cellular prion protein is present in dopaminergic neurons and modulates the dopaminergic system

MOLECULAR AND SYNAPTIC MECHANISMS

Cellular prion protein is present in dopaminergic neuronsand modulates the dopaminergic system

Daniel Rial,1,2 Fabr�ıcio A. Pamplona,1,3 Eduardo L. G. Moreira,1,4 Karin M. Moreira,5 D�ebora Hipolide,5

Diana I. Rodrigues,2 Patr�ıcia A. Dombrowski,6 Claudio Da Cunha,6 Paula Agostinho,2,7 Reinaldo N. Takahashi,1

Roger Walz,4,8 Rodrigo A. Cunha2,7 and Rui D. Prediger1,41Departamento de Farmacologia, Centro de Ciencias Biol�ogicas, Universidade Federal de Santa Catarina, UFSC, Florian�opolis,Brazil2Center for Neuroscience and Cell Biology, Faculty of Medicine, Rua Larga University of Coimbra, 3004-504, Coimbra, Portugal3D’Or Institute for Research and Education, Rio de Janeiro, Brazil4Centro de Neurociencias Aplicadas (CeNAp), Hospital Universitario, Universidade Federal de Santa Catarina, UFSC,Florian�opolis, SC, Brazil5Departamento de Psicobiologia, Universidade Federal de Sao Paulo, UNIFESP, Sao Paulo, SP, Brazil6Departamento de Farmacologia, Universidade Federal do Parana, UFPR, Curitiba, PR, Brazil7FMUC – Faculty of Medicine, University of Coimbra, Coimbra, Portugal8Centro de Epilepsia do Estado de Santa Catarina, Hospital Governador Celso Ramos, Florian�opolis, SC, Brazil

Keywords: behavior, cellular prion protein, dopamine, dopamine receptor, striatum

Abstract

Cellular prion protein (PrPC) is widely expressed in the brain. Although the precise role of PrPC remains uncertain, it has beenproposed to be a pivotal modulator of neuroplasticity events by regulating the glutamatergic and serotonergic systems. Here wereport the existence of neurochemical and functional interactions between PrPC and the dopaminergic system. PrPC was found toco-localize with dopaminergic neurons and in dopaminergic synapses in the striatum. Furthermore, the genetic deletion of PrPC

down-regulated dopamine D1 receptors and DARPP-32 density in the striatum and decreased dopamine levels in the prefrontalcortex of mice. This indicates that PrPC affects the homeostasis of the dopaminergic system by interfering differently in differentbrain areas with dopamine synthesis, content, receptor density and signaling pathways. This interaction between PrPC and thedopaminergic system prompts the hypotheses that the dopaminergic system may be implicated in some pathological features ofprion-related diseases and, conversely, that PrPC may play a role in dopamine-associated brain disorders.

Introduction

Cellular prion protein (PrPC) is a cell-surface glycosylphosphatidyli-nositol-anchored protein expressed across the brain with higher lev-els in the olfactory bulb (OB), striatum (STR), hippocampus (HIP)and prefrontal cortex (PFC) (Fournier et al., 1995; Sales et al.,1998). The neuronal distribution of PrPC includes presynaptic andpostsynaptic sites (Sales et al., 1998). Functionally it was suggestedthat PrPC controls synaptic plasticity (Collinge et al., 1994), epi-lepsy (Walz et al., 1999), cognition (Criado et al., 2005) and aging(Rial et al., 2009). Ford et al. (2002) showed that the expression ofPrPC is high in GABAergic neurons, but the protein is also presentin noradrenergic, glutamatergic, cholinergic and serotonergicneurons. Interestingly, in this same study, the authors reported thattyrosine hydroxylase (TH)-positive neurons (representative of dopa-minergic neurons) displayed a very low PrPC expression (Ford

et al., 2002). This is of particular interest as the dopaminergic sys-tem also has a particular importance in adaptive changes in severalareas abundantly expressing PrPC such as the STR, PFC and OB(Tritsch & Sabatini, 2012).The actions of dopamine (DA) are mediated through two main

classes of receptor subtypes, termed D1-like receptors (D1 and D5)and D2-like receptors (D2, D3 and D4), that induce opposite intracel-lular responses – D1 receptors stimulate and D2 receptors inhibitcyclic adenosine monophosphate (cAMP) formation (Amenta et al.,1987). One of the most important signaling transduction pathwaysoperated by DA receptors is mediated by dopamine- and cAMP-reg-ulated phosphoprotein, 32 kDa (DARPP-32) (Svenningsson et al.,2004). Accordingly, striatal DARPP-32 has been established as acrucial mediator of the biochemical, electrophysiological, transcrip-tional and behavioral effects of DA (Fienberg et al., 1998).In keeping with the major relevance of the dopaminergic system

for several physiological and pathological adaptive changes andbehavior responses and considering that PrPC modulates diverseaspects of behavior and neuroplasticity (Linden et al., 2008), the aimof the present study was to investigate the PrPC–DA interactions in

Correspondence: Dr D. Rial, 2CNC, as above.E-mail: [email protected]

Received 29 October 2013, revised 12 March 2014, accepted 27 March 2014

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd

European Journal of Neuroscience, Vol. 40, pp. 2479–2486, 2014 doi:10.1111/ejn.12600

mice using pharmacological and genetic approaches combined withbehavioral and neurochemical assays.

