Reactive astrocytes associated with prion disease impair the blood brain barrier.

BACKGROUND: Impairment of the blood-brain barrier (BBB) is considered to be a common feature among neurodegenerative diseases, including Alzheimer's, Parkinson's and prion diseases. In prion disease, increased BBB permeability was reported 40 years ago, yet the mechanisms behind the loss of BBB integrity have never been explored. Recently, we showed that reactive astrocytes associated with prion diseases are neurotoxic. The current work examines the potential link between astrocyte reactivity and BBB breakdown. RESULTS: In prion-infected mice, the loss of BBB integrity and aberrant localization of aquaporin 4 (AQP4), a sign of retraction of astrocytic endfeet from blood vessels, were noticeable prior to disease onset. Gaps in cell-to-cell junctions along blood vessels, together with downregulation of Occludin, Claudin-5 and VE-cadherin, which constitute tight and adherens junctions, suggested that loss of BBB integrity is linked with degeneration of vascular endothelial cells. In contrast to cells isolated from non-infected adult mice, endothelial cells originating from prion-infected mice displayed disease-associated changes, including lower levels of Occludin, Claudin-5 and VE-cadherin expression, impaired tight and adherens junctions, and reduced trans-endothelial electrical resistance (TEER). Endothelial cells isolated from non-infected mice, when co-cultured with reactive astrocytes isolated from prion-infected animals or treated with media conditioned by the reactive astrocytes, developed the disease-associated phenotype observed in the endothelial cells from prion-infected mice. Reactive astrocytes were found to produce high levels of secreted IL-6, and treatment of endothelial monolayers originating from non-infected animals with recombinant IL-6 alone reduced their TEER. Remarkably, treatment with extracellular vesicles produced by normal astrocytes partially reversed the disease phenotype of endothelial cells isolated from prion-infected animals. CONCLUSIONS: To our knowledge, the current work is the first to illustrate early BBB breakdown in prion disease and to document that reactive astrocytes associated with prion disease are detrimental to BBB integrity. Moreover, our findings suggest that the harmful effects are linked to proinflammatory factors secreted by reactive astrocytes.

Inflammatory response of microglia to prions is controlled by sialylation of PrPSc.

Neuroinflammation is recognized as one of the obligatory pathogenic features of neurodegenerative diseases including Alzheimer's, Parkinson's or prion diseases. In prion diseases, space and time correlations between deposition of disease-associated, pathogenic form of the prion protein or PrPSc and microglial-mediated neuroinflammation has been established. Yet, it remains unclear whether activation of microglia is triggered directly by a contact with PrPSc, and what molecular features of PrPSc microglia sense and respond to that drive microglia to inflammatory states. The current study asked the questions whether PrPSc can directly trigger activation of microglia and whether the degree of microglia response depends on the nature of terminal carbohydrate groups on the surface of PrPSc particles. PrPSc was purified from brains of mice infected with mouse-adapted prion strain 22L or neuroblastoma N2a cells stably infected with 22L. BV2 microglial cells or primary microglia were cultured in the presence of purified 22L. We found that exposure of BV2 cells or primary microglia to purified PrPSc triggered proinflammatory responses characterized by an increase in the levels of TNFα, IL6, nitric oxide (NO) and expression of inducible Nitric Oxide Synthase (iNOS). Very similar patterns of inflammatory response were induced by PrPSc purified from mouse brains and neuroblastoma cells arguing that microglia response is independent of the source of PrPSc. To test whether the microglial response is mediated by carbohydrate epitopes on PrPSc surface, the levels of sialylation of PrPSc N-linked glycans was altered by treatment of purified PrPSc with neuraminidase. Partial cleavage of sialic acid residues was found to boost the inflammatory response of microglia to PrPSc. Moreover, transient degradation of Iκßα observed upon treatment with partially desialylated PrPSc suggests that canonical NFκB activation pathway is involved in inflammatory response. The current study is the first to demonstrate that PrPSc can directly trigger inflammatory response in microglia. In addition, this work provides direct evidence that the chemical nature of the carbohydrate groups on PrPSc surface is important for microglial activation.

Post-conversion sialylation of prions in lymphoid tissues.

