Loss of Prion Protein in a Transgenic Model of Amyotrophic Lateral Sclerosis

MCN

Molecular and Cellular Neuroscience 19, 216 –224 (2002) doi:10.1006/mcne.2001.1049, available online at http://www.idealibrary.com on

Loss of Prion Protein in a Transgenic Model of Amyotrophic Lateral Sclerosis Luc Dupuis, Corinne Mbebi, Jose´-Luis Gonzalez de Aguilar, Fre´de´rique Rene, Andre´ Muller, Marc de Tapia, and Jean-Philippe Loeffler 1 Laboratoire de Signalisations Mole´culaires et Neurode´ge´ne´rescence, EA 3433 Faculte´ de Me´decine, Universite´ Louis Pasteur, 11 rue Humann, 67085 Strasbourg, France

Amyotrophic lateral sclerosis (ALS) is a motor neuron degenerative disorder caused in a proportion of cases by missense mutations in the gene encoding Cu/Zn superoxide dismutase (Cu/Zn-SOD) which result in unknown, lethal enzymatic activity. Based on a differential screening approach, we show here that the gene encoding the cellular prion protein (PrP C) was specifically repressed in a transgenic model of ALS overexpressing the mutant G86R Cu/Zn-SOD. Analysis by Northern blot, semiquantitative RT–PCR, and Western blot revealed that PrP C down-regulation, which appeared early in the asymptomatic phase of the pathology, occurred preferentially in those tissues primarily affected by the disease (spinal cord, sciatic nerve, and gastrocnemius muscle). This down-regulation was not accompanied by refolding of the aberrant PrP Sc isoform, the agent which causes transmissible spongiform encephalopathies. Furthermore, modification of PrP C expression was specifically linked to the presence of the G86R mutant since no changes were observed in transgenic mice overexpressing wild-type Cu/Zn-SOD. PrP C has been shown to play a role in the protection against oxidative stress, and we therefore propose that its down-regulation may contribute at least in part to ALS pathogenesis.

INTRODUCTION Amyotrophic lateral sclerosis (ALS) is a lethal neurological disorder affecting middle-aged individuals. It appears as a weakness and muscle atrophy of the extremities, caused by the selective loss of large motor neurons in the spinal cord, brainstem, and motor cortex 1

To whom correspondence and reprint requests should be addressed. Fax: ⫹33-(0)3-90-243065. E-mail: [email protected].

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(Mulder et al., 1986). The precise mechanisms underlying motor neuron degeneration remain unclear, and multiple factors have been proposed to be involved. Many studies agree that oxidative stress plays an important role in the pathogenesis of the disease (refer to Robberecht, 2000). It was observed by Rosen et al. (1993) that a subset of patients, with autosomal dominantly inherited ALS, harbor point mutations in the gene encoding Cu/Zn superoxide dismutase (Cu/Zn-SOD), which is a free radical scavenging enzyme. Overexpression of ALS-linked Cu/Zn-SOD in transgenic mice allows the development of a neurological disorder that resembles human ALS without affecting SOD enzymatic activity. This strongly suggests that mutations confer an unknown gain of function to the enzyme (Gurney, 1994; Ripps et al., 1995; Wong et al., 1995). How this deleterious enzymatic activity provokes neuronal degeneration is a matter of controversy. Some studies have proposed that the mutant Cu/Zn-SOD is able to generate highly toxic hydroxyl radicals that can damage essential cellular constituents (Wiedau-Pazos et al., 1996). Alternatively, another hypothesis has emerged from the observation that Cu/Zn-SOD catalyzes peroxynitrite radicals to form a nitronium-like intermediate, which in turn induces nitration of tyrosine residues and subsequent cell injury (Beckman, 1996). The cellular prion protein (PrP C) is a glycophosphatidylinositol-anchored glycoprotein that binds copper and possesses SOD activity (Brown et al., 1997, 1999). However, its precise physiological role remains elusive. Studies involving PrP C knockout mice (Prnp 0/0) support a protective role in the cellular resistance to oxidative stress (Brown and Besinger, 1998) and to apoptosis (Kuwahara et al., 1999). In addition, its expression seems to influence, through modulation of copper traf1044-7431/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

