Frequencies of PrP genotypes and their implication for breeding against scrapie susceptibility in nine Pakistani sheep breeds

Prion protein (PrP) gene of 308 sheep was genotyped to investigate polymorphisms at scrapie-associated codons 136, 154 and 171 to assess the resistance of nine different Pakistani sheep breeds to natural/typical scrapie. As a result six genotypes were established on the basis of polymorphic codons 154 and 171. The most scrapie-susceptible codon 136 (A/V) was monomorphic (A) in all breeds. Wild-type genotype ARQ/ARQ was detected with maximum prevalence ranging from 63.2% in crossbred Pak-karakul to 100% in native Buchi, Kachi and Thalli breeds. The most frequent of typical scrapie-associated genotypes was ARQ/ARR as indicated by five of nine breeds. The coding region of PrP gene of 49 animals from the total sampled was also sequenced to ascertain additional polymorphisms. Polymorphism was found in 13 animals of the six breeds in codons 101(Q/R), 112(M/T), 146(N/S) and 189(Q/L) and ten genotypes were established on the basis of these polymorphic codons. Only Hissardale possessed five of the ten genotypes. The most frequent genotype was M¹¹²ARQ/T¹¹²ARQ detected in Hissardale, Pak-karakul and Awassi, whereas genotypes ARQr²³¹/ARQr²³¹ and ARQR²³¹/ARQr²³¹ (established on the basis of silent polymorphism agg/cgg-R/R) were detected in all breeds. Some animals consisted of three polymorphisms at different PrP codons that are not common in European breeds. An infrequent double heterozygosity (c/c a/g g/t) for codon 171 resulting in a genotype R/H was also detected in three animals each one from Kajli, Hissardale and Pak-karakul. This study concludes that all native sheep breeds are poor in scrapie-resistant PrP genotypes and could contract scrapie if exposed to prions.     

Involvement of lysosomal degradation in VEGF-C-induced down-regulation of VEGFR-3

The vascular endothelial growth factor (VEGF)-C-induced down-regulation of VEGF receptor (VEGFR)-3 is important in lymphangiogenesis. Here, we demonstrate that VEGF-C, -D, and -C156S, but not VEGF-A, down-regulate VEGFR-3. VEGF-C stimulates VEGFR-3 tyrosyl phosphorylation and transient phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinases in lymphatic endothelial cells. VEGF-C-induced down-regulation of VEGFR-3 was blocked by a VEGF-C trap, tyrosine kinase inhibitor, and leupeptin, pepstatin, and E64 (LPE), but was unaffected by Notch 1 activator and γ-secretase inhibitors. Our findings indicate that VEGF-C down-regulates VEGFR-3 in lymphatic endothelial cells through VEGFR-3 kinase activation and, in part, via lysosomal degradation.     

Piperazine derivatives inhibit PrP/PrP propagation in vitro and in vivo

Prion diseases are fatal neurodegenerative disorders, which are not curable and no effective treatment exists so far. The major neuropathological change in diseased brains is the conversion of the normal cellular form of the prion protein PrPc^C into a disease-associated isoform PrP^Sc. PrP^Sc accumulates into multimeres and fibrillar aggregates, which leads to the formation of amyloid plaques. Increasing evidence indicates a fundamental role of PrP^Sc species and its aggregation in the pathogenesis of prion diseases, which initiates the pathological cascade and leads to neurodegeneration accompanied by spongiform changes. In search of compounds that have the potential to interfere with PrP^Sc formation and propagation, we used a cell based assay for the screening of potential aggregation inhibitors. The assay deals with a permanently prion infected cell line that was adapted for a high-throughput screening of a compound library composed of 10,000 compounds (DIVERset 2, ChemBridge).     

