Biochemical and Neuropathological Findings in a Creutzfeldt-Jakob Disease Patient with the Rare Val180Ile-129Val Haplotype in the Prion Protein Gene.

Genetic Creutzfeldt-Jakob disease (gCJD) associated with the V180I mutation in the prion protein (PrP) gene (PRNP) in phase with residue 129M is the most frequent cause of gCJD in East Asia, whereas it is quite uncommon in Caucasians. We report on a gCJD patient with the rare V180I-129V haplotype, showing an unusually long duration of the disease and a characteristic pathological PrP (PrPSc) glycotype. Family members carrying the mutation were fully asymptomatic, as commonly observed with this mutation. Neuropathological examination showed a lesion pattern corresponding to that commonly reported in Japanese V180I cases with vacuolization and gliosis of the cerebral cortexes, olfactory areas, hippocampus and amygdala. PrP was deposited with a punctate, synaptic-like pattern in the cerebral cortex, amygdala and olfactory tract. Western blot analyses of proteinase-K-resistant PrP showed the characteristic two-banding pattern of V180I gCJD, composed of mono- and un-glycosylated isoforms. In line with reports on other V180I cases in the literature, Real-Time Quaking Induced Conversion (RT-QuIC) analyses did not demonstrate the presence of seeding activity in the cerebrospinal fluid and olfactory mucosa, suggesting that this haplotype also may result in a reduced seeding efficiency of the pathological PrP. Further studies are required to understand the origin, penetrance, disease phenotype and transmissibility of 180I-129V haplotype in Caucasians.

Differentiation-Dependent Effects of a New Recombinant Manganese Superoxide Dismutase on Human SK-N-BE Neuron-Like Cells.

We have recently isolated a new isoform of recombinant manganese superoxide dismutase (rMnSOD) which provides a potent antitumor activity and strongly counteracts the occurrence of oxidative stress and tissue inflammation. This isoform, in addition to the enzymatic action common to all SODs, also shows special functional and structural properties, essentially due to the presence of a first leader peptide that allows the protein to enter easily into cells. Among endogenous antioxidants, SOD constitutes the first line of natural defence against pathological effects induced by an excess of free radicals. Here, we firstly describe the effects of our rMnSOD administration on the proliferant and malignant undifferentiated human neuroblastoma SK-N-BE cell line. Moreover, we also test the effects of rMnSOD in the all trans retinoic-differentiated SK-N-BE neuron-like cells, a quiescent "not malignant" model. While rMnSOD showed an antitumor activity on proliferating cells, a poor sensitivity to rMnSOD overload in retinoid-differentiated neuron-like cells was observed. However, in the latter case, in presence of experimental-induced oxidative stress, overcharge of rMnSOD enhanced the oxidant effects, through an increase of H2O2 due to low activity of both catalase and glutathione peroxidase. In conclusion, our data show that rMnSOD treatment exerts differential effects, which depend upon both cell differentiation and redox balance, addressing attention to the potential use of the recombinant enzyme on differentiated neurons. These facts ultimately pave the way for further preclinical studies aimed at evaluation of rMnSOD effects in models of neurodegenerative diseases.

Prion Strain Characterization of a Novel Subtype of Creutzfeldt-Jakob Disease.

In 2007, we reported a patient with an atypical form of Creutzfeldt-Jakob disease (CJD) heterozygous for methionine-valine (MV) at codon 129 who showed a novel pathological prion protein (PrPTSE) conformation with an atypical glycoform (AG) profile and intraneuronal PrP deposition. In the present study, we further characterize the conformational properties of this pathological prion protein (PrPTSE MVAG), showing that PrPTSE MVAG is composed of multiple conformers with biochemical properties distinct from those of PrPTSE type 1 and type 2 of MV sporadic CJD (sCJD). Experimental transmission of CJD-MVAG to bank voles and gene-targeted transgenic mice carrying the human prion protein gene (TgHu mice) showed unique transmission rates, survival times, neuropathological changes, PrPTSE deposition patterns, and PrPTSE glycotypes that are distinct from those of sCJD-MV1 and sCJD-MV2. These biochemical and experimental data suggest the presence of a novel prion strain in CJD-MVAGIMPORTANCE Sporadic Creutzfeldt-Jakob disease is caused by the misfolding of the cellular prion protein, which assumes two different major conformations (type 1 and type 2) and, together with the methionine/valine polymorphic codon 129 of the prion protein gene, contribute to the occurrence of distinct clinical-pathological phenotypes. Inoculation in laboratory rodents of brain tissues from the six possible combinations of pathological prion protein types with codon 129 genotypes results in the identification of 3 or 4 strains of prions. We report on the identification of a novel strain of Creutzfeldt-Jakob disease isolated from a patient who carried an abnormally glycosylated pathological prion protein. This novel strain has unique biochemical characteristics, does not transmit to humanized transgenic mice, and shows exclusive transmission properties in bank voles. The identification of a novel human prion strain improves our understanding of the pathogenesis of the disease and of possible mechanisms of prion transmission.

