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1.  Prion Pathogenesis in the Absence of NLRP3/ASC Inflammasomes 
PLoS ONE  2015;10(2):e0117208.
The accumulation of the scrapie prion protein PrPSc, a misfolded conformer of the cellular prion protein PrPC, is a crucial feature of prion diseases. In the central nervous system, this process is accompanied by conspicuous microglia activation. The NLRP3 inflammasome is a multi-molecular complex which can sense heterogeneous pathogen-associated molecular patterns and culminates in the activation of caspase 1 and release of IL 1β. The NLRP3 inflammasome was reported to be essential for IL 1β release after in vitro exposure to the amyloidogenic peptide PrP106-126 and to recombinant PrP fibrils. We therefore studied the role of the NLRP3 inflammasome in a mouse model of prion infection. Upon intracerebral inoculation with scrapie prions (strain RML), mice lacking NLRP3 (Nlrp3-/-) or the inflammasome adaptor protein ASC (Pycard-/-) succumbed to scrapie with attack rates and incubation times similar to wild-type mice, and developed the classic histologic and biochemical features of prion diseases. Genetic ablation of NLRP3 or ASC did not significantly impact on brain levels of IL 1β at the terminal stage of disease. Our results exclude a significant role for NLRP3 and ASC in prion pathogenesis and invalidate their claimed potential as therapeutic target against prion diseases.
doi:10.1371/journal.pone.0117208
PMCID: PMC4324825  PMID: 25671600
2.  Neurodegeneration and Unfolded-Protein Response in Mice Expressing a Membrane-Tethered Flexible Tail of PrP 
PLoS ONE  2015;10(2):e0117412.
The cellular prion protein (PrPC) consists of a flexible N-terminal tail (FT, aa 23–128) hinged to a membrane-anchored globular domain (GD, aa 129–231). Ligation of the GD with antibodies induces rapid neurodegeneration, which is prevented by deletion or functional inactivation of the FT. Therefore, the FT is an allosteric effector of neurotoxicity. To explore its mechanism of action, we generated transgenic mice expressing the FT fused to a GPI anchor, but lacking the GD (PrPΔ141–225, or “FTgpi”). Here we report that FTgpi mice develop a progressive, inexorably lethal neurodegeneration morphologically and biochemically similar to that triggered by anti-GD antibodies. FTgpi was mostly retained in the endoplasmic reticulum, where it triggered a conspicuous unfolded protein response specifically activating the PERK pathway leading to phosphorylation of eIF2α and upregulation of CHOP ultimately leading to neurodegeration similar to what was observed in prion infection.
doi:10.1371/journal.pone.0117412
PMCID: PMC4319788  PMID: 25658480
3.  ASC has extracellular and prionoid activities that propagate inflammation 
Nature immunology  2014;15(8):727-737.
Microbes or danger signals trigger inflammasome sensors, which induce polymerization of the adapter ASC and assembly of an ASC speck. ASC specks recruit and activate caspase-1, which induces IL-1β cytokine maturation and pyroptotic cell death. Here we show that after pyroptosis ASC specks accumulate in the extracellular space, where they promote further IL-1β maturation. In addition, phagocytosis of ASC specks induces lysosomal damage, nucleation of soluble ASC as well as caspase-1 and IL-1β activation in the recipient cell. ASC specks appear in bodily fluids from inflamed tissues and autoantibodies against ASC specks develop in patients and animals with autoimmune pathologies. Together, these findings reveal extracellular functions of ASC specks and a novel form of cell-to-cell communication.
doi:10.1038/ni.2913
PMCID: PMC4116676  PMID: 24952505
4.  The Role of the NADPH Oxidase NOX2 in Prion Pathogenesis 
PLoS Pathogens  2014;10(12):e1004531.
Prion infections cause neurodegeneration, which often goes along with oxidative stress. However, the cellular source of reactive oxygen species (ROS) and their pathogenetic significance are unclear. Here we analyzed the contribution of NOX2, a prominent NADPH oxidase, to prion diseases. We found that NOX2 is markedly upregulated in microglia within affected brain regions of patients with Creutzfeldt-Jakob disease (CJD). Similarly, NOX2 expression was upregulated in prion-inoculated mouse brains and in murine cerebellar organotypic cultured slices (COCS). We then removed microglia from COCS using a ganciclovir-dependent lineage ablation strategy. NOX2 became undetectable in ganciclovir-treated COCS, confirming its microglial origin. Upon challenge with prions, NOX2-deficient mice showed delayed onset of motor deficits and a modest, but significant prolongation of survival. Dihydroethidium assays demonstrated a conspicuous ROS burst at the terminal stage of disease in wild-type mice, but not in NOX2-ablated mice. Interestingly, the improved motor performance in NOX2 deficient mice was already measurable at earlier stages of the disease, between 13 and 16 weeks post-inoculation. We conclude that NOX2 is a major source of ROS in prion diseases and can affect prion pathogenesis.
