Differentially targeted seeding reveals unique pathological alpha-synuclein propagation patterns.

Parkinson's disease is a progressive neurodegenerative disorder characterized by the intracellular accumulation of insoluble alpha-synuclein aggregates into Lewy bodies and neurites. Increasing evidence indicates that Parkinson's disease progression results from the spread of pathologic alpha-synuclein through neuronal networks. However, the exact mechanisms underlying the propagation of abnormal proteins in the brain are only partially understood. The objective of this study was first to describe the long-term spatiotemporal distributions of Lewy-related pathology in mice injected with alpha-synuclein preformed fibrils and then to recreate these patterns using a computational model that simulates in silico the spread of pathologic alpha-synuclein. In this study, 87 2-3-month-old non-transgenic mice were injected with alpha-synuclein preformed fibrils to generate a comprehensive post-mortem dataset representing the long-term spatiotemporal distributions of hyperphosphorylated alpha-synuclein, an established marker of Lewy pathology, across the 426 regions of the Allen Mouse Brain Atlas. The mice were injected into either the caudoputamen, nucleus accumbens or hippocampus, and followed over 24 months with pathologic alpha-synuclein quantified at seven intermediate time points. The pathologic patterns observed at each time point in this high-resolution dataset were then compared to those generated using a Susceptible-Infected-Removed (SIR) computational model, an agent-based model that simulates the spread of pathologic alpha-synuclein for every brain region taking simultaneously into account the effect of regional brain connectivity and Snca gene expression. Our histopathological findings showed that differentially targeted seeding of pathological alpha-synuclein resulted in unique propagation patterns over 24 months and that most brain regions were permissive to pathology. We found that the SIR model recreated the observed distributions of pathology over 24 months for each injection site. Null models showed that both Snca gene expression and connectivity had a significant influence on model fit. In sum, our study demonstrates that the combination of normal alpha-synuclein concentration and brain connectomics contributes to making brain regions more vulnerable to the pathological process, providing support for a prion-like spread of pathologic alpha-synuclein. We propose that this rich dataset and the related computational model will help test new hypotheses regarding mechanisms that may alter the spread of pathologic alpha-synuclein in the brain.

Differential α-synuclein expression contributes to selective vulnerability of hippocampal neuron subpopulations to fibril-induced toxicity.

The accumulation of misfolded α-synuclein (aSyn) and neuron loss define several neurodegenerative disorders including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). However, the precise relationship between pathology and neurotoxicity and why these processes disproportionately affect certain neuron subpopulations are poorly understood. We show here that Math2-expressing neurons in the hippocampal Cornu ammonis (CA), a region significantly affected by aSyn pathology in advanced PD and DLB, are highly susceptible to pathological seeding with pre-formed fibrils (PFFs), in contrast to dentate gyrus neurons, which are relatively spared. Math2+ neurons also exhibited more rapid and severe cell loss in both in vitro and in vivo models of synucleinopathy. Toxicity resulting from PFF exposure was dependent on endogenous aSyn and could be attenuated by N-acetyl-cysteine through a glutathione-dependent process. Moreover, aSyn expression levels strongly correlate with relative vulnerability among hippocampal neuron subtypes of which Math2+ neurons contained the highest amount. Consistent with this, antisense oligonucleotide (ASO)-mediated knockdown of aSyn reduced the neuronal pathology in a time-dependent manner. However, significant neuroprotection was observed only with early ASO intervention and a substantial reduction of aSyn pathology, indicating toxicity occurs after a critical threshold of pathological burden is exceeded in vulnerable neurons. Together, our findings reveal considerable heterogeneity in endogenous aSyn levels among hippocampal neurons and suggest that this may contribute to the selective vulnerability observed in the context of synucleinopathies.

4-hydroxytamoxifen leads to PrPSc clearance by conveying both PrPC and PrPSc to lysosomes independently of autophagy.

