Activation of Fas and caspase 3 precedes PrP accumulation in 87V scrapie.

The sequence of events involved in the neurodegeneration caused by transmissible spongiform encephalopathies is not yet known. Using a murine scrapie model in which neurodegeneration in the hippocampus is restricted to the CA2, we show an up-regulation of the proapoptotic markers Fas and caspase 3 early in the incubation period prior to disease-specific prion protein (PrP) deposition and clinical signs. These results suggest that activation of Fas and caspase 3 are involved in the early pathological sequence of events during murine scrapie, and that these proapoptotic markers may be a specific method for early detection of neurodegeneration.     

PrP Deposition, Microglial Activation, and Neuronal Apoptosis in Murine Scrapie

The present study investigated the relationship among PrP deposition, microglial activation, vacuolation, and neuronal death in the hippocampus of the 301V/VM murine scrapie model (mean incubation period 117 ± 1 days). PrP deposition was first detected after 30 days and microglial activation after 60 days. Vacuolation in the CA1 and CA2 pyramidal layer was present from 90 days onward. Only occasionalin situend labeling (ISEL)-positive neurons were present in the hippocampus of scrapie-infected mice from 75 days postinoculation (d.p.i.), except at 105 d.p.i. when relatively large numbers of apoptotic, ISEL-positive neurons in the CA1 hippocampal region were observed. Terminally ill animals showed almost complete loss of CA1 pyramidal neurons. Electron microscopy of the CA1 region at 105 days confirmed that these neurons were dying by apoptosis. These data suggest that microglial activation in scrapie is a response to abnormal PrP deposition rather than a response to neuronal cell loss.     

The truncated 23-230 form of the prion protein localizes to the nuclei of inducible cell lines independently of its nuclear localization signals and is not cytotoxic

The mechanisms of prion-induced neurological dysfunction observed in prion diseases are poorly understood. Transgenic mice expressing a truncated form of the prion protein (23-230 PrP) acquire cerebellar degeneration (Ma and Lindquist, Science, 2002). To decipher the mechanisms of neurodegeneration induced by 23-230 PrP, we established inducible cell lines expressing this truncated form of PrP. We found that 23-230 PrP, expected to be cytosolic, accumulated mostly in the nucleus of the cells and was not cytotoxic. Nuclear localization of this mutant form of PrP is independent of its predicted nuclear localization signals. In contrast to what we previously described for PrPSc, nuclear accumulation of 23-230 PrP does not require a functional microtubule network. We observed that 23-230 PrP interacts with chromatin in vivo, as already described for recombinant PrP and for PrPSc. Our data demonstrate that the 23-230 PrP model does not reflect the situation of a cytosolic PrP but could represent a very useful tool to understand the consequences of the accumulation of the prion protein in the nucleus.     

Using Protein Misfolding Cyclic Amplification Generates a Highly Neurotoxic PrP Dimer Causing Neurodegeneration

Under the “protein-only” hypothesis, prion-based diseases are proposed to result from an infectious agent that is an abnormal isoform of the prion protein in the scrapie form, PrP^Sc. However, since PrP^Sc is highly insoluble and easily aggregates in vivo, this view appears to be overly simplistic, implying that the presence of PrP^Sc may indirectly cause neurodegeneration through its intermediate soluble form. We generated a neurotoxic PrP dimer with partial pathogenic characteristics of PrP^Sc by protein misfolding cyclic amplification in the presence of 1-palmitoyl-2-oleoylphosphatidylglycerol consisting of recombinant hamster PrP (23–231). After intracerebral injection of the PrP dimer, wild-type hamsters developed signs of neurodegeneration. Clinical symptoms, necropsy findings, and histopathological changes were very similar to those of transmissible spongiform encephalopathies. Additional investigation showed that the toxicity is primarily related to cellular apoptosis. All results suggested that we generated a new neurotoxic form of PrP, PrP dimer, which can cause neurodegeneration. Thus, our study introduces a useful model for investigating PrP-linked neurodegenerative mechanisms.     

