On the biology of prions

Summary Prions cause scrapie and Creutzfeldt-Jakob disease (CJD); these infectious pathogens are composed largely, if not entirely, of protein molecules. No prion-specific polynucleotide has been identified. Purified preparations of scrapie prions contain high titers (10^9.5 ID₅₀/ml), one protein (PrP 27-30) and amyloid rods (10–20 nm in diameter ×100–200 nm in length). Considerable evidence indicates that PrP 27-30 is required for and inseparable from scrapie infectivity. PrP 27-30 is encoded by a cellular gene and is derived from a larger protein, denoted PrP^Sc or PrP 33-35^Sc, by protease digestion. A cellular isoform, designated PrP^C or PrP 33-35^C, is encoded by the same gene as PrP^Sc and both proteins appear to be translated from the same 2.1 kb mRNA. Monoclonal antibodies to PrP 27-30, as well as antisera to PrP synthetic peptides, specifically react with both PrP^C and PrP^Sc, establishing their relatedness. PrP^C is digested by proteinase K, while PrP^Sc is converted to PrP 27-30 under the same conditions. Prion proteins are synthesized with signal peptides and are integrated into membranes. Detergent extraction of microsomal membranes isolated from scrapie-infected hamster brains solubilizes PrP^C but induces PrP^Sc to polymerize into amyloid rods. This procedure allows separation of the two prion protein isoforms and the demonstration that PrP^Sc accumulates during scrapie infection, while the level of PrP^C does not change. The prion amyloid rods generated by detergent extraction are identical morphologically, except for length, to extracellular collections of prion amyloid filaments which form plaques in scrapie- and CJD-infected brains. The prion amyloid plaques stain with antibodies to PrP 27-30 and PrP peptides. PrP 33-35^C does not accumulate in the extracellular space. Prion rods composed of PrP 27-30 can be dissociated into phospholipid vesicles with full retention of scrapie infectivity. The murine PrP gene (Prn-p) is linked to thePrn-i gene which controls the length of the scrapie incubation period. Prolonged incubation times are a cardinal feature of scrapie and CJD. While the central role of PrP^Sc in scrapie pathogenesis is well established, the chemical as well as conformational differences between PrP^C and PrP^Sc are unknown but probably arise from post-translational modifications.Supported by research grants from the National Institutes of Health (AG 02132 and NS 14069) and a Senator Jacob Javits Center of Excellence in Neuroscience (NS 22786) as well as by gifts from RJR-Nabisco, Inc. and Sherman Fairchild FoundationThis review is based upon a plenary lecture entitled Biology and Neuropathology of Prions presented at the Xth International Congress of Neuropathology, Stockholm, Sweden, September 11, 1986, and is dedicated to the memory of Peter Wilhelm Lampert (1929–1986)     

A neurotoxic prion protein fragment enhances proliferation of microglia but not astrocytes in culture

The scrapie isoform of the prion protein (PrP^Sc) induces pathological changes in the central nervous system including neurodegeneration and gliosis. A synthetic prion protein (PrP) peptide corresponding to amino acid residues 106–126 has been shown to be toxic to neurons that express PrP^C, the cellular isoform of PrP. Here we show that in mixed glial cultures PrP106–126 induces astroglial proliferation that is dependent on cellular PrP^c expression. In purified cultures of glial subtypes only microglia proliferated in response to PrP106–126. This effect was independent of PrP expression. Destruction of microglia in mixed glial cultures by L-leucine methyl ester (LLME) treatment abolished enhanced proliferation caused by PrP106–126. This proliferative effect can be restored by co-culturing LLME-treated astrocytes with microglia. Microglia therefore seem to mediate the proliferative effect exerted by PrP106–126 on astrocytes. © 1996 Wiley-Liss, Inc.     

