Ubiquitin, cellular inclusions and their role in neurodegeneration

Covalent binding of ubiquitin to proteins marks them for degradation by the ubiquitin/ATP-dependent pathway. This pathway plays a major role in the breakdown of abnormal proteins that result from oxidative stress, neurotoxicity and mutations. Failure to eliminate ubiquitinated proteins disrupts cellular homeostasis, causing degeneration. Inclusions containing ubiquitinated proteins are commonly detected in many neurological disorders. These aggregates are mostly cytosolic; nevertheless, ubiquitinated inclusions are found in endosomes/lysosomes in Alzheimer's disease and prion encephalopathies, and in nuclei in disorders associated with CAG/polyglutamine repeats, such as Huntington's disease and spinocerebellar ataxias. Ubiquitinated aggregates must result from a malfunction or overload of the ubiquitin/ATP-dependent pathway or from structural changes in the protein substrates, halting their degradation. Prevention of protein aggregation in these diseases might offer new therapeutic leads.     

Prion protein immunocytochemistry: reliable protocols for the investigation of Creutzfeldt–Jakob disease

Current criteria for the histological diagnosis of Creutzfeldt–Jakob disease (CJD) include features such as spongiform change, neuronal loss and reactive gliosis which are shared to a varying extent with other neurodegenerative disorders. Reliable visualization of prion protein (PrP) has substantial potential value in diagnostic practice and as a research tool, since accumulation of the disease–associated isoform of this protein is apparently specific for spongiform encephalopathies. A number of antisera against PrP have previously been employed in conjunction with a range of pre–treatments designed to optimize the specificity of immunostaining; such varied usage makes the comparison and interpretation of results difficult. This study was undertaken to identify optimal combinations of each of three PrP antisera and five pre–treatments designed to specifically demonstrate disease–specific PrP in a series of seven CJD cases, six cases of Alzheimer–type dementia and six non–demented control cases. Specific staining of amyloid plaques, spongiform neuropil, neurons and, occasionally, astrocytes was achieved in CJD cases. Alzheimer and control cases were unstained. Use of formic acid with guanidine thiocyanate, and hydrolytic autoclaving with IB 3 and SP30 antisera proved most effective and can be recommended for future immunocytochemical studies. PrP immunocytochemistry revealed a greater extent of subcortical neural involvement than routine histological techniques in CJD; the relationship between classical neuropathology in CJD and PrP accumulation as revealed by immunocytochemistry is not clear cut and requires further investigation. These findings may help to broaden our understanding of human spongiform encephalopathies, and have implications for diagnostic practices in neuropathology.     

Amyloid and prions: some biochemical investigations of cerebral amyloidosis in mice

Prion-like transmission of protein aggregates or amyloid in several neurodegenerative diseases, such as Parkinson's disease, Huntington's disease and Alzheimer's disease, in addition to the transmissible spongiform encephalopathies (or prion diseases), has been proposed recently. This is a controversial idea and, in this paper, we consider what we mean by a "prion", and by "amyloid", and present some biochemical investigations of cerebral prion amyloidosis in mice.     

Lack of TAR-DNA binding protein-43 (TDP-43) pathology in human prion diseases

Aims: TAR-DNA binding protein-43 (TDP-43) is the major ubiquitinated protein in the aggregates in frontotemporal dementia with ubiquitin-positive, tau-negative inclusions and motor neurone disease. Abnormal TDP-43 immunoreactivity has also been described in Alzheimer's disease, Lewy body diseases and Guam parkinsonism–dementia complex. We therefore aimed to determine whether there is TDP-43 pathology in human prion diseases, which are characterised by variable deposition of prion protein (PrP) aggregates in the brain as amyloid plaques or more diffuse deposits. Material and methods: TDP-43, ubiquitin and PrP were analysed by immunohistochemistry and double-labelling immunofluorescence, in sporadic, acquired and inherited forms of human prion disease. Results: Most PrP plaques contained ubiquitin, while synaptic PrP deposits were not associated with ubiquitin. No abnormal TDP-43 inclusions were identified in any type of prion disease case, and TDP-43 did not co-localize with ubiquitin-positive PrP plaques or with diffuse PrP aggregates. Conclusions: These data do not support a role for TDP-43 in prion disease pathogenesis and argue that TDP-43 inclusions define a distinct group of neurodegenerative disorders.     

