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Developmental expression of cellular prion protein and apoptotic molecules in the rat cerebellum: Effects of platinum compounds
Journal of Chemical Neuroanatomy 46 (2012) 19–29
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Developmental expression of cellular prion protein and apoptotic molecules in the rat cerebellum: Effects of platinum compounds Maria Grazia Bottone a,b, Veronica Dal Boa, Valeria Maria Piccolini a, Giovanni Bottiroli b, Sandra Angelica De Pascali c, Francesco Paolo Fanizzi c, Graziella Bernocchi a,* a b c
Dipartimento di Biologia e Biotecnologie ‘‘Lazzaro Spallanzani’’, Universita` di Pavia, via Ferrata 9, 27100 Pavia, Italy Istituto di Genetica Molecolare del CNR, Sezione di Istochimica e Citometria, via Ferrata 9, 27100 Pavia, Italy Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita` del Salento, 73100 Lecce, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 May 2012 Received in revised form 7 September 2012 Accepted 14 September 2012 Available online 24 September 2012
Programmed cell death is regulated by prototypes of a large family of Bcl-2-like proteins such as Bax and Bcl-2. A neuroprotective role for cellular prion protein (PrPc) on programmed cell death has been reported, although the cytosolic accumulation of PrPc correlates with toxicity and death of some neurons by apoptosis. In order to understand the signalling function of PrPc in promoting survival or suppressing cell death, we analyzed the expression and co-localization of PrPc, Bax and Bcl-2 proteins in the developing cerebellum of rats treated at PD10 (postnatal day 10) with the chemotherapeutic drug cisplatin (cisPt) or the new platinum (Pt) compound [Pt(O,O0 -acac)(g-acac)(DMS)] (PtAcacDMS). Differences in the expression of PrPc, Bax and Bcl-2 were found in proliferating cells and immature Purkinje neurons. One day after administration (PD11), cisPt markedly increased the apoptosis of the proliferating cells of the EGL (external granular layer); at the same time, several apoptotic bodies with strong Bax immunoreactivity were noticed. After PtAcacDMS, changes in PrPc and apoptotic proteins, with respect to the controls, were found but Bax immunopositive apoptotic bodies were not detectable, which could mean that apoptotic cell death of proliferating cells is preserved. Co-localization was clearly detected in the Purkinje cell population and may explain better the mechanisms by which PrPc and the apoptotic proteins function, and particularly the role of PrPc. Considering the reactivity of Purkinje neurons to these proteins at PD11 and Pd17, at least PrPc expression increased after cisPt and PtAcacDMS treatments or, if PrPc decreased, balanced itself with Bcl-2. The noteworthiness of this finding is that it emphasizes that most of the post-mitotic Purkinje cells need to be rescued, otherwise they undergo degeneration and are not replaced. Based on the effects of both Pt compounds on Purkinje cell differentiation, it should be emphasized that PrPc, together with the synergistic action of the co-localized anti-apoptotic protein, acts as a neuroprotective protein countering cytotoxicity in the postnatal critical phases of cerebellum development. ß 2012 Elsevier B.V. All rights reserved.
Keywords: Cellular prion protein Bcl-2 Bax Platinum compounds Neuroprotection
1. Introduction Prion diseases are neurodegenerative disorders that affect animals and humans. The prion pathogen infection consists in an abnormal prion protein which is an altered isoform of a normal
Abbreviations: cisPt, cisplatin; EGL, external granular layer; IGL, internal granular layer; ML, molecular layer; PCL, Purkinje cell layer; PD, postnatal day; PrPc, cellular prion protein; Pt, platinum; PtAcacDMS, [Pt(O,O0 -acac)(g-acac)(DMS)]. * Corresponding author at: Dipartimento di Dipartimento di Biologia e Biotecnologie ‘‘Lazzaro Spallanzani’’, Laboratori di Biologia Animale, Via Ferrata 9, I-27100 Pavia, Italy. Tel.: +39 0382 986327; fax: +39 0382 986325. E-mail addresses: [email protected] (M.G. Bottone), [email protected] (D.B. Veronica), [email protected] (V.M. Piccolini), [email protected] (G. Bottiroli), [email protected] (S.A. De Pascali), [email protected] (F.P. Fanizzi), [email protected] (G. Bernocchi). 0891-0618/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchemneu.2012.09.003
cellular prion protein (PrPc), a sialoglycoprotein widely expressed at the cell surface via a glycosyl phosphatidyl inositol anchor (Prusiner, 1996,1998; Stahl and Prusiner, 1991; review: Chiesa and Harris, 2009); cytosolic localized PrPc has also been detected (Mironov et al., 2003). It is now clear, however, that alterations in the normal function of PrPc may play an important role in causing or contributing to the disease phenotype; for this reason, elucidating the physiological activity of PrPc has become a major priority in prion research. PrPc function remains enigmatic, due to its ubiquitous distribution; not only it is expressed most abundantly in the brain, but has been also detected in non-neuronal tissues (Horiuchi et al., 1995). Some possible biological functions for PrPc have been proposed. A generalized physiological function in the brain as antioxidant (Brown et al., 1997) involvement in copper homeostasis (Pauly and Harris, 1998), signal transduction
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(Mouillet-Richard et al., 2000), neurotransmission and synapse formation (Carleton et al., 2001; Collinge et al., 1994; ZomosaSignoret et al., 2008), and adhesion to the extracellular matrix (Schmitt-Ulms et al., 2001). The accumulation of PrPc in the cytosol seems to correlate with toxicity in some neuronal cells (Harris and True, 2006; Wollmann and Lindquist, 2002) but, interestingly, a subset of neurons in the hippocampus, neocortex and thalamus of mouse brain appeared healthy even though cytosol-localized PrPc has been detected in these neurons (Barmada and Harris, 2005; Mironov et al., 2003). Recently, evidences emerged suggesting that PrPc may be cytoprotective, particularly against internal and environmental stimuli that initiate an apoptotic programme (review: Westergard et al., 2007). PrPc over-expression rescues cultured neurons and some mammalian cell lines, from several kinds of death-inducing stimuli (Kuwahara et al., 1999; Li et al., 2007a; Roucou and LeBlanc, 2005; Solforosi et al., 2004). At present, neuronal roles for PrPc as an anti- and conversely pro-apoptotic protein have been postulated (Kim et al., 2004). It may be speculated is possible that PrPc is a member of the Bcl-2 family, since a similarity between the Bcl-2 homology domain of the Bcl-2 family members and the octapeptide repeats in Nterminal region of PrPc has been noticed (review: Zomosa-Signoret et al., 2008). Investigations on the pro-apoptotic molecule Bax, which plays a major role in regulating neuronal death in the CNS (central nervous system) both during development and after injury (Akhtar et al., 2004; Yuan and Yankner, 2000), indicate that neurodegeneration involves both Bax-dependent and Bax-independent pathways. PrPc could inhibit Bax directly or, indirectly by acting as a signalling molucule (Roucou and LeBlanc, 2005). Bax deletion in mice expressing the neurotoxic form of PrPc slows apoptosis of cerebellar granule cells (Li et al., 2007a,b). In parallel, a protective effect of PrPc has been proposed which depends on signalling function of PrPc to promote neuronal survival or suppress neuronal death (Li et al., 2007a,b; Solforosi et al., 2004). In the present study, immunocytochemical techniques were used to investigate the trend and the role of PrPc in the developing rat cerebellum in relation to the involvement of the pro- and antiapoptotic proteins Bax or Bcl-2 before and after treatment with platinum (Pt) compounds cisplatin (cisPt) and a new platinum complex [Pt(O,O0 -acac)(gamma-acac)(DMS)] (PtAcacDMS). Particularly, high levels of PrPc (Laine´ et al., 2001; Sale`s et al., 1998), Bcl2 and Bax (review: Vogel, 2002) are expressed in the Purkinje neurons. CisPt, the most commonly used therapeutic agent (review: McWhinney et al., 2009), is a highly effective anticancer drug which has an important role also in the treatment of childish malignancies such as neuroblastoma, osteosarcoma and some brain tumours (Prestayko et al., 1979). Mechanisms of action of cisPt are principally based on binding of DNA of mitotic cells (Ahmad, 2010; Eastman, 1991). In proliferating cells, Pt atoms form covalent bonds to purine bases whose result is to block DNA synthesis. The DNA damage triggers cell-cycle arrest, activation of the tumour-suppressor p53 and apoptosis (Qin et al., 2002). Moreover, cisPt toxicity can also induce death in non mitotic cells by interacting with cytoplasmic proteins (Mandic et al., 2003). Among the new Pt complexes, PtAcacDMS, which has two acetylacetonate ligands, one O,O0 -chelate and the other sigmalinked by metine in the gamma position, was found to be the most active in rapidly producing a sustained apoptotic response (Muscella et al., 2008). Differently from cisPt, whose activity appears to be at the cellular level and DNA linking, the cytotoxicity of the new complex is associated solely with intracellular accumulation. Moreover, PtAcacDMS has shown low reactivity with nucleobases and a specific reactivity with sulphur ligands. This suggests that its preferred cell target could be amino acid