Dual Modulation of ERK1/2 and p38 MAP Kinase Activities Induced by Minocycline Reverses the Neurotoxic Effects of the Prion Protein Fragment 90–231

Several in vitro and in vivo studies addressed the identification of molecular determinants of the neuronal death induced by PrP^Sc or related peptides. We developed an experimental model to assess PrP^Sc neurotoxicity using a recombinant polypeptide encompassing amino acids 90–231 of human PrP (hPrP90–231) that corresponds to the protease-resistant core of PrP^Sc identified in prion-infected brains. By means of mild thermal denaturation, we can convert hPrP90–231 from a PrP^C-like conformation into a PrP^Sc-like structure. In virtue of these structural changes, hPrP90–231 powerfully affected the survival of SH-SY5Y cells, inducing caspase 3 and p38-dependent apoptosis, while in the native α-helix-rich conformation, hPrP90–231 did not induce cell toxicity. The aim of this study was to identify drugs able to block hPrP90–231 neurotoxic effects, focusing on minocycline, a tetracycline with known neuroprotective activity. hPrP90–231 caused a caspase 3-dependent apoptosis via the blockade of ERK1/2 activation and the subsequent activation of p38 MAP kinase. We propose that hPrP90–231-induced apoptosis is dependent on the inhibition of ERK1/2 responsiveness to neurotrophic factors, removing a tonic inhibition of p38 activity and resulting in caspase 3 activation. Minocycline prevented hPrP90–231-induced toxicity interfering with this mechanism: the pretreatment with this tetracycline restored ERK1/2 activity and reverted p38 and caspase 3 activities. The effects of minocycline were not mediated by the prevention of hPrP90–231 structural changes or cell internalization (differently from Congo Red). In conclusion, minocycline elicits anti-apoptotic effects against the neurotoxic activity of hPrP90–231 and these effects are mediated by opposite modulation of ERK1/2 and p38 MAP kinase activities.     

Molecular biology and pathology of prion strains in sporadic human prion diseases

Prion diseases are believed to propagate by the mechanism involving self-perpetuating conformational conversion of the normal form of the prion protein, PrP^C, to the misfolded, pathogenic state, PrP^Sc. One of the most intriguing aspects of these disorders is the phenomenon of prion strains. It is believed that strain properties are fully encoded in distinct conformations of PrP^Sc. Strains are of practical relevance to human prion diseases as their diversity may explain the unusual heterogeneity of these disorders. The first insight into the molecular mechanisms underlying heterogeneity of human prion diseases was provided by the observation that two distinct disease phenotypes and their associated PrP^Sc conformers co-distribute with distinct PrP genotypes as determined by the methionine/valine polymorphism at codon 129 of the PrP gene. Subsequent studies identified six possible combinations of the three genotypes (determined by the polymorphic codon 129) and two common PrP^Sc conformers (named types 1 and 2) as the major determinants of the phenotype in sporadic human prion diseases. This scenario implies that each 129 genotype–PrP^Sc type combination would be associated with a distinct disease phenotype and prion strain. However, notable exceptions have been found. For example, two genotype–PrP^Sc type combinations are linked to the same phenotype, and conversely, the same combination was found to be associated with two distinct phenotypes. Furthermore, in some cases, PrP^Sc conformers naturally associated with distinct phenotypes appear, upon transmission, to lose their phenotype-determining strain characteristics. Currently it seems safe to assume that typical sporadic prion diseases are associated with at least six distinct prion strains. However, the intrinsic characteristics that distinguish at least four of these strains remain to be identified.     

An autopsied case of MM1 + MM2‐cortical with thalamic‐type sporadic Creutzfeldt‐Jakob disease presenting with hyperintensities on diffusion‐weighted MRI before clinical onset

