Copper binding to PrPC may inhibit prion disease propagation

Although it has been well established that PrP(C), the normal isoform of PrP(Sc), is a copper-binding protein, the role of this metal in the function of PrP(C) as well as in prion disease pathology remains unclear. Here, we show that when scrapie-infected neuroblastoma cells were cultured in the presence of copper, the accumulation of PrP(Sc) in these cells was markedly reduced. In addition, our results indicate that when normal neuroblastoma cells were cultured in the presence of copper ions, they could no longer bind and internalize PrP(Sc). In another set of experiments, copper was added to the drinking water of normal and scrapie-infected hamsters. Our results show that administration of copper to normal hamsters induced cerebellar PrP(C) accumulation. Most important, a significant delay in prion disease onset was observed when scrapie-infected hamsters were treated with copper. As shown before for neuroblastoma cells, also in vivo most of the copper-induced accumulation of PrP(C) was intracellular. We hypothesized that PrP(C) internalization by copper may hinder PrP(Sc) interaction with this molecule, and thereby affect prion disease propagation.     

GFP-tagged prion protein is correctly localized and functionally active in the brains of transgenic mice

Prion diseases result from conversion of PrPC, a neuronal membrane glycoprotein of unknown function, into PrPSc, an abnormal conformer that is thought to be infectious. To facilitate analysis of PrP distribution in the brain, we have generated transgenic mice in which a PrP promoter drives expression of PrP-EGFP, a fusion protein consisting of enhanced green fluorescent protein inserted adjacent to the glycolipid attachment site of PrP. We find that PrP-EGFP in the brain is glycosylated and glycolipid-anchored and is localized to the surface membrane and the Golgi apparatus of neurons. Like endogenous PrP, PrP-EGFP is concentrated in synapse-rich regions and along axon tracts. PrP-EGFP is functional in vivo, since it ameliorates the cerebellar neurodegeneration induced by a truncated form of PrP. These observations clarify uncertainties in the cellular localization of PrPC in brain, and they establish PrP-EGFP transgenic mice as useful models for further studies of prion biology.     

Synaptosomal glutamate release and uptake in mice lacking the cellular prion protein

Glutamate plays a central role in the fast excitatory synaptic transmission and is a key neurotransmitter involved in several neurophysiological processes. Glutamate levels on the synaptic cleft are related to neural excitability, neuroplasticity, and neuronal damage associated with excitotoxicity. Mice lacking the cellular prion protein (PrP(c)) gene (Prnp) present a decreased astrocytic glutamate uptake in cultures, higher neuronal excitability in vitro and sensitivity to pro-convulsant drugs in vivo, and age-dependent memory impairment. Here, we investigate if PrP(c) might be involved in neuronal uptake and release of glutamate. For this purpose, we compared synaptosomal preparations from the cerebral cortex, entorhinal cortex, hippocampus, cerebellum, and olfactory bulb of 3- or 9-month-old PrP(c) null mice and with respective wild-type controls. Although we observed differences in synaptosomal glutamate release and uptake regarding the age of mice and the brain structure studied, these differences were similar for PrP(c) null mice and their respective wild-type controls. Therefore, despite a possible correlation between neuronal glutamate transporters, excitability, and neuronal damage, our results suggest that PrP(c) expression is not critical for neuronal glutamate transport.     

Tubulovesicular structures are not labeled using antibodies to prion protein (PrP) with the immunogold electron microscopy techniques

