Protein & Peptide Letters, 2009, 16, 000-000 1

0929-8665/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.

Confocal Microscopy Evidence of Prion Protein Fragment hPrP[173-195] Internalization in Rat B104 Neuroblastoma Cell Line

Emanuela Urso1, Raffaele Acierno

1, Maria Giulia Lionetto

2, Antonia Rizzello

1, Andrea Papa

1,

Trifone Schettino2 and Michele Maffia

1,*

1Lab. of Adaptive Physiology, Department of Biological and Environmental Science and Technology, University of

Lecce, prov.le Lecce-Monteroni, 73100 Lecce, Italy; 2Lab. of General and Environmental Physiology, Department of

Biological and Environmental Science and Technology, University of Lecce, prov.le Lecce-Monteroni, 73100 Lecce,

Italy

Abstract: The cytotoxicity of hPrP[173-195] prion peptide against a neuroblastoma cell model was found independent of

its tendency to aggregate over time. Cytosolic and nuclear inclusions of peptide were highlighted by confocal microscopy,

suggesting a role as a transcription factor in activating signal transduction pathways involved in cell toxicity.

Keywords: Prion disease; cellular prion protein (PrPC); helix-2; structural ambivalence; synthetic prion peptides; confocal mi-

croscopy.

INTRODUCTION

The cellular prion protein PrPC is a glycosylphosphatidy-

linositol (GPI)-linked protein of the surface of a variety of cell types including neurones, glial cells and leukocytes. In humans it’s encoded by the PRNP gene located on chromo-some 20 [1, 2]. Solution Nuclear Magnetic Resonance (NMR) experiments on prion protein folding in several spe-cies (Syrian hamster, mouse, bovine and human) evidenced a well-conserved structure, with a high -helical content and high susceptibility to proteolytic digestion [3]. PrP

C consists

of a globular domain encompassing residues from 120 to 231 and a N-terminal unstructured "tail" of about 100 amino ac-ids. The former is folded into three alpha-helices and a two-stranded, anti-parallel -sheet [4, 5], while the latter is com-posed of a highly conserved repeat of four octapeptide units with the consensus sequence PHGGGWGQ, proposed to selectively bind copper(II) ions under physiological condi-tions [6, 7]. Although the physiological function of PrP

C is

undefined, it was proposed to drive copper delivery across plasma membrane by endocytosis [8] and to hold a neuropro-tective role achieved through its anti-oxidant activity [9, 10] and activation of cAMP/PKA dependent transduction path-ways [11]. A widespread expression throughout the brain tissues suggested a possible role of PrP

C in synaptic trans-

mission [12, 13].

The structural transition of the cellular prion protein to the aberrant isoform PrP

Sc (prion) is commonly considered a

critical event in the pathogenesis of a large group of neu-rodegenerative diseases known as Transmissible Spongiform Encephalopathies (TSEs). The abnormal protein displays a higher percentage of -sheet structure and an increased resis-tance to proteolysis respect to PrP

C, although sharing with it

the primary sequence [14, 15]. The most accepted current

*Author correspondence to this author at the Department of Biological and Environmental Science and Technology, University of Salento, Lecce, Italy;

E-mail: m.maffia@fisiologia.unile.it; michele.maffia@unile.it

theory explaining PrPC conversion leading to PrP

Sc formation

states that structural transition takes place through a physical interaction between the two isoforms, where PrP

Sc would act

as a template [15].

Several and detailed studies have been addressed to gain insight into the structural determinants affecting the prion conversion. Nevertheless, neither the mechanism of confor-mational change nor the tertiary structure of PrP

Sc have been

clarified yet because of the aggregative properties of the scrapie isoform, which make it difficult to isolate the PrP

Sc

molecule [16, 17]. A bulk of experimental evidences, aimed to define structural prerequisites for PrP

CPrP

Sc conversion,

has been collected so far, with controversial outcomes [18-21]. The examination of monomeric and dimeric foldings of the carboxy-terminal domain of PrP

C evidenced some re-

gions possibly driving the structural transition of prion pro-tein [4, 22-24]. It’s noteworthy how the crystalline dimeric conformer represents a link between the PrP

C isoform and

the pathogenous one [24, 25]. In this rearrangement the C-terminal region of helix-2 is converted into a strand con-formation while the helix-3 swaps between the two globular domains. The putative full conversion of helix-2 together with its immediate neighbourhood into three adjacent strands could be a prerequisite for the emergence of the PrP

Sc

structure [25].