A 78‐year‐old Japanese man presented with rapidly progressive dementia and gait disturbances. Eight months before the onset of clinical symptoms, diffusion‐weighted magnetic resonance imaging (DWI) demonstrated hyperintensities in the right temporal, right parietal and left medial occipital cortices. Two weeks after symptom onset, DWI showed extensive hyperintensity in the bilateral cerebral cortex, with regions of higher brightness that existed prior to symptom onset still present. Four weeks after clinical onset, periodic sharp wave complexes were identified on an electroencephalogram. Myoclonus was observed 8 weeks after clinical onset. The patient reached an akinetic mutism state and died 5 months after onset. Neuropathological examination showed widespread cerebral neocortical involvement of fine vacuole‐type spongiform changes with large confluent vacuole‐type spongiform changes. Spongiform degeneration with neuron loss and hypertrophic astrocytosis was also observed in the striatum and medial thalamus. The inferior olivary nucleus showed severe neuron loss with hypertrophic astrocytosis. Prion protein (PrP) immunostaining showed widespread synaptic‐type PrP deposition with perivacuolar‐type PrP deposition in the cerebral neocortex. Mild to moderate PrP deposition was also observed extensively in the basal ganglia, thalamus, cerebellum and brainstem, but it was not apparent in the inferior olivary nucleus. PrP gene analysis showed no mutations, and polymorphic codon 129 showed methionine homozygosity. Western blot analysis of protease‐resistant PrP showed both type 1 scrapie type PrP (PrP^Sc) and type 2 PrP^Sc. Based on the relationship between the neuroimaging and pathological findings, we speculated that cerebral cortical lesions with large confluent vacuoles and type 2 PrP^Sc would show higher brightness and continuous hyperintensity on DWI than those with fine vacuoles and type 1 PrP^Sc. We believe the present patient had a combined form of MM1 + MM2‐cortical with thalamic‐type sporadic Creutzfeldt‐Jakob disease (sCJD), which suggests a broader spectrum of sCJD clinicopathological findings.     

Novel Compounds Identified by Structure-Based Prion Disease Drug Discovery Using In Silico Screening Delay the Progression of an Illness in Prion-Infected Mice

The accumulation of abnormal prion protein (PrP^Sc) produced by the structure conversion of PrP (PrP^C) in the brain induces prion disease. Although the conversion process of the protein is still not fully elucidated, it has been known that the intramolecular chemical bridging in the most fragile pocket of PrP, known as the “hot spot,” stabilizes the structure of PrP^C and inhibits the conversion process. Using our original structure-based drug discovery algorithm, we identified the low molecular weight compounds that predicted binding to the hot spot. NPR-130 and NPR-162 strongly bound to recombinant PrP in vitro, and fragment molecular orbital (FMO) analysis indicated that the high affinity of those candidates to the PrP is largely dependent on nonpolar interactions, such as van der Waals interactions. Those NPRs showed not only significant reduction of the PrP^Sc levels but also remarkable decrease of the number of aggresomes in persistently prion-infected cells. Intriguingly, treatment with those candidate compounds significantly prolonged the survival period of prion-infected mice and suppressed prion disease-specific pathological damage, such as vacuole degeneration, PrP^Sc accumulation, microgliosis, and astrogliosis in the brain, suggesting their possible clinical use. Our results indicate that in silico drug discovery using NUDE/DEGIMA may be widely useful to identify candidate compounds that effectively stabilize the protein.Electronic supplementary materialThe online version of this article (10.1007/s13311-020-00903-9) contains supplementary material, which is available to authorized users.     

Accumulation of prion protein in the vagus nerve in creutzfeldt–jakob disease

Disease‐associated proteins are thought to propagate along neuronal processes in neurodegenerative diseases. To detect disease‐associated prion protein (PrP^Sc) in the vagus nerve in different forms and molecular subtypes of Creutzfeldt–Jakob disease (CJD), we applied 3 different anti‐PrP antibodies. We screened the vagus nerve in 162 sporadic and 30 genetic CJD cases. Four of 31 VV‐2 type sporadic CJD and 7 of 30 genetic CJD cases showed vagal PrP^Sc immunodeposits with distinct morphology. Thus, PrP^Sc in CJD affects the vagus nerve analogously to α‐synuclein in Parkinson disease. The morphologically diverse deposition of PrP^Sc in genetic and sporadic CJD argues against uniform mechanisms of propagation of PrP^Sc. Ann Neurol 2019;85:782–787     

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.     

