Currently, no effective therapeutics exist for the treatment of incurable neurodegenerative diseases such as Alzheimer’s disease (AD). The cellular prion protein (PrP
C
) acts as a high-affinity ...receptor for amyloid beta oligomers (AβO), a main neurotoxic species mediating AD pathology. The interaction of AβO with PrP
C
subsequently activates Fyn tyrosine kinase and neuroinflammation. Herein, we used our previously developed peptide aptamer 8 (PA8) binding to PrP
C
as a therapeutic to target the AβO–PrP–Fyn axis and prevent its associated pathologies. Our in vitro results indicated that PA8 prevents the binding of AβO with PrP
C
and reduces AβO-induced neurotoxicity in mouse neuroblastoma N2a cells and primary hippocampal neurons. Next, we performed in vivo experiments using the transgenic 5XFAD mouse model of AD. The 5XFAD mice were treated with PA8 and its scaffold protein thioredoxin A (Trx) at a 14.4 µg/day dosage for 12 weeks by intraventricular infusion through Alzet
®
osmotic pumps. We observed that treatment with PA8 improves learning and memory functions of 5XFAD mice as compared to Trx-treated 5XFAD mice. We found that PA8 treatment significantly reduces AβO levels and Aβ plaques in the brain tissue of 5XFAD mice. Interestingly, PA8 significantly reduces AβO–PrP interaction and its downstream signaling such as phosphorylation of Fyn kinase, reactive gliosis as well as apoptotic neurodegeneration in the 5XFAD mice compared to Trx-treated 5XFAD mice. Collectively, our results demonstrate that treatment with PA8 targeting the AβO–PrP–Fyn axis is a promising and novel approach to prevent and treat AD.
Full text
Available for:
EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Alzheimer's disease (AD) is an incurable, progressive and devastating neurodegenerative disease. Pathogenesis of AD is associated with the aggregation and accumulation of amyloid beta (Aβ), a major ...neurotoxic mediator that triggers neuroinflammation and memory impairment. Recently, we found that cellulose ether compounds (CEs) have beneficial effects against prion diseases by inhibiting protein misfolding and replication of prions, which share their replication mechanism with Aβ. CEs are FDA-approved safe additives in foods and pharmaceuticals. Herein, for the first time we determined the therapeutic effects of the representative CE (TC-5RW) in AD using in vitro and in vivo models. Our in vitro studies showed that TC-5RW inhibits Aβ aggregation, as well as neurotoxicity and immunoreactivity in Aβ-exposed human and murine neuroblastoma cells. In in vivo studies, for the first time we observed that single and weekly TC-5RW administration, respectively, improved memory functions of transgenic 5XFAD mouse model of AD. We further demonstrate that TC-5RW treatment of 5XFAD mice significantly inhibited Aβ oligomer and plaque burden and its associated neuroinflammation via regulating astrogliosis, microgliosis and proinflammatory mediator glial maturation factor beta (GMFβ). Additionally, we determined that TC-5RW reduced lipopolysaccharide-induced activated gliosis and GMFβ in vitro. In conclusion, our results demonstrate that CEs have therapeutic effects against Aβ pathologies and cognitive impairments, and direct, potent anti-inflammatory activity to rescue neuroinflammation. Therefore, these FDA-approved compounds are effective candidates for developing therapeutics for AD and related neurodegenerative diseases associated with protein misfolding.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
A hallmark of Alzheimer's disease (AD) is the accumulation of extracellular amyloid-β (Aβ) plaques in the brains of patients. N-terminally truncated pyroglutamate-modified Aβ (pEAβ) has been ...described as a major compound of Aβ species in senile plaques. pEAβ is more resistant to degradation, shows higher toxicity and has increased aggregation propensity and β-sheet stabilization compared to non-modified Aβ. Here we characterized recombinant pEAβ(3-40) in aqueous trifluoroethanol (TFE) solution regarding its aggregation propensity and structural changes in comparison to its non-pyroglutamate-modified variant Aβ(1-40). Secondary structure analysis by circular dichroism spectroscopy suggests that pEAβ(3-40) shows an increased tendency to form β-sheet-rich structures in 20% TFE containing solutions where Aβ(1-40) forms α-helices. Aggregation kinetics of pEAβ(3-40) in the presence of 20% TFE monitored by thioflavin-T (ThT) assay showed a typical sigmoidal aggregation in contrast to Aβ(1-40), which lacks ThT positive structures under the same conditions. Transmission electron microscopy confirms that pEAβ(3-40) aggregated to large fibrils and high molecular weight aggregates in spite of the presence of the helix stabilizing co-solvent TFE. High resolution NMR spectroscopy of recombinantly produced and uniformly isotope labeled U-15N-pEAβ(3-40) in TFE containing solutions indicates that the pyroglutamate formation affects significantly the N-terminal region, which in turn leads to decreased monomer stability and increased aggregation propensity.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
...PrP100–120 harbors one high affinity binding site for toxic amyloid β-oligomers (Aβo) which are associated with pathogenesis of AD. ...proteolytic processing of the cellular prion protein is not ...totally understood. ...three main proteolytic cleavage events have been described: physiological α-cleavage at amino acids 110–111/112 Figure 1, β-cleavage at amino acids 89/90 executed by calpains upon oxidative stress, and shedding at amino acids 228/229 mainly by the zinc metalloproteinase ADAM10 (Béland and Roucou, 2014). Notably, it cannot be converted to PrPSc and moreover, acts as a dominant-negative inhibitor of PrPSc formation. ...enhancing α-cleavage represents a valuable treatment target for prion diseases and possibly other neurodegenerative diseases that benefit from high levels of neuroprotective PrPN1 and/or proteolysis of PrPC at the hydrophobic domain (Béland and Roucou, 2014). ...high levels of PrPC1 act as a negative inhibitor for prion conversion.