Assessment of prion reduction filters in decreasing infectivity of ultracentrifuged 263K scrapie-infected brain homogenates in "spiked" human blood and red blood cells.

BACKGROUND: The safety of red blood cells (RBCs) is of concern because of the occurrence of four transfusion-transmitted variant Creutzfeldt-Jakob disease (vCJD) cases in the United Kingdom. The absence of validated screening tests requires the use of procedures to remove prions from blood to minimize the risk of transmission. These procedures must be validated using infectious prions in a form that is as close as possible to one in blood. STUDY DESIGN AND METHODS: Units of human whole blood (WB) and RBCs were spiked with high-speed supernatants of 263K scrapie-infected hamster brain homogenates. Spiked samples were leukoreduced and then passed through prion-removing filters (Pall Corporation). In another experiment, RBCs from 263K scrapie-infected hamsters were treated as above, and residual infectivity was measured by bioassay. RESULTS: The overall removal of infectivity by the filters from prion-spiked WB and RBCs was approximately two orders of magnitude. No infectivity was detected in filtered hamster RBCs endogenously infected with scrapie. CONCLUSION: The use of prion-removing filters may help to reduce the risk of transfusion-transmitted vCJD. To avoid overestimation of prion removal efficiency in validation studies, it may be more appropriate to use supernates from ultracentrifugation of scrapie-infected hamster brain homogenate rather than the current standard brain homogenates.

Chronic wasting disease and atypical forms of bovine spongiform encephalopathy and scrapie are not transmissible to mice expressing wild-type levels of human prion protein.

The association between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) has demonstrated that cattle transmissible spongiform encephalopathies (TSEs) can pose a risk to human health and raises the possibility that other ruminant TSEs may be transmissible to humans. In recent years, several novel TSEs in sheep, cattle and deer have been described and the risk posed to humans by these agents is currently unknown. In this study, we inoculated two forms of atypical BSE (BASE and H-type BSE), a chronic wasting disease (CWD) isolate and seven isolates of atypical scrapie into gene-targeted transgenic (Tg) mice expressing the human prion protein (PrP). Upon challenge with these ruminant TSEs, gene-targeted Tg mice expressing human PrP did not show any signs of disease pathology. These data strongly suggest the presence of a substantial transmission barrier between these recently identified ruminant TSEs and humans.

Comparison of nanofiltration efficacy in reducing infectivity of centrifuged versus ultracentrifuged 263K scrapie-infected brain homogenates in "spiked" albumin solutions.

BACKGROUND: The safety of plasma-derived products is of concern for possible transmission of variant Creutzfeldt-Jakob disease. The absence of validated screening tests requires the use of procedures to remove or inactivate prions during the manufacture of plasma-derived products to minimize the risk of transmission. These procedures need proper validation studies based on spiking human plasma or intermediate fractions of plasma fractionation with prions in a form as close as possible to that present in blood. STUDY DESIGN AND METHODS: Human albumin was spiked with low-speed or high-speed supernatants of 263K scrapie-infected hamster brain homogenates. Spiked albumin was then passed through a cascade of filters from 100 nm down to 20 to 15 nm. Residual infectivity was measured by bioassay. RESULTS: The overall removal of infectivity spiked into albumin through serial nanofiltration steps was 4 to 5 logs using low-speed supernatant and 2 to 3 logs with high-speed supernatant. CONCLUSION: These findings confirm the utility of nanofiltration in removing infectivity from plasma (or other products) spiked with scrapie brain homogenate supernatants. However, efficiency is diminished using supernatants that have been ultracentrifuged to reduce aggregated forms of the infectious agent. Thus, filtration removal data based on experiments using "standard" low-speed centrifugation supernatants might overestimate the amount of prion removal in plasma or urine-derived therapeutic products.

