cAMP binding to closed pacemaker ion channels is cooperative.

The cooperative action of the subunits in oligomeric receptors enables fine-tuning of receptor activation, as demonstrated for the regulation of voltage-activated HCN pacemaker ion channels by relating cAMP binding to channel activation in ensemble signals. HCN channels generate electric rhythmicity in specialized brain neurons and cardiomyocytes. There is conflicting evidence on whether binding cooperativity does exist independent of channel activation or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides. Here, we show positive cooperativity in ligand binding to closed HCN2 channels in native cell membranes by following the binding of individual fluorescence-labeled cAMP molecules. Kinetic modeling reveals that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in closed HCN2 channels not pre-activated by voltage is already cooperative. Hence, cooperativity is not causally linked to channel activation by voltage. Our analysis also shows that single-molecule binding measurements at equilibrium can quantify cooperativity in ligand binding to receptors in native membranes.

Latozinemab, a novel progranulin-elevating therapy for frontotemporal dementia.

BACKGROUND: Heterozygous loss-of-function mutations in the progranulin (PGRN) gene (GRN) cause a reduction in PGRN and lead to the development of frontotemporal dementia (FTD-GRN). PGRN is a secreted lysosomal chaperone, immune regulator, and neuronal survival factor that is shuttled to the lysosome through multiple receptors, including sortilin. Here, we report the characterization of latozinemab, a human monoclonal antibody that decreases the levels of sortilin, which is expressed on myeloid and neuronal cells and shuttles PGRN to the lysosome for degradation, and blocks its interaction with PGRN. METHODS: In vitro characterization studies were first performed to assess the mechanism of action of latozinemab. After the in vitro studies, a series of in vivo studies were performed to assess the efficacy of a mouse-cross reactive anti-sortilin antibody and the pharmacokinetics, pharmacodynamics, and safety of latozinemab in nonhuman primates and humans. RESULTS: In a mouse model of FTD-GRN, the rodent cross-reactive anti-sortilin antibody, S15JG, decreased total sortilin levels in white blood cell (WBC) lysates, restored PGRN to normal levels in plasma, and rescued a behavioral deficit. In cynomolgus monkeys, latozinemab decreased sortilin levels in WBCs and concomitantly increased plasma and cerebrospinal fluid (CSF) PGRN by 2- to threefold. Finally, in a first-in-human phase 1 clinical trial, a single infusion of latozinemab caused a reduction in WBC sortilin, tripled plasma PGRN and doubled CSF PGRN in healthy volunteers, and restored PGRN to physiological levels in asymptomatic GRN mutation carriers. CONCLUSIONS: These findings support the development of latozinemab for the treatment of FTD-GRN and other neurodegenerative diseases where elevation of PGRN may be beneficial. Trial registration ClinicalTrials.gov, NCT03636204. Registered on 17 August 2018, https://clinicaltrials.gov/ct2/show/NCT03636204 .

Uncoupling of Voltage- and Ligand-Induced Activation in HCN2 Channels by Glycine Inserts.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetramers that generate electrical rhythmicity in special brain neurons and cardiomyocytes. The channels are activated by membrane hyperpolarization. The binding of cAMP to the four available cyclic nucleotide-binding domains (CNBD) enhances channel activation. We analyzed in the present study the mechanism of how the effect of cAMP binding is transmitted to the pore domain. Our strategy was to uncouple the C-linker (CL) from the channel core by inserting one to five glycine residues between the S6 gate and the A'-helix (constructs 1G to 5G). We quantified in full-length HCN2 channels the resulting functional effects of the inserted glycines by current activation as well as the structural dynamics and statics using molecular dynamics simulations and Constraint Network Analysis. We show functionally that already in 1G the cAMP effect on activation is lost and that with the exception of 3G and 5G the concentration-activation relationships are shifted to depolarized voltages with respect to HCN2. The strongest effect was found for 4G. Accordingly, the activation kinetics were accelerated by all constructs, again with the strongest effect in 4G. The simulations reveal that the average residue mobility of the CL and CNBD domains is increased in all constructs and that the junction between the S6 and A'-helix is turned into a flexible hinge, resulting in a destabilized gate in all constructs. Moreover, for 3G and 4G, there is a stronger downward displacement of the CL-CNBD than in HCN2 and the other constructs, resulting in an increased kink angle between S6 and A'-helix, which in turn loosens contacts between the S4-helix and the CL. This is suggested to promote a downward movement of the S4-helix, similar to the effect of hyperpolarization. In addition, exclusively in 4G, the selectivity filter in the upper pore region and parts of the S4-helix are destabilized. The results provide new insights into the intricate activation of HCN2 channels.

