Development of a Quantitative FMRP Assay for Mouse Tissue Applications.

Fragile X syndrome results from the absence of the FMR1 gene product-Fragile X Mental Retardation Protein (FMRP). Fragile X animal research has lacked a reliable method to quantify FMRP. We report the development of an array of FMRP-specific monoclonal antibodies and their application for quantitative assessment of FMRP (qFMRPm) in mouse tissue. To characterize the assay, we determined the normal variability of FMRP expression in four brain structures of six different mouse strains at seven weeks of age. There was a hierarchy of FMRP expression: neocortex > hippocampus > cerebellum > brainstem. The expression of FMRP was highest and least variable in the neocortex, whereas it was most variable in the hippocampus. Male C57Bl/6J and FVB mice were selected to determine FMRP developmental differences in the brain at 3, 7, 10, and 14 weeks of age. We examined the four structures and found a developmental decline in FMRP expression with age, except for the brainstem where it remained stable. qFMRPm assay of blood had highest values in 3 week old animals and dropped by 2.5-fold with age. Sex differences were not significant. The results establish qFMRPm as a valuable tool due to its ease of methodology, cost effectiveness, and accuracy.

Anti-prion Protein Antibody 6D11 Restores Cellular Proteostasis of Prion Protein Through Disrupting Recycling Propagation of PrPSc and Targeting PrPSc for Lysosomal Degradation.

PrPSc is an infectious and disease-specific conformer of the prion protein, which accumulation in the CNS underlies the pathology of prion diseases. PrPSc replicates by binding to the cellular conformer of the prion protein (PrPC) expressed by host cells and rendering its secondary structure a likeness of itself. PrPC is a plasma membrane anchored protein, which constitutively recirculates between the cell surface and the endocytic compartment. Since PrPSc engages PrPC along this trafficking pathway, its replication process is often referred to as "recycling propagation." Certain monoclonal antibodies (mAbs) directed against prion protein can abrogate the presence of PrPSc from prion-infected cells. However, the precise mechanism(s) underlying their therapeutic propensities remains obscure. Using N2A murine neuroblastoma cell line stably infected with 22L mouse-adapted scrapie strain (N2A/22L), we investigated here the modus operandi of the 6D11 clone, which was raised against the PrPSc conformer and has been shown to permanently clear prion-infected cells from PrPSc presence. We determined that 6D11 mAb engages and sequesters PrPC and PrPSc at the cell surface. PrPC/6D11 and PrPSc/6D11 complexes are then endocytosed from the plasma membrane and are directed to lysosomes, therefore precluding recirculation of nascent PrPSc back to the cell surface. Targeting PrPSc by 6D11 mAb to the lysosomal compartment facilitates its proteolysis and eventually shifts the balance between PrPSc formation and degradation. Ongoing translation of PrPC allows maintaining the steady-state level of prion protein within the cells, which was not depleted under 6D11 mAb treatment. Our findings demonstrate that through disrupting recycling propagation of PrPSc and promoting its degradation, 6D11 mAb restores cellular proteostasis of prion protein.

The role of reduced expression of fragile X mental retardation protein in neurons and increased expression in astrocytes in idiopathic and syndromic autism (duplications 15q11.2-q13).

Fragile X syndrome (FXS), caused by lack of fragile X mental retardation protein (FMRP), is associated with a high prevalence of autism. The deficit of FMRP reported in idiopathic autism suggests a mechanistic overlap between FXS and autism. The overall goal of this study is to detect neuropathological commonalities of FMRP deficits in the brains of people with idiopathic autism and with syndromic autism caused by dup15q11.2-q13 (dup15). This study tests the hypothesis based on our preliminary data that both idiopathic and syndromic autism are associated with brain region-specific deficits of neuronal FMRP and structural changes of the affected neurons. This immunocytochemical study revealed neuronal FMRP deficits and shrinkage of deficient neurons in the cerebral cortex, subcortical structures, and cerebellum in subjects with idiopathic and dup(15)/autism. Neuronal FMRP deficit coexists with surprising infiltration of the brains of autistic children and adults with FMRP-positive astrocytes known to be typical only for the fetal and short postnatal periods. In the examined autistic subjects, these astrocytes selectively infiltrate the border between white and gray matter in the cerebral and cerebellar cortex, the molecular layer of the cortex, part of the amygdala and thalamus, central cerebellar white matter, and dentate nucleus. Astrocyte pathology results in an additional local loss of FMRP in neurons and their shrinkage. Neuronal deficit of FMRP and shrinkage of affected neurons in structures free of FMRP-positive astrocytes and regions infiltrated with FMRP-expressing astrocytes appear to reflect mechanistic, neuropathological, and functional commonalities of FMRP abnormalities in FXS and autism spectrum disorder. Autism Res 2018, 11: 1316-1331. © 2018 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Immunocytochemistry reveals a deficit of fragile X mental retardation protein (FMRP) in neurons of cortical and subcortical brain structures but increased FMRP expression in astrocytes infiltrating gray and white matter. The detected shrinkage of FMRP-deficient neurons may provide a mechanistic explanation of reported neuronal structural and functional changes in autism. This study contributes to growing evidence of mechanistic commonalities between fragile X syndrome and autism spectrum disorder.