Small Molecules with Anti-Prion Activity.

Prion pathologies are fatal neurodegenerative diseases caused by the misfolding of the physiological Prion Protein (PrPC) into a ß-structure-rich isoform called PrPSc. To date, there is no available cure for prion diseases and just a few clinical trials have been carried out. The initial approach in the search of anti-prion agents had PrPSc as a target, but the existence of different prion strains arising from alternative conformations of PrPSc, limited the efficacy of the ligands to a straindependent ability. That has shifted research to PrPC ligands, which either act as chaperones, by stabilizing the native conformation, or inhibit its interaction with PrPSc. The role of transition-metal mediated oxidation processes in prion misfolding has also been investigated. Another promising approach is the indirect action via other cellular targets, like membrane domains or the Protein- Folding Activity of Ribosomes (PFAR). Also, new prion-specific high throughput screening techniques have been developed. However, so far no substance has been found to be able to extend satisfactorily survival time in animal models of prion diseases. This review describes the main features of the Structure-Activity Relationship (SAR) of the various chemical classes of anti-prion agents.

Ring trial of 2nd generation RT-QuIC diagnostic tests for sporadic CJD.

OBJECTIVE: Real-time quaking-induced conversion (RT-QuIC) assays detect prion-seeding activity in a variety of human biospecimens, including cerebrospinal fluid and olfactory mucosa swabs. The assay has shown high diagnostic accuracy in patients with prion disorders. Recently, advances in these tests have led to markedly improved diagnostic sensitivity and reduced assay times. Accordingly, an algorithm has been proposed that entails the use of RT-QuIC analysis of both sample types to diagnose sporadic Creutzfeldt-Jakob disease with nearly 100% accuracy. Here we present a multi-center evaluation (ring trial) of the reproducibility of these improved "second generation" RT-QuIC assays as applied to these diagnostic specimens. METHODS: Cerebrospinal fluid samples were analyzed from subjects with sporadic Creutzfeldt-Jakob (n = 55) or other neurological diseases (n = 45) at multiple clinical centers. Olfactory mucosa brushings collected by multiple otolaryngologists were obtained from nine sporadic Creutzfeldt-Jakob disease cases and 19 controls. These sample sets were initially tested blindly by RT-QuIC by a coordinating laboratory, recoded, and then sent to five additional testing laboratories for blinded ring trial testing. RESULTS: Unblinding of the results by a third party indicated 98-100% concordance between the results obtained by the testing of these cerebrospinal fluid and nasal brushings at the six laboratories. INTERPRETATION: This second-generation RT-QuIC assay is highly transferrable, reproducible, and therefore robust for the diagnosis of sporadic Creutzfeldt-Jakob disease in clinical practice.

Proteomic profiling of PrP27-30-enriched preparations extracted from the brain of hamsters with experimental scrapie.

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders characterized by the accumulation in the CNS of a pathological conformer (PrP(TSE)) of the host-encoded cellular prion protein (PrP(C)). PrP(TSE) has a central role in the pathogenesis of the disease but other factors are likely involved in the pathological process. In this work we employed a multi-step proteomic approach for the identification of proteins that co-purify with the protease-resistant core of PrP(TSE) (PrP27-30) extracted from brains of hamsters with experimental scrapie. We identified ferritin, calcium/calmodulin-dependent protein kinase alpha type II, apolipoprotein E, and tubulin as the major components associated with PrP27-30 but also trace amounts of actin, cofilin, Hsp90alpha, the gamma subunit of the T-complex protein 1, glyceraldehyde 3-phosphate dehydrogenase, histones, and keratins. Whereas some of these proteins (tubulin and ferritin) are known to bind PrP, other proteins (calcium/calmodulin-dependent protein kinase alpha type II, Hsp90alpha) may associate with PrP(TSE) fibrils during disease. Apolipoprotein E and actin have been previously observed in association with PrP(TSE), whereas cofilin and actin were shown to form abnormal rods in the brain of patients with Alzheimer disease. The roles of these proteins in the development of brain lesions are still unclear and further work is needed to explain their involvement in the pathogenesis of TSEs.

PrPTSE in muscle-associated lymphatic tissue during the preclinical stage of mice infected orally with bovine spongiform encephalopathy.