Author Summary
The deposition of misfolded, aggregated prion protein in the brain causes transmissible spongiform encephalopathies (TSE), a group of disorders including Creutzfeldt–Jakob disease and mad cow disease. TSE are characterized by neurodegeneration and progressive, lethal neurological dysfunction. Signs of oxidative damage are found in TSE, implying excessive production of reactive oxygen species (ROS), yet their source is unclear. Here, we analyzed the role of the NADPH oxidase enzyme, NOX2, in prion pathogenesis. NOX2 is a membrane-bound electrochemical pump that generates ROS. We found that NOX2 is upregulated in the brains of patients with Creutzfeldt-Jakob disease and of prion-infected mice. Interestingly, NOX2 ablation led to abrogation of ROS production in mice inoculated with prions, and was associated with a milder clinical course of the disease and increased life expectancy. We conclude that NOX2 is a relevant contributor to the excessive production of ROS. This study spawns the possibility that inhibiting NOX2 activation might help attenuate prion disease progression – a legitimate and important goal even if there is little reason to expect anti-NOX2 therapies to be curative.
doi:10.1371/journal.ppat.1004531
PMCID: PMC4263757  PMID: 25502554
5.  Superresolution Imaging of Amyloid Fibrils with Binding-Activated Probes 
ACS Chemical Neuroscience  2013;4(7):1057-1061.
Protein misfolding into amyloid-like aggregates underlies many neurodegenerative diseases. Thus, insights into the structure and function of these amyloids will provide valuable information on the pathological mechanisms involved and aid in the design of improved drugs for treating amyloid-based disorders. However, determining the structure of endogenous amyloids at high resolution has been difficult. Here we employ binding-activated localization microscopy (BALM) to acquire superresolution images of α-synuclein amyloid fibrils with unprecedented optical resolution. We propose that BALM imaging can be extended to study the structure of other amyloids, for differential diagnosis of amyloid-related diseases and for discovery of drugs that perturb amyloid structure for therapy.
doi:10.1021/cn400091m
PMCID: PMC3715833  PMID: 23594172
Alzheimer’s disease; Parkinson’s disease; amyloid; superresolution; neurodegenerative diseases; binding-activated; localization microscopy; diagnosis; alpha-synuclein
6.  Prion Transmission Prevented by Modifying the β2-α2 Loop Structure of Host PrPC 
The Journal of Neuroscience  2014;34(3):1022-1027.
Zoonotic prion transmission was reported after the bovine spongiform encephalopathy (BSE) epidemic, when >200 cases of prion disease in humans were diagnosed as variant Creutzfeldt-Jakob disease. Assessing the risk of cross-species prion transmission remains challenging. We and others have studied how specific amino acid residue differences between species impact prion conversion and have found that the β2-α2 loop region of the mouse prion protein (residues 165–175) markedly influences infection by sheep scrapie, BSE, mouse-adapted scrapie, deer chronic wasting disease, and hamster-adapted scrapie prions. The tyrosine residue at position 169 is strictly conserved among mammals and an aromatic side chain in this position is essential to maintain a 310-helical turn in the β2-α2 loop. Here we examined the impact of the Y169G substitution together with the previously described S170N, N174T “rigid loop” substitutions on cross-species prion transmission in vivo and in vitro. We found that transgenic mice expressing mouse PrP containing the triple-amino acid substitution completely resisted infection with two strains of mouse prions and with deer chronic wasting disease prions. These studies indicate that Y169 is important for prion formation, and they provide a strong indication that variation of the β2-α2 loop structure can modulate interspecies prion transmission.
doi:10.1523/JNEUROSCI.4636-13.2014
PMCID: PMC3891945  PMID: 24431459
conversion; transmission; amyloid; TSE; prions
7.  SIRPα polymorphisms, but not the prion protein, control phagocytosis of apoptotic cells 
The Journal of Experimental Medicine  2013;210(12):2539-2552.