Detection and Quantification of the Fragile X Mental Retardation Protein 1 (FMRP).

The final product of FMR1 gene transcription, Fragile X Mental Retardation Protein 1 (FMRP), is an RNA binding protein that acts as a repressor of translation. FMRP is expressed in several tissues and plays important roles in neurogenesis, synaptic plasticity, and ovarian functions and has been implicated in a number of neuropsychological disorders. The loss of FMRP causes Fragile X Syndrome (FXS). In most cases, FXS is due to large expansions of a CGG repeat in FMR1-normally containing 6-54 repeats-to over 200 CGGs and identified as full mutation (FM). Hypermethylation of the repeat induces FMR1 silencing and lack of FMRP expression in FM male. Mosaic FM males express low levels of FMRP and present a less severe phenotype that inversely correlates with FMRP levels. Carriers of pre-mutations (55-200 CGG) show increased mRNA, and normal to reduced FMRP levels. Alternative splicing of FMR1 mRNA results in 24 FMRP predicted isoforms whose expression are tissues and developmentally regulated. Here, we summarize the approaches used by several laboratories including our own to (a) detect and estimate the amount of FMRP in different tissues, developmental stages and various pathologies; and (b) to accurately quantifying FMRP for a direct diagnosis of FXS in adults and newborns.

Fragile X protein in newborn dried blood spots.

BACKGROUND: The fragile X syndrome (FXS) results from mutation of the FMR1 gene that prevents expression of its gene product, FMRP. We previously characterized 215 dried blood spots (DBS) representing different FMR1 genotypes and ages with a Luminex-based immunoassay (qFMRP). We found variable FMRP levels in the normal samples and identified affected males by the drastic reduction of FMRP. METHODS: Here, to establish the variability of expression of FMRP in a larger random population we quantified FMRP in 2,000 anonymous fresh newborn DBS. We also evaluated the effect of long term storage on qFMRP by retrospectively assaying 74 aged newborn DBS that had been stored for 7-84 months that included normal and full mutation individuals. These analyses were performed on 3 mm DBS disks. To identify the alleles associated with the lowest FMRP levels in the fresh DBS, we analyzed the DNA in the samples that were more than two standard deviations below the mean. RESULTS: Analysis of the fresh newborn DBS revealed a broad distribution of FMRP with a mean approximately 7-fold higher than that we previously reported for fresh DBS in normal adults and no samples whose FMRP level indicated FXS. DNA analysis of the lowest FMRP DBS showed that this was the low extreme of the normal range and included a female carrying a 165 CGG repeat premutation. In the retrospective study of aged newborn DBS, the FMRP mean of the normal samples was less than 30% of the mean of the fresh DBS. Despite the degraded signal from these aged DBS, qFMRP identified the FXS individuals. CONCLUSIONS: The assay showed that newborn DBS contain high levels of FMRP that will allow identification of males and potentially females, affected by FXS. The assay is also an effective screening tool for aged DBS stored for up to four years.

Altered lymphocyte proliferation and innate immune function in scrapie 139A- and ME7-infected mice.