The involvement of muscles in the pathogenesis of transmissible spongiform encephalopathies (TSEs) is irregular and unpredictable. We show that the TSE-specific protein (PrP(TSE)) is present in muscles of mice fed with a mouse-adapted strain of bovine spongiform encephalopathy as early as 100 days post-infection, corresponding to about one-third of the incubation period. The proportion of mice with PrP(TSE)-positive muscles and the number of muscles involved increased as infection progressed, but never attained more than a limited distribution, even at the clinical stage of disease. The appearance of PrP(TSE) in muscles during the preclinical stage of disease was probably due to the haematogenous/lymphatic spread of infectivity from the gastrointestinal tract to lymphatic tissues associated with muscles, whereas in symptomatic animals, the presence of PrP(TSE) in the nervous system, in neuromuscular junctions and in muscle fibres suggests a centrifugal spread from the central nervous system, as already observed in other TSE models.

Neuroinvasion of the 263K scrapie strain after intranasal administration occurs through olfactory-unrelated pathways.

The olfactory system has been implicated in the pathogenesis of transmissible spongiform encephalopathies (TSEs). To examine this issue and identify the pattern of TSE agent spread after intranasal administration, we inoculated a high-infectious dose of neurotropic scrapie strain 263K into the nasal cavity of Syrian hamsters. All animals allowed to survive became symptomatic with a mean incubation period of 162.4 days. Analysis at different time points revealed deposition of the pathological prion protein (PrP(TSE)) in nasal-associated lymphoid tissues in the absence of brain involvement from 80 days post-infection (50% of the incubation period). Olfactory-related structures and brainstem nuclei were involved from 100 days post-inoculation (62% of the incubation period) when animals were still asymptomatic. Intriguingly, vagal or trigeminal nuclei were identified as early sites of PrP(TSE) deposition in some pre-symptomatic animals. These findings indicate that the 263K scrapie agent is unable to effectively spread from the olfactory neuroepithelium to the olfactory-related structures and that, after intranasal inoculation, neuroinvasion occurs through olfactory-unrelated pathways.

Megalencephalic Leukoencephalopathy with Subcortical Cysts Protein-1 (MLC1) Counteracts Astrocyte Activation in Response to Inflammatory Signals.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 (MLC1) is a membrane protein expressed by perivascular astrocytes. MLC1 mutations cause MLC, an incurable leukodystrophy characterized by macrocephaly, brain edema, cysts, myelin vacuolation, and astrocytosis, leading to cognitive/motor impairment and epilepsy. Although its function is unknown, MLC1 favors regulatory volume decrease after astrocyte osmotic swelling and down-regulates intracellular signaling pathways controlling astrocyte activation and proliferation. By combining analysis of human brain tissues with in vitro experiments, here we investigated MLC1 role in astrocyte activation during neuroinflammation, a pathological condition exacerbating patient symptoms. MLC1 upregulation was observed in brain tissues from multiple sclerosis, Alzheimer's, and Creutzfeld-Jacob disease, all pathologies characterized by strong astrocytosis and release of inflammatory cytokines, particularly IL-1ß. Using astrocytoma lines overexpressing wild-type (WT) or mutated MLC1 and astrocytes from control and Mlc1 knock-out (KO) mice, we found that IL-1ß stimulated WT-MLC1 plasma membrane expression in astrocytoma cells and control primary astrocytes. In astrocytoma, WT-MLC1 inhibited the activation of IL-1ß-induced inflammatory signals (pERK, pNF-kB) that, conversely, were constitutively activated in mutant expressing cells or abnormally upregulated in KO astrocytes. WT-MLC1+ cells also expressed reduced levels of the astrogliosis marker pSTAT3. We then monitored MLC1 expression timing in a demyelinating/remyelinating murine cerebellar organotypic culture model where, after the demyelination and release of inflammatory cytokines, recovery processes occur, revealing MLC1 upregulation in these latter phases. Altogether, these findings suggest that by modulating specific pathways, MLC1 contributes to restore astrocyte homeostasis after inflammation, providing the opportunity to identify drug target molecules to slow down disease progression.

Efficient transmission and characterization of Creutzfeldt-Jakob disease strains in bank voles.

