Cryptic epitopes in N-terminally truncated prion protein are exposed in the full-length molecule: dependence of conformation on pH

Prion diseases are diseases of protein conformation. Structure-dependent antibodies have been sought to probe conformations of the prion protein (PrP) resulting from environmental changes, such as differences in pH. Despite the absence of such antibodies for full-length PrP, a recombinant Fab (D13) and a Fab derived from mAb 3F4 showed pH-dependent reactivity toward epitopes within the N-terminus of N-terminally truncated PrP(90-231). Refolding and maintaining this protein at pH > or =5.2 before immobilization on an ELISA plate inhibited reactivity relative to protein exposed to pH < or =4.7. The reactivity was not affected by pH changes after immobilization, showing retention of conformation after binding to the plate surface, although guanidine hydrochloride at 1.5-2 M was able to expose the cryptic epitopes after immobilization at pH > or =5.2. The alpha-helical CD spectrum of PrP(90-231) refolded at pH 5.5 was reduced somewhat by these pH changes, with a minor shift toward beta-sheet at pH 4 and then toward coil at pH 2. No covalent changes were caused by the pH differences. This pH dependence suggests titration of an acidic region that might inhibit the N-terminal epitopes. A similar pH dependence for a monoclonal antibody reactive to the central region identified an acidic region incorporating Glu152 as a significant participant.     

Impairment of superoxide dismutase activation by N-terminally truncated prion protein (PrP) in PrP-deficient neuronal cell line

Previous studies have reported a neuroprotective role for cellular prion protein (PrP(C)) against apoptosis induced by serum deprivation in an immortalized prion protein gene (Prnp)-deficient neuronal cell line, but the mechanisms remain unclear. In this study, to investigate the mechanisms by which PrP(C) prevents apoptosis, the authors compared apoptosis of Prnp(-/-) cells with that of Prnp(-/-) cells expressing the wild-type PrP(C) or PrP(C) lacking N-terminal octapeptide repeat region under serum-free conditions. Re-introduction of Prnp rescued cells from apoptosis, upregulated superoxide dismutase (SOD) activity, enhanced superoxide anion elimination, and inhibited caspase-3/9 activation. On the other hand, N-terminally truncated PrP(C) enhanced apoptosis accompanied by potentiation of superoxide production and caspase-3/9 activation due to inhibition of SOD. These results suggest that PrP(C) protects Prnp(-/-) cells from apoptosis via superoxide- and caspase-3/9-dependent pathways by upregulating SOD activity. Furthermore, the octapeptide repeat region of PrP(C) plays an essential role in regulating apoptosis and SOD activity.     

Biochemical fingerprints of prion diseases: scrapie prion protein in human prion diseases that share prion genotype and type

The phenotype of human prion diseases is influenced by the prion protein (PrP) genotype as determined by the methionine (M)/valine (V) polymorphism at codon 129, the scrapie PrP (PrPSc) type and the etiology. To gain further insight into the mechanisms of phenotype determination, we compared two-dimensional immunoblot profiles of detergent insoluble and proteinase K-resistant PrP species in a type of sporadic Creutzfeldt-Jakob disease (sCJDMM2), variant CJD (vCJD) and sporadic fatal insomnia (sFI). Full-length and truncated PrP forms present in the insoluble fractions were also separately analyzed. These three diseases were selected because they have the same M/M PrP genotype at codon 129 and the same type 2 PrPSc, but different etiologies, also sCJDMM2 and sFI are sporadic, whereas vCJD is acquired by infection. We observed minor differences in the PrP detergent-insoluble fractions between sCJDMM2 and vCJD, although both differ in the corresponding fractions from sFI. We detected more substantial heterogeneity between sCJDMM2 and vCJD in the two-dimensional blots of the proteinase K-resistant PrP fraction suggesting that different PrP species are selected for conversion to proteinase K-resistant PrP in sCJDMM2 and vCJD. These differences are mostly, but not exclusively, due to variations in the type of the N-linked glycans. We also show that the over-representation of the highly glycosylated forms distinctive of the proteinase K-resistant PrPSc of vCJD in one-dimensional blots is due to differences in both the amount and the natures of the glycans. Overall, these findings underline the complexity of phenotypic determination in human prion diseases.     

