Role of Microglia in Neuronal Cell Death in Prion Disease

To elucidate the role played by the prion protein in scrapie pathogenesis, we performed experiments with PrP27–30 isolated from scrapie-infected hamster brains in cell culture and studied in vivo the temporal and spatial correlation between deposition of the disease-associated isoform of the prion protein (PrP^Sc), microglial activation and neuronal cell death in mice infected with scrapie strains 79A, ME7 and RML. The results presented here show that cellular expression of PrP^c and the presence of microglia are necessary for the neurotoxicity of PrP^Sc in vitro. In vivo , accumulation of protease-resistant prion protein was detected early in the incubation period using the histoblot technique. Microglial activation was also detected early in the incubation period of all models studied. Both the time course and the spatial distribution of microglial activation closely resembled the pattern of PrP^Sc deposition. Microglial activation clearly preceded the detection of apoptotic neuronal cell death which was assessed using the in situ end-labeling technique (ISEL). Taken together, our results indicate that microglial activation is involved in the neurotoxicity of PrP^Sc both in vitro and in vivo.     

Effects of agent strain and host genotype on PrP accumulation in the brain of sheep naturally and experimentally affected with scrapie

Different cellular and neuroanatomical types of disease-specific prion protein (PrP(d)) accumulation in the brain were identified in sheep of different breeds and PrP genotypes exposed to experimental or natural scrapie infection. Immunohistochemical examination of the brains of 43 sheep with clinical signs compatible with scrapie revealed 12 different PrP(d)types, which were subjectively quantified in eight different brain regions. The PrP(d)types were grouped into four PrP(d)patterns, the relative magnitude of which provided the PrP(d)profile of each sheep examined. The analysis of the differences in magnitude and relative proportion of each of these PrP(d)types and patterns indicated (1) an effect of the scrapie strain on the PrP(d)profile, and (2) a possible effect of the host genotype on the magnitude of PrP(d)accumulation in the brain, apparently related to the incubation period. Furthermore, intraneuronal deposition of PrP(d)was the type most closely associated with the development of clinical disease. We conclude that different scrapie strains can be distinguished by PrP immunohistochemical examination of brains of affected animals.     

Evidence for co-infection of ovine prion strains in classical scrapie isolates

The diversity of strains of ovine prions within classical scrapie isolates was investigated by transmission studies in wild type mice. To determine the maximum diversity of prion strains present in each ovine scrapie isolate examined, isolates from mice having the shortest and longest incubation times for terminal disease after primary inoculation were passaged serially. Serial passage of ARQ/ARQ scrapie isolates in RIII mice revealed the ME7 prion strain in mice with short incubation times for terminal prion disease and the 87A strain in those mice with long incubation times. Serial passage of VRQ/VRQ scrapie isolates in RIII mice led to emergence of the 221C prion strain in mice with short incubation times and a variant of the 221C strain in those mice with long incubation times. RIII mice with short incubation times had higher levels of total and proteinase K-resistant PrP(Sc) compared with those RIII mice with long incubation times, while mice with long incubation times had large aggregates and plaques of PrP(Sc). ME7 PrP(Sc) differed in stability compared with the 87A prion strain, while PrP(Sc) associated with 221C had similar stability to that of the 221C variant. Serial passage in VM mice led to identification of ME7 and 87V in the same scrapie isolate. The data show that different prion strains can emerge from the same ovine scrapie isolate following serial passage in wild type mice and that the transmission properties of these strains correlate with distinct patterns of PrP(Sc) deposition.     

Prion replication alters the distribution of synaptophysin and caveolin 1 in neuronal lipid rafts