Conformational tendencies exhibited by prion protein helices have been explored by NMR investigations and se-quence alignments in the attempt to individuate regions sus-ceptible to conformational fluctuations. A comparative analysis of short-range contacts in PrP

C and other non-

amyloidogenic proteins from a databank (Protein Data Bank) revealed that they are unusually distributed within the prion protein and localised around the helix-2 [26]. The network of contacts involving this prion protein (PrP) region induces it to assume a forced -helical conformation within the protein, in disagreement with predicted secondary structure (phe-

2 Protein & Peptide Letters, 2009, Vol. 16, No. 11 Urso et al.

nomenon of discordance), suggesting that the helix-2 region easily undergoes structural rearrangements [26].

Synthetic variants of PrP helix-2 have been structurally characterized also by Circular Dichroism (CD) analysis, in-dicating a conformational ambivalence of the variants due to the low free energy barrier between and structure [16]. This characteristic is confirmed by the structural fragility of the synthetic variant 180-195, which assumes a -conformation under a wide variety of conditions [16]. As a corollary, also the overall rearrangement of prion protein could be affected by microenvironmental factors such as pH or the presence of metal ions (CuII) [16, 27, 28].

In support of an involvement of the helix-2 region in PrP

CPrP

Sc transition, Kallberg and co-workers (2001)

demonstrated by bioinformatic tools that the prion protein sequence 179-191 (helix-2) actually exhibits an -helical structure in discordance with the theoretical predictions of a

-sheet rearrangement [29]. The same study showed that PrP helices 1 and 3 are -helical structured according to compu-tational methods. The discrepancy found for the helix-2 se-quence is common to several amyloid-forming proteins, sug-gesting a role for this region in PrP

Sc nucleation and oli-

gomerization [29]. In the discordant nucleus of PrPC helix-2

the cysteine residue is connected to helix-3 through a disul-phide bond and asparagine-178 is glycosylated. Conse-quently, alterations of this region can have some repercus-sions on the overall structure of the protein [29]. Interest-ingly, structural predictions obtained by computational methods gave conflicting information about secondary struc-ture propensities of helix-2, consistent with the possibility that prion protein can adopt more than one conformation. This implies that PrP

C could be flexible independently of

external factors and susceptible of structural transitions [30-32]. Lastly, of more than 20 different mutations in the PRNP gene associated with inherited human TSEs [33] such as familial Creutzfeldt Jacob disease (fCJD) [34], Gerstmann-Sträussler-Scheinker syndrome (GSS) [35] and Fatal Famil-ial Insomnia (FFI) [36], seven of them (D178N, V180I, T183A, H187R, T188R, T188K, T188A) are located in the helix-2 region [26].

Given the emerging idea of helix-2 as an anchor-point in the PrP

CPrP

Sc transition, we tested on neuroblastoma cell

cultures two synthetic model peptides, hPrP[173-195]AcAm, which represents the entire helix-2 region, and the shorter hPrP[180-195]AcAm, found to be a site of structural insta-bility in the helix-2 sequence. In addition, the intracellular intake of hPrP[173-195]AcAm peptide was detected by con-focal microscopy using a fluoresceinated version of the fragment.

MATERIALS AND METHODS

Peptide Synthesis and Characterization

Three peptides corresponding to the helix-2 of prion pro-tein were synthesized in batch by standard Fmoc chemistry protocol on Rink-amide MBHA resins.

The acetylated-N-terminus and amidated-C-terminus peptides with sequences Ac-NNFVHDCVNITIKQHTVTT TTKG-NH2 and Ac-VNITIKQHTVTTTTKG-NH2 (hPrP

[173-195]AcAm and hPrP[180-195]AcAm), after peptide

assembling, were acetylated with 1 M acetic anhydride in dimethylformamide (DMF) containing 5% diisopropylethy-lamine (DIEA). In order to clarify the nature of interaction between cells and the helix-2, the fluoresceinated-N-terminus and amidated-C-terminus peptide Fl-NNFVHDC (Me)VNITIKQHTVTTTTKG-NH2 (hPrP[173-195]FlAm

