Glimepiride Reduces the Expression of PrPC,Prevents PrPSc Formation and Protects against Prion Mediated Neurotoxicity

Background

A hallmark of the prion diseases is the conversion of the host-encoded cellular prion protein (PrP^C) into a disease related, alternatively folded isoform (PrP^Sc). The accumulation of PrP^Sc within the brain is associated with synapse loss and ultimately neuronal death. Novel therapeutics are desperately required to treat neurodegenerative diseases including the prion diseases.

Principal Findings

Treatment with glimepiride, a sulphonylurea approved for the treatment of diabetes mellitus, induced the release of PrP^C from the surface of prion-infected neuronal cells. The cell surface is a site where PrP^C molecules may be converted to PrP^Sc and glimepiride treatment reduced PrP^Sc formation in three prion infected neuronal cell lines (ScN2a, SMB and ScGT1 cells). Glimepiride also protected cortical and hippocampal neurones against the toxic effects of the prion-derived peptide PrP82–146. Glimepiride treatment significantly reduce both the amount of PrP82–146 that bound to neurones and PrP82–146 induced activation of cytoplasmic phospholipase A₂ (cPLA₂) and the production of prostaglandin E₂ that is associated with neuronal injury in prion diseases. Our results are consistent with reports that glimepiride activates an endogenous glycosylphosphatidylinositol (GPI)-phospholipase C which reduced PrP^C expression at the surface of neuronal cells. The effects of glimepiride were reproduced by treatment of cells with phosphatidylinositol-phospholipase C (PI-PLC) and were reversed by co-incubation with p-chloromercuriphenylsulphonate, an inhibitor of endogenous GPI-PLC.

Conclusions

Collectively, these results indicate that glimepiride may be a novel treatment to reduce PrP^Sc formation and neuronal damage in prion diseases.     

Ginkgolides protect against amyloid-β1–42-mediated synapse damage in vitro

Background

The early stages of Alzheimer's disease (AD) are closely associated with the production of the Aβ_1–42 peptide, loss of synapses and gradual cognitive decline. Since some epidemiological studies showed that EGb 761, an extract from the leaves of the Ginkgo biloba tree, had a beneficial effect on mild forms of AD, the effects of some of the major components of the EGb 761 extract (ginkgolides A and B, myricetin and quercetin) on synapse damage in response to Aβ_1–42 were examined.

Results

The addition of Aβ_1–42 to cortical or hippocampal neurons reduced the amounts of cell associated synaptophysin, a pre-synaptic membrane protein that is essential for neurotransmission, indicating synapse damage. The effects of Aβ_1–42 on synapses were apparent at concentrations approximately 100 fold less than that required to kill neurons; the synaptophysin content of neuronal cultures was reduced by 50% by 50 nM Aβ_1–42. Pre-treatment of cortical or hippocampal neuronal cultures with ginkgolides A or B, but not with myrecitin or quercetin, protected against Aβ_1–42-induced loss of synaptophysin. This protective effect was achieved with nanomolar concentrations of ginkgolides. Previous studies indicated that the ginkgolides are platelet-activating factor (PAF) receptor antagonists and here we show that Aβ_1–42-induced loss of synaptophysin from neuronal cultures was also reduced by pre-treatment with other PAF antagonists (Hexa-PAF and CV6209). PAF, but not lyso-PAF, mimicked the effects Aβ_1–42 and caused a dose-dependent reduction in the synaptophysin content of neurons. This effect of PAF was greatly reduced by pre-treatment with ginkgolide B. In contrast, ginkgolide B did not affect the loss of synaptophysin in neurons incubated with prostaglandin E₂.

Conclusion

Pre-treatment with ginkgolides A or B protects neurons against Aβ_1–42-induced synapse damage. These ginkgolides also reduced the effects of PAF, but not those of prostaglandin E₂, on the synaptophysin content of neuronal cultures, results consistent with prior reports that ginkgolides act as PAF receptor antagonists. Such observations suggest that the ginkgolides are active components of Ginkgo biloba preparations and may protect against the synapse damage and the cognitive loss seen during the early stages of AD.     