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.     

The role of platelet activating factor in prion and amyloid-beta neurotoxicity

In the prion diseases, neurodegeneration is preceded by the accumulation of the disease-associated isoform of the prion protein (PrP). In the present study, neurones treated with three different phospholipase A2 inhibitors were resistant to the toxic effects of PrP peptides or a synthetic miniprion (sPrP106). Phospholipase A2 inhibitors also protected neurones against a toxic peptide found in Alzheimer's disease (amyloid-beta1-42). Further studies showed that neurones pre-treated with platelet activating factor (PAF) antagonists were equally resistant to PrP peptides or amyloid-beta1-42. Moreover, both phospholipase A2 inhibitors and PAF antagonists reduced the activation of caspase-3, a marker of apoptosis, and the production of prostaglandin E2 that is closely associated with neuronal death in prion or Alzheimer's diseases.     

Elk with a long incubation prion disease phenotype have a unique PrPd profile

The transmissible spongiform encephalopathies (TSEs) invariably result in fatal neurodegeneration and accumulation of PrP, an abnormal form of the host prion protein PrP, encoded by the PRNP gene. A naturally occurring polymorphism (methionine/valine) at PRNP codon 129 is associated with variation in relative disease susceptibility, incubation time, clinical presentation, neuropathology, and/or PrP biochemical characteristics in a range of human TSEs. A methionine/leucine polymorphism at the corresponding site in the Rocky Mountain elk PRNP gene is associated with variation in relative susceptibility and incubation time in the cervid TSE chronic wasting disease. We now report that elk lacking the predisposing 132-methionine allele develop chronic wasting disease after a long incubation period and display a novel PrP folding pattern.     

The metabolism of glycosaminoglycans is impaired in prion diseases

It is well established that the conversion of PrP(C) to PrP(Sc) is the key event in prion disease biology. In addition, several lines of evidence suggest that glycosaminoglycans (GAGs) and in particular heparan sulfate (HS) may play a role in the PrP(C) to PrP(Sc) conversion process. It has been proposed that PrP(Sc) accumulation in prion diseases may induce aberrant activation of lysosomal activity, which has been shown to result in neurodegeneration in a number of diseases, especially lysosomal storage disorders. Among such diseases, only the ones resulting from defects in GAGs degradation are accompanied by secretion of large amounts of GAG metabolites in urine. In this work, we show that GAGs are secreted in the urine of prion-infected animals and humans, and surprisingly, also in the urine of mice ablated for the PrP gene. We hypothesize that both the presence of PrP(Sc) or the absence of PrP(C) may alter the metabolism of GAGs.     

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.     

p38 MAP kinase mediates the cell death induced by PrP106-126 in the SH-SY5Y neuroblastoma cells

Prion diseases are neurodegenerative pathologies characterized by the accumulation in the brain of a protease-resistant form of the prion protein (PrP(c)), named PrP(Sc). A synthetic peptide homologous to residues 106-126 of PrP (PrP106-126) maintains many PrP(Sc) characteristics. We investigated the intracellular signaling responsible for the PrP106-126-dependent cell death of SH-SY5Y, a cell line derived from a human neuroblastoma. In this cell line, PrP106-126 induced apoptotic cell death and caused activation of caspase-3, although the blockade of this enzyme did not inhibit cell death. The p38 MAP kinase blockers, SB203580 and PD169316, prevented the apoptotic cell death evoked by PrP106-126 and Western blot analysis revealed that the exposure of the cells to the peptide induced p38 phosphorylation. Taken together, our data suggest that the p38 MAP kinase pathway can mediate the SH-SY5Y cell death induced by PrP106-126.     