C179C(Me)) was also prepared with the Cys179 methy-lathed to prevent oxidative phenomena. At the N-terminus of this peptide a -alanine residue was firstly introduced, to avoid fluoresceine elimination by Edman degradation mechanism, and then the fluoresceine probe, using 2 eq. of Fluoresceine Isothiocyanate (FITC) and 2 eq. of diisopro-pylethylamine (DIEA) in dimethylformamide (DMF), for 2 hours at room temperature. Cleavage from the solid support was performed by treatment with a trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/water (90:5:5, v/v/v) mixture for 90 min at room temperature. Then, peptides were precipi-tated in cold ether, dissolved in water/acetonitrile (1:1, v/v), lyophilized and purified by RP-HPLC using a C18 Jupiter (250x22 mm) column applying a linear gradient of 0.1% TFA acetonitrile in 0.1% TFA water. Peptide purity and in-tegrity were confirmed by RP-HPLC analysis and by MALDI-TOF mass measurements (Voyager-DE Biospec-trometry Workstation, PerSeptive Biosystems).

Cell Culture

B104 neuroblastoma cells derived from rat central nerv-ous system [37] were grown at 37 °C, 5% CO2, in Dul-becco’s modified Eagle’s medium (Euroclone Life Science, Milano, Italy), 1g/l glucose, supplemented with 10% fetal bovine serum (FBS), 1 mM Na-piruvate, 2 mM glutamine and antibiotics (penicillin/streptomycin 5000 U/ml/5 mg/ml) [38].

MTT Test

Neuroblastoma cells were plated into 96-well trays and, after a day, hPrP[173-195]AcAm peptide was added to the culture medium with increasing concentrations from 0 to 55

M. Cell survival was determined after 18, 24, 48 h of incu-bation. Rising concentrations (0-55 M) of hPrP[180-195]AcAm peptides were tested on cell culture, with cell survival detected after 48 h exposure.

MTT ([3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetra-zolium bromide] 5mg/ml; SIGMA-Aldrich, St. Louis, MO, USA) was added to cell culture for 3 h at 37 °C. The MTT formazan product was released from cells by addition of dymethilsulfoxide and measured spectrophotometrically at 570 nm [39]. Percent of survival was assessed by compari-son with untreated cultures (control). The LC50 values (de-fined as the 50% lethal concentration) after 48 h exposure were estimated by testing peptides over a broad range of concentrations from 0 to 240 M. LC50 values were deter-mined by Hill plot analysis.

Co-Treatment with Tetracycline

Tetracycline is a commonly employed antibiotic known for its ability to decrease resistance of PrP

Sc isoforms to pro-

teinase K digestion by interfering with the conformational rearrangement of proteins [40]. To test if this compound was able to revert the toxic effect found for hPrP[173-195]AcAm

Confocal Microscopy Evidence of Prion Protein Fragment Protein & Peptide Letters, 2009, Vol. 16, No. 11 3

and hPrP[180-195]AcAm peptides, cultured cells were sepa-rately exposed for 48 h to increasing concentrations (0-55

M) of both prion fragments alone or in addition to tetracy-cline hydrochloride (SIGMA-Aldrich, St. Louis, MO, USA) in a 1:0.5 molar ratio. Cell viability was estimated by MTT test.

Confocal Microscopy

To get information about the way of action of the wild-type prion peptide (hPrP[173-195]AcAm), we preliminarily tried to inquire into its interaction with cultured cells by con-focal microscopy visualization. At this aim a fluoresceinated version of peptide was synthesized, whose toxicity was found to be unaltered respect to the unmarked fragment (data not shown).