Prion diseases are fatal neurodegenerative disorders involving the abnormal folding of a native cellular protein, named PrP(C), to a malconformed aggregation-prone state, enriched in beta sheet secondary structure, denoted PrP(Sc). Recently, autophagy has garnered considerable attention as a cellular process with the potential to counteract neurodegenerative diseases of protein aggregation such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. Stimulation of autophagy by chemical compounds has also been shown to reduce PrP(Sc) in infected neuronal cells and prolong survival times in mouse models. Consistent with previous reports, we demonstrate that autophagic flux is increased in chronically infected cells. However, in contrast to recent findings we show that autophagy does not cause a reduction in scrapie burden. We report that in infected neuronal cells different compounds known to stimulate autophagy are ineffective in increasing autophagic flux and in reducing PrP(Sc). We further demonstrate that tamoxifen and its metabolite 4-hydroxytamoxifen lead to prion degradation in an autophagy-independent manner by diverting the trafficking of both PrP and cholesterol to lysosomes. Our data indicate that tamoxifen, a well-characterized, widely available pharmaceutical, may have applications in the therapy of prion diseases.

Identification of an intracellular site of prion conversion.

Prion diseases are fatal, neurodegenerative disorders in humans and animals and are characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrP(C)), denoted PrP(Sc), which represents the major component of infectious scrapie prions. Characterization of the mechanism of conversion of PrP(C) into PrP(Sc) and identification of the intracellular site where it occurs are among the most important questions in prion biology. Despite numerous efforts, both of these questions remain unsolved. We have quantitatively analyzed the distribution of PrP(C) and PrP(Sc) and measured PrP(Sc) levels in different infected neuronal cell lines in which protein trafficking has been selectively impaired. Our data exclude roles for both early and late endosomes and identify the endosomal recycling compartment as the likely site of prion conversion. These findings represent a fundamental step towards understanding the cellular mechanism of prion conversion and will allow the development of new therapeutic approaches for prion diseases.

Seminal plasma of brown trout, Salmo trutta fario (L.) contains a factor able to retain iron at acid pH, typical feature of lactoferrin.

Blood and seminal plasma of brown trout Salmo trutta fario were analyzed for their iron binding potential adopting two different methods. Seminal plasma showed an iron binding capacity that was retained even if samples were exposed at acid pH, similarly to mammalian lactoferrin that binds ferric iron also at acid pH. This suggests that the iron binding capacity is determined by a factor having a lactoferrin-like activity. Moreover, trout seminal plasma proteins were also analyzed in their pattern by sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membrane. When seminal plasma was subjected to immunoblotting using goat anti-bovine lactoferrin antibodies as a probe, only a single band having an apparent molecular weight of around 80 kDa was specifically detected, showing that this protein has homology with bovine lactoferrin.

Doppel and PrPC co-immunoprecipitate in detergent-resistant membrane domains of epithelial FRT cells.

Dpl (doppel) is a paralogue of the PrPC (cellular prion protein), whose misfolded conformer (the scrapie prion protein, PrPSc) is responsible for the onset of TSEs (transmissible spongiform encephalopathies) or prion diseases. It has been shown that the ectopic expression of Dpl in the brains of some lines of PrP-knockout mice provokes cerebellar ataxia, which can be rescued by the reintroduction of the PrP gene, suggesting a functional interaction between the two proteins. It is, however, still unclear where, and under which conditions, this event may occur. In the present study we addressed this issue by analysing the intracellular localization and the interaction between Dpl and PrPC in FRT (Fischer rat thyroid) cells stably expressing the two proteins separately or together. We show that both proteins localize prevalently on the basolateral surface of FRT cells, in both singly and doubly transfected clones. Interestingly we found that they associate with DRMs (detergent-resistant membranes) or lipid rafts, from where they can be co-immunoprecipitated in a cholesterol-dependent fashion. Although the interaction between Dpl and PrPC has been suggested before, our results provide the first clear evidence that this interaction occurs in rafts and is dependent on the integrity of these membrane microdomains. Furthermore, both Dpl and PrPC could be immunoprecipitated with flotillin-2, a raft protein involved in endocytosis and cell signalling events, suggesting that they share the same lipid environment.

Snca-GFP Knock-In Mice Reflect Patterns of Endogenous Expression and Pathological Seeding.

