Metabolism of minor isoforms of prion proteins Cytosolic prion protein and transmembrane prion protein

Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein confi...     

Developmental expression of prion protein and its ligands stress‐inducible protein 1 and vitronectin

Prion protein (PrP^C) is the normal isoform of PrP^Sc, a protein involved in neurodegenerative disorders. PrP^C participates in neuritogenesis, neuroprotection, and memory consolidation through its interaction with the secreted protein stress‐inducible protein 1 (STI1) and the extracellular matrix protein vitronectin (Vn). Although PrP^C mRNA expression has been documented during embryogenesis, its protein expression patterns have not been evaluated. Furthermore, little is known about either Vn or STI protein expression. In this study, PrP^C, STI1, and Vn protein expression was explored throughout mouse embryonic life. We found that the distributions of the three proteins were spatiotemporally related. STI1 and Vn expression became evident at E8, earlier than PrP^C, in the nervous system and heart. At E10, we observed, in the spinal cord, a gradient of expression of the three proteins, more abundant in the notochord and floor plate, suggesting that they can have a role in axonal growth. As development proceeded, the three proteins were detected in other organs, suggesting that they may play a role in the development of nonneural tissues as well. Finally, although STI1 and Vn are PrP^C ligands, their expression was not altered in PrP^C‐null mice. J. Comp. Neurol. 517:371–384, 2009. © 2009 Wiley‐Liss, Inc.     

Subtyping of human cellular prion proteins and their differential solubility

A human form of a prion disorder is the Creutzfeldt-Jakob disease. A hallmark of the disease is the accumulation of misfolded prion proteins (PrP^Sc), which exist as heterogeneous subtypes. PrP^Sc is formed by protein conversion from the host-encoded cellular prion (PrP^C), which is expressed and modified to various isoforms. Little is known about variation in PrP^C; however, it is assumed that PrP^C types play important roles in the formation of PrP^Sc. In this study, we separated distinct human PrP^C subtypes on the basis of differential protein solubilities in detergent solutions. Single and sequential application of the detergents Triton X-100, octyl-glucopyranoside and CHAPS facilitated high solubility of glycosylated PrP^C isoforms, whereas high proportions of nonglycosylated PrP^C remained non-soluble. Most proteins became highly soluble with laurylsarcosine and sodium dodecyl sulphate. Our findings demonstrate that the solubility characteristics of heterogeneous PrP^C overlap in human brains and convey distinct solubility subtypes. Differentiation by solubility experiments can therefore provide valuable information on prion protein composition, facilitate the separation of subtypes, and offer new prospects for conversion specificity of distinct isoforms.     

Interplay between 20S proteasomes and prion proteins in scrapie disease

Scrapie is a transmissible spongiform encephalopathy affecting the central nervous system in sheep. The key event in such neurodegeneration is the conversion of the normal prion protein (PrP^C) into the pathological isoform (PrP^Sc). Misfolded prion proteins are normally degraded by the proteasome. This work, analyzing models of scrapie disease, describes the in vivo relationship between the proteasome and prions. We report that the disease is associated with an increase of proteasome functionality, most likely as a means of counteracting the increased levels of oxidative stress. Here, we show that prions coprecipitate with the 20S proteasome and that they colocalize within the same neuron, thus raising the possibility that PrP interacts with the proteasome in both normal and diseased brain, affecting substrate trafficking and proteasome functionality. This interaction, inducing proteasome activation, leads to different neuronal alterations and triggers apoptosis. Furthermore, testing the effects of isolated PrP^C on purified 20S proteasomes, we obtain a concentration‐ and proteasome composition‐dependent decrease in the complex activity. © 2009 Wiley‐Liss, Inc.     