A Quantitative and Qualitative Analysis of Prion Protein Immunohistochemical Staining in Creutzfeldt-Jakob Disease Using Four Anti Prion Protein Antibodies

Creutzfeldt-Jakob disease (CJD) is the most common spongiform encephalopathy affecting humans. Prion protein (PrP) immunohistochemistry may be useful for studying the localization of prion protein and assessing its role in CJD, the accumulation of a specific protease resistant PrP isoform being apparently pathognomic to the spongiform encephalopathies. However, a number of factors influence the results of immunostaining, making interpretation and comparisons between the staining of different PrP antisera difficult. This study has examined qualitatively and quantitatively the staining produced by four antisera raised to a variety of prion protein homologues in two cases of CJD and two age-matched controls. Quantitative analysis was provided through the use of custom designed image analysis software. Kuru, granular and multicentric plaques, cellular, perivacuolar and white matter PrP deposits were observed in CJD cases with all four antisera. No significant immunostaining was seen in the control tissue. Some antibody specific staining patterns were observed qualitatively; however, quantitative analysis showed statistically significant correlations between all the antisera on the diseased brain tissue. Prion protein immunohistochemistry is thus useful in interpreting patterns of protein distribution in diseased brain but care may be required in interpreting the results of a single antibody.     

Increased clusterin (apolipoprotein J) expression in human and mouse brains infected with transmissible spongiform encephalopathies

Clusterin (apolipoprotein J), a multifunctional protein involved in amyloidogenesis in Alzheimer's disease, was studied immunohistochemically in both human transmissible spongiform encephalopathies (TSEs) and a mouse model of human TSE. Clusterin immunoreactivity was co-localized with plaque-type deposits but not with punctate-type prion protein (PrP) deposits in human TSEs. On the other hand, clusterin-positive astrocytes were readily demonstrated in the regions of punctate PrP deposits, but not around plaque PrP deposits despite the presence of surrounding astrocytes. Clusterin expression in astrocytes was not disease specific, but the punctate immunoreactivity for clusterin was more prominently demonstrated in TSEs with punctate PrP deposits. Serial analysis in the mouse model of human TSE revealed that clusterin expression in astrocytes was enhanced in the lesions with punctate-type PrP deposits during the disease progression. Thus, the induction of clusterin expression in astrocytes could be more enhanced by punctate-type PrP deposits than by plaque-type deposits. The clusterin molecules co-localized in plaque PrP deposits might be derived not from surrounding astrocytes but from other resources such as cerebrospinal fluid and blood plasma, both of which contain clusterin in significant amounts. Taken together with previously reported findings of the anti-amyloidogenic property in clusterin, our findings suggest that clusterin may be induced as one of the important molecules participating in the neurodegeneration caused by abnormally deposited PrP.     

Prion protein genotype and pathological phenotype studies in sporadic Creutzfeldt-Jakob disease

A comparative semi-automated morphometric study was performed on the distribution of prion protein, spongiform change and astrocytosis in the brains of nine cases of sporadic Creutzfeldt-Jakob disease of differing genotype at the methionine-valine polymorphism at codon 129 of the prion protein gene. Custom-designed image analysis software was used to produce objective figures for each of the different pathological features throughout 13 different areas of the brain used for analysis. A significant positive correlation was observed between prion protein deposition and astrocytosis in all cases and no significant correlation was observed between spongiform change and prion protein deposition. Different patterns of pathology were found to relate to codon 129 genotype; valine homozygosity favoured the targeting of pathology to deep grey matter structures, while methionine homozygosity favoured cortical targeting of pathology. These results provide evidence that prion protein deposition is closely associated with an astrocytic reaction and suggest that codon 129 genotype may influence the pathological phenotype.     

A neuropathological subset of Alzheimer's disease with concomitant Lewy body disease and spongiform change

Summary The neuropathological heterogeneity of Alzheimer's disease (AD) is increasingly recognized. Diffuse Lewy body disease, for example, most frequently occurs in cases fulfilling histopathological criteria for AD, and these patients usually present with dementia rather than parkinsonism. We report five cases of concomitant AD and diffuse Lewy body disease with still another coexistent neuropathological feature: localized and stereotyped spongiform change in the neuropil. This spongiform change was most striking in the superior and inferior temporal, entorhinal, and insular cortex and the amygdala and was virtually indistinguishable from that seen in Creutzfeldt-Jakob disease. Electron microscopic study on one case revealed membrane-containing vacuoles in close association with neuritic plaques and plaired helical filament-filled processes. Immunocytochemistry using antibodies to prion proteins (PrP_sc or PrP_27–30) failed to label plaque or vascular amyloid in the five cases. Four primates inoculated with brain tissue from one case have not evidenced neurological disease in the 3 years since the transmission experiment. We conclude that these cases represent a neuropathological subset of AD with relatively widespread Lewy bodies and a localized spongiform change, predominantly involving the medial temporal region. Despite the light and electron microscopic commonality with Creutzfeldt-Jakob disease, there is no clear evidence that these cases represent a form of transmissible spongiform encephalopathy.Supported by Veterans Administration Merit Award 5747004 (SM). The contributions made to this study by the UCSD group (LH, EM, and RDT) were supported by National Institutes of Health grants AGO5131 and AGO8201 and the McKnight Foundation     

