Cleavage site-directed antibodies reveal the prion protein in humans is shed by ADAM10 at Y226 and associates with misfolded protein deposits in neurodegenerative diseases.

Proteolytic cell surface release ('shedding') of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, for the human body neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and apparently strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimer`s disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregated deposits of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.

Probing Alzheimer's pathology: Exploring the next generation of FDDNP analogues for amyloid ß detection.

Fluorescent probes are a powerful tool for imaging amyloid ß (Aß) plaques, the hallmark of Alzheimer's disease (AD). Herein, we report the synthesis and comprehensive characterization of 21 novel probes as well as their optical properties and binding affinities to Aß fibrils. One of these dyes, 1Ae, exhibited several improvements over FDDNP, an established biomarker for Aß- and Tau-aggregates. First, 1Ae had large Stokes shifts (138-213 nm) in various solvents, thereby reducing self-absorption. With a high quantum yield ratio (φ(dichloromethane/methanol) = 104), 1Ae also ensures minimal background emission in aqueous environments and high sensitivity. In addition, compound 1Ae exhibited low micromolar binding affinity to Aß fibrils in vitro (Kd = 1.603 µM), while increasing fluorescence emission (106-fold) compared to emission in buffer alone. Importantly, the selective binding of 1Ae to Aß1-42 fibrils was confirmed by an in cellulo assay, supported by ex vivo fluorescence microscopy of 1Ae on postmortem AD brain sections, allowing unequivocal identification of Aß plaques. The intermolecular interactions of fluorophores with Aß were elucidated by docking studies and molecular dynamics simulations. Density functional theory calculations revealed the unique photophysics of these rod-shaped fluorophores, with a twisted intramolecular charge transfer (TICT) excited state. These results provide valuable insights into the future application of such probes as potential diagnostic tools for AD in vitro and ex vivo such as determination of Aß1-42 in cerebrospinal fluid or blood.

Ibogaine-Mediated ROS/Antioxidant Elevation in Isolated Rat Uterus Is ß-Adrenergic Receptors and KATP Channels Mediated.

Ibogaine effects are mediated by cellular receptors, ATP depletion followed by ROS production and antioxidant enzyme activity elevation in a dose and time dependent manner. Since the role of KATP channels and ß-adrenoceptors in ROS cellular circuit was established here we explored their role in ibogaine pro-antioxidant effectiveness. Single dose of ibogaine (10 mg/L i.e., 28.8 µmol/L) was applied to isolated rat uterus (spontaneous and Ca2+-stimulated) and contractility and antioxidant enzymes activity were monitored during 4 h. Ibogaine increased amplitude and frequency of spontaneous active uteri immediately after addition that was prevented by propranolol (ß1 and ß2 adrenoceptors selective antagonists) and glibenclamide (KATP sensitive channels inhibitor; only frequency) pre-treatment. In Ca2+-stimulated uteri, ibogaine decreased both amplitude and frequency after 4 h. Pre-treatment with propranolol abolished ibogaine induced amplitude lowering, while glibenclamide had no effect. In both types of active uterus, ibogaine induced a decrease in SOD1 and an increase in CAT activity after 2 h. In Ca2+-stimulated uterus, there was also a decrease of SOD2 activity after 2 h. After 4 h, SOD1 activity returned to the baseline level, but GSH-Px activity increased. Pre-treatment with both propranolol and glibenclamide abolished observed changes of antioxidant enzymes activity suggesting that ibogaine pro-antioxidative effectiveness is ß-adrenergic receptors and KATP channels mediated.

Ibogaine Has Sex-Specific Plasma Bioavailability, Histopathological and Redox/Antioxidant Effects in Rat Liver and Kidneys: A Study on Females.

