Increased Expression of Prion Protein Is Associated with Changes in ...

Journal of Molecular Neuroscience Copyright © 1999 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN0895-8696/99/13:121–126/$11.50

Increased Expression of Prion Protein Is Associated with Changes in Dopamine Metabolism and MAO Activity in PC12 Cells Hyoung-Gon Lee,1 Seok-Joo Park,1 Eun-Kyoung Choi,1 Richard I. Carp,3 and Yong-Sun Kim* ,1,2 1

Institute of Environment & Life Science, Hallym Academy of Sciences, and 2Department of Microbiology, College of Medicine, Hallym University, Chuncheon 200-702, Korea; and 3New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314

Abstract Prion diseases of humans and animals occur following infection with infectious agents containing PrPSc or in situations in which there is a mutation of the prion protein (PrP) gene. The cellular prion protein (PrPC) is a sialoglycoprotein that is expressed predominantly in neurons. PrPC is converted into a pathogenic form of PrP (PrPSc), which is distinguishable from PrPC by its relative resistance to protease digestion. A number of postulates have been advanced for the function of normal PrP (PrPC), but this issue has not been resolved. To investigate the function(s) of PrPC, we established clonal PC12 cell lines, which have elevated PrPC expression. The results show that there were alterations in dopamine metabolism and in monoamine oxidase (MAO) activity in transfected PC12 cells that overexpress PrPC. There was an increase in concentration of DOPAC, a metabolite of dopamine, and in MAO activity in cells overexpressing PrPC. MAO is involved in oxidative degradation of dopamine (DA). Our data suggest that PrPC plays a role in DA metabolism by regulating MAO activity. Index Entries: PrPC; PC12 cells; dopamine; DOPAC; monoamine oxidase (MAO).

*Author to whom all correspondence and reprint requests should be addressed.

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Introduction

Materials and Methods

Prion protein (PrPC) is a glycoprotein constitutively expressed on the neuronal cell surface (Prusiner, 1991). A protease-resistant isoform of PrPC (PrPSc) is implicated in the pathogenesis of a series of transmissible spongiform encephalopathies (TSEs), including scrapie, CreutzfeldtJakob disease (CJD), Gerstmann-SträusslerSheinker disease (GSS), and bovine spongiform encephalopathy (BSE). The importance of PrPC in the susceptibility and in the incubation time of these diseases has been demonstrated, but is still unexplained (Prusiner, 1998). The discovery that PrPC is bound to the cell surface by a glycosylphosphatidylinositol (GPI) anchor suggests that PrPC may function as a receptor or adhesion molecule (Stahl et al., 1987). Furthermore, PrPC does not seem to be essential since disruption of the PrP gene has not caused any detectable abnormalities in the nervous system, at least not in young PrPC knock-out mice (Büeler et al., 1992). However, electrophysiological studies of hippocampal slices from PrP knock-out mice suggest that the absence of PrPC may alter synapse formation (Collinge et al., 1994). By immunocytochemistry, PrPC has been localized to the synapse (Salès et al., 1998). A wide range of brain functions are mediated by catecholamines (i.e., dopamine [DA], norepinephrine), and it has been shown that there is damage to the catecholaminergic neurotransmitter system in scrapie (Bassant et al., 1984; Yun et al., 1998). Scrapie agent infection also affects the chollinergic neurotransmitter system in pheochromocytoma (PC12) cells, which were derived from rat adrenal medullary tumors (Rubenstein et al., 1991). In humans, destruction of specific sets of catecholaminergic neurons contributes to the symptoms of schizophrenia, Huntington’s disease, and Parkinson’s disease (Richards et al., 1998). These cells synthesize and release DA (Greene and Rein, 1977). DA is synthesized from tyrosine and degraded by monoamine oxidase (MAO) (Cooper et al., 1996). From the above reports, it appears that there might be a relationship between PrPC expression and neurotransmitter systems. The present study examined DA metabolism and release in clonal PC12 cells, which have increased expression of PrPC.

Preparation of PrP Expression Construct


The entire open reading frame (ORF) of mouse PrP was amplified by polymerase chain reaction (PCR) from the plasmid N2TBX1 (ATCC 63010) using the following primers: 5’-CTGCCGCAGCCCCTGCCATATGCTTCATGTTGGTTTTTGGTTTGC-3’ (primer 1), 5’-GCAAACCAAAAACCAACATGAAGCATATGGCAGGGGCTGCGGCAG-3’ (primer 2), 5’-GACCAGAAGCTTATGGCGAACCTTGGCTACTGG-3’ (primer 3; complementary to 5’ end of PrP ORF, including HindIII cleavage site) and 5’GACCAGGGGCCCTCATCCCACGATCAGGAAGATG-3’ (primer 4; complementary to 3’ end of PrP ORF including ApaI cleavage site). To create the mouse PrP tagged with the 3F4 antibody epitope, an epitope present in human and hamster PrPC (Kascsak et al., 1987), N2TBX1 was first amplified with primer 1 and primer 4, and with primer 2 and primer 3, respectively. The 3F4 antibody epitope regions of primers 1 and 2 have been underlined. These two PCR products were then annealed with each other and amplified again with primer 3 and primer 4. After cleavage with HindIII and ApaI, the final PCR product was cloned into pcDNA3 (Invitrogen), which had been cleaved with the same two enzymes. The DNA sequence of the PrP gene was confirmed by sequencing.