B104 cells were grown on laminin (BD Biosciences, Cowley, UK; 10 g/mL) coated glass cover slips for 24 hours. Then cells were incubated with 10 M hPrP[173-195]FlAm C179C(Me) for 5 min, 30 min, 6 hours. We chose to test the lower concentration of peptide found to induce the maximum toxicity to neuroblastoma cells, as shown by pre-liminary MTT test. After the incubation, cell monolayers were fixed in 2% paraformaldehyde for 10 min at room tem-perature and then 30 min on ice. This step was followed by three washes in TBS (20 mM Tris-HCl, pH 7.4, 137 mM NaCl) supplemented with 0.1% Na-azide. To label cell sur-face the fixed cell monolayers were incubated for 1 min in 0.02 mg/mL Wheat Germ Agglutinin (WGA)-conjugated tetramethylrhodamine (Molecular Probes, Eugene, OR, USA) dissolved in TBS containing 1% BSA. Wheat Germ Agglutinin is a widely characterized lectin consisting of two identical subunits (18 KDa). It possesses binding sites spe-cific for cell surface-associated carbohydrates, allowing the fluorescent probe to attach to the plasmamembrane [41]. Cover slips were finally rinsed twice with MOPS buffer (10 mM MOPS, 5 mM EGTA, 20 mM K2HPO4, 2 mM MgSO4, pH 6.9) and mounted on glass slides using DABCO (1,4-diazabicyclo[2.2.2]octane; SIGMA-Aldrich, St. Louis, MO, USA) mounting medium. The cells were observed using a 40X/1.4 NA plan apochromat objective mounted on a TE300 (Nikon, Tokyo, Japan) fluorescence microscope equipped with a Nikon C1 confocal laser scanning unit. Excitation sources were Argon-488 for Fluoresceine signal and Helium-Neon-543 for tetramethylrhodamine. The vertical cross-sectional images of the cells were visualized by using repeti-tive z-optical scanning with a step distance of 1 m. Unla-beled preparations were found to exhibit no fluorescence under the conditions used. Where double labeling was em-ployed, laser intensity settings and emission wavelengths were optimized for minimal bleed through between the two channels.

To estimate the peptide cellular intake for each time con-dition the green signal intensity was quantitatively analysed by ImageJ software and expressed as density arbitrary units after background correction.

Statistical Analysis

Results of MTT tests were reported as means±SEM of three experiments with three replicates for each one and ana-lysed by GraphPad Prism 4 software. Comparison of treated

cultures versus control condition was performed by means of two way-ANOVA (Bonferroni post-test). A p value <0.05 (*) was considered statistically significant.

RESULTS

Effect of Peptide Treatment on Cell Viability

The first synthetic peptide we tested (hPrP[173-195]AcAm) encompasses the full-length helix-2 of the hu-man prion protein, a region proposed to play a key role in PrP

CPrP

Sc conformational transition because of its struc-

tural ambivalence. To detect its effect on cell viability, we exposed B104 neuroblastoma cells to increasing concentra-tions of prion fragment from 0 to 55 M for 18, 24 and 48 hours. Loss of cell viability evidenced by the MTT test on our pure neuronal culture treated with increasing concentra-tions of hPrP[173-195]AcAm (within 0-55 M range) re-sulted approximately the same for each incubation time (p>0.05), with the highest cell death percentage comprised within 40% for 18 h treatment and about 50% for 24 and 48 h treatment Fig. (1). After 18 and 24 h cell exposure the syn-thetic prion fragment produced a dose-dependent toxic effect within the 0-10 M range. Dose-dependence was extended to 20 M after 48 h exposure Fig. (1). LC50 value for hPrP[173-195]AcAm after 48 h treatment (calculated over a broader range of peptide concentrations, 0-240 μM) was es-timated to be 67.7 M. Exposure of B104 cells to increasing concentrations of hPrP[180-195]AcAm peptide for 48 hours resulted in a more pronounced effect on cell viability with respect to the biological response evoked by the full-length helix-2 fragment, as proved by LC50 value of 10.7 M Fig. (2).

Figure 1. Neurotoxicity of hPrP[173-195]AcAm peptide on B104

neuroblastoma cell line after 18, 24 and 48 hours exposure to in-

creasing concentrations of prion peptide.

For each exposure time, cell survival was significantly decreased

(**p<0.01) respect to untreated cultures already at the lower con-

centration tested (2 μM; N=3).

Effect of Tetracycline

Growing concentrations of hPrP[173-195]AcAm and hPrP[180-195]AcAm fragments (0-55 μM) were tested on

4 Protein & Peptide Letters, 2009, Vol. 16, No. 11 Urso et al.

B104 cultured cells in the presence of tetracycline in a 1:0.5 molar ratio. Co-treatment with the antibiotic significantly reversed the cytotoxicity of hPrP[173-195]AcAm, totally abolishing cell death within the 2-20 μM range (Fig. 3A). Tetracycline exhibited a mild effect on the biological behav-iour of the shorter fragment hPrP[180-195]AcAm, inducing only a slight recovery of cell viability for most of fragment concentrations (Fig. 3B).

Figure 2. Neurotoxic effect of hPrP[180-195]AcAm peptide on

B104 neuroblastoma cell line after exposure to increasing concen-

trations of prion peptide.