Ibogaine induces rapid changes in cellular energetics followed by the elevation of antioxidant activities. As shown earlier in male rats, ibogaine treatment with both 1 and 20 mg/kg b.w. per os led to significant glycogenolytic activity in the liver. In this work, female rats treated with the same doses of ibogaine per os displayed lower liver glycogenolytic activity relative to males, dilatation of the central vein and branches of the portal vein, and increased concentration of thiols 6 h after treatment. These changes were followed by increased catalase activity and lipid peroxidation, and decreased xanthine oxidase activity after 24 h. In kidneys, mild histopathological changes were found in all treated animals, accompanied by a decrease of glutathione reductase (after 6 and 24 h at both doses) and an increase of catalase (6 h) and xanthine oxidase activity (6 and 24 h). Ibogaine did not affect antioxidant enzymes activity in erythrocytes. Bioavailability of ibogaine was two to three times higher in females than males, with similar kinetic profiles. Compared to previous results in males, ibogaine showed sex specific effect at the level of antioxidant cellular system. Effects of ibogaine in rats are sex- and tissue-specific, and also dose- and time-dependent.

Lay Public View of Neuroscience and Science-Based Brain Health Recommendations in Slovenia.

Background: Brain health is one of the cornerstones of a long and full life. Active care for brain health and reduction of lifestyle-related risks for brain disorders may be a key strategy in tackling the growing prevalence of mental and neurological illnesses. Public knowledge, perception, and preventive behavior need to be considered in the planning of effective strategies for brain health promotion. Our research is the first effort aimed at assessing Slovenian lay public knowledge, search and use of scientific information about the brain, and care for brain health. Methods: An online survey was used to gather data for descriptive and associative statistical analyses of a sample of the Slovenian public (n = 2568) in August 2017. Participants with formal brain-related education were excluded, leaving the remaining sample of the lay public (n = 1012). Demographic characteristics and information regarding the perceived importance and knowledge of brain health and engagement in preventive behaviors of participants were collected, and key associative analyses were carried out. Results: The majority of respondents (89%) considered brain health to be important. Over one-third (39%) considered their knowledge of the brain as sufficient relative to their needs. Most of the respondents identified science-recommended practices to be important for brain health. No recommendation was followed daily by the majority of the respondents, primarily due to declared lack of time (59%), and lack of information (32%). Information was obtained primarily from television (38%), followed by newspapers and magazines (31%), the Internet (31%), and direct conversations (27%). However, the highest-rated, preferred source of information was lectured by experts. One-third of our sample struggled with the trustworthiness of information sources. Female gender and older age were associated with a higher frequency of healthy practices. Personal or familial diagnoses of brain disorders were not associated with a higher frequency of the behavior in favor of brain health, but did affect available time and perceived value of preventive practices. Conclusions: Our research provides an initial insight into the perceptions, knowledge, and brain health-promoting behavior of the Slovenian lay public. Our findings can inform future strategies for science communication, public education and engagement, and policy-making to improve lifelong active care for brain health.

Curcumin binds to the alpha-helical intermediate and to the amyloid form of prion protein - a new mechanism for the inhibition of PrP(Sc) accumulation.

Conversion of the native, predominantly alpha-helical conformation of prion protein (PrP) into the beta-stranded conformation is characteristic for the transmissible spongiform encephalopathies such as Creutzfeld-Jakob disease. Curcumin, an extended planar molecule and a dietary polyphenol, inhibits in vitro conversion of PrP and formation of protease resistant PrP in neuroblastoma cell lines. Curcumin recognizes the converted beta-form of the PrP both as oligomers and fibrils but not the native form. Curcumin binds to the prion fibrils in the left-handed chiral arrangement as determined by circular dichroism. We show that curcumin labels the plaques of the brain sections of variant Creutzfeld-Jakob disease cases and stains the same structures as antibodies against the PrP. In contrast to thioflavin T, curcumin also binds to the alpha-helical intermediate of PrP present at acidic pH at stoichiometry of 1 : 1. Congo red competes with curcumin for binding to the alpha-intermediate as well as to the beta-form of PrP but is toxic and binds also to the native form of PrP. We therefore show that the partially unfolded structural intermediate of the PrP can be targeted by non-toxic compound of natural origin.

The Effects of Ibogaine on Uterine Smooth Muscle Contractions: Relation to the Activity of Antioxidant Enzymes.