The regulation of phagocytosis previously ascribed to prion protein PrPC is found to be controlled by the linked locus encoding SIRPα.
Prnp−/− mice lack the prion protein PrPC and are resistant to prion infections, but variable phenotypes have been reported in Prnp−/− mice and the physiological function of PrPC remains poorly understood. Here we examined a cell-autonomous phenotype, inhibition of macrophage phagocytosis of apoptotic cells, previously reported in Prnp−/− mice. Using formal genetic, genomic, and immunological analyses, we found that the regulation of phagocytosis previously ascribed to PrPC is instead controlled by a linked locus encoding the signal regulatory protein α (Sirpa). These findings indicate that control of phagocytosis was previously misattributed to the prion protein and illustrate the requirement for stringent approaches to eliminate confounding effects of flanking genes in studies modeling human disease in gene-targeted mice. The plethora of seemingly unrelated functions attributed to PrPC suggests that additional phenotypes reported in Prnp−/− mice may actually relate to Sirpa or other genetic confounders.
doi:10.1084/jem.20131274
PMCID: PMC3832919  PMID: 24145514
8.  Prions and lymphoid organs 
Prion  2013;7(2):157-163.
Prion colonization of secondary lymphoid organs (SLOs) is a critical step preceding neuroinvasion in prion pathogenesis. Follicular dendritic cells (FDCs), which depend on both tumor necrosis factor receptor 1 (TNFR1) and lymphotoxin β receptor (LTβR) signaling for maintenance, are thought to be the primary sites of prion accumulation in SLOs. However, prion titers in RML-infected TNFR1−/− lymph nodes and rates of neuroinvasion in TNFR1−/− mice remain high despite the absence of mature FDCs. Recently, we discovered that TNFR1-independent prion accumulation in lymph nodes relies on LTβR signaling. Loss of LTβR signaling in TNFR1−/− lymph nodes coincided with the de-differentiation of high endothelial venules (HEVs)—the primary sites of lymphocyte entry into lymph nodes. These findings suggest that HEVs are the sites through which prions initially invade lymph nodes from the bloodstream. Identification of HEVs as entry portals for prions clarifies a number of previous observations concerning peripheral prion pathogenesis. However, a number of questions still remain: What is the mechanism by which prions are taken up by HEVs? Which cells are responsible for delivering prions to lymph nodes? Are HEVs the main entry site for prions into lymph nodes or do alternative routes also exist? These questions and others are considered in this article.
doi:10.4161/pri.23536
PMCID: PMC3609124  PMID: 23357827
prions; lymph nodes; high endothelial venules; lymphotoxin beta receptor; tumor necrosis factor receptor 1; peripheral prion replication; neuroinvasion; follicular dendritic cell
9.  Genetic Depletion of Complement Receptor CD21/35 Prevents Terminal Prion Disease in a Mouse Model of Chronic Wasting Disease1 
The Complement System has been shown to facilitate peripheral prion pathogenesis. Mice lacking Complement receptors CD21/35 partially resist terminal prion disease when infected intraperitoneally with mouse-adapted scrapie prions. Chronic wasting disease (CWD) is an emerging prion disease of captive and free-ranging cervid populations that, like scrapie, has been shown to involve the immune system, which probably contributes to their relatively facile horizontal and environmental transmission. Here we show that mice overexpressing the cervid prion protein and susceptible to CWD (Tg(cerPrP)5037 mice) but lack CD21/35 expression completely resist clinical CWD upon peripheral infection. CD21/35 deficient Tg5037 mice exhibit greatly impaired splenic prion accumulation and replication throughout disease, similar to CD21/35 deficient murine PrP mice infected with mouse scrapie. TgA5037;CD21/35-/- mice exhibited little or no neuropathology and deposition of misfolded, protease-resistant PrP associated with CWD. CD21/35 translocates to lipid rafts and mediates a strong germinal center response to prion infection that we propose provides the optimal environment for prion accumulation and replication. We further propose a potential role for CD21/35 in selecting prion quasi-species present in prion strains that may exhibit differential zoonotic potential compared to the parental strains.
doi:10.4049/jimmunol.1201579
PMCID: PMC3478448  PMID: 23002439
ChronicWasting Disease; Complement; Prions
10.  Crystallization and preliminary X-ray diffraction analysis of prion protein bound to the Fab fragment of the POM1 antibody 
The complex of MoPrP(120–232) and Fab POM1 has been crystallized (space group C2, unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å, β = 95.6°). Diffraction data to 2.30 Å resolution have been collected using synchrotron radiation.
