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.     

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 (GH(3) 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 GH(3) cells, the same treatment causes a blockade of these channels causing a toxic effect.     

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.     

Involvement of the 5-lipoxygenase pathway in the neurotoxicity of the prion peptide PrP106-126

Transmissible spongiform encephalopathies are characterised by the transformation of the normal cellular prion protein (PrP(C)) into an abnormal isoform (PrP(TSE)). Previous studies have shown that N-methyl-D-aspartate (NMDA) receptor antagonists can inhibit glutathione depletion and neurotoxicity induced by PrP(TSE) and a toxic prion protein peptide, PrP106-126, in vitro. NMDA receptor activation is known to increase intracellular accumulation of Ca(2+), resulting in up-regulation of arachidonic acid (AA) metabolism. This can stimulate the lipoxygenase pathways that may generate a number of potentially neurotoxic metabolites. Because of the putative relationship between AA breakdown and PrP106-126 neurotoxicity, we investigated AA metabolism in primary cerebellar granule neuron cultures treated with PrP106-126. Our studies revealed that PrP106-126 exposure for 30 min significantly up-regulated AA release from cerebellar granule neurons. PrP106-126 neurotoxicity was mediated through the 5-lipoxygenase (5-LOX) pathway, as shown by abrogation of neuronal death with the 5-LOX inhibitors quinacrine, nordihydroguaiaretic acid, and caffeic acid. These inhibitors also prevented PrP106-126-induced caspase 3 activation and annexin V binding, indicating a central role for the 5-LOX pathway in PrP106-126-mediated proapoptosis. Interestingly, inhibitors of the 12-lipoxygenase pathway had no effect on PrP106-126 neurotoxicity or proapoptosis. These studies clearly demonstrate that AA metabolism through the 5-LOX pathway is an important early event in PrP106-126 neurotoxicity and consequently may have a critical role in PrP(TSE)-mediated cell loss in vivo. If this is so, therapeutic intervention with 5-LOX inhibitors may prove beneficial in the treatment of prion disorders.     

Humanin rescues cortical neurons from prion-peptide-induced apoptosis

We recently demonstrated that a soluble oligomeric prion peptide, the putative 118-135 transmembrane domain of prion protein (PrP), exhibited membrane fusogenic properties and induced apoptotic cell death both in vitro and in vivo. A recently discovered rescue factor humanin (HN) was shown to protect neuronal cells from various insults involved in human neurodegenerative diseases. We thus addressed the question of whether HN might modulate the apoptosis induced by the soluble PrP(118-135) fragment. We found that the incubation of rat cortical neurons with 10 microM HN prevented soluble PrP(118-135) fragment-induced cell death concomitantly with inhibition of apoptotic events. An HN variant, termed HNG, exhibited a 500-fold increase in the protective activity in cortical neurons, whereas the HNA variant displayed no protective effect. The effects of HN and HNG peptides did not require a preincubation with the PrP(118-135) fragment, strongly suggesting that these peptides rescue cells independently of a direct interaction with the prion peptide. By contrast, and in agreement with a previous study, HN had no effect on the fibrillar PrP(106-126) peptide-induced cell death. This protective effect for neurons from PrP(118-135)-induced cell death strongly suggests that PrP(118-135) and PrP(106-126) peptides may trigger different pathways leading to neuronal apoptosis.     

Ionic mechanisms of action of prion protein fragment PrP(106–126) in rat basal forebrain neurons

