Brains Of Prion-infected Mice Research Articles

Aberrance of GAP43/p-GAP43 Closely Associates with the Pathology of Neuron Loss in Prion-Infected Rodent Models.

Prion diseases are fatal neurodegenerative disorders characterized by neuron damage and loss. Growth-associated protein 43 (GAP43) functions in neuronal plasticity and synaptic function, but its role in prion diseases is not fully elucidated. In this study, we investigated the changes of GAP43 in the central nerve system (CNS) of several prion-infected rodent models and explored the potential relationship of GAP43 with PrPSc deposit and neuron loss using various methods. We found that GAP43 levels were significantly decreased in the brain tissues of scrapie-infected rodent models at the terminal stage of the disease. Immunohistochemical analysis showed that GAP43 colocalized with NeuN-positive cells morphologically, indicating the presence of GAP43 in mature neurons. On contrary, the levels of GAP43 and p-GAP43 increased in a prion-infected cell line SMB-S15 in vitro, accompanying with the increase of intracellular calcium. Stimulation of lipopolysaccharide (LPS) upregulated while removal of PrPSc propagation downregulated the level of GAP43 in SMB-S15 cells. Morphological colocalization and molecular interaction between GAP43 and PrPSc have been addressed in the brains of prion-infected rodents and prion-infected cell line. Histological assays of the serial sections of the whole brains of prion-infected mice proposed that the reduced GAP43 level correlated with large amount of PrPSc deposits and notable neuron damage and loss showing cell crumpled and nuclear pyknosis. The impairment of GAP43 signaling and disturbance of calcium homeostasis by aberrance of brain GAP43/p-GAP43 not only reflect but also likely contribute to the pathology of severe neuron loss at the end of prion disease.

Aberrant Enhanced NLRP3 Inflammasomes and Cell Pyroptosis in the Brains of Prion-Infected Rodent Models Are Largely Associated with the Proliferative Astrocytes.

Neuroinflammation is a common pathological feature in a number of neurodegenerative diseases, which is mediated primarily by the activated glial cells. Nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) inflammasome-associated neuroinflammatory response is mostly considered. To investigate the situation of the NLRP3-related inflammation in prion disease, we assessed the levels of the main components of NLRP3 inflammasome and its downstream biomarkers in the scrapie-infected rodent brain tissues. The results showed that the transcriptional and expressional levels of NLRP3, caspase-1, and apoptosis-associated speck-like protein (ASC) in the brains of scrapie-infected rodents were significantly increased at terminal stage. The increased NLPR3 overlapped morphologically well with the proliferated GFAP-positive astrocytes, but little with microglia and neurons. Using the brain samples collected at the different time-points after infection, we found the NLRP3 signals increased in a time-dependent manner, which were coincidental with the increase of GFAP. Two main downstream cytokines, IL-1β and IL-18, were also upregulated in the brains of prion-infected mice. Moreover, the gasdermin D (GSDMD) levels, particularly the levels of GSDMD-NT, in the prion-infected brain tissues were remarkably increased, indicating activation of cell pyroptosis. The GSDMD not only co-localized well with the astrocytes but also with neurons at terminal stage, also showing a time-dependent increase after infection. Those data indicate that NLRP3 inflammasomes were remarkably activated in the infected brains, which is largely mediated by the proliferated astrocytes. Both astrocytes and neurons probably undergo a pyroptosis process, which may help the astrocytes to release inflammatory factors and contribute to neuron death during prion infection.

Hydrogen Peroxide Gas Plasma Sterilizer Combined with Dielectric Barrier Discharge and Corona Discharge Inactivates Prions