Apoptotic cell death and impairment of L-type voltage-sensitive calcium channel activity in rat cerebellar granule cells treated with the prion protein fragment 106-126

Prion diseases are neurodegenerative pathologies characterized by the accumulation, in the brain, of altered forms of the prion protein (PrP), named PrP(Sc). A synthetic peptide homologous to residues 106-126 of PrP (PrP106-126) was reported to maintain the neurodegenerative characteristics of PrP(Sc). We investigated the intracellular mechanisms involved in PrP106-126-dependent degeneration of primary cultures of cerebellar granule neurons. Prolonged exposure of such neurons to PrP106-126 induced apoptotic cell death. The L-type voltage-sensitive calcium channel blocker nicardipine reproduced this effect, suggesting that blockade of Ca(2+) entry through this class of calcium channels may be responsible for the granule cell degeneration. Microfluorometric analysis showed that PrP106-126 caused a reduction in cytosolic calcium levels, elicited by depolarizing K(+) concentrations in these neurons. Electrophysiological studies demonstrated that PrP106-126 and nicardipine selectively reduce the L-type calcium channel current. These data demonstrate that PrP106-126 alters the activity of L-type voltage-sensitive calcium channels in rat cerebellar granule cells and suggest that this phenomenon is related to the cell death induced by the peptide.     

Adult human microglia secrete cytokines when exposed to neurotoxic prion protein peptide: no intermediary role for prostaglandin E2

Prion diseases are characterized by accumulation of protease resistant isoforms of prion protein (termed PrP(SC)), glial activation and neurodegeneration. The time course of PrP deposition, appearance of activated microglia, and of neuronal apoptosis in experimentally-induced prion disease suggests that microglial activation precedes the process of neuronal loss. Activated microglia and inflammatory mediators, including cytokines and prostaglandin E2 (PGE2) co-localize with PrP deposits. In vitro, mouse microglia secrete neurotoxic agents and interleukins (IL)-1 and IL-6, when exposed to synthetic peptides representing the neurotoxic fragment of PrP. In this study, adult human microglia were found to secrete IL-6 and TNF-alpha upon exposure to synthetic fibrillar PrP105-132, the putative transmembrane domain of PrP. Little cytokine release occurred following exposure of microglia to C-terminally amidated, nonfibrillar PrP105-132, suggesting that the degree of fibrillarity of PrP peptides affects their biological properties. Non-steroidal anti-inflammatory drugs (NSAIDs) are thought to exert beneficial effects in neurodegenerative disorders through suppressive effects on microglial activation and on cyclooxygenase (COX) activity. Since microglial COX-2 expression and PGE(2) synthesis are increased in human and experimental prion diseases, we investigated the effects of the NSAIDs indomethacin and BF389, an experimental COX-2 selective inhibitor, on the PrP105-132-induced microglial IL-6 and TNF-alpha synthesis in vitro. No inhibitory effects of the NSAIDs were observed. Furthermore, PrP105-132 did not stimulate microglial PGE(2) synthesis. We conclude that, unlike IL-1beta-induced IL-6 synthesis in astrocytes, the PrP-induced IL-6 synthesis in human adult microglia is not PGE2 mediated.     

Prion protein heterogeneity in sporadic but not variant Creutzfeldt-Jakob disease: UK cases 1991-2002

Human prion diseases can occur as an idiopathic disorder (sporadic Creutzfeldt-Jakob disease) or can be acquired, as is the case for variant Creutzfeldt-Jakob disease. These disorders are characterized by the accumulation of a protease-resistant form of the host-encoded prion protein termed PrP(Sc) in the brains of affected individuals. PrP(Sc) has been proposed to be the principal, if not sole, component of the infectious agent, with its accumulation in the central nervous system the primary event leading to neurodegeneration. A major question remains as to whether self-propagating structural differences in PrP(Sc) might account for the clinicopathological diversity evident in Creutzfeldt-Jakob disease and whether different prion protein types underlie the existence of different strains of causative agent. Here, we describe the results of a large-scale biochemical study of PrP(Sc) from autopsy-proved cases of variant Creutzfeldt-Jakob disease (n = 59) and compare these with cases of sporadic Creutzfeldt-Jakob disease (n = 170) in the United Kingdom over the period 1991 to 2002. The results show PrP(Sc) in variant Creutzfeldt-Jakob disease to be remarkably stereotyped. In contrast, considerable heterogeneity in PrP(Sc) exists both between and within cases of sporadic Creutzfeldt-Jakob disease.     