Transmission of prions between species is limited by the "species barrier," which hampers a full characterization of human prion strains in the mouse model. We report that the efficiency of primary transmission of prions from Creutzfeldt-Jakob disease patients to a wild rodent species, the bank vole (Clethrionomys glareolus), is comparable to that reported in transgenic mice carrying human prion protein, in spite of a low prion protein-sequence homology between man and vole. Voles infected with sporadic and genetic Creutzfeldt-Jakob disease isolates show strain-specific patterns of spongiform degeneration and pathological prion protein-deposition, and accumulate protease-resistant prion protein with biochemical properties similar to the human counterpart. Adaptation of genetic Creutzfeldt-Jakob disease isolates to voles shows little or no evidence of a transmission barrier, in contrast to the striking barriers observed during transmission of mouse, hamster, and sheep prions to voles. Our results imply that in voles there is no clear relationship between the degree of homology of the prion protein of the donor and recipient species and susceptibility, consistent with the view that the prion strain gives a major contribution to the species barrier. The vole is therefore a valuable model to study human prion diversity and, being susceptible to a range of animal prions, represents a unique tool for comparing isolates from different species.

Intranasal oxytocin administration promotes emotional contagion and reduces aggression in a mouse model of callousness.

Deficits in empathy, the ability to share an emotion of another individual, constitute a hallmark of several psychopathological conditions, including conduct disorder. The co-occurrence of excess rates of aggression, general violation of societal norms and callous-unemotional traits confers specific risk for adult psychopathy. In the present study, we relied on a recently devised experimental model of conduct disorder in mice to test the potential efficacy of intranasal oxytocin administration. Two subgroups of BALB/cJ male mice exhibiting opposite profiles in emotional contagion (i.e. socially transmitted adoption of another's emotional states) underwent a series of tests mapping onto reactive aggression, information processing, perseverative behaviour, punishment-related emotional memory, physiological arousal and hormonal stress reactivity, with or without intranasal oxytocin administration (5.0 or 20.0 µg/kg). Collectively, our data indicate that a trait of markedly reduced emotional contagion is associated with a behavioural syndrome of sensorimotor gating deficits, impaired emotional memory, increased aggression and stereotyped behaviours, dysregulations in the circadian rhythms of activity and body temperature and dampened physiological reactivity to external stressors. Moreover, in the absence of changes in oxytocin receptor density in the neural network involved in empathy-like behaviour, we showed that oxytocin administration normalised emotional contagion, aggression and behavioural stereotypies, thereby ameliorating the phenotype of mice characterised by deficient empathy-like behaviour. Besides, oxytocin led to a lower, more prolonged neuroendocrine response of the HPA-axis to stress in all mice. Ultimately, current data support the notion that oxytocin may constitute a valid therapeutic approach in disturbances characterised by abnormal aggression and excess callousness.

Scrapie infectivity is quickly cleared in tissues of orally-infected farmed fish.

BACKGROUND: Scrapie and bovine spongiform encephalopathy (BSE) belongs to the group of animal transmissible spongiform encephalopathy (TSE). BSE epidemic in the UK and elsewhere in Europe has been linked to the use of bovine meat and bone meals (MBM) in the feeding of cattle. There is concern that pigs, poultry and fish bred for human consumption and fed with infected MBM would eventually develop BSE or carry residual infectivity without disease. Although there has been no evidence of infection in these species, experimental data on the susceptibility to the BSE agent of farm animals other than sheep and cow are limited only to pigs and domestic chicken. In the framework of a EU-granted project we have challenged two species of fish largely used in human food consumption, rainbow trout (Oncorhynchus mykiss) and turbot (Scophthalmus maximus), with a mouse-adapted TSE strain (scrapie 139A), to assess the risk related to oral consumption of TSE contaminated food. In trout, we also checked the "in vitro" ability of the pathological isoform of the mouse prion protein (PrPSc) to cross the intestinal epithelium when added to the mucosal side of everted intestine. RESULTS: Fish challenged with a large amount of scrapie mouse brain homogenate by either oral or parenteral routes, showed the ability to clear the majority of infectivity load. None of the fish tissues taken at different time points after oral or parenteral inoculation was able to provoke scrapie disease after intracerebral inoculation in recipient mice. However, a few recipient mice were positive for PrPSc and spongiform lesions in the brain. We also showed a specific binding of PrPSc to the mucosal side of fish intestine in the absence of an active uptake of the prion protein through the intestinal wall. CONCLUSION: These results indicate that scrapie 139A, and possibly BSE, is quickly removed from fish tissues despite evidence of a prion like protein in fish and of a specific binding of PrPSc to the mucosal side of fish intestine.

Migration of dendritic cells into the brain in a mouse model of prion disease.

