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The neurochemical nature of PrPc-containing cells in the rat brain
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
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Research Report
The neurochemical nature of PrP c -containing cells in the rat brain Francisco J. Moleres⁎, José L. Velayos⁎ Department of Anatomy, Faculty of Medicine, University of Navarra, Irunlarrea s/n, 31080 Pamplona, Spain
A R T I C LE I N FO
AB S T R A C T
Article history:
The cellular prion protein (PrPC) is a membrane-bound glycoprotein abundantly expressed in
Accepted 20 July 2007
neurons and glial cells within the CNS. The scrapie prion protein (PrPSc) is a conformationally
Available online 10 August 2007
altered isoform of PrPC that is responsible for prion diseases, also termed transmissible spongiform encephalopathies (TSE), a group of neurodegenerative diseases that affect a wide
Keywords:
variety of mammal species, including humans. The presence of the cellular isoform of PrP is
Neurotransmitters
necessary for the establishment and further evolution of prion diseases and the physiological
Perineuronal nets
conditions where PrPC is present seems to modulate the alterations in TSE. In this work, the
Prion proteins
presence of PrPC in GABAergic, glutamatergic, nitrergic, cholinergic, serotoninergic and
Transmissible spongiform
orexinergic populations of cells within the rat brain is examined. Our observations show
encephalopathies
that PrPC is widely expressed in a subset of neurons that contain markers of inhibitory populations of cells throughout the rat brain. The presence of PrPC in other cells types containing important neurotransmitters for the overall brain function is congruent with the imbalances reported for some of them in TSE. Within the cerebral cortex, PrPC is scarcely located in a subset of cells expressing the laminin receptor precursor (LRP) to such a low extent that suggests that other LRP-independent mechanisms actively participate during the pathogenic process. Taken together, our data demonstrate that investigation of the chemical partners of PrPC within cells gives a rational basis for the interpretation of the histopathological alterations in TSE and might help analyze some pathogenic mechanisms of PrPSc. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Transmissible spongiform encephalopathies (TSE) represent a heterogeneous group of neurodegenerative conditions that affect both animals and humans (Prusiner, 1998). They are produced by the appearance of the scrapie isoform of the prion protein (PrPSc, also termed prions), a pathogenic and posttranslationally altered isoform of the cellular form of the prion protein (PrPC), a membrane-bound glycoprotein attached to the
cell surface by a GPI anchor (Stahl et al., 1987) that is ubiquitously expressed throughout many tissues and cell types in all the animal species where TSE occur, but especially abundant in neurons (Bendheim et al., 1992; Kretzschmar et al., 1986). In spite of the multiorganic presence of PrPC within mammals, its function remains to be determined as the study of knockout mice for PrP (PrP−/−) has not shed sufficient light on this issue (Aguzzi and Polymenidou, 2004). Other experimental approaches have shown that PrPC might be involved in neurogenesis and
⁎ Corresponding authors. Fax: +34 948425649. E-mail addresses: [email protected] (F.J. Moleres), [email protected] (J.L. Velayos). Abbreviations: CB, calbindin; CBP, calcium-binding proteins; CJD, Creutzfeldt-Jakob disease; CR, calretinin; FFI, Fatal Familial Insomnia; PNN, perineuronal nets; PrPC, cellular prion protein; PrPSc, scrapie prion protein; PV, parvalbumin; TSEs, transmissible spongiform encephalophaties 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.07.069
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differentiation, neuroprotection and cell signaling (Steele et al., 2006; Vassallo and Herms, 2003), although the complete knowledge to fully understand the unified function of this protein needs further examination. PrPC has been shown to be necessary for the establishment and further evolution of TSE (Bueler et al., 1993). According to the “protein-only hypothesis”, postulated by Standley Prusiner in 1982 (Prusiner, 1982), PrPSc interacts with PrPC and acts as a template for the conversion of PrPC into a de novo generated PrPSc within the cell that had exogenously acquired that first molecule of PrPSc. The appearance of this molecule of PrPSc in the organism is used to classify TSE as: (i) acquired TSE (mainly due to dietary intake of PrPSc or contamination through different exposures to the agent), such as kuru, new variant Creutzfeldt–Jakob disease (vCJD), or iatrogenic CJD (iCJD); (ii) familial TSE (mutations in the coding region of PRNP, the gene that encodes PrPC, that under these circumstances generates mutant, abnormally folded, PrP), such as Gerstmann– Sträussler–Scheinker (GSS), familial CJD (fCJD), or fatal familial insomnia (FFI); (iii) sporadic TSE (unknown origin, a rare stochastic event that produces the abnormal folding of PrP), such as sporadic CJD (sCJD, the most common form of human TSE) and sporadic fatal insomnia (sFI) (Prusiner, 1998). Each TSE is produced by a different strain of PrPSc, which determines its phenotype (Parchi et al., 2000). TSE can be confirmed by the different physicochemical properties of the two isoforms of PrP (Meyer et al., 1986; Oesch et al., 1985) and by several neuropathological features as spongiosis and PrPSc deposition. Other non-TSE histological features like gliosis and neuronal loss are generally present (Budka, 2003). Although the presence and extension of these histological features are variable, the neuronal loss seems to be specific to some particular subsets of cells within the affected brains. Hence, a decrease in the number of a population of inhibitory neurons has been consistently described in human and experimental TSE (Belichenko et al., 1999; Bouzamondo-Bernstein et al., 2004; Bouzamondo et al., 2000; Durand-Gorde et al., 1984, 1985; Ferrer et al., 1993; Gregoire et al., 1993; Guentchev et al., 1998, 1997, 1999; Lu et al., 1995; Tschampa et al., 2002). Since the expression of PrPC is necessary but not sufficient for PrPSc replication, the question of what makes PrPSc damage some subsets of cells and leave others unchanged remains to be experimentally answered. Hence, as the neuronal vulnerability to PrPSc seems to be influenced by the chemical nature of a particular neuron, its investigation will set a basis for the explanation of the pathogenic mechanisms in TSE. Parvalbumin (PV), calbindin (CB) and calretinin (CR) are three types of calcium-binding proteins (CBP) that are physiologically expressed in non-overlapping populations of GABAergic cells within the cerebral cortex (Celio, 1986). PV-positive neurons are particularly affected in most forms of TSE (Ferrer et al., 1993), mainly those surrounded by perineuronal nets (PNN) (Belichenko et al., 1999; Guentchev et al., 1998), a specialized form of extracellular matrix which surrounds a subset of PV-containing cells (Celio and Blumcke, 1994). In addition to the prominent loss of inhibitory cells within the brain of TSE affected subjects, the alteration of other systems has also been demonstrated, as shown by the alteration of serotoninergic (Ledoux, 2005), glutamatergic (Rodríguez et al., 2005, 2006) and cholinergic systems (McDermott et al., 1978; Rubenstein et al., 1991). Currently there is no explanation for this selective neuronal loss, although several hypotheses have emerged in an effort to determine the
molecular basis of this cellular decrease. However, some of these models of PrPSc pathogenesis are based on neuropathological recordings and do not take into account the neuronal substrate where PrPC is expressed, a limiting factor stated by the “protein-only hypothesis”. Therefore, the investigation of the chemical nature of the neurons that contain PrPC is a key step for the correct interpretation and understanding of the histolopathological aspects of TSE. In a previous work, we quantified the levels of expression of PrPC, analyzed its localization and described the distribution pattern of PrPC in CBP-containing inhibitory cells of the cerebral cortex (Moleres and Velayos, 2005). This study helped us propose a pathogenic mechanism of PrPSc that can explain the inhibitory cellular loss in TSE, hence demonstrating that the characterization of PrPC-positive cells is a key step to interpret, within a physiological background, the histological findings of TSE (Moleres and Velayos, 2005). In the present work, we have increased the number of neuronal markers for the characterization of cortical PrPC and have also analyzed the biochemical nature of the cells where PrPC is located.