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TABLE 1 Down-Regulated Genes in G86R Mice Group

Number of clones

Axonal growth Cytoskeleton Electrical properties of neurons Metabolism Gene expression machinery Miscellaneous Unknown genes and/or function

2 4 8 14 7 8 12

Examples Myelin proteins (2) Neurofilament light subunit Ligand-gated ion channels (2), signaling proteins (3), receptors (2), ion channels (1) Intermediary metabolic enzymes (3), mitochondrially encoded genes (11) Splicing factor (1), ribosomal proteins (3), transcription factor (1) molecular chaperones (2) Prion protein

ficking, the activity of other cuproenzymes, such as Cu/Zn-SOD (Brown and Besinger, 1998; Pauly and Harris, 1998). At present, considerable effort has been made to understand the role of the aberrant proteaseresistant isoform of prion protein in animal and human transmissible spongiform encephalopathies (PrP Sc) (Prusiner, 1982, 1991). The established view is that the fatal accumulation of this misfolded isoform in the central nervous system results from its ability to induce the refolding of the native PrP C into the abnormal configuration (Nguyen et al., 1995). Nevertheless, growing evidence supports the view that, in addition to the intrinsic toxic properties of PrP Sc, a loss of the normal function of PrP C may also cause a problem for the injured cells (Brown, 2001). At this point, one can ask whether the likely loss of normal PrP C function in prion diseases and the aberrant Cu/Zn-SOD activity in ALS share common neurotoxic mechanisms involving oxidative stress. In the present study, we used a transgenic model with the missense mutation G86R in the Cu/Zn SOD gene that corresponds to the same mutation at position 85 in some ALS patients (Ripps et al., 1995). In this model, mutant Cu/Zn-SOD overexpression is associated with an age-related decline of motor function accompanied by motor neuron loss, axonal degeneration, and muscle atrophy (Dupuis et al., 2000). In order to systematically identify differentially expressed genes relevant to the initiation stage of ALS pathology, we have recently developed a differential screening approach based on a subtractive suppressive hybridization (SSH) technique (see Dupuis et al., 2000, for more details). Using this approach, we have isolated cDNAs of genes that are either overexpressed or repressed in the lumbar spinal cord of G86R mice prior to disease onset. We show here additional data concerning one of the isolated cDNAs that corresponds to the prion protein gene. Our findings reveal that PrP C is specifically down-regulated in G86R mice.

RESULTS SSH and high-throughput screening reveal a specific repression of the gene Prnp in G86R mice. To address the question whether specific genes are down-regulated during the asymptomatic phase in transgenic mice with ALS-like pathology, a library enriched in underexpressed cDNAs from 75-day-old mouse lumbar spinal cords was constructed and subsequently screened. Fifty-five clones, showing at least a twofold repression, were further analyzed by sequencing. The corresponding genes were classified into seven major groups according to their related functions, as listed in Table 1. Among these genes, the decreased expression of the neurofilament light subunit agreed with that reported by Bergeron et al. (1994) in ALS patients. In contrast, the implication of the other identified genes is at present unknown and, in some cases, unexpected. The R.37 clone, showing a differential expression ratio of 2.5 as determined by reverse Northern blot analysis (Fig. 1A), revealed 100% identity within the 3⬘ end of the coding determining sequence and part of the 3⬘ UTR of mouse PrP mRNA (Fig. 1B). Therefore, the present data show the early repression of Prnp in animals harboring an ALS-associated Cu/Zn-SOD mutation. PrP mRNA levels are persistently down-regulated in G86R mice. Our initial observation was confirmed by Northern blot analysis using R.37 clone cDNA as a probe. PrP mRNA levels were significantly reduced in the lumbar spinal cord of asymptomatic G86R mice when compared to control littermates (Fig. 2A, left). This reduction was also noticeable in animals 105 days old without any apparent pathological sign (Fig. 2A right). The degree of repression was identical in both 75- and 105-day-old mice, which strongly suggest that Prnp down-regulation is constant and persists during the asymptomatic phase of the disease. In addition, such repression seems specific to the tissue primarily