Clustering of sialylated glycosylphosphatidylinositol anchors mediates PrP-induced activation of cytoplasmic phospholipase A2 and synapse damage

Precisely how the accumulation of PrP^Sc causes the neuronal degeneration that leads to the clinical symptoms of prion diseases is poorly understood. Our recent paper showed that the clustering of specific glycosylphosphatidylinositol (GPI) anchors attached to PrP proteins triggered synapse damage in cultured neurons. First, we demonstrated that small, soluble PrP^Sc oligomers caused synapse damage via a GPI-dependent process. Our hypothesis, that the clustering of specific GPIs caused synapse damage, was supported by observations that cross-linkage of PrP^C, either chemically or by monoclonal antibodies, also triggered synapse damage. Synapse damage was preceded by an increase in the cholesterol content of synapses and activation of cytoplasmic phospholipase A₂ (cPLA₂). The presence of a terminal sialic acid moiety, a rare modification of mammalian GPI anchors, was essential in the activation of cPLA₂ and synapse damage induced by cross-linked PrP^C. We conclude that the sialic acid modifies local membrane microenvironments (rafts) surrounding clustered PrP molecules resulting in aberrant activation of cPLA₂ and synapse damage. A recent observation, that toxic amyloid-β assemblies cross-link PrP^C, suggests that synapse damage in prion and Alzheimer diseases is mediated via a common molecular mechanism, and raises the possibility that the pharmacological modification of GPI anchors might constitute a novel therapeutic approach to these diseases.     

Clustering of sialylated glycosylphosphatidylinositol anchors mediates PrP-induced activation of cytoplasmic phospholipase A2 and synapse damage

Precisely how the accumulation of PrP^Sc causes the neuronal degeneration that leads to the clinical symptoms of prion diseases is poorly understood. Our recent paper showed that the clustering of specific glycosylphosphatidylinositol (GPI) anchors attached to PrP proteins triggered synapse damage in cultured neurons. First, we demonstrated that small, soluble PrP^Sc oligomers caused synapse damage via a GPI-dependent process. Our hypothesis, that the clustering of specific GPIs caused synapse damage, was supported by observations that cross-linkage of PrP^C, either chemically or by monoclonal antibodies, also triggered synapse damage. Synapse damage was preceded by an increase in the cholesterol content of synapses and activation of cytoplasmic phospholipase A₂ (cPLA₂). The presence of a terminal sialic acid moiety, a rare modification of mammalian GPI anchors, was essential in the activation of cPLA₂ and synapse damage induced by cross-linked PrP^C. We conclude that the sialic acid modifies local membrane microenvironments (rafts) surrounding clustered PrP molecules resulting in aberrant activation of cPLA₂ and synapse damage. A recent observation, that toxic amyloid-β assemblies cross-link PrP^C, suggests that synapse damage in prion and Alzheimer diseases is mediated via a common molecular mechanism, and raises the possibility that the pharmacological modification of GPI anchors might constitute a novel therapeutic approach to these diseases.     

Cholesterol ester hydrolase inhibitors reduce the production of synaptotoxic amyloid-β oligomers

The production of amyloid-β (Aβ) is the key factor driving pathogenesis in Alzheimer's disease (AD). Increasing concentrations of Aβ within the brain cause synapse degeneration and the dementia that is characteristic of AD. Here the factors that affect the release of disease-relevant forms Aβ were studied in a cell model. 7PA2 cells expressing the human amyloid precursor protein released soluble Aβ oligomers that caused synapse damage in cultured neurons. Supernatants from 7PA2 cells treated with the cholesterol synthesis inhibitor squalestatin contained similar concentrations of Aβ₄₂ to control cells but did not cause synapse damage in neuronal cultures. These supernatants contained reduced concentrations of Aβ₄₂ oligomers and increased concentrations of Aβ₄₂ monomers. Treatment of 7PA2 cells with platelet-activating factor (PAF) antagonists had similar effects; it reduced concentrations of Aβ₄₂ oligomers and increased concentrations of Aβ₄₂ monomers in cell supernatants. PAF activated cholesterol ester hydrolases (CEH), enzymes that released cholesterol from stores of cholesterol esters. Inhibition of CEH also reduced concentrations of Aβ₄₂ oligomers and increased concentrations of Aβ₄₂ monomers in cell supernatants. The Aβ monomers produced by treated cells protected neurons against Aβ oligomer-induced synapse damage. These studies indicate that pharmacological manipulation of cells can alter the ratio of Aβ monomer:oligomer released and consequently their effects on synapses.     