The main event in the pathogenesis of prion diseases is the conversion of the cellular prion protein (PrP(C)) into the abnormal, protease-resistant prion protein (PrP(res)). PrP(C) is a GPI-anchored protein located in lipid rafts or detergent-resistant membranes (DRMs). Here we describe the association of PrP with DRMs in neuronal cell bodies and axons during the course of murine scrapie and its relation with the distribution of the PrP-interacting proteins caveolin 1 and synaptophysin. Scrapie infection triggered the accumulation of PrP(res) in DRMs from retinas and optic nerves from early stages of the disease before evidence of neuronal cell loss. Most of the PrP(res) remained associated with lipid rafts throughout different stages in disease progression. In contrast to PrP(res), caveolin 1 and synaptophysin in retina and optic nerves shifted to non-DRM fractions during the course of scrapie infection. The accumulation of PrP(res) in DRMs was not associated with a general alteration in their composition, because no change in the total protein distribution across the sucrose gradient or in the flotation characteristics of the glycosphingolipid GM1 or Thy-1 were observed until advanced stages of the disease. However, an increase in total cholesterol levels was observed in optic nerve and retinas. Only during late stages of the disease was a decrease in the number of neuronal cell bodies observed, suggesting that synaptic abnormalities are the earliest sign of neuronal dysfunction that ultimately results in neuronal death. These results indicate that prion replication triggers an abnormal localization of caveolin 1 and synaptophysin, which in turn may alter neuronal function.     

Propagation of ovine prions from "poor" transmitter scrapie isolates in ovine PrP transgenic mice

Ovine prion strains have typically been identified by their transmission properties, which include incubation time and lesion profile, in wild type mice. The existence of scrapie isolates that do not propagate in wild type mice, defined here as "poor" transmitters, are problematic for conventional prion strain typing studies as no incubation time or neuropathology can be recorded. This may arise because of the presence of an ovine prion strain within the original inoculum that does not normally cross the species barrier into wild type mice or the presence of a low dose of an infectious ovine prion strain that does. Here we have used tg59 and tg338 mouse lines, which are transgenic for ovine ARQ or VRQ PrP, respectively, to strain type "poor" transmitter ovine scrapie isolates. ARQ and VRQ homozygous "poor" transmitter scrapie isolates were successfully propagated in both ovine PrP transgenic mouse lines. We have used secondary passage incubation time, PrPSc immunohistochemistry and molecular profile, to show that different prion strains can be isolated from different "poor" transmitter samples during serial passage in ovine PrP transgenic mice. Our observations show that poor or inadequate transmissibility of some classical scrapie isolates in wild type mice is associated with unique ovine prion strains in these particular sheep scrapie samples. In addition, the analysis of the scrapie isolates used here revealed that the tg338 mouse line was more versatile and more robust at strain typing ovine prions than tg59 mice. These novel observations in ovine PrP transgenic mice highlight a new approach to ovine prion strain typing.     

Efficient in vitro amplification of a mouse-adapted scrapie prion protein

Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie prion protein (PrP(Sc)), a major protein component of the infectious agents associated with prion diseases. Although exponential in vitro amplification of hamster scrapie PrP(Sc) has been established, the PMCA used was unsuccessful in achieving good amplification of PrP(Sc) from other animals. Here, we have investigated the cause of the insufficient PrP(Sc) amplification in mice and have developed an improved method suitable for amplification of the PrP(Sc) of the mouse-adapted scrapie prion strain Chandler. Mouse PrP(C), the cellular form of the prion protein, tends to become resistant to proteases during incubation independent of sonication. By adding digitonin to the reaction buffer as a lipid detergent, accumulation of the protease-resistant PrP(C) was inhibited; hence, mouse PrP(Sc) could be amplified to infinite levels. The present study is the first report describing effective amplification of PrP(Sc) of the mouse-adapted scrapie prion and this improved PMCA technique will contribute to prion research that uses mice as experimental animals.     

An antibody to the aggregated synthetic prion protein peptide (PrP106-126) selectively recognizes disease-associated prion protein (PrP) from human brain specimens

Human prion diseases are characterized by the conversion of the normal host cellular prion protein (PrP^C) into an abnormal misfolded form [disease-associated prion protein (PrP^Sc)]. Antibodies that are capable of distinguishing between PrP^C and PrP^Sc may prove to be useful, not only for the diagnosis of these diseases, but also for a better understanding of the molecular mechanisms involved in disease pathogenesis. In an attempt to produce such antibodies, we immunized mice with an aggregated peptide spanning amino acid residues 106 to 126 of human PrP (PrP106–126). We were able to isolate and single cell clone a hybridoma cell line (P1:1) which secreted an IgM isotype antibody [monoclonal antibody (mAb P1:1)] that recognized the aggregated, but not the monomeric form of the immunogen. When used in immunoprecipitation assays, the antibody did not recognize normal PrP^C from non-prion disease brain specimens, but did selectively immunoprecipitate full-length PrP^Sc from cases of variant and sporadic Creutzfeldt–Jakob disease and Gerstmann–Straussler–Scheinker disease. These results suggest that P1:1 recognizes an epitope formed during the structural rearrangement or aggregation of the PrP that is common to the major PrP^Sc types found in the most common forms of human prion disease.     