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FIG. 1. Differential screening in G86R mice. (A) Screening of a subtracted cDNA library by reverse Northern blot identified the R.37 clone as a confirmed positive (circle). Chosen clones of the subtractive cDNA library were PCR-amplified. PCR products were dot blotted on duplicated nylon membranes and further hybridized with randomprimed cDNA probes synthesized with either wild-type (Wt) or littermate G86R mouse total RNA (extracted from seven pooled animals each). Selection of confirmed positive clones was based on the lower signal obtained with the G86R probe compared to that obtained with the Wt probe. The corresponding G3PDH cDNA dots, used as internal controls, are shown in the top left insets. (B) Computational analysis of the R.37 clone revealed perfect homology with the PrP cDNA sequence. A search in databases indicated that the R.37 clone matched the 3⬘ end of the coding determining sequence (CDS) and part of the 3⬘ UTR of mouse PrP cDNA, as indicated in the figure. Positions of the primers designated in the PrP CDS used for RT–PCR experiments are indicated by arrows.

affected by the pathology since no differences were observed in brain PrP mRNA levels at the same ages (Fig. 2B). To obtain independent evidence of these findings, semiquantitative RT–PCR analyses were performed using primers in the coding determining sequence of Prnp outside the region overlapped by the R.37 clone (see small arrows in Fig. 1B). As illustrated in Fig. 3, a marked decrease in PrP mRNA levels was observed in the lumbar spinal cord of G86R mice, thereby reinforcing the specific down-regulation of Prnp in asymptomatic ALS-affected animals. PrP C levels are reduced in the motor unit of G86R mice. To better determine the expression pattern of Prnp in the tissues that constitute the motor unit, and are primarily affected by the progression of the ALSlike pathology in G86R mice (Dupuis et al., 2000), we carried out Western blot analyses using two different PrP C antibodies. The amount of PrP C was reduced in the spinal cord, sciatic nerve, and gastrocnemius muscle of 75 (Fig. 4A)- and 105-day-old animals (Fig. 4B). Conversely, brain PrP C levels remained unchanged, which clearly supports the specific down-regulation of Prnp in the motor unit at both the transcriptional and the translational levels. In addition, the absence of modification in the brain excludes the possibility that PrP C downregulation could result from alteration of gene expres-

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sion at the integration site of the mutant Cu/Zn-SOD transgene. The screening results also provided evidence for the down-regulation of two chaperones in asymptomatic G86R mice. Indeed, R.45 and R.46 clones overlapped the cDNAs of the chaperones Hsj2 and CCT2, respectively (Table 2). Hsj2 is one of the murine homologues of the bacterial dnaJ/Hsp40 which regulates the chaperoning activity of members of the dnaK/Hsp70 family (Cummings et al., 1998). CCT2 forms part of the eukaryotic cytosolic complex chaperone-containing TCP1 (CCT) (Llorca et al., 1999). These findings strongly suggest the implication of chaperones in mutant Cu/ZnSOD-induced motor neuron degeneration, likely due to inadequate protein folding mechanisms. In this respect, we explored whether the concomitant appearance of the misfolded PrP Sc isoform could accompany the altered PrP C expression in G86R mice. Because PrP Sc is partially resistant to enzymatic degradation (Baldwin et al., 1995), protein extracts were submitted to proteinase K digestion prior to Western blot analysis. As shown in Fig. 4C, loss of PrP immunoreactivity was digestion time-dependent but no differences were observed between wild-type and G86R mice (see graph in Fig. 4C). Therefore, these findings clearly exclude the implication of the aberrant PrP Sc in this ALS transgenic model. PrP mRNA and protein levels are not modified in transgenic mice overexpressing wild-type Cu/Zn-SOD. To determine the specificity of modified PrP C expression, semiquantitative RT–PCR and Western blot analyses were performed in transgenic C57B1/6xDBA/2 mice overexpressing the human wild-type Cu/Zn-SOD gene (Ceballos-Picot et al., 1991). As illustrated in Fig. 5, PrP mRNA and protein levels in individual lumbar spinal cords showed no differences between wild-type Cu/Zn-SOD mice and control littermates. In contrast, a decrease in such levels was observed in G86R Cu/ZnSOD mice, which strongly suggests that the early alteration of PrP C expression is specifically linked to the presence of the ALS-associated mutant enzyme.