Sialylated glycans on the surface of mammalian cells act as part of a "self-associated molecular pattern," helping the immune system to recognize "self" from "altered self" or "nonself." To escape the host immune system, some bacterial pathogens have evolved biosynthetic pathways for host-like sialic acids, whereas others recruited host sialic acids for decorating their surfaces. Prions lack nucleic acids and are not conventional pathogens. Nevertheless, prions might use a similar strategy for invading and colonizing the lymphoreticular system. Here we show that the sialylation status of the infectious, disease-associated state of the prion protein (PrP(Sc)) changes with colonization of secondary lymphoid organs (SLOs). As a result, spleen-derived PrP(Sc) is more sialylated than brain-derived PrP(Sc). Enhanced sialylation of PrP(Sc) is recapitulated in vitro by incubating brain-derived PrP(Sc) with primary splenocytes or cultured macrophage RAW 264.7 cells. General inhibitors of sialyltranserases (STs), the enzymes that transfer sialic acid residues onto terminal positions of glycans, suppressed extrasialylation of PrP(Sc). A fluorescently labeled precursor of sialic acid revealed ST activity associated with RAW macrophages. This study illustrates that, upon colonization of SLOs, the sialylation status of prions changes by host STs. We propose that this mechanism is responsible for camouflaging prions in SLOs and has broad implications.

Loss of Cellular Sialidases Does Not Affect the Sialylation Status of the Prion Protein but Increases the Amounts of Its Proteolytic Fragment C1.

The central molecular event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrPC), which is a sialoglycoprotein, into the disease-associated, transmissible form denoted PrPSc. Recent studies revealed a correlation between the sialylation status of PrPSc and incubation time to disease and introduced a new hypothesis that progression of prion diseases could be controlled or reversed by altering the sialylation level of PrPC. Of the four known mammalian sialidases, the enzymes that cleave off sialic acid residues, only NEU1, NEU3 and NEU4 are expressed in the brain. To test whether cellular sialidases control the steady-state sialylation level of PrPC and to identify the putative sialidase responsible for desialylating PrPC, we analyzed brain-derived PrPC from knockout mice deficient in Neu1, Neu3, Neu4, or from Neu3/Neu4 double knockouts. Surprisingly, no differences in the sialylation of PrPC or its proteolytic product C1 were noticed in any of the knockout mice tested as compared to the age-matched controls. However, significantly higher amounts of the C1 fragment relative to full-length PrPC were detected in the brains of Neu1 knockout mice as compared to WT mice or to the other knockout mice. Additional experiments revealed that in neuroblastoma cell line the sialylation pattern of C1 could be changed by an inhibitor of sialylatransferases. In summary, this study suggests that targeting cellular sialidases is apparently not the correct strategy for altering the sialylation levels of PrPC, whereas modulating the activity of sialylatransferases might offer a more promising approach. Our findings also suggest that catabolism of PrPC involves its α-cleavage followed by desialylation of the resulting C1 fragments by NEU1 and consequent fast degradation of the desialylated products.

A new mechanism for transmissible prion diseases.

The transmissible agent of prion disease consists of prion protein (PrP) in ß-sheet-rich state (PrP(Sc)) that can replicate its conformation according to a template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide accurately reproduces that of the PrP(Sc) template. Here, three conformationally distinct amyloid states were prepared in vitro using Syrian hamster recombinant PrP (rPrP) in the absence of cellular cofactors. Surprisingly, no signs of prion infection were found in Syrian hamsters inoculated with rPrP fibrils that resembled PrP(Sc), whereas an alternative amyloid state, with a folding pattern different from that of PrP(Sc), induced a pathogenic process that led to transmissible prion disease. An atypical proteinase K-resistant, transmissible PrP form that resembled the structure of the amyloid seeds was observed during a clinically silent stage before authentic PrP(Sc) emerged. The dynamics between the two forms suggest that atypical proteinase K-resistant PrP (PrPres) gave rise to PrP(Sc). While no PrP(Sc) was found in preparations of fibrils using protein misfolding cyclic amplification with beads (PMCAb), rPrP fibrils gave rise to atypical PrPres in modified PMCAb, suggesting that atypical PrPres was the first product of PrP(C) misfolding triggered by fibrils. The current work demonstrates that a new mechanism responsible for prion diseases different from the PrP(Sc)-templated or spontaneous conversion of PrP(C) into PrP(Sc) exists. This study provides compelling evidence that noninfectious amyloids with a structure different from that of PrP(Sc) could lead to transmissible prion disease. This work has numerous implications for understanding the etiology of prion and other neurodegenerative diseases.

Genesis of tramsmissible protein states via deformed templating.