Functional and structural characterization of interactions between opposite subunits in HCN pacemaker channels.

Hyperpolarization-activated and cyclic nucleotide (HCN) modulated channels are tetrameric cation channels. In each of the four subunits, the intracellular cyclic nucleotide-binding domain (CNBD) is coupled to the transmembrane domain via a helical structure, the C-linker. High-resolution channel structures suggest that the C-linker enables functionally relevant interactions with the opposite subunit, which might be critical for coupling the conformational changes in the CNBD to the channel pore. We combined mutagenesis, patch-clamp technique, confocal patch-clamp fluorometry, and molecular dynamics (MD) simulations to show that residue K464 of the C-linker is relevant for stabilizing the closed state of the mHCN2 channel by forming interactions with the opposite subunit. MD simulations revealed that in the K464E channel, a rotation of the intracellular domain relative to the channel pore is induced, which is similar to the cAMP-induced rotation, weakening the autoinhibitory effect of the unoccupied CL-CNBD region. We suggest that this CL-CNBD rotation is considerably involved in activation-induced affinity increase but only indirectly involved in gate modulation. The adopted poses shown herein are in excellent agreement with previous structural results.

The cAMP effector PKA mediates Moody GPCR signaling in Drosophila blood-brain barrier formation and maturation.

The blood-brain barrier (BBB) of Drosophila comprises a thin epithelial layer of subperineural glia (SPG), which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions (SJs). Previously, we identified a novel Gi/Go protein-coupled receptor (GPCR), Moody, as a key factor in BBB formation at the embryonic stage. However, the molecular and cellular mechanisms of Moody signaling in BBB formation and maturation remain unclear. Here, we identify cAMP-dependent protein kinase A (PKA) as a crucial antagonistic Moody effector that is required for the formation, as well as for the continued SPG growth and BBB maintenance in the larva and adult stage. We show that PKA is enriched at the basal side of the SPG cell and that this polarized activity of the Moody/PKA pathway finely tunes the enormous cell growth and BBB integrity. Moody/PKA signaling precisely regulates the actomyosin contractility, vesicle trafficking, and the proper SJ organization in a highly coordinated spatiotemporal manner. These effects are mediated in part by PKA's molecular targets MLCK and Rho1. Moreover, 3D reconstruction of SJ ultrastructure demonstrates that the continuity of individual SJ segments, and not their total length, is crucial for generating a proper paracellular seal. Based on these findings, we propose that polarized Moody/PKA signaling plays a central role in controlling the cell growth and maintaining BBB integrity during the continuous morphogenesis of the SPG secondary epithelium, which is critical to maintain tissue size and brain homeostasis during organogenesis.

Thermodynamic profile of mutual subunit control in a heteromeric receptor.