DISCUSSION One of the most important challenges of ALS pathology is the elucidation of the molecular mechanisms that provoke the progressive and selective degeneration of motor neurons. In an attempt to address this question, we concentrated our studies on the asymptomatic phase of the disease. To this aim, the use of transgenic mice overexpressing ALS-linked Cu/Zn-SOD muta-

Prion Protein Loss in an ALS Model

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FIG. 2. Northern blot analysis of PrP mRNA down-regulation in G86R mice. Northern blots were performed with total RNA extracted from lumbar spinal cord (A) or brain (B) of 75- and 105-day-old G86R mice or wild-type (Wt) littermates. To limit interindividual variations, lumbar spinal cords from four animals were pooled prior to RNA extraction. Lower panels show inverted images of methylene blue-stained membranes, attesting to equal amounts of loaded RNA. Quantification of PrP mRNA levels was accomplished by measuring band intensities with PhosphorImager software. Data were normalized relative to the corresponding methylene blue-stained 28S band and are expressed as percentages of Wt values. They are means ⫾ SE (n ⫽ 3). Student’s t test, *P ⬍ 0.05, ns, nonsignificant differences.

tions represents a very valuable potential since these animals develop ALS-like neurological disorders with established patterns of progression. In the present study we show evidence for an early altered expression of the prion protein gene in one of these transgenic lines, the G86R mice. Based on a differential screening approach, a cDNA that matched part of the sequence of the gene encoding PrP C was found to be specifically repressed in the lumbar spinal cord of G86R mice. This repression was significant 1 month before the appearance of histological and/or pathological signs characteristic of the disease

(Dupuis et al., 2000). In addition, it persisted in animals very close to disease onset, thus suggesting a constant down-regulation during the asymptomatic phase. Analysis of PrP mRNA levels by either Northern blot or RT–PCR corroborated these findings. Moreover, when the amount of PrP C present in the motor unit was evaluated by Western blot, a clear reduction in protein levels was observed at the two ages tested. Interestingly, such modifications in Prnp expression appeared to be particularly restricted, at least during the asymptomatic phase, to the tissues that would be primarily affected later at the onset of the disease (i.e., lumbar

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FIG. 3. RT–PCR analysis of PrP mRNA down-regulation in G86R mice. Semiquantitative RT–PCR was performed with total RNA extracted from lumbar spinal cord of 75 (A)- or 105-day-old (B) G86R mice and wild-type (Wt) littermates, using PrP (left panels)- or G3PDH (right panels)-specific primer pairs. To ensure a quantifiable relationship between cDNA input and PCR product output, various numbers of PCR cycles were assayed for both PrP and the internal control G3PDH. In each case, a control experiment in which reverse transcriptase was omitted was performed (indicated by ⫺RT). To limit interindividual variations, lumbar spinal cords from four animals were pooled prior to RNA extraction.

spinal cord, sciatic nerve, and gastrocnemius muscle). Even though degeneration of upper motor neurons also occurs in ALS (Hirano, 1991), the fact that global levels of PrP mRNA and PrP C in the G86R mouse brain were similar to those found in wild-type littermates ensures a normal rate of Prnp expression in neuronal structures not affected by the disease. It should also be stressed that the altered expression of Prnp was specifically linked to the presence of the mutant enzyme since no modifications were observed when comparing transgenic mice overexpressing wild-type Cu/Zn-SOD with their control littermates. A recent study has reported that the chaperoning activity involved in proper protein folding was decreased in mice overexpressing the ALS-linked Cu/ZnSOD mutation G93A (Bruening et al., 1999). Screening of our repressed G86R cDNA library revealed the down-regulation of two chaperones, confirming the im-