residues of enzymes and other proteins involved in apoptotic induction, thus characterizing it as a compound with non-genomic targets. Recently, the low neurotoxicity of PtAcacDMS was supported by findings on cerebellum postnatal ontogenesis (Bernocchi et al., 2011; Cerri et al., 2011). The trend of the PrPc, Bax and Bcl-2 markers was followed at two crucial stages of cerebellum postnatal histogenesis, i.e. during the active cell proliferation in the external granular layer (EGL) and during the maturation phase of Purkinje cells (review: Altman, 1972a,b). Therefore, particular attention was paid to changes in the layers of differentiating Purkinje cells and precursors of granule cells. 2. Materials and methods 2.1. Animal and treatments Ten-day-old Wistar rats were given a single subcutaneous injection of cisPt (0.5 mg/ml; Teva Pharma, Italy) or PtAcacDMS in the nape of the neck at a dose of 5 mg/g b.w. (corresponding to the therapeutic dose suggested by Bodenner et al. (1986) and Dietrich et al. (2006)) or 10 mg/g b.w. Throughout the experiment, the rats were kept in an artificial 12 h light:12 h dark cycle and provided rat chow and tap water ad libitum. One day (PD11), 7 days (PD17) and 20 days (PD30) after drug administration, treated (4 per stage) and untreated control rats (4 per stage) of the same age were deeply anesthetized with an intraperitoneal injection of 35% chloral hydrate (100 ml/100 g b.w.; Sigma, St. Louis, MO, USA); the brains were quickly removed, fixed in Carnoy’s solution (6 absolute ethanol/3 chloroform/1 acetic acid) for 48 h, then placed in absolute ethanol and in acetone, and embedded in Paraplast X-tra (Sigma). Sections (8 mm thick) of cerebellar vermis were cut serially in the sagittal plane and collected on silan-coated slides. The slides were then processed for immunohistochemical procedures (as described later). To avoid possible staining differences due to small changes in the procedure, each reaction was carried out simultaneously on slides from treated and untreated animals at different stages. All experiments were performed according to the guidelines for care and use of laboratory animals as published by the Italian Ministry of Health (DDL 116/92). All efforts were made to minimize the number of animals used and their suffering. 2.2. Single immunoperoxidase stainings The immunoreaction was performed on cerebellum sections of 11, 17 and 30 day-old-rats. Sections were deparaffinized in xylene, rehydrated through a series of graded alcohol treatments and rinsed in PBS. The endogenous peroxidases were suppressed by incubation of sections with 3% H2O2 in 10% methanol in phosphatebuffered saline (PBS; Sigma) for 7 min. Subsequently, the sections were washed in PBS and incubated for 20 min in normal serum (15 ml normal serum/1000 ml PBS) at room temperature in order to block nonspecific antigen binding sites. The serum for blocking is prepared from the same species in which the biotinylated secondary antibody is made. Localization of PrPc was achieved by applying on brain sections a monoclonal mouse antibody anti-PrPc (Sigma) diluted 1:100 in PBS overnight in a dark moist chamber. Thereafter, the sections were sequentially incubated with diluted biotinylated secondary antibody solution (1:200; Vector Laboratories, Burlingame, CA, USA) for 30 min. Sections were washed in PBS and incubated for 30 min at room temperature with Vectastain Elite ABC reagent (Vector Laboratories). Then, 0.05% 3,30 -diaminobenzidine tetrahydrochloride (DAB; Sigma) with 0.01% H2O2 in Tris–HCl buffer (0.05 M, pH 7.6) was used as a chromogen. After each reaction step, sections were washed thoroughly in PBS (two changes of 5 min each). Sections were dehydrated in ethanol, cleared in xylene, and mounted in Eukitt (Kindler, Freiburg, Germany). For control staining, some sections were incubated with PBS instead of the primary antibody. No immunoreactivity was present in this condition. The slides were observed with an Olympus BX51 microscope, and the images were recorded with an Olympus Camedia C-5050 digital camera and stored on a PC. Corrections to brightness and contrast were made with Paint Shop Pro 7 (Jasc Software Inc.). 2.3. Double immunofluorescence reactions Localization of PrPc/Bcl-2 and PrPc/Bax was achieved by applying on cerebellum sections of 11 and 17 day-old-rats, respectively: a mouse monoclonal anti-PrPc (1:100; Sigma) and a rabbit polyclonal anti-Bcl-2 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and a mouse monoclonal anti-PrPc (1:100; Sigma) and a rabbit polyclonal anti-Bax (1:100; Santa Cruz Biotechnology),
in PBS overnight in a dark moist chamber. Sections were washed in PBS and incubated with the secondary antibodies, respectively: Alexa-Fluor 488 goat anti-mouse (1:100, Molecular Probes, Milan, Italy) and Alexa-Fluor 594 donkey anti-rabbit (1:100, Molecular Probes), in PBS for
M.G. Bottone et al. / Journal of Chemical Neuroanatomy 46 (2012) 19–29 1 h. After washing in PBS, the nuclei were counterstained with 0.1 mg/ml Hoechst 33258 for 6 min, and coverslips were lastly mounted in a drop of Mowiol (Calbiochem, San Diego, CA, USA). For control staining, some sections were incubated with PBS instead of the primary antibodies. No immunoreactivity was present in these sections. The slides were viewed by fluorescence microscopy with an Olympus BX51 equipped with a 100W mercury lamp used under the following conditions: 330– 385 nm excitation filter (excf), 400-nm dichroic mirror (dm), and 420-nm barrier filter (bf), for Hoechst 33258 and 450-480 nm excf, 500 nm dm, and 515 nm bf for Alexa 488. Images were recorded with an Olympus Camedia C-5050 digital camera and stored on a PC. Images were optimized for colour, brightness, and contrast by using Paint Shop Pro 7 software (Jasc Software Inc.). 2.4. Evaluation of fluorescence intensity The extent of the labelling was evaluated on digitized images of sections acquired under exposure time avoiding any pixel saturation effect. The labelling intensity was measured by means of densitometric analysis (Image-J 1.46p; NIH, Bethesda, MA, USA). The mask shape was adjusted depending on the spatial distribution of the tissue layer or cell under measurement; the labelling was measured as the intensity mean value over the area (EGL, ML and Purkinje cell cytoplasm). The immunocytochemical fluorescence intensity for Bcl-2, PrPc and Bax were evaluated in a total of 40 fields (10 fields per animal) for controls and treated animals, at each stage. Results were recorded on Microsoft Office Excel Software spreadsheets and are expressed as the means SD (standard deviation). Statistical differences between control and treated animals were evaluated by Student’s t-test.
3. Results Studies were performed on control untreated and cisPt- or PtAcacDMS-treated rats of the same age, i.e. at 11, 17 and 30 days of postnatal life. Since the Pt compounds were injected at 10 days (PD10), treated rats were sacrificed 1, 7 and 20 days after treatment. The observations were performed on lobules VI–VIII of the cerebellar cortex, i.e. in the lobules that showed several changes in the EGL and Purkinje cell layer after cisPt treatment (Avella et al., 2006; Pisu et al., 2005). 3.1. Light microscopy Immunoreaction for PrPc on PD11, control rats (Fig. 1a) showed labelling for PrPc in all the cerebellar layers. In the EGL, the staining was also found in the precursors of granule cells. Purkinje cells showed weak labelling in the cytoplasm. The molecular layer (ML) was very weakly stained and the interneurons were not clearly identifiable; the internal granular layer (IGL) showed intense staining, ascribable to granule cells and glomeruli. At this age, one day after 5 mg/g cisPt treatment (Fig. 1b), a general decreased reactivity was revealed, except for several Purkinje cells with labelled cytoplasm; when cisPt was injected at the dose of 10 mg/g (Fig. 1c) increased immunoreactivity was found in the EGL and IGL. Apoptotic cell bodies were observed in the EGL. At the dose of 5 mg/ g PtAcacDMS (Fig. 1d), there were no evident changes in the EGL immunopositivity, while a decreased immunolabelling was found in the IGL in comparison with controls; the higher dose, 10 mg/g (Fig. 1e), induced strong labelling in the Purkinje cells only. On PD17 (Fig. 1f–j), weak immunoreactivity was observed in all cerebellum layers of control rats (Fig. 1f), except for EGL, which is strongly labelled. Generally, in cisPt- (Fig. 1g-h) and PtAcacDMStreated rats (Fig. 1i and j), intense labelling for PrPc was seen in the EGL; the IGL was more strongly, non-homogeneously labelled, the ML and Purkinje cell layer presented a diffuse immunoreactivity. However, after 10 mg/g cisPt (Fig. 1 h), only the Purkinje cells were more intensely immunostained than in controls, but the cerebellar cortex cytoarchitecture was altered due to the presence of haemorrhagic foci in all the layers; the EGL and IGL appeared weakly immunopositive.
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On PD30, no changes were found in controls (not shown) in comparison with the previous developmental stages and in the cerebellar layers of treated rats (not shown). Weak labelling was observed in the ML and Purkinje cell layer; there were labelled granule cells and glomeruli in the IGL. 3.2. Fluorescence microscopy 3.2.1. Double immunoreaction for Bcl-2/PrPc and Bax/PrPc The double immunostaining for Bcl-2 and PrPc showed labelling in all layers of cerebellum cortex of control rats at PD11 (Fig. 2a–c). Immunostained cells in the EGL and IGL were identifiable as precursors of granule cells and granule cells themselves. The Purkinje cells showed a very strong immunopositivity for Bcl-2, but not for PrPc, as better seen in the merge (Fig. 2c). The 5 mg/g cisPt dose (Fig. 2d–f) induced an evident increased labelling for PrPc (Fig. 2e) but not for Bcl-2 (Fig. 2d) in the Purkinje cells both in the soma and dendrite primary tree, as better seen in the merge (Fig. 2f). Increased immunoreactivity was revealed both for Bcl-2 and PrPc after treatment with 10 mg of cisPt (Fig. 2g–i). At this dose, the labelling for Bcl-2 (Fig. 2g) was stronger than in controls also in the EGL, while PrPc (Fig. 2h) appeared to be generally weaker. After PtAcacDMS treatment, at both doses (Fig. 2j–o), Bcl-2 (Fig. 2j and m) and PrPc immunoreactivity (Fig. 2k and n) were still detectable in all the layers, especially in the Purkinje cells, as better seen in the merge (Fig. 2l and o). In the EGL, the immunocytochemical expression for Bcl-2 and PrPc appeared to be the same as in controls at the dose of 5 mg/g, while it was lowered at the dose of 10 mg/g. The double immunoreaction for Bax and PrPc indicated the distribution of the two proteins in the cerebellar cortex at PD11 (Fig. 3). In control animals (Fig. 3a–c), the labelling for Bax (Fig. 3a) was present in all the cerebellar layers, including the Purkinje cells, where, on the other hand, weak PrPc immunoreactivity was shown (Fig. 3b). After cisPt injection (Fig. 3d–i), Bax labelling in the Purkinje cells did not change in general, while PrPc increased in intensity. In the proliferating EGL, bodies with strong immunopositivity for Bax were observed, especially after the 10 mg/g dose (Fig. 3g–i). These structures are identifiable as apoptotic bodies (Fig. 4a–d), likely formed from macrophages and apoptotic cells, as better seen by the merge (Fig. 4c). The treatment with PtAcacDMS (Fig. 3j–o) maintained immunolabelling for Bax (Fig. 3j and m) and PrPc (Fig. 3k and n) in the Purkinje cells, while in the EGL, Bax and PrPc labelling appeared to be lower in comparison with controls, at the dose of 10 mg/g (Fig. 3a–c), but not Bax immunopositive apoptotic bodies were found. The fluorescence intensity values at PD11 are reported in the histograms of Fig. 5. In comparison with the age-matched controls, the fluorescence intensity for Bcl-2 decreased or increased in the EGL after cisPt treatment at the dose of 5 or 10 mg/g, respectively; in parallel, the fluorescence intensity for PrPc did not change or was decreased. The immuno-expression of Bax in the EGL was decreased, or maintained steady levels, although, as previously reported, strongly immunopositive – Bax apoptotic bodies were scattered among the proliferating cells. After PtAcacDMS, at the low and high dose, Bcl-2 fluorescence intensity of the EGL increased or decreased, while PrPc did not change or was decreased, respectively; Bax intensity values increased or decreased. Purkinje cells of cisPt-treated rats showed small but significant changes in Bcl-2 intensity, and PrPc increased in fluorescence intensity; generally Bax values did not change. In PtAcacDMStreated rats PrPc increase contrasted Bcl-2 drop, while Bax immunofluorescence appeared unalterated.