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).     

Neonatal Iron Treatment Increases Apoptotic Markers in Hippocampal and Cortical Areas of Adult Rats

Oxidative stress, cellular damage, and neuronal apoptosis are believed to underlie the progressive cognitive decline that accompanies natural aging and to be exacerbated in neurodegenerative diseases. Over the years, we have consistently demonstrated that iron neonatal treatment induces oxidative stress and memory deficits in adult rats, but the mechanisms underlying these effects remained undefined. The purpose of this study was to examine whether neonatal iron overload was associated with apoptotic cell death in adult and old rats. We analyzed Par-4 and caspase-3 immunoreactivity in specific brain areas including the hippocampus CA1, CA3 and dentate gyrus (DG), the adjacent cortex and the striatum in adult (3 months-old) and aged (24 months-old) rats from control (vehicle-treated) and neonatally iron-treated groups. Neonatal iron treatment consisted of a daily oral administration of 10 mg/kg of Fe⁺², for three consecutive days, from post-natal 12–14. Control aged animals showed increased levels of both markers when compared to untreated adult animals. When adults were compared, iron-treated animals presented significantly higher Par-4 and caspase-3 immunoreactivities in CA1, CA3 and cortex. In the DG, this effect was statistically significant only for Par-4. Interestingly, when control and iron-treated aged animals were compared, a significant decrease in both apoptotic markers was observed in the later groups in the same areas. These results may be interpreted as an acceleration of aging progressive damages caused by iron overload and may contribute to a better understanding of the damaging potential of iron accumulation to brain function and the resulting increased susceptibility to neurodegeneration.     

The release of calcium from the endoplasmic reticulum induced by amyloid-beta and prion peptides activates the mitochondrial apoptotic pathway

In this study, we analyzed whether ER Ca²⁺ release, induced by amyloid-β (Aβ) and prion (PrP) peptides activates the mitochondrial-mediated apoptotic pathway. In cortical neurons, addition of the synthetic Aβ1–40 or PrP106–126 peptides depletes ER Ca²⁺ content, leading to cytosolic Ca²⁺ overload. The Ca²⁺ released through ryanodine (RyR) and inositol 1,4,5-trisphosphate (IP₃R) receptors was shown to be involved in the loss of mitochondrial membrane potential, Bax translocation to mitochondria and apoptotic death. Our data further demonstrate that Ca²⁺ released from the ER leads to the depletion of endogenous GSH levels and accumulation of reactive oxygen species, which were also involved in the depolarization of the mitochondrial membrane. These results illustrate that the early Aβ- and PrP -induced perturbation of ER Ca²⁺ homeostasis affects mitochondrial function, activating the mitochondrial-mediated apoptotic pathway and help to clarify the mechanism implicated in neuronal death that occurs in AD and PrD.     

The cellular prion protein (PrPC) prevents apoptotic neuronal cell death and mitochondrial dysfunction induced by serum deprivation

Prion diseases are transmissible neurodegenerative disorders that are invariably fatal in humans and animals. Although the nature of the infectious agent and pathogenic mechanisms of prion diseases are not clear, it has been reported that prion diseases may be associated with aberrant metabolism of cellular prion protein (PrP(C)). In various reports, it has been postulated that PrP(C) may be involved in one or more of the following: neurotransmitter metabolism, cell adhesion, signal transduction, copper metabolism, antioxidant activity or programmed cell death. Despite suggestive results supporting each of these mechanisms, the physiological function(s) of PrP(C) is not known. To investigate whether PrP(C) can prevent apoptotic cell death in prion diseases, we established the cell lines stably expressing PrP(C) from PrP knockout (PrP(-/-)) neuronal cells and examined the role of PrP(C) under apoptosis and/or serum-deprived condition. We found that PrP(-/-) cells were vulnerable to apoptotic cell death and that this vulnerability was rescued by the expression of PrP(C). The expression levels of apoptosis-related proteins including p53, Bax, caspase-3, poly(ADP-ribose) polymerase (PARP) and cytochrome c were significantly increased in PrP(-/-) cells. In addition, Ca(2+) levels of mitochondria were increased, whereas mitochondrial membrane potentials were decreased in PrP(-/-) cells. These results strongly suggest that PrP(C) may play a central role as an effective anti-apoptotic protein through caspase-dependent apoptotic pathways in mitochondria, supporting the concept that disruption of PrP(C) and consequent reduction of anti-apoptotic capacity of PrP(C) may be one of the pathogenic mechanisms of prion diseases.     