Tubulovesicular structures (TVS) are disease-specific, intraneuronal particles found by thin-section electron microscopy in all of the transmissible spongiform encephalopathies. We used immunogold (both 10 nm immunogold and 1 nm immunogold silver enhanced) methods for ultrastructural localization of prion protein (PrP). In all scrapie models examined (263 K and 22CH in hamsters and 87V and ME7 in mice), TVS-containing processes were readily detected but neither these processes nor TVS themselves were decorated with gold particles. Even when amyloid plaques were observed in a close contact with TVS-containing neuronal processes, the processes remained unstained, while the plaques were decorated with gold particles. TVS located in areas adjacent to plaques in the 87V model and in areas of diffuse PrP immunolabelling in ME7 were also unlabelled with anti-PrP sera. Using immunogold techniques we were unable to label TVS with anti-PrP antibodies. As these technique proved to be sensitive enough to immunolabel not only amyloid plaques but also pre-amyloid accumulations of PrP, we strongly believe that the absence of staining reflects the structure of TVS and that they are not composed of PrP. That TVS are PrP negative may have several important implications for hypotheses about their nature. Principally, it does not support the suggestion that TVS are cross-sections of “thick tubules” visualized by touch-preparations of scrapie-affected mouse and hamster brains. If PrP is the infectious agent, as suggested by the prion hypothesis, the absence of stainable PrP in TVS would indicate that these are not the ultrastructural correlate of the agent. If, however, TVS turn out to be more than merely a useful ultrastructural marker for the whole group of transmissible spongiform encephalopathies, it may suggest that PrP and the agent are two separate entities. Received: 11 March 1996 / Revised: 9 May 1996 / Accepted: 26 September 1996     

PrP<Superscript>Sc</Superscript>-like prion protein conformer in sudden infant death syndrome brain

Nothing is known about the pathophysiology of sudden infant death syndrome (SIDS). Here we show the presence of misfolded prion protein (PrP^Sc-like) in extracts of various sections of the brains of two SIDS victims. DNA sequence information for one of these (death at 12 days) revealed two nucleotide variants in the protein coding region of the PrP gene. This may be a key finding in the understanding of SIDS pathology, and may suggest ways for identifying risk factors for SIDS in newborn infants.     

Successful transmission of Creutzfeldt-Jakob disease from human to mouse verified by prion protein accumulation in mouse brains

The accumulation of prion protein (PrP) was revealed in the brains of mice inoculated with the brain homogenate from seven patients with Creutzfeldt-Jakob disease (CJD) by immunohistochemistry using hydrolytic autoclaving. It was not found in the brains of mice inoculated with material from either two patients with Gerstmann-Str?ussler syndrome or two with other dementing illnesses. PrP accumulation took the forms of diffuse neuropil accumulation in the gray matter and plaque-like accumulation in the white matter and was observed in particular areas in the supratentorial structure. Its distribution was narrower than that in the brains of mice infected with a mouse-adapted CJD strain. PrP accumulation was found not only in all histopathologically positive mice, but also in some histopathologically negative mice. In all groups of mice inoculated with the material from each CJD patient, the percentage of mice with PrP accumulation was equal to or exceeded that of mice with the histopathological findings. PrP immunohistochemistry using formic acid pretreatment stained such plaque-like accumulation less intensely than that using hydrolytic autoclaving and did not stain diffuse neuropil accumulation. Therefore, PrP accumulation which can be revealed in the brains of first-passage CJD mice by this new immunohistochemical method may be the most sensitive hallmark of successful transmission.     

Deposition of the prion protein (PrP) during the evolution of experimental Creutzfeldt-Jakob disease

We studied the immunocytochemical distribution of the prion or proteinase-resistant protein (PrP) during the evolution of experimental Creutzfeldt-Jakob disease (CJD) in mice. Fifty-one brains were collected up to 22 weeks following intracerebral inoculation with the Fujisaki strain of the CJD agent. Slides were also immunostained for apolipoprotein E (apoE) and glial fibrillary acidic protein. Vacuolar changes with focal astrocytosis first occurred around the needle track at week 2 and later spread along white matter tracks. Until week 12, changes were asymmetrical, affecting more the side of inoculation. Spongiform change and astrogliosis spread subsequently to the gray matter. Time course and intensity of spongiform change and immunocytochemistry for PrP were discrepant: in most brain regions, severe vacuolation preceded immunocytochemically detectable PrP accumulation. PrP deposits in form of small dots were first detectable at week 6 in the area surrounding the needle track. After week 7, plaque-like amorphous PrP deposits were observed in white matter pathways. Finally, PrP was detectable also in basal ganglia and in the dorsal hippocampus (week 13) and in the neocortex (week 17), as the synaptic type of PrP immunopositivity. In the hippocampus, diffuse PrP deposits paralleled spongiform change, while in the cortex severe vacuolation was accompanied only by weak synaptic PrP deposits. Immunocytochemically detectable apoE was restricted to compact plaque-type PrP deposits after week 15. We conclude that disease-specific neuropathology spreads from the needle track along white matter pathways towards the gray matter; in this model, there is some discrepancy between development of tissue pathology and immunocytochemically detectable deposition of PrP. Immunocytochemically detectable apoE deposition follows PrP accumulation. Received: 22 December 1998 / Revised, accepted: 6 April 1999     