Amyloid-β-induced synapse damage is mediated via cross-linkage of cellular prion proteins

The cellular prion protein (PrP(C)), which is highly expressed at synapses, was identified as a receptor for the amyloid-β (Aβ) oligomers that are associated with dementia in Alzheimer disease. Here, we report that Aβ oligomers secreted by 7PA2 cells caused synapse damage in cultured neurons via a PrP(C)-dependent process. Exogenous PrP(C) added to Prnp knock-out((0/0)) neurons was targeted to synapses and significantly increased Aβ-induced synapse damage. In contrast, the synapse damage induced by a phospholipase A(2)-activating peptide was independent of PrP(C). In Prnp wild-type((+/+)) neurons Aβ oligomers activated synaptic cytoplasmic phospholipase A(2) (cPLA(2)). In these cells, the addition of Aβ oligomers triggered the translocation of cPLA(2) in synapses to cholesterol dense membranes (lipid rafts) where it formed a complex also containing Aβ and PrP(C). In contrast, the addition of Aβ to Prnp((0/0)) neurons did not activate synaptic cPLA(2), which remained in the cytoplasm and was not associated with Aβ. Filtration assays and non-denaturing gels demonstrated that Aβ oligomers cross-link PrP(C). We propose that it is the cross-linkage of PrP(C) by Aβ oligomers that triggers abnormal activation of cPLA(2) and synapse damage. This hypothesis was supported by our observation that monoclonal antibody mediated cross-linkage of PrP(C) also activated synaptic cPLA(2) and caused synapse damage.     

Channels formed with a mutant prion protein PrP(82-146) homologous to a 7-kDa fragment in diseased brain of GSS patients

A major prion protein (PrP) mutant that forms amyloid fibrils in the diseased brain of patients with Gerstmann-Sträussler-Scheinker syndrome (GSS) is a fragment of 7 kDa spanning from residues 81-82 to 144-153 of PrP. Analysis of ionic membrane currents, recorded with a libid bilayer technique, revealed that the wild-type fragment PrP(82-146) WT and the partially scrambled PrP(82-146) (127-146) SC are capable of forming heterogenous ion channels that are similar to those channels formed with PrP(106-126). In contrast, PrP(82-146) peptides in which the region from residue 106 to 126 had been scrambled (SC) showed a reduction in interaction with lipid membranes and did not form channels. The PrP(82-146) WT- and PrP(82-146) (127-146) SC-formed cation channels with fast kinetics are Cu²⁺ sensitive and rifampicin (RIF) insensitive, whereas the time-dependent inactivating channels formed by these same peptides are both Cu²⁺ and RIF insensitive. The presence of RIF in the solution before the addition of PrP(82-146) WT or PrP(82-146) (127-146) SC affected their incorporation into the lipid bilayers. PrP(82-146) WT and PrP(82-146) (127-146) SC fast cation channels formed in the presence of RIF appeared in an electrically semisilent state or an inactivated state. Increasing [Cd²⁺]_cis enhanced the incorporation of PrP(82-146) WT and PrP(82-146) (127-146) SC channels formed in the presence of RIF. We conclude that the major PrP mutant fragment in the diseased brain of GSS patients is prone to form channels in neuronal membranes, causing their dysfunction. We propose that Cd²⁺ may accentuate the neurotoxicity of this channel-forming PrP fragment by enhancing its incorporation into the membrane. prion diseases; prion channels; amyloids; neurodegenerative diseases; membrane-linked pathologies; vacuolation; cytotoxic proteins     

Impairment of Glucose and Glutamate Transport and Induction of Mitochondrial Oxidative Stress and Dysfunction in Synaptosomes by Amyloid β-Peptide: Role of the Lipid Peroxidation Product 4-Hydroxynonenal