朊蛋白在神经系统作用机制研究进展

细胞型朊蛋白(PrP~C)作为一种跨膜糖蛋白在哺乳动物中广泛存在。基因敲除的研究显示PrP~C在神经系统的活动中的关键作用包括周围神经髓鞘的形成以及对神经毒素刺激的保护。PrP~C在不同的细胞类型中也有不同的生物学作用。如PrP~C模块化结构、多种结合伴侣以及与脂质筏的密切关联的特性,使其具有组装多组分复合物的能力,从而触发不同的信号通路,调节细胞分化。PrP~C在大脑中参与的病理性作用仍然没有一致的定论,其错误折叠产生的异构体PrP~(SC)是朊病毒疾病的主要致病因素。但有证据指出PrP~C在朊病毒疾病中发挥的致病作用独立于羊瘙痒病朊蛋白亚型(PrP~(SC)),在朊病毒感染过程中,朊病毒疾病的临床和神经病理症状与大脑中PrP~C而不是PrP~(SC)的表达水平成正比。另外,PrP~C可能还是一种与神经退行性病变相关的蛋白,参与β淀粉样蛋白(Aβ)等聚集性蛋白的神经毒素信号转导,还充当α-突触核蛋白的细胞受体,促进其在细胞吸收以及大脑中传播。虽然朊病毒的研究已经取得很大的进展,但PrP~C在大脑中的作用仍然没有明确,因此探索PrP~C在细胞中作用具有十分重要的意义。     

In vivo toxicity of prion protein in murine scrapie: ultrastructural and immunogold studies

Prion protein (PrP) is a cell surface, host coded, sialoglycoprotein which accumulates in excess in scrapie, Creutzfeldt‐Jakob disease, bovine spongiform encephalopathy and other transmissible spongiform encephalopathies. Infection of mice with the 87 V or ME7 scrapie strains results in distinctive and very different light microscopical patterns of vacuolation and disease specific PrP accumulation. In both of these scrapie strains immunogold electron microscopy was used to locate PrP to the plasmalemma of neurons from where it was released into the neuropil. Initial PrP accumulation around neurons and in early plaques lacking amyloid fibrils was generally not associated with morphological changes either of the neuron or dendrite releasing the PrP or in the adjacent neuropil in which excess PrP accumulated. However, accumulation of pre‐amyloid PrP in some brain areas was associated with specific degeneration of dendritic spines and axon terminals. Initial PrP aggregation into fibrils was also associated with tissue damage with both ME7 and 87 V plaques and diffuse accumulations. Tissue damage associated with fibrillogenesis was localized and would not be expected to have clinical significance. We conclude that pre‐amyloid PrP release and accumulation is not invariably toxic, either to the neuron releasing PrP or to the neuropil into which it is released. However, axon terminal degeneration and dendritic spine loss in some neuroanatomical areas may be indicative of specific PrP toxicity and may be the main cause of neurological dysfunction in murine scrapie.     

Correlation between prion protein gene codon 129 polymorphism and late-onset Alzheimer's disease