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plication of these important molecular effectors in ALS pathology. Because PrP Sc, the pathogenic agent in transmissible spongiform encephalopathies, is a misfolded isoform of the native PrP C (Prusiner, 1998), we asked whether PrP Sc was present in our ALS transgenic model. Both isoforms possess the same primary sequence and have no detectable posttranslational differences. However, PrP Sc is rich in ␤-sheet structures, insoluble in nondenaturing detergents, and resistant to protease digestion (Baldwin et al., 1995). In our hands, treatment of lumbar spinal cord extracts with proteinase K and subsequent Western blot analysis revealed no differences in the time-dependent sensitivity of PrP C to protease digestion between G86R and wild-type mice. Therefore, we can exclude the presence of the aberrant PrP Sc in this animal model. The findings presented here address the question of how PrP C down-regulation and mutant Cu/Zn-SOD overexpression are interconnected. Studies dealing with either Prnp 0/0 mice or mice overexpressing PrP C have reported that levels of Cu/Zn-SOD activity correlate positively with Prnp expression (Brown and Besinger, 1998). In addition, several lines of evidence suggest that PrP C, through its ability to bind copper (Brown et al., 1997), controls the transfer of the metal ion to appropriate protein targets and particularly Cu/Zn-SOD (Brown and Besinger, 1998). These observations, together with the fact that Prnp 0/0 mice are characterized by decreased copper content (Brown et al., 1997), suggest that the PrP C down-regulation found in G86R mice might be interpreted as an attempt to reduce copper availability and prevent its utilization by the mutant Cu/Zn-SOD. Indeed, it has been postulated that the catalytic copper of mutant Cu/Zn-SOD would be responsible for the generation of highly toxic free radicals that would activate the cascade of oxidative reactions leading to motor neuron degeneration (Beckman et al., 1994; Yim et al., 1996). Furthermore, we found that altered Prnp expression occurred long before animals presented any pathological signs, which suggests the early activation of a compensatory mechanism in which PrP C would be repressed to avoid, at least for a given period, copper-linked toxicity. Alternatively, PrP C down-regulation could also activate, or at least reinforce, other specific pathogenic events in ALS. Since PrP C possesses by itself SOD activity and protects cells against oxidative injury (Brown et al., 1999; Wong et al., 2000), we can expect that its reduction might exacerbate the oxidative stress initiated by the ubiquitous and constant overexpression of mutant Cu/Zn-SOD. This notion would be of special relevance if we take into account that PrP C is preferentially located in synapses

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FIG. 4. Western blot analysis of PrP C content in G86R mice. Western blots were performed with protein extracts from different tissues of 75 (A)- or 105-day-old (B) G86R mice and wild-type (Wt) littermates, using two different antibodies to PrP (Pri917 and Pri8G814). The figure shows representative blots of pooled tissues from four animals immunolabeled with the Pri917 antibody. Identical results were observed using the Pri8G814 antibody (data not shown). (C) Western blot analysis of limited proteinase K digestion performed on lumbar spinal cord extracts. Note that two different exposure times were used to avoid weak PrP immunoreactivity in G86R mice. Quantitative analysis of the digestion profiles is shown on the graph.