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Fig. 1. Immunoperoxidase staining for PrPc on PD11 and PD17. At PD11 (a–e) all the cerebellar layers are labelled for PrPc. EGL and IGL of control rats (a) cells are immunostained; Purkinje cells show weak labelling in the cytoplasm (arrows). After 5 mg/g cisPt treatment (b), a decreased reactivity is present, except for several Purkinje cells with labelled cytoplasm (arrows); at the dose of 10 mg/g and (c) apoptotic cell bodies are shown in the EGL and IGL are intensely labelled (arrowheads). After 5 mg/g PtAcacDMS (d), strong labelling of Purkinje cells was shown after the high dose injection (e, arrows). At PD17 (f–j), weak immunoreactivity is shown in all layers of control rats (f), except for EGL. Intense labelling for PrPc is shown in the EGL of cisPt (g and h) and PtAcacDMS treated rats (i and j); the IGL is labelled, the molecular and Purkinje cell layer show a diffuse immunoreactivity. After 10 mg/g cisPt, Purkinje cells are strongly immunostained (h; arrows), the EGL and IGL is almost immunonegative (asterisk); presence of haemorrhagic foci (hf) are visible. EGL, external granular layer; IGL, internal granular layer; ML, molecular layer; and PL, Purkinje cell layer. Scale bar: 60 mm.
M.G. Bottone et al. / Journal of Chemical Neuroanatomy 46 (2012) 19–29
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Fig. 2. Double fluorescence immunoreaction for Bcl-2 (red fluorescence) and PrPc (green fluorescence) on PD11. Labelling is present in all layers of controls (a–c). Immunopositive cells are identifiable in the EGL and IGL. Strong immunopositivity for Bcl-2 (a), but not for PrPc (b), as better seen in the merge (c), is shown in the Purkinje cells (arrows) of controls. After cisPt treatment at the doses of 5 mg/g (d–f) and 10 mg/g (g–i), increased labelling for PrPc (e and h; arrows), but not for Bcl-2 (d and g) is observed in the Purkinje cells, both in the soma and dendrite, as better seen in the merge (f and i). At the higher dose, labelling for Bcl-2 (g) is strong in the EGL, while that for PrPc (h) is weaker. Also after PtAcacDMS treatment at both doses (j–o), PrPc immunostaining (k and n), and not Bcl-2 labelling (j and m), is more intense in the Purkinje cell layer (arrows). EGL, external granular layer; IGL, internal granular layer; ML, molecular layer; and PL, Purkinje cell layer. Scale bar: 60 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
At PD17 (Fig. 6), in comparison with controls (Fig. 6a–c), Bcl-2 and PrPc labelling appeared to be maintained in the EGL, which is heavier in the cisPt-treated rats (Fig. 6d–f) Haemorrhagic foci were found following cisPt 10 mg/g (Fig. 6d–f). After PtAcacDMS (Fig. 6g–l), similar results were obtained as regards Bcl-2 and PrPc in the EGL. After treatments, Purkinje cells were labelled in the soma for all three proteins. The immunoreactivity for Bcl-2 and especially for PrPc was strong in the ML (Fig. 6d–l) after cisPt and PtAcacDMS.
At PD17, control rats showed immunolabelling for both Bax and PrPc in all the cerebellar layers (not shown). No evident changes were detected for Bax immunoreactivity after cisPt, while after PtAcacDMS injection at both doses, Bax labelling was intense in the EGL, did not change in the Purkinje cell layer and increased in the ML (not shown), as indicated by the fluorescence intensity values (Fig. 7). The fluorescence intensity values at PD17 are reported in the bar charts of Fig. 7. With respect tp controls, in the 17 day-old-rats the values decreased in the EGL and Purkinje cell layer. In
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Fig. 3. Double fluorescence immunoreaction for Bax (red fluorescence) and PrPc (green fluorescence) on PD11. The distribution of PrPc immunopositivity within the cerebellar cortex of controls (b and c) is similar to Fig. 2. Bax labelling (a and c) is present in all the cerebellar layers, including the Purkinje cells. After cisPt injection (d and f), increased labelling in the Purkinje cells (arrows) and immunopositive bodies for Bax and PrPc are observed in the proliferating EGL, especially after the 10 mg/g dose (g–i, arrowhead). After treatment with PtAcacaDMS (j–o) unaltered immunostaining for Bax (j and m) and increased labelling for PrPc (k and n) are clearly visible in the Purkinje cell layer (arrows); in the other cerebellar layers Bax and PrPc labelling is the same as in controls (a–c). EGL, external granular layer; IGL, internal granular layer; ML, molecular layer; and PL, Purkinje cell layer. Scale bar: 60 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
comparison with controls, the increased fluorescence intensity of Bcl-2 in the EGL of cisPt-treated rats added to an increased PrPc intensity; Bax fluorescence values did not change. After PtAcacDMS, there were similar results for Bcl-2 and PrPc; Bax intensity values increased. As regards Purkinje cells, in cisPttreated rats, and mainly in PtAcacDMS-treated rats, at both the doses, the decreased Bcl-2 intensity values were balanced by the increased PrPc values. In addition, the fluorescence intensity of Bcl-2 and PrPc in the ML, where Purkinje dendrite branches extend, was higher in the PtAcacDMS-treated rats in comparison with the cisPt-treated ones while Bax intensity values strongly increased.
4. Discussion The normal cellular function of PrPc remains unclear. Studies conducted in transgenic and PrPc knockout mice implicate PrPc in synaptic transmission, neuronal differentiation and generally in CNS maturation (review: Collinge et al., 1994). Cell death by apoptosis, a critical event of neuronal development in the CNS, may be associated with PrPc (review: Westergard et al., 2007). PrPc is synthesized in the neuronal soma, transported down the axon and attached to the cell surface by glycosylphosphatidylinositol anchored proteins (GPI linkage) (Borchelt et al., 1994; Rodolfo et al., 1999; Sale`s et al., 1998; Stahl et al., 1987). While it is to be
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Fig. 4. Double fluorescence immunoreaction for Bax (red fluorescence) and PrPc (green fluorescence), and counterstaining with Hoechst 33258 (blue fluorescence) on PD11. In the proliferating EGL, after the treatment with 10 mg cisPt dose, immunopositive bodies for Bax (a) and PrPc (b, arrows) are found. These structures are identifiable as apoptotic bodies, likely made by macrophages and apoptotic cells, as better seen by the merge (c, arrows) and Hoechst 33258 staining (d, arrows). Scale bar: 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 5. Fluorescence intensity values in the developing cerebellar cortex on PD11. The bar charts indicate the trend of the fluorescence intensity values for Bcl-2, Bax and PrPc in the EGL and in the Purkinje cell cytoplasm. These data (means SD) show changes during normal histogenesis and after Pt compounds treatment. Number of evaluation: n = 40. EGL, external granular layer; PC, Purkinje cell layer. Significance of differences: *p < 0.05; **p < 0.01; and ***p < 0.001.
noted that most brain morphological studies report that PrPc is expressed in the cytoplasm in neuronal perikarya, with scarce expression in the neuropil; however, these data are contradictory and do not fit with biochemical and cell biology results. Using ultrastructural immunocytochemistry, the protein, in fact, appeared to be located on the outer surface of the cell membrane and in Golgi and endosomal intracytoplasmic organelles of all neuronal and glial cell types in rat cerebellum (Laine´ et al., 2001). On the other hand, differences in PrPc expression have been described among brain regions (review: Bailly et al., 2004). The differential expression provides a putative anatomical basis for the differential vulnerability of brain neurons to prion (Vorberg and Priola, 2002), as well as for some specific limitations of the detection methods employed. In transgenic mice, by using an immunogold technique, neuronal PrPc has been observed mainly bound to the cell surface and to presynaptic sites; dictyosomes and recycling organelles in most of the major neuron types of several brain areas also exhibited PrPc antigen (Bailly et al., 2004). In the early postnatal brain, PrPc is initially distributed along many fibre tracts, at the surface of elongating axons; PrPc increases markedly at the time of synapse formation, suggesting a role in the function of neuronal contacts (Sale`s et al., 2002). In this study, we were able to exploit our earlier work on the neurotoxicity of Pt compounds (Avella et al., 2006; Pisu et al., 2005), to study the possible involvement of PrPc in cell proliferation/death, and cell differentiation. Developmental alterations are induced by platinum compounds in lobules VI–VIII of cerebellum during the histogenetic process between PD11 and at PD17, i.e. a crucial period for proliferation, differentiation and synaptogenesis (Altman, 1972a,b). In this study we found that PrPc was clearly detectable in untreated rats by fluorescence and immunoperoxidase labelling, although at different intensity, in cells of all the cerebellar layers, i.e. the EGL, Purkinje cell cytoplasm and dendrites, and IGL; no PrPcpositive interneurons were identified in the ML. The pattern fits well with the distribution of PrPc at this postnatal phase of cerebellar development as has been reported by immunoreaction at light microscopy (Sale`s et al., 2002).
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Fig. 6. Double fluorescence immunoreaction for Bcl-2 (red fluorescence) and PrPc (green fluorescence) on PD17. In comparison with controls (a–c), the immunoreactivity for Bcl-2 (d, g and j) and especially for PrPc (e, h and k) increases strongly in the ML, mainly at the dose of 10 mg/g of cisPt (d–f) and PtAcacDMS (j–l). PrPc labelling was shown in the EGL, that is thicker in cisPt-treated rats (d–f, asterisks) respect to control ones (b and c). Note haemorragic foci (hf) after cisPt treatment (d). EGL, external granular layer; IGL, internal granular layer; ML, molecular layer; and PL, Purkinje cell layer. Scale bar: 60 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
CisPt and PtAcacDMS treatments induced differential changes in the PrPc expression at light and fluorescence microscopy. Special attention was paid to cerebellar layers shown to be strongly affected by cisPt, i.e. EGL and Purkinje cell layers comprising somata and dendrites (Avella et al., 2006; Pisu et al., 2005; Scherini and Bernocchi, 1994). The most evident effects of cisPt were: an increased expression of PrPc in the Purkinje cell somata and main proximal dendrites at PD11, together with a general weak labelling of the granule cells progenitors of EGL, where, as previously described (Cerri et al., 2011; Pisu et al., 2005), a marked increase of apoptotic bodies was found at higher 10 mg/g cisPt dose; a similar pattern was noticed in the IGL. This trend was followed by an increased and diffuse intensity in the ML where Purkinje cells extend their dendrite tree, at PD17. These findings have to be considered within the framework of the major changes previously found after cisPt. In the developing cerebellum, a single dose of cisPt administered at PD10 affected the differentiation of post-mitotic Purkinje neurons, as well as the formation of synaptic contacts on these neurons (Avella et al., 2006); some (20%) Purkinje cells even underwent to degeneration and death (Scherini and Bernocchi, 1994). The altered expression of PrPc in the light of above-mentioned findings
is consistent with a role of the protein in neuronal maturation and synapse formation, as suggested by Sale`s et al. (2002). The increased expression of PrPc after cisPt in the soma of the Purkinje cells at PD11 could protect these neurons from degeneration and death or assist their recovery (Avella et al., 2006). On the other hand, after treatment with PtAcacDMS, PrPc immunoreactivity increased in the Purkinje cell soma, whereas no relevant cytoarchitectural changes were detectable after this treatment; the EGL proliferating cells did not undergo apoptotic cell death over the normal trend and Purkinje cell dendrite growth was not altered significantly (Bernocchi et al., 2011; Cerri et al., 2011). Based on the cisPt- and PtAcacDMS-induced changes, the intriguing role of PrPc may be further sustained by the trend and distribution mainly of Bcl-2 protein, but also Bax. The homology between the PrPc and Bcl-2 proteins possibly indicates that PrPc is a member of the Bcl-2 family of proteins (Roucou et al., 2005; Westergard et al., 2007). Bcl-2 and related proteins are implicated in the regulation of apoptosis. Bcl-2 protects neurons from apoptotic stimuli such as deprivation of NGF, toxic Ca2+ levels, and oxidative stress (Allsopp et al., 1993; Farlie et al., 1995; Martinou et al., 1994; Reed, 1994; Yang and
M.G. Bottone et al. / Journal of Chemical Neuroanatomy 46 (2012) 19–29
Fig. 7. Fluorescence intensity values in the developing cerebellar cortex on PD17. The histograms show the immunoreactivity variations for Bcl-2, Bax and PrPc in the EGL, ML and in the Purkinje cell cytoplasm. These data (means SD) display alterations during normal histogenesis and after Pt compounds treatment. Number of evaluation: n = 40. EGL, external granular layer; PC, Purkinje cell layer; and ML, molecular layer. Significance of differences: *p < 0.05; **p < 0.01; and ***p < 0.001.