Synthetic fibril peptide promotes clearance of scrapie prion protein by lysosomal degradation

Transmissible spongiform encephalopathies are infectious and neurodegenerative disorders that cause neural deposition of aggregates of the disease-associated form of PrP(Sc). PrP(Sc) reproduces by recruiting and converting the cellular PrP(C), and ScN2a cells support PrP(Sc) propagation. We found that incubation of ScN2a cells with a fibril peptide named P9, which comprises an intrinsic sequence of residues 167-184 of mouse PrP(C), significantly reduced the amount of PrP(Sc) in 24 hr. P9 did not affect the rates of synthesis and degradation of PrP(C). Interestingly, immunofluorescence analysis showed that the incubation of ScN2a cells with P9 induced colocalization of the accumulation of PrP with cathepsin D-positive compartments, whereas the accumulation of PrP in the cells without P9 colocalized mainly with lysosomal associated membrane proteins (LAMP)-1-positive compartments but rarely with cathepsin D-positive compartments in perinuclear regions. Lysosomal enzyme inhibitors attenuated the anti-PrP(Sc) activity; however, a proteasome inhibitor did not impair P9 activity. In addition, P9 neither promoted the ubiquitination of cellular proteins nor caused the accumulation of LC3-II, a biochemical marker of autophagy. These results indicate that P9 promotes PrP(Sc) redistribution from late endosomes to lysosomes, thereby attaining PrP(Sc) degradation.     

Differential expression of Prnp and Sprn in scrapie infected sheep also reveals Prnp genotype specific differences

The central role for PrP in the pathogenesis of the transmissible spongiform encephalopathies (TSEs) is illustrated by the resistance of Prnp^0/0 mice to disease and by the inverse association of Prnp gene dosage with incubation period. Understanding the role of PrP^C in TSEs necessitates knowledge of expression levels of the Prnp gene during the development of disease. SSBP/1 scrapie shows a defined pattern of disease progression and here we show that Prnp and shadow of PrP (Sprn) are differentially expressed in different brain areas and lymphoid tissues. Counter-intuitively we found that there is no positive correlation between expression of Prnp or Sprn and patterns of disease progression. Prnp and Sprn expression levels are both influenced by Prnp genotype; although the scrapie-sensitive VRQ/VRQ sheep did not express the highest level of either. In addition, infection with SSBP/1 scrapie seems to have little effect on either PrP or Shadoo expression levels.     

Scrapie-infected mice and PrP knockout mice share abnormal localization and activity of neuronal nitric oxide synthase

PrP(Sc), the only identified component of the scrapie prion, is a conformational isoform of PrPc. The physiological role of PrPc, a glycolipid-anchored glycoprotein, is still unknown. We have shown previously that neuronal nitric oxide synthase (nNOS) activity is impaired in the brains of mice sick with experimental scrapie as well as in scrapie-infected neuroblastoma cells. In this work we investigated the cell localization of nNOS in brains of wild-type and scrapie-infected mice as well as in mice in which the PrP gene was ablated. We now report that whereas in wild-type mice, nNOS, like PrPc, is associated with detergent-insoluble cholesterol-rich membranous microdomains (rafts), this is not the case in brains of scrapie-infected or in those of adult PrP(0/0) mice. Also, adult PrP(0/0), like scrapie-infected mice, show reduced nNOS activity. We suggest that PrPc may play a role in the targeting of nNOS to its proper subcellular localization. The similarities of nNOS properties in PrP(0/0) as compared with scrapie-infected mice suggest that at least this role of PrPc may be impaired in scrapie-infected brains.     

Comparative profiling of highly enriched 22L and Chandler mouse scrapie prion protein preparations

Transmissible spongiform encephalopathies (TSEs) or prion diseases are characterized by the accumulation of an aggregated isoform of the prion protein (PrP). This pathological isoform, termed PrP^Sc, appears to be the primary component of the TSE infectious agent or prion. However, it is not clear to what extent other protein cofactors may be involved in TSE pathogenesis or whether there are PrP^Sc‐associated proteins which help to determine TSE strain‐specific disease phenotypes. We enriched PrP^Sc from the brains of mice infected with either 22L or Chandler TSE strains and examined the protein content of these samples using nanospray LC‐MS/MS. These samples were compared with “mock” PrP^Sc preparations from uninfected brains. PrP was the major component of the infected samples and ferritin was the most abundant impurity. Mock enrichments contained no detectable PrP but did contain a significant amount of ferritin. Of the total proteins identified, 32% were found in both mock and infected samples. The similarities between PrP^Sc samples from 22L and Chandler TSE strains suggest that the non‐PrP^Sc protein components found in standard enrichment protocols are not strain specific.     