Prion diseases are neurodegenerative disorders that are characterized by the presence of the misfolded prion protein (PrP). Neurotoxicity in these diseases may result from prion‐induced modulation of ion channel function, changes in neuronal excitability, and consequent disruption of cellular homeostasis. We therefore examined PrP effects on a suite of potassium (K⁺) conductances that govern excitability of basal forebrain neurons. Our study examined the effects of a PrP fragment [PrP(106–126), 50 nM] on rat neurons using the patch clamp technique. In this paradigm, PrP(106–126) peptide, but not the “scrambled” sequence of PrP(106–126), evoked a reduction of whole‐cell outward currents in a voltage range between –30 and +30 mV. Reduction of whole‐cell outward currents was significantly attenuated in Ca²⁺‐free external media and also in the presence of iberiotoxin, a blocker of calcium‐activated potassium conductance. PrP(106–126) application also evoked a depression of the delayed rectifier (I_K) and transient outward (I_A) potassium currents. By using single cell RT‐PCR, we identified the presence of two neuronal chemical phenotypes, GABAergic and cholinergic, in cells from which we recorded. Furthermore, cholinergic and GABAergic neurons were shown to express K_v4.2 channels. Our data establish that the central region of PrP, defined by the PrP(106–126) peptide used at nanomolar concentrations, induces a reduction of specific K⁺ channel conductances in basal forebrain neurons. These findings suggest novel links between PrP signalling partners inferred from genetic experiments, K⁺ channels, and PrP‐mediated neurotoxicity. © 2010 Wiley‐Liss, Inc.     

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.     

PrP and β-Amyloid Fragments Activate Different Neurotoxic Mechanisms in Cultured Mouse Cells

Alzheimer's disease and prion diseases such as Creutzfeldt-Jakob disease are caused by as yet undefined metabolic disturbances of normal cellular proteins, the amyloid precursor protein and the prion protein (PrP). Synthetic fragments of both proteins, β-amyloid 25–35 (βA25–35) and PrP106–126, have been shown to be toxic to neurons in culture. Cell death in both cases occurs by apoptosis. Here we show that there are considerable differences in the mechanisms involved. Thus, PrP106–126 is not toxic to cortical cell cultures of PrP knockout mouse neurons whereas PA25–35 is. The toxicity of both peptides involves Ca²⁺ uptake through voltage-sensitive Ca²⁺ channels but only PrP106–126 toxicity involves the activity of NMDA receptors. The toxicity of PA25–35, but not PrP106–126, is attenuated by the action of forskolin. These results indicate that PrP106–126 and βA25–35 induce neuronal apoptosis through different mechanisms.     

The neurotoxicity of prion protein (PrP) peptide 106-126 is independent of the expression level of PrP and is not mediated by abnormal PrP species

A synthetic peptide homologous to region 106-126 of the prion protein (PrP) is toxic to cells expressing PrP, but not to PrP knockout neurons, arguing for a specific role of PrP in mediating the peptide's activity. Whether this is related to a gain of toxicity or a loss of function of PrP is not clear. We explored the possibility that PrP106-126 triggered formation of PrP(Sc) or other neurotoxic PrP species. We found that PrP106-126 did not induce detergent-insoluble and protease-resistant PrP, nor did it alter its membrane topology or cellular distribution. We also found that neurons expressing endogenous or higher level of either wild-type PrP or a nine-octapeptide insertional mutant were equally susceptible to PrP106-126, and that sub-physiological PrP expression was sufficient to restore vulnerability to the peptide. These results indicate that PrP106-126 interferes with a PrP function that requires only low protein levels, and is not impaired by a pathogenic insertion in the octapeptide region.     

Sublethal concentrations of prion peptide PrP106-126 or the amyloid beta peptide of Alzheimer's disease activates expression of proapoptotic markers in primary cortical neurons