Abstract: Deposits of amyloid β-peptide (Aβ), reduced glucose uptake into brain cells, oxidative damage to cellular proteins and lipids, and excitotoxic mechanisms have all been suggested to play roles in the neurodegenerative process in Alzheimer's disease. Synapse loss is closely correlated with cognitive impairments in Alzheimer's disease, suggesting that the synapse may be the site at which degenerative mechanisms are initiated and propagated. We report that Aβ causes oxyradical-mediated impairment of glucose transport, glutamate transport, and mitochondrial function in rat neocortical synaptosomes. Aβ induced membrane lipid peroxidation in synaptosomes that occurred within 1 h of exposure; significant decreases in glucose transport occurred within 1 h of exposure to Aβ and decreased further with time. The lipid peroxidation product 4-hydroxynonenal conjugated to synaptosomal proteins and impaired glucose transport; several antioxidants prevented Aβ-induced impairment of glucose transport, indicating that lipid peroxidation was causally linked to this adverse action of Aβ. FeSO₄ (an initiator of lipid peroxidation), Aβ, and 4-hydroxynonenal each induced accumulation of mitochondrial reactive oxygen species, caused concentration-dependent decreases in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, and reduced cellular ATP levels significantly. Aβ also impaired glutamate transport, an effect blocked by antioxidants. These data suggest that Aβ induces membrane lipid peroxidation, which results in impairment of the function of membrane glucose and glutamate transporters, altered mitochondrial function, and a deficit in ATP levels; 4-hydroxynonenal appears to be a mediator of these actions of Aβ. These data suggest that oxidative stress occurring at synapses may contribute to the reduced glucose uptake and synaptic degeneration that occurs in Alzheimer's disease patients. They further suggest a sequence of events whereby oxidative stress promotes excitotoxic synaptic degeneration and neuronal cell death in a variety of different neurodegenerative disorders.     

The basics of preclinical drug development for neurodegenerative disease indications

Background

Because of the emerging intersections of HIV infection and Alzheimer's disease, we examined cerebrospinal fluid (CSF) biomarkers related of amyloid and tau metabolism in HIV-infected patients.

Methods

In this cross-sectional study we measured soluble amyloid precursor proteins alpha and beta (sAPPα and sAPPβ), amyloid beta fragment 1-42 (Aβ_1-42), and total and hyperphosphorylated tau (t-tau and p-tau) in CSF of 86 HIV-infected (HIV+) subjects, including 21 with AIDS dementia complex (ADC), 25 with central nervous system (CNS) opportunistic infections and 40 without neurological symptoms and signs. We also measured these CSF biomarkers in 64 uninfected (HIV-) subjects, including 21 with Alzheimer's disease, and both younger and older controls without neurological disease.

Results

CSF sAPPα and sAPPβ concentrations were highly correlated and reduced in patients with ADC and opportunistic infections compared to the other groups. The opportunistic infection group but not the ADC patients had lower CSF Aβ_1-42 in comparison to the other HIV+ subjects. CSF t-tau levels were high in some ADC patients, but did not differ significantly from the HIV+ neuroasymptomatic group, while CSF p-tau was not increased in any of the HIV+ groups. Together, CSF amyloid and tau markers segregated the ADC patients from both HIV+ and HIV- neuroasymptomatics and from Alzheimer's disease patients, but not from those with opportunistic infections.

Conclusions

Parallel reductions of CSF sAPPα and sAPPβ in ADC and CNS opportunistic infections suggest an effect of CNS immune activation or inflammation on neuronal amyloid synthesis or processing. Elevation of CSF t-tau in some ADC and CNS infection patients without concomitant increase in p-tau indicates neural injury without preferential accumulation of hyperphosphorylated tau as found in Alzheimer's disease. These biomarker changes define pathogenetic pathways to brain injury in ADC that differ from those of Alzheimer's disease.     

Gerstmann-Sträussler-Scheinker disease amyloid protein polymerizes according to the "dock-and-lock" model

Prion protein (PrP) amyloid formation is a central feature of genetic and acquired prion diseases such as Gerstmann-Str?ussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob disease. The major component of GSS amyloid is a PrP fragment spanning residues approximately 82-146, which when synthesized as a peptide, readily forms fibrils featuring GSS amyloid. The present study employed surface plasmon resonance (SPR) to characterize the binding events underlying PrP82-146 oligomerization at the first stages of fibrillization, according to evidence suggesting a pathogenic role of prefibrillar oligomers rather than mature amyloid fibrils. We followed in real time the binding reactions occurring during short term (seconds) addition of PrP82-146 small oligomers (1-5-mers, flowing species) onto soluble prefibrillar PrP82-146 aggregates immobilized on the sensor surface. SPR data confirmed very efficient aggregation/elongation, consistent with the hypothesis of nucleation-dependent polymerization process. Much lower binding was observed when PrP82-146 flowed onto the scrambled sequence of PrP82-146 or onto prefibrillar Abeta42 aggregates. As previously found with Abeta40, SPR data could be adequately fitted by equations modeling the "dock-and-lock" mechanism, in which the "locking" step is due to sequential conformational changes, each increasing the affinity of the monomer for the fibril until a condition of irreversible binding is reached. However, these conformational changes (i.e. the locking steps) appear to be faster and easier with PrP82-146 than with Abeta40. Such differences suggest that PrP82-146 has a greater propensity to polymerize and greater stability of the aggregates.     