Mutational analysis of topological determinants in prion protein (PrP) and measurement of transmembrane and cytosolic PrP during prion infection

The prion protein (PrP) can adopt multiple membrane topologies, including a fully translocated form (SecPrP), two transmembrane forms (NtmPrP and CtmPrP), and a cytosolic form. It is important to understand the factors that influence production of these species, because two of them, CtmPrP and cytosolic PrP, have been proposed to be key neurotoxic intermediates in certain prion diseases. In this paper, we perform a mutational analysis of PrP synthesized using an in vitro translation system in order to further define sequence elements that influence the formation of CtmPrP. We find that substitution of charged residues in the hydrophobic core of the signal peptide increases synthesis of CtmPrP and also reduces the efficiency of translocation into microsomes. Combining these mutations with substitutions in the transmembrane domain causes the protein to be synthesized exclusively with the CtmPrP topology. Reducing the spacing between the signal peptide and the transmembrane domain also increases CtmPrP. In contrast, topology is not altered by mutations that prevent signal peptide cleavage or by deletion of the C-terminal signal for glycosylphosphatidylinositol anchor addition. Removal of the signal peptide completely blocks translocation. Taken together, our results are consistent with a model in which the signal peptide and transmembrane domain function in distinct ways as determinants of PrP topology. We also present characterization of an antibody that selectively recognizes CtmPrP and cytosolic PrP by virtue of their uncleaved signal peptides. By using this antibody, as well as the distinctive gel mobility of CtmPrP and cytosolic PrP, we show that the amounts of these two forms in cultured cells and rodent brain are not altered by infection with scrapie prions. We conclude that CtmPrP and cytosolic PrP are unlikely to be obligate neurotoxic intermediates in familial or infectiously acquired prion diseases.     

Comparison of the expression of Bacillus thuringiensis full-length and N-terminally truncated vip3A gene in Escherichia coli

AIMS: Studies were performed to demonstrate the function of the putative signal peptide of Vip3A proteins in Escherichia coli. METHODS AND RESULTS: The full-length vip3A-S184 gene was isolated from a soil-isolated Bacillus thuringiensis, and the vip3AdeltaN was constructed by deleting 81 nucleotides at the 5'-terminus of vip3A-S184. Both were transformed and expressed in E. coli. About 19.2% of Vip3A-S184 proteins secreted soluble proteins and others formed inclusion bodies in the periplasmic space. In contrast, the Vip3AdeltaN was insoluble and formed inclusion bodies in the cytoplasm. Bioassay indicated that Vip3A-S184 showed different toxicity against Spodoptera exigua, Helicoverpa armigera and S. litura, but Vip3AdeltaN showed no toxicity to either of them because of the deletion of the first 27 amino acids at the N-terminus. CONCLUSIONS: The results suggest that the deleted N-terminal sequences were essential for the secretion of Vip3A-S184 protein in E. coli and might be required for toxicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The function of the putative signal peptide of Vip3A protein in E. coli was investigated. These would be helpful to make clear the unknown secretion pathway of Vip3A protein in B. thuringiensis and determine the receptor-binding domain or toxic fragment of Vip3A-S184 protein.     

Expression of Bacillus thuringiensis full-length and N-terminally truncated vip184 gene in an acrystalliferous strain of subspecies kurstaki

Vip3A is an 89-kDa protein secreted by Bacillus thuringiensis during vegetative growth. The 3.5 kb full-length vip184 gene was cloned from a wild-type isolate of B. thuringiensis, and the vip184S gene was constructed by deletion of the putative signal peptide encoding sequence. Both genes were expressed in the acrystalliferous strain Cry^–B of B. thuringiensis. Vip184 protein was observed mainly in the centrifuged pellets of B. thuringiensis Cry^–B(pHPT3), which contains the vip184 gene, and was less abundant in the concentrated supernatant. However, Vip184S proteins were not detected in the concentrated supernatant, but only in the pellets of Cry^–B(pHPT3S), which contains vip184S gene. This indicated that Vip184S proteins were not secreted into the culture medium and that the putative signal peptides were essential for the secretion of Vip184. The toxicity of Cry^–B(pHPT3) and Cry^–B(pHPT3S) were demonstrated against the neonate larvae of Spodoptera exigua and S. litura. Pellets and concentrated supernatant of Cry^–B(pHPT3) showed high activity against S. exigua and S. litura, but the Cry^–B(pHPT3S) strain was not toxic to either because of the deletion of N-terminal putative signal peptides. Therefore, this may suggest that the putative signal peptides are required for lethality.     

