Enhanced CD9 expression in the mouse and human brains infected with transmissible spongiform encephalopathies

A tetraspan protein CD9, normally expressed in the myelin sheath of the central and peripheral nervous system, was identified to be up-regulated in mouse brains infected with transmissible spongiform encephalopathy (TSE), by mRNA differential display screening. To elucidate its role in the neurodegeneration process observed in TSE, CD9 expression was examined in the murine disease model and in the human disease materials. Up-regulation of CD9 gene expression in the TSE-infected mouse brains was detected as early as a preclinical stage, when abnormal prion protein deposition and vacuolation were obviously manifested in the internal capsule and thalamus. In contrast, other myelin protein genes showed a reverse pattern of CD9 gene expression. Enhanced CD9 expression was immunohistochemically detected in the astrocytes of such pathological regions. In human specimens of TSE, enhanced CD9 immunoreactivity was observed in the astrocytes and some oligodendrocytes in the brains, but no relevant alteration in CD9 immunoreactivity was observed in the other organs or tissues. Positive CD9 immunoreactivity in astrocytes was also manifest in other neurological disorders in a less prominent manner. The findings indicate that up-regulated CD9 plays a role in glial cells in pathological conditions, especially in such a devastating condition as TSE.     

Tissue plasminogen activator in brain tissues infected with transmissible spongiform encephalopathies

Prion propagation involves conversion of host PrP(C) to a disease-related isoform, PrP(Sc), which accumulates during disease and is the principal component of the transmissible agent. Proteolysis seems to play an important role in PrP metabolism. Plasminogen, a serine protease precursor, has been shown to interact with PrP(Sc). Plasminogen can be proteolytically activated by tissue plasminogen activator (tPA). Recent reports imply a crosstalk between tPA-mediated plasmin activation and PrP. In our study, both tPA activity and tPA gene expression were found elevated in TSE-infected brains as compared to their normal counterparts. Furthermore, it was proved that PrP(Sc), in contrast to PrP(C), could not be degraded by plasmin. In addition, it was observed that TSE symptoms and subsequent death of plasminogen-deficient and tPA-deficient scrapie challenged mice preceded that of wild-type controls. Our data imply that enhanced tPA activity observed in prion infected brains may reflect a neuro-protective response.     

Cerebrospinal fluid biomarkers in human genetic transmissible spongiform encephalopathies

The 14-3-3 protein test has been shown to support the clinical diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) when associated with an adequate clinical context, and a high differential potential for the diagnosis of sporadic CJD has been attributed to other cerebrospinal fluid (CSF) proteins such as tau protein, S100b and neuron specific enolase (NSE). So far there has been only limited information available about biochemical markers in genetic transmissible spongiform encephalopathies (gTSE), although they represent 10–15% of human TSEs. In this study, we analyzed CSF of 174 patients with gTSEs for 14-3-3 (n = 166), tau protein (n = 78), S100b (n = 46) and NSE (n = 50). Levels of brain-derived proteins in CSF varied in different forms of gTSE. Biomarkers were found positive in the majority of gCJD (81%) and insert gTSE (69%), while they were negative in most cases of fatal familial insomnia (13%) and Gerstmann-Sträussler-Scheinker syndrome (10%). Disease duration and codon 129 genotype influence the findings in a different way than in sporadic CJD.     

Cell death and autophagy in prion diseases (transmissible spongiform encephalopathies)

Neuronal autophagy, like apoptosis, is one of the mechanisms of programmed cell death. In this review, we summarize current information about autophagy in naturally occurring and experimentally induced scrapie, Creutzfeldt-Jakob disease and Gerstmann-Str?ussler-Scheinker syndrome against the broad background of neural degenerations in transmissible spongiform encephalopathies (TSEs). Typically a sequence of events is observed: from a part of the neuronal cytoplasm sequestrated by concentric arrays of double membranes (phagophores); through the enclosure of the cytoplasm and membrane proliferation; to a final transformation of the large area of the cytoplasm into a collection of autophagic vacuoles of different sizes. These autophagic vacuoles form not only in neuronal perikarya but also in neurites and synapses. On the basis of ultrastructural studies, we suggest that autophagy may play a major role in transmissible spongiform encephalopathies and may even participate in the formation of spongiform change.     

