Musical and Multilingual Experience Are Related to Healthy Aging: Better Some Than None But Even Better Together.

OBJECTIVES: Life experiences that are complex, sustained, and intense, such as active participation in music and speaking multiple languages, have been suggested to contribute to maintaining or improving cognitive performance and mental health. The current study focuses on whether lifetime musical and multilingual experiences differentially relate to cognition and well-being in older adults, and tests whether there is a cumulative effect of both experiences. METHODS: A total of 11,335 older adults from the population-based Lifelines Cohort Study completed a musical and multilingual background and experience questionnaire. Latent class analysis was used to categorize individuals into subgroups according to their various musical and multilingual experiences resulting in a (1) nonmusical, low-multilingual group; (2) nonmusical, high-multilingual group; (3) musical, low-multilingual group; and (4) musical high-multilingual group. To determine whether the groups differed in terms of cognition or emotional affect, differences in Ruff Figural Fluency Test (RFFT) and Positive and Negative Affect Schedule scores were investigated by means of multinomial logistic regression analysis. RESULTS: Having high-multilingual, and not musical, experience was related to better RFFT performance compared to no experience, but not to more positive affect. Having both musical and high-multilingual experiences is related to better RFFT performance and more positive affect in advanced age compared to having only one experience or none. Importantly, these results were found independently of age, level of education, and socioeconomic status. DISCUSSION: Musical and multilingual experiences are related to healthy aging, especially when combined, which supports the suggestion that a broader spectrum of lifetime experiences relates to cognitive reserve.

Foreign Language Learning as Cognitive Training to Prevent Old Age Disorders? Protocol of a Randomized Controlled Trial of Language Training vs. Musical Training and Social Interaction in Elderly With Subjective Cognitive Decline.

Introduction: With aging comes a reduction of cognitive flexibility, which has been related to the development of late-life depression and progression of general cognitive decline. Several factors have been linked to attenuating such decline in cognitive flexibility, such as education, physical exercise and stimulating leisure activities. Speaking two or more languages has recently received abundant attention as another factor that may build up cognitive reserve, thereby limiting the functional implications of compromised cognition that accompany old age. With the number of older adults reaching record levels, it is important to attenuate the development of old-age disorders. Learning to speak a foreign language might offer a powerful tool in promoting healthy aging, but up to date effect studies are sparse. Here, the protocol that forms the foundation of the current study is presented. The present study aims to: (1) examine the effects of a foreign language training on cognitive flexibility and its neural underpinnings, and on mental health; and (2) assess the unique role of foreign language training vs. other cognitive or social programs. Method: One-hundred and ninety-eight Dutch elderly participants reporting subjective cognitive decline are included and randomized to either a language intervention, a music intervention, or a social control intervention. During 3 to 6 months, the language group learns English, the music group learns to play the guitar and the social group participates in social meetings where art workshops are offered. At baseline, at a 3-month follow-up, and at 6 months after termination of the training program, clinical, cognitive and brain activity measurements (combined EEG and fNIRS methods) are taken to assess cognitive flexibility and mental health. Discussion: This is the first trial addressing combined effects of language learning in elderly on cognition, language proficiency, socio-affective measures, and brain activity in the context of a randomized controlled trial. If successful, this study can provide insights into how foreign language training can contribute to more cognitively and mentally healthy years in older adulthood. Clinical Trial Registration: The trial is registered at the Netherlands Trial Register, July 2, 2018, trial number NL7137. https://www.trialregister.nl/trial/7137.

Differential Role of Serines and Threonines in Intracellular Loop 3 and C-Terminal Tail of the Histamine H4 Receptor in ß-Arrestin and G Protein-Coupled Receptor Kinase Interaction, Internalization, and Signaling.