Prion diseases are neurodegenerative diseases that are characterized by the con­version of the cellular prion protein PrPc to the pathogenic isoform PrPsc. Several antibodies are known to interact with the cellular prion protein and to inhibit this transition. An antibody Fab fragment, Fab POM1, was produced that recognizes a structural motif of the C-terminal domain of mouse prion protein. To study the mechanism by which Fab POM1 recognizes and binds the prion molecule, the complex between Fab POM1 and the C-terminal domain of mouse prion (residues 120–232) was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group C2, with unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å, β = 95.6°.
doi:10.1107/S1744309111026273
PMCID: PMC3212364  PMID: 22102029
prions; antibodies; POM1
11.  Follicular Dendritic Cells Emerge from Ubiquitous Perivascular Precursors 
Cell  2012;150(1):194-206.
Summary
The differentiation of follicular dendritic cells (FDC) is essential to the remarkable microanatomic plasticity of lymphoid follicles. Here we show that FDC arise from ubiquitous perivascular precursors (preFDC) expressing platelet-derived growth factor receptor β (PDGFRβ). PDGFRβ-Cre-driven reporter gene recombination resulted in FDC labeling, whereas conditional ablation of PDGFRβ+-derived cells abolished FDC, indicating that FDC originate from PDGFRβ+ cells. Lymphotoxin-α-overexpressing prion protein (PrP)+ kidneys developed PrP+ FDC after transplantation into PrP mice, confirming that preFDC exist outside lymphoid organs. Adipose tissue-derived PDGFRβ+ stromal-vascular cells responded to FDC maturation factors and, when transplanted into lymphotoxin β receptor (LTβR) kidney capsules, differentiated into Mfge8+CD21/35+ FcγRIIβ+PrP+ FDC capable of trapping immune complexes and recruiting B cells. Spleens of lymphocyte-deficient mice contained perivascular PDGFRβ+ FDC precursors whose expansion required both lymphoid tissue inducer (LTi) cells and lymphotoxin. The ubiquity of preFDC and their strategic location at blood vessels may explain the de novo generation of organized lymphoid tissue at sites of lymphocytic inflammation.
doi:10.1016/j.cell.2012.05.032
PMCID: PMC3704230  PMID: 22770220
12.  BSE-associated Prion-Amyloid Cardiomyopathy in Primates 
Emerging Infectious Diseases  2013;19(6):985-988.
Prion amyloidosis occurred in the heart of 1 of 3 macaques intraperitoneally inoculated with bovine spongiform encephalopathy prions. This macaque had a remarkably long duration of disease and signs of cardiac distress. Variant Creutzfeldt-Jakob disease, caused by transmission of bovine spongiform encephalopathy to humans, may manifest with cardiac symptoms from prion-amyloid cardiomyopathy.
doi:10.3201/eid1906.120906
PMCID: PMC3713817  PMID: 23735198
BSE; cardiomyopathy; prions; PrPSc; vCJD; variant Creutzfeldt-Jakob disease; bovine spongiform encephalopathy; primates
13.  The complex PrPc-Fyn couples human oligomeric Aβ with pathological tau changes in Alzheimer’s disease 
Amidst controversy, the cellular form of the prion protein PrPc has been proposed to mediate oligomeric Aβ-induced deficits. In contrast, there is consistent evidence that the Src kinase Fyn is activated by Aβ oligomers and leads to synaptic and cognitive impairment in transgenic animals. However, the molecular mechanism by which soluble Aβ activates Fyn remains unknown. Combining the use of human and transgenic mouse brain tissue as well as primary cortical neurons, we demonstrate that soluble Aβ binds to PrPc at neuronal dendritic spines in vivo and in vitro where it forms a complex with Fyn, resulting in the activation of the kinase. Using the antibody 6D11 to prevent oligomeric Aβ from binding to PrPc, we abolished Fyn activation and Fyn-dependent tau hyperphosphorylation induced by endogenous oligomeric Aβ in vitro. Finally we showed that gene dosage of Prnp regulates Aβ-induced Fyn/tau alterations. Altogether, our findings identify a complete signaling cascade linking one specific endogenous Aβ oligomer, Fyn alteration and tau hyperphosphorylation in cellular and animal models modeling aspects of the molecular pathogenesis of Alzheimer’s disease.
doi:10.1523/JNEUROSCI.1858-12.2012
PMCID: PMC3568961  PMID: 23175838
amyloid-beta; oligomer; prion protein; Fyn; Alzheimer’s disease; transgenic
14.  Prions, prionoids and pathogenic proteins in Alzheimer disease 
Prion  2013;7(1):55-59.