Ibogaine is an indole alkaloid originally extracted from the root bark of the African rainforest shrub Tabernanthe iboga. It has been explored as a treatment for substance abuse because it interrupts drug addiction and relieves withdrawal symptoms. However, it has been shown that ibogaine treatment leads to a sharp and transient fall in cellular ATP level followed by an increase of cellular respiration and ROS production. Since contractile tissues are sensitive to changes in the levels of ATP and ROS, here we investigated an ibogaine-mediated link between altered redox homeostasis and uterine contractile activity. We found that low concentrations of ibogaine stimulated contractile activity in spontaneously active uteri, but incremental increase of doses inhibited it. Inhibitory concentrations of ibogaine led to decreased SOD1 and elevated GSH-Px activity, but doses that completely inhibited contractions increased CAT activity. Western blot analyses showed that changes in enzyme activities were not due to elevated enzyme protein concentrations but posttranslational modifications. Changes in antioxidant enzyme activities point to a vast concentration-dependent increase in H2O2 level. Knowing that extracellular ATP stimulates isolated uterus contractility, while H2O2 has an inhibitory effect, this concentration-dependent stimulation/inhibition could be linked to ibogaine-related alterations in ATP level and redox homeostasis.

Design, Syntheses, and in Vitro Evaluation of New Fluorine-18 Radiolabeled Tau-Labeling Molecular Probes.

Deposition of aggregates of hyperphosphorylated tau protein is a hallmark of tauopathies like Alzheimer and many other neurodegenerative diseases. A sensitive and selective method of in vivo detection of tau-aggregate presence and distribution could provide the means of an early diagnosis of tau-associated diseases. Furthermore, the use of selective molecular probes that enable histochemical differentiation of protein aggregates post-mortem would be advantageous for the insight into the properties of tau protein aggregates. We chose to design new molecular probes based on the structure of 2-(1-(6-((2-[18F]fluoroethyl)(methyl)amino)-2-naphthyl)ethylidene)malononitrile to investigate their likelihood of fitting into VQIVYK tau protein binding channel model. In a modular approach, using cross-coupling reactions, we synthesized a series of candidates, radiolabeled them with fluorine-18 radioisotope, and determined their physicochemical and in vitro binding properties. Herein we report the synthesis of a series of molecular probes capable of detection of tau protein deposits in vitro.

The 2,6-disubstituted naphthalene derivative FDDNP labeling reliably predicts Congo red birefringence of protein deposits in brain sections of selected human neurodegenerative diseases.

Deposition of conformationally altered proteins prominently characterizes pathogenesis and pathomorphology of a number of neurodegenerative disorders. 2-(1-{6-[(2-[F-18]fluoroethyl) (methyl)amino]-2-naphthyl} ethylidene) malononitrile ([F-18]FDDNP), a hydrophobic, viscosity-sensitive, solvent-sensitive, fluorescent imaging probe has been used with positron emission tomography to visualize brain pathology in the living brain of Alzheimer disease (AD) patients. Its non-radiofluorinated analog FDDNP was shown to label senile plaques and neurofibrillary tangles (NFTs) in brain tissue sections. This work aimed at evaluating FDDNP labeling of various protein deposits in fixed, paraffin-embedded brain tissue sections of selected neurodegenerative disorders: AD, cerebral amyloid angiopathy (CAA), transmissible spongiform encephalopathies, progressive supranuclear palsy (PSP), Pick disease (PiD), Parkinson disease, dementia with Lewy bodies, multiple system atrophy (MSA). Cerebral hypertensive vascular hyalinosis (HVH) was used as negative control. Significant agreement between amyloid histochemical properties and FDDNP labeling of the deposits was established. FDDNP labeling showed high positive predictive value for birefringence in senile plaques and NFTs in AD, prion plaques and amyloid deposits in CAA. No FDDNP labeled structures were observed in HVH, PSP, PiD or MSA tissue sections. Our findings may be of significant value for the detection of neuropathological aggregates with [F-18]FDDNP in some of these disorders in the living brain of human subjects.