Amino acid inhibition of the autophagic/lysosomal pathway of protein degradation in isolated rat hepatocytes

Protein degradation in isolated rat hepatocytes, as measured by the release of [¹⁴C]valine from pre-labelled protein, is partly inhibited by a physiologically balanced mixture of amino acids. The inhibition is largely due to the seven amino acids leucine, phenylalanine, tyrosine, tryptophan, histidine, asparagine and glutamine.When the amino acids are tested individually at different concentrations, asparagine and glutamine are the strongest inhibitors. However, when various combinations are tested, a mixture of the first five amino acids as well as a combination of leucine and asparagine inhibit protein degradation particularly strongly.The inhibition brought about by asparagine plus leucine is not additive to the inhibition by propylamine, a lysosomotropic inhibitor; thus indicating that the amino acids act exclusively upon the lysosomal pathway of protein degradation.Following a lag of about 15 min the effect of asparagine plus leucine is maximal and equal to the effect of propylamine, suggesting that their inhibition of the lysosomal pathway is complete as well as specific.Degradation of endocytosed ¹²⁵I-labelled asialofetuin is not affected by asparagine plus leucine, indicating that the amino acids do not affect lysosomes directly, but rather inhibit autophagy at a step prior to the fusion of autophagic vacuoles with lysosomes.The aminotransferase inhibitor, aminooxyacetate, does not prevent the inhibitory effect of any of the amino acids, i.e. amino acid metabolites are apparently not involved.     

Down-regulation of brain-derived neurotrophic factor and its signaling components in the brain tissues of scrapie experimental animals

Prion is a unique nucleic acid-free pathogen that causes human and animal fatal neurodegenerative diseases. Brain-derived neurotrophic factor (BDNF) is a prototypic neurotrophin that helps to support the survival of existing neurons, and encourage the growth and differentiation of new neurons and synapses through axonal and dendritic sprouting. There are two distinct classes of glycosylated receptors, neurotrophin receptor p75 (p75NTR) and tropomyosin-related kinase (Trk), that can bind to BDNF. To obtain insights into the possible alterations of brain BDNF and its signaling pathway in prion disease, the levels of BDNF and several molecules in the BDNF pathway in the brain tissues of scrapie agents 263K-infected hamsters were separately evaluated. Western blots and/or immunohistochemical (IHC) assays revealed that BDNF, TrkB, GRB2 and p75NTR, were significantly downregulated in the brain tissues of scrapie-infected rodents at terminal stage. Double-stained immunofluorescent assay (IFA) demonstrated that BDNF and phospho-TrkB predominately expressed in neurons. Dynamic analyses of the brain samples collected at the different time-points during the incubation period illustrated continuous decreases of BDNF, TrkB, phospho-TrkB, GRB2 and p75NTR, which correlated well with neuron loss. However, these proteins remained almost unchanged in the prion infected cell line SMB-S15 compared with those of its normal cell line SMB-PS. These data suggest that the BDNF signaling pathway is severely hindered in the brains of prion disease, which may contribute, at least partially, to the neuron death.     

Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity.

The scrapie amyloid (prion) protein (PrP27-30) is the protease-resistant core of a larger precursor (PrPSc) and a component of the infectious scrapie agent; the potential to form amyloid is a result of posttranslational event or conformational abnormality. The conformation, heat stability, and solvent-induced conformational transitions of PrP27-30 were studied in the solid state in films by CD spectroscopy and correlated with the infectivity of rehydrated and equilibrated films. The exposure of PrP27-30 in films to 60 degrees C, 100 degrees C, and 132 degrees C for 30 min did not change the beta-sheet secondary structure; the infectivity slightly diminished at 132 degrees C and correlated with a decreased solubility of PrP27-30 in sodium dodecyl sulfate (SDS), probably due to cross-linking. Exposing PrP27-30 films to formic acid (FA), trifluoroacetic acid (TFA), trifluoroethanol (TFE), hexafluoro-2-propanol (HFIP), and SDS transformed the amide CD band, diminished the mean residue ellipticity of aromatic bands, and inactivated scrapie infectivity. The convex constraint algorithm (CAA) deconvolution of the CD spectra of the solvent-exposed and rehydrated solid state PrP27-30 identified five common spectral components. The loss of infectivity quantitatively correlated with a decreasing proportion of native, beta-pleated sheet-like secondary structure component, an increasing amount of alpha-helical component, and an increasingly disordered tertiary structure. The results demonstrate the unusual thermal stability of the beta-sheet secondary structure of PrP27-30 protein in the solid state. The conformational perturbations of PrP27-30 parallel the changes in infectivity and suggest that the beta-sheet structure plays a key role in the physical stability of scrapie amyloid and in the ability to propagate and replicate scrapie.     