Neurodegenerative disorders such as prion diseases and Alzheimer's disease (AD) are characterized by neuronal dysfunction and accumulation of amyloidogenic protein. In vitro studies have demonstrated that these amyloidogenic proteins can induce cellular oxidative stress and therefore may contribute to the neuronal dysfunction observed in these illnesses. Although the neurotoxic pathways are not fully elucidated, recent studies in AD have demonstrated up-regulation of caspases in neurons treated with amyloid beta (Abeta) peptide, suggesting involvement of apoptotic processes. To examine the role of proapoptotic pathways in prion diseases we treated primary mouse cortical neurons with the toxic prion protein peptide PrP106-126 and measured caspase activation and annexin V binding. We found that PrP106-126 induced a rapid and marked elevation in caspase 3, 6, and 8-like activity in neuronal cultures. Increased annexin V binding was observed predominantly on cortical cell neurites in peptide-treated cultures. Interestingly, these effects were induced by sublethal (5-50 microM) or lethal (100-200 microM) concentrations of PrP106-126. Sublethal concentrations of PrP106-126 maintained elevated caspase activation for at least 10 days with no loss of cell viability. Abeta1-40 also up-regulated caspase 3 activity and annexin V binding at both sublethal (5 microM) and lethal (25 microM) concentrations. There were no changes to proapoptotic marker expression in cultures treated with scrambled PrP106-126 (200 microM) or Abeta1-28 (25 microM) peptides. These studies demonstrate that amyloidogenic peptides can induce prolonged activation of proapoptotic marker expression in cultured neurons even at sublethal concentrations. These effects could contribute to chronic neuronal dysfunction and increase susceptibility to additional metabolic insults in neurodegenerative disorders. If so, targeting of therapeutic strategies against neuronal caspase activation early in the disease course could be beneficial in AD and prion diseases.     

Prion protein peptides: optimal toxicity and peptide blockade of toxicity

In prion disease neurodegeneration requires deposition of the abnormal isoform of the prion protein (PrP(Sc)) within nervous tissue. In vitro PrP(Sc) has neurotoxicity that can be mimicked by peptides based on part of its sequence. In this investigation the region of the protein required for maximal neurotoxicity was precisely determined. The optimal neurotoxic peptide was found to contain amino acids 112-126 of the human sequence. The sequence AGAAAAGA was found to be necessary but not sufficient for a neurotoxic effect. The AGAAAAGA peptide blocked the toxicity of PrP106-126, suggesting that this sequence is necessary for the interaction of PrP106-126 with neurons. These results suggest that targeting or use of the AGAAAAGA peptide may represent a therapeutic opportunity for controlling prion disease.     

Aspirin inhibits cytotoxicity of prion peptide PrP106-126 to neuronal cells associated with microglia activation in vitro

The synthetic peptide consisting of amino acid residues 106-126 of the human prion protein PrP106-126 has been demonstrated to generate fibrils, which damage neurons either directly by interacting with components of the cell surface to trigger cell apoptosis signaling or indirectly by activating microglia to produce inflammatory mediators. In our study, rat microglia cells were treated with PrP106-126 or scramble PrP106-126 (Scr PrP). Activated nuclear factor kappaB (NF-kappaB) was determined using immunofluorescence staining and the expression of TNF-alpha and IL-1beta mRNA was measured by quantitative RT-PCR. Inhibitory activity of aspirin on neurotoxicity of PrP106-126 associated with microglia activation was determined using an apoptosis detection kit. Treatment of microglia with 25 microM PrP106-126, but not Scr PrP, resulted in activation and translocation of NF-kappaB, which peaked after 20 min of treatment. The activation of NF-kappaB was followed by increased mRNA expression of TNF-alpha and IL-1beta peaking at about 20 h. In the presence of microglia, aspirin significantly inhibited neuro-2a cell death induced by PrP106-126. The number of neuro-2a cells in apoptosis and necrosis with 5 mM aspirin was about 3-fold lower than the cell culture without aspirin (P<0.05). These data suggest that increased production of cytokines by microglia cells in prion disease is probably regulated by NF-kappaB translocation and may contribute to neurotoxicity of prions, and neurotoxicity of PrP106-126 may be inhibited by aspirin.     