The histamine H4 receptor (H4R) activates Gαi-mediated signaling and recruits ß-arrestin2 upon stimulation with histamine. ß-Arrestins play a regulatory role in G protein-coupled receptor (GPCR) signaling by interacting with phosphorylated serine and threonine residues in the GPCR C-terminal tail and intracellular loop 3, resulting in receptor desensitization and internalization. Using bioluminescence resonance energy transfer (BRET)-based biosensors, we show that G protein-coupled receptor kinases (GRK) 2 and 3 are more quickly recruited to the H4R than ß-arrestin1 and 2 upon agonist stimulation, whereas receptor internalization dynamics toward early endosomes was slower. Alanine-substitution revealed that a serine cluster at the distal end of the H4R C-terminal tail is essential for the recruitment of ß-arrestin1/2, and consequently, receptor internalization and desensitization of G protein-driven extracellular-signal-regulated kinase (ERK)1/2 phosphorylation and label-free cellular impedance. In contrast, alanine substitution of serines and threonines in the intracellular loop 3 of the H4R did not affect ß-arrestin2 recruitment and receptor desensitization, but reduced ß-arrestin1 recruitment and internalization. Hence, ß-arrestin recruitment to H4R requires the putative phosphorylated serine cluster in the H4R C-terminal tail, whereas putative phosphosites in the intracellular loop 3 have different effects on ß-arrestin1 versus ß-arrestin2. Mutation of these putative phosphosites in either intracellular loop 3 or the C-terminal tail did not affect the histamine-induced recruitment of GRK2 and GRK3 but does change the interaction of H4R with GRK5 and GRK6, respectively. Identification of H4R interactions with these proteins is a first step in the understanding how this receptor might be dysregulated in pathophysiological conditions.

Classification, Nomenclature, and Structural Aspects of Adhesion GPCRs.

Representation of the nine distinct aGPCR subfamilies and their unique N-terminal domain architecture. The illustration also shows the extracellular structural feature shared by all aGPCRs (except ADGRA1), known as the GPCR autoproteolysis-inducing (GAIN) domain, that mediates autoproteolysis and subsequent attachment of the cleaved NTF and CTF fragments The adhesion family of G protein-coupled receptors (aGPCRs) is unique among all GPCR families with long N-termini and multiple domains that are implicated in cell-cell and cell-matrix interactions. Initially, aGPCRs in the human genome were phylogenetically classified into nine distinct subfamilies based on their 7TM sequence similarity. This phylogenetic grouping of genes into subfamilies was found to be in congruence in closely related mammals and other vertebrates as well. Over the years, aGPCR repertoires have been mapped in many species including model organisms, and, currently, there is a growing interest in exploring the pharmacological aspects of aGPCRs. Nonetheless, the aGPCR nomenclature has been highly diverse because experts in the field have used different names for different family members based on their characteristics (e.g., epidermal growth factor-seven-span transmembrane (EGF-TM7)), but without harmonization with regard to nomenclature efforts. In order to facilitate naming of orthologs and other genetic variants in different species in the future, the Adhesion-GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposed a unified nomenclature for aGPCRs. Here, we review the classification and the most recent/current nomenclature of aGPCRs and as well discuss the structural topology of the extracellular domain (ECD)/N-terminal fragment (NTF) that is comparable with this 7TM subfamily classification. Of note, we systematically describe the structural domains in the ECD of aGPCR subfamilies and highlight their role in aGPCR-protein interactions.

7TM Domain Structure of Adhesion GPCRs.