Creutzfeldt-Jakob disease and other human transmissible spongiform encephalopathies. Part II

The second part of this work presents the neuropathological problems of the Creutzfeldt-Jakob disease and basic informations about other human prion diseases. General problems of prion diseases and clinical symptoms of Creutzfeldt-Jakob disease were presented in the first part. Prion diseases are also known as transmissible cerebral amyloidoses (TCA) or transmissible (subacute) spongiform encephalopathies (TSE, SSE). There are following human TSE's: Creutzfeldt-Jakob disease (CJD)--the most frequent TSE, and its new variant (vCJD)--a result of BSE's transmission into human, sometimes treated as a separate disease; also: Gerstmann-Str?ussler-Scheinker syndrome (GSS) that may be a variant of familial CJD, kuru--probably a result of sporadic CJD's transmission by cannibalism, and fatal familial insomnia (FFI). Their clinical symptoms (and especially of the CJD), are nonspecific and sometimes variable. The imaging, EEG and other laboratory tests are not specific either. Neuropathological studies are needed but their interpretation may be equivocal. TSE's are characterised by the neurodegenerative process with characteristic spongiosis. However, vacuolisation--similar as in TSE-spongiosis--may occur in some CNS's disorders and in the case of putrescent brain tissue. In some cases of CJD, particularly those of long duration, the neuronal loss and astrocyte proliferation can mask the presence of spongiform changes, especially when vacuoles are not numerous. The only certain diagnostic marker for TSE is PrP(Sc), prion protein, presently believed to be a direct cause for all TSEs (TCAs). The PrP(Sc) has a dominant beta-sheet amyloid structure which makes its detection by immunohistochemical procedure possible only with special pretreatment, e.c.: hydrolytic autoclaving, hydrated autoclaving, incubations: formic acid (or guanidine thiocyanate) pretreatment, also combined pretreatments. These methods are standard diagnostic procedures for transmissible cerebral amyloidoses.     

Creutzfeldt-Jakob disease and other human transmissible spongiform encephalopathies. Part I

In the first part of this work the main problems of prion diseases--also called transmissible cerebral amyloidoses (TCA) or subacute (transmissible) encephalopathies (SSE, TSE)--and clinical symptoms of Creutzfeldt-Jakob disease are presented. Some problems of neuropathology of Creutzfeldt-Jakob disease and basic informations about other human prion diseases will be presented in the second part. The growth of the interest in prion diseases during last years is caused by the problem of bovine spongiform encephalopathy (BSE or "mad cow disease") and its transmission into a human. The new variant of Creutzfeldt-Jakob disease (nvCJD) has appeared. Prion diseases: Gerstmann-Str?ussler-Scheinker syndrome (GSS), kuru, fatal familial insomnia (FFI) and particularly the most frequent of them--Creutzfeldt-Jakob disease (CJD)--have nonspecific, sometimes variable clinical (psychopathological and neurological) symptoms. The imaging, EEG, cerebrospinal fluid tests and other laboratory tests are not specific either and their diagnostic value is limited. Neuropathological studies are needed but their interpretation is often difficult. The only certain diagnostic marker for TSE is the presence of PrP(Sc), the prion protein, which is presently believed to be a direct cause for all transmissible cerebral amyloidoses (TCA).     

Increased brain synthesis of prostaglandin E2 and F2-isoprostane in human and experimental transmissible spongiform encephalopathies

The levels of 2 arachidonic acid metabolites formed either by enzymatic activity of cyclooxygenase, i.e. prostaglandin E2 (PGE2), or by free radical-catalyzed peroxidation, i.e. F2-isoprostane 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha), were measured in the CSF of subjects with sporadic and familial Creutzfeldt-Jakob disease (CJD) and in brain homogenates of scrapie-infected mice. The CSF levels of both metabolites were increased in sporadic CJD (n = 52) and familial CJD (n = 10) patients when compared with a group of patients with noninflammatory disorders. Similarly, PGE2 and 8-epi-PGF2alpha levels were higher in brain homogenates obtained from C57BL/6J mice infected with the ME7 scrapie strain than in brain homogenates from control animals. As PGE2 is 1 of the most abundant prostaglandins released during inflammation and 8-epi-PGF2alpha is a quantitative marker of lipid peroxidation, our results provide in vivo biochemical evidence for the occurrence of inflammation and oxidative stress in human and experimental transmissible spongiform encephalopathies (TSEs), a concept so far based mainly on histopathological and in vitro evidence. Interestingly, in sporadic CJD patients, high CSF levels of PGE2, but not 8-epi-PGF2alpha, correlated with short survival time, suggesting that the inflammatory response correlates with the clinical duration of disease.     

Oxidative stress and the prion protein in transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic or genetic in origin. These diseases are believed to be the consequence of the conformational conversion of the prion protein into an abnormal isoform. Their exact pathogenic mechanism remains uncertain, but it is believed that oxidative stress plays a central role. In this article, we will first review in detail the data supporting the latter hypothesis. Subsequently, we will discuss the relationship between the prion protein and the cellular response to oxidative stress, attempting ultimately to link PrP function and neurodegeneration in these disorders.     

