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,1
* Cellular and Molecular Basis of Neurodegeneration and Neurorepair, Department of Cell Biology, University of Barcelona, Barcelona, Spain;
Developmental Neurobiology and Regeneration, Institute for Research in Biomedicine (IRB Barcelona), Department of Cell Biology, University of Barcelona, Barcelona, Spain; and
Institute of Neuropathology, University Hospital of Zürich, Zurich, Switzerland
1Correspondence: Department of Cell Biology, Institute for Research in Biomedicine, University of Barcelona, Josep Samitier 1–5, E-08028 Barcelona, Spain. E-mail: jadelrio{at}pcb.ub.es, jadelrio{at}ub.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PrP) are ataxic and display cerebellar cell loss and premature death. Studies on the molecular and intracellular events that trigger cell death in these mutants may contribute to elucidate the functions of PrPc and to the design of treatments for prion disease. Here we examined the effects of Bcl-2 overexpression in neurons on the development of the neurological syndrome and cerebellar pathology of PrP. We show that PrP overexpression activates the stress-associated kinases ERK1–2 in reactive astroglia, p38 and the phosphorylation of p53, which leads to the death of cerebellar neurons in mutant mice. We found that the expression of PrP in cell lines expressing very low levels of PrPc strongly induces the activation of apoptotic pathways, thereby leading to caspase-3 activation and cell death, which can be prevented by coexpressing Bcl-2. Finally, we corroborate in vivo that neuronal-directed Bcl-2 overexpression in PrP mice (PrP Bcl-2) markedly reduces caspase-3 activation, glial activation, and neuronal cell death in cerebellum by improving locomotor deficits and life expectancy.—Nicolas, O., Gavín, R., Braun, N., Ureña, J. M., Fontana, X., Soriano, E., Aguzzi, A., del Río,. J. A. Bcl-2 overexpression delays caspase-3 activation and rescues cerebellar degeneration in prion-deficient mice that overexpress amino-terminally truncated prion.
Key Words: cerebellar syndrome • granule cells • oxidative damage • prion protein
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Although many studies have examined the putative roles of PrPc in the CNS, a full repertory of PrPc functions remains to be determined. PrPc is involved in copper metabolism, and cell homeostasis and is considered a neuroprotective molecule (2)
. Particular attention has been devoted to determine the functions of the OR and HR domains of PrPc. For example, the OR participates in copper binding (3)
and its modification increases susceptibility to oxidative attack (e.g., 4
). The study of numerous PrPc transgenic mice has provided a relevant advance toward the identification of PrPc functions (5)
. Several Prnpo/o mouse lines exhibit ataxia and Purkinje cell (PC) degeneration as a result of the overexpression of PrP-like Doppel (Dpl; see ref 5
for review). In addition, the transgenic expression of amino truncated PrPc (e.g., F35), lacking most of the OR and HR in Prnpo/o mice (termed PrP in the present study), induces severe ataxia because of extensive cerebellar granule cell (GC) death (6
7
8)
. In both PrP- and Dpl-transgenic mice, neurodegenerative effects are largely counteracted by Prnp expression. This observation indicates that PrP and Dpl interfere with normal PrPc functions (9)
; however, they also suggest that the neuroprotective properties of PrPc may reside in the N-terminal region. Recent studies in Prno/o cells demonstrate that Dpl overexpression becomes neurotoxic by increasing oxidative damage (e.g., 10
) and that the overexpression of a C-terminal fragment of the PrPc (C1 fragment) sensitizes the cell to caspase-3 activation (11)
. However, no data are available on PrP-mediated neurotoxicity.