Schematic presentation of the overall adhesion G Protein-Coupled Receptor (aGPCR) structure and functional domains, covering an extracellular N-terminal fragment (NTF), a membrane-spanning C-terminal fragment (CTF) and a GPCR proteolysis site (GPS). (Left side) aGPCR model constructed based on the seven-transmembrane (7TM) structure (blue) of secretin family glucagon receptor (GCGR) (PDB, 4L6R) [11] and the GPCR autoproteolysis inducing (GAIN) domain (magenta) structure of latrophilin 1 (PDB, 4DLQ) [9]. The ß-13 strand residues are depicted in green. (Right side) The experimentally validated full-length secretin family GCGR structure combining structural and experimental information from the GCGR 7TM crystal structure (PDB, 4L6R) (blue), the GCGR extracellular domain (ECD) structure (PDB, 4ERS) (magenta) and the ECD structure of glucagon-like peptide-1 (GLP-1)-bound glucagon-like peptide-1 receptor (GLP-1R) (PDB, 3IOL) (green), complemented by site-directed mutagenesis, electron microscopy (EM), hydrogen-deuterium exchange (HDX) and cross-linking studies [11-13]) Despite the recent breakthroughs in the elucidation of the three-dimensional structures of the seven transmembrane (7TM) domain of the G protein-coupled receptor (GPCR) superfamily, a corresponding structure of a member of the adhesion GPCR (aGPCR) family has not yet been solved. In this chapter, we give an overview of the current knowledge of the 7TM domain of aGPCRs by comparative structure-based sequence similarity analyses between aGPCRs and GPCRs with known crystal structure. Of the GPCR superfamily, only the secretin family shares some sequence similarity with aGPCRs. This chapter will therefore emphasize on the comparison of these two GPCR families. Two 7TM domain structures of secretin family GPCRs are known that provide insight into the structure-function relationships of conserved sequence motifs that play important roles and are also present in most aGPCRs. This suggests that the 7TM domains of aGPCRs and secretin family GPCRs share a similar structural fold and that the conserved residues in both families may be involved in similar intermolecular interaction networks and facilitate similar conformational changes. Comparison of the residues that line the large peptide hormone binding pocket in the 7TM domain of secretin family GPCRs with corresponding residues in aGPCRs indicates that in the latter, the corresponding pocket in the 7TM domain is relatively hydrophobic and may be even larger. Improved knowledge on these conserved sequence motifs will help to understand the interactions of the aGPCR 7TM domain with ligands and gain insight into the activation mechanism of aGPCRs.

Adhesion GPCRs in immunology.

Adhesion GPCRs (aGPCRs) form a subfamily of the large GPCR super family. Most aGPCRs are characterised by a non-covalent bipartite structure that consists of a large extracellular domain and a membrane-spanning 7 transmembrane domain. Typically, aGPCRs can combine cell adhesion by the large extracellular domain with intracellular signalling by the 7 transmembrane domain. Immune responses rely on cellular communication and subsequent defence reactions. Indeed, aGPCR ADGRB1 and members of the ADGRE class have been linked to processes like phagocytosis, leucocyte activation and migration. Nevertheless, research is hampered by absence of endogenous ligands, unknown activity of generated antibodies and non-identified signalling pathways. Yet, based on their membrane localisation and important function, aGPCRs could be novel drug targets to modulate leucocyte function.

Adhesion G Protein-Coupled Receptors: From In Vitro Pharmacology to In Vivo Mechanisms.

The adhesion family of G protein-coupled receptors (aGPCRs) comprises 33 members in humans. aGPCRs are characterized by their enormous size and complex modular structures. While the physiologic importance of many aGPCRs has been clearly demonstrated in recent years, the underlying molecular functions have only recently begun to be elucidated. In this minireview, we present an overview of our current knowledge on aGPCR activation and signal transduction with a focus on the latest findings regarding the interplay between ligand binding, mechanical force, and the tethered agonistic Stachel sequence, as well as implications on translational approaches that may derive from understanding aGPCR pharmacology.

The Viral G Protein-Coupled Receptor ORF74 Hijacks ß-Arrestins for Endocytic Trafficking in Response to Human Chemokines.

Kaposi's sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of the angioproliferative tumor Kaposi's sarcoma. Whereas G protein-dependent signaling of ORF74 has been the subject of several studies, the interaction of this viral GPCR with ß-arrestins has hitherto not been investigated. Bioluminescence resonance energy transfer experiments demonstrate that ORF74 recruits ß-arrestins and subsequently internalizes in response to human CXCL1 and CXCL8, but not CXCL10. Internalized ORF74 traffics via early endosomes to recycling and late endosomes. Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for ß-arrestin recruitment and subsequent endocytic trafficking. Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74.

Combinatorial Consensus Scoring for Ligand-Based Virtual Fragment Screening: A Comparative Case Study for Serotonin 5-HT(3)A, Histamine H(1), and Histamine H(4) Receptors.