α-Synuclein (aSyn) participates in synaptic vesicle trafficking and synaptic transmission but its misfolding is also strongly implicated in Parkinson's disease (PD) and other neurodegenerative synucleinopathies in which misfolded aSyn accumulates in different regions of the central and peripheral nervous systems. Although increased aSyn expression levels or altered aggregation propensities likely underlie familial PD with SNCA amplification or mutations, the majority of synucleinopathies arise sporadically, indicating that disease can develop under normal levels of wild-type (wt) aSyn. We report here the development and characterization of a mouse line expressing an aSyn-green fluorescence protein (GFP) fusion protein under the control of native Snca regulatory elements. Regional and subcellular localization of the aSyn-GFP fusion protein in brains and peripheral tissues of knock-in (KI) mice are indistinguishable from that of wt littermates. Importantly, similar to wt aSyn, aSyn-GFP disperses from synaptic vesicles on membrane depolarization, indicating that the tag does not alter normal aSyn dynamics at synapses. In addition, intracerebral injection of aSyn pre-formed fibrils into KI mice induced the formation of aSyn-GFP inclusions with a distribution pattern similar to that observed in wt mice, albeit with attenuated kinetics because of the GFP-tag. We anticipate that this new mouse model will facilitate in vitro and in vivo studies requiring in situ detection of endogenous aSyn, thereby providing new insights into aSyn function in health and disease.

Immune-modulating and anti-vascular activities of two xanthenone acetic acid analogues: A comparative study to DMXAA.

In order to proliferate, solid tumours require the development and continuous expansion of an organised host-derived vascular network. The anti-vascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) emerged as derivative of the flavone-8-acetic acid (FAA) and xanthenone-4-acetic acid (XAA). Its anti-vascular activity is not based on direct cytotoxic effects, but is characterized by an immune-mediated component, through the activation of NF-kappaB pathway, and a direct anti-vascular action, involving the induction of endothelial cell apoptosis and changes in tumour vessel permeability. Despite promising pre-clinical results, DMXAA showed moderate anti-tumour activity in clinical trials. In this study, we compared to DMXAA the in vitro immune-modulating and the anti-vascular properties of two XAA analogues, AP/1649 and AP/1897. Their immune-stimulating activities were evaluated on a human monocyte cell line and their anti-vascular activities were studied by measuring the induction of HUVECs apoptosis and using DCE-MRI to determine tumour perfusion following drug treatment. Although the two molecules exerted an immune stimulation comparable to that produced by DMXAA, they showed reduced (AP/1649) or minimal (AP/1897) anti-vascular activity in vitro, and no anti-vascular effects in vivo. These results endorse the current theories concerning two independent actions exerted by DMXAA.

Serum disposition of bovine lactoferrin after oral and anal administration and its proteolytic cleavage by gastric transit in rainbow trout (Oncorhynchus mykiss W.).

Several studies have shown an immunomodulatory effect of orally administered bovine lactoferrin (LF) in fish, but the process of digestion was not characterized. In the present study, we investigated the fate of bovine LF after oral and anal administration, and studied the appearance of intact LF in the bloodstream and its proteolytic attack during the gastric transit in rainbow trout (Oncorhynchus mykiss) held at 9 degrees C and 18 degrees C. Data obtained showed the presence of intact bovine LF in the bloodstream only after anal administration in fish held at 18 degrees C and the presence of several peptides derived from bovine LF in the gastric content. Immunoblotting analysis showed that only a part of bovine LF-derived peptides reacted with the applied anti-bovine LF antibody. The concentration of intact bovine LF, after 30 min of administration, in the gastric content of fish reared at 18 degrees C, being extremely low, if any, led us to suspect that the immunoregulatory effect of dietary bovine LF shown in fish by several authors is not due to the intact form but to bioactive fragments, originated by the proteolytic attack during the gastric transit, as demonstrated in higher vertebrates.

The ability of coumarin-, flavanon- and flavonol-analogues of flavone acetic acid to stimulate human monocytes.