The Bcl-2 protein is crucial for the regulation of cell death since it interacts with the proapoptotic protein Bax to prevent mitochondrial permeability and release of proapoptotic molecules (12)
. Bax-mediated apoptosis can be triggered by oxidative stress, while Bax synthesis is activated by the tumor suppressor protein p53 (13)
. The hypothesis of the present study is that tight regulation of Bcl-2/Bax functions suppresses neurodegeneration induced by F35 overexpression in vitro and in vivo. We tested this hypothesis by analyzing the effects of Bcl-2 overexpression on F35-transfected cells and on the onset of the neurological syndrome and cerebellar pathology in PrP mice by generating a transgenic mouse that also overexpresses Bcl-2 under the neuronal specific enolase (NSE)-promoter (PrP Bcl-2). In contrast to PrP animals, in most PrP Bcl-2 mice, neuronal-directed Bcl-2 overexpression reduced p53 activation, caspase-3 activation, and cerebellar degeneration. These alterations correlated with clear improvements in gait and behavior deficits in these mutant mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
Prnpo/o (Zürich I) and Tg20/Tga20 mice were purchased from EMMA (Monterotondo, Italy). Prnpo/o mice overexpressing N-terminal truncated (lacking aa32–134) PrPc protein (called PrP mice in our study) were generated previously (6)
. NSEa-Bcl-2 transgenic mice were a gift from M. Barbacid (CNIO, Madrid, Spain) and I. Silos-Santiago (The Scripps Research Institute, La Jolla, CA, USA). All experimental procedures were performed in accordance with the guidelines of the Spanish Ministry of Science and Technology, following European Standards.
Breeding and PCR genotyping of mice
Wild-type and neomycin-targeted Prnp loci and the F35 transgene were detected as described previously (6
, 14)
. Bcl-2 transgene detection under Neuronal Specific Enolase (NSE) promoter was performed as described (15)
. NSEa-Bcl-2 mice present many Bcl-2 copies in several genome alleles (13)
. Prnpo/o F35+/o mice overexpressing Bcl-2 (called PrPBcl-2, see below) were generated by mating Prnpo/o with NSEa-Bcl-2 mice to obtain Prnp+/0 Bcl-2 F1 offspring. Finally, these mice were crossed with Prnp+/0 F35+/o to obtain Prnpo/o F35+/o Bcl-2, termed PrP Bcl-2 in this study. To ensure that Bcl-2 overexpression was maintained during breeding, we performed RT-PCR and WB techniques, which revealed that Bcl-2 expression and protein levels in PrP Bcl-2 mice were higher than in wild-type, Prnpo/o and PrP mice (not shown, see
Fig. 4
G for WB). A total of 136 animals were used to produce the transgenic mice. PrP and PrP Bcl-2 mice were examined every 2 to 5 days for gait deficit.
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Histological methods
Mice were perfused with 0.1 M PBS buffered paraformaldehyde pH 7.3 and cryoprotected in 30% sucrose. Coronal sections (30 µm thick) were obtained in a freezing microtome. Sections from individual animals were processed in parallel. Free-floating sections were rinsed in 0.1 M PBS, and endogenous peroxidases were blocked in 3% H2O2 and 10% methanol dissolved in 0.1 M PBS. After being rinsed, sections were incubated in 0.1 M PBS containing 0.2% gelatin, 10% normal serum, 0.2% glycine, and 0.5% Triton-X 100 for 1 h at room temperature. Sections were then incubated overnight at 4°C with the different primary antibodies. Tissue-bound primary antibody was detected by the ABC method, following the manufacturer’s instructions (Vector Labs, Burlingame, CA, USA), and revealed with 0.03% diaminobenzidine and 0.003% H2O2. Revealed sections were mounted onto gelatinized slides and coverslipped with Eukitt (Merck, Darmstadt, Germany). Selected sections were processed for double immunofluorescence (e.g., phospho-ERK1–2 + GFAP) using Alexa-Fluor 488- and Alexa-Fluor 568-tagged secondary antibodies (Molecular Probes, Eugene, OR, USA). Sections were mounted on Fluoromount (Vector Labs) and analyzed on an Olympus Fluoview SV 500 confocal microscope. All images were obtained in sequential scanning laser mode to avoid fluorochrome cross-excitation.
Cell culture transfection and propidium iodide labeling
EBNA-293T cells were cultured on 12 mm Ø coverslips in a 6-well plate (Nunc, Denmark) to obtain 50–60% confluence before transfection using Lipofectamine Plus (Invitrogen, Carlsbad, CA, USA). Transfection was performed with F35-, PrPc-, and Bcl-2-pcDNA3-encoding plasmids following the manufacturer’s instructions. Two days after transfection, cells were incubated with propidium iodide (PI, 1 µg/ml, Sigma-Aldrich, Andover, UK) or collected for caspase-3 assay (see below) or WB techniques. PI-treated cultures were rinsed in PBS 0.1 M, fixed with 2% buffered paraformaldehyde, counterstained with Bisbenzimide, mounted on Fluoromount, and analyzed on a Nikon Eclipse Te200 inverted microscope. For quantification, the percentage of PI-labeled cells with respect to bisbenzimide labeled cells was determined using the Quantity one Image Software Analysis (Bio-Rad, Hercules, CA, USA).