In the current study we have evaluated the applicability of ligand-based virtual screening (LBVS) methods for the identification of small fragment-like biologically active molecules using different similarity descriptors and different consensus scoring approaches. For this purpose, we have evaluated the performance of 14 chemical similarity descriptors in retrospective virtual screening studies to discriminate fragment-like ligands of three membrane-bound receptors from fragments that are experimentally determined to have no affinity for these proteins (true inactives). We used a complete fragment affinity data set of experimentally determined ligands and inactives for two G protein-coupled receptors (GPCRs), the histamine H1 receptor (H1R) and the histamine H4 receptor (H4R), and one ligand-gated ion channel (LGIC), the serotonin receptor (5-HT3AR), to validate our retrospective virtual screening studies. We have exhaustively tested consensus scoring strategies that combine the results of multiple actives (group fusion) or combine different similarity descriptors (similarity fusion), and for the first time systematically evaluated different combinations of group fusion and similarity fusion approaches. Our studies show that for these three case study protein targets both consensus scoring approaches can increase virtual screening enrichments compared to single chemical similarity search methods. Our cheminformatics analyses recommend to use a combination of both group fusion and similarity fusion for prospective ligand-based virtual fragment screening.

Bispyrimidines as potent histamine H(4) receptor ligands: delineation of structure-activity relationships and detailed H(4) receptor binding mode.

The basic methylpiperazine moiety is considered a necessary substructure for high histamine H4 receptor (H4R) affinity. This moiety is however also the metabolic hot spot for various classes of H4R ligands (e.g., indolcarboxamides and pyrimidines). We set out to investigate whether mildly basic 2-aminopyrimidines in combination with the appropriate linker can serve as a replacement for the methylpiperazine moiety. In the series of 2-aminopyrimidines, the introduction of an additional 2-aminopyrimidine moiety in combination with the appropriate linker lead to bispyrimidines displaying pKi values for binding the human H4R up to 8.2. Furthermore, the methylpiperazine replacement results in compounds with improved metabolic properties. The attempt to transfer the knowledge generated in the class of bispyrimidines to the indolecarboxamides failed. Combining the derived structure-activity relationships with homology modeling leads to new detailed insights in the molecular aspects of ligand-H4R binding in general and the binding mode of the described bispyrimidines in specific.

Small and colorful stones make beautiful mosaics: fragment-based chemogenomics.

Smaller stones with a wide variety of colors make a higher resolution mosaic. In much the same way, smaller chemical entities that are structurally diverse are better able to interrogate protein binding sites. This feature article describes the construction of a diverse fragment library and an analysis of the screening of six representative protein targets belonging to three diverse target classes (G protein-coupled receptors ADRB2, H1R, H3R, and H4R, the ligand-gated ion channel 5-HT3R, and the kinase PKA) using chemogenomics approaches. The integration of experimentally determined bioaffinity profiles across related and unrelated protein targets and chemogenomics analysis of fragment binding and protein structure allow the identification of: (i) unexpected similarities and differences in ligand binding properties, and (ii) subtle ligand affinity and selectivity cliffs. With a wealth of fragment screening data being generated in industry and academia, such approaches will contribute to a more detailed structural understanding of ligand-protein interactions.

Virtual fragment screening: discovery of histamine H3 receptor ligands using ligand-based and protein-based molecular fingerprints.

Virtual fragment screening (VFS) is a promising new method that uses computer models to identify small, fragment-like biologically active molecules as useful starting points for fragment-based drug discovery (FBDD). Training sets of true active and inactive fragment-like molecules to construct and validate target customized VFS methods are however lacking. We have for the first time explored the possibilities and challenges of VFS using molecular fingerprints derived from a unique set of fragment affinity data for the histamine H(3) receptor (H(3)R), a pharmaceutically relevant G protein-coupled receptor (GPCR). Optimized FLAP (Fingerprints of Ligands and Proteins) models containing essential molecular interaction fields that discriminate known H(3)R binders from inactive molecules were successfully used for the identification of new H(3)R ligands. Prospective virtual screening of 156,090 molecules yielded a high hit rate of 62% (18 of the 29 tested) experimentally confirmed novel fragment-like H(3)R ligands that offer new potential starting points for the design of H(3)R targeting drugs. The first construction and application of customized FLAP models for the discovery of fragment-like biologically active molecules demonstrates that VFS is an efficient way to explore protein-fragment interaction space in silico.