Prion diseases are infectious and fatal neurodegenerative diseases affecting humans and animals. Transmission is possible within and between species with zoonotic potential. Currently, no prophylaxis ...or treatment exists. Prions are composed of the misfolded isoform PrP
Sc
of the cellular prion protein PrP
C
. Expression of PrP
C
is a prerequisite for prion infection, and conformational conversion of PrP
C
is induced upon its direct interaction with PrP
Sc
. Inhibition of this interaction can abrogate prion propagation, and we have previously established peptide aptamers (PAs) binding to PrP
C
as new anti-prion compounds. Here, we mapped the interaction site of PA8 in PrP and modeled the complex in silico to design targeted mutations in PA8 which presumably enhance binding properties. Using these PA8 variants, we could improve PA-mediated inhibition of PrP
Sc
replication and de novo infection of neuronal cells. Furthermore, we demonstrate that binding of PA8 and its variants increases PrP
C
α-cleavage and interferes with its internalization. This gives rise to high levels of the membrane-anchored PrP-C1 fragment, a transdominant negative inhibitor of prion replication. PA8 and its variants interact with PrP
C
at its central and most highly conserved domain, a region which is crucial for prion conversion and facilitates toxic signaling of Aβ oligomers characteristic for Alzheimer’s disease. Our strategy allows for the first time to induce α-cleavage, which occurs within this central domain, independent of targeting the responsible protease. Therefore, interaction of PAs with PrP
C
and enhancement of α-cleavage represent mechanisms that can be beneficial for the treatment of prion and other neurodegenerative diseases.
Full text
Available for:
EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Conversion of the intrinsically disordered protein alpha-synuclein (alpha-syn) into amyloid aggregates is a key process in Parkinson's disease. The sequence region 35-59 contains beta-strand segments ...beta1 and beta2 of alpha-syn amyloid fibril models and most disease-related mutations. beta1 and beta2 frequently engage in transient interactions in monomeric alpha-syn. The consequences of beta1-beta2 contacts are evaluated by disulfide engineering, biophysical techniques, and cell viability assays. The double-cysteine mutant alpha-synCC, with a disulfide linking beta1 and beta2, is aggregation-incompetent and inhibits aggregation and toxicity of wild-type alpha-syn. We show that alpha-syn delays the aggregation of amyloid-beta peptide and islet amyloid polypeptide involved in Alzheimer's disease and type2 diabetes, an effect enhanced in the alpha-synCC mutant. Tertiary interactions in the beta1-beta2 region of alpha-syn interfere with the nucleation of amyloid formation, suggesting promotion of such interactions as a potential therapeutic approach.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The interconversion of monomers, oligomers, and amyloid fibrils of the amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer disease. The determination of the kinetics of the ...individual association and dissociation reactions is hampered by the fact that forward and reverse reactions to/from different aggregation states occur simultaneously. Here, we report the kinetics of dissociation of Aβ monomers from protofibrils, prefibrillar high molecular weight oligomers previously shown to possess pronounced neurotoxicity. An engineered binding protein sequestering specifically monomeric Aβ was employed to follow protofibril dissociation by tryptophan fluorescence, precluding confounding effects of reverse or competing reactions. Aβ protofibril dissociation into monomers follows exponential decay kinetics with a time constant of ∼2 h at 25 °C and an activation energy of 80 kJ/mol, values typical for high affinity biomolecular interactions. This study demonstrates the high kinetic stability of Aβ protofibrils toward dissociation into monomers and supports the delineation of the Aβ folding and assembly energy landscape.
Background: Protofibrils of the amyloid-β peptide (Aβ) are neurotoxic oligomers implicated in development and progression of Alzheimer disease.
Results: The dissociation of Aβ protofibrils into their monomeric subunits is a slow process, occurring on the time scale of hours.
Conclusion: Aβ protofibrils possess a high kinetic stability toward dissociation into monomers.