Extracellular chaperones prevent Aβ42-induced toxicity in rat brains

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterised by cognitive decline, formation of the extracellular amyloid β (Aβ₄₂) plaques, neuronal and synapse loss, and activated microglia and astrocytes. Extracellular chaperones, which are known to inhibit amyloid fibril formation and promote clearance of misfolded aggregates, have recently been shown to reduce efficiently the toxicity of HypF-N misfolded oligomers to immortalised cell lines, by binding and clustering them into large species. However, the role of extracellular chaperones on Aβ oligomer toxicity remains unclear, with reports often appearing contradictory. In this study we microinjected into the hippocampus of rat brains Aβ₄₂ oligomers pre-incubated for 1 h with two extracellular chaperones, namely clusterin and α₂-macroglobulin. The chaperones were found to prevent Aβ₄₂-induced learning and memory impairments, as assessed by the Morris Water Maze test, and reduce Aβ₄₂-induced glia inflammation and neuronal degeneration in rat brains, as probed by fluorescent immunohistochemical analyses. Moreover, the chaperones were able to prevent Aβ₄₂ colocalisation with PSD-95 at post-synaptic terminals of rat primary neurons, suppressing oligomer cytotoxicity. All such effects were not effective by adding pre-formed oligomers and chaperones without preincubation. Molecular chaperones have therefore the potential to prevent the early symptoms of AD, not just by inhibiting Aβ₄₂ aggregation, as previously demonstrated, but also by suppressing the toxicity of Aβ₄₂ oligomers after they are formed. These findings elect them as novel neuroprotectors against amyloid-induced injury and excellent candidates for the design of therapeutic strategies against AD.     

The role of activity in synaptic degeneration in a protein misfolding disease, prion disease

In chronic neurodegenerative diseases associated with aggregates of misfolded proteins (such as Alzheimer''s, Parkinson''s and prion disease), there is an early degeneration of presynaptic terminals prior to the loss of the neuronal somata. Identifying the mechanisms that govern synapse degeneration is of paramount importance, as cognitive decline is strongly correlated with loss of presynaptic terminals in these disorders. However, very little is known about the processes that link the presence of a misfolded protein to the degeneration of synapses. It has been suggested that the process follows a simple linear sequence in which terminals that become dysfunctional are targeted for death, but there is also evidence that high levels of activity can speed up degeneration. To dissect the role of activity in synapse degeneration, we infused the synaptic blocker botulinum neurotoxin A (BoNT/A) into the hippocampus of mice with prion disease and assessed synapse loss at the electron microscopy level. We found that injection of BoNT/A in naïve mice caused a significant enlargement of excitatory presynaptic terminals in the hippocampus, indicating transmission impairment. Long-lasting blockade of activity by BoNT/A caused only minimal synaptic pathology and no significant activation of microglia. In mice with prion disease infused with BoNT/A, rates of synaptic degeneration were indistinguishable from those observed in control diseased mice. We conclude that silencing synaptic activity neither prevents nor enhances the degree of synapse degeneration in prion disease. These results challenge the idea that dysfunction of synaptic terminals dictates their elimination during prion-induced neurodegeneration.     

Conformational plasticity of the Gerstmann-Sträussler-Scheinker disease peptide as indicated by its multiple aggregation pathways

