A Comparison of Three Fluorophores for the Detection of Amyloid Fibers and Prefibrillar Oligomeric Assemblies. ThT (Thioflavin T); ANS (1-Anilinonaphthalene-8-sulfonic Acid); and bisANS (4,4 '-Dianilino-1,1 '-binaphthyl-5,5 '-disulfonic Acid)

Amyloid fiber formation is a key event in many misfolding disorders. The ability to monitor the kinetics of fiber formation and other prefibrillar assemblies is therefore crucial for understanding these diseases. Here we compare three fluorescent probes for their ability to monitor fiber formation, ANS (1-anilinonaphthalene-8-sulfonic acid) and bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid) along with the more widely used thioflavin T (ThT). For this, we have used two highly amyloidogenic peptides: amyloid-β (Aβ) from Alzheimer’s disease and islet amyloid polypeptide (IAPP) associated with type II diabetes. Using a well-plate reader, we show all three fluorophores can report the kinetics of fiber formation. Indeed, bis-ANS is markedly more sensitive to fiber detection than ThT and has a submicromolar affinity for Aβ fibers. Furthermore, we show that fluorescence detection is very sensitive to the presence of excess fluorophore. In particular, beyond a 1:1 stoichiometry these probes demonstrate marked fluorescence quenching, for both Aβ and IAPP. Indeed, the fiber-associated fluorescence signal is almost completely quenched in the presence of excess ThT. There is also intense interest in the detection of prefibrillar amyloid assemblies, as oligomers and protofibrils are believed to be highly cytotoxic. We generate stable, fiber-free, prefibrillar assemblies of Aβ and survey their fluorescence with ANS and bis-ANS. Fluorescence from ANS has often been used as a marker for oligomers; however, we show ANS can fluoresce more strongly in the presence of fibers and should therefore be used as a probe for oligomers with caution

N-Terminally Truncated Amyloid-beta((11-40/42)) Cofibrillizes with its Full-Length Counterpart: Implications for Alzheimer's Disease

Biotechnology and Biological Sciences Research Council. Grant Number: BB/M023877/

Prion protein stabilizes amyloid-β (Aβ) oligomers and enhances Aβ neurotoxicity in a Drosophila model of Alzheimer's disease

The cellular prion protein (PrPC) can act as a cell-surface receptor for β-amyloid (Aβ) peptide; however, a role for PrPC in the pathogenesis of Alzheimer's disease (AD) is contested. Here, we expressed a range of Aβ isoforms and PrPC in the Drosophila brain. We found that co-expression of Aβ and PrPC significantly reduces the lifespan, disrupts circadian rhythms, and increases Aβ deposition in the fly brain. In contrast, under the same conditions, expression of Aβ or PrPC individually did not lead to these phenotypic changes. In vitro studies revealed that substoichiometric amounts of PrPC trap Aβ as oligomeric assemblies and fragment-preformed Aβ fibers. The ability of membrane-anchored PrPC to trap Aβ as cytotoxic oligomers at the membrane surface and fragment inert Aβ fibers suggests a mechanism by which PrPC exacerbates Aβ deposition and pathogenic phenotypes in the fly, supporting a role for PrPC in AD. This study provides a second animal model linking PrPC expression with Aβ toxicity and supports a role for PrPC in AD pathogenesis. Blocking the interaction of Aβ and PrPC represents a potential therapeutic strategy

Mesenchymal Stromal Cells Improve Salivary Function and Reduce Lymphocytic Infiltrates in Mice with Sjögren's-Like Disease

Non-obese diabetic (NOD) mice develop Sjögren's-like disease (SS-like) with loss of saliva flow and increased lymphocytic infiltrates in salivary glands (SGs). There are recent reports using multipotent mesenchymal stromal cells (MSCs) as a therapeutic strategy for autoimmune diseases due to their anti-inflammatory and immunomodulatory capabilities. This paper proposed a combined immuno- and cell-based therapy consisting of: A) an injection of complete Freund's adjuvant (CFA) to eradicate autoreactive T lymphocytes, and B) transplantations of MSCs to reselect lymphocytes. The objective of this was to test the effectiveness of CD45(-)/TER119(-) cells (MSCs) in re-establishing salivary function and in reducing the number of lymphocytic infiltrates (foci) in SGs. The second objective was to study if the mechanisms underlying a decrease in inflammation (focus score) was due to CFA, MSCs, or CFA+MSCs combined.Donor MSCs were isolated from bones of male transgenic eGFP mice. Eight week-old female NOD mice received one of the following treatments: insulin, CFA, MSC, or CFA+MSC (combined therapy). Mice were followed for 14 weeks post-therapy. CD45(-)/TER119(-) cells demonstrated characteristics of MSCs as they were positive for Sca-1, CD106, CD105, CD73, CD29, CD44, negative for CD45, TER119, CD11b, had high number of CFU-F, and differentiated into osteocytes, chondrocytes and adipocytes. Both MSC and MSC+CFA groups prevented loss of saliva flow and reduced lymphocytic infiltrations in SGs. Moreover, the influx of T and B cells decreased in all foci in MSC and MSC+CFA groups, while the frequency of Foxp3(+) (T(reg)) cell was increased. MSC-therapy alone reduced inflammation (TNF-α, TGF-β), but the combination of MSC+CFA reduced inflammation and increased the regenerative potential of SGs (FGF-2, EGF).The combined use of MSC+CFA was effective in both preventing saliva secretion loss and reducing lymphocytic influx in salivary glands