Lymphoid organs play an important role in prion disease development and progression. While the role of lymphoid organs and changes in immune-related genes have been extensively investigated in scrapie-infected animals, innate immunity has not. Previous studies examined lymphocyte function in scrapie-infected C3H/HeJ mice, which exhibit defects in lipopolysaccharide (LPS) response now known to result from a mutation in Toll-like receptor (TLR) 4. We examined immune function in scrapie-infected CD1 mice, which are LPS responders. Lymphocyte proliferation from CD1 mice infected with either 139A or ME7 scrapie was measured in response to concanavalin (Con) A or LPS at 1 and 3 months after infection. Following LPS exposure, mice infected 3 months with ME7, but not 139A, demonstrated significantly decreased lymphocyte proliferation compared to controls. After Con A exposure, lymphocyte proliferation in scrapie-infected mice did not differ from controls. Gender-specific comparison of lymphocyte proliferation showed significant decreases in mitogenic responses in females infected 3 months with either 139A or ME7, compared to controls. Males infected for 3 months with ME7, but not 139A, showed significantly decreased proliferation after lymphocyte exposure to LPS, but not Con A. Neither gender showed changes in lymphocyte proliferation after 1 month of scrapie infection. Innate immune activation of peritoneal macrophages was determined via production of nitric oxide (NO), IL-6, and TNF-α after exposure to TLR ligands. TNF-α and IL-6 production were reduced in macrophages from females infected with either scrapie strain for 3 months, while NO production after TLR agonist plus IFN-γ exposure was decreased in both females and males infected for 3 months with 139A, compared to ME7. These data demonstrated altered innate immunity, suggesting hormonal and/or other gender-specific regulation may contribute to gender differences in some immune functions. Our data demonstrate lymphocyte proliferation and innate immune functioning in scrapie-infected mice deteriorate with disease progression.

Fragile X screening by quantification of FMRP in dried blood spots by a Luminex immunoassay.

Fragile X is the most common inherited cause of intellectual disability and is frequently associated with autism. The syndrome is due to mutations of the FMR1 gene that result in the absence of fragile X mental retardation protein (FMRP). We have developed a rapid, highly sensitive method for quantifying FMRP from dried blood spots and lymphocytes. This assay uses two new antibodies, a bacterially expressed abbreviated FMRP standard, and a Luminex platform to quantify FMRP. The assay readily distinguished between samples from males with fragile X full mutations and samples from normal males. It also differentiated mosaic from nonmosaic full-mutation male samples. This assay, because of its methodology and minimal cost, could be the basis for newborn or population screening.

Inflammatory mediators are inhibited by a taurine metabolite in CpG oligodeoxynucleotide and IFN-r activated macrophage cell line.

Taurine plays an important role in brain and retinal development, and has an antiinflammatory and antioxidant function. Taurine chloramine (Tau-Cl) is produced in polymorphonuclear leukocytes via the myeloperoxidase/halide system. We previously demonstrated that Tau-Cl inhibits the production of nitric oxide (NO) and TNF-α in human and murine macrophages activated with IFN-γ in combination with individual Toll-like receptor (TLR) ligands including those for TLR2 and/or TLR4. In the current study, we further explored the effects of Tau-Cl in RAW 264.7 cells stimulated with the TLR9 ligand CpG oligodeoxynucleotide (ODN). Specifically, we examined the effect of CpG ODN plus IFN-γ on the production of NO and TNF-α, and the effect of Tau-Cl on this process. Our findings show that CpG ODN plus IFN-γ-activated RAW 264.7 cells secrete high levels of NO and TNF-α, and that Tau-Cl (0.8 mM) inhibits this effect in a dose-dependent manner, more potently inhibiting the production of NO (99% inhibition) than that of TNF-α (48% inhibition). Nitric oxide synthase (iNOS) protein was also induced by CpG ODN plus IFN-γ, and was also inhibited by Tau-Cl. Furthermore, while CpG ODN plus IFN-γ induced TNF-α and iNOS mRNAs, Tau-Cl transiently suppressed this effect. Taurine itself had no effects on any of these processes. Our findings in a macrophage cell line demonstrate that Tau-Cl inhibits proinflammatory mediators resulting from TLR9 activation, and have implications for the utility of Tau-Cl in scenarios where such activation is deleterious such as in autoimmune conditions or infections in which overwhelming inflammation may occur. CpG ODNs and Tau-Cl both have potential for topical treatment of autoimmune conditions, including psoriasis, vitiligo, and alopecia areata. As CpG ODNs may, under some conditions, up-regulate Tregs, addition of Tau-Cl to CpG ODN topical formulations has potential for improving cancer immunotherapy.

