Rita Moos

Prion protein and Aβ-related synaptic toxicity impairment

Alzheimers disease (AD), the most common neurodegenerative disorder, goes along with extracellular amyloid‐β (Aβ) deposits. The cognitive decline observed during AD progression correlates with damaged spines, dendrites and synapses in hippocampus and cortex. Numerous studies have shown that Aβ oligomers, both synthetic and derived from cultures and AD brains, potently impair synaptic structure and functions. The cellular prion protein (PrPC) was proposed to mediate this effect. We report that ablation or overexpression of PrPC had no effect on the impairment of hippocampal synaptic plasticity in a transgenic model of AD. These findings challenge the role of PrPC as a mediator of Aβ toxicity.

The POM Monoclonals: A Comprehensive Set of Antibodies to Non-Overlapping Prion Protein Epitopes

PrPSc, a misfolded and aggregated form of the cellular prion protein PrPC, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrPC in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrPC and PrPSc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrPC. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrPC. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrPSc. Other antibodies immunoprecipitate PrPC, but not PrPSc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrPC and PrPSc. Amino-proximal antibodies were found to react with repetitive PrPC epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays.

The Comprehensive Native Interactome of a Fully Functional Tagged Prion Protein

The enumeration of the interaction partners of the cellular prion protein, PrPC, may help clarifying its elusive molecular function. Here we added a carboxy proximal myc epitope tag to PrPC. When expressed in transgenic mice, PrPmyc carried a GPI anchor, was targeted to lipid rafts, and was glycosylated similarly to PrPC. PrPmyc antagonized the toxicity of truncated PrP, restored prion infectibility of PrPC-deficient mice, and was physically incorporated into PrPSc aggregates, indicating that it possessed all functional characteristics of genuine PrPC. We then immunopurified myc epitope-containing protein complexes from PrPmyc transgenic mouse brains. Gentle differential elution with epitope-mimetic decapeptides, or a scrambled version thereof, yielded 96 specifically released proteins. Quantitative mass spectrometry with isotope-coded tags identified seven proteins which co-eluted equimolarly with PrPC and may represent component of a multiprotein complex. Selected PrPC interactors were validated using independent methods. Several of these proteins appear to exert functions in axomyelinic maintenance.

Canine MDCK cell lines are refractory to infection with human and mouse prions

Influenza vaccine production in embryonated eggs is associated with many disadvantages, and production in cell culture systems is a viable alternative. Madin Darby canine kidney (MDCK) cells are permissive for a variety of orthomyxoviruses and have proven particularly suitable for vaccine mass production. However, mammalian cells harboring the Prnp gene can theoretically acquire prion infections. Here, we have attempted to infect MDCK cells and substrains thereof with prions. We found that MDCK cells did not produce any protease-resistant PrP(Sc) upon exposure to brain homogenates derived from humans suffering from Creutzfeldt-Jakob disease (CJD) or from mice infected with Rocky Mountain Laboratory (RML) scrapie prions. Further, transmission of MDCK lysates to N2aPK1 cells did not induce formation of PrP(Sc) in the latter. PrP(C) biogenesis and processing in MDCK cells were similar to those of prion-sensitive N2aPK1 cells. However, steady-state levels of PrP(C) were very low, and PrP(C) did not partition with detergent-resistant membranes upon density gradient analysis. These factors may account for their resistance to infection. Alternatively, prion resistance may be related to the specific sequence of canine Prnp, as suggested by the lack of documented prion diseases in dogs.

Human herpesvirus type 6 and cytomegalovirus in AIDS-associated Kaposi's sarcoma: No evidence for an etiological association☆

Epidemiological studies indicate that acquired immune deficiency syndrome (AIDS)-associated Kaposis sarcoma (KS) may be caused by an infectious, preferentially sexually transmitted agent. Herpesviruses infections are common sexually transmitted diseases in homosexual men, who are also the main risk group for developing Kaposis sarcoma. To evaluate a possible role of human herpesvirus-6 (HHV-6) and cytomegalovirus (CMV) in the development of AIDS-associated KS, we investigated cutaneous AIDS-associated KS in 26 AIDS patients using the polymerase chain reaction (PCR) and immunohistochemistry (IHC) to detect the presence of HHV-6 and CMV. Human herpesvirus-6 was detected in nine of 26 Kaposis sarcoma specimens (all cases were HHV-6 subtype B) and in eight of 27 normal skin specimens from human immunodeficiency virus (HIV) seropositive and HIV seronegative patients (one case was HHV-6 subtype A and seven cases were HHV-6 subtype B). In two of four patients showing HHV-6 in KS of the skin, the virus also was detected in other investigated tissues, such as heart, lung, liver, kidney, and adrenals. Cytomegalovirus was detected only in AIDS-associated KS (seven of 26 KS specimens) and not in normal skin tissues of HIV-seropositive and HIV-seronegative patients. Cytomegalovirus was detected in other organs of those patients showing CMV in Kaposis sarcoma. Our data indicate that the presence of HHV-6 and CMV in AIDS-associated KS most likely reflects disseminated viral infection. Human herpesvirus-6 and CMV may be cofactors but not the only causative agents for the development of AIDS-associated KS.