Differential effects of proteinase inhibitors and amines on the lysosomal and non-lysosomal pathways of protein degradation in isolated rat hepatocytes

Ammonia, which like other lysosomotropic amines inhibits protein degradation in isolated rat hepatocytes by 70–80%, was utilized as a diagnostic tool to distinguish between the relative effects of various proteinase inhibitors on the lysosomal and non-lysosomal pathways of intracellular protein degradation.Leupeptin was found to inhibit lysosomal protein degradation by 80–85%, and non-lysosomal degradation by about 15%. Antipain had a similar, but somewhat weaker effect. Pepstain, bestatin and aprotinin (Traysylol) produced minor inhibitory effects (possibly on both degradation, pathways), whereas bacitracin and soybean trypsin inhibitor wre ineffective.Chymostatin inhibited lysosomal protein degradation by about 45%, whereas the non-lysosomal pathway was inhibited by more than 50%. Chymostatin was unique among the inhibitors tested in causing such a pronounced effect on non-lysosomal protein degradation, and appeared to selectively inhibit the energy-dependent portion of this pathway.The effects of the various inhibitors were additive to the extent expected on the basis of their kwown actions on lysosomal and non-lysosomal protein degradation. Thus, a combination of methylamine, leupeptine and chymostatin inhibited overall protein degradation by about 90%, resulting in a substantial improvement of the cellular nitrogen balance.The degradation inhibitors caused a partial inhibition of protein synthesis, apparently mainly by shutting down the supply of amino acids from the lysosome. The inhibitory effects of leupeptin and antipain were completely reversed by amino acid addition, whereas some inhibition remained in the case of chymostatin and the lysosomotropic amines, possibly reflecting a certain nonspecific toxicity.     

Scrapie infection activates the replication of ecotropic, xenotropic, and polytropic murine leukemia virus (MuLV) in brains and spinal cords of senescence-accelerated mice: implication of MuLV in progression of scrapie pathogenesis

Senescence-accelerated mice (SAMP8) have a short life span, whereas SAMR1 mice are resistant to accelerated senescence. Previously it has been reported that the Akv strain of ecotropic murine leukemia virus (E-MuLV) was detected in brains of SAMP8 mice but not in brains of SAMR1 mice. In order to determine the change of MuLV levels following scrapie infection, we analyzed the E-MuLV titer and the RNA expression levels of E-MuLV, xenotropic MuLV, and polytropic MuLV in brains and spinal cords of scrapie-infected SAM mice. The expression levels of the 3 types of MuLV were increased in scrapie-infected mice compared to control mice; E-MuLV expression was detected in infected SAMR1 mice, but only in the terminal stage of scrapie disease. We also examined incubation periods and the levels of PrPSc in scrapie-infected SAMR1 (sR1) and SAMP8 (sP8) mice. We confirmed that the incubation period was shorter in sP8 (210+/-5 days) compared to sR1 (235+/-10 days) after intraperitoneal injection. The levels of PrPSc in sP8 were significantly greater than sR1 at 210+/-5 days, but levels of PrPSc at the terminal stage of scrapie in both SAM strains were virtually identical. These results show the activation of MuLV expression by scrapie infection and suggest acceleration of the progression of scrapie pathogenesis by MuLV.     