WB techniques and measurement of caspase-3 activity
Tissue samples from cerebella were homogenized (10% w/v) in RIPA buffer containing protease and phosphatase inhibitors, using a motor-driven glass-Teflon homogenizer in ice. Transfected EBNA-293T cells were collected using 150 µl of RIPA buffer per well. After protein quantification, tissue and cell extracts (20 µg) were boiled in Laemmli sample buffer at 100°C for 10 min, followed by 12% SDS-PAGE electrophoresis. They were then electrotransferred to nitrocellulose membranes and processed for immunoblotting using primary antibodies and the ECL-plus kit (Amersham-Pharmacia Biotech, Buckinghamshire, UK). In our experiments, each nitrocellulose membrane was used for detecting both phosphorylated and total kinase or protein levels. For quantification, revealed films were scanned at 1200 x 1200 dpi (HP 5530 photo scanner) and the densitometric analysis was performed using the Quantity one Image Software Analysis (Bio-Rad). The caspase-3 activity assay was performed using Ac-DEVD-AFC (Sigma-Aldrich) as substrate (see (16)
for details).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PrP micePrP mice have a life expectancy around of 90–95 days (6
, 7)
. The first signs of locomotor deficits, ascertained as reduced exploratory activities and slight staggering gait (score +), were observed around 35–40 days of age (Table 1
). In the following days, these mice displayed emerging ataxia with increased trembling gait and progressive paralysis in hind legs (scores ++ and +++, Table 1
and see Supplemental Video 2 for gait symptoms of score ++). At three months of age, the deteriorated condition of the mice precluded further maintenance and survival since they were unable to stand alone and displayed profound paralysis in hind legs (score ++++, Table 1
and Supplemental Video 3). Correlation of gait deficits with the histological analysis of the cerebellum showed that the first signs of GC pyknosis was also detected in the anterior cerebellar folia (vermis region) of affected mice around 35–40 days (not shown). Degeneration progressed with emerging disorganization of cerebellar lamination as the result of a reduction in GC cells, numerous macrophage inclusions, and relevant vacuolization. These effects extended in a rostro-caudal gradient, first affecting rostral then posterior cerebellar folia (Fig. 1
). Staining using Calbindin or GFAP antibodies showed relevant PC disorganization and death, accompanied by general gliosis (Fig. 1G-I
). At 
90 days of age, cerebellar cytoarchitecture was lost in PrP mice and the shrinkage of the cerebellar layers was striking (Fig. 1C-F
).
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Activation of stress-associated kinases (ERK1–2 and p38), p53 phosphorylation, and caspase-3 activity in PrP cerebellum
In postmitotic neurons, p53 may mediate cell death by caspase activation under conditions of oxidative stress or DNA damage (13)
. p53 Activity can be induced, among other factors, by p38 through serine phosphorylation. p38 Activity also down-regulates murine double minute-2 (MDM2) protein, which has key roles in phospho-p53 degradation (17)
. Thus, we examined the activation of ERK1–2 and p38 and levels of phospho-p53 in PrP cerebellum at several stages of neurodegeneration (Fig. 2
; see also Fig. 4H-I
). First, our results corroborated previous studies illustrating endogenous activation of ERK1–2 in Prnp0/0 mice compared to controls (18)
and decreased levels of total p53 in the former compared to the latter (18
, 19
; Fig. 2A
). Moreover, WB analysis of cerebellar protein extracts and further quantification of revealed films showed a strong increase in activated ERK1–2, p38, and phospho-p53 in PrP compared to Prnp+/+ and Prnp0/0 mice (Fig. 2A-B
). In contrast to Prnpo/o mice, ERK1–2 activation in the cerebellum of PrP animals increased through postnatal life (Fig. 2C
), which may imply a progressive oxidative insult to cerebellar cells with age. Immunohistochemical detection showed the expression of phospho-ERK1–2 in GFAP-positive reactive astroglia in white matter and especially in reactive Bergmann glia (Fig. 2F
). In parallel experiments, high levels of caspase-3 activation were observed in PrP cerebellum at 76 days of age compared to age-matched wild-type or Prnp0/0 mice (see Fig. 4J
).