Materials and methods

Animals

Female knockout mice homozygous for a disruption of the Prnpgene (Prnp null mice, designated as Prnp0/0 mice), produced as pre-viously described (Bueler et al., 1992) and wild-type mice (Prnp+/+,which are direct littermates of the Prnp0/0 with the same mixed129Sv and C57/Bl6 background) were donated by Dr Vilma R.Martins (International Research Center, A.C. Camargo Hospital, SãoPaulo) and were used when they were 2–3 months old.We also used female Edinburgh genetically modified mice (EP-

rnp+/+, EPrnp+/� and EPrnp�/�) where a neomycin cassette wasinserted in the Kpn-1 enzyme restriction site present in Prnp openreading frame section. These mice also have a mixed background(129Sv and C57Bl/10) generating 25% recessive homozygotic mice(EPrnp�/�), 50% heterozygotic mice (EPrnp+/�) and 25% dominanthomozygotic mice (EPrnp+/+). Wistar rats (8 weeks old, fromCharles River, Barcelona, Spain) were used for the synaptic detec-tion of PrPC. All animals were kept four per cage under a 12-hlight/dark cycle (lights on at 07:00 h) with free access to food andwater. All procedures were approved by the Local Ethics Committeeon the Use of Animals (Protocol number/UFSC PP 00452), whichfollows the NIH guidelines, published in “Principles of LaboratoryAnimal Care”.

Climbing behavior

Prnp+/+ and Prnp0/0 mice were individually placed into a wire meshcage (30 9 15 9 18 cm) and observed for 15 min to record thetime spent in climbing behavior (all four paws on the wire mesh).Climbing activity is indicative of central dopaminergic function andit is widely used to test the efficacy of antipsychotic drugs (Fetskoet al., 2003). When tested, the DA D1 receptor antagonist SCH-23390 (Sigma-Aldrich, St Louis, MO, USA) (0.1 mg/kg) or the DAD2 receptor antagonist sulpiride (RBI, Natick, MA, USA) (80 mg/kg) were administered intraperitoneally (i.p.) 30 min before the eval-uation of climbing behavior.

Western blot

Mice were anesthetized and killed by decapitation and the dissectedbrain tissues (OB, PFC, STR and HIP) were homogenized in an ice-cold buffer containing 10 mM HEPES [4-(2-hydroxyethyl)-1-piperaz-ineethanesulfonic acid, pH 7.4), 1.5 mM MgCl2, 10 mM KCl, 1 mM

phenylmethylsulfonyl fluoride, 5 lg/mL leupeptin, 5 lg/mL pepsta-tin A, 10 lg/mL aprotinin, 1 mM sodium orthovanadate, 10 mM b-glycerophosphate, 50 mM sodium fluoride and 0.5 mM dithiothreitol(all from Sigma-Aldrich). The homogenates were chilled on ice for15 min, vigorously shaken for 15 min in the presence of 0.1% Tri-ton X-100 and then centrifuged at 10 000 g for 30 min. The super-natant was collected as the cytosolic fraction (Medeiros et al., 2007)and stored at �80 °C until use. Protein concentration was deter-mined using a Bio-Rad protein assay kit (Bio-Rad, Mississauga,ON, Canada). Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) with 12% gels anddetected immunologically following electrotransfer onto nitrocellu-lose membranes (Amersham-Pharmacia Biotechnology, Piscataway,NJ, USA). The membranes were blocked in phosphate-buffered

saline (PBS; 140 mM, NaCl, 3 mM KCl, 20 mM NaH2PO4 and15 mM KH2PO4, pH 7.4) containing 5% powdered milk and 0.05%Tween-20 for 1 h at 25 °C. Membranes were then incubated over-night at 4 °C with an anti-tyrosine hydroxylase antibody (1 : 1000;MAB1423 from R&D, Minneapolis, MN, USA) in blocking solutionthen with an horseradish peroxidase-conjugated goat anti-mouseimmunoglobulin G (IgG) (1 : 1000; from Sigma-Aldrich) for 1 h.After exposure to peroxidase, the blots were visualized using a Perk-inElmer enhanced chemiluminescence system. Finally, the proteinsand molecular weight standards (BioRad, Hercules, CA, USA) wererevealed by Ponceau Red staining to confirm that similar amountsof protein were loaded in each lane.

