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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.

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. 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. 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.

Background The cellular prion protein (PrPC) plays a key role in the pathogenesis of Transmissible Spongiform Encephalopathies in which the protein undergoes post-translational conversion to the infectious form (PrPSc). Although endocytosis appears to be required for this conversion, the mechanism of PrPC 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 PrPC 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 PrPC 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 PrPC endocytosis. PrPC 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, PrPC co-immunoprecipitates with clathrin and remains associated with detergent-insoluble microdomains during internalization thus indicating that PrPC 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 PrPC can be crucial for the ability of different prion strains to infect and to replicate in different cell lines.

The effects of the dietary inclusion of a polyherbal formulation based on three powdered herbs (W. somnifera, T. cordifolia, O. sanctum) on some physiological and immune parameters were studied in healthy and stressed laying hens. The effects of the dietary polyherbal formulation were also compared with those of dietary ascorbic acid (AA) supplement, nowadays considered one of the most potent immunostimulant substances widely used as a food supplement.Experimental data did not show any positive effects, or very low ones, on the assessed parameters in healthy hens as a consequence of the two dietary supplementations. On the contrary, the dietary inclusion of the polyherbal mixture or AA partially counteracted the adverse effects in hens subjected to a moderate and transient dexamethasone-induced stress, when ameliorating effects on natural IgM antibody level, specific antibody response, total immunoglobulin content, respiratory burst activity and total antioxidant capacity were shown. The obtained results justify the ethnomedical use of this polyherbal mixture in stressed laying hens in which faster recovery has been demonstrated, whereas healthy specimens did not seem to substantially benefit from the dietary integration, neither with the polyherbal product nor with AA. Thus, the presence of nutraceutical compounds in several herbal plants exerting no side-effects might be useful for exploring them as an alternative to allopathic substances for preventive or therapeutic purposes in poultry.

Tunnelling nanotubes provide a means of intercellular communication. They are now shown to facilitate spreading of prions between neuronal cells, as well as their propagation from dentritic cells to primary neurons. In variant Creutzfeldt–Jakob disease, prions (PrP Sc ) enter the body with contaminated foodstuffs and can spread from the intestinal entry site to the central nervous system (CNS) by intercellular transfer from the lymphoid system to the peripheral nervous system (PNS) 1 . Although several means 2 , 3 , 4 and different cell types 5 , 6 , 7 have been proposed to have a role, the mechanism of cell-to-cell spreading remains elusive. Tunnelling nanotubes (TNTs) have been identified between cells 8 , 9 , 10 , 11 , 12 , both in vitro and in vivo 10 , 11 , 13 , and may represent a conserved means of cell-to-cell communication 14 , 15 , 16 . Here we show that TNTs allow transfer of exogenous and endogenous PrP Sc between infected and naive neuronal CAD cells 17 . Significantly, transfer of endogenous PrP Sc aggregates was detected exclusively when cells chronically infected with the 139A mouse prion strain were connected to mouse CAD cells by means of TNTs, identifying TNTs as an efficient route for PrP Sc spreading in neuronal cells. In addition, we detected the transfer of labelled PrP Sc from bone marrow-derived dendritic cells to primary neurons connected through TNTs. Because dendritic cells can interact with peripheral neurons in lymphoid organs, TNT-mediated intercellular transfer would allow neurons to transport prions retrogradely to the CNS 1 . We therefore propose that TNTs are involved in the spreading of PrP Sc within neurons in the CNS and from the peripheral site of entry to the PNS by neuroimmune interactions with dendritic cells.

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.

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.

The aim of this study, carried out within the NOVOROD project (PSR Campania 2007-2013 Misura 124 HC), was to evaluate the aromatic profile of milk and dairy products of dairy cows supplemented with destoned olive cake (DOC). Two techniques for the headspace aroma profile analysis: the thermal desorption by gas chromatography with a mass selective (GC/MS) detector and the electronic nose were compared. The trial was performed into a dairy cow farm. Ten dairy cows were allotted into two homogenous groups: control and experimental. Animals received the same diet, while in the experimental group the concentrate supplementation (15% dry matter) was replaced with DOC. After two weeks of adaptation, animals were fed with the experimental diet for other 15 days. During the experimental period, the milk was collected and cumulatively processed in Caciotta cheese (a soft cheese, 25 days ripened) and in Semicotto cheese (hard cheese, 3 months ripened) for 13 times for each group. Milk and cheese volatile organic compounds (VOCs) were extracted and concentrated with dynamic headspace method and determined by GC/MS. Statistical analysis of data, expressed in arbitrary units (a.u.=peak area × 10–6) and allotted in compound classes, was carried out by ANOVA. All data collected of VOCs were processed with principal component analysis (PCA). Milk and cheese samples were comparatively analysed with the electronic nose, fitted with a tenmetal oxide sensors electronic device. PCA has been used for the evaluation of the pattern data. Our results show that the use of DOC, as an unconventional feed for livestock, has no effect on the aromatic profile of both milk and dairy products. In fact, no significant differences were found in both milk and dairy products between the two groups using the GC/MS method. Also the discriminating analysis carried out by electronic nose did not show significant differences between the two groups. The two compared techniques seem to lead to the same result.

The present experiment was carried out in order to evaluate the potential adjuvant effect of bovine lactoferrin (bLF) in modulating the specific antibody (Ab) response to an exogenous protein, the human-γ-globulins (HγG), injected intraperitoneally in rainbow trout Oncorhynchus mykiss (Walbaum 1792). Moreover, serum natural antibody (NatAb) level, serum total immunoglobulin (IgM) level and specific Ab response to bLF were also analysed. Experimental data showed that HγG is able to stimulate a specific Ab response when not adjuvanted with Freund’s complete adjuvant (FCA), whereas bLF induces a specific Ab response only if adjuvanted with FCA. Statistical analyses revealed that neither bLF or FCA administered singly had significant effects on serum NatAb and IgM levels, whereas the specific immune response observed against HγG when adjuvanted with bLF was comparable to that achieved when FCA was used as the adjuvant. These results provide evidence of the potential for bLF to be used as an adjuvant for salmonids.

The accumulation of misfolded [alpha]-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.sup.+ 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.sup.+ 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.