Cell Culture PC12 cells (ATCC) were grown in RPMI-1640 supplemented with 10% horse serum, 5% fetal bovine serum, 50 U penicillin/mL, and 100 µg streptomycin/mL. Cells were grown at 37°C in 5% CO2, and the media were changed every 3–4 d.

Expression of PrP Construct in PC12 Cells The pcDNA3-PrP construct described above was transfected into PC12 cells using calcium phosphate precipitation (Kim et al., 1994). To select stable clonal transfectants, the cells were grown in the presence of 400 µg/mL G418 for selection. After 3 wk, each surviving cell colony was picked and cultured. As a negative control, pcDNA3 vector without insert was transfected separately into PC12 cells. Expression levels of PrP in the transfectants were

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examined by Western blot using anti-3F4 antibody (Kascsak et al., 1987).

Dopamine and DOPAC Analysis For measuring basal release of DA and dihydroxyphenylacetic acid (DOPAC), PC12 cells were plated at 2 × 106 cells/60 mm dish and incubated for 24 h at 37°C. The cells were washed with phosphate-buffered saline (PBS) twice and incubated for 15 min at 37°C in serum-free RPMI-1640. At the end of incubation, medium was collected and mixed with the same volume of 0.2 M perchloric acid (PCA) and the cells were scraped with a rubber policeman in 500 µL of 0.1 M PCA. An aliquot of cell lysate was assayed for protein concentration by BCAassay (Pierce). Cell lysates were centrifuged at 10,000g for 10 min, and the level of DA and DOPAC in the supernatant was determined by reverse-phase, high-pressure liquid chromatography (HPLC) coupled with an electrochemical detector (Waters) (Morier-Teissier and Rips, 1987). One liter of mobile phase (pH 4.3) contained: 50 mM potassium phosphate monobasic, 0.1 mM ethylenediamine tetra-acetic acid (EDTA), 0.36 mM octane sulfonic acid, and 6.5% acetonitrile. The column was a Supelcosil LC-18S (250 × 4.6 mm, 5 µm), reverse phase (Supelco). The mobile phase (filtered and degassed) was delivered at a flow rate of 1 mL/min; the applied potential was set to 650 mV. DA and DOPAC released in the medium were expressed as ng/min/mg of protein, and the intracellular levels of DA and DOPAC were expressed as ng/mg of protein.

MAO Assay MAO activity was measured fluorimetrically using kynuramine as a substrate by a previously reported method (Morinan and Garratt, 1985). Briefly, the cells were gathered by centrifugation at 800g and washed with PBS twice, and suspended in PBS. After the cells were sonicated for 30 s and centrifuged at 7000g for 10 min, total protein concentration in supernatant was measured by BCA assay (Pierce). Thirty micrograms of protein were incubated in reaction solution containing kynuramine for 30 min. Finally, fluorescent intensity was measured at wavelength of 315 nm (for excitation) and wavelength 380 nm (for emis-


Fig. 1. Expression of PrPC in PC12-cells. Western blot showing PrPC protein in three different clonal lines of PC12 cells overexpressing mouse PrPC containing the 3F4 epitope. PC12 clonal lines transfected with vector alone (V1 and V8) are also shown. Equivalent amounts of protein (40 µg/lane) from cell lysates of the indicated cell lines were used. Protein molecular-size markers indicated on the left side of panel represent 48, 33, 28, and 19 kDa from the top.

sion) using a fluorescence spectrophotometer (Kontron instruments).

Statistical Analysis Results were typically expressed as mean ± standard deviation (SD). Data were analyzed by student’s t-test and values of p < 0.01 were taken as being statistically significant.

Results PC12 cells were stably transfected with a DNA construct encoding mouse PrP C containing the human-, hamster- and feline-specific epitope of 3F4; additional lines were transfected with vector alone (V). Western blots using anti-3F4 antibody demonstrated that there was increased expression of PrPC at various levels in each of the cell lines transfected with PrPC (Fig. 1). Three clonal lines termed PC123F4-n8, n14, and n15 that expressed relatively high levels of PrPC and two PC12-V clonal lines that had been transfected with vector alone were used in the present study. PrPC protein levels of PC12-3F4-n14 and n15 were about four- to nine-fold higher than that of PC12-3F4-n8.