Acute cellular uptake of abnormal prion protein is cell type and scrapie-strain independent

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that include Creutzfeldt-Jakob disease, bovine spongiform encephalopathy and sheep scrapie. Although one of the earliest events during TSE infection is the cellular uptake of protease resistant prion protein (PrP-res), this process is poorly understood due to the difficulty of clearly distinguishing input PrP-res from either PrP-res or protease-sensitive PrP (PrP-sen) made by the cell. Using PrP-res tagged with a unique antibody epitope, we examined PrP-res uptake in neuronal and fibroblast cells exposed to three different mouse scrapie strains. PrP-res uptake was rapid and independent of scrapie strain, cell type, or cellular PrP expression, but occurred in only a subset of cells and was influenced by PrP-res preparation and aggregate size. Our results suggest that PrP-res aggregate size, the PrP-res microenvironment, and/or host cell-specific factors can all influence whether or not a cell takes up PrP-res following exposure to TSE infectivity.     

Severe, Early and Selective Loss of a Subpopulation of GABAergic Inhibitory Neurons in Experimental Transmissible Spongiform Encephalopathies

Little is known about the pathogenetic basis of characteristic symptoms in transmissible spongiform encephalopathies (TSEs) such as myoclonus and characteristic EEG hyperactivity. We investigated the GABAergic system and its subpopulations in mice inoculated with experimental scrapie (ME7, RML, 22A strains) and Creutzfeldt-Jakob disease (CJD; Fujisaki strain), to study damage to inhibitory neurons. Since recent studies have shown electrophysiological changes in prion protein (PrP) knockout mice, we also studied mice lacking or overexpressing the PrP gene. Antibodies against glutamic acid decarboxylase (GAD), parvalbumin (PV), calbindin (CB), and calretinin (CR) were used to stain GABAergic neurons, and isolectin-B₄ to stain perineuronal nets around PV+ neurons. In scrapie infected mice, cortical PV+ neurons were severely reduced while CB+ and CR+ neurons were well preserved. In CJD inoculated mice, loss of PV+ neurons was severe and occurred very early after inoculation. PrP^-/- and tg20 mice showed normal appearance of PV, CB, CR, GAD+ neurons and their neuropil, and of isolectin-B₄+ perineuronal nets. The early, severe and selective loss of cortical PV+ neurons in experimental scrapie and CJD suggest selective loss of PV+ GABAergic neurons as important event during disease development, possibly as one basis of excitatory symptoms in TSEs.     

Increased immunohistochemical labelling for prion protein occurs in diverse neurological disorders of sheep: relevance for normal cellular PrP function

The classical prion diseases (e.g. scrapie of sheep and goats and bovine spongiform encephalopathy of cattle) are characterized by the accumulation of abnormal forms of the prion protein (PrP), usually recognized by their relative resistance to proteolysis compared with the physiological cellular forms of PrP. However, novel prion diseases have been detected in sheep, cattle and man, in which the abnormal PrP has less resistance to proteolysis than identified previously. These more subtle differences between abnormal and normal forms of PrP can be problematic in routine diagnostic tests and raise questions in respect of the range of PrP disorders. Abnormal accumulations of PrP in atypical and classical prion diseases can be recognized by immunohistochemistry. To determine whether altered PrP expression or trafficking might occur in nosological entities not previously connected with prion disease, the brains of sheep affected with diverse neurological conditions were examined for evidence of altered PrP labelling. Such altered immunolabelling was detected in association with either basic lesions or specific diseases. Some reactive glial cells and degenerate neurons found in several different recognized disorders and non-specific inflammatory processes were associated with abnormal PrP labelling, which was absent from brains of healthy, age-matched sheep. The results agree with previous indications that normal PrP function may be linked with the oxidative stress response, but the data also suggest that PrP functions are more extensive than simple protective responses against stress insults.     