BACKGROUND:Studies addressing the correlation between prion protein gene codon 129 polymorphism,Alzheimer's disease,and cognitive disorders have mainly focused on Caucasians.However,prion protein gene codon 129 polymorphism is thought to also affect the Chinese Han and Wei populations.OBJECTIVE:To analyze the differences of prion protein gene codon 129 distribution among the elderly Chinese Han,East Asian,and Caucasian populations,and to study the correlation between prion protein gene codon 129 distribution and late-onset Alzheimer's disease.DESIGN,TIME AND SETTING:A gene polymorphism analysis was performed in the Institute of Geriatrics,General Hospital of Chinese PLA between January 2006 and January 2007.PARTICIPANTS:A total of 152 elderly Chinese Han people were selected from the Beijing Troop Cadre's Sanitarium.Among them,60 patients with late-onset Alzheimer's disease,with a mean age of (82±7) years (range 67-94 years) and disease course of (5.9±4.4) years,comprising 44 males with a mean age of (83±7) years and 16 females with a mean age of (78±7) years,were selected for the case group.An additional 92 healthy elderly subjects,with a mean of (76±9) years (range 60-94 years),comprising 76 males with a mean age of (77±9) years and 16 females with a mean age of (70±8) years,were selected for the control group.There were no significant differences in age and gender between the two groups (P>0.05).METHODS:DNA was extracted from peripheral blood leukocytes using routine phenol/chloroform methodology.Prion protein gene codon 129 polymorphism and ApoE polymorphism were measured using PCR-restriction fragment length polymorphism.The ApoEε allele was considered the standard for analyzing correlations between prion protein gene codon 129 polymorphism and late-onset Alzheimer's disease.MAIN OUTCOME MEASURES:Prion protein gene codon 129 distribution;correlation between genotypic frequency and allele frequency of prion protein gene codon 129 with Alzheimer's disease;relationship between methionine/methionine genotype of prion protein gene,ApoEε4 allele,gender,and age of Alzheimer's disease patients.RESULTS:Methionine/methionine genotypic frequency of prion protein gene codon 129 was 94.08% in the Chinese elderly population,and the methionine/valine genotypic frequency was 5.92%.However,valine/valine homozygotes were not determined.There was no significant difference in prion protein gene codon 129 polymorphism between the Chinese elderly and East Asian populations (P>0.05).However,there was a significant difference between the Chinese elderly and the Caucasian population (P<0.05).The methionine/methionine genotype for the positive and negative ApoEε4 alleles was a risk factor for increased incidence of Alzheimer's disease,but there was no significant difference between the positives and the negatives (odds ratio=1.33,95% confidence interval=0.32-5.49,P>0.05).CONCLUSION:Prion protein gene codon 129 distribution in the Chinese elderly was different from the Caucasian population,which suggested that the methionine/methionine genotype of prion protein gene codon 129 negatively correlated with late-onset Alzheimer's disease.     

Cellular and subcellular morphological localization of normal prion protein in rodent cerebellum

Normal cellular prion protein, a necessary protagonist in fatal neurodegenerative prion diseases, was mapped in rodent cerebellum to establish its cellular and ultrastuctural localization. Existing morphological data about native prion protein distribution in brain tissues remain, indeed, contradictory and do not fit with biochemical and cell biological results. Using ultrastructural preembedding immunocytochemistry and a monoclonal anti-mouse prion protein antibody, this report shows that cellular prion protein is present in all cortico-cerebellar and deep nuclei neuronal cell types, as well as in all glial cell types. The heaviest expression appears on parallel fibres and astrocytic processes. The protein is exclusively located on the outer cell membrane and in Golgi and endosomal intracytoplasmic organelles, with no cytoplasmic or synaptic vesicle labelling. Most important, and in contrast with previous ultrastructural data, cellular prion protein is shown to be distributed on all portions of neurons, without any preferential synaptic targeting. The present morphological report shows, for the first time in vivo, that the cellular prion protein is present on the entire cell surface membrane of all neuronal and glial cell types of the rat cerebellum. This ubiquitous presence supports the notion that prion protein has a generalized cellular function in brain tissue rather than a specialized role restricted to synaptic transmission.     

Deletion of the amino-terminal domain of the prion protein does not impair prion protein-dependent neuronal differentiation and neuritogenesis

The cellular prion protein (PrP(C)) is a highly conserved glycoprotein of unknown biological function. To gain insight into the physiological role of PrP(C), we generated a novel PrP knockout cell line, named PrP(o/o) ML, by immortalization of neuroepithelial precursor cells derived from the cerebellum of PrP-knockout mice using the temperature-sensitive simian virus 40 (SV40) large T antigen. We demonstrated that the PrP(o/o) ML cell line is a unipotent precursor line with glutamatergic properties, which can acquire neuronal features when cultivated under specific conditions. The role of the prion protein in the process of neuronal differentiation was then analyzed in the PrP(o/o) ML cells reconstituted with either the full-length or an amino-terminally deleted form of the prion protein. We show that the expression of PrP(C) facilitates the processes of neuronal differentiation and neuritogenesis and that the deletion of its amino-terminal domain reduces the efficiency, but does not suppress this activity. This cell line represents a useful tool for studying PrP-dependent signal transduction pathways during differentiation of neuronal stem/precursor cells.     