Bcl-2 overexpression in F35-transfected EBNA-293T cells decreases caspase-3 activation and cell death
The expression of F35 in EBNA-293T cells displaying very low levels of endogenous PrPc expression induced cellular pyknosis, nuclei fragmentation, and cell death (Fig. 3
B). Cell degeneration was first determined by staining F35-transfected cells with PI, and the percentage of PI-labeled cells with respect to total cell number was ascertained with nuclear counterstaining with bisbenzimide (Fig. 3F-G
). Next, we explored whether the antiapoptotic protein Bcl-2 can reverse the cytotoxic effects observed after F35 overexpression. The proto-oncogene Bcl-2 protects cells from apoptotic cell death in several lesion models by inactivating Bax via heterodimerization and/or preventing the release of cytochrome C from mitochondria and further caspase activation (12)
. Bcl-2 expression did not impair F35 expression in double transfected EBNA-293T cells (Fig. 3M
). The percentage of EBNA-293T cells stained with PI (Fig. 3F-G
) was markedly reduced after cotransfection with F35 and Bcl-2-encoding plasmids (Fig. 3H-I
). The decreased cytotoxicity was further confirmed by measuring caspase-3 activation levels, which were significantly reduced (50%) but not fully abolished in the F35-Bcl-2-cotransfected cells compared to F35-transfected ones (Fig. 3L
). Furthermore, our experiments showed that PrPc-overexpression in EBNA-293T also increases the number of PI-labeled cells and caspase-3 activity compared to controls (Fig. 3J-L
). These latter data corroborate previous studies that report a reduced viability of HEK293 cells stably transfected with PrPc compared to nontransfected cells (19
, 20)
. Detailed microscopic examination of PrP-transfected (6H4-positive) displayed disrupted morphology with condensed nuclei in contrast to controls. Surprisingly, although degenerating PrPc-transfected EBNA-293T cells also displayed disrupted morphology (not shown), most transfected cells showed increased filopodia and were often observed forming cell aggregates (Fig. 3C
). The latter observation confirms previous results described by Mangé et al., who reported an increased aggregation in PrPc-transfected neuroblastoma cells and proposed a possible implication of PrPc in cell adhesion (21)
and neurite/filopodial extension as a trans-interacting protein as suggested in (22)
.
Bcl-2 overexpression delays caspase-3 activation, reactive astrogliosis, and cell death in PrP transgenic mice
Next, we examined whether Bcl-2 overexpression also protects cerebellar cells from neurodegeneration in PrP mice. We obtained transgenic mice overexpressing Bcl-2 under the NSE promoter carrying the PrP transgene in a Prnpo/o background (see Materials and Methods; Fig. 4
). None of the these mice showed any gait deficits before 50 days of age and most survived more than 90 days without apparent deficits until sacrifice (Table 1
, see below). At the time of death (from 50 to 214 days), 13 out of the 14 PrP Bcl-2 transgenic mice did not show altered gait and displayed normal behavior (Suppl. video 1) or very few gait symptoms (score +). When minor gait alterations were present, a delay of around 33–35 days in the onset of the first gait symptoms was observed between PrP Bcl-2 and PrP mice and only one mouse showed score ++ deficits with trembling gait and considerable paralysis in hind legs at 108 days (Table 1
, Supplemental Video 2). Histological study of PrP Bcl-2 transgenic mice with normal gait (7 out of 14 mice, 50%) correlated with healthy cerebellar cytoarchitecture, with all folia and cellular lamination clearly defined as in adult Prnpo/o or wild-type mice (Fig. 4)
. In contrast, affected PrP Bcl-2 mice (score +, 6 out of 14, 42%; death between 76 and 111 days) showed signs of GC loss only in anterior cerebellar folia (Supplemental Figure 1), which correlated with degenerative stages in PrP mice of 45 to 55 days old. This observation indicates that Bcl-2 overexpression delays the onset of clinical illness and increases the life expectancy of PrP mice. Histological analysis of healthy PrP Bcl-2 mice revealed normal Bergmann glia and PC distribution with dendrites expanding the molecular layer, as ascertained with GFAP and Calbindin antibodies (Fig. 4E-F
).