Prions are highly resistant to physical or chemical damage, although previous studies have shown that STERRAD®, a hydrogen gas plasma sterilizer using radiofrequency (RF) discharge, has an inactivation effect. Here, the effect of hydrogen peroxide gas combined with dielectric barrier discharge (DBD) plasma and corona discharge plasma using a RENO-S130 sterilizer on scrapie prions was examined. Scrapie prion-infected mouse brain homogenate was air-dried on a cover glass, sealed in a Tyvek pouch, and subjected to RENO-S130 treatment using either non-lumen mode (28 min) or Eco mode (45 min) with hydrogen peroxide gas derived from 50% hydrogen peroxide. Control (untreated) samples were prepared on a cover glass using the same procedure but without exposure to RENO-S130. PrPres (proteinase K (PK)-resistant prion protein), an index of the conformational variant of prion protein (PrPSc), was decreased by treatment with RENO-S130 under both modes of operation. Specifically, PrPres was identified after the 1st and 2nd cycles of protein misfolding cyclic amplification (PMCA) in control samples but was below the detection limit in RENO-S130-treated samples. A bioassay showed that treatment of prions with RENO-S130 (non-lumen or Eco mode) significantly prolonged mouse survival time. Taken together, these findings show hydrogen peroxide gas combined with DBD/corona discharge plasma can inactivate prions by reducing prion propagation and prion infectivity. This treatment is potentially applicable to the sterilization of prion-contaminated heat-sensitive medical devices.

Oral administration of repurposed drug targeting Cyp46A1 increases survival times of prion infected mice

Prion diseases are fatal, infectious, and incurable neurodegenerative disorders caused by misfolding of the cellular prion protein (PrPC) into the infectious isoform (PrPSc). In humans, there are sporadic, genetic and infectious etiologies, with sporadic Creutzfeldt-Jakob disease (sCJD) being the most common form. Currently, no treatment is available for prion diseases. Cellular cholesterol is known to impact prion conversion, which in turn results in an accumulation of cholesterol in prion-infected neurons. The major elimination of brain cholesterol is achieved by the brain specific enzyme, cholesterol 24-hydroxylase (CYP46A1). Cyp46A1 converts cholesterol into 24(S)-hydroxycholesterol, a membrane-permeable molecule that exits the brain. We have demonstrated for the first time that Cyp46A1 levels are reduced in the brains of prion-infected mice at advanced disease stage, in prion-infected neuronal cells and in post-mortem brains of sCJD patients. We have employed the Cyp46A1 activator efavirenz (EFV) for treatment of prion-infected neuronal cells and mice. EFV is an FDA approved anti-HIV medication effectively crossing the blood brain barrier and has been used for decades to chronically treat HIV patients. EFV significantly mitigated PrPSc propagation in prion-infected cells while preserving physiological PrPC and lipid raft integrity. Notably, oral administration of EFV treatment chronically at very low dosage starting weeks to months after intracerebral prion inoculation of mice significantly prolonged the lifespan of animals. In summary, our results suggest that Cyp46A1 as a novel therapeutic target and that its activation through repurposing the anti-retroviral medication EFV might be valuable treatment approach for prion diseases.

The role of macrophage scavenger receptor 1 (Msr1) in prion pathogenesis

The progression of prion diseases is accompanied by the accumulation of prions in the brain. Ablation of microglia enhances prion accumulation and accelerates disease progression, suggesting that microglia play a neuroprotective role by clearing prions. However, the mechanisms underlying the phagocytosis and clearance of prion are largely unknown. The macrophage scavenger receptor 1 (Msr1) is an important phagocytic receptor expressed by microglia in the brain and is involved in the uptake and clearance of soluble amyloid-β. We therefore asked whether Msr1 might play a role in prion clearance and assessed the scavenger function of Msr1 in prion pathogenesis. We found that Msr1 expression was upregulated in prion-infected mouse brains. However, Msr1 deficiency did not change prion disease progression or lesion patterns. Prion deposition in Msr1 deficient mice was similar to their wild-type littermates. In addition, prion-induced neuroinflammation was not affected by Msr1 ablation. We conclude that Msr1 does not play a major role in prion pathogenesis.Key messagesMsr1 expression is upregulated in prion-infected mouse brains at the terminal stageMsr1 deficiency does not affect prion disease progressionMsr1 does not play a major role in prion clearance or prion pathogenesisMicroglia-mediated phagocytosis and clearance of Aβ and prion may adopt distinct molecular pathways