High performance liquid chromatography (HPLC) analysis ofmonoamines

We used a previously validated HPLC separation method to deter-mine the levels of DA and its metabolites, 3,4-dihydroxyphenylace-tic acid (DOPAC) and homovallinic acid (HVA), as well asnorepinephrine and serotonin (Tadaiesky et al., 2008). Briefly, micewere killed by decapitation, their brains were removed immediatelyand the structures of interest (OB, PFC, STR and HIP) were dis-sected, frozen in liquid nitrogen and stored at �70 °C. The HPLCsystem consisted an LC-20AT pump (Shimadzu, São Paulo, Brazil)equipped with a manual Rheodyne 7725 injector with a 20-lL loopfeeding a Synergi Fusion-RP C-18 reversed-phase column(150 9 4.6 mm inner diameter with 4-lm particle size) protectedby a 4 9 3.0-mm pre-column (SecurityGuard Cartridges Fusion-RP), both maintained inside a temperature-controlled oven (25 °C;Shimadzu). The monoamines were identified with an electrochemicaldetector (ESA Coulochem III Electrochemical Detector) equippedwith a guard cell (ESA 5020) with the electrode set at 350 mV anda dual electrode analytical cell (ESA 5011A) with two chambers inseries – each chamber includes a porous graphite colorimetric elec-trode, a double counter electrode and a double reference electrode.Oxidizing potentials were set at 100 mV for the first electrode andat 450 mV for the second electrode. The tissue samples werehomogenized with an ultrasonic cell disrupter (Sonics, Newtown,CT, USA) in 0.1 M perchloric acid containing 0.02% sodium meta-bisulfite and internal standard. After centrifugation at 10 000 g for30 min at 4 °C, 20 lL of the supernatant was injected into the chro-matograph. The mobile phase, used at a flow rate of 1 mL/min, hadthe following composition – 20 g citric acid monohydrated (Merck,Darmstadt, Germany), 200 mg octane-1-sulfonic acid sodium salt(Merck), 40 mg EDTA (ethylenediaminetetraacetic acid; Sigma), in10% (v/v) methanol (Merck) to a final volume of 1000 mL withHPLC-grade water. pH was adjusted to 4.0 and the mobile phasewas filtered through a 0.45-lm filter before use. The peak areas ofthe external standards were used to quantify the metabolites of inter-est in the tested samples.

Autoradiography

Prnp+/+ and Prnp0/0 mice were killed by decapitation and their brainswere immediately removed, frozen over dry ice and stored at�80 °C until cryostat sectioning. Serial 20-lm coronal sectionswere cut on a Leica cryostat at �20 °C, collected onto glass slidesand stored at �80 °C until the day of the assays. DA D1 receptorbinding was probed with [3H]-SCH-23390, DA D2 receptor bindingwas detected using [3H]-raclopride, and the density of DA trans-porter (DAT) was evaluated with [3H]-WIN-35248. Briefly, the sec-tions were brought to room temperature and then pre-incubated in

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons LtdEuropean Journal of Neuroscience, 40, 2479–2486

2480 D. Rial et al.

50 mM Tris buffer (pH 7.4) containing 120 mM NaCl, 4 mM MgCl2,1 mM EDTA and 1.5 mM CaCl2, for 30 min (D1 receptor and DAT)or 15 min (D2 receptor). Sections were then incubated in Tris buffercontaining either 2 nM [3H]-SCH-23390 (86.0 Ci/mmol, PerkinElmer, Boston, USA) for 90 min at 37 °C (D1 receptor), or 2 nM[3H]-raclopride (76.0 Ci/mmol, Perkin Elmer) for 2 h at room tem-perature (D2 receptor) or 10 nM [3H]-WIN-35248 (85.0 Ci/mmol,Perkin-Elmer) for 2 h at room temperature (DAT). Sections werethen washed once (D1 receptor) or twice (D2 receptor) in ice-coldTris buffer for 5 min, followed by one quick dip rinse of ice-colddistilled water before drying. Slices were exposed to Kodak BiomaxMR-1 in tungsten cassettes together with calibrated standards for4 weeks. All groups were represented in each film. Films weredeveloped and densitometry analyses were performed using theMCID system (Imaging Research, St. Catherine’s, ON, Canada). Atleast five sections were measured to obtain values for each mousefor each region. Anatomical regions were defined according to themouse brain atlas of Paxinos and Franklin (2001).

Striatal DARPP-32 immunohistochemistry

Total DARPP-32 (tDARPP-32) density was evaluated in the STR ofsections (prepared as described above) from PrPC control (Prnp+/+)and PrPC null (Prnp0/0) mice using an anti-DARPP-32 antibody(1 : 500; from Cell Signaling, Danvers, MA, USA). Eight striata(four Prnp+/+ and four Prnp0/0) were used. DAPI (40,6-diamidino-2-phenylindole) was purchased from Cell Signaling. The sections werevisualized and images were acquired in a Zeiss LSM510 META(Carl Zeiss, Gottingen, Germany) confocal laser-scanning micro-scope using a 63 9 Plan-ApoChromat objective (1.4 numericalaperture) with LSM510 software. Quantification was made as theratio between tDARPP-32 and DAPI immunostaining in order tonormalize the labeling per number of cells. Note that we only quan-tified the total density of DARPP-32, which provides a measure ofthe potential transducing impact of dopaminergic inputs onto striatalmedium spiny neurons, rather the phosphorylation pattern of DAR-PP-32, which is an actual integrative measure of glutamatergic anddopaminergic signaling in medium spiny neurons (Svenningssonet al., 2004).