Neurotoxicity of reactive aldehydes: the concept of "aldehyde load" as demonstrated by neuroprotection with hydroxylamines

The concept of "oxidative stress" has become a mainstay in the field of neurodegeneration but has failed to differentiate critical events from epiphenomena and sequalae. Furthermore, the translation of current concepts of neurodegenerative mechanisms into effective therapeutics for neurodegenerative diseases has been meager and disappointing. A corollary of current concepts of "oxidative stress" is that of "aldehyde load". This relates to the production of reactive aldehydes that covalently modify proteins, nucleic acids, lipids and carbohydrates and activate apoptotic pathways. However, reactive aldehydes can also be generated by mechanisms other than "oxidative stress". We therefore hypothesized that agents that can chemically neutralize reactive aldehydes should demonstrate superior neuroprotective actions to those of free radical scavengers. To this end, we evaluated hydroxylamines as aldehyde-trapping agents in an in vitro model of neurodegeneration induced by the reactive aldehyde, 3-aminopropanal (3-AP), a product of polyamine oxidase metabolism of spermine and spermidine. In this model, the hydroxylamines N-benzylhydroxylamine, cyclohexylhydroxylamine and t-butylhydroxylamine were shown to protect, in a concentration-dependent manner, against 3-AP neurotoxicity. Additionally, a therapeutic window of 3 h was demonstrated for delayed administration of the hydroxylamines. In contrast, the free radical scavengers TEMPO and TEMPONE and the anti-oxidant ascorbic acid were ineffective in this model. Extending these tissue culture findings in vivo, we examined the actions of N-benzylhydroxylamine in the trimethyltin (TMT) rat model of hippocampal CA3 neurodegeneration. This model involves augmented polyamine metabolism resulting in the generation of reactive aldehydes that compromise mitochondrial integrity. In the rat TMT model, NBHA (50 mg/kg, sc, daily) provided 100% protection against neurodegeneration, as reflected by measurements of KCl-evoked glutamate release from hippocampal brain slices and septal high affinity glutamate uptake. In contrast, ascorbic acid (100 mg/kg, sc, daily) failed to protect CA3 neurons from TMT toxicity. In summary, our data support further evaluation of the concept of "aldehyde load" in neurodegeneration and the potential clinical investigation of agents that are effective traps for reactive aldehydes.     

Neuroprotective functions of prion protein

The normal function of prion protein (PrP) is usually disregarded at the expense of the more fascinating role of PrP in transmissible prion diseases. However, the normal PrP may play an important role in cellular function in the central nervous system, since PrP is highly expressed in neurons and motifs in the sequence of PrP are conserved in evolution. The finding that prion null mice do not have a significant overt phenotype suggests that the normal function of PrP is of minor importance. However, the absence of PrP in cells or in vivo contributes to an increased susceptibility to oxidative stress or apoptosis-inducing insults. An alternative explanation is that the PrP normal function is so important that it is redundant. Probing into the characteristics of PrP has revealed a number of features that could mediate important cellular functions. The neuroprotective actions so far identified with PrP are initiated through cell surface signaling, antioxidant activity, or anti-Bax function. Here, we review the characteristics of the PrP and the evidence that PrP protects against neurodegeneration and neuronal cell death.