The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods.

Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.

The Nervous System Relevance of the Calcium Sensing Receptor in Health and Disease.

The calcium sensing receptor (CaSR) was first identified in parathyroid glands, and its primary role in controlling systemic calcium homeostasis by the regulation of parathyroid hormone (PTH) secretion has been extensively described in literature. Additionally, the receptor has also been investigated in cells and tissues not directly involved in calcium homeostasis, e.g., the nervous system (NS), where it plays crucial roles in early neural development for the differentiation of neurons and glial cells, as well as in the adult nervous system for synaptic transmission and plasticity. Advances in the knowledge of the CaSR's function in such physiological processes have encouraged researchers to further broaden the receptor's investigation in the neuro-pathological conditions of the NS. Interestingly, pre-clinical data suggest that receptor inhibition by calcilytics might be effective in counteracting the pathomechanism underlying Alzheimer's disease and ischemia, while a CaSR positive modulation with calcimimetics has been proposed as a potential approach for treating neuroblastoma. Importantly, such promising findings led to the repurposing of CaSR modulators as novel pharmacological alternatives for these disorders. Therefore, the aim of this review article is to critically appraise evidence which, so far, has been yielded from the investigation of the role of the CaSR in physiology of the nervous system and to focus on the most recent emerging concepts which have reported the receptor as a therapeutic target for neurodegeneration and neuroblastic tumors.

Calcilytic NPS 2143 Reduces Amyloid Secretion and Increases sAßPPα Release from PSEN1 Mutant iPSC-Derived Neurons.

Despite numerous efforts and studies over the last three decades, Alzheimer's disease (AD) remains a disorder not fully understood and incurable so far. Development of induced pluripotent stem cell (iPSC) technology to obtain terminally differentiated neurons from adult somatic cells revolutionized the study of AD, providing a powerful tool for modelling the disease and for screening candidate drugs. Indeed, iPSC reprogramming allowed generation of neurons from both sporadic and familial AD patients with the promise to recapitulate the early pathological mechanisms in vitro and to identify novel targets. Interestingly, NPS 2143, a negative allosteric modulator of the calcium sensing receptor, has been indicated as a possible therapeutic for AD. In the present study, we assessed the potential of our iPSC-based familial AD cellular model as a platform for drug testing. We found that iPSC-derived neurons respond to treatment with γ-secretase inhibitor, modifying the physiological amyloid-ß protein precursor (AßPP) processing and amyloid-ß (Aß) secretion. Moreover, we demonstrated the expression of calcium sensing receptor (CaSR) protein in human neurons derived from healthy and familial AD subjects. Finally, we showed that calcilytic NPS 2143 induced a changing of Aß and sAßPPα secreted into conditioned media and modulation of CaSR and PSEN1 expression at the plasma membrane of AD neurons. Overall, our findings suggest that NPS 2143 affects important AD processes in a relevant in vitro system of familial AD.

Loop F of the GABAA receptor alpha subunit governs GABA potency.

Gamma-amino butyric acid (GABA) is an abundant neurotransmitter in the CNS. GABAergic interneurons orchestrate pyramidal neurons in the cerebral cortex, and thus control learning and memory. Ionotropic receptors for GABA (GABAAR) are heteropentameric complexes of α, ß and γ integral membrane-protein subunits forming Cl- -channels operated by GABA, which are vital for brain function and are important drug targets. However, knowledge on how GABAAR bind GABA is controversial. Structural biology versus functional modelling combined with site-directed mutagenesis suggest markedly different roles for loop F of the extracellular domain of the α-subunit when complexed with GABA. Here, we report that contrary to the results of structural studies, loop F of the α-subunit controls the potency of GABA on GABAAR. We examined the effect of replacing a short, variable segment of loop F of the GABAA α5-subunit with the corresponding segment of the α2-subunit (GABAA5_LF2) and vice versa (GABAA2-LF5). When compared with their respective wild-type counterparts, GABAA5_LF2 receptors displayed enhanced sensitivity towards GABA, whilst in GABAA2-LF5 sensitivity was diminished. Mice homozygous for the genetic knock-in of the GABAA5_LF2 subunit showed a marked deficit in long- but not short-term object recognition memory. Working memory in place learning, spontaneous alternation and the rewarded T-maze were all normal. The deficit in long-term recognition memory was reversed by an α5-GABAA negative allosteric modulator compound. The data show that loop F governs GABA potency in a receptor isoform-specific manner in vitro. Moreover, this mechanism of ligand recognition appears to be operative in vivo and impacts cognitive performance.

Loop-F of the α-subunit determines the pharmacologic profile of novel competitive inhibitors of GABAA receptors.