2.
Results
2.1. Biochemical characterization of the PrPC-containing neurons in the cerebral cortex In our previous work, we demonstrated that PrP and CBP displayed a prominent collocation within the cerebral cortex. Additionally, we found that PrP was present in a subset of PNNsurrounded cells (Moleres and Velayos, 2005). In this work, further characterization of these PrP- and PNN-immunopositive cells reveals that both markers are co-expressed in cells that also contained LRP, a protein present in a subset of cells surrounded by PNN (Fig. 1). The use of additional markers such as vGLUT demonstrates that, in addition to this widespread presence in inhibitory cortical cells, there is a subset of PrP-positive cells surrounded by the presynaptic terminals labeled by this marker of excitatory neurons (Fig. 2A). This collocation is more abundant in the frontal cortex, although it is also present in other cortical territories, likely representing projection neurons and being clearly differentiated from the inhibitory (PNN-surrounded) interneurons previously shown in this and in our previous work (Moleres and Velayos, 2005). In addition to GABAergic and glutamatergic markers, we have also analyzed the presence of PrP in glial cells and in nitrergic neurons. Thus, we have noted the presence of some astrocytes and oligodendrocites positively labeled for PrP throughout the different layers of the cerebral cortex where PrP is expressed (Fig. 2B). Within nitrergic neurons, PrP is present in some nNOSimmunoreactive cells throughout the different layers of the cerebral cortex, although this co-expression is slightly higher in layers II–III of the visual cortex (Fig. 2C), layers where the expression of PrP is more prominent within this cortical region.
2.2.
Cortically derived regions and subcortical areas
The study of the hippocampus displays that PrP is abundantly expressed within inhibitory cells containing either PV (some of
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Fig. 1 – Expression of PrP in LRP-containing neurons within the cerebral cortex of the rat brain. Triple immunofluorescent analysis for the presence of PrP (green channel) in LRP- (red channel) and in PNN- (blue channel) surrounded cortical neurons within the rat brain. Panels D and E correspond to merged images for the three markers. The arrowhead in E points to a cortical neuron located in deep layers of the cerebral cortex which is positively labeled for PrP, PNN and LRP, whereas the arrow notes the expression of LRP in a PNN-surrounded cell that lacks PrP. Note that LRP is expressed only in a subset of PNN, as noted by the prominent number of PNN-surrounded neurons that lack LRP (asterisks). Scale bars: 50 μm.
them surrounded by PNN) or CB (Fig. 3A), both in CA1 and CA3, whereas its localization in the dentate gyrus (DG) is predominantly around PNN-surrounded neurons (Fig. 3B). In addition, nitrergic neurons within this brain structure are also positively labeled for PrP (Fig. 3C). Several nuclei within the basal prosencephalon, such as the ventral pallidum (VP) and the horizontal limb of the diagonal band of Broca (HDB), express PrP in GABAergic neurons that contain PV, some of them surrounded by PNN. Inhibitory CB-positive cells also carry a pool of cells with the cellular isoform of PrP in the horizontal limb of the diagonal band (HDB) (Fig. 3D). The expression of PrP in other immunopositive cell types such as nitrergic neurons in the accumbens nucleus has been also noted, together with the presence of PrP in some cholinergic neurons of the basal magnocellular nucleus of Meynert (B) (Fig. 3E). Within the striatum, the lateral globus pallidus expresses PrP in PVergic cells (Fig. 3F), whereas some immunopositive labeled neurons of the caudate putamen are cholinergic. The distribution of PrP within the thalamus and hypothalamus has been also examined. In the thalamus PrP is mainly present in GABAergic cells within the mediodorsal (MD) and the reticular thalamic nucleus (Rt). PrP-containing neurons within the zona incerta (ZI) result immunopositive for nNOS. Within the hypothalamus, PrPC appears confined to CB-
positive cells within the supraoptic nucleus (SO) and to orexinergic and PNN-surrounded cells in the lateral hypothalamic area (LH). A subset of cells in the lateral hypothalamic area (LH) and the vast majority in the retrochiasmatic part of the supraoptic nucleus (SOR) are immunoreactive for nNOS (Figs. 3G and H, respectively).
2.3.
Brainstem and cerebellum
In the brainstem, the presence of PrP has been analyzed within populations of GABAergic, nitrergic and serotoninergic cells. On the one hand, the anterior pretectal nucleus (APT), the substantia nigra (SN), the deep mesencephalic nucleus (DpMe), the inferior colliculus (IC), the oral part of the reticular formation of the pons (PnO), the trapezoid body (Tz), the gigantocellular reticular nucleus (Gi), the inferior olive (OID) and the nucleus of the solitary tract (SoI) are areas where PrP is present within populations of cells immunoreactive for one or more of the GABAergic markers herein considered (Figs. 4A–C). On the other hand, PrPC-immunoreactive neurons in the substantia nigra (SN), the oral pontine reticular nucleus (PnO), the pariaqueductal gray (PAG), the laterodorsal tegmental nucleus (LDTg), the central gray of the pons (CG), the trapezoid body (Tz) and the gigantocellular reticular nucleus (Gi) are immunostained for nNOS (Figs. 4E–H).
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Fig. 2 – Characterization of the cortical cells immunolabeled for PrP. Double immunofluorescent studies for the presence of PrP (green channel) in glutamatergic (A, red channel), glial (B, red channel) and nitrergic (C, red channel) cells within the cerebral cortex of the rat brain. Images A1–A3 show a high magnification of the area squared in panel A to demonstrate the presence of vGLUT-immunoreactive terminals in the cell that contain PrP (see the yellow areas at the bottom of the neuron, A3). Note in panel B that, although more abundantly expressed in neurons (green cells), PrP is also present in glial cells, as shown by its presence within astrocytes (arrows). Arrow in C points to a nitrergic neuron immunolabeled for PrP. Cortical layers in panel C appear in Roman numbers. Scale bars: 50 μm.
PrP-containing neurons located in the raphe nuclei (RLi) contain serotonin. In the cerebellum, PrPC appears located within the Purkinje cell layer (hence, PV-and CB-positive) and in the molecular layer of the cerebellum, in cells that are also immunolabeled for PV and CB (Fig. 4D).
3.