Intracellular mechanisms mediating the neuronal death and astrogliosis induced by the prion protein fragment 106–126

Prion encephalopathies include fatal diseases of the central nervous system of men and animals characterized by nerve cell loss, glial proliferation and deposition of amyloid fibrils into the brain. During these diseases a cellular glycoprotein (the prion protein, PrP^C) is converted, through a not yet completely clear mechanism, in an altered isoform (the prion scrapie, PrP^Sc) that accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism. PrP^Sc is believed to be responsible for the neuronal loss that is observed in the prion disease. The PrP 106–126, a synthetic peptide that has been obtained from the amyloidogenic portion of the prion protein, represents a suitable model for studying the pathogenic role of the PrP^Sc, retaining, in vitro, some characteristics of the entire protein, such as the capability to aggregate in fibrils, and the neurotoxicity. In this work we present the results we have recently obtained regarding the action of the PrP 106–126 in different cellular models. We report that the PrP 106–126 induces proliferation of cortical astrocytes, as well as degeneration of primary cultures of cortical neurons or of neuroectodermal stable cell lines (GH₃ cells). In particular, these two opposite effects are mediated by the same attitude of the peptide to interact with the L-type calcium channels: in the astrocytes, the activity of these channels seems to be activated by PrP 106–126, while, in the cortical neurons and in the GH₃ cells, the same treatment causes a blockade of these channels causing a toxic effect.     

Polyunsaturated Fatty Acids Protect Against Prion-Mediated Synapse Damage In Vitro

A loss of synapses is characteristic of the early stages of the prion diseases. Here we modelled the synapse damage that occurs in prion diseases by measuring the amount of synaptophysin, a pre-synaptic membrane protein essential for neurotransmission, in cortical or hippocampal neurones incubated with the disease associated isoform of the prion protein (PrP^Sc), or with the prion-derived peptide PrP82-146. The addition of PrP^Sc or PrP82-146 caused a dose-dependent reduction in the synaptophysin content of PrP wildtype neurones indicative of synapse damage. They did not affect the synaptophysin content of PrP null neurones. The loss of synaptophysin in PrP wildtype neurones was preceded by the accumulation of PrP82-146 within synapses. Since supplements containing polyunsaturated fatty acids (PUFA) are frequently taken for their perceived health benefits including reported amelioration of neurodegenerative conditions, the effects of some common PUFA on prion-mediated synapse damage were examined. Pre-treatment of cortical or hippocampal neurones with docosahexaenoic (DHA) or eicosapentaenoic acids (EPA) protected neurones against the loss of synaptophysin induced by PrP82-146 or PrP^Sc. This effect of DHA and EPA was selective as they did not alter the loss of synaptophysin induced by a snakevenom neurotoxin. The effects of DHA and EPA were associated with a significant reduction in the amount of FITC-PrP82-146 that accumulated within synapses. Such observations raise the possibility that supplements containing PUFA may protect against the synapse damage and cognitive loss seen during the early stages of prion diseases.     

Virus-induced alterations of membrane lipids affect the incorporation of PrP Sc into cells

Prion diseases are fatal neurodegenerative disorders characterized by long incubation periods. To investigate whether concurrent diseases can modify the clinical outcome of prion‐affected subjects, we tested the effect of viral infection on the binding and internalization of PrP^Sc, essential steps of prion propagation. To this effect, we added scrapie brain homogenate or purified PrP^Sc to fibroblasts previously infected with minute virus of mice (MVM), a mouse parvovirus. We show here that the rate of incorporation of PrP^Sc into MVM‐infected cells was significantly higher than that observed for naïve cells. Immunostaining of cells and immunoblotting of subcellular fractions using antibodies recognizing PrP and LysoTracker, a lysosomal marker, revealed that in both control and MVM‐infected cells the incorporated PrP^Sc was associated mostly with lysosomes. Interestingly, floatation gradient analysis revealed that the majority of the PrP^Sc internalized into MVM‐infected cells shifted toward raft‐containing low‐density fractions. Concomitantly, the MVM‐infected cells demonstrated increased levels of the glycosphingolipid GM1 (an essential raft lipid component) throughout the gradient and a shift in caveolin 1 (a raft protein marker) toward lighter membrane fractions compared with noninfected cells. Our results suggest that the effect of viral infection on membrane lipid composition may promote the incorporation of exogenous PrP^Sc into rafts. Importantly, membrane rafts are believed to be the conversion site of PrP^C to PrP^Sc; therefore, the association of exogenous PrP^Sc with such membrane microdomains may facilitate prion infection. © 2008 Wiley‐Liss, Inc.     