Significance: The longevity of Aβ protofibrils permits sustained toxic effects.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The aggregation of Tau into paired helical filaments is involved in the pathogenesis of several neurodegenerative diseases, including Alzheimer disease. The aggregation reaction is characterized by ...conformational conversion of the repeat domain, which partially adopts a cross-β-structure in the resulting amyloid-like fibrils. Here, we report the selection and characterization of an engineered binding protein, β-wrapin TP4, targeting the Tau repeat domain. TP4 was obtained by phage display using the four-repeat Tau construct K18ΔK280 as a target. TP4 binds K18ΔK280 as well as the longest isoform of human Tau, hTau40, with nanomolar affinity. NMR spectroscopy identified two alternative TP4-binding sites in the four-repeat domain, with each including two hexapeptide motifs with high β-sheet propensity. Both binding sites contain the aggregation-determining PHF6 hexapeptide within repeat 3. In addition, one binding site includes the PHF6* hexapeptide within repeat 2, whereas the other includes the corresponding hexapeptide Tau(337–342) within repeat 4, denoted PHF6**. Comparison of TP4-binding with Tau aggregation reveals that the same regions of Tau are involved in both processes. TP4 inhibits Tau aggregation at substoichiometric concentration, demonstrating that it interferes with aggregation nucleation. This study provides residue-level insight into the interaction of Tau with an aggregation inhibitor and highlights the structural flexibility of Tau.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In this study, we characterized unexpected side-products in a commercially synthesized peptide with the sequence RPRTRLHTHRNR. This so-called peptide D3 was selected by mirror phage display against ...low molecular weight amyloid-β-peptide (Aβ) associated with Alzheimer’s disease. Capillary electrophoresis (CE) was the method of choice for structure analysis because the extreme hydrophilicity of the peptide did not allow reversed-phase liquid chromatography (RPLC) and hydrophilic interaction stationary phases (HILIC). CE-MS analysis, applying a strongly acidic background electrolyte and different statically adsorbed capillary coatings, provided fast and efficient analysis and revealed that D3 unexpectedly showed strong ion-pairing with sulfuric acid. Moreover, covalent O-sulfonation at one or two threonine residues was identified as a result of a side reaction during peptide synthesis, and deamidation was found at either the asparagine residue or at the C-terminus. In total, more than 10 different species with different m/z values were observed. Tandem-MS analysis with collision induced dissociation (CID) using a CE-quadrupole-time-of-flight (QTOF) setup predominantly resulted in sulfate losses and did not yield any further characteristic fragment ions at high collision energies. Therefore, direct infusion Fourier transform ion cyclotron resonance (FT-ICR) MS was employed to identify the covalent modification and discriminate O-sulfonation from possible O-phosphorylation by using an accurate mass analysis. Electron transfer dissociation (ETD) was used for the identification of the threonine O-sulfation sites. In this work, it is shown that the combination of CE-MS and FT-ICR-MS with ETD fragmentation was essential for the full characterization of this extremely basic peptide with labile modifications.
Full text
Available for:
DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Background
Alzheimer’s disease (AD) is a major cause of dementia and age‐related neurodegenerative disease with no current treatment. Pathologically, AD is characterized primarily by accumulation of ...amyloid‐β (Aβ) in the brain. The interaction of Aβ‐oligomers (Aβo) with the cellular prion protein (PrPC) subsequently mediated AD pathologies. The interference with Aβo‐PrPC interaction is a valuable strategy for developing therapeutics for AD. Previously, we developed peptide aptamers (PAs) binding to the PrPC, partially covering the binding site of Aβo . In this study, we aimed to investigate the PAs effects on preventing Aβo‐PrPC interaction and toxicity in vitro and pathologies in AD models.
Method
Wild type mouse neuroblastoma N2a cells (overexpressing mouse PrP) were treated with Aβ (1 mM) and PAs (10 µg/ml), and after 24 hr performed the MTT assay. Following these in vitro experiments, we carried out in vivo experiments using transgenic 5xFAD (expressing human APP and PSEN1 transgenes harboring in total 5 mutations associated with familial AD) mice. The 5xFAD mice were treated with PAs at a 14.4 ug /day dosage for 6 weeks by intraventricular infusion using Alzet® osmotic pumps. After completion of treatment, fear conditioning test (FCT) was performed for consolidated leaning and memory functions of the 5xFAD mice.
Result
The MTT results indicated that PA treatment significantly reduced Aβo‐induced toxicity. Overall, our in vitro results indicated that PAs reduced Aβ‐induced neurotoxicity. In the FCT, we observed that PAs increased the time of freezing percentage as comparted to the non‐treated 5xFAD mice. The FCT results represented that PAs significantly improved consolidated learning and memory functions of 5xFAD mice as compared to the non‐treated 5xFAD mice.
Conclusion
Our results demonstrate that treatment with PAs targeting Aβo‐PrPC interaction would be valuable and novel strategy to treat AD.
Full text
Available for:
FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
PDF
![Contact between the [beta]1...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/1433-7851/small "Contact between the [beta]1 and [beta]2 Segments of [alpha]-Synuclein that Inhibits Amyloid Formation")