CSF analysis in patients with sporadic CJD and other transmissible spongiform encephalopathies

Patients with suspected Creutzfeldt–Jakob disease (CJD) often have routine cerebrospinal fluid (CSF) analysis performed to exclude treatable inflammatory conditions; however, little information is available about the range of results obtained for CSF tests in patients with sporadic CJD and other transmissible spongiform encephalopathies (TSE). Data from 450 patients with sporadic CJD and 47 patients with other TSEs were collected as part of an EC-supported multinational study. Raised white cell counts of >5 cells/ μ l were found in three of 298 patients with sporadic CJD, with two cell counts of 7 cells/ μ l and one of 20 cells/ μ l. Total protein concentrations of >0.9 g/l were found in five of 438 patients with sporadic CJD, although none had a concentration of >1 g/l. CSF oligoclonal IgG was detected in eight of 182 sporadic CJD patients. Of the patients with other TSEs, six had elevated cell counts ranging from 6 to 14 cells/ μ l but none had total protein concentrations of >0.9 g/l and one patient had detectable oligoclonal IgG. None of the patients with sporadic CJD or other TSEs had abnormalities in all three tests.     

Disease associated prion protein may deposit in the peripheral nervous system in human transmissible spongiform encephalopathies

There is increasing evidence indicating involvement of the peripheral nervous system (PNS) in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Immunocytochemically detectable deposits of TSE-specific abnormal prion protein (PrP^sc) are considered as a surrogate marker for infectivity. We used anti-PrP immunocytochemistry to trace PrP^sc deposition in spinal and enteric ganglia, and peripheral nerve in Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), and fatal familial insomnia. Discrete PrP^sc deposits were detectable only in a few posterior root nerve fibers in an adaxonal location in one of nine CJD and the one GSS patients examined. Follicular dendritic cells of the gut and enteric nervous system were not labeled. Thus, PrP^sc may spread to the PNS in different forms of human prion disease. In contrast to our observations in experimental scrapie (Groschup et al., Acta Neuropathol, this issue), the deposits were scant. Possible explanations for this discrepancy comprise strain difference, or centripetal (experimental scrapie) versus centrifugal (sporadic and genetic human prion diseases) spread of PrP^sc, resulting in different patterns and amounts of PrP^sc accumulation in the PNS.     

Prion protein immunohistochemical staining in the brains of monkeys with transmissible spongiform encephalopathy

Prion protein (PrP) immunohistochemical staining of the brains of common marmosets (Callithrix jacchus) with experimental transmissible spongiform encephalopathy is described. The monkeys ( n =17) had been injected, intracerebrally, 17–49  months previously with homogenates of brain tissue taken post mortem from a cow with BSE ( n =2 monkeys), a sheep with natural scrapie ( n =2 monkeys), human cases of growth hormone related Creutzfeldt–Jakob disease (CJD) ( n =2 monkeys), sporadic CJD ( n =5 monkeys), or Gerstmann–Sträussler–Scheinker disease (GSS) ( n =4 monkeys), or from monkeys with spongiform encephalopathy resulting from injection with brain tissue from these last two cases ( n =1 monkey from each case). Only diffuse PrP-staining was seen in monkeys injected with CJD-material whereas more aggregated deposits of PrP were seen in monkeys injected with BSE-, scrapie-and GSS-brain tissue. There were no patterns of staining specific to the brains injected with BSE-material that could be used to identify the origin of that inoculum. BSE-and scrapie-injected monkey brains could be distinguished from each other because in BSE-injected monkey brain the spongiform vacuolation was largely confined to subcortical structures whereas in scrapie-injected monkey brain the spongiform vacuolation was also prominent in the neocortex. The patterns of PrP deposition differed markedly between those seen in monkey brains injected with BSE-material or CJD-material, but the patterns of PrP staining seen in monkey brains injected with BSE-material were also seen in monkey brains injected with scrapie-or GSS-material. Overall there was a correlation between the length of the incubation period and the amount of aggregated PrP-staining, but no correlation between the neuropathological picture and the clinical presentation of neurological signs.     

