Defining the microglia response during the time course of chronic neurodegeneration

Inflammation has been proposed as a major component of neurodegenerative diseases, although the precise role it plays has yet to be defined. We examined the role of key contributors to this inflammatory process, microglia, the major resident immune cell population of the brain, in a prion disease model of chronic neurodegeneration. Initially, we performed an extensive reanalysis of a large study of prion disease, where the transcriptome of mouse brains had been monitored throughout the time course of disease. Our analysis has provided a detailed classification of the disease-associated genes based on cell type of origin and gene function. This revealed that the genes upregulated during disease, regardless of the strain of mouse or prion protein, are expressed predominantly by activated microglia. In order to study the microglia contribution more specifically, we established a mouse model of prion disease in which the 79A murine prion strain was introduced by an intraperitoneal route into BALB/cJ(Fms-EGFP/−) mice, which express enhanced green fluorescent protein under the control of the c-fms operon. Samples were taken at time points during disease progression, and histological analysis of the brain and transcriptional analysis of isolated microglia was carried out. The analysis of isolated microglia revealed a disease-specific, highly proinflammatory signature in addition to an upregulation of genes associated with metabolism and respiratory stress. This study strongly supports the growing recognition of the importance of microglia within the prion disease process and identifies the nature of the response through gene expression analysis of isolated microglia. IMPORTANCE Inflammation has been proposed as a major component of neurodegenerative diseases. We have examined the role of key contributors to this inflammatory process, microglia, the major resident immune cell population of the brain, in a murine prion disease model of chronic neurodegeneration. Our study demonstrates that genes upregulated throughout the disease process are expressed predominantly by microglia. A disease-specific, highly proinflammatory signature was observed in addition to an upregulation of genes associated with metabolism and respiratory stress. This study strongly supports the growing recognition of the important contribution of microglia to a chronic neurodegenerative disease process

Trans-Dominant Inhibition of Prion Propagation In Vitro Is Not Mediated by an Accessory Cofactor

Previous studies identified prion protein (PrP) mutants which act as dominant negative inhibitors of prion formation through a mechanism hypothesized to require an unidentified species-specific cofactor termed protein X. To study the mechanism of dominant negative inhibition in vitro, we used recombinant PrPC molecules expressed in Chinese hamster ovary cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. Bioassays confirmed that the products of these reactions are infectious. Using this system, we find that: (1) trans-dominant inhibition can be dissociated from conversion activity, (2) dominant-negative inhibition of prion formation can be reconstituted in vitro using only purified substrates, even when wild type (WT) PrPC is pre-incubated with poly(A) RNA and PrPSc template, and (3) Q172R is the only hamster PrP mutant tested that fails to convert into PrPSc and that can dominantly inhibit conversion of WT PrP at sub-stoichiometric levels. These results refute the hypothesis that protein X is required to mediate dominant inhibition of prion propagation, and suggest that PrP molecules compete for binding to a nascent seeding site on newly formed PrPSc molecules, most likely through an epitope containing residue 172

N-Glycans and Glycosylphosphatidylinositol-Anchor Act on Polarized Sorting of Mouse PrPC in Madin-Darby Canine Kidney Cells

The cellular prion protein (PrPC) plays a fundamental role in prion disease. PrPC is a glycosylphosphatidylinositol (GPI)-anchored protein with two variably occupied N-glycosylation sites. In general, GPI-anchor and N-glycosylation direct proteins to apical membranes in polarized cells whereas the majority of mouse PrPC is found in basolateral membranes in polarized Madin-Darby canine kidney (MDCK) cells. In this study we have mutated the first, the second, and both N-glycosylation sites of PrPC and also replaced the GPI-anchor of PrPC by the Thy-1 GPI-anchor in order to investigate the role of these signals in sorting of PrPC in MDCK cells. Cell surface biotinylation experiments and confocal microscopy showed that lack of one N-linked oligosaccharide leads to loss of polarized sorting of PrPC. Exchange of the PrPC GPI-anchor for the one of Thy-1 redirects PrPC to the apical membrane. In conclusion, both N-glycosylation and GPI-anchor act on polarized sorting of PrPC, with the GPI-anchor being dominant over N-glycans

Propagation of RML Prions in Mice Expressing PrP Devoid of GPI Anchor Leads to Formation of a Novel, Stable Prion Strain