Korsmeyer, 1996; Zhong et al., 1993). Furthermore Bcl-2 interacts with the pro-apoptotic protein Bax. In a cell, Bcl-2 heterodimerizes with Bax and rescues cells from apoptosis. The ratio of Bcl-2–Bax heterodimers to Bax–Bax homodimers determines the susceptibility of a cell to apoptotic stimuli such as those mentioned above (Oltvai and Korsmeyer, 1994; Yin et al., 1994). Li et al. (2007b) have shown that transgenic (Tg) mice expressing PrPc with a deletion of the N-terminal tail encompassing amino acid residues 32–134 spontaneously resulted in degeneration of cerebellar granule cells. They further showed that Bax deletion in the transgenic mice expressing this neurotoxic form of PrPc slowed apoptosis of the cerebellar granule cells as determined by the TUNEL reaction and caspase-3 staining. The results indicated that the neurotoxicity of a PrPc deleted of aminoacids 32–134 depends in part on activation of a Bax-dependent, mitochondrial pathway of apoptosis. The most interesting finding to explain the role of PrPc as a neuroprotective agent is its interaction with Bcl-2 and Bax, two key modulators of apoptosis. Here, for the first time, we have shown that PrPc co-localizes with Bcl-2 and Bax in certain cell layers or
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cell types of cerebellum. Interestingly, co-localization appeared to be induced by injury; however, differential effects of cisPt and PtAcacDMS were found. In particular, after the higher dose of the Pt compounds, the expression of anti-apoptotic molecule Bcl-2 increased after cisPt and decreased after PtAcacDMS in the EGL, but PrPc immunoexpression was decreased. Therefore, the trend of the two proteins was not the same in the EGL but they seemed to have a dose-dependent compensatory reaction in cisPt-treated rats. However, at PD11, cisPt greatly activated Bax protein in the apoptotic bodies of EGL as a sign of marked cell death (Bernocchi et al., 2011; Pisu et al., 2005). Intriguingly, in these bodies, Bax and PrPc coexist. On the contrary, after PtAcacDMS, changes in PrPc and Bcl-2 with respect to the controls were found, but apoptotic bodies with strong Bax immunoreactivity were not detectable, which could mean that apoptotic cell death of proliferating cells is preserved (Bernocchi et al., 2011; Cerri et al., 2011). More interesting are the data on PD17, when Bcl-2 and PrPc synergistically counter cell death in the surviving EGL proliferating cells; in parallel, Bax intensity values decreased after cisPt, while they increased after PtAcacDMS (see Section 5). The abovementioned knowledge suggests that PrPc may be implicated in regulating apoptosis through the balance between Bcl-2 and Bax, but a prerequisite for the interaction of PrPc with these apoptotic proteins is that all three must co-localize in the same cell. Co-localization was clearly detected in the Purkinje cell population and may explain better the mechanisms by which PrPc and the apoptotic proteins function, and particularly the role of PrPc. Considering the reactivity of Purkinje neurons to these proteins at PD11, at least PrPc expression increased after cisPt and PtAcacDMS treatments or, if PrPc decreased, balanced itself with Bcl-2. PrPc may be involved with the apoptotic proteins to rescue, as much as possible, Purkinje neurons from death. This finding is noteworthy in that it is to consider in the light that Purkinje cells are post-mitotic and immature in postnatal life (Altman, 1972b), and need to be rescued; otherwise they degenerated and cannot be replaced. Consistent with this idea, the immunoreactivity for PrPc and Bcl-2 increased in the ML, seven days after cisPt, where the dendrites of Purkinje cells embark on the recovery phase within the reorganization/remodelling of the cerebellar cytoarchitecture (Avella et al., 2006; Pisu et al., 2005). In this context, Bax immunoexpression in the ML decreased, or was not changed, especially after Pt AcacDMS; therefore, Bcl-2, as well as PrPc, prevailed over the pro-apoptotic protein Bax, supporting the notion that for a correct neuronal differentiation, the balance between Bax and Bcl-2/PrPc is fundamental in order to be able to cope with injury and ensure normal dendrite growth. 5. Conclusions Focusing mainly on the effects of Pt compounds on EGL proliferation and Purkinje cell differentiation, it is emphasized that PrPc combined with the synergistic action of the co-localized antiapoptotic protein Bcl-2, is involved in neuroprotection to rescue cytotoxicity in the postnatal critical phases of cerebellum development. As regards a comparison between the two Pt compounds, cisPt appeared to have neurotoxic effects on normal proliferating granule cells of the EGL, provoking their cell death (Bernocchi et al., 2011; Cerri et al., 2011), as seen by the presence of apoptotic bodies. Instead, after PtAcacDMS treatment, synergistic increased or decreased expression of all molecules, including Bax, was maintained without damages to normal cell genesis. Interestingly, it must be emphasized that the lowering of the two proteins (PrPc and Bcl-2) in the EGL after PtAcacDMS, especially at the dose of 10 mg/g, follows the normal trend of these proteins, as indicated by intensity values in 17 day-old-rats. Nevertheless, it is surprising
M.G. Bottone et al. / Journal of Chemical Neuroanatomy 46 (2012) 19–29
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that Bax intensity was increased in the EGL after PtAcacDMS at PD17. Phenomena other than cell death could explain this finding, comparing this result with data on cisPt treatment which demonstrated a recovery of EGL proliferation (Pisu et al., 2005). Actually, the increase of Bax values could be ascribed to the presence of small elements immunopositive to Bax (Bernocchi et al., 2011). The study of causes and involvement of these cells is now in progress. In conclusion, it is to be kept in mind the demonstration that both cisPt and PtAcacDMS are able to reach the developing brain tissue once injected (review: Cerri et al., 2011). Interestingly, the brain Pt content after PtAcacDMS administration was up to 4-fold higher than that after cisPt. This might mean that the novel Pt complex is less bound to plasma proteins than cisPt and can cross the blood brain barrier more readily. The presence of Pt in the brain tissue after PtAcacDMS administration fits in with data obtained from in vitro studies on HeLa and MCF-7 cells (Muscella et al., 2007, 2008). Since it is known that the cytostatic effect is closely associated with the accumulation of Pt in the cell, this could represent an advantage for PtAcacDMS inasmuch as it would permit the use of lower doses of this Pt complex reducing, at the same time, the risk of side effects and drug resistance. Conflicts of interest The authors declared no conflict of interest. Acknowledgements The research was supported by grants from Fondi di Ateneo per la Ricerca (FAR, University of Pavia), Fondazione Banca del Monte di Lombardia (Italy) and Programmi di Ricerca di Rilevante Interesse Nazionale (PRIN, 2009ZFPSPW). References Ahmad, S., 2010. Platinum–DNA interactions and subsequent cellular processes controlling sensitivity to anticancer platinum complexes. Chemistry & Biodiversity 7, 543–566. Akhtar, R.S., Ness, J.M., Roth, K.A., 2004. Bcl-2 family regulation of neuronal development and neurodegeneration. Biochimica et Biophysica Acta 1644, 189–203. Allsopp, T.E., Wyatt, S., Paterson, H.F., Davies, A.M., 1993. The proto-oncogene bcl-2 can selectively rescue neurotrophic factor-dependent neurons from apoptosis. Cell 73, 295–307. Altman, J., 1972a. Postnatal development of the cerebellar cortex in the rat. I. The external granular layer and the transitional molecular layer. Journal of Comparative Neurology 145, 353–398. Altman, J., 1972b. Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer. Journal of Comparative Neurology 145, 399–464. Avella, D., Pisu, M.B., Roda, E., Gravati, M., Bernocchi, G., 2006. Reorganization of the rat cerebellar cortex during postnatal development following cisplatin treatment. Experimental Neurology 201, 131–143. Bailly, Y., Haeberle´, A.M., Blanquet-Grossard, F., Chasserot-Golaz, S., Grant, N., Schultze, T., Bombarde, G., Grazzi, J., Cesbron, J.Y., Lemaire-Vieille, C., 2004. Prion protein (PrPc) immunocytochemistry and expression of the green fluorescent protein reporter gene under control of the bovine PrP gene promoter in the mouse brain. Journal of Comparative Neurology 473, 244–269. Barmada, S.J., Harris, D.A., 2005. Visualization of prion infection in transgenic mice expressing green fluorescent protein-tagged prion protein. Journal of Neuroscience 25, 5824–5832. Bernocchi, G., Bottone, M.G., Piccolini, V.M., Dal Bo, V., Santin, G., De Pascali, S.A., Migoni, D., Fanizzi, F.P., 2011. Developing central nervous system and vulnerability to platinum compounds. Chemotherapy Research and Practice 2011, 315418. Bodenner, D.L., Dedon, P.C., Keng, P.C., Borch, R.F., 1986. Effect of diethyldithiocarbamate on cis-diamminedichloroplatinum(II)-induced cytotoxicity, DNA crosslinking, and gamma-glutamyl transpeptidase inhibition. Cancer Research 46, 2745–2750. Borchelt, D.R., Koliatsos, V.E., Guarnieri, M., Pardo, C.A., Sisodia, S.S., Price, D.L., 1994. Rapid anterograde axonal transport of the cellular prion glycoprotein in the peripheral and central nervous systems. Journal of Biological Chemistry 269, 14711–14714.