Prion protein is ubiquitinated after developing protease resistance in the brains of scrapie-infected mice

Although the key event in the pathology of prion diseases is thought to be the conversion of cellular prion protein (PrP(C)) to the protease-resistant scrapie species termed PrP(Sc), the factors that contribute to neurodegeneration in scrapie-infected animals are poorly understood. One probable determinant could be when the accumulation of PrP(Sc) in infected brain overwhelms the ubiquitin-proteasome system and triggers the degenerative cascade. In the present study, it was found that in mouse brains infected with the ME7 scrapie strain, the level of ubiquitin protein conjugates increased significantly at approximately 144 days post-infection (pi) when clinical signs first become apparent. This elevation correlated with the detection of protease-resistant PrP(Sc) and a decline in two endopeptidase activities associated with proteasome function. However, ubiquitination of PrP was only detected at the terminal stage, 3 weeks after the development of clinical symptoms (approximately 165 days pi). These results suggest that ubiquitination of PrP is a late event phenomenon and this conjugation occurs after the formation of protease-resistant PrP(Sc). Whether this post-translational modification and the impairment of proteasome function are pivotal events in the pathogenesis of prion diseases remains to be determined.     

Mucosal vaccination delays or prevents prion infection via an oral route

In recent years major outbreaks of prion disease linked to oral exposure of the prion agent have occurred in animal and human populations. These disorders are associated with a conformational change of a normal protein, PrP(C) (prion protein cellular), to a toxic and infectious form, PrP(Sc) (prion protein scrapie). None of the prionoses currently have an effective treatment. A limited number of active immunization approaches have been shown to slightly prolong the incubation period of prion infection. Active immunization in wild-type animals is hampered by auto-tolerance to PrP and potential toxicity. Here we report that mucosal vaccination with an attenuated Salmonella vaccine strain expressing the mouse PrP, is effective at overcoming tolerance to PrP and leads to a significant delay or prevention of prion disease in mice later exposed orally to the 139A scrapie strain. This mucosal vaccine induced gut anti-PrP immunoglobulin (Ig)A and systemic anti-PrP IgG. No toxicity was evident with this vaccination approach. This promising finding suggests that mucosal vaccination may be a useful method for overcoming tolerance to PrP and preventing prion infection among animal and potentially human populations at risk.     

High titers of mucosal and systemic anti-PrP antibodies abrogate oral prion infection in mucosal-vaccinated mice

Significant outbreaks of prion disease linked to oral exposure of the prion agent have occurred in animal and human populations. These disorders are associated with a conformational change of a normal protein, PrP^C (C for cellular), to a toxic and infectious form, PrP^Sc (Sc for scrapie). None of the prionoses currently have an effective treatment. Some forms of prion disease are thought to be spread by oral ingestion of PrP^Sc, such as chronic wasting disease and variant Creutzfeldt-Jakob disease. Attempts to obtain an active immunization in wild-type animals have been hampered by auto-tolerance to PrP and potential toxicity. Previously, we demonstrated that it is possible to overcome tolerance and obtain a specific anti-PrP antibody response by oral inoculation of the PrP protein expressed in an attenuated Salmonella vector. This past study showed that 30% of vaccinated animals were free of disease more than 350 days post-challenge. In the current study we have both optimized the vaccination protocol and divided the vaccinated mice into low and high immune responder groups prior to oral challenge with PrP^Sc scrapie strain 139A. These methodological refinements led to a significantly improved therapeutic response. 100% of mice with a high mucosal anti-PrP titer immunoglobulin (Ig) A and a high systemic IgG titer, prior to challenge, remained without symptoms of PrP infection at 400 days (log-rank test P<0.0001 versus sham controls). The brains from these surviving clinically asymptomatic mice were free of PrP^Sc infection by Western blot and histological examination. These promising findings suggest that effective mucosal vaccination is a feasible and useful method for overcoming tolerance to PrP and preventing prion infection via an oral route     