Starting from 2 M the neurotoxic effect was statistically signifi-

cant (N=3, **p<0.01).

Cellular Intake of Peptide

We employed a confocal microscopy method to detect the intracellular intake of the peptide. Cells were incubated with 10 M hPrP[173-195]FlAm C179C(Me) for 5 min, 30 min and 6 hours. Following fixation, monolayers were treated with the fluorescent probe WGA-conjugated tetram-

ethylrhodamine for plasmamembrane labelling. Fig. (4) shows the confocal optical sections of the middle of cell bod-ies, with the red fluorescent signal evidencing cell mem-branes while the peptide is visualised in green. Confocal doubled stained pictures show a diffuse and remarkable green emission signal inside the cells already after 5 min of incubation (Fig. 4A). Fluorescence intensity doesn’t increase with longer exposures to the peptide (Fig. 4B, C, D).

In order to better clarify the intracellular localization of the peptide, the red and green fluorescence signals were de-tected separately in the same cells (Fig. (5)). In Fig. 5A, where only tetramethylrhodamine red fluorescence is shown, an intracellular red circled region is clearly evident inside the cells. This region shows a spherical shape in serial z-cross-sectional images of the cells (data not shown), indicating that the fluorescent dye WGA-tetramethylrhodamine enters the cell and binds to the carbohydrate residues attached to the nuclear membrane in addition to those exposed on the plas-mamembrane. Internal membrane compartments also ap-peared labelled. By overlapping peptide green fluorescence to red fluorescence, it was possible to show the peptide local-ization both in the cytoplasm and in the nucleus of neuro-blastoma cells (Fig. 5B, C). Rare peptide aggregates are de-tectable close to cell membranes and inside the cells behind the nuclear border (Fig. 5B; head arrow). Prion peptide lo-calisation suggests an interaction of the prion fragment with not defined cytoskeleton components, as already revealed by a similar approach for another prion peptide, PrP[106-126], undoubtedly retained the cytotoxic core of PrP

Sc [42].

DISCUSSION

Neurodegenerative diseases known as Transmissible Spongiform Encephalopathies (or prion diseases) represent one of the most intriguing contemporary challenges to bio-medical research. According to the currently accepted "pro-tein-only" hypothesis, the development of these pathologies

Figure 3. Effect of tetracycline on B104 cell line survival under hPrP[173-195]AcAm (A) and hPrP[180-195]AcAm (B) treatment.

Cultured cells were incubated for 48 h in the presence of increasing concentrations of peptides alone (black bar) or in addition to tetracycline

(1:0.5 molar ratio; white bar). The effect of co-incubation on cell viability was evaluated by MTT test (N=3; *p<0.05, **p<0.01 versus treat-

ment with single peptides).

Confocal Microscopy Evidence of Prion Protein Fragment Protein & Peptide Letters, 2009, Vol. 16, No. 11 5

Figure 4. Confocal imaging of fluoresceinated hPrP[173-195]FlAm

C179C(Me) cellular intake by B104 neuroblastoma cell line (40x

objective magnification).

Red fluorescence individuates cell borders while green signal marks

peptide localisation. Co-localisation is evidenced by yellow areas.

Incubation times: (A) 5 min; (B) 30 min; (C) 6 h. Densitometric

analysis showed that the maximum intake of peptide can be de-

tected after 5 min of incubation (N=3; D). Peptide intake was calcu-

lated as mean±SEM of intracellular immunofluorescence signal

intensities (expressed by arbitrary units) of at least 30 cells for each

time exposure condition.

would be due to accumulation in the nervous tissue of the pathologic isoform (PrP

Sc) of the physiologic protein PrP

C

[1, 43, 44]. PrPSc

shares the same amino acid sequence of its normal counterpart, although it differs because of a higher -sheet content [7, 14]. The abnormal form also shows higher resistance to proteolysis and the ability to aggregate extracel-lularly and to form amyloid plaques [45]. Despite a general confidence in PrP

Sc involvement in the aetiology of prion

diseases, the events leading to prion rearrangement and the

ultimate causes of the associated neurodegeneration are poorly understood. Investigations on PrP-deficient cell lines revealed slight alterations in metabolism [9, 46], and PRNP knock-out mouse strains evidenced moderate disturbances in development, but they didn’t manifest disease later [47]. These findings suggest that loss of PrP

C function could be a

marginal component of neuronal damage, supporting a direct action of PrP

Sc or its metabolic fragments [12].