Like patients with prion disease, Alzheimer patients suffer from a fatal, progressive form of dementia. There is growing evidence that amyloid-β (Aβ) aggregates may be transmissible similar to prions, at least under extreme experimental conditions. However, unlike mice infected with prion protein (PrP) prions, those inoculated with Aβ do not die. The transmission of Aβ and PrP thus differs conspicuously in the neurological effects they induce in their hosts, the difference being no less than a matter of life and death. Far from being a mere academic nuance, this distinction between Aβ and PrP begs the crucial questions of what, exactly, controls prion toxicity and how prion toxicity relates to prion infectivity.
doi:10.4161/pri.23061
PMCID: PMC3609051  PMID: 23208281
Alzheimer’s disease; PrP; amyloid-β; pathogenic proteins; prionoids; prions; tau
15.  Prion Pathogenesis Is Faithfully Reproduced in Cerebellar Organotypic Slice Cultures 
PLoS Pathogens  2012;8(11):e1002985.
Prions cause neurodegeneration in vivo, yet prion-infected cultured cells do not show cytotoxicity. This has hampered mechanistic studies of prion-induced neurodegeneration. Here we report that prion-infected cultured organotypic cerebellar slices (COCS) experienced progressive spongiform neurodegeneration closely reproducing prion disease, with three different prion strains giving rise to three distinct patterns of prion protein deposition. Neurodegeneration did not occur when PrP was genetically removed from neurons, and a comprehensive pharmacological screen indicated that neurodegeneration was abrogated by compounds known to antagonize prion replication. Prion infection of COCS and mice led to enhanced fodrin cleavage, suggesting the involvement of calpains or caspases in pathogenesis. Accordingly, neurotoxicity and fodrin cleavage were prevented by calpain inhibitors but not by caspase inhibitors, whereas prion replication proceeded unimpeded. Hence calpain inhibition can uncouple prion replication from its neurotoxic sequelae. These data validate COCS as a powerful model system that faithfully reproduces most morphological hallmarks of prion infections. The exquisite accessibility of COCS to pharmacological manipulations was instrumental in recognizing the role of calpains in neurotoxicity, and significantly extends the collection of tools necessary for rigorously dissecting prion pathogenesis.
Author Summary
Transmissible spongiform encephalopathies (TSEs) are a group of fatal protein misfolding diseases causing neurodegeneration in vivo. TSEs are unique in that the infectious agent termed ‘prion’ consists of a misfolded protein lacking sequence specific nucleic acids. Prion-infected cultured cells do not develop visible pathological changes, and this has hampered mechanistic studies of prion-induced neurodegeneration. Here, we have developed a prion-induced neurodegeneration model that uses cultured slices of living brain tissue. Such slices display all the classical hallmark of prion disease, namely prion replication, inflammation, spongiform changes and neurodegeneration. Neurotoxicity is blocked by anti-prion drugs by reducing prion replication. We demonstrate for the first time an involvement of calcium-regulated cysteine proteases called calpains in driving neurotoxicity. We find that the proteolytic processing of the calpain substrate is induced by prion infection and blocked by calpain inhibitors without prion replication being affected. The assay system developed here allows for precise dissection of the mechanisms of prion-induced degeneration with pharmacological means.
doi:10.1371/journal.ppat.1002985
PMCID: PMC3486912  PMID: 23133383
16.  Genetic Depletion of Complement Receptors CD21/35 Prevents Terminal Prion Disease in a Mouse Model of Chronic Wasting Disease 
The complement system has been shown to facilitate peripheral prion pathogenesis. Mice lacking complement receptors CD21/35 partially resist terminal prion disease when infected i.p. with mouse-adapted scrapie prions. Chronic wasting disease (CWD) is an emerging prion disease of captive and free-ranging cervid populations that, similar to scrapie, has been shown to involve the immune system, which probably contributes to their relatively facile horizontal and environmental transmission. In this study, we show that mice overexpressing the cervid prion protein and susceptible to CWD (Tg(cerPrP)5037 mice) but lack CD21/35 expression completely resist clinical CWD upon peripheral infection. CD21/35-deficient Tg5037 mice exhibit greatly impaired splenic prion accumulation and replication throughout disease, similar to CD21/35-deficient murine prion protein mice infected with mouse scrapie. TgA5037;CD21/35−/− mice exhibited little or no neuropathology and deposition of misfolded, protease-resistant prion protein associated with CWD. CD21/35 translocate to lipid rafts and mediates a strong germinal center response to prion infection that we propose provides the optimal environment for prion accumulation and replication. We further propose a potential role for CD21/35 in selecting prion quasi-species present in prion strains that may exhibit differential zoonotic potential compared with the parental strains.
