The Hidden Role of Non-Canonical Amyloid ß Isoforms in Alzheimer's Disease.

Recent advances have placed the pro-inflammatory activity of amyloid ß (Aß) on microglia cells as the focus of research on Alzheimer's Disease (AD). Researchers are confronted with an astonishing spectrum of over 100 different Aß variants with variable length and chemical modifications. With the exception of Aß1-42 and Aß1-40, the biological significance of most peptides for AD is as yet insufficiently understood. We therefore aim to provide a comprehensive overview of the contributions of these neglected Aß variants to microglia activation. First, the impact of Aß receptors, signaling cascades, scavenger mechanisms, and genetic variations on the physiological responses towards various Aß species is described. Furthermore, we discuss the importance of different types of amyloid precursor protein processing for the generation of these Aß variants in microglia, astrocytes, oligodendrocytes, and neurons, and highlight how alterations in secondary structures and oligomerization affect Aß neurotoxicity. In sum, the data indicate that gene polymorphisms in Aß-driven signaling pathways in combination with the production and activity of different Aß variants might be crucial factors for the initiation and progression of different forms of AD. A deeper assessment of their interplay with glial cells may pave the way towards novel therapeutic strategies for individualized medicine.

Amyloid beta and its naturally occurring N-terminal variants are potent activators of human and mouse formyl peptide receptor 1.

Formyl peptide receptors (FPRs) may contribute to inflammation in Alzheimer's disease through interactions with neuropathological Amyloid beta (Aß) peptides. Previous studies reported activation of FPR2 by Aß1-42, but further investigation of other FPRs and Aß variants is needed. This study provides a comprehensive overview of the interactions of mouse and human FPRs with different physiologically relevant Aß-peptides using transiently transfected cells in combination with calcium imaging. We observed that, in addition to hFPR2, all other hFPRs also responded to Aß1-42, Aß1-40, and the naturally occurring variants Aß11-40 and Aß17-40. Notably, Aß11-40 and Aß17-40 are very potent activators of mouse and human FPR1, acting at nanomolar concentrations. Buffer composition and aggregation state are extremely crucial factors that critically affect the interaction of Aß with different FPR subtypes. To investigate the physiological relevance of these findings, we examined the effects of Aß11-40 and Aß17-40 on the human glial cell line U87. Both peptides induced a strong calcium flux at concentrations that are very similar to those obtained in experiments for hFPR1 in HEK cells. Further immunocytochemistry, qPCR, and pharmacological experiments verified that these responses were primarily mediated through hFPR1. Chemotaxis experiments revealed that Aß11-40 but not Aß17-40 evoked cell migration, which argues for a functional selectivity of different Aß peptides. Together, these findings provide the first evidence that not only hFPR2 but also hFPR1 and hFPR3 may contribute to neuroinflammation in Alzheimer's disease through an interaction with different Aß variants.

Emerging contributions of formyl peptide receptors to neurodegenerative diseases.

Inflammation is a central element of many neurodegenerative diseases. Formyl peptide receptors (FPRs) can trigger several receptor-dependent signal transduction pathways that play a key role in neuroinflammation and neurodegeneration. They are chemotactic receptors that help to regulate pro- and anti-inflammatory responses in most mammals. FPRs are primarily expressed in the immune and nervous systems where they interact with a complex pattern of pathogen-derived and host-endogenous molecules. Mounting evidence points towards a contribution of FPRs - via neuropathological ligands such as Amyloid beta, and neuroprotective ligands such as Humanin, Lipoxin A4, and Annexin A1 - to multiple pathological aspects of neurodegenerative diseases. In this review, we aim to summarize the interplay of FPRs with neuropathological and neuroprotective ligands. Next, we depict their capability to trigger a number of ligand-dependent cell signaling pathways and their potential to interact with additional intracellular cofactors. Moreover, we highlight first studies, demonstrating that a pharmacological inhibition of FPRs helps to ameliorate neuroinflammation, which may pave the way towards novel therapeutic strategies.

Chemosensory Cell-Derived Acetylcholine Drives Tracheal Mucociliary Clearance in Response to Virulence-Associated Formyl Peptides.

Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.

Bacterial MgrB peptide activates chemoreceptor Fpr3 in mouse accessory olfactory system and drives avoidance behaviour.