Scrapie infection and endogenous retroviral expression in sheep lymphoid tissues

Transmissible spongiform encephalopathies, or prion diseases, are fatal neurodegenerative diseases affecting humans and animals. Although many host tissues express PrPC (essential for prion replication), relatively few cell types accumulate significant levels of infectivity, including neurons and other cell types in the nervous system, and follicular dendritic cells in secondary lymphoid organs. This suggests that tissue or cell-specific receptors or cofactors could play a role in controlling differential susceptibility to infection. Endogenous retroviruses (ERV), the remnants of ancient retroviral integration into the host germline, may represent one such cofactor. We examined the effect of scrapie infection on expression of three ovine ERV families (enJSRV/β1-OERV, γ1-OERV, γ2-OERV) in secondary lymphoid tissues of sheep at different time points following subcutaneous inoculation, using RT-qPCR. These OERVs were constitutively expressed in the prescapular lymph node and spleen of uninfected sheep. However, we were unable to find convincing evidence of specific differential expression of OERV in the same tissues following scrapie infection, in contrast to previous studies of ERV expression in brains of prion-infected mice and macaques. This study is the first to quantify the expression of potentially functional OERV transcripts in sheep lymphoid tissues, opening up interesting questions about the consequences for host immune function.

RNA-seq and network analysis reveal unique glial gene expression signatures during prion infection

BackgroundPrion diseases and prion-like disorders, including Alzheimer’s disease and Parkinson’s disease, are characterized by gliosis and accumulation of misfolded aggregated host proteins. Ablating microglia in prion-infected brain by treatment with the colony-stimulating factor-1 receptor (CSF-1R) inhibitor, PLX5622, increased accumulation of misfolded prion protein and decreased survival time.MethodsTo better understand the role of glia during neurodegeneration, we used RNA-seq technology, network analysis, and hierarchical cluster analysis to compare gene expression in brains of prion-infected versus mock-inoculated mice. Comparisons were also made between PLX5622-treated prion-infected mice and untreated prion-infected mice to assess mechanisms involved in disease acceleration in the absence of microglia.ResultsRNA-seq and network analysis suggested that microglia responded to prion infection through activation of integrin CD11c/18 and did not adopt the expression signature associated with other neurodegenerative disease models. Instead, microglia acquired an alternative molecular signature late in the disease process. Furthermore, astrocytes expressed a signature pattern of genes which appeared to be specific for prion diseases. Comparisons were also made with prion-infected mice treated with PLX5622 to assess the impact of microglia ablation on astrocyte gene expression during prion infection. In the presence of microglia, a unique mix of transcripts associated with A1- and A2-reactive astrocytes was increased in brains of prion-infected mice. After ablation of microglia, this reactive astrocyte expression pattern was enhanced. Thus, after prion infection, microglia appeared to decrease the overall A1/A2-astrocyte responses which might contribute to increased survival after infection.ConclusionsRNA-seq analysis indicated dysregulation of over 300 biological processes within the CNS during prion disease. Distinctive microglia- and astrocyte-associated expression signatures were identified during prion infection. Furthermore, astrogliosis and the unique astrocyte-associated expression signature were independent of microglial influences. Astrogliosis and the unique astrocyte-associated gene expression pattern were increased when microglia were ablated. Our findings emphasize the potential existence of alternative pathways for activating the A1/A2 paradigm in astrocytes during neurodegenerative disease.

Toll-like receptor 2 confers partial neuroprotection during prion disease.

Neuroinflammation and neurodegeneration are common during prion infection, but the mechanisms that underlie these pathological features are not well understood. Several components of innate immunity, such as Toll-like receptor (TLR) 4 and Complement C1q, have been shown to influence prion disease. To identify additional components of innate immunity that might impact prion disease within the central nervous system (CNS), we screened RNA from brains of pre-clinical and clinical 22L-infected mice for alterations in genes associated with innate immunity. Transcription of several genes encoding damage-associated molecular pattern (DAMP) proteins and receptors were increased in the brains of prion-infected mice. To investigate the role of some of these proteins in prion disease of the CNS, we infected mice deficient in DAMP receptor genes Tlr2, C3ar1, and C5ar1 with 22L scrapie. Elimination of TLR2 accelerated disease by a median of 10 days, while lack of C3aR1 or C5aR1 had no effect on disease tempo. Histopathologically, all knockout mouse strains tested were similar to infected control mice in gliosis, vacuolation, and PrPSc deposition. Analysis of proinflammatory markers in the brains of infected knockout mice indicated only a few alterations in gene expression suggesting that C5aR1 and TLR2 signaling did not act synergistically in the brains of prion-infected mice. These results indicate that signaling through TLR2 confers partial neuroprotection during prion infection.

Identification of Alprenolol Hydrochloride as an Anti-prion Compound Using Surface Plasmon Resonance Imaging.