doi:10.4049/jimmunol.1201579
PMCID: PMC3478448  PMID: 23002439
17.  Cerebrospinal fluid biomarker supported diagnosis of Creutzfeldt–Jakob disease and rapid dementias: a longitudinal multicentre study over 10 years 
Brain  2012;135(10):3051-3061.
To date, cerebrospinal fluid analysis, particularly protein 14-3-3 testing, presents an important approach in the identification of Creutzfeldt–Jakob disease cases. However, one special point of criticism of 14-3-3 testing is the specificity in the differential diagnosis of rapid dementia. The constant observation of increased cerebrospinal fluid referrals in the national surveillance centres over the last years raises the concern of declining specificity due to higher number of cerebrospinal fluid tests performed in various neurological conditions. Within the framework of a European Community supported longitudinal multicentre study (‘cerebrospinal fluid markers’) we analysed the spectrum of rapid progressive dementia diagnoses, their potential influence on 14-3-3 specificity as well as results of other dementia markers (tau, phosphorylated tau and amyloid-β1–42) and evaluated the specificity of 14-3-3 in Creutzfeldt–Jakob disease diagnosis for the years 1998–2008. A total of 29 022 cerebrospinal fluid samples were analysed for 14-3-3 protein and other cerebrospinal fluid dementia markers in patients with rapid dementia and suspected Creutzfeldt–Jakob disease in the participating centres. In 10 731 patients a definite diagnosis could be obtained. Protein 14-3-3 specificity was analysed for Creutzfeldt–Jakob disease with respect to increasing cerebrospinal fluid tests per year and spectrum of differential diagnosis. Ring trials were performed to ensure the comparability between centres during the reported time period. Protein 14-3-3 test specificity remained high and stable in the diagnosis of Creutzfeldt–Jakob disease during the observed time period across centres (total specificity 92%; when compared with patients with definite diagnoses only: specificity 90%). However, test specificity varied with respect to differential diagnosis. A high 14-3-3 specificity was obtained in differentiation to other neurodegenerative diseases (95–97%) and non-neurological conditions (91–97%). We observed lower specificity in the differential diagnoses of acute neurological diseases (82–87%). A marked and constant increase in cerebrospinal fluid test referrals per year in all centres did not influence 14-3-3 test specificity and no change in spectrum of differential diagnosis was observed. Cerebrospinal fluid protein 14-3-3 detection remains an important test in the diagnosis of Creutzfeldt–Jakob disease. Due to a loss in specificity in acute neurological events, the interpretation of positive 14-3-3 results needs to be performed in the clinical context. The spectrum of differential diagnosis of rapid progressive dementia varied from neurodegenerative dementias to dementia due to acute neurological conditions such as inflammatory diseases and non-neurological origin.
doi:10.1093/brain/aws238
PMCID: PMC3470713  PMID: 23012332
rapid dementia; Creutzfeldt–Jakob disease; cerebrospinal fluid; 14-3-3; specificity; neurodegeneration; differential diagnosis in dementia
18.  Evaluation of OPEN Zinc Finger Nucleases for Direct Gene Targeting of the ROSA26 Locus in Mouse Embryos 
PLoS ONE  2012;7(9):e41796.