Discussion
One of the most important neuropathological features of TSE is a selective neuronal loss that mainly affects several populations of cells. Since PrPC is necessary for PrPSc-mediated pathogenesis in TSE, the chemical nature of the PrPC-containing cells must be considered as an important factor to investigate this selective cellular vulnerability in TSE. In a previous work we had determined the chemical nature of the cortical cells that contain PrPC in the rat brain, showing that this protein was mainly expressed in PVergic cells located in deep layers of the cerebral cortex and in CB-positive neurons of layer II (Moleres and Velayos, 2005) (see Fig. 5). Additionally, PrPC was found to be present in a subset of cells
surrounded by PNN, although the extent and implications of those collocations had not been studied in detail. In the present work, we have analyzed those PrP–PNN collocations in an effort to determine the co-expression pattern among these two markers and estimate the pathophysiological implications based on this coexistence within the same populations of cells. Additionally, we have expanded the number of areas and markers to more precisely characterize the chemical nature of the cells that contain PrPC within the rat brain (see Fig. 5). In sCJD, the most common form of human TSE, the prominent loss of PVergic cells in the cerebral cortex, is accompanied by a decrease in the number of PNN (Belichenko et al., 1999). In this work, we have observed that PrPC-positive cells are surrounded by a subset of PNN mainly in deep layers of the cerebral cortex, whereas the co-expression of both markers in superficial layers was not generally observed. Interestingly, we have noted that those PNN-surrounded, PrPC-immunopositive cells were also labeled for LRP, a protein that has been shown to interact in vivo with PrP and, therefore, it has been proposed to act as a receptor for the internalization of PrPSc from the extracellular space to the cells that will be infected (Vana et al., 2006).
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Fig. 3 – Characterization of the PrP-immunopositive neurons in cortically derived and subcortical structures. Double immunofluorescent analysis for the presence of PrP (green channel) in GABAergic (A, D and F, red channels; B, blue channel), nitrergic (C, G and H, red channels) and cholinergic (E, red channel) cells. Figures correspond to areas of the hippocampus (CA3 in A, dentate gyrus in B and CA1 in C), horizontal limb of the diagonal band of Broca (D), basal magnocellular nucleus of Meynert (E), lateral globus pallidus (F), lateral hypothalamic area (G) and the supraoptic nucleus (H). Framed areas in panels A and D show the prominent collocation for PrP and CB. Arrowheads in panels B, C, E and F point to double-labeled neurons (yellow) for PrP and the marker highlighted at the top right of each image. Note in panels G and H the widespread collocation (yellow cells) for PrP and nNOS. Scale bars: 50 μm.
LRP is a protein almost exclusively expressed in a subset of PNN located in both superficial (II–III) and deep (V) layers of the cerebral cortex and in some hippocampal neurons (Baloui et al., 2004). The fact that PNN only surrounds GABA cells that express PV, but not CB or CR (Luth et al., 1992), together with the almost exclusive collocation of PrPC in CB-positive cells of cortical layer II (Moleres and Velayos, 2005) is congruent with our observation that PrPC, PNN and LRP are mainly collocated in deep, but not in superficial layers of the cerebral cortex, supporting the role of LRP in PrPSc-mediated pathogenesis. The low coexistence of PrPC and PNN (only restricted to a subset of those expressing LRP) casts doubts on the exclusive role of LRP in prion pathogenesis (Vana et al., 2006) as it cannot completely explain: (i) the high decrease of PNN-surrounded neurons in prion diseases (Belichenko et al., 1999), (ii) the
excitatory symptoms and the EEG pattern attributed to the selective loss of GABAergic cells in prion diseases (Ferrer et al., 1993), (iii) the global intake of PrPSc within neurons in TSE (Campana et al., 2005). Therefore, other possibilities must be considered. A recently published model of prion pathogenesis has proposed that, during the early stages of TSE, PrPSc might hijack a factor that could be necessary for the survival of PNN (Moleres and Velayos, 2005). Although the nature of that factor is unknown so far, it is congruent with the commonly accepted idea that the conversion of PrPC into PrPSc is mediated by a factor or a protein that has been named “protein X” (Telling et al., 1995). In contrast to the “LRP-dependent model” of prion pathogenesis, this “factor-dependent model” does not require PrPSc– PNN interaction. However, far from underestimating the in-
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Fig. 4 – Determination of the chemical nature of the PrP-containing cells in the brainstem and cerebellum. Double immunofluorescent studies for PrP (green channel) in GABAergic (A, C and D, red channels), nitrergic (E and G, red channels) and cholinergic (H, red channel) neurons. Figures correspond to deep mesencephalic nucleus (A), gigantocellular reticular nucleus (B, C), cerebellum (D), trapezoid body (E) and gigantocellular reticular nucleus (F–H). Arrows in panels A, E and H point to double-labeled neurons (yellow) for PrP and the marker highlighted at the top right of each image. Note the widespread expression of PrP in PVergic (B and C), CB-positive (D) and nitrergic (F and G) cells in the areas shown in the figure. Within the cerebellum, PrP appears located in the CB-positive Purkinje cell layer. Scale bars: 50 μm.
volvement of LRP in prion pathogenesis, this model helps complement the former “interaction-dependent” mechanism to explain the prominent and widespread loss of PNN in the brain areas primarily damaged by prions and the subsequent clinical signs that have been attributed to this loss of inhibitory cells. Concerning the uptake of prions from the extracellular space, it is worth noting that, in addition to LRP, other mechanisms participate in the acquisition of PrPSc molecules, both from the extracellular space and from cells located at variable distances. Exosomes are membranous vesicles whose content is secreted to the extracellular space after multivesicular bodies come into contact with the plasma membrane and they are also involved in the interchange of particles, including prions, between neighbor or distant cells (Fevrier et al., 2004). There are other systems more specifically involved in the transfer of GPI-anchored proteins. One of them is retrotranslocation, which requires cell-to-cell contact and an intact GPI anchor (Liu et al., 2002). Additionally, other systems have been involved in the uptake of PrPSc from the extracellular space such as caveolae-, clatrine- or raft-mediated endocytosis, pinocytosis and transcytosis (Campana et al., 2005; Magalhaes et al., 2005). Thus, similarly to the integration of pathogenic models explained above, the combination of all these intercellular and extracellular mechanisms can explain the uptake of PrPSc molecules in TSE. This work shows a prominent association between PrPC and GABAergic cells throughout the rat brain, mainly within those
inhibitory neurons containing PV. Hence, the widespread presence of PrPC molecules in neurons expressing CBP suggests that the cellular isoform of PrP might play a role in calcium signaling (Krebs et al., 2007; Moleres and Velayos, 2005). Several studies in prion-deficient neuronal cells have indicated that PrPC is involved in calcium metabolism, particularly in the regulation of intracellular free calcium levels. It might act as a sensor of calcium flux depending on the levels of copper and/or reactive oxygen species. Thus, PrPC binds copper, enables redox reactions and initiates calcium-activated signaling cascades. By regulating calcium homeostasis, PrPC could influence neuronal survival and synaptic physiology (Vassallo and Herms, 2003). In addition to GABA, other neurotransmitters such as glutamate are altered in TSE. Several studies underlined the alteration of the glutamatergic system in human and experimental TSE (Rodríguez et al., 2005, 2006) and in animal models of prion disease. In line with these findings, we have detected excitatory cells in the cerebral cortex, mainly in the prefrontal cortex, which resulted immunostained for PrPC. Therefore, the direct interaction between PrPC and PrPSc might be the cause to explain an imbalance in the metabolism of glutamate within the brains of TSE subjects. In addition to the excitatory system in sCJD, the pathogenic process in animal and human TSE also targets the serotoninergic (Ledoux, 2005) and, although less intensely, the cholinergic systems (McDermott et al., 1978; Rubenstein et al., 1991), which is in agreement with the presence of PrPC in those cell types.