Immunomodulation targeting abnormal protein conformation reduces pathology in a mouse model of Alzheimer's disease.

Many neurodegenerative diseases are characterized by the conformational change of normal self-proteins into amyloidogenic, pathological conformers, which share structural properties such as high ß-sheet content and resistance to degradation. The most common is Alzheimer's disease (AD) where the normal soluble amyloid ß (sAß) peptide is converted into highly toxic oligomeric Aß and fibrillar Aß that deposits as neuritic plaques and congophilic angiopathy. Currently, there is no highly effective treatment for AD, but immunotherapy is emerging as a potential disease modifying intervention. A major problem with most active and passive immunization approaches for AD is that both the normal sAß and pathogenic forms are equally targeted with the potential of autoimmune inflammation. In order to avoid this pitfall, we have developed a novel immunomodulatory method that specifically targets the pathological conformations, by immunizing with polymerized British amyloidosis (pABri) related peptide which has no sequence homology to Aß or other human proteins. We show that the pABri peptide through conformational mimicry induces a humoral immune response not only to the toxic Aß in APP/PS1 AD transgenic mice but also to paired helical filaments as shown on AD human tissue samples. Treated APP/PS1 mice had a cognitive benefit compared to controls (p<0.0001), associated with a reduction in the amyloid burden (p = 0.0001) and Aß40/42 levels, as well as reduced Aß oligomer levels. This type of immunomodulation has the potential to be a universal ß-sheet disrupter, which could be useful for the prevention or treatment of a wide range of neurodegenerative diseases.

Anti-PrPC monoclonal antibody infusion as a novel treatment for cognitive deficits in an Alzheimer's disease model mouse.

BACKGROUND: Alzheimer's Disease (AD) is the most common of the conformational neurodegenerative disorders characterized by the conversion of a normal biological protein into a ß-sheet-rich pathological isoform. In AD the normal soluble Aß (sAß) forms oligomers and fibrils which assemble into neuritic plaques. The most toxic form of Aß is thought to be oligomeric. A recent study reveals the cellular prion protein, PrPC, to be a receptor for Aß oligomers. Aß oligomers suppress LTP signal in murine hippocampal slices but activity remains when pretreated with the PrP monoclonal anti-PrP antibody, 6D11. We hypothesized that targeting of PrPC to prevent Aß oligomer-related cognitive deficits is a potentially novel therapeutic approach. APP/PS1 transgenic mice aged 8 months were intraperitoneally (i.p.) injected with 1 mg 6D11 for 5 days/week for 2 weeks. Two wild-type control groups were given either the same 6D11 injections or vehicle solution. Additional groups of APP/PS1 transgenic mice were given either i.p. injections of vehicle solution or the same dose of mouse IgG over the same period. The mice were then subjected to cognitive behavioral testing using a radial arm maze, over a period of 10 days. At the conclusion of behavioral testing, animals were sacrificed and brain tissue was analyzed biochemically or immunohistochemically for the levels of amyloid plaques, PrPC, synaptophysin, Aß40/42 and Aß oligomers. RESULTS: Behavioral testing showed a marked decrease in errors in 6D11 treated APP/PS1 Tg mice compared with the non-6D11 treated Tg groups (p < 0.0001). 6D11 treated APP/PS1 Tg mice behaved the same as wild-type controls indicating a recovery in cognitive learning, even after this short term 6D11 treatment. Brain tissue analysis from both treated and vehicle treated APP/PS1 groups indicate no significant differences in amyloid plaque burden, Aß40/42, PrPC or Aß oligomer levels. 6D11 treated APP/PS1 Tg mice had significantly greater synaptophysin immunoreactivity in the dentate gyrus molecular layer of the hippocampus compared to vehicle treated APP/PS1 Tg mice (p < 0.05). CONCLUSIONS: Even short term treatment with monoclonal antibodies such as 6D11 or other compounds which block the binding of Aß oligomers to PrPC can be used to treat cognitive deficits in aged AD transgenic mice.