Fatal Familial Insomnia and Familial Creutzfeldt-Jakob Disease: Clinical, Pathological and Molecular Features

Fatal familial insomnia (FFI) and a subtype of familial Creutzfeldt-Jakob disease (CJD¹⁷⁸) are two prion diseases that have different clinical and pathological features, the same aspartic acid to asparagine mutation (D178N) at codon 178 of the prion protein (PrP) gene, but distinct genotypes generated by the methionine-valine polymorphism at codon 129 (129M or 129V) in the mutant allele of the PrP gene. The D178N, 129M allele segregates with FFI while the D178N, 129V allele segregates with CJD¹⁷⁸. The proteinase K resistant PrP (PrP^res) isoforms present in FFI and CJD¹⁷⁸ differ in degree of glycosylation and size. Thus, the amino acid, methionine or valine, at position 129 of the mutant allele, in conjunction with D178N mutation results in significant alterations of PrP^res in FFI and CJD¹⁷⁸. The 129 polymorphic site also exerts influence through the normal allele: the course of the disease is shorter in the patients homozygous at codon 129 and other minor but consistent phenotypic differences occur between homozygous and heterozygous FFI patients. The comparative study of PrP^res distribution in FFI homozygotes and heterozygotes at codon 129 has lead to the conclusion that the phenotypic differences observed between these two FFI patient populations may be the result of different rates of conversion of normal PrP into PrP^res, at least in some brain regions.     

Newly discovered forms of prion diseases in ruminants

Transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative diseases caused by unconventional agents, the prions. They are characterised by the accumulation in infected tissues of an abnormally folded form of the host-encoded prion protein (PrP). This pathological form is partially resistant to protease digestion, leading to the production of so-called PrP(res) fragments. Different isolates from the same host species may show different eletrophoretic profiles, reflecting the existence of different prion strains. The active surveillance of ruminant TSEs implemented in European countries, based on a large-scale biochemical testing of brain tissue samples from carcasses, has revealed PrP(res) profiles unnoticed so far. Experimental transmission of these atypical cases to various transgenic mouse lines has led to the recognition of a novel scrapie strain in sheep and goats, called Nor98, and of two variant strains of spongiform encephalopathy in cattle. This review is aimed at summarising the current knowledge on these newly recognised forms of ruminants TSEs, and at discussing their possible origin and potential implications in terms of animal and human health.     

A domain responsible for spontaneous conversion of bank vole prion protein

Bank vole is a small rodent that shows high susceptibility to infection with diverse prion strains. To determine whether the increased susceptibility of bank voles to prion diseases can be attributed to the intrinsic nature of bank vole prion protein (PrP) or to host factors other than PrP, we produced transgenic mice overexpressing bank vole PrP. These transgenic mice spontaneously developed neurological illness with spongiform changes and the accumulation of abnormal PrP in the brain. Then, we produced transgenic mice overexpressing chimeric mouse/bank vole PrP, which differs from mouse PrP only at two residues located at the C‐terminus, to determine the minimum essential domain for the induction of spontaneous generation of abnormal PrP. These transgenic mice also developed spontaneous neurological illness with spongiform changes and the accumulation of abnormal PrP in the brain. In addition, knock‐in mice expressing bank vole PrP at the same level as that of wild‐type mice did not develop spontaneous disease but showed high susceptibility to infection with diverse prion strains, similarly to bank voles. Taken together, these findings show that bank vole PrP has a high propensity for the conformational conversion both in spontaneous disease and in prion infection, probably due to the characteristic structural properties of the C‐terminal domain.     