Prion replication occurs via a template-assisted mechanism, which postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a template. The concept of prion-like template-assisted propagation of an abnormal protein conformation has been expanded to amyloidogenic proteins associated with Alzheimer, Parkinson, Huntington diseases, amyotrophic lateral sclerosis and others. Recent studies demonstrated that authentic PrP (Sc)  and transmissible prion disease could be generated in wild type animals by inoculation of recombinant prion protein amyloid fibrils, which are structurally different from PrP (Sc) and lack any detectable PrP (Sc)  particles. Here we discuss a new replication mechanism designated as "deformed templating," according to which fibrils with one cross-ß folding pattern can seed formation of fibrils or particles with a fundamentally different cross-ß folding pattern. Transformation of cross-ß folding pattern via deformed templating provides a mechanistic explanation behind genesis of transmissible protein states induced by amyloid fibrils that are considered to be non-infectious. We postulate that deformed templating is responsible for generating conformationally diverse amyloid populations, from which conformers that are fit to replicate in a particular cellular environment are selected. We propose that deformed templating represents an essential step in the evolution of transmissible protein states.

Genesis of mammalian prions: from non-infectious amyloid fibrils to a transmissible prion disease.

The transmissible agent of prion disease consists of a prion protein in its abnormal, ß-sheet rich state (PrP(Sc)), which is capable of replicating itself according to the template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a PrP(Sc) template. Here we report that authentic PrP(Sc) and transmissible prion disease can be generated de novo in wild type animals by recombinant PrP (rPrP) amyloid fibrils, which are structurally different from PrP(Sc) and lack any detectable PrP(Sc) particles. When induced by rPrP fibrils, a long silent stage that involved two serial passages preceded development of the clinical disease. Once emerged, the prion disease was characterized by unique clinical, neuropathological, and biochemical features. The long silent stage to the disease was accompanied by significant transformation in neuropathological properties and biochemical features of the proteinase K-resistant PrP material (PrPres) before authentic PrP(Sc) evolved. The current work illustrates that transmissible prion diseases can be induced by PrP structures different from that of authentic PrP(Sc) and suggests that a new mechanism different from the classical templating exists. This new mechanism designated as "deformed templating" postulates that a change in the PrP folding pattern from the one present in rPrP fibrils to an alternative specific for PrP(Sc) can occur. The current work provides important new insight into the mechanisms underlying genesis of the transmissible protein states and has numerous implications for understanding the etiology of neurodegenerative diseases.

The polybasic N-terminal region of the prion protein controls the physical properties of both the cellular and fibrillar forms of PrP.

Individual variations in structure and morphology of amyloid fibrils produced from a single polypeptide are likely to underlie the molecular origin of prion strains and control the efficiency of the species barrier in the transmission of prions. Previously, we observed that the shape of amyloid fibrils produced from full-length prion protein (PrP 23-231) varied substantially for different batches of purified recombinant PrP. Variations in fibril morphology were also observed for different fractions that corresponded to the highly pure PrP peak collected at the last step of purification. A series of biochemical experiments revealed that the variation in fibril morphology was attributable to the presence of miniscule amounts of N-terminally truncated PrPs, where a PrP encompassing residue 31-231 was the most abundant of the truncated polypeptides. Subsequent experiments showed that the presence of small amounts of recombinant PrP 31-231 (0.1-1%) in mixtures with full-length PrP 23-231 had a dramatic impact on fibril morphology and conformation. Furthermore, the deletion of the short polybasic N-terminal region 23-30 was found to reduce the folding efficiency to the native alpha-helical forms and the conformational stability of alpha-PrP. These findings are very surprising considering that residues 23-30 are very distant from the C-terminal globular folded domain in alpha-PrP and from the prion folding domain in the fibrillar form. However, our studies suggest that the N-terminal polybasic region 23-30 is essential for effective folding of PrP to its native cellular conformation. This work also suggests that this region could regulate diversity of prion strains or subtypes despite its remote location from the prion folding domain.

Amyloid fibrils of mammalian prion protein induce axonal degeneration in NTERA2-derived terminally differentiated neurons.

Defects in axonal transport and synaptic dysfunctions are associated with early stages of several neurodegenerative diseases including Alzheimer's, Huntington's, Parkinson's, and prion diseases. Here, we tested the effect of full-length mammalian prion protein (rPrP) converted into three conformationally different isoforms to induce pathological changes regarded as early subcellular hallmarks of prion disease. We employed human embryonal teratocarcinoma NTERA2 cells (NT2) that were terminally differentiated into neuronal and glial cells and co-cultured together. We found that rPrP fibrils but not alpha-rPrP or soluble beta-sheet rich oligomers caused degeneration of neuronal processes. Degeneration of processes was accompanied by a collapse of microtubules and aggregation of cytoskeletal proteins, formation of neuritic beads, and a dramatic change in localization of synaptophysin. Our studies demonstrated the utility of NT2 cells as valuable human model system for elucidating subcellular events of prion pathogenesis, and supported the emerging hypothesis that defects in neuronal transport and synaptic abnormalities are early pathological hallmarks associated with prion diseases.