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Lee et al. Table 1 The Effect of Increased Expression of PrPC on the Concentration of DA and DOPAC and the DOPAC/DA Ratio in PC12 Cells Basal release (ng/min/mg protein)

Cellular content (ng/mg protein)

Cell

DA

DOPAC

DOPAC/DA ratio

DA

DOPAC

DOPAC/DA ratio

V1 V8 n8 n14 n15

2.51 ± 0.34 2.61 ± 0.65 0.96 ± 0.12a 1.31 ± 0.33a 0.42 ± 0.11a

0.15 ± 0.03 0.11 ± 0.05 0.19 ± 0.04 0.37 ± 0.17a 0.81 ± 0.21a

0.063 ± 0.016 0.040 ± 0.014 0.194 ± 0.027a 0.275 ± 0.092a 1.950 ± 0.27a

1666.5 ± 465.5 1990.1 ± 370.1 1138.4 ± 93.0 1374.3 ± 402.7 995.4 ± 198.8

1.58 ± 0.45 1.03 ± 0.22 2.93 ± 0.55a 5.25 ± 0.96a 12.73 ± 3.34a

0.00096 ± 0.00022 0.00052 ± 0.00012 0.00257 ± 0.00039a 0.00405 ± 0.0013a 0.01275 ± 0.00151a

p < 0.01 compared with values of vector-transfected cell lines (V1, V8). Values are mean ± SD of determinations made in five separate cultures. a

DAis a major catecholamine that is released from PC12 cells. Levels of DA and DOPAC in PC12-3F4 cell lines were measured. The basal release and the intracellular content of DOPAC were increased in PC12-3F4 cell lines (Table 1), and they were dependent on PrPC expression levels. DA release levels were significantly decreased (p < 0.01) in all PC123F4 cell lines compared to either V1 or V8 cells. However, with regard to DA cellular content, some comparisons were significant, e.g., n15 vs V8 or n8 vs V8, whereas other comparisons (n8 vs V1) did not yield significantly different values. Since DOPAC is made from degradation of DA, we analyzed DOPAC/DA ratio; the DOPAC/DA ratio increased in PC12-3F4 cell lines in a PrPC expression-dependent manner (Table 1). Release of DOPAC was much higher than DA in the PC123F4-n15 cell line that expresses the highest level of PrPC (Fig. 1). DA levels in PC12-3F4 cell lines were not significantly decreased vs the V1 cell line, whereas the DOPAC/DA ratio was significantly increased in all PC12-3F4 cell lines. DOPAC is made from DA metabolism by oxidative degradation of DA, and this reaction is mediated by MAO. To determine whether MAO is involved in the increase of DOPAC in PC12-3F4 cell lines, we analyzed MAO activity. There was an increase in MAO activity in PC12-3F4 cell lines similar to the increase in the DOPAC/DA ratio (Fig. 2). In PC12-3F4 cell lines, MAO activity was approx 4 times (n8) to 10 times (n15) greater than that of cell lines transfected with vector alone (V1, V8).


Fig. 2. MAO activity in PC12 clonal cell lines transfected with vector alone (V1 and V8) or with PrPC containing the 3F4 epitope (n8, n14, n15). Data represent means ± SD values from triplicate samples from at least three separate experiments. MAO activity of all PC123F4 cell lines was significantly higher than vector-transfected cell lines (p < 0.01).

Discussion The objective of this study was to test the effect of overexpression of PrPC on the release and metabolism of DA in PC12 cells. Our results demonstrate that there was an alteration of DA metabolism in PC12 cells overexpressing PrPC. In PrPC-transfected cell lines, MAO activity was increased 4 to 10 times compared to cell lines transfected with vector alone, and the DAmetabolite, DOPAC, was also increased. The change in DAlevels may be caused by increased