Rabies: Spongiform lesions in the brain

Summary Striped skunks (Mephitis mephitis) experimentally infected with street rabies virus developed spongiform lesions that light- and electron-microscopically were indistinguishable from those found in the transmissible spongiform encephalopathies of man and animals. These previously unreported lesions were also detected in naturally occurring cases of rabies. The spongiform lesions consisted of round or oval vacuoles in the neuropil, rarely in neuronal perikarya. The most severely affected areas were the thalamus and cerebral cortex. The implications of this finding include similarities in the pathogenetic mechanisms of rabies and the traditional spongiform encephalopathies and the possibility of lesion variation due to differences in rabies viral strains. The spongiform lesions of rabies will require consideration in differential diagnosis.     

Spongiform encephalopathy in transgenic mice expressing a point mutation in the β2-α2 loop of the prion protein

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases attributed to misfolding of the cellular prion protein, PrP(C), into a β-sheet-rich, aggregated isoform, PrP(Sc). We previously found that expression of mouse PrP with the two amino acid substitutions S170N and N174T, which result in high structural order of the β2-α2 loop in the NMR structure at pH 4.5 and 20°C, caused transmissible de novo prion disease in transgenic mice. Here we report that expression of mouse PrP with the single-residue substitution D167S, which also results in a structurally well ordered β2-α2 loop at 20°C, elicits spontaneous PrP aggregation in vivo. Transgenic mice expressing PrP(D167S) developed a progressive encephalopathy characterized by abundant PrP plaque formation, spongiform change, and gliosis. These results add to the evidence that the β2-α2 loop has an important role in intermolecular interactions, including that it may be a key determinant of prion protein aggregation.     

Distribution of the cellular prion protein (PrPC) in brains of livestock and domesticated species

In transmissible spongiform encephalopathies (TSEs) the prion protein (PrP) plays a central role in pathogenesis. The PrP gene (Prnp) has been described in a number of mammalian and avian species and its expression product, the cellular prion protein (PrP^C), has been mapped in brains of different laboratory animals (rodent and non-human primates). However, mapping of PrP^C expression in mammalian species suffering from natural (bovine and ovine) and experimental (swine) TSE or in species in which prion disease has never been reported (equine and canine) deserves further attention. Thus, localising the cellular prion protein (PrP^C) distribution in brain may be noteworthy for the understanding of prion disease pathogenesis since lesions seem to be restricted to particular brain areas. In the present work, we analysed the distribution of PrP^C expression among several brain structures of the above species. Our results suggest that the expression of PrP^C, within the same species, differs depending on the brain structure studied, but no essential differences between the PrP^C distribution patterns among the studied species could be established. Positive immunoreaction was found mainly in the neuropil and to a lesser extent in neuronal bodies which occasionally appeared strongly stained in discrete regions. Overall, the expression of PrP^C in the brain was significantly higher in grey matter areas than in white matter, where accumulation of PrP^Sc is first observed in prion diseases. Therefore, other factors besides the level of expression of cellular PrP may account for the pathogenesis of TSEs     

Gene knockout of tau expression does not contribute to the pathogenesis of prion disease

Prion diseases or transmissible spongiform encephalopathies are a group of fatal and transmissible disorders affecting the central nervous system of humans and animals. The principal agent of prion disease transmission and pathogenesis is proposed to be an abnormal protease-resistant isoform of the normal cellular prion protein. The microtubule-associated protein tau is elevated in patients with Creutzfeldt-Jakob disease. To determine whether tau expression contributes to prion disease pathogenesis, tau knockout and control wild-type mice were infected with the M1000 strain of mouse-adapted human prions. Immunohistochemical analysis for total tau expression in prion-infected wild-type mice indicated tau aggregation in the cytoplasm of a subpopulation of neurons in regions associated with spongiform change. Western immunoblot analysis of brain homogenates revealed a decrease in total tau immunoreactivity and epitope-specific changes in tau phosphorylation. No significant difference in incubation period or other disease features were observed between tau knockout and wild-type mice with clinical prion disease. These results demonstrate that, in this model of prion disease, tau does not contribute to the pathogenesis of prion disease and that changes in the tau protein profile observed in mice with clinical prion disease occurs as a consequence of the prion-induced pathogenesis.     