Immunocytochemical analysis of PrpC in dopaminergiccultured neurons

Embryos were removed at embryonic day 16 from pregnant Wistarrats (Charles River) that had been anesthetized, to prepare mesence-phalic neuronal cultures, as previously described (Fath et al., 2009).Briefly, the ventral mesencephalon was dissected in ice cold Hank’sbalanced salt solution, diced, and incubated for 15 min at 37 °C inHEPES-buffered minimum essential medium containing 0.05%Trypsin and 0.001% DNase I. The tissue was then washed oncewith minimum essential medium containing 0.052% trypsin inhibitorand gently dissociated with a 5-mL pipette to yield a suspension ofdispersed single cells. Cells were plated at a density of ~ 85 000cells/cm2 in Neurobasal medium (containing 10% heat-inactivatedfetal calf serum, 2% B27, 1% glutamax and 1% penicillin-strepto-mycin). After 24 h, the medium was changed and cultures weremaintained for up to 7 days in vitro in Neurobasal medium at 37 °Cin a humidified 5% CO2 incubator. Cells were then fixed with 4%paraformaldehyde for 30 min and washed twice in PBS (140 mM

NaCl, 3 mM KCl, 20 mM Na2HPO4, 1.5 mM KH2PO4). The cellswere permeabilized in PBS with 0.2% Triton X-100 for 10 min,blocked for 1 h in PBS with 3% bovine serum albumin and 5%

normal horse serum, washed twice with PBS and incubated over-night at 4 °C with different mixtures of primary antibodies, namely– mouse anti-PrPC (1 : 100; 8H4 from Sigma-Aldrich) or mouseanti-PrPC (1 : 1000; 6D11 from Sigma-Aldrich) and either rat anti-DAT (1 : 500; from Millipore, Billerica, MA, USA) or rabbit anti-TH (1 : 1000; MAB1423 from R&D) antibodies. The cells werethen washed three times with PBS with 3% bovine serum albuminand incubated for 1 h at room temperature with AlexaFluor-598(red)-labelled donkey anti-rabbit or anti-rat IgG antibodies and withAlexaFluor-488 (green)-labelled donkey anti-mouse IgG antibody(1 : 200 for each; from Invitrogen, Carlsbad, CA, USA). The cellswere washed three times in PBS, and then incubated with DAPI for15 min. We confirmed that none of the secondary antibodies pro-duced any signal in preparations to which the addition of the corre-sponding primary antibody was omitted. After washing andmounting on slides with Prolong Antifade, the preparations werevisualized and images where acquired in a Zeiss LSM510 META(Carl Zeiss) confocal laser-scanning microscope using a 63 9 Plan-ApoChromat objective (1.4 numerical aperture) with the LSM510software.

Preparation of membranes and purified nerve terminals fromthe rat striatum

Total membranes and synaptosomal (synaptic) membranes were pre-pared from the same animals, essentially as previously describedand validated (Rebola et al., 2003). Briefly, the striatum from onerat was homogenized at 4 °C in sucrose solution (0.32 M sucrose,1 mM EDTA, 10 mM HEPES, 1 mg/mL bovine serum albumin; pH7.4), supplemented with a protease inhibitor, phenylmethylsulfonylfluoride (PMSF, 0.1 mM), a cocktail of inhibitors of proteases(CLAP 1%, Sigma-Aldrich) and the antioxidant dithiothreitol(1 lM). The homogenate was centrifuged at 3000 g for 10 min at4 °C, and the resulting supernatant was then divided to prepare totalmembranes and synaptosomes. To prepare the total membranes, thesupernatant was further centrifuged at 25 000 g for 60 min at 4 °C.The supernatants were discarded and the pellet, corresponding tototal membranes from different brain cell types, was resuspended in5% (w/v) SDS with 0.1 mM PMSF and CLAP (1%), for subsequentWestern blot analysis. To prepare synaptosomal membranes, thesupernatant was further centrifuged at 14 000 g for 12 min at 4 °C.The resulting pellet (P2 fraction) was resuspended in 1 mL of a45% (v/v) Percoll solution in HEPES buffer medium (140 mM