Effects of a naturally occurring amino acid substitution in bovine PrP: a model for inherited prion disease in a natural host species

Objective

The most common hereditary prion disease is human Creutzfeldt-Jakob disease (CJD), associated with a mutation in the prion gene resulting in a glutamic acid to lysine substitution at position 200 (E200K) in the prion protein. Models of E200K CJD in transgenic mice have proven interesting but have limitations including inconsistencies in disease presentation, requirement for mixed species chimeric protein constructs, and the relatively short life span and time to disease onset in rodents. These factors limit research on the mechanism by which the mutation drives disease development. Therefore, our objective was to provide the first assessment of cattle carrying the homologous mutation, E211K, as a system for investigating longer-term disease mechanisms. The E211K substitution was associated with a case of bovine spongiform encephalopathy from 2006.

Results

We assessed the molecular properties of bovine E211K prion protein, characterized the molecular genetics of a population of cattle E211K carriers (offspring of the original EK₂₁₁ cow) in relation to findings in humans, and generated preliminary evidence that the impacts of copper-induced oxidative stress may be different in cattle as compared to observations in transgenic mouse models. The cattle E211K system provides the opportunity for future analysis of physiological changes over time.

     

The cellular prion protein (PrP(C)): its physiological function and role in disease

Prion diseases are caused by conversion of a normal cell-surface glycoprotein (PrP(C)) into a conformationally altered isoform (PrP(Sc)) that is infectious in the absence of nucleic acid. Although a great deal has been learned about PrP(Sc) and its role in prion propagation, much less is known about the physiological function of PrP(C). In this review, we will summarize some of the major proposed functions for PrP(C), including protection against apoptotic and oxidative stress, cellular uptake or binding of copper ions, transmembrane signaling, formation and maintenance of synapses, and adhesion to the extracellular matrix. We will also outline how loss or subversion of the cytoprotective or neuronal survival activities of PrP(C) might contribute to the pathogenesis of prion diseases, and how similar mechanisms are probably operative in other neurodegenerative disorders.     

Reversal of misfolding: prion disease behavioral and physiological impairments recover following postnatal neuronal deletion of the PrP gene

The prionoses are fatal neurodegenerative diseases caused by a pathogenic protein, PrP scrapie, that derives from misfolding of a normal form, PrP(c). These diseases progress through stages. A new study by Mallucci et al. in this issue of Neuron shows that prion disease may be reversed in mice by selective removal of the gene in neurons after early physiological, cognitive, and pathological features have developed.     

Different isoforms of the non-integrin laminin receptor are present in mouse brain and bind PrP

The prion protein (PrP) plays a central role in prion diseases, and identifying its cellular receptor appears to be of crucial interest. We previously showed in the yeast two-hybrid system that PrP interacts with the 37 kDa precursor (LRP) of the high affinity 67 kDa laminin receptor (LR), which acts as the cellular receptor of PrP in cellular models. However, among the various isoforms of the receptor that have been identified so far, those which are present in the central nervous system and which bind PrP are still unknown. In this study, we have purified mouse brain fractions enriched in the laminin receptor and have performed overlay assays in order to identify those isoforms that interact with the prion protein. We demonstrate (i) the presence, in mouse brain, of several isoforms of the LRP/LR corresponding to different maturation states of the receptor (44, 60, 67 and 220 kDa) and (ii) the binding of all of these isoforms to PrP. Our data strongly support a physiological role of the laminin receptor/PrP interaction in the brain and highlight its relevance for transmissible spongiform encephalopathies.     