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Fig. 5 – Schematic representation of the GABAergic, glutamatergic, nitrergic, cholinergic, serotoninergic, orexinergic and glial cells that contain PrP in rat brain. Image corresponds to figure 81 (lateral 0.90 mm) of the Paxinos and Watson rat brain atlas (Paxinos and Watson, 1998). Each collocation is represented by dots of a different color. Note that the density of the dots is orientative and does not represent the number or relative amount of immunopositive cells for PrP in a particular area. Additionally, some of the neurons here represented as independent neuronal populations immunolabeled for PrP might correspond to a single group of cells as those neurons can simultaneously express more than one marker analyzed in this work, with the exception of PV-, CB- and CR-immunopositive neurons in the cerebral cortex, together with GABAergic and glutamatergic cells that represent non-overlapping populations throughout the rat brain. Abbreviations: Acb: accumbens nucleus; VP: ventral pallidum; CPu: caudate putamen; LS: lateral septum; LGP: lateral globus pallidus; HDB: nucleus of the horizontal limb of the diagonal band; SO: supraoptic nucleus; SOR: supraoptic nucleus, retrochiasmatic part; B: basal nucleus of Meynert; Rt: reticular thalamic nucleus; ZI: zona incerta; LH: lateral hypothalamic area; DG: dentate gyrus; APT: anterior pretectal nucleus; DpMe: deep mesencephalic nucleus; SN: substantia nigra; RLi: rostral linear nucleus of the raphe; IC: inferior colliculus; PAG: periaqueductal gray; PnO: reticular formation of the pons, oral part; Tz: trapezoid body; LDTg: laterodorsal tegmental nucleus; CG: central gray of the pons; GI: gigantocellular reticular nucleus; IOD: inferior olives; SoI: nucleus of the solitary tract; Pk: Purkinje cells of the cerebellum; Mol: molecular layer of the cerebellum. The different layers of the cerebral cortex appear in Roman numbers.
Other less studied systems that might play an important role in the pathogenic process are the nitrergic and the orexinergic systems. The alteration of the latter has not been found yet in TSE although the role of orexins (also termed hypocretins, involved in the sleep/wake cycle) in FFI must be further investigated (Tafti et al., 2005). On the other hand, nitrergic alterations have been also demonstrated in animal and human TSE (Freixes et al., 2006; Ovadia et al., 1996). Due to the prominent collocation of PrPC in those cell types, the contribution or the specific role of this system in the pathophysiology of prion diseases seems to be worth examining.
4.
Experimental procedures
The experiments were carried out in accordance with the guidelines of the European Communities Council Directive
(86/609/EEC) and the Animal Care Committee of the University of Navarra. Great care was taken to avoid animal suffering. Twenty male wistar rats weighting 250–300 g (2–3 months of age) were used for immunohistochemistry. Animals were anesthetized with chloral hydrate (1 ml/100 g) and transcardially perfused with Ringer's saline rinsing solution in H2Od to clean blood from vessels. Brains were removed, fixed for 24 h in 10% formalin at room temperature and embedded in paraffin. Brains were then sagittally (n = 15) or coronally (n = 5) cut at 3 μm. Tissue was deparaffinized at 60 °C for 30 min and rehydrated from xylene to decreasing gradients of ethanol. The endogenous peroxidase was inactivated at 3% H2O2 in H2Od for 9 min. In order to retrieve tissue antigenicity, slides were then microwaved for 30 min in 0.01 M citrate buffer (pH 6.0) at room temperature to block non-specific binding.
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An antibody to the N-terminal region of PrP (residues 23–37, Assay Dessigns, Ann Arbor, MI, USA) was used for the detection of the protein in the rat brain. The specificity of the antibody had been previously tested by its preabsorption with a synthetic peptide corresponding to residues 23–37 of PrP and omission of the different layers of the technique (Moleres and Velayos, 2005). Additionally, its specificity has been further tested by other groups showing that it specifically binds PrP expressed by several cell types even in different animal species, showing a similar but more remarkable labeling than other antibodies such as 6H4 (Prionics, Zurich, Switzerland) (Marcos et al., 2005a,b, 2004). Double immunofluorescent studies for PrP were performed as previously described (Moleres and Velayos, 2005). Briefly, the anti-PrP antibody was colocated with the monoclonals to detect PV- (1:4000; SIGMA, St. Louis, MO, USA), CB- (1:3000; SIGMA, St. Louis, MO, USA), laminin receptor precursor- (LRP, 1:100, Serotek, Oxford, UK), glial fibrillary acidic protein- (GFAP, 1:500; Chemicon, Temecula, CA, USA), 2′,3′-cyclic nucleotide 3′-phosphodiesterase- (CNPase, 1:500; SIGMA, St. Louis, MO, USA) immunoreactive cells, along with a lectin from Wisteria floribunda (1:500; SIGMA, St. Louis, MO, USA) that labeled PNN and polyclonals produced in goat to detect CR- and choline acetyltransferase- (ChAT) (1:2500 and 1:100, respectively; Chemicon, Temecula, CA), nitric oxide synthase- (nNOS, 1:2000, kindly provided by Dr. José Rodrigo, CSIC, Madrid), 5tryptophan hydroxylase- (5-HT, 1:4000, Diasorin, Stillwater, USA) and orexin- (1:100, Santa Cruz Biotechnology, Santa Cruz, USA) immunoreactive neurons. Primary antibodies were incubated o/n at 4 °C. The secondary detection was performed by incubating with goat anti-rabbit Alexa 488 or donkey antirabbit Alexa 488 (for the detection of PrP) and either goat antimouse Alexa 546, streptavidin Alexa 546 or donkey anti-goat Alexa 546 (Molecular Probes, Eugene, OR, USA) for 60 min at 1:100 in TBS (to determine the chemical nature of the labeled neurons). Sections were then covered with an aqueous solution of PBS–Glycerol (SIGMA, St. Louis, MO, USA) and visualized with the aid of an Eclipse E800M microscope (Nikon) equipped with a mercury bulb, appropriate filters and a digital camera ColorView I followed by digitalization with the software supplied by the manufacturer. When triple immunofluorescence studies were performed, the secondary antibody streptavidin Alexa 633 (Molecular Probes, Eugene, OR, USA) was added and these sections were visualized with a laser confocal microscope equipped with the appropriate lasers for the visualization of the three fluorochromes. At least 5 sagittally and 10 coronally cut slices of each animal were studied for each of the double/triple labeling experiments described above. All the areas of the rat brain were represented on these sections, which contained fields where the expression of PrP and the corresponding markers was clearly and faithfully detectable. Omission of primaries and/or secondaries was performed as control.