Topographic distribution of scrapie amyloid-immunoreactive plaques in chronic wasting disease in captive mule deer (Odocoileus hemionus hemionus)

Summary Chronic wasting disease (CWD), a progressive neurological disorder of captive mule deer, blacktailed deer, hybrids of mule deer and white-tailed deer and Rocky Mountain elk, is characterized neuropathologically by widespread spongiform change of the neuropil, intracytoplasmic vacuolation in neuronal perikarya and astrocytic hypertrophy and hyperplasia. We report the topographic distribution of amyloid plaques reactive to antibodies prepared against scrapie amyloid in CWD-affected captive mule deer (Odocoileus hemionus hemionus). Scrapie amyloid-immunoreactive plaques were found in the cerebral gray and white matter, in deep subcortical nuclei, in isolation or in clusters in areas of vacuolation, and perivascularly, in subpial and subependymal regions. In the cerebellum, immunoreactive amyloid plaques were observed in the molecular, pyramidal and granular layers. Scrapie amyloid-immunoreactive deposits were also seen in neuronal perikarya. Furthermore, amyloid plaques in CWD-affected captive mule deer were alcianophilic at 0.3 M magnesium chloride indicating the presence of weakly to moderately sulfated glycosaminoglycans. Our data corroborate that CWD in captive mule deer belongs to the subacute virus spongiform encephalopathies.     

Heparan Sulfate Proteoglycan Is Associated with Amyloid Plaques and Neuroanatomically Targeted PrP Pathology throughout the Incubation Period of Scrapie-Infected Mice

Heparan sulfate proteoglycan (HSPG) has been found to be associated with amyloid deposits in a number of diseases including the cerebral amyloid plaques of Alzheimer's disease and the transmissible spongiform encephalopathies (TSEs). The role of HSPG in amyloid formation and the neurodegenerative pathology of these diseases have not been established. We have addressed these questions using a scrapie mouse model which exhibits both amyloid and nonamyloid deposition of abnormal PrP protein, the protein marker of TSE infection. The distribution of HSPG was examined throughout the course of the disease in the brains of experimentally infected mice and compared with the distribution of abnormal PrP. Abnormally high levels of HSPG were associated with most types of PrP pathology including all plaque types and diffuse neuroanatomically targeted forms. Scrapie-associated HSPG was present from 70 days after infection, the earliest time-point examined, in the same target areas as abnormal PrP. The association with amyloid plaques may indicate that HSPG is involved in amyloid plaque formation and/or persistence but involvement with early diffuse forms of PrP suggests a more fundamental role in scrapie pathogenesis.     

Microglia and the pathogenesis of spongiform encephalopathies

Alterations in the phenotype and function of microglia, the resident mononuclear phagocytes of the central nervous system, are among the earliest indications of pathology within the brain and spinal cord. The prion diseases, also known as spongiform encephalopathies, are fatal neurodegenerative disorders with sporadic, genetic or acquired infectious manifestations. A hallmark of all prion diseases is the aberrant metabolism and resulting accumulation of the prion protein. Conversion of the normal cellular protein [PrP^c] into the abnormal pathogenic (or disease-causing) isoform [PrP^Sc] involves a conformational alteration whereby the α-helical content is transformed into β-sheet. The histological characteristics of these disorders are spongiform change, astrocytosis, neuronal loss and progressive accumulation of the protease-resistant prion isoform. An additional upregulation in microglial response has been reported in Kuru, Creutzfeldt–Jakob disease (CJD), Gerstmann–Sträussler–Scheinker syndrome (GSS), scrapie, in transgenic murine models and in culture, where microglial activation often accompanies prion protein deposition and neuronal loss. This article will review the roles of microglia in spongiform encephalopathies.     

Decreased cell surface prion protein in mouse models of prion disease

Transmissible spongiform encephalopathies are infectious neurodegenerative diseases caused by prions, composed of ordered aggregates of misfolded cellular prion protein. Neural antigen density of prion protein, Thy-1 and glial fibrillary acidic protein was analyzed using flow cytometry of dissociated mouse brain cells after inoculation with mouse-adapted transmissible spongiform encephalopathy agents. Transmissible spongiform encephalopathy gliosis was demonstrated by increased intracellular immunoreactivity for glial fibrillary acidic protein compared with controls. Immunoreactivity for cell surface prion protein was reduced 2.8-3.8-fold compared with control brain cells, whereas surface Thy-1 protein was reduced 1.5-4-fold. Double-staining protocols revealed loss of brain cells highly immunoreactive for prion protein and Thy-1, with a preferential reduction of prion protein, suggesting that prion protein expression, trafficking or consumption may be affected early in disease.     