(Herms et al., 1999; Sales et al., 1998) and in the subsynaptic sarcoplasm of the neuromuscular junction (Gohel et al., 1999). In this context, the diminished PrP C content in the gastrocnemius muscle of G86R mice would con-

tribute to degeneration of the motor axis through specific, local oxidative damage. The findings presented here open interesting avenues for further research. Although most studies have fo-

TABLE 2 Chaperones Underexpressed in G86R Mice Isolated during the Screening Clone number

Homologous chaperone

Associated chaperones

Accession number

Fold repression

R.45 (216 bp) R.46 (190 bp)

Hsj2 CCT2

dnaJ/Hsp40, regulators of Hsp70/dnaK Cytosolic eukaryotic “chaperonin containing TCP-1” (CCT)

NM008298 NM007636

4 2

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FIG. 5. PrP mRNA and protein levels in transgenic mice overexpressing G86R (G86R-SOD) or wild-type Cu/Zn-SOD (Wt-SOD). (A) Semiquantitative RT–PCR was performed with total RNA extracted from lumbar spinal cord of 105-day-old transgenic mice (Tg) and the corresponding non-transgenic littermates (Ct). Specific primer pairs for PrP and the internal control G3PDH were used. To ensure the linearity of the amplification, different dilutions of cDNA template were tested. In each case, the experiment was performed in duplicate and a control experiment in which reverse transcriptase was omitted is shown (indicated by ⫺RT); Ld, 100-bp molecular weight marker. (B) Western blot analysis of PrP C content in lumbar spinal cord of animals as above. A representative blot of individual samples is shown. Quantification of PrP C protein levels was accomplished by measuring band intensities with MultiAnalyst software (Bio-Rad). Data are expressed as a percentage of Ct values. They are means ⫾ SE (n ⫽ 3). One-way ANOVA followed by Neuman–Keuls multiple comparison test; *P ⬍ 0.05, ns, nonsignificant differences.

cused on the adverse effects of the aberrant PrP Sc, it is probable that the consequences of the loss of normal PrP C have been underestimated (Brown, 2001). However, it should be stressed that the observed PrP C downregulation cannot completely account for the pathological features of ALS, since Prnp 0/0 mice do not develop the characteristic ALS-like pathology that is seen in G86R mice (Bueler et al., 1993). Obviously, further functional studies are needed to assess the precise contribution of PrP C down-regulation in the molecular mechanisms leading to the selective motor neuron degeneration observed in human ALS.

EXPERIMENTAL METHODS Animals and tissue preparation. Transgenic mice with the missense mutation Gly86 3 Arg (G86R) in the Cu/Zn-SOD gene were used (Ripps et al., 1995). Propagation of the strain, animal care, and genotyping were performed as previously described (Lutz-Bucher et al.,

1999). As determined in our previous work (Dupuis et al., 2000), asymptomatic mice 75 and 105 days old without visible hind limb paralysis were selected for the present study. Samples of lumbar spinal cord, sciatic nerve, gastrocnemius muscle, and brain were dissected from male mice, immediately frozen in liquid nitrogen, and stored at ⫺80°C until use. Adult C57B1/6xDBA/2 mice heterozygous for the human Cu/Zn-SOD gene were used as transgenic control mice overexpressing wild-type Cu/Zn-SOD (Ceballos-Picot et al., 1991). All animal experiments were performed under the supervision of authorized investigators. RNA extraction. To perform SSH, Northern blot, and RT–PCR, total RNA was isolated from tissues by acid guanidinium thiocyanate–phenol– chloroform extraction (Chomczynski and Sacchi, 1987). RNA recovery was determined by UV spectrophotometry at 260 and 280 nm and denaturing agarose gel electrophoresis. cDNA subtraction library construction and highthroughput screening. SSH and subsequent screening were carried out as previously described in detail else-