Astrocytosis and amyloid deposition in scrapie-infected hamsters

In scrapie infection, prion protein (PrP^Sc) is localized in areas where there is neurodegeneration and astrocytosis. It is thought that PrP^Sc is toxic to neurons and trophic for astrocytes. In our study, paraffin sections from scrapie infected (263K and 139H) and control hamsters were examined with histological and immunocytochemical staining. We found that PrP^Sc was present in the ependymal cells of both 263K- and 139H-infected hamsters. In 139H-infected hamsters, PrP^Sc was found in the cytoplasm of neurons in cerebral cortex and in hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. In contrast, neuronal cytoplasm and nuclei, were positive for PrP^Sc in most areas such as cortex, hippocampus, and thalamus in 263K-infected hamsters. Many aggregations of PrP^Sc could be seen in the cortex, hippocampus, substantia nigra and around the Pia mater, corpus callosum, fimbria, ventricles, and blood vessels in sections from 139H- and/or 263K-positive animals. Furthermore, PrP^Sc was also co-localized with glial fibrillary acidic protein (GFAP) in many reactive astrocytes (approximately 90%) in certain areas such as the hippocampus in 263K-infected hamsters, but not 139H-infected hamsters. The patterns of astrocytosis and PrP^Sc formation were different between 139H- and 263K-infected hamsters, which may be used for a diagnosis purpose. Our results suggest a hypothesis that multiple cell-types are capable of PrP^Sc production. Our results also confirm that reactive astrocytes can produce and/or accumulate PrP^Sc during some scrapie strain infections. The findings suggest a `snowball effect', that is: astrocytosis might play an important role in amyloidosis, while amyloidosis may induce further astrocytosis at least in 263K-infected hamsters.     

Mutant prion protein D202N associated with familial prion disease is retained in the endoplasmic reticulum and forms ‘curly’ intracellular aggregates

Transmissible Spongiform Encephalopathies are fatal neurodegenerative disorders of humans and animals that are familial, sporadic, and infectious in nature. Familial disorders of humans include Gerstmann–Straussler–Scheinker disease (GSS), familial Creutzfeldt–Jakob disease (CJD), and fatal familial insomnia, and result from point mutations in the prion protein gene. Although neurotoxicity in familial cases is believed to result from a spontaneous change in conformation of mutant prion protein (PrP) to the pathogenic PrP-scrapie (PrP^Sc) form, emerging evidence indicates otherwise. We have investigated the processing and metabolism of mutant PrP D202N (PrP^202N) in cell models to elucidate possible mechanisms of cytotoxicity. In this report, we demonstrate that PrP^202N expressed in human neuroblastoma cells fails to achieve a mature conformation following synthesis and accumulates in the endoplasmic reticulum as ‘curly’ aggregates. In addition, PrP^202N cells show increased sensitivity to free radicals, indicating that neuronal susceptibility to oxidative damage may account for the neurotoxicity observed in cases of GSS resulting from PrP D202N mutation. Yaping Gu and Susamma Verghese contributed equally.     

Prion protein fragment 106–126 induces apoptotic cell death and impairment of L-type voltage-sensitive calcium channel activity in the GH3 cell line