Acknowledgments This work was supported by BMH4-CT96-856 (EU) and PIUNA. F.J. Moleres is sponsored by a FPU fellowship (AP2003-5033). Special thanks are due to María Ángeles Erdozain, Pedro
García, Ainhoa Moreno, Beatriz Paternain and José Miguel Maruri for excellent technical counseling. REFERENCES
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
The neurochemical nature of PrP c -containing cells in the rat brain Francisco J. Moleres⁎, José L. Velayos⁎ Department of Anatomy, Faculty of Medicine, University of Navarra, Irunlarrea s/n, 31080 Pamplona, Spain
A R T I C LE I N FO
AB S T R A C T
Article history:
The cellular prion protein (PrPC) is a membrane-bound glycoprotein abundantly expressed in
Accepted 20 July 2007
neurons and glial cells within the CNS. The scrapie prion protein (PrPSc) is a conformationally
Available online 10 August 2007
altered isoform of PrPC that is responsible for prion diseases, also termed transmissible spongiform encephalopathies (TSE), a group of neurodegenerative diseases that affect a wide
Keywords:
variety of mammal species, including humans. The presence of the cellular isoform of PrP is
Neurotransmitters
necessary for the establishment and further evolution of prion diseases and the physiological
Perineuronal nets
conditions where PrPC is present seems to modulate the alterations in TSE. In this work, the
Prion proteins
presence of PrPC in GABAergic, glutamatergic, nitrergic, cholinergic, serotoninergic and
Transmissible spongiform
orexinergic populations of cells within the rat brain is examined. Our observations show
encephalopathies
that PrPC is widely expressed in a subset of neurons that contain markers of inhibitory populations of cells throughout the rat brain. The presence of PrPC in other cells types containing important neurotransmitters for the overall brain function is congruent with the imbalances reported for some of them in TSE. Within the cerebral cortex, PrPC is scarcely located in a subset of cells expressing the laminin receptor precursor (LRP) to such a low extent that suggests that other LRP-independent mechanisms actively participate during the pathogenic process. Taken together, our data demonstrate that investigation of the chemical partners of PrPC within cells gives a rational basis for the interpretation of the histopathological alterations in TSE and might help analyze some pathogenic mechanisms of PrPSc. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Transmissible spongiform encephalopathies (TSE) represent a heterogeneous group of neurodegenerative conditions that affect both animals and humans (Prusiner, 1998). They are produced by the appearance of the scrapie isoform of the prion protein (PrPSc, also termed prions), a pathogenic and posttranslationally altered isoform of the cellular form of the prion protein (PrPC), a membrane-bound glycoprotein attached to the
cell surface by a GPI anchor (Stahl et al., 1987) that is ubiquitously expressed throughout many tissues and cell types in all the animal species where TSE occur, but especially abundant in neurons (Bendheim et al., 1992; Kretzschmar et al., 1986). In spite of the multiorganic presence of PrPC within mammals, its function remains to be determined as the study of knockout mice for PrP (PrP−/−) has not shed sufficient light on this issue (Aguzzi and Polymenidou, 2004). Other experimental approaches have shown that PrPC might be involved in neurogenesis and
⁎ Corresponding authors. Fax: +34 948425649. E-mail addresses: [email protected] (F.J. Moleres), [email protected] (J.L. Velayos). Abbreviations: CB, calbindin; CBP, calcium-binding proteins; CJD, Creutzfeldt-Jakob disease; CR, calretinin; FFI, Fatal Familial Insomnia; PNN, perineuronal nets; PrPC, cellular prion protein; PrPSc, scrapie prion protein; PV, parvalbumin; TSEs, transmissible spongiform encephalophaties 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.07.069
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differentiation, neuroprotection and cell signaling (Steele et al., 2006; Vassallo and Herms, 2003), although the complete knowledge to fully understand the unified function of this protein needs further examination. PrPC has been shown to be necessary for the establishment and further evolution of TSE (Bueler et al., 1993). According to the “protein-only hypothesis”, postulated by Standley Prusiner in 1982 (Prusiner, 1982), PrPSc interacts with PrPC and acts as a template for the conversion of PrPC into a de novo generated PrPSc within the cell that had exogenously acquired that first molecule of PrPSc. The appearance of this molecule of PrPSc in the organism is used to classify TSE as: (i) acquired TSE (mainly due to dietary intake of PrPSc or contamination through different exposures to the agent), such as kuru, new variant Creutzfeldt–Jakob disease (vCJD), or iatrogenic CJD (iCJD); (ii) familial TSE (mutations in the coding region of PRNP, the gene that encodes PrPC, that under these circumstances generates mutant, abnormally folded, PrP), such as Gerstmann– Sträussler–Scheinker (GSS), familial CJD (fCJD), or fatal familial insomnia (FFI); (iii) sporadic TSE (unknown origin, a rare stochastic event that produces the abnormal folding of PrP), such as sporadic CJD (sCJD, the most common form of human TSE) and sporadic fatal insomnia (sFI) (Prusiner, 1998). Each TSE is produced by a different strain of PrPSc, which determines its phenotype (Parchi et al., 2000). TSE can be confirmed by the different physicochemical properties of the two isoforms of PrP (Meyer et al., 1986; Oesch et al., 1985) and by several neuropathological features as spongiosis and PrPSc deposition. Other non-TSE histological features like gliosis and neuronal loss are generally present (Budka, 2003). Although the presence and extension of these histological features are variable, the neuronal loss seems to be specific to some particular subsets of cells within the affected brains. Hence, a decrease in the number of a population of inhibitory neurons has been consistently described in human and experimental TSE (Belichenko et al., 1999; Bouzamondo-Bernstein et al., 2004; Bouzamondo et al., 2000; Durand-Gorde et al., 1984, 1985; Ferrer et al., 1993; Gregoire et al., 1993; Guentchev et al., 1998, 1997, 1999; Lu et al., 1995; Tschampa et al., 2002). Since the expression of PrPC is necessary but not sufficient for PrPSc replication, the question of what makes PrPSc damage some subsets of cells and leave others unchanged remains to be experimentally answered. Hence, as the neuronal vulnerability to PrPSc seems to be influenced by the chemical nature of a particular neuron, its investigation will set a basis for the explanation of the pathogenic mechanisms in TSE. Parvalbumin (PV), calbindin (CB) and calretinin (CR) are three types of calcium-binding proteins (CBP) that are physiologically expressed in non-overlapping populations of GABAergic cells within the cerebral cortex (Celio, 1986). PV-positive neurons are particularly affected in most forms of TSE (Ferrer et al., 1993), mainly those surrounded by perineuronal nets (PNN) (Belichenko et al., 1999; Guentchev et al., 1998), a specialized form of extracellular matrix which surrounds a subset of PV-containing cells (Celio and Blumcke, 1994). In addition to the prominent loss of inhibitory cells within the brain of TSE affected subjects, the alteration of other systems has also been demonstrated, as shown by the alteration of serotoninergic (Ledoux, 2005), glutamatergic (Rodríguez et al., 2005, 2006) and cholinergic systems (McDermott et al., 1978; Rubenstein et al., 1991). Currently there is no explanation for this selective neuronal loss, although several hypotheses have emerged in an effort to determine the
molecular basis of this cellular decrease. However, some of these models of PrPSc pathogenesis are based on neuropathological recordings and do not take into account the neuronal substrate where PrPC is expressed, a limiting factor stated by the “protein-only hypothesis”. Therefore, the investigation of the chemical nature of the neurons that contain PrPC is a key step for the correct interpretation and understanding of the histolopathological aspects of TSE. In a previous work, we quantified the levels of expression of PrPC, analyzed its localization and described the distribution pattern of PrPC in CBP-containing inhibitory cells of the cerebral cortex (Moleres and Velayos, 2005). This study helped us propose a pathogenic mechanism of PrPSc that can explain the inhibitory cellular loss in TSE, hence demonstrating that the characterization of PrPC-positive cells is a key step to interpret, within a physiological background, the histological findings of TSE (Moleres and Velayos, 2005). In the present work, we have increased the number of neuronal markers for the characterization of cortical PrPC and have also analyzed the biochemical nature of the cells where PrPC is located.