Transgenic mice expressing bovine PrP with a four extra repeat octapeptide insert mutation show a spontaneous, non-transmissible, neurodegenerative disease and an expedited course of BSE infection

Transgenic (Tg) mice carrying four extra octapeptide repeats (OR) in the bovine PrP gene (10OR instead of 6) have been generated. In these mice, neuropathological changes were observed depending upon the level of transgene expression. These changes primarily involved a slowly advancing neurological disorder, characterized clinically by ataxia, and neuropathologically, by vacuolization in different brain areas, gliosis, and loss of cerebellar granule cells. Accumulation of insoluble bovine 10OR-PrP (bo10OR-PrP) was observed depending on the level of expression but no infectivity was found associated with this insoluble form. We also compared the behavior of bo6OR-PrP and bo10OR-PrP Tg mouse lines in response to BSE infection. BSE-inoculated bo10ORTg mice showed an altered course of BSE infection, reflected by reduced incubation times when compared to bo6ORTg mice expressing similar levels of the wild type 6OR-PrP. In BSE-inoculated mice, it was possible to detect PrP(res) in 100% of the animals. While insoluble bo10OR-PrP from non-inoculated bo10ORTg mice was non-infectious, brain homogenates from BSE-inoculated bo10ORTg mice were highly infectious in all the Tg mouse lines tested. This Tg mouse model constitutes a new way of understanding the pathobiology of bovine transmissible spongiform encephalopathy. Its potential applications include the assessment of new therapies against prion diseases.     

The effect of chloroquine on lysosomal function and cell-coat glycoprotein transport in the absorptive cells of cultured human small-intestinal tissue

Summary The effect of chloroquine, an inhibitor of intralysosomal catabolism, on the synthesis, transport, and degradation of cell-coat glycoproteins in absorptive cells of cultured human small-intestinal tissue was investigated by morphometrical, autoradiographical, and biochemical methods. Neither synthesis nor transport of cell-coat material was affected by the drug, but culturing of the absorptive cells in the presence of chloroquine led to a dose- and time-dependent enlargement of the dense bodies; other cell structures showed no alterations. ³H-fucose-labelled material accumulated in the dense bodies of the absorptive cells of these cultures. Since no increase of -glucuronidase and acid phosphatase activity (both lysosomal enzymes of glycoprotein nature) was found, this accumulation of radiolabelled material can be explained as a chloroquine-mediated inhibition of the degradation of cell-coat glycoproteins. These macromolecules probably enter the lysosome-like bodies by a crinophagic mechanism, i.e., fusion of these organelles with the apical vesicles and tubules involved in intracellular transport. These findings suggest that the lysosome-like bodies have a function in the regulation of cell-coat glycoprotein transport in human intestinal absorptive cells, i.e., the degradation of excess cell-coat material.     

Caprine PrP variants harboring Asp-146, His-154 and Gln-211 alleles display reduced convertibility upon interaction with pathogenic murine prion protein in scrapie infected cells

Scrapie, the prion disease of sheep and goats, is a devastating malady of small ruminants. Due to its infectious nature, epidemic outbreaks may occur in flocks/herds consisting of highly susceptible animals. Field studies identified scrapie-protective caprine PrP variants, harboring specific single amino acid changes (Met-142, Arg-143, Asp-146, Ser-146, His-154, Gln-211 and Lys-222). Their effects are under further evaluation, and aim to determine the most protective allele. We assessed some of these variants (Asp-146, His-154, Gln-211 and Lys-222), after their exogenous expression as murine-caprine chimeras in a scrapie- infected murine cell line. We report that exogenously expressed PrPs undergo conformational conversion upon interaction with the endogenous pathological murine prion protein (PrP^SC), which results in the detection of goat-specific and partially PK-resistant moieties. These moieties display a PK-resistance pattern distinct from the one detected in natural goat scrapie cases. Within this cellular model, distinct conformational conversion potentials were assigned to the tested variants. Molecules carrying the Asp-146, His-154 and Gln-211 alleles showed significantly lower conversion levels compared to wild type, confirming their protective effects against scrapie. Although we utilized a heterologous conversion system, this is to our knowledge, the first study of caprine PrP variants in a cellular context of scrapie, that confirms the protective effects of some of the studied alleles.     