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Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu
Developmental expression of cellular prion protein and apoptotic molecules in the rat cerebellum: Effects of platinum compounds Maria Grazia Bottone a,b, Veronica Dal Boa, Valeria Maria Piccolini a, Giovanni Bottiroli b, Sandra Angelica De Pascali c, Francesco Paolo Fanizzi c, Graziella Bernocchi a,* a b c
Dipartimento di Biologia e Biotecnologie ‘‘Lazzaro Spallanzani’’, Universita` di Pavia, via Ferrata 9, 27100 Pavia, Italy Istituto di Genetica Molecolare del CNR, Sezione di Istochimica e Citometria, via Ferrata 9, 27100 Pavia, Italy Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita` del Salento, 73100 Lecce, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 May 2012 Received in revised form 7 September 2012 Accepted 14 September 2012 Available online 24 September 2012
Programmed cell death is regulated by prototypes of a large family of Bcl-2-like proteins such as Bax and Bcl-2. A neuroprotective role for cellular prion protein (PrPc) on programmed cell death has been reported, although the cytosolic accumulation of PrPc correlates with toxicity and death of some neurons by apoptosis. In order to understand the signalling function of PrPc in promoting survival or suppressing cell death, we analyzed the expression and co-localization of PrPc, Bax and Bcl-2 proteins in the developing cerebellum of rats treated at PD10 (postnatal day 10) with the chemotherapeutic drug cisplatin (cisPt) or the new platinum (Pt) compound [Pt(O,O0 -acac)(g-acac)(DMS)] (PtAcacDMS). Differences in the expression of PrPc, Bax and Bcl-2 were found in proliferating cells and immature Purkinje neurons. One day after administration (PD11), cisPt markedly increased the apoptosis of the proliferating cells of the EGL (external granular layer); at the same time, several apoptotic bodies with strong Bax immunoreactivity were noticed. After PtAcacDMS, changes in PrPc and apoptotic proteins, with respect to the controls, were found but Bax immunopositive apoptotic bodies were not detectable, which could mean that apoptotic cell death of proliferating cells is preserved. Co-localization was clearly detected in the Purkinje cell population and may explain better the mechanisms by which PrPc and the apoptotic proteins function, and particularly the role of PrPc. Considering the reactivity of Purkinje neurons to these proteins at PD11 and Pd17, at least PrPc expression increased after cisPt and PtAcacDMS treatments or, if PrPc decreased, balanced itself with Bcl-2. The noteworthiness of this finding is that it emphasizes that most of the post-mitotic Purkinje cells need to be rescued, otherwise they undergo degeneration and are not replaced. Based on the effects of both Pt compounds on Purkinje cell differentiation, it should be emphasized that PrPc, together with the synergistic action of the co-localized anti-apoptotic protein, acts as a neuroprotective protein countering cytotoxicity in the postnatal critical phases of cerebellum development. ß 2012 Elsevier B.V. All rights reserved.
Keywords: Cellular prion protein Bcl-2 Bax Platinum compounds Neuroprotection
1. Introduction Prion diseases are neurodegenerative disorders that affect animals and humans. The prion pathogen infection consists in an abnormal prion protein which is an altered isoform of a normal
Abbreviations: cisPt, cisplatin; EGL, external granular layer; IGL, internal granular layer; ML, molecular layer; PCL, Purkinje cell layer; PD, postnatal day; PrPc, cellular prion protein; Pt, platinum; PtAcacDMS, [Pt(O,O0 -acac)(g-acac)(DMS)]. * Corresponding author at: Dipartimento di Dipartimento di Biologia e Biotecnologie ‘‘Lazzaro Spallanzani’’, Laboratori di Biologia Animale, Via Ferrata 9, I-27100 Pavia, Italy. Tel.: +39 0382 986327; fax: +39 0382 986325. E-mail addresses: [email protected] (M.G. Bottone), [email protected] (D.B. Veronica), [email protected] (V.M. Piccolini), [email protected] (G. Bottiroli), [email protected] (S.A. De Pascali), [email protected] (F.P. Fanizzi), [email protected] (G. Bernocchi). 0891-0618/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchemneu.2012.09.003
cellular prion protein (PrPc), a sialoglycoprotein widely expressed at the cell surface via a glycosyl phosphatidyl inositol anchor (Prusiner, 1996,1998; Stahl and Prusiner, 1991; review: Chiesa and Harris, 2009); cytosolic localized PrPc has also been detected (Mironov et al., 2003). It is now clear, however, that alterations in the normal function of PrPc may play an important role in causing or contributing to the disease phenotype; for this reason, elucidating the physiological activity of PrPc has become a major priority in prion research. PrPc function remains enigmatic, due to its ubiquitous distribution; not only it is expressed most abundantly in the brain, but has been also detected in non-neuronal tissues (Horiuchi et al., 1995). Some possible biological functions for PrPc have been proposed. A generalized physiological function in the brain as antioxidant (Brown et al., 1997) involvement in copper homeostasis (Pauly and Harris, 1998), signal transduction
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(Mouillet-Richard et al., 2000), neurotransmission and synapse formation (Carleton et al., 2001; Collinge et al., 1994; ZomosaSignoret et al., 2008), and adhesion to the extracellular matrix (Schmitt-Ulms et al., 2001). The accumulation of PrPc in the cytosol seems to correlate with toxicity in some neuronal cells (Harris and True, 2006; Wollmann and Lindquist, 2002) but, interestingly, a subset of neurons in the hippocampus, neocortex and thalamus of mouse brain appeared healthy even though cytosol-localized PrPc has been detected in these neurons (Barmada and Harris, 2005; Mironov et al., 2003). Recently, evidences emerged suggesting that PrPc may be cytoprotective, particularly against internal and environmental stimuli that initiate an apoptotic programme (review: Westergard et al., 2007). PrPc over-expression rescues cultured neurons and some mammalian cell lines, from several kinds of death-inducing stimuli (Kuwahara et al., 1999; Li et al., 2007a; Roucou and LeBlanc, 2005; Solforosi et al., 2004). At present, neuronal roles for PrPc as an anti- and conversely pro-apoptotic protein have been postulated (Kim et al., 2004). It may be speculated is possible that PrPc is a member of the Bcl-2 family, since a similarity between the Bcl-2 homology domain of the Bcl-2 family members and the octapeptide repeats in Nterminal region of PrPc has been noticed (review: Zomosa-Signoret et al., 2008). Investigations on the pro-apoptotic molecule Bax, which plays a major role in regulating neuronal death in the CNS (central nervous system) both during development and after injury (Akhtar et al., 2004; Yuan and Yankner, 2000), indicate that neurodegeneration involves both Bax-dependent and Bax-independent pathways. PrPc could inhibit Bax directly or, indirectly by acting as a signalling molucule (Roucou and LeBlanc, 2005). Bax deletion in mice expressing the neurotoxic form of PrPc slows apoptosis of cerebellar granule cells (Li et al., 2007a,b). In parallel, a protective effect of PrPc has been proposed which depends on signalling function of PrPc to promote neuronal survival or suppress neuronal death (Li et al., 2007a,b; Solforosi et al., 2004). In the present study, immunocytochemical techniques were used to investigate the trend and the role of PrPc in the developing rat cerebellum in relation to the involvement of the pro- and antiapoptotic proteins Bax or Bcl-2 before and after treatment with platinum (Pt) compounds cisplatin (cisPt) and a new platinum complex [Pt(O,O0 -acac)(gamma-acac)(DMS)] (PtAcacDMS). Particularly, high levels of PrPc (Laine´ et al., 2001; Sale`s et al., 1998), Bcl2 and Bax (review: Vogel, 2002) are expressed in the Purkinje neurons. CisPt, the most commonly used therapeutic agent (review: McWhinney et al., 2009), is a highly effective anticancer drug which has an important role also in the treatment of childish malignancies such as neuroblastoma, osteosarcoma and some brain tumours (Prestayko et al., 1979). Mechanisms of action of cisPt are principally based on binding of DNA of mitotic cells (Ahmad, 2010; Eastman, 1991). In proliferating cells, Pt atoms form covalent bonds to purine bases whose result is to block DNA synthesis. The DNA damage triggers cell-cycle arrest, activation of the tumour-suppressor p53 and apoptosis (Qin et al., 2002). Moreover, cisPt toxicity can also induce death in non mitotic cells by interacting with cytoplasmic proteins (Mandic et al., 2003). Among the new Pt complexes, PtAcacDMS, which has two acetylacetonate ligands, one O,O0 -chelate and the other sigmalinked by metine in the gamma position, was found to be the most active in rapidly producing a sustained apoptotic response (Muscella et al., 2008). Differently from cisPt, whose activity appears to be at the cellular level and DNA linking, the cytotoxicity of the new complex is associated solely with intracellular accumulation. Moreover, PtAcacDMS has shown low reactivity with nucleobases and a specific reactivity with sulphur ligands. This suggests that its preferred cell target could be amino acid
residues of enzymes and other proteins involved in apoptotic induction, thus characterizing it as a compound with non-genomic targets. Recently, the low neurotoxicity of PtAcacDMS was supported by findings on cerebellum postnatal ontogenesis (Bernocchi et al., 2011; Cerri et al., 2011). The trend of the PrPc, Bax and Bcl-2 markers was followed at two crucial stages of cerebellum postnatal histogenesis, i.e. during the active cell proliferation in the external granular layer (EGL) and during the maturation phase of Purkinje cells (review: Altman, 1972a,b). Therefore, particular attention was paid to changes in the layers of differentiating Purkinje cells and precursors of granule cells. 2. Materials and methods 2.1. Animal and treatments Ten-day-old Wistar rats were given a single subcutaneous injection of cisPt (0.5 mg/ml; Teva Pharma, Italy) or PtAcacDMS in the nape of the neck at a dose of 5 mg/g b.w. (corresponding to the therapeutic dose suggested by Bodenner et al. (1986) and Dietrich et al. (2006)) or 10 mg/g b.w. Throughout the experiment, the rats were kept in an artificial 12 h light:12 h dark cycle and provided rat chow and tap water ad libitum. One day (PD11), 7 days (PD17) and 20 days (PD30) after drug administration, treated (4 per stage) and untreated control rats (4 per stage) of the same age were deeply anesthetized with an intraperitoneal injection of 35% chloral hydrate (100 ml/100 g b.w.; Sigma, St. Louis, MO, USA); the brains were quickly removed, fixed in Carnoy’s solution (6 absolute ethanol/3 chloroform/1 acetic acid) for 48 h, then placed in absolute ethanol and in acetone, and embedded in Paraplast X-tra (Sigma). Sections (8 mm thick) of cerebellar vermis were cut serially in the sagittal plane and collected on silan-coated slides. The slides were then processed for immunohistochemical procedures (as described later). To avoid possible staining differences due to small changes in the procedure, each reaction was carried out simultaneously on slides from treated and untreated animals at different stages. All experiments were performed according to the guidelines for care and use of laboratory animals as published by the Italian Ministry of Health (DDL 116/92). All efforts were made to minimize the number of animals used and their suffering. 