To mimic in vitro the behaviour of the scrapie isoform, various peptides reproducing short sequences of the prion protein have been used [27, 48-50], among which the PrP[106-126] fragment was the most extensively studied [48, 51, 52]. This peptide encompasses the 112-114 PrP region, susceptible to metabolic cleavage, which acquires protease resistance after PrP

CPrP

Sc transition [53]. The conforma-

tional change confers to the peptide several characteristics of PrP

Sc, such as -sheet conformation, propensity to aggregate

and protease resistance [48, 53]. PrP[106-126] was found to directly interact with the cellular prion protein by binding to the homologue 112-119 residues and to compromise the PrP

C superoxide dismutase activity in cerebellar cell cultures

[54]. The reported mechanisms of action include mitochon-drial depolarization [55], activation of the cell death pathway through the caspase enzymes [56] and disruption of calcium homeostasis [57, 58]. According to the evidence of an affin-ity to tubulin [51], PrP[106-126] was demonstrated to inter-act with cytoskeleton. Consequently, the toxicity of that fragment was attributed to the destabilization of microtu-bules, which, in turn, influences the function of L-type cal-cium channels [59]. There’s large evidence that oxidative stress and disturbance in the expression of antioxidants can participate to the neurological damage [60].

Daniels et al. (2001) focused on the intrinsic toxicity of the C-terminal region of prion protein (PrP[121-231]), whose sequence was "dissected" to synthesize five peptides des-tined to be separately tested on cultured neurones [61]. The results suggested that toxicity is particularly associated with 163-184 amino acid residues, although the further N-terminal residues also participate to the phenomena. Simul-taneous addition of PrP[121-231] and PrP[112-125] frag-ments to cell cultures suppressed the toxicity of the former, suggesting the co-existence of either a toxic domain and a neutralising one in the intact prion protein [61]. Another PrP fragment, PrP[118-135], was found to exhibit membrane destabilising properties and to determine apoptotic cell death in a time- and dose-dependent manner [50]. The toxicity induced in cultured cortical neurones was correlated to oxi-dative stress, since antioxidant molecules were able to pro-tect these cells against prion fragment action [50].

Following a similar approach, we studied the biological effects of a synthetic prion protein fragment denoted as hPrP[173-195]AcAm (N- and C-terminus were blocked), whose sequence corresponds to the whole hPrP helical re-gion two. Our choice was encouraged by recent reports about a high probable role of helix-2 in promoting the process of PrP oligomerization and then fibrillization [21, 24, 27, 62].

At this regard, synthetic peptides reproducing the -helical regions of PrP

C were previously characterized by CD

spectroscopy to assess their conformational and aggregation properties [27]. While the helix-1 fragment PrP[144-154]

6 Protein & Peptide Letters, 2009, Vol. 16, No. 11 Urso et al.

was unable to assume a -sheet conformation under a variety of pH and time conditions, PrP[178-193] (helix-2) revealed a tendency to form a -sheet structure and visible aggregates over time, independently of the establishment of disulphide bonds [27]. More recently Tizzano et al. (2005) confirmed the time-dependent aggregation properties of hPrP[173-195] peptide both in the blocked (N and C-terminus acetylated and amidated) and in the free form [16]. CD analysis allowed to attribute a remarkable conformational lability to this por-tion of the prion protein due to the little free energy differ-ence between and folding and the site of local instability identified with the short C-terminal sequence of threonines (190-195). All these characteristics strongly support the in-volvement of helix-2 as a crucial site in the direct interaction between the normal and aberrant isoforms of prion protein during infection [16] and make the hPrP[173-195] fragment an optimal model to investigate the PrP

C/PrP

Sc theory model

on cell cultures.