Expression of full-length and truncated trkB in human striatum and substantia nigra neurons: implications for Parkinson’s disease

Brain derived neurotrophic factor (BDNF) is a potent mediator of cell survival and differentiation and can reverse neuronal injury associated with Parkinson’s disease (PD). Tropomyosin receptor kinase B (trkB) is the high affinity receptor for BDNF. There are two major trkB isoforms, the full-length receptor (trkB.tk⁺) and the truncated receptor (trkB.t1), that mediate the diverse, region specific functions of BDNF. Both trkB isoforms are widely distributed throughout the brain, but the isoform specific distribution of trkB.t1 and trkB.tk⁺ to human neurons is not well characterized. Therefore, we report the regional and neuronal distribution of trkB.tk⁺ and trkB.t1 in the striatum and substantia nigra pars compacta (SNpc) of human autopsy tissues from control and PD cases. In both PD and control tissues, we found abundant, punctate distribution of trkB.tk⁺ and trkB.t1 proteins in striatum and SNpc neurons. In PD, trkB.tk⁺ is decreased in striatal neurites, increased in striatal somata, decreased in SNpc somata and dendrites, and increased in SNpc axons. TrkB.t1 is increased in striatal somata, decreased in striatal axons, and increased in SNpc distal dendrites. We believe changes in trkB isoform distribution and expression levels may be markers of pathology and affect the neuronal response to BDNF.     

High prion and PrPSc levels but delayed onset of disease in scrapie-inoculated mice heterozygous for a disrupted PrP gene.

BACKGROUND: It has been proposed that the prion, the infectious agent of transmissible spongiform encephalopathies, is PrPSc, a post-translationally modified form of the normal host protein PrPC. We showed previously that mice devoid of PrPC (Prn-p0/0) are completely resistant to scrapie. We now report on the unexpected response of heterozygous (Prn-p0/+) mice to scrapie infection. MATERIALS AND METHODS: Prn-p0/+, Prn-p0/0 and Prn-p+/+ mice were obtained from crosses of Prn-p0/+ mice. Mice were inoculated intracerebrally with mouse-adapted scrapie agent and the clinical progression of the disease recorded. Mice were sacrificed at intervals, PrPSc was determined as protease-resistant PrP and the prion titer by the incubation time assay. RESULTS: Prn-p0/+ mice, which have about half the normal level of PrPC in their brains, show enhanced resistance to scrapie, as manifested by a significant delay in onset and progression of clinical disease. However, while in wild type animals an increase in prion titer and PrPSc levels is followed within weeks by scrapie symptoms and death, heterozygous Prn-p0/+ mice remain free of symptoms for many months despite similar levels of scrapie infectivity and PrPSc. CONCLUSIONS: Our findings extend previous reports showing an inverse relationship between PrP expression level and incubation time for scrapie. However, contrary to expectation, overall accumulation of PrPSc and prions to a high level do not necessarily lead to clinical disease. These findings raise the question whether high titers of prion infectivity could also persist for long periods under natural circumstances in the absence of clinical symptoms.     

Progesterone signals through membrane progesterone receptors (mPRs) in MDA-MB-468 and mPR-transfected MDA-MB-231 breast cancer cells which lack full-length and N-terminally truncated isoforms of the nuclear progesterone receptor

The functional characteristics of membrane progesterone receptors (mPRs) have been investigated using recombinant mPR proteins over-expressed in MDA-MB-231 breast cancer cells. Although these cells do not express the full-length progesterone receptor (PR), it is not known whether they express N-terminally truncated PR isoforms which could possibly account for some progesterone receptor functions attributed to mPRs. In the present study, the presence of N-terminally truncated PR isoforms was investigated in untransfected and mPR-transfected MDA-MB-231 cells, and in MDA-MB-468 breast cancer cells. PCR products were detected in PR-positive T47D Yb breast cancer cells using two sets of C-terminus PR primers, but not in untransfected and mPR-transfected MDA-MB-231 cells, nor in MDA-MB-468 cells. Western blot analysis using a C-terminal PR antibody, 2C11F1, showed the same distribution pattern for PR in these cell lines. Another C-terminal PR antibody, C-19, detected immunoreactive bands in all the cell lines, but also recognized α-actinin, indicating that the antibody is not specific for PR. High affinity progesterone receptor binding was identified on plasma membranes of MDA-MB-468 cells which was significantly decreased after treatment with siRNAs for mPRα and mPRβ. Plasma membranes of MDA-MB-468 cells showed very low binding affinity for the PR agonist, R5020, ≤1% that of progesterone, which is characteristic of mPRs. Progesterone treatment caused G protein activation and decreased production of cAMP in MDA-MB-468 cells, which is also characteristic of mPRs. The results indicate that the progestin receptor functions in these cell lines are mediated through mPRs and do not involve any N-terminally truncated PR isoforms.     