Next, we explored kinase activation in PrP and PrP Bcl-2 mice over time to ascertain whether Bcl-2 overexpression alters this activation (Fig. 4)
. WB analysis of cerebellar extracts revealed that phospho-ERK1–2 remained constant in PrP cerebella during neurodegeneration. In contrast, phospho-p38 levels decreased at advanced stages of cerebellar neurodegeneration in PrP mice (Fig. 4H
). This decrease may be associated with the increased neuronal damage (GC and PC) observed in older PrP mice. Densitometric analysis of revealed films demonstrated that phospho-ERK1–2 activation was similar between PrP Bcl-2 and PrP, and phospho-p38 and phospho-p53 followed a similar evolution in PrP Bcl-2, being reduced in PrP Bcl-2 mice aged 55 days but progressively increased in older PrP Bcl-2 (>90 days; Fig. 4H-I
). However, the levels of p38 and p53 activation reached in PrP Bcl-2 were lower (25–50% reduction, depending on the age) than in PrP age-matched animals. Moreover, levels of p38 and p53 activation were similar in PrP mice of 46 days and PrP Bcl-2 of >90 days, thereby indicating protracted kinase activation (Fig. 4H-I
). However, the level of caspase-3 activity was strongly reduced in PrP Bcl-2 mice (80% reduction) compared to age-matched PrP animals (Fig. 4J
). These data indicate that Bcl-2 overexpression largely delays phospho-p38 and phospho-p53 activation in PrP mice but strongly reduces caspase-3 activation. On the other hand, Tg20 transgenic PrPc-overexpressing mice (23)
showed increased caspase-3 activity compared to wild-type, Prnp0/0, Bcl-2, and Prnp0/0-Bcl-2 mice but lower than PrP. These data are similar to those observed previously in EBNA-293T cells after PrPc overexpression (Fig. 3)
. In parallel experiments, we observed that overexpression of the Bcl-2 transgene did not preclude F35 expression (not shown). Taken together, our results indicate that although F35-expressing cerebella showed relevant cellular stress (as ascertained by the phospho-ERK1–2, phospho-p38, and phospho-p53 levels), the neuronal-directed overexpression of Bcl-2 largely reduced caspase-3 activation and rescued cerebellar degeneration and gait deficits in most PrP Bcl-2 mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. However, mouse lines with extensive deletions in Prnp show severe ataxia and PC death caused by Dpl overexpression (5)
. The overall structure of Dpl is similar to the F35 protein, both lack the OR, the CC, and the HR but preserve the C-terminal domain of PrPc (9)
. The overexpression of these two PrPc variants in Prnpo/o mice induces cell death in several cerebellar cell types (GC or PC) as the result of their cell-specific expression (23
, 24)
. However, it is plausible that Dpl and F35 induce cell death by the same or similar mechanisms since the targeted expression of F35 or Dpl in PC causes similar degenerative phenotypes (25
, 26)
. Dpl-mediated neurotoxicity is produced by increasing oxidative stress (10)
. Our results demonstrate that F35 overexpression is toxic in non-neuronal cells with low PrPc levels and induces relevant activation of the stress-inducible kinases ERK1–2 and p38 in PrP cerebellum, both indicating an oxidative insult. Truncated forms of PrPc lacking the N-terminal polybasic regions and the OR interfere with PrPc endocytosis via clathrin-coated vesicles and beta-cleavage of PrPc, respectively, thereby impairing the antioxidative functions of PrPc (27
28
29)
. Moreover, the HR binds to the stress-inducible protein 1 (STI1), which plays a crucial role in preventing oxidative cell damage (30)
. Thus, the absence of the OR and HR regions in PrP may induce the loss of the natural protective functions of PrPc, thereby leading to increased intracellular oxidative damage and cell death. The fusion of the OR and the most N-terminal portion of the HR region to Dpl overcomes cell death when serum is removed (31)
. However, F35 may alternatively arrest a survival ligand, which transduces antiapoptotic signals through PrPc signaling (6
, 32
, 33)
. In this regard, a 37KD/67/KD laminin receptor or the adhesion molecule NCAM bind PrPc can trigger cell survival signals (34
, 35)
.