Cyclic nucleotide-gated (CNG) ion channels of olfactory neurons are tetrameric membrane receptors that are composed of two A2 subunits, one A4 subunit, and one B1b subunit. Each subunit carries a cyclic nucleotide-binding domain in the carboxyl terminus, and the channels are activated by the binding of cyclic nucleotides. The mechanism of cooperative channel activation is still elusive. Using a complete set of engineered concatenated olfactory CNG channels, with all combinations of disabled binding sites and fit analyses with systems of allosteric models, the thermodynamics of microscopic cooperativity for ligand binding was subunit- and state-specifically quantified. We show, for the closed channel, that preoccupation of each of the single subunits increases the affinity of each other subunit with a Gibbs free energy (ΔΔG) of ∼-3.5 to ∼-5.5 kJ ⋅ mol-1, depending on the subunit type, with the only exception that a preoccupied opposite A2 subunit has no effect on the other A2 subunit. Preoccupation of two neighbor subunits of a given subunit causes the maximum affinity increase with ΔΔG of ∼-9.6 to ∼-9.9 kJ ⋅ mol-1 Surprisingly, triple preoccupation leads to fewer negative ΔΔG values for a given subunit as compared to double preoccupation. Channel opening increases the affinity of all subunits. The equilibrium constants of closed-open isomerizations systematically increase with progressive liganding. This work demonstrates, on the example of the heterotetrameric olfactory CNG channel, a strategy to derive detailed insights into the specific mutual control of the individual subunits in a multisubunit membrane receptor.

Allosteric signaling in C-linker and cyclic nucleotide-binding domain of HCN2 channels.

Opening of hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels is controlled by membrane hyperpolarization and binding of cyclic nucleotides to the tetrameric cyclic nucleotide-binding domain (CNBD), attached to the C-linker (CL) disk. Confocal patch-clamp fluorometry revealed pronounced cooperativity of ligand binding among protomers. However, by which pathways allosteric signal transmission occurs remained elusive. Here, we investigate how changes in the structural dynamics of the CL-CNBD of mouse HCN2 upon cAMP binding relate to inter- and intrasubunit signal transmission. Applying a rigidity-theory-based approach, we identify two intersubunit and one intrasubunit pathways that differ in allosteric coupling strength between cAMP-binding sites or toward the CL. These predictions agree with results from electrophysiological and patch-clamp fluorometry experiments. Our results map out distinct routes within the CL-CNBD that modulate different cAMP-binding responses in HCN2 channels. They signify that functionally relevant submodules may exist within and across structurally discernable subunits in HCN channels.

TREM2 activation on microglia promotes myelin debris clearance and remyelination in a model of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS) triggered by autoimmune mechanisms. Microglia are critical for the clearance of myelin debris in areas of demyelination, a key step to allow remyelination. TREM2 is expressed by microglia and promotes microglial survival, proliferation, and phagocytic activity. Herein we demonstrate that TREM2 was highly expressed on myelin-laden phagocytes in active demyelinating lesions in the CNS of subjects with MS. In gene expression studies, macrophages from subjects with TREM2 genetic deficiency displayed a defect in phagocytic pathways. Treatment with a new TREM2 agonistic antibody promoted the clearance of myelin debris in the cuprizone model of CNS demyelination. Effects included enhancement of myelin uptake and degradation, resulting in accelerated myelin debris removal by microglia. Most importantly, antibody-dependent TREM2 activation on microglia increased density of oligodendrocyte precursors in areas of demyelination, as well as the formation of mature oligodendrocytes thus enhancing remyelination and axonal integrity. These results are relevant as they propose TREM2 on microglia as a potential new target to promote remyelination.

Shifting paradigms: The central role of microglia in Alzheimer's disease.

Recent human genetic studies have challenged long standing hypotheses about the chain of events in Alzheimer's disease (AD), as the identification of genetic risk factors in microglial genes supports a causative role for microglia in the disease. Parallel transcriptome and histology studies at the single-cell level revealed a rich palette of microglial states affected by disease status and genetic risk factors. Taken together, those findings support microglia dysfunction as a central mechanism in AD etiology and thus the therapeutic potential of modulating microglial activity for AD treatment. Here we review how human genetic studies discovered microglial AD risk genes, such as TREM2, CD33, MS4A and APOE, and how experimental studies are beginning to decipher the cellular functions of some of these genes. Our review also focuses on recent transcriptomic studies of human microglia from postmortem tissue to critically assess areas of similarity and dissimilarity between human and mouse models currently in use in order to better understand the biology of innate immunity in AD.