Heterogeneity of normal prion protein in two- dimensional immunoblot: presence of various glycosylated and truncated forms

The common use of one-dimensional (1-D) immunoblot with a single monoclonal antibody (Mab) engenders the notion that the normal or cellular prion protein (PrP(C) ) comprises few and simple forms. In this study we used two-dimensional (2-D) immunoblot with a panel Mabs to various regions of the prion protein to demonstrate the complexity of the PrP(C) present in human brain. We distinguished over 50 immunoblot spots, each representing a distinct PrP(C) species based on combinations of different molecular weights and isoelectric points (pIs). The PrP(C) heterogeneity is due to the presence of a full-length and two major truncated forms as well as to the diversity of the glycans linked to most of these forms. The two major truncated forms result from distinct cleavage sites located at the N-terminus. In addition, enzymatic removal of sialic acid and lectin binding studies indicate that the glycans linked to the full-length and truncated PrP(C) forms differ in their structure and ratios of the glycoforms. The truncation of PrP(C) and the heterogeneity of the linked glycans may play a role in regulating PrP(C) function. Furthermore, the presence of relatively large quantities of different PrP(C) species may provide additional mechanisms by which the diversity of prion strains could be generated.     

Presence and seeding activity of pathological prion protein (PrP(TSE)) in skeletal muscles of white-tailed deer infected with chronic wasting disease

Chronic wasting disease (CWD) is a contagious, rapidly spreading transmissible spongiform encephalopathy (TSE), or prion disease, occurring in cervids such as white tailed-deer (WTD), mule deer or elk in North America. Despite efficient horizontal transmission of CWD among cervids natural transmission of the disease to other species has not yet been observed. Here, we report for the first time a direct biochemical demonstration of pathological prion protein PrP(TSE) and of PrP(TSE)-associated seeding activity, the static and dynamic biochemical markers for biological prion infectivity, respectively, in skeletal muscles of CWD-infected cervids, i. e. WTD for which no clinical signs of CWD had been recognized. The presence of PrP(TSE) was detected by Western- and postfixed frozen tissue blotting, while the seeding activity of PrP(TSE) was revealed by protein misfolding cyclic amplification (PMCA). Semi-quantitative Western blotting indicated that the concentration of PrP(TSE) in skeletal muscles of CWD-infected WTD was approximately 2000-10,000-fold lower than in brain tissue. Tissue-blot-analyses revealed that PrP(TSE) was located in muscle-associated nerve fascicles but not, in detectable amounts, in myocytes. The presence and seeding activity of PrP(TSE) in skeletal muscle from CWD-infected cervids suggests prevention of such tissue in the human diet as a precautionary measure for food safety, pending on further clarification of whether CWD may be transmissible to humans.     

Dynamics of a truncated prion protein,PrP(113–231), from 15N NMR relaxation: Order parameters calculated and slow conformational fluctuations localized to a distinct region

Prion diseases are associated with the misfolding of the prion protein (PrP^C) from a largely α‐helical isoform to a β‐sheet rich oligomer (PrP^Sc). Flexibility of the polypeptide could contribute to the ability of PrP^C to undergo the conformational rearrangement during PrP^C–PrP^Sc interactions, which then leads to the misfolded isoform. We have therefore examined the molecular motions of mouse PrP^C, residues 113–231, in solution, using ¹⁵N NMR relaxation measurements. A truncated fragment has been used to eliminate the effect of the 90‐residue unstructured tail of PrP^C so the dynamics of the structured domain can be studied in isolation. ¹⁵N longitudinal (T₁) and transverse relaxation (T₂) times as well as the proton‐nitrogen nuclear Overhauser effects have been used to calculate the spectral density at three frequencies, 0, ω_N, and 0.87ω_H. Spectral densities at each residue indicate various time‐scale motions of the main‐chain. Even within the structured domain of PrP^C, a diverse range of motions are observed. We find that removal of the tail increases T₂ relaxation times significantly indicating that the tail is responsible for shortening of T₂ times in full‐length PrP^C. The truncated fragment of PrP has facilitated the determination of meaningful order parameters (S²) from the relaxation data and shows for the first time that all three helices in PrP^C have similar rigidity. Slow conformational fluctuations of mouse PrP^C are localized to a distinct region that involves residues 171 and 172. Interestingly, residues 170–175 have been identified as a segment within PrP that will form a steric zipper, believed to be the fundamental amyloid unit. The flexibility within these residues could facilitate the PrP^C–PrP^Sc recognition process during fibril elongation.     