Identification of inter-species transmission of prion strains

The concern of the potential transmission of animal spongiform encephalopathies to humans, which arose as soon as the interspecies transmission of these diseases was recognized, has been reinforced with the emergence of bovine spongiform encephalopathy (BSE) in cattle. Recent experimental findings suggest that the infectious agent causing BSE in cattle can lead to the occurrence of a new form of Creutzfeldt-Jakob disease in humans. These findings help us understand how the transmission to humans of an animal disease may be recognized. This can involve an indirect approach through the analysis of neurodegeneration, either in the disease host, or more specifically, in genetically well-defined experimental hosts to which the disease can be transmitted. Recent experimental studies have also shown that the different molecular features of the abnormal form of the prion protein, which accumulates in the infected tissues, can provide important clues to the relationships between different spongiform encephalopathies. However, a better understanding of the molecular features associated with the specific pathogenic behavior of different strains is required. Complex relationships between the infectious agents involved in spongiform encephalopathies and the disease host can make the recognition of a link between animal prion strains and the human disease difficult to establish.     

Selection of distinct strain phenotypes in mice infected by ovine natural scrapie isolates similar to CH1641 experimental scrapie

A few cases of transmissible spongiform encephalopathies in sheep have been described in France in which the protease-resistant prion protein (PrP(res)) exhibited some features in Western blot of experimental bovine spongiform encephalopathy in sheep. Their molecular characteristics were indistinguishable from those produced in the CH1641 experimental scrapie isolate. Four of these CH1641-like isolates were inoculated intracerebrally into wild-type C57Bl/6 mice. In striking contrast to previous results in ovine transgenic mice, CH1641 transmission in wild-type mice was efficient. Several components of the strain signature, that is, PrP(res) profile, brain distribution, and morphology of the deposits of the disease-associated prion protein, had some similarities with "classical" scrapie and clearly differed from both bovine spongiform encephalopathy in sheep and CH1641 transmission in ovine transgenic mice. These results on CH1641-like isolates in wild-type mice may be consistent with the presence in these isolates of mixed conformers with different abilities to propagate and mediate specific disease phenotypes in different species.     

Scrapie-specific neuronal lesions are independent of neuronal PrP expression

In the transmissible spongiform encephalopathies (TSE), accumulation of the abnormal disease-specific prion protein is associated with neurodegeneration. Previous data suggested that abnormal prion protein (PrP) could induce neuronal pathology only when neurons expressed the normal form of PrP, but conflicting evidence also has been reported. Understanding whether neuronal PrP expression is required for TSE neuropathological damage in vivo is essential for determining the mechanism of TSE pathogenesis. Therefore, these experiments were designed to study scrapie pathogenesis in vivo in the absence of neuronal PrP expression. Hamster scrapie (strain 263K) was used to infect transgenic mice expressing hamster PrP in the brain only in astrocytes. These mice previously were shown to develop clinical scrapie, but it was unclear whether the brain pathology was caused by damage to astrocytes, neurons, or other cell types. In this electron microscopic study, neurons demonstrated TSE-specific pathology despite lacking PrP expression. Abnormal PrP was identified around astrocytes, primarily in the extracellular spaces of the neuropil, but astrocytes showed only reactive changes and no damage. Therefore, in this model the pathogenesis of the disease appeared to involve neuronal damage associated with extracellular astrocytic accumulation of abnormal PrP acting upon nearby PrP-negative neurons or triggering the release of non-PrP neurotoxic factors from astrocytes.     

Scrapie pathogenesis in brain and retina: Effects of prion protein expression in neurons and astrocytes

Brain damage in the transmissible spongiform encephalopathies or prion diseases is associated with the conversion of normal host prion protein to an abnormal protease-resistant isoform, and expression of prion protein is required for susceptibility to these diseases. This article reviews the data on studies using transgenic mice expressing prion protein in specific individual cell types to study the roles of these cell types in prion disease pathogenesis. Surprisingly damage to neurons in brain and retina appeared to require different prion protein-expressing cells, suggesting that different pathogenic mechanisms operate in these two neuronal tissues.