Species-dependent differences in cofactor utilization for formation of the protease-resistant prion protein in vitro.

The cofactor preferences for in vitro propagation of the protease-resistant isoforms of the prion protein (PrP(Sc)) from various rodent species were investigated using the serial protein misfolding cyclic amplification (sPMCA) technique. Whereas RNA molecules facilitate hamster PrP(Sc) propagation, RNA and several other polyanions do not promote the propagation of mouse and vole PrP(Sc) molecules. Pretreatment of crude Prnp(0/0) (PrP knockout) brain homogenate with RNase A or micrococcal nuclease inhibited hamster but not mouse PrP(Sc) propagation in a reconstituted system. Mouse PrP(Sc) propagation could be reconstituted by mixing PrP(C) substrate with homogenates prepared from either brain or liver, but not from several other tissues that were tested. These results reveal species-specific differences in cofactor utilization for PrP(Sc) propagation in vitro and also demonstrate the existence of an endogenous cofactor present in brain tissue not composed of nucleic acids.

Anti-PrP Mab 6D11 suppresses PrP(Sc) replication in prion infected myeloid precursor line FDC-P1/22L and in the lymphoreticular system in vivo.

The pathogenesis of prion diseases is related to conformational transformation of cellular prion protein (PrP(C)) into a toxic, infectious, and self-replicating conformer termed PrP(Sc). Following extracerebral inoculation, the replication of PrP(Sc) is confined for months to years to the lymporeticular system (LRS) before the secondary CNS involvement results in occurrence of neurological symptoms. Therefore, replication of PrP(Sc), in the early stage of infection can be targeted by therapeutic approaches, which like passive immunization have limited blood-brain-barrier penetration. In this study, we show that 6D11 anti-PrP monoclonal antibody (Mab) prevents infection on a FDC-P1 myeloid precursor cell line stably infected with 22L mouse adapted scrapie strain. Passive immunization of extracerebrally infected CD-1 mice with Mab 6D11 resulted in effective suppression of PrP(Sc) replication in the LRS. Although, a rebound of PrP(Sc) presence occurred when the Mab 6D11 treatment was stopped, passively immunized mice showed a prolongation of the incubation period by 36.9% (pb0.0001) and a significant decrease in CNS pathology compared to control groups receiving vehicle or murine IgG. Our results indicate that antibody-based therapeutic strategies can be used, even on a short-term basis, to delay or prevent disease in subjects accidentally exposed to prions.

Induction of toll-like receptor 9 signaling as a method for ameliorating Alzheimer's disease-related pathology.

The pathogenesis of Alzheimer's disease (AD) is thought to be related to the accumulation of amyloid beta (Abeta) in amyloid deposits and toxic oligomeric species. Immunomodulation is emerging as an effective means of shifting the equilibrium from Abeta accumulation to clearance; however, excessive cell mediated inflammation and cerebral microhemorrhages are two forms of toxicity which can occur with this approach. Vaccination studies have so far mainly targeted the adaptive immune system. In the present study, we have stimulated the innate immune system via the Toll-like receptor 9 (TLR9) with cytosine-guanosine-containing DNA oligodeoxynucleotides in Tg2576 AD model transgenic mice. This treatment produced a 66% and 80% reduction in the cortical (p = 0.0001) and vascular (p = 0.0039) amyloid burden, respectively, compared with nontreated AD mice. This was in association with significant reductions in Abeta42, Abeta40, and Abeta oligomer levels. We also show that treated Tg mice performed similarly to wild-type mice on a radial arm maze. Our data suggest that stimulation of innate immunity via TLR9 is highly effective at reducing the parenchymal and vascular amyloid burden, along with Abeta oligomers, without apparent toxicity.

Accelerated prion disease pathogenesis in Toll-like receptor 4 signaling-mutant mice.