Innate immune chemoreceptors of the formyl peptide receptor (Fpr) family are expressed by vomeronasal sensory neurons (VSNs) in the accessory olfactory system. Their biological function and coding mechanisms remain unknown. We show that mouse Fpr3 (Fpr-rs1) recognizes the core peptide motif f-MKKFRW that is predominantly present in the signal sequence of the bacterial protein MgrB, a highly conserved regulator of virulence and antibiotic resistance in Enterobacteriaceae. MgrB peptide can be produced and secreted by bacteria, and is selectively recognized by a subset of VSNs. Exposure to the peptide also stimulates VSNs in freely behaving mice and drives innate avoidance. Our data shows that Fpr3 is required for neuronal detection and avoidance of peptides derived from a conserved master virulence regulator of enteric bacteria.

Trpc5 deficiency causes hypoprolactinemia and altered function of oscillatory dopamine neurons in the arcuate nucleus.

Dopamine neurons of the hypothalamic arcuate nucleus (ARC) tonically inhibit the release of the protein hormone prolactin from lactotropic cells in the anterior pituitary gland and thus play a central role in prolactin homeostasis of the body. Prolactin, in turn, orchestrates numerous important biological functions such as maternal behavior, reproduction, and sexual arousal. Here, we identify the canonical transient receptor potential channel Trpc5 as an essential requirement for normal function of dopamine ARC neurons and prolactin homeostasis. By analyzing female mice carrying targeted mutations in the Trpc5 gene including a conditional Trpc5 deletion, we show that Trpc5 is required for maintaining highly stereotyped infraslow membrane potential oscillations of dopamine ARC neurons. Trpc5 is also required for eliciting prolactin-evoked tonic plateau potentials in these neurons that are part of a regulatory feedback circuit. Trpc5 mutant females show severe prolactin deficiency or hypoprolactinemia that is associated with irregular reproductive cyclicity, gonadotropin imbalance, and impaired reproductive capabilities. These results reveal a previously unknown role for the cation channel Trpc5 in prolactin homeostasis of female mice and provide strategies to explore the genetic basis of reproductive disorders and other malfunctions associated with defective prolactin regulation in humans.

A calcium optimum for cytotoxic T lymphocyte and natural killer cell cytotoxicity.

KEY POINTS: Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to eliminate cancer cells. We analysed the Ca2+ dependence of CTL and NK cell cytotoxicity and found that in particular CTLs have a very low optimum of [Ca2+ ]i (between 122 and 334 nm) and [Ca2+ ]o (between 23 and 625 µm) for efficient cancer cell elimination, well below blood plasma Ca2+ levels. As predicted from these results, partial down-regulation of the Ca2+ channel Orai1 in CTLs paradoxically increases perforin-dependent cancer cell killing. Lytic granule release at the immune synapse between CTLs and cancer cells has a Ca2+ optimum compatible with this low Ca2+ optimum for efficient cancer cell killing, whereas the Ca2+ optimum for CTL migration is slightly higher and proliferation increases monotonously with increasing [Ca2+ ]o . We propose that a partial inhibition of Ca2+ signals by specific Orai1 blockers at submaximal concentrations could contribute to tumour elimination. ABSTRACT: Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to protect the human body against cancer. Ca2+ is a key metabolic factor for lymphocyte function and cancer homeostasis. We analysed the Ca2+ dependence of CTL and NK cell cytotoxicity against cancer cells and found that CTLs have a bell-shaped Ca2+ dependence with an optimum for cancer cell elimination at rather low [Ca2+ ]o (23-625 µm) and [Ca2+ ]i (122-334 nm). This finding predicts that a partial inhibition of Orai1 should increase (rather than decrease) cytotoxicity of CTLs at [Ca2+ ]o higher than 625 µm. We tested this hypothesis in CTLs and indeed found that partial down-regulation of Orai1 by siRNA increases the efficiency of cancer cell killing. We found two mechanisms that may account for the Ca2+ optimum of cancer cell killing: (1) migration velocity and persistence have a moderate optimum between 500 and 1000 µm [Ca2+ ]o in CTLs, and (2) lytic granule release at the immune synapse between CTLs and cancer cells is increased at 146 µm compared to 3 or 800 µm, compatible with the Ca2+ optimum for cancer cell killing. It has been demonstrated in many cancer cell types that Orai1-dependent Ca2+ signals enhance proliferation. We propose that a decrease of [Ca2+ ]o or partial inhibition of Orai1 activity by selective blockers in the tumour microenvironment could efficiently reduce cancer growth by simultaneously increasing CTL and NK cell cytotoxicity and decreasing cancer cell proliferation.

Organization and Plasticity of Sodium Channel Expression in the Mouse Olfactory and Vomeronasal Epithelia.