Nonpolar substitution at the C-terminus of the prion protein, a mimic of the glycosylphosphatidylinositol anchor, partially impairs amyloid fibril formation.

In contrast to most amyloidogenic proteins or peptides that do not contain any significant posttranslational modifications, the prion protein (PrP) is modified with either one or two polysaccharides and a GPI anchor which attaches PrP to the plasma membrane. Like other amyloidogenic proteins, however, PrP adopts a fibrillar shape when converted to a disease-specific conformation. Therefore, PrP polymerization offers a unique opportunity to examine the effects of biologically relevant nonpeptidic modifications on conversion to the amyloid conformation. To test the extent to which a long hydrophobic chain at the C-terminus affects the intrinsic amyloidogenic propensity of PrP, we modified recombinant PrP with an N-myristoylamidomaleimidyl group, which can serve as a membrane anchor. We show that while this modification increases the affinity of PrP for the cell membrane, it does not alter the structure of the protein. Myristoylation of PrP affected amyloid formation in two ways: (i) it substantially decreased the extent of fibrillation, presumably due to off-pathway aggregation, and (ii) it prohibited assembly of filaments into higher order fibrils by preventing their lateral association. The negative effect on lateral association was abolished if the myristoylated moiety at the C-terminus was replaced by a polar group of similar size or by a hydrophobic group of smaller size. When preformed PrP fibrils were provided as seeds, myristoylated PrP supported fibril elongation and formation of higher order fibrils composed of several filaments. Our studies illustrate that, despite a bulky hydrophobic moiety at C-terminus, myristoylated PrP can still incorporate into fibrillar structure and that the C-terminal hydrophobic substitution does not affect the size of the proteinase K resistant core but controls the mode of lateral assembly of filaments into higher order fibrils.

Site-specific conformational studies of prion protein (PrP) amyloid fibrils revealed two cooperative folding domains within amyloid structure.

Despite the ability of most proteins to form amyloid, very little is know about amyloid fibril structures and the factors that govern their stability. Using amyloid fibrils produced from full-length prion protein (PrP), we describe a reliable approach for determining both site-specific and global conformational stability of the fibrillar form. To measure site-specific stability, we produced six variants of PrP by replacing the residues at positions 88, 98, 127, 144, 196, and 230 with cysteine, labeled the new cysteines with the fluorescent dye acrylodan, and investigated their conformational status within the amyloid form in guanidine hydrochloride-induced denaturation experiments. We found that the fibrils labeled at positions 127, 144, 196, and 230 displayed cooperative unfolding and showed a very high C1/2 value similar to that observed for the global unfolding of the amyloid structure. The unfolding at residue 98 was also cooperative; however, it showed a C1/2 value substantially lower than that of global unfolding, whereas the unfolding of fibrils labeled at residue 88 was non-cooperative. These data illustrate that there are at least two independent cooperative folding domains within the amyloid structure of the full-length PrP. In addition, kinetic experiments revealed only a partial overlap between the region that constituted the fibrillar cross-beta core and the regions that were involved in nucleation. This result illustrates that separate PrP regions accounted for the nucleation and for the formation of the conformationally most stable fibrillar core.

Annealing prion protein amyloid fibrils at high temperature results in extension of a proteinase K-resistant core.

Amyloids are highly ordered, rigid beta-sheet-rich structures that appear to have minimal dynamic flexibility in individual polypeptide chains. Here, we demonstrate that substantial conformational rearrangements occur within mature amyloid fibrils produced from full-length mammalian prion protein. The rearrangement results in a substantial extension of a proteinase K-resistant core and is accompanied by an increase in the beta-sheet-rich conformation. The conformational rearrangement was induced in the presence of low concentrations of Triton X-100 either by brief exposure to 80 degrees C or, with less efficacy, by prolonged incubation at 37 degrees C at pH 7.5 and is referred to here as "annealing." Upon annealing, amyloid fibrils acquired a proteinase K-resistant core identical to that found in bovine spongiform encephalopathy-specific scrapie-associated prion protein. Annealing was also observed when amyloid fibrils were exposed to high temperatures in the absence of detergent but in the presence of brain homogenate. These findings suggest that the amyloid fibrils exist in two conformationally distinct states that are separated by a high energy barrier and that yet unknown cellular cofactors may facilitate transition of the fibrils into thermodynamically more stable state. Our studies provide new insight into the complex behavior of prion polymerization and highlight the annealing process, a previously unknown step in the evolution of amyloid structures.