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PrP Expression and Dopamine Metabolism MAO activity. MAOs (EC 1.4.3.4) are integral proteins of the outer mitochondrial membranes. Isoenzymes (MAO-A and MAO-B) occur in various cells (both neuronal and nonneuronal in the central nervous system [CNS] and in peripheral organs) where they oxidatively deaminate biogenic and xenobiotic amines. In the CNS, they play a physiological role in the metabolic inactivation of monoamine neurotransmitters (Richards et al., 1998). MAO-A and MAO-B metabolize different substrates, but DA is a common substrate of both subtypes. Although MAO subtype was not determined in the present study, previous reports show that PC12 cells have mainly MAO-A, not MAO-B (Naoi et al., 1987). Transgenic mice expressing four- to sixfold higher MAO-B activity in brains compared to nontransgenic littermates showed alteration of DA metabolism (Andersen et al., 1994). Analysis of these brains demonstrated a 30–40% increase in the basal levels of DOPAC. Furthermore, PC12 cells that have elevated MAO activity (over threefold that of control) resulting from transfection of MAO-B show an increase of DOPAC level (Wei et al., 1997). Therefore, the increase in MAO activity in PC12-3F4 cell lines may be sufficient to cause the alteration of DOPAC/DA ratio. Although the cellular functions of PrPC are not fully determined, some previous investigations have suggested several possible roles. First, PrPC plays a role in synaptic functions. PrP knock-out mice exhibit reduced long-term potentiation and reduced γ-aminobutyric acid (GABA) (Collinge et al., 1994). Second, PrP knock-out mice produced by another research group have altered circadian rhythms (Sakaguchi et al., 1996). Third, PrPC expression regulates Cu/Zn superoxide dismutase (SOD) and may regulate copper metabolism through binding to copper (Brown and Besinger, 1998). There is no direct evidence that copper can regulate MAO activity. However, flavin adenine dinucleotide (FAD) binding to MAO is essential to MAO activity and a metal cofactor may contribute to this mechanism (Sourkes, 1972). Furthermore, it is unknown whether concentration of intracellular copper affects MAO activity or not. Therefore, it is possible that elevation of copper concentration by overexpression of PrPC affects MAO activity, probably through the action of Cu/Zn SOD. Abnormality of DA system has been reported in neurodegenerative diseases, such as Parkinson’s


125 disease and Alzheimer’s disease (Richards et al., 1998). Additionally, an abnormally low concentration of DAhas been found in the striatum of scrapieinoculated animals (Bassant et al., 1984; Yun et al., 1998). Since various impairments of motor function, e.g., ataxia and tremor, are present in naturally occurring and experimental scrapie, it is not surprising to observe a fall in striatal DA. Moreover, it is interesting that transgenic mice harboring high copy numbers of wild-type PrP transgenes exhibited spongiform degeneration in the brain and scrapie-like symptoms (Westaway et al., 1994). DA metabolism and MAO activity were not determined in these transgenic mice; our results suggest that elevation of MAO activity and degradation of DA caused by PrPC overexpression may play a role in neuronal degeneration in these transgenic mice. In conclusion, the increased expression of PrPC in PC12 cells was accompanied by an increase in MAO activity, and the elevation of MAO activity correlated with an increase of DOPAC derived from DA. These results suggest that PrPC may regulate MAO activity in PC12 cells.

Acknowledgments We thank Richard Kascsak for providing 3F4 antibody and Seung-Il Choi, Jae-Il Kim, and Richard Rubenstein for critical reading of the manuscripts. The authors wish to acknowledge the financial support of the Korea Research Foundation made in the program (genetic engineering) year of 1998.

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126 opment and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 356, 577–582. Collinge J., Whittington M. A., Sidle K. C. L., Smith C. J., Palmer M. S., Clarke A. R., et al. (1994) Prion protein is necessary for normal synaptic function. Nature 370, 295–297. Cooper J. R., Bloom F. E., and Roth R. H. (1996) Dopamine, in The Biochemical Basis of Neuropharmacology. Oxford University Press, New York, pp. 293–351. Greene L. A. and Rein G. (1977) Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor (NGF) responsive pheochromocytoma cells. Brain Res. 129, 247–263. Kascsak R. J., Rubenstein R., Merz P. A., Tonna-DeMasi M., Fersko R., Carp R. I., et al. (1987) Mouse polyclonal and monoclonal antibody to scrapieassociated fibril proteins. J. Virol. 61, 3688–3693. Kim J. H., Johansen F. E., Catino J. J., Prywes R., and Kumar C. C. (1994) Suppression of Ras transformation by serum response factor. J. Biol. Chem. 269, 13,740–13,743. Morier-Teissier E. and Rips R. (1987) Catecholamine metabolite measurements in mouse brain using high-performance liquid chromatography and electrochemical detection: comparison of a onecolumn technique with a two-column switching technique. Methods Enzymol. 142, 535–549. Morinan A. and Garratt H. M. (1985) An improved fluorimetric assay for brain monoamine oxidase. J. Phamacol. Methods. 13, 213–223. Naoi M., Suzuki H., Takahashi T., Shibahara K., and Nagatsu T. (1987) Ganglioside GM1 causes expression of type B monoamine oxidase in a rat clonal pheochromocytoma cell line, PC12h. J. Neurochem. 49, 1602–1605. Prusiner S. B. (1991) Molecular biology of prion diseases. Science 252, 1515–1522. Prusiner S. B. (1998) Prions. Proc. Natl. Acad. Sci. USA 95, 13,363–13,383.


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