To understand the molecular basis of neuronal excitation in the mammalian olfactory system, we conducted a systematic analysis of the organization of voltage-gated sodium (Nav) channel subunits in the main olfactory epithelium (MOE) and vomeronasal organ (VNO) of adult mice. We also analyzed changes in Nav channel expression during development in these two systems and during regeneration of the MOE. Quantitative PCR shows that Nav1.7 is the predominant isoform in both adult MOE and VNO. We detected pronounced immunoreactivity for Nav1.7 and Nav1.3 in axons of olfactory and vomeronasal sensory neurons (VSNs). Analysis of Nav1.2 and Nav1.6 revealed an unexpected subsystem-specific distribution. In the MOE, these Nav channels are absent from olfactory sensory neurons (OSNs) but present in non-neuronal olfactory cell types. In the VNO, Nav1.2 and Nav1.6 are confined to VSNs, with Nav1.2-immunoreactive somata solely present in the basal layer of the VNO. The subcellular localization of Nav1.3 and Nav1.7 in OSNs can change dramatically during periods of heightened plasticity in the MOE. During the first weeks of development and during regeneration of the olfactory epithelium following chemical lesion, expression of Nav1.3 and Nav1.7 is transiently enhanced in the somata of mature OSNs. Our results demonstrate a highly complex organization of Nav channel expression in the mouse olfactory system, with specific commonalities but also differences between the MOE and the VNO. On the basis of their subcellular localization, Nav1.3 and Nav1.7 should play major roles in action potential propagation in both MOE and VNO, whereas Nav1.2 and Nav1.6 are specific to the function of VSNs. The plasticity of Nav channel expression in OSNs during early development and recovery from injury could reflect important physiological requirements in a variety of activity-dependent mechanisms.

Trpm5 expression in the olfactory epithelium.

The Ca2+-activated monovalent cation channel Trpm5 is a key element in chemotransduction of taste receptor cells of the tongue, but the extent to which Trpm5 channels are expressed in olfactory sensory neurons (OSNs) of the main olfactory epithelium (MOE) of adult mice as part of a specific pheromonal detection system is debated. Here, we used a novel Trpm5-IRES-Cre knockin strain to drive Cre recombinase expression, employed previously validated Trpm5 antibodies, performed in situ hybridization experiments to localize Trpm5 RNA, and searched extensively for Trpm5 splice variants in genetically-labeled, Trpm5-expressing MOE cells. In contrast to previous reports, we find no evidence for the existence in adult mouse OSNs of the classical Trpm5 channel known from taste cells. We show that Trpm5-expressing adult OSNs express a novel Trpm5 splice variant, Trpm5-9, that is unlikely to form a functional cation channel by itself. We also demonstrate that Trpm5 is transiently expressed in a subpopulation of mature OSNs in the embryonic olfactory epithelium, indicating that Trpm5 channels could play a specific role in utero during a narrow developmental time window. Ca2+ imaging with GCaMP3 under the control of the Trpm5-IRES-Cre allele using a newly developed MOE wholemount preparation of the adult olfactory epithelium reveals that Trpm5-GCaMP3 OSNs comprise a heterogeneous group of sensory neurons many of which can detect general odorants. Together, these studies are essential for understanding the role of transient receptor potential channels in mammalian olfaction.

The sensing of bacteria: emerging principles for the detection of signal sequences by formyl peptide receptors.

The ability to detect specific chemical signatures released by bacteria and other microorganisms is a fundamental feature of immune defense against pathogens. There is increasing evidence that chemodetection of such microorganism-associated molecular patterns (MAMPs) occurs at many places in the body including specific sets of chemosensory neurons in the mammalian nose. Formyl peptide receptors (FPRs) are a unique family of G protein-coupled receptors (GPCRs) that can detect the presence of bacteria and function as chemotactic receptors. Here, we highlight the recent discovery of a vast family of natural FPR agonists, the bacterial signal peptides (or signal sequences), thus providing new insight into the molecular mechanisms of bacterial sensing by human and mouse FPRs. Signal peptides in bacteria are formylated, N-terminal protein signatures required for directing the transfer of proteins through the plasma membrane. After their cleavage and release, signal peptides are available for FPR detection and thus provide a previously unrecognized MAMP. With over 170 000 predicted sequences, bacterial signal peptides represent one of the largest families of GPCR ligands and one of the most complex classes of natural activators of the innate immune system. By recognizing a conserved three-dimensional peptide motif, FPRs employ an unusual detection mechanism that combines structural promiscuity with high specificity and sensitivity, thus solving the problem of detecting thousands of distinct sequences yet maintaining selectivity. How signal peptides are released by bacteria and sensed by GPCRs and how these processes shape the responses of other cells and whole organisms represents an important topic for future research.

Strain-specific Loss of Formyl Peptide Receptor 3 in the Murine Vomeronasal and Immune Systems.