Flavone acetic acid (FAA) is a semi-synthetic flavonoid characterised by potent immune-modulatory and antivascular activity on mice but not in humans. Previously, the synthesis and cytotoxic activity on a human adenocarcinoma cell line of coumarin-, flavanon- and flavonol-derivatives of FAA were described. These analogues were able to induce the reduction of lysosomal neutral red uptake at 5 x 10(-5) M concentration and some of them were more effective than FAA. Some of these derivatives were selected to investigate their ability to exert immune-modulation on a human model, by using the most potent analogue that has emerged thus far, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), as a reference compound. We investigated the cytotoxicity of the selected derivatives on two human ovarian adenocarcinoma cell lines and their ability to activate the immune system by inducing lytic properties, TNF-alpha and nitric oxide in human monocytes. The immune-modulating activity was assessed by treating a cell line of human monocytes (Mono Mac 6, MM6) with FAA-derivatives alone or in association with lipopolysaccharide (LPS). None of the tested molecules showed any significant ability to directly affect tumor cell proliferation, whereas they were able to induce the lytic properties of MM6 cells. In particular, two coumarin derivatives, a and d, and the flavonol acetic acid, l, showed comparable results to DMXAA. The combination with LPS did not lead to synergistic interactions in the induction of the lytic properties of MM6, but it significantly increased the release of TNF-alpha, especially after 4 h of treatment. Instead, the maximum release of nitric oxide (NO) was detected after 24 h of treatment and after exposure to the FAA derivatives alone. Derivative a combined with LPS and analogue d alone were able to induce a higher TNF-alpha and NO release, respectively, whereas flavonol acetic acid was characterised by a strictly similar activity to DMXAA.

Evaluation of the effects of dexamethasone-induced stress on levels of natural antibodies in immunized laying hens.

Natural antibodies (NAb) are an important humoral component of innate immunity, playing a pivotal role as first line of defence against pathogens even without prior antigen-specific activation or antigen-driven selection. The levels of NAb in plasma of young laying hens were explored in more detail and identified 2,4,6-trinitrophenyl bovine serum albumin (TNP-BSA), as the non-self antigen showing the highest levels of IgΥ- and IgM-NAb. Subsequently, the relation between specific antibody (SpAb) levels and NAb levels, and the effect of dexamethasone (DEX)-induced stress on the acquired Ab response and on NAb levels were examined. According to obtained results, the affinity of NAb and SpAb, measured using the thiocyanate elution method, resulted higher in SpAb than in NAb. After stress induction, IgM-NAb and SpAb levels showed a transient decrease, whereas the levels of IgΥ-NAb were not changed. Moreover, statistical analysis showed positive correlations between IgΥ- and IgM-NAb levels and between IgM-NAb and SpAb levels that are lost as stress has been induced, whereas no correlation was observed between IgΥ-NAb and SpAb levels, neither before nor after the DEX-administration. This indicates that IgM-NAb assessment could be a valid tool to estimate the potential of the acquired Ab response and that the dexamethasone-induced stress condition causes depression of IgM-NAb levels and the acquired Ab response, but it has no evaluable effects on IgΥ-NAb levels.

Mono- or di-fluorinated analogues of flavone-8-acetic acid: synthesis and in vitro biological activity.

BACKGROUND: Previously, the antitumour activity of some flavone-8-acetic acid (FAA) derivatives substituted with an acid function in position 2 of the benzene ring was evaluated. The most active compound resulted the one bearing a fluorine atom in position 7 of the flavone nucleus. In this paper, we evaluated new mono- or di-fluorinated FAA derivatives. MATERIALS AND METHODS: The cytotoxicity towards two human ovarian adenocarcinoma cell lines, the capability to stimulate human mononuclear cells and murine macrophages' lytic properties were evaluated by MTT. Moreover, the potentiation of lipopolysaccharide (LPS) activity was studied by ELISA analysis of TNF-alpha release. RESULTS: The analogues showed a direct cytotoxicity comparable to that of 5,6-dimethyl-xanthen-9-one-4-acetic acid (DMXAA), at present in clinical trials. None of the tested compounds was able to stimulate human mononuclear cells' lytic properties after either 4- or 24-h treatment, while after 4-h treatment, the derivative 5a was more able to stimulate murine macrophages with respect to DMXAA. Moreover, a significant increase of 5c and 5d activation was obtained with LPS association, reflected by TNF-alpha production as well. CONCLUSION: Like FAA, the new fluorinated derivatives 5a, 5c and 5d showed remarkable activity in murine cells, but this was not confirmed in human models.

Homopterocarpanes as bridged triarylethylene analogues: synthesis and antagonistic effects in human MCF-7 breast cancer cells.