Anti-human TREM2 induces microglia proliferation and reduces pathology in an Alzheimer's disease model.

TREM2 is a receptor for lipids expressed in microglia. The R47H variant of human TREM2 impairs ligand binding and increases Alzheimer's disease (AD) risk. In mouse models of amyloid ß (Aß) accumulation, defective TREM2 function affects microglial response to Aß plaques, exacerbating tissue damage, whereas TREM2 overexpression attenuates pathology. Thus, AD may benefit from TREM2 activation. Here, we examined the impact of an anti-human TREM2 agonistic mAb, AL002c, in a mouse AD model expressing either the common variant (CV) or the R47H variant of TREM2. Single-cell RNA-seq of microglia after acute systemic administration of AL002c showed induction of proliferation in both CV- and R47H-transgenic mice. Prolonged administration of AL002c reduced filamentous plaques and neurite dystrophy, impacted behavior, and tempered microglial inflammatory response. We further showed that a variant of AL002c is safe and well tolerated in a first-in-human phase I clinical trial and engages TREM2 based on cerebrospinal fluid biomarkers. We conclude that AL002 is a promising candidate for AD therapy.

Relating ligand binding to activation gating in P2X2 receptors using a novel fluorescent ATP derivative.

Ionotropic purinergic receptors (P2X receptors) are non-specific cation channels that are activated by the binding of ATP at their extracellular side. P2X receptors contribute to multiple functions, including the generation of pain, inflammation, or synaptic transmission. The channels are trimers and structural information on several of their isoforms is available. In contrast, the cooperation of the subunits in the activation process is poorly understood. We synthesized a novel fluorescent ATP derivative, 2-[DY-547P1]-AET-ATP (fATP) to unravel the complex activation process in P2X2 and mutated P2X2 H319K channels with enhanced apparent affinity by characterizing the relation between ligand binding and activation gating. fATP is a full agonist with respect to ATP that reports the degree of binding by bright fluorescence. For quantifying the binding, a fast automated algorithm was employed on human embryonic kidney cell culture images. The concentrations of half maximum occupancy and activation as well as the respective Hill coefficients were determined. All Hill coefficients exceeded unity, even at an occupancy <10%, suggesting cooperativity of the binding even for the first and second binding step. fATP shows promise for continuative functional studies on other purinergic receptors and, beyond, any other ATP-binding proteins.

N6-modified cAMP derivatives that activate protein kinase A also act as full agonists of murine HCN2 channels.

cAMP acts as a second messenger in many cellular processes. Three protein types mainly mediate cAMP-induced effects: PKA, exchange protein directly activated by cAMP (Epac), and cyclic nucleotide-modulated channels (cyclic nucleotide-gated or hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels). Discrimination among these cAMP signaling pathways requires specific targeting of only one protein. Previously, cAMP modifications at position N6 of the adenine ring (PKA) and position 2'-OH of the ribose (Epac) have been used to produce target-selective compounds. However, cyclic nucleotide-modulated ion channels were usually outside of the scope of these previous studies. These channels are widely distributed, so possible channel cross-activation by PKA- or Epac-selective agonists warrants serious consideration. Here we demonstrate the agonistic effects of three PKA-selective cAMP derivatives, N6-phenyladenosine-3',5'-cyclic monophosphate (N6-Phe-cAMP), N6-benzyladenosine-3',5'-cyclic monophosphate (N6-Bn-cAMP), and N6-benzoyl-adenosine-3',5'-cyclic monophosphate (N6-Bnz-cAMP), on murine HCN2 pacemaker channels. Electrophysiological characterization in Xenopus oocytes revealed that these derivatives differ in apparent affinities depending on the modification type but that their efficacy and effects on HCN2 activation kinetics are similar to those of cAMP. Docking experiments suggested a pivotal role of Arg-635 at the entrance of the binding pocket in HCN2, either causing stabilizing cation-π interactions with the aromatic ring in N6-Phe-cAMP or N6-Bn-cAMP or a steric clash with the aromatic ring in N6-Bnz-cAMP. A reduced apparent affinity of N6-Phe-cAMP toward the variants R635A and R635E strengthened that notion. We conclude that some PKA activators also effectively activate HCN2 channels. Hence, when studying PKA-mediated cAMP signaling with cAMP derivatives in a native environment, activation of HCN channels should be considered.