The shifting biology of prions

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are rare fatal neurodegenerative diseases of humans and animals. Although some TSEs, like scrapie in sheep, have been known to exist for centuries, bovine spongiform encephalopathy (BSE) was recognized only 15 years ago. New variant Creutzfeldt–Jakob disease (nvCJD) of humans is probably caused by consumption of BSE-infected materials. The nature of the infectious agent is not fully elucidated, but substantial evidence suggests that it is devoid of nucleic acids and consists at least in part of an abnormal form of a host protein termed PrP^C. Despite their rarity, prion diseases have become an important topic in public health and basic research because of the connection between nvCJD and BSE and also because of the unusual biological attributes of the infectious agent.     

Immunohistochemical expression of prion protein (PrPC) in the human forebrain during development

The cellular prion protein (PrPC) is a ubiquitous protein whose expression in the adult brain occurs mainly in synapses. We used monoclonal antibodies to study fetal and perinatal PrPC expression in the human forebrain. Double immunofluorescence and confocal microscopy with GFAP, Iba1, MAP2, doublecortin, synaptophysin, and GAP-43 were used to localize PrPC. PrPC immunoreactivity was observed in axonal tracts and fascicles from the 11th week to the end of gestation. Synapses expressed PrPC at increasing levels throughout synaptogenesis. At midgestation, a few PrPC-labeled neurons were detected in the cortical anlage and numerous ameboid and intermediate microglial cells were PrPC-positive. In contrast, at the end of gestation, microglial PrPC expression decreased to almost nothing, whereas neuronal PrPC expression increased, most notably in ischemic areas. In adults, PrPC immunoreactivity was restricted to the synaptic neuropil of the gray matter. At all ages, choroid plexus, ependymal, and endothelial cells were labeled, whereas astrocytes were only occasionally immunoreactive. In conclusion, the early expression of PrPC in the axonal field may suggest a specific role for this molecule in axonal growth during development. Moreover, PrPC may play a role in early microglial cell development.     

Phenotypic variability in the brains of a family with a prion disease characterized by a 144-base pair insertion in the prion protein gene

The use of prion protein (PrP) immunohistochemistry in neuropathology has allowed identification of prion diseases with otherwise atypical histological features. The brains from family members with familial prion diseases can show marked histological variation. A histological and immunohistochemical study was performed on 10 brains of patients with a familial prion disease caused by a 144-base pair (bp) insertion in the prion protein gene. The histology from the cases showed variability in the severity of spongiform change and astrocytosis in both the cerebellum and the cerebrum. There was also variability in the density of microglial cells. The PrP immunohistochemistry revealed that in nine cases there was a similar patch-like deposition of PrP within the molecular layer of the cerebellum. Although in the cerebellum there did seem to be some correlation between the severity of spongiform change, astrocytosis and the density of microglial cells, there was no such correlation between any of these three parameters and the density of PrP staining. There was deposition of beta-amyloid precursor protein (beta-APP) in the cerebellum, suggesting that disrupted axonal transport had a possible role in the evolution of the disease. The cases illustrate the histological variability that can occur in familial prion diseases despite similarity in PrP staining. They also reveal that the relationship between PrP deposition and cerebral or cerebellar damage might be complex.     