Analysis of multiple histamine H4 receptor compound classes uncovers Gαi protein- and ß-arrestin2-biased ligands.

After the recent description of ß-arrestin2 recruitment to the human histamine H4 receptor (hH4R) in response to the well known H4R antagonist 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methyl-piperazine (JNJ 7777120), we evaluated in this study the efficacy of 31 known hH4R ligands to induce Gα(i) protein signaling and ß-arrestin2 recruitment by the hH4R. The selected hH(4)R ligands belong to nine different structural classes that partly cover (pre)clinical trial candidates. We have identified hH4R ligands with a significant bias for the Gα(i) protein or ß-arrestin2 pathway on the basis of efficacy differences. In addition, hH4R antagonists that did not show positive efficacy in either functional readouts were found. A common trend in pathway preference for the nine different ligand classes could not be observed. In particular, the isothiourea class shows very diverse results, varying from Gα(i) protein-biased or ß-arrestin2-biased to nonbiased antagonists upon minor structural changes. The identified biased hH4R ligands are important pharmacological tools to unravel the significance of biased hH4R signaling in H4R pharmacology.

Development of a profiling strategy for metabolic mixtures by combining chromatography and mass spectrometry with cell-based GPCR signaling.

In this study, we developed an in-line methodology that combines analytical with pharmacological techniques to characterize metabolites of human histamine H(4) receptor (hH(4)R) ligands. Liquid chromatographic separation of metabolic mixtures is coupled to high-resolution fractionation into 96- or 384-well plates and directly followed by a cell-based reporter gene assay to measure receptor signaling. The complete methodology was designed, optimized, validated, and ultimately miniaturized into a high-density well plate format. Finally, the methodology was demonstrated in a metabolic profiling setting for three hH(4)R lead compounds and the drug clozapine. This new methodology comprises integrated analytical separations, mass spectrometry, and a cell-based signal transduction-driven reporter gene assay that enables the implementation of comprehensive metabolic profiling earlier in the drug discovery process.

Molecular pharmacology of histamine H4 receptors.

The histamine H4 receptor (H4R) is the youngest member of the histamine receptor family. Based on its predominant expression pattern in hematopoietic cells, the H4R is considered to be an interesting drug target for inflammatory disorders such as allergy and asthma. Since the identification and cloning of the H4R in 2000, drug discovery programs boosted the development of various H4R (specific) ligands. Differences between H4R orthologs in combination with available three-dimensional G protein-coupled receptor (GPCR) models have guided site-directed mutagenesis studies to gain insight in ligand binding and receptor activation. In addition, ongoing characterization of H4R-mediated signaling in transfected and native cells contributes to further unravel the (patho-) physiological functions of H4Rs.

Molecular determinants of ligand binding modes in the histamine H(4) receptor: linking ligand-based three-dimensional quantitative structure-activity relationship (3D-QSAR) models to in silico guided receptor mutagenesis studies.

The histamine H(4) receptor (H(4)R) is a G protein-coupled receptor (GPCR) that plays an important role in inflammation. Similar to the homologous histamine H(3) receptor (H(3)R), two acidic residues in the H(4)R binding pocket, D(3.32) and E(5.46), act as essential hydrogen bond acceptors of positively ionizable hydrogen bond donors in H(4)R ligands. Given the symmetric distribution of these complementary pharmacophore features in H(4)R and its ligands, different alternative ligand binding mode hypotheses have been proposed. The current study focuses on the elucidation of the molecular determinants of H(4)R-ligand binding modes by combining (3D) quantitative structure-activity relationship (QSAR), protein homology modeling, molecular dynamics simulations, and site-directed mutagenesis studies. We have designed and synthesized a series of clobenpropit (N-(4-chlorobenzyl)-S-[3-(4(5)-imidazolyl)propyl]isothiourea) derivatives to investigate H(4)R-ligand interactions and ligand binding orientations. Interestingly, our studies indicate that clobenpropit (2) itself can bind to H(4)R in two distinct binding modes, while the addition of a cyclohexyl group to the clobenpropit isothiourea moiety allows VUF5228 (5) to adopt only one specific binding mode in the H(4)R binding pocket. Our ligand-steered, experimentally supported protein modeling method gives new insights into ligand recognition by H(4)R and can be used as a general approach to elucidate the structure of protein-ligand complexes.