Zinc finger nucleases (ZFNs) enable precise genome modification in a variety of organisms and cell types. Commercial ZFNs were reported to enhance gene targeting directly in mouse zygotes, whereas similar approaches using publicly available resources have not yet been described. Here we report precise targeted mutagenesis of the mouse genome using Oligomerized Pool Engineering (OPEN) ZFNs. OPEN ZFN can be constructed using publicly available resources and therefore provide an attractive alternative for academic researchers. Two ZFN pairs specific to the mouse genomic locus gt(ROSA26)Sor were generated by OPEN selections and used for gene disruption and homology-mediated gene replacement in single cell mouse embryos. One specific ZFN pair facilitated non-homologous end joining (NHEJ)-mediated gene disruption when expressed in mouse zygotes. We also observed a single homologous recombination (HR)-driven gene replacement event when this ZFN pair was co-injected with a targeting vector. Our experiments demonstrate the feasibility of achieving both gene ablation through NHEJ and gene replacement by HR by using the OPEN ZFN technology directly in mouse zygotes.
doi:10.1371/journal.pone.0041796
PMCID: PMC3435328  PMID: 22970113
19.  Lymphotoxin, but Not TNF, Is Required for Prion Invasion of Lymph Nodes 
PLoS Pathogens  2012;8(8):e1002867.
Neuroinvasion and subsequent destruction of the central nervous system by prions are typically preceded by a colonization phase in lymphoid organs. An important compartment harboring prions in lymphoid tissue is the follicular dendritic cell (FDC), which requires both tumor necrosis factor receptor 1 (TNFR1) and lymphotoxin β receptor (LTβR) signaling for maintenance. However, prions are still detected in TNFR1−/− lymph nodes despite the absence of mature FDCs. Here we show that TNFR1-independent prion accumulation in lymph nodes depends on LTβR signaling. Loss of LTβR signaling, but not of TNFR1, was concurrent with the dedifferentiation of high endothelial venules (HEVs) required for lymphocyte entry into lymph nodes. Using luminescent conjugated polymers for histochemical PrPSc detection, we identified PrPSc deposits associated with HEVs in TNFR1−/− lymph nodes. Hence, prions may enter lymph nodes by HEVs and accumulate or replicate in the absence of mature FDCs.
Author Summary
Prions are unique infectious agents thought to be composed entirely of an abnormal conformer of the endogenous prion protein. Prions cause a severe neurological disorder in humans and other animals known as prion disease. Though prion disease can arise spontaneously or from genetic mutations in the gene encoding the prion protein, many cases of prion disease arise due to peripheral exposure to the infectious agent. In these cases, prions must journey from the gastrointestinal tract and/or the bloodstream to the brain. Prions often colonize secondary lymphoid organs prior to invading the nervous system via neighboring peripheral nerves. Prion accumulation in follicular dendritic cells found in germinal centers of lymphoid organs is thought to be a crucial step in this process. However, prion colonization of lymph nodes, in contrast to spleen, does not depend on follicular dendritic cells, indicating that other mechanisms must exist. Here, we identify the signaling pathway required for follicular dendritic cell-independent prion colonization of lymph nodes, which also controls the differentiation of high endothelial venules, the primary entry point for lymphocytes into lymph nodes. Importantly, prions could be found within these structures, indicating that high endothelial venules are required for prion entry and accumulation in lymph nodes.
doi:10.1371/journal.ppat.1002867
PMCID: PMC3415451  PMID: 22912582
20.  Aerosols 
Prion  2011;5(3):138-141.
We and others have recently reported that prions can be transmitted to mice via aerosols. These reports spurred a lively public discussion on the possible public-health threats represented by prion-containing aerosols. Here we offer our view on the context in which these findings should be placed. On the one hand, the fact that nebulized prions can transmit disease cannot be taken to signify that prions are airborne under natural circumstances. On the other hand, it appears important to underscore the fact that aerosols can originate very easily in a broad variety of experimental and natural environmental conditions. Aerosols are a virtually unavoidable consequence of the handling of fluids; complete prevention of the generation of aerosols is very difficult. While prions have never been found to be transmissible via aerosols under natural conditions, it appears prudent to strive to minimize exposure to potentially prion-infected aerosols whenever the latter may arise—for example in scientific and diagnostic laboratories handling brain matter, cerebrospinal fluids, and other potentially contaminated materials, as well as abattoirs. Equally important is that prion biosafety training be focused on the control of, and protection from, prion-infected aerosols.
doi:10.4161/pri.5.3.16851
PMCID: PMC3226037  PMID: 21778819
prion; prion transmission; scrapie; chronic wasting diseases; CWD; Creutzfeldt-Jacob-disease; CJD; TSE; aerosol; pathogens; allergens
21.  ZyFISH: A Simple, Rapid and Reliable Zygosity Assay for Transgenic Mice 
PLoS ONE  2012;7(5):e37881.