Characterization of a chemical anoxia model in cerebellar granule neurons using sodium azide: Protection by nifedipine and MK-801

Induction of chemical anoxia, using sodium azide in cerebellar granule cells maintained in primary culture, was evaluated as an in vitro assay for screening of potential neuroprotective compounds. The purpose of this study was to evaluate sodium azide as an alternative to cyanide salts, compounds which, despite their unfavorable characteristics, are often used in assays for chemical anoxia. The viability of neuronal cultures after treatment with azide, with or without preincubation with calcium channel blockers, tetrodotoxin (TTX), or glutamate receptor antagonists, was monitored by subsequent incubation with the tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), followed by isopropanol extraction and spectrophotometric quantification of cellularly reduced MTT. The azide-induced degeneration of neurons was shown to be dependent on the concentration as well as on the duration of incubation with submaximal concentrations of azide. Incubation of the neurons with nifedipine, a blocker of L-type voltage-sensitive calcium channels (L-VSCC), or with the noncompetitive N-methyl-D-aspartate (NMDA) subtype glutamate receptor antagonist MK-801, prior to addition of submaximal concentrations of azide, significantly attenuated azide-induced neuronal death. Blockers of N-type and Q-type VSCC (o-conotoxin MVIIA and MVIIC, respectively) and the P-type VSCC blocker o-agatoxin IVA had no effect in this assay. The sodium channel blocker TTX was without effect when added to neurons under depolarizing conditions, but potently and effectively protected cells when experiments were performed in a nondepolarizing buffer. The results show that chemical anoxia induced by incubation of cultured neurons with azide leads to detrimental effects, which may be quantitatively monitored by the capability of the cells to reduce MTT. This procedure is a suitable method for screening of compounds for possible protective effects against neuronal death induced by energy depletion. In addition, the results suggest involvement of L-type VSCC as well as of glutamate receptors in the pathways leading to neuronal degradation induced by energy depletion in cerebellar granule neurons. This would further support the notion that these pathways might be important in neurodegeneration induced by cerebral ischemia or anoxia. © 1996 Wiley-Liss, Inc.     

Quinacrine acts as an antioxidant and reduces the toxicity of the prion peptide PrP106-126

The accumulation of protein aggregates in the brain is a central feature of several different neurodegenerative diseases. We have recently shown that Abeta and alpha-synuclein, associated with Alzheimer's disease, Parkinson's disease and related disorders, can both induce the formation of hydroxyl radicals following incubation in solution, upon addition of Fe(II). PrP106-126, a model peptide for the study of prion protein-mediated cell death, shares the same property. In this study we show that quinacrine (an anti-malarial drug and inhibitor of prion replication) acts as an effective antioxidant, readily scavenging hydroxyl radicals formed from hydrogen peroxide via the Fenton reaction or generated during incubation of the PrP106-126 peptide. Furthermore, the toxicity of PrP106-126 to cultured cells was significantly inhibited by quinacrine.     

Study on the toxic mechanism of prion protein peptide 106-126 in neuronal and non neuronal cells

A synthetic peptide corresponding to the 106-126 amyloidogenic region of the cellular human prion protein (PrP(c)) is useful for in vitro study of prion-induced neuronal cell death. The aim of the present work was to examine the implication of the cellular prion protein in the toxicity mechanism induced by PrP 106-126. The effect of PrP 106-126 was investigated both on human neuroblastoma SH-SY5Y cells and on SH-SY5Y overexpressing murine cellular prions (wtPrP). We show by metabolic assay tests and ATP assays that PrP(c) expression does not modulate the toxicity of the prion peptide. Moreover, we investigated the effect of this peptide on an established non neuronal model, rabbit kidney epithelial A74 cells that express a doxycycline-inducible murine PrP(c) gene. We show for the first time that the prion peptide 106-126 does not exert any toxic effect on this cell line in the presence or absence of doxycycline. Our results show that the PrP 106-126-induced cell alteration is independent of PrP(c) expression. Rather, it seems to act via an interaction with lipidic components of the plasma membrane as strengthened by our results showing the differential susceptibility of neuronal and non neuronal cell lines that significantly differ by their membrane fatty acid composition.     