Fragment library screening reveals remarkable similarities between the G protein-coupled receptor histamine H4 and the ion channel serotonin 5-HT3A.

A fragment library was screened against the G protein-coupled histamine H(4) receptor (H(4)R) and the ligand-gated ion channel serotonin 5-HT(3A) (5-HT(3A)R). Interestingly, significant overlap was found between H(4)R and 5-HT(3A)R hit sets. The data indicates that dual active H(4)R and 5 HT(3A)R fragments have a higher complexity than the selective compounds which has important implications for chemical genomics approaches. The results of our fragment-based library screening study illustrate similarities in ligand recognition between H(4)R and 5-HT(3A)R and have important consequences for selectivity profiling in ongoing drug discovery efforts on H(4)R and 5-HT(3A)R. The affinity profiles of our fragment screening studies furthermore match the chemical properties of the H(4)R and 5-HT(3A)R binding sites and can be used to define molecular interaction fingerprints to guide the in silico prediction of protein-ligand interactions and structure.

Real-time visualization of heterotrimeric G protein Gq activation in living cells.

BACKGROUND: Gq is a heterotrimeric G protein that plays an important role in numerous physiological processes. To delineate the molecular mechanisms and kinetics of signalling through this protein, its activation should be measurable in single living cells. Recently, fluorescence resonance energy transfer (FRET) sensors have been developed for this purpose. RESULTS: In this paper, we describe the development of an improved FRET-based Gq activity sensor that consists of a yellow fluorescent protein (YFP)-tagged Gγ2 subunit and a Gαq subunit with an inserted monomeric Turquoise (mTurquoise), the best cyan fluorescent protein variant currently available. This sensor enabled us to determine, for the first time, the kon (2/s) of Gq activation. In addition, we found that the guanine nucleotide exchange factor p63RhoGEF has a profound effect on the number of Gq proteins that become active upon stimulation of endogenous histamine H1 receptors. The sensor was also used to measure ligand-independent activation of the histamine H1 receptor (H1R) upon addition of a hypotonic stimulus. CONCLUSIONS: Our observations reveal that the application of a truncated mTurquoise as donor and a YFP-tagged Gγ2 as acceptor in FRET-based Gq activity sensors substantially improves their dynamic range. This optimization enables the real-time single cell quantification of Gq signalling dynamics, the influence of accessory proteins and allows future drug screening applications by virtue of its sensitivity.

Triazole ligands reveal distinct molecular features that induce histamine H4 receptor affinity and subtly govern H4/H3 subtype selectivity.

The histamine H(3) (H(3)R) and H(4) (H(4)R) receptors attract considerable interest from the medicinal chemistry community. Given their relatively high homology yet widely differing therapeutic promises, ligand selectivity for the two receptors is crucial. We interrogated H(4)R/H(3)R selectivities using ligands with a [1,2,3]triazole core. Cu(I)-assisted "click chemistry" was used to assemble diverse [1,2,3]triazole compounds (6a-w and 7a-f), many containing a peripheral imidazole group. The imidazole ring posed some problems in the click chemistry putatively due to Cu(II) coordination, but Boc protection of the imidazole and removal of oxygen from the reaction mixture provided effective strategies. Pharmacological studies revealed two monosubstituted imidazoles (6h,p) with <10 nM H(4)R affinities and >10-fold H(4)R/H(3)R selectivity. Both compounds possess a cycloalkylmethyl group and appear to target a lipophilic pocket in H(4)R with high steric precision. The use of the [1,2,3]triazole scaffold is further demonstrated by the notion that simple changes in spacer length or peripheral groups can reverse the selectivity toward H(3)R. Computational evidence is provided to account for two key selectivity switches and to pinpoint a lipophilic pocket as an important handle for H(4)R over H(3)R selectivity.