Prion diseases are transmissible neurodegenerative disorders of humans and animals, which are characterized by the aggregation of abnormal prion protein (PrPSc) in the central nervous system. Although several small compounds that bind to normal PrP (PrPC) have been shown to inhibit structural conversion of the protein, an effective therapy for human prion disease remains to be established. In this study, we screened 1200 existing drugs approved by the US Food and Drug Administration (FDA) for anti-prion activity using surface plasmon resonance imaging (SPRi). Of these drugs, 31 showed strong binding activity to recombinant human PrP, and three of these reduced the accumulation of PrPSc in prion-infected cells. One of the active compounds, alprenolol hydrochloride, which is used clinically as a β-adrenergic blocker for hypertension, also reduced the accumulation of PrPSc in the brains of prion-infected mice at the middle stage of the disease when the drug was administered orally with their daily water from the day after infection. Docking simulation analysis suggested that alprenolol hydrochloride fitted into the hotspot within mouse PrPC, which is known as the most fragile structure within the protein. These findings provide evidence that SPRi is useful in identifying effective drug candidates for neurodegenerative diseases caused by abnormal protein aggregation, such as prion diseases.

Flow Cytometric Detection of PrPSc in Neurons and Glial Cells from Prion-Infected Mouse Brains.

In prion diseases, an abnormal isoform of prion protein (PrPSc) accumulates in neurons, astrocytes, and microglia in the brains of animals affected by prions. Detailed analyses of PrPSc-positive neurons and glial cells are required to clarify their pathophysiological roles in the disease. Here, we report a novel method for the detection of PrPSc in neurons and glial cells from the brains of prion-infected mice by flow cytometry using PrPSc-specific staining with monoclonal antibody (MAb) 132. The combination of PrPSc staining and immunolabeling of neural cell markers clearly distinguished neurons, astrocytes, and microglia that were positive for PrPSc from those that were PrPSc negative. The flow cytometric analysis of PrPSc revealed the appearance of PrPSc-positive neurons, astrocytes, and microglia at 60 days after intracerebral prion inoculation, suggesting the presence of PrPSc in the glial cells, as well as in neurons, from an early stage of infection. Moreover, the kinetic analysis of PrPSc revealed a continuous increase in the proportion of PrPSc-positive cells for all cell types with disease progression. Finally, we applied this method to isolate neurons, astrocytes, and microglia positive for PrPSc from a prion-infected mouse brain by florescence-activated cell sorting. The method described here enables comprehensive analyses specific to PrPSc-positive neurons, astrocytes, and microglia that will contribute to the understanding of the pathophysiological roles of neurons and glial cells in PrPSc-associated pathogenesis.IMPORTANCE Although formation of PrPSc in neurons is associated closely with neurodegeneration in prion diseases, the mechanism of neurodegeneration is not understood completely. On the other hand, recent studies proposed the important roles of glial cells in PrPSc-associated pathogenesis, such as the intracerebral spread of PrPSc and clearance of PrPSc from the brain. Despite the great need for detailed analyses of PrPSc-positive neurons and glial cells, methods available for cell type-specific analysis of PrPSc have been limited thus far to microscopic observations. Here, we have established a novel high-throughput method for flow cytometric detection of PrPSc in cells with more accurate quantitative performance. By applying this method, we succeeded in isolating PrPSc-positive cells from the prion-infected mouse brains via fluorescence-activated cell sorting. This allows us to perform further detailed analysis specific to PrPSc-positive neurons and glial cells for the clarification of pathological changes in neurons and pathophysiological roles of glial cells.

Age and Environment Influences on Mouse Prion Disease Progression: Behavioral Changes and Morphometry and Stereology of Hippocampal Astrocytes.

Because enriched environment (EE) and exercise increase and aging decreases immune response, we hypothesized that environmental enrichment and aging will, respectively, delay and increase prion disease progression. Mice dorsal striatum received bilateral stereotaxic intracerebral injections of normal or ME7 prion infected mouse brain homogenates. After behavior analysis, animals were euthanized and their brains processed for astrocyte GFAP immunolabeling. Our analysis related to the environmental influence are limited to young adult mice, whereas age influence refers to aged mice raised on standard cages. Burrowing activity began to reduce in ME7-SE two weeks before ME7-EE, while no changes were apparent in ME7 aged mice (ME7-A). Object placement recognition was impaired in ME7-SE, NBH-A, and ME7-A but normal in all other groups. Object identity recognition was impaired in ME7-A. Cluster analysis revealed two morphological families of astrocytes in NBH-SE animals, three in NBH-A and ME7-A, and four in NBH-EE, ME7-SE, and ME7-EE. As compared with control groups, astrocytes from DG and CA3 prion-diseased animals show significant numerical and morphological differences and environmental enrichment did not reverse these changes but induced different morphological changes in GFAP+ hippocampal astroglia. We suggest that environmental enrichment and aging delayed hippocampal-dependent behavioral and neuropathological signs of disease progression.