During recent months, several studies have focused on delimiting the region of the PrPc implicated in premature neuronal death in mice overexpressing N-truncated variants of PrPc (11
, 32
, 33)
. For example, Sunyach and coworkers (11)
demonstrated that the C-terminal product derived from the endoproteolitic processing of PrPc (C1 fragment), which lacks all the N-terminal region of the PrPc until the 110 residue, positively controls p53 transcription and activity. Moreover, this C1 portion potentiates staurosporine-induced caspase-3 activation through a p53-mechanism in HEK293 cells stably overexpressing C1. In contrast, these effects are not elicited by a C2 fragment that contains the amino acids 90–110, which are missing in C1 (11)
. In a broad sense, the C1 fragment and the product of F35 construct are similar, both lack the most N-terminal portion of the PrPc, including the CC flanked by the OR region and the HR respectively. Our present findings demonstrate that p53 and caspase-3 are activated in the degenerating cerebella of PrP mice and that F35 overexpression in EBNA-293T cells increases cell death (accessed by PI-uptake), which correlates with enhanced caspase-3 activity. Taken together, these results open up the interesting question of whether the neurotoxic mechanisms observed in PrP mice or F35-transfected cells are similar to those derived from C1 overproduction, as proposed by Sunyach and coworkers. In this regard, a recent study by Baumann et al. (33)
showed that mice overexpressing a PrPc variant lacking 94–134 amino acids (PrPCD), comprising the CC and the HR of the CD, displayed a severe and fast neurodegeneration. Surprisingly, transgenic mice lacking only 114–121 residues (PrPpHC) corresponding to the HR but preserving the CC did not elicit any pathological phenotype in Prnpo/o mice (33)
. Moreover, the neurodegenerative phenotype of the PrPCD mice was completely counteracted by coexpressing high levels of full-length PrPc and partially overcome by coexpressing a PrPc variant lacking all octarepeats (33)
. A parallel study by Li et al. (32)
illustrated that mice lacking the 105–125 amino acids of the CD [Tg(PrP105–125)] in a Prnp0/0 background showed premature death (few days after birth). Taken together, the findings of these studies suggest that the CC of the PrPc molecule is involved in both neuroprotective and neurodegenerative function. However, additional experiments including those performed with Prnp0/0 deficient mice overexpressing the C1 fragment, together with additional biochemical experiments in vitro in cellular models overexpressing PrPCD and PrPpHC and PrP105–125 constructs, are required to ascertain this hypothesis.
The cellular prion protein: a protective or proapoptotic molecule?
We observed that PrPc overexpression in EBNA-293T cells increased cell death and caspase-3 activity and caused relevant changes in cell morphology. Enhanced caspase-3 activity was also observed in Tg20 transgenic mice overexpressing PrPc. Our observations confirm previous studies by Paitel and coworkers, which demonstrated that the stable overexpression of PrPc sensitizes HEK293 cells to a proapoptotic phenotype described by Paitel et al. (19
, 36)
and by Westaway and coworkers (37)
, which reported that older mice harboring high copy numbers of wild-type PrPc showed, among other symptoms, cell degeneration in the nervous system. However, Tg20 mice are described without clinical illness (23)
, and the 4-fold expression of PrPc in transgenic mice (Tg(WT-E1) never causes the development of clinical symptoms (38)
.
Although considerable effort has been devoted to determining the function of PrPc, the full repertory of physiological functions in healthy neurons is still unknown. PrPc has been implicated in copper homeostasis (39
, 40)
, neuronal plasticity (41)
, cell proliferation (42)
, and neurite outgrowth (22
, 35)
. In addition, several studies also point to a neuroprotective role of PrPc (22
, 43)
. Indeed, PrPc is overexpressed in ischemic brain and correlates with neuroprotective effects (44
, 45)
. PrPc also protects human primary neurons in culture against bax-mediated apoptosis (46)
. Moreover, although the first descriptions of Prnp0/0 mice indicate normal behavior and few deficits (14)
, several studies in recent years have described several phenotypic alterations in Prnp mutant mice (reviewed in ref 5
).