Microinjection of DNA constructs into fertilized mouse oocytes typically results in random transgene integration at a single genomic locus. The resulting transgenic founders can be used to establish hemizygous transgenic mouse lines. However, practical and experimental reasons often require that such lines be bred to homozygosity. Transgene zygosity can be determined by progeny testing assays which are expensive and time-consuming, by quantitative Southern blotting which is labor-intensive, or by quantitative PCR (qPCR) which requires transgene-specific design. Here, we describe a zygosity assessment procedure based on fluorescent in situ hybridization (zyFISH). The zyFISH protocol entails the detection of transgenic loci by FISH and the concomitant assignment of homozygosity using a concise and unbiased scoring system. The method requires small volumes of blood, is scalable to at least 40 determinations per assay, and produces results entirely consistent with the progeny testing assay. This combination of reliability, simplicity and cost-effectiveness makes zyFISH a method of choice for transgenic mouse zygosity determinations.
doi:10.1371/journal.pone.0037881
PMCID: PMC3362593  PMID: 22666404
22.  Five Questions on Prion Diseases 
PLoS Pathogens  2012;8(5):e1002651.
doi:10.1371/journal.ppat.1002651
PMCID: PMC3342938  PMID: 22570608
23.  Spongiform encephalopathy in transgenic mice expressing a point mutation in the β2–α2 loop of the prion protein 
Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases attributed to misfolding of the cellular prion protein, PrPC, into a β-sheet-rich, aggregated isoform, PrPSc. We previously found that expression of mouse PrP with the two amino acid substitutions S170N and N174T, which result in high structural order of the β2–α2 loop in the NMR structure at pH 4.5 and 20 °C, caused transmissible de novo prion disease in transgenic mice. Here we report that expression of mouse PrP with the single-residue substitution D167S, which also results in a structurally well-ordered β2–α2 loop at 20 °C, elicits spontaneous PrP aggregation in vivo. Transgenic mice expressing PrPD167S developed a progressive encephalopathy characterized by abundant PrP plaque formation, spongiform change, and gliosis. These results add to the evidence that the β2–α2 loop has an important role in intermolecular interactions, including that it may be a key determinant of prion protein aggregation.
doi:10.1523/JNEUROSCI.3504-11.2011
PMCID: PMC3205959  PMID: 21957246
24.  Microglial ablation and lipopolysaccharide preconditioning affects pilocarpine-induced seizures in mice 
Neurobiology of disease  2010;39(1):85-97.
Activated microglia have been associated with neurodegeneration in patients and in animal models of Temporal Lobe Epilepsy (TLE), however their precise functions as neurotoxic or neuroprotective is a topic of significant investigation. To explore this, we examined the effects of pilocarpine induced seizures in transgenic mice where microglia/macrophages were conditionally ablated. We found that unilateral ablation of microglia from the dorsal hippocampus did not alter acute seizure sensitivity. However, when this procedure was coupled with lipopolysaccharide (LPS) preconditioning (1 mg/kg given 24 hours prior to acute seizure), we observed a significant pro-convulsant phenomenon. This effect was associated with lower metabolic activation in the ipsilateral hippocampus during acute seizures, and could be attributed to activity in the mossy fiber pathway. These findings reveal that preconditioning with LPS 24 hours prior to seizure induction may have a protective effect which is abolished by unilateral hippocampal microglia/macrophage ablation.
doi:10.1016/j.nbd.2010.04.001
PMCID: PMC2875881  PMID: 20382223
Microglia; seizure; epilepsy; lipopolysaccharide; status epilepticus; glucose; metabolism; positron emission tomography; pilocarpine; inflammation; mouse; FDG; imaging; immune system
25.  Sheep with Scrapie and Mastitis Transmit Infectious Prions through the Milk▿  
Journal of Virology  2010;85(2):1136-1139.
Prions are misfolded proteins that are infectious and naturally transmitted, causing a fatal neurological disease in humans and animals. Prion shedding routes have been shown to be modified by inflammation in excretory organs, such as the kidney. Here, we show that sheep with scrapie and lentiviral mastitis secrete prions into the milk and infect nearly 90% of naïve suckling lambs. Thus, lentiviruses may enhance prion transmission, conceivably sustaining prion infections in flocks for generations. This study also indicates a risk of prion spread to sheep and potentially to other animals through dietary exposure to pooled sheep milk or milk products.
doi:10.1128/JVI.02022-10
PMCID: PMC3020009  PMID: 21084475

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