2.
Results
2.1. Biochemical characterization of the PrPC-containing neurons in the cerebral cortex In our previous work, we demonstrated that PrP and CBP displayed a prominent collocation within the cerebral cortex. Additionally, we found that PrP was present in a subset of PNNsurrounded cells (Moleres and Velayos, 2005). In this work, further characterization of these PrP- and PNN-immunopositive cells reveals that both markers are co-expressed in cells that also contained LRP, a protein present in a subset of cells surrounded by PNN (Fig. 1). The use of additional markers such as vGLUT demonstrates that, in addition to this widespread presence in inhibitory cortical cells, there is a subset of PrP-positive cells surrounded by the presynaptic terminals labeled by this marker of excitatory neurons (Fig. 2A). This collocation is more abundant in the frontal cortex, although it is also present in other cortical territories, likely representing projection neurons and being clearly differentiated from the inhibitory (PNN-surrounded) interneurons previously shown in this and in our previous work (Moleres and Velayos, 2005). In addition to GABAergic and glutamatergic markers, we have also analyzed the presence of PrP in glial cells and in nitrergic neurons. Thus, we have noted the presence of some astrocytes and oligodendrocites positively labeled for PrP throughout the different layers of the cerebral cortex where PrP is expressed (Fig. 2B). Within nitrergic neurons, PrP is present in some nNOSimmunoreactive cells throughout the different layers of the cerebral cortex, although this co-expression is slightly higher in layers II–III of the visual cortex (Fig. 2C), layers where the expression of PrP is more prominent within this cortical region.
2.2.
Cortically derived regions and subcortical areas
The study of the hippocampus displays that PrP is abundantly expressed within inhibitory cells containing either PV (some of
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Fig. 1 – Expression of PrP in LRP-containing neurons within the cerebral cortex of the rat brain. Triple immunofluorescent analysis for the presence of PrP (green channel) in LRP- (red channel) and in PNN- (blue channel) surrounded cortical neurons within the rat brain. Panels D and E correspond to merged images for the three markers. The arrowhead in E points to a cortical neuron located in deep layers of the cerebral cortex which is positively labeled for PrP, PNN and LRP, whereas the arrow notes the expression of LRP in a PNN-surrounded cell that lacks PrP. Note that LRP is expressed only in a subset of PNN, as noted by the prominent number of PNN-surrounded neurons that lack LRP (asterisks). Scale bars: 50 μm.
them surrounded by PNN) or CB (Fig. 3A), both in CA1 and CA3, whereas its localization in the dentate gyrus (DG) is predominantly around PNN-surrounded neurons (Fig. 3B). In addition, nitrergic neurons within this brain structure are also positively labeled for PrP (Fig. 3C). Several nuclei within the basal prosencephalon, such as the ventral pallidum (VP) and the horizontal limb of the diagonal band of Broca (HDB), express PrP in GABAergic neurons that contain PV, some of them surrounded by PNN. Inhibitory CB-positive cells also carry a pool of cells with the cellular isoform of PrP in the horizontal limb of the diagonal band (HDB) (Fig. 3D). The expression of PrP in other immunopositive cell types such as nitrergic neurons in the accumbens nucleus has been also noted, together with the presence of PrP in some cholinergic neurons of the basal magnocellular nucleus of Meynert (B) (Fig. 3E). Within the striatum, the lateral globus pallidus expresses PrP in PVergic cells (Fig. 3F), whereas some immunopositive labeled neurons of the caudate putamen are cholinergic. The distribution of PrP within the thalamus and hypothalamus has been also examined. In the thalamus PrP is mainly present in GABAergic cells within the mediodorsal (MD) and the reticular thalamic nucleus (Rt). PrP-containing neurons within the zona incerta (ZI) result immunopositive for nNOS. Within the hypothalamus, PrPC appears confined to CB-
positive cells within the supraoptic nucleus (SO) and to orexinergic and PNN-surrounded cells in the lateral hypothalamic area (LH). A subset of cells in the lateral hypothalamic area (LH) and the vast majority in the retrochiasmatic part of the supraoptic nucleus (SOR) are immunoreactive for nNOS (Figs. 3G and H, respectively).
2.3.
Brainstem and cerebellum
In the brainstem, the presence of PrP has been analyzed within populations of GABAergic, nitrergic and serotoninergic cells. On the one hand, the anterior pretectal nucleus (APT), the substantia nigra (SN), the deep mesencephalic nucleus (DpMe), the inferior colliculus (IC), the oral part of the reticular formation of the pons (PnO), the trapezoid body (Tz), the gigantocellular reticular nucleus (Gi), the inferior olive (OID) and the nucleus of the solitary tract (SoI) are areas where PrP is present within populations of cells immunoreactive for one or more of the GABAergic markers herein considered (Figs. 4A–C). On the other hand, PrPC-immunoreactive neurons in the substantia nigra (SN), the oral pontine reticular nucleus (PnO), the pariaqueductal gray (PAG), the laterodorsal tegmental nucleus (LDTg), the central gray of the pons (CG), the trapezoid body (Tz) and the gigantocellular reticular nucleus (Gi) are immunostained for nNOS (Figs. 4E–H).
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Fig. 2 – Characterization of the cortical cells immunolabeled for PrP. Double immunofluorescent studies for the presence of PrP (green channel) in glutamatergic (A, red channel), glial (B, red channel) and nitrergic (C, red channel) cells within the cerebral cortex of the rat brain. Images A1–A3 show a high magnification of the area squared in panel A to demonstrate the presence of vGLUT-immunoreactive terminals in the cell that contain PrP (see the yellow areas at the bottom of the neuron, A3). Note in panel B that, although more abundantly expressed in neurons (green cells), PrP is also present in glial cells, as shown by its presence within astrocytes (arrows). Arrow in C points to a nitrergic neuron immunolabeled for PrP. Cortical layers in panel C appear in Roman numbers. Scale bars: 50 μm.
PrP-containing neurons located in the raphe nuclei (RLi) contain serotonin. In the cerebellum, PrPC appears located within the Purkinje cell layer (hence, PV-and CB-positive) and in the molecular layer of the cerebellum, in cells that are also immunolabeled for PV and CB (Fig. 4D).
3.