A series of new compounds structurally derived from 6a,12a-dihydro-6H,7H-[1]-benzopyran-[4,3-b]-benzopyran (homopterocarpane) was efficiently synthesized by reduction of the corresponding pyrilium salts obtained by treatment of selected flavanones and aldehydes with anhydrous HClO4. Cytotoxic effects on the human breast cancer cell line MCF-7 and antiestrogenic activity (only for compounds which resulted more active than tamoxifen (TAM)) on MCF-7 cells stimulated by 17beta-estradiol were evaluated. In vivo antiestrogenic activity and the relative binding affinity were also assessed. Some of the new compounds (4c, 4h, 4i and 4l) showed a biological activity in the micromolar range, and were more potent than TAM taken as the reference.

Engineering cortical neuron polarity with nanomagnets on a chip.

Intra- and extracellular signaling play critical roles in cell polarity, ultimately leading to the development of functional cell-cell connections, tissues, and organs. In the brain, pathologically oriented neurons are often the cause for disordered circuits, severely impacting motor function, perception, and memory. Aside from control through gene expression and signaling pathways, it is known that nervous system development can be manipulated by mechanical stimuli (e.g., outgrowth of axons through externally applied forces). The inverse is true as well: intracellular molecular signals can be converted into forces to yield axonal outgrowth. The complete role played by mechanical signals in mediating single-cell polarity, however, remains currently unclear. Here we employ highly parallelized nanomagnets on a chip to exert local mechanical stimuli on cortical neurons, independently of the amount of superparamagnetic nanoparticles taken up by the cells. The chip-based approach was utilized to quantify the effect of nanoparticle-mediated forces on the intracellular cytoskeleton as visualized by the distribution of the microtubule-associated protein tau. While single cortical neurons prefer to assemble tau proteins following poly-L-lysine surface cues, an optimal force range of 4.5-70 pN by the nanomagnets initiated a tau distribution opposed to the pattern cue. In larger cell clusters (groups comprising six or more cells), nanoparticle-mediated forces induced tau repositioning in an observed range of 190-270 pN, and initiation of magnetic field-directed cell displacement was observed at forces above 300 pN. Our findings lay the groundwork for high-resolution mechanical encoding of neural networks in vitro, mechanically driven cell polarization in brain tissues, and neurotherapeutic approaches using functionalized superparamagnetic nanoparticles to potentially restore disordered neural circuits.

Synthesis and antitumor activity of new derivatives of xanthen-9-one-4-acetic acid.

Xanthen-9-one-4-acetic acid (XAA) analogues in which the substituents in positions 5 and 6 are included in cyclic structures are described. Direct in vitro toxicity of the synthesized compounds against four tumor cell lines was evaluated, and their ability to stimulate mouse peritoneal macrophages and human monocytes in culture to become tumoricidal (indirect toxicity) was also studied. Despite low direct toxicity, almost all the compounds proved to be able to significantly enhance the lytic properties of both murine macrophages and human monocytes as well as the parent compound XAA and its most active derivative DMXAA taken as reference. In particular, compounds 4a, 5a, 7a, 13a,b, and 16a,b showed higher activity than the lead compound on human monocytes, compound 7a being 2.5 times more active than DMXAA, which is the most potent compound synthesized so far. Moreover, compounds 4a, 5a, 7a, 13a, 16a, and 16b proved to be able to induce TNF production in human immune cells.

Synthesis and biological evaluation of 3-alkoxy analogues of flavone-8-acetic acid.

New analogues of flavone-8-acetic acid were synthesized, bearing an alkoxy group in position 3 and different substituents on the benzene ring in position 2 of the flavone nucleus. The compounds were tested for direct cytotoxicity against four human tumor cell lines and for indirect antitumor effects by measuring their ability to enhance lytic properties of murine macrophages and human monocytes. Though direct toxicity was very low, the compounds were able to induce significant indirect toxicity. Notably, most of them (4c, 4d, 4e, 4f, 4h, 4i, 4m,4n, and 4o) showed important activity on human monocytes and could be regarded as the first flavone derivatives endowed with such activity. Particularly interesting seem to be compounds 4m and 4n, which showed IC(50) values up to 7 times higher than DMXAA, which has now completed phase I clinical trials.

Puumala virus pulmonary syndrome in a Romanian immigrant.