Transmission of chronic wasting disease identifies a prion strain causing cachexia and heart infection in hamsters

Chronic wasting disease (CWD) is an emerging prion disease of free-ranging and captive cervids in North America. In this study we established a rodent model for CWD in Syrian golden hamsters that resemble key features of the disease in cervids including cachexia and infection of cardiac muscle. Following one to three serial passages of CWD from white-tailed deer into transgenic mice expressing the hamster prion protein gene, CWD was subsequently passaged into Syrian golden hamsters. In one passage line there were preclinical changes in locomotor activity and a loss of body mass prior to onset of subtle neurological symptoms around 340 days. The clinical symptoms included a prominent wasting disease, similar to cachexia, with a prolonged duration. Other features of CWD in hamsters that were similar to cervid CWD included the brain distribution of the disease-specific isoform of the prion protein, PrP(Sc), prion infection of the central and peripheral neuroendocrine system, and PrP(Sc) deposition in cardiac muscle. There was also prominent PrP(Sc) deposition in the nasal mucosa on the edge of the olfactory sensory epithelium with the lumen of the nasal airway that could have implications for CWD shedding into nasal secretions and disease transmission. Since the mechanism of wasting disease in prion diseases is unknown this hamster CWD model could provide a means to investigate the physiological basis of cachexia, which we propose is due to a prion-induced endocrinopathy. This prion disease phenotype has not been described in hamsters and we designate it as the 'wasting' or WST strain of hamster CWD.     

Dactylogyrids on the gills of roach in central Finland: features of infection and species composition.

Monogenean parasites were examined from the gills of 660 roach (Rutilus rutilus) in four interconnected lakes in Central Finland between February and November 1986 and in three of the same lakes between February and December 1988. One of the lakes is eutrophic and polluted due to a paper and pulp mill, one is oligotrophic and in a natural state, and the other two lakes are eutrophic. The prevalence of Dactylogyrus infection was always high. Differences between the lakes and the years were observed in the intensity of infection, which was significantly higher in the polluted lake. The intensity was also higher in older fish. Nine Dactylogyrus species were found, and of these D. crucifer and D. nanus were numerically dominant in all of the lakes studied, especially in the oligotrophic lake. In 1986 D. fallax, D. similis and D. suecicus constituted significant proportions of the fauna in the polluted lake. D. micracanthus was most common in the eutrophic lakes. In 1988 the species composition in the polluted lake was most similar to that in the eutrophic lake.     

Early behavioral changes and quantitative analysis of neuropathological features in murine prion disease: Stereological analysis in the albino Swiss mice model

Behavioral and neuropathological changes have been widely investigated in murine prion disease but stereological based unbiased estimates of key neuropathological features have not been carried out. After injections of ME7 infected (ME7) or normal brain homogenates (NBH) into dorsal CA1 of albino Swiss mice and C57BL6, we assessed behavioral changes on hippocampal-dependent tasks. We also estimated by optical fractionator at 15 and 18 weeks post-injections (w.p.i.) the total number of neurons, reactive astrocytes, activated microglia and perineuronal nets (PN) in the polymorphic layer of dentate gyrus (PolDG), CA1 and septum in albino Swiss mice. On average, early behavioral changes in albino Swiss mice start four weeks later than in C57BL6. Cluster and discriminant analysis of behavioral data in albino Swiss mice revealed that four of nine subjects start to change their behavior at 12 w.p.i. and reach terminal stage at 22 w.p.i and the remaining subjects start at 22 w.p.i. and reach terminal stage at 26 w.p.i. Biotinylated dextran-amine BDA-tracer experiments in mossy fiber pathway confirmed axonal degeneration and stereological data showed that early astrocytosis, microgliosis and reduction in the perineuronal nets are independent of a change in the number of neuronal cell bodies. Statistical analysis revealed that the septal region had greater levels of neuroinflammation and extracellular matrix damage than CA1. This stereological and multivariate analysis at early stages of disease in an outbred model of prion disease provided new insights connecting behavioral changes and neuroinflammation and seems to be important to understand the mechanisms of prion disease progression.Key words: prion disease, optical fractionator, neuropathology, behavioral changes, albino Swiss mice