Formyl peptide receptor 3 (Fpr3, also known as Fpr-rs1) is a G protein-coupled receptor expressed in subsets of sensory neurons of the mouse vomeronasal organ, an olfactory substructure essential for social recognition. Fpr3 has been implicated in the sensing of infection-associated olfactory cues, but its expression pattern and function are incompletely understood. To facilitate visualization of Fpr3-expressing cells, we generated and validated two new anti-Fpr3 antibodies enabling us to analyze acute Fpr3 protein expression. Fpr3 is not only expressed in murine vomeronasal sensory neurons but also in bone marrow cells, the primary source for immune cell renewal, and in mature neutrophils. Consistent with the notion that Fpr3 functions as a pathogen sensor, Fpr3 expression in the immune system is up-regulated after stimulation with a bacterial endotoxin (lipopolysaccharide). These results strongly support a dual role for Fpr3 in both vomeronasal sensory neurons and immune cells. We also identify a large panel of mouse strains with severely altered expression and function of Fpr3, thus establishing the existence of natural Fpr3 knock-out strains. We attribute distinct Fpr3 expression in these strains to the presence or absence of a 12-nucleotide in-frame deletion (Fpr3Δ424-435). In vitro calcium imaging and immunofluorescence analyses demonstrate that the lack of four amino acids leads to an unstable, truncated, and non-functional receptor protein. The genome of at least 19 strains encodes a non-functional Fpr3 variant, whereas at least 13 other strains express an intact receptor. These results provide a foundation for understanding the in vivo function of Fpr3.

Recognition of bacterial signal peptides by mammalian formyl peptide receptors: a new mechanism for sensing pathogens.

Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system.

Formyl peptide receptors from immune and vomeronasal system exhibit distinct agonist properties.

The formyl peptide receptor (Fpr) family is well known for its contribution to immune defense against pathogens in human and rodent leukocytes. Recently, several structurally related members of these receptors were discovered in sensory neurons of the mouse vomeronasal organ (VNO), key detectors of pheromones and related semiochemicals. Although the biological role of vomeronasal Fprs is not yet clear, the known contribution of other Fprs to host immune defense suggested that they could contribute to vomeronasal pathogen sensing. Precise knowledge about the agonist properties of mouse Fprs is required to determine their function. We expressed all seven mouse and three human Fprs using an in vitro system and tested their activation with 32 selected compounds by conducting high throughput calcium measurements. We found an intriguing functional conservation between human and mouse immune Fprs that is most likely a consequence of closely similar biological constraints. By contrast, our data suggest a neofunctionalization of the vomeronasal Fprs. We show that the vomeronasal receptor mFpr-rs1 can be activated robustly by W-peptide and structural derivatives but not by other typical ligands of immune Fprs. mFpr-rs1 exhibits a stereo-selective preference for peptides containing d-amino acids. The same peptide motifs are contained in pathogenic microorganisms. Thus, the ligand profile of mFpr-rs1 is consistent with a role in vomeronasal pathogen sensing.

Mammalian-specific OR37 receptors are differentially activated by distinct odorous fatty aldehydes.

The capacity of the mammalian olfactory system to detect an enormous collection of different chemical compounds is based on a large repertoire of odorant receptors (ORs). A small group of these ORs, the OR37 family, is unique due to a variety of special features. Members of this subfamily are exclusively found in mammals, they share a high degree of sequence homology and are highly conserved during evolution. It is still elusive which odorants may activate these atypical receptors. We have reasoned that compounds from skin, hairs, or skin glands might be potential candidates. We have exposed mice to such compounds and monitored activation of glomeruli through the expression of the activity marker c-fos in juxtaglomerular cells surrounding ventrally positioned glomeruli in the olfactory bulb (OB). Employing this methodology it was found that stimulation with long-chain alkanes elicits activation in the ventral part of the OB, however, none of the OR37 glomeruli. Analyses of long-chain hydrocarbon compounds with different functional groups revealed that long-chain aliphatic aldehydes elicited an activation of defined OR37 glomeruli, each of them responding preferentially to an aldehyde with different chain lengths. These results indicate that OR37 receptors may be tuned to distinct fatty aldehydes with a significant degree of ligand specificity.

G protein G(alpha)o is essential for vomeronasal function and aggressive behavior in mice.