NaCl, 5 mM KCl, 25 mM HEPES, 1 mM EDTA, 10 mM glucose; pH7.4). After centrifugation at 14 000 g for 2 min at 4 °C, the whitetop layer was collected (synaptosomal fraction), resuspended in1 mL HEPES buffer and further centrifuged at 20 800 g for 2 minat 4 °C. The supernatant was discarded and the pellet was resus-pended in 5% (w/v) SDS supplemented with 0.1 mM PMSF andCLAP (1%), for Western blot analysis.Purified nerve terminals from the striatum were obtained as previ-

ously described (Rodrigues et al., 2008). Briefly, one striatum washomogenized in medium containing 0.25 M sucrose and 10 mM HE-PES (pH 7.4). The homogenate was spun for 3 min at 2000 g at4 °C and the supernatant spun again at 9500 g for 13 min. The pel-let was re-suspended in 2 mL of 0.25 M sucrose and 10 mM HE-PES (pH 7.4) and 2 mL was placed onto 3 mL of Percolldiscontinuous gradient containing 0.32 M sucrose, 1 mM EDTA,0.25 mM dithiothreitol and 3, 10 or 23% Percoll, pH 7.4, and thegradients were centrifuged at 25 000 g for 11 min at 4 °C. Thenerve terminals were collected between the 10 and 23% Percollbands, diluted in 15 mL of a saline buffer (140 mM NaCl, 5 mM


PrPC regulates dopaminergic neurotransmission 2481

KCl, 5 mM NaHCO3, 1.2 mM NaH2PO4, 1 mM MgCl2, 10 mM glu-cose, and 10 mM HEPES, pH 7.4) and washed by centrifugation at22 000 g for 11 min at 4 °C. The purity of the nerve terminals wasconfirmed by immunocytochemistry (Rodrigues et al., 2008) show-ing that over 95% of visualized elements were immunopositive forsynaptophysin and < 3% were immunopositive for glial fibrillaryacidic protein (GFAP, data not shown). The nerve terminals wereplaced onto coverslips previously coated with poly-D-lysine (Sigma-Aldrich), fixed in PBS with 4% paraformaldehyde for 15 min andwashed twice with PBS. The nerve terminals were permeabilized inPBS with 0.2% Triton X-100 for 10 min and then blocked for 1 hin PBS with 3% bovine serum albumin and 5% normal bovineserum and the double immunocytochemical labeling of PrPC withDAT or TH was carried out as described for cultured neurons. Eachcoverslip was analysed using a Zeiss Imager Z2 fluorescence micro-scope equipped with an AxioCam HRm and 63 9 Plan-ApoChro-mat oil objective (1.4 numerical aperture) by counting six differentfields and in each field a total of 500 individualized elements, aspreviously described (Canas et al., 2014). The images, acquired ineach color channel using identical masks, were quantified using theIMAGEJ 1.37v software (NIH, Bethesda, MD, USA), to determine theco-localization of the different fluorophores in the platted nerve ter-minals by calculating the co-localization coefficients from the redand green two color-channel scatter plots (Costes et al., 2004) usinga macro routine developed by our group to automatically evaluatethe Pearson’s correlation between first and the second color channelwith a significance level > 95% (Rodrigues et al., 2008).

Statistical analysis

All data are expressed as mean � standard error of the mean andthe statistical analysis was carried out using two-tailed Student’st-test for independent samples for comparison of two groups

(usually genotype); and two-way analysis of variance was used formultifactorial comparison (testing the impact of drug treatment anddifferences between genotypes, i.e. between Prnp0/0 mice and theirrespective wild-type controls), provided that they passed the Shap-iro–Wilk’s W normality test. Following significant analyses of vari-ance, multiple post-hoc comparisons were performed using theNewman–Keuls test. The accepted level of significance for the testswas P ≤ 0.05. All tests were performed using the STATISTICA soft-ware package (StatSoft Inc., Tulsa, OK, USA).

Results

PrPC knockout mice display increased spontaneous climbingbehavior that is blocked by DA D1 and D2 receptorantagonists

In preliminary observations, we noted that PrPC-null (Prnp0/0) micedisplayed atypical climbing behavior, suggesting a modified dopami-nergic system (Fetsko et al., 2003). Thus, we systematically com-pared wild-type (Prnp+/+) and Prnp0/0 mice in the climbing behaviortest and concluded that Prnp0/0 mice spent significantly more timethan wild-type mice engaged in climbing behavior (differencebetween the two genotypes – F1,17 = 18.5, P < 0.05) (Fig. 1A). Thisfinding was confirmed using a different PrPC knockout mouse line,known as Edinburgh (difference between the two genotypesF2,11 = 11.9, P < 0.05; Fig. 1B). To assess the relative involvementof the DA D1 and D2 receptors in the expression of this altered phe-notype in Prnp0/0 mice, we administered Prnp+/+ and Prnp0/0 micewith D1 or D2 receptor antagonists, SCH-23390 (0.1 mg/kg, i.p.) andsulpiride (80 mg/kg, i.p.), respectively. Figure 1c and d show thatthe pretreatment with sulpiride decreased the spontaneous climbingbehavior in Prnp+/+ mice (treatment factor – F2,23 = 4.4, P < 0.05),which was abolished by the pretreatment with SCH-23390. More