Detection and typing of human papillomavirus in biopsy and cytological specimens by polymerase chain reaction and restriction enzyme analysis: A method suitable for semiautomation

Screening for high‐risk human papillomavirus (HPV) types allows the detection of women at a high risk of cervical squamous carcinomas, thereby defining a subset of patients targeted for more intensive screening and follow‐up. Thirty‐four cervical biopsy specimens and isolated cells from cervical smears of normal women or women diagnosed with high‐grade intraepithelial lesion (HGSIL) were screened for the presence of HPV by in situ hybridization (ISH) and/or by polymerase chain reaction (PCR). The exact HPV type was determined using a novel restriction typing method. The detection of HPV was facilitated greatly by the use of a PCR‐enzyme‐linked immunosorbent assay (ELISA)‐based method. HPV was detected by PCR in 32% of the biopsy specimens, whereas only 23% had a positive staining by ISH. In one case, a double infection was detected by ISH as well as by PCR. In two cases, the presence of HPV was detected by both methods but the exact type was different. Analyzing cells isolated from cervical smears by the PCR‐ELISA technique or by PCR followed by agarose gel electrophoresis, HPV was detected only in patients with HGSIL and not in the control group. The PCR system is more sensitive than conventional ISH, and the PCR‐ELISA system presented in this study is efficient in screening large series of cytological samples. Furthermore, this system allows exact HPV typing on the microtiter plate. These innovations may allow the application of HPV detection and typing as a routine screening method to identify patients with a high risk of developing cervical neoplasia.

Atypical Prion Protein Conformation in Familial Prion Disease with PRNP P105T Mutation

Protease‐resistant prion protein (PrPSc) is diagnostic of prion disease, yet its detection is frequently difficult. Here, we describe a patient with a PRNP P105T mutation and typical familial prion disease. Brain PrPSc was undetectable by conventional Western blotting and barely detectable after phosphotungstate precipitation, where it displayed an atypical pattern suggestive of noncanonical conformation. Therefore, we used a novel misfolded protein assay (MPA) that detects PrP aggregates independently of their protease resistance. The MPA revealed the presence of aggregated PrP in similar amounts as in typical sporadic Creutzfeldt‐Jakob disease. These findings suggest that measurements of PrP aggregation with the MPA may be potentially more sensitive than protease‐based methodologies.

Triggering receptor expressed on myeloid cells-2 is involved in prion-induced microglial activation but does not contribute to prion pathogenesis in mouse brains

Dysfunctional variants of the innate immune cell surface receptor TREM2 (triggering receptor expressed on myeloid cells-2) were identified as major genetic risk factors for Alzheimers disease and other neurodegenerative conditions. Here we assessed a possible involvement of TREM2 in prion disease. We report that TREM2 expression by microglia is significantly up-regulated upon prion infection. However, depletion of TREM2 did not affect disease incubation time and survival after intracerebral prion infection. Interestingly, markers of microglial activation were attenuated in prion-infected TREM2(-/-) mice, suggesting an involvement of TREM2 in prion-induced microglial activation. Further phenotype profiling of microglia revealed that TREM2 deficiency did not change microglial phenotypes. We conclude that TREM2 is involved in prion-induced microglial activation but does not noticeably modulate the pathogenesis of experimental prion infections.

Modern diagnostic methods in the Ewing's sarcoma family: Six patients with histologic soft tissue tumors*

Background: Soft tissue tumors often present a major diagnostic challenge for the pathologist. The correct diagnosis has important prognostic and therapeutic consequences. In recent years significant progress has been made in identifying characteristic chromosomal abnormalities associated with certain solid tumors. More than 85% of tumors in the Ewings sarcoma (ES) family contain a specific t(11;22) (q24;q12) translocation. Methods and Results: We present six patients with a soft tissue tumor of which only four were diagnosed primarily as belonging to the ES family. All cases were further examined by the following methods: immunohistochemistry with MIC2, cytogenetics, nested reverse transcription-polymerase chain reaction of the t(11;22), using fresh-frozen or formalin-fixed, paraffin-embedded archival material. Conclusions: This method clearly allowed the diagnosis of a tumor of the ES family in all six cases.

Cystatin F is a biomarker of prion pathogenesis in mice.

Misfolding of the cellular prion protein (PrPC) into the scrapie prion protein (PrPSc) results in progressive, fatal, transmissible neurodegenerative conditions termed prion diseases. Experimental and epidemiological evidence point toward a protracted, clinically silent phase in prion diseases, yet there is no diagnostic test capable of identifying asymptomatic individuals incubating prions. In an effort to identify early biomarkers of prion diseases, we have compared global transcriptional profiles in brains from pre-symptomatic prion-infected mice and controls. We identified Cst7, which encodes cystatin F, as the most strongly upregulated transcript in this model. Early and robust upregulation of Cst7 mRNA levels and of its cognate protein was validated in additional mouse models of prion disease. Surprisingly, we found no significant increase in cystatin F levels in both cerebrospinal fluid or brain parenchyma of patients with Creutzfeldt-Jakob disease compared to Alzheimer’s disease or non-demented controls. Our results validate cystatin F as a useful biomarker of early pathogenesis in experimental models of prion disease, and point to unexpected species-specific differences in the transcriptional responses to prion infections.