The rodent vomeronasal organ (VNO) mediates the regulation of species-specific and interspecies social behaviors. We have used gene targeting to examine the role of the G protein Gαo, encoded by the gene Gnao1, in vomeronasal function. We used the Cre-loxP system to delete Gαo in those cells that express olfactory marker protein, which includes all vomeronasal sensory neurons of the basal layer of the VNO sensory epithelium. Using electrophysiology and calcium imaging, we show that the conditional null mice exhibit strikingly reduced sensory responses in V2R receptor-expressing vomeronasal sensory neurons to specific molecular cues, including MHC1 antigens, major urinary proteins, and exocrine gland-secreting peptide. Gαo is also vital for vomeronasal sensing of two N-formylated mitochondrially encoded peptides derived from NADH dehydrogenase 1. Furthermore, we show that Gαo is an essential requirement for the display of male-male territorial aggression as well as maternal aggression in mice. Finally, we show that Gαo-dependent maternal aggression can be induced by major urinary proteins. These cellular and behavioral phenotypes identify Gαo as the primary G-protein α-subunit mediating the detection of peptide and protein pheromones by sensory neurons of the VNO.

Genomic, genetic and functional dissection of bitter taste responses to artificial sweeteners.

Bitter taste perception is initiated by TAS2R receptors, which respond to agonists by triggering depolarization of taste bud cells. Mutations in TAS2Rs are known to affect taste phenotypes by altering receptor function. Evidence that TAS2Rs overlap in ligand specificity suggests that they may also contribute joint effects. To explore this aspect of gustation, we examined bitter perception of saccharin and acesulfame K, widely used artificial sweeteners with aversive aftertastes. Both substances are agonists of TAS2R31 and -43, which belong to a five-member subfamily (TAS2R30-46) responsive to a diverse constellation of compounds. We analyzed sequence variation and linkage structure in the ∼140 kb genomic region encoding TAS2R30-46, taste responses to the two sweeteners in subjects, and functional characteristics of receptor alleles. Whole-gene sequences from TAS2R30-46 in 60 Caucasian subjects revealed extensive diversity including 34 missense mutations, two nonsense mutations and high-frequency copy-number variants. Thirty markers, including non-synonymous variants in all five genes, were associated (P< 0.001) with responses to saccharin and acesulfame K. However, linkage disequilibrium (LD) in the region was high (D', r(2) > 0.95). Haplotype analyses revealed that most associations were spurious, arising from LD with variants in TAS2R31. In vitro assays confirmed the functional importance of four TAS2R31 mutations, which had independent effects on receptor response. The existence of high LD spanning functionally distinct TAS2R loci predicts that bitter taste responses to many compounds will be strongly correlated even when they are mediated by different genes. Integrative approaches combining phenotypic, genetic and functional analysis will be essential in dissecting these complex relationships.

Loss-of-function mutations in sodium channel Nav1.7 cause anosmia.

Loss of function of the gene SCN9A, encoding the voltage-gated sodium channel Na(v)1.7, causes a congenital inability to experience pain in humans. Here we show that Na(v)1.7 is not only necessary for pain sensation but is also an essential requirement for odour perception in both mice and humans. We examined human patients with loss-of-function mutations in SCN9A and show that they are unable to sense odours. To establish the essential role of Na(v)1.7 in odour perception, we generated conditional null mice in which Na(v)1.7 was removed from all olfactory sensory neurons. In the absence of Na(v)1.7, these neurons still produce odour-evoked action potentials but fail to initiate synaptic signalling from their axon terminals at the first synapse in the olfactory system. The mutant mice no longer display vital, odour-guided behaviours such as innate odour recognition and avoidance, short-term odour learning, and maternal pup retrieval. Our study creates a mouse model of congenital general anosmia and provides new strategies to explore the genetic basis of the human sense of smell.

Oligomerization of TAS2R bitter taste receptors.

A family of 25 G protein-coupled receptors, TAS2Rs, mediates bitter taste in humans. Many of the members of this family are coexpressed in a subpopulation of taste receptor cells on the tongue, thereby allowing the possibility of receptor-receptor interactions with potential influences on their function. In this study, we used several experimental approaches to investigate whether TAS2Rs can form oligomers and if this has an effect on receptor function. Coimmunoprecipitations clearly demonstrated that TAS2Rs can physically interact in HEK293T cells. Further bioluminescence resonance energy transfer analysis of all 325 possible binary combinations of TAS2Rs established that the vast majority of TAS2R pairs form oligomers, both homomers and heteromers. Subsequent screenings of coexpressed bitter receptors with 104 different tastants did not reveal any heteromer-specific agonists. Additional studies also showed no obvious influence of TAS2R hetero-oligomerization on plasma membrane localization or pharmacological properties of the receptors. Thus, our results show that receptor oligomerization occurs between TAS2R bitter taste receptors; however, functional consequences of hetero-oligomerization were not obvious.