A B

C D

Fig. 1. Effects of Prnp genetic deletion on the spontaneous climbing behavior in mice. (A) Time (s) spent by Prnp+/+ and Prnp0/0 mice in climbing behaviorduring a 15-min session of observation. *P < 0.05 compared with Prnp+/+ control group. (B) Spontaneous climbing behavior expressed by Edinburgh knockoutmice strain (EPrnp�/�) vs. heterozygotic mice (EPrnp+/�) and control wild-type group (EPrnp+/+). *P < 0.05 compared with EPrnp+/+ control group. Effects ofthe dopamine receptor antagonists SCH-23390 (D1 receptor antagonist, 0.1 mg/kg, i.p.) or sulpiride (D2 receptor antagonist, 80 mg/kg, i.p.) on the climbingbehavior of Prnp+/+ (C) and Prnp0/0 mice (D) [*P < 0.05 compared with saline-treated group (first bar from left); &P < 0.05 compared with sulpiride-treatedgroup] tested in a different cohort of mice. The results are presented as mean � SEM of 8–10 mice per group.


2482 D. Rial et al.

importantly, SCH-23390 reduced significantly the climbing behaviorin Prnp0/0 mice (treatment factor F2,24 = 15.1, P < 0.05) while sulpi-ride only partially reduced the climbing behavior (treatment factorF2,24 = 4.1, P < 0.05). Note that there was some variability in theclimbing behavior of Prnp+/+ and Prnp0/0 across experiments (cf.Fig. 1a, c and d), but there was always a preserved trend of signifi-cantly more time spent by Prnp0/0 than wild-type mice in climbingbehavior in the different tested cohorts of mice.

Deletion of PrPC imbalances the mesolimbic dopaminergicsystem

To probe whether the deletion of PrPC affects the DA system, wefirst compared in Prnp+/+ and Prnp0/0 mice the immunoreactivity ofTH, the rate-limiting enzyme of DA synthesis. We focused on DA-rich brain regions, namely the OB, PFC, STR and HIP. Interest-ingly, a lower density of TH was found in the OB (n = 3–4;t2,0.05 = 6.2, P < 0.05) and PFC (n = 3–4; t2,0.05 = 6.0, P < 0.05)of Prnp0/0 compared with wild-type mice, while TH density wasunchanged in the STR (n = 3–4; t2,0.05 = 2.1, P > 0.05) and HIP(n = 3–4; t2,0.05 = 0.1, P > 0.05) (Fig. 2A and B). Consistent withthis reduced TH immunoreactivity, Prnp0/0 mice displayed lowerDA levels in the OB (difference between the two genotypes –F1,6 = 11.9, P < 0.05) and PFC (difference between the two geno-types – F1,7 = 6.6, P < 0.05) whereas no significant alterations wereobserved in the STR (difference between the two genotypes –F1,8 = 1.6, P > 0.05) and HIP (difference between the twogenotypes F1,8 = 2.6, P > 0.05) (Fig. 2C and E). The levels of nor-epinephrine and serotonin were not significantly altered in the testedbrain areas of Prnp0/0 mice (data not shown).

Effects of the PrPC deletion on striatal dopaminergic signaling

Despite the lack of alterations of the DA content in the STR ofPrnp0/0 mice, we tested the possibility that PrPC deletion mightinstead modify the density of DA D1 and D2 receptors or of

DAT focusing on the STR, which is the main brain region wherechanges in the dopaminergic system have been related to climb-ing behavior (Protais et al., 1976). Autoradiography analysisshowed that PrPC-null mice displayed a lower density of D1

receptors (difference between the two genotypes – F1,10 = 6.8,P < 0.05) (Fig. 3A–C), but similar densities of D2 receptors andDAT (data not shown) in the STR in comparison with wild-typeanimals.We also probed the density of DARPP-32, a main signaling mole-

cule operated by DA, in the STR. The striatum of Prnp0/0 mice dis-played a lower immunoreactivity for total DARPP-32 (differencebetween the two genotypes – F1,9 = 7.9, P < 0.05) when comparedwith Prnp+/+ mice (Fig. 3D–F).

Presence of PrPC in DA neurons

Our behavioral and neurochemical studies indicate that PrPC dele-tion disrupts the homeostasis of the dopaminergic system. However,a previous study failed to localize PrPC in dopaminergic neurons(Ford et al., 2002), and we now re-evaluated this question usingpreparations with enriched dopaminergic components and comple-mentary methods of protein detection. Western blot analysis(Fig. 4A) showed that PrPC immunoreactivity was present in thenerve terminal fraction, as well as in whole membranes preparedfrom the STR. The localization of PrPC in nerve endings was re-enforced by the immunohistochemical detection of PrPC in purifiednerve terminals from the striatum of Prnp+/+ mice (n = 3–4;Fig. 4B). We confirmed the selectivity of the two anti-PrPC antibod-ies used (which recognize different PrPC epitopes) by the absence ofsignal in striatal nerve terminals from Prnp0/0 mice (n = 3, Fig. 4B).Quantitative immunocytochemistry revealed a co-localization ofPrPC in about 25% of TH-positive and DAT-positive striatal nerveterminals of Prnp+/+ mice (n = 3–4; Fig. 4C and D). In addition, inmesencephalic neuronal cultures from Prnp+/+ mice, we confirmedthe co-localization between PrPC and TH- and DAT-positiveneurons (n = 3; Fig. 4E–G).

