP/ADAM enzymes uncoupled with dopamine receptors.

Stimulation of dopamine D1-like receptors triggers intracellular calcium ion influx and activates protein kinase C or A, which are implicated in the enzyme activation of ADAMs (Kansra et al. 1995; Surmeier et al. 1995; Izumi et al. 1998; Merlos-Suarez et al. 2001; Le Gall et al. 2003). In agreement, an intracellular calcium scavenger as well as a protein kinase C inhibitor attenuated the dopamine D1-like agonist-dependent EGF release from striatal cultures. These findings suggest the contribution of calcium ion influx to ectodomain shedding. However, we failed to observe EGF release or ectodomain shedding with the dopamine D2-like receptor agonist that must increase intracellular calcium ion concentrations in post-mitotic striatal neurons (Yan et al. 1999). This discrepancy is potentially caused by the involvement of other EGF-homologues or cell type differences between D2-like receptor-expressing and EGF precursor-expressing populations. Further investigation is required to clarify the molecular mechanism leading to ADAM activation in these neurons.

Epidermal growth factor, a retrograde neurotrophic signal for midbrain dopaminergic neurons

Our previous study revealed that the production of EGF in the striatum in vivo depends on its dopaminergic afferent innervation (Iwakura et al. 2005). Parkinson’s disease, which is caused by neurodegeneration of midbrain dopaminergic neurons, specifically decreases tissue content of free EGF in postmortem striatum of its patients. A dopaminergic lesion of this neuronal population with 6-hydoxydopamine similarly results in EGF reduction in rat striatum (Iwakura et al. 2005). Both results corroborate our present observation that dopaminergic neurotransmission is responsible for the production of mature EGF in the target brain region – the striatum. Of note, chronic schizophrenia elevates protein levels of ErbB1 in the striatum, suggesting the higher sensitivity of dopaminergic terminals to this neurotrophic signal (Futamura et al. 2002). Conversely, Parkinson’s disease lowers ErbB1 levels, suggesting the lower sensitivity to EGF. In the accompanying paper, we demonstrated that EGF acts on midbrain dopaminergic neurons and promote their differentiation and maturation during development (Iwakura et al. 2011). These findings reveal tight neurotrophic interactions between the afferent nerves of nigrostriatal neurons and their targets of the striatum, which are mediated by dopamine and EGF, respectively (Fig. 9). Thus, we assume that these interactions might be impaired in Parkinson’s disease and/or schizophrenia. Future studies should illustrate how the feed forward interaction between dopamine and EGF contributes to the emergence or progression of dopaminergic neuropathology in these brain disorders.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This work was supported by a grant-in-aid for Basic Scientific Research B, grants for Promotion Research Projects and Young Investigators of Niigata University, Core Research for Evolutional Science and Technology from the JST Corporation, and a grant-in-aid from the Ministry of Health, Labor and Welfare, Japan. We thank Dr Shunji Kozaki for botulinum neurotoxin A and Dr Masami Takahashi for the SNAP-25 antibody.

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  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Figure S1. Characterization of neuronal cell-enriched cultures from embryonic striatal tissues. Cultures were immunostained with anti-MAP2 antibody (a), anti-NeuN antibody (b), anti-GFAP antibody (c) and anti-nestin antibody (d) followed by anti-mouse or anti-rabbit immunoglobulin fluorescent secondary antibodies. Left panels represent the phase contrasted microscopic views corresponding to the right panels. The frequency of individual cell types is shown in Table 1 (= 4 sister cultures). Representative pictures are displayed. Scale bar, 100 μm.

Figure S2. Protein samples were prepared from neuron-enriched and non-neuronal cell-enriched culture, subjected to immunoblotting with the anti-dopamine receptor antibodies, and compared with the levels in the striatum of postnatal rats (PND7). Alternatively, the immunoblots were probed with anti-neuron-specific enolase (NSE) antibody or anti-glial fibrillary acidic protein (GFAP) antibody. Representative two lanes of the immunoblot are shown for figure display.

Figure S3. Characterization of non-neuronal cell-enriched cultures from embryonic striatal tissues. Cultures were immunostained with the anti-A2B5 antibody (a), anti-GFAP antibody (b), anti-MAP2 antibody (c) and anti-nestin antibody (d) followed by anti-mouse or anti-rabbit immunoglobulin fluorescent secondary antibodies. Left panels represent the phase contrasted microscopic views corresponding to the right panels. The frequency of individual cell types is shown in Table 2 (= 4 sister cultures). Representative pictures are displayed. Scale bars, 100 μm.

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