Rapid detection of Creutzfeldt-Jakob disease and scrapie prion proteins

Creutzfeldt-Jakob disease (CJD) and Gerstmann-Str?ussler syndrome (GSS) of humans as well as scrapie of animals are caused by prions. The scrapie prion protein isoform (PrPSc) is the only macromolecule identified to date which is a component of the infectious prion particle. PrPSc is converted to PrP 27-30 by limited proteolysis while the cellular isoform, designated PrPC, is completely digested under the same conditions. ELISA studies demonstrated that native PrP 27-30 bound to plastic surfaces resisted proteolysis and exhibited little or no immunoreactivity but after denaturation with guanidinium thiocyanate (GdnSCN), immunoreactivity was greatly enhanced. PrPSc bound to nitrocellulose also exhibited enhanced immunoreactivity after denaturation. PrPSc was readily detected in brain extracts from scrapie-infected hamsters, mice, and sheep by dot-blot immunoassays using limited proteolysis followed by GdnSCN denaturation. The high sensitivity and specificity of the immunoassay allowed detection of regional differences in PrPSc in sheep brain. CJD prion protein isoform (PrPCJD) was also detected in the brains of all 10 patients tested with neuropathologically confirmed CJD and in 1 patient with GSS. Enhanced immunoreactivity of PrPSc or PrPCJD after denaturation cannot only be used for immunodiagnosis of prion diseases but may also form the basis of new assays in experimental studies directed at the chemical structure of the prion particle.     

Ultrastructural pathology of prion diseases revisited: brain biopsy studies

We report here a detailed ultrastructural comparison of brain biopsies from 13 cases of Creutzfeldt-Jakob disease (CJD) and from one case of fatal familial insomnia (FFI). The latter disease has not heretofore benefited from ultrastructural study. In particular, we searched for tubulovesicular structures (TVS), 35-nm particles regarded as the only disease-specific structures at the level of thin-section electron microscopy. Our material consisted of brain biopsies obtained by open surgery from one FFI case from a new French family, one case of variant CJD (vCJD), nine cases of sporadic CJD (sCJD), two cases of iatrogenic (human growth hormone) CJD and one case of hereditary CJD (Val203Iso). The ultrastructural picture of the cerebral cortex of the FFI patient was virtually indistinguishable from that of CJD. TVS were found, albeit only after prolonged search. Typical spongiform change was observed, consisting of intracellular membrane-bound vacuoles containing secondary chambers (vacuoles within vacuoles) and amorphous material. Neuronal degeneration was widespread: some processes contained degenerating mitochondria and lysosomal electron-dense bodies and these met the criteria for neuroaxonal dystrophy. Other processes contained branching cisterns; still others were filled with electron-dense masses and amorphous vesicles. The overall ultrastructural appearance of variant CJD was similar to that of FFI cerebral cortex, except for a much higher number of cellular processes containing TVS. We detected TVS in the majority of sCJD cases that, in addition to typical spongiform change and robust astrocytic reaction, showed widespread neuritic and synaptic degeneration and autophagic vacuoles. We conclude that TVS are readily found in FFI, vCJD and sCJD and that widespread neuritic degeneration is a part of ultrastructural pathology in prion diseases.     

Heterogeneity and regulation of cellular prion protein glycoforms in neuronal cell lines

The normal cellular prion protein is a small sialoglycoprotein highly expressed in neurons, the physiological function of which is largely unknown. Due to extensive N-glycosylations with a wide range of oligosaccharides, the prion protein displays a complex glycosylation pattern that could be of relevance for its function. The cellular prion protein patterns in adult mouse and rat brain, and in neuronal cell lines, appeared highly heterogeneous, as distinct levels and glycoforms of cellular prion protein were revealed by immunoblotting of corresponding samples. Amongst neuronal cell lines, mouse N2a neuroblastoma cells expressed low levels of endogenous prion protein. Mouse hypothalamic GT1-7 cells and rat pheochromocytoma PC-12 cells expressed highly glycosylated forms of cellular prion protein that were found neither in adult mouse and rat brain, nor in mouse brain during development. In contrast, rat B104 neuroblastoma cells abundantly expressed N-glycosylated cellular prion protein forms similar to those observed in mouse and rat brain. In all these cell lines, the prion protein was normally exported to and expressed at the outer cell membrane. Our results suggest that B104 cells may represent an appropriate cell model to investigate the physiological role of cellular prion protein in further detail as they highly express the normal 'brain-like' cellular prion protein glycoforms. In addition, we observed that the various prion glycoforms in B104 cells were tightly regulated both as a function of cell density and during neuronal differentiation, implying a potential role of cellular prion protein in cell-cell interactions and differentiation.     