The in vitro activity of hPrP[173-195]AcAm synthetic fragment was investigated in B104 rat neuroblastoma cell model [37]. When exposed to increasing concentrations of the helix-2 peptide for growing time periods up to 48 hours, cells displayed a dose-dependent reduction of cell viability up to 10 M as regards 18 and 24 h cell treatment. After 48 h exposure, we detected a dose-dependent loss of cell viability extended up to 20 M, not increased by higher concentra-tions of fragment. In the light of the structural determinations performed by Tizzano et al. (2005) [16], it appears that cell toxicity doesn’t require the peptide to be in an aggregate state. Fragment we analysed assumes a random conformation below 21 M concentration and its folding into aggregates, observed above 21 M, doesn’t significantly accentuate the lethal effect of fragment, indicating that the ability to rear-range in a similar way is not relevant for the neurotoxic ac-tion. Moreover, the 173-195 peptide, lacking the N-linked glycosylation observed in the intact prion protein, exhibits an increased tendency to form fibrils as a consequence of re-duced steric impediments to intermolecular bindings [62]. Consistent with our concept on the non-essentiality of aggre-

gation properties to cytotoxicity, Thompson et al. (2000) showed that 6 day treatment of mouse neuronal cultures with helix-2 derived peptides (PrP[178-193] and PrP[180-193]) didn’t affect cell viability, despite their ability to form -sheet rich aggregates and fibrils [27].

The cytotoxicity of hPrP[180-195]AcAm peptide to B104 cells was shown to be higher respect to the whole he-lix-2 fragment, as proven by the lower LC50 value, so con-firming the hypothesis that the C-terminus residues of PrP helix-2 are critical for the induction of the biological effects. In agreement, the relevance of the 180-195 sequence for the neuronal toxicity exhibited by helix-2 derived peptides, in-dependently of their structural organization, was recently reported [63].

In the attempt to individuate appropriate approaches to limit the advancing of prion infection, a variety of molecules have been tested, including Congo Red [64, 65] and iodo-doxorubicin [66]. These compounds are considered strong antagonists of the onset of prion infection in that they con-tain hydrophobic aromatic rings allowing the binding to the lipophilic regions of PrP

Sc [67, 68]. Among them, the com-

monly employed antibiotic tetracycline has emerged, as it exhibits the ability to decrease the resistance of PrP

Sc iso-

forms to proteinase K digestion by interfering with the con-formational rearrangement of proteins while having a low toxicity [40]. Studies about the effectiveness of this com-pound demonstrated that co-treatment of neuronal cultures with the prion fragment PrP[106 126] and tetracycline totally abolished the citotoxic effect of the former [40].

We demonstrated that the simultaneous exposure of B104 cells to hPrP[173-195]AcAm and tetracycline significantly reduced the toxic effect evoked by the prion fragment indi-vidually tested. Tetracycline was less effective, but anyway functioning, when cells were treated with hPrP[180-195]AcAm prion peptide. We concluded that the antibiotic evidently interacts with both peptides, so hindering their disruptive action. As suggested by the less pronounced effect of tetracycline under the N-terminal truncated peptide treat-

Figure 5. Delivery of fluoresceinated hPrP[173-195]FlAm C179C(Me) peptide into the nucleus of neuroblastoma cells.

Pictures show the cell membrane labelling (red, A), the peptide localisation (green, B) and the superimposition of the emission signals

(merged, C). The head arrow in panel B shows a peptide aggregate close to the nuclear membrane.

Confocal Microscopy Evidence of Prion Protein Fragment Protein & Peptide Letters, 2009, Vol. 16, No. 11 7

ment, amino acidic residues located in that portion are im-portant but presumably not essential. A recent report demon-strated that tetracycline strongly interacts with the helix-2 derived prion peptides used in the present study and the binding involves amino acid residues clustered into the C-terminal region of the helix (i.e. Thr183, Gln186, His187, Thr190-193, Lys194), independently of its secondary con-formation [69]. The binding of tetracycline to helix-2 pep-tides could hinder them from changing their conformation: as previously discussed, destabilization toward a -sheet structure could be a factor indirectly promoting the cytotoxic action of prion fragments. Anyway, this could represent only one of the mechanisms of cell protection carried out by the antibiotic. It could otherwise prevent the helix-2 peptides from reaching and destabilizing the cellular prion protein by a direct interaction with it. We consider not so plausible that tetracycline acts by interfering with aggregation processes of helix-2 prion fragments since we previously argued that this way of folding doesn’t influence toxicity.

