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.

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.

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

PrP is an infectious and disease-specific conformer of the prion protein, which accumulation in the CNS underlies the pathology of prion diseases. PrP replicates by binding to the cellular conformer of the prion protein (PrP) expressed by host cells and rendering its secondary structure a likeness of itself. PrP is a plasma membrane anchored protein, which constitutively recirculates between the cell surface and the endocytic compartment.

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

The final product of 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).

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.

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.

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.

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.

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 β-sheet content and resistance to degradation. The most common is Alzheimer's disease (AD) where the normal soluble amyloid β (sAβ) peptide is converted into highly toxic oligomeric Aβ and fibrillar Aβ 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.

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 β-sheet-rich pathological isoform. In AD the normal soluble Aβ (sAβ) forms oligomers and fibrils which assemble into neuritic plaques. The most toxic form of Aβ is thought to be oligomeric.

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.

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.

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.

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.

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.

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.

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.

Removal of exogenous (spiked) and endogenous prion infectivity from red cells with a new prototype of leukoreduction filter.

Background: Two recent probable cases of transmission of a variant of human Creutzfeldt-Jakob disease (vCJD) through blood transfusion suggest that the disease can be transmitted through transfusion of blood components from presymptomatic blood donors. In the absence of a preclinical screening test, removal of the infectious agent by processing is the only means by which risk to recipients of blood from donors with inapparent vCJD infections can be eliminated.

Study Design And Methods: In the endogenous infectivity study, a pool of 500 mL of whole blood was collected into CP2D anticoagulant from 263K-strain scrapie-infected hamsters, processed into 300 mL of red cells (RBCs), and then passed through a prion removal filter.

Protease-resistant prion protein amplification reconstituted with partially purified substrates and synthetic polyanions.

Little is currently known about the biochemical mechanism by which induced prion protein (PrP) conformational change occurs during mammalian prion propagation. In this study, we describe the reconstitution of PrPres amplification in vitro using partially purified and synthetic components. Overnight incubation of purified PrP27-30 and PrPC molecules at a molar ratio of 1:250 yielded approximately 2-fold baseline PrPres amplification.

Nonenzymatic glycation at the N terminus of pathogenic prion protein in transmissible spongiform encephalopathies.

Transmissible spongiform encephalopathies (TSEs) are transmissible neurodegenerative diseases characterized by the accumulation of an abnormally folded prion protein, termed PrPSc, and the development of pathological features of astrogliosis, vacuolation, neuronal cell loss, and in some cases amyloid plaques. Although considerable structural characterization of prion protein has been reported, neither the method of conversion of cellular prion protein, PrPC, into the pathogenic isoform nor the post-translational modification processes involved is known. We report that in animal and human TSEs, one or more lysines at residues 23, 24, and 27 of PrPSc are covalently modified with advanced glycosylation end products (AGEs), which may be carboxymethyl-lysine (CML), one of the structural varieties of AGEs.

Small, highly structured RNAs participate in the conversion of human recombinant PrP(Sen) to PrP(Res) in vitro.

We have identified a small, highly structured (shs)RNA that binds human recombinant prion protein (hrPrP) with high affinity and specificity under physiological conditions (e.g. 10% bovine calf serum (BCS), neutral pH, nanomolar concentrations of RNA and hrPrP).