The immune system plays a key role in the dissemination of prion infections from the periphery to the central nervous system (CNS). While follicular dendritic cells are critical for prion replication in lymphoid tissue and subsequent neuroinvasion, myeloid dendritic cells (DCs) have been implicated in both the clearance and propagation of pathological prion protein. Since nothing is known on the ability of DCs to migrate to the CNS during prion diseases, we investigated the immunohistochemical localization of CD205(+) DCs in the brain of C57BL/6 mice intraperitoneally infected with the mouse-adapted KFu strain of Gerstmann-Sträussler-Scheinker syndrome, a human genetic prion disorder. In normal brain, CD205(+) cells were present in the meninges and choroid plexus, whereas in the majority of mice sacrificed between 120 and 300 days post infection, CD205(+) DCs were also detected in the cerebral cortex, subcortical white matter, thalamus and medulla oblongata. These findings demonstrate that DCs can enter the CNS of prion-infected mice, suggesting a possible role for these cells in the pathogenesis of prion disorders.

Differential responses to acute administration of a new 5-HT7-R agonist as a function of adolescent pre-treatment: phMRI and immuno-histochemical study.

LP-211 is a new, selective agonist of serotonin (5-hydroxytryptamine, 5-HT) receptor 7 (5-HT7-R), which is part of a neuro-transmission system with a proposed role in neural plasticity and in mood, cognitive and sleep regulation. Adolescent subchronic LP-211 treatment produces some persisting changes in rats' forebrain structural and functional parameters. Here, using pharmacological MRI (phMRI), we investigated the effect of acute administration with LP-211 (10 mg/kg i.p.), or vehicle, to adult rats previously exposed to the same drug (0.25 mg/kg/day for 5 days), or vehicle, during adolescence (44-48 post-natal days); histology and immuno-histochemistry were performed ex vivo to evaluate neuro-anatomical and physiological long-term adaptation to pharmacological pre-treatment. The phMRI signal reveals forebrain areas (i.e., hippocampus, orbital prefrontal cortex), activated in response to LP-211 challenge independently of adolescent pre-treatment. In septum and nucleus accumbens, sensitized activation was found in adolescent pre-treated rats but not in vehicle-exposed controls. Immuno-histochemical analyses showed marked differences in septum as long-term consequence of the adolescent pre-treatment: increased level of 5-HT7-R, increased number of 5-HT7-R positive cells, and enhanced astrocyte activation. For nucleus accumbens, immuno-histochemical analyses did not reveal any difference between adolescent pre-treated rats and vehicle-exposed controls. In conclusion, subchronic LP-211 administration during adolescence is able to induce persistent physiological changes in the septal 5-HT7-R expression and astrocyte response that can still be observed in adulthood. Data shed new insights into roles of 5-HT7-R for normal and pathologic behavioral regulations.

Different prion conformers target the olfactory pathway in sporadic Creutzfeldt-Jakob disease.

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are mammalian neurodegenerative diseases that occur as sporadic, inherited, or iatrogenic forms. Human TSEs exhibit a wide spectrum of phenotypic variability, which is influenced by (1) the conformation of the pathologic prion protein, or PrP(Sc); (2) the polymorphic codon 129 of the prion protein gene (PRNP), involving synonymous or nonsynonymous expression of Met or Val; and (3) the site of formation or entry of the self-replicating PrP(Sc). Brain deposition of PrP(Sc) occurs in a phenotype-specific regional pattern, either as extracellular amyloid plaques and plaque-like aggregates, or as fine granular immunoreactivity at intracellular sites and presynaptic and postsynaptic locations, including dendrites. We previously demonstrated PrP(Sc) deposition in ciliated dendrites of olfactory sensory neurons in sporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease. PrP(Sc) immunoreactivity was not limited to the olfactory neuroepithelium. But additionally involved the central olfactory pathway. More recently, we have found that the pathology of the olfactory pathway occurs early in the disease course, either in the myoclonic or classic sCJD or in the ataxic variant. Intriguingly, in the ataxic or cerebellar variant, mainly observed in patients with the Met/Val polymorphism (2) carrying PrP(Sc) type 2, olfactory involvement is accompanied by pathologic changes in the dorsal motor nucleus of the vagus and other brainstem nuclei. These findings suggest that different molecular events and distinct routes of PrP(Sc) spread contribute to the prominent heterogeneity of sCJD, conceivably providing support to the olfactory pathogenesis theory of neurodegenerative diseases.