The neurotransmitter γ-amino butyric acid (GABA) has a fundamental role in CNS function and ionotropic (GABAA) receptors that mediate many of the actions of GABA are important therapeutic targets. This study reports the mechanism of action of novel GABAA antagonists based on a tricyclic oxazolo-2,3-benzodiazepine scaffold. These compounds are orthosteric antagonists of GABA on heteropentameric GABAA receptors of αxß2γ2 configuration expressed in HEK293 cells. In silico modelling predicted that the test compounds docked in the GABA binding-pocket and would interact with amino-acid residues in the α- and ß-subunit interface that are known to be important for the binding of GABA. Intriguingly, optimal docking also required an interaction with the non-conserved amino-terminal segment of Loop-F of the α-subunit. Testing of a compound with altered regiochemistry of the oxazolone moiety supported the model with respect to the conserved GABA-interacting residues in vitro as well as in vivo. The prediction regarding loop-F was examined by replacing the amino-terminal variable segment of loop-F of the α5-subunit with the corresponding residues in the α1- and α2-subunits. When tested with the novel inhibitors, the receptors formed by the modified α5-subunits displayed the pharmacologic phenotype of the source of loop-F. In summary, these data show that the variable amino-terminal segment of loop-F of the α-subunit determines the pharmacologic selectivity of the novel tricyclic inhibitors of GABAA receptors.

Neurons derived from sporadic Alzheimer's disease iPSCs reveal elevated TAU hyperphosphorylation, increased amyloid levels, and GSK3B activation.

BACKGROUND: Alzheimer's disease (AD) is the most common type of dementia, affecting one in eight adults over 65 years of age. The majority of AD cases are sporadic, with unknown etiology, and only 5% of all patients with AD present the familial monogenic form of the disease. In the present study, our aim was to establish an in vitro cell model based on patient-specific human neurons to study the pathomechanism of sporadic AD. METHODS: We compared neurons derived from induced pluripotent stem cell (iPSC) lines of patients with early-onset familial Alzheimer's disease (fAD), all caused by mutations in the PSEN1 gene; patients with late-onset sporadic Alzheimer's disease (sAD); and three control individuals without dementia. The iPSC lines were differentiated toward mature cortical neurons, and AD pathological hallmarks were analyzed by RT-qPCR, enzyme-linked immunosorbent assay, and Western blotting methods. RESULTS: Neurons from patients with fAD and patients with sAD showed increased phosphorylation of TAU protein at all investigated phosphorylation sites. Relative to the control neurons, neurons derived from patients with fAD and patients with sAD exhibited higher levels of extracellular amyloid-ß 1-40 (Aß1-40) and amyloid-ß 1-42 (Aß1-42). However, significantly increased Aß1-42/Aß1-40 ratios, which is one of the pathological markers of fAD, were observed only in samples of patients with fAD. Additionally, we detected increased levels of active glycogen synthase kinase 3 ß, a physiological kinase of TAU, in neurons derived from AD iPSCs, as well as significant upregulation of amyloid precursor protein (APP) synthesis and APP carboxy-terminal fragment cleavage. Moreover, elevated sensitivity to oxidative stress, as induced by amyloid oligomers or peroxide, was detected in both fAD- and sAD-derived neurons. CONCLUSIONS: On the basis of the experiments we performed, we can conclude there is no evident difference except secreted Aß1-40 levels in phenotype between fAD and sAD samples. To our knowledge, this is the first study in which the hyperphosphorylation of TAU protein has been compared in fAD and sAD iPSC-derived neurons. Our findings demonstrate that iPSC technology is suitable to model both fAD and sAD and may provide a platform for developing new treatment strategies for these conditions.

Selective inhibition of extra-synaptic α5-GABAA receptors by S44819, a new therapeutic agent.