Hantaviruses belong to the Bunyaviridae family, which is comprised of Bunyavirus, Phlebovirus, Nairovirus, and Hantavirus. Euroasiatic Hantaviruses belong to two distinct subfamilies: Murinae (comprising Hantaan, Dobrava, and Seoul viruses), which are responsible for hemorrhagic fever with renal syndrome (HFRS), and Arvicolinae (comprising Puumala virus), responsible for nephropathia epidemica (NE) and HFRS. On the contrary, the New World Hantavirus belongs to the Sigmodontinae subfamily and includes the North American viruses: Sin Nombre, Monongahela; New York, Bayou, Black Creek Canal, and the South American, which comprise the Andes, Oran, Lechiguanas, Laguna Negra, Juquitiba; both groups are responsible for the hantavirus pulmonary syndrome (HPS).

Antioxidant Potential of the Polyherbal Formulation "ImmuPlus": A Nutritional Supplement for Horses.

In order to counteract harmful effects of oxidative stress due to pathological conditions or physical exercise, horses are often administered dietary supplements having supposed high antioxidant activities. The aim of the present study was to identify the in vitro antioxidant potential of "ImmuPlus", a polyherbal formulation (Global Herbs LTD, Chichester, West Sussex, Great Britain), containing three medicinal plants (Withania somnifera, Tinospora cordifolia, and Emblica officinalis), known in Ayurveda for their use in human disease treatment. Extracts obtained by different solvents (water, methanol, ethanol, acetone, and hexane) were tested for total antioxidant capacity, total reducing power, scavenging activity against DPPH radical, and total polyphenol and flavonoid contents. Our results showed that, except as regards hexane, all the used solvents are able to extract compounds having high antioxidant activity, even when compared to ascorbic acid. Regression analysis showed significant correlations between antioxidant properties and polyphenol/flavonoid contents, indicating the latter, known for their beneficial effects on health of human and animal beings, as major components responsible for the strong antioxidant capacities. Moreover, obtained results suggest the effective role of the polyherbal mixture as good source of antioxidants in horses.

Synaptoneurosome micromethod for fractionation of mouse and human brain, and primary neuronal cultures.

Brain and primary neuron fractions enriched in synaptic terminals are important tools for neuroscientists in biochemical, neuroanatomical and physiological studies. We describe an annotated updated micro-method for preparing synaptoneurosomes (SNs) enriched in presynaptic and postsynaptic elements. An easy to follow, step-by-step, protocol is provided for making SNs from small amounts of mammalian brain tissue. This includes novel applications for material obtained from human neurosurgical procedures and primary rat neuronal cultures. Our updated method for preparing SNs using smaller amounts of tissue provides a valuable new tool and expands the capabilities of neuroscientists.

Lipid rafts and clathrin cooperate in the internalization of PrP in epithelial FRT cells.

BACKGROUND: The cellular prion protein (PrP(C)) plays a key role in the pathogenesis of Transmissible Spongiform Encephalopathies in which the protein undergoes post-translational conversion to the infectious form (PrP(Sc)). Although endocytosis appears to be required for this conversion, the mechanism of PrP(C) internalization is still debated, as caveolae/raft- and clathrin-dependent processes have all been reported to be involved. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the mechanism of PrP(C) endocytosis in Fischer Rat Thyroid (FRT) cells, which lack caveolin-1 (cav-1) and caveolae, and in FRT/cav-1 cells which form functional caveolae. We show that PrP(C) internalization requires activated Cdc-42 and is sensitive to cholesterol depletion but not to cav-1 expression suggesting a role for rafts but not for caveolae in PrP(C) endocytosis. PrP(C) internalization is also affected by knock down of clathrin and by the expression of dominant negative Eps15 and Dynamin 2 mutants, indicating the involvement of a clathrin-dependent pathway. Notably, PrP(C) co-immunoprecipitates with clathrin and remains associated with detergent-insoluble microdomains during internalization thus indicating that PrP(C) can enter the cell via multiple pathways and that rafts and clathrin cooperate in its internalization. CONCLUSIONS/SIGNIFICANCE: These findings are of particular interest if we consider that the internalization route/s undertaken by PrP(C) can be crucial for the ability of different prion strains to infect and to replicate in different cell lines.