Mossy fibre reorganization in the hippocampus of prion protein null mice

Mice lacking prion protein (PrP-null) are resistant to transmissible spongiform encephalopathies. However, the normal functions of this highly conserved protein remain controversial. This study examines whether PrP-null mice develop normal neuronal pathways, specifically the mossy fibre pathway, within the hippocampus. Timm stained hippocampal sections from the PrP-null group had more granules than the controls in: the granule cell layer, the inner molecular layer of the dentate gyrus, and the infrapyramidal region of CA3. This resembles the mossy fibre collateral and terminal sprouting seen in certain epilepsies. The abnormal connectivity might be predicted to promote epileptiform activity, but extracellular electrophysiological recordings from the granule cell layer revealed a reduced excitability in the PrP-null group, both with and without blockade of GABA_A receptor-mediated inhibition. We propose that reorganization of neuronal circuity is a feature of PrP-null mice.     

Expression and localization of the prion protein PrPC in the olfactory system of the mouse

The normal physiological function of the prion protein PrP^C remains elusive despite its widespread expression, particularly throughout the nervous system. A critical step toward identifying its function is to precisely localize its pattern of expression. Historically, the immunolocalization of PrP^C has proved to be notoriously difficult and nonconsensual. We have thus undertaken a detailed expression analysis by means of a combination of in situ hybridization, knockout mice, and immunohistochemistry, using recently generated highly specific antibodies. We have attempted to accurately localize PrP^C expression in a tissue that is highly structured and of crucial behavioral importance to mice, the olfactory system. We found that PrP^C was expressed in both peripheral and central neurons of the olfactory system and that its distribution was axonal‐specific in both olfactory sensory neurons of the olfactory epithelium and mitral cells of the olfactory bulb. Our detailed expression analysis and the axonal localization we observed may provide important hints toward potential functions of PrP^C. J. Comp. Neurol. 508:487–499, 2008. © 2008 Wiley‐Liss, Inc.     

Diffuse deposition of immunohistochemically labeled prion protein in the granular layer of the cerebellum in a patient with Creutzfeldt-Jakob disease

Summary Amyloid plaques in Creutzfeldt-Jakob discase, kuru, and Gerstmann-Sträussler-Scheinker syndrome are known to contain an abnormal isoform of a cellular protein, the prion protein (PrP). The prion protein in its normal cellular isoform is a membranebound glycoprotein of unknown function. The mechanisms causing a modification of PrP and accumulation in amyloid plaques are unknown. Here we present a case of Creutzfeldt-Jakob disease with widespread deposition of immunohistochemically labeled PrP in the internal granular layer of the cerebellum. Immunohistochemically labeled PrP was deposited in delicate granules, which often were associated with cellular processes or the cytoplams of undefined cells, or diffusely deposited in the neuropil.Supported by research grants from the Wilhelm-Sander-Stiftung (89.036.1) and the Friedrich-Baur-Stiftung (H. Kretzschmar)     

Cellular prion protein modulates defensive attention and innate fear-induced behaviour evoked in transgenic mice submitted to an agonistic encounter with the tropical coral snake Oxyrhopus guibei

The cellular prion protein (PrP^C) is a neuronal anchored glycoprotein that has been associated with distinct functions in the CNS, such as cellular adhesion and differentiation, synaptic plasticity and cognition. Here we investigated the putative involvement of the PrP^C in the innate fear-induced behavioural reactions in wild-type (WT), PrP^C knockout (Prnp^0/0) and the PrP^C overexpressing Tg-20 mice evoked in a prey versus predator paradigm. The behavioural performance of these mouse strains in olfactory discrimination tasks was also investigated. When confronted with coral snakes, mice from both Prnp^0/0 and Tg-20 strains presented a significant decrease in frequency and duration of defensive attention and risk assessment, compared to WT mice. Tg-20 mice presented decreased frequency of escape responses, increased exploratory behaviour, and enhancement of interaction with the snake, suggesting a robust fearlessness caused by PrP^C overexpression. Interestingly, there was also a discrete decrease in the attentional defensive response (decreased frequency of defensive alertness) in Prnp^0/0 mice in the presence of coral snakes. Moreover, Tg-20 mice presented an increased exploration of novel environment and odors. The present findings indicate that the PrP^C overexpression causes hyperactivity, fearlessness, and increased preference for visual, tactile and olfactory stimuli-associated novelty, and that the PrP^c deficiency might lead to attention deficits. These results suggest that PrP^c exerts an important role in the modulation of innate fear and novelty-induced exploration.     