A B

C D

Fig. 2. Effects of Prnp genetic deletion on DA synthesis and content. (A) The left panel shows representative Western blots of tyrosine hydroxylase (TH) den-sity in the olfactory bulb (OB), prefrontal cortex (PFC), striatum (STR) and hippocampus (HIP) of Prnp+/+ and Prnp0/0 mice, and the loading controls carriedout using a Ponceau staining are shown in the right panel. (B) Percentage TH immunoreactivity relative to the Prnp+/+ control group in Western blot analysis offour mice (*P < 0.05). (C) DA levels in the OB and PFC of Prnp+/+ and Prnp0/0 mice (*P < 0.05) compared with Prnp+/+ control group (Newman–Keuls post-hoc test). (D) DA levels in the STR and HIP of Prnp+/+ and Prnp0/0 mice. Levels are presented as mean � SEM of 3–4 mice per group.



Discussion

The present study provides the first evidence that the genetic manip-ulation of PrPC interferes with the endogenous DA machinery andmodulates DA-dependent phenotype in mice. Additionally, we alsoobtained direct neurochemical evidence for the presence of PrPC inDA neurons. This is in agreement with recent reports suggesting arole of PrPC in the functioning of the dopaminergic system in thebrain, namely: (1) PrPC controls DA metabolism through the regula-tion of monoamino-oxidase activity (Lee et al., 1999) (2) PrPC wasproposed to be associated with Parkinson’s disease, a neurodegener-ative disease associated with the progressive death of dopaminergicneurons (Olanow & Prusiner, 2009; Plate et al., 2013) (3) patientswith Creutzfeldt–Jakob disease (CJD), a prion disease, have abnor-mally high levels of a-synuclein (a highly expressed protein in Par-kinson’s disease) (Mollenhauer et al., 2008) (4) patients withschizophrenia, which according to the “DA theory of schizophrenia”is caused by an overactive DA system in the brain, displaydecreased levels of PrPC (Weis et al., 2008).The observation of increased spontaneous climbing behavior in

Prnp0/0 mice was our first clue for a modification of the DA system,motivating a more detailed investigation of the changes of the DAsystem caused by deletion of PrPC. Climbing behavior depends onthe synergistic activation of D1 and D2 receptors, and the indepen-dent blockade of each one of these receptor sub-types alone is suffi-cient to abrogate climbing behavior (Fetsko et al., 2003). Althoughthe STR is a key structure mediating climbing behavior (Protaiset al., 1976), we did not find alterations in DA levels in this brain

structure. However, we observed a significant reduction of D1

receptors (but not D2 receptors or DAT) in the striatum of Prnp0/0

mice. These findings indicate that PrPC can modulate dopaminergictransmission in the STR through changes of DA receptors, ratherthan of DA levels. This is re-enforced by the different pharmacolog-ical profile of climbing behavior in Prnp0/0 and Prnp+/+ mice,whereby the activation of D1 receptors played a pivotal role inPrnp0/0, but not in wild-type mice. However, we also observedchanges (see below) that suggest altered D2 receptor signaling inthe STR, indicating that the modification of climbing behavior inPrnp0/0 mice might actually involve an imbalanced dopaminergicsignaling in the STR. Indeed, we also observed that PrPC deletiondecreased DARPP-32 levels in the striatum, which suggests anincreased resistance to the inhibitory effects of DA (operatedthrough the D2 receptor) in this brain region, as observed in DAR-PP-32 knockout mice, where higher concentrations of raclopride (aselective D2 receptor antagonist) are required to induce catalepsy(Lindgren et al., 2003). Interestingly, PrPC levels are decreased inschizophrenic patients, suggesting that the integrative role of neuro-transmitters operating through DARPP-32 might also be altered inthose subjects (Kunii et al., 2014).Our overall conclusion that the lack of PrPC differently modulates

DA synthesis, content, the density of DA receptors and their signal-ing pathways in different brain areas does not allow us to concludethat PrPC directly affects the dopaminergic system. However, theevidence showing that PrPC is present in DA neurons strengthensthis contention. In fact, using complementary methods applied to

A

D

E F

B C

Fig. 3. Effects of Prnp genetic deletion on dopaminergic signaling. (A–C) Comparison of the density of D1 receptors in the striatum of Prnp+/+ and Prnp0/0

mice. Results are presented as mean � SEM of four mice per group, with *P < 0.05 when compared with PrPC control mice. Images at 5 9 magnification.Acc, nucleus accumbens; DLCPu, dorsolateral caudate putamen. (D) Representative confocal microscopy images of tDARPP-32 immunoreactivity in the stria-tum of Prnp+/+ and Prnp0/0 mice. Scale bar = 10 lm. (E) Measurement of the tDARPP-32/DAPI ratio. The results are presented as mean � SEM of 3–4 miceper group with *P < 0.05 when compared with PrPC control mice. (F) Representative 2.5D representation of the tDARPP-32/DAPI measurements.