The existence of several prion strains and their capacity of overcoming species barriers seem to point to a high conformational adaptability of the prion protein. To investigate this structural plasticity, we studied here the aggregation pathways of the human prion peptide PrP82-146, a major component of the Gerstmann-Sträussler-Scheinker amyloid disease.By Fourier transform infrared (FT-IR) spectroscopy, electron microscopy, and atomic force microscopy (AFM), we monitored the time course of PrP82-146 fibril formation. After incubation at 37 °C, the unfolded peptide was found to aggregate into oligomers characterized by intermolecular β-sheet infrared bands. At a critical oligomer concentration, the emergence of a new FT-IR band allowed to detect fibril formation. A different intermolecular β-sheet interaction of the peptides in oligomers and in fibrils is, therefore, detected by FT-IR spectroscopy, which, in addition, suggests a parallel orientation of the cross β-sheet structures of PrP82-146 fibrils. By AFM, a wide distribution of PrP82-146 oligomer volumes—the smallest ones containing from 5 to 30 peptides—was observed. Interestingly, the statistical analysis of AFM data enabled us to detect a quantization in the oligomer height values differing by steps of ∼ 0.5 nm that could reflect an orientation of oligomer β-strands parallel with the sample surface. Different morphologies were also detected for fibrils that displayed high heterogeneity in their twisting periodicity and a complex hierarchical assembly.Thermal aggregation of PrP82-146 was also investigated by FT-IR spectroscopy, which indicated for these aggregates an intermolecular β-sheet interaction different from that observed for oligomers and fibrils. Unexpectedly, random aggregates, induced by solvent evaporation, were found to display a significant α-helical structure as well as several β-sheet components.All these results clearly point to a high plasticity of the PrP82-146 peptide, which was found to be capable of undergoing several aggregation pathways, with end products displaying different secondary structures and intermolecular interactions.     

Paradoxical Role of Prion Protein Aggregates in Redox-Iron Induced Toxicity

Background

Imbalance of iron homeostasis has been reported in sporadic Creutzfeldt-Jakob-disease (sCJD) affected human and scrapie infected animal brains, but the contribution of this phenotype to disease associated neurotoxicity is unclear.

Methodology/Principal Findings

Using cell models of familial prion disorders, we demonstrate that exposure of cells expressing normal prion protein (PrP^C) or mutant PrP forms to a source of redox-iron induces aggregation of PrP^C and specific mutant PrP forms. Initially this response is cytoprotective, but becomes increasingly toxic with time due to accumulation of PrP-ferritin aggregates. Mutant PrP forms that do not aggregate are not cytoprotective, and cells show signs of acute toxicity. Intracellular PrP-ferritin aggregates induce the expression of LC3-II, indicating stimulation of autophagy in these cells. Similar observations are noted in sCJD and scrapie infected hamster brains, lending credence to these results. Furthermore, phagocytosis of PrP-ferritin aggregates by astrocytes is cytoprotective, while culture in astrocyte conditioned medium (CM) shows no measurable effect. Exposure to H₂O₂, on the other hand, does not cause aggregation of PrP, and cells show acute toxicity that is alleviated by CM.

Conclusions/Significance

These observations suggest that aggregation of PrP in response to redox-iron is cytoprotective. However, subsequent co-aggregation of PrP with ferritin induces intracellular toxicity unless the aggregates are degraded by autophagosomes or phagocytosed by adjacent scavenger cells. H₂O₂, on the other hand, does not cause aggregation of PrP, and induces toxicity through extra-cellular free radicals. Together with previous observations demonstrating imbalance of iron homeostasis in prion disease affected brains, these observations provide insight into the mechanism of neurotoxicity by redox-iron, and the role of PrP in this process.     

Phospholipase A2 inhibitors or platelet-activating factor antagonists prevent prion replication

A key feature of prion diseases is the conversion of the cellular prion protein (PrP(C)) into disease-related isoforms (PrP(Sc)), the deposition of which is thought to lead to neurodegeneration. In this study a pharmacological approach was used to determine the metabolic pathways involved in the formation of protease-resistant PrP (PrP(res)) in three prion-infected cell lines (ScN2a, SMB, and ScGT1 cells). Daily treatment of these cells with phospholipase A(2) (PLA(2)) inhibitors for 7 days prevented the accumulation of PrP(res). Glucocorticoids with anti-PLA(2) activity also prevented the formation of PrP(res) and reduced the infectivity of SMB cells. Treatment with platelet-activating factor (PAF) antagonists also reduced the PrP(res) content of cells, while the addition of PAF reversed the inhibitory effect of PLA(2) inhibitors on PrP(res) formation. ScGT1 cells treated with PLA(2) inhibitors or PAF antagonists for 7 days remained clear of detectable (PrPres) when grown in control medium for a further 12 weeks. Treatment of non-infected cells with PLA(2) inhibitors or PAF antagonists reduced PrP(C) levels suggesting that limiting cellular PrP(C) may restrict prion formation in infected cells. These data indicate a pivotal role for PLA(2) and PAF in controlling PrP(res) formation and identify them as potential therapeutic agents.     