Brain biopsy in Creutzfeldt-Jakob disease: evolution of pathological changes by prion protein immunohistochemistry

The formation of protease-resistant prion protein (PrPsc) is considered to be an early event in the pathogenesis of Creutzfeldt-Jakob disease (CJD) and hence its demonstration in brain biopsies by immunohistochemistry is considered diagnostic. We analysed eight brain biopsies from the frontal cortex collected from different parts of India from cases diagnosed as CJD on clinical and pathological grounds for the expression of prion protein (PrP). The duration of illness in these cases varied from 2 months to 1 year. Immunohistochemistry was carried out on paraffin sections using two different clones (KG9 and 3F4) of monoclonal antibodies to PrP. Although all eight cases showed classical features of spongiform encephalopathy of varying severity, only five of the eight cases revealed PrP(sc) in the brain tissue. The immunolabelling was focal and all areas with spongiform change were not labelled. A temporal evolution in the staining pattern was evident - particulate diffuse labelling (synaptic type) in early stages (2 months), perivacuolar deposits in intermediate stages (5-6 months), and dense plaques in late stages (12 months).     

Cellular prion protein localization in rodent and primate brain

The presence of an abnormal, protease-resistant form of the prion protein (PrP) is the hallmark of various forms of transmissible spongiform encephalopathies (TSE) which can affect a number of mammalian species, including humans. The normal, cellular form of this protein, PrP^c, while abundant in brain is also present in many tissues and a number of species. In order to address the unresolved question of the precise localization of normal cerebral PrP^c, we used a free-floating immunohistochemistry procedure to localize the protein at both the light and the electron microscopic levels in the brain of three TSE-sensitive species: hamster, macaque and humans. This method shows that PrP^c is abundant in synaptic terminal fields in olfactory bulb, limbic-associated structures and in the striato-nigral complex, whereas many other regions of the hamster brain are essentially devoid of immunoreactivity. With the striking exception of the olfactory nerve, in which axons are continually growing throughout life, PrP^c is not abundant in fibre pathways. PrP^c distribution in the primate hippocampus and cortex is very similar to the distribution observed in hamster. PrP^c was present at synaptic profiles as shown by immunoelectron microscopy, but was not detectable in neuronal perikaryon either by light or electron microscopy. Our results show that PrP^c is abundant in a number of brain structures known for ongoing plasticity, and are consistent with the hypothesis that the protein also plays a role in synaptic function.     

Prion proteins: a biological role beyond prion diseases

The biological role of the scrapie isoform of prion protein (PrP(Sc)) as an infectious agent in numerous human and non-human disorders of the central nervous system is well established. In contrast, and despite decades of intensive research, the physiological function of the endogenous cellular form of the prion protein (PrP(C)) remains elusive. In mammals, the ubiquitous expression of PrP(C) suggests biological functions other than its pathological role in propagating the accumulation of its misfolded isotype. Other functions that have been attributed to PrP(C) include signal transduction, synaptic transmission and protection against cell death through the apoptotic pathway. More recently, immunoregulatory properties of PrP(C) have been reported. We review accumulating in vitro and in vivo evidence regarding physiological functions of PrP(C).     