Taken together, our results indicate that PrPc is involved directly or indirectly in numerous cellular functions and that its absence or overexpression sensitizes neurons to cell death. Thus, it is tempting to speculate that basal PrPc expression may play a crucial role in controlling neural homeostatic equilibrium by acting through several processes and that these profound changes in basal PrPc expression (e.g., complete absence or high overexpression) may predispose neurons to cell death under certain physiological situations.
Delayed caspase-3 activation by Bcl-2, a putative clue to reversing cerebellar degeneration in PrP mice
Our findings also demonstrate that the overexpression of Bcl-2 under the NSE promoter blocked or greatly delayed caspase-3 activation and rescued most (50%) PrP mice from global GC loss and cerebellar degeneration. In vitro experiments also corroborated that caspase-3 activation and cell death induced by F35 overexpression were blocked by Bcl-2 in a similar manner. Moreover, in PrP Bcl-2 mice, activated p53 and p38 were decreased; however, phosphorylated levels of ERK1–2 were similar to PrP mice. These results indicate that oxidative insults occur in PrP Bcl-2 cerebellar neurons, although GC and PC cell death is largely blocked by Bcl-2 overexpression in most of these mice.
Taken together, our findings also suggest that F35 overexpression in the absence of PrPc triggers cell death pathways that differ to those associated with Bax activation, since the overexpression of Bcl-2 in vitro and in vivo is unable to completely overcome cell death in F35-expressing cells. This hypothesis has also been recently illustrated by Li and coworkers (8)
using Bax0/0 PrP transgenic mice. In these mice, Bax deletion delays the development of clinical illness and slows apoptosis of cerebellar GC but has no effect on white matter degeneration, which also suggests that PrP-induced neurodegeneration involves Bax-independent pathways. In a previous study by Radovanovic et al. (7)
, NSE-Prnp expression rescued GC degeneration in PrP mice but these animals showed myelin defects as a result of the absence of PrPc in oligodendrocytes. Although healthy PrP Bcl-2 mice displayed normal gait and no relevant changes were observed at the stages analyzed, we cannot exclude a delayed myelin alteration in older mice. However, neither macrophage infiltration nor apparent leucoencephalopathy was detected in healthy PrP Bcl-2 animals.
Most approaches used to overcome neurodegeneration in PrP and Dpl mice have addressed Prnp expression in distinct cerebellar cell types to abrogate neuronal degeneration (e.g., 7
). Bcl-2 overexpression or Bax suppression has been used to ameliorate neurodegenerative processes in transgenic mice overexpressing PrPc with 14 octapeptide repeats (47)
or recently in PrP mice (8)
. However, our study is the first to report the use of neuronal-directed Bcl-2 overexpression to overcome cerebellar degeneration in PrP mice. However, it has been reported that Bcl-2 may interact with PrPc (aa 115–156) by its C-terminal domain and that this interaction can be proapoptotic for several isoforms of cytosolic PrP (48)
. Prnpo/o cerebella showed higher levels of Bcl-2 protein, which may putatively interact with PrP as in wild-type mice (18)
. Although this putative interaction was not addressed in the present study, a recent report indicates that PrPc lacking the OR is not endocyted and remains in lipid rafs (28)
. Thus a putative interaction of PrP and Bcl-2 in these conditions is unlikely, and in PrP Bcl-2 mice, Bcl-2 acts as an antiapoptotic protein, probably by a direct effect on Bax activation and mitochondrial homeostasis.
Bax deletion prevents the granule cell apoptosis loss but not other neurological symptoms (synaptic loss) in the cerebella of Tg(PG14) mice (47)
. In this study the neurological symptoms (synaptic loss) observed in the Bax0/0 Tg(PG14) mice are likely to be associated with PrP accumulation (47)
. Taken together, the results obtained in the present study and those reported in others (8
, 47)
indicate that the antiapoptotic therapies alone have the capacity to rescue neuronal degeneration but require other therapies such as pharmacological intervention to completely overcome neurological symptoms in PrP mice or Tg(PG14) mice. However, it would be of interest to establish whether the regulation of the antiapoptotic mechanisms exerted by Bcl-2 are useful to abrogate PC degeneration in Dpl mice.
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ACKNOWLEDGMENTS |
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Received for publication January 25, 2007. Accepted for publication April 12, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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