Prion diseases such as scrapie involve the accumulation of disease-specific prion protein, PrP(Sc), in the brain. Toll-like receptors (TLRs) are a family of proteins that recognize microbial constituents and are central players in host innate immune responses. The TLR9 agonist unmethylated CpG DNA was shown to prolong the scrapie incubation period in mice, suggesting that innate immune activation interferes with prion disease progression. Thus, it was predicted that ablation of TLR signaling would result in accelerated pathogenesis. C3H/HeJ (Tlr4(Lps-d)) mice, which possess a mutation in the TLR4 intracellular domain preventing TLR4 signaling, and strain-matched wild-type control (C3H/HeOuJ) mice were infected intracerebrally or intraperitoneally with various doses of scrapie inoculum. Incubation periods were significantly shortened in C3H/HeJ compared with C3H/HeOuJ mice, regardless of the route of infection or dose administered. At the clinical phase of disease, brain PrP(Sc) levels in the two strains of mice showed no significant differences by Western blotting. In addition, compared with macrophages from C3H/HeOuJ mice, those from C3H/HeJ mice were unresponsive to fibrillogenic PrP peptides (PrP residues 106 to 126 [PrP(106-126)] and PrP(118-135)) and the TLR4 agonist lipopolysaccharide but not to the TLR2 agonist zymosan, as measured by cytokine production. These data confirm that innate immune activation via TLR signaling interferes with scrapie infection. Furthermore, the results also suggest that the scrapie pathogen, or a component(s) thereof, is capable of stimulating an innate immune response that is active in the central nervous system, since C3H/HeJ mice, which lack the response, exhibit shortened incubation periods following both intraperitoneal and intracerebral infections.

A sensitive and quantitative assay for normal PrP in plasma.

Transmissible spongiform encephalopathies can be transmitted by blood transfusion. The risk of spreading the disease among the human population could be mitigated with the implementation of a blood screening assay. We developed a two-antibody assay for PrP detection in plasma using the ORIGEN technology with a protocol modification to improve the limit of detection and to increase the sample volume assayed. In the standard 200 microL format, the assay had a detection limit of 7-10 pg of recombinant PrP and 3 pg in 1 mL final volume implementation. PrP concentration measured in normal and scrapie-infected hamster brains was 7.5+/-0.9 and 57.3+/-9.6 microg/g, respectively. After a concentration step with an immuno-affinity resin, plasma PrP(c) was detected by Western blot and its concentration was measured at 3.5+/-0.8 ng/mL. From these data and assuming that blood has the same specific infectivity as brain, we estimated the concentration of abnormal PrP in hamster-infected plasma to be 32 f g/mL. The assay also detected abnormal brain PrP spiked into plasma although the limit of detection was affected. This is a novel and sensitive assay for the detection of PrP in plasma that could be developed into a platform for a plasma-based TSE test.

CpG oligodeoxynucleotide-enhanced humoral immune response and production of antibodies to prion protein PrPSc in mice immunized with 139A scrapie-associated fibrils.

Prion diseases are characterized by conversion of the cellular prion protein (PrP(C)) to a protease-resistant conformer, the srapie form of PrP (PrP(Sc)). Humoral immune responses to nondenatured forms of PrP(Sc) have never been fully characterized. We investigated whether production of antibodies to PrP(Sc) could occur in PrP null (Prnp(-/-)) mice and further, whether innate immune stimulation with the TLR9 agonist CpG oligodeoxynucleotide (ODN) 1826 could enhance this process. Whether such stimulation could raise anti-PrP(Sc) antibody levels in wild-type (Prnp(+/+)) mice was also investigated. Prnp(-/-) and Prnp(+/+) mice were immunized with nondenatured 139A scrapie-associated fibrils (SAF), with or without ODN 1826, and were tested for titers of PrP-specific antibodies. In Prnp(-/-) mice, inclusion of ODN 1826 in the immunization regime increased anti-PrP titers more than 13-fold after two immunizations and induced, among others, antibodies to an N-terminal epitope, which were only present in the immune repertoire of mice receiving ODN 1826. mAb 6D11, derived from such a mouse, reacts with the N-terminal epitope QWNK in native and denatured forms of PrP(Sc) and recombinant PrP and exhibits a K(d) in the 10(-)(11) M range. In Prnp(+/+) mice, ODN 1826 increased anti-PrP levels as much as 84% after a single immunization. Thus, ODN 1826 potentiates adaptive immune responses to PrP(Sc) in 139A SAF-immunized mice. These results represent the first characterization of humoral immune responses to nondenatured, infectious PrP(Sc) and suggest methods for optimizing the generation of mAbs to PrP(Sc), many of which could be used for diagnosis and treatment of prion diseases.