Structure-based drug design identifies polythiophenes as antiprion compounds.

Prions cause transmissible spongiform encephalopathies for which no treatment exists. Prions consist of PrP(Sc), a misfolded and aggregated form of the cellular prion protein (PrP(C)). We explore the antiprion properties of luminescent conjugated polythiophenes (LCPs) that bind and stabilize ordered protein aggregates. By administering a library of structurally diverse LCPs to the brains of prion-infected mice via osmotic minipumps, we found that antiprion activity required a minimum of five thiophene rings bearing regularly spaced carboxyl side groups. Solid-state nuclear magnetic resonance analyses and molecular dynamics simulations revealed that anionic side chains interacted with complementary, regularly spaced cationic amyloid residues of model prions. These findings allowed us to extract structural rules governing the interaction between LCPs and protein aggregates, which we then used to design a new set of LCPs with optimized binding. The new set of LCPs showed robust prophylactic and therapeutic potency in prion-infected mice, with the lead compound extending survival by >80% and showing activity against both mouse and hamster prions as well as efficacy upon intraperitoneal administration into mice. These results demonstrate the feasibility of targeted chemical design of compounds that may be useful for treating diseases of aberrant protein aggregation such as prion disease.

Interaction between misfolded PrP and the ubiquitin-proteasome system in prion-mediated neurodegeneration

Prion diseases are associated with the conformational conversion of cellular prion protein (PrP(C)) to pathological β-sheet isoforms (PrP(Sc)), which is the infectious agent beyond comprehension. Increasing evidence indicated that an unknown toxic gain of function of PrP(sc) underlies neuronal death. Conversely, strong evidence indicated that cellular prion protein might be directly cytotoxic by mediating neurotoxic signaling of β-sheet-rich conformers independent of prion replication. Furthermore, the common properties of β-sheet-rich isoform such as PrP(Sc) and β amyloid protein become the lynchpin that interprets the general pathological mechanism of protein misfolding diseases. Dysfunction of the ubiquitin-proteasome system (UPS) has been implicated in various protein misfolding diseases. However, the mechanisms of this impairment remain unknown in many cases. In prion disease, prion-infected mouse brains have increased levels of ubiquitin conjugates, which correlate with decreased proteasome function. Both PrP(C) and PrP(Sc) accumulate in cells after proteasome inhibition, which leads to increased cell death. A direct interaction between 20S core particle and PrP isoforms was demonstrated. Here we review the ability of misfolded PrP and UPS to affect each other, which might contribute to the pathological features of prion-mediated neurodegeneration.

Prions disturb post-Golgi trafficking of membrane proteins

Conformational conversion of normal cellular prion protein PrP(C) into pathogenic PrP(Sc) is central to the pathogenesis of prion diseases. However, the pathogenic mechanism remains unknown. Here we show that post-Golgi vesicular trafficking is significantly delayed in prion-infected N2a cells. Accordingly, cell surface expression of membrane proteins examined, including PrP(C), insulin receptor involved in neuroprotection, and attractin, whose mutation causes prion disease-like spongiform neurodegeneration, is reduced. Instead, they accumulate in the Golgi apparatus. PrP(Sc) is detected throughout endosomal compartments, being particularly abundant in recycling endosome. We also show reduced surface expression of PrP(C) and insulin receptor in prion-infected mouse brains well before the onset of disease. These results suggest that prion infection might impair post-Golgi trafficking of membrane proteins to the cell surface in neurons via PrP(Sc) accumulated in recycling endosome, and eventually induce neuronal dysfunctions associated with prion diseases.