The prion diseases are transmissible neurodegenerative pathologies characterized by the accumulation of altered forms of the prion protein (PrP), termed PrP^Sc, in the brain. Previous studies have shown that a synthetic peptide homologous to residues 106–126 of PrP (PrP 106–126) maintains many characteristics of PrP^Sc, i.e., the ability to form amyloid fibrils and to induce apoptosis in neurons. We have investigated the intracellular mechanisms involved in the cellular degeneration induced by PrP 106–126, using the GH3 cells as a model of excitable cells. When assayed in serum-deprived conditions (48 hr), PrP 106–126 (50 μM) induced cell death time-dependently, and this process showed the characteristics of the apoptosis. This effect was specific because a peptide with a scrambled sequence of PrP 106–126 was not effective. Then we performed microfluorimetric analysis of single cells to monitor intracellular calcium concentrations and showed that PrP 106–126 caused a complete blockade of the increase in the cytosolic calcium levels induced by K⁺ (40 mM) depolarization. Conversely, the scrambled peptide was ineffective. The L-type voltage-sensitive calcium channel blocker nicardipine (1 μM) also induced apoptosis in GH3 cells, suggesting that the blockade of Ca²⁺ entry through this class of calcium channels may cause GH3 apoptotic cell death. We thus analyzed, by means of electrophysiological studies, whether Prp 106–126 modulate L-type calcium channels activity and demonstrated that the apoptotic effect of PrP 106–126 was due to a dose-dependent inactivation of the L-type calcium channels. These data demonstrate that the prion protein fragment 106–126 induces a GH3 apoptotic cell death inducing a selective inhibition of the activity of the L-type voltage-sensitive calcium channels. J. Neurosci. Res. 54:341–352, 1998. © 1998 Wiley-Liss, Inc.     

Excitotoxicity Through NMDA Receptors Mediates Cerebellar Granule Neuron Apoptosis Induced by Prion Protein 90-231 Fragment

Prion diseases recognize, as a unique molecular trait, the misfolding of CNS-enriched prion protein (PrP^C) into an aberrant isoform (PrP^Sc). In this work, we characterize the in vitro toxicity of amino-terminally truncated recombinant PrP fragment (amino acids 90-231, PrP90-231), on rat cerebellar granule neurons (CGN), focusing on glutamatergic receptor activation and Ca²⁺ homeostasis impairment. This recombinant fragment assumes a toxic conformation (PrP90-231^TOX) after controlled thermal denaturation (1 h at 53 °C) acquiring structural characteristics identified in PrP^Sc (enrichment in β-structures, increased hydrophobicity, partial resistance to proteinase K, and aggregation in amyloid fibrils). By annexin-V binding assay, and evaluation of the percentage of fragmented and condensed nuclei, we show that treatment with PrP90-231^TOX, used in pre-fibrillar aggregation state, induces CGN apoptosis. This effect was associated with a delayed, but sustained elevation of [Ca²⁺]_i. Both CGN apoptosis and [Ca²⁺]_i increase were not observed using PrP90-231 in PrP^C-like conformation. PrP90-231^TOX effects were significantly reduced in the presence of ionotropic glutamate receptor antagonists. In particular, CGN apoptosis and [Ca²⁺]_i increase were largely reduced, although not fully abolished, by pre-treatment with the NMDA antagonists APV and memantine, while the AMPA antagonist CNQX produced a lower, although still significant, effect. In conclusion, we report that CGN apoptosis induced by PrP90-231^TOX correlates with a sustained elevation of [Ca²⁺]_i mediated by the activation of NMDA and AMPA receptors.     

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.     

A Prion Protein Fragment Primes Type 1 Astrocytes to Proliferation Signals from Microglia

Gliosis is a hallmark of prion disease. A neurotoxic prion peptide (PrP106-126) induces astrocyte proliferation in the presence of microglia. This peptide also directly enhances microglial proliferation in culture. We have investigated this further to understand the method by which factors released by microglia and PrP106-126 work together to enhance astrocyte proliferation. PrP106-126 in the presence of microglia specifically enhanced type 1 astrocyte proliferation but not Type 2. Astrocytes that do not express the prion protein were more sensitive to oxidative stress and the toxicity of cytosine arabinoside. In the presence of cytosine arabinoside, PrP106-126 was toxic to pure astrocyte cultures. Using conditioned medium from microglia we have shown that PrP^c-expressing astrocytes proliferate in response to factors released by microglia stimulated by granulocyte/macrophage colony-stimulating factor. This response is enhanced in the presence of PrP106-126. PrP^c-deficient astrocytes do not show this response. These results suggest that astrocytes are primed by PrP106-126 to respond more to factors released by proliferating microglia. Astrocytes may proliferate in this system to escape entering the cell suicide pathway.