Stepwise activation of a class C GPCR begins with millisecond dimer rearrangement.

G protein-coupled receptors (GPCRs) are key biological switches that transmit both internal and external stimuli into the cell interior. Among the GPCRs, the "light receptor" rhodopsin has been shown to activate with a rearrangement of the transmembrane (TM) helix bundle within ∼1 ms, while all other receptors are thought to become activated within ∼50 ms to seconds at saturating concentrations. Here, we investigate synchronous stimulation of a dimeric GPCR, the metabotropic glutamate receptor type 1 (mGluR1), by two entirely different methods: (i) UV light-triggered uncaging of glutamate in intact cells or (ii) piezo-driven solution exchange in outside-out patches. Submillisecond FRET recordings between labels at intracellular receptor sites were used to record conformational changes in the mGluR1. At millimolar ligand concentrations, the initial rearrangement between the mGluR1 subunits occurs at a speed of τ1 ∼ 1-2 ms and requires the occupancy of both binding sites in the mGluR1 dimer. These rapid changes were followed by significantly slower conformational changes in the TM domain (τ2 ∼ 20 ms). Receptor deactivation occurred with time constants of ∼40 and ∼900 ms for the inter- and intrasubunit conformational changes, respectively. Together, these data show that, at high glutamate concentrations, the initial intersubunit activation of mGluR1 proceeds with millisecond speed, that there is loose coupling between this initial step and activation of the TM domain, and that activation and deactivation follow a cyclic pathway, including-in addition to the inactive and active states-at least two metastable intermediate states.

All four subunits of HCN2 channels contribute to the activation gating in an additive but intricate manner.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetramers that elicit electrical rhythmicity in specialized brain neurons and cardiomyocytes. The channels are dually activated by voltage and binding of cyclic adenosine monophosphate (cAMP) to their four cyclic nucleotide-binding domains (CNBDs). Here we analyze the effects of cAMP binding to different concatemers of HCN2 channel subunits, each having a defined number of functional CNBDs. We show that each liganded CNBD promotes channel activation in an additive manner and that, in the special case of two functional CNBDs, functionality does not depend on the arrangement of the subunits. Correspondingly, the reverse process of deactivation is slowed by progressive liganding, but only if four and three ligands as well as two ligands in trans position (opposite to each other) are bound. In contrast, two ligands bound in cis positions (adjacent to each other) and a single bound ligand do not affect channel deactivation. These results support an activation mechanism in which each single liganded CNBD causes a turning momentum on the tetrameric ring-like structure formed by all four CNBDs and that at least two liganded subunits in trans positions are required to maintain activation.

Dynamic analysis of the mesenchymal-epithelial transition of blood-brain barrier forming glia in Drosophila.

During development, many epithelia are formed by a mesenchymal-epithelial transition (MET). Here, we examine the major stages and underlying mechanisms of MET during blood-brain barrier formation in Drosophila We show that contact with the basal lamina is essential for the growth of the barrier-forming subperineurial glia (SPG). Septate junctions (SJs), which provide insulation of the paracellular space, are not required for MET, but are necessary for the establishment of polarized SPG membrane compartments. In vivo time-lapse imaging reveals that the Moody GPCR signaling pathway regulates SPG cell growth and shape, with different levels of signaling causing distinct phenotypes. Timely, well-coordinated SPG growth is essential for the uniform insertion of SJs and thus the insulating function of the barrier. To our knowledge, this is the first dynamic in vivo analysis of all stages in the formation of a secondary epithelium, and of the key role trimeric G protein signaling plays in this important morphogenetic process.