Tottering mouse motor dysfunction is abolished on the Purkinje cell degeneration (pcd) mutant background

Tottering (tg) mice inherit a recessive mutation of the calcium channel alpha 1A subunit gene, which encodes the pore-forming protein of P/Q-type voltage-sensitive calcium channels and is predominantly expressed in cerebellar granule and Purkinje neurons. The phenotypic consequences of the tottering mutation include ataxia, polyspike discharges, and an intermittent motor dysfunction best described as paroxysmal dystonia. These dystonic episodes induce c-fos mRNA expression in the cerebellar circuitry, including cerebellar granule and Purkinje neurons, deep cerebellar nuclei, and the postsynaptic targets of the deep nuclei. Cellular abnormalities associated with the mutation include hyperarborization of brainstem nucleus locus ceruleus axons and abnormal expression of L-type calcium channels in cerebellar Purkinje cells. Here, the role of these two distinct neural pathways in the expression of tottering mouse intermittent dystonia was assessed. Lesion of locus ceruleus axons with the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzyl-amine (DSP-4) did not affect the frequency of tottering mouse dystonic episodes. In contrast, removal of cerebellar Purkinje cells with the Purkinje cell degeneration (pcd) mutation by generation of tg/tg; pcd/pcd double mutant mice completely eliminated tottering mouse dystonia. Further, the c-fos expression pattern of tg/tg; pcd/pcd double mutants following restraint was indistinguishable from that of wild-type mice, suggesting that the pcd lesion eliminated an essential link in this abnormal neural network. These data suggest that the cerebellar cortex, where the mutant gene is abundantly expressed, contributes to the expression of tottering mouse dystonic episodes.     

Scrapie-infected GT1-1 cells show impaired function of voltage-gated N-type calcium channels (Ca(v) 2.2) which is ameliorated by quinacrine treatment

Prions are transmissible pathogens that cause neurodegenerative diseases, although the mechanisms behind the nervous system dysfunctions are unclear. To study the effects of a prion infection on voltage-gated calcium channels, scrapie-infected gonadotropin-releasing hormone neuronal cells (ScGT1-1) in culture were depolarized by KCl and calcium responses recorded. Lower calcium responses were observed in infected compared to uninfected cells. This effect was still observed when L-type calcium channels were blocked by nimodipine. After inhibition of N-type calcium channels with omega-conotoxin GVIA, there was no difference in calcium responses. The calcium responses after nimodipine treatment became progressively lower during infection, but there was no major loss of the cellular prion protein (PrP(C)) or marked increase in accumulation of the abnormal prion protein (PrP(Sc)) in the cultures. These results indicate that scrapie infection causes a dysfunction of voltage-gated N-type calcium channels, which is exacerbated slowly over time. Quinacrine treatment cleared PrP(Sc) and restored calcium responses in the ScGT1-1 cultures.     

Activation of microglial cells by PrP and β-amyloid fragments raises intracellular calcium through L-type voltage sensitive calcium channels

The prion protein (PrP) and the amyloid β (Aβ) precursor protein (APP) are two normal proteins constitutively synthesised in human brain. An altered form of PrP accumulates in Creutzfeldt–Jakob disease, while Aβ is involved in the pathogenesis of Alzheimer's disease. Synthetic fragments of both proteins, PrP106–126 and β25–35 (β25–35), have been demonstrated to induce neurodegeneration and microglia activation. This study was undertaken to compare PrP106–126 and β25–35 capability of activating human resting microglial cells. Our results show that both peptides are able to induce microglial activation and to elicit an increase in [Ca²⁺]_i levels in cells loaded with calcium-green 1. Inhibitors of L-type voltage-sensitive calcium channels (verapamil, nifedipine and diltiazem) prevented the increase in [Ca²⁺]_i concentration as observed after treatment with PrP106–126 and β25–35, thus indicating a transmembrane calcium influx through these channels. In addition, verapamil abolished the proliferative effect of both PrP106–126 and β25–35.