A traceback phenomenon can reveal the origin of prion infection

The transmission of prions to animals with incongruent prion protein (PrP) gene (referred to as cross‐sequence transmission) results in a relatively long incubation period and can generate a new prion strain with unique transmissibility designated as a traceback phenomenon. For example, cross‐sequence transmission of bovine spongiform encephalopathy (BSE) prions to human generated variant Creutzfeldt‐Jakob disease (vCJD) prions which retained the transmissibility to mice expressing bovine PrP. This finding suggests that traceback studies could enable us to identify the origin of prions. There are two distinct phenotypes in dura mater graft‐associated Creutzfeldt‐Jakob disease (dCJD), with the majority represented by a non‐plaque‐type of dCJD (np‐dCJD) and the minority by a plaque‐type of dCJD (p‐dCJD). To identify the origin of p‐dCJD, we performed a traceback study using mice expressing human PrP with methionine homozygosity (129M/M) or valine homozygosity (129V/V) at polymorphic codon 129. The characteristics of p‐dCJD such as the accumulation of abnormal isoform of PrP (PrP^Sc) intermediate in size between type 1 and type 2, and plaque‐type PrP deposition in the brain were maintained after transmission to the 129M/M mice. Furthermore, the 129V/V mice were more susceptible to p‐dCJD prions than the 129M/M mice and produced type 2 PrP^Sc that were identical in size to those from the 129V/V mice inoculated with sporadic CJD prions from a patient with 129V/V and type 2 PrP^Sc (sCJD‐VV2). In addition, we performed intracerebral transmission of sCJD‐VV2 prions to the 129M/M mice as an experimental model for p‐dCJD. These 129M/M mice showed the accumulation of the intermediate type PrP^Sc and plaque‐type PrP deposition in the brain. These results suggest that p‐dCJD could be caused by cross‐sequence transmission of sCJD‐VV2 prions to individuals with the 129M/M genotype.     

Cellular prion protein is present in dopaminergic neurons and modulates the dopaminergic system

Cellular prion protein (PrP^C) is widely expressed in the brain. Although the precise role of PrP^C remains uncertain, it has been proposed to be a pivotal modulator of neuroplasticity events by regulating the glutamatergic and serotonergic systems. Here we report the existence of neurochemical and functional interactions between PrP^C and the dopaminergic system. PrP^C was found to co‐localize with dopaminergic neurons and in dopaminergic synapses in the striatum. Furthermore, the genetic deletion of PrP^C down‐regulated dopamine D₁ receptors and DARPP‐32 density in the striatum and decreased dopamine levels in the prefrontal cortex of mice. This indicates that PrP^C affects the homeostasis of the dopaminergic system by interfering differently in different brain areas with dopamine synthesis, content, receptor density and signaling pathways. This interaction between PrP^C and the dopaminergic system prompts the hypotheses that the dopaminergic system may be implicated in some pathological features of prion‐related diseases and, conversely, that PrP^C may play a role in dopamine‐associated brain disorders.     

Advances in the detection of prion protein in peripheral tissues of variant Creutzfeldt-Jakob disease patients using paraffin-embedded tissue blotting

The accumulation of PrP(Sc), an abnormal and disease-associated form of the normal prion protein (PrP(c)), within the central nervous system (CNS) is a key pathological feature of Creutzfeldt-Jakob disease (CJD). Following limited proteolytic digestion of PrP(Sc), the detection of PrP(res) within lymphoid tissues is a unique characteristic of variant CJD in comparison with other human prion diseases, raising fears of an increased risk of iatrogenic spread. Because levels of PrP(res) in lymphoid tissues are lower than those found in CNS tissue, there is concern that other peripheral tissues may harbour infectivity at levels that current detection systems cannot demonstrate PrP(res). We have modified the paraffin-embedded tissue blot (PET blot), a technique combining immunohistochemistry (IHC), histoblot and Western blotting, for the detection of PrP(res) in paraffin sections in peripheral tissues in variant CJD. Five cases of variant CJD were examined, using a panel of anti-PrP antibodies. In each of these five cases, spleen, tonsil, lymph nodes and dorsal root ganglia showed an increase in the sensitivity and specificity of labelling using the PET blot when compared with optimized PrP(res) IHC methods. Control cases showed no evidence of PrP accumulation in either peripheral or CNS tissues. Autopsy and biopsy brain material from sporadic CJD cases also showed an increased sensitivity of PrP(res) detection with the PET blot, confirming its value as an important diagnostic and research tool in human prion diseases.