Transgenic and Knockout Mice in Research on Prion Diseases

Since the discovery of the prion protein (PrP) gene more than a decade ago, transgenetic investigations on the PrP gene have shaped the field of prion biology in an unprecedented way. Many questions regarding the role of PrP in susceptibility of an organism exposed to prions have been elucidated. For example mice with a targeted disruption of the PrP gene have allowed the demonstration that an organism that lacks PrP^c is resistant to infection by prions. Reconstitution of these mice with mutant PrP genes allowed investigations on the structure-activity relationship of the PrP gene with regard to scrapie susceptibility. Unexpectedly, transgenic mice expressing PrP with specific amino-proximal truncations spontaneously develop a neurologic syndrome presenting with ataxia and cerebellar lesions. A distinct spontaneous neurologic phenotype was observed in mice with internal deletions in PrP. Using ectopic expression of PrP in PrP knockout mice has turned out to be a valuable approach towards the identification of host cells that are capable of replicating prions. Transgenic mice have also contributed to our understanding of the molecular basis of the species barrier for prions. Finally, the availability of PrP knockout mice and transgenic mice overexpressing PrP allows selective reconstitution experiments aimed at expressing PrP in neurografts or in specific populations of hemato- and lymphopoietic cells. Such studies have shed new light onto the mechanisms of prion spread and disease pathogenesis.     

Chimeric porcine reproductive and respiratory syndrome viruses reveal full function of genotype 1 envelope proteins in the backbone of genotype 2

Earlier studies indicated that transgenic (tg) mice engineered to express prion protein (PrP) lacking the glycophosphatidylinositol (GPI^−/−) membrane anchor formed abnormal proteinase-resistant prion (PrPsc) amyloid deposits in their brains and hearts when infected with the RML strain of murine scrapie. In contrast, RML scrapie infection of normal mice with a GPI-anchored PrP did not deposit amyloid with PrPsc in the brain or the heart. Here we report that scrapie-infected GPI^−/− PrP tg mice also deposit PrP and transmissible infectious material in the gut, kidneys, and islets of Langerhans. Similar to previously reported amyloid deposits in the brain and heart, amyloid deposits were found in the gut; however, no amyloid deposited in the islets. By high-resolution electron microscopy, we show PrP is located primarily in α cells and also β cells. Islets contain abundant insulin and there is no abnormality in glucose metabolism in infected GPI^−/− PrP tg mice.     

Characteristics of prion protein (PrP(Sc)) in the brains of hamsters inoculated serially with a mouse-passaged scrapie strain

Syrian hamsters were inoculated intracerebrally with a mouse-passaged scrapie strain. After serial passage, the incubation period decreased, but the vacuolar lesion profiles remained unchanged. In immunoblot analysis, accumulated prion protein (PrP) showed hamster PrP characteristics from the first passage. However, immunohistochemical examination revealed a changing pattern of accumulated PrP with each passage. In particular, there were many PrP plaques in the subpial and subependymal region at the third passage, no such plaques having been observed in the same region at the first passage. These results suggest that the species barrier influences not only the incubation period but also the pattern of accumulation of PrP in affected brains.     

Autophagy and Cell Death of Purkinje Cells Overexpressing Doppel in Ngsk Prnp-deficient Mice

In Ngsk prion protein (PrP)-deficient mice ( NP^0/0 ), ectopic expression of PrP-like protein Doppel (Dpl) in central neurons induces significant Purkinje cell (PC) death resulting in late-onset ataxia. NP^0/0 PC death is partly prevented by either knocking-out the apoptotic factor BAX or overexpressing the anti-apoptotic factor BCL-2 suggesting that apoptosis is involved in Dpl-induced death. In this study, Western blotting and immunohistofluorescence show that both before and during significant PC loss, the scrapie-responsive gene 1 ( Scrg1 )—potentially associated with autophagy—and the autophagic markers LC3B and p62 increased in the NP^0/0 PCs whereas RT-PCR shows stable mRNA expression, suggesting that the degradation of autophagic products is impaired in NP^0/0 PCs. At the ultrastructural level, autophagic-like profiles accumulated in somatodendritic and axonal compartments of NP^0/0 , but not wild-type PCs. The most robust autophagy was observed in NP^0/0 PC axon compartments in the deep cerebellar nuclei suggesting that it is initiated in these axons. Our previous and present data indicate that Dpl triggers autophagy and apoptosis in NP^0/0 PCs. As observed in amyloid neurodegenerative diseases, upregulation of autophagic markers as well as extensive accumulation of autophagosomes in NP^0/0 PCs are likely to reflect a progressive dysfunction of autophagy that could trigger apoptotic cascades.