MicroRNA 146a (miR-146a) Is Over-Expressed during Prion Disease and Modulates the Innate Immune Response and the Microglial Activation State

Increasing evidence supports the involvement of microRNAs (miRNAs) in inflammatory and immune processes in prion neuropathogenesis. MiRNAs are small, non-coding RNA molecules which are emerging as key regulators of numerous cellular processes. We established miR-146a over-expression in prion-infected mouse brain tissues concurrent with the onset of prion deposition and appearance of activated microglia. Expression profiling of a variety of central nervous system derived cell-lines revealed that miR-146a is preferentially expressed in cells of microglial lineage. Prominent up-regulation of miR-146a was evident in the microglial cell lines BV-2 following TLR2 or TLR4 activation and also EOC 13.31 via TLR2 that reached a maximum 24–48 hours post-stimulation, concomitant with the return to basal levels of transcription of induced cytokines. Gain- and loss-of-function studies with miR-146a revealed a substantial deregulation of inflammatory response pathways in response to TLR2 stimulation. Significant transcriptional alterations in response to miR-146a perturbation included downstream mediators of the pro-inflammatory transcription factor, nuclear factor-kappa B (NF-κB) and the JAK-STAT signaling pathway. Microarray analysis also predicts a role for miR-146a regulation of morphological changes in microglial activation states as well as phagocytic mediators of the oxidative burst such as CYBA and NOS3. Based on our results, we propose a role for miR-146a as a potent modulator of microglial function by regulating the activation state during prion induced neurodegeneration.

Role of Cyclophilin A from Brains of Prion-infected Mice in Stimulation of Cytokine Release by Microglia and Astroglia in Vitro

Prion diseases or transmissible spongiform encephalopathy diseases are typically characterized by deposition of abnormally folded partially protease-resistant host-derived prion protein (PrPres), which is associated with activated glia and increased release of cytokines. This neuroinflammatory response may play a role in transmissible spongiform encephalopathy pathogenesis. We previously reported that brain homogenates from prion-infected mice induced cytokine protein release in primary astroglial and microglial cell cultures. Here we measured cytokine release by cultured glial cells to determine what factors in infected brain contributed to activation of microglia and astroglia. In assays analyzing IL-12p40 and CCL2 (MCP-1), glial cells were not stimulated in vitro by either PrPres purified from infected mouse brains or prion protein amyloid fibrils produced in vitro. However, significant glial stimulation was induced by clarified scrapie brain homogenates lacking PrPres. This stimulation was greatly reduced both by antibody to cyclophilin A (CyPA), a known mediator of inflammation in peripheral tissues, and by cyclosporine A, a CyPA inhibitor. In biochemical studies, purified truncated CyPA fragments stimulated a pattern of cytokine release by microglia and astroglia similar to that induced by scrapie-infected brain homogenates, whereas purified full-length CyPA was a poor stimulator. This requirement for CyPA truncation was not reported in previous studies of stimulation of peripheral macrophages, endothelial cell cardiomyocytes, and vascular smooth muscle cells. Therefore, truncated CyPA detected in brain following prion infection may have an important role in the activation of brain-derived primary astroglia and microglia in prion disease and perhaps other neurodegenerative or neuroinflammatory diseases.

Accumulation and aberrant composition of cholesteryl esters in Scrapie-infected N2a cells and C57BL/6 mouse brains

ObjectiveCholesterol changes have been described in prion-cell models and in experimental rodent scrapie; yet, the pattern of this association is still controversial.MethodsTo shed light on the matter, we analysed and compared cholesterol variations in ScN2a cells and in brains of Scrapie-infected C57Bl/6 mice, using two different methods: a fluorimetric-enzymatic cholesterol assay, and high performance liquid chromatography-mass spectroscopy (HPLC-MS).ResultsCompared to uninfected controls, similar cholesterol metabolism anomalies were observed in infected cells and brains by both methods; however, only HPLC-MS revealed statistically significant cholesterol variations, particularly in the cholesteryl esters (CE) fraction. HPLC-MS analyses also revealed different fatty acid composition of the CE fraction in cells and brains. In N2a cells, their profile reflected that of serum, while in normal brains cholesteryl-linoleate only was found at detectable levels. Following prion infection, most CE species were increased in the CE pool of ScN2a cells, whereas a conspicuous amount of cholesteryl-arachidonate only was found to contribute to the cerebral increase of CE. Of interest, oral pravastatin administration to Scrapie-infected mice, was associated with a significant reduction of cerebral free cholesterol (FC) along with a concomitant further increase of the CE pool, which included increased amounts of both cholesteryl-linoleate and cholesteryl-arachidonate.ConclusionAlthough mechanistic studies are needed to establish the pathophysiological relevance of changes in cerebral CE concentrations, to the best of our knowledge this is the first report to provide evidence of increased cholesterol esterification in brains of prion-infected mice, untreated and treated with pravastatin.