2.2. Single immunoperoxidase stainings The immunoreaction was performed on cerebellum sections of 11, 17 and 30 day-old-rats. Sections were deparaffinized in xylene, rehydrated through a series of graded alcohol treatments and rinsed in PBS. The endogenous peroxidases were suppressed by incubation of sections with 3% H2O2 in 10% methanol in phosphatebuffered saline (PBS; Sigma) for 7 min. Subsequently, the sections were washed in PBS and incubated for 20 min in normal serum (15 ml normal serum/1000 ml PBS) at room temperature in order to block nonspecific antigen binding sites. The serum for blocking is prepared from the same species in which the biotinylated secondary antibody is made. Localization of PrPc was achieved by applying on brain sections a monoclonal mouse antibody anti-PrPc (Sigma) diluted 1:100 in PBS overnight in a dark moist chamber. Thereafter, the sections were sequentially incubated with diluted biotinylated secondary antibody solution (1:200; Vector Laboratories, Burlingame, CA, USA) for 30 min. Sections were washed in PBS and incubated for 30 min at room temperature with Vectastain Elite ABC reagent (Vector Laboratories). Then, 0.05% 3,30 -diaminobenzidine tetrahydrochloride (DAB; Sigma) with 0.01% H2O2 in Tris–HCl buffer (0.05 M, pH 7.6) was used as a chromogen. After each reaction step, sections were washed thoroughly in PBS (two changes of 5 min each). Sections were dehydrated in ethanol, cleared in xylene, and mounted in Eukitt (Kindler, Freiburg, Germany). For control staining, some sections were incubated with PBS instead of the primary antibody. No immunoreactivity was present in this condition. The slides were observed with an Olympus BX51 microscope, and the images were recorded with an Olympus Camedia C-5050 digital camera and stored on a PC. Corrections to brightness and contrast were made with Paint Shop Pro 7 (Jasc Software Inc.). 2.3. Double immunofluorescence reactions Localization of PrPc/Bcl-2 and PrPc/Bax was achieved by applying on cerebellum sections of 11 and 17 day-old-rats, respectively: a mouse monoclonal anti-PrPc (1:100; Sigma) and a rabbit polyclonal anti-Bcl-2 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and a mouse monoclonal anti-PrPc (1:100; Sigma) and a rabbit polyclonal anti-Bax (1:100; Santa Cruz Biotechnology),
in PBS overnight in a dark moist chamber. Sections were washed in PBS and incubated with the secondary antibodies, respectively: Alexa-Fluor 488 goat anti-mouse (1:100, Molecular Probes, Milan, Italy) and Alexa-Fluor 594 donkey anti-rabbit (1:100, Molecular Probes), in PBS for
M.G. Bottone et al. / Journal of Chemical Neuroanatomy 46 (2012) 19–29 1 h. After washing in PBS, the nuclei were counterstained with 0.1 mg/ml Hoechst 33258 for 6 min, and coverslips were lastly mounted in a drop of Mowiol (Calbiochem, San Diego, CA, USA). For control staining, some sections were incubated with PBS instead of the primary antibodies. No immunoreactivity was present in these sections. The slides were viewed by fluorescence microscopy with an Olympus BX51 equipped with a 100W mercury lamp used under the following conditions: 330– 385 nm excitation filter (excf), 400-nm dichroic mirror (dm), and 420-nm barrier filter (bf), for Hoechst 33258 and 450-480 nm excf, 500 nm dm, and 515 nm bf for Alexa 488. Images were recorded with an Olympus Camedia C-5050 digital camera and stored on a PC. Images were optimized for colour, brightness, and contrast by using Paint Shop Pro 7 software (Jasc Software Inc.). 2.4. Evaluation of fluorescence intensity The extent of the labelling was evaluated on digitized images of sections acquired under exposure time avoiding any pixel saturation effect. The labelling intensity was measured by means of densitometric analysis (Image-J 1.46p; NIH, Bethesda, MA, USA). The mask shape was adjusted depending on the spatial distribution of the tissue layer or cell under measurement; the labelling was measured as the intensity mean value over the area (EGL, ML and Purkinje cell cytoplasm). The immunocytochemical fluorescence intensity for Bcl-2, PrPc and Bax were evaluated in a total of 40 fields (10 fields per animal) for controls and treated animals, at each stage. Results were recorded on Microsoft Office Excel Software spreadsheets and are expressed as the means SD (standard deviation). Statistical differences between control and treated animals were evaluated by Student’s t-test.
3. Results Studies were performed on control untreated and cisPt- or PtAcacDMS-treated rats of the same age, i.e. at 11, 17 and 30 days of postnatal life. Since the Pt compounds were injected at 10 days (PD10), treated rats were sacrificed 1, 7 and 20 days after treatment. The observations were performed on lobules VI–VIII of the cerebellar cortex, i.e. in the lobules that showed several changes in the EGL and Purkinje cell layer after cisPt treatment (Avella et al., 2006; Pisu et al., 2005). 3.1. Light microscopy Immunoreaction for PrPc on PD11, control rats (Fig. 1a) showed labelling for PrPc in all the cerebellar layers. In the EGL, the staining was also found in the precursors of granule cells. Purkinje cells showed weak labelling in the cytoplasm. The molecular layer (ML) was very weakly stained and the interneurons were not clearly identifiable; the internal granular layer (IGL) showed intense staining, ascribable to granule cells and glomeruli. At this age, one day after 5 mg/g cisPt treatment (Fig. 1b), a general decreased reactivity was revealed, except for several Purkinje cells with labelled cytoplasm; when cisPt was injected at the dose of 10 mg/g (Fig. 1c) increased immunoreactivity was found in the EGL and IGL. Apoptotic cell bodies were observed in the EGL. At the dose of 5 mg/ g PtAcacDMS (Fig. 1d), there were no evident changes in the EGL immunopositivity, while a decreased immunolabelling was found in the IGL in comparison with controls; the higher dose, 10 mg/g (Fig. 1e), induced strong labelling in the Purkinje cells only. On PD17 (Fig. 1f–j), weak immunoreactivity was observed in all cerebellum layers of control rats (Fig. 1f), except for EGL, which is strongly labelled. Generally, in cisPt- (Fig. 1g-h) and PtAcacDMStreated rats (Fig. 1i and j), intense labelling for PrPc was seen in the EGL; the IGL was more strongly, non-homogeneously labelled, the ML and Purkinje cell layer presented a diffuse immunoreactivity. However, after 10 mg/g cisPt (Fig. 1 h), only the Purkinje cells were more intensely immunostained than in controls, but the cerebellar cortex cytoarchitecture was altered due to the presence of haemorrhagic foci in all the layers; the EGL and IGL appeared weakly immunopositive.
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On PD30, no changes were found in controls (not shown) in comparison with the previous developmental stages and in the cerebellar layers of treated rats (not shown). Weak labelling was observed in the ML and Purkinje cell layer; there were labelled granule cells and glomeruli in the IGL. 3.2. Fluorescence microscopy 3.2.1. Double immunoreaction for Bcl-2/PrPc and Bax/PrPc The double immunostaining for Bcl-2 and PrPc showed labelling in all layers of cerebellum cortex of control rats at PD11 (Fig. 2a–c). Immunostained cells in the EGL and IGL were identifiable as precursors of granule cells and granule cells themselves. The Purkinje cells showed a very strong immunopositivity for Bcl-2, but not for PrPc, as better seen in the merge (Fig. 2c). The 5 mg/g cisPt dose (Fig. 2d–f) induced an evident increased labelling for PrPc (Fig. 2e) but not for Bcl-2 (Fig. 2d) in the Purkinje cells both in the soma and dendrite primary tree, as better seen in the merge (Fig. 2f). Increased immunoreactivity was revealed both for Bcl-2 and PrPc after treatment with 10 mg of cisPt (Fig. 2g–i). At this dose, the labelling for Bcl-2 (Fig. 2g) was stronger than in controls also in the EGL, while PrPc (Fig. 2h) appeared to be generally weaker. After PtAcacDMS treatment, at both doses (Fig. 2j–o), Bcl-2 (Fig. 2j and m) and PrPc immunoreactivity (Fig. 2k and n) were still detectable in all the layers, especially in the Purkinje cells, as better seen in the merge (Fig. 2l and o). In the EGL, the immunocytochemical expression for Bcl-2 and PrPc appeared to be the same as in controls at the dose of 5 mg/g, while it was lowered at the dose of 10 mg/g. The double immunoreaction for Bax and PrPc indicated the distribution of the two proteins in the cerebellar cortex at PD11 (Fig. 3). In control animals (Fig. 3a–c), the labelling for Bax (Fig. 3a) was present in all the cerebellar layers, including the Purkinje cells, where, on the other hand, weak PrPc immunoreactivity was shown (Fig. 3b). After cisPt injection (Fig. 3d–i), Bax labelling in the Purkinje cells did not change in general, while PrPc increased in intensity. In the proliferating EGL, bodies with strong immunopositivity for Bax were observed, especially after the 10 mg/g dose (Fig. 3g–i). These structures are identifiable as apoptotic bodies (Fig. 4a–d), likely formed from macrophages and apoptotic cells, as better seen by the merge (Fig. 4c). The treatment with PtAcacDMS (Fig. 3j–o) maintained immunolabelling for Bax (Fig. 3j and m) and PrPc (Fig. 3k and n) in the Purkinje cells, while in the EGL, Bax and PrPc labelling appeared to be lower in comparison with controls, at the dose of 10 mg/g (Fig. 3a–c), but not Bax immunopositive apoptotic bodies were found. The fluorescence intensity values at PD11 are reported in the histograms of Fig. 5. In comparison with the age-matched controls, the fluorescence intensity for Bcl-2 decreased or increased in the EGL after cisPt treatment at the dose of 5 or 10 mg/g, respectively; in parallel, the fluorescence intensity for PrPc did not change or was decreased. The immuno-expression of Bax in the EGL was decreased, or maintained steady levels, although, as previously reported, strongly immunopositive – Bax apoptotic bodies were scattered among the proliferating cells. After PtAcacDMS, at the low and high dose, Bcl-2 fluorescence intensity of the EGL increased or decreased, while PrPc did not change or was decreased, respectively; Bax intensity values increased or decreased. Purkinje cells of cisPt-treated rats showed small but significant changes in Bcl-2 intensity, and PrPc increased in fluorescence intensity; generally Bax values did not change. In PtAcacDMStreated rats PrPc increase contrasted Bcl-2 drop, while Bax immunofluorescence appeared unalterated.