Discussion
One of the most important neuropathological features of TSE is a selective neuronal loss that mainly affects several populations of cells. Since PrPC is necessary for PrPSc-mediated pathogenesis in TSE, the chemical nature of the PrPC-containing cells must be considered as an important factor to investigate this selective cellular vulnerability in TSE. In a previous work we had determined the chemical nature of the cortical cells that contain PrPC in the rat brain, showing that this protein was mainly expressed in PVergic cells located in deep layers of the cerebral cortex and in CB-positive neurons of layer II (Moleres and Velayos, 2005) (see Fig. 5). Additionally, PrPC was found to be present in a subset of cells
surrounded by PNN, although the extent and implications of those collocations had not been studied in detail. In the present work, we have analyzed those PrP–PNN collocations in an effort to determine the co-expression pattern among these two markers and estimate the pathophysiological implications based on this coexistence within the same populations of cells. Additionally, we have expanded the number of areas and markers to more precisely characterize the chemical nature of the cells that contain PrPC within the rat brain (see Fig. 5). In sCJD, the most common form of human TSE, the prominent loss of PVergic cells in the cerebral cortex, is accompanied by a decrease in the number of PNN (Belichenko et al., 1999). In this work, we have observed that PrPC-positive cells are surrounded by a subset of PNN mainly in deep layers of the cerebral cortex, whereas the co-expression of both markers in superficial layers was not generally observed. Interestingly, we have noted that those PNN-surrounded, PrPC-immunopositive cells were also labeled for LRP, a protein that has been shown to interact in vivo with PrP and, therefore, it has been proposed to act as a receptor for the internalization of PrPSc from the extracellular space to the cells that will be infected (Vana et al., 2006).
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Fig. 3 – Characterization of the PrP-immunopositive neurons in cortically derived and subcortical structures. Double immunofluorescent analysis for the presence of PrP (green channel) in GABAergic (A, D and F, red channels; B, blue channel), nitrergic (C, G and H, red channels) and cholinergic (E, red channel) cells. Figures correspond to areas of the hippocampus (CA3 in A, dentate gyrus in B and CA1 in C), horizontal limb of the diagonal band of Broca (D), basal magnocellular nucleus of Meynert (E), lateral globus pallidus (F), lateral hypothalamic area (G) and the supraoptic nucleus (H). Framed areas in panels A and D show the prominent collocation for PrP and CB. Arrowheads in panels B, C, E and F point to double-labeled neurons (yellow) for PrP and the marker highlighted at the top right of each image. Note in panels G and H the widespread collocation (yellow cells) for PrP and nNOS. Scale bars: 50 μm.
LRP is a protein almost exclusively expressed in a subset of PNN located in both superficial (II–III) and deep (V) layers of the cerebral cortex and in some hippocampal neurons (Baloui et al., 2004). The fact that PNN only surrounds GABA cells that express PV, but not CB or CR (Luth et al., 1992), together with the almost exclusive collocation of PrPC in CB-positive cells of cortical layer II (Moleres and Velayos, 2005) is congruent with our observation that PrPC, PNN and LRP are mainly collocated in deep, but not in superficial layers of the cerebral cortex, supporting the role of LRP in PrPSc-mediated pathogenesis. The low coexistence of PrPC and PNN (only restricted to a subset of those expressing LRP) casts doubts on the exclusive role of LRP in prion pathogenesis (Vana et al., 2006) as it cannot completely explain: (i) the high decrease of PNN-surrounded neurons in prion diseases (Belichenko et al., 1999), (ii) the
excitatory symptoms and the EEG pattern attributed to the selective loss of GABAergic cells in prion diseases (Ferrer et al., 1993), (iii) the global intake of PrPSc within neurons in TSE (Campana et al., 2005). Therefore, other possibilities must be considered. A recently published model of prion pathogenesis has proposed that, during the early stages of TSE, PrPSc might hijack a factor that could be necessary for the survival of PNN (Moleres and Velayos, 2005). Although the nature of that factor is unknown so far, it is congruent with the commonly accepted idea that the conversion of PrPC into PrPSc is mediated by a factor or a protein that has been named “protein X” (Telling et al., 1995). In contrast to the “LRP-dependent model” of prion pathogenesis, this “factor-dependent model” does not require PrPSc– PNN interaction. However, far from underestimating the in-
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Fig. 4 – Determination of the chemical nature of the PrP-containing cells in the brainstem and cerebellum. Double immunofluorescent studies for PrP (green channel) in GABAergic (A, C and D, red channels), nitrergic (E and G, red channels) and cholinergic (H, red channel) neurons. Figures correspond to deep mesencephalic nucleus (A), gigantocellular reticular nucleus (B, C), cerebellum (D), trapezoid body (E) and gigantocellular reticular nucleus (F–H). Arrows in panels A, E and H point to double-labeled neurons (yellow) for PrP and the marker highlighted at the top right of each image. Note the widespread expression of PrP in PVergic (B and C), CB-positive (D) and nitrergic (F and G) cells in the areas shown in the figure. Within the cerebellum, PrP appears located in the CB-positive Purkinje cell layer. Scale bars: 50 μm.
volvement of LRP in prion pathogenesis, this model helps complement the former “interaction-dependent” mechanism to explain the prominent and widespread loss of PNN in the brain areas primarily damaged by prions and the subsequent clinical signs that have been attributed to this loss of inhibitory cells. Concerning the uptake of prions from the extracellular space, it is worth noting that, in addition to LRP, other mechanisms participate in the acquisition of PrPSc molecules, both from the extracellular space and from cells located at variable distances. Exosomes are membranous vesicles whose content is secreted to the extracellular space after multivesicular bodies come into contact with the plasma membrane and they are also involved in the interchange of particles, including prions, between neighbor or distant cells (Fevrier et al., 2004). There are other systems more specifically involved in the transfer of GPI-anchored proteins. One of them is retrotranslocation, which requires cell-to-cell contact and an intact GPI anchor (Liu et al., 2002). Additionally, other systems have been involved in the uptake of PrPSc from the extracellular space such as caveolae-, clatrine- or raft-mediated endocytosis, pinocytosis and transcytosis (Campana et al., 2005; Magalhaes et al., 2005). Thus, similarly to the integration of pathogenic models explained above, the combination of all these intercellular and extracellular mechanisms can explain the uptake of PrPSc molecules in TSE. This work shows a prominent association between PrPC and GABAergic cells throughout the rat brain, mainly within those
inhibitory neurons containing PV. Hence, the widespread presence of PrPC molecules in neurons expressing CBP suggests that the cellular isoform of PrP might play a role in calcium signaling (Krebs et al., 2007; Moleres and Velayos, 2005). Several studies in prion-deficient neuronal cells have indicated that PrPC is involved in calcium metabolism, particularly in the regulation of intracellular free calcium levels. It might act as a sensor of calcium flux depending on the levels of copper and/or reactive oxygen species. Thus, PrPC binds copper, enables redox reactions and initiates calcium-activated signaling cascades. By regulating calcium homeostasis, PrPC could influence neuronal survival and synaptic physiology (Vassallo and Herms, 2003). In addition to GABA, other neurotransmitters such as glutamate are altered in TSE. Several studies underlined the alteration of the glutamatergic system in human and experimental TSE (Rodríguez et al., 2005, 2006) and in animal models of prion disease. In line with these findings, we have detected excitatory cells in the cerebral cortex, mainly in the prefrontal cortex, which resulted immunostained for PrPC. Therefore, the direct interaction between PrPC and PrPSc might be the cause to explain an imbalance in the metabolism of glutamate within the brains of TSE subjects. In addition to the excitatory system in sCJD, the pathogenic process in animal and human TSE also targets the serotoninergic (Ledoux, 2005) and, although less intensely, the cholinergic systems (McDermott et al., 1978; Rubenstein et al., 1991), which is in agreement with the presence of PrPC in those cell types.