PrPC, a host protein which in prion-infected animals is converted to PrPSc, is linked to the cell membrane by a GPI anchor. Mice expressing PrPC without GPI anchor (tgGPI- mice), are susceptible to prion infection but accumulate anchorless PrPSc extra-, rather than intracellularly. We investigated whether tgGPI− mice could faithfully propagate prion strains despite the deviant structure and location of anchorless PrPSc. We found that RML and ME7, but not 22L prions propagated in tgGPI− brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA). Surprisingly, the levels of proteinase K-resistant PrPSc (PrPres) in RML- or ME7-infected tgGPI− brain were 25–50 times higher than in wild-type brain. When returned to wild-type brain, ME7 prions recovered their original properties, however RML prions had given rise to a novel prion strain, designated SFL, which remained unchanged even after three passages in wild-type mice. Because both RML PrPSc and SFL PrPSc are stably propagated in wild-type mice we propose that the two conformations are separated by a high activation energy barrier which is abrogated in tgGPI− mice

Distinct Type of Transmission Barrier Revealed by Study of Multiple Prion Determinants of Rnq1

Prions are self-propagating protein conformations. Transmission of the prion state between non-identical proteins, e.g. between homologous proteins from different species, is frequently inefficient. Transmission barriers are attributed to sequence differences in prion proteins, but their underlying mechanisms are not clear. Here we use a yeast Rnq1/[PIN+]-based experimental system to explore the nature of transmission barriers. [PIN+], the prion form of Rnq1, is common in wild and laboratory yeast strains, where it facilitates the appearance of other prions. Rnq1's prion domain carries four discrete QN-rich regions. We start by showing that Rnq1 encompasses multiple prion determinants that can independently drive amyloid formation in vitro and transmit the [PIN+] prion state in vivo. Subsequent analysis of [PIN+] transmission between Rnq1 fragments with different sets of prion determinants established that (i) one common QN-rich region is required and usually sufficient for the transmission; (ii) despite identical sequences of the common QNs, such transmissions are impeded by barriers of different strength. Existence of transmission barriers in the absence of amino acid mismatches in transmitting regions indicates that in complex prion domains multiple prion determinants act cooperatively to attain the final prion conformation, and reveals transmission barriers determined by this cooperative fold

Evaluation of a near patient test and ELISA-based assays for detecting anti-herpes simplex virus type-2 antibodies

Several type-specific serologic assays for herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), based on glycoprotein G1 (gG1) and gG2, have recently been developed. These include immunodot (POCkit HSV-2) and enzyme-linked immunosorbent assay (ELISA). The diagnostic value of POCkit HSV-2, a near-patient test, and of 2 immunoenzymatic, type-specific assays was evaluated on 122 patients attending an STD clinic. Western blot was used as the reference test. The sensitivity of POCkit HSV-2 was good but the specificity was poor, so that in a population with low seroprevalence, a positive result is likely to be a false positive. Analysis of 2 currently available HSV type-specific ELISAs yielded results suggesting that the sensitivity of these tests may also be suboptima

The catalytic subunit of herpes simplex virus type 1 DNA polymerase contains a nuclear localization signal in the UL42-binding region

The herpes simplex virus type 1 DNA polymerase consists of a catalytic subunit (POL or UL30) and a processivity factor (UL42). The POL/UL42 interaction, which occurs through the extreme C-terminus of POL, is essential for HSV-1 replication and thus represents a valid target for drug inhibition. We recently showed (A. Loregian et al. (1999) Proc. Natl. Acad. Sci. USA 96, 5221-5226) that an oligopeptide corresponding to the 27 C-terminal amino acids of POL, when delivered into herpes simplex virus type 1-infected cells by a protein carrier, was able to localize into the nucleus and to inhibit viral replication by disruption of the POL/UL42 interaction. In this report, to further characterize the 27 mer (Pol peptide), we investigated whether its nuclear localization was due to the presence of a nuclear localization signal. By testing the ability of the Pol peptide to localize the beta-galactosidase, a normally cytoplasmic protein, to the nucleus, we confirmed that the Pol peptide contained a functional nuclear localization signal, corresponding to the RRMLHR motif. This sequence proved not only necessary but also sufficient for nuclear localization, because its substitution with a six-alanine stretch prevented nuclear translocation of the beta-galactosidase-Pol peptide fusion. Site-directed mutagenesis experiments on this revealed that both the three basic arginines and the two hydrophobic residues Met and Leu were crucial for nuclear targeting. Finally, functionally equivalent sequences were also found in the C-terminus of the catalytic subunits of human cytomegalovirus (RRLHL) and of equine herpesvirus-1 DNA polymerase (RRILH)