The non-octarepeat copper binding site of the prion protein is a key regulator of prion conversion

The conversion of the prion protein (PrP(C)) into prions plays a key role in transmissible spongiform encephalopathies. Despite the importance for pathogenesis, the mechanism of prion formation has escaped detailed characterization due to the insoluble nature of prions. PrP(C) interacts with copper through octarepeat and non-octarepeat binding sites. Copper coordination to the non-octarepeat region has garnered interest due to the possibility that this interaction may impact prion conversion. We used X-ray absorption spectroscopy to study copper coordination at pH 5.5 and 7.0 in human PrP(C) constructs, either wild-type (WT) or carrying pathological mutations. We show that mutations and pH cause modifications of copper coordination in the non-octarepeat region. In the WT at pH 5.5, copper is anchored to His96 and His111, while at pH 7 it is coordinated by His111. Pathological point mutations alter the copper coordination at acidic conditions where the metal is anchored to His111. By using in vitro approaches, cell-based and computational techniques, we propose a model whereby PrP(C) coordinating copper with one His in the non-octarepeat region converts to prions at acidic condition. Thus, the non-octarepeat region may act as the long-sought-after prion switch, critical for disease onset and propagation

Propagation of RML Prions in Mice Expressing PrP Devoid of GPI Anchor Leads to Formation of a Novel, Stable Prion Strain

PrPC, a host protein which in prion-infected animals is converted to PrPSc, is linked to the cell membrane by a GPI anchor. Mice expressing PrPC without GPI anchor (tgGPI- mice), are susceptible to prion infection but accumulate anchorless PrPSc extra-, rather than intracellularly. We investigated whether tgGPI− mice could faithfully propagate prion strains despite the deviant structure and location of anchorless PrPSc. We found that RML and ME7, but not 22L prions propagated in tgGPI− brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA). Surprisingly, the levels of proteinase K-resistant PrPSc (PrPres) in RML- or ME7-infected tgGPI− brain were 25–50 times higher than in wild-type brain. When returned to wild-type brain, ME7 prions recovered their original properties, however RML prions had given rise to a novel prion strain, designated SFL, which remained unchanged even after three passages in wild-type mice. Because both RML PrPSc and SFL PrPSc are stably propagated in wild-type mice we propose that the two conformations are separated by a high activation energy barrier which is abrogated in tgGPI− mice

Cu2+ accentuates distinct misfolding of A beta((1-40)) and A beta((1-42)) peptides, and potentiates membrane disruption

Central to Alzheimer's disease is the misfolding of amyloid-beta (Aβ) peptide, which generates an assorted population of amorphous aggregates, oligomers and fibres. Metal ion homoeostasis is disrupted in the brains of sufferers of Alzheimer's disease and causes heightened Alzheimer's disease phenotype in animal models. In the present study, we demonstrate that substochiometric Cu2+ affects the misfolding pathway of Aβ(1–40), and the more toxic Aβ(1–42), in markedly different ways. Cu2+ accelerates Aβ(1–40) fibre formation. In contrast, for Aβ(1–42), substoichiometric levels of Cu2+ almost exclusively promote the formation of oligomeric and protofibrillar assemblies. Indeed, mature Aβ(1–42) fibres are disassembled into oligomers when Cu2+ is added. These Cu2+ stabilized oligomers of Aβ(1–42) interact with the lipid bilayer, disrupting the membrane and increasing permeability. Our investigation of Aβ(1–40)/Aβ(1–42) mixtures with Cu2+ revealed that Aβ(1–40) neither contributed to nor perturbed formation of Aβ(1–42) oligomers, although Cu2+–Aβ(1–42) does frustrate Cu2+–Aβ(1–40) fibre growth. Small amounts of Cu2+ accentuate differences in the propensity of Aβ(1–40) and Aβ(1–42) to form synaptotoxic oligomers, providing an explanation for the connection between disrupted Cu2+ homoeostasis and elevated Aβ(1–42) neurotoxicity in Alzheimer's disease.</jats:p

Prion protein stabilizes amyloid-beta (A beta) oligomers and enhances A beta neurotoxicity in a Drosophila model of Alzheimer's disease

This research was originally published in the Journal of Biological Chemistry. Younan, N. D., et al. (2018). "Prion protein stabilizes amyloid-β (Aβ) oligomers and enhances Aβ neurotoxicity in a Drosophila model of Alzheimer's disease." Journal of Biological Chemistry 293(34): 13090-13099. © the Author(s).This work was supported by Wellcome Trust Grant 093241/Z/10/Z and Biotechnology and Biological Sciences Research Council Grant BB/M023877/

The Effects of Hyperthermic Intraperitoneal Chemoperfusion on Colonic Anastomosis: An Experimental Study in a Rat Model

Introduction: Cytoreductive surgery (CRS) with subsequent hyperthermic intraperitoneal chemotherapy (HIPEC) is a promising modality to treat and prevent peritoneal metastases. However, this treatment is associated with signficant morbidity and mortality. Whether or not CRS with HIPEC interferes with anastomotic healing has also been debated. This study was designed to investigate the effects of mitomycin C, cisplatin, oxaliplatin, and doxorubicin used in HIPEC treatment on colonic anastomosis healing in a rat model