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Fig. 1. Immunoperoxidase staining for PrPc on PD11 and PD17. At PD11 (a–e) all the cerebellar layers are labelled for PrPc. EGL and IGL of control rats (a) cells are immunostained; Purkinje cells show weak labelling in the cytoplasm (arrows). After 5 mg/g cisPt treatment (b), a decreased reactivity is present, except for several Purkinje cells with labelled cytoplasm (arrows); at the dose of 10 mg/g and (c) apoptotic cell bodies are shown in the EGL and IGL are intensely labelled (arrowheads). After 5 mg/g PtAcacDMS (d), strong labelling of Purkinje cells was shown after the high dose injection (e, arrows). At PD17 (f–j), weak immunoreactivity is shown in all layers of control rats (f), except for EGL. Intense labelling for PrPc is shown in the EGL of cisPt (g and h) and PtAcacDMS treated rats (i and j); the IGL is labelled, the molecular and Purkinje cell layer show a diffuse immunoreactivity. After 10 mg/g cisPt, Purkinje cells are strongly immunostained (h; arrows), the EGL and IGL is almost immunonegative (asterisk); presence of haemorrhagic foci (hf) are visible. EGL, external granular layer; IGL, internal granular layer; ML, molecular layer; and PL, Purkinje cell layer. Scale bar: 60 mm.
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Fig. 2. Double fluorescence immunoreaction for Bcl-2 (red fluorescence) and PrPc (green fluorescence) on PD11. Labelling is present in all layers of controls (a–c). Immunopositive cells are identifiable in the EGL and IGL. Strong immunopositivity for Bcl-2 (a), but not for PrPc (b), as better seen in the merge (c), is shown in the Purkinje cells (arrows) of controls. After cisPt treatment at the doses of 5 mg/g (d–f) and 10 mg/g (g–i), increased labelling for PrPc (e and h; arrows), but not for Bcl-2 (d and g) is observed in the Purkinje cells, both in the soma and dendrite, as better seen in the merge (f and i). At the higher dose, labelling for Bcl-2 (g) is strong in the EGL, while that for PrPc (h) is weaker. Also after PtAcacDMS treatment at both doses (j–o), PrPc immunostaining (k and n), and not Bcl-2 labelling (j and m), is more intense in the Purkinje cell layer (arrows). EGL, external granular layer; IGL, internal granular layer; ML, molecular layer; and PL, Purkinje cell layer. Scale bar: 60 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
At PD17 (Fig. 6), in comparison with controls (Fig. 6a–c), Bcl-2 and PrPc labelling appeared to be maintained in the EGL, which is heavier in the cisPt-treated rats (Fig. 6d–f) Haemorrhagic foci were found following cisPt 10 mg/g (Fig. 6d–f). After PtAcacDMS (Fig. 6g–l), similar results were obtained as regards Bcl-2 and PrPc in the EGL. After treatments, Purkinje cells were labelled in the soma for all three proteins. The immunoreactivity for Bcl-2 and especially for PrPc was strong in the ML (Fig. 6d–l) after cisPt and PtAcacDMS.
At PD17, control rats showed immunolabelling for both Bax and PrPc in all the cerebellar layers (not shown). No evident changes were detected for Bax immunoreactivity after cisPt, while after PtAcacDMS injection at both doses, Bax labelling was intense in the EGL, did not change in the Purkinje cell layer and increased in the ML (not shown), as indicated by the fluorescence intensity values (Fig. 7). The fluorescence intensity values at PD17 are reported in the bar charts of Fig. 7. With respect tp controls, in the 17 day-old-rats the values decreased in the EGL and Purkinje cell layer. In
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Fig. 3. Double fluorescence immunoreaction for Bax (red fluorescence) and PrPc (green fluorescence) on PD11. The distribution of PrPc immunopositivity within the cerebellar cortex of controls (b and c) is similar to Fig. 2. Bax labelling (a and c) is present in all the cerebellar layers, including the Purkinje cells. After cisPt injection (d and f), increased labelling in the Purkinje cells (arrows) and immunopositive bodies for Bax and PrPc are observed in the proliferating EGL, especially after the 10 mg/g dose (g–i, arrowhead). After treatment with PtAcacaDMS (j–o) unaltered immunostaining for Bax (j and m) and increased labelling for PrPc (k and n) are clearly visible in the Purkinje cell layer (arrows); in the other cerebellar layers Bax and PrPc labelling is the same as in controls (a–c). EGL, external granular layer; IGL, internal granular layer; ML, molecular layer; and PL, Purkinje cell layer. Scale bar: 60 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
comparison with controls, the increased fluorescence intensity of Bcl-2 in the EGL of cisPt-treated rats added to an increased PrPc intensity; Bax fluorescence values did not change. After PtAcacDMS, there were similar results for Bcl-2 and PrPc; Bax intensity values increased. As regards Purkinje cells, in cisPttreated rats, and mainly in PtAcacDMS-treated rats, at both the doses, the decreased Bcl-2 intensity values were balanced by the increased PrPc values. In addition, the fluorescence intensity of Bcl-2 and PrPc in the ML, where Purkinje dendrite branches extend, was higher in the PtAcacDMS-treated rats in comparison with the cisPt-treated ones while Bax intensity values strongly increased.
4. Discussion The normal cellular function of PrPc remains unclear. Studies conducted in transgenic and PrPc knockout mice implicate PrPc in synaptic transmission, neuronal differentiation and generally in CNS maturation (review: Collinge et al., 1994). Cell death by apoptosis, a critical event of neuronal development in the CNS, may be associated with PrPc (review: Westergard et al., 2007). PrPc is synthesized in the neuronal soma, transported down the axon and attached to the cell surface by glycosylphosphatidylinositol anchored proteins (GPI linkage) (Borchelt et al., 1994; Rodolfo et al., 1999; Sale`s et al., 1998; Stahl et al., 1987). While it is to be
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Fig. 4. Double fluorescence immunoreaction for Bax (red fluorescence) and PrPc (green fluorescence), and counterstaining with Hoechst 33258 (blue fluorescence) on PD11. In the proliferating EGL, after the treatment with 10 mg cisPt dose, immunopositive bodies for Bax (a) and PrPc (b, arrows) are found. These structures are identifiable as apoptotic bodies, likely made by macrophages and apoptotic cells, as better seen by the merge (c, arrows) and Hoechst 33258 staining (d, arrows). Scale bar: 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 5. Fluorescence intensity values in the developing cerebellar cortex on PD11. The bar charts indicate the trend of the fluorescence intensity values for Bcl-2, Bax and PrPc in the EGL and in the Purkinje cell cytoplasm. These data (means SD) show changes during normal histogenesis and after Pt compounds treatment. Number of evaluation: n = 40. EGL, external granular layer; PC, Purkinje cell layer. Significance of differences: *p < 0.05; **p < 0.01; and ***p < 0.001.
noted that most brain morphological studies report that PrPc is expressed in the cytoplasm in neuronal perikarya, with scarce expression in the neuropil; however, these data are contradictory and do not fit with biochemical and cell biology results. Using ultrastructural immunocytochemistry, the protein, in fact, appeared to be located on the outer surface of the cell membrane and in Golgi and endosomal intracytoplasmic organelles of all neuronal and glial cell types in rat cerebellum (Laine´ et al., 2001). On the other hand, differences in PrPc expression have been described among brain regions (review: Bailly et al., 2004). The differential expression provides a putative anatomical basis for the differential vulnerability of brain neurons to prion (Vorberg and Priola, 2002), as well as for some specific limitations of the detection methods employed. In transgenic mice, by using an immunogold technique, neuronal PrPc has been observed mainly bound to the cell surface and to presynaptic sites; dictyosomes and recycling organelles in most of the major neuron types of several brain areas also exhibited PrPc antigen (Bailly et al., 2004). In the early postnatal brain, PrPc is initially distributed along many fibre tracts, at the surface of elongating axons; PrPc increases markedly at the time of synapse formation, suggesting a role in the function of neuronal contacts (Sale`s et al., 2002). In this study, we were able to exploit our earlier work on the neurotoxicity of Pt compounds (Avella et al., 2006; Pisu et al., 2005), to study the possible involvement of PrPc in cell proliferation/death, and cell differentiation. Developmental alterations are induced by platinum compounds in lobules VI–VIII of cerebellum during the histogenetic process between PD11 and at PD17, i.e. a crucial period for proliferation, differentiation and synaptogenesis (Altman, 1972a,b). In this study we found that PrPc was clearly detectable in untreated rats by fluorescence and immunoperoxidase labelling, although at different intensity, in cells of all the cerebellar layers, i.e. the EGL, Purkinje cell cytoplasm and dendrites, and IGL; no PrPcpositive interneurons were identified in the ML. The pattern fits well with the distribution of PrPc at this postnatal phase of cerebellar development as has been reported by immunoreaction at light microscopy (Sale`s et al., 2002).
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Fig. 6. Double fluorescence immunoreaction for Bcl-2 (red fluorescence) and PrPc (green fluorescence) on PD17. In comparison with controls (a–c), the immunoreactivity for Bcl-2 (d, g and j) and especially for PrPc (e, h and k) increases strongly in the ML, mainly at the dose of 10 mg/g of cisPt (d–f) and PtAcacDMS (j–l). PrPc labelling was shown in the EGL, that is thicker in cisPt-treated rats (d–f, asterisks) respect to control ones (b and c). Note haemorragic foci (hf) after cisPt treatment (d). EGL, external granular layer; IGL, internal granular layer; ML, molecular layer; and PL, Purkinje cell layer. Scale bar: 60 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
CisPt and PtAcacDMS treatments induced differential changes in the PrPc expression at light and fluorescence microscopy. Special attention was paid to cerebellar layers shown to be strongly affected by cisPt, i.e. EGL and Purkinje cell layers comprising somata and dendrites (Avella et al., 2006; Pisu et al., 2005; Scherini and Bernocchi, 1994). The most evident effects of cisPt were: an increased expression of PrPc in the Purkinje cell somata and main proximal dendrites at PD11, together with a general weak labelling of the granule cells progenitors of EGL, where, as previously described (Cerri et al., 2011; Pisu et al., 2005), a marked increase of apoptotic bodies was found at higher 10 mg/g cisPt dose; a similar pattern was noticed in the IGL. This trend was followed by an increased and diffuse intensity in the ML where Purkinje cells extend their dendrite tree, at PD17. These findings have to be considered within the framework of the major changes previously found after cisPt. In the developing cerebellum, a single dose of cisPt administered at PD10 affected the differentiation of post-mitotic Purkinje neurons, as well as the formation of synaptic contacts on these neurons (Avella et al., 2006); some (20%) Purkinje cells even underwent to degeneration and death (Scherini and Bernocchi, 1994). The altered expression of PrPc in the light of above-mentioned findings
is consistent with a role of the protein in neuronal maturation and synapse formation, as suggested by Sale`s et al. (2002). The increased expression of PrPc after cisPt in the soma of the Purkinje cells at PD11 could protect these neurons from degeneration and death or assist their recovery (Avella et al., 2006). On the other hand, after treatment with PtAcacDMS, PrPc immunoreactivity increased in the Purkinje cell soma, whereas no relevant cytoarchitectural changes were detectable after this treatment; the EGL proliferating cells did not undergo apoptotic cell death over the normal trend and Purkinje cell dendrite growth was not altered significantly (Bernocchi et al., 2011; Cerri et al., 2011). Based on the cisPt- and PtAcacDMS-induced changes, the intriguing role of PrPc may be further sustained by the trend and distribution mainly of Bcl-2 protein, but also Bax. The homology between the PrPc and Bcl-2 proteins possibly indicates that PrPc is a member of the Bcl-2 family of proteins (Roucou et al., 2005; Westergard et al., 2007). Bcl-2 and related proteins are implicated in the regulation of apoptosis. Bcl-2 protects neurons from apoptotic stimuli such as deprivation of NGF, toxic Ca2+ levels, and oxidative stress (Allsopp et al., 1993; Farlie et al., 1995; Martinou et al., 1994; Reed, 1994; Yang and
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Fig. 7. Fluorescence intensity values in the developing cerebellar cortex on PD17. The histograms show the immunoreactivity variations for Bcl-2, Bax and PrPc in the EGL, ML and in the Purkinje cell cytoplasm. These data (means SD) display alterations during normal histogenesis and after Pt compounds treatment. Number of evaluation: n = 40. EGL, external granular layer; PC, Purkinje cell layer; and ML, molecular layer. Significance of differences: *p < 0.05; **p < 0.01; and ***p < 0.001.