The thiol proteinase inhibitors,Z-Phe-PheCHN2 and Z-Phe-AlaCHN2, inhibit lysosomal protein degradation in isolated rat hepatocytes

The effects on protein metabolism of Z-Phe-PheCHN₂ and Z-Phe-AlaCHN₂ were examined in isolated rat hepatocytes. The two thiol proteinase inhibitors caused a drastic reduction in the degradation of both endogenous adn endocytosed (asialo-fetuin) protein. The inhibition was not additive to that of the lysosomotropic base methylamine, indicating that Z-Phe-PheCHN₂ and Z-Phe-AlaCHN₂ only affect lysosomal degradation. At high concentrations (0.1–1 mM) both inhibitors reduced protein synthesis strongly. This finding indicates non-specific/toxic effects, which may limit the usefulness of the inhibitors.     

Proteasomal and lysosomal clearance of faulty secretory proteins: ER-associated degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD) pathways

About 40% of the eukaryotic cell’s proteins are inserted co- or post-translationally in the endoplasmic reticulum (ER), where they attain the native structure under the assistance of resident molecular chaperones and folding enzymes. Subsequently, these proteins are secreted from cells or are transported to their sites of function at the plasma membrane or in organelles of the secretory and endocytic compartments. Polypeptides that are not delivered within the ER (mis-localized proteins, MLPs) are rapidly destroyed by cytosolic proteasomes, with intervention of the membrane protease ZMPSTE24 if they remained trapped in the SEC61 translocation machinery. Proteins that enter the ER, but fail to attain the native structure are rapidly degraded to prevent toxic accumulation of aberrant gene products. The ER does not contain degradative devices and the majority of misfolded proteins generated in this biosynthetic compartment are dislocated across the membrane for degradation by cytosolic 26S proteasomes by mechanisms and pathways collectively defined as ER-associated degradation (ERAD). Proteins that do not engage ERAD factors, that enter aggregates or polymers, are too large, display chimico/physical features that prevent dislocation across the ER membrane (ERAD-resistant misfolded proteins) are delivered to endo-lysosome for clearance, by mechanisms and pathways collectively defined as ER-to-lysosomes-associated degradation (ERLAD). Emerging evidences lead us to propose ERLAD as an umbrella term that includes the autophagic and non-autophagic pathways activated and engaged by ERAD-resistant misfolded proteins generated in the ER for delivery to degradative endo-lysosomes.     

Glucocorticoids inhibit precursor incorporation into protein in splenic lymphocytes by stimulating protein degradation and expanding intracellular amino acid pools

In the presence of tracer concentrations of extracellular leucine (5 μM), treatment of rat splenic lymphocyte suspensions in vitro with 1 μM dexamethasone for 2.5–4 h caused a 30–35% inhibition of [³H]leucine incorporation into protein. As the extracellular leucine concentration was raised to 5 mM, this inhibition was progressively reduced to 0–12%. This phenomenon correlated with a marked dependence on extracellular leucine concentration of the dexamethasone-dependent enlargement of free intracellular leucine pools in splenic lymphocytes: a 123% increase in pool size with tracer extracellular leucine; a 10% increase with 5 mM leucine. Varying extracellular leucine had no effect on: (1) nuclear [³H]dexamethasone binding by the cells; (2) the concentration of dexamethasone needed for half-maximal inhibition of [³H]leucine incorporation; (3) the time course of onset and maximal expression of the hormonal inhibition of [³H]leucine incorporation; or (4) the magnitude of dexamethasone-dependent inhibition of [³H]uridine incorporation into RNA by these cells. There was no detectable effect of dexamethasone on uptake and retention of [³H]leucine by the cells, regardless of the extracellular leucine concentration. Treatment of splenic lymphocytes for 4 h in vitro with 1 μM dexamethasone caused a small shift of ribosomes from larger aggregate polysomes to smaller forms. Thus, glucocorticoid-induced inhibition of amino acid incorporation in splenic lymphocytes is a multicomponent response, of which an actual decrease in protein synthesis is only a small part. Enlargement of free intracellular amino acid pools, probably resulting from increased protein degradation, is the major contributing factor to the hormonal inhibition of amino acid incorporation.