Passage of chronic wasting disease prion into transgenic mice expressing Rocky Mountain elk (Cervus elaphus nelsoni) PrPC.

Chronic wasting disease (CWD) of elk (Cervus elaphus nelsoni) and mule deer (Odocoileus hemionus) is one of three naturally occurring forms of prion disease, the others being Creutzfeldt-Jakob disease in humans and scrapie in sheep. In the last few decades, CWD has spread among captive and free-ranging cervids in 13 US states, two Canadian provinces and recently in Korea. The origin of the CWD agent(s) in cervids is not known. This study describes the development of a transgenic mouse line (TgElk) homozygous for a transgene array encoding the elk prion protein (PrP(C)) and its use in propagating and simulating CWD in mice. Intracerebral injection of one mule deer and three elk CWD isolates into TgElk mice led to disease with incubation periods of 127 and 95 days, respectively. Upon secondary passage, the incubation time was reduced to 108 and 90 days, respectively. Upon passage into TgElk mice, CWD prions (PrP(Sc)) maintained the characteristic Western blot profiles seen in CWD-affected mule deer and elk and produced histopathological modifications consistent with those observed in the natural disease. The short incubation time observed on passage from cervid to mouse with both mule deer and elk CWD brain homogenates and the demonstrated capacity of the animals to propagate (mouse to mouse) CWD agents make the TgElk line a valuable model to study CWD agents in cervid populations. In addition, these results with this new transgenic line suggest the intriguing hypothesis that there could be more than one strain of CWD agent in cervids.

Clearance and prevention of prion infection in cell culture by anti-PrP antibodies.

Prion diseases are transmissible and invariably fatal neurodegenerative disorders associated with a conformational transformation of the cellular prion protein (PrP(C)) into a self-replicating and proteinase K (PK)-resistant conformer, scrapie PrP (PrP(Sc)). Humoral immunity may significantly prolong the incubation period and even prevent disease in murine models of prionoses. However, the mechanism(s) of action of anti-PrP monoclonal antibodies (Mabs) remain(s) obscure. The murine neuroblastoma N2a cell line, infected with the 22L mouse-adapted scrapie strain, was used to screen a large library of Mabs with similar binding affinities to PrP, to identify those antibodies which could clear established infection and/or prevent infection de novo. Three Mabs were found capable of complete and persistent clearing of already-infected N2a cells of PrP(Sc). These antibodies were 6D11 (generated to PK-resistant PrP(Sc) and detecting PrP residues 93-109), and 7H6 and 7A12, which were raised against recombinant PrP and react with neighbouring epitopes of PrP residues 130-140 and 143-155, respectively. Mabs were found to interact with PrP(Sc) formation both on the cell surface and after internalization in the cytosol. Treatment with Mabs was not associated with toxicity nor did it result in decreased expression of PrP(C). Both preincubation of N2a cells with Mabs prior to exposure to 22L inoculum and preincubation of the inoculum with Mabs prior to infecting N2a cells resulted in a significant reduction in PrP(Sc) levels. Information provided in these studies is important for the rational design of humoral immune therapy for prion infection in animals and eventually in humans.

Copper (II) ions potently inhibit purified PrPres amplification.

The structural conversion of a host protein, PrP(C), into a protease-resistant isoform, PrPres, is the central event in the pathogenesis of infectious prion diseases. Purification of native PrP(C) molecules from hamster brain by either cation exchange or immobilized chelator chromatographic resins yielded preparations that supported efficient amplification of scrapie-induced PrPres in vitro. Using these purified preparations, we determined that in vitro PrPres amplification was inhibited by CuCl2 and ZnCl2 at IC50 concentrations of approximately 400 nm and 10 microM, respectively. In contrast, 100 microM MnCl2 did not directly inhibit PrPres amplification or block Cu2+-mediated inhibition. Additionally, the inhibition of PrPres amplification by Cu2+ ions could be reversed by addition of either neocuproine or imidazole. Cu2+ inhibited PrPres amplification in both the presence and absence of stimulatory polyanion molecules. These biochemical findings support the hypothesis that Cu2+ ions might regulate the pathogenesis of prion diseases in vivo.