To individuate the cellular sites interested in the interac-tion with the whole 173-195 fragment, we employed a con-focal microscopy method, finding that the prion peptide ex-tensively localised both in the cytoplasm and in the nucleus of neuroblastoma cells, with the maximal intake detectable after 5 min exposure. We are confident that the wide nuclear peptide translocation is not due to artifactual permeabiliza-tion activity of fixative. As previously demonstrated [70], paraformaldehyde highly preserves the internal structures of cells, while fixation with ethanol/acetic acid mixture, metha-nol or acetone appears to disrupt cell membranes, promoting the nuclear intake of test compounds. The accumulation of 173-195 prion fragment in the nucleus of B104 cells is inter-estingly reminiscent of PrP

Sc [71, 72] and pathogenic PrP

C

mutants [73, 74] nuclear localisation observed in infected cell lines.

It has been reported that in prion-infected cells the prion protein is retrotranslocated from the cell membrane to the ER, where PrP

C to PrP

Sc conformational change is likely to

take place [75]. Here, misfolded prion proteins would be subject to mislocation in the cytosol and subsequent degrada-tion by ubiquitin-proteasome system [75-78]. Resulting PrP fragments showing N-terminal nuclear localisation signals (NLSI, II) have been described to be translocated into the nucleus [78].

The 173-195 prion fragment lacks sequences needed for targeting to the nuclear compartment, but alternative possible pathways, such as a passive diffusion mechanism [79], would explain the widespread localisation observed through-out the cytoplasm.

An alternative speculation is that the pathway of PrPSc

nuclear intake could direct the cellular localisation of 173-195 peptide by an interaction between the cellular prion pro-tein and the fragment mimicking the abnormal isoform, pos-sibly retained until getting into the nucleus. The possibility of a direct interaction between the helix 2 fragment and the cellular prion protein is made plausible by the hypothesis that PrP

C can act as a receptor functional to PrP

Sc internalisa-

tion during infection [80]. In support of that, a comparative NMR study on the recombinant Syrian Hamster PrP(29-231) polypeptide and the analogous PrP(90-231) deprived of the

N-terminal sequence evidenced in the longer molecule tran-sient interactions between the unstructured N-terminus and the helix-2 region, with probable involvement of 187-193 residues [81]. In the same report, authors observed that the energy barrier separating a random structured region from a

-sheet conformation is likely lower than expected for a se-quence with a stable secondary structure, thus supporting an involvement of PrP N-terminus in the earlier phases of PrP

C

conversion to a -sheet-rich isoform [81]. Solution NMR studies of human and bovine prion proteins confirmed the conformational lability of this region revealing that aminoac-ids 23-121 corresponding to the PrP N-terminal tail are in an unstructured rearrangement [3, 82, 83], with the flexible "disorder" appearing to be accentuated by lipid membrane environment [84].

The nuclear localisation of 173-195 peptide seems an unusual and interesting phenomenon, especially if we con-sider that PrP[106-126], a gold standard peptide of PrP

Sc-

induced neurodegeneration, was strictly detectable in the cytoplasm of treated cells [42].

Our immunofluorescence results triggered us to hypothe-size for hPrP[173-195] peptide a role in modulating tran-scriptional activities promoting the events leading to oxida-tive stress, a recognised hallmark of neurodegenerative dam-age [85]. On the same thread, a high affinity of murine PrP

C

and other prion peptides for DNA sequences was assessed [86]. Particularly, the PrP

Sc conformer was found to co-

localise with euchromatin, a region highly activated in tran-scription [72].

In summary, we demonstrated that the helix-2 fragments hPrP[173-195]AcAm and hPrP[180-195]AcAm, whose se-quences partially overlap, are toxic to cultured B104 neuro-blastoma cells. At the same time we assessed the capacity of neuroblastoma cells to sequester the wild-type 173-195 fragment into the cytosolic and nuclear compartments. Lastly, the ability of tetracycline to partially revert toxicity of hPrP[173-195]AcAm and hPrP[180-195]AcAm fragments was ascertained.

The experimental evidences point to the likely involve-ment of the helix-2 region as an anchor-point in the PrP

C

conversion, contradicting the current models describing prevalently the unstructured amino terminus of prion protein as crucial in that process.

ACKNOWLEDGEMENTS

This work was supported by MIUR FIRB-project n. RBNE03FMCJ_003. We are grateful to Dr P. Palladino and Dr L. Ronga (Department of Biological Science-Biostructure section, University of Naples "Federico II") for synthesizing and providing synthetic peptides. We thank Dr. Maria Elena Giordano for help with confocal microscope.

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Received: May 02, 2008 Revised: September 08, 2008 Accepted: November 13, 2008