In the mammalian central nervous system (CNS) GABAA receptors (GABAARs) mediate neuronal inhibition and are important therapeutic targets. GABAARs are composed of 5 subunits, drawn from 19 proteins, underpinning expression of 20-30 GABAAR subtypes. In the CNS these isoforms are heterogeneously expressed and exhibit distinct physiological and pharmacological properties. We report the discovery of S44819, a novel tricyclic oxazolo-2,3-benzodiazepine-derivative, that selectively inhibits α5-subunit-containing GABAARs (α5-GABAARs). Current α5-GABAAR inhibitors bind to the "benzodiazepine site". However, in HEK293 cells expressing recombinant α5-GABAARs, S44819 had no effect on 3H-flumazenil binding, but displaced the GABAAR agonist 3H-muscimol and competitively inhibited the GABA-induced responses. Importantly, we reveal that the α5-subunit selectivity is uniquely governed by amino acid residues within the α-subunit F-loop, a region associated with GABA binding. In mouse hippocampal CA1 neurons, S44819 enhanced long-term potentiation (LTP), blocked a tonic current mediated by extrasynaptic α5-GABAARs, but had no effect on synaptic GABAARs. In mouse thalamic neurons, S44819 had no effect on the tonic current mediated by δ-GABAARs, or on synaptic (α1ß2γ2) GABAARs. In rats, S44819 enhanced object recognition memory and reversed scopolamine-induced impairment of working memory in the eight-arm radial maze. In conclusion, S44819 is a first in class compound that uniquely acts as a potent, competitive, selective antagonist of recombinant and native α5-GABAARs. Consequently, S44819 enhances hippocampal synaptic plasticity and exhibits pro-cognitive efficacy. Given this profile, S44819 may improve cognitive function in neurodegenerative disorders and facilitate post-stroke recovery.

A novel GABA(A) alpha 5 receptor inhibitor with therapeutic potential.

Novel 2,3-benzodiazepine and related isoquinoline derivatives, substituted at position 1 with a 2-benzothiophenyl moiety, were synthesized to produce compounds that potently inhibited the action of GABA on heterologously expressed GABAA receptors containing the alpha 5 subunit (GABAA α5), with no apparent affinity for the benzodiazepine site. Substitutions of the benzothiophene moiety at position 4 led to compounds with drug-like properties that were putative inhibitors of extra-synaptic GABAA α5 receptors and had substantial blood-brain barrier permeability. Initial characterization in vivo showed that 8-methyl-5-[4-(trifluoromethyl)-1-benzothiophen-2-yl]-1,9-dihydro-2H-[1,3]oxazolo[4,5-h][2,3]benzodiazepin-2-one was devoid of sedative, pro-convulsive or motor side-effects, and enhanced the performance of rats in the object recognition test. In summary, we have discovered a first-in-class GABA-site inhibitor of extra-synaptic GABAA α5 receptors that has promising drug-like properties and warrants further development.

Endocytosis of the AT1A angiotensin receptor is independent of ubiquitylation of its cytoplasmic serine/threonine-rich region.

Agonist-induced internalisation of the rat type 1A (AT(1A)) angiotensin II receptor is associated with phosphorylation of a serine/threonine-rich region in its cytoplasmic tail. In yeast, hyperphosphorylation of the alpha-factor pheromone receptor regulates endocytosis of the receptor by facilitating the monoubiquitylation of its cytoplasmic tail on lysine residues. The role of receptor ubiquitylation in AT(1A) receptor internalisation was evaluated by deletion or replacement of lysine residues in its agonist-sensitive serine/threonine-rich region. Expression of such receptor mutants in CHO cells showed that these modifications had no detectable effect on the angiotensin II-induced endocytosis of the AT(1A) receptor. Furthermore, fusion of ubiquitin in-frame to an internalisation-deficient AT(1A) receptor mutant with a truncated carboxyl-terminal tail did not restore the endocytosis of the resulting chimeric receptor. No impairment of receptor internalisation was observed after substitution of all lysine residues in the serine/threonine-rich region at saturating angiotensin II concentrations, where endocytosis occurs by a beta-arrestin and dynamin independent mechanism. Taken together, these data demonstrate that ubiquitylation of the cytoplasmic serine/threonine-rich region of the AT(1A) receptor on lysine residues is not required for its agonist-induced internalisation, and suggest that endocytosis of mammalian G protein-coupled receptors (GPCRs) occurs by a different mechanism than that of yeast GPCRs.

Optical waveguide lightmode spectroscopic techniques for investigating membrane-bound ion channel activities.

Optical waveguide lightmode spectroscopic (OWLS) techniques were probed for monitoring ion permeation through channels incorporated into artificial lipid environment. A novel sensor set-up was developed by depositing liposomes or cell-derived membrane fragments onto hydrophilic polytetrafluoroethylene (PTFE) membrane. The fibrous material of PTFE membrane could entrap lipoid vesicles and the water-filled pores provided environment for the hydrophilic domains of lipid-embedded proteins. The sensor surface was kept clean from the lipid holder PTFE membrane by a water- and ion-permeable polyethylene terephthalate (PET) mesh. The sensor set-up was tested with egg yolk lecithin liposomes containing gramicidin ion channels and with cell-derived membrane fragments enriched in GABA-gated anion channels. The method allowed monitoring the move of Na(+) and organic cations through gramicidin channels and detecting the Cl(-)-channel functions of the (α5ß2γ2) GABAA receptor in the presence or absence of GABA and the competitive GABA-blocker bicuculline.