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Fig. 5 – Schematic representation of the GABAergic, glutamatergic, nitrergic, cholinergic, serotoninergic, orexinergic and glial cells that contain PrP in rat brain. Image corresponds to figure 81 (lateral 0.90 mm) of the Paxinos and Watson rat brain atlas (Paxinos and Watson, 1998). Each collocation is represented by dots of a different color. Note that the density of the dots is orientative and does not represent the number or relative amount of immunopositive cells for PrP in a particular area. Additionally, some of the neurons here represented as independent neuronal populations immunolabeled for PrP might correspond to a single group of cells as those neurons can simultaneously express more than one marker analyzed in this work, with the exception of PV-, CB- and CR-immunopositive neurons in the cerebral cortex, together with GABAergic and glutamatergic cells that represent non-overlapping populations throughout the rat brain. Abbreviations: Acb: accumbens nucleus; VP: ventral pallidum; CPu: caudate putamen; LS: lateral septum; LGP: lateral globus pallidus; HDB: nucleus of the horizontal limb of the diagonal band; SO: supraoptic nucleus; SOR: supraoptic nucleus, retrochiasmatic part; B: basal nucleus of Meynert; Rt: reticular thalamic nucleus; ZI: zona incerta; LH: lateral hypothalamic area; DG: dentate gyrus; APT: anterior pretectal nucleus; DpMe: deep mesencephalic nucleus; SN: substantia nigra; RLi: rostral linear nucleus of the raphe; IC: inferior colliculus; PAG: periaqueductal gray; PnO: reticular formation of the pons, oral part; Tz: trapezoid body; LDTg: laterodorsal tegmental nucleus; CG: central gray of the pons; GI: gigantocellular reticular nucleus; IOD: inferior olives; SoI: nucleus of the solitary tract; Pk: Purkinje cells of the cerebellum; Mol: molecular layer of the cerebellum. The different layers of the cerebral cortex appear in Roman numbers.
Other less studied systems that might play an important role in the pathogenic process are the nitrergic and the orexinergic systems. The alteration of the latter has not been found yet in TSE although the role of orexins (also termed hypocretins, involved in the sleep/wake cycle) in FFI must be further investigated (Tafti et al., 2005). On the other hand, nitrergic alterations have been also demonstrated in animal and human TSE (Freixes et al., 2006; Ovadia et al., 1996). Due to the prominent collocation of PrPC in those cell types, the contribution or the specific role of this system in the pathophysiology of prion diseases seems to be worth examining.
4.
Experimental procedures
The experiments were carried out in accordance with the guidelines of the European Communities Council Directive
(86/609/EEC) and the Animal Care Committee of the University of Navarra. Great care was taken to avoid animal suffering. Twenty male wistar rats weighting 250–300 g (2–3 months of age) were used for immunohistochemistry. Animals were anesthetized with chloral hydrate (1 ml/100 g) and transcardially perfused with Ringer's saline rinsing solution in H2Od to clean blood from vessels. Brains were removed, fixed for 24 h in 10% formalin at room temperature and embedded in paraffin. Brains were then sagittally (n = 15) or coronally (n = 5) cut at 3 μm. Tissue was deparaffinized at 60 °C for 30 min and rehydrated from xylene to decreasing gradients of ethanol. The endogenous peroxidase was inactivated at 3% H2O2 in H2Od for 9 min. In order to retrieve tissue antigenicity, slides were then microwaved for 30 min in 0.01 M citrate buffer (pH 6.0) at room temperature to block non-specific binding.
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An antibody to the N-terminal region of PrP (residues 23–37, Assay Dessigns, Ann Arbor, MI, USA) was used for the detection of the protein in the rat brain. The specificity of the antibody had been previously tested by its preabsorption with a synthetic peptide corresponding to residues 23–37 of PrP and omission of the different layers of the technique (Moleres and Velayos, 2005). Additionally, its specificity has been further tested by other groups showing that it specifically binds PrP expressed by several cell types even in different animal species, showing a similar but more remarkable labeling than other antibodies such as 6H4 (Prionics, Zurich, Switzerland) (Marcos et al., 2005a,b, 2004). Double immunofluorescent studies for PrP were performed as previously described (Moleres and Velayos, 2005). Briefly, the anti-PrP antibody was colocated with the monoclonals to detect PV- (1:4000; SIGMA, St. Louis, MO, USA), CB- (1:3000; SIGMA, St. Louis, MO, USA), laminin receptor precursor- (LRP, 1:100, Serotek, Oxford, UK), glial fibrillary acidic protein- (GFAP, 1:500; Chemicon, Temecula, CA, USA), 2′,3′-cyclic nucleotide 3′-phosphodiesterase- (CNPase, 1:500; SIGMA, St. Louis, MO, USA) immunoreactive cells, along with a lectin from Wisteria floribunda (1:500; SIGMA, St. Louis, MO, USA) that labeled PNN and polyclonals produced in goat to detect CR- and choline acetyltransferase- (ChAT) (1:2500 and 1:100, respectively; Chemicon, Temecula, CA), nitric oxide synthase- (nNOS, 1:2000, kindly provided by Dr. José Rodrigo, CSIC, Madrid), 5tryptophan hydroxylase- (5-HT, 1:4000, Diasorin, Stillwater, USA) and orexin- (1:100, Santa Cruz Biotechnology, Santa Cruz, USA) immunoreactive neurons. Primary antibodies were incubated o/n at 4 °C. The secondary detection was performed by incubating with goat anti-rabbit Alexa 488 or donkey antirabbit Alexa 488 (for the detection of PrP) and either goat antimouse Alexa 546, streptavidin Alexa 546 or donkey anti-goat Alexa 546 (Molecular Probes, Eugene, OR, USA) for 60 min at 1:100 in TBS (to determine the chemical nature of the labeled neurons). Sections were then covered with an aqueous solution of PBS–Glycerol (SIGMA, St. Louis, MO, USA) and visualized with the aid of an Eclipse E800M microscope (Nikon) equipped with a mercury bulb, appropriate filters and a digital camera ColorView I followed by digitalization with the software supplied by the manufacturer. When triple immunofluorescence studies were performed, the secondary antibody streptavidin Alexa 633 (Molecular Probes, Eugene, OR, USA) was added and these sections were visualized with a laser confocal microscope equipped with the appropriate lasers for the visualization of the three fluorochromes. At least 5 sagittally and 10 coronally cut slices of each animal were studied for each of the double/triple labeling experiments described above. All the areas of the rat brain were represented on these sections, which contained fields where the expression of PrP and the corresponding markers was clearly and faithfully detectable. Omission of primaries and/or secondaries was performed as control.
Acknowledgments This work was supported by BMH4-CT96-856 (EU) and PIUNA. F.J. Moleres is sponsored by a FPU fellowship (AP2003-5033). Special thanks are due to María Ángeles Erdozain, Pedro
García, Ainhoa Moreno, Beatriz Paternain and José Miguel Maruri for excellent technical counseling. REFERENCES
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