Korsmeyer, 1996; Zhong et al., 1993). Furthermore Bcl-2 interacts with the pro-apoptotic protein Bax. In a cell, Bcl-2 heterodimerizes with Bax and rescues cells from apoptosis. The ratio of Bcl-2–Bax heterodimers to Bax–Bax homodimers determines the susceptibility of a cell to apoptotic stimuli such as those mentioned above (Oltvai and Korsmeyer, 1994; Yin et al., 1994). Li et al. (2007b) have shown that transgenic (Tg) mice expressing PrPc with a deletion of the N-terminal tail encompassing amino acid residues 32–134 spontaneously resulted in degeneration of cerebellar granule cells. They further showed that Bax deletion in the transgenic mice expressing this neurotoxic form of PrPc slowed apoptosis of the cerebellar granule cells as determined by the TUNEL reaction and caspase-3 staining. The results indicated that the neurotoxicity of a PrPc deleted of aminoacids 32–134 depends in part on activation of a Bax-dependent, mitochondrial pathway of apoptosis. The most interesting finding to explain the role of PrPc as a neuroprotective agent is its interaction with Bcl-2 and Bax, two key modulators of apoptosis. Here, for the first time, we have shown that PrPc co-localizes with Bcl-2 and Bax in certain cell layers or
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cell types of cerebellum. Interestingly, co-localization appeared to be induced by injury; however, differential effects of cisPt and PtAcacDMS were found. In particular, after the higher dose of the Pt compounds, the expression of anti-apoptotic molecule Bcl-2 increased after cisPt and decreased after PtAcacDMS in the EGL, but PrPc immunoexpression was decreased. Therefore, the trend of the two proteins was not the same in the EGL but they seemed to have a dose-dependent compensatory reaction in cisPt-treated rats. However, at PD11, cisPt greatly activated Bax protein in the apoptotic bodies of EGL as a sign of marked cell death (Bernocchi et al., 2011; Pisu et al., 2005). Intriguingly, in these bodies, Bax and PrPc coexist. On the contrary, after PtAcacDMS, changes in PrPc and Bcl-2 with respect to the controls were found, but apoptotic bodies with strong Bax immunoreactivity were not detectable, which could mean that apoptotic cell death of proliferating cells is preserved (Bernocchi et al., 2011; Cerri et al., 2011). More interesting are the data on PD17, when Bcl-2 and PrPc synergistically counter cell death in the surviving EGL proliferating cells; in parallel, Bax intensity values decreased after cisPt, while they increased after PtAcacDMS (see Section 5). The abovementioned knowledge suggests that PrPc may be implicated in regulating apoptosis through the balance between Bcl-2 and Bax, but a prerequisite for the interaction of PrPc with these apoptotic proteins is that all three must co-localize in the same cell. Co-localization was clearly detected in the Purkinje cell population and may explain better the mechanisms by which PrPc and the apoptotic proteins function, and particularly the role of PrPc. Considering the reactivity of Purkinje neurons to these proteins at PD11, at least PrPc expression increased after cisPt and PtAcacDMS treatments or, if PrPc decreased, balanced itself with Bcl-2. PrPc may be involved with the apoptotic proteins to rescue, as much as possible, Purkinje neurons from death. This finding is noteworthy in that it is to consider in the light that Purkinje cells are post-mitotic and immature in postnatal life (Altman, 1972b), and need to be rescued; otherwise they degenerated and cannot be replaced. Consistent with this idea, the immunoreactivity for PrPc and Bcl-2 increased in the ML, seven days after cisPt, where the dendrites of Purkinje cells embark on the recovery phase within the reorganization/remodelling of the cerebellar cytoarchitecture (Avella et al., 2006; Pisu et al., 2005). In this context, Bax immunoexpression in the ML decreased, or was not changed, especially after Pt AcacDMS; therefore, Bcl-2, as well as PrPc, prevailed over the pro-apoptotic protein Bax, supporting the notion that for a correct neuronal differentiation, the balance between Bax and Bcl-2/PrPc is fundamental in order to be able to cope with injury and ensure normal dendrite growth. 5. Conclusions Focusing mainly on the effects of Pt compounds on EGL proliferation and Purkinje cell differentiation, it is emphasized that PrPc combined with the synergistic action of the co-localized antiapoptotic protein Bcl-2, is involved in neuroprotection to rescue cytotoxicity in the postnatal critical phases of cerebellum development. As regards a comparison between the two Pt compounds, cisPt appeared to have neurotoxic effects on normal proliferating granule cells of the EGL, provoking their cell death (Bernocchi et al., 2011; Cerri et al., 2011), as seen by the presence of apoptotic bodies. Instead, after PtAcacDMS treatment, synergistic increased or decreased expression of all molecules, including Bax, was maintained without damages to normal cell genesis. Interestingly, it must be emphasized that the lowering of the two proteins (PrPc and Bcl-2) in the EGL after PtAcacDMS, especially at the dose of 10 mg/g, follows the normal trend of these proteins, as indicated by intensity values in 17 day-old-rats. Nevertheless, it is surprising
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that Bax intensity was increased in the EGL after PtAcacDMS at PD17. Phenomena other than cell death could explain this finding, comparing this result with data on cisPt treatment which demonstrated a recovery of EGL proliferation (Pisu et al., 2005). Actually, the increase of Bax values could be ascribed to the presence of small elements immunopositive to Bax (Bernocchi et al., 2011). The study of causes and involvement of these cells is now in progress. In conclusion, it is to be kept in mind the demonstration that both cisPt and PtAcacDMS are able to reach the developing brain tissue once injected (review: Cerri et al., 2011). Interestingly, the brain Pt content after PtAcacDMS administration was up to 4-fold higher than that after cisPt. This might mean that the novel Pt complex is less bound to plasma proteins than cisPt and can cross the blood brain barrier more readily. The presence of Pt in the brain tissue after PtAcacDMS administration fits in with data obtained from in vitro studies on HeLa and MCF-7 cells (Muscella et al., 2007, 2008). Since it is known that the cytostatic effect is closely associated with the accumulation of Pt in the cell, this could represent an advantage for PtAcacDMS inasmuch as it would permit the use of lower doses of this Pt complex reducing, at the same time, the risk of side effects and drug resistance. Conflicts of interest The authors declared no conflict of interest. Acknowledgements The research was supported by grants from Fondi di Ateneo per la Ricerca (FAR, University of Pavia), Fondazione Banca del Monte di Lombardia (Italy) and Programmi di Ricerca di Rilevante Interesse Nazionale (PRIN, 2009ZFPSPW). References Ahmad, S., 2010. Platinum–DNA interactions and subsequent cellular processes controlling sensitivity to anticancer platinum complexes. Chemistry & Biodiversity 7, 543–566. Akhtar, R.S., Ness, J.M., Roth, K.A., 2004. Bcl-2 family regulation of neuronal development and neurodegeneration. Biochimica et Biophysica Acta 1644, 189–203. Allsopp, T.E., Wyatt, S., Paterson, H.F., Davies, A.M., 1993. The proto-oncogene bcl-2 can selectively rescue neurotrophic factor-dependent neurons from apoptosis. Cell 73, 295–307. Altman, J., 1972a. Postnatal development of the cerebellar cortex in the rat. I. The external granular layer and the transitional molecular layer. Journal of Comparative Neurology 145, 353–398. Altman, J., 1972b. Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer. Journal of Comparative Neurology 145, 399–464. Avella, D., Pisu, M.B., Roda, E., Gravati, M., Bernocchi, G., 2006. Reorganization of the rat cerebellar cortex during postnatal development following cisplatin treatment. Experimental Neurology 201, 131–143. Bailly, Y., Haeberle´, A.M., Blanquet-Grossard, F., Chasserot-Golaz, S., Grant, N., Schultze, T., Bombarde, G., Grazzi, J., Cesbron, J.Y., Lemaire-Vieille, C., 2004. Prion protein (PrPc) immunocytochemistry and expression of the green fluorescent protein reporter gene under control of the bovine PrP gene promoter in the mouse brain. Journal of Comparative Neurology 473, 244–269. Barmada, S.J., Harris, D.A., 2005. Visualization of prion infection in transgenic mice expressing green fluorescent protein-tagged prion protein. Journal of Neuroscience 25, 5824–5832. Bernocchi, G., Bottone, M.G., Piccolini, V.M., Dal Bo, V., Santin, G., De Pascali, S.A., Migoni, D., Fanizzi, F.P., 2011. Developing central nervous system and vulnerability to platinum compounds. Chemotherapy Research and Practice 2011, 315418. Bodenner, D.L., Dedon, P.C., Keng, P.C., Borch, R.F., 1986. Effect of diethyldithiocarbamate on cis-diamminedichloroplatinum(II)-induced cytotoxicity, DNA crosslinking, and gamma-glutamyl transpeptidase inhibition. Cancer Research 46, 2745–2750. Borchelt, D.R., Koliatsos, V.E., Guarnieri, M., Pardo, C.A., Sisodia, S.S., Price, D.L., 1994. Rapid anterograde axonal transport of the cellular prion glycoprotein in the peripheral and central nervous systems. Journal of Biological Chemistry 269, 14711–14714.
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