Evaluation of rapid tests for the diagnosis of transmissible spongiform encephalopathies in sheep and goats

In accordance with EU Regulation 999/2001, rapid tests already adopted for bovine spongiform encephalopathy (BSE; Prionics Check Western, Platelia-BSE and Enfer TSE) are to be applied in all European countries to a sub-population of over 18-month-old slaughtered or dead sheep and goats to improve Scrapie surveillance and to determine the possible presence of BSE in sheep; however, the three tests have thus far been evaluated only for BSE and no official data are available about their performances on Scrapie. We evaluated the accuracy of these methods for TSE diagnosis in Italian sheep and goats, using a pre-homogenisation protocol on brain-stem samples to obtain comparable data from the three tests. Our results show that the tests can be considered reliable tools for active surveillance in the small ruminants population.     

The phenotypic expression of different mutations in transmissible human spongiform encephalopathy.

Clinical, pathological, and experimental transmission characteristics are reviewed for each of the known mutations in the amyloid precursor gene (PRNP) associated with familial spongiform encephalopathies. All mutation groups show an earlier age at onset and longer duration of illness than sporadic disease, and more or less distinctive patterns of illness can be recognized for each mutation, although much variability may occur even among affected members of the same family. Experimental transmission of disease has been accomplished for most of the mutations, with shortened incubation periods in the inoculated animals that parallel the earlier age at onset of human illness in these cases, implying a shortened pre-clinical phase of disease rather than an earlier 'infecting event'. Mutations thus not only predispose to spongiform encephalopathy, but also accelerate its pathogenetic tempo and influence its phenotypic expression.     

Technical communication Immunogold light and electron microscopic detection of amyloi plaques in transmissible spongiform encephalopathies

The antigenicity of the ‘prion’ protein amyloid fibrils was shown to be preserved after glutaraldehyde/O_sO₄ fixation in uranyl acetate- stained brain tissue blocks from patients with Gerstmann-Straussler syndrome (GSS) and from mice infected with Creutzfeldt-Jakob disease (CJD). Amyloid plaques were demonstrated by light microscopy in immunogold silver-intensified semithin sections. Under the electron microscope, the amyloid fibrils were labelled in immunogold-reacted ultrathin sections using an antiserum prepared against GSS amyloid plaque cores and mouse amyloid fibrils respectively. The influence of various oxidizing agents (hydrogen peroxide, sodium metaperiodate) on the tissue preservation and the immunohistochemical detection was tested.     

Lesions akin to transmissible spongiform encephalopathy in the brains of rats inoculated with immature cerebellum

Summary Fourteen BD IX rats were inoculated intracerebrally with a homogenate prepared from the immature cerebellar cortex of 10-day-old rats, when synaptogenesis is at its peak in this species. Eight controls were inoculated with mature cerebellar cortex. Transient ultrastructural changes were observed between 2 and 23 weeks' incubation in those animals which had received an inoculum of immature cerebellum. These changes pointed to a re-activation of embryonic or neo-natal growth mechanisms and were identical to those occurring in kuru-inoculated spider monkeys. With longer incubation histopathological lesions such as intracytoplasmic vacuolation, chromatolysis and neuronophagia appeared in neurons of the brain stem reticular formation. Such features are common in all the spongiform encephalopathies. All controls were negative. It is suggested that the transmissible agent in these diseases might be the factor which influences the various stages of normal neuronal maturation. A hypothesis is developed which would reconcile the infectious character of these diseases with a genetic factor and explain the unconventional behaviour of the agent as well as the mode of its transmission.Preliminary results of this work were included in a paper read at the 28th meeting of the Deutsche Gesellschaft für Neuropathologie und Neuroanatomie in October 1983 [3]. The work is dedicated to the memory of Herbert Butler (James) Parry 1912–1980     

Drug therapy in human and experimental transmissible spongiform encephalopathy

During the past 30 years, over 60 different chemical compounds have been used to treat experimental animals infected with transmissible spongiform encephalopathies (TSE), including a wide variety of anti-infectious agents, immunomodulating drugs, and chemicals interacting with the lympho-reticular system. Some compounds achieved a prolongation of the incubation period, but this effect decreased or disappeared when they were administered at or near the onset of symptomatic disease. Recent in vitro and tissue culture studies support earlier speculation about the importance of a chemical structure containing both water-soluble and lipid-soluble components, evidently as a means of interaction with the misfolded membrane-bound 'prion' protein. A number of compounds shown to eliminate the protein (or infectivity) in TSE-infected tissue cultures are the subject of ongoing studies in animals, and are under consideration for human drug trials. As with other recalcitrant infections, combinations of drugs with different modes of action are likely to be necessary for any effective therapy. Also, very recent work in developing antibodies that can neutralize in vitro infection (and, in conjunction with genetic engineering, in vivo infection) has renewed interest in the strategies of both active and passive immunization.