Differential adhesion determines the organization of synaptic fascicles in the Drosophila visual system.

BACKGROUND: Neuronal circuits in worms, flies, and mammals are organized so as to minimize wiring length for a functional number of synaptic connections, a phenomenon called wiring optimization. However, the molecular mechanisms that establish optimal wiring during development are unknown. We addressed this question by studying the role of N-cadherin in the development of optimally wired neurite fascicles in the peripheral visual system of Drosophila. RESULTS: Photoreceptor axons surround the dendrites of their postsynaptic targets, called lamina cells, within a concentric fascicle called a cartridge. N-cadherin is expressed at higher levels in lamina cells than in photoreceptors, and all genetic manipulations that invert these relative differences displace lamina cells to the periphery and relocate photoreceptor axon terminals into the center. CONCLUSIONS: Differential expression of a single cadherin is both necessary and sufficient to determine cartridge structure because it positions the most-adhesive elements that make the most synapses at the core and the less-adhesive elements that make fewer synapses at the periphery. These results suggest a general model by which differential adhesion can be utilized to determine the relative positions of axons and dendrites to establish optimal wiring.

A network of cadherin-mediated interactions polarizes growth cones to determine targeting specificity.

Neuronal growth cones select synaptic partners through interactions with multiple cell surfaces in their environment. Many of these interactions are adhesive, yet it is unclear how growth cones integrate adhesive cues to direct their movements. Here, we examine the mechanisms that enable photoreceptors in the Drosophila visual system to choose synaptic partners. We demonstrate that the classical cadherin, N-cadherin, and an atypical cadherin, Flamingo, act redundantly to instruct the targeting choices made by every photoreceptor axon. These molecules gradually bias the spatial distribution of growth cone filopodia, polarizing each growth cone toward its future synaptic target before direct contact with the target occurs. We demonstrate that these molecules are localized to distinct subcellular domains and create a network of adhesive interactions distributed across many growth cones. Because this network comprises multiple redundant interactions, a complex wiring diagram can be constructed with extraordinary fidelity, suggesting a general principle.

Axon trapping: constructing the visual system one layer at a time.

The molecular mechanisms that instruct the formation of synaptic layers are only incompletely understood. In this issue of Neuron, Timofeev et al. (2012) describe an instructive role for the guidance molecule Netrin and its receptor Frazzled in mediating layer-specific targeting of one photoreceptor cell type in the Drosophila visual system.

The cytoskeletal regulator Genghis khan is required for columnar target specificity in the Drosophila visual system.

A defining characteristic of neuronal cell type is the growth of axons and dendrites into specific layers and columns of the brain. Although differences in cell surface receptors and adhesion molecules are known to cause differences in synaptic specificity, differences in downstream signaling mechanisms that determine cell type-appropriate targeting patterns are unknown. Using a forward genetic screen in Drosophila, we identify the GTPase effector Genghis khan (Gek) as playing a crucial role in the ability of a subset of photoreceptor (R cell) axons to innervate appropriate target columns. In particular, single-cell mosaic analyses demonstrate that R cell growth cones lacking Gek function grow to the appropriate ganglion, but frequently fail to innervate the correct target column. Further studies reveal that R cell axons lacking the activity of the small GTPase Cdc42 display similar defects, providing evidence that these proteins regulate a common set of processes. Gek is expressed in all R cells, and a detailed structure-function analysis reveals a set of regulatory domains with activities that restrict Gek function to the growth cone. Although Gek does not normally regulate layer-specific targeting, ectopic expression of Gek is sufficient to alter the targeting choices made by another R cell type, the targeting of which is normally Gek independent. Thus, specific regulation of cytoskeletal responses to targeting cues is necessary for cell type-appropriate synaptic specificity.