2484 D. Rial et al.

enriched dopaminergic preparations, we showed that dopaminergicmesencephalic neurons (TH- or DAT-positive) are also endowedwith PrPC immunoreactivity and that about 25% of TH-positive andDAT-positive nerve terminals (i.e. dopaminergic nerve terminals) inthe STR are co-localized with the PrPC. Although we cannotexclude that the modifications observed in the dopaminergic systemmight indirectly result from the deletion of PrPC in non-dopaminer-gic components, the most parsimonious explanation is the possibilitythat PrPC might directly affect the functioning of DA neurons. Irre-spective of the cellular and molecular mechanisms, this ability ofPrPC to control the homeostasis of the dopaminergic system sug-gests that PrPC might be related to physiopathological conditionsassociated with DA dysfunction. Thus, the DA system, especially inthe STR (Tritsch & Sabatini, 2012), and PrPC (Khosravani et al.,2008; Rial et al., 2009) seem to be parallel and inter-twinned sys-tems involved in neuroplastic adaptive changes. In particular, it istempting to speculate that PrPC could play a role in neurodegenera-tive diseases affecting the DA system, namely in Parkinson’s dis-ease, which is in line with the identification of parkinsoniansymptoms in patient with the D202N Gerstmann–Str€aussler–Schein-ker PrPC mutation (Plate et al., 2013). Furthermore, there is a paral-lel between the trans-cellular propagation of misfolded prionproteins and the new concept of a-synuclein cell-to-cell transmissi-bility (Freundt et al., 2012), suggesting that a-synuclein might usethe misfolded prion proteins as vectors to subsequently damageneighboring neurons. Certainly, additional studies are necessary tounderstand to what extent the binomial PrPC–DA interaction isimportant to control the plastic events underlying the demise ofbrain disorders.

In summary, we provide pioneering evidence suggesting that PrPC

is an important modulator of the DA neurotransmitter system andthat the genetic deletion of Prnp alters behavioral and neurochemicalhallmarks of DA function. Taken together, the results of the presentstudy indicate that PrPC is relevant for the homeostasis of the meso-corticolimbic DA transmission, driving adaptive fluctuations in thelevels of D1 receptors. However, the molecular pathways relatingPrPC to adaptive changes of the DA system remain to be uncovered.

Acknowledgements

This work was supported by grants from the Conselho Nacional de Desen-volvimento Cient�ıfico e Tecnol�ogico (CNPq), Coordenac�ão de Aperfeic�oa-mento de Pessoal de N�ıvel Superior (CAPES), Programa de Apoio aosN�ucleos de Excelência (PRONEX- NENASC Project), Fundac�ão de Apoio �aPesquisa do Estado de Santa Catarina (FAPESC), Fundac�ão para a Ciência eTecnologia (PTDC/SAU-NMC/114810/2009) and CAPES/FCT. D.R.received scholarships from CNPq. R.W., R.N.T. and R.D.P. are supported byresearch fellowships from CNPq. We thank the Advanced Light MicroscopyUnit of IBMC (Institute for Molecular and Cell Biology) for technical help.The authors declare no conflict of interest.

Abbreviations

cAMP, cyclic adenosine monophosphate; DA, dopamine; DARPP-32,dopamine- and cAMP-regulated phosphoprotein 32 kDa; DAT, DA transporter;EDTA, ethylenediaminetetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HIP, hippocampus; HPLC, high-performanceliquid chromatography; IgG, immunoglobulin G; i.p., intraperitoneally; OB,olfactory bulb; PBS, phosphate-buffered saline; PFC, prefrontal cortex; PMSF,phenylmethylsulfonyl fluoride; PrPC, cellular prion protein; SDS, sodium dode-cyl sulfate; STR, striatum; tDARPP, total DARPP; TH, tyrosine hydroxylase.

A

C

E F G

D

B

Fig. 4. Localization of PrPC in dopaminergic neurons. (A) Comparison of the density of PrPC in synaptossomal (SYN) and total membranes (TM), detectedwith the PrP6D11 antibody. (B) The disappearance of PrPC immunolabeling when using either PrP6D11 or PrP8H4 antibodies in neurons from Prnp0/0 mice tes-tifies to the selectivity of both antibodies for PrPC. Scale bar = 5 lm. (C) Localization of PrPC in dopaminergic (i.e. TH-positive and DAT-positive) nerveterminals. Scale bar = 5 lm. The results are representative of 3–4 rats per group. (D) Quantification of PrPC in TH and DAT-positive neurons. (E–G) Co-locali-zation of PrPC and dopaminergic markers in mesencephalic neuronal cultures, as assessed using the two antibodies against different PrPC epitopes. Scalebar = 10 lm (E, F) and 5 lm (G). The results are representative of three independent cultures.



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cellular prion protein is present in dopaminergic neurons and modulates the dopaminergic system

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