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where (Dupuis et al., 2000). All PCR and screening procedures were performed using a 96-well Hybaid PCR-express Thermocycler and a vacuum 96-well dot blot apparatus. Selected clones were sequenced on both strands using an automated sequencer (DNA Sequencer, Applied Biosystems). Vector sequences were removed using the “BLAST 2 sequences” algorithm (National Institutes of Health). The acquired sequence data were then aligned against the GenBank nucleotide database at the National Center for Biotechnology Information (National Institutes of Health) using the Blast program to search for sequence matches. Northern blot. Following denaturation, 10 ␮g of total RNA was loaded on a 1.2% denaturing agarose gel and electrophoresed on a SubCell GT apparatus (BioRad) at 100 V in 1⫻ Mops buffer for 4 h. RNAs were then transferred overnight on a ZetaProbe GT membrane (Bio-Rad) using capillary transfer (Sambrook et al., 1989) and further fixed by alkali treatment. Equal loading was ensured by staining membranes with methylene blue. Membranes were then prehybridized in 50% formamide, 6⫻ SSC, 10⫻ Denhardt’s solution, 0.1% SDS, and 500 ␮g/mL of salmon sperm DNA (Sigma). Hybridization was performed in 10 mL of fresh prehybridization mixture containing the cDNA probe previously labeled with [␣- 32P]dCTP (NEN Research) by the random priming method (Nonaprimer kit, Appligene). Membranes were finally washed in 0.1⫻ SSC plus 0.1% SDS and autoradiographed using a PhosphorImager (Fuji). RT–PCR. Two micrograms of total RNA was reverse transcribed using 200 U of MoMuLV reverse transcriptase and 0.5 ␮g of random primers (Promega) under standard reaction conditions (Sambrook et al., 1989). After PCR amplification, products were separated on a 2% agarose gel and visualized by ethidium bromide staining. Quantification was performed using a GelDoc 2000 apparatus (Bio-Rad). To determine quantifiable PCR conditions, several dilutions of the initial cDNA were tested for each primer pair using various numbers of cycles. RT–PCR analysis of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA levels was used as internal control. PCR primers were as follows: PrP 5⬘ primer, 5⬘-GCCAGTGGATCAGTACAGCA-3⬘; PrP 3⬘ primer, 5⬘-GAGAATGCGAAGGAACAAGC-3⬘; G3PDH 5⬘ primer, 5⬘-ACCACAGTCCATGCCATCAC-3⬘; and G3PDH 3⬘ primer, 5⬘-TCCACCACCCTGTTGCTGTA-3⬘. Western blot. Tissues were homogenized in PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.5% aprotinine, and 5 mM DTT. After boiling for 5 min, extracts were sonicated for 30 s, and 1 mM PMSF was added. After centrifugation, su-

pernatants were collected, and equal amounts of protein, according to the Bradford protein assay (Bio-Rad), were electrophoresed through a 13% SDS–polyacrylamide gel. Separated proteins were then electrotransferred to nitrocellulose membranes (Bio-Rad) and immunostained with two different specific monoclonal antibodies to PrP, Pri917, and Pri8G814 (Demart et al., 1999; a kind gift from Dr. J. Grassi), used at a final dilution of 1/5000. Then, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Pierce) diluted 1/2500 and developed by enhanced chemiluminescence detection. Limited proteinase K digestion. Twenty micrograms of total protein extract was digested with 30 ␮g/mL of proteinase K (Sigma). After digestion, extracts were boiled for 5 min and analyzed by Western blot as described above. Statistics. For statistical purposes, Northern blot, RT–PCR, and Western blot analyses were performed three times, and, unless otherwise indicated, four mice were pooled in each experiment. Representative images and blots are shown. Data were expressed as means ⫾ SE. Statistical significance was determined either by Student’s t test or by one-way ANOVA followed by Neuman–Keuls multiple comparison test. Differences were considered significant at P ⬍ 0.05.

ACKNOWLEDGMENTS This work was supported by grants from the French Ministry of Research (G.I.S. Infection Prion) and the American Association for Amyotrophic Lateral Sclerosis (ALSA). L.D. is supported by a grant from the Ministe`re de la Recherche. Our thanks to Mr. M. Rene´ and to Drs. A. Nicole and I. Ce´ ballos-Picot (Hoˆ pital Necker, Paris, France) for providing C57B1/6xDBA/2 mice and Dr. J. Grassi (CEA/Saclay, Gif-sur-Yvette, France) for PrP antibodies. Thanks to Mr. N. Wilson for additional review of the manuscript. We are grateful to Mrs. C. Nelson for skillful technical assistance.

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