Astrocytes with previous chronic exposure to amyloid β‐peptide fragment 1–40 suppress excitatory synaptic transmission

Synaptic dysfunction and neuronal death are responsible for cognitive and behavioral deficits in Alzheimer's disease (AD). It is well known that such neurological abnormalities are preceded by long‐term exposure of amyloid β‐peptide (Aβ) and/or hyperphosphorylated tau prior. In addition to the neurological deficit, astrocytes as a major glial cell type in the brain, significantly participate in the neuropathogenic mechanisms underlying synaptic modulation. Although astrocytes play a significant key role in modulating synaptic transmission, little is known on whether astrocyte dysfunction caused by such long‐term Aβ exposure affects synapse formation and function. Here, we show that synapse formation and synaptic transmission are attenuated in hippocampal‐naïve neurons co‐cultured with astrocytes that have previously experienced chronic Aβ_1‐40 exposure. In this abnormal astrocytic condition, hippocampal neurons exhibit decrements of evoked excitatory post‐synaptic currents (EPSCs) and miniature EPSC frequency. Furthermore, size of readily releasable synaptic pools and number of excitatory synapses were also significantly decreased. Contrary to these negative effects, release probability at individual synapses was significantly increased in the same astrocytic condition. Taken together, our data indicate that lower synaptic transmission caused by astrocytes previously, and chronically, exposed to Aβ1–40 is attributable to a small number of synapses with higher release probability.

     

Specific amyloid-β42 deposition in the brain of a Gerstmann-Sträussler-Scheinker disease patient with a P105L mutation on the prion protein gene

ABSTRACT

Although colocalization of amyloid β (Aβ) with prion protein (PrP) in the kuru plaque has previously been observed in the brain of prion diseases patients, the participating Aβ species has not been identified. Here, we present an immunohistochemical assessment of the brain and spinal cord of a 69-year-old Japanese female patient with Gerstmann-Sträussler-Scheinker disease with a P105L mutation on the PRNP gene (GSS-P105L). Immunohistochemical assessment of serial brain sections was performed using anti-PrP and -Aβ antibodies in the hippocampus, frontal and occipital lobes. She died 69 years after a 21-year clinical course. Immunohistochemistorical examination revealed that ~50% of the kuru plaques in the cerebrum were colocalized with Aβ, and Aβ42 was predominantly observed to be colocalized with PrP-plaques. The Aβ deposition patterns were unique, and distinct from diffuse plaques observed in the normal aging brain or Alzheimer’s disease brain. The spinal cord exhibited degeneration in the lateral corticospinal tract, posterior horn, and fasciculus gracilis. We have demonstrated for the first time that Aβ42, rather than Aβ40, is the main Aβ component associated with PrP-plaques, and also the degeneration of the fasciculus gracilis in the spinal cord in GSS-P105L, which could be associated with specific clinical features of GSS-P105L.     

Brain-derived proteins in the CSF, do they correlate with brain pathology in CJD?

Background

Brain derived proteins such as 14-3-3, neuron-specific enolase (NSE), S 100b, tau, phosphorylated tau and Aβ_1–42 were found to be altered in the cerebrospinal fluid (CSF) in Creutzfeldt-Jakob disease (CJD) patients. The pathogenic mechanisms leading to these abnormalities are not known, but a relation to rapid neuronal damage is assumed. No systematic analysis on brain-derived proteins in the CSF and neuropathological lesion profiles has been performed.

Methods

CSF protein levels of brain-derived proteins and the degree of spongiform changes, neuronal loss and gliosis in various brain areas were analyzed in 57 CJD patients.

Results

We observed three different patterns of CSF alteration associated with the degree of cortical and subcortical changes. NSE levels increased with lesion severity of subcortical areas. Tau and 14-3-3 levels increased with minor pathological changes, a negative correlation was observed with severity of cortical lesions. Levels of the physiological form of the prion protein (PrP^c) and Aβ_1–42 levels correlated negatively with cortical pathology, most clearly with temporal and occipital lesions.

Conclusion

Our results indicate that the alteration of levels of brain-derived proteins in the CSF does not only reflect the degree of neuronal damage, but it is also modified by the localization on the brain pathology. Brain specific lesion patterns have to be considered when analyzing CSF neuronal proteins.     

Cell type-specific neuroprotective activity of untranslocated prion protein

Background

A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP). However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions.

Principal Findings

Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells.

Significance

These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function.