Size frequency distributions of the florid prion protein aggregates in variant Creutzfeldt-Jakob disease follow a power-law function

Abstract The objective was to test the hypothesis that the size frequency distributions of the prion protein (PrP) plaques in cases of variant Creutzfeldt-Jakob disease (vCJD) follow a power-law function. The design was a retrospective neuropathological study. The patients were 11 cases of clinically and neuropathologically verified vCJD. Size distributions of the diffuse and florid-type plaques were measured in several areas of the cerebral cortex and hippocampus from each case and a power-law function fitted to each distribution. The size distributions of the florid and diffuse plaques were fitted successfully by a powerlaw function in 100% and 42% of brain areas investigated respectively. Processes of aggregation/disaggregation may be more important than surface diffusion in the pathogenesis of the florid plaques. By contrast, surface diffusion may be a more significant factor in the development of the diffuse plaques.     

Deposition of disease-associated prion protein involves the peripheral nervous system in experimental scrapie

There is some evidence that the peripheral nervous system (PNS) is involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). The TSE-specific abnormal prion protein (PrP^sc) is considered as surrogate marker for infectivity. We traced the deposition of PrP^sc by immunocytochemistry in sheep and hamsters inoculated intraperitoneally with scrapie. The trigeminal, dorsal root, celiac, thoracic, and nodose ganglia contained ganglion cells and fewer satellite cells with prominent granular PrP^sc deposition. As a novel deposition pattern, punctate deposits in adaxonal location were seen along nerve fibers of peripheral nerve adjacent to ganglia. Such prominent involvement of the PNS in two different experimental scrapie models emphasizes the need to consider the PNS in natural scrapie and other TSEs including bovine spongiform encephalopathy as potential source of infectivity. Received: 5 May 1999 / Revised, accepted: 5 July 1999     

Divalent transition-metal ions (Cu and Zn) in the brains of epileptogenic and normal mice

The concentrations of Cu²⁺ and Zn²⁺ in 3 strains of mice were determined spectrophotometrically. The brain of the inborn audiogenic mouse (DBA/2J) contains higher levels of Zn²⁺ and Cu²⁺ than those found in the normal mouse (CBA/Ca or Parkes). Small differences in the metallic content in the whole brains of audiogenic and normal mice are accentuated in the hippocampus and the colliculus.     

Plaque-type deposition of prion protein in the damaged white matter of sporadic Creutzfeldt-Jakob disease MM1 patients

Plaque-type deposition of prion protein (PrP) in the brain has been extremely rare in sporadic Creutzfeldt-Jakob disease patients with methionine homozygosity at polymorphic codon 129 of the PrP gene and type 1 abnormal isoform of PrP (sCJD-MM1). Here we report three sCJD-MM1 patients who showed prominent PrP-positive amyloid plaques in the cerebral and cerebellar white matter. All three patients showed clinical courses of long duration (2 years ≤), particularly at the end-stage. The white matter of these patients was severely damaged because of the prolonged disease duration. Furthermore, Alzheimer’s amyloid precursor protein, which accumulates within the axonal swellings under pathological conditions, co-accumulated with the PrP-amyloid plaques. These findings suggest that the axonal damage reflecting the prolonged disease duration causes the deposition of PrP-amyloid plaques in the white matter. The present study shows that PrP-amyloid plaques can occur in the white matter of sCJD-MM1 cases.     

Codon 178 mutation of the human prion protein gene in a German family (Backer family): sequencing data from 72-year-old celloidin-embedded brain tissue

Familial Creutzfeldt-Jakob disease was first described in a family from northern Germany in the 1920s (Backer family). PCR amplification of DNA extracted from brain tissue embedded in celloidin 72 years ago shows a GAC to AAC substitution at codon 178 of the prion protein gene. This mutation is associated with fatal familial insomnia and familial Creutzfeldt-Jakob disease in a number of families of diverse ethnic background.