Identification of Chemoattractive Factors Involved in the Migration of Bone Marrow-Derived Mesenchymal Stem Cells to Brain Lesions Caused by Prions

Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to migrate to brain lesions of neurodegenerative diseases; however, the precise mechanisms by which MSCs migrate remain to be elucidated. In this study, we carried out an in vitro migration assay to investigate the chemoattractive factors for MSCs in the brains of prion-infected mice. The migration of immortalized human MSCs (hMSCs) was reduced by their pretreatment with antibodies against the chemokine receptors, CCR3, CCR5, CXCR3, and CXCR4 and by pretreatment of brain extracts of prion-infected mice with antibodies against the corresponding ligands, suggesting the involvement of these receptors, and their ligands in the migration of hMSCs. In agreement with the results of an in vitro migration assay, hMSCs in the corpus callosum, which are considered to be migrating from the transplanted area toward brain lesions of prion-infected mice, expressed CCR3, CCR5, CXCR3, and CXCR4. The combined in vitro and in vivo analyses suggest that CCR3, CCR5, CXCR3, and CXCR4, and their corresponding ligands are involved in the migration of hMSCs to the brain lesions caused by prion propagation. In addition, hMSCs that had migrated to the right hippocampus of prion-infected mice expressed CCR1, CX3CR1, and CXCR4, implying the involvement of these chemokine receptors in hMSC functions after chemotactic migration. Further elucidation of the mechanisms that underlie the migration of MSCs may provide useful information regarding application of MSCs to the treatment of prion diseases.

Misfolded PrP impairs the UPS by interaction with the 20S proteasome and inhibition of substrate entry

Prion diseases are associated with the conversion of cellular prion protein (PrP(C)) to toxic β-sheet isoforms (PrP(Sc)), which are reported to inhibit the ubiquitin-proteasome system (UPS). Accordingly, UPS substrates accumulate in prion-infected mouse brains, suggesting impairment of the 26S proteasome. A direct interaction between its 20S core particle and PrP isoforms was demonstrated by immunoprecipitation. β-PrP aggregates associated with the 20S particle, but did not impede binding of the PA26 complex, suggesting that the aggregates do not bind to its ends. Aggregated β-PrP reduced the 20S proteasome's basal peptidase activity, and the enhanced activity induced by C-terminal peptides from the 19S ATPases or by the 19S regulator itself, including when stimulated by polyubiquitin conjugates. However, the 20S proteasome was not inhibited when the gate in the α-ring was open due to a truncation mutation or by association with PA26/PA28. These PrP aggregates inhibit by stabilising the closed conformation of the substrate entry channel. A similar inhibition of substrate entry into the proteasome may occur in other neurodegenerative diseases where misfolded β-sheet-rich proteins accumulate.

Alteration of the endocannabinoid system in mouse brain during prion disease

Prion diseases are neurodegenerative disorders characterized by deposition of the pathological prion protein (PrPsc) within the brain of affected humans and animals. Microglial cell activation is a common feature of prion diseases; alterations of various neurotransmitter systems and neurotransmission have been also reported. Owing to its ability to modulate both neuroimmune responses and neurotransmission, it was of interest to study the brain endocannabinoid system in a prion-infected mouse model. The production of the endocannabinoid, 2-arachidonoyglycerol (2-AG), was enhanced 10 weeks post-infection, without alteration of the other endocannabinoid, anandamide. The CB2 receptor expression was up-regulated in brains of prion-infected mice as early as 10 weeks and up to 32 weeks post-infection whereas the mRNAs of other cannabinoid receptors (CBRs) remain unchanged. The observed alterations of the endocannabinoid system were specific for prion infection since no significant changes were observed in the brain of prion-resistant mice, that is, mice devoid of the Prnp gene. Our study highlights important